WO2013013103A1

WO2013013103A1 - Sample assay for detecting patient compliance and/or health monitoring

Info

Publication number: WO2013013103A1
Application number: PCT/US2012/047493
Authority: WO
Inventors: Josiah SEALE; Brian Belmont; Angela KILBY
Original assignee: Adherean, Inc.
Priority date: 2011-07-20
Filing date: 2012-07-19
Publication date: 2013-01-24

Abstract

The present invention relates to devices, kits, instruments, and methods to detect patient compliance, overmedication, undermedication, and/or untreated conditions. The device detects medication metabolites, unmetabolized medication, indicators of treatment failure such as hepatotoxicity, and/or markers of undertreated or untreated disease in a sample provided by the patient.

Description

P A T E N T

Docket 699302000240

SAMPLE ASSAY FOR DETECTING PATIENT COMPLIANCE AND/OR HEALTH

MONITORING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application Serial No. 61/509,929, filed July 20, 2011. This application also relates to U.S. provisional application Serial No. 61/509,910, filed July 20, 2011. The contents of both applications are incorporated by reference in their entireties.

TECHNICAL FIELD

[0002] The present invention relates to devices, kits, instruments, and methods to detect patient compliance, overmedication, undermedication, and/or untreated conditions. The device detects medication metabolites, unmetabolized medication, indicators of treatment failure such as hepatotoxicity, and/or markers of undertreated or untreated disease in a sample provided by the patient.

BACKGROUND OF THE INVENTION

[0003] Medication adherence and the lack thereof is a global problem of significant magnitude. For example, a large portion of hospitalizations are attributable to patients failing to fully complete their medication regimens as prescribed.

[0004] From a strictly objective perspective, the problem of medication adherence is a puzzling one. If the marginal cost of taking a medication is low or non-existent (i.e., the patient can obtain the medication at low or no cost), and not taking the medication might result in health consequences for the patient, it would seem that patients would take their medication. However, the quantity of patients who fully adhere to their treatment regimen is much lower than this logic would imply.

[0005] Subjects often respond to small incentives as an effective means of changing behavior. However, these small incentives must be combined with an effective sd-595478 1 P A T E N T

Docket 699302000240 monitoring mechanism, because subjects tend to respond poorly to systems that are easily fooled.

[0006] To address this problem, several solutions have been proposed. Some solutions opt to ignore the fact that patients may dissimulate having taken the medication and reward based on refill rate or patients asserting they have taken their medications. Other solutions opt to use some other behavior as a proxy for patients actually taking their medication. Pill boxes that count, register, and/or transmit the occasions on which they have been opened and closed, for example, use opening and closing the pill box as a proxy for the patient having taken the medication. Another proposed solution is to tag medications with a substance, compound, or digestible device that can be detected by some other means.

[0007] All of these existing solutions fall short on some level. Solutions that reward patients based on refill rate or assertions of adherence may be vulnerable to patients who now have an incentive to be disingenuous about their actual behavior. Solutions that rely on indirect measures of adherence, such as patients opening and closing pill bottles, fall short on at least three points, firstly in that they are ultimately incentivizing the behavior that is rewarded (opening and closing the pill bottle, for example) as opposed to the patient actually taking the medication, secondly because they do not provide a means that translates well across different ways of packaging and delivering medications (such as blister packs or liquid medication), and thirdly because they quickly become cumbersome for those patients taking multiple medications. Solutions that try to create a new metric of adherence by adding a detectable substance, compound, or digestible device to the medication may face an immensely challenging regulatory framework in which the detectable element must be separately shown to not interact negatively with each targeted medication, and face a further uphill battle in that they do not provide an easy solution for capturing the adherence patterns of patients using multiple medications at the same time: if more than one medication is tagged with the same substance, compound, or digestible device it becomes impossible to distinguish which of those medications the patient might be taking. sd-595478 2 P A T E N T

Docket 699302000240

[0008] Up to date, a system that would rely on direct measures of adherence (e.g. , the presence of medication, medication metabolite, or other health markers in the patient's blood, urine, saliva, sweat, etc.) has not been practical for a home-based setting, as these tests have generally been expensive, invasive, and/or required laboratory training to be carried out. Solution-based assays marketed for at-home usage that provide a direct measure of adherence may be cumbersome in format, not generalizable across conditions, expensive to produce, and/or difficult to interpret effectively.

[0009] The present invention addresses this and the related need in the art by providing an easy to use, often disposable, direct measure that can be used to convey information corresponding to the presence, absence, or amount of medication indicator present in a subject. Because it provides a direct measure, it can further be more effectively used in a program to incentivize or encourage patients to submit their results and/or take their medication as prescribed.

BRIEF SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention is directed to a test device for assessing medication status in a subject, which device comprises a porous matrix that comprises a test reagent at a test location on said porous matrix, wherein a liquid sample derived from a subject flows laterally along said test device and passes said test location to form a detectable signal to indicate presence, absence and/or amount of a medication indicator in said subject that can be used to assess medication status in said subject.

[0011] In another aspect, the present invention is directed to a method for assessing medication status in a subject, which method comprises: a) contacting a liquid sample derived from a subject with the above test device, wherein the liquid sample is applied to a site of the test device upstream of the test location; b) transporting a medication indicator, if present in the liquid sample, and a labeled reagent to the test location; and c) assessing a detectable

sd-595478 3 P A T E N T

Docket 699302000240 signal at the test location that indicates presence, absence and/or amount of the medication indicator in the subject to assess medication status in the subject.

