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A particularly efficient design for a nonbibulous lateral flow one step assay for an analyte in a biological sample is disclosed. In the improved device of the invention, three zones which are in nonbibulous lateral flow contact are employed: a sample receiving zone, a labeling zone, and a capture zone. The sample containing analyte is carried through the labeling zone and interacts with an assay label comprising visible moieties, preferably particles, which are coupled to specific binding reagent for analyte or to a competitor with analyte for a capture reagent. The flow continues into the capture zone where the visible moieties to which analyte or competitor are coupled are captured. Excess fluid is absorbed into an absorbent zone in contact with the capture zone. A positive result is obtained by visualizing the visible moieties in the capture zone. An additional improvement comprises obtaining the nonbibulous lateral flow by converting a bibulous support to a nonbibulous support through coating with a blocking agent, such a methylated BSA. Control label comprising visible moieties (preferably, visually distinguishable from those of the assay label) may also be included in the labeling zone and captured in a separate control portion of the capture zone to verify that the flow of liquid is as expected.
1. An assay device for detection of the presence or absence of an analyte in a sample, said assay device providing lateral flow contact between three separately prepared zones, said zones comprising:
(a) a sample receiving zone prepared by lyophilizing a first matrix through which all dissolved or dispersed components in a liquid sample flow at substantially equal rates, said sample receiving zone being in lateral flow contact with
(b) a labeling zone prepaired by reversibly coupling a label
which label comprises visible moieties coupled either to a specific binding partner reactive with the analyte or to a ligand competitive with analyte for binding to capture reagent
to a second matrix in the presence of a methylated BSA blocking agent so that all dissolved or dispersed components in a liquid sample will flow at substantially equal rates through said second matrix, followed by lyophilizing said second matrix said labeling zone being in lateral flow contact with
(c) a capture zone prepared by irreversibly coupling a capture reagent which binds to analyte to at least a portion of a third matrix followed by treating said third matrix with a methylated BSA blocking agent so that all dissolved or dispersed components in a liquid sample will flow in substantially equal rates said capture zone being in fluid contact with an absorbent;
wherein the presence or absence of analyte in the sample is detectable in the capture zone within one minute of adding the sample to the sample receiving zone.
2. The device of claim 1 wherein said capture reagent is selected from the group consisting of an antibody and an immunologically reactive fragment thereof specific for analyte.
3. The device of claim 1 wherein said assay label comprises visible moieties coupled to a specific binding partner for said analyte so as to bind said analyte to said specific binding partner.
4. The device of claim 3 wherein said specific binding partner for analyte is selected from the group consisting of an antibody and an immunologically reactive fragment thereof.
5. The device of claim 1 wherein said assay label comprises visible moieties coupled to a competitor of said analyte for said capture reagent.
6. The device of claim 5 wherein said competitor of said analyte is additional analyte or similarly immunologically active analog thereof.
7. The device of claim 1 wherein said capture zone matrix is selected from the group consisting of nitrocellulose, nitrocellulose blends with polyester or cellulose, and nylon.
8. The device of claim 1 wherein the labeling zone further includes a control label comprising visible moieties coupled to a control binding reagent, and wherein said capture zone further includes a specific binding partner for said control binding reagent in a portion of said capture zone different from that wherein said capture reagent for said assay label is located.
9. The device of claim 8 wherein said control reagent is avidin or a derivative thereof and wherein said specific binding partner for the control reagent is biotin.
10. The device of claim 8 wherein said assay label and control label are visually distinguishable.
11. The device of claim 10 wherein said assay label and control label are of different colors.
12. The device of claim 1 wherein said visible moieties are particles.
13. The device of claim 12 wherein said particles are selected from the group consisting of colored latex beads, red blood cells, metal sols and liposomes containing dye.
14. A method to determine the presence or absence of analyte in a sample, which method comprises applying said sample to the sample receiving zone of the device of claim 1, so as to permit said sample to flow through the labeling zone and the capture zone into the absorbent zone, and
detecting the presence or absence of visible moieties in the capture zone in said portion thereof containing the capture reagent capable of binding said assay label.
15. The method of claim 14 wherein said analyte is human chorionic gonadotropin (hCG).
16. The method of claim 15 wherein the specific binding partner for analyte is a monoclonal antibody immunoreactive with hCG.
17. The method of claim 16 wherein the capture reagent is rabbit anti-hCG.
18. The method of claim 14 wherein the visible moieties are particles.
19. The method of claim 14 wherein the particles are selected from the group consisting of colored latex beads, red blood cells, metal sols and liposomes containing dye.
20. The device of claim 1, wherein said matrices of said sample receiving zone, labeling zone and capture zone have been converted from a form in which all dissolved or dispersed components in a liquid sample do not flow at substantially equal rates to a form in which all dissolved or dispersed components in a liquid sample flow at substantially equal rates.
21. The method of claim 14, wherein said matrices of said sample receiving zone, labeling zone and capture zone have been converted from a form in which all dissolved or dispersed components in a liquid sample do not flow at substantially equal rates to a form in which all dissolved or dispersed components in a liquid sample flow at substantially equal rates.
This application is a continuation of application Ser. No. 07/639,967, filed Jan. 11, 1991.
The invention relates to immunological and related assay methods and apparatus, especially to those for testing biological samples using sandwich or competitive assays.
The literature on various forms of specific binding assays, especially immunoassays, is extensive and commercial products are numerous. A large number of simplified and conveniently packaged assays are currently marketed, and patent protection has been sought for a large number of these.
Of particular interest in connection with the invention herein are those assays which are conducted using a lateral flow protocol. U.S. Pat. No. 4,943,522 issued 24 Jul., 1990,incorporated herein by reference, sets forth citations which describe such devices and methods. This patent itself is directed to a lateral flow device which relies on a nonbibulous support to conduct liquids from one portion of the device to another. The present invention represents an improvement on this method and device wherein nonbibulous lateral flow is used to conduct visible moieties, especially labeled particles, e.g., dyed latex, red blood cells or liposomes capable of reacting with analyte or a competitor thereto into a capture zone for detection, using a bibulous support made nonbibulous by treatment with a blocking agent. The result is a one-step assay which can be conducted in a very short period of time (typically, within 60 seconds), and wherein the readout is usually available instantaneously upon the sample contacting a capture zone.
Other disclosures of lateral flow assays have also appeared. For example, U.S. Pat. No. 4,861,711, issued 29 Aug., 1989 describes a lateral flow assay wherein all components needed for the detection of an analyte are embedded in a single sheet. The lateral flow is referred to as chromatographic behavior. European patent application 306,772 published 15 Mar., 1989 describes a lateral flow device which comprises a chromatographic medium wherein the zone for application of sample and the reaction zone with an immobilized reagent capable of binding the analyte or a label-specific binding material are separated. British application 2,204,398 published 9 Nov., 1988 and assigned to Unilever describes a lateral flow device wherein sample applied to the device picks up labeled reagent and permeates into a detection zone. Labels include gold sols and colored particles. U.S. Pat. No. 4,168,146 describes lateral flow through a test strip; the presence of an analyte is determined by addition of a suitable color indicator.
European application 276,152 published 27 Jul., 1988 and assigned to Symbiotics Corp. describes a bibulous matrix lateral flow device which operates on two separate planes. U.S. Pat. Nos. 4,094,647; 4,235,601 and 4,361,537, all assigned to Thyroid Diagnostics Inc. describe chromatographic strips which, when placed in developing fluid, move a sample through a series of zones for binding to reagent and detection. U.S. Pat. No. 4,857,453 assigned to Syntex describes a device wherein reagents are supplied in breakable containers which are integral to the device.
EP 158,746 and U.S. Pat. No. 4,775,636 assigned to Janssen describe the use of metal sols as visible detecting labels for specific binding assays.
U.S. Pat. No. 4,703,017, assigned to Becton Dickinson, describes test strip devices wherein binders for an analyte and/or a visible tracer are applied to defined areas of a solid support by adsorption or covalent coupling. After application of the binder to one or more test areas of the substrate, the residual binding capacity of the test substrate is saturated or blocked by treatment with one or more types of proteins which do not specifically bind the materials to be employed in the assay. The tracer, when bound under assay conditions to the binder or to the analyte bound to the binder, is visible on the support without further treatment. The test strip is contacted and incubated with a sample containing (or suspected of containing) analyte; a sample strip may be provided with a plurality of test areas. U.S. Pat. No. 4,855,240, also assigned to Becton Dickinson, describes an assay wherein a sample and a tracer as described in U.S. Pat. No. 4,703,017 are applied at disparate locations on a flat lateral flow device.
With the exception of the first mentioned U.S. Pat. No. 4,943,522, all of the lateral flow methods heretofore discussed employ bibulous supports. The bibulous nature of the support increases the time of the assay and permits obtaining a result only after 5 minutes or more. The advantages of immediate availability of results conferred by the methods and devices of the present invention result from the conversion of the bibulous to a nonbibulous support in all regions of the matrix and from the design and miniaturization of the device as a whole.
The invention provides rapid and accurate methods for assessing the presence or absence of analytes in biological samples and devices for the conduct of these methods.
Thus, in one aspect, the invention is directed to an assay device for determination of the presence or absence of an analyte in a biological sample wherein the device comprises a sample receiving zone comprising a solid support capable of conducting non-bibulous lateral flow of sample in contact with a labeling zone capable of conducting nonbibulous lateral flow and containing visible moieties coupled to either a specific binding partner reactive with the analyte or a ligand competitive with analyte for binding to a capture reagent, which labeling zone is in turn in nonbibulous lateral flow contact with a capture zone, also capable of conducting nonbibulous lateral flow and containing a specific binding partner for the visible moiety to which analyte has been attached or which is attached to competitor. The zones can be on the same solid support or on contiguous separate supports or matrices. The three zones are in nonbibulous lateral flow contact in the sense that they permit late al nonbibulous liquid flow between them. The sample application, labeling and capture zones also permit liquid to flow through the zones themselves by nonbibulous lateral flow. The capture zone is contiguous with a means for absorbing liquid and particles that have flowed through all three zones. The sample receiving zone may further serve to remove debris or interfering substances from the sample by physical entrapment without impeding the nonbibulous lateral flow.
