Datasets:

dheerajpai
/

uspatents

Dataset card Data Studio Files Files and versions Community

Split (1)

train · 1.94M rows

text stringlengths 0 1.67M
Tc and re labeler radioactive glycosylated octreotide derivatives
Improved sst-receptor binding peptidic ligands for diagnostic and therapeutic applications in nuclear medicine are provided. The improved ligands contain either natural or unnatural amino acids or peptidomimetic structures that are modified at either the N-terminal or the C-terminal end or at both termini, a carbohydrate unit and a chelator or prosthetic group to provide a complexation of a radioisotope binding or holding the radioisotope. The sst- or SSTR-receptor binding peptidic ligands may also contain one or more multifunctional linker units optionally coupling the peptide, and/or the sugar moiety and/or the chelator and/or the prosthetic group. Upon administering the ligand to a mammal through the blood system the ligand provides improved availability, clearance kinetics, sst-receptor targeting and internalization over the non-carbohydrated ligands.
1. A somatostatin receptor (sst) binding peptidic ligand comprising natural or unnatural amino acids or a peptidomimetic structure, at least one carbohydrate and at least one chelating group allowing mono- or multidentate complexation of a radioisotope selected from Tc and Re. 2. The ligand of claim 1 wherein the radioisotope is an isotope of Tc. 3. The ligand of claim 1 wherein the chelating group is bound to the N-terminal of the peptide and the carbohydrate is bound to the chelating group. 4. The ligand of claim 1 wherein the carbohydrate is bound to the N-terminal of the peptide and the chelating group is bound to the carbohydrate. 5. A ligand of claim 1 wherein the carbohydrate is a sugar. 6. A ligand of claim 5 wherein the sugar is a mono- or polysaccharide. 7. A ligand of claim 1 further comprising at least one multifunctional linker unit. 8. A ligand of claim 7 wherein the sugar is coupled to the receptor binding structure, to the multifunctional linker unit or to the chelator via a glycosidic bond 9. A ligand of claim 7 wherein the sugar is a coupling product between an amino group in the receptor binding structure, the multifunctional linker unit or the chelator and an aldose via Amadori Reaction. 10. A ligand of claim 7 wherein the sugar selected from the group consisting of glucose, maltose and maltotriose. 11. A ligand of claim 7 wherein the ligand is glycosylated at the N-terminal. 12. The ligand of claim 7 wherein the radioisotope is selected form the group consisting of isotopes Tc and Re. 13. A ligand of claim 7 wherein the multifunctional linker unit is combined with the N terminus of the peptide, and the carbohydrate and the chelating group are bound to the multifunctional linker unit. 14. A ligand of claim 7 wherein the multifunctional linker unit is bound to the N-terminal of the ligand, the chelating group is bound to the multifunctional linker unit and the carbohydrate is bound to the chelating group. 15. The ligand of claim 1 further comprising at least one multifunctional linker unit and at least two sst-receptor binding structures peptide groups. 16. The ligand of claim 14 wherein the two peptide groups are bound to the multifunctional linker unit and both the chelating group and the carbohydrate are bound to the multifunctional linker. 17. The ligand of claim 14 wherein there are two carbohydrate units, each bound to the N-terminal of one of the peptide units, each of said carbohydrate units bound to a multifunctional linker unit and the chelating group is bound to the multifunctional linker unit. 18. The ligand of claim 14 wherein there are two multifunctional linker units, each bound to the N-terminal of the peptide, one chelating group linking the multifunctional linker units and two carbohydrate groups, each bound to one of the multifunctional linker units. 19. The ligand of claim 14 wherein the radioisotope is selected from the group consisting of isotopes of Tc and Re. 20. A ligand of claim 14 wherein the carbohydrate is the sugar is a coupling product between an amino group in the receptor binding structure, the multifunctional linker unit or the chelator and an aldose via Amadori Reaction. 21. A ligand of claim 14 wherein the sugar is coupled to the receptor binding structure, to the multifunctional linker unit or to the chelator via a glycosidic bond. 22. A pharmaceutical composition comprising a composition of claim 1 and a pharmaceutically acceptable carrier. 23. A pharmaceutical composition comprising a composition of claim 7 and a pharmaceutically acceptable carrier. 24. A pharmaceutical composition comprising a composition of claim 14 and a pharmaceutically acceptable carrier. 25. A method of imaging selected from the group consisting of sst-imaging which comprises administering to a patient a composition of claim 22. 26. A method of imaging selected from the group consisting of sst-imaging which comprises administering to a patient a composition of claim 23. 27. A method of imaging selected from the group consisting of sst-maging which comprises administering to a patient a composition of claim 24. 28. The compound [99mTc]Gluc-K0(H)TOC. 29. The compound [99mTc]Mtr-K0(H)TOC.



Communication methods and apparatus
This invention generally relates to methods and apparatus for radio frequency communications over mains power cabling and other wiring, in particular communications at microwave frequencies. Embodiments of the invention are particularly suitable for ultra wideband (UWB) communications. A method of communicating a microwave signal having a frequency of 1 GHz or higher using a cable comprising at least one conductor, the method comprising: positioning a transmit antenna at a transmission point on said cable at a distance from said cable to couple said microwave signal into said cable; driving said transmit antenna with said microwave signal to induce onto said cable a propagating wave to propagate along said cable; positioning a receive antenna to receive an electromagnetic signal generated by said propagating wave; receiving a version of said microwave signal using said receive antenna
1. A method of communicating a microwave signal having a frequency of 1 GHz or higher using a cable comprising at least one conductor, the method comprising: positioning a transmit antenna at a transmission point on said cable at a distance from said cable to couple said microwave signal into said cable; driving said transmit antenna with said microwave signal to induce onto said cable a propagating wave to propagate along said cable; positioning a receive antenna to receive an electromagnetic signal generated by said propagating wave; receiving a version of said microwave signal using said receive antenna. 2. A method as claimed in claim 1 wherein said distance is equal to or less than an average free space wavelength of said microwave signal. 3. A method as claimed in claim 1 wherein said distance is such that a capacitative impedance between said antenna and said cable is less than the impedance of free space. 4. A method as claimed in claim 1, 2 or 3 wherein said propagating wave comprises a single-ended signal. 5. A method as claimed in any preceding claim wherein one of said transmit antenna and said receive antenna comprises a monopole antenna. 6. A method as claimed in any preceding claim wherein one of said transmit antenna and said receive antenna comprises a magnetic loop antenna. 7. A method as claimed in any one of claims 1 to 6 wherein at least one of said transmit antenna and said receive antenna is substantially resistively isolated from said at least one conductor. 8. A method as claimed in any preceding claim wherein said driving comprises driving said transmit antenna with respect to a ground. 9. A method as claimed in claim 8 wherein said ground comprises a connection to free space. 10. A method as claimed in claim 8 wherein said ground comprises a local ground having a capacitative coupling to a ground for said propagating wave. 11. A method as claimed in claim 10 wherein said ground comprises a portion of power wiring. 12. A method as claimed in any preceding claim wherein said driving comprises inducing said propagating wave to propagate preferentially in one direction along said cable. 13. A method as claimed in any preceding claim wherein said cable has a plurality of substantially electrically separate conductors, and wherein said driving comprises inducing said propagating wave onto said plurality of conductors. 14. A method as claimed in any preceding claim wherein said cable comprises an alternating current power cable. 15. A method of transmitting a radio frequency signal using a cable comprising at least one conductor, the method comprising: positioning a transmit antenna at a transmission point on said cable at a distance from said cable to couple said microwave signal into said cable; driving said transmit antenna with said signal to induce onto said cable a propagating wave to propagate along said cable. 16. A method of transmitting a radio frequency signal using a cable comprising a bundle of substantially electrically separate conductors, the method comprising: coupling said radio frequency signal to said conductors to drive said bundle of conductors with said rf signal such that each said conductor carries substantially the same signal; and driving said bundle of conductors with said rf signal to generate a propagating rf signal associated with said cable. 17. A method as claimed in claim 16 wherein said driving comprises driving with respect to a ground, and wherein said ground is coupled to a ground for said propagating rf signal. 18. A radio frequency (rf) signal transmission system for transmitting a signal of at least 1 GHz guided by an electrical conductor, the system comprising: an electrical conductor for guiding said signal; a transmit antenna positioned at a distance from said conductor to couple said microwave signal into said cable, said antenna being substantially resistively isolated from said conductor; and an input, coupled to said transmit antenna, to receive said rf signal and to provide an rf drive corresponding to said signal to said antenna to launch a propagating wave corresponding to said signal on said electrical conductor. 19. A system as claimed in claim 18 wherein said distance is less than a wavelength of said rf signal from said electrical conductor. 20. A system as claimed in claim 18 wherein said distance is such that a capacitative impedance between said antenna and said cable is less than the impedance of free space. 21. A signal transmission system as claimed in claim 18, 19 or 20 wherein said propagating wave comprises a single-ended voltage. 22. A signal transmission system as claimed in claim 18, 19, 20 or 21 wherein said rf drive is referenced to a reference level connection. 23. A signal transmission system as claimed in claim 22 wherein said reference level connection comprises a ground for said transmission system coupled to a ground for said propagating wave. 24. A signal transmission system as claimed in claim 23 wherein said coupling between said transmission system ground and said ground for said propagating wave has an impedance substantially equal to or less than the impedance of free space. 25. A signal transmission system as claimed in claim 22, 23 or 24 wherein said reference level connection comprises a portion of power wiring for said transmission system which, for said rf signal, is substantially isolated, from said electrical conductor. 26. A signal transmission system as claimed in claim 22, 23 or 24 wherein said reference level connection comprises a connection to free space. 27. A signal transmission system as claimed in claim 26 wherein said connection to free space comprises a second antenna and a second antenna driver to drive said second antenna with an inverted version of said rf signal. 28. A signal transmission system as claimed in any one of claims 18 to 27 wherein said transmit antenna comprises a pair of transmit antennas, the system further comprising a transmit antenna driver configured to drive said pair of transmit antennas such that a signal transmitted from one of said pair of antennas has a phase delay with respect to a signal transmitted from the other of said pair of antennas. 29. A signal transmission system as claimed in any one of claims 18 to 28 wherein said transmit antenna comprises a monopole antenna. 30. A signal transmission system as claimed in any one of claims 18 to 28 wherein said transmit antenna comprises a magnetic loop antenna. 31. A signal transmission system as claimed in any one of claims 18 to 28 wherein said transmit antenna comprises a broadband antenna. 32. A signal transmission system as claimed in any one of claims 18 to 28 wherein said transmit antenna comprises a helical conductor. 33. A signal transmission system as claimed in claim 32 wherein said helical conductor encircles said electrical conductor. 34. A signal transmission system as claimed in any one of claims 29 to 33 wherein said transmit antenna is positioned such that a capacitative impedance between said transmit antenna and said conductor is less than the impedance of free space. 35. A signal transmission system as claimed in any one of claims 18 to 34 wherein said conductor comprises one conductor of an electrical power cable or a conductor configured for connection to an electrical power cable. 36. An rf signal transmission system for transmitting an rf signal guided by one or more electrical conductors of an electrical cable, the rf signal having a frequency of 1 GHz or greater, the system comprising: a signal transducer to couple said rf signal into said electrical cable; an input, coupled to said transducer to receive said rf signal and to provide an rf drive corresponding to said signal to said transducer to launch a propagating wave corresponding to said signal on said one or more conductors; and means for making an electrical connection at a frequency of said rf signal to an effective ground for said propagating wave, said effective ground having an indirect connection to earth for said propagating wave, said indirect connection having an impedance at an average frequency of said signal of substantially equal to or less than the impedance of free space. 37. A system as claimed in claim 36 wherein said effective ground comprises a local ground plane for said transmission system having a capacitative impedance to earth for said propagating wave. 38. A system as claimed in claim 36 wherein said effective ground comprises a ground antenna. 39. A system as claimed in claim 36, 37 or 38 wherein said power cable comprises a bundle of electrically substantially separate conductors, and wherein said transducer comprises a common mode driver for said bundle of conductors. 40. A system as claimed in claim 36, 37, 38 or 39 wherein said signal transducer comprises a transmit antenna configured to be positioned less than a wavelength of said rf signal from said electrical power cable and substantially resistively isolated from said one or more conductors. 41. A system as claimed in claim 36, 37, 38 or 39 wherein said signal transducer comprises a transmit antenna configured to be positioned at a distance from said cable such that a capacitative impedance between said antenna and said cable is substantially equal to or less than the impedance of free space. 42. An rf signal reception system for receiving a signal guided by one or more electrical conductors of an electrical cable, the signal having a frequency of greater than 1 GHz, the system comprising: a receive antenna for receiving said guided signal; and means for making an electrical connection at a frequency of said rf signal to an effective ground for said guided signal, said effective ground having an indirect connection to earth for said propagating wave, said indirect connection having an impedance at an average frequency of said signal of substantially equal to or less than the impedance of free space. 43. A system or method as claimed in any preceding claim wherein said signal comprises a UWB signal. 44. A method of distributing an ultrawideband (UWB) communications signal through a building, the method comprising: generating a UWB signal; and coupling the UWB signal to at least one electrical conductor of a mains power supply circuit of the building to distribute the UWB signal. 45. A method as any claim 44 further comprising: generating a plurality of UWB signals at a plurality of UWB transmitters; and coupling said plurality of UWB signals onto said electrical conductor at a plurality of different points within said building. 46. A method as claim 45 further comprising establishing a common timing between at least a subset of said UWB signals. 47. A method as claim 44,45 or 46 further comprising receiving said UWB signal at a plurality of points within said building. 48. A method as claimed in any one of claims 44 to 47 wherein said coupling comprises capacitative coupling. 49. A method as claimed in any one of claims 44 to 48 comprising coupling said UWB signal to two electrical conductors of said mains power supply circuit. 50. A method as claimed in any one of claims 44 to 49 wherein said coupling is performed at a central distribution point of said mains power supply circuit of said building. 51. A data communications network configured to use the method of any preceding method claim. 52. Apparatus for distributing an ultrawideband (UWB) communications signal through a building, the apparatus comprising: means for generating a UWB signal; and means for coupling the UWB signal to at least one electrical conductor of a mains power supply circuit of the building to distribute the UWB signal. 53. A consumer electronics device incorporating the apparatus of claim 52. 54. A consumer electronics device as claimed in claim 53 wherein the consumer electronics device is mains powered.



Method and device for compressing square bales for stalk material
In a method and a device for compressing square large bales for stalk material on large baling presses by using twines that engage across longitudinal sides and end faces of bales and are tied together, wherein compressed large bales can be formed by a number of adjacently positioned, individually compressed and tied small bales, a first tying unit and a second tying unit at a pressing channel of a large baling press are provided, wherein the first and second tying units have knotters. The first tying unit for tying individual small bales can be activated. Alternatively, the first and second tying units can be activated individually together for simultaneously tying several small bales to form a large bale, wherein the first and second tying units interact with the same knotters.
1.-9. (canceled) 10. A method for compressing square large bales for stalk material on large baling presses by using twines that engage across longitudinal sides and end faces of bales and are tied together, wherein compressed large bales can be formed by a number of adjacently positioned, individually compressed and tied small bales; the method comprising the steps of: providing a first tying unit and a second tying unit at a pressing channel of a large baling press, wherein the first and second tying units have knotters; alternatively activating the first tying unit for tying individual small bales and activating the first and second tying units individually together for simultaneously tying several small bales to form a large bale, wherein the first and second tying units interact with the same knotters. 11. The method according to claim 10, wherein the first and second tying units are single knotting devices or double knotting devices. 12. A device for performing the method according to claim 10, the device comprising: a first tying unit and a second tying unit, wherein the tying units are configured as single knotting devices having first and second knotters that are switchable on and off and are provided with twines that permanently contact bales to be formed; wherein the first and second tying units have first and second needles; wherein the first knotters serve for intermediate tying of small bales and are separately and independently activatable and interact with the first needles pivotable into engagement with the first knotters; wherein the second needles remain in a rest position while the first needles carry out intermediate tying of the small bales. 13. A device according to claim 12, further comprising: an outer shaft having an inner toothing and an inner shaft having an outer toothing, wherein the inner shaft is arranged inside the outer shaft and the outer toothing meshes with the inner toothing; a length measuring wheel contacting the bales to be formed; an electromagnetic control device connected to the length measuring wheel; a pressure cylinder engaging the inner shaft and acted on by the electromagnetic control device; wherein the first and the second knotters together serve for intermediate tying and main tying of the bales and are arranged on the outer shaft; a coupling member arranged between the inner shaft and the outer shaft and configured to couple the inner and outer shafts to one another or to separate the inner and outer shafts from one another; wherein a relative movement between the inner shaft and the outer shaft switches the first knotters on and off for intermediate tying; wherein, for controlling the inner shaft, the length measuring wheel positions the pressure cylinder via the electromagnetic control device. 14. The device according to claim 13, wherein the coupling is a star coupling, a friction coupling, a magnet coupling, or a wheel coupling. 15. The device according to claim 12, wherein the tying units are double knotting devices, wherein the first and second knotters are separately activatable and are without twines permanently contacting the bales to be formed, wherein the first and second needles provided with twines have a common support correlated with the first and second knotters, wherein the first knotters and the first needles are used for tying small bales and wherein the first and the second knotters interact simultaneously together with the first needles for intermediate tying and the second needles for forming large bales. 16. The device according to claim 12, further comprising a first support and a second support, wherein the first needles for intermediate tying are arranged on the first support and the second needles for a main tying action are arranged on the second support, wherein the first and second supports are arranged adjacent and axis-parallel to one another and are pivotable, wherein the second support advances the second needles separately to the second knotters and the first and second supports, when locked hydraulically or mechanically with one another, supply the first and second needles simultaneously to the first and second knotters for the intermediate and main tying actions. 17. The device according to claim 16, wherein the first and second supports are pipes or profiled rods. 18. The device according to claim 16, wherein the first needles or the second needles are moved by a rotary movement of the first and second supports, respectively, to the first and second knotters and wherein the first support or the second support has through openings, recesses or steps for the second needles or the first needles, respectively, for an unimpeded movement of the second or first needles. 19. The device according to claim 12, further comprising a shaft, wherein the first and second needles are arranged together on the shaft adjacent to one another, wherein the first needles are drivable separately and independently relative to the second needles for intermediate tying of the small bales or driven simultaneously and together with the second needles for tying the large bales. 20. The device according to claim 12, comprising first and second needle supports for the first and second needles, respectively, wherein the first and second needle supports are pivotable by swings for interaction with the first and second knotters together or separately individually. 21. The device according to claim 12, comprising first and second needle supports for the first and second needles, respectively, wherein the first and second needle supports are pivotable and moveable by swings for interaction with the first and second knotters together or separately individually. 22. The device according to claim 12, comprising first and second needle supports for the first and second needles, respectively, wherein the first and second needle supports are moveable by swings for interaction with the first and second knotters together or separately individually.