[0012] The principles of the present test devices and methods can be applied, or can be adapted to apply, to the lateral flow test devices and assays known in the art. For example, the principles of the present test devices and methods can be applied, or can be adapted to apply, to the lateral flow test devices and assays disclosed and/or claimed in the U.S. patent Nos. 3,641,235, 3,959,078, 3,966,897, 4,094,647, 4,168,146, 4,299,916,

4,347,312, 4,366,241, 4,391,904, 4,425,438, 4,517,288, 4,960,691, 5,141,875, 4,857,453, 5,073,484, 4,695,554, 4,703,017, 4,743,560, 5,075,078, 5,591,645, 5,656,448, RE 38,430 E, 5,602,040, 6,017,767, 6,319,676, 6,352,862, 6,485,982, 5,120,643, 4,956,302, RE 39,664 E, 5,252,496, 5,514,602, 7,238,538 B2, 7,175,992 B2, 6,770,487 B2, 5,712,170, 5,275,785, 5,504,013, 6,156,271, 6,187,269, 6,399,398, 7,317,532, EP 0,149,168 Al, EP 0,323,605 Al, EP 0,250,137 A2, GB 1,526,708 and WO99/40438.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows a top view of an exemplary test device. The rectangular portion of the drawing marked as "A" represents a hydrophobic or hydrophilic membrane in the exemplary test device. The portion of the drawing marked as "B" represents the first location as described in connection with the exemplary test device. The portion of the drawing marked as "C" represents the second location as described in connection with the exemplary test device. The portion of the drawing marked as "D" represents the optional control location downstream from the second location as described in connection with the exemplary test device.

[0014] Figure 2 illustrates construction of a lateral flow strip.

[0015] Figure 3 illustrates an exemplary kanamycin test. Two identical lateral flow strips, with a capture line containing Kanamycin-BSA conjugate, were used to detect the

sd-595478 4 P A T E N T

Docket 699302000240 presence or absence of kanamycin in the applied sample (as noted). The capture line region is indicated by the arrow.

DETAILED DESCRIPTION OF THE INVENTION

[0016] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.

A. Definitions

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entireties. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is

incorporated herein by reference.

[0018] As used herein, "a" or "an" means "at least one" or "one or more."

[0019] As used herein, a "binding reagent" refers to any substance that binds to an analyte with desired affinity and/or specificity. Non-limiting examples of the binding reagent include cells, cellular organelles, viruses, particles, microparticles, molecules, or an aggregate or complex thereof, or an aggregate or complex of molecules. Exemplary binding reagents can be an amino acid, a peptide, a protein, e.g., an antibody or receptor, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, e.g., DNA or RNA, a vitamin, a

monosaccharide, an oligosaccharide, a carbohydrate, a lipid, an aptamer and a complex thereof.

[0020] As used herein, the term "specifically binds" refers to the specificity of a binding reagent, e.g., an antibody, such that it preferentially binds to a defined analyte.

Recognition by a binding reagent or an antibody of a particular analyte in the presence of sd-595478 5 P A T E N T

Docket 699302000240 other potential targets or interfering substances is one characteristic of such binding. In some embodiments, a binding reagent that specifically binds to an analyte avoids binding to other interfering moiety or moieties in the sample to be tested.

[0021] As used herein the term "avoids binding" refers to the specificity of particular binding reagents, e.g., antibodies or antibody fragments. Binding reagents, antibodies or antibody fragments that avoid binding to a particular moiety generally contain a specificity such that a large percentage of the particular moiety would not be bound by such binding reagents, antibodies or antibody fragments. This percentage generally lies within the acceptable cross reactivity percentage with interfering moieties of assays utilizing the binding reagents or antibodies directed to detecting a specific target. Frequently, the binding reagents, antibodies or antibody fragments of the present disclosure avoid binding greater than about 90% of an interfering moiety, although higher percentages are clearly

contemplated and preferred. For example, binding reagents, antibodies or antibody fragments of the present disclosure avoid binding about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 99% or more of an interfering moiety. Less occasionally, binding reagents, antibodies or antibody fragments of the present disclosure avoid binding greater than about 70%, or greater than about 75%, or greater than about 80%, or greater than about 85% of an interfering moiety.

[0022] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule, and can be an immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD and IgE. IgY, which is the major antibody type in avian species such as chicken, is also included within the definition. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric sd-595478 6 P A T E N T

Docket 699302000240 antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.

[0023] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts. As used herein, a "monoclonal antibody" further refers to functional fragments of monoclonal antibodies.

[0024] As used herein, the term "antigen" refers to a target molecule that is specifically bound by an antibody through its antigen recognition site. The antigen may be monovalent or polyvalent, i.e., it may have one or more epitopes recognized by one or more antibodies. Examples of kinds of antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, etc.

[0025] As used herein, "mammal" refers to any of the mammalian class of species. Frequently, the term "mammal," as used herein, refers to humans, human subjects or human patients. Also frequently, the term "mammal," as used herein, refers to non-human mammalian subjects.

