Novel backbone-cyclized BPI peptide analogs and methods of making the same by the use of bridging groups attached via the alpha nitrogens of amino acid derivatives to provide novel non-peptidic linkages. Novel building units used in the synthesis of these backbone-cyclized peptide analogs are Nxcex1-functionalized amino acids constructed to include a spacer and a terminal functional group. The reactive terminal functional groups are protected by specific protecting groups that can be selectively removed to effect either backbone-to-backbone or backbone-to-side chain cyclizations. A plurality of these Nxcex1xcfx89-functionalized amino acids are incorporated into a library of peptide sequences, preferably during solid phase peptide synthesis.

This invention relates to novel conformationally constrained backbone cyclized Bactericidal/Permeability Increasing Protein (xe2x80x9cBPIxe2x80x9d) peptidomimetics, to libraries of the same, to methods for the production of such peptidomimetics libraries and to methods of using such materials to screen for biologically active compounds.
Classically, the pharmaceutical industry has screened a wide variety of compounds derived from natural sources to yield potential drug candidates or lead compounds for the development of new drugs. These laborious screening efforts have relied on the random testing of a vast number of chemical entities. In recent years, various strategies have been adopted for the generation of libraries of compounds that are subsequently screened as a novel, rational approach to drug discovery and development.
It has become apparent that a variety of methodologies can be applied to the problem of generating a diverse group of candidate compounds, based on the known principles of peptide chemistry and/or molecular biology. Peptides are a convenient class of molecules for the generation of combinatorial libraries, since they are composed of a finite set of amino acid building units, which can be efficiently assembled either by chemical synthesis or transcription/translation of DNA. Combinatorial libraries are discussed by Gallop et al., J. Med. Chem., 37, 1233-1251 (1994); Gordon et al., J. Med. Chem., 37, 1385-1401 (1994); Pinilla et al., Biopolymers (Peptide Science), 37, 221-240, (1995); and Lebl et al., Biopolymers (Peptide Science), 37, 177-198 (1995). The set of amino acid building units can include only the naturally encoded amino acids, when the libraries are encoded by oligonucleotides on a plasmid, phage, or any other vector. This set can be expanded to include both D and L amino acids and/or non-natural amino acids in synthetic libraries.
Linear peptides suffer from several serious drawbacks as potential drugs, inasmuch as they are notoriously unstable in vivo, often lack high affinity of binding to their receptor, frequently lack selectivity to one kind of receptor, and generally have poor oral bioavailability. In efforts to overcome such problems, it is also possible to utilize the methodologies developed in connection with synthetic peptide libraries to generate collections of cyclic peptides, novel biopolymers and even novel branched oligomeric compounds, reviewed by Zuckermann, Current Opinion in Structural Biology, 3, 580-584 (1993).
One of the most significant synthetic technologies that facilitate the generation and screening of diverse chemical libraries is the resin-splitting method, which is a polymer supported multiple synthesis procedure that allows a high degree of control over the composition of a peptide mixture. Mixtures are generated by dividing a solid support into individual portions, and coupling a different amino acid to each portion, and then recombining the portions. These steps may be performed in an iterative fashion to provide the required degree of diversity.
Totally random libraries generated by these types of methods are disclosed in WO92/00091 and WO92/09300. Each individual bead will contain a unique peptide sequence, which can be probed for activity with a soluble receptor or antibody. Positive beads can be isolated and sequenced using Edman sequencing chemistry. WO92/00091 further discloses methods to provide selectively cleavable linkers between peptide and resin, such that part of the peptide can be liberated from the resin and assayed for activity in soluble form, while another part can be sequenced. In addition, it is also possible to generate random libraries in which each bead carries more than one peptide, by coupling of mixtures of amino acids to the beads, as disclosed by Homik et al., Reactive Polymers, 22, 213-220 (1994).
Another methodology is disclosed by Geysen et al., J. Immunol. Meth., 102, 259-274 (1987), which involves the synthesis of peptides on derivatized polystyrene pins which are arranged in such a fashion that they correspond to the arrangement of wells in a 96-well microtiter plate. Individual chemical reactions can be performed in each well, thereby yielding individual peptides on each pin. The pins are typically probed using an enzyme linked immunoassay (ELISA) or radioimmunoassay (RIA), carried out in the microtiter wells, or the peptides may be released from the pins and tested in solution. The mimotope approach of Geysen et al. generates diverse peptides that are probed for activity in situ. The best dipeptide sequence is selected for elongation to diverse tripeptides, the best tripeptide is selected for elongation to a tetrapeptide and so on.
Ideally, chemistries that are amenable to combinatorial library synthesis would have the following characteristics: be polymer-supported to facilitate the resin splitting technique; be assembled in high yield with automatable chemistry; and allow the incorporation of a wide variety of chemical functionalities.
Cyclic peptides are generally recognized as possessing enhanced bioavailability due to increased metabolic stability, as well as a relatively constrained conformation when compared to the same sequence in a linear form. The enhanced metabolic stability should allow diminished doses at longer intervals. The restricted conformation should improve the drug selectivity, thereby potentially preventing side-effects. All of these properties are desirable in conjunction with the quest for new drug candidates.
The generation of libraries of cyclic peptides requires, in addition to any previously stated considerations, that the cyclization reaction be performed in a high yield and with a minimum of additional manipulations. Unfortunately, classical cyclization reactions are highly sequence dependent in terms of the expected yields, making the uniform cyclization of a peptide mixture unreliable.
Recent advances in the cyclization of peptides directly on the solid support have improved the synthetic procedure, and even allowed the automation of cyclization reactions based on known cyclization schemes. In the past, cyclizations were typically performed in solution under conditions of high dilution. Polymer-supported cyclizations can both avoid potential side reactions such as oligomerization and facilitate product purification. For example, on-resin cyclization methods have recently been used to prepare cyclopeptides with bridges formed of thioethers, disulfides, or lactams between two side chains, lactams between the amino terminus and a side chain, and lactams between the amino and carboxy termini (reviewed by Zuckermann, Current Opinion in Structural Biology, 3, 580-584 (1993)).
The use of resin-bound cyclic peptides and free cyclic peptides in combinatorial libraries is disclosed in WO 92/00091. However, these cyclic peptides do not contain any conformationally constraining element, and in cases where cyclization is achieved, these peptides may still adopt a number of conformations and suffer many of the same shortcomings as linear peptides.
Cyclic semi-random peptide libraries, which are disclosed in WO 95/01800, are exclusively cyclic penta-peptide and hexa-peptide libraries containing one or more randomized amino acids and a conformationally constraining element in the form of an amino acid residue such as proline which fixes the beta turn angles of the adjacent amino acid residues. The advantages of such conformationally constraining elements is stressed by the inventors of this approach. However, inclusion of such elements via incorporation of a particular amino acid residue into the peptide sequence may have detrimental effects on those residues required for receptor recognition or other biological activity. Furthermore, in WO 95/01800, the cyclization reaction is merely another coupling reaction in which the terminal amino group of the linear peptide is coupled to the terminal carboxy group of the peptide.
Backbone cyclized peptides are generally known, as discussed, for instance, in Gilon et al., Biopolymers, 31, 745-750 (1991) and in EPO 564,739 A2 and EPO 564,739 A3. Such compounds have not been used for constructing libraries for screening purposes.
In addition, methods are known for combining amino acids and peptides. U.S. Pat. No. 5,010,175 describes another method of incorporating random amino acids into a peptide. According to that method, a mixture of amino acids is incorporated by coupling a mixture in which the individual amino acids are present in varying proportions depending upon their relative rates of reaction in the coupling, e.g., the amount of amino acid is inversely proportional to its rate of coupling.
It is an object of this invention to provide backbone-cyclic BPI peptide analogs that are novel and that are suited for screening for bioactive molecules. To this end, the present invention provides backbone-cyclized BPI peptide analogs that contain a peptide sequence having at least one building unit comprising an Nxcex1-derivative of an amino acid, with at least one backbone nitrogen in each peptide sequence linked to a side chain of at least one other amino acid in the peptide sequence or to at least one other backbone nitrogen in the peptide sequence by a bridging group comprising a disulfide, amide, thioether, thioester, imine, ether, or alkene bridge to form a backbone-cyclized peptide analog. At least one of the building units is preferably located other than at the end of the peptide sequence, and more preferably, none of the building units is located at the end of the peptide sequence.
