WO2008089435A1 - Methods of identifying pain modulators - Google Patents

Abstract

The invention relates to methods of identifying modulators of a cationic amino acid transporter protein, or modulators of CAT-regulated nitric oxide production, which can be used to alleviate pain or to treat pain insensitivity.

Description

METHODS OF IDENTIFYING PAIN MODULATORS

Priority is claimed to US Prov. Patent Appl. No. 60/885,529, filed January 18, 2007, which is incorporated herein.

Field of the Invention

The present invention generally relates to methods and compositions for alleviating or modulating pain. More particularly, the present invention relates to methods of identifying modulators of a cationic amino acid transporter (CAT) and the use of such modulators for treating pain-related disorders.

Background of the Invention

Acute pain, chronic pain, and ongoing spontaneous pain are disabling conditions characterized by increased sensitivity to normally non-noxious stimuli (allodynia) and/or painful stimuli (hyperalgesia). Conversely, people born with congenital insensitivity to pain typically have shorter life spans and suffer numerous ailments such as broken bones, bed sores, and chronic infection. See, e.g., Lilley et al., Pharmacology and the Nursing Process, 2005, 4th ed., pp. 147. St. Louis, Mosby.

Nitric oxide synthase (NOS) is suggested to play a role in the pathophysiology of inflammatory and neuropathic pain (Naik et al., Eur. J. Pharmacol. 2006, 530: 56-69; Mayer et al., Proc. Natl. Acad. Sci. USA. 1999, 96: 7731-7736). Mice lacking the inducible form of nitric oxide synthase (iNOS), one of three isoenzymes that catalyzes the conversion of L-arginine to nitric oxide (NO) and citrulline, demonstrate reduced thermal hyperalgesia after administration of an irritant. The iNOS-deficient mice also display an increase in prostaglandin E₂ (PGE₂) formation and enhanced cyclo- oxygenase-2 (COX-2) activity, which contribute to nociceptive sensitization. See Guhring et al., J. Neurosci.. 2000, 20(18): 6714-6720.

The cationic amino acid transporter (CAT) family of transporters mediates most L-arginine uptake in mammalian cells. CAT-2 and iNOS are co-induced after cytokine stimulation in numerous cell types, suggesting that CAT-2 may regulate NO production by controlling L-arginine transport. This hypothesis was confirmed using a CAT-2 knockout mouse. Macrophages from CAT-2 deficient mice synthesized significantly reduced amounts of NO after appropriate stimulation, demonstrating that CAT-2 arginine transport function is required for sustained NO production in activated macrophages (Nicholson et al., J. Biol. Chem.. 2001 , 276(19): 15881-15885). CAT-2 arginine transport deficiency similarly reduced NO production in astrocytes (Manner et al., J. Neurochem., 2003, 85: 476-482).

Cationic amino acid transporters are also implicated in a wide variety of other biological responses, including inflammation, asthma and other pulmonary conditions, cardiovascular dysfunction, conditions associated with oxidative damage, such as ischemia-reperfusion and sepsis, renal disease, etc. See e.g., Gill et al., J. Biol. Chem.. 1996, 271 (19):1 1280-1 1283; Schwartz et al., J. Lab. Clin. Med.. 2001 , 137(5): 356-362; Oyadomah et al., Nitric Oxide, 2001 , 5(3):252-260; NeNn et al., Am. J. Physiol. Lung Cell MoI. Physiol., 2001 , 281 (5):L1232-1239; NeNn et al., Am. J. Respir. Cell MoI. Biol., 2002, 2693): 348-355; Schwartz et al., Kidney Int., 2002, 62(5):1700-1706; Huang et al., Resuscitation, 2004, 63(2): 203-212; Chu et al., Acta Anaesthesiol. Taiwan, 2005, 43(1 ):23-32; and McCord et al., Hypertension, 2006, 47(1 ): 109-1 15. Therefore, notwithstanding the role of CAT-2 in NO induction, prior to the instant invention, the role of cationic amino acid transporters in pain pathophysiology remained unclear.

Many current pain therapies provide limited, temporary relief, and therefore treatments which target alternative molecules within the iNOS pathway may afford greater efficacy. To meet this need, the present invention demonstrates a functional relationship between CAT-2, NO production, and nocioception. Based thereon, also provided are methods of identifying modulators of CAT and/or components of CAT-regulated NO production, and methods of using the same to alleviate or modulate pain.