[0026] As used herein, the term "subject" is not limited to a specific species or sample type. For example, the term "subject" may refer to a patient, and frequently a human patient. However, this term is not limited to humans and thus encompasses a variety of mammalian or non-mammal species.

[0027] As used herein the term "sample" refers to anything which may contain an analyte for which an analyte assay is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like. Biological tissues are aggregate of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle sd-595478 7 P A T E N T

Docket 699302000240 and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).

[0028] As used herein the term "isolated" refers to material removed from its original environment, and/or is altered from its natural state. For example, an isolated polypeptide could be coupled to a carrier, and still be "isolated" because that polypeptide is not in its original environment.

[0029] As used herein, high-throughput screening (HTS) refers to processes that test a large number of samples, such as samples of diverse chemical structures against disease targets to identify "hits" (see, e.g., Broach, et al., High throughput screening for drug discovery, Nature, 384: 14-16 (1996); Janzen, et al., High throughput screening as a discovery tool in the pharmaceutical industry, Lab Robotics Automation: 8261-265 (1996); Fernandes, P.B., Letter from the society president, /. Biomol. Screening, 2: 1 (1997); Burbaum, et al., New technologies for high-throughput screening, Curr. Opin. Chem. Biol., 7:72-78 (1997)). HTS operations are highly automated and computerized to handle sample preparation, assay procedures and the subsequent processing of large volumes of data.

[0030] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more amino acids. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0031] The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" are used interchangeably herein to refer to a polymeric form of nucleotides of sd-595478 8 P A T E N T

Docket 699302000240 any length, e.g., at least 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more nucleotides, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single- stranded deoxyribonucleic acid ("DNA"), as well as triple-, double- and single- stranded ribonucleic acid ("RNA"). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide {e.g. , peptide nucleic acids ("PNAs")) and polymorpholino

(commercially available from the Anti-Virals, Inc., Corvallis, OR., as Neugene) polymers, and other synthetic sequence- specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. Thus, these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' to P5' phosphoramidates, 2'-0-alkyl-substituted RNA, hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, "caps," substitution of one or more of the nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages {e.g. , methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages {e.g. , phosphorothioates,

phosphorodithioates, etc.), and with positively charged linkages {e.g.,

aminoalkylphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including enzymes {e.g. nucleases), toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators {e.g., acridine, psoralen, etc.), those containing chelates (of, e.g. , metals, radioactive metals, boron, oxidative sd-595478 9 P A T E N T

Docket 699302000240 metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.

[0032] It will be appreciated that, as used herein, the terms "nucleoside" and "nucleotide" will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, e.g. , wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like. The term "nucleotidic unit" is intended to encompass nucleosides and nucleotides.

[0033] "Nucleic acid probe" and "probe" are used interchangeably and refer to a structure comprising a polynucleotide, as defined above, that contains a nucleic acid sequence that can bind to a corresponding target. The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.

[0034] As used herein, "complementary or matched" means that two nucleic acid sequences have at least 50% sequence identity. Preferably, the two nucleic acid sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. "Complementary or matched" also means that two nucleic acid sequences can hybridize under low, middle and/or high stringency condition(s).

[0035] As used herein, "substantially complementary or substantially matched" means that two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively, "substantially complementary or substantially matched" means that two nucleic acid sequences can hybridize under high stringency condition(s).

[0036] In general, the stability of a hybrid is a function of the ion concentration and temperature. Typically, a hybridization reaction is performed under conditions of lower sd-595478 10 P A T E N T

Docket 699302000240 stringency, followed by washes of varying, but higher, stringency. Moderately stringent hybridization refers to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule. The hybridized nucleic acid molecules generally have at least 60% identity, including for example at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity. Moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhardt' s solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS, at 42°C. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5x Denhardt' s solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0. lx SSPE, and 0.1% SDS at 65°C. Low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5x

Denhardt' s solution, 6x SSPE, 0.2% SDS at 22°C, followed by washing in lx SSPE, 0.2% SDS, at 37°C. Denhardt' s solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M EDTA. Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989); and Ausubel et al., Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons (1999).

[0037] Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary. See Kanehisa (1984) Nucleic Acids Res. 12:203-215.

[0038] As used herein, "biological sample" refers to any sample obtained from a living or viral source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid or protein or other macromolecule can sd-595478 Π P A T E N T

Docket 699302000240 be obtained. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. For example, isolated nucleic acids that are amplified constitute a biological sample. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom. Also included are soil and water samples and other environmental samples, viruses, bacteria, fungi, algae, protozoa and components thereof.

[0039] It is understood that aspects and embodiments of the invention described herein include "consisting" and/or "consisting essentially of aspects and embodiments.

[0040] Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0041] Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

B. Test devices for assessing medication status in a subject

[0042] In one aspect, the present disclosure provides for a test device for assessing medication status in a subject, which device comprises a porous matrix that comprises a test reagent at a test location on said porous matrix, wherein a liquid sample derived from a subject flows laterally along said test device and passes said test location to form a detectable sd-595478 12 P A T E N T

Docket 699302000240 signal to indicate presence, absence and/or amount of a medication indicator in said subject that can be used to assess medication status in said subject.