Methods for analysis of spectral data and their applications
This invention pertains to chemometric methods for the analysis of chemical, biochemical, and biological data, for example, spectral data, for example, nuclear magnetic resonance (NMR) spectra, and their applications, including, e.g., classification, diagnosis, prognosis.
1. A method of classifying a sample, said method comprising the step of relating NMR spectral intensity at one or more predetermined diagnostic spectral windows for said sample with a predetermined condition. 2-182. (canceled) 183. A method of classifying a sample, said method comprising the step of relating NMR spectral intensity at one or more predetermined diagnostic spectral windows for said sample with a predetermined condition. 184. A method according to claim 183, wherein said sample is a sample from a subject and said predetermined condition is a predetermined condition of said subject. 185. A method according to claim 183, wherein said relating with a predetermined condition is relating with the presence or absence of a predetermined condition. 186. A method according to claim 184, wherein said relating with a predetermined condition is relating with the presence or absence of a predetermined condition. 187. A method according to claim 183, wherein said relating NMR spectral intensity is relating a modulation of NMR spectral intensity, relative to a control value. 188. A method according to claim 184, wherein said relating NMR spectral intensity is relating a modulation of NMR spectral intensity, relative to a control value. 189. A method according to claim 185, wherein said relating NMR spectral intensity is relating a modulation of NMR spectral intensity, relative to a control value. 190. A method according to claim 186, wherein said relating NMR spectral intensity is relating a modulation of NMR spectral intensity, relative to a control value. 191. A method according to claim 183, wherein said one or more predetermined diagnostic spectral windows is: a single predetermined diagnostic spectral window. 192. A method according to claim 183, wherein said one or more predetermined diagnostic spectral windows is: a plurality of predetermined diagnostic spectral windows. 193. A method according to claim 183, wherein said one or more predetermined diagnostic spectral windows is: a plurality of diagnostic spectral windows, and, said NMR spectral intensity at one or more predetermined diagnostic spectral windows is: a combination of a plurality of NMR spectral intensities, each of which is NMR spectral intensity for one of said plurality of predetermined diagnostic spectral windows. 194. A method according to claim 193, wherein said combination is a linear combination. 195. A method according to claim 183, wherein said one or more, predetermined diagnostic spectral windows are associated with one or more diagnostic species. 196. A method according to claim 183, wherein at least one of said one or more predetermined diagnostic spectral windows encompasses a chemical shift value for an NMR resonance of a diagnostic species. 197. A method according to claim 183, each of a plurality of said one or more predetermined diagnostic spectral windows encompasses a chemical shift value for an NMR resonance of a diagnostic species. 198. A method of classifying a sample, said method comprising the step of relating the amount of, or relative amount of one or more diagnostic species present in said sample with a predetermined condition. 199. A method according to claim 198, wherein said sample is a sample from a subject and said predetermined condition is a predetermined condition of said subject. 200. A method, according to claim 198, wherein said relating with a predetermined condition is relating with the presence or absence of a predetermined condition. 201. A method, according to claim 199, wherein said relating with a predetermined condition is relating with the presence or absence of a predetermined condition. 202. A method according to claim 198, wherein said relating the amount of, or relative amount of one or more diagnostic species is relating a modulation of the amount of, or relative amount of one or more diagnostic species. 203. A method according to claim 199, wherein said relating the amount of, or relative amount of one or more diagnostic species is relating a modulation of the amount of, or relative amount of one or more diagnostic species. 204. A method according to claim 200, wherein said relating the amount of, or relative amount of one or more diagnostic species is relating a modulation of the amount of, or relative amount of one or more diagnostic species. 205. A method according to claim 201, wherein said relating the amount of, or relative amount of one or more diagnostic species is relating a modulation, of the amount of, or relative amount of one or more diagnostic species. 206. A method according to claim 198, wherein said classification is performed on the basis of an amount, or a relative amount, of a single diagnostic species. 207. A method according to claim 198, wherein said classification is performed on the basis of an amount, or a relative amount, of a plurality of diagnostic species. 208. A method according to claim 198, wherein said classification is performed on the basis of a total amount, or a relative total amount, of a plurality of diagnostic species. 209. A method according to claim 198, wherein: said one or more diagnostic species is: a plurality of diagnostic specie; and, said amount of, or relative amount of one or more diagnostic species is: a combination of a plurality of amounts, or relative amounts, each of which is the amount of, or relative amount of one of said plurality of diagnostic species. 210. A method according to claim 209, wherein said combination is a linear combination. 211. A method of classifying a test sample, said method comprising the step of: using a predictive mathematical model; wherein said model is formed by applying a modelling method to modelling data; to classify said test sample. 212. A method according to claim 211, wherein said modelling data comprises a plurality of data sets for modelling samples of known class; and said classifying is classifying said test sample as being a member of one of said known classes. 213. A method according to claim 211, wherein said modelling data comprises at least one data set for each of a plurality of modelling samples; wherein said modelling samples define a class group consisting of a plurality of classes; wherein each of said modelling samples is of a known class selected from said class group; and said model is used with a data set for said test sample; and said classifying is classifying said test sample as being a member of one class selected from said class group. 214. A method according to claim 213, wherein said class group comprises classes associated with said predetermined condition. 215. A method according to claim 211, wherein said modelling method is a multivariate statistical analysis modelling method. 216. A method according to claim 211, wherein said modelling method is a multivariate statistical analysis modelling method which employs a pattern recognition method. 217. A method according to claim 211, wherein said modelling method is, or employs PCA, PLS, or PLS-DA. 218. A method according to claim 211, wherein said modelling method includes a step of data filtering, orthogonal data filtering, or OSC. 219. A method according to claim 211, wherein said modelling data comprise NMR spectral data. 220. A method according to claim 211, wherein said modelling data comprise both NMR spectral data and non-NMR spectral data. 221. A method according to claim 211, wherein said modelling data comprises at least one data set for each of a plurality of modelling samples. 222. A method according to claim 221, wherein each of said data sets comprises NMR spectral data. 223. A method according to claim 221, wherein each of said data sets comprises both NMR spectral data and non-NMR spectral data. 224. A method of classifying a subject comprising classifying a sample from said subject by a method according to claim 183. 225. A method of diagnosis of a predetermined condition of a subject comprising classifying a sample from said subject by a method according to claim 183. 226. A method of prognosis of a subject which employs a method according to claim 183. 227. A method of therapeutic monitoring of a subject undergoing therapy which employs a method according to claim 183. 228. A method of evaluating drug therapy and/or drug efficacy which employs a method according to claim 183. 229. A method of identifying a diagnostic species, or a combination of a plurality of diagnostic species, for a predetermined condition, said method comprising the steps of: (a) applying a multivariate statistical analysis method to experimental data; wherein said experimental data comprises at least one data comprising experimental parameters measured for each of a plurality of experimental samples; wherein said experimental samples define a class group consisting of a plurality of classes; wherein at least one of said plurality of classes is a class associated with said predetermined condition, e.g., a class associated with the presence of said predetermined condition; wherein at least one of said plurality of classes is a class not associated with said predetermined condition, e.g., a class associated with the absence of said predetermined condition; wherein each of said experimental samples is of known class selected from said class group; and: (b) identifying one or more critical experimental parameters; wherein each of said critical experimental parameters is statistically significantly different for classes of said class group, e.g., is statistically significant for discriminating between classes of said class group; and, (c) matching each of one or more of said one or more critical experimental parameters with said diagnostic species; or: (b) identifying a combination of a plurality of critical experimental parameters; wherein said combination of a plurality of critical experimental parameters is statistically significantly different for classes of said class group, e.g., is statistically significant for discriminating between classes of said class group; and, (c) matching each of one or more of said plurality of critical experimental parameters with said combination of a plurality of diagnostic species. 230. A method, according to claim 229, wherein: one or more of said critical experimental parameters is a spectral parameter, and said identifying and matching steps are: (b) identifying one or more critical experimental spectral parameters; and, (c) matching each of one or more of said one or more critical experimental spectral parameters with a spectral feature, e.g., a spectral peak; and matching one or more of said spectral peaks with said diagnostic species; or: (b) identifying a combination of a plurality of critical experimental spectral parameters; and, (c) matching each of a plurality of said plurality of critical experimental spectral parameters with a spectral feature, e.g., a spectral peak; and matching one or more of said spectral peaks with said combination of a plurality of diagnostic species. 231. A method according to claim 229, wherein said multivariate statistical analysis method is a multivariate statistical analysis method which employs a pattern recognition method. 232. A method according to claim 229, wherein said multivariate statistical analysis method is, or employs PCA, PLS or PLS-DA. 233. A method according to claim 229, wherein said multivariate statistical analysis method includes a step of data filtering, a step of orthogonal data filtering, or a step of OSC. 234. A method according to claim 229, wherein said experimental parameters comprise NMR spectral data. 235. A method according to claim 229, wherein said experimental parameters comprise both NMR spectral data and non-NMR spectral data. 236. A method according to claim 229, wherein said class group comprises classes associated with said predetermined condition. 237. A method according to claim 236, said method further comprising the additional step of: (d) confirming the identity of said diagnostic species. 238. A diagnostic species identified by a method according to claim 229. 239. A method of classification which employs or relies upon one or more diagnostic species identified by a method according to claim 229. 240. An assay for use in a method of classification, which assay relies upon one or more diagnostic species identified by a method according to claim 229. 241. A computer program, optionally embodied on a computer readable medium, comprising computer program means adapted to perform a method according to claim 183, when said program is run on a computer. 242. A system comprising: (a) a first component comprising a device for obtaining NMR spectral intensity data for a sample; and, (b) a second component comprising computer system or device, such as a computer or linked computers, operatively configured to implement a method according to claim 183, and operatively linked to said first component.
<SOH> BACKGROUND <EOH>Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Biosystems Biosystems can conveniently be viewed at several levels of bio-molecular organisation based on biochemistry, i.e., genetic and gene expression (genomic and transcriptomic), protein and signalling (proteomic) and metabolic control and regulation (metabonomic). There are also important cellular ionic regulation variations that relate to genetic, proteomic and metabolic activities, and systematic studies on these even at the cellular and sub-cellular level should also be investigated to complete the full description of the bio-molecular organisation of a bio-system. Significant progress has been made in developing methods to determine and quantify the biochemical processes occurring in living systems. Such methods are valuable in the diagnosis, prognosis and treatment of disease, the development of drugs, for improving therapeutic regimes for current drugs, and the like. Many diseases of the human or animal body (such as cancers, degenerative diseases, autoimmune diseases and the like) have an underlying basis in alterations in the expression of certain genes. The expressed gene products, proteins, mediate effects such as abnormal cell growth, cell death or inflammation. Some of these effects are caused directly by protein-protein interactions; other are caused by proteins acting on small molecules (e.g. “second messengers”) which trigger effects including further gene expression. Likewise, disease states caused by external agents such as viruses and bacteria provoke a multitude of complex responses in infected host. In a similar manner, the treatment of disease through the administration of drugs can result in a wide range of desired effects and unwanted side effects in a patient. In recent years, it has been appreciated that the reaction of human and animal subjects to disease and treatments for them can vary according to the genomic makeup of an individual. This has led to the development of the field of “pharmacogenomics.” A fuller understanding of how an individual's own genome reacts to a particular disease and/or drug treatment will allow the development of new therapies, as well as the refinement of existing ones. At the genetic level, methods for examining gene expression in response to these types of events are often referred to as “genomic methods,” and are concerned with the detection and quantification of the expression of an organism's genes, collectively referred to as its “genome,” usually by detecting and/or quantifying genetic molecules, such as DNA and RNA. Genomic studies often exploit proprietary “gene chips,” which are small disposable devices encoded with an array of genes that respond to extracted mRNAs produced by cells (see, for example, Klenk et al., 1997). Many genes can be placed on a chip array and patterns of gene expression, or changes therein, can be monitored rapidly, although at some considerable cost. However, the biological consequences of gene expression, or altered gene expression following perturbation, are extremely complex. This has led to the development of “proteomic methods” which are concerned with the semi-quantitative measurement of the production of cellular proteins of an organism, collectively referred to as its “proteome” (see, for example, Geisow, 1998). Proteomic measurements utilise a variety of technologies, but all involve a protein separation method, e.g., 2D gel-electrophoresis, allied to a chemical characterisation method, usually, some form of mass spectrometry. At present, genomic methods have a high associated operational cost and proteomic methods require investment in expensive capital cost equipment and are labour intensive, but both have the potential to be powerful tools for studying biological response. The choice of method is still uncertain since careful studies have sometimes shown a low correlation between the pattern of gene expression and the pattern of protein expression, probably due to sampling for the two technologies at inappropriate time points. See, e.g., Gygi et al., 1999. Even in combination, genomic and proteomic methods still do not provide the range of information needed for understanding integrated cellular function in a living system, since they do not take account of the dynamic metabolic status of the whole organism. For example, genomic and proteomic studies may implicate a particular gene or protein in a disease or a xenobiotic response because the level of expression is altered, but the change in gene or protein level may be transitory or may be counteracted downstream and as a result there may be no effect at the cellular and/or biochemical level. Conversely, sampling tissue for genomic and proteomic studies at inappropriate time points may result in a relevant gene or protein being overlooked. Gene-based prognosis has yet to become a clinical reality for any major prevalent disease, almost all of which have multi-gene modes of inheritance and significant environmental impact making it difficult to identify the gene panels responsible for susceptibility. While genomic and proteomic methods may be useful aids, for example, in drug development, they do suffer from substantial limitations. For example, while genomic and proteomic methods may ultimately give profound insights into toxicological mechanisms and provide new surrogate biomarkers of disease, at present it is very difficult to relate genomic and proteomic findings to classical cellular or biochemical indices or endpoints. One simple reason for this is that with current technology and approach, the correlation of the time-response to drug exposure is difficult. Further difficulties arise with in vitro cell-based studies. These difficulties are particularly important for the many known cases where the metabolism of the compound is a prerequisite for a toxic effect and especially true where the target organ is not the site of primary metabolism. This is particularly true for pro-drugs, where some aspect of in situ chemical (e.g., enzymatic) modification is required for activity. Metabonomics A new “metabonomic” approach has been developed which is aimed at augmenting and complementing the information provided by genomics and proteomics. “Metabonomics” is conventionally defined as “the quantitative measurement of the multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification” (see, for example, Nicholson et al., 1999). This concept has arisen primarily from the application of 1 H NMR spectroscopy to study the metabolic composition of biofluids, cells, and tissues and from studies utilising pattern recognition (PR), expert systems and other chemoinformatic tools to interpret and classify complex NMR-generated metabolic data sets. Metabonomic methods have the potential, ultimately, to determine the entire dynamic metabolic make-up of an organism. As outlined above, each level of bio-molecular organisation requires a series of analytical bio-technologies appropriate to the recovery of the individual types of bio-molecular data. Genomic, proteomic and metabonomic technologies by definition generate massive data sets which require appropriate multi-variate statistical tools (chemometrics, bio-informatics) for data mining and to extract useful biological information. These data exploration tools also allow the inter-relationships between multivariate data sets from the different technologies to be investigated, they facilitate dimension reduction and extraction of latent properties and allow multidimensional visualization. This leads to the concept of “bionomics”, the quantitative measurement and understanding of the integrated function (and dysfunction)of biological systems at all major levels of bio-molecular organisation. In the study of altered gene expression, (known as transcriptomics), the variables are mRNA responses measured using gene chips, in proteomics, protein synthesis and associated post-translational modifications are typically measured using (mainly) gel-electrophoresis coupled to mass spectrometry. In both cases, thousands of variables can be measured and related to biological end-points using statistical methods. In metabolic (metabonomic) studies, only NMR (especially 1 H) and mass spectrometry has been used to provide this level of data density on bio-materials although these data can be supplemented by conventional biochemical assays. For in vivo mammalian studies, the ability to perform metabonomic studies on biofluids such as plasma, CSF and urine is very important because it gives integrated systems-based information on the whole organism. Furthermore, in clinical settings, for the full utilization of functional genomic knowledge in patient screening, diagnostics and prognostics, it is much more practical and ethically-acceptable to analyze biofluid samples than to perform human tissue biopsies and measure gene responses. A pathological condition or a xenobiotic may act at the pharmacological level only and hence may not affect gene regulation or expression directly. Alternatively significant disease or toxicological effects may be completely unrelated to gene switching. For example, exposure to ethanol in vivo may cause many changes in gene expression but none of these events explains drunkenness. In cases such as these, genomic and proteomic methods are likely to be ineffective. However, all disease or drug-induced pathophysiological perturbations result in disturbances in the ratios and concentrations, binding or fluxes of endogenous biochemicals, either by direct chemical reaction or by binding to key enzymes or nucleic acids that control metabolism. If these disturbances are of sufficient magnitude, effects will result which will affect the efficient functioning of the whole organism. In body fluids, metabolites are in dynamic equilibrium with those inside cells and tissues and, consequently, abnormal cellular processes in tissues of the whole organism following a toxic insult or as a consequence of disease will be reflected in altered biofluid compositions. Fluids secreted, excreted, or otherwise derived from an organism (“biofluids”) provide a unique window into its biochemical status since the composition of a given biofluid is a consequence of the function of the cells that are intimately concerned with the fluid's manufacture and secretion. For example, the composition of a particular fluid (e.g., urine, blood plasma, milk, etc.) can carry biochemical information on details of organ function (or dysfunction), for example, as a result of xenobiotics, disease, and/or genetic modification. Similarly, the composition and condition of an organism's tissues are also indicators of the organism's biochemical status. In general, a xenobiotic is a substance (e.g., compound, composition) which is administered to an organism, or to which the organism is exposed. In general, xenobiotics are chemical, biochemical or biological species (e.g., compounds) which are not normally present in that organism, or are normally present in that organism, but not at the level obtained following administration/exposure. Examples of xenobiotics include drugs, formulated medicines and their components (e.g., vaccines, immunological stimulants, inert carrier vehicles), infectious agents, pesticides, herbicides, substances present in foods (e.g. plant compounds administered to animals), and substances present in the environment. In general, a disease state pertains to a deviation from the normal healthy state of the organism. Examples of disease states include, but are not limited to, bacterial, viral, and parasitic infections; cancer in all its forms; degenerative diseases (e.g., arthritis, multiple sclerosis); trauma (e.g., as a result of injury); organ failure (including diabetes); cardiovascular disease (e.g., atherosclerosis, thrombosis); and, inherited diseases caused by genetic composition (e.g., sickle-cell anaemia). In general, a genetic modification pertains to alteration of the genetic composition of an organism. Examples of genetic modifications include, but are not limited to: the incorporation of a gene or genes into an organism from another species; increasing the number of copies of an existing gene or genes in an organism; removal of a gene or genes from an organism; and, rendering a gene or genes in an organism non-functional. Biofluids often exhibit very subtle changes in metabolite profile in response to external stimuli. This is because the body's cellular systems attempt to maintain homeostasis (constancy of internal environment), for example, in the face of cytotoxic challenge. One means of achieving this is to modulate the composition of biofluids. Hence, even when cellular homeostasis is maintained, subtle responses to disease or toxicity are expressed in altered biofluid composition. However, dietary, diurnal and hormonal variations may also influence biofluid compositions, and it is clearly important to differentiate these effects if correct biochemical inferences are to be drawn from their analysis. Metabonomics offers a number of distinct advantages (over genomics and proteomics) in a clinical setting: firstly, it can often be performed on standard preparations (e.g., of serum, plasma, urine, etc.), circumventing the need for specialist preparations of cellular RNA and protein required for genomics and proteomics, respectively. Secondly, many of the risk factors already identified (e.g., levels of various lipids in blood) are small molecule metabolites which will contribute to the metabonomic dataset. Application of NMR to Metabonomics One of the most successful approaches to biofluid analysis has been the use of NMR spectroscopy (see, for example, Nicholson et al., 1989); similarly, intact tissues have been successfully analysed using magic-angle-spinning 1 H NMR spectroscopy (see, for example, Moka et al., 1998; Tomlins et al., 1998). The NMR spectrum of a biofluid provides a metabolic fingerprint or profile of the organism from which the biofluid was obtained, and this metabolic fingerprint or profile is characteristically changed by a disease, toxic process, or genetic modification. For example, NMR spectra may be collected for various states of an organism (e.g., pre-dose and various times post-dose, for one or more xenobiotics, separately or in combination; healthy (control) and diseased animal; unmodified (control) and genetically modified animal). For example, in the evaluation of undesired toxic side-effects of drugs, each compound or class of compound produces characteristic changes in the concentrations and patterns of endogenous metabolites in biofluids that provide information on the sites and basic mechanisms of the toxic process. 1 H NMR analysis of biofluids has successfully uncovered novel metabolic markers of organ-specific toxicity in the laboratory rat, and it is in this “exploratory” role that NMR as an analytical biochemistry technique excels. However, the biomarker information in NMR spectra of biofluids is very subtle, as hundreds of compounds representing many pathways can often be measured simultaneously, and it is this overall metabonomic response to toxic insult that so well characterises the lesion. Another important advantage of NMR-based metabonomics over genomics or proteomics is the intrinsic analytical accuracy of NMR spectroscopy. Reanalysis of the same sample by 1H NMR spectroscopy results in a typical coefficient of variation for the measurement of peak intensities in a spectrum of less than 5% across the whole range of peaks. Thus if the appropriate experiments are undertaken, on average the value of each peak intensity will lie in the range 0.95 to 1.05 of the true value. In addition, it is possible using NMR spectroscopy to measure absolute amounts or concentrations of a number of analytes whereas using gene chip technology only fold changes can be determined. The best available accuracy achieved using gene chips is a two fold change, i.e., the value for each parameter lies in the range 0.50 to 2.00 fold of the “true” value) and proteomic technology is even less intrinsically accurate. A similar limitation also applies to proteomic studies. Although, undoubtedly, technology is improving at a rapid rate the gap between the intrinsic accuracies of NMR spectroscopy and gene chip technology is so wide that it will require a revolutionary rather than evolutionary improvement in gene expression quantification methodology before it can rival the accuracy of NMR spectroscopy. The intrinsic accuracy of NMR provides a distinct advantage when applying pattern recognition techniques. The multivariate nature of the NMR data means that classification of samples is possible using a combination of descriptors even when one descriptor is not sufficient, because of the inherently low analytical variation in the data. All biological fluids and tissues have their own characteristic physico-chemical properties, and these affect the types of NMR experiment that may be usefully employed. One major advantage of using NMR spectroscopy to study complex biomixtures is that measurements can often be made with minimal sample preparation (usually with only the addition of 5-10% D 2 O) and a detailed analytical profile can be obtained on the whole biological sample. Sample volumes are small, typically 0.3 to 0.5 mL for standard probes, and as low as 3 μL for microprobes. Acquisition of simple NMR spectra is rapid and efficient using flow-injection technology. It is usually necessary to suppress the water NMR resonance. Many biofluids are not chemically stable and for this reason care should be taken in their collection and storage. For example, cell lysis in erythrocytes can easily occur. If a substantial amount of D 2 O has been added, then it is possible that certain 1 H NMR resonances will be lost by H/D exchange. Freeze-drying of biofluid samples also causes the loss of volatile components such as acetone. Biofluids are also very prone to microbiological contamination, especially fluids, such as urine, which are difficult to collect under sterile conditions. Many biofluids contain significant amounts of active enzymes, either normally or due to a disease state or organ damage, and these enzymes may alter the composition of the biofluid following sampling. Samples should be stored deep frozen to minimise the effects of such contamination. Sodium azide is usually added to urine at the collection point to act as an antimicrobial agent. Metal ions and or chelating agents (e.g., EDTA) may be added to bind to endogenous metal ions (e.g., Ca 2+ , Mg 2+ and Zn 2+ ) and chelating agents (e.g., free amino acids, especially glutamate, cysteine, histidine and aspartate; citrate) to intentionally alter and/or enhance the NMR spectrum. In all cases the analytical problem usually involves the detection of “trace” amounts of analytes in a very complex matrix of potential interferences. It is, therefore, critical to choose a suitable analytical technique for the particular class of analyte of interest in the particular biomatrix which could be, for example, a biofluid or a tissue. High resolution NMR spectroscopy (in particular 1 H NMR) appears to be particularly appropriate. The main advantages of using 1 H NMR spectroscopy in this area are the speed of the method (with spectra being obtained in 5 to 10 minutes), the requirement for minimal sample preparation, and the fact that it provides a non-selective detector for all metabolites in the biofluid regardless of their structural type, provided only that they are present above the detection limit of the NMR experiment and that they contain non-exchangeable hydrogen atoms. The speed advantage is of crucial importance in this area of work as the clinical condition of a patient may require rapid diagnosis, and can change very rapidly and so correspondingly rapid changes must be made to the therapy provided. NMR studies of body fluids should ideally be performed at the highest magnetic field available to obtain maximal dispersion and sensitivity and most 1 H NMR studies have been performed at 400 MHz or greater. With every new increase in available spectrometer frequency the number of resonances that can be resolved in a biofluid increases and although this has the effect of solving some assignment problems, it also poses new ones. Furthermore, there are still important problems of spectral interpretation that arise due to compartmentation and binding of small molecules in the organised macromolecular domains that exist in some biofluids such as blood plasma and bile. All this complexity need not reduce the diagnostic capabilities and potential of the technique, but demonstrates the problems of biological variation and the influence of variation on diagnostic certainty. The information content of biofluid spectra is very high and the complete assignment of the 1 H NMR spectrum of most biofluids is usually not possible (even using 900 MHz NMR spectroscopy). However, the assignment problems vary considerably between biofluid types. Some fluids have near constant composition and concentrations and in these the majority of the NMR signals have been assigned. In contrast, urine composition can be very variable and there is enormous variation in the concentration range of NMR-detectable metabolites; consequently, complete analysis is much more difficult. Those metabolites present close to the limits of detection for 1-dimensional (1D) NMR spectroscopy (typically ca. 100 nM at 800 MHz) pose severe NMR spectral assignment problems. (In absolute terms, the detection limit may be ca. 4 nmol, e.g., 1 μg of a 250 g/mol compound in a 0.5 mL sample volume.) Even at the present level of technology in NMR, it is not yet possible to detect many important biochemical substances (e.g. hormones, some proteins, nucleic acids) in body fluids because of problems with sensitivity, line widths, dispersion and dynamic range and this area of research will continue to be technology-limited. In addition, the collection of NMR spectra of biofluids may be complicated by the relative water intensity, sample viscosity, protein content, lipid content, and low molecular weight peak overlap. Usually in order to assign 1 H NMR spectra, comparison is made with spectra of authentic materials and/or by standard addition of an authentic reference standard to the sample. Additional confirmation of assignments is usually sought from the application of other NMR methods, including, for example, 2-dimensional (2D) NMR methods, particularly COSY (correlation spectroscopy), TOCSY (total correlation spectroscopy), inverse-detected heteronuclear correlation methods such as HMBC (heteronuclear multiple bond correlation), HSQC (heteronuclear single quantum coherence), and HMQC (heteronuclear multiple quantum coherence), 2D J-resolved (JRES) methods, spin-echo methods, relaxation editing, diffusion editing (e.g., both ID NMR and 2D NMR such as diffusion-edited TOCSY), and multiple quantum filtering. Detailed 1 H NMR spectroscopic data for a wide range of metabolites and biomolecules found in biofluids have been published (see, for example, Lindon et al., 1999) and supplementary information is available in several literature compilations of data (see, for example, Fan, 1996; Sze et al., 1994). For example, the successful application of 1 H NMR spectroscopy of biofluids to study a variety of metabolic diseases and toxic processes has now been well established and many novel metabolic markers of organ-specific toxicity have been discovered (see, for example, Nicholson et al., 1989; Lindon et al., 1999). For example, NMR spectra of urine is identifiably altered in situations where damage has occurred to the kidney or liver. It has been shown that specific and identifiable changes can be observed which distinguish the organ that is the site of a toxic lesion. Also it is possible to focus in on particular parts of an organ such as the cortex of the kidney and even in favourable cases to very localised parts of the cortex. It is also possible to deduce the biochemical mechanism of the xenobiotic toxicity, based on a biochemical interpretation of the changes in the urine. A wide range of toxins has now been investigated including mostly kidney toxins and liver toxins, but also testicular toxins, mitochondrial toxins and muscle toxins. Pattern Recognition However, a limiting factor in understanding the biochemical information from both 1D and 2D-NMR spectra of tissues and biofluids is their complexity. The most efficient way to investigate these complex multiparametric data is employ the 1D and 2D NMR metabonomic approach in combination with computer-based “pattern recognition” (PR) methods and expert systems. These statistical tools are similar to those currently being explored by workers in the fields of genomics and proteomics. Pattern recognition (PR) methods can be used to reduce the complexity of data sets, to generate scientific hypotheses and to test hypotheses. In general, the use of pattern recognition algorithms allows the identification, and, with some methods, the interpretation of some non-random behaviour in a complex system which can be obscured by noise or random variations in the parameters defining the system. Also, the number of parameters used can be very large such that visualisation of the regularities, which for the human brain is best in no more than three dimensions, can be difficult. Usually the number of measured descriptors is much greater than three and so simple scatter plots cannot be used to visualise any similarity between samples. Pattern recognition methods have been used widely to characterise many different types of problem ranging for example over linguistics, fingerprinting, chemistry and psychology. In the context of the methods described herein, pattern recognition is the use of multivariate statistics, both parametric and non-parametric, to analyse spectroscopic data, and hence to classify samples and to predict the value of some dependent variable based on a range of observed measurements. There are two main approaches. One set of methods is termed “unsupervised” and these simply reduce data complexity in a rational way and also produce display plots which can be interpreted by the human eye. The other approach is termed “supervised” whereby a training set of samples with known class or outcome is used to produce a mathematical model and this is then evaluated with independent validation data sets. Unsupervised PR methods are used to analyse data without reference to any other independent knowledge, for example, without regard to the identity or nature of a xenobiotic or its mode of action. Examples of unsupervised pattern recognition methods include principal component analysis (PCA), hierarchical cluster analysis (HCA), and non-linear mapping (NLM). One of the most useful and easily applied unsupervised PR techniques is principal components analysis (PCA) (see, for example, Kowalski et al, 1986). Principal components (PCs) are new variables created from linear combinations of the starting variables with appropriate weighting coefficients. The properties of these PCs are such that (i) each PC is orthogonal to (uncorrelated with) all other PCs, and (ii) the first PC contains the largest part of the variance of the data set (information content) with subsequent PCs containing correspondingly smaller amounts of variance. PCA, a dimension reduction technique, takes m objects or samples, each described by values in K dimensions (descriptor vectors), and extracts a set of eigenvectors, which are linear combinations of the descriptor vectors. The eigenvectors and eigenvalues are obtained by diagonalisation of the covariance matrix of the data. The eigenvectors can be thought of as a new set of orthogonal plotting axes, called principal components (PCs). The extraction of the systematic variations in the data is accomplished by projection and modelling of variance and covariance structure of the data matrix. The primary axis is a single eigenvector describing the largest variation in the data, and is termed principal component one (PC1). Subsequent PCs, ranked by decreasing eigenvalue, describe successively less variability. The variation in the data that has not been described by the PCs is called residual variance and signifies how well the model fits the data. The projections of the descriptor vectors onto the PCs are defined as scores, which reveal the relationships between the samples or objects. In a graphical representation (a “scores plot” or eigenvector projection), objects or samples having similar descriptor vectors will group together in clusters. Another graphical representation is called a loadings plot, and this connects the PCs to the individual descriptor vectors, and displays both the importance of each descriptor vector to the interpretation of a PC and the relationship among descriptor vectors in that PC. In fact, a loading value is simply the cosine of the angle which the original descriptor vector makes with the PC. Descriptor vectors which fall close to the origin in this plot carry little information in the PC, while descriptor vectors distant from the origin (high loading) are important in interpretation. Thus a plot of the first two or three PC scores gives the “best” representation, in terms of information content, of the data set in two or three dimensions, respectively. A plot of the first two principal component scores, PC1 and PC2 provides the maximum information content of the data in two dimensions. Such PC maps can be used to visualise inherent clustering behaviour, for example, for drugs and toxins based on similarity of their metabonomic responses and hence mechanism of action. Of course, the clustering information might be in lower PCs and these have also to be examined. Hierarchical Cluster Analysis, another unsupervised pattern recognition method, permits the grouping of data points which are similar by virtue of being “near” to one another in some multidimensional space. Individual data points may be, for example, the signal intensities for particular assigned peaks in an NMR spectrum. A “similarity matrix,” S, is constructed with elements s ij =1−r ij /r ij max , where r ij is the interpoint distance between points i and j (e.g., Euclidean interpoint distance), and r ij max is the largest interpoint distance for all points. The most distant pair of points will have s ij equal to 0, since r ij then equals r ij max . Conversely, the closest pair of points will have the largest s ij . For two identical points, s ij is 1. The similarity matrix is scanned for the closest pair of points. The pair of points are reported with their separation distance, and then the two points are deleted and replaced with a single combined point. The process is then repeated iteratively until only one point remains. A number of different methods may be used to determine how two clusters will be joined, including the nearest neighbour method (also known as the single link method), the furthest neighbour method, and the centroid method (including centroid link, incremental link, median link, group average link, and flexible link variations). The reported connectivities are then plotted as a dendrogram (a treelike chart which allows visualisation of clustering), showing sample-sample connectivities versus increasing separation distance (or equivalently, versus decreasing similarity). The dendrogram has the property in which the branch lengths are proportional to the distances between the various clusters and hence the length of the branches linking one sample to the next is a measure of their similarity. In this way, similar data points may be identified algorithmically. Non-linear mapping (NLM) is a simple concept which involves calculation of the distances between all of the points in the original K dimensions. This is followed by construction of a map of points in 2 or 3 dimensions where the sample points are placed in random positions or at values determined by a prior principal components analysis. The least squares criterion is used to move the sample points in the lower dimension map to fit the inter-point distances in the lower dimension space to those in the K dimensional space. Non-linear mapping is therefore an approximation to the true inter-point distances, but points close in K-dimensional space should also be close in 2 or 3 dimensional space (see, for example, Brown et al., 1996; Farrant et al., 1992). In this simple metabonomic approach, a sample from an animal treated with a compound of unknown toxicity is compared with a database of NMR-generated metabolic data from control and toxin-treated animals. By observing its position on the PR map relative to samples of known effect, the unknown toxin can often be classified. The same approach can be used for human samples for classification according to disease. However, such data are often more complex, with time-related biochemical changes detected by NMR. Also, it is more rigorous to compare effects of xenobiotics in the original K-dimensional NMR metabonomic space. Alternatively, and in order to develop automatic classification methods, it has proved efficient to use a “supervised” approach to NMR data analysis. Here, a “training set” of NMR metabonomic data is used to construct a statistical model that predicts correctly the “class” of each sample. This training set is then tested with independent data (referred to as a test or validation set) to determine the robustness of the computer-based model. These models are sometimes termed “expert systems,” but may be based on a range of different mathematical procedures. Supervised methods can use a data set with reduced dimensionality (for example, the first few principal components), but typically use unreduced data, with all dimensionality. In all cases the methods allow the quantitative description of the multivariate boundaries that characterise and separate each class, for example, each class of xenobiotic in terms of its metabolic effects. It is also possible to obtain confidence limits on any predictions, for example, a level of probability to be placed on the goodness of fit (see, for example, Kowalski et al., 1986). The robustness of the predictive models can also be checked using cross-validation, by leaving out selected samples from the analysis. Expert systems may operate to generate a variety of useful outputs, for example, (i) classification of the sample as “normal” or “abnormal” (this is a useful tool in the control of spectrometer automation, e.g., using sequential flow injection NMR spectroscopy); (ii) classification of the target organ for toxicity and site of action within the tissue where in certain cases, mechanism of toxic action may also be classified; and, (iii) identification of the biomarkers of a pathological disease condition or toxic effect for the particular compound under study. For example, a sample can be classified as belonging to a single class of toxicity, to multiple classes of toxicity (more than one target organ), or to no class. The latter case would indicate deviation from normality (control) based on the training set model but having a dissimilar metabolic effect to any toxicity class modelled in the training set (unknown toxicity type). Under (ii), a system could also be generated to support decisions in clinical medicine (e.g., for efficacy of drugs) rather than toxicity. Examples of supervised pattern recognition methods include the following: soft independent modelling of class analysis (SIMCA) (see, for example, Wold, 1976); partial least squares analysis (PLS) (see, for example, Wold, 1966; Joreskog, 1982; Frank, 1984; Bro, R., 1997); linear descriminant analysis (LDA) (see, for example, Nillson, 1965); K-nearest neighbour analysis (KNN) (see, for example, Brown et al., 1996); artificial neural networks (ANN) (see, for example, Wasserman, 1989; Anker et al., 1992; Hare, 1994); probabilistic neural networks (PNNs) (see, for example, Parzen, 1962; Bishop, 1995; Speckt, 1990; Broomhead et al., 1988; Patterson, 1996); rule induction (RI) (see, for example, Quinlan, 1986); and, Bayesian methods (see, for example, Bretthorst, 1990a, 1990b, 1988). As the size of metabonomic databases increases together with improvements in rapid throughput of NMR samples (>300 samples per day per spectrometer is now possible with the first generation of flow injection systems), more subtle expert systems may be necessary, for example, using techniques such as “fuzzy logic” which permit greater flexibility in decision boundaries. Application to Metabonomics Pattern recognition methods have been applied to the analysis of metabonomic data. See, for example, Lindon et al., 2001. A number of spectroscopic techniques have been used to generate the data, including NMR spectroscopy and mass spectrometry. Pattern recognition analysis of such data sets has been succesful in some cases. The successful studies include, for example, complex NMR data from biofluids, (see, for example, Anthony et al., 1994; Anthony et al., 1995; Beckwith-Hall et al., 1998; Gartland et al., 1990a; Gartland et al., 1990b; Gartland et al., 1991; Holmes et al., 1998a; Holmes et al., 1998b; Holmes et al., 1992; Holmes et al., 1994; Spraul et al., 1994; Tranter et al., 1999) conventional NMR spectra from tissue samples (Somorjai et al., 1995), magic-angle-spinning (MAS) NMR spectra of tissues (Garrod et al., 2001), in vivo NMR spectra (Morvan et al., 1990; Howells et al., 1993; Stoyanova et al., 1995; Kuesel et al., 1996; Confort-Gouny et al., 1992; Weber et al., 1998), wines (Martin et al., 1998, 1999) and plant tissues (Kopka et al., 2000). Although the utility of the metabonomic approach is well established, its full potential has not yet been exploited. The metabolic variation is often subtle, and powerful analysis methods are required for detection of particular analytes, especially when the data (e.g., NMR spectra) are so complex. For example, all that has been previously proposed is still not generally sufficient to achieve clinically useful diagnosis of disease. New methods to extract useful metabolic information from biofluids are needed. The inventors have developed novel methods (which employ multivariate statistical analysis and pattern recognition (PR) techniques, and optionally data filtering techniques) of analysing data (e.g., NMR spectra) from a test population which yield accurate mathematical models which may subsequently be used to classify a test sample or subject, and/or in diagnosis. Unlike methods previously described, the methods described herein have the power to provide clinically useful and accurate diagnostic and prognostic information in a medical setting. The methods described herein represent a significant advance over chemometric methodologies described previously. Although chemometrics has been able to provide some classification of types previously, the studies have required that the classification be done under a series of restrictions which limit the ability to apply the method to analysis of complex datasets as would be required to apply the method for the practical diagnosis/prognosis of diseases that could be useful clinically. For example, several studies have reported on the classification of animals on the basis of an NMR spectrum of urine or plasma. Although these studies clearly demonstrate the potential of the technique, they are limited because the animals which compose each class are genetically homogenous (in-bred populations). As a result, these methods have been demonstrated to be able to detect patterns but only against “low noise” backgrounds. Application of metabonomics to “real” populations (e.g., in human clinical practice) requires the ability to detect patterns against the substantial noise due to the genetic variation of outbred populations and also due to dietary and hormonal differences. Similarly, many of the studies described to date have examined relatively major differences between groups, for example, the ability to differentiate renally acting toxins from liver acting toxins. The two groups under study differed in a broad spectrum of metabolites making the pattern relatively easy to detect. In conjugation with the restriction of using in-bred populations of animals, most studies published to date have only demonstrated metabonomics to be practicable under conditions of high “signal to noise” ratio, conditions which are very different from the human clinical environment. Some studies have begun to attempt classifications of out-bred human populations where the data variation is high. However, to date, all these studies have simplified the system substantially to focus in on specific molecules: for example, some studies have looked specifically at the resonances associated with lipoproteins. Since lipoproteins are major constituents of plasma, the variance they contribute readily exceeds the background variance due to genetic and environmental differences between individuals. Unfortunately, such an approach is insufficiently powerful to identify weak patterns against the background biochemical noise, and could not be used, for example, to determine the extent of coronary heart disease or to distinguish identical from non-identical twins. Identification of such low “signal to noise” ratio patterns requires the application of the methods of this invention, which represent a significant advance over what has been previously reported.
<SOH> SUMMARY OF THE INVENTION <EOH>One aspect of the present invention pertains to a method of classifying a sample, as described herein. One aspect of the present invention pertains to a method of classifying a subject as described herein. One aspect of the present invention pertains to a method of diagnosing a subject as described herein. One aspect of the present invention pertains to a method of identifying a diagnostic species, or a combination of a plurality of diagnostic species, for a predetermined condition, as described herein. One aspect of the present invention pertains to a diagnostic species identified by a method as described herein. One aspect of the present invention pertains to a diagnostic species identified by a method as described herein, for use in a method of classification. One aspect of the present invention pertains to a method of classification which employs or relies upon one or more diagnostic species identified by a method as described herein One aspect of the present invention pertains to use of one or more diagnostic species identified by a method of classification as described herein. One aspect of the present invention pertains to an assay for use in a method of classification, which assay relies upon one or more diagnostic species identified by a method as described herein. One aspect of the present invention pertains to use of an assay in a method of classification, which assay relies upon one or more diagnostic species identified by a method as described herein. One aspect of the present invention pertains to a method of therapeutic monitoring of a subject undergoing therapy which employs a method of classification as described herein. One aspect of the present invention pertains to a method of evaluating drug therapy and/or drug efficacy which employs a method of classification, as described herein. One aspect of the present invention pertains to a computer system or device, such as a computer or linked computers, operatively configured to implement a method as described herein; and related computer code computer programs, data carriers carrying such code and programs, and the like. These and other aspects of the present invention are described herein. As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the present invention will also pertain to other aspects of the present invention.