In another aspect, the invention is directed to a method to conduct one-step assays which comprises applying a sample containing or suspected of containing an analyte to the sample receiving zone of the invention apparatus. In still another aspect, the invention is directed to methods to prepare the apparatus of the invention, and to improved nonbibulous matrices obtained by treating bibulous matrices with blocking agents.
(A more detailed description of the drawings follows in the Examples section.)
FIG. 1 shows steps in the assembly of one embodiment of the lateral flow portion of the apparatus of the invention.
FIG. 2 shows the assembly of an invention device placing the lateral flow plate in a casing.
The invention concerns improvements in assays which are conducted on supports which conduct nonbibulous lateral flow of fluids. As defined in the above-referenced patent U.S. Pat. No. 4,943,522, "nonbibulous" lateral flow refers to liquid flow in which all of the dissolved or dispersed components of the liquid which are not permanently entrapped or "filtered out" are carried at substantially equal rates and with relatively unimpaired flow laterally through the membrane or support. This is distinguished from preferential retention of one or more components as would occur, for example, in materials capable of absorbing or "imbibing" one or more components, as occurs in chromatographic separations. "Bibulous" materials include untreated forms of paper, nitrocellulose and the like which effect chromatographic separation of components contained in liquids which are passed therethrough.
Bibulous materials, however, can be converted to those which exhibit nonbibulous flow characteristics by the application of blocking agents, in particular certain detergents and proteins, which obscure the interactive forces that account for the bibulous nature of the supports per se. Thus, nonbibulous materials include those which are intrinsically capable of conducting nonbibulous flow, such as porous polyethylene sheets or other inert materials or can be comprised of bibulous materials which have been blocked. Preferred blocking agents include bovine serum albumin, either per se or in methylated or succinylated form, whole animal sera, such as horse serum or fetal calf serum, and other blood proteins. Other protein blocking agents include casein and non-fat dry milk.
Detergent-based blocking agents can also be used. The types of detergents which are appropriate are selected from nonionic, cationic, anionic and amphoteric forms, and the selection is based on the nature of the membrane being blocked. Considerations which govern the selection of the appropriate detergent blocking agent are well understood in the art. It is preferred to use detergents in combination with protein-based blocking agents. Suitable detergents which can be used either alone or in admixture with the protein blocking agents include polyoxyethylene sorbitan alcohol detergents (i.e., the Tween series), polyoxyethylene alcohols such as Nonidet P-40 or polyoxyethylene ethers such as Triton X-100. The selection of blocking agent and formulation of the blocking composition is important, as the blocking must be sufficient to effect nonbibulous flow, but the modified surface must not interfere with analyte-label-capture interaction.
One improvement which is the basis for the present invention is the use of, in the assay devices herein or in assay devices which utilize nonbibulous lateral flow in general, matrices or supports that are formed by the conversion of membranes or other supports with bibulous characteristics to nonbibulous membranes or supports. This is effected through application of blocking solutions. While intrinsically nonbibulous supports, such as the polyethylene sheet material manufactured by Porex Technologies Corporation (described in the above-referenced U.S. Pat. No. 4,943,522), can be employed in lateral flow assays, the use of converted microporous bibulous supports is preferred and has some advantages, such as more efficient immobilization of a capture binding reagent and resulting increased sensitivity, improved lateral flow, and enhanced speed of detection.
To convert a bibulous support such as paper or nitrocellulose to a support capable of effecting nonbibulous lateral flow, the original support is treated with a solution of the blocking agent in an effective concentration to dispose of unwanted reactivities at the surface. In general, this treatment is conducted with a blocking solution, such as a protein solution of 1-20 mg/ml protein at approximately room temperature for several minutes-several hours. The resulting coated material is then permanently adsorbed to the surface by air-drying, lyophilization, or other drying methods.
A further improvement in the assay devices of the invention lies in the design of the assay itself. The invention assay devices comprise four distinct zones, at least three of which conduct nonbibulous lateral flow. The device is designed so that the sample containing analyte is applied to a sample receiving zone and then flowed through a labeling zone into a capture zone. The capture zone is in turn in contact with a means for removing excess liquid sample. In general, this will consist of an absorbent, such as filter paper or glass fiber filter, which is generally bibulous in nature.
Selection of the carrier porous material and its treatment is done with consideration of the specific function each zone performs in the assay device.
The sample-receiving zone serves simply to begin the flow of analyte-containing sample and for this reason should be constructed of a material of low analyte retention. One means to impart this property is to impregnate the sample receiving zone with a neutral protein-blocking reagent, followed by treatment to immobilize the blocking agent (e.g., lyophilization). An additional advantage of this treatment is increased wetability and wicking action, speeding the transfer of the sample into the labeling zone. The sample-receiving zone may also function as a mechanical filter, entrapping any undesirable particulates present in the sample.
The labeling zone contains visible moieties which can be detected if accumulated in the capture zone. The visible moieties can be simply dyes or dye polymers which are visible when present in sufficient quantity, or can be, and are preferred to be, particles such as dyed latex beads, liposomes, or metallic, organic, inorganic or dye sols, dyed or colored cells or organisms, red blood cells and the like. Means for including various dyes within liposomes are well known, and have been disclosed, for example, in U.S. Pat. No. 4,695,554 as utilized in the examples below.
The visible moieties used in the assay provide the means for detection of the nature of and quantity of result, and accordingly, their appearance in the capture zone must be a function of the analyte in the sample. In general, this can be provided for by coupling the visible moieties to a ligand which binds specifically to analyte, or which competes with analyte for a capture reagent in the capture zone. In the first approach, the visible moieties are coupled to a specific binding partner which binds the analyte specifically. For example, if the analyte is an antigen, an antibody specific for this antigen may be used; immunologically reactive fragments of the antibody, such as F(ab')2, Fab or Fab' can also be used. These visible moieties, or "test" visible moieties, then bind to analyte in the sample as the sample passes through the labeling zone and are carried into the capture zone by the liquid flow. When the complexes reach the capture zone, a capture reagent, which is then specific for analyte, such as an antibody or fragment thereof as set forth above, retains those coupled conjugates to which analyte has been bound, and permits those which do not contain analyte to pass into the absorbent zone. In the second approach, the visible moieties are coupled to a ligand which is competitive with analyte for a capture reagent in the capture zone, most typically, other molecules of the analyte itself. Both the analyte from the sample and the competitor bound to the visible moieties are then carried into the capture zone. Both analyte and its competitor then react with the capture reagent, which in this instance is also typically specifically reactive with analyte (and its competitor). The unlabeled analyte thus is able to reduce the quantity of competitor-conjugated visible moieties which are retained in the capture zone. This reduction in retention of the visible moieties becomes a measure of the analyte in the sample.
The labeling zone may also include "control" visible moieties which do not contain the specific binding agent or analyte competitor and which are also carried through to a control area of the capture zone by the liquid flow. These control visible moieties are coupled to a control reagent which binds to a capture partner specific for it and can then be captured in a separate "control" portion of the capture zone to verify that the flow of liquid is as expected. The visible moieties used in the control may be the same or different color than those used for the test moieties. If different colors are used, ease of reading the results is enhanced.
In all of the above, employment of the selected blocking agent together with colored moieties in the labeling zone followed by the immobilization of the blocking agent on the support (by, e.g., a freeze-drying process) is of utmost importance for improved performance of the device. It is well known that visible moieties, especially particles, aggregate upon air-drying and do not readily rehydrate in contact with a liquid sample. Therefore, absent conversion to the nonbibulous surface, instead of being transported to the capture zone with the sample, the visible moieties will remain trapped in the labeling zone.
As the analyte labeled with the visible moieties or the labeled competitor pass through the capture zone, the labeled analyte or competitor are bound to at least a portion of the membrane in the capture zone (the "test" portion) through the interaction of a specific reagent applied therein which reacts directly with the analyte or competitor bound to the test visible moieties. If control visible moieties are used, provision is made for their capture in the capture zone in an area separate from that where the test visible moieties will be bound. The control visible moieties, preferably of a different color from their "test" counterparts, are provided with a control binding agent which is specific not for analyte or the test capture reagent, but for a material in a capture area in the capture zone which represents a control band. Thus they are directly detected in a separate "control" portion. For example, in the procedures exemplified below, the labeling control beads include streptavidin and their control band portion in the capture zone contains biotin, which couples to avidin specifically. Other "irrelevant" binding pairs can also be used-such as antigen/antibody reactions unrelated to analyte.
The experimental results are read in the capture zone by noting the presence or absence of a visible signal at the location of the capture zone for the test visible moieties. The use of a control region is helpful in that appearance of a color in the control region signals the time at which the test result can be read, even for a negative result. Thus, when the expected color appears in the control region, the presence or absence of a color in the test region can be noted. The use of different colors for test and control regions aids in this process.
The use of the matrix which is bibulous inherently, but convertible to a nonbibulous flow characteristic is particularly useful in the creation of this capture zone. Capture reagents can be applied to the matrix before the application of blocking agent and immobilized in situ, obviating the need for an activation treatment as frequently required for attachment of capture reagents to bibulous supports. At this stage, the bibulous nature of the matrix during the coupling of the capture reagents may be advantageous. However, the blocking/washing treatment which converts the bibulous membrane to nonbibulous support provides unimpaired and speedy flow of all components of the system.
The speed of the assay, which requires less than one minute and provides in many instances essentially an instantaneous result as the sample flows through the capture zone, is attainable because of the nonbibulous nature of the zones and of the short distance the sample must traverse in each zone. Miniaturization of the diagnostic device results in the remarkable speed of the assay, well below the times disclosed in similar lateral flow assays, such as those described in U.S. Pat. No. 4,361,637; U.S. Pat. No. 4,861,711; U.S. Pat. No. 4,740,468; and European patent 0 306 772 A. Miniaturization permits instantaneous results which are observable as soon as the sample contacts the capture zone and which occur almost immediately or within 60 seconds of the addition of the sample to the sample receiving zone. The speed of appearance and intensity of the positive visible reaction seen depends on the concentration of analyte in the sample. The speed of appearance of the positive visual reaction can be adjusted to provide the optimal visual result with concentrations of analyte of clinical importance and adjusted to suit the timing needs of the end-user.