Summary of the Invention

The present invention provides methods of identifying a pain modulator, the method characterized by (a) providing a cationic amino acid transporter (CAT) protein; (b) providing one or more test agents to the cationic amino acid transporter (CAT) protein under conditions sufficient for binding; (c) assaying binding of said one or more test agents to the cationic amino acid transporter (CAT) protein; and (d) selecting a test agent which demonstrates specific binding to the cationic amino acid transporter (CAT) protein, and which modulates pain sensation. Also provided are methods of identifying a pain modulator by (a) providing a CAT protein under conditions for production of nitric oxide (NO); (b) providing one or more test agents or a control agent to the CAT protein of (a); (c) assaying NO production; and (d) selecting a test agent that shows altered NO production in the presence of the test agent as compared to said control agent.

Brief Description of the Drawings

Figures 1A-1 B are bar graphs showing that CAT-2 knockout mice withstand acute visceral pain. Age-matched cohorts of male and female CAT- 2 knockout and wild type mice were evaluated in a mouse para- phenylquinone (PPQ) model of acute visceral pain, as described in Example 1. In the PPQ model, both male and female CAT-2 knockout mice exhibited significantly less PPQ-induced abdominal stretches compared to CAT-2 wild type mice.

Figures 2A-2B are bar graphs showing that CAT-2 knockout mice withstand acute inflammatory pain. Age-matched cohorts of male and female CAT-2 knockout and wild type mice were evaluated in a mouse carrageenan model of acute inflammatory pain, as described in Example 2. Significant differences in carrageenan-induced thermal hyperalgesia were observed such that both male and female CAT-2 knockout mice exhibited less hyperalgesia, compared to their wild type littermates.

Detailed Description of the Invention The present invention discloses methods of identifying pain modulators which attenuate or enhance NO production. Methods of identifying modulators that enhance nocioception, for example, agents useful for treating congential pain insensitivity, are also disclosed. Further provided are therapeutic methods that employ pain modulators identified by performing the disclosed methods.

As described herein, animals lacking a functional CAT-2 gene show increased tolerance of acute pain (see Examples 1-2), suggesting that antagonists of CAT and/or other components of CAT-regulated NO production can be used to alleviate pain. The CAT family includes at least four members: CAT-1 (e.g., GenBank® Accession Nos. AAC27721 NP_003036, AAH63303, AAM 5408, AAH69358, P30825), CAT-2 (e.g., GenBank® Accession No. P52569), including two isoforms produced by differential splicing, CAT-2a (e.g., GenBank® Accession Nos. NP_001008539 AAH69648, AAI04906) and CAT-2b (e.g., GenBank® Accession No. NPJD03037), CAT-3 (e.g., GenBank® Accession Nos. NP_1 16192 AAH33816, NP_001041629, Q8WY07), and CAT-4 (e.g., GenBank® Accession Nos. NP_004164 AAH62565, AAI07161 , AAI07162, AAH08814, 043246).

The disclosed methods encompass identification agents that specifically bind to and modulate any of the above-noted CAT proteins, or other CAT proteins, to thereby alter NO production.

L Methods of Identifying CAT Modulators

CAT modulators, i.e., agonists/activators and antagonists/inhibitors, are agents that alter chemical and biological activities or properties of a CAT protein. Methods for identifying modulators involve assaying a reduced or enhanced level or quality of CAT function in the presence of one or more test agents as compared to a control level. Representative CAT modulators include excess or variant CAT proteins that mimic CAT activity or that elicit a dominant negative phenotype, i.e., antagonism of CAT function. Additional molecules which comprise CAT modulators are described herein below under the subheading "Test Agents." A control level or quality of CAT activity refers to a level or quality of wild type CAT activity, for example, when using a recombinant expression system comprising expression of a CAT protein. When evaluating the modulating capacity of a test agent, a control level or quality of CAT activity comprises a level or quality of activity in the absence of the test agent.

Significantly changed activity of a CAT protein refers to a quantifiable change in a measurable quality that is larger than the margin of error inherent in the measurement technique. For example, significant inhibition refers to CAT activity that is reduced by about 2-fold or greater relative to a control measurement, or an about 5-fold or greater reduction, or an about 10-fold or greater reduction. Similarly, significant activation or agonism refers to CAT activity that is enhanced by about 2-fold or greater relative to a control measurement, or an about 5-fold or greater enhancement, or an about 10-fold or greater enhancement.