Brake actuator apparatus and method for actuating a brake
A brake actuating apparatus includes a force transmission element operable to transmit a brake actuating force and an abutment member moveable to abut the force transmission element. A piezo-electric device operable on expansion applies a force between the abutment member and the force transmission element to move the force transmission element in a direction of brake actuation. A support member moveable independently of the abutment member supports the force transmission element in the new position following expansion of the piezo-electric device.
1. A brake actuating apparatus comprising: a force transmission element that transmits a brake actuating force; an abutment member that selectively abuts and disengages the force transmission element; a piezo-electric device that is expandable to apply a force between the abutment member and the force transmission element to move the force transmission element in a direction of brake actuation; and a support member moveable independently of the abutment member to support the force transmission element stationary in a new position following expansion of the piezo-electric device. 2. The brake actuating apparatus according to claim 1 wherein the piezo-electric device is attached to the abutment member. 3. The brake actuating apparatus according to claim 1 wherein the piezo-electric device is attached to the force transmission element. 4. The brake actuating apparatus according to claim 1 further including a first body member, and the abutment member has a threaded portion in threaded engagement with the first body member and is moveable by relative rotation between the abutment member and the first body member. 5. The brake actuating apparatus according to claim 1 further including a second body member, and the support member includes a threaded portion in threaded engagement with the second body member and is axially moveable by relative rotation between the support member and the second body member. 6. The brake actuating apparatus according to claim 1 wherein at least one of the abutment member and the support member is rotatable by a rotation device. 7. The brake actuating apparatus according to claim 1 further including a first body member and a second body member, and the abutment member has a first threaded portion in threaded engagement with the first body member and the abutment member is moveable by relative rotation between the abutment member and the first body member, and the support member has a second threaded portion in threaded engagement with the second body member and the support member is moveable by relative rotation between the support member and the second body member, wherein at least one of the first body member and the second body member is rotatable by a rotation device. 8. The brake actuating apparatus according to claim 6 wherein the rotation device is an electric motor. 9. The brake actuating apparatus according to claim 1 wherein the support member supports the force transmission element for movement of the abutment member, with the piezo-electric device contracted, to an expanded position wherein expansion of the piezoelectric device causes the abutment member to abut the force transmission element, and the support member is moveable to a contraction position wherein contraction of the piezo-electric device allows the force transmission element to move into abutment with the support member. 10. A method for actuating a brake comprising the steps of: a) moving an abutment member into abutment with a force transmission element operable for transmitting a brake actuating force; b) expanding a piezo-electric device to apply a force between the abutment member and the force transmission element to move the force transmission element in a direction of brake actuation into a new position; c) moving a support member into abutment with the force transmission element to support the force transmission element in said new position; d) contracting the piezo-electric device; and e) repeating steps a) to d) to actuate the brake. 11. The method for actuating a brake according to claim 10 further comprising the steps of: f) moving the abutment member out of abutment with the force transmission element while the force transmission element is supported by the support member and the piezo-electric device is contracted; g) expanding the piezo-electric device to abut the abutment member on the force transmission element; h) moving the support member away from the force transmission element; i) contracting the piezo-electric device to cause the support member to support the force transmission element; and j) repeating steps f) to i) to release the brake. 12. (CANCELLED) 13. The brake actuating apparatus according to claim 7 wherein the rotation device is an electric motor.



Ordering by hamming value
Apparatus and methods are disclosed for treating an input set of weightless binary tuples to order the Hamming values thereof. In the general case, the tuples are thermometer coded in a common direction (left or right) and thereafter an orthogonal thermocoding operation is applied in which orthogonal tuples, each taking a bit from a respective bit position of the thermocoded tuples, is subjected to a thermocoding operation in a common direction (up or down). At the completion of this operation the tuples represent the Hamming values of the input tuples, ranked in Hamming value order. The first thermocoding operation may be omitted where the input tuples are already in thermometer code. Also described is a scheme, which uses a tagging procedure to give the result in terms of the original weightless binary tuples, whereby the input pattern information is preserved.
1. A method of treating an input set of weightless binary thermometer code tuples (t1 to t5) each with the set bits grouped at a given end of each tuple, to order the Hamming values of said weightless binary thermometer code tuples, which comprises performing an orthogonal thermocoding operation equal or corresponding to one in which: (i) the set of weightless binary thermometer code tuples is assembled into a matrix with said weightless binary tuples stacked in a given direction (e.g. vertically) to define a set of orthogonal tuples (c1 to c6) each extending in said given direction (e.g. vertically) and each made up of a bit taken from each of said weightless binary tuples at a respective bit position, and (ii) each of said orthogonal tuples (c1 to c6) is subjected to a thermocoding step to group the set bits at a given end of each tuple, thereby to order the Hamming values of said weightless binary thermometer code tuples. 2. A method of treating an input set of weightless binary tuples (t1 to t5) to order the Hamming values of said weightless binary tuples, which comprises performing a thermocoding operation on each of said weightless binary tuples (t1 to t5) to group the set bits at a given end of each tuple to provide a set of weightless binary thermometer code tuples, and thereafter performing an orthogonal thermocoding operation corresponding to one in which: (i) the set of weightless binary thermometer code tuples is assembled into a matrix with said weightless binary tuples stacked in a given direction (e.g. vertically) to define a set of orthogonal tuples (c1 to c6) each extending in said given direction (e.g. vertically) and each made up of a bit taken from each of said weightless binary tuples at a respective bit position, and (ii) each of said orthogonal tuples (c1 to c6) is subjected to a thermocoding step to group the set bits at a given end of each tuple, thereby to order the Hamming values of said weightless binary tuples. 3. A method according to claim 1, wherein the input set (t1 to t5) comprises an odd number of tuples, and which further comprises determining the median of said set by identifying the central value of said ordered Hamming values. 4. A method according to claim 1 wherein the input set comprises an even number (2n) of tuples, and which further comprises determining a median by identifying in said ordered set of Hamming value a value corresponding to one of the nth or the (n+1)th values. 5. A method according to claim 1 wherein the input set comprises an even number (2n) of tuples, and which further comprises determining a median of said set by introducing a further tuple into said set before performing said orthogonal thermocoding operation so that the set comprises an odd number of tuples. 6. A method according to claim 5, wherein said further tuple is produced by duplicating one of the other tuples in said input set. 7. A method according to claim 1, which further comprise determining at least one weightless outlier value by determining at least one of the first and last of said ordered Hamming values. 8. A method of applying a filtering operation to a two- or higher-dimensional array of tuples, which comprises: performing the method of any of the preceding claims on a set of values in a predetermined window in said array in a filtering step, using at least a selected one of said ordered Hamming values as the filtered result of said filtering step, and incrementing said window relative to said array, to provide a series of filtered results. 9. A method according to claim 8, wherein for each filtering step, a median Hamming value is taken as the filtered result. 10. A method according to claim 8, wherein said array of tuples is two-dimensional and represents a two-dimensional image. 11. A method according to claim 8, wherein said array of tuples is three-dimensional and represents a three dimensional volume. 12. A method according to claim 1 wherein, having ordered the Hamming values of said weightless binary tuples, and after said orthogonal thermocoding operation, the bit pattern of at least one of said tuples corresponding to a given ranking is compared to the bit patterns of the tuples before said orthogonal thermocoding operation to identify an or the equivalent tuple thereto, and the location of said equivalent tuple is used to determine a corresponding input tuple, thereby to identify or present the input tuple with said given ranking. 13. A method according to claim 12, wherein the bit patterns of all of the tuples after said orthogonal coding operation are compared to the bit patterns of the tuples before said orthogonal thermocoding operation thereby to identify equivalents to each thereof and thereby to identify a ranking of each of said input tuples. 14. A method according to claim 13, wherein said comparison is done asynchronously and in parallel. 15. A method of filtering a stream of weightless binary tuples comprises selecting from said stream a plurality of sample tuples and applying to said sample tuples a method according to claim 1, to order the Hamming values of said tuples and selecting at least one of the Hamming values according to its order and ouputting said selected Hamming value as the filtered output. 16. A method according to claim 15, which comprises selecting a median Hamming value for the filtered output. 17. Apparatus for treating an input set of weightless binary thermometer code tuples (t1 to t5) with the set bits grouped at a given end of each tuple, thereby to order the Hamming value of said weightless binary thermometer code tuples, said apparatus comprising: transformation means (14) for performing an orthogonal thermocoding operation corresponding to one in which: (i) the set of weightless binary thermometer code tuples is assembled into a matrix with said weightless binary tuples stacked in a given direction (e.g. vertically) to define a set of orthogonal tuples (c1 to c6) each extending in said given direction (e.g. vertically) and each made up of a bit taken from each of said weightless binary tuples at a respective bit position, and (ii) each of said orthogonal tuples (c1 to c6) is subjected to a thermocoding step to group the set bits at a given end of each tuple, thereby to order the Hamming values of said weightless binary thermometer code tuples. 18. Apparatus for treating an input set of weightless binary tuples, which comprises: first stage thermocode converter means (12) for converting each of said weightless binary tuples into a weightless binary thermometer code tuple to provide a set thereof, and transformation means (14) for performing an orthogonal thermocoding operation corresponding to one in which: (i) the set of weightless binary thermometer code tuples is assembled into a matrix with said weightless binary tuples stacked in a given direction (e.g. vertically) to define a set of orthogonal tuples each extending in said given direction (e.g. vertically) and each made up of a bit taken from each of said weightless binary tuples at a respective given bit position, and (ii) each of said orthogonal tuples is subjected to a thermocoding step to group the set bits at a given end of each tuple, thereby to order the Hamming values of said weightless binary thermometer code tuples. 19. Apparatus according to claim 18, wherein said first stage thermometer code converter means (12) comprises respective thermometer code converter means (12) for each of said weightless binary tuples making up the input set. 20. Apparatus according to claim 17, wherein said transformation means includes orthogonal tuple defining means for defining a set of orthogonal tuples (c1 to c6), each said orthogonal tuple being made up of bits taken from a respective bit position of said weightless binary thermometer code tuples, and respective thermometer coding means (14) for thermocoding each of said orthogonal tuples. 21. Apparatus according to claim 20, including means for analysing a matrix made up of said thermocoded orthogonal tuples to determine the or a central tuple (16) orthogonal to said orthogonal tuples, said central tuple (16) representing to the median Hamming value of said weightless binary thermometer code tuples. 22. Apparatus according to claim 19, including means for analysing a matrix made up of said thermocoded orthogonal tuples to determine at least one of the first (18) and the last (20) of said orthogonal set of tuples, which represents at least one outlier. 23. Apparatus according to claim 19, wherein at least one of thermometer coding means (12, 14) comprises an asynchronous thermometer coding means (22) using a modular structure (26). 24. Apparatus according to claim 19, wherein at least one of the thermometer coding means (14) comprises a pipelined synchronous thermometer coding means. 25. Apparatus for treating an input set of weightless binary thermometer code tuples (t1 to t5) each with the set bits grouped at a given end of the tuple, said apparatus comprising: respective thermometer code converter means (14) for each bit position of the input tuples, each thermometer coding means (14) receiving a bit from each of the weightless binary thermometer code tuples and performing a thermometer coding operation on said bits, collectively to apply an orthogonal coding operation on said weightless binary tuple code tuples, to provide a set of orthogonally thermocoded tuples (c1 to c6), which when assembled side-by-side in a matrix make up, an ordered set of tuples representing the Hamming value of said weightless binary thermometer code tuples. 26. Apparatus for treating an input set of weightless binary tuples (t1 to t5) which comprises:— respective first stage thermometer code converter means (12) for converting each of said weightless binary tuples (t1 to t5) into a weightless binary thermometer code tuple; respective second stage thermometer code converter means (14) for each bit position of the input tuples, each thermometer coding means receiving a bit from each of the weightless binary thermometer code tuples and performing a thermometer coding operation on said bits, collectively to apply an orthogonal coding operation on said weightless binary tuple code tuples, to provide a set of orthogonally thermocoded tuples (c1 to c6), which, when assembled in a matrix, make up in the direction orthogonal to said orthogonally thermocoded tuples (c1 to c6), an ordered set of tuples representing the Hamming value of said input weightless binary thermometer code tuples (t1 to t5). 27. Apparatus according to claim 17, which includes bit pattern comparison means for comparing the bit pattern, after said orthogonal thermocoding step, of at least one of said tuples corresponding to a given Hamming value ranking, with each of the bit patterns of the tuples before said orthogonal thermocoding operation to determine an equivalent thereto, and pattern selection means responsive to said bit pattern comparison means to output a tuple having a bit pattern identical to that of the input tuple with said given Hamming value ranking. 28. Apparatus according to claim 25, wherein said bit pattern comparison means comprises respective logic modules (49′, 49″, 49′″) each for comparing the bit pattern of a given single tuple (VHT1, VHT2 or VHT3) resulting from said orthogonal coding step with a respective one of the tuples input to said orthogonal thermocoding step (HT1, HT2 and HT3). 29. Apparatus according to claim 26, wherein said apparatus further includes respective logic modules (49′, 49″, 49′″) each for comparing the bit patterns of the remaining tuples (VHT1, VHT2, VHT3) resulting from said orthogonal coding step with the tuples input to said orthogonal thermocoding step (HT1, HT2, HT3). 30. A filter for filtering a stream of weightless binary tuples comprising input means (32) for selecting from an input stream of weightless tuples a predetermined plurality of sample tuples and for supplying said sample tuples to apparatus (36) according to claim 15 to order the Hamming values of said weightless tuples, and selection means (36) for selecting at least one of said Hamming values according to its order and for outputting said selected Hamming value as the filtered output. 31. A filter according to claim 30, wherein said input means comprises a plurality of sample holding registers (32) arranged in series and clock means (34) for clocking said stream of weightless tuples through said registers (32). 32. A filter according to claim 30, wherein said selection means (36) selects a Hamming value having a median Hamming value for the filtered output. 33. A filter according to claim 30, for filtering data representing a two dimensional image, wherein said selection means comprises a plurality of sample holding registers (32), line delay means and clock means for defining a rolling filter window.