According to one aspect of the invention, at least one pair of backbone nitrogens in the peptide sequence is linked together to form a BPI peptide analog having the general formula (I):
wherein: d, e, and f each independently designates 0 or an integer from 1to 10; each {AA} designates an amino acid residue or the residue of a plurality of amino acids linked together through peptide bonding, wherein each {AA} may be the same or different; Q represents H or an acyl group; E represents a hydroxyl group, a carboxyl protecting group or an amino group, or the carboxy terminal group COxe2x80x94E, wherein the CO is part of {AA}, can be reduced to CH2xe2x80x94OH or CHO; each of R and Rxe2x80x2 is independently hydrogen or an amino acid side-chain optionally bound with a specific protecting group; and the line designates a bridging group of the formula:
(i) xe2x80x94Xxe2x80x94Mxe2x80x94Yxe2x80x94Wxe2x80x94Zxe2x80x94; or (ii) xe2x80x94Xxe2x80x94Mxe2x80x94Zxe2x80x94
wherein: M and W are independently selected from the group consisting of disulfide, amide, thioether, thioester, imine, ether, and alkene; and X, Y and Z are each independently selected from the group consisting of alkylene, substituted alkylene, arylene, homo- or hetero-cycloalkylene and substituted cycloalkylene.
In another aspect of the invention, the backbone of the analog is cyclized to a side-chain of an amino acid to form a peptide analog of the general formula (II):
wherein the variables are as disclosed above.
A further backbone-cyclized bicyclic BPI peptide analog includes an Nxcex1-derivative of an amino acid and has formula (III):
wherein each BU represents an Nxcex1xcfx89-functionalized derivative of amino acids of formula (IV):
wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; Rxe2x80x2 is an amino acid side chain, optionally bound with a specific protecting group; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, and alkyl halides; and the other variables are as disclosed above. The BU groups are incorporated into the peptide sequence and may subsequently be selectively cyclized via the functional group G with one of the side chains of the amino acids in said peptide sequence or with another xcfx89-functionalized amino acid derivative.
It is preferred that libraries of these peptide analogs be prepared for screening to determine biologically active compounds. In accordance with the present invention, these libraries have at least four members. In a preferred embodiment, the library as described above comprises two or more sublibraries, each containing a plurality of related peptide analogs.
The present invention also provides methods for the preparation of these peptide analogs and to libraries of such analogs. The methods comprise the steps of providing peptide sequences having a plurality of building units containing amino acids and linked nitrogen atoms and incorporating into each peptide sequence at least one Nxcex1xcfx89-functionalized derivative of an amino acid of formula (IV) by selectively cyclizing a functional group G with another xcfx89-functionalized amino acid derivative or with one of the side chains of the amino acids in said peptide sequence to form backbone-cyclized BPI peptide analogs.
Preferred embodiments for G in formula (IV) include amine, thiol, and carboxyl groups. Preferred embodiments for R and Rxe2x80x2 in formulas (I)-(III) include CH3xe2x80x94, (CH3)2CHxe2x80x94, (CH3)2CHCH2xe2x80x94, CH3CH2CH(CH3)xe2x80x94, CH3S(CH2)2xe2x80x94, HOCH2xe2x80x94, CH3CH(OH)xe2x80x94, HSCH2xe2x80x94, NH2C(xe2x95x90O)CH2xe2x80x94, NH2C(xe2x95x90O)(CH2)2xe2x80x94, NH2(CH2)3xe2x80x94, HOC(xe2x95x90O)CH2xe2x80x94, HOC(xe2x95x90O)(CH2)2xe2x80x94, NH2(CH2)4xe2x80x94, C(NH2)2NH(CH2)3xe2x80x94, HO-phenyl-CH2xe2x80x94, benzyl, methylindole, and methylimidazole.
A particularly useful embodiment of the present invention involves providing the BPI peptide analogs as described above covalently coupled to insoluble polymeric supports.
The present invention likewise provides a method of screening compounds which comprises forming a library of backbone cyclized BPI peptide analogs as described herein and screening the analogs for BPI activity.
In order to fully describe the present invention, the following definitions will be used:
A xe2x80x9clibraryxe2x80x9d of backbone cyclized BPI peptide analogs indicates a collection of BPI peptide analogs wherein at least one conformational constraint consisting of a bridge linking novel building units via modified side chains attached to the nitrogens of the amide bonds is present. Typically the amino acids in other positions of the peptide will be xe2x80x9cvariablexe2x80x9d or xe2x80x9cconstantxe2x80x9d. Each library is characterized by its building units, its constant amino acid residues and its variable amino acid residues. Each library may be composed of xe2x80x9csub-librariesxe2x80x9d which are synthesized in parallel, using a divergent or convergent synthetic scheme.
A xe2x80x9cvariablexe2x80x9d position or amino acid residue may have more than one amino acid in the specified position of the peptide. Typically, in a set of sub-libraries, each sub-library differs from the other in the identity of at least one of its defined amino acid(s) (e.g., the defined amino acid(s) will be constant throughout a single sub-library, yet differ between sub-libraries within the set). A xe2x80x9cconstantxe2x80x9d amino acid or sequence is one whose identity and position are invariant throughout the peptides of the library, and across a set of sub-libraries.
The conformation of a peptide backbone is determined by the three dihedral angles xcfx86(C-N-Cxcex1-C), "psgr"(N-Cxcex1-C-N) and xcfx89(Cxcex1-C-N-Cxcex1), which not only specify the position of the peptide backbone atoms, but also the angle of projection of the amino acid side chains (Ca-Cb vector) from the peptide backbone. A peptide with a xe2x80x9cconformationally constrained backbonexe2x80x9d will either be rigid, existing in only a single conformer characterized by specific values of xcfx86, "psgr", and xcfx89 for each residue, or will exist as an equilibrium mixture of a relatively few discrete conformers, the backbone torsional angles of all residues for each conformer being well described. Thus, a backbone-cyclic peptide with a conformational constraint indicates one in which the atoms and bonds which constitute the ring are energetically able to assume only a limited number of positions in space relative to one another at or around room temperature, and these positions may be well defined by conventional techniques of molecular modeling and crystallography.
An xe2x80x9coptimizedxe2x80x9d conformer is that which has the greatest activity (e.g., biological response, binding or inhibition of biological response or binding) when a library having a defined amino acid sequence is screened for a given target activity. Preferably, only a single bridge will confer optimal activity. An optimized bridge is characterized by its chemical structure and position in the peptide sequence
The xe2x80x9camino acid setxe2x80x9d comprises all amino acids which are to be varied within the peptide at a particular position. Typically the amino acid set will comprise 2-50 different amino acid residues. The amino acid set may be varied in the number of amino acid residues and types of residues for each position in the peptide, or the same set may by used for all positions in the peptide.
The term xe2x80x9camino acidxe2x80x9d refers to compounds which have an amino terminus and carboxy terminus, preferably in a 1,2- 1,3-, or 1,4-substitution pattern on a carbon backbone. xcex1-Amino acids are most preferred, and include the 20 natural amino acids (which are L-amino acids except for glycine), which are found in proteins, the corresponding D-amino acids, the biosynthetically available amino acids which are not found in proteins (e.g., 4-hydroxy-proline, 5-hydroxy-lysine, citrulline, ornithine, canavanine, djenkolic acid, xcex2-cyanolanine), and synthetically derived xcex1-amino acids, such as amino-isobutyric acid, norleucine, norvaline, homocysteine and homoserine. xcex2-Alanine and xcex3-aminobutyric acid are examples of 1,3- and 1,4-amino acids, and many others are well known to the art. Statine-like isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CHOH), hydroxyethylene isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CHOHCH2), reduced amide isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CH2NH linkage) and thioamide isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CSNH linkage) are also useful residues for this invention.