Suitable analytes to which the method of the invention can be applied are any for which a specific binding partner can be found. In general, most analytes of medical and biological significance can find specific binding partners in antibodies prepared against them or fragments of these antibodies. Suitable analytes include soluble analytes such as hormones, enzymes, lipoproteins, bacterial or viral antigens, immunoglobulins, lymphokines, cytokines, drugs, soluble cancer antigens, and the like. These analytes include various proteins such as protamines, histones, porphorylated proteins, nucleoproteins, and so forth such as, for example, transcortin, erythropoietin, transferrin, various globulins, thyroxin-binding globulin, the immunoglobulins of various subclasses A, G, D, E, and M, various complement factors, blood clotting factors such as fibrinogen, Factor VIII, tissue thromboplastin, and thrombin.
Also included are hormones such as insulin, glucagon, relaxin, thyrotropin, somatotropin, gonadotropin, follicle-stimulating hormone, gastrin, bradykinin, vasopressin, and various releasing factors. A wide range or antigenic polysaccharides can also be determined such as those from Chlamydia, Neisseria gonorrheae, Pasteurella pestis, Shigella dysentereae, and certain fungi such as Mycosporum and Aspergillus. Another major group comprises oligonucleotide sequences which react specifically with other oligonucleotides or protein targets. An extensive list of soluble analytes determinable in the method of the invention is found in U.S. Pat. No. 3,996,345, which is incorporated herein by reference.
The following examples are intended to illustrate but not to limit the invention.
The test device has three active components--a sample receiving zone, a labeling zone containing a particulate label and a capture zone. The three active portions, which communicate through nonbibulous liquid flow, are assembled so as to take advantage of a means to absorb or remove excess liquid which has been transported through these three zones, and to support the active components of the device. The construction is as follows.
Preparation of the Sample Receiving Zone
The sample receiving zone is prepared from Sontara O-100 DuPont Orlon spunlace fabric. The fabric is rendered nonbibulous by saturating with methylated bovine serum albumin (methylated BSA). The conversion to nonbibulous material is achieved by treatment at 47.6 μl/cm2 with a 10 mg/ml solution of the methylated BSA at room temperature at five minutes. The pad of Sontara is then frozen at -70� C. along with a lyophilization flask for at least an hour. The Sontara membrane is then lyophilized overnight on a Virtis Freezemobile. The treated sample receiving zone is then nonbibulous, and is cut into 10�7.5 mm rectangles with the spunlace fibers being parallel to the longer side of the pad.
Preparation of the Labeling Zone
To prepare the labeled control beads for inclusion in the labeling zone, 0.5 ml of blue latex beads initially suspended at a weight/vol concentration of 2.5% are washed twice with glycine-buffered saline (GBS) (100 mM glycine, 171 mM NaCl). The beads are sonicated for 10 minutes in contact with the GBS and then microcentrifuged for three minutes.
The pelleted beads are treated with 0.5 ml coupling solution which consists of 0.4 mg/ml streptavidin and 0.2 mg/ml methylated BSA in GBS. The pelleted beads are resuspended and sonicated for 10 minutes, and then rotated overnight at room temperature.
The bead preparation is then centrifuged for three minutes and the supernatant removed by aspiration. The bead pellet is then resuspended in 0.5 ml of the 10 mg/ml methylated BSA. This mixture is rotated end over end for four hours at room temperature, and the bead preparation centrifuged to recover the pellet. The supernatant is aspirated and the pellet is washed three times with bead storage solution which consists of 1 mg/ml methylated BSA in 50 mM Tris, pH 8. The final bead preparation is prepared in a bead storage solution at a concentration of 1% solids. The resulting beads will be conducted by nonbibulous flow through the capture zone described below. They have been reacted so as to bind to a biotin-containing capture band.
"Test" beads for labeling analyte, containing monoclonal anti-hCG, are prepared in similar manner as follows:
One-half (0.5) ml of red latex beads (it is preferred that the control and test beads are of different colors) are washed twice with 1 ml GBS as described above by sonicating for 10 minutes and recovered by microcentrifugation for three minutes. To the pelleted beads is added 0.5 ml of coupling solution, in this case consisting of 0.8 mg/ml monoclonal anti-hCG and 0.2 mg/ml methylated BSA in GBS. The pellet is resuspended and sonicated for 10 minutes, and then rotated overnight at room temperature. After centrifugation for three minutes, the supernatant is aspirated and the bead pellet is resuspended in 0.5 ml of the 10 mg/ml methylated BSA and rotated end over end for four hours at room temperature. After centrifugation and removal of the supernatant, the pellet is washed three times with bead storage solution as described above, and the final bead preparation is at a concentration of 1% solids.
Finally, to prepare the labeling zone containing both test and control beads the test beads are diluted into methylated BSA at a concentration of 0.06% solids and the control beads into the same solution at 0.02% solids. The resultant mixture is stirred and poured onto a Sontara O-100 DuPont spunlace fabric membrane at 47.6 μl/cm2 The labeled pad is then kept at room temperature for five minutes and frozen at -70� C., along with the lyophilizing flask for at least an hour. The resulting membranes are lyophilized overnight on Virtis Freezemobile. The label-containing pads are then cut into 10�7.5 mm rectangles with the spunlace fibers parallel to the longer side of the pad.
Preparation of Capture Zone Membrane
To prepare the capture zone membrane, nitrocellulose is rendered nonbibulous as follows. Nitrocellulose obtained from Schleicher and Schuell, having a pore size of 8-12 μm, is affixed to a chart recorder and hCG capture bands are dispensed as 2-cm-spaced parallel lines using a solution of 2 mg/ml rabbit anti-hCG (purified by protein A) in Tris buffer using the Tri-Dac dispense system operated in the manual mode with valve pressure at 70 psi, container pressure setting at 5 psi, fluid flow at 15 sec/drop, chart speed at 500 mm/minute. These are lines reactive with the test beads. The membrane is then spotted in parallel lines 0.3 cm above the previously-spotted anti-hCG capture zone with a 0.6 mg/ml biotinylated rabbit gamma globulin solution dissolved in Tris buffer. These are lines reactive with the streptavidin on the control beads. After air drying for 10-30 minutes, the membrane is placed onto a tray containing blocking buffer (10 mg/ml methylated BSA in 50 mM Tris) for 15 minutes at room temperature. The membrane is removed and blotted and then transferred to a tray containing wash buffer (50 mM Tris maleate, pH 5.4) for 5 minutes at room temperature. The blocked and washed membrane is then blotted, allowed to air dry, stored in a desiccator at room temperature until assembly of the device.
Assembly of the Device A 20�7.5 mm strip of the capture zone membrane, shown as component 1 in FIG. 1, is affixed centrally on an adhesive transparency strip shown as component 5 in FIG. 1. The transparency strip is a 700�17 mm strip of P 2200 3M transparency made adhesive with double-sided adhesive tape 444 (3M).
The pad containing visible label, shown as component 2 in FIG. 1, is then affixed next to the capture zone pad with a 1 mm overlap as shown in FIG. 1. The sample receiving pad, shown as component 3, is then placed next to label-containing pad with 1 mm overlap, as shown in the figure.
The device is then provided with an absorbent pad, which is a 20�7.5 mm rectangle of ED No. 939 absorbent, which is affixed to the distal end of the capture zone membrane with a 1 mm overlap, as shown in the figure.
The resulting test strip on the transparency backing is then covered with a plastic top, shown as component 6 in FIG. 2, with the length of the strip being centrally within the groove of the plastic top. Both sets of capture lines are exposed in the viewing window, and the sample application hole is immediately above the sample receiving pad. Finally, the bottom of the device, which is a 700�17 mm bottom strip of 1 mm thick opaque white plastic made adhesive with double-sided adhesive tape (3M) is attached to the other side of the transparency strip.
In a manner analogous to that set forth in Example 1, the counterpart liposome-containing device is prepared. The preparation of the sample receiving pad, the capture zone membrane, and the assembly of the components on backing is identical to that set forth in Example 1. The labeled liposome-containing zone pad is prepared as follows:
Liposomes are prepared according to the procedures set forth in U.S. Pat. No. 4,695,554 and contain sulforhodamine dye. Different colored dyes are used for control and test liposomes.
For test liposomes, monoclonal anti-hCG, which has been purified from ascites, is used. Ascites is 50% SAS cut at 4� C. and desalted on a G-25 column into 10 mM Tris. The antibodies are then further purified on a Q-Sepharose Fast Flow column using a salt gradient of 0-0.5 M sodium chloride in 10 mM Tris. The fractions are monitored at 280 nm, and the peaks collected and buffered exchanged into 1M sodium phosphate. The antibody is diluted to 3 mg/ml and 8.5 molar equivalents of SPDP is added. The solution is incubated for 30 minutes at 27� C. and buffer exchanged on G25 and 0.1M sodium acetate. The material is reduced with 1M DTT in sodium acetate to give a final concentration of 50 mM and incubated at 27� for 25 minutes and then buffer exchanged on G25 with Tris acetate buffer. The relative amounts of liposomes and antibody are determined assuming 8 μmol of phosphorus per mg of antibody and 1 μmol of phosphorus is supplied for 1 ml of uncoupled liposomes. The liposomes are then titrated to pH 8 with 1M Tris and coupled with antibody for two hours at 27� C.
The resulting antibody-conjugated liposomes are sized on a C6 Fast Flow column diluted in liposome storage buffer to an absorbance of 0.25 units at 565 nm.
The liposome-containing pad is then prepared by saturating a Sontara pad with a liquid solution of anti-hCG conjugated liposomes diluted to an absorbance at 565 nm of 0.06 at 35 μl to a 10�7.5 mm assay pad. The liposome storage buffer for this dilution contains 10% sucrose and 10 mg/ml methylated BSA. The labeling pads are kept at room temperature for 5 minutes and frozen at -70� C. along with the lyophilizing flask for at least an hour. The membranes are then lyophilized overnight in a Virtis Freezemobile.