<SOH> SUMMARY OF THE INVENTION <EOH>According to one aspect of this invention there is provided a method of treating an input set of weightless binary thermometer code tuples each with the set bits grouped at a given end of each tuple, to order the Hamming values of said weightless binary thermometer code tuples, which comprises performing an orthogonal thermocoding operation equal or corresponding to one in which: (i) the set of weightless binary thermometer code tuples is assembled into a matrix with said weightless binary tuples stacked in a given direction (e.g. vertically) to define a set of orthogonal tuples each extending in said given direction (e.g. vertically) and each made up of a bit taken from each of said weightless binary tuples at a respective bit position, and (ii) each of said orthogonal tuples is subjected to a thermocoding step to group the set bits at a given end of each tuple, thereby to order the Hamming values of said weightless binary thermometer code tuples. The orthogonal thermocoding operation may be performed in various different ways as to be described below and it is not essential for the weightless binary thermometer code tuples actually to be assembled into a matrix provided that an analogous thermocoding operation is achieved. Surprisingly, we have found that performing an orthogonal thermocoding operation on an input set of weightless binary thermometer code tuples orders the Hamming values of the tuples without altering the Hamming values. If the input data is not already in the form of weightless binary thermometer code tuples then there may be appropriate encoding and decoding upstream and downstream of the orthogonal thermocoding operation to convert input data (e.g. weighted binary, weightless binary etc.) into thermometer code and to return it to the desired coding. Accordingly, in another aspect, where the input is in the form of weightless binary tuples, this invention provides a method of treating an input set of weightless binary tuples to order the Hamming values of said weightless binary tuples, which comprises performing a thermocoding operation on each of said weightless binary tuples to group the set bits at a given end of each tuple to provide a set of weightless binary thermometer code tuples, and thereafter performing an orthogonal thermocoding operation corresponding to one in which: (i) the set of weightless binary thermometer code tuples is assembled into a matrix with said weightless binary tuples stacked in a given direction (e.g. vertically) to define a set of orthogonal tuples each extending in said given direction (e.g. vertically) and each made up of a bit taken from each of said weightless binary tuples at a respective bit position, and (ii) each of said orthogonal tuples is subjected to a thermocoding step to group the set bits at a given end of each tuple, thereby to order the Hamming values of said weightless binary tuples. Having ordered the set of Hamming values one or more selected Hamming values of the set may be determined. For example, where the input set comprises an odd number of tuples a weightless median of said set may be determined by identifying the central value of the ordered set. Where the input set comprises an even number (2n) of tuples, a median may be determined by identifying in the ordered set a value corresponding to the n th or the n+1 th value. Alternatively, where the input set comprises an even number of tuples, a further tuple may be introduced into the set (for example by duplicating one of the other tuples of the input set) to make an odd number of tuples before performing the orthogonal thermocoding operation. At least one weightless outlier value may be determined by identifying at least one of the first and last of said ordered Hamming values. Where the input bit pattern corresponding to one of the ordered Hamming values is required, in one embodiment having ordered the Hamming values of said weightless binary tuples, and after said orthogonal thermocoding operation, the bit pattern of at least one of said tuples corresponding to a given ranking is compared to the bit patterns of the tuples before said orthogonal thermocoding operation to identify an or the equivalent tuple thereto, and the location of said equivalent tuple is used to determine a corresponding input tuple, thereby to identify or present the input tuple with said given ranking. The invention extends to a method of applying a filtering operation to a two- or—higher dimensional array of tuples in which the method as defined above is performed on a set of values in a pre-determined window in said array in a filtering step and at least a selected one of the ordered Hamming values is used as the filtered result, with the window being incremented relative to the array to provide a series of filtered results. In one instance, a median Hamming value is taken as the filtered result. The filtering operation may be applied to a two-dimensional array of tuples, for example representing a two-dimensional image. Alternatively, the filtering operation may be applied to a three-dimensional array of tuples, representing a three-dimensional volume of air traffic space. The invention also extends to filter apparatus for applying the filtering operation described above. In another aspect, this invention provides apparatus for treating an input set of weightless binary thermometer code tuples with the set bits grouped at a given end of each tuple, thereby to order the Hamming value of said weightless binary thermometer code tuples, said apparatus comprising: transformation means for performing an orthogonal thermocoding operation corresponding to one in which: (i) the set of weightless binary thermometer code tuples is assembled into a matrix with said weightless binary tuples stacked in a given direction (e.g. vertically) to define a set of orthogonal tuples each extending in said given direction (e.g. vertically) and each made up of a bit taken from each of said weightless binary tuples at a respective bit position, and (ii) each of said orthogonal tuples is subjected to a thermocoding step to group the set bits at a given end of each tuple, thereby to order the Hamming values of said weightless binary thermometer code tuples. The above apparatus is intended to receive an input set of weightless binary tuples in thermometer code. In another aspect, intended for treating an input set of weightless binary tuples not already in thermometer code, there is provided apparatus for treating an input set of weightless binary tuples, which comprises: first stage thermocode converter means for converting each of said weightless binary tuples into a weightless binary thermometer code tuple to provide a set thereof, and transformation means for performing an orthogonal thermocoding operation corresponding to one in which: (i) the set of weightless binary thermometer code tuples is assembled into a matrix with said weightless binary tuples stacked in a given direction (e.g. vertically) to define a set of orthogonal tuples each extending in said given direction (e.g. vertically) and each made up of a bit taken from each of said weightless binary tuples at a respective given bit position, and (ii) each of said orthogonal tuples is subjected to a thermocoding step to group the set bits at a given end of each tuple, thereby to order the Hamming values of said weightless binary thermometer code tuples. Preferably, the first stage thermocode converter means comprises respective thermocode converter means for each of said weightless binary tuples making up the input set. The transformation means preferably includes orthogonal tuple defining means for defining a set of orthogonal tuples, each said orthogonal tuple being made up of bits taken from a respective bit position of said weightless binary thermometer code tuples, and respective thermocode converter means for thermocoding each of said orthogonal tuples. The orthogonal tuple defining means typically comprises a mapping of interconnects mapping the bits of the thermometer code tuples to the appropriate thermocode converter means. Preferably, said apparatus includes means for analysing a matrix made up of said thermocoded orthogonal tuples to determine the or a central tuple orthogonal to said orthogonal tuples, said central tuple representing to the median Hamming value of said weightless binary thermometer code tuples. Likewise the apparatus may include means for determining the first and the last of the tuples orthogonal to said orthogonal tuples, representing at least one outlier. The thermocode means may take many forms. For example it may comprise an asynchronous thermometer coding means using a modular structure. Alternatively, at least one of the thermocode means may comprise a pipelined synchronous thermocode means. Examples of such devices are given in our earlier published International Patent Application No. WO 99/33184. Yet further, the thermocode converter means may comprise a thermometer code converter which operates on an input stream of weightless binary data, as described in our copending application of even date Patent Application No. PCT/GB03/______(our reference XA1619). In another aspect, this invention provides apparatus for treating an input set of weightless binary thermometer code tuples each with the set bits grouped at a given end of the tuple, said apparatus comprising: respective thermocode converter means for each bit position of the input tuples, each thermocode converter means receiving a bit from each of the weightless binary thermometer code tuples and performing a thermometer coding operation on said bits, collectively to apply an orthogonal coding operation on said weightless binary tuple code tuples, to provide a set of orthogonally thermocoded tuples, which when assembled side-by-side in a matrix make up an ordered set of tuples representing the Hamming values of said weightless binary thermometer code tuples. In yet another aspect this invention provides apparatus for treating an input set of weightless binary tuples which comprises:— respective first stage thermocode converter means for converting each of said weightless binary tuples into a weightless binary thermometer code tuple; respective second stage thermocode converter means for each bit position of the input tuples, each thermocode converter means receiving a bit from each of the weightless binary thermometer code tuples and performing a thermometer coding operation on said bits, collectively to apply an orthogonal coding operation on said weightless binary tuple code tuples, to provide a set of orthogonally thermocoded tuples, which when assembled in a matrix make up an ordered set of tuples representing the Hamming value of said weightless binary thermometer code tuples. Whilst the invention has been described above, it extends to any inventive combination of the features set out above or in the following description. The invention may be performed in various ways, and an embodiment thereof will now be described by way of example-only, reference being made to the accompanying drawings, in which:— FIG. 1 is a schematic view of a first embodiment of a weightless median evaluator in accordance with this invention; FIG. 2 is an example of a known thermometer code converter of modular form for use as a component in the embodiment of FIG. 1 ; FIG. 3 is a schematic diagram of a weightless median filter in accordance with the invention and using a weightless median evaluator of the form shown in FIG. 1 ; FIG. 4 is a chart showing the weightless binary inputs, the results of a first stage horizontal thermocode conversion and the median filter result for a series of samples 8 bits wide and with a window size of three successive samples; FIG. 5 is a reference image (512×512 pixel 8 bit weighted grey-scale); FIG. 6 is the image of FIG. 5 but with 10% added salt-and-pepper noise; FIG. 7 is the image of FIG. 5 using the weightless median filter, and FIG. 8 is a graph showing the trend in filtration improvement as the noise density is increased. FIG. 9 is a top level block diagram of a second embodiment in accordance with this invention, which orders a set of weightless binary tuples according to their Hamming value, and FIGS. 10 to 12 are logic diagrams of Boolean logic implementations of the tag compare modules and the pattern select module of the second embodiment. detailed-description description="Detailed Description" end="lead"? The embodiments disclosed herein describe a new mechanism for Hamming value ordering and weightless median extraction from a set of weightless binary tuples. Obviously the input data needs to be converted into weightless binary if it is not already in this form. Initially the bit manipulation steps performed to achieve Hamming value ordering and weightless median extraction will be described with reference to the five weightless tuples t1 to t5, each 6 bits wide, described in the introduction, on pages 3 and 4 of the text as filed. Having described the bit manipulation steps required apparatus for implementing the invention will be described in further detail. Given the earlier example of, t1 = [ 0 ⁢ 0 ⁢ 1 ⁢ 0 ⁢ 0 ⁢ 0 ] t2 = [ 1 ⁢ 0 ⁢ 0 ⁢ 1 ⁢ 0 ⁢ 0 ] t3 = [ 0 ⁢ 0 ⁢ 1 ⁢ 0 ⁢ 0 ⁢ 0 ] t4 = [ 0 ⁢ 0 ⁢ 1 ⁢ 1 ⁢ 0 ⁢ 1 ] t5 = [ 1 ⁢ 1 ⁢ 0 ⁢ 0 ⁢ 0 ⁢ 0 ] ⁢ c1 c2 c3 c4 c5 ⁢ c6 t1 to t5 are the input tuples, and columns c1 to c6 are imposed orthogonal thermocoding is now performed; in a first stage, each tuple t1 to t5 is thermocoded. So the rows t1 to t5 are thermocoded in either direction—to the left or the right. This gives, thermocoding to the left, [ 1 0 0 0 0 0 ] [ 1 1 0 0 0 0 ] [ 1 0 0 0 0 0 ] [ 1 1 1 0 0 0 ] [ 1 1 0 0 0 0 ] Then thermocoding is performed along the columns, either upwards or downwards. This gives, thermocoding upwards, the desired result:— [ 1 1 1 0 0 0 ] ⁢ Highest ⁢ ⁢ weightless ⁢ ⁢ outlier [ 1 1 0 0 0 0 ] [ 1 1 0 0 0 0 ] Weightless ⁢ ⁢ median [ 1 0 0 0 0 0 ] [ 1 0 0 0 0 0 ] Lowest ⁢ ⁢ weightless ⁢ ⁢ outlier The orthogonal thermocoding process yields the weightless median in thermocode as the central value i.e. row t5. This process may be visualised as assembling the input tuples into a matrix, then performing a first thermocoding step in which each tuple is thermocoded along the rows to the left, and a second thermocoding step in which the columns are thermocoded upwards. In this visualisation the tuples could be stacked vertically or horizontally, provided the first thermocoding is done in one sense along the direction of the tuples. In practice of course there may be no matrix as such because the tuples may be supplied to individual first stage thermocoders whose outputs are hardwired in a preset mapping to the inputs of individual second stage thermocoders, with selected outputs of the second stage thermocoders hardwired to an output interface. It should be noted that, in the described embodiment, the two stage orthogonal process must be performed in order, namely a horizontal operation along the direction of the tuple and then a vertical operation orthogonal thereto. Naturally if the input data is already thermocoded it is only required to perform the vertical thermocoding to obtain the weightless median. It will be appreciated that this provides a unique weightless median only if there is an odd number of tuples. When presented with an even set of tuples, one of the tuples (for example, the first) is duplicated to obtain an odd set. Alternatively the tuple one above or below the non-existent median may be taken as an approximation; such an approximation obtained in either manner is also referred to herein for convenience as a weightless median. The above method is technology independent; it may be implemented electronically, optically, magnetically etc. Referring now to FIG. 1 , this shows a block diagram for a weightless median evaluator which also determines the weightless outliers. Referring to the Figure, the weightless median evaluator 10 is designed to receive and evaluate the median of 5, 5-bit wide weightless binary tuples. The evaluator 10 comprises a first layer of thermocoders 12 designed to thermocode to the left, i.e. to group the set bits at the left hand end of the thermocode output. The inputs to each first layer thermocoder are here shown above the thermocoder box, whilst the thermocode output is shown within the box. An orthogonal set of five second layer thermocoders 14 receives as inputs the outputs of the first layer of thermocoders 12 . Each second layer thermocoder 14 takes the output bits from a respective bit position of the thermocoded output of the first layer; thus the leftmost second layer thermocoder 14 takes the leftmost bits of the thermocode output of the first layer of thermocoders, the rightmost second layer thermocoder 14 takes the rightmost bits of the thermocoded output of the first layer and so on. Thus if the five input tuples (0 0 1 1 0), (0 1 0 1 1), (0 0 0 1 0), (1 1 0 1 1) and (0 0 0 1 0) are visualised as a matrix made up of these tuples stacked vertically, thus 0 0 1 1 0 0 1 0 1 1 0 0 0 1 0 1 1 0 1 1 0 0 0 1 0 the first layer of thermocoders thermocode the rows to the left, to provide the following array: 1 1 0 0 0 1 1 1 0 0 1 0 0 0 0 1 1 1 1 0 1 0 0 0 0 the orthogonal thermocoding operation effects the transformation to the following: 1 1 1 1 0 1 1 1 0 0 1 1 0 0 0 1 0 0 0 0 1 0 0 0 0 Referring back to FIG. 1 , the central output bit of each second layer thermocoder is output as a tuple at 16 to represent the weightless median. Likewise the first and last bits from each of the second layer thermocoders may be output at 18 and 20 respectively to indicate the highest and lowest outliers. FIG. 2 is a diagram representing a suitable form of asynchronous modular thermometer coder 22 which could be used to implement the arrangement of FIG. 1 . Five such decoders would be required in the first layer and five in the second layer. The thermometer code converter of FIG. 2 has six inputs 24 but the sixth input could be set at zero in each case. The decoder of FIG. 2 is described in more detail in International Published Application WO 99/33184 but briefly is made up of a series of interconnected modules or “bit manipulation cells” 26 each made up of an AND gate 28 and an OR gate 30 interconnected as shown in the Figure. It is emphasised that this is just one form of thermometer code converter and many others may be used. The orthogonal median evaluator of FIG. 1 may be used to form a weightless median filter as shown in FIG. 3 . In the arrangement of FIG. 3 a stream of n-bit weightless tuples is supplied to a chain of three sample holding registers 32 through which the samples are clocked under the control of a clock 34 . The chain is tapped and three samples are provided to a weightless median evaluator 36 . The sample holding registers 32 effectively define a rolling three sample window which supplies three weightless samples at each clock cycle. Here the weightless evaluator 36 has a first layer of three thermocode generators (one for each sample) (not shown) with each first layer thermocode generator being n bits wide. The second layer will have n thermocode generators (not shown) each three bits wide. FIG. 4 shows a series of 14 input samples (labelled “Weightless Binary Input”), the result of the first thermocode conversion (labelled “Horizontal Thermocode”) and the median filter result output defined by the tuple made up from the output bits at the centre bit position of each of the n second level thermocode generators. In particular it will be noted that the potentially suspect sample 5 has been attenuated in the filtered output. In another example, a MATLAB® simulation of a two-dimensional, three-by-three sample space (defining a nine-element “window”) operated on the same principles as the block diagram shown in FIG. 3 . The nine-element window is set up in known fashion using line delays and sample holding registers controlled by a clock signal to make available the current nine samples to a weightless median evaluator. In this example, a test image consisting of a 512×512 pixel 8-bit weighted grey-scale image was used as a reference. The 8-bit binary code converts into 255 bits of thermometer code. Quantisation noise was excluded from the simulation. As before, the number of thermocoders in the first stage is dictated by the number of samples in the window (i.e. nine) with the number of second stage orthogonal thermometer code converters corresponding to the digital width of the samples. FIG. 5 is a representation of the image used in this example, before the addition of noise. A noise density of 10% was used to add impulsive salt-and-pepper noise to the reference image to form a suitable image for processing using the weightless median filter. FIG. 6 shows the image with the noise added. The mean square error introduced by the noise was 2121 and the peak signal-to-noise ratio was 14.9 dB. The filtered image is shown in FIG. 7 . The mean square error has been reduced by the filtration to 71 and the peak signal-to-noise ratio has increased to 29.6 dB. FIG. 8 shows the trend in filtration improvement as the noise densities increase. The embodiment of FIG. 1 provides an ordered set of tuples which are thermocoded versions of the input tuples rather than the tuples themselves. Whilst this is useful in many applications such as the median filter illustrated in FIG. 3 , there are other applications in which it is desirable to output, say, the actual tuple having the median value in a set, rather than a thermocoded version thereof. Accordingly, the further embodiment illustrated and described with reference to FIGS. 9 to 12 receives a set of weightless binary input tuples and uses a technique similar to the above technique of orthogonal thermocoding to order thermocoded versions of the input tuples, but with a modification to give the result in terms of the original weightless binary tuples whereby the input pattern information is preserved. FIG. 9 is a top level block diagram of this further embodiment, which will be described in conjunction with a worked example. The input pattern, which in this example is three input tuples T1 to T3, e.g. T1 0 1 0 1 T2 0 1 0 0 T3 1 1 0 1 , is applied to an input device 40 . The tuples from the input device pass to a horizontal thermocoder 42 where the tuples are horizontally thermocoded (e.g. to the left) to yield: HT1 1 1 0 0 HT2 1 0 0 0 HT3 1 1 1 0 , where HT1 denotes a tuple obtained by horizontally thermocoding T1 etc. The bits from the horizontal thermocoder 42 are then passed to a vertical thermocoder 44 where the bits in vertical tuples, corresponding to the columns of an array obtained by vertically stacking the tuples HT1, HT2, HT3, are vertically thermocoded (e.g. vertically downwards) to give: VHT ⁢ ⁢ 1 1 0 0 0 VHT ⁢ ⁢ 2 1 1 0 0 VHT ⁢ ⁢ 3 1 1 1 0 , where the prefix “V” denotes a vertical thermocode operation and correspondingly the prefix “VH” denotes an orthogonal thermocode operation. At this stage, the tuples have been rearranged or orthogonally thermocoded as in the embodiment of FIG. 1 . However in this further embodiment of FIG. 9 , the values of the orthogonally thermocoded tuples VHT1 to VHT3 (i.e. 1 0 0 0), (1 1 0 0), (1 1 1 0) are used as “tags” which are compared logically with HT1 to HT3 to determine the respective mappings, with the resulting comparison being used to select the corresponding input tuple T1 to T3. In the above example, the vertical thermocoding has been done downwards which mean that VHT2 corresponds to the weightless median and VHT1 and VHT3 correspond to the lowest and highest outliers respectively. To determine the lowest outlier, VHT1's tag is 1 0 0 0 in this example. This tag (1 0 0 0) is compared with HT1 to HT3 and here corresponds to HT2, and HT2 selects T2, i.e. (0 1 0 0) Similarly for the median, VHT2's tag is (1 1 0 0) which corresponds to HT1 which selects T1 i.e. (0 1 0 1). For the highest outlier VHT3's tag is (1 1 1 0) which corresponds to HT3 which selects T3 i.e. (1 1 0 1). To implement this, the results of the horizontal thermocoding by the horizontal thermocoder 42 and the results of the vertical thermocoding at the vertical thermocoder 44 pass to a tag compare module 46 which performs the tag comparison described above and outputs to a pattern select module 48 a select signal which selects from the original non-thermocoded input pattern the lower outlier (referred to as new T1), the median having the median Hamming value (referred to as New T2) and the highest outlier (referred to as New T3). Although in this example (and in the circuits of FIGS. 10 to 12 ) only three input and three output tuples are provided, it will be immediately apparent to one skilled in the art that the technique may easily be expanded to process larger numbers of input tuples and to provide an output in which the tuples are ordered according to their Hamming values, with the original patterns in the tuples preserved. In this illustrated example the output from the pattern select module 48 is thus:— New ⁢ ⁢ T ⁢ ⁢ 1 = 0 1 0 0 ( outlier ⁢ ⁢ with ⁢ ⁢ lower ⁢ ⁢ Hamming ⁢ ⁢ value ) New ⁢ ⁢ T ⁢ ⁢ 2 = 0 1 0 1 ( median ) New ⁢ ⁢ T ⁢ ⁢ 3 = 1 1 0 1 ( outlier ⁢ ⁢ with ⁢ ⁢ highest ⁢ ⁢ Hamming ⁢ ⁢ value ) , although the technique could be pruned to generate one, or more, or all the outliers or medians. FIGS. 10 to 12 are logic diagrams of Boolean logical implementations of the tag compare module 46 and the pattern select module 48 of FIG. 9 . In this example three operations are performed in parallel using three generally similar arrangements to determine the lowest outlier tuple T1, the weightless median T2, and the highest outlier tuple T3, using the dedicated logic circuits in FIGS. 10, 11 and 12 respectively. Each logic circuit receives the output bits from the entire result of the thermocoding by the horizontal thermocoder 42 , and a respective one of the rows of the bits from the result of the thermocoding by the vertical thermocoder 44 . Referring initially to FIG. 10 , at the foot thereof is shown the coordinate system for identifying the bits in the tuple input pattern 50 , the horizontal thermocoding output pattern 52 , and the vertical thermocoding output pattern 54 , and this coordinate system is used in FIGS. 11 and 12 also. Each tag compare module 46 in the three circuits comprises three sub modules 49 , 49 ″ and 49 ′″, each of which comprises four 2-input EXNOR gates 56 , the outputs of which are passed to a respective 4-input AND gate 58 ′, 58 ″ and 58 ′″. The outputs from the AND gates 58 ′ to 58 ′″ are prioritised by a two input AND gate 60 with the input from the first sub module 49 ′ inverted, and a three input AND gate 62 with the inputs from the sub modules 41 ′ and 49 ″ inverted, such that a high output from the first AND gate 58 ′ ensures that the outputs from the second and third AND gates 58 ″ and 58 ′″ are inhibited, and likewise a high output from the second AND gate 58 ″ inhibits the output from the third AND gate 58 ′″. Consequently the output of each tag compare module 46 is a single ‘1’ on one of the three output lines 64 ′ to 64 ′″ thereof, which is passed to the associated pattern select module 48 to select a corresponding one of the input tuples. Each pattern select module 48 is made up of a 4×3 array of AND gates 66 each of which receives a corresponding bit from the input pattern 50 (i.e. the three 4-bit tuples T 1 to T 3 in this example), with the input lines 64 ′, 64 ″ and 64 ′″ connected to respective rows of the AND gates. The outputs from each of the columns of the AND gates are ORed together by respective OR gates 68 , such that a logic 1 on one of the lines 64 ′, 64 ″ and 64 ′″ selects that row which is presented on the output of the OR gates 68 . The tag compare module 46 of FIG. 10 determines the lowest outlier. The first sub module 49 ′ compares bitwise the bits of the first row of the output of the vertical thermocoder (i.e. VHT1) with the bits of the first row of the output of the horizontal thermocoder (i.e. HT1). The second sub module 49 ″ compares VHT1 with HT2, and the third submodule 49 ′″ compares VHT2 with HT3. The tag compare modules 46 of FIGS. 10 and 11 are generally similar except in FIG. 10 , the submodules 49 ′, 49 ″, 49 ′″ compare the second row of the output of the vertical thermocoder i.e. VHT2 with HT1, HT2 and HT3 respectively and in FIG. 12 the submodules 49 ′, 49 ″, 49 ′″ compare the third row of the vertical thermocoder, i.e. VHT3 with HT1, HT2 and HT3 respectively. Thus the outputs from the circuits of FIGS. 10 to 12 may be reassembled to provide a bit pattern corresponding to the input bit pattern reordered according to the Hamming values of the input tuples. This embodiment deals automatically with cases where several or all the bit patterns are the same. It will be appreciated that the architecture is readily extensible in terms of the numbers of tuples and bit width. The number of tuples does not have to be odd. The implementation is entirely asynchronous. This technique preserves the original bit patterns and their Hamming value ratings (ordering) but does not always preserve their order. For example 1 0 1 1 0 1 0 1 1 0 1 0 0 1 0 0 ⁢ ⁢ may ⁢ ⁢ yield ⁢ ⁢ 1 0 1 1 1 0 1 0 0 1 0 1 0 1 0 0 detailed-description description="Detailed Description" end="tail"?