[0043] In one specific embodiment, the medication indicator to be detected comprises or is an antigen, the binding reagent on the test device comprises or is an antibody. Preferably, the antibody specifically binds to the medication indicator.

[0044] In one example, the test device is used in a sandwich assay format, in which a binding reagent, e.g. , an antibody, is used as a reagent at the test location, and another binding reagent having a detectable label is also used to form a labeled binding reagent- medication indicator - binding reagent or antibody sandwich at the test location to generate readout signals. Alternatively, a binding reagent is used as a reagent at the test location, and an antibody having a detectable label is also used to form a labeled antibody- medication indicator - binding reagent sandwich at the test location to generate readout signals. In one example, the sandwich assay uses two antibodies, one as the capture reagent and the other as the labeled reagent.

[0045] The test device can also be used in a competition assay format. In one example, a binding reagent, e.g., an antibody, is used as a capture reagent at the test location. A medication indicator or a medication indicator analog having a detectable label, either added in a liquid or previously dried on the test device and redissolved or resuspended by a liquid, will compete with a medication indicator in a sample to bind to the capture reagent at the test location. In another example, a medication indicator or a medication indicator analog is used as a capture reagent at the test location. A binding reagent, e.g. , an antibody, having a detectable label, is either added in a liquid or previously dried on the test device and redissolved or resuspended by a liquid. A medication indicator in a sample will compete with the medication indicator or the medication indicator analog at the test location for binding to the binding reagent, e.g., an antibody, having a detectable label.

[0046] The test reagent can be any suitable substance. In some embodiments, the test reagent is capable of binding to a medication indicator or another binding reagent that is sd-595478 13 P A T E N T

Docket 699302000240 capable of binding to a medication indicator. Preferably, the test reagent is capable of specifically binding to a medication indicator or another binding reagent that is capable of binding or specific binding to a medication indicator. In other embodiments, the test reagent is a medication indicator or a medication indicator analog that competes with a medication indicator in the liquid sample for binding to a binding reagent for the medication indicator.

[0047] In some embodiments, the test reagent is an inorganic molecule, an organic molecule or a complex thereof. Exemplary organic molecules include an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and a complex thereof. In some embodiments, the protein is an antigen or an antibody.

[0048] The matrix can comprise or be made of any suitable material. In some embodiments, the matrix comprises nitrocellulose, glass fiber, polypropylene, polyethylene (preferably of very high molecular weight), polyvinylidene flouride, ethylene vinylacetate, acrylonitrile and/or polytetrafluoro-ethylene. See e.g., U.S. patent No. 6,187,598. It can be advantageous to pre-treat the membrane with a surface-active agent during manufacture, as this can reduce any inherent hydrophobicity in the membrane and therefore enhance its ability to take up and deliver a moist sample rapidly and efficiently. The matrix can also be made from paper or other cellulosic materials. In some embodiments, the matrix comprises or is made of nitrocellulose or glass fiber.

[0049] The matrix can also be in any suitable form or shape. In some

embodiments, the matrix is in the form a strip or a circle. The matrix can also comprise or be made of any suitable number of element. In some embodiments, the matrix is a single element or comprises multiple elements.

[0050] The present test devices can comprise any suitable additional elements. In some embodiments, the test device can further comprise a sample application element upstream from and in fluid communication with the matrix. In other embodiments, the test device can further comprise a liquid absorption element downstream from and in fluid sd-595478 14 P A T E N T

Docket 699302000240 communication with the matrix. In still other embodiments, the test device can further comprise a control zone comprising means for indicating proper flow of the liquid sample and/or a valid test result. In yet other embodiments, at least a portion of the matrix is supported by a solid backing. In yet other embodiments, the entire matrix is supported by a solid backing.

[0051] In some embodiments, a labeled reagent can be dried on the test device and the dried labeled reagent can be redissolved or resuspended by a liquid, e.g., a sample liquid and/or additional liquid, and transported laterally through the test device to generate readout, control and/or other signals. For example, a portion of the matrix, upstream from the test location, can comprise a dried, labeled reagent, the labeled reagent capable of being moved by a liquid sample and/or a further liquid to the test location and/or a control location to generate a detectable signal.

[0052] The dried, labeled reagent can be located at any suitable places on the test device. In one example, the dried, labeled reagent is located downstream from a sample or liquid application place on the test device. In another example, the dried, labeled reagent is located upstream from a sample or liquid application place on the test device.

[0053] The type of the labeled reagent can be determined based on the intended assay formats. For example, if the test device is to be used in a sandwich assay, the labeled reagent should be capable of binding, and preferably capable of specifically binding, to the analyte or a target, or another substance that binds to the analyte or the target. The same labeled reagent can also be used for certain competitive binding assays. For other types of the competitive binding assays, the labeled reagent should be an analyte or an analyte analog linked to a detectable label.

[0054] In some embodiments, a portion of the matrix, upstream from the test location, comprises a dried, labeled reagent, the labeled reagent being capable of being moved by a liquid sample and/or a further liquid to the test location and/or a control location to generate a detectable signal. The dried, labeled reagent can be located downstream from a sd-595478 15 P A T E N T

Docket 699302000240 sample application place on the test device. Alternatively, the dried, labeled reagent can be located upstream from a sample application place on the test device.