The device of Example 1 is placed flat on a benchtop and two drops of sample at approximately 30 μl per drop are applied to the sample receiving zone. The liquid is allowed to flow through the three zones in nonbibulous lateral flow contact to the absorbent zone, with a blue control band appearing in the upper portion of the viewing window in less than a minute. If hCG is present in the sample at least 50 mIU/ml, an additional red band in the hCG capture region is visible.
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L-arginine conjugated to a polymeric matrix is disclosed. The matrix can be used as, for example, a coating for an implantable medical device such as a stent.
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10. A coating for a prosthetic device comprising a composition, the composition comprising an oligomer of L-arginine, L-arginine, an L-arginine analog, or an L-arginine analog oligomer linked through a labile bond to a polymeric matrix.
11. The coating according to
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14. A method of coating a prosthesis comprising coating the prosthesis with a composition comprising an oligomer of L-arginine, L-arginine, L-arginine analog, or L-arginine analog oligomer linked through a labile bond to a polymeric matrix.
15. The method of
16. The method of
17. A method of inhibiting restenosis of a blood vessel comprising depositing into a designated region of the blood vessel the composition according to
18. A stent coated with a coating comprising a composition for inhibiting the narrowing of a blood vessel, the composition comprising an oligomer of L-arginine, L-arginine, an L-arginine analog, or an L-arginine analog oligomer bonded linked through a labile bond to a polymeric matrix.
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1. Field of the Invention
This invention relates to a biocompatible carrier containing L-arginine and L-arginine oligomers for introduction to a certain target cell population in a vascular region, such as smooth muscle cells or inflammatory cells, requiring modulation to ameliorate a diseased state, particularly for the treatment of stenosis or restenosis following a vascular trauma or disease. Moreover, the invention is directed to a composition containing L-arginine, or L-arginine oligomer thereof combined with a coating on an implantable device.
2. General Background and State of the Art
Percutaneous transluminal coronary angioplasty (PTCA) is a procedure for treating heart disease. A catheter assembly having a balloon portion is introduced percutaneously into the cardiovascular system of a patient via the brachial or femoral artery. The catheter assembly is advanced through the coronary vasculature until the balloon portion is positioned across the occlusive lesion. Once in position across the lesion, the balloon is inflated to a predetermined size to radially compress against the atherosclerotic plaque of the lesion to remodel the lumen wall. The balloon is then deflated to a smaller profile to allow the catheter to be withdrawn from the patient's vasculature.
A problem associated with the above procedure includes formation of intimal flaps or torn arterial linings which can collapse and occlude the conduit after the balloon is deflated. Moreover, thrombosis and restenosis of the artery may develop over several months after the procedure, which may require another angioplasty procedure or a surgical by-pass operation. To reduce the partial or total occlusion of the artery by the collapse of arterial lining and to reduce the chance of the development of thrombosis and restenosis, a stent is implanted in the lumen to maintain the vascular patency.
Stents are used not only as a mechanical intervention but also as a vehicle for providing biological therapy. As a mechanical intervention, stents act as scaffoldings, functioning to physically hold open and, if desired, to expand the wall of the passageway. Typically, stents are capable of being compressed, so that they can be inserted through small vessels via catheters, and then expanded to a larger diameter once they are at the desired location. Examples in patent literature disclosing stents which have been successfully applied in PTCA procedures include stents illustrated in U.S. Pat. No. 4,733,665 issued to Palmaz, U.S. Pat. No. 4,800,882 issued to Gianturco, and U.S. Pat. No. 4,886,062 issued to Wiktor.
Biological therapy can be achieved by medicating the stents. Medicated stents provide for the local administration of a therapeutic substance at the diseased site. In order to provide an efficacious concentration to the treated site, systemic administration of such medication often produces adverse or toxic side effects for the patient. Local delivery is a preferred method of treatment in that smaller total levels of medication are administered in comparison to systemic dosages, but are concentrated at a specific site. Local delivery thus produces fewer side effects and achieves more favorable results. One proposed method for medicating stents involves the use of a polymeric carrier coated onto the surface of a stent. A solution which includes a solvent, a polymer dissolved in the solvent, and a therapeutic substance dispersed in the blend is applied to the stent. The solvent is allowed to evaporate, leaving on the stent surface a coating of the polymer and the therapeutic substance impregnated in the polymer.
Local administration of therapeutic agents via stents has shown some favorable results in reducing restenosis. However, development of restenosis remains a persistent problem which has not been significantly alleviated by therapeutic substances which are currently used in the market. Accordingly, there is a great need for better and more effective therapeutic compositions, and methods of administering the compositions, for the effective treatment of restenosis.
Nitric oxide (NO) has many beneficial effects, including vasodilation and inhibiting smooth muscle cell hyperproliferation. However, the temporal, optical and hydrolytic instability of nitric oxide donor moieties has rendered them inefficient for use in reducing restenosis. For example, it is conventionally known that NO is a free radical, has a very short half life and is fast quenching. As a result, long term benefits of reducing restenosis would be difficult to achieve with the use of conventional NO donors.
L-arginine is an amino acid that can be loaded to a higher total effective dose of NO than a conventional NO donor. One of the advantages of using L-arginine as an NO donor is that the rate of NO release from L-arginine is controlled and sustained, which is an improvement over using conventional NO donors. In addition, several molecular components are required in the biosynthesis of nitric oxide from L-arginine including the enzymes iNOS and eNOS, with L-arginine often being the limiting reagent in this reaction scheme. When L-arginine is absorbed by a cell in the presence of eNOS, nitric oxide is produced. In view of the beneficial effects of NO in reducing restenosis, there is a need in the art for a sustained delivery of L-arginine or L-arginine oligomer released from a stent.
The present invention has met the need for a more efficient and sustained method of delivering nitric oxide to a localized area by providing a method of sustained delivery of L-arginine or L-arginine oligomer from the hydrolysis of a polymer matrix from a prosthesis such as a stent.
Accordingly, the present invention is directed to a composition for inhibiting the narrowing of a blood vessel, comprising an oligomer of L-arginine, L-arginine, L-arginine analog, or L-arginine analog oligomer linked through a labile bond to a polymeric matrix, wherein the composition is used for coating an implantation device to inhibit the narrowing of the blood vessel. In this method, the composition may further comprise L-arginine, L-arginine analog, L-arginine oligomer, or L-arginine analog oligomer that is phase dispersed in the polymeric matrix. The oligomer may comprise more than 2 residues of L-arginine or L-arginine analog, or from 2 to 20 residues, from 2 to 15, from 2 to 9, from 2 to 7, or 7 residues. In the composition, the labile bond may be hydrolyzable by water. And the labile bond may be, but not limited to, an anhydride bond, ester bond, or imine bond.
The polymeric matrix may comprise poly(ethylene-co-vinyl alcohol), polyethylene glycol, acryloyl polyethylene glycol, poly(ethylene-co-glycidylmethacrylate), poly(ethylene-co-acrylic acid), poly(ethylene-co-maleic anhydride), hyaluronic acid, heparin, albumin, or glycosaminoglycan. The composition may also comprise a non-linking polymer. The non-linking polymer may be poly-n-butylmethacrylate, polyvinylidenefluoride, polyhexafluoropropylene, polycyclohexylmethacrylate, polyvinyledenefluoride-co-polyhexafluoropropylene, or hyaluronic acid.
The present invention is also directed to a coating for a prosthetic device, comprising a composition for inhibiting the narrowing of a blood vessel, comprising an oligomer of L-arginine, L-arginine, L-arginine analog, or L-arginine analog oligomer linked through a labile bond to a polymeric matrix. The prosthesis may be selected from a group of balloon-expandable stents, self-expandable stents, and grafts. The coating additionally may comprise a therapeutically active ingredient to be used in combination with the composition. The therapeutically active ingredient may be selected from a group of antineoplastic, anti-inflammatory, antiplatelet, anticoagulant, antifibrin, antithrombin, antimitotic, antiproliferative, antibiotic, antioxidant substances, and combinations thereof.
In another aspect, the invention is directed to a method of coating a surface of a prosthesis with a coating that comprises a composition for inhibiting the narrowing of a blood vessel, comprising an oligomer of L-arginine or L-arginine analog linked through a labile bond to a polymeric matrix, which method comprises spraying the coating on to a prosthesis or immersing the prosthesis in a solution of the coating.
In another embodiment, the invention is directed to a method of inhibiting restenosis of a blood vessel comprising depositing into a designated region of the blood vessel the L-arginine composition discussed above, wherein the composition is coated on to a prosthetic device.
The present invention is also directed to a stent coated with a coating comprising a composition for inhibiting the narrowing of a blood vessel, comprising an oligomer of L-arginine, L-arginine, L-arginine analog, or L-arginine analog oligomer linked through a labile bond to a polymeric matrix. And the stent further comprises a therapeutically active ingredient to be used in combination with the composition.
These and other embodiments of the invention will be more fully understood from the following referenced drawing attached hereto, description of the invention, and the claims appended hereto.
As used herein, "abnormal" or "inappropriate" proliferation means division, growth or migration of cells occurring more rapidly or to a significantly greater extent than which typically occurs in a normally functioning cell of the same type, i.e., hyperproliferation.
As used herein, "analogs" or "derivatives" refers to L-arginine or any variation of it that retains the biological activity of L-arginine, so long as it is capable of acting as a substrate of nitric oxide production.