Anti-alpha3(IV)nc1 monoclonal antibodies and animal model for human anti-glomerular basement membrane autoantibody disease
The present invention relates to methods of making a mouse model of human anti-GBM disease, to mice produced by such methods, and to human antibodies and antigen-binding portions thereof that specifically bind to α3(IV) NC1 collagen. The present invention also relates to compositions comprising the above antibodies or portions thereof and methods of using such compositions for diagnosis, and to nucleic acid molecules encoding the above antibodies or portions thereof. The present invention further relates to methods of isolating compounds/peptides that specifically binds anti-α3(IV) NC1 antibodies, pharmaceutical compositions comprising these compounds/peptides and methods of using such compositions for diagnosis and treatment.
1. An isolated human monoclonal antibody or an antigen-binding portion thereof that specifically binds α3(IV) NC1 collagen. 2. The antibody or an antigen-binding portion according to claim 1, wherein said antibody or antigen-binding portion competitively inhibits the binding of an anti-glomerular basement membrane (anti-GBM) auto-antibody from a patient with anti-GBM disease. 3. The antibody or an antigen-binding portion according to claim 1, wherein said antibody or antigen-binding portion, when administered to a mouse, causes human-like anti-GBM disease in said mouse. 4. The antibody or an antigen-binding portion according to any one of claims 1 to 3, wherein said antibody or antigen-binding portion is produced by immunizing a mouse capable of producing fully human antibodies with α3(IV) NC1 collagen. 5. The antibody or an antigen-binding portion according to claim 4, wherein said mouse is a XenoMouse mouse. 6. The antibody or an antigen-binding portion according to any one of claims 1 to 5, wherein said α3(IV) NC1 collagen is human. 7. A hybridoma cell line that produces the F1.1 monoclonal antibody, wherein said cell line has ATCC deposit No. PTA-4237. 8. The F1.1 monoclonal antibody produced by the cell line according to claim 7. 9. An antibody or an antigen-binding portion thereof comprising a heavy chain and a light chain, wherein the heavy chain amino acid sequence comprises the CDR1 through CDR3 amino acid sequence in SEQ ID NO: 2 and wherein the light chain amino acid sequence comprises the CDR1 through CDR3 amino acid sequence of SEQ ID NO: 4. 10. The antibody or an antigen-binding portion thereof according to claim 9, wherein said heavy chain amino acid sequence comprises the amino acid sequence in SEQ ID NO: 2 and wherein the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 4. 11. An antibody or an antigen-binding portion thereof comprising a heavy chain and a light chain, wherein the heavy chain is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 and wherein the light chain is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3. 12. An antibody or an antigen-binding portion thereof comprising a heavy chain, wherein the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 2. 13. A nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1. 14. An antibody or an antigen-binding portion thereof comprising a light chain, wherein the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 4. 15. A nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3. 16. A host cell transformed with a the nucleic acid according to claim 13 or 15. 17. A The nucleic acid according to claim 13 or 15, operably linked to an expression control sequence. 18. A host cell transformed with a the nucleic acid according to claim 17. 19. An antibody or an antigen-binding portion thereof comprising a heavy chain, wherein the heavy chain comprises the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 shown in SEQ ID NOS: 6, 8, and 10, respectively. 20. An antibody or an antigen-binding portion thereof comprising a heavy chain, wherein the heavy chain utilizes the human VH gene, human D gene and human JH gene and the utilized by the antibody according to claim 8. 21. The antibody or an antigen-binding portion thereof according to claim 20, further comprising a kappa light chain, wherein said light chain utilizes the human Vκ gene and human Jκ gene utilized in the antibody according to claim 8. 22. A method for producing a mouse model for human anti-GBM disease, comprising the steps of: a. providing a mouse capable of producing a fully human antibody; and b. immunizing said mouse with α3(IV) NC1 collagen. 23. The method according to claim 22, wherein said mouse is a XenoMouse® mouse. 24. A method of inducing anti-GBM disease in a mouse, comprising administering to said mouse an antibody selected from the group consisting of: an antibody according to any one of claims 3 to 5 or 8 or an anti-α3(IV) NC1 collagen auto-antibody from a patient suffering from anti-GBM disease. 25. The method according to claim 22 or claim 24, wherein said α3(IV) NC1 collagen is selected from the group consisting of: baculovirus expressed recombinant α3(IV) NC1 collagen, bovine α3(IV) NC1 collagen dimers, E. coli expressed recombinant α3(IV) NC1collagen and human fetal 293-kidney cell expressed α3(IV) NC1 collagen. 26. The mouse produced by the method according to any one of claims 22 to 25. 27. A method for screening or identifying compositions for use in treating or preventing one or more symptoms of anti-GBM disease, comprising the steps of: a. administering a composition to a mouse according to claim 26; and b. detecting a decrease in said one or more symptoms.
<SOH> BACKGROUND OF THE INVENTION <EOH>Anti-GBM disease is a human autoimmune disease mediated by the spontaneous production of antibodies against α3(IV) NC1 collagen (Kalluri et al., J. Biol. Chem. 266:24018-24024 (1991)). This typically results in linear deposition of antibodies within the glomerular basement membrane (GBM) leading to rapidly progressive glomerulonephritis. When associated with lung hemorrhage, it is termed Goodpasture syndrome (Couser, Am. J. Kidney Dis. 11:449-464 (1988)). Identification and isolation of α3(IV) NC1 collagen as the principal target of the human autoantibody response has provided the unique opportunity to study the major antigenic epitopes in this disease (Kalluri et al., Proc. Assoc. Am. Physicians 108:134-139 (1996); Hellmark et al., J. Biol. Chem. 274:25862-25868 (1999); Hellmark et al., Kidney Int. 55:936-944 (1999)). Most of the pathogenic antibodies derived from serum and kidney eluates of patients react with this protein (Kalluri et al., J. Am. Soc. Nephrol. 6:1178-1185 (1995)). Furthermore, following immunization of normal mice with α3(IV) NC1 collagen, the animals develop disease that closely resembles the human form of the disease (Kalluri et al., J. Clin. Invest. 100:2263-2275 (1997)). Investigation of these antibodies indicates that the antibody production is antigen driven and is consistent with the notion that the antigen is hidden with defective or inefficient peripheral tolerance to anti-α3(IV) NC1 autoantibody producing B cells. Nevertheless further evaluation of the molecular basis of the human autoimmune response has been limited by lack of access to disease-relevant immune cells and individual pathogenic antibodies. The present invention provides fully human monoclonal antibodies specific for α3(IV) NC1 and methods and compositions comprising such monoclonal antibodies. The invention further provides an animal model for human anti-GBM disease.
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides methods of producing an animal model for human anti-GBM disease comprising the steps of immunizing a non-human animal with α3(IV) NC1 and testing the animal for the production of antibodies that bind α3(IV) NC1 and for phenotypic characteristics of anti-GBM disease. In a preferred embodiment, the animal is a XenoMouse® animal genetically engineered to produce human IgG in response to antigenic challenge. In another preferred embodiment, the animal is a XenoMouse® animal genetically engineered to produce human IgG2 (γ 2 k) in response to antigenic challenge. In one aspect of the invention, the animal is immunized with the NC1 domain of α3 strand of type IV collagen (α3(IV) NC1). In another aspect of the invention, the animal is immunized with bovine α3(IV) NC1. In yet another aspect of the invention, the animal is immunized with recombinantly produced human α3(IV) NC1. Recombinantly produced human α3(IV) NC1 may be expressed in a variety of cells, both prokaryotic and eukaryotic, e.g., E. coli, baculovirus, or human fetal 293 kidney cells. The invention also provides methods of producing an animal model for human anti-GBM disease comprising the steps of passively immunizing the animal with a monoclonal antibody specific for α3(IV) NC1, and testing the animal for phenotypic characteristics of anti-GBM disease. In a preferred embodiment, the animal is a mouse. In a more preferred embodiment, the animal is a XenoMouse® animal. In a even more preferred embodiment, the animal is XenoMouse II® animal. Preferably, the monoclonal antibody is specific to an epitope bound by Mab F 1.1. More preferably, the monoclonal antibody is Mab F1.1. The present invention also provides the animals produced by the above methods of making an animal disease model for human anti-GBM disease. Such animal models for human anti-GBM disease are useful for further investigation of anti-GBM disease in vivo as well as the testing of therapies to treat this disease. The invention also provides methods for evaluating strategies and/or compounds for preventing or treating anti-GBM disease using the mouse model of human anti-GBM disease. This unique anti-GBM disease model is directly applicable to the human form of the disease and should provide the means for evaluating the human α3(IV) NC1 autoantibody response. In preferred embodiments, the mouse model is used to test specific therapies aimed at modulation of either B cells producing human autoantibodies or the human pathogenic antibodies themselves, in vivo, prior to trial in patients with the spontaneous form of the disease. In addition to its use for evaluating the efficacy of candidate compounds or other therapeutic interventions, the model can be used to investigate the etiology of the disease. This new model of anti-GBM disease therefore provides both the means and unique reagents to decipher further the molecular basis of the human anti-GBM autoantibody response. The present invention provides antibodies or antigen-binding portions thereof that specifically bind α3(IV) NC1. In certain embodiments, the antibodies or antigen-binding portions are isolated and may be polyclonal or monoclonal. In preferred embodiments, the antibodies are human monoclonal antibodies. The present invention includes antibodies that comprise a human heavy chain and/or human light chain, the entire human variable region or any portion thereof, including individual CDRs of an antibody provided herein. In some embodiments, the antibody comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO: 2. In other embodiments, the antibody comprises a heavy chain comprising the CDR1, CDR2 and CDR3 shown in Table 2 (SEQ ID NO: 2). In another embodiment, the antibody heavy chain comprises a portion of the amino acid sequence shown in Table 2 (SEQ ID NO: 2) from CDR1 through CDR3. In other embodiments, any of the above-described antibodies further comprises a light chain comprising the amino acid sequence shown in SEQ ID NO: 4 (see also Table 3), CDR1 through CDR3 or a portion thereof or CDR1, CDR2 and CDR3 of the amino acid sequence of the light chain variable region sequence shown in Table 3 (SEQ ID NO: 4). The antibodies or portion thereof of the invention may be an immunoglobulin G (IgG), an IgM, an IgE, an IgA or an IgD molecule. In a preferred embodiment, the human antibody is an IgG and is an IgG1, IgG2, IgG3 or IgG4 subtype. In another preferred embodiment, the human antibody is an IgG2 subtype. In a more preferred embodiment, the human antibody is Mab F1.1. In another embodiment, the antibody or antigen-binding portion thereof is derived from an Fab fragment, an F(ab′) 2 fragment, an Fv fragment, a single chain antibody or a chimeric antibody. In another embodiment, the antibody or antigen-binding portion thereof forms part of a fusion protein. According to another object, the invention provides a human anti-α3(IV) NC1 antibody or antigen-binding portion thereof that is labeled or derivatized. In preferred embodiments, the labeled or derivatized antibody or portions thereof is used in diagnostic methods or in methods of screening for compounds/peptides that bind the antibody or portions thereof. In another aspect, the invention provides polynucleotide molecules comprising sequences encoding the heavy and light chain immunoglobulin molecules of the invention or portions thereof, particularly nucleotide sequences encoding the heavy and light chain variable regions, contiguous heavy and light chain amino acid sequences from CDR1 through CDR3 and individual CDR's. In one embodiment, the invention provides vectors and host cells comprising the nucleic acid molecule(s). In another embodiment, the invention provides a method of recombinantly producing the heavy and/or light chain, the antigen-binding portions thereof or derivatives thereof, including production by an immortalized cell, synthetic means, recombinant expression, or phage display. In another aspect, the invention provides an immortalized cell line, such as a hybridoma that produces human anti-α3(IV) NC1 monoclonal antibody. In another aspect, the invention provides a method for identifying a compound/peptide that specifically binds a anti-α3(IV) NC1 antibody of the invention or fragments thereof. The screening method comprises the steps of providing an anti-α3(IV) NC1 antibody or fragment thereof, providing a test compound/peptide, incubating the antibody or fragment thereof with the test compound/peptide, and determining the ability of the test compound to bind the antibody or fragment thereof. In a preferred embodiment, the isolated compound/peptide inhibits the binding of the anti-α3(IV) NC1 antibody to α3(IV) NC1. This can be determined in a competition assay wherein both α3(IV) NC1 and the compound/peptide are incubated with the anti-α3(IV) NC1 antibody or fragment thereof. In one embodiment, the test compound/peptide is a member of a library of small molecules or peptides. In another embodiment, the peptide library is a phage-display library. Preferably, the library is derived from cDNA, genomic DNA, semi-synthetic or fully synthetic, semi-random or random nucleic acid sequences. In another preferred embodiment, the anti-α3(IV) NC1 antibody used in the screening is labeled or derivatized. In another aspect, the present invention provides anti-idiotype (“anti-Id”) antibodies directed against human anti-GBM antibodies. In certain embodiments, the anti-Id antibodies or antigen-binding portions thereof are isolated and may be polyclonal or monoclonal. In preferred embodiments, the anti-Id antibodies are human monoclonal antibodies. In certain embodiments, the human anti-Id antibodies specifically bind anti-GBM antibody or fragments thereof isolated from patients with anti-GBM disease or from an animal model of anti-GBM disease of the current invention. In certain embodiments, said human anti-GBM antibody or fragment thereof is isolated from a XenoMouse® animal, e.g., a XenoMouse II® animal. In one embodiment, the anti-Id antibody specifically binds Mab F1.1. In a related aspect, the present invention provides a method for producing said anti-Id antibody. Said method comprises the step of immunizing a non-human animal with an anti-GBM antibody. In certain embodiments, said method further comprises isolating antibody-producing cells from said animal. In preferred embodiments, said non-human animal is a mouse, more preferably a XenoMouse® mouse, e.g., a XenoMouse II® mouse. In accordance with another aspect, the invention provides pharmaceutical compositions and kits comprising the anti-α3(IV) NC1 antibody-binding compounds/peptides identified by the screening methods of the current invention and a pharmaceutically acceptable carrier, or the anti-Id antibodies of the invention and a pharmaceutically acceptable carrier. In a preferred embodiment, the pharmaceutical composition or kit further comprises another component, such as an imaging reagent or therapeutic agent. In preferred embodiments, the pharmaceutical composition or kit is used in diagnostic or therapeutic methods. Another aspect of the invention comprises diagnostic methods. In one embodiment, the invention provides a method for diagnosing the presence and/or location of anti-α3(IV) NC 1 antibody in a sample, comprising contacting the sample with a diagnostic agent. The diagnostic agent can be immobilized on a solid support or be in solution. In certain embodiments, the method uses purified α3(IV) NC1 as the diagnostic agent. In other embodiments, the method uses as the diagnostic agent a compound/peptide identified by the screening methods of the invention or an anti-Id antibody of the invention that specifically binds to anti-α3(IV) NC1 antibody. In a preferred embodiment, an anti-α3(IV) NC1 antibody (e.g., Mab F1.1) or an antigen-binding portion thereof of the current invention is used as a positive control. In a preferred embodiment, α3(IV) NC1 or the compound/peptide or anti-Id antibody is labeled. The diagnostic methods may be used in vivo or in vitro. In another embodiment, there is provided a diagnostic method that comprises determining whether said compound/peptide or anti-Id antibody inhibits or decreases the level of anti-α3(IV) NC1 antibody in a subject (and/or alleviate the symptoms of anti-GBM disease in a subject). Another object of the invention comprises therapeutic methods of using the pharmaceutical compositions of the invention. In one embodiment, the therapeutic method comprises administering an effective amount of the composition to a subject in need thereof. In a preferred embodiment, the subject is suffering from anti-GBM disease. In a more preferred embodiment, the method inhibits or decreases the binding of anti-α3(IV) NC1 antibody to α3(IV) NC1. In another embodiment, the method is performed along with other therapies (e.g., antibody removal by plasmapheresis). In a still further embodiment, the compound/peptide or anti-Id antibody is labeled with a radiolabel, a drug conjugate, an immunotoxin or a toxin, or is a fusion protein comprising a toxic peptide. In another aspect, the present invention provides methods, vectors and/or host cells comprising the appropriate nucleic acid molecule(s) for producing a peptide identified by the screening methods of the invention that specifically binds an anti-α3(IV) NC1 antibody, including production by an immortalized cell, synthetic means, recombinant expression or phage display. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Sealing apparatus and method
Apparatus (20) and methods for segmenting a blood-filled tube (42) into multiple, blood-containing, sealed segment are disclosed. The apparatus (20) includes a tubing locating surface (22) for mounting the tubing and sealing means (24) cooperatively associated with the tubing locating surface (22).
1. Sealing apparatus comprising: a tubing locating surface for placing a length of blood-containing tubing thereon; and sealing means cooperatively associated with said surface for concurrently providing multiple, sealed, blood-containing tubing segments. 2. Apparatus of claim 1 wherein said tubing locating surface comprises means for configuring said length of tubing in a desired configuration. 3. Apparatus of claim 2 wherein said configuring means comprises a series of spaced apart pins. 4. Apparatus of claim 3, further comprising a longitudinal axis and said tubing locating surface pins are spaced from said axis of said tubing locating surface to provide the tubing with a wave configuration. 5. Apparatus of claim 4 wherein said pins are located near said axis of said tubing locating surface. 6. Apparatus of claim 5 wherein said pins are moveable from a position near the said axis of said tubing receiving surface to position across and spaced from the said axis. 7. Apparatus of claim 1 wherein said sealing means comprises a cover including a sealing element on the interior surface of said cover. 8. Apparatus of claim 7 wherein said cover comprises a plurality of sealing elements on the interior surface of said cover. 9. Apparatus of claim 7 wherein said cover is hingedly attached to said tubing locating surface. 10. Apparatus of claim 4 wherein said sealing element is parallel to said axis of said tubing locating surface. 11. Apparatus of claim 4 wherein said sealing elements are perpendicular to said axis of said tubing locating surface. 12. Apparatus of claim 1 further comprising a pair of facing and movable sealing means disposed at 90 degree angles to said tubing locating surface. 13. Apparatus of claim 1 wherein said sealing means comprises a single sealing element. 14. Apparatus of claim 2 comprising a pre-programmed controller to allow for configuration of different tubing lengths and different tubing thicknesses. 15. Apparatus of claim 2 wherein said tubing locating surface comprises means for configuring said length of tubing to provide a packet of sealed tubing segments, each segment being separated from each other by a seal and said seal being attached to an adjacent seal. 16. Apparatus of claim 1 wherein said sealing means comprises means for providing radio frequency energy. 17. Apparatus of claim 1 wherein said sealing means comprises a laser generating means. 18. A method for providing multiple, sealed, blood-containing tubing segments comprising: providing a length of blood-containing tubing; mounting said tubing on a tubing locating surface; sealing said tubing to concurrently provide a plurality of sealed, blood-containing, tubing segments. 19. The method of claim 18 comprising configuring said length of tubing in a desired configuration prior to sealing. 20. The method of claim 19 comprising mounting said tubing on a tubing locating surface having means for configuring said tubing in said desired configuration. 21. The method of claim 20 comprising mounting said tubing on a tubing locating surface having a series of pins spaced from a longitudinal axis of said surface whereby said tubing is configured in the shape of a wave. 22. The method of claim 21 comprising mounting said tubing on a tubing locating surface having a plurality of pins disposed near said axis of said surface and slots for lateral movement of said pins, said method further comprising: placing said tubing between said pins; laterally moving said pins across said axis to a position spaced from said axis; contacting said tubing at the locations where said tubing intersects said axis. 23. The method of claim 18 comprising mounting said tubing along a longitudinal axis of said tubing locating surface and contacting said tubing at multiple locations of said tubing.
<SOH> BACKGROUND <EOH>In a typical blood donation procedure, blood is collected from a donor using a plastic, disposable blood collection kit. In its most basic form, the blood collection kit includes a venipuncture needle, one or more plastic collection containers and a length of plastic tubing connected to the needle and the container(s) to provide a flow path therebetween. After collection, the blood may be further processed or stored until it is ready to be transfused to a patient. Before it is used, however, the collected blood must be tested to establish compatibility with the patient's (recipient's) blood type (e.g. A, B, AB, O) and/or possibly for other purposes as well. Thus, samples of the donated blood must be collected for the required testing. The blood that remains in the plastic tubing after the blood donation may be used for such samples. Recently, sealing devices have been developed to allow the plastic tubing to be sealed into one or more blood-containing segments. The segmented tubing portions can then be separated from the rest of the tube by cutting or tearing at the seal line. The blood in the detached segments can then be used for the tests that may be required. Presently, it is common to prepare up to as many as 16 separate blood-containing tubing segments which can then be used for the required tests. The sealing devices currently in use operate on the principle of heat-sealing. The device typically includes a sealing head with heatable jaws for compressing the tubing. The tubing is captured within a slot between the jaws, which compress the wall of the tubing, melts and fuses it. There are many commercially available sealing devices. One such sealing device is the Hematron II available from Baxter Healthcare Corporation of Deerfield, Ill. The Hematron II is a portable, dielectric sealing device that includes a single sealing head. Multiple tubing segments can be provided by manually advancing the tube through the sealing head and sealing the tubing at the desired locations. Another manufacturer of sealing devices is Starstedt of Newton, N.C. which provides a stationary device (as contrasted to a hand-held device of the type described below). Several modules of this device, each having a single sealing head, can be placed in a series to provide multiple (up to 6) seals in the tubing. More recently, hand-held sealing devices have been introduced. Examples of such hand-held devices include the Auto-Seal, Handy-Seal and the Hematron III, all available from Baxter Healthcare Corporation. Other hand-held and/or stationary devices are available from other suppliers/manufacturers. Although the devices currently available have generally worked satisfactorily, further improvements in the field of sealing blood tubing are desirable. For example, the sealing devices described above require either manual location of the tubing in the sealing head, manual movement or advancement of the tubing between the sealing heads or the combination of multiple sealing heads (which require greater energy, resulting in increased cost of the device). Thus, it would be desirable to provide a sealing device that limits the amount of manual involvement and quickly provides the required number of tubing segments. It would also be desirable to provide a low-cost, energy efficient apparatus.
<SOH> SUMMARY <EOH>In one aspect, the present invention is directed to an apparatus for providing multiple tubing segments. The apparatus includes a tubing locating surface for placing a length of blood-containing tubing thereon. The apparatus also includes sealing means cooperatively associated with the tubing locating surface for concurrently providing multiple, sealed, blood-containing tubing segments. In another aspect, the present invention is directed to a method for providing multiple, sealed, blood-containing, tubing segments. The method includes providing a length of blood-containing tubing, mounting the tubing on a tubing locating surface. The method further includes sealing the tubing to concurrently provide a plurality of sealed, blood-containing, tubing segments.

Polymeric material, molded article, and processes for producing these
Disclosed is a polymeric material obtained by melt-kneading, using a kneading machine, a resin composition which contains organic polymers having metal alkoxy groups. An organic-inorganic hybrid polymeric material which is suitable for use in high-performance and high-function polymeric materials, was provided.
1. A polymeric material obtained by melt-kneading, using a kneading machine, a resin composition which contains organic polymers having metal alkoxy groups, wherein the organic polymers having metal alkoxy groups have a main backbone of a thermoplastic resin which is polycarbonate, polyarylate, polysulfone, polyamide, polyacetal, polyethylene terephthalate, or polybutylene terephthalate. 2. A polymeric material obtained by melt-kneading, using a kneading machine, a resin composition which contains organic polymers having metal alkoxy groups and other organic polymers. 3. A polymeric material obtained by melt-kneading, using a kneading machine, a resin composition which contains organic polymers having metal alkoxy groups, other organic polymers and metal alkoxide compounds (which comprise partial hydrolyzates and polycondensates of metal alkoxide compounds). 4. A polymeric material obtained by melt-kneading, using a kneading machine, a resin composition which contains organic polymers having metal alkoxy groups, other organic polymers and metal oxides. 5. The polymeric material according to claim 1 obtained by allowing the organic polymers having metal alkoxy groups to react in the melt-kneading. 6. The polymeric material according to claim 2 obtained by allowing the organic polymers having metal alkoxy groups to react in the melt-kneading. 7. The polymeric material according to claim 3 obtained by allowing the organic polymers having metal alkoxy groups and the metal alkoxide compounds to react in the melt-kneading. 8. The polymeric material according to claim 4 obtained by allowing the organic polymers having metal alkoxy groups and the metal oxides to react in the melt-kneading. 9. The polymeric material according to any one of claims 1 to 4 wherein the kneading machine is a single screw extruder or a twin screw extruder. 10. (canceled) 11. The polymeric material according to claim 2 wherein other organic polymers have a main backbone of a thermoplastic resin. 12. The polymeric material according to claim 11 wherein the thermoplastic resin is polycarbonate, polyarylate, polysulfone, polyamide, polyacetal, polyethylene terephthalate, or polybutylene terephthalate. 13. The polymeric material according to claim 11 wherein the thermoplastic resin is a methacrylic resin, an acrylic resin, polystyrene, an AS resin (acrylonitrile/styrene copolymer), an ABS resin (acrylonitrile/butadiene/styrene copolymer), a vinyl chloride resin, or a polyphenylene sulfide. 14. The polymeric material according to any one of claims 1 to 8 wherein a metal element of the metal alkoxy groups is at least one selected from the group consisting of Si, Ti and Zr. 15. The polymeric material according to claim 3 or 7 wherein a metal element of the metal alkoxide compounds is at least one selected from the group consisting of Si, Ti and Zr. 16. The polymeric material according to claim 4 or 8 wherein a metal element of the metal oxides is at least one selected from the group consisting of Si, Ti and Zr. 17. The polymeric material according to any one of claims 1 to 8 wherein a metal element of the metal alkoxy groups is Si. 18. The polymeric material according to claim 3 or 7 wherein a metal element of the metal alkoxide compounds is Si. 19. The polymeric material according to claim 4 or 8 wherein a metal element of the metal oxides is Si. 20. A molded product obtained by molding the polymeric material according to claim 1 using a molding machine. 21. The molded product according to claim 20 wherein the molding machine is an injection molding machine or an extrusion molding machine. 22. A process for producing a polymeric material comprising: melt-kneading, using a kneading machine, a resin composition which contains organic polymers having metal alkoxy groups, wherein the organic polymers having metal alkoxy groups have a main backbone of a thermoplastic resin which is polycarbonate, polyarylate, polysulfone, polyamide, polyacetal, polyethylene terephthalate, or polybutylene terephthalate. 23. A process for producing a polymeric material comprising: melt-kneading, using a kneading machine, a resin composition which contains organic polymers having metal alkoxy groups and other organic polymers. 24. A process for producing a polymeric material comprising: melt-kneading, using a kneading machine, a resin composition which contains organic polymers having metal alkoxy groups, other organic polymers and metal alkoxide compounds (which comprise partial hydrolyzates and polycondensates of metal alkoxide compounds). 25. A process for producing a polymeric material comprising: melt-kneading, using a kneading machine, a resin composition which contains organic polymers having metal alkoxy groups, other organic polymers and metal oxides. 26. The process according to any one of claims 22 to 25 wherein the kneading machine is a single screw extruder or a twin screw extruder. 27. (canceled) 28. The process according to any one of claims 23 to 25 wherein other organic polymers have a main backbone of a thermoplastic resin. 29. The process according to claim 28 wherein the thermoplastic resin is polycarbonate, polyarylate, polysulfone, polyamide, polyacetal, polyethylene terephthalate, or polybutylene terephthalate. 30. The process according to claim 28 wherein the thermoplastic resin is a methacrylic resin, an acrylic resin, polystyrene, an AS resin (acrylonitrile/styrene copolymer), an ABS resin (acrylonitrile/butadiene/styrene copolymer), or a vinyl chloride resin, or a polyphenylene sulfide. 31. The process according to any one of claims 22 to 25 wherein a metal element of the metal alkoxy groups is at least one selected from the group consisting of Si, Ti and Zr. 32. The process according to claim 24 wherein a metal element of the metal alkoxide compounds is at least one selected from the group consisting of Si, Ti and Zr. 33. The process according to claim 25 wherein a metal element of the metal oxides is at least one selected from the group consisting of Si, Ti and Zr. 34. The process according to any one of claims 22 to 25 wherein a metal element of the metal alkoxy groups is Si. 35. The process according to claim 24 wherein a metal element of the metal alkoxide compounds is Si. 36. The process according to claim 25 wherein a metal element of the metal oxides is Si. 37. A process for producing a molded product comprising: molding the polymeric material according to claim 1 or 11 using a molding machine. 38. The process according to claim 37 wherein the molding machine is an injection molding machine or an extrusion molding machine.
<SOH> BACKGROUND ART <EOH>Plastics are substituting for existing materials, such as metal, glass, wood, and paper, due to their molding processabilities, high productivities, light weights, flexibilities, excellent mechanical or electrical properties, etc. Their application range is wide and they are used for a variety of applications such as construction materials, structural or mechanical parts of electric or electronic products, exterior or interior parts of automobiles, vehicles, aircraft and ships, miscellaneous goods and packing materials. For this reason, there are many kinds of plastics and those of various types are marketed. However, there is a great demand from the market for improvement in various characteristics or cost, and alloying of different plastics and compounding with other ingredients are performed briskly. For example, about the improvement in mechanical property, heat resistance, dimensional stability and the like, organic-inorganic composite materials in which a solid inorganic material typified by glass fiber and carbon fiber has been blended were studied. This technique has improved strength, thermal deformation resistance in a short period of time, dimensional stability and the like. However, a plastic and an inorganic material are generally incompatible and it is difficult to finely disperse both materials and, consequently, the size of dispersed particles of an inorganic material in an organic-inorganic composite material is generally up to the order of micrometers. Since the size of particles have great effects on strength such as tensile strength and the strength is reduced as particles becomes larger (see L. E. Nielsen, Dynamic Properties of Polymer and Composite Material, p. 253), it is natural that there are limitations to the improvement in strength of organic-inorganic composite materials described above. Further, for some types of plastics, e.g., ABS resin, polyamide 6-6, polycarbonate, polyacetal and fully aromatic polyester, there have been raised new problems such as reduction in impact strength caused by decrease in interfacial strength. On the other hand, organic-inorganic hybrid polymeric materials containing inorganic elements such as Si, Ti and Zr introduced into their backbone have been studied for the purpose of improvement in various physical properties of plastics including surface hardness, luster, antifouling property, strength, heat resistance, weather resistance, chemical resistance and the like. The size of dispersed particles of each component of an organic-inorganic hybrid polymeric material is up to the orders of sub-microns to nanometers and it is possible to disperse the components at the molecular level. As a method for the preparation thereof, for example, there have been known a method subjecting an organic monomer or an organic polymer and an inorganic backbone-containing compound to radical copolymerization and a method bonding an inorganic functional group such as alkoxysilane as a side chain to an organic polymer and thereafter cross-linking it. For example, Japanese Patent Kokai Publication No. H5-43679 and Japanese Patent Kokai Publication No. H5-86188 disclose a method for obtaining an organic-inorganic hybrid polymeric material by allowing a vinyl polymer and a silicon compound to react and thereafter cross-linking them by a sol-gel method. Japanese Patent Kokai Publication No. H8-104710 and Japanese Patent Kokai Publication No. H8-104711 disclose a method for obtaining an organic-inorganic hybrid polymeric material by subjecting vinyl monomers to radical polymerization with an alkoxysilyl group-terminated azo-type initiator and hydrolyzing and condensing the resulting alkoxysilyl group-terminated vinyl polymer. Further, we reported in Japanese Patent Kokai Publication No. H11-209596, etc., a method for obtaining an organic-inorganic hybrid polymeric material by synthesizing an alkoxysilyl group-terminated polycarbonate or polyarylate and thereafter hydrolyzing and polycondensing it by a sol-gel method. However, most of the conventional organic-inorganic hybrid polymeric materials are produced by methods in a solution system using a sol-gel method. The sol-gel method is a method for molding glass or ceramic by starting from a solution, passing a state of sol containing fine particles and further passing a state of gel containing a liquid or the air in a space defined by the frames of a solid (see Sumio SAKKA, Science of the Sol-Gel Method, Introduction). Accordingly, although simple structures such as films and rods can be produced, it is very difficult to produce molded products of complex shape. The methods carried out in a solution system are disadvantageous also in terms of productivity and cost and, therefore, are not practical except for specific applications. Japanese Patent Kokai Publication No. 2000-327930 discloses a method for producing an organic-inorganic hybrid polymeric material by heat-treating an organic polymer, an organic polymer having a metal alkoxy group, a metal alkoxide compound or a metal oxide with a mixer such as a Brabender. However, such a mixer has only a poor kneading ability since its mixing portion is constituted of a pair of blades having a short shaft. In addition, one pair of blades are fixed so that it is impossible to set the conditions of the kneading portion depending upon a material to be employed. Such a mixer, therefore, is difficult to finely disperse an organic polymer and an inorganic component such as metal oxide, which are of great incompatibility, with each other and is not suitable for the preparation of organic-inorganic hybrid polymeric materials. Furthermore, such a mixer has many problems with respect to steps and productivity for its industrial use since it is a batch-type instrument.
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention solves the conventional problems and the objective is to provide an organic-inorganic hybrid polymeric material suitable for high-performance and high-function plastics or a molded product containing the material as a component, and simple and practical methods for producing these with high productivity and with low cost. The present invention provides a polymeric material obtained by melt-kneading, using a kneading machine, a resin composition that contains organic polymers having metal alkoxy groups, a molded product obtained by molding the polymeric material using a molding machine, a process for producing a polymeric material comprising: melt-kneading, using a kneading machine, a resin composition that contains organic polymers having metal alkoxy groups and other organic polymers, and a process for producing a molded product comprising: processing the polymeric material using a molding machine. The object is achieved thereby.