As used herein xe2x80x9cpeptidexe2x80x9d indicates a sequence of amino acids linked by peptide bonds. The peptide analogs of this invention comprise a sequence of amino acids of 4 to 12 amino acid residues, preferably 6 to 10 residues, each residue being characterized by having an amino and a carboxy terminus.
A xe2x80x9cbuilding unitxe2x80x9d indicates an Nxcex1-derivatized xcex1-amino acid of the general Formula (IV):
wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; Rxe2x80x2 is an amino acid side chain, optionally bound with a specific protecting group; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, and alkyl halides; which is incorporated into the peptide sequence and subsequently selectively cyclized via the functional group G with one of the side chains of the amino acids in said peptide sequence or with another w-functionalized amino acid derivative.
The methodology for producing the building units is described in U.S. Pat. No. 5,874,529, which is expressly incorporated herein by reference thereto. The building units are abbreviated by the three letter code of the corresponding modified amino acid followed by the type of reactive group (N for amine, C for carboxyl), and an indication of the number of spacing methylene groups. For example, Gly-C2 describes a modified Gly residue with a carboxyl reactive group and two methylene spacer, and Phe-N3 designates a modified phenylalanine group with a amino reactive group and three methylene spacer.
As used herein xe2x80x9clinear peptidexe2x80x9d denotes the peptide sequence that is constructed only of amino acid residues and is devoid of any building units.
As used herein xe2x80x9cbackbone cyclic peptidexe2x80x9d denotes an analog of a linear peptide which contains at least one building unit that has been linked to form a bridge via the alpha nitrogen of the peptide backbone to another building unit, or to another amino acid in the sequence.
As used herein xe2x80x9cpre-cyclic peptidexe2x80x9d denotes an analog identical to the cyclic analog except that it is retained in the non-cyclized form to serve as control during the biological or other screening assays.
xe2x80x9cPre-cyclic peptide libraryxe2x80x9d denotes the portion of the peptide analog library, containing the building units identical to those of the backbone cyclized library, but is devoid of the conformational constraint of the latter.
Certain abbreviations are used herein to describe this invention and the manner of making and using it. For instance, AcOH refers to acetic acid, Ada refers to adamantanacetyl, Adac refers to adamantanecarbonyl, Alloc refer to allyloxycarbonyl, BCIP refers to 5-bromo-4-chloro-3-indolyl phosphate, Boc refers to the t-butyloxycarbonyl radical, BOP refers to benzotriazol-1-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate, BPI refers to Bactericidal/permeability increasing protein, BSA refers to bovine serum albumin, Cbz refers to the carbobenzyloxy radical, DCC refers to dicyclohexylcarbodiimide, DCM refers to Dichloromethane, Dde refers to 1-(4,4-dimethyl2,6-dioxocyclohex-1-ylidene-ethyl, DIEA refers to diisopropyl-ethyl amine, DMF refers to dimethyl formamide, DPPA refers to diphenylphosphoryl azide, Dtc refers to 5,5-dimethylthiazolidine-4-carboxylic acid, EDC refers to N-ethyl-Nxe2x80x2(dimethylaminopropyl)-carbodiimide, EDT refers to ethanedithiol, Fmoc refers to the fluorenylmethoxycarbonyl radical, HATU refers to [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate, HBTU refers to 1-hydroxybenztriazolyltetramethyl-uronium hexafluorophosphate, HF refers to hydrofluoric acid, HOBT refers to 1-hydroxybenzotriazole, HPLC refers to high pressure liquid chromatography, MALDI-TOF MS refers to matrix-assisted laser desorption, time-of-flight mass spectrometry, Mts refers to the 4-methoxy-2,3,6-trimethylbenzenzsulfonyl, NBT refers to nitro blue tetrazolium, NMM refers to N-methylmorpholine, NMP refers to 1-methyl-2-pyrolidonone, PBS refers to Phosphate buffered saline, Pmc refers to pentamethylchroman-6-sulfonyl, PNPP refers to p-nitrophenyl phosphate, PPA refers to 1-propanephosphoric acid cyclic anhydride, PyBOP refers to Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, PyBrOP refers to Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, RT refers to room temperature, SMPS refers to simultaneous multiple peptide synthesis, TBTU refers to 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, t-Bu refers to the tertiary butyl radical, TFA refers to trifluoroacetic acid, TIS refers to triisopropylsilane, Tpr refers to thiazolidine-4-carboxylic acid, Trt refers to trityl, Ts refers to toluenesulfonyl.
The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and either sequential, divergent and convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used. The D isomers are indicated by xe2x80x9cDxe2x80x9d before the residue abbreviation. List of Non-coded amino acids: Abu refers to 2-aminobutyric acid, Aib refers to 2-amino-isobutyric acid, Cha refers to cyclohexylalanine, Hcys refer to homocysteine, Hyp refers to S-trans-4-hydroxyproline, 1Nal refers to 1-naphtylalanine, 2Nal refers to 2-naphtylalanine, Nva refers to norvaline, Oic refers to octahydroindolecarboxylic acid, Phg refers to phenylglycine, pCIPhe refers to p-chloro-phenylalanine, pFPhe refers to p-fluoro-phenylalanine, pNO2Phe refers to p-nitro-phenylalanine, Thi refers to thienylalanine.
According to the present invention, the principles of cyclic peptide libraries have now been successfully applied to the generation of novel mixtures of peptidomimetic compounds, which are characterized in that they incorporate novel building units with modified side chains attached to the alpha nitrogens of alpha amino acids. These novel building units permit the generation of BPI peptidomimetics that are backbone-to-backbone cyclized and conformationally constrained.
The most striking advantages of this approach are:
1) The method enables cyclization of the peptide sequence without compromising the side chains of the peptide sequence that are involved in biological recognition and functionality.
2) The method allows optimization of the peptide conformation by allowing permutation of the bridge length, direction, and bond type (e.g., amide, disulfide, thioether, thioester, etc.) and position of the bond in the ring. 3) When applied to cyclization of linear peptides of known activity, the bridge is expected not to be involved in target recognition, thereby creating a site suitable for attachment of tags such as radioactive tracers, cytotoxic drugs, light capturing substances, or any other desired label.
The peptides of the invention can be collected and placed in libraries to enable screening for varying degrees of conformational constraint, in order to find the optimal backbone conformation of the peptide in performing its role as an agonist or antagonist. This is accomplished by varying both the position of the bridgeheads (i.e., the positions in the linear sequence of residues that are to be cyclized), as well as varying the length, the direction and the bond type of the bridge between these units.
The general methodology for preparing the cyclic peptides of this invention involves solid phase peptide synthesis using an orthogonal protection scheme which allows for chain elongation, selective removal of the protecting groups, cyclization of the protected peptides and removal of all side-chains protecting groups with or without cleavage from the resin. It is desirable that the various peptide sequences be present in the libraries in substantially equal amount.
The coupling reactions are performed by methods to create amide or ester bonds and are performed by methods familiar in the art as described herein. Typical coupling reagents are carbodiimides, activated anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPA, PPA, BOP, PyBOP, PyBrop, HATU, HBTU, TBTU, HOBT, N-hydroxysuccinimide and oxalyl chloride are typical.
Synthesis of peptide libraries containing more than one building unit, bridge type or amino acid at one or more positions can be performed by different synthetic schemes, as known in the art of peptide synthesis. Preferred methods of generating libraries include the following:
Partitioning, Coupling, and Recombination Scheme
1. The resin is partitioned into a number of aliquots corresponding to the number of amino acids or building units used for the defined set used at each position.
2. Each aliquot is coupled exhaustively to a single building unit or amino acid using solid phase methodologies.
3. The synthesis subsequently proceeds by recombining of all resin portions before the next coupling step is performed.
4. Steps 1 and 2 may be repeated as necessary, depending on whether a constant or variable residue is being coupled.
Alternatively, in a divergent synthetic scheme, at any given point in the synthesis each resin aliquot may be treated individually from that point on until the end of the synthesis, thus generating sub-libraries. The synthesis may be carried out in parallel for part or all of the remaining synthetic process, up to and including the cyclization and cleavage steps.