An assay of CAT function may comprise determining a level of CAT gene expression; determining binding activity of a recombinantly expressed CAT protein; determining an active conformation of a CAT protein; determining activation of signaling events in response to binding of a CAT modulator; determining transport activity in response to binding of a CAT modulator, or determining a change in pain sensation.

In accordance with the present invention there is also provided a rapid and high throughput screening method that relies on the methods described herein. This screening method comprises separately contacting a CAT protein with a plurality of test agents. In such a screening method the plurality of target agents preferably comprises more than about 10⁴ samples, or more preferably comprises more than about 10⁵ samples, and still more preferably more than about 10⁶ samples.

The in vitro and cellular assays of the invention may comprise soluble assays, or may further comprise a solid phase substrate for immobilizing one or more components of the assay. For example, a CAT protein, or a cell expressing a CAT protein, may be bound directly to a solid state component via a covalent or non-covalent linkage. Optionally, the binding may include a linker molecule or tag that mediates indirect binding of a CAT protein to a substrate. LA. Test Agents

A test agent refers to any agent that potentially interacts with a CAT protein or other component of CAT-regulated NO production, including any synthetic, recombinant, or natural product or composition. A test agent suspected to interact with a CAT protein, or other component of CAT- regulated NO production, may be evaluated for such an interaction using the methods disclosed herein.

Representative test agents include but are not limited to peptides, proteins, nucleic acids (e.g., aptamers), small molecules (e.g., chemical compounds), antibodies or fragments thereof, nucleic acid-protein fusions, any other affinity agent, and combinations thereof. A test agent may be a purified molecule, a homogenous sample, or a mixture of molecules or compounds.

A small molecule refers to a compound, for example an organic compound, with a molecular weight of less than about 1 ,000 daltons, more preferably less than about 750 daltons, still more preferably less than about 600 daltons, and still more preferably less than about 500 daltons. A small molecule also preferably has a computed log octanol-water partition coefficient in the range of about -4 to about +14, more preferably in the range of about -2 to about +7.5.

Test agents may be obtained or prepared as a library or collection of molecules. A library may contain a few or a large number of different molecules, varying from about ten molecules to several billion molecules or more. A plurality of test agents in a library may be assayed simultaneously. Optionally, test agents derived from different libraries may be pooled for simultaneous evaluation.

Representative libraries include but are not limited to a peptide library (U.S. Patent Nos. 6,156,51 1 , 6,107,059, 5,922,545, and 5,223,409), an oligomer library (U.S. Patent Nos. 5,650,489 and 5,858,670), an aptamer library (U.S. Patents No. 6,180,348 and 5,756,291 ), a small molecule library (U.S. Patent Nos. 6,168,912 and 5,738,996), a library of antibodies or antibody fragments (U.S. Patent Nos. 6,174,708, 6,057,098, 5,922,254, 5,840,479, 5,780,225, 5,702,892, and 5,667988), a library of nucleic acid- protein fusions (U.S. Patent No. 6,214,553), and a library of any other affinity agent that may potentially bind to a CAT protein (e.g., U.S. Patent Nos. 5,948,635, 5,747,334, and 5,498,538).

A library may comprise a random collection of molecules. Alternatively, a library may comprise a collection of molecules having a bias for a particular sequence, structure, or conformation. See e.g., U.S. Patent Nos. 5,264,563 and 5,824,483. Methods for preparing libraries containing diverse populations of various types of molecules are known in the art, for example as described in U.S. Patents cited herein above. Numerous libraries are also commercially available.

Test agents also include antibodies capable of specifically binding cationic amino acid transporters, such as, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, human monoclonal antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein, Nature, 1975, 256:495-497; and U.S. Patent No. 4,376,1 10), the human B-cell hybridoma technique (Kosbor et al., Immunology Today, 1983, 4:72; Cote et al., Proc. Natl. Acad. Sci. USA, 1983, 80:2026-2030), and the EBV-hybridoma technique (Cole et al., in Monoclonal Antibodies And Cancer Therapy, 1995, Alan R. Liss, Inc., New York, pp. 77- 96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Hybridomas producing monoclonal antibodies may be cultivated in vitro or in vivo.