Medicinal table with prolonged release of the active principle
The invention concerns a medicinal tablet to be sucked made with boiled sugar of solid consistency designed to dissolve in the buccal cavity, comprising at least an active principle. The invention is characterised in that it further comprises at least a matrix agent for slowing down the release of the active principle(s) which therefore remain in prolonged contact with the region of the mouth and the pharynx the dissolving time in the buccal cavity being at least 15 minutes.
1. A medicinal cooked sugar pastille to be sucked, of solid consistency, intended to dissolve in the buccal cavity, comprising at least one active principle, characterized in that it further comprises at least one matrix agent allowing the release of the active principle(s) to be slowed, which active principle(s) then remain(s) in prolonged contact with the buccopharyngeal area, the time of dissolution in the buccal cavity being at least 15 minutes. 2. The pastille as claimed in claim 1, characterized in that the matrix agent is an agent capable of slowing the dissolution of the cooked sugar in the mouth. 3. The pastille as claimed in either one of claims 1 and 2, characterized in that the matrix agent further confers on the pastille an increased resistance, lasting even on contact with the saliva, so that the patient cannot bite this pastille and swallow bits of it. 4. The pastille as claimed in any one of claims 1 to 3, characterized in that the time of dissolution of the pastille in the buccal cavity is from 25 to 35 minutes, preferably 30 minutes. 5. The pastille as claimed in any one of claims 1 to 4, characterized in that the matrix agent is chosen from the group formed by noncellulosic polysaccharides, cellulosic derivatives, acrylic acid polymers, fatty substances and polyvinylpyrrolidone, these substances being used alone or as a mixture and representing 1 to 10% by weight of the pastille, typically 1 to 5%. 6. The pastille as claimed in any one of claims 1 to 5, characterized in that the matrix agent is chosen from the group formed by: guar gum, carob gum, sodium and potassium alginates, agar-agar, carrageenan, gum arabic, sterculia gum, gum tragacanth. 7. The pastille as claimed in any one of claims 1 to 5, characterized in that the matrix agent is chosen from the group formed by hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylcellulose. 8. The pastille as claimed in any one of claims 1 to 5, characterized in that the acrylic acid polymer is a carbomer, a polymethacrylate or a copolymer of vinyl acetate. 9. The pastille as claimed in any one of claims 1 to 5, characterized in that the fatty substance is chosen from the group formed of waxes, “Gelucire” products, glyceryl behenate, glyceryl palmitostearate. 10. The pastille as claimed in any one of claims 1 to 5, characterized in that the matrix agent is polyvinylpyrrolidone. 11. The pastille as claimed in any one of claims 1 to 10, characterized in that it further comprises a major sugary excipient, or diluent, chosen from sucrose, fructose, lactose, sorbitol, mannitol, lactitol, glucose, maltitol, isomaltol, polydextrose and malto-dextrins, used alone or as a mixture, representing 80 to 99% by weight of the pastille. 12. The pastille as claimed in any one of claims 1 to 11, characterized in that it further comprises at least one auxiliary substance chosen from sweeteners, antioxidants, colorants and flavors. 13. The pastille as claimed in any one of claims 1 to 11, characterized in that it comprises nicotine as active principle. 14. The pastille as claimed in claim 13, characterized in that it comprises isomaltol, methocel, nicotine and aspartame. 15. A process for production of a medicinal pastille as claimed in any one of claims 1 to 13, characterized in that it comprises successively: a step of boiling a syrup of sugary diluent substance; a step of cooking at a higher temperature, of the order of 100° C. to 160° C.; a step of mixing with incorporation of the active principle(s) and of the auxiliary substance(s); a step of production of the pastille; the matrix agent being incorporated either in the the course of the boiling step, or in the course of the cooking step, or during mixing.



Method for conducting solid phase synthesis of molecule libraries using combinatorial sealing matrices
The invention relates to a method for producing molecule libraries at predetermined locations on a substrate surface by means of sequential chemical reactions involving the use of combinatorial sealing matrices. The topological structures applied to the sealing matrices cover individual areas of the substrate surface in a defined order thereby preventing different partial areas of the substrate surface from undergoing chemical conversions. Several sealing matrices having different topological relief structures can be used in a number of reaction cycles for the synthesis of molecule libraries consisting of complex chemical compounds. The sealing matrices are made of an elastic material such as polydimethylsiloxane. The synthesis involves the use of simple and highly optimized standard methods and standard chemicals of the solid phase synthesis. The reaction rate is accelerated by carrying out the reaction steps in a microfluidic flow-through system. The inventive method can be used for easily and rapidly producing molecule libraries in the microarray format.
1. A method for producing geometrically arranged molecule libraries comprising chemical compounds on a substrate surface, which method is characterized by the following steps: preparing at least one sealing matrix whose relief-like topological structures ensure a sealing contact with said substrate surface at predefined sites; contacting said sealing matrix with said substrate surface; conducting a chemical reaction on the substrate surface areas not covered by said sealing matrix; separating said sealing matrix from said substrate surface. 2. The method as claimed in claim 1, characterized in that two or more different or identical sealing matrices are used for conducting two or more chemical reactions on the substrate surface. 3. The method as claimed in claim 1, characterized in that the molecule library synthesized on the substrate surface forms an array of different groups of chemical compounds whose composition and location are known. 4. The method as claimed in claim 1, characterized in that the topological structures on the sealing matrices and the chemical compounds synthesized at the predefined sites of the substrate surface are arranged in the form of a dot matrix, a circular, helical, strip-shaped, linear or other geometrical structure. 5. The method as claimed in claim 1, characterized in that the molecule library synthesized on the substrate surface can be used for conducting parallel binding reactions. 6. The method as claimed in claim 1, characterized in that the chemical reaction is covalent linking, chemical or enzymic modification, coupling, cleavage, hydrolysis, noncovalent bonding or another chemical reaction. 7. The method as claimed in claim 1, characterized in that the sealing matrix comprises, at least partially, an elastic material, preferably, polydimethylsiloxane. 8. The method as claimed claim 1, characterized in that different parts of the sealing matrix comprise different materials. 9. The method as claimed claim 1, characterized in that the sealing matrix is made of a material transparent for at least one particular wavelength and thus enables photochemical reactions or near-field optical processes to be carried out via location-selective light conduction. 10. The method as claimed in claim 8, characterized in that the sealing matrix transparent for a particular wavelength enables relative positioning of said sealing matrix on the substrate surface to be controlled via location-selective light conduction. 11. The method as claimed in claim 1, characterized in that the sealing matrix has an electrically conducting surface, in particular for controlling electrochemical reactions. 12. The method as claimed in claim 1, characterized in that a topological structure on the sealing matrix covers a plurality of areas designed for the synthesis of chemical compounds. 13. The method as claimed in claim 1, characterized in that individual areas on the substrate surface, designed for the synthesis of chemical compounds, have a spatial dimension of preferably less than 10 μm, in particular less than 2 μm. 14. The method as claimed in claim 1, characterized in that the substrate surface has activated, geometrically arranged areas on which chemical compounds are synthesized. 15. The method as claimed in claim 14, characterized in that the areas designed for the synthesis of chemical compounds are activated by location-selective silanization with ω-functionalized ethoxy- or methoxy-or chlorosilanes or by applying functionalized thiols or disulfides to noble metal films. 16. The method as claimed in claim 1, characterized in that the contact faces of the topological structures on the sealing matrix are larger than the substrate surface areas designed for the synthesis of chemical compounds. 17. The method as claimed in claim 1, characterized in that the surface areas outside the areas designed for the synthesis of chemical compounds are passivated. 18. The method as claimed in claim 17, characterized in that the areas on which no chemical compounds are to be synthesized are passivated by coating with polymers, quenchers or proteins or by location-selectively deactivating or removing active groups. 19. The method as claimed in claim 1, characterized in that the chemical compounds synthesized on the substrate surface are DNA, RNA, aptamers or their, in particular nuclease-resistant, derivatives such as PNA or thioRNA. 20. The method as claimed in claim 1, characterized in that the chemical compounds synthesized on the substrate surface are peptides, proteins or their derivatives. 21. The method as claimed claim 1, characterized in that the chemical compounds synthesized on the substrate surface are carbohydrates. 22. The method as claimed claim 1, characterized in that the chemical compounds synthesized on the substrate surface are dendrimers or other organic or inorganic macromolecules. 23. An apparatus for preparing the sealing matrices and molecule libraries on a substrate surface according to claim 1, characterized in that individual steps are carried out semi-automatically or automatically. 24. A kit comprising the essential substances for producing molecule libraries according to any of the methods as defined in claim 1. 25. A kit comprising the essential substances for carrying out binding assays on molecule libraries according to any of the methods as defined in claim 1. 26. A sealing matrix having relief-like topological structures, in particular for carrying out the methods as claimed in claim 1. 27. The sealing matrix as claimed in claim 26, characterized by at least one feature of of the following: (a) that the topological structures on the sealing matrices and the chemical compounds synthesized at the predefined sites of the substrate surface are arranged in the form of a dot matrix, a circular, helical, strip-shaped, linear or other geometrical structure; (b) the sealing matrix comprises, at least partially, an elastic material, preferably, polydimethylsiloxane; ©) different parts of the sealing matrix comprise different materials; (d) the sealing matrix is made of a material transparent for at least one particular wavelength and thus enables photochemical reactions or near-field optical processes to be carried out via location-selective light conduction; (e) the sealing matrix transparent for a particular wavelength enables relative positioning of said sealing matrix on the substrate surface to be controlled via location-selective light conduction; (f) the sealing matrix has an electrically conducting surface, in particular for controlling electrochemical reactions; (g) a topological structure on the sealing matrix covers a plurality of areas designed for the synthesis of chemical compounds; (h) the contact faces of the topological structures on the sealing matrix are larger than the substrate surface areas designed for the synthesis of chemical compounds. 28. A device for carrying out the method as claimed in claim 1, preferably in the form of a cassette, characterized in that it has at least one sealing matrix or is designed for received such a sealing matrix.



Pkb-3564 substance with neovascularization inhibitory activity
A compound represented by formula (I) wherein R1 represents hydrogen atom and R2 represents hydroxyl group, or R1 and R2 may combine together to represent oxo group or oxime group; R3 represents hydrogen atom and R4 represents hydroxyl group, or R3 and R4 may combine together to represent oxo group or oxime group; R5 represents hydrogen atom and R6 represents hydroxyl group, or R5 and R6 may combine together to represent oxo group or oxime group; R7 represents hydrogen atom and R8 represents hydrogen atom, or R7 and R8 may combine together to represent oxo group or oxime group; R9 and R10 represent hydrogen atom, an alkyl group, or an alkenyl group. The compound is useful as an active ingredient of a medicament having angiogenesis suppressive activity, apoptosis suppressive activity, and cell cycle inhibitory activity.
1. A compound represented by the general formula (I): wherein R1 represents hydrogen atom and R2 represents hydroxyl group, or R1 and R2 may combine together to represent oxo group or oxime group; R3 represents hydrogen atom and R4 represents hydroxyl group, or R3 and R4 may combine together to represent oxo group or oxime group; R5 represents hydrogen atom and R6 represents hydroxyl group, or R5 and R6 may combine together to represent oxo group or oxime group; R7 represents hydrogen atom and R8 represents hydrogen atom, or R7 and R8 may combine together to represent oxo group or oxime group; and R9 and R10 independently represent hydrogen atom, an optionally substituted alkyl group, or an optionally substituted alkenyl group. 2. The compound according to claim 1, wherein R1 is hydrogen atom, R2 is hydroxyl group, R3 and R4 combine together to represent oxo group, R5 is hydrogen atom, R6 is hydroxyl group, R7 and R8 combine together to represent oxo group, and R9 and R10 are both methyl groups. 3. The compound according to claim 1, wherein R1 and R2 combine together to represent oxo group, R3 and R4 combine together to represent oxo group, R5 and R6 combine together to represent oxo group, R7 and R8 combine together to represent oxo group, and R9 and R10 are both methyl groups. 4. A compound represented by the general formula (II): wherein R11 represents hydrogen atom and R12 represents hydroxyl group, or R11 and R12 may combine together to represent oxo group or oxime group; R13 represents hydrogen atom and R14 represents hydroxyl group, or R13 and R14 may combine together to represent oxo group or oxime group; R15 represents hydrogen atom and R16 represents hydroxyl group, or R15 and R16 may combine together to represent oxo group or oxime group; R17 represents hydrogen atom and R18 represents hydrogen atom, or R17 and R18 may combine together to represent oxo group or oxime group; and R19 and R20 independently represent hydrogen atom, an optionally substituted alkyl group, or an optionally substituted alkenyl group. 5. The compound according to claim 4, wherein R11 is hydrogen atom, R12 is hydroxyl group, R13 and R14 combine together to represent oxo group, R15 is hydrogen atom, R16 is hydroxyl group, R17 and R18 combine together to represent oxo group, and R19 and R20 are both methyl groups. 6. A medicament which comprises the compound according to any one of claims 1 to 5 as an active ingredient. 7. The medicament according to claim 6, which is an angiogenesis inhibitor. 8. The medicament according to claim 6, which is an apoptosis suppressant. 9. The medicament according to claim 6, which is a cell cycle inhibitor. 10. The medicament according to claim 6, which is an antitumor agent. 11. The medicament according to claim 6, which is used for preventive and/or therapeutic treatment of a disease in which abnormal acceleration of apoptosis is involved.
<SOH> BACKGROUND ART <EOH>Apoptosis, a mode of cell death reported in the 1970s, differs from necrosis and is a cell death induced through a specific intracellular signal transduction system [Cell, 88, 355-365 (1997)]. Apoptosis is involved in pathologic conditions of variety of diseases. A control of apoptosis may possibly contribute to progresses of therapeutic treatments of these diseases. For example, a deviation from a normal apoptosis control mechanism and a resulting acceleration of cell death are believed to induce articular rheumatism, hepatitis with viral infection such as hepatitis B and C, fulminant hepatitis, diabetes, myocardial infarction, ulcerative colitis, brightism, alopecia, neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, ischemic brain damage, acquired immune deficiency syndrome, ecstatic cardiomyopathy and the like [Science, 267, 1456-1462 (1995)]. Examples of physiological stimulation inducing apoptosis include Fas/Apo-1/CD95 ligand, Fas antibody, TNF (tumor necrosis factor) and the like [Cell, 88, 355-365 (1997)]. As compounds suppressing apoptosis, various peptide caspase inhibitors as cysteine protease inhibitors and the like are known; however, the compounds have problems in instability inherent to peptide compounds and insufficient potency. Antitumor agents clinically used at present can achieve temporal retraction or disappearance of cancers on the basis of their cytotoxicity. However, as they act on healthy cells to cause serious adverse effects, their applications are much limited. In addition, among gastric cancers, colon cancers, pancreatic cancers and the like, there are a number of naturally resistant cancers to which anticancer agents are almost ineffective, or appearance of acquired resistant cancer cells to which primarily effective anticancer agents become ineffective, which causes a serious problem. Recently, so-called tumor angiogenesis for supplying nutriment and oxygen to cancer cells has been focused as a mechanism of solid cancer proliferation beyond a certain size. An idea of “a therapy by inhibition of tumor angiogenesis” has been being established in which cancer proliferation is suppressed by inhibiting tumor angiogenesis [European Journal of Cancer, 32A, 2534-2539 (1996)]. Although a number of compounds have already been practically developed as antitumor agents, clinical uses of an angiogenesis inhibitors as early as possible have been desired, because they have advantages that conventional antitumor agents do not have. Some angiogenesis inhibitors have been developed and studied so far; however, novel compounds as potential lead compounds have always been desired strongly. A recruit mechanism of vascular endothelial cells into a cancer tissue has a number of similarities to a recruit mechanism of leukocytes and the like into inflammatory sites, and therefore, medicaments inhibiting chemotaxis of vascular endothelial cells are expected to have potentials as anti-inflammatory agents as well as angiogenesis inhibitors [Immunity, 12, 121-127 (2000)].


Structure determination of materials using electron microscopy
The invention includes methods for automating acquisition of electron microscopic images. Methods use in the invention include development of search algorithms, including spiral search algorithms for the automated determination of areas suitable for imaging in vitrified specimens at liquid nitrogen temperatures, development of criteria to image areas that meet user-specific needs for the thickness of vitreous ice in which the proteins are embedded, automated setting of key imaging parameters such as extent of defocus and magnification required for recording images, automated assessment of most suitable conditions such as thermal and mechanical stability of specimen stage immediately before recording of the image, recording a “low-dose” image of radiation sensitive biological specimens by carrying out all of the setting and assessment steps on an area immediately adjacent to the area of interest, thereby avoiding pre-exposure of the final imaged area to electrons, creation of a seamless interface to transfer the images recorded on a camera directly to computers capable of processing the recorded images, and carrying Automated out the entire process of data collection from a remote computer either within the network, or connected to the network through a telephone modem from any remote location.
1. An automated method for the structural determination of a sample using electron microscopy, said method comprising the steps of: (a) defining one or more imaging parameters; (b) selecting one or more sample sites for imaging based upon said imaging parameters; and (c) imaging said one or more sample sites. 2. The method of claim 1, wherein said imaging parameters comprise the chemical composition of said sample. 3. The method of claim 1, wherein said imaging parameters comprise the buffer conditions at sample preparation. 4. The method of claim 1, additionally comprising the step of preparing said sample in a liquid medium. 5. The method of claim 4, additionally comprising the step of freezing said sample and said liquid medium. 6. The method of claim 5, wherein the frozen thickness of said sample comprises an imaging parameter. 7. The method of claim 5, wherein the sample is frozen using ethane. 8. The method of claim 5, wherein said sample is frozen to a temperature of at least about −1700° C. 9. The method of claim 1, wherein more than one sample site is imaged. 10. The method of claim 9, wherein said samples are identified by the electron microscope which undertakes a patterned search routine. 11. The method of claim 10, wherein said search routine pattern is spiral. 12. An automated method for the structural determination of a sample using electron microscopy, said method comprising the steps of: (a) setting imaging parameters; (b) setting operating conditions of said microscope wherein said operating conditions are set based on said imaging parameters, and wherein said microscope comprises an electron beam and a microscope stage; (c) locating at least one specific sample site to image; (d) adjusting focus settings; (e) centering said electron beam within said sample site; and (f) taking a picture. 13. The method of claim 12, wherein said sample comprises a frozen solution. 14. The method of claim 13, wherein said imaging parameters comprise the thickness of said frozen sample. 15. The method of claim 12, wherein said step of locating at least one sample site to image further comprises the step of sending said microscope stage to a specific location. 16. The method of claim 15, wherein said step of locating at least one sample site to image further comprises the step of irradiating a portion of said frozen sample log enough to melt a portion of said frozen sample. 17. The method of claim 16, wherein said step of locating at least one sample site to image further comprises the step of estimating the thickness of said frozen sample at said sample site. 18. The method of claim 12, wherein said step of taking a picture comprises taking a picture on a charge coupled device camera. 19. The method of claim 12, wherein said step of taking a picture comprises taking a picture on photographic film. 20. The method of claim 12, wherein said step of locating at least one sample site to image further comprises the step of allowing users to store x,y locations of regions suitable for imaging.
<SOH> BACKGROUND OF THE INVENTION <EOH>Over the last three decades, the three-dimensional structures of a variety of biologically interesting macromolecular complexes with helical, icosahedral, octahedral or no symmetry have been determined using images recorded with an electron microscope. High resolution electron microscopic analyses of two-dimensional crystals have in a few instances resulted in structures of proteins with atomic resolution. While analysis of non-crystalline “single particle” suspensions has resulted in the determination of many structures at medium (7 Å-15 Å) resolutions. Although the last decade has seen considerable improvements in the speed of image analysis, the availability of sufficient numbers of high quality images continues to be the rate limiting step for almost all biological structure determination projects. An obvious approach to increase the throughput in structure determination efforts is to introduce automated data collection routines that emulate the manual data recording strategies employed by experienced users. Several reports have already appeared in the literature with this aim in mind, and have been pioneered by researchers interested in structure determination using electron tomography, single molecule and helical reconstruction and two-dimensional crystallography. The availability of the partially computerized customized microscopy (“CM”) series of microscopes which allowed the construction of efficient hardware and software interfaces to access key microscope controls has been an important element in the success of these efforts. A few recent reports have reported the introduction of significant levels of automation in the data collection process using CM series microscopes, however, these microscopes are no longer manufactured. For example, Carragher et al., disclose partially automated methods for obtaining electron micrographs in “Leginon: An Automated System for Acquisition of Images from Vitreous Ice Specimens,” Journal of Structural Biology, (132, 33-45 (2000)). Similarly, Fung discloses processes for elucidating the three-dimensional architecture of large biological complexes and subcellular organelles in “Toward Fully Automated High-Resolution Electron Tomography,” Journal of Structural Biology, (116, 181-189 (1996)). Fung also describes systems that automate the various steps necessary for data collection in tomography. Kisseberth et al. disclose tools that may be used in the control of a remotely situated electron microscope in “emScope: A Tool Kit for Control and Automation of a Remote Electron Microscope”, Journal of Structural Biology (120, 309-319(1997)). However, much further work is needed in order to eliminate interfaces that decrease image clarity, to allow for more efficient data collection, and to decrease the need for operator interface with the microscope.
<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with one aspect of the invention, there is provided an automated method for the structural determination of a sample using electron microscopy including the steps of defining one or more imaging parameters, selecting one or more sample sites for imaging based upon the imaging parameters, and imaging the one or more sample sites. In accordance with another aspect of the invention, there is provided an automated method for the structural determination of a sample using electron microscopy including the steps of setting imaging parameters, setting operating conditions of the microscope where the operating conditions are set based on the imaging parameters, and where the microscope includes an electron beam and a microscope stage, locating at least one specific sample site to image, adjusting focus settings, centering the electron beam within the sample site, and taking a picture. With the advent of computerized microscopes such as the Tecnai series from FEI (Hillsboro, Oreg.), it is now possible to envision general methods for automated data collection that not only completely do away with the requirement of specialized microscope-specific hardware interfaces, but are also accessible to any user via a standardized Windows-based or equivalent desktop interface. The prospect of invoking and monitoring automated data collection on a Tecnai microscope from anywhere in the world by working from a personal computer is a highly attractive one that offers potentially powerful opportunities for high-throughput data collection. The process of three-dimensional structure determination of a material by electron microscopy can be broken into three distinct stages: (i)specimen preparation, which for example can encompass protein purification and transfer of the sample onto a specimen holder and into the electron microscope, (ii)data collection, which is the process of recording images at the required electron optical settings onto either film or a charge coupled device (“CCD”) camera and (iii)image analysis and three-dimensional reconstruction, which computationally processes the information contained in a collection to obtain a three-dimensional structure of the imaged material. The time necessary to collect the required number of images is an important parameter in all microscopic experiments, but is especially crucial and rate-limiting in high-resolution electron microscopic approaches for biological structure determination. Methods to speed up data collection can be very useful, especially in “single molecule” microscopy of large multi-protein and protein-nucleic acid complexes, where many thousands of individual molecular images have to be averaged to determine the three-dimensional structure. The claimed invention provides methods for automated low-dose image acquisition procedures on microscopic equipment such as a Tecnai 12 electron microscope (manufactured by FEI, formerly Philips Electron Optics Inc.). In a typical semi-automated session, the user inserts the specimen into the microscope, and quickly selects regions of interest to be imaged. All subsequent steps of image acquisition are carried out automatically to record high resolution images on either film or CCD, at desired defocus values, and under conditions that satisfy user-specified limits for drift rates of the specimen stage. In the fully automated procedure, the initial determination of regions suitable for imaging is also carried out automatically. The claimed methods also automate all steps following insertion of the specimen in the microscope. These steps can be carried out on a remote personal computer connected to the microscope computer by way of the Internet. Both features are implemented using Windows NT and web-based tools, and in principle, provide tools for automated data collection on any Tecnai microscope by any user from any location. The invention allows automation of the steps of structure determination of a variety of entities of fundamental interest in biological, medical and veterinary research, with further application to materials science and semiconductor-related materials. Specifically, the invention allows for automated data collection from protein complexes frozen in vitreous ice. Once the specimen is inserted into the microscope, the remaining steps required for data collection can be fully automated. Knowledge of the three-dimensional structures of biological macromolecules and subcellular assemblies is fundamental both to understanding their function in cells and in rational drug design efforts. The methods of the invention are especially suitable for structural analysis of cellular components that are not easily studied by traditional structural tools such as X-ray crystallography and NMR spectroscopy. The methods may become useful as routine and powerful diagnostic tools in the emerging area of molecular medicine.