Coupling of Mixtures
Synthesis is performed using a mixture of amino acids that are coupled in a certain position to one resin aliquot. The use of exactly one equivalent of total amino acid and the long coupling time serves partially to correct for the different rates of coupling of the individual amino acids in the mixture and to help ensure that an equimolar mixture of amino acids is obtained at each position. The procedure of U.S. Pat. No. 5,010,175 can also be used.
After completion of the solid phase peptide elongation, by any scheme, portions of the peptide are cyclized, via the bridging groups attached to the backbone amide bond nitrogens of the building units. It is preferable that a portion is retained in the non-cyclized form to serve as control during the biological or other screening assays. This portion of the peptide analog library, which contains the building units identical to those of the backbone cyclized library, but is devoid of the conformational constraint of the latter, is referred to as the xe2x80x9cpre-cyclicxe2x80x9d. Alternatively, in any of the synthesis schemes, the backbone cyclization step may be performed and additional coupling cycles of amino acid residues may then be carried out.
Portions of the peptide may be cleaved from the resin and protecting groups removed, as required prior to assay of biological activity. The peptides are cleaved from the resin support by methods known in the art, the precise method being dependent upon the characteristics of the resin. It will be understood by those skilled in the art that the removal of certain protecting groups may occur simultaneously with cleavage of the peptide from the resin.
Typically the coupling between the resin and the first amino acid will form an ester bond, which will yield a carboxylic acid group on the peptide when it is cleaved from the resin. HMPB, Rink, PAM, Hycram and hydroxymethyl resins are exemplary. In addition, the carboxy terminal amino acid group may be converted to an amide, an ester or reduced to a terminal alcohol.
The reactive functional groups of the side chains of each amino acid or peptide are suitably protected as known in the peptide art. For example, the Boc, Cbz or Fmoc group may be used for protection of an amino group, especially an xcex1-amino group. An alkyl (e.g., t-Bu, Me), cHex, benzyl or allyl ester may be used for the protection of the side chain carboxyl of Asp or Glu. A benzyl, or suitably substituted benzyl, trityl, Alloc or t-Bu group is used to protect the mercapto group of cysteine, or other thiol containing residues; or the hydroxyl of Tyr, Ser or Thr, Cys and other sulfur-containing amino acids may also be protected by the Acm group or by formation of a disulfide with a thioalkyl (e.g., ethyl mercaptan) or thioaryl group. The benzyl/benzyloxymethyl, or a suitably substituted benzyl/benzyloxymethyl, Boc or formyl group may be used for protection of the imidazolyl group of His; and the Pmc, nitro or a suitably substituted benzene-sulfonyl group (e.g., Ts, Mts) for protection of the guanidino nitrogen of Arg. The phthalamido, Boc, Fmoc, Alloc carbobenzyloxy or benzyl group, or suitably substituted benzyl or benzyloxy group, may be used for protecting the (-amino group of lysine. Suitable substitution of the carbobenzyloxy or benzyl protecting groups is substitution with one to five chloro, bromo, nitro, methoxy or methyl groups, usually ortho and/or para, and is used to modify the reactivity of the protective group. These protective groups are removed by such methods as catalytic hydrogenation, sodium in liquid ammonia, hydrazine, base, TFA or HF treatment, as known in the art. The choice of side chain protecting groups is chosen so that they will not be removed under conditions which are used to deprotect the reactive functional group used in the coupling reaction (e.g., generally the (-amino group) to form the peptide backbone of the peptide chain. The protective group of the reactive functional group is removed prior to coupling each successive amino acid.
The bridging groups of the building units (i.e., G in Formula IV) are used according to the present invention with an orthogonal protection scheme, such that these protecting groups can be removed selectively, under conditions which do not affect the protecting groups on the side chains or cleavage of the peptide from the resin. This enables backbone cyclization on the resin, which is preferred synthetically. Alternatively, the fully protected peptide may be removed from the resin, and cyclization performed in solution after selective removal of the protecting groups of the building units.
The cyclization reaction is carried out by means of selective coupling the bridging group of one building unit to a bridging group of another building unit or amino acid side chain. By way of example, the PyBOP is particularly useful reagent for conducting the coupling reaction, in case of formation of an amide bond. To form a disulfide bridge oxidative conditions are used.
A typical scheme for preparing libraries according to the invention involves using resin such as TentaGel or Rink resin as the support, Fmoc as the (-amino protecting group, t-butyl based protecting groups for the side chains, and allyl/Alloc for the side chain of building unit. Other schemes of orthogonal protection known to those skilled in the art are obviously applicable as well. Generally, one will calculate the number of amino acids in the amino acid set for each position in the peptide, and will use sufficient resin so that there is at least a five-fold molecular excess of reactive sites on the resin to the number of possible peptide sequences.
When the C-terminal amino acid is variable, it is convenient to begin the synthesis using a mixture of individual aminoacyl peptide resins with an equimolar distribution of the amino acids used. An equimolar mixture of the same protected amino acids can also be prepared. An aliquot of the protected amino acid mixture corresponding to exactly one equivalent of total amino acid is allowed to couple to the resin mixture. The use of exactly one equivalent of total amino acid and the long coupling time serves partially to correct for the different rates of coupling of the individual amino acids in the mixture and to help ensure that an equimolar mixture of amino acids is obtained at each position. At this point, a Kaiser test may be performed to assess the completeness of coupling and recoupling with one equivalent of the equimolar mixture can be performed as necessary.
The amino acid sequence scaffold is based on known active sequences from BPI protein. It will thus be possible to further improve the activity of such known sequences, such as by rigidification of the active conformer.
The application of the present invention is particularly suitable for peptides of 3 up to 14 amino acid residues. However, it is also useful to define peptide fragments that compete with larger polypeptides having up to 45 to 70 residues. These methods can be used to produce both conformationally constrained agonists and antagonists. They can either optimize the properties of known sequences or generate novel analogs.
Amino acids in certain positions are replaced by Backbone-Cyclization Building-Units or by natural and non-natural trifunctional amino acids such as Asp, Glu, Cys, Hcys, Lys, Orn and their D counterparts. Thus positional as well as structural scans are performed by changing the position of cyclization, the link of the ring to the backbone, the chirality at the position of cyclization, the ring forming bond, the ring size and the exact placement of the bond within the ring. These variations may also be performed in conjunction with changing the amino acids sequence of the peptide.
In one preferred embodiment of the present invention, backbone-cyclic BPI peptide libraries were prepared by Simultaneous Multiple Peptide Synthesis (SMPS, Houghten, Proc. Natl. Acad. Sci. USA, 82, 5131-5135, 1985). Resin portions were kept separate by capturing them in polypropylene bags, and coupling and deprotection cycles were performed on all bags together in a polypropylene box, except for steps of coupling of different amino acids at the same sites of the peptides. In each bag there was only one (crude) compound by the end of the synthesis.
After completion of the synthesis, samples of resin-peptide were taken form each bag to make mixtures based on structural homology (e.g. a mixture could be comprised of all peptides with a D-amino acid at a certain position or all peptides with the same ring size etc.). The peptides were cleaved from the resin and screened for biological activity as crude mixtures. Then peptides from the most active mixtures were cleaved separately from the resin, purified by preparative HPLC, characterized by mass-spectrometry and amino-acid-analysis and assayed for their biological activity.