As used herein, "inhibiting" cellular activity means reducing, delaying or eliminating smooth muscle cell hyperplasia, restenosis, vascular occlusions, platelet activation, or inflammatory response, particularly following biologically or mechanically mediated vascular injury or trauma or under conditions that would predispose a mammal to suffer such a vascular injury or trauma. As used herein, the term "reducing" means decreasing the intimal thickening that results from stimulation of smooth muscle cell proliferation. "Delaying" means retarding the progression of the hyper-proliferative vascular disease or delaying the time until onset of visible intimal hyperplasia, as observed, for example, by histological or angiographic examination. "Elimination" of restenosis following vascular trauma or injury means completely "reducing" and/or completely "delaying" intimal hyperplasia in a patient to an extent which makes it no longer necessary to surgically intervene, i.e., to re-establish a suitable blood flow through the vessel by, for example, repeat angioplasty, atheroectomy, or coronary artery bypass surgery. The effects of reducing, delaying, or eliminating restenosis may be determined by methods known to one of ordinary skill in the art, including, but not limited to, angiography, ultrasonic evaluation, fluoroscopy imaging, fiber optic visualization, or biopsy and histology. Biologically mediated vascular injury includes, but is not limited to injury caused by or attributed to autoimmune disorders, alloimmune related disorders, infectious disorders including endotoxins and herpes viruses such as cytomegalovirus, metabolic disorders such as atherosclerosis, and vascular injury resulting from hypothermia and irradiation. Mechanical mediated vascular injury includes, but is not limited to vascular injury caused by catheterization procedures or vascular scraping procedures such as percutaneous transluminal coronary angioplasty, vascular surgery, stent placement, transplantation surgery, laser treatment, and other invasive procedures which disrupted the integrity of the vascular intima or endothelium. The active ingredient of the invention is not restricted in use for therapy following vascular injury or trauma; rather, the usefulness of the active ingredient will also be determined by the ingredient's ability to inhibit cellular activity of smooth muscle cells or inhibit the development of restenosis.
As used herein, "L-arginine" includes L-arginine that may be isolated from nature or chemically synthesized. Various forms of L-arginine may be also indicated where L-arginine may be in either a monomeric or an oligomeric form. By derivatives or analogs of "L-arginine," it is meant to refer to modifications of the compound that do not affect its ability to act as a substrate for nitric oxide production. L-arginine is used hereinafter without explicitly referring to those derivatives and analogs which are modifications. It is understood that by "L-arginine," those derivatives and analogs that act as a substrate for nitric oxide production may be included.
As used herein, "linking polymer" or "linking type polymer" refers to those polymers, copolymers, etc. that have the capability to bind to L-arginine or oligomers of L-arginine through a bond, e.g., labile bond. Such a labile bond may be generally cleaved by hydrolysis, thus releasing L-arginine or L-arginine oligomer.
As used herein, "migration" of smooth muscle cells means movement of these cells in vivo from the medial layers of a vessel into the intima, such as may also be studied in vitro by following the motion of a cell from one location to another, e.g., using time-lapse cinematography or a video recorder and manual counting of smooth muscle cell migration out of a defined area in the tissue culture over time.
As used herein, "non-linking polymer" or "non-linking type polymer" refers to those polymers that do not generally bind to L-arginine or oligomers of L-arginine.
As used herein, "oligomer" of L-arginine refers to at least two residues of L-arginine linked together. In particular, "R7" refers to a seven residue L-arginine oligomer. Following the same pattern, "R3" refers to a three residue L-arginine oligomer.
As used herein, "proliferation" of smooth muscle cells means increase in cell number.
As used herein, "smooth muscle cells" include those cells derived from the medial and adventitia layers of the vessel which proliferate in intimal hyperplastic vascular sites following vascular trauma or injury. Under light microscopic examination, characteristics of smooth muscle cells include a histological morphology of a spindle shape with an oblong nucleus located centrally in the cell with nucleoli present and myofibrils in the sarcoplasm. Under electron microscopic examination, smooth muscle cells have long slender mitochondria in the juxtanuclear sarcoplasm, a few tubular elements of granular endoplasmic reticulum, and numerous clusters of free ribosomes. A small Golgi complex may also be located near one pole of the nucleus.
L-arginine Linked Polymeric Matrix
L-arginine and oligomers thereof may be covalently linked by a hydrolyzable bond to various polymers so that sustained release of nitric oxide can be effectuated. In the practice of the invention, various polymers possessing the capability to form hydrolyzable bonds with L-arginine or derivatives thereof may be used. In this regard, L-arginine may be linked to large biological molecules such as albumin, hyaluronic acid and glycosaminoglycans, as well as other large molecules such as heparin. Moreover, the L-arginine and L-arginine oligomers may bind to natural or synthetic polymers.
Examples of such polymers may be ones that form esters, anhydrides, or imines. It is understood that any polymer may be used in linking the L-arginine or L-arginine derivative to the polymer so long as the bond that is formed is hydrolyzable. Specifically useful polymers may include poly (ethylene-co-vinyl alcohol), commercially known as EVAL, and poly(ethylene-co- glycidylmethacrylate (EGMA). Both of these polymers exemplify polymers that may form ester bonds with L-arginine or oligomers thereof. Another useful polymer is poly(ethylene-co-acrylic acid) (EAA) and poly(ethylene-co-maleic anhydride) (EMA), which may form an anhydride linkage with L-arginine or an L-arginine oligomer. In the instance where anhydride bonds are formed, the resultant stable polymer may undergo hydrolysis, thus releasing L-arginine.
In yet another embodiment of the invention, polymers having no functional groups to form any linkage with L-arginine may be also included in the polymeric matrix. Such polymers may include, but not limited to, poly-n-butylmethacrylate (PBMA), polyvinylidenefluoride (KYNAR) (PVDF), polyhexafluoropropylene (HFP), polycyclohexylmethacrylate (PCMA), poly(vinyledenefluoride-co-hexafluoropropylene), hyaluronic acid, and combinations thereof.
Active Agent
Sufficient amounts of an active agent can also be included in the matrix. The active agent should inhibit the activity of vascular smooth muscle cells. More specifically, the active agent is aimed at inhibiting abnormal or inappropriate migration and proliferation of smooth muscle cells. Examples of the active or therapeutic agents that may be used in the invention in addition to L-arginine include antiproliferative substances such as actinomycin D, or derivatives and analogs thereof (manufactured by Sigma-Aldrich 1001 West Saint Paul Avenue, Milwaukee, Wis. 53233; or COSMEGEN available from Merck). Synonyms of actinomycin D include dactinomycin, actinomycin IV, actinomycin I1, actinomycin X1, and actinomycin C1. The active agent can also fall under the genus of antineoplastic, antiinflammatory, antiplatelet, anticoagulant, antifibrin, antithrombin, antimitotic, antibiotic, antiallergic and antioxidant substances. Examples of such antineoplastics and/or antimitotics include paclitaxel (e.g. TAXOL� by Bristol-Myers Squibb Co., Stamford, Conn.), docetaxel (e.g. Taxotere�, from Aventis S.A., Frankfurt, Germany) methotrexate, azathioprine, vincristine, vinblastine, fluorouracil, doxorubicin hydrochloride (e.g. Adriamycin� from Pharmacia & Upjohn, Peapack N.J.), and mitomycin (e.g. Mutamycin� from Bristol-Myers Squibb Co., Stamford, Conn.) Examples of such antiplatelets, anticoagulants, antifibrin, and antithrombins include sodium heparin, low molecular weight heparins, heparinoids, hirudin, argatroban, forskolin, vapiprost, prostacyclin and prostacyclin analogues, dextran, D-phe-pro-arg-chloromethylketone (synthetic antithrombin), dipyridamole, glycoprotein IIb/IIIa platelet membrane receptor antagonist antibody, recombinant hirudin, and thrombin inhibitors such as Angiomax™ (Biogen, Inc., Cambridge, Mass.) Examples of such cytostatic or antiproliferative agents include angiopeptin, angiotensin converting enzyme inhibitors such as captopril (e.g. Capoten� and Capozide� from Bristol-Myers Squibb Co., Stamford, Conn.), cilazapril or lisinopril (e.g. Prinivil� and Prinzide� from Merck & Co., Inc., Whitehouse Station, N.J.); calcium channel blockers (such as nifedipine), colchicine, fibroblast growth factor (FGF) antagonists, fish oil (omega 3-fatty acid), histamine antagonists, lovastatin (an inhibitor of HMG-CoA reductase, a cholesterol lowering drug, brand name Mevacor� from Merck & Co., Inc., Whitehouse Station, N.J.), monoclonal antibodies (such as those specific for Platelet-Derived Growth Factor (PDGF) receptors), nitroprusside, phosphodiesterase inhibitors, prostaglandin inhibitors, suramin, serotonin blockers, steroids, thioprotease inhibitors, triazolopyrimidine (a PDGF antagonist), and nitric oxide. An example of an antiallergic agent is permirolast potassium. Other therapeutic substances or agents which may be appropriate include alpha-interferon, genetically engineered epithelial cells, rapamycin, dexamethasone and functional analogs or structural derivatives thereof.
The dosage or concentration of the active ingredient required to produce a favorable therapeutic effect should be less than the level at which the active ingredient produces toxic effects and greater than the level at which non-therapeutic results are obtained. The dosage or concentration of the active ingredient required to inhibit the desired cellular activity of the vascular region can depend upon factors such as the particular circumstances of the patient; the nature of the trauma; the nature of the therapy desired; the time over which the ingredient administered resides at the vascular site; and if other therapeutic agents are employed, the nature and type of the substance or combination of substances. Therapeutic effective dosages can be determined empirically, for example by infusing vessels from suitable animal model systems and using immunohistochemical, fluorescent or electron microscopy methods to detect the agent and its effects, or by conducting suitable in vitro studies. Standard pharmacological test procedures to determine dosages are understood by one of ordinary skill in the art.
The use of the matrix is not limited to stents and the coating can also be used with a variety of other medical devices. Examples of implantable medical devices, that can be used in conjunction with the embodiments of this invention include stent-grafts, grafts (e.g., aortic grafts), artificial heart valves, cerebrospinal fluid shunts, pacemaker electrodes, axius coronary shunts and endocardial leads (e.g., FINELINE and ENDOTAK, available from Guidant Corporation). The underlying structure of the device can be of virtually any design. The device can be made of a metallic material or an alloy such as, but not limited to, cobalt-chromium alloys (e.g., ELGILOY), stainless steel (316L), "MP35N," "MP20N," ELASTINITE (Nitinol), tantalum, tantalum-based alloys, nickel-titanium alloy, platinum, platinum-based alloys such as, e.g., platinum-iridium alloy, iridium, gold, magnesium, titanium, titanium-based alloys, zirconium-based alloys, or combinations thereof. Devices made from bioabsorbable or biostable polymers can also be used with the embodiments of the present invention. "MP35N" and "MP20N" are trade names for alloys of cobalt, nickel, chromium and molybdenum available from Standard Press Steel Co. of Jenkintown, Pa. "MP35N" consists of 35% cobalt, 35% nickel, 20% chromium, and 10% molybdenum. "MP20N" consists of 50% cobalt, 20% nickel, 20% chromium, and 10% molybdenum.