[0055] In some embodiments, the test can further comprise, upstream from the test location, a conjugate element that comprises a dried, labeled reagent, the labeled reagent being capable of moved by a liquid sample and/or a further liquid to the test location and/or a control location to generate a detectable signal. The conjugate element can be located downstream from a sample application place on the test device. Alternatively, the conjugate element can be located upstream from a sample application place on the test device.

[0056] In some embodiments, the labeled reagent binds, and preferably specifically binds, to a medication indicator in the liquid sample. In other embodiments, the labeled reagent competes with a medication indicator in the liquid sample for binding to a binding reagent for the medication indicator at the test location.

[0057] Any suitable label can be used. The label can be a soluble label, such as a colorimetric, radioactive, enzymatic, luminescent or fluorescent label. The label can also be a particle or particulate label, such as a particulate direct label, or a colored particle label. Exemplary particle or particulate labels include colloidal gold label, latex particle label, nanoparticle label and quantum dot label. Depending on the specific configurations, the labels such as colorimetric, radioactive, enzymatic, luminescent or fluorescent label, can be either a soluble label or a particle or particulate label.

[0058] In some embodiments, the labeled reagent is dried in the presence of a material that stabilizes the labeled reagent, facilitates solubilization or resuspension of the labeled reagent in a liquid, and/or facilitates mobility of the labeled reagent. Any suitable material can be used. For example, the material can be a protein, e.g., a meta-soluble protein, a peptide, a polysaccharide, a sugar, e.g., sucrose, a polymer, a gelatin or a detergent. See e.g., U.S. patent Nos. 5,120,643 and 6,187,598.

[0059] The present test devices can be used with any suitable liquid. In one example, a sample liquid alone is used to transport a medication indicator and/or the labeled sd-595478 16 P A T E N T

Docket 699302000240 reagent to the test location. In another example, a developing liquid is used to transport a medication indicator and/or the labeled reagent to the test location.

[0060] In some embodiments, the test device can further comprise a housing that covers at least a portion of the test device, wherein the housing comprises a sample or liquid application port to allow sample or liquid application upstream from or to the test location and an optic opening around the test location and/or the control location to allow signal detection at the test location and/or the control location. The optic opening can be achieved in any suitable way. For example, the optic opening can simply be an open space.

Alternatively, the optic opening can be a transparent cover.

[0061] In other embodiments, the housing can cover the entire test device. In still other embodiments, at least a portion of the sample receiving portion of the matrix or the sample application element is not covered by the housing and a sample or liquid is applied to the portion of the sample receiving portion of the matrix or the sample application element outside the housing and then transported to the test location and/or control location. The housing can comprise any suitable material. For example, the housing can comprise a plastic material.

[0062] The present test devices can be used to assess medication status in any suitable subject. In some embodiments, the present test devices can be used to assess medication status in a human. In other embodiments, the present test devices can be used to assess medication status in an animal, e.g., a non-human mammal.

[0063] The present test devices can be used to assess medication status in a suitable subject by assessing any suitable medication indicator from the subject. In some

embodiments, the medication indicator is a medication metabolite, an unmetabolized medication, or an indicator of over medication, under medication or medication failure.

[0064] In some embodiments, the present test devices can be used to assess medication status in a subject of the exemplary medications listed in the Orange Book:

Approved Drug Products with Therapeutic Equivalence Evaluations (Current through March sd-595478 17 P A T E N T

Docket 699302000240

2012) published by the U.S. Food and Drug Administration, the exemplary medications listed in The Merck Index (a U.S. publication, the printed 14th Edition, Whitehouse Station, N.J., USA) and its online version (The Merck Index Online^SM, Last Loaded on Web: Tuesday, May 01, 2012), and the exemplary medications listed in Biologies Products & Establishments published by the U.S. Food and Drug Administration.

[0065] In some embodiments, the present invention provides for a test device wherein the liquid or sample has moved laterally along the test device to generate a detectable signal at the test location.

C. Methods for assessing medication status in a subject

[0066] In another aspect, the present disclosure provides for a method for assessing medication status in a subject, which method comprises: a) contacting a liquid sample derived from a subject with the test device described in above Section B, wherein the liquid sample is applied to a site of the test device upstream of the test location; b) transporting a medication indicator, if present in the liquid sample, and a labeled reagent to the test location; and c) assessing a detectable signal at the test location that indicates presence, absence and/or amount of the medication indicator in the subject to assess medication status in the subject.

[0067] In some embodiments, the liquid and the labeled reagent are premixed to form a mixture and the mixture is applied to the test device. For example, the labeled reagent can be provided or stored in a liquid and then can be premixed with a sample to form a mixture and the mixture is applied to the test device. In another example, the labeled reagent can be dried in a location or container not in fluid communication with the test device, e.g., in a test tube or well such as a microtiter plate well. In use, the sample liquid can be added to the container, e.g., the test tube or well, to form the mixture and the mixture can then be applied to the test device.

[0068] In other embodiments, the test device comprises a dried labeled reagent before use and the dried labeled reagent is solubilized or resuspended, and transported to the test location by the liquid sample. The dried labeled reagent can be located at any suitable sd-595478 18 P A T E N T

Docket 699302000240 location on the test device. For example, the dried labeled reagent can be located

downstream from the sample application site, and the dried labeled reagent is solubilized or resuspended, and transported to the test location by the liquid sample. In another example, the dried labeled reagent can be located upstream from the sample application site, and the dried labeled reagent is solubilized or resuspended, and transported to the test location by another liquid.