Backbone-cyclic peptides containing a disulfide bond in the ring were prepared by inclusion of protected xcfx89-thiol building units in the sequence. Backbone to backbone cyclization was performed by disulfide bond formation between two such building units in a given sequence. Alternatively were backbone to side-chain cyclic peptides prepared by closing a disulfide ring between the w-thiol group of a building unit and the thiol group of Cys or HCys. Formation of the disulfide bond on the resin was obtained by adaptation of the diphenylsulfoxide-silyl chloride method (Akaji et. al., J. Amer. Chem. Soc., 114, 4137, 1992). This is done as was presented in the literature (Camarero et. al., Tetrahedron Lett., 36, 1137-1140, 1995) for prolonged periods (16-24 h) at room temperature. The yields were not high but were sufficient for biological screening. A backbone-bicyclic peptide library was prepared by combining lactam and disulfide cyclizations. Analogs of [Arg6]SP6-11 where Met11 was replaced by an w-thiol containing building unit, Cys or HCys, and Gly9 was replaced by an w-amine building unit were synthesized manually in SMPS bags using Fmoc chemistry. The w-amino group of the building unit in position 9 was protected by Boc. After coupling of this building unit the Boc group was removed and to the w-amine was coupled a second w-thiol building unit or amino acid with its a-amine protected with Boc. Then the Fmoc group was removed from the a-amine of the building unit in position 9 and the synthesis of the peptides was continued. After completion of the synthesis of the hexapeptide chain, a dicarboxylic acid was coupled to the amino terminus and then the Boc group protecting the xcex1-amino group of the xcfx89-thiol containing building unit or amino acid was removed and the lactam ring was closed. Then the disulfide ring was closed by the above mentioned solid-phase diphenylsulfoxide-silyl chloride method. Since the yields of these peptides were relatively low due to side-reactions in the disulfide formation step, the peptides comprising the most active mixtures were re-synthesized and cyclized separately after cleavage from the resin by the normal solution diphenylsulfoxide-silyl chloride method.
In another preferred embodiment, libraries are synthesized by the portioning-mixing method (Furka et al., Int. J. Pep. Protein Res., 37, 487-493, 1991). Typically, in each variable position the resin is split into the appropriate number of aliquots, and different amino acids or building units are coupled to each. Any appropriate reaction vessel may be used to contain these aliquots; in a preferred embodiment it is very convenient to use an individual column for each portion of the resin. After the coupling is completed and the coupling mixture is washed out, all or part of the resin portions are recombined. Removal of xcex1-N protecting groups (typically Fmoc) is performed on the re-combined resin. Further cycles of coupling, and the other steps, are carried out similarly with or without portioning and mixing of the resin. Preferably, in this scheme of production, the library consists of several sub-libraries which differ in one or more amino acid residue, building unit and/or bridge. The final resin portions (sub-libraries) are cyclized to yield backbone cyclized mixtures or left as pre-cyclic mixtures. After removal of side-chain protecting groups and optional cleavage of the sub-library peptides from the resin, screening of the sub-libraries set leads to identification of an optimized sub-library. Further synthesis and screening cycles lead to the optimized backbone cyclized peptide. In each successive synthetic cycles, the complexity of the mixture is smaller.
In another preferred embodiment libraries are synthesized on non-cleavable resins to yield solid-phase supported libraries. Diversity of bridges and amino acids sequence is achieved by the positional scanning method (reviewed by Pinilla et. al. ibid.).


A pumpkin carving kit includes first and second sets of different types of cutting tools, preferably drill elements and saw elements, at least one pattern sheet having a decorative design and an instruction book having first instructions for securing the pattern sheet and transferring the design onto the surface of the pumpkin, second instructions for using the first set of cutting tools on selected design features and third instructions for using the second set of cutting tools on other selected design features. The instruction book mounts an envelope that receives the tool sets which are organized on a tree. Preferably, a plurality of differently sized pattern sheets are included and are each provided with cut lines for slits allowing slit-adjacent portions to overlap whereby the shape of the pattern sheet conforms to the round shape of the pumpkin.

The present invention is directed to a pumpkin carving kit useful for carving decorative designs in the fleshy shell of a pumpkin. The kit includes a combination of tools, decorative patterns and an instruction book containing instructions that correlate design features of the pattern to the cutting tools.
Pumpkin carving has long been one of the several ways in which Halloween is celebrated. Traditionally, pumpkin carving involves the removal of a portion of the pumpkin shell surrounding the stem, removal of the seeds and fibers contained in the pumpkin and carving humorous, grotesque or other decorative features in the pumpkin shell by removing fleshy portions of the shell to obtain the desired appearance. Internal illumination is then provided either by a candle or by a battery-operated light. This illumination results in a glowing decorative pattern.
In the past, pumpkin carving was typically accomplished through the use of pocket knives, paring knives and the like. Due to the size of these cutting instruments, though, the typical carver was only able to obtain crude decorative features. Since the cutting knives used in pumpkin carving are difficult to control, any attempt at carving detailed, intricate design features usually met with failure since the carver often unintentionally removed portions of the pumpkin shell which destroyed the design.
The development of intricately carved pumpkin designs is believed to originated with Paul John Bardeen, the father of the inventor of the present pumpkin carving kit. His innovation used small hand-held saws and spiral drill bits to produce intricately and exotically carved pumpkins. The present invention is directed to an improvement over this early system, and the presentation of improved cutting tools and materials in a kit form for carving pumpkins.


A safety syringe comprising a casing having a neck and a plunger having a distal portion, a weak portion, and a coupling portion covered by a hollow stopper having a membrane. The plunger is channeled in the casing and is moveable between an extended position and a compressed position. A needle holder, in the neck, contains a distally facing arrowhead and a proximally facing needle. Moving the plunger from an extended position to a compressed position, causes the arrowhead to puncture the membrane and engage the coupling portion. If the plunger is returned to the extended position, the needle is drawn into the casing and the distal portion of the plunger can be removed. If the plunger is re-compressed, the needle encounters a wall in the neck preventing the needle from exiting the casing. The syringe is easy to manufacture and prevents manufacturing burrs from being injected into patients.

This application claims priority to, and incorporates by reference, Chinese Patent Application No. 00205827.8 filed on Feb. 24, 2000.
1. Field of the Invention
The present invention relates to a syringe, particularly, to a safety syringe, belonging to the medical device technical field. Safety syringes are designed for one-time use and are, therefore, not re-useable.
2. Background of the Art
In the medical field, there is a great need to avoid cross-contamination or infection which may be caused when a syringe is used more than once. Single use syringes have long been thought as being one way to avoid these potential problems. However, current single use syringes present a variety of problems. Some syringes which claim to be xe2x80x9csingle usexe2x80x9d in actuality can be reused with user intervention. Some syringes are designated as xe2x80x9csingle usexe2x80x9d solely because they are to be discarded after use pursuant to a regulation; nothing prevents their reuse. Some syringe designs expose the drug or bodily fluid contained therein to reactive components of the syringe (e.g. a spring used to retract the plunger). In addition, some syringes, although not capable of being reused, present health risks to the medical personnel handling them. Accordingly, there is a need for a single use syringe which is: (a) easy to use; (b) incapable of being reused; (c) presents little or no danger to medical personnel after use; (d) designed in such a way as to prevent contact between reactive syringe components and the fluid contained within the syringe; and (e) capable of being manufactured at low cost.
Currently, there are mainly two classes of safety syringes. One class of syringes, which employs a spring to retract a needle into the syringe, is represented in U.S. Pat. Nos. 5,769,822, 5,980,494, and 6,039,713. Although these syringes are effective and easy to use, the also have inherent disadvantages. First, the spring-loaded structure is complicated because of the number of parts involved creates difficulties in constructing the syringe. Second, due to the complicated structure, the costs of manufacturing are high making marketing difficult. Third, due to the location of the spring at the needle end of the syringe, it is difficult to position the fluid close to the inlet to the needle; this increases fluid residue and makes removal of air bubbles more difficult.
The second class of syringes employs a locking mechanism on the plunger which engages a corresponding structure at the base of the needle when the plunger is completely compressed. A subsequent withdrawal of the plunger pulls the needle into the syringe barrel. The structure of these syringes is simpler and the cost of production lower in comparison to the former class. However, this class of syringes also has disadvantages. For example, in Chinese Patent CN2270509Y, the corresponding parts of the locking mechanism between the needle and the plunger are exposed to fluid contained within the syringe. Accordingly, any burrs on the parts which result from manufacturing may fall into the fluid and be thereafter accidentally transmitted to the patient giving rise to a potentially dangerous situation. In addition, in many of these syringes, the locking mechanism can be disengaged and the syringe reused.