L-arginine may be present in the matrix as a phase dispersion, or it may be linked to a polymer described above through a hydrolyzable bond. L-arginine may be present as a monomer, or it may be present as an oligomer. Thus, either of the monomeric or oligomeric form of L-arginine may be phase dispersed or linked to a polymer through a labile bond. It is also understood that a combination of phase dispersed monomer and/or oligomer of L-arginine may be mixed with polymer bound monomer and/or oligomer of L-arginine. The oligomeric version of L-arginine has a particularly advantageous feature. Without being bound by theory, it is believed that the oligomeric form is readily absorbed by the tissue, hence this form is effective in delivering nitric oxide to vessel walls.
L-arginine oligomer may comprise 2 or more L-arginine residues. Usefully, the oligomeric form may comprise from 2 to 20 L-arginine residues. Or, the oligomeric form may comprise from 2 to 15 residues. Still yet, the oligomeric form may comprise from 2 to 9 residues. Also, the oligomeric form may comprise from 2 to 7 residues. In particular, the oligomer has 7 residues. However, it is understood that the number of residues in the oligomer may be modified and optimized depending on the degree of control of the release rate that is desired.
It is also understood that multiple layers of coating may be used, so long as at least one of the layers contains a form of L-arginine linked through a hydrolyzable bond to the linking polymer. For example, a layer may be added to the L-arginine bearing layer and may include an active ingredient mixed with a non-linking polymer or a linking polymer linked to L-arginine. Alternatively, the layer may not have any active ingredient at all, and instead may comprise various polymers, which in effect would be used either as a primer layer or a barrier layer. By way of example only, and without limiting the scope of the invention, in one embodiment of the invention, the first coating layer may contain an active ingredient and a linking type polymer linked with L-arginine or an oligomer thereof. The second coating layer may contain either the same linking type polymer used in the first coating layer, or it may contain a different linking polymer linked with the L-arginine or oligomer thereof, with or without the presence of the active ingredient.
Another exemplary configuration may be that the first coating layer may contain an active ingredient along with a linking type polymer bound through a hydrolytically degradable bond with L-arginine or an oligomer thereof. The second coating layer may contain a non-linking type polymer with either the presence or absence of an active ingredient. In still another example, the first coating layer may contain an active ingredient and a linking type polymer linked to L-arginine, or an oligomer thereof, and the second coating layer may contain a mixture of non-linking polymers and linking polymers linked to various forms of L-arginine.
Further, in another configuration, the first coating layer may contain an active ingredient together with a non-linking polymer. The second coating layer may contain either the same non-linking type polymer used in the first coating layer, or it may contain a different non-linking polymer, with or without the presence of the active ingredient. Phase dispersed L-arginine may be interspersed in either or both of the layers.
Another exemplary configuration may be that the first coating layer may contain an active ingredient along with a non-linking type polymer. The second coating layer may contain a linking type polymer bound through a hydrolytically degradable bond with L-arginine or an oligomer thereof in either the presence or absence of an active ingredient. In still another example, the first coating layer may contain an active ingredient and a non-linking type polymer, and the second coating layer may contain a mixture of non-linking polymers and linking polymers linked to L-arginine and/or oligomers thereof.
It is understood that while in the above exemplifications, where only two coating layers are discussed, it is contemplated that many layers of coating comprising variations in the amount of active ingredient, non-linking polymers, linking polymers, and L-arginine and oligomers thereof, may be used in the practice of the invention as is desired, according to the nitric oxide release profile that is desired.
In one embodiment, it is contemplated that various forms of L-arginine, and in particular, the R7 oligomeric form of L-arginine, are phase dispersed in a polymeric matrix so that they are released first from the polymeric matrix upon contact with vessel walls. Thereafter, the L-arginine forms that are linked to the linking type polymers are released in a controlled manner as a result of cleavage of the labile bonds.
It is also contemplated that L-arginine or an oligomer thereof bound to linking polymers through various labile bonds may be released from the polymers in correlation to the type of labile bond that is being broken. Thus, a rate of release profile may be obtained according to the type of labile bonds that are present in the polymeric matrix. For instance, anhydride labile bonds are quickly broken upon contact with water. Imine bonds have an intermediate level of stability in the presence of water. Among the three exemplified labile bond types—anhydride, imine and ester bond—ester bonds are the most resistant to breakage in the presence of water. Thus, depending on the desires of the practitioner, a polymeric matrix may be formed comprising a mixture of these types of bonds to provide controlled release of L-arginine or oligomers thereof or any other active ingredient associated in the polymeric matrix. For example, if a quick release of the ligands from the polymeric matrix is desired, then the polymeric matrix should be composed of mostly anhydride bonds with the ligands. If a slow release of the ligands is desired, then the polymeric matrix should be comprised of mostly ester bonds.
An example of a polymeric matrix system would include a polymeric matrix comprising non-linking polymers and/or linking polymers. In particular, a copolymer such as acrylic-PEG-L-arginine may be blended with a matrix polymer such as EVAL, EVAL-PEG blend, or polyurethane. In addition, various forms of unbound L-arginine may be dispersed into the matrix so that a fast initial release rate of L-arginine is effected. This initial fast release rate may be followed by a more sustained and controlled delivery of L-arginine by hydrolytic cleavage of the labile bond according to the order of degradability of the bonds, resulting in the release of the ligands from the coupled copolymers.
Method of Use
In accordance with the above described method, the active ingredient and L-arginine or L-arginine oligomer can be applied to a prosthesis, e.g., a stent, retained on the stent during delivery and expansion of the stent, and released at a desired control rate and for a predetermined duration of time at the site of implantation. The release rate of the active ingredient can be controlled by modifying release parameters such as the amount of the comonomer content of the copolymer and the initial active ingredient content in the copolymer. The rate of release can also be adjusted by the addition of a second polymeric layer, with or without the active ingredient. A stent having the above described medicated coating is useful for a variety of medical procedures, including, by way of example, treatment of obstructions caused by tumors in bile ducts, an esophagus, trachea/bronchi and other biological passageways. A stent having the above described medicated coating is particularly useful for treating occluded regions of blood vessels caused by abnormal or inappropriate migration and proliferation of smooth muscle cells, thrombosis, and restenosis. Stents may be placed in a wide array of blood vessels, which include both arteries and veins. Representative examples of sites include the iliac, renal, and coronary arteries.
Briefly, an angiogram is first performed to determine the appropriate positioning for stent therapy. Angiography is typically accomplished by injecting a radiopaque contrasting agent through a catheter inserted into an artery or vein as an x-ray is taken. A guidewire is then advanced through the lesion or proposed site of treatment. Over the guidewire is passed a delivery catheter which allows a stent in its collapsed configuration to be inserted into the passageway. The delivery catheter is inserted either percutaneously or by surgery into the femoral artery, brachial artery, femoral vein, or brachial vein, and advanced into the appropriate blood vessel by steering the catheter through the vascular system under fluoroscopic guidance. A stent having the above described coating may then be expanded at the desired area of treatment. A post insertion angiogram may also be utilized to confirm appropriate positioning.
To a 100 ml roundbottom flask is added a stirbar, condenser and nitrogen bubbler. Nitrogen is bubbled through the reaction flask. The EVAL is added to the flask and purged with nitrogen. Anhydrous DMAC is added to the flask using a cannula and nitrogen to push the nitrogen into the reaction flask. The flask is heated to 60� C. for 3 hours to dissolve the EVAL polymer. The polymer solution is then cooled to room temperature and the Nα-9-fluorenylmethoxycarbonyl (FMOC)-L-arginine is added and after about 2 minutes of stirring went into solution. The solution is then cooled to 0� C. and the 1 M dicyclohexylcarbodiimide (DCC) solution in methylenechloride (MeCl2) is added dropwise. After the DCC is added, a crystal of dimethylaminopyridine (DMAP) is added to catalyze the esterification process. The solution is then stirred at 0� C. for 2 hours and allowed to warm to room temperature. The solution is then stirred under nitrogen for overnight. After stirring overnight, a significant concentration of white precipitate is formed in the reaction media. The reactants and amounts are shown in Table 1.
The suspension is then added to a separatory funnel. To a flask is added 450 ml of water, 450 ml of methanol with 25 ml of 1N HCI. To the flask is then added the precipitate suspension in a dropwise manner. A white polymer is precipitated from the acidic methanol/water media, which is stirred for 15 minutes and then filtered under vacuum. The polymer is then re-dissolved in DMSO, filtered through a glass frit filter and re-precipitated into methanol/water forming a white fluffy precipitate. The sample is then dried under vacuum for overnight. The sample is extracted with methanol to remove residual DMAP and urea and dried under vacuum.
To a roundbottom flask with a condenser and nitrogen source is added anhydrous N,N-dimethylacetamide, EVAL-(FMOC-L-arginine) ester and tetrabutylammonium fluoride. In the reaction of the deprotection, it is assumed that the yield for the esterification step is 100%. The calculations are shown in Table 2.
To a reaction flask is added the EVAL-(FMOC-L-arginine) ester and anhydrous DMAC under nitrogen. The sample is stirred and heated to 60� C. for 3 hours until the derivatized EVAL polymer went into solution. The solution is then cooled to room temperature. After the solution is cooled to room temperature, tetrabutylammonium fluoride is then added at room temperature, after which the solution turned a pink-red color. The solution is then stirred for 30 minutes.