A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies may be produced (Morrison et al., Proc. Natl. Acad. Sci. USA, 1984, 81 :6851-6855; Takeda et al., Nature, 1985, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity.

A humanized antibody is a type of chimeric antibody, wherein variable region residues responsible for antigen binding (i.e., residues of a complementarity determining region and any other residues that participate in antigen binding) are derived from a non-human species, while the remaining variable region residues (i.e., residues of the framework regions) and constant regions are derived, at least in part, from human antibody sequences. Residues of the variable regions and constant regions of a humanized antibody may also be derived from non-human sources. Variable regions of a humanized antibody are also described as humanized (i.e., a humanized light or heavy chain variable region). The non-human species is typically that used for immunization with antigen, such as mouse, rat, rabbit, non-human primate, or other non-human mammalian species. Humanized antibodies may be prepared using any one of a variety of methods including veneering, grafting of complementarity determining regions (CDRs), grafting of abbreviated CDRs, grafting of specificity determining regions (SDRs), and Frankenstein assembly, as described below. These general approaches may be combined with standard mutagenesis and synthesis techniques to produce an anti-CAT antibody of any desired sequence.

Test antibodies may also be human monoclonal antibodies, for example those produced by immortalized human cells, by SCID-hu mice or other non-human animals capable of producing human antibodies. Human antibodies may also be isolated from antibody phage libraries, for example, as described by Marks et al., J. MoI. Biol.. 1991 , 222:581 -597. Chain shuffling and recombination techniques may be used to produce phage libraries having increased antibody diversity, e.g., libraries including antibodies with increased binding affinity. See Marks et al., Biotechnology, 1992, 10:779-783 and Waterhouse et al., Nuc. Acids. Res., 1993, 21 :2265-2266.

Test antibodies can also comprise single chain antibodies, which are typically formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. See e.g., U.S. Patent No. 4,946,778; Bird, Science, 1988, 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85:5879-83; and Ward et al., Nature, 1989, 334:544-546).

Antibody fragments that recognize specific CAT epitopes may be generated by known techniques. For example, such fragments include F(ab')₂ fragments that can be produced by pepsin digestion of the antibody molecule and Fab fragments that can be generated by reducing the disulfide bridges of the F(ab') ₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., Science, 1989, 246:1275-1281 ) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Specific binding of an antibody or antibody fragment to a CAT protein (or other component of CAT-regulated NO production) refers to preferential binding of an antibody to a CAT protein in a heterogeneous sample comprising multiple different antigens. Substantially lacking binding describes a level of binding of an antibody to a control protein or sample, i.e., a level of binding characterized as non-specific or background binding. The binding of an antibody to an antigen is specific if the binding affinity is at least about 10^"7 M or higher, such as at least about 10^"8 M or higher, including at least about 10^"9 M or higher, at least about 10^"11 M or higher, or at least about 10^"12 M or higher.

Test antibodies described herein above may be recombinantly prepared using standard techniques. See e.g., Harlow & Lane, Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York and U.S. Patent Nos. 4,196,265; 4,946,778; 5,091 ,513; 5,132,405; 5,260,203; 5,677,427; 5,892,019; 5,985,279; 6,054,561. Tetravalent antibodies (H₄L₄) comprising two intact tetramehc antibodies, including homodimers and heterodimers, may be prepared, for example, as described in PCT International Publication No. WO 02/096948. Antibody dimers may also be prepared via introduction of cysteine residue(s) in the antibody constant region, which promote interchain disulfide bond formation, using heterobifunctional cross-linkers (Wolff et al., Cancer Res., 1993, 53: 2560-2565), or by recombinant production to include a dual constant region (Stevenson et al., Anticancer Drug Pes.. 1989, 3: 219-230). Other agents that may be used as test agents include the CAT gene, its expression product(s) and functional fragments thereof. Such agents include antisense, hbozyme, and triple helix molecules. Techniques for the production and use of such molecules are well known to those of skill in the art.

Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the -10 and +10 regions of the CAT gene nucleotide sequence of interest, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of hbozyme action involves sequence-specific hybridization of the hbozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. The composition of hbozyme molecules must include one or more sequences complementary to the CAT mRNA, and should include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference herein in its entirety. As such within the scope of the invention are engineered hammerhead motif hbozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding CAT proteins.