U.S. Pat. No. 5,601,534 discloses a one-time-use safety syringe comprising a simple structure which reliably releases gases and retains a very small amount of residual fluid. However, it also presents disadvantages. The structure employs an inward facing needle extension which is bent by an arrowhead attached to the plunger. The extension is bent into a cavity in the plunger such that when the plunger is pulled away from the needle, the needle is pulled into the syringe barrel.
A first disadvantage is that the size of the extension must correspond to the size of the needle; a large needle requires a large extension which is not as easily bent thereby degrading the integrity of the engagement with the plunger.
A second disadvantage is that the rubber stopper, which houses the cavity, is solid. Due to the solid nature of the stopper, the extension must completely pierce the stopper to create a strong engagement with the cavity. To create this strong engagement requires a strong compression of the plunger at the completion of the injection; this can be difficult when the needle and needle extension are large.
A third disadvantage results with large needles. A large compressive force needs to be applied to the plunger to complete an injection with a large needle. This force counteracts the force necessary for the needle extension to puncture the plunger. To address this problem, the needle""s engagement with the barrel head needs to enhanced. However, by enhancing the needle""s engagement with the barrel head, it becomes more difficult to withdraw the needle into the syringe barrel after the injection is complete.
Fourth, the engagement of the needle extension and the plunger is such that upon withdrawal of the needle into the syringe barrel, residual fluid in the barrel can leak out of the syringe.
Fifth, the size of the plunger cavity corresponds to the size of the needle. Accordingly, one plunger can not be used with a variety of needle sizes. Therefore, medical personnel must maintain an inventory of various sizes of plungers which is costly and which occupies precious storage space.
A solution to the aforementioned deficiencies in the art are resolved by the safety syringe herein described. Specifically, the presently described syringe has the following advantages over the prior art: (a) the hooks within the stopper which engage the arrowhead are not in contact with the fluid contained in the syringe thereby reducing the potential for manufacturing burrs to fall into the fluid and thereby be injected into the patient. The structure is possible because the engagement of the needle holder and the plunger occurs when the arrowhead pierces the stopper membrane. After the completion of the injection thereby establishing this engagement, the syringe can not be reused. Moreover, the puncturing of the membrane requires a force which is much smaller than the force necessary to compromise the o-ring seal in the plunger neck; accordingly the counteractive force problems in the prior art are eliminated; (b) the o-ring positioned in the syringe neck prevents fluid from escaping when the needle is initially pulled toward the syringe barrel; (c) the syringe has a simple structure which is both easy to use and manufacture; (d) the cavity in the stopper and the arrowhead can be used with any size needle (i.e. the size of the cavity and the arrowhead is not dependent on the size of the needle); and (e) the size of the needle does not compromise the integrity of the locking between the plunger and the needle holder.
These and other advantages are made possible by a safety syringe comprising a syringe casing having a distal end and a proximal end. A plunger is channeled in the syringe casing and is capable of moving from an extended position to a compressed position. The plunger has a weak portion, a distal portion, and a coupling portion. A stopper, having a membrane, is affixed to the coupling portion of the plunger; the membrane is between the proximal end of the syringe casing and the plunger. A needle holder, initially coupled to the proximal end of the syringe casing, has a distally facing arrowhead and a proximally facing needle. When the plunger is moved from the extended position to the compressed position, the arrowhead punctures the membrane and lockingly engages the coupling portion of the plunger. Subsequently, when the plunger is returned to the extended position, the needle holder and needle are drawn into an interior of the syringe casing.
In the safety syringe above-described, the weak part of the plunger may include a plurality of tines. In this situation, when the plunger is returned to the extended position from the compressed position, the distal portion of the plunger may be disengaged from the weak portion by breaking the tines.
In a preferred embodiment, the safety syringe includes a plurality of o-rings formed on an outer surface of the stopper. The plurality of o-rings frictionally engage an inner surface of the syringe casing when the plunger is moved between the extended position and the compressed position.
In the safety-syringe above-described, the proximal end of the syringe casing comprises a cylindrical neck portion which houses the needle holder. The cylindrical neck portion comprises a circumferential channel formed on an inner surface thereof. The needle holder comprises a circumferential o-ring on an outer surface thereof which is sized to substantially fill the channel.
The safety-syringe may also include a safety mechanism. The safety mechanism comprises a check plate which is affixed to an outer surface of the syringe casing and which is adapted to move from an unlocked position to a locked position. The check plate is designed to engage a notch in a side of the distal portion of the plunger. When the check plate is in the locked position, it engages the notch thereby preventing the plunger from moving from the extended position to the compressed position.
In the presently described safety syringe, the needle and the syringe casing have longitudinal axes. Before engagement of the arrowhead and the coupling portion, the needle axis and the syringe casing axis are substantially parallel. By way of contrast, after the needle holder and the needle are drawn into the interior of the syringe casing, a mechanism alters the orientation of the needle axis with respect to the syringe axis so that they are not substantially parallel. Additionally, the proximal end of the syringe casing comprises a cylindrical neck portion which houses the needle holder; a circumferential wall extends from an interior end of the cylindrical neck portion to an outer surface of the proximal end of syringe casing. Due to the misalignment of the needle axis and the syringe casing axis, the needle will contact the wall if the plunger is moved toward the compressed position.
Ideally, the stopper substantially covers the coupling portion of the plunger when the plunger is in the extended position. Moreover, when the plunger is in the compressed position (i.e., after engagement of the arrowhead and the coupling portion housed within the stopper), the stopper substantially covers the arrowhead and the coupling portion.
A second embodiment of a safety syringe includes a syringe barrel and a needle holder, having an arrowhead at a rear portion thereof, sealingly installed onto a front end of the syringe barrel. The arrowhead has a circumferential groove disposed at its base. A needle is sleeved onto the needle holder. A cylindrical rubber stopper, coupled to a plunger, has a membrane at on one end and a bore at the other end. The plunger has an coupling portion dimensioned to engage the arrowhead. The rubber stopper is hollow and is sleeved over the plunger coupling portion. Moreover, the plunger is moveable between an extended position and an compressed position at which an injection is completed. The rubber stopper and the plunger are sequentially installed into the syringe barrel.
In the syringe of this type, the membrane end of the rubber stopper faces the arrowhead; at the completion of the injection, the arrowhead pierces the membrane and engages the coupling portion. After this engagement, when the plunger is drawn backwards towards the extended position, the needle holder and the needle are drawn into the syringe barrel.
Preferably, the syringe also includes an inner sealing channel on an internal side of the front end of the syringe barrel. Correspondingly, a sealing ring sleeved onto the needle holder is preferably dimensioned to match the inner sealing channel. A rear portion of the inner sealing channel should have a smaller arc than an arc of the sealing ring. Moreover, a frontal portion of the inner sealing channel should have an inclined face having an arc larger than the arc of the rear portion. In a syringe having these channel characteristics, the needle holder can be slideably engaged in the front end of the syringe barrel by means of the sealing ring and can be moved in the direction of the syringe barrel when the plunger is withdrawn are engagement of the arrowhead and coupling portion.
The plunger in a safety syringe of this nature preferably comprises a weak portion and a resistance plate fixedly connected to the coupling portion. Moreover, the plunger preferably has a xe2x80x9c+xe2x80x9d shaped formed by a vertical rib plate and a horizontal cross rib plate. The vertical rib plate is weakly connected to the resistance plate by means of a plurality of tines. After completion of the injection and after the needle holder and needle are drawn into the syringe barrel, the tines of the weak portion can be broken upon application of a bending moment.
Similar to the first embodiment, the safety syringe may also include a mechanism by which premature use of the syringe is prevented. Specifically, the syringe may include a safety mechanism having a wing plate, a check plate, and a flexible film connecting the wing plate and the check plate. The safety mechanism is positioned on an exterior surface of the syringe barrel at a position proximate to an insertion bore into which the plunger is inserted. The check plate and one rib of the vertical rib plate have corresponding grooves which may be engaged if the check plate is rotated toward the vertical rib plate by means of the flexible film. If the grooves of the check plate and the vertical rib plate are engaged, premature completion of the injection is prevented.