The reaction solution is then added to 350 ml of deionized water while being vigorously stirred. The white polymer precipitate is then filtered and dried under vacuum. The polymer is re-dissolved into DMAC and then precipitated into methanol:ethanol (75:25). The suspension was then filtered and dried under vacuum. See
A method of obtaining an anhydride linkage between poly(butyl methacrylate-co-acrylic acid) and L-arginine or L-arginine oligomer is described. L-arginine is reacted with protecting groups to block the amino group of arginine. L-arginine is then reacted with thallium alkoxide to form a thallium salt to which polyethylene-co-acryloyl chloride is added. Then, the carboxylic acid moiety of poly (ethylene-co-acrylic acid) is converted to the acid chloride with thionyl chloride. After the completion of these reactions, the modified polymer is precipitated with anhydrous methanol, and then the L-arginine amine groups is are deprotected. After the deprotection of the amine, the polymer is extracted with anhydrous methanol to remove trace contaminants. See the reaction scheme in
An acryloyl-PEG-OH group is reacted with the carboxy group of L-arginine to produce a polymer with a hydrolytically labile ester bond. This product is then further processed by copolymerizing with ethylmethacrylate, hydroxyethylmethacrylate, methylmethacrylate, or butylmethacrylate. The reaction product is then mixed with a matrix linking polymer and polymerized in situ on the stent to form an interpenetrating network, or alternatively, the acrylate group is polymerized separately in solution and mixed with a coating matrix and then applied to the stent.
EVAL is linked to L-arginine or an L-arginine oligomer by forming an ester linkage. EVAL-L-arginine is then blended with a matrix polymer coating such as EVAL, EVAL-PEG blend, polyurethane and the like.
EVAL-L-arginine copolymer is blended with coating matrices comprising a PEG-superoxide dismutase donor or an EVAL-superoxide dismutase conjugate. EVAL-L-arginine copolymer may be also blended with coating matrices comprising an active ingredient.
Glycidol is used to modify polyethyleneimine (PEI), an amine-containing compound. A diamine spacer is first lengthened through the epoxide coupling of glycidol. So modified, the compound contains a spacer that terminates in vicinal diols. Periodate oxidation provides a formyl function at the end of the spacer.
Step 1. Dissolve 0.13 grams EVAL in 3.6 grams DMAC.
Step 2. Dissolve 0.085 grams R7 in 1.6 grams methanol.
Step 3. While stirring the EVAL solution with a magnetic stir bar, add the R7/Methanol solution drop-wise. A stable, clear solution results. When sprayed onto a stent and dried, the resultant coating is clear and has high gloss, indicating a lack of gross phase separation in the dried film.
Step 1. Dissolve 0.13 grams EVAL in 3.6 grams DMAC
Step 2. Dissolve 0.065 grams R7 in 1.5 grams methanol.
Step 3. While stirring the EVAL solution with a magnetic stir bar, add the R7/Methanol solution drop-wise. A stable, clear solution results. When sprayed onto a stent and dried, the resultant coating is clear and has high gloss, indicating a lack of gross phase separation in the dried film.
Step 1. Dissolve 0.2 grams PBMA in 3.3 grams DMAC.
Step 2. Dissolve 0.1 grams R7 in 1.4 grams methanol.
Step 3. While stirring the EVAL solution with a magnetic stir bar, add the R7/Methanol solution drop-wise. A stable, clear solution results. When sprayed onto a stent and dried, the resultant coating is clear and has high gloss, indicating a lack of gross phase separation in the dried film.
While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications can be made without departing from this invention in its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as fall within the true spirit and scope of this invention.
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New Metolius Hand Repair Balm�6/�10, added Apr/2011, see all Metolius news & reviews
announcement by Metolius
This announcement has been read 3,570 times
Suffering from sore tips? New from Metolius is the Hand Repair Balm, available in 0.5oz and 1oz tubs.
A specific climber's hand repair balm prepared exclusively for Metolius by Miller Creek Farms in Bend, Oregon.
Ingredients include Beeswax, Almond Oil, Shea Butter, Calendula, Camomile an Aloe Vera.
SRP 0.5oz £6, 1oz £10
You can follow the UK team here: http://climbersblogs.blogspot.com/
For more information visit Beyond Hope
Gear Forum ( Read more | 1 comments, 12 Apr 2011 )
This announcement has been read 3,570 times
Gear News and Outdoor Industry News at UKC and UKH presents climbing, walking and mountaineering equipment and stories that will be of interest to UKClimbing.com's readers. They are not gear reviews and are provided by companies that advertise with UKClimbing Limited. Please feel free to comment about the stories and products on the associated thread.


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Athena Pool Cue Cases
If you're looking for a durable yet stylish option for transporting your pool cues, check out the pool cue cases by Athena. Athena cases feature awesome designs, plus they feature a carabineer clip and an elastic mesh compartment inside the small pocket. These billiards cases also feature a separate extension pouch inside the long pocket big enough to hold most standard sized jumper butts.
All Athena pool cue cases include a 1 year manufacturer's warranty to protect against defects.
Athena Pink Zebra 3 Butt 5 Shaft Hard Case
Our Price: $169.15
Save 15%
Why would anyone buy a generic black case when they can have something as uniquely awesome as the Athena Pink Zebra. This case easily holds 3 butts and 5 shafts and features some of the best design work we've ever seen on an Athena cue or case.Learn More
Athena Purple Tribal Heart 2 Butt 2 Shaft Hard Case
Our Price: $75.65
Save 15%The Athena Purple Tribal Heart case is the perfect choice for women who want to protect there pool cues and look good doing it. This case holds two butts and two shafts so there is plenty of room for a Athena Cue and a Breaking Cue. The large front pocket also has an internal pouch that is perfect for storing the 10" Athena extension. What else do you need? This case has it all! Learn More
Athena Heartburn 2 Butt 2 Shaft Hard Case
Our Price: $75.65
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Just like the ATH38 pool cue, this Athena 2x2 case features the wildly popular "Heartburn" design with black tribal curls trailing the tribal hearts to give an "on fire" look. The case itself is extremely sturdy, utilizing heavy duty vinyl throughout the exterior and poured molds for the interior. The case will hold two complete pool cues (shafts and butts) and includes two generous pockets to hold all your accessories.Learn More
Athena Pink Tribal Rose 2 Butt 2 Shaft Hard Case
Our Price: $75.65
Save 15%
This Athena case is just what women are looking for. This case is the perfect size to fit your Athena pool cue along with an extra spot for the breaking cue. It also features two nice pockets one of which includes a pouch to store the 10" Athena extension. Learn More
Athena Towel
Our Price: $9.95
Save 17%
Believe it or not, towels play a huge role in playing pool well. Over time your hands get sweaty. That sweat creates sticky shafts which can create an inconsistent stroke and ruin your game. Sure, you could go with a basic towel, but why would you when you can get a towel as spectacularly awesome as this one?Learn More


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The current population of Weston Lakes is 499 with the median age of 51. Weston Lakes's median household income is $118,860, and the average household net worth is $909,243. 27% of Weston Lakes's population are long term residents having lived in their homes for more than 5 years, while 14% of Weston Lakes's population have moved in the last year.
|Weston Lakes
|United States
|Median Household Income
|Average Household Net Worth
|Long-term Residents (5+ years)
|Annual Turnover
|Median Age
|51 years
|33 years
|37 years
Age distribution represents the distribution of the population in Weston Lakes by age group. In Weston Lakes, the male-to-female ratio is 246:253, which is 1% higher than the ratio of Fort Bend County, which is 312,140:324,379. Age demographic data is sourced from census, 2015.
Crime rate index represents the average crime rate for a local area in comparison to nearby areas and the national average. A crime index of 100 represents the national average. Fort Bend County, has 62% less property crime than Texas, and is 53% below the nation's average. Fort Bend County, has 63% less personal crime than Texas and when compared to that of United States, Fort Bend County is 59% below the national average.
Marital status represents the percent of residents in Weston Lakes that are single, married, widowed and divorced. In Weston Lakes, 92% of people are married and 8% of people are single. The percent of people married is 13% higher than the ratio of Fort Bend County, which is 79%. Marital data is sourced from census, 2015.
Household income distribution represents the distribution of Weston Lakes income brackets at the household level. Overall, the median household income for Weston Lakes is $118,860, which is 41% higher than that of Fort Bend County ($84,305). Income data for Weston Lakes is sourced from census, 2015.
Education data represents the highest level of education attained by residents in Weston Lakes. In Weston Lakes, 4% of residents 18 and over graduated from high school and 50% completed a Bachelor's degree or higher, which is 10% higher than that of Fort Bend County. Education statistics is sourced from census, 2015.


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Main Page | Risk Factors | Reducing Your Risk | Screening | Symptoms | Diagnosis | Treatment Overview | Chemotherapy | Radiation Therapy | Surgical Procedures | Other Treatments | Lifestyle Changes | Living With Bladder Cancer | Talking to Your Doctor | Resource Guide
Screening is a way to evaluate people without symptoms to determine if they are at risk for cancer or have already developed cancer. Screening involves:
There are no official screening guidelines for bladder cancer. Testing is only recommended for people who are experiencing symptoms that may suggest bladder cancer. People who smoke should be advised to stop. They should also be informed of their increased risk of bladder cancer and other serious disease.
Some experts think that people with a higher-than-normal risk of bladder cancer should be periodically screened. These include the following groups of people:
If you are at increased risk, you may be advised to have periodic testing:
Screening is not 100% effective in diagnosing or excluding cancer. If you develop symptoms that suggest cancer, even after a negative screening test, you should contact your doctor for an evaluation.
Bladder cancer. American Cancer Society website. Available at: http://www.cancer.org/cancer/bladdercancer/ . Accessed June 5, 2013.
Bladder cancer. Urology Care Foundation website. Available at: http://www.urologyhealth.org/urology/index.cfm?article=100 . Updated March 2013. Accessed June 5, 2013.
US Preventive Services Task Force. Screening for bladder cancer. Agency for Healthcare Research and Quality website. Available at http://www.uspreventiveservicestaskforce.org/uspstf/uspsblad.htm . Updated August 2011. Accessed June 6, 2013.
What you need to know about bladder cancer. National Cancer Institute website. Available at http://www.cancer.gov/cancerinfo/wyntk/bladder . Updated August 30, 2010. Accessed June 5, 2013.