[0069] In some embodiments, the labeled reagent is solubilized or resuspended, and transported to the test location by the liquid sample alone. In other embodiments, the medication indicator and/or labeled reagent is solubilized or resuspended, and transported to the test location by another liquid.

[0070] The present methods can be used to assess medication status in a suitable subject by assessing a medication indicator in any suitable sample. In some embodiments, the liquid sample is a body fluid sample, e.g., a whole blood, a serum, a plasma and a urine sample.

[0071] In some embodiments, the present methods can be used to assess medication status in a subject by assessing presence or absence of a medication indicator in a sample. In other embodiments, the present methods can be used to assess medication status in a subject by quantifying or semi-quantifying the amount of a medication indicator in a liquid sample. In still other embodiments, the present methods can be used to assess medication status in a subject by assessing multiple medication indicators in a liquid sample.

[0072] In some embodiments, the present methods can be used to assess medication status in a subject of the exemplary medications listed in the Orange Book:

Approved Drug Products with Therapeutic Equivalence Evaluations (Current through March 2012) published by the U.S. Food and Drug Administration, the exemplary medications listed in The Merck Index (a U.S. publication, the printed 14th Edition, Whitehouse Station, N.J., USA) and its online version (The Merck Index Online^SM, Last Loaded on Web: Tuesday,

sd-595478 19 P A T E N T

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May 01, 2012), and the exemplary medications listed in Biologies Products & Establishments published by the U.S. Food and Drug Administration.

Exemplary embodiments

[0073] In one aspect, the present invention provides for devices to detect medication metabolites, unmetabolized medication, indicators of treatment failure such as hepatotoxicity, and/or markers of undertreated or untreated disease in a sample provided by the patient.

[0074] In one embodiment, the present invention provides a device for detecting at least an analyte in a sample in the form of a competitive lateral flow assay, which device comprises: at least a naturally hydrophobic membrane comprising at least a first location and a second location, wherein said first location comprises a dried particle labeled binding reagent capable of binding to an analyte, if present in a sample to be tested, to form a first complex comprising said labeled binding reagent and said analyte; said second location, downstream from said first location, comprises an immobilized substance capable of competing with analyte that might be present in the sample, to form a second complex comprising said first labeled binding reagent and said immobilized substance; whereby the addition of a liquid, either as part of the sample or independently from it, for transporting said sample and said first labeled binding reagent to said second location to form said second complex allows the presence, absence and/or amount of said analyte in said sample to be determined by assessing response or lack thereof at said second complex at said second location.

[0075] Any suitable naturally hydrophobic membrane can be used. In some embodiments, the naturally hydrophobic membrane comprises nitrocellulose membrane. The first and second locations can take any suitable form. For example, the first location, the second location or both locations can be in the form of a zone or zones.

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[0076] Any suitable label, or labeled particle, can be used. In some embodiments, the labeled particle comprises a quantum dot or a colored particle. In other embodiments, the label, or labeled particle can be dried before use, either on the test device or on a location off the test device. The label, or labeled particle, can be dried in any suitable manner, e.g. , air dried or lyophilized. In some embodiments, the particle labeled binding reagent is dried in the presence of a material that: a) stabilizes the particle labeled binding reagent; b) facilitates resuspension of the particle labeled binding reagent in the liquid; and/or c) facilitates mobility of the particle labeled binding reagent. Such exemplary material can be a protein, a peptide, a polysaccharide, a sugar, a polymer, a gelatin or a detergent.

[0077] In some embodiments, the first labeled binding reagent binds specifically to the analyte. The first labeled binding reagent can be any suitable substance, e.g. , an antibody to the analyte, and/or an aptamer, aptazyme, and/or ribozyme that specifically binds with the analyte.

[0078] The substance can be immobilized at the second location in any suitable manner. In some embodiments, the substance can be immobilized by absorption, adsorption, or covalent binding to the naturally hydrophobic membrane; or attached to another substance or particle that is immobilized to the naturally hydrophobic membrane.

[0079] In some embodiments, the naturally hydrophobic membrane can be supported by a solid backing. In other embodiments, the device can further comprise a housing that covers at least the first and second locations on the membrane, wherein the housing comprises a sample application site to allow sample application upstream from or to the first location on the membrane and an opening around the second location to allow response detection at the second location. The housing can be made of any suitable material. In some embodiments, the housing can comprise a plastic material.

[0080] In some embodiments, the device can further comprise a control location downstream from, but in fluid communication with, the second location, wherein the control location comprises means for indicating a valid test result. sd-595478 21 P A T E N T

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[0081] In another aspect, the present invention provides for a method for detecting an analyte in a sample, comprising a) contacting a sample with the above device, wherein the sample is applied to a site of the membrane upstream of the second site; b) transporting an analyte, if present in said sample, and the dried first particle labeled binding reagent capable of binding to an analyte, by a liquid to the second location to form the second complex comprising said first labeled binding reagent and the immobilized substance capable of competing with the analyte and binding with the first labeled binding reagent at the second location if the first labeled binding reagent has not bound with analyte that might be present in the sample; and c) determining the presence, absence and/or amount of said analyte in said sample by assessing behavior in said first labeled binding reagent in said second complex at said second location.