Ideally, a plane forming the base of the arrowhead is not perpendicular to a central axis of the syringe barrel. In this fashion, after the needle is drawn into the syringe barrel, the axis of the needle will be directed toward an inner wall of the syringe barrel; a subsequent re-compression of the plunger toward the compressed position will cause the needle to abut the inner wall of the syringe barrel.
The present invention also contemplates a method for preventing reuse of a syringe. The method uses a syringe having a syringe casing which, in turn, has a longitudinal axis, a distal end, and a proximal end having a cylindrical neck portion. In the syringe used by the method, a circumferential wall extends from an interior end of the cylindrical neck portion to an outer surface of the proximal end of syringe casing. A plunger having a weak portion, a distal portion, and a coupling portion, is channeled in the syringe casing and is moveable from an extended position to a compressed position. A stopper having a membrane is affixed to the coupling portion of the plunger; the membrane is between the proximal end of the syringe casing and the plunger. A needle holder, housed in the cylindrical neck portion of the syringe casing, has a distally facing arrowhead and a proximally facing needle having an axis and a needle tip.
The method includes: (I) moving the plunger from the extended position to the compressed position; (II) puncturing the membrane with the arrowhead when the plunger is in the compressed position; (III) coupling the arrowhead to the coupling portion; (IV) returning the plunger to the extended position and thereby drawing the needle holder and the needle into an interior of the syringe casing; and (V) preventing reuse of the needle by either (a) altering the orientation of the needle axis with respect to the syringe casing axis from an orientation in which the axes were substantially parallel to an orientation in which the needle tip will encounter the circumferential wall if the plunger is moved back toward the compressed position or (b) disengaging the distal portion of the plunger by breaking the weak part.
In the method in which the preventing reuse of the needle is preformed by altering the orientation of the needle axis with respect to the syringe casing, a further step is possible. This method may also include bending the needle when the needle tip encounters the circumferential wall when the plunger is subsequently moved toward the compressed position.
In addition, in the method in which the preventing reuse of the needle is performed by disengaging the distal portion of the plunger from the weak part, the disengagement may occur if the weak part of the plunger is comprised of a plurality of tines which are easily broken upon application of a bending moment applied to the plunger when the plunger is in the extended position.

No. 1-7, Building 14, Capital Normal University


A method for modifying the design of a component with regard to one or more criteria using a virtual model of the component, wherein a virtual model of the component is produced by combination of the component from basic objects with the aid of Boolean operators, and each basic object is assigned an information element in which attributes of the basic object are stored. Each basic object is decomposed into primitive objects which are combined with aid of Boolean operators, the primitive objects being surfaces or bodies which can be decomposed into rasters. Each geometrical contact surface is assigned a connecting element in which information for coordinating the objects is stored, and the rastering of the primitive objects is performed by a conventional gridding method. A modification of the geometrical shape of objects is undertaken with the information elements defining the limits of possible modifications.

This application claims the priority of Application No. 100 43 737.0, filed Sep. 5, 2000 in Germany, and PCT DE01/03350, filed on Sep. 4, 2001.
The invention relates to a method for modifying, in particular for optimizing, the design of a component with regard to one or more criteria using a virtual model of the component.
The current development processes are developing from stepwise development towards simultaneous development, the disciplines of technical development and analysis being in closer cooperation, and knowledge of all relevant technical disciplines being introduced in early phases of product development. Simultaneous development is found to be necessary in order to develop products with improved technology and of higher quality in a shorter development time, something which also leads to reduced costs.
The probability of the development of better and less expensive components is rising in principle through the application of high performance analysis tools and the knowledge of experts in the various fields of engineering science. A possible impediment to success is, however, the normally limited time in which the components have to be developed.
With this in mind, the multidisciplinary design optimization (MDO) is regarded as an effective method for improving quality and performance of products in conjunction with reducing costs. This happens chiefly through shortening the evaluation time required overall for a design cycle, and by creating an automated analysis tool which searches for the best solution or performs specific tasks in a limited time.
Various publications provide a good overview of the state of the art in multidisciplinary design optimization. To name only a few, there are the articles by Alexandrov edit. [Natalia Alexandrov and M. Y. Hussaini, editors, Multidisciplinary Design Optimization—State of the Art, SIAM 1997], Bartholomew [Peter Bartholomew, The role of MDO within aerospace design and progress towards an MDO capability; in 7th AIAA/USA/NASA/ISSIMO Symposium on Multidisciplinary Analysis and Optimization, p. 2157-2165; AIAA, 1998], Kroo [Ilan Kroo; Mdo for large-scale design; in Natalia M. Aleandrov and M. Y. Hussaini, editors, Multidisciplinary Design Optimization—State of the Art, pages 22-44; SIAM, 1997], Sobieski and Haftka [Jaroslaw Sobieszczanski-Sobieski and Raphael T. Haftka; Multidisciplinary aerospace design optimization: Survey of recent developments, Conference Paper 96-0711, AIAA, 1996. Presented at the 34th Aerospace Sciences Meeting and Exhibit; Jan. 15-18, 1996] and Vanderplaats [G. N. Vanderplaats; Structural design optimization status and direction; Journal of Aircraft, 36(1):11-20, January-February 1999].
Sobieski and Haftka distinguish between three categories of multidisciplinary design optimization, which place different requirements on EDP and organization.                The first category comprises only a few technical disciplines. An expert can process all the required information and all necessary knowledge.        The second category comprises all methods which achieve the multidisciplinary object, all the required technical disciplines being introduced at a low degree of complexity. This optimization method is mostly applied to the design concept of a system, the analysis tools being used as individual modules. If these methods are, applied to tasks in the continuing design process, they are confronted with some of the organizational challenges set by multidisciplinary design optimization.        The third category is described as a stage of the challenge in terms of computing and organization. In order to permit the analysis in a modular environment, specific techniques, for example, decomposition, approximation and comprehensive sensor technologies are used as tools in order to organize the modules themselves and the exchange of data.
Similar classifications are proposed in other publications, for example by Bartholomew and Kroo. They distinguish between the various degrees of complexity and accuracy of the applied analysis programs and the optimization architecture.
The publications described above lead to a modular analysis environment, something which necessitates the use of decomposition and approximation techniques in order to be able to solve relatively complex design tasks. Kroo explains the necessity to develop the parameterized geometry at different degrees of detail and abstraction in order to be able to apply the multidisciplinary optimization to realistic, large-scale design tasks which comprise many disciplines and analysis tools from the state of the art.
In order to permit the fully automated design optimization with the use of a parameterized geometry at different degrees of detail, the geometry modelling must be more strongly linked to the rastering. Samareh [Jamshid A. Samareh; Status and future of geometry modelling and grid generation for design and optimization; Journal of Aircraft, 36(1):97-104, January-February 1999] compiles an overview of the requirements to be fulfilled and the problems to be solved in order to create such a fully automated geometry model and a rastering method for multidisciplinary optimization applications.
Another aspect of multidisciplinary design optimization is discussed by Wood and Bauer in [Richard M. Wood and Steven X. S. Bauer; A discussion of knowledge based design; Conference Paper 98-4944, AIAA, 1998; Presented at the 7th AIAA/USAF/NASA/ISSMO Symposium on Multidisciplinary Analysis and Optimization; Sep. 2-4, 1998]. Wood and Bauer point out the need to allow expert knowledge to flow into the optimization process in order to improve the technology and quality of the products. They say that knowledge from previous design cycles must flow into the early phase of the design process.
None of the solutions known from the prior art permits a completely automated design optimization.
It is an object of the present invention to propose a completely automated method for multidisciplinary design optimization of the two-, quasi- three- or three-dimensional component which permits small-scale changes to be transferred to the complete component.