Last reviewed May 2014 by Mohei Abouzied, MD
Please be aware that this information is provided to supplement the care provided by your physician. It is neither intended nor implied to be a substitute for professional medical advice. CALL YOUR HEALTHCARE PROVIDER IMMEDIATELY IF YOU THINK YOU MAY HAVE A MEDICAL EMERGENCY. Always seek the advice of your physician or other qualified health provider prior to starting any new treatment or with any questions you may have regarding a medical condition.
Copyright © 2012 EBSCO Publishing All rights reserved.
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text:
Today's hard economic times have forced many manufacturing companies to work smarter to stay competitive in the current market.
A.C.T. Tooling
has developed a cutting tool which has greatly improved set-up times, run times and accuracy of machining which equates into better pricing and faster turn around times. This tool, the collet-less boring bar has improved production times and profitability.
Our Cutting Tool Solution
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Improves Production Efficiency
Saves Tooling Costs
Saves Set-Up Times
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Applicable for Most Projects
Works on Essentially all Materials
A.C.T. Mission Statement -
Save time and money in manufacturing, allowing the United States to compete globally.
Augusta Cutting Tools
A.C.T. Collet-Less Boring Bar
A.C.T. has designed a collet-less boring bar, enabling the manufacturer to quickly change boring bars as manufacturing demands and requirements change. It gives more rigidity and machining accuracy for the manufacturer.
Manufactured in the U.S.A. Patent Pending.
The A.C.T. Collet-Less Boring Bar is as simple as
1, 2, 3 - Clamp, Touch and Go
Website Designed
for Your Business


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Israeli archeologists uncover ancient church
MOSHAV ALUMA, Israel - Israeli archeologists say they have uncovered the remnants of a 1,500-year-old church dating back to the Byzantine era.
The Israel Antiquities Authority says the site was found during typical excavation work that took place before a planned construction of a new neighborhood in southern Israel.
Among the finds were a colorful mosaic and five inscriptions that attest to a once-vibrant Christian community in the region.
Baptists to pay $12.5 million in abuse case
ORLANDO, Fla. - A Florida jury has awarded $12.5 million in damages to a young man who was sexually abused by a Baptist minister when he was a child.
But an attorney for the Florida Baptist Convention said Tuesday that the organization would appeal the ruling from a jury in Lake County, northwest of Orlando.
Attorney Gary Yeldell says the convention is confident that an appellate court will overturn the ruling.
Vatican to cap costs for sainthood cases
VATICAN CITY - How much does it cost to become a saint? The Vatican said last week it has put a cap of sorts on the often-spiraling budgets for sainthood cases, another nod to Pope Francis' call for sobriety.
Cardinal Angelo Amato, head of the Vatican's saint-making office, said officials must now abide by a "reference price" to curb overall expenditure and make the process more equitable for less well-funded cases.
Associated Press
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AT&T offers Christmas deals on Blackberrys and a free Solstice
Christmas will be here before you know it so you had better get your holiday shopping done. If the people on your shopping list are still rocking that old flip phone that was cool two years ago, it's time for an upgrade. AT&T is offering some deals on popular smartphones and feature phones for the holidays including several half off Blackberry devices and a free Samsung Solstice.


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The stylish 22-year-old centre-back has been hampered by a string of injuries since arriving at Celtic Park three-and-a-half years ago.
And the Norway U21 international has turned down a couple of offers from Celtic to extend his stay in Glasgow.
Rogne is in Israel with his country for the U21 European Championship, where he hopes to start against the host nation, Italy and England in the group stages of the competition.
When asked about his future, Rogne said: "The only thing to do is to play U21 Championship for Norway. The rest we will take later.
"It's basically a perfect showcase. Doing well down there, it's a very nice window. But it can go both ways, you could also make a bad championship."


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Wednesday, August 6, 2008
Lego Paper Freebie
7 comments:
- Maria said...
Thank you! Your blog post will be advertised on the DigiFree Digital Scrapbooking Freebie search engine today (look for timestamp: 07 Aug [LA 12:00am, NY 02:00am, UK 07:00am, OZ 05:00pm] ).
- August 7, 2008 at 2:42 AM
- Vedette said...
Thank you so much for the papers!!
PS: If it isn't the false adress and phone No. you might want to change/remove your personal info on the invitation, just for the blog.. You never know who's on the net seeing it... There are predetors everywhere...
Good luck o the Bday! Sounds like a fun party!
- August 7, 2008 at 5:08 AM
- CactusFreek said...
Very nice! Thanks :o)
- August 7, 2008 at 5:19 AM
- JT said...
Creative Imaginations will be introducing a line of Lego scrapbook products in October. They have already begun taking orders from retailers.
- August 8, 2008 at 9:14 AM
- Vedette said...
Thank you for your nice omments about my LOs! I've only been scrapping since Feb. (I think), so I'm working on it... LOL!
I'm using Paint Shop Pro X, which is not quite compatible with Vista. (Ugh! I hate Vista!) For every operation I make on PSPX, it freezes. Took me 4 hrs to make a double sided LO today, and I'nm still not finished... I've tried PSE, but I just don't get that program at all. I've used PSP for 4 years now, and just starting to feel like master it... LOL!
They say PSP XII is supposed to be compatible with Vista, well, I think they're lying.... I downladed the trial version, but it was all just the same old...
Oh well, enough of my rambling..
Have a nice evening/ morning/ day/ night! =)
(depending on the time zone you in)
- August 9, 2008 at 11:21 AM
- Danielle said...
Thanks so much!! I have been looking for lego paper!
- August 9, 2008 at 1:48 PM
- Mags said...
Great paper!
Thanks so much!
:) Mags
- August 21, 2008 at 6:35 PM


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Domestic Violence Prevention Funds
By KRWG News
New Mexico – The National Latino Alliance for the Elimination of Domestic Violence has been awarded $516,204 in a U.S. Department of Justice grant aimed at combating domestic violence.
The Albuquerque nonprofit, known as Alianza, will use the funding to provide training and technical assistance to domestic violence and sexual assault coalitions.
Sen. Jeff Bingaman, D-N.M., said the funding comes through the Justice Department's Office on Violence Against Women, which provides direct support to domestic violence and sexual assault organizations.
The office provides funding, training and technical assistance while promoting public awareness activities and public policy advocacy.
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|Henry Earl Scott (updated)
|Sunday, 24 November 2013 16:28
Henry Earl Scott 57 of Horse Cave, KY died Saturday, November 23rd at his home. The Edmonson County native was the son of the late Henry and Evelyn Norris Scott.
He is survived by his wife Bethney Key Scott; 1 son Joey Scott (Nakole) of Horse Cave; 3 daughters Christina Denise Scott of Mt. Pleasant, IA, Laura Whitlow (Brandon) and Kelly Lindsey (Raymond) of Cave City; 3 brothers Phillip Scott (Connie) of Hardyville, Mike Scott of Glasgow and Ted Scott of Scottsville; 11 grandchildren Shelby, Brett, Miranda, Katilyn, Becca, Josh, Matt, Madison, Luke, Payton and Jackson; 5 great-grandchildren Emma, Allie, Skyler, Brayden and Kylie; 1 niece Carla and 2 great-nephews Andrew and Wyatt. In addition to his parents he was preceded in death by his grandparents Clarence and Dimple Scott and Willard and Lydia Norris and a niece Heather Scott.
Funeral services will be 1pm Tuesday at the A. F. Crow & Son Funeral Home with burial in the Campground Cemetery. Visitation will begin at 2pm Monday at the funeral home.


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Winning a Warnie is so graciously received these days that it must cause non-bloggers to wonder. After all a Warnie merely represents me feeling that a particular blog is worthy of recognition. Previous Warnie winners do nominate new members of this exclusive club, but it is I who has the final say and should take the credit or blame! Some have wondered why most Warnie winners are non-Americans. Well, I suspect this may be because Warnies tend to be issued to non-political blogs, and many of my American Blogging friends focus much more on politics than I am comfortable with.
Eternal Perspectives has posted his Warnie acceptance speech
May 23, 2005 by Adrian Warnock
Filed Under: Warnie Awards
Related posts from Adrian Warnock:
About Adrian Warnock
Adrian Warnock is a medical doctor, and a writer. Since 1995 he has been a member of Jubilee Church London which has sites in Enfield, Wood Green and Ilford. Adrian serves as part of Jubilee's leadership team alongside Tope Koleoso. Together they have written Hope Reborn - How to Become a Christian and Live for Jesus, published by Christian Focus. Adrian is also the author of Raised With Christ - How The Resurrection Changes Everything, published by Crossway. Read more about Adrian Warnock or connect with him on Twitter, Facebook or Google+.
You are warmly invited to comment on this blog. By doing so you demonstrate that you accept Adrian's comment policy.


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You may need to study a bit more. Local property taxes go to fund these schools too. If kids from North Olmsted go to charter schools then your N.O. taxes pay for them.
Thank you.
Are you going to do it, or just post about it?
WHO WAS THERE? Please, PD. Post the names.
I knew I liked her!
It's about time. Buh bye.
No. It won't help with economic development. It will help to expand the power base for this 'council' who will then be fighting it out with each other for a tiny slice of the pie. It's a bad idea.
It creates new, overlapping, confusing 'school districts' that will primarily benefit Bellefaire. Will mean a new levy, confusing to the average voter. The article said it all.
Hahahaha, byebye4now. Just what I was thinking about tonyfontaine.
ConfusedTeen said in another post that she's from Maple Heights. Must have a long study hall, with computer access, at the High School there.
It's you're. Not your. It's called a contraction. I just went over contractions last night with my second grader.
I thought that you were leaving ConfusedTeen.
You're an idiot teaparty.
It's they're, not their. It's called a contraction. 'They're' is a contraction for 'they are'.
Who's the master of the English language now?
"the rude comment meant to get people fired up"? You're talking about your own comment. You are sick.
Unbelievable. Even when the girl and the police say he's white you don't want to believe it?
Politicians share apartments ALL THE TIME. It's what they do in Columbus, and in Washington. They have houses back home and then share apartments with others during the week. There is nothing strange about that.
There IS something strange about Dann having his scheduler come to that apartment, whether she was in her PJ's or not.