Magnetic sensor and method for analysing a fluid
An apparatus and method for analysing a fluid including means for generating a non-uniform magnetic field in a space and means for measuring the strength of the magnetic field arranged to enable a change in the field strength to be measured when a sample of fluid to be analysed is introduced into the space.
1. An apparatus for analysing a fluid, comprising: means for generating a non-uniform magnetic field in a space, the field varying with linear distance in the space; and means for measuring a magnetic field strength over a linear distance to enable a change in the magnetic field strength to be measured when a sample of fluid to be analysed is introduced into the space. 2. The apparatus as claimed in claim 1, wherein the means for generating the non-uniform magnetic field comprises two opposed, spaced apart, permanent magnets arranged to generate said non-uniform magnetic field between each other. 3. The apparatus as claimed in claim 1, wherein the means for generating the non-uniform magnetic field comprises two opposed, spaced apart, permanent magnets each mounted on a yoke and arranged to generate said non-uniform magnetic field between each other. 4. Apparatus as claimed in claim 1, wherein the means for generating the non-uniform magnetic field comprises two opposed, spaced apart, permanent magnets arranged to generate said non-uniform magnetic field between each other, wherein each permanent magnet is fitted with a shaped pole piece operative to introduce non-uniformity into the field. 5. The apparatus as clamed in claim 1, wherein the means for measuring the magnetic field strength comprises a linear array of magnetic field detectors. 6. The apparatus as claimed in claim 1, wherein the means for measuring the magnetic field strength comprises a linear array of magnetic field detectors extending in a direction in which the field strength varies. 7. The apparatus as claimed in claim 1, wherein the means for measuring the magnetic field strength comprises a single field detector mounted on a positional apparatus operative to move the field detector to scan a field over a linear distance in which the magnetic field strength varies. 8. The apparatus as claimed in claim 1, wherein the means for measuring the magnetic field strength comprises a linear array of magnetic field detectors and each of said detectors produces an output which depends upon the magnetic field strength measured by each detector and the output from each detector is transmitted to a processing means arranged to determine a change in the magnetic field strength sensed by each detector on introduction of a sample to be analysed into the space. 9. The apparatus as claimed in claim 1, wherein the means for measuring the magnetic field strength comprises a single field detector that produces an output which depends upon the magnetic field strength measured by the field detector, wherein the field detector is mounted on a positional apparatus operative to move the field detector to scan a field over a linear distance in which the magnetic field strength varies and the output from the field detector is transmitted to a processing means arranged to determine a change in magnetic field strength sensed by the field detector on introduction of a sample to be analysed into the space. 10. The apparatus as claimed in claim 1, wherein the means for measuring the magnetic field strength comprises a linear array of magnetic field detectors, wherein each of said detectors produces an output that depends upon the magnetic field strength measured by each detector, the output from each detector is transmitted to a processing means arranged to determine a change in magnetic field strength sensed by each detector on introduction of a sample to be analysed into the space, and the processing means is arranged to annul the output from each detector such that when the space is empty the output from each detector is zero and that on introduction of a sample into the space a positive or negative output from each detector represents the change in magnetic field strength brought about by introduction of the sample. 11. The apparatus as claimed in claim 1, wherein the means for measuring the magnetic field strength comprises a single field detector that produces an output which depends upon the magnetic field strength measured by the field detector, the field detector is mounted on a positional apparatus operative to move the field detector to scan a field over a linear distance, the output from the detector is transmitted to a processing means arranged to determine a change in magnetic field strength sensed by the field detector on introduction of a sample to be analysed into the space, and the processing means is arranged to annul the output from the field detector such that when the space is empty the output from each detector is zero and that on introduction of a sample into the space, a positive or negative output from each detector represents the change in magnetic field strength brought about by introduction of the sample. 12. The apparatus as claimed in claim 1 wherein, the means for measuring the magnetic strength comprises a linear array of magnetic field detectors, wherein each detector produces an output which depends upon the strength of the magnetic field measured by each detector and the output from each detector is transmitted to a processing means arranged to determine a change in magnetic field strength sensed by each detector on introduction of a sample to be analysed into the space, and wherein the processing means is arranged to compare the measured change in magnetic field strength with stored information and to provide an indication of contents of a sample being analysed based upon said comparison. 13. The apparatus as claimed in claim 1, wherein the means for measuring comprises a single field detector that produces an output which depends upon the magnetic field strength measured by the field detector, the field detector is mounted on a positional apparatus operative to move the field detector to scan a field over a linear distance, and the output from the detector is transmitted to a processing means arranged to determine a change in magnetic field strength sensed by the field detector on introduction of a sample to be analysed into the space, and wherein the processing means is arranged to compare the change in magnetic field strength with stored information and to provide an indication of contents of a sample being analysed based upon said comparison. 14. The apparatus as claimed in claim 1, wherein the means for measuring the magnetic field strength comprises a linear array of magnetic field detectors, wherein each detector produces an output which depends upon the strength of the magnetic field strength measured by each detector and the output from each detector is transmitted to a processing means arranged to determine the change in magnetic field strength sensed by each detector on introduction of a sample to be analysed into the space, and wherein the processing means further comprises a control unit including a means for outputting information to a user. 15. The apparatus as claimed in claim 1, wherein the means for measuring the magnetic field strength comprises a single field detector that produces an output which depends upon the magnetic field strength measured by the field detector, the field detector is mounted on a positional apparatus operative to move the field detector to scan a field over a linear distance, and the output from the field detector is transmitted to a processing means arranged to determine a change in magnetic field sensed by the field detector on introduction of a sample to be analysed into the space, and wherein the processing means further comprises a control unit including a means for outputting information to a user. 16. The apparatus as claimed in claim 1, wherein the means for generating a non-uniform magnetic field and the means for detecting the magnetic field strength are adapted to be worn on a human or animal body. 17. A method for analysing fluid comprising the steps of: applying a non-uniform magnetic field that varies with linear distance in space to a sample of fluid to be analysed; and measuring a magnetic field strength over a linear distance to determine a change in the magnetic field strength caused by the sample of fluid. 18. The method as claimed in claim 17, wherein the magnetic field strength measured is compared with stored information to provide an indication of the contents of the sample of fluid being analysed. 19. The method as claimed in claim 17, wherein the sample of fluid is blood.
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to an apparatus and method for analysing a fluid, particularly, although not exclusively, body fluids such as blood, to determine the presence and concentration of various substances in blood. 2. Description of the Related Art Analysis of blood is widely practised in the medical treatment and diagnosis of humans and animals. A plurality of methods are known for analysing blood. Embodiments of the present invention seek to provide an alternative method for analysing blood. Conventionally it is necessary for a sample of blood to be removed from a living body for analysis outside the body. This can be unpleasant and inconvenient, especially if frequent analysis of blood is required such as can be the case for a sufferer of diabetes where frequent analysis of the concentration of glucose in their blood is necessary. Embodiments of the present invention seek to provide an apparatus and method for non-invasive analysis of blood in the body.
<SOH> SUMMARY OF THE INVENTION <EOH>According to a first aspect of the present invention there is provided apparatus for analysing a fluid comprising means for generating a non-uniform magnetic field in a space, the field varying with linear distance in the space, and means for measuring the strength of the field over a linear distance to enable a change in the strength of the field to be measured when a sample of fluid to be analysed is introduced into the space. According to a second aspect of the present invention there is provided a method for analysing fluid comprising the steps of: applying a non-uniform magnetic field which varies with linear distance in space to a sample of fluid to be analysed and measuring the field strength over a linear distance to determine a change in the strength of the field caused by the presence of the sample. All materials become magnetized to some extent when placed in a magnetic field. The extent and sign of magnetisation depends upon the nature of the material. When a material becomes magnetised in a magnetic field it experiences a force. Where a fluid containing a number of materials which respond differently to a magnetic field is placed into a non-uniform magnetic field the different materials are subjected to different forces and will thus tend to migrate to different regions of the field. The different magnetic properties of the materials present in the fluid will influence the field in different ways. With a knowledge of the way in which different materials influence the field it is possible to infer their presence and also concentration in a fluid subjected to a non-uniform magnetic field, by measuring the way in which the presence of the fluid influences the magnetic field. The means for generating the non-uniform magnetic field preferably comprises two opposed, spaced apart permanent magnets, such as rare earth magnets, arranged to generate a field between each other. The magnets may be mounted on a yoke, for example a soft iron yoke. The magnets are each preferably fitted with a shaped pole piece operative to introduce non-uniformity into the field. The means for measuring the strength of the field may comprise a linear array of magnetic field detectors, such as Hall effect devices. Any Hall effect devices are preferably of high sensitivity, especially of sensitivity of at least 30 mVmAkG −1 . Any Hall effect devices preferably comprise a ternary material. The array preferably extends in a direction in which the field strength varies. Each detector in the array preferably produces an output that depends upon the strength of the magnetic field measured by the detector. The output from each detector is preferably transmitted to a processing means. The processing means, which is preferably an electric or electronic processing means and may comprise a computer, is preferably arranged to determine the change in magnetic field sensed by each detector on introduction of a sample to be analysed into the space. One way in which this can be achieved is for the processing means to annul the output from each detector such that when the space is empty the output from each detector is zero. Then, on introduction of a sample into the space, the positive or negative output from each detector represents the change in field brought about by introduction of the sample. Alternatively, the means for measuring strength of magnetic field may comprise a single field detector mounted on positional apparatus operative to move the detector to near the field over a linear distance. The change in field strength along the direction in which the field is measured will be indicative of the constituents of a sample being analysed. The processing means is preferably arranged to compare the measured change in field strength with stored information and to provide an indication of the contents of a sample being analysed, based upon the comparison. The processing means is preferably comprised in a control unit including a means for outputting information to a user, for example a display. The apparatus is suitable for analysing blood whilst in the body. For this it is preferable that the means for generating a magnetic field and the means for detecting the magnetic field are adapted to be worn on the body. Conveniently, they may be comprised in a clip adapted to be worn on an ear lobe. The apparatus and method enable rapid analysis of fluids, particularly blood, which may be analysed non-invasively. Various other purposes and advantages of the invention will become clear from its description in the specification that follows and from the novel features particularly pointed out in the appended claims. Therefore, to the accomplishment of the objectives described above, this invention consists of the features hereinafter illustrated in the drawings, fully described in the detailed description of the preferred embodiment and particularly pointed out in the claims. However, such drawings and description disclose but one of the various ways in which the invention may be practiced.

Nucleic acid coding for the cgl1 polypeptide and diagnostic and therapeutic application of said nucleic acid and of the cgl1 polypeptide
The invention provides a nucleic acid coding for the CGL1 polypeptide with the SEQ ID No 1 amino acid sequence or also fragments or variants of the CGL1 polypeptide. The invention also relates to the use of a nucleic acid such as defined hereinabove for producing a nucleotide probe or primer specific for the normal cgl1 gene or the mutated cgl1 gene. Another object of the invention is also to provide methods for screening a candidate compound interacting with the CGL1 polypeptide or modulating the expression of the cgl1 gene as well as sets or kits for screening such candidate compounds.
1. A nucleic acid encoding for a CGL1 polypeptide or for a fragment or a variant of the CGL1 polypeptide with a SEQ ID No 1 amino acid sequence. 2. A nucleic acid according to claim 1, characterized in that the variant of the CGL1 polypeptide is selected amongst polypeptides with amino acid sequences from SEQ ID No 2 to SEQ ID No 10. 3. A nucleic acid according to claim 1, characterized in that the fragment of the CGL1 polypeptide is selected amongst polypeptides with amino acid sequences from SEQ ID No 11 to SEQ ID No 13. 4. A nucleic acid according to claim 1, characterized in that it comprises the polynucleotide ranging from the nucleotide in position 345 up to the nucleotide in position 1541 of the SEQ ID No 14 nucleotide sequence. 5. A nucleic acid according to claim 1, characterized in that it comprises the cgl1 gene contained in the BAC vector referred to as RP 11-831H9 with its nucleotide sequences being referenced in the GenBank data base under the access number No AP001458, or No AC 090306. 6. A probe or a nucleotide primer that hybridises specifically with a nucleic acid according to any one of claims 1 and 3. 7. (canceled) 8. A probe or a nucleotide primer hybridising specifically with the mutated cgl1 gene. 9. A probe or a nucleotide primer hybridizing specifically with the mutated cgl1 gene, which further hybridizes specifically with a nucleic acid according to claim 2. 10. A probe or nucleotide primer according to claim 8, characterized in that it hybridizes specifically with the cgl1 gene carrying a mutation selected amongst the F63fsX75, F100fsX111, F105fsX112, F105fsX111, F108fsX113, R138X, A212P, F213fsX232, del/ins E5-6 et del E4-6 mutations. 11. A nucleotide primer couple for detecting a mutation within the cgl1 gene, characterized in that it is selected amongst the nucleotide sequences SEQ ID No 15 and SEQ ID NO 16, and SEQ ID No 17 and SEQ ID No 18. 12. A method for detecting a mutation within a nucleic acid coding for the CGL1 polypeptide, characterized in that it comprises the following steps of: a) contacting one or more nucleotide probes according to any one of claims 8 to 10 with the sample to be tested; b) detecting the complex optionally formed between the probe(s) and the nucleic acid present in the sample. 13. A detecting method according to claim 12, characterized in that the probe(s) is/are immobilized on a substrate. 14. A set or a kit for detecting a mutation in the cgl1 gene, characterized in that it comprises: a) one or more nucleotide probes according to any one of claims 8 to 10; b) if need be, the reactants required for the hybridization reaction. 15. A method for detecting a mutation in a nucleic acid coding for the CGL1 polypeptide, characterized in that it comprises the following steps of: a) contacting the sample to be tested with one or more primers according to any one of claims 8 to 11; b) detecting the amplified nucleic acids. 16. A set or a kit for detecting a mutation in a nucleic acid coding for the CGL1 polypeptide, characterized in that it comprises: a) one or more nucleotide primers according to any one of claims 8 to 11; b) if need be, the reactants agents required for the amplification reaction. 17. A recombinant vector comprising a nucleic acid according to any one of claims 1 to 5. 18. A recombinant host cell which is transfected or transformed by a nucleic acid according to any one of claims 1 to 5 or by a recombinant vector comprising a nucleic acid according to any one of claims 1 to 5. 19. A preventive or curative therapeutic method which comprises administering to a subject prone to or afflicted with a lipodystrophia, a Lawrence syndrome or obesity an effective amount of a nucleic acid according to any one of claims 1 and 3 to 5, a recombinant vector comprising a nucleic acid according to any one of claims 1 and 3 to 5, or a recombinant host cell which is transfected or transformed by a) a nucleic acid according to any one of claims 1 and 3 to 5 or by b) a recombinant vector comprising a nucleic acid according to any one of claims 1 and 3 to 5. 20. A preventive or curative therapeutic method which comprises administering to a subject prone to or afflicted with diabetes or an insulin-resistant syndrome an effective amount of a nucleic acid according to any one of claims 1 and 3 to 5, a recombinant vector comprising a nucleic acid according to any one claims 1 and 3 to 5, or a recombinant host cell which is transfected or transformed by a) a nucleic according to any one of claims 1 and 3 to 5 or by b) a recombinant vector comprising a nucleic acid according to any one of claims 1 and 3 to 5. 21. A pharmaceutical composition for preventing or treating a lipodystrophia, a Lawrence syndrome or obesity comprising a nucleic acid, a recombinant vector comprising a nucleic acid, or a recombinant host cell which is transfected or transformed by a nucleic acid or by a recombinant vector comprising a nucleic acid, in association with one or more physiologically compatible excipients, and wherein the nucleic acid is a nucleic acid according to any one of claims 1 and 3 to 5. 22. A pharmaceutical composition for preventing or treating diabetes or an insulin-resistant syndrome, comprising a nucleic acid, a recombinant vector comprising a nucleic acid, or a recombinant host cell which is transfected or transformed by a nucleic acid or by a recombinant vector comprising a nucleic acid, in association with one or more physiologically compatible excipients, and wherein the nucleic acid is a nucleic acid according to any one of claims 1 and 3 to 5. 23. A polypeptide characterized in that it is the CGL1 polypeptide with a SEQ ID No 1 sequence or a fragment or a variant of the CGL1 polypeptide. 24. A polypeptide according to claim 23, characterized in that the variant of the CGL1 polypeptide is selected amongst the polypeptides with amino acid sequences from SEQ ID No 2 to SEQ ID No 10. 25. A polypeptide according to claim 23, characterized in that the fragment of the CGL1 polypeptide is selected amongst the polypeptides with amino acid sequences from SEQ ID No 11 to SEQ ID No 13. 26. A preventive or curative therapeutic method which comprises administering to a subject prone to or afflicted with a lipodystrophia, a Lawrence syndrome or obesity an effective amount of a drug comprising CGL1 polypeptide with the SEQ ID No. 1 sequence. 27. A preventive or curative therapeutic method which comprises administering to a subject prone to or afflicted with diabetes or an insulin-resistant syndrome an effective amount of a drug comprising a CGL1 polypeptide with the SEQ ID No. 1 sequence. 28. A pharmaceutical composition for preventing or treating a lipodystrophia, a Lawrence syndrome or obesity, comprising a CGL1 polypeptide with a SEQ ID No 1 sequence, in association with one or more physiologically compatible excipients. 29. A pharmaceutical composition for preventing or treating a diabetes or an insulin-resistance syndrome, comprising a CGL1 polypeptide with a SEQ ID No 1 sequence, in association with one or more physiologically compatible excipients. 30. An antibody raised against a polypeptide according to any one of claims 23 to 25. 31. A method for detecting the presence of a polypeptide according to any one of claims 23 to 25 in a sample, comprising the following steps of: a) contacting an antibody raised against a polypeptide according to any one of claims 23 to 25 with the sample to be tested; b) detecting the optionally formed polypeptide-antibody complexes. 32. A set or kit for detecting a polypeptide according to any one of claims 23 to 25 in a sample, comprising: a) an antibody raised against the polypeptide according to any one of claims 23 to 25; b) if need be, the reactants required for detecting any optionally formed polypeptide-antibody complex. 33. A method for screening a candidate compound interacting with the CGL1 polypeptide with a SEQ ID No 1 sequence or with a fragment of the CGL1 polypeptide, characterized in that it comprises the following steps of: a) contacting the candidate compound with the CGL1 polypeptide or the fragment of the CGL1 polypeptide; b) detecting the optionally formed complexes between the CGL1 polypeptide or the fragment of the CGL1 polypeptide, on the one hand, and the candidate compound, on the other hand. 34. A set or kit for screening a candidate compound interacting with the CGL1 polypeptide with the SEQ ID No 1 sequence or a fragment of the CGL1 polypeptide, characterized in that it comprises: a) a CGL1 polypeptide or a fragment of the CGL1 polypeptide; b) if need be, the reactants required for detecting the optionally formed complexes between the CGL1 polypeptide and the fragment of CGL1 polypeptide, on the one hand, and the candidate compound, on the other hand. 35. A method for screening a candidate compound modulating the expression of the cgl1 gene, characterized in that it comprises the following steps of: a) culturing a host cell expressing, naturally or after a genetic recombination, the cgl1 gene; b) contacting the host cell cultured in step a) with a candidate compound; c) determining the ability of the candidate compound to modulate the expression of the cgl1 gene by the host cell. 36. A set or kit for screening a candidate compound modulating the expression of the cgl1 gene, characterized in that it comprises: a) a host cell expressing, naturally of after a genetic recombination, the cgl1 gene; b) if need be, the means required for determining the ability of the candidate compound to modulate the expression of the cgl1 gene by the host cell. 37. A set or kit according to claim 36, characterized in that the means required for determining the ability of the candidate compound to modulate the expression of the cgl1 gene by the host cell are one or more probes specific for the cgl1 gene. 38. A non human transgenic animal having its somatic and/or germinal cells being transformed by a un nucleic acid according to claim 1. 39. A non human transgenic animal having its somatic and/or germinal cells being transformed by a nucleic acid being inserted into the genome so as to inactivate the gene corresponding to cgl1, in said transgenic animal. 40. A method for in vivo screening a candidate molecule or a substance modulating the expression of a nucleic acid according to any one of claims 1 to 5 comprising the following steps of: a) administering the candidate substance or molecule to a non-human transgenic animal having its somatic and/or germinal cells transformed by an un nucleic acid according to any one of claims 1 to 5; b) detecting the expression level of the nucleic acid according to any one of claims 1 to 5; c) comparing the results obtained in b) with the results obtained in a transgenic animal which has not received the candidate substance or molecule. 41. A kit or set for in vivo screening a candidate molecule or substance modulating the expression of a nucleic acid according to any one of claims 1 to 5 comprising: a) a non-human transgenic animal having its somatic and/or germinal cells transformed by a un nucleic acid according to any one of claims 1 to 5; b) if need be, means for detecting the expression level of the nucleic acid according to any one of claims 1 to 5. 42. A method for in vivo screening a candidate molecule or substance modulating the expression or the activity of the CGL1 polypeptide, comprising the steps consisting of: a) administering the candidate substance or molecule to a non-human transgenic animal having its somatic and/or germinal cells transformed by an un nucleic acid according to any one of claims 1 to 5 or a non-human transgenic animal having its somatic and/or germinal cells transformed by a nucleic acid inserted into the genome so as to inactivate the gene corresponding to cgl1, in said transgenic animal; b) detecting the expression location and level of the CGL1 polypeptide using an antibody raised against the polypeptide wherein the polypeptide is the CGL1 polypeptide with a SEQ ID No. 1 sequence or a fragment or a variant of the CGL1 polypeptide, such a polypeptide wherein the variant of the CGL1 polypeptide is selected amongst the polypeptides with amino acid sequences from SEQ ID No. 2 to SEQ ID No. 10, or such a polypeptide wherein the fragment of the CGL1 polypeptide is a member selected from the group consisting of those having an amino acid sequence from SEQ ID No. 11 to SEQ ID No. 13; and c) comparing the obtained results with those obtained in an animal which has not received the candidate substance or molecule. 43. A kit or set for in vivo screening a candidate molecule or substance modulating the expression or the activity of the CGL1 polypeptide, comprising: a) a transgenic animal according to any one of claims 38 and 39; b) if need be, the means for detecting the expression location or level of the CGL1 polypeptide.

<SOH> SUMMARY OF THE INVENTION <EOH>The Applicant has presently identified a causal gene for the congenital generalized lipodystrophia (CGL) syndrome, located on the 11q13 locus of the human chromosome 11. The CGL causal gene has been referred to as cgl1 by the Applicant, who also identified the protein coded by such a gene, referred to as CGL1. The pathologies being preferably aimed at according to the invention are as follows: generalized congenital lipodystrophia, or lipoatrophic diabetes, or Berardinelli-Seip syndrome or Berardinelli-Seip congenital lipodystrophia, Lawrence syndrome, acquired lipodystrophia or secondary lipodystrophia, partial lipodystrophia, secondary (or acquired) lipodystrophya to the antiretroviral treatment of HIV infected patients, type 1 diabetes, type 2 diabetes, obesity, metabolic syndrome or insulin-resistant syndrome to the X syndrome. According to the invention, the cgl1 gene may also be referred to as BSCL2 . According to the invention, the CGL1 protein may also be referred to as Seipine . It has therefore been provided according to the invention a nucleic acid coding for CGL1 polypeptide of an amino acid sequence SEQ ID No 1 or also fragments or variants of CGL1 polypeptide. The invention also relates to the use of a nucleic acid such as defined herein above for manufacturing a specific probe or nucleotide primer for the normal cgl1 gene or the mutated cgl1 gene. The invention also relates to methods for detecting a mutation within a nucleic acid coding for CGL1 polypeptide, said methods implementing one or more specific nucleotide probes or primers for the mutated cgl1 gene, as well as sets or kits for detecting a mutation within the cgl1 gene. The invention also relates to recombinant vectors comprising a nucleic acid coding for CGL1 polypeptide, more particularly recombinant vectors useful in genic therapy methods, as well as recombinant host cells being transfected or transformed by such a nucleic acid or by a vector such as defined herein above. Another object of the invention is the use of a nucleic acid coding for CGL1 polypeptide for manufacturing a drug for preventing or treating a lipodystrophia, a diabetes or an obesity, and in particular a lipodystrophia associated with the CGL syndrome. It also relates to pharmaceutical compositions for preventing or treating a lipodystrophia or a diabetes, characterized in that they comprise a nucleic acid coding for CGL1 polypeptide or a recombinant vector or a recombinant host cell as defined herein above, in association with one or more physiologically compatible excipients. Another object of the invention is also the CGL1 polypeptide with a sequence SEQ ID No 1 or also a fragment or variant of the CGL1 polypeptide. It also relates to the use of a CGL1 polypeptide for manufacturing a drug for preventing or treating a lipodystrophia or a diabetes as well as a pharmaceutical composition comprising a CGL1 polypeptide. The invention also relates to an antibody raised against the CGL1 polypeptide, or also a fragment or a variant of the CGL1 polypeptide, as well as detection methods and sets or kits implementing such an antibody. Another object of the invention is also to provide methods for screening a candidate compound interacting with the CGL1 polypeptide as well as sets or kits for screening such candidate compounds. It also relates to methods for screening a candidate compound modulating the expression of the cgl1 gene as well as to sets or kits for screening such candidate compounds.