The inventive method can be used to modify a component, in particular to optimize it, by applying modifications to different stages of details of the component. The first stage is to create a virtual model of the component. For this purpose, the component is combined from basic objects with the aid of solid modelling Boolean operators, the basic objects being, in particular, surfaces or rotationally symmetrical bodies. Each of the basic objects is assigned an information element in which the attributes of the respective basic object, in particular parameter values, interval limits and rules, are stored. These attributes determine and delimit the respective basic object.
It has hereby become possible to render accessible to multidisciplinary design optimization geometries and/or basic objects to which knowledge and/or information is allocated.
Each basic object is then constructed from further basic objects or one or more primitive objects which are combined with the aid of Boolean operators. The primitive objects are surfaces or bodies of different complexity and geometrical multiplicity which can be decomposed into rasters. Each further basic object or primitive object can again be assigned an information element in which the attributes of the further basic object or the primitive object, in particular parameter values, interval limits and rules, can be stored. These attributes determine and delimit the respective primitive object.
The Boolean combination of the component from individual basic objects yields geometrical contact surfaces or contact lines between two or more objects. The same also holds for the primitive objects. Each of these geometrical contact surfaces and contact lines is assigned a connecting element in which information for coordinating the objects is stored.
These so-called connecting or coordination elements can also be allocated as additional knowledge to each basic object or primitive object in order to obtain a better subdivision into rasterable surfaces in the further process.
The primitive objects are rastered in the next step, specifically by a conventional griding method.
The modification of the component can be undertaken subsequently. This is performed by modifying individual or a group of objects, the information of the information elements defining the limits of possible modifications, the connecting elements defining the coordination of the objects. Customary raster techniques are applied in this case.
The proposed model is a feature-based geometry model which permits multidisciplinary optimization in different degrees of detail. It uses an object-oriented access in order to produce the geometry and to integrate all the information required during the design process. The geometry at different degrees of detail permits starting with a two-dimensional geometry, then with rotationally symmetrical or stretched bodies, and finally with a realistic, three dimensional geometry.
The basic objects can be constructed from one or more further basic objects or primitive objects which can be combined by Boolean operators. Each basic object is again assigned an information element in this case, in which attributes of the basic object, in particular parameter values, interval limits and rules, are stored. Each geometrical contact surface or contact line which is produced in the case of the Boolean operation between two or more objects is assigned a connecting element in which information for coordinating the objects is stored.
The primitive objects can also be constructed from one or more further primitive objects, as a result of which it is possible to modify the degree of detail or the degree of construction of the model can be modified.
The primitive objects can, in turn, be combined by Boolean operators. They can be assigned in each case an information element in which attributes of the primitive object, in particular parameter values, interval limits and rules, are stored. Each geometrical contact surface or contact line which is produced in the case of the Boolean operation between two or more objects can be assigned a connecting element in which information for coordinating the objects is stored.
The modification of the geometrical shape is preferably performed exclusively or predominantly on the lowest object plane, that is to say the primitive objects, it being possible to raster the objects to be modified.
The inventive method proposed permits the multidisciplinary optimization of a component by modifying the degree of detail. Each technical discipline can start with the optimization at the stage optimum for it. The details of this design can be changed during the optimization process without the need for a complete change to the structure and topology of the previously designed geometry.
All the information required in later design steps can be taken from the information elements assigned to the individual objects, or be derived from the information. The information contained can be employed or passed to other tools.
The inventive modification method is preferably applied to optimize a component. It is preferably used after termination of the basic conception with a lower level of detail, and increases the variety of detail and the complexity of the topology as the process progresses.
The degree of the level of detail of the geometry of the model can be modified by changing the type of the primitive objects, by adding further objects to the preceding model, that is to say the latter is constructed from further basic objects and/or primitive objects.
A feature of the component can be represented by combining a primitive object with an information element which contains attributes of the object. These can be, in particular, parameter values, interval limits or rules. The rules reflect the knowledge of a person skilled in the art of the respective fields. It is thus therefore possible to define a feature of the component as a geometrical object which contains all the specific information which is required to determine and to delimit the geometry.
It is also possible to derive from the information stored in the information element further information and rules which are required in later design phases.
In order to render automated geometry modelling and rastering possible, it is desirable to maintain the principle features of each primitive object during the assembly of the topology of the overall component. The modelling technique is expanded for this purpose by creating additional curves or surfaces at the points at which objects are connected to one another or separated from one another.
These additional curves or surfaces are termed connecting elements. The connecting elements are used in order to decompose the geometry, which is constructed from rasterable constituents, into these rasterable constituents. These elements can both be produced automatically during the modelling with the aid of Boolean operators, and be allocated to each object as additional knowledge. It is then easy to produce a raster for the rasterable primitive objects.
Boolean operators produce an object which includes two or more primitive objects and one or more connecting elements between the primitive objects. Which type of object is selected for the primitive object depends on the degree of detail. With each further combination of objects, the degree of assembly and thus the degree of detail is increased.
Each connecting element includes curves or surfaces which cut the primitive objects such that they do not overlap, but meet precisely along this curve or surface. By using this technique, the topology of each primitive object can remain substantially the same during the optimization process. The production of the connecting elements corresponds to the customary raster methods, in which specific connecting conditions are determined for each object.
In another version of the inventive method, the Boolean operations are combined with an optional generation of intersection sets of neighbouring objects which are introduced into the connecting element. These intersection sets are created where the two objects intersect. The connecting elements then contain information on possible connecting surfaces or connecting lines between neighbouring objects. The connecting elements can then be positioned for the rastering such that they obey as well as possible the rules for producing rasters.
The primitive objects are preferably standardized, rasterable parts such as triangles, rectangles, pentagons, tetrahedra, pentahedra or hexahedra.
The primitive objects are preferably produced from surfaces or bodies which use lower geometrical objects, and which are combined with the aid of Boolean operators or by rotation, stitching or stretching. Primitive objects of the lowest degree of detail are objects such as circular or rectangular surfaces or bodies. The degree of detail grows through the use of objects such as B-spline curves or B-spline surfaces in order to describe the contours or surfaces. The degree of detail is characterized by the degree of the geometrical variety. If the number of the parameters rises, the objects become more general and the variety of geometrical shapes rises.
The resulting primitive objects are preferably standardized. They have the same input and output parameters, in order to permit a simple exchange between primitive objects of different degrees of detail.
An automated rastering is rendered possible by the assembly of rasterable primitive objects and the insertion of the connecting elements. The production of the connecting elements falls back on knowledge from the decomposition of geometry using the finite element method.
The actual rastering by a commercially available raster generator can be preceded by a preparatory step which makes available the data which are required by the raster generator.
Geometrical information such as, for example vertices, corners, surfaces, bodies, and specific raster information such as, for example, the number of elements, the type of the elements, the number of nodes, can be made available in the preparatory step. The entire geometry is decomposed at the connecting elements in the case of the method of rastering preferably used.
The result is individual surfaces for the production of a two-dimensional raster, or individual bodies for the production of a three-dimensional raster.
The advantage of the joining of geometrical modelling and rastering in one system is the high degree of accuracy in the geometry, which is then transferred to a commercially available raster generator. Processes such as the connection of edges which adjoin one another at their end points, the filtering out of corners which are shorter than a prescribed tolerance, or the closing of gaps between edges, can be carried out by using the information from the geometrical model.
Boolean operations which are applied during the production of the geometrical model can cause a change in the contours of the original primitive objects. The contour edges can be shortened, or new edges can be added. The recognition of which edges can be connected is thereby implemented. Both the raster surface or the raster body, and the edges of surfaces which belong to the sides of an element can be filtered out by means of this recognition.
In addition to this recognition process, it is possible to carry out other operations such as, for example, filtering and closing edges. All the information used for commercial raster generation can be derived from the objects recognized.
Additional information which is relevant for the production of rasters can be input in to the module or determined during the raster production.
The production of connecting curves between two objects is explained in more detail below with the aid of FIGS. 1 to 3:
FIGS. 1 to 3 show the production of connecting curves for surfaces. The method is the same for bodies, except that curves are used instead of points, and surfaces are used instead of curves in order to produce the connecting surfaces.

Method for modifying the design of a structural part