[0082] The sample can be applied to any suitable location of the test device. In some embodiments, the sample is applied to the first location of the membrane. In other embodiments, the sample is applied to a site of the membrane upstream of the first location of the membrane.

[0083] The present methods can be used to assess a medication indicator in any suitable sample. In some embodiments, the sample is whole blood, a serum, a plasma, or a urine sample.

[0084] The present methods can be used to assess any suitable medication indicators. Exemplary medication indicators include medication metabolites, unmetabolized medication, ketones, glucose, transaminases, albumin, sarcosine, cancer markers,

hepatotoxicity markers, and nephrotoxicity markers.

Example

Description

[0085] Lateral flow strips were constructed in order to detect the presence or absence of the medication kanamycin in a sample. The test was a competitive assay, in sd-595478 22 P A T E N T

Docket 699302000240 which a suitable concentration of kanamycin in a sample would prevent the binding of the detection DNA-gold nanoparticle conjugates to the capture line. In the absence of a suitable concentration of kanamycin present in the sample, the DNA-gold nanoparticle conjugates are predicted to bind to the capture line reagents and form a visually detectable signal. The capture line was created by the immobilization of Kanamycin-BSA conjugate onto the lateral flow membrane. The detection particles were composed of a gold nanoparticle conjugated to a DNA oligonucleotide containing a kanamycin-binding sequence.

[0086] When liquid sample not containing kanamycin was applied to the test strip, a visually detectable band formed at the capture line. When liquid sample containing kanamycin was applied to the test strip, no detectable band formed at the capture line. (See Figure 3.)

Method

[0087] A lateral flow assay strip was constructed using a cellulose sample pad (Millipore, # CFSP223000), conjugate pad (Millipore, # GFCP203000), HiFlow Plus HFB09004 membrane (Millipore, # HF09004XSS), absorbent pad (Millipore, #

CFSP223000), and laminate card (Millipore, #HF000MC100), as shown in Figure 2.

[0088] For detection, 40nm diameter gold nanoparticles (Cytodiagnostics) were functionalized by thiol-conjugation of a DNA oligonucleotide containing a

kanamycin-binding aptamer sequence. The oligonucleotide had sequence

5 ' -TTTTTTTTTTTTTTTTGGGGGTTGAGGCTAAGCCGA-3 ' (SEQ ID NO: l), where the thiol is located on the 5' end of the aptamer and the kanamycin binding aptamer sequence is underlined. After conjugation, the DNA-gold nanoparticle conjugate was resuspended in 2 mM borate, pH 7 to a final OD = 4.

[0089] The capture region of the lateral flow strip was prepared by dispensing 1 microliter of a 2 mg/mL solution of Kanamycin-B ovine Serum Albumin conjugate

(Bio-World), dissolved in 10 mM sodium phosphate buffer, 3% methanol, pH 7.4., and

sd-595478 23 P A T E N T

Docket 699302000240 allowed to dry for 24 hours. Five microliters of DNA-conjugated gold nanoparticle, as described above, was dispensed onto the conjugate pad region of the strip.

[0090] Liquid sample (10 mM sodium phosphate, with or without 0.5 mg/mL kanamycin) was applied to two identical strips, and the tests were allowed to develop.

sd-595478 24

Claims

P A T E N T Docket 699302000240 CLAIMS The claimed invention is:

1. A test device for assessing medication status in a subject, which device comprises a porous matrix that comprises a test reagent at a test location on said porous matrix, wherein a liquid sample derived from a subject flows laterally along said test device and passes said test location to form a detectable signal to indicate presence, absence and/or amount of a medication indicator in said subject that can be used to assess medication status in said subject.

2. The test device of claim 1, wherein the matrix comprises nitrocellulose, glass fiber, polypropylene, polyethylene (preferably of very high molecular weight),

polyvinylidene flouride, ethylene vinylacetate, acrylonitrile and/or polytetrafluoro-ethylene.

3. The test device of claim 1 or 2, wherein the test reagent is capable of binding to a medication indicator or another binding reagent that is capable of binding to a medication indicator.

4. The test device of claim 3, wherein the test reagent is capable of specifically binding to a medication indicator or another binding reagent that is capable of binding to a medication indicator.

5. The test device of claim 1 or 2, wherein the test reagent is a medication indicator or a medication indicator analog that competes with a medication indicator in the liquid sample for binding to a binding reagent for the medication indicator.

6. The test device of any of the claims 1-5, wherein the test reagent is an inorganic molecule, an organic molecule or a complex thereof. sd-595478 25 P A T E N T

Docket 699302000240

7. The test device of claim 6, wherein the organic molecule is selected from the group consisting of an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and a complex thereof.

8. The test device of claim 7, wherein the protein is an antigen or an antibody.

9. The test device of any of the exemplary claims 1-8, wherein the matrix is in the form a strip or a circle.

10. The test device of any of the claims 1-8, wherein the matrix is a single element or comprises multiple elements.

11. The test device of any of the claims 1-10, which further comprises a sample application element upstream from and in fluid communication with the matrix.

12. The test device of any of the claims 1-11, which further comprises a liquid absorption element downstream from and in fluid communication with the matrix.