Specific hbozyme cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for hbozyme cleavage sites that include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the CAT gene containing the cleavage site may be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence unsuitable. The suitability of candidate sequences may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription should be single stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyhmidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-hch molecules provide base complementarity to a purine-hch region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen that are purine-hch, for example, containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

It is possible that the antisense, ribozyme, and/or triple helix molecules described herein may reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by both normal and mutant CAT gene alleles. In order to ensure that substantially normal levels of CAT gene activity are maintained, nucleic acid molecules that encode and express CAT gene polypeptides exhibiting normal activity may be introduced into cells that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized. Alternatively, it may be preferable to co-administer normal CAT protein into the cell or tissue in order to maintain the requisite level of cellular or tissue CAT activity.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyhbonucleotides and oligohbonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Various well-known modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyhbonucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyhbonucleotide backbone.

I.B. Binding Assays

In one aspect of the invention, a method for identifying a CAT modulator comprises determining specific binding of a test agent to a CAT protein. For example, a method for identifying a CAT binding partner may comprise: (a) providing a CAT protein; (b) providing a one or more test agents to the CAT protein under conditions sufficient for binding; (c) assaying binding of a test agent to the isolated CAT protein; and (d) selecting a test agent that demonstrates specific binding to the CAT protein.

Specific binding refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biological materials. The binding of a test agent to a CAT protein may be considered specific if the binding affinity is about 1 x10⁴M^"1 to about 1x10⁶M^"1 or greater. Specific binding also refers to saturable binding. To demonstrate saturable binding of a test agent to a CAT protein, Scatchard analysis may be carried out as described, for example, by Mak et al. J. Biol. Chem., 1989, 264:21613-21618. Specific binding may also encompass a quality or state of mutual action such that binding of a test agent to a CAT protein is modulatory.

The principle of the assays used to identify compounds that bind to the CAT protein involves preparing a reaction mixture of the CAT protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways, including liquid or solid phase reaction formats.

For example, one method to conduct such an assay would involve anchoring the CAT protein or the test substance onto a solid phase and detecting target protein/test substance complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, CAT may be anchored onto a solid surface, and a test compound, which is not anchored, may be labeled, either directly or indirectly.

An anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored. To test for specific binding, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre- labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously non- immobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-lg antibody).

A binding assay may also be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected, e.g., using an immobilized antibody specific for CAT gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes. In addition to the above-mentioned techniques, which require some knowledge of the characteristics of the candidate binding agent, several techniques may be used to detect interactions between a CAT protein and an unknown test agent. Representative methods include, but are not limited to, Fluorescence Correlation Spectroscopy, Surface-Enhanced Laser Desorption/lonization Time-Of-Flight Spectroscopy, and BIACORE® technology, each technique described herein below. These methods are amenable to automated, high-throughput screening.

Fluorescence Correlation Spectroscopy (FCS) measures the average diffusion rate of a fluorescent molecule within a small sample volume (Tallgren, 1980). The sample size may be as low as 10³ fluorescent molecules and the sample volume as low as the cytoplasm of a single bacterium. The diffusion rate is a function of the mass of the molecule and decreases as the mass increases. FCS may therefore be applied to protein- ligand interaction analysis by measuring the change in mass and therefore in diffusion rate of a molecule upon binding. In a typical experiment, the target to be analyzed (e.g., a CAT protein) is expressed as a recombinant protein with a sequence tag, such as a poly-histidine sequence, inserted at the N- terminus or C-terminus. The expression is mediated in a host cell, such as E.coli, yeast, Xenopus oocytes, or mammalian cells. The protein is purified using chromatographic methods. For example, the poly-histidine tag may be used to bind the expressed protein to a metal chelate column such as Ni²⁺ chelated on iminodiacetic acid agarose. The protein is then labeled with a fluorescent tag such as carboxytetramethylrhodamine or BODI PY™ reagent (available from Molecular Probes of Eugene, Oregon). The protein is then exposed in solution to the potential ligand, and its diffusion rate is determined by FCS using instrumentation available from Carl Zeiss, Inc. (Thornwood, New York). Ligand binding is determined by changes in the diffusion rate of the protein.