Trinuclear copper-based compound and ligand for nucleic acid scission and anticancer treatment
The present invention is related to a novel method for splitting nucleic acids at specific points on a complementary nucleic acid segment using a trinuclear copper-based compound of formula (I). Additionally, the present invention is related to a novel treatment of cancer, tumors, and cancer cells using a trinuclear copper-based compound of formula (I) or a naked ligand of formula (II).
1. A method of treating a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, ovarian cancer, cancer of the head and neck, bladder cancer, small cell cancer of the lung, squamous-cell carcinomas of the head, neck, esophagus, skin, and the genitourinary tract, including the cervix, vulva, scrotum, and penis, prostate cancer, and breast cancer, in a patient in need thereof, said method comprising administering to a patient a cancer-treating effective amount of a compound of formula I wherein R1-R6 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linkage L3 through a nitrogen atom of the heterocycle, and wherein the heterocycle is linked to a respective linkage L1 through any of the nitrogen or carbon atoms of the heterocycle other than the nitrogen atom that links to linkage L3; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; R7-R11 are each independently an anion or uncharged species; each L1 is independently an ethyl, methyl, or ether linkage; each L3 is a direct bond; and each L2 is independently (a) a C1-C6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C1-C6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C5-C8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide, and (d) an aromatic or cycloalkyl C9-C13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. 2. A method of treating a cancer tumor, wherein the cancer tumor is selected from the group consisting of leukemia tumor, non-small cell lung tumor, colon cancer tumor, central nervous system cancer tumor, melanoma tumor, ovarian cancer tumor, renal cancer tumor, ovarian cancer tumor, cancer tumors of the head and neck, bladder cancer tumor, small cell cancer tumor of the lung, squamous-cell carcinoma tumors of the head, neck, esophagus, skin, and the genitourinary tract, including the cervix, vulva, scrotum, and penis, prostate cancer tumor, and breast cancer tumor, said method comprising administering to the cancer tumor a cancer tumor-treating effective amount of a compound of formula I wherein R1-R6 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linkage L3 through a nitrogen atom of the heterocycle, and wherein the heterocycle is linked to a respective linkage L1 through any of the nitrogen or carbon atoms of the heterocycle other than the nitrogen atom that links to linkage L3; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; R7-R11 are each independently an anion or uncharged species; each L1 is independently an ethyl, methyl, or ether linkage; each L3 is a direct bond; and each L2 is independently (a) a C1-C6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C1-C6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C5-C8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide, and (d) an aromatic or cycloalkyl C9-C13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. 3. A method of treating cancer cells, wherein the cancer cells selected from the group consisting of leukemia cells, non-small cell lung cancer cells, colon cancer cells, central nervous system cancer cells, melanoma cells, ovarian cancer cells, renal cancer cells, ovarian cancer cells, cancer cells of the head and neck, bladder cancer cells, small cell cancer cells of the lung, squamous-cell carcinoma cells of the head, neck, esophagus, skin, and the genitourinary tract, including the cervix, vulva, scrotum, and penis, prostate cancer cells, and breast cancer cells, said method comprising administering to the cancer cells a cancer cell-treating effective amount of a compound of formula I wherein R1-R6 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linkage L3 through a nitrogen atom of the heterocycle, and wherein the heterocycle is linked to a respective linkage L1 through any of the nitrogen or carbon atoms of the heterocycle other than the nitrogen atom that links to linkage L3; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; R7-R11 are each independently an anion or uncharged species; each L1 is independently an ethyl, methyl, or ether linkage; each L3 is a direct bond; and each L2 is independently (a) a C1-C6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C1-C6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C5-C8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide, and (d) an aromatic or cycloalkyl C9-C13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. 4. (Cancelled) 5. (Cancelled) 6. A method of splitting a nucleic acid segment at a specific position thereon, wherein said method comprises (a) providing a first nucleic acid segment having (i) an n position, wherein said n position is occupied by a first purine nucleotide that is non-complementary to a corresponding position x on a second nucleic acid segment, and (ii) an n+1 position which is occupied by a guanine residue, wherein said n+1 position is located directly adjacent to the n position upstream from the 5′ end of the first nucleic acid segment, and a second nucleic acid segment which is complementary to the first nucleic acid segment upstream from the position x, wherein the second nucleic acid segment is located either on a different or the same nucleic acid strand as the first nucleic acid segment; (b) contacting at least the second nucleic acid segment with a compound of formula I for a time sufficient to split the nucleic acid at the position x of the second nucleic acid segment wherein R1-R6 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linkage L3 through a nitrogen atom of the heterocycle, and wherein the heterocycle is linked to a respective linkage L1 through any of the nitrogen or carbon atoms of the heterocycle other than the nitrogen atom that links to linkage L3; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; R7-R11 are each independently an anion or uncharged species; each L1 is independently an ethyl, methyl, or ether linkage; each L3 is a direct bond; and each L2 is independently (a) a C1-C6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C1-C6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C5-C8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide, and (d) an aromatic or cycloalkyl C9-C13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. 7. (Cancelled) 8. A method of treating a cancer selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, ovarian cancer, cancer of the head and neck, bladder cancer, small cell cancer of the lung, squamous-cell carcinomas of the head, neck, esophagus, skin, and the genitourinary tract, including the cervix, vulva, scrotum, and penis, prostate cancer, and breast cancer, in a patient in need thereof, said method comprising administering to a patient a cancer-treating effective amount of a compound of formula II wherein R21-R26 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linker L21 through a carbon or a nitrogen atom of the heterocycle; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide; each L21 is independently an ethyl, methyl, or ether linkage; and each L22 is independently (a) a C1-C6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C1-C6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C5-C8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; and (d) an aromatic or cycloalkyl C9-C13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. 9. A method of treating a cancer tumor, wherein the cancer tumor is selected from the group consisting of leukemia tumor, non-small cell lung cancer tumor, colon cancer tumor, central nervous system cancer tumor, melanoma tumor, ovarian cancer tumor, renal cancer tumor, ovarian cancer tumor, cancer tumors of the head and neck, bladder cancer tumor, small cell cancer tumor of the lung, squamous-cell carcinoma tumors of the head, neck, esophagus, skin, and the genitourinary tract, including the cervix, vulva, scrotum, and penis, prostate cancer tumor, and breast cancer tumor, said method comprising administering to the cancer tumor a cancer tumor-treating effective amount of a compound of formula II wherein R21-R26 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linker L21 through a carbon or a nitrogen atom of the heterocycle; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide; each L21 is independently an ethyl, methyl, or ether linkage; and each L22 is independently (a) a C1-C6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C1-C6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C5-C8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; and (d) an aromatic or cycloalkyl C9-C13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. 10. A method of treating cancer cells, wherein the cancer cells selected from the group consisting of leukemia cells, non-small cell lung cancer cells, colon cancer cells, central nervous system cancer cells, melanoma cells, ovarian cancer cells, renal cancer cells, ovarian cancer cells, cancer cells of the head and neck, bladder cancer, small cell cancer cells of the lung, squamous-cell carcinoma cells of the head, neck, esophagus, skin, and the genitourinary tract, including the cervix, vulva, scrotum, and penis, prostate cancer cells, and breast cancer cells, said method comprising administering to the cancer cells a cancer cell-treating effective amount of a compound of formula II wherein R21-R26 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linker L21 through a carbon or a nitrogen atom of the heterocycle; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide; each L21 is independently an ethyl, methyl, or ether linkage; and each L22 is independently (a) a C1-C6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C1-C6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C5-C8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; and (d) an aromatic or cycloalkyl C9-C13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. 11. (Cancelled) 12. (Cancelled) 13. The method of claim 6, wherein the complementary nucleic acid segment is located on the same nucleic acid strand as the nucleic acid segment. 14. The method of claim 6, wherein the complementary nucleic acid segment is located on a different nucleic acid strand as the nucleic acid segment. 15. A pharmaceutical composition containing a pharmaceutically effective amount of at least one compound of formula I wherein R1-R6 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linkage L3 through a nitrogen atom of the heterocycle, and wherein the heterocycle is linked to a respective linkage L1 through any of the nitrogen or carbon atoms of the heterocycle other than the nitrogen atom that links to linkage L3; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; R7-R11 are each independently an anion or uncharged species; each L1 is independently an ethyl, methyl, or ether linkage; each L3 is a direct bond; and each L2 is independently (a) a C1-C6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C1-C6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C5-C8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide, and (d) an aromatic or cycloalkyl C9-C13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. 16. A pharmaceutical composition containing a pharmaceutically effective amount of at least one compound of formula II wherein R21-R26 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linker L21 through a carbon or a nitrogen atom of the heterocycle; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide; each L21 is independently an ethyl, methyl, or ether linkage; and each L22 is independently (a) a C1-C6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C1-C6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C5-C8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; and (d) an aromatic or cycloalkyl C9-C13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C1-C4 alkoxy, C1-C4 alkyl, benzyl, nitro, C1-C4 acylamino, formyl, formamido, thioformamido, C1-C4 alkoxycarbonylamino, C1-C4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. 17. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is pharmaceutically effective against leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, ovarian cancer, cancer of the head and neck, bladder cancer, small cell cancer of the lung, squamous-cell carcinomas of the head, neck, esophagus, skin, and the genitourinary tract, including the cervix, vulva, scrotum, and penis, prostate cancer, and breast cancer. 18. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition is pharmaceutically effective against leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, ovarian cancer, cancer of the head and neck, bladder cancer, small cell cancer of the lung, squamous-cell carcinomas of the head, neck, esophagus, skin, and the genitourinary tract, including the cervix, vulva, scrotum, and penis, prostate cancer, and breast cancer.
<SOH> BACKGROUND OF INVENTION <EOH>A number of transition metal complexes have been found to be able to differentiate between double vs. single-stranded DNA or B vs. Z helical forms of DNA through noncovalent recognition. This selectivity is primarily due to the binding of the transition metal complex in either the major or minor groove of duplex structures or in association with the nucleobases in unpaired strands. The electron-rich character of the nucleobases often makes them strong ligands for metals and efficient targets of oxidation. Guanine has been shown to have the highest affinity for coordination to transition metal ions and it is also the most easily oxidized, followed by adenine, cytosine and thymine (in order of ease of oxidation). Although base oxidation can be highly specific and directed to one site, strand scission has been shown to result from base oxidation only after treatment with subsequent heat and alkaline conditions. However, direct strand scission does not necessarily require any special treatment to detect the sites of reaction. Some complexes that exhibit direct strand cleavage in conjunction with sequence specificity are bleomycin.Fe(II) and the metallointercalator, [Rh(phen) 2 phi] 3+ . Although there is both a structural and a sequence requirement in each of these cases, the recognition criteria are not sufficiently unique to limit the number of target sites in DNA. Scission may be targeted specifically to one site by incorporating known DNA recognition elements into the ligand suprastructure of a well-characterized nucleolytic agent such as EDTA.Fe(II), which, when underivatized, promotes oxidative cleavage of DNA in a random fashion without nucleotide sequence selectivity. While this approach localizes cleavage to a site where the recognition element binds to DNA, the reaction is rarely constrained to a single nucleotide. Strand cleavage frequently extends over more than 5 bases. A longstanding goal of considerable interest has been to construct transition metal complexes that can mediate direct and specific strand scission targeted to a single base with a significantly high level of recognition such that cleavage occurs at a limited number of sites along a target polynucleotide. Most investigations focusing on oxidative strand scission of DNA by transition metals have typically relied on mononuclear complexes. Among these complexes, bis(1,10-phenanthroline)copper, [Cu(OP) 2 ] 2+ , has been studied extensively due to its high nucleolytic efficiency. The cleavage pattern induced by [Cu(OP) 2 ] 2+ is predominantly sequence-neutral, although some variability in intensity due to local perturbations of DNA structure affects its efficiency. Also a slight, but distinct, preference for cleavage at 5′-AT-3′ and 5′-GT-3′ sites has been observed. Otherwise, [Cu(OP) 2 ] 2+ like EDTA.Fe(II) may be conjugated to binding elements such as proteins and complementary sequences of RNA or DNA that possess affinity for specific sites on DNA. Still, multiple sites adjacent to the locus of recognition are typically oxidized by these complexes even when tethered to a DNA recognition element. An example of research into oxidative strand scission of DNA by transition metals is detailed in the article entitled “A new trinuclear complex and its reactions with plasmid DNA” by Steven T. Frey, Helen H. J. Sun, Narasimha N. Murthy, and Kenneth D. Karlin, Inorganica Chimica Acta 242 (1996) 329-338 (hereinafter referred to as “Frey”). This article discusses the synthesis of a novel trinuclear copper(II) complex and its reactivity with plasmid pBR322. While Frey discloses a trinuclear compound within the disclosure of formulas I and II of the present invention, it is noted that Frey does not disclose that these compounds possess the surprising ability to treat cancer or to split nucleic acids at specific locations adjacent to a complementary nucleic acid segment. In fact, on page 335 of the article, Frey teaches away from the splitting of nucleic acids at a specific position by noting “that cleavage [with the trinuclear compound] is not sequence specific.” Therefore, Frey is not relevant to the novelty or nonobviousness of the present invention.
<SOH> SUMMARY OF INVENTION <EOH>The present invention is based on the discovery that certain trinuclear copper-based compounds possess the ability to recognize and promote scission of a nucleic acid at specific positions. Additionally, it has been discovered that the trinuclear copper-based compounds and the naked ligand possess the ability to treat cancer. Thus, the invention is directed towards a method of treating cancer in a patient in need thereof, comprising administering to a patient a cancer-treating effective amount of a compound of formula I, wherein R 1 -R 6 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linkage L 3 through a nitrogen atom of the heterocycle, and wherein the heterocycle is linked to a respective linkage L 1 through any of the nitrogen or carbon atoms of the heterocycle other than the nitrogen atom that links to linkage L 3 ; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C 1 -C 4 alkoxy, C 1 -C 4 alkyl, benzyl, nitro, C 1 -C 4 acylamino, formyl, formamido, thioformamido, C 1 -C 4 alkoxycarbonylamino, C 1 -C 4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; R 7 -R 11 are each independently an anion or uncharged species; each L 1 is independently an ethyl, methyl, or ether linkage; each L 3 is a direct bond; and each L 2 is independently (a) a C 1 -C 6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C 1 -C 6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C 1 -C 4 alkoxy, benzyl, nitro, C 1 -C 4 acylamino, formyl, formamido, thioformamido, C 1 -C 4 alkoxycarbonylamino, C 1 -C 4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C 5 -C 8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C 1 -C 4 alkoxy, C 1 -C 4 alkyl, benzyl, nitro, C 1 -C 4 acylamino, formyl, formamido, thioformamido, C 1 -C 4 alkoxycarbonylamino, C 1 -C 4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide, and (d) an aromatic or cycloalkyl C 9 -C 13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C 1 -C 4 alkoxy, C 1 -C 4 alkyl, benzyl, nitro, C 1 -C 4 acylamino, formyl, formamido, thioformamido, C 1 -C 4 alkoxycarbonylamino, C 1 -C 4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. The invention is further directed towards a method of treating a cancer tumor, said method comprising administering to the cancer tumor a cancer tumor-treating effective amount of a compound of formula I. The invention is additionally directed towards a method of treating cancer cells, said method comprising administering to the cancer cells a cancer cell-treating effective amount of a compound of formula I. The invention is further directed towards a use of a compound of formula I to treat cancer. The invention is further directed towards the use of a compound of formula I to prepare a medicament suitable for treating cancer. The invention is further directed towards a method of splitting a nucleic acid segment at a specific position thereon, wherein said method comprises (a) providing a first nucleic acid segment having (i) an n position, wherein said n position is occupied by a first purine nucleotide that is non-complementary to a corresponding position x on a second nucleic acid segment, and (ii) an n+1 position which is occupied by a guanine residue, wherein said n+1 position is located directly adjacent to the n position and upstream towards the 5′ end of the first nucleic acid segment, and a second nucleic acid segment which is complementary to the first nucleic acid segment upstream from the position x, wherein the second nucleic acid segment is located either on a different or the same nucleic acid strand as the first nucleic acid segment; and (b) contacting at least the second nucleic acid segment with a compound of formula I for a time sufficient to split the nucleic acid at the position x of the second nucleic acid segment. The invention is further directed towards the use of a compound of formula I to split a nucleic acid segment at a specific position x thereon, wherein the specific position x is located on a second nucleic acid segment which is complementary to a first nucleic acid segment upstream from the position x, wherein the position x is non-complementary to an n position on the first nucleic acid segment, wherein the n position is occupied by a first purine nucleotide, and the n position is directly adjacent to an n+1 position and is located upstream towards the 5′ end of the first nucleic acid segment, wherein said n+1 position is occupied by a guanine. The invention is further directed towards a method of treating cancer in a patient in need thereof, said method comprising administering to a patient a cancer-treating effective amount of a compound of formula II, wherein R 21 -R 26 are each independently a 5 to 6 membered heterocycle containing 1-3 nitrogen atoms and optionally one oxygen atom, with the remainder of the atoms being carbon atoms, wherein the heterocycle is linked to a respective linker L 21 through a carbon or nitrogen atom of the heterocycle; and wherein the 5 to 6 membered heterocycle is unsubstituted or substituted with halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C 1 -C 4 alkoxy, C 1 -C 4 alkyl, benzyl, nitro, C 1 -C 4 acylamino, formyl, formamido, thioformamido, C 1 -C 4 alkoxycarbonylamino, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide; each L 21 is independently an ethyl, methyl, or ether linkage; and each L 22 is independently (a) a C 1 -C 6 alkyl which may optionally be interrupted with one or more ether linkages, wherein the C 1 -C 6 alkyl is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C 1 -C 4 alkoxy, benzyl, nitro, C 1 -C 4 acylamino, formyl, formamido, thioformamido, C 1 -C 4 alkoxycarbonylamino, C 1 -C 4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide; (b) an ether linkage; (c) an aromatic or cycloalkyl C 5 -C 8 monocycle which is unsubstituted or substituted with 1 to 3 substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C 1 -C 4 alkoxy, C 1 -C 4 alkyl, benzyl, nitro, C 1 -C 4 acylamino, formyl, formamido, thioformamido, C 1 -C 4 alkoxycarbonylamino, C 1 -C 4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl and acetamide, and (d) an aromatic or cycloalkyl C 9 -C 13 bicycle which is unsubstituted or substituted with 1 to 3 substituents, said substituents each independently selected from the group consisting of halogen, hydroxy, formyloxy, azido, carboxyl, cyano, amino, C 1 -C 4 alkoxy, C 1 -C 4 alkyl, benzyl, nitro, C 1 -C 4 acylamino, formyl, formamido, thioformamido, C 1 -C 4 alkoxycarbonylamino, C 1 -C 4 alkoxycarbonyl, phenyloxycarbonylamino, naphthyloxycarbonylamino, semicarbazido, heteroaryl, 4-acetoxyphenyloxy, phenyl or acetamide. The invention is further directed towards a method of treating a cancer tumor, said method comprising administering to the cancer tumor a cancer tumor-treating effective amount of a compound of formula II. The invention is further directed towards a method of treating cancer cells, said method comprising administering to the cancer cells a cancer cell-treating effective amount of a compound of formula II. The invention is further directed towards a use of a compound of formula II to treat cancer. The invention is further directed towards the use of a compound of formula II to prepare a medicament suitable for treating cancer. The invention is also directed towards a pharmaceutical composition containing a pharmaceutically effective amount of at least one compound of formula I. The invention is also directed towards a pharmaceutical composition containing a pharmaceutically effective amount of at least one compound of formula II.

Folate mimetics and folate-receptor binding conjugates thereof
A cell population expressing folate receptors is selectively targeted with a folate mimetic. The folate mimetic is conjugated to a diagnostic or therapeutic agent to enable selective delivery of the agent to the targeted cell population.
1. A compound having the formula wherein X and Y are each-independently selected from the group consisting of halo, R2, OR2, SR3, and NR4R5; U, V, and W represent divalent moieties each independently selected from the group consisting of —(R6′)C═, —N═, —(R6′)C(R7′)—, and —N(R4′)—; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N and —C═C— such that the ring structure of which T is a member is aromatic; A1 and A2 are each independently selected from the group consisting of —C(Z)-, —C(Z)O—, —OC(Z)-, —N(R4″)—, —C(Z)-N(R4″)—, —N(R4″)—C(Z)-, —O—C(Z)-N(R4″)—, —N(R4″)—C(Z)-O—, —N(R4″)—C(Z)-N(R5″)—, —O—, —S—, —S(O)—, —S(O)2—, —N(R4′)S(O)2—, —C(R6″)(R7″)—, —N(C≡CH)—, —N(CH2—C≡CH)—, C1-C12 alkyl and C1-C12 alkoxy; where Z is oxygen or sulfur provided that A2 does not represent —C(O)NH—; R1is selected-from the group consisting of hydrogen, halo, C1-C12 alkyl, and C1-C12 alkoxy; R2, R3, R4, R4′, R4″, R5, R5″, R6″ and R7″ are each independently selected from the group consisting of hydrogen, halo, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C1-C12 alkenyl, C1-C12 alkynyl, (C1-C12 alkoxy)carbonyl, and (C1-C12 alkylamino)carbonyl; R6 and R7 are each independently selected from the group consisting of hydrogen, halo, C1-C12 alkyl, and C1-C12 alkoxy; or, R6 and R7 are taken together to form O═; R6′ and R7′ are each independently selected from the group consisting of hydrogen, halo, C1-C12 alkyl, and C1-C12 alkoxy; or, R6′ and R7′ are taken together to form O═; L is a divalent linker; n, p, r and s are each independently either 0 or 1 provided that when n=1, then r=1; and B is hydrogen or a leaving group; provided that the linker L does not include a naturally occurring amino acid covalently linked to A2 at its α-amino group through an amide bond. 2. The compound of claim 1 having binding affinity for a folate receptor molecule. 3. The compound of claim 1 wherein X and Y are each independently selected from the group consisting of hydrogen, halo, CH3, OH, SH and NH2; U, V and W represent divalent moieties each independently selected from the group consisting of —CH═ and —N═; Q represents C; A1 is selected from the group consisting of —C(Z)-, —NH—, —N(CH3)—, —O—, —S—, —S(O)—, —S(O)2—, —CH2—, —CH(CH3)—, —C(CH3)2—, —N(CH2—C≡CH)— and —N(C≡CH)— where Z is oxygen or sulfur; R1 is selected from the group consisting of hydrogen, halo and methyl; R6 and R7 are each independently selected from the group consisting- of hydrogen, halo, CH3, OH, SH and NH2; or, R6 and R7 are taken together to form O═; A2 is selected from the group consisting of —C(Z)-, —C(Z)O—, —OC(Z)-, —N(R4″)—, —C(Z)-N(R4″)—, —N(R4″)—C(Z)-, —O—C(Z)-N(R4″)—, —N(R4″)—C(Z)-O—, —N(R4″)—C(Z)-N(R5″)—, —O—, —S—, —S(O)—, —S(O)2—, —N(R4″)S(O)2—, —C(R6″)(R7″), C1-C6 alkyl; C1-C6 alkoxy; where Z is oxygen or sulfur provided that A2 does not represent —C(O)NH—; and p and r are each 1. 4. The compound of claim 3 wherein T is —C═C—. 5. The compound of claim 3 wherein X is OH. 6. The compound of claim 3 having binding affinity for a folate receptor molecule. 7. The compound of claim 1 wherein X is OH; Y is NH2; U and W are each —N═; V is —CH═; Q is C; T is —C═C—; A1 is —NH—; R1 is hydrogen; A2 is —C(O)— or —C(O)O— and is para to A1; R6 and R7 are each H; and p, r and s are each 1. 8. The compound of claim 7 having binding affinity for a folate receptor molecule. 9. A compound that is isosteric with the compound of claim 7 and that has binding affinity for a folate receptor molecule. 10. A ligand-agent conjugate having the formula wherein X, Y, U, V, W, Q, T, A1, A2, R1, R6, R7, L, n, p, r and s are as defined in claim 1; q is an integer ≧1; and, D is a diagnostic agent or a therapeutic agent. 11. The ligand-agent conjugate of claim 10 that has binding affinity for a folate receptor molecule. 12. The ligand-agent conjugate of claim 10 comprising a metabolically labile linker L. 13. The ligand-agent conjugate of claim 12 wherein the metabolically labile linker L is hydrolytically or reductively cleaved in the cell to release the diagnostic or therapeutic agent Z. 14. The ligand-agent conjugate of claim 12 wherein the metabolically labile linker L comprises a disulfide or an ester. 15. A pharmaceutical composition comprising the ligand agent conjugate of claim 10 and at least one component selected from the group consisting of a pharmaceutically acceptable carrier, excipient, or diluent. 16. A method for delivering a diagnostic agent or a therapeutic agent to a target cell population comprising a folate receptor, the method comprising: providing a ligand-agent conjugate having the formula wherein X, Y, U, V, W, Q, T, A1, A2, R1, R6, R7, L, n, p, r and s are as defined in claim 1, q is an integer ≧1; and D is a diagnostic agent or a therapeutic agent; and contacting the target cell population with an effective amount of the ligand-agent conjugate to permit binding of the ligand-conjugate to the folate receptor. 17. The method of claim 16 wherein D is a diagnostic agent comprising a contrast agent for use in medical imaging. 18. The method of claim 16 wherein the ligand-agent conjugate binds to the cell surface and is not internalized by the cells of the cell population. 19. The method of claim 16 wherein the diagnostic or therapeutic agent D is internalized by the cells of the cell population.
<SOH> BACKGROUND OF THE INVENTION <EOH>A number of methods are known for selectively targeting cells in a patient for delivery of diagnostic or therapeutic agents. Selective targeting has led to the introduction of various diagnostic agents for visualization of tissues, such as contrast agents useful in Magnetic Resonance Imaging (MRI), radiodiagnostic compositions, and the like. Introduction of therapeutic agents, such as compositions for radiotherapy or for neutron capture therapy, compositions for chemotherapy, various proteins, peptides, and nucleic acids, protein toxins, antisense oligonucleotides, liposomes, analgesics, antibiotics, antihypertensive agents, antiviral agents, antihistamines, expectorants, vitamins, plasmids, and the like, has also been demonstrated. Folate conjugates have been used for the selective targeting of cell populations expressing folate receptors or other folate binding proteins to label or deliver bioactive compounds to such cells. The relative populations of these receptors and binding proteins have been exploited in achieving selectivity in the targeting of certain cells and tissues, such as the selective targeting of tumors expressing elevated levels of high-affinity folate receptors. The following publications, the disclosures of which are incorporated herein by reference, illustrate the nature and use of folate conjugates for diagnosis or delivery of biologically significant compounds to selected cell populations in patients in need of such diagnosis or treatment: (a) Leamon and Low, “Cytotoxicity of Momordin-folate Conjugates in Cultured Human Cells” in J. Biol. Chem., 1992, 267, 24966-24967. (b) Leamon et al., “Cytotoxicity of Folate-pseudomonas Exotoxin Conjugates Towards Tumor Cells” in J. Biol. Chem., 1993, 268, 24847-24854. (c) Lee and Low, “Delivery of Liposomes into Cultured Kb Cells via Folate Receptor-mediated Endocytosis” in J. Biol. Chem., 1994, 269, 3198-3204. (d) Wang et al., “Delivery of Antisense Oligonucleotides Against the Human Epidermal Growth Factor Receptor into Cultured Kb Cells with Liposomes Conjugated to Folate via Polyethyleneglycol” in Proc. Natl. Acad. Sci. USA., 1995, 92, 3318-3322. (e) Wang et al., “Synthesis, Purification and Tumor Cell Uptake of Ga-67-deferoxamine-folate, a Potential Radiopharmaceutical for Tumor Imaging” in Bioconj. Chem., 1996, 7, 56-63. (f) Leamon et al., “Delivery of Macromolecules into Living Cells: a Method That Exploits Folate Receptor Endocytosis” in Proc. Natl. Acad. Sci., U.S.A., 1991, 88, 5572-5576. (g) Krantz et al., “Conjugates of Folate Anti-Effector Cell Antibodies” in U.S. Pat. No. 5,547,668. (h) Wedeking el al., “Metal Complexes Derivatized with Folate for Use in Diagnostic and Therapeutic Applications” in U.S. Pat. No. 6,093,382. (i) Low et al., “Method for Enhancing Transmembrane Transport of Exogenous Molecules” in U.S. Pat. No. 5,416,016. (j) Miotti et al., “Characterization of Human Ovarian Carcinoma-Associated Antigens Defined by Novel Monoclonal Antibodies with Tumor-Restricted Specificity”in Int. J. Cancer, 1987, 39,297-303. (k) Campell et al., “Folate-Binding Protein is a Marker for Ovarian Cancer”in Cancer Res., 1991, 51, 5329-5338. (l) Jansen et al., “Identification of a Membrane-Associated Folate-Binding Protein in Human Leukemic CCRF-CEM Cells with Transport-Related Methotrexate Resistance”in Cancer Res., 1989, 49, 2455-2459. Multiple types of folate recognition sites present on cells, such as α-folate receptors, β-folate receptors, folate binding proteins, and the like, have been shown to recognize and bind the conjugates described above. The primary pathway for entry of folate derivatives into cells is through a facilitated transport mechanism mediated by a membrane transport protein. However, when folate is covalently conjugated to certain small molecules and macromolecules, the transport system can fail to recognize the folate molecule. Advantageously, in addition to the facilitated transport protein, some cells possess a second membrane-bound receptor, folate binding protein (FBP), that allows folate uptake via receptor-mediated endocytosis. At physiological plasma concentrations (nanomolar range), folic acid binds to cell surface receptors and is internalized via an endocytic process. Receptor-mediated endocytosis is the movement of extracellular ligands bound to cell surface receptors into the interior of the cells through invagination of the membrane, a process that is initiated by the binding of a ligand to its specific receptor. The uptake of substances by receptor-mediated endocytosis is a characteristic ability of some normal, healthy cells such as macrophages, hepatocytes, fibroblasts, reticulocytes, and the like, as well as abnormal or pathogenic cells, such as tumor cells. Notably, folate binding proteins involved in endocytosis are less sensitive to modification of the folate molecule than the membrane transport proteins, and often recognize folate conjugates. Both targeting and uptake of conjugated diagnostic and therapeutic agents are enhanced. Following endosome acidification, the folate receptor changes conformation near its ligand-binding domain and releases the folic acid molecule. Folate receptors are known to recycle back to the membrane surface for additional rounds of ligand-mediated internalization. However, a significant fraction of the internalized receptor-folic acid complex has been shown to return back to the cell surface shortly after endocytosis. This suggests that the acid-triggered ligand release mechanism does not proceed to completion, at least after the first round of internalization (Kamen et al., 1988, J. Biol. Chem. 263, 13602-13609). Pteroic acid, which is essentially folic acid lacking the distal glutamyl residue ( FIG. 1 ), does not bind to the high-affinity folate receptor to any appreciable extent (Kamen et al., 1986, Proc. Natl. Acad. Sci., USA. 83, 5983-5987); in fact, 2 μM pteroic acid (100-fold excess) had absolutely no effect on the binding of folate to the folate receptor. Thus, the glutamyl residue of folic acid, or some portion thereof, was generally thought to be required for efficient, specific receptor recognition. However, recent studies have revealed that the glutamyl residue of folic acid could be replaced with a lysyl residue without disturbing the binding affinity of the ligand (McAlinden et al., 1991, Biochemistry 30, 5674-5681.; Wu et al., 1997, J. Membrane Biol. 159, 137-147), that the glutamyl residue can be replaced with a glycyl residue without substantially altering cellular uptake, and that no selective isomeric (i.e., α-glutamyl vs. γ-glutamyl) conjugation requirement necessarily exists (Leamon et al., J. Drug Targeting 7:157-169 (1999); Linder et al., J. Nuclear Med. 41(5):470 Suppl. 2000). Efforts to improve the selectivity of targeting or increase the diversity of the agents delivered to the cell or tissue have been hampered by a number of complications, including the complex syntheses required for the preparation of these conjugates. Such synthetic schemes are not only time consuming, but may also preclude the use of certain conjugates due to synthetic incompatibilities. A folic acid analog capable of expanding the number or diversity of agents, via the conjugates of such agents and these folic acid analogs, presentable to target cells would be advantageous.

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.