13. The test device of any of the claims 1-12, which further comprises a control location comprising means for indicating proper flow of the liquid sample and/or a valid test result.

14. The test device of any of the claims 1-13, wherein at least a portion of the matrix is supported by a solid backing.

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15. The test device of any of the claims 1-14, wherein a portion of the matrix, upstream from the test location, comprises a dried, labeled reagent, the labeled reagent being capable of being moved by a liquid sample and/or a further liquid to the test location and/or a control location to generate a detectable signal.

16. The test device of claim 15, wherein the dried, labeled reagent is located downstream from a sample application place on the test device.

17. The test device of claim 15, wherein the dried, labeled reagent is located upstream from a sample application place on the test device.

18. The test device of any of the claims 1-17, which further comprises, upstream from the test location, a conjugate element that comprises a dried, labeled reagent, the labeled reagent being capable of moved by a liquid sample and/or a further liquid to the test location and/or a control location to generate a detectable signal.

19. The test device of claim 18, wherein the conjugate element is located downstream from a sample application place on the test device.

20. The test device of claim 18, wherein the conjugate element is located upstream from a sample application place on the test device.

21. The test device of any of the claims 15-20, wherein the labeled reagent binds, and preferably specifically binds, to a medication indicator in the liquid sample.

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22. The test device of any of the claims 15-20, wherein the labeled reagent competes with a medication indicator in the liquid sample for binding to a binding reagent for the medication indicator at the test location.

23. The test device of any of the claims 15-22, wherein the label is a soluble label.

24. The test device of any of the claims 15-22, wherein the label is a particle label, e.g. , a gold or latex particle label.

25. The test device of any of the claims 15-24, wherein the labeled reagent is dried in the presence of a material that: a) stabilizes the labeled reagent; b) facilitates solubilization or resuspension of the labeled reagent in a liquid; and/or c) facilitates mobility of the labeled reagent.

26. The test device of claim 25, wherein the material is selected from the group consisting of a protein, a peptide, a polysaccharide, a sugar, a polymer, a gelatin and a detergent.

27. The test device of any of the claims 1-26, wherein a sample liquid alone is used to transport a medication indicator and/or the labeled reagent to the test location.

28. The test device of any of the claims 1-26, wherein a developing liquid is used to transport a medication indicator and/or the labeled reagent to the test location.

29. The test device of any of the claims 1-28, which further comprises a housing that covers at least a portion of the test device, wherein the housing comprises a sample

sd-595478 28 P A T E N T

Docket 699302000240 application port to allow sample application upstream from or to the test location and an optic opening around the test location to allow signal detection at the test location.

30. The test device of claim 29, wherein the housing covers the entire test device.

31. The test device of claim 29, wherein at least a portion of the sample receiving portion of the matrix or the sample application element is not covered by the housing and a sample is applied to the portion of the sample receiving portion of the matrix or the sample application element outside the housing and is then transported to the test location.

32. The test device of any of the claims 29-31, wherein the housing comprises a plastic material.

33. The test device of any of the claims 1-32, wherein the subject is a human.

34. The test device of any of the claims 1-32, wherein the subject is an animal.

35. The test device of any of the claims 1-34, wherein the medication indicator is a medication metabolite, an unmetabolized medication, or an indicator of over medication, under medication or medication failure.

36. The test device of any of the claims 1-35, wherein the liquid sample has moved laterally along the test device to generate a detectable signal at the test location.

37. A method for assessing medication status in a subject, which method comprises:

sd-595478 29 P A T E N T

Docket 699302000240 a) contacting a liquid sample derived from a subject with the test device of any of the claims 1-36, wherein the liquid sample is applied to a site of the test device upstream of the test location;

b) transporting a medication indicator, if present in the liquid sample, and a labeled reagent to the test location; and

c) assessing a detectable signal at the test location that indicates presence, absence and/or amount of the medication indicator in the subject to assess medication status in the subject.

38. The method of claim 37, wherein the liquid sample and the labeled reagent are premixed to form a mixture and the mixture is applied to the test device.

39. The method of claim 37, wherein the test device comprises a dried labeled reagent before use and the dried labeled reagent is solubilized or resuspended, and transported to the test location by the liquid sample.

40. The method of claim 39, wherein the dried labeled reagent is located

downstream from the sample application site, and the dried labeled reagent is solubilized or resuspended, and transported to the test location by the liquid sample.

41. The method of claim 39, wherein the dried labeled reagent is located upstream from the sample application site, and the dried labeled reagent is solubilized or resuspended, and transported to the test location by another liquid.

42. The method of claim 39, wherein the labeled reagent is solubilized or resuspended, and transported to the test location by the liquid sample alone.

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43. The method of claim 39, wherein the medication indicator and/or labeled reagent is solubilized or resuspended, and transported to the test location by another liquid.

44. The method of any of the claims 39-43, wherein the liquid sample is a body fluid sample.

45. The method of claim 44, wherein the body fluid sample is selected from the group consisting of a whole blood, a serum, a plasma and a urine sample.

46. The method of any of the claims 37-45, which is used to quantify or

semi-quantify the amount of a medication indicator in a liquid sample.

47. The method of any of the claims 37-46, which is used to detect multiple medication indicators in a liquid sample.

sd-595478 31