Surface-Enhanced Laser Desorption/lonization (SELDI) was developed by Hutchens & Yip, Rapid Commun. Mass Spectrom., 1993, 7:576-580. When coupled to a time-of-flight mass spectrometer (TOF), SELDI provides a technique to rapidly analyze molecules retained on a chip. It may be applied to ligand-protein interaction analysis by covalently binding the target protein, or portion thereof, on the chip and analyzing by mass spectrometry the small molecules that bind to this protein (Worrall et al., 1998). In a typical experiment, a target protein (e.g., a CAT protein) is recombinantly expressed and purified. The target protein is bound to a SELDI chip either by utilizing a poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction. A chip thus prepared is then exposed to the potential ligand via, for example, a delivery system able to pipet the ligands in a sequential manner (autosampler). The chip is then washed in solutions of increasing stringency, for example a series of washes with buffer solutions containing an increasing ionic strength. After each wash, the bound material is analyzed by submitting the chip to SELDI-TOF. Ligands that specifically bind a target protein are identified by the stringency of the wash needed to elute them.

BIACORE® relies on changes in the refractive index at the surface layer upon binding of a ligand to a target protein (e.g., a CAT protein) immobilized on the layer. In this system, a collection of small ligands is injected sequentially in a 2-5 microliter cell, wherein the target protein is immobilized within the cell. Binding is detected by surface plasmon resonance (SPR) by recording laser light refracting from the surface. In general, the refractive index change for a given change of mass concentration at the surface layer is practically the same for all proteins and peptides, allowing a single method to be applicable for any protein (Liedberg et al., 1983; Malmquist, 1993). In a typical experiment, a target protein is recombinantly expressed, purified, and bound to a BIACORE® chip. Binding may be facilitated by utilizing a poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction. A chip thus prepared is then exposed to one or more potential ligands via the delivery system incorporated in the instruments sold by Biacore (Uppsala, Sweden) to pipet the ligands in a sequential manner (autosampler). The SPR signal on the chip is recorded and changes in the refractive index indicate an interaction between the immobilized target and the ligand. Analysis of the signal kinetics of on rate and off rate allows the discrimination between non-specific and specific interaction. See also Homola et al., Sensors and Actuators, 1999, 54:3-15 and references therein. I.C. Functional Assays

An assay of CAT activity may further comprise determining activation of signaling events in response to binding of a CAT modulator or determining transport activity in the presence of a CAT modulator. For example, agents may be tested for modulation of CAT function by assaying NO production or other CAT-regulated activity in cells expressing a functional CAT protein. Test agents may also be assessed in animal models subjected to experimental pain conditions. Representative methods for determining test agents that function as CAT antagonists are described in Examples 3-6.

As one example, a method for identifying a CAT inhibitor may comprise (a) providing a CAT protein under conditions for production of NO; (b) providing one or more test agents or a control agent to the CAT protein (a); (c) allowing sufficient time for the production of NO; (d) assaying for the production of NO; and (e) selecting a test agent that shows (i) diminished NO production in the presence of the agent as compared to a control agent.

I.C.1. Recombinant Systems for CAT Expression

To conduct cell-based assays of CAT function, the present invention further provides systems for expression of a recombinant CAT protein, which may be used for identification of CAT modulators. An expression system refers to a host cell comprising a heterologous nucleic acid and the protein encoded by the heterologous nucleic acid. For example, a heterologous expression system may comprise a host cell transfected with a construct comprising a recombinant CAT nucleic acid, a host cell transfected with CAT cRNA, a host cell transfecting with a construct comprising a vector and a nucleic acid molecule encoding a CAT protein operatively linked to a promoter, or a cell line produced by introduction of heterologous nucleic acids into a host cell genome. The system may further comprise one or more additional heterologous nucleic acids relevant to CAT function, such as components of iNOS signaling. These additional nucleic acids may be expressed as a single construct or multiple constructs. Isolated proteins and recombinantly produced proteins may be purified and characterized using a variety of standard techniques that are known to the skilled artisan. See e.g., Schroder & Lϋbke, The Peptides, 1965, Academic Press, New York; Schneider & Eberle, Peptides, 1992: Proceedings of the Twenty-Second European Peptide Symposium, September 13-19, 1992, Interlaken, Switzerland. Escom, Leiden; Bodanszky, Principles of Peptide Synthesis, 1993, 2nd rev. ed. Springer-Verlag, Berlin/ New York; Ausubel (ed.), Short Protocols in Molecular Biology, 1995, 3rd ed. Wiley, New York.

A construct for expression of a CAT protein may include a vector and a CAT nucleotide sequence, wherein the CAT nucleotide sequence is operatively linked to a promoter sequence. A construct for recombinant CAT expression may also comprise transcription termination signals and sequences required for proper translation of the nucleotide sequence. Preparation of an expression construct, including addition of translation and termination signal sequences, is known to one skilled in the art.

Recombinant production of a CAT protein may be directed by a constitutive promoter or an inducible promoter. Representative promoters that may be used in accordance with the present invention include Simian virus 40 early promoter, a long terminal repeat promoter from retrovirus, an actin promoter, a heat shock promoter, and a metallothien protein.

Suitable vectors that may be used to express a CAT protein include but are not limited to viruses such as vaccinia virus or adenovirus, baculovirus vectors, yeast vectors, bacteriophage vectors (e.g., lambda phage), plasmid and cosmid DNA vectors, transposon-mediated transformation vectors, and derivatives thereof.

Constructs are introduced into a host cell using a transfection method compatible with the vector employed. Standard transfection methods include electroporation, DEAE-Dextran transfection, calcium phosphate precipitation, liposome-mediated transfection, transposon-mediated transformation, infection using a retrovirus, particle-mediated gene transfer, hyper-velocity gene transfer, and combinations thereof.

Host cells are cells into which a heterologous nucleic acid molecule may be introduced. Representative host cells include eukaryotic hosts such as mammalian cells (e.g., HEK-293 cells, HeLa cells, CV-1 cells, COS cells), amphibian cells (e.g., Xenopus oocytes), insect cells (e.g., Sf9 cells), as well as prokaryotic hosts such as E.coli and Bacillus subtilis. Preferred host cells for functional assays that employ a heterologous expression system substantially or completely lack endogenous expression of a CAT protein.

A host cell strain may be chosen which modulates the expression of the recombinant sequence, or modifies and processes the gene product in a specific fashion desired. For example, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins). Appropriate cell lines or host cells may be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system may be used to produce a non-glycosylated core protein product, and expression in yeast will produce a glycosylated product.

Assays employing cells that express recombinant CAT may additionally employ control cells that are substantially devoid of native CAT and proteins substantially similar to a CAT protein. When using transiently transfected cells, a control cell may comprise, for example, an untransfected host cell. When using a stable cell line expressing a CAT protein, a control cell may comprise, for example, a parent cell line used to derive the CAT-expressing cell line.

Assays of CAT activity that employ transiently transfected cells may include a marker that distinguishes transfected cells from non-transfected cells. A marker may be encoded by or otherwise associated with a construct for CAT expression, such that cells are simultaneously transfected with a nucleic acid molecule encoding CAT and the marker. Representative detectable molecules that are useful as markers include but are not limited to a heterologous nucleic acid, a protein encoded by a transfected construct (e.g., an enzyme or a fluorescent protein), a binding protein, and an antigen.

The present invention further encompasses recombinant expression of a CAT protein in a stable cell line. Methods for generating a stable cell line following transformation of a heterologous construct into a host cell are known in the art. See e.g., Joyner, Gene Targeting: A Practical Approach, 1993, Oxford University Press, Oxford/New York. Thus, transformed cells, tissues, or non-human organisms are understood to encompass not only the end product of a transformation process, but also transgenic progeny or propagated forms thereof.

Transiently transfected cells and cells of a stable cell line expressing CAT may be frozen and stored for later use. Frozen cells may be readily transported for use at a remote location. Methods for preparation and handling of frozen cells may be found in Freshney, Culture of Animal Cells: A Manual of Basic Technique, 1987, 2nd ed. A.R. Liss, New York and in U.S. Patent Nos. 6,176,089; 6,140,123; 5,629,145; and 4,455,842; among other places.

I.C.2. Transgenic Animals

The present invention also provides transgenic animals comprising a disruption of a CAT locus, for example a CAT-2 locus as described by Nicholson et al., J. Biol. Chem., 2001 , 276(19): 15881 -15885, herein incorporated by reference in its entirety. A disrupted gene may result in an altered level of a CAT gene or expression of a mutated variant of a CAT gene. Mice with complete or partial functional inactivation of the CAT gene in all somatic cells may be generated using standard techniques of site-specific recombination in embryonic stem cells. See Capecchi, Science, 1989, 244:1288-1292; Thomas & Capecchi, Nature. 1990, 346:847-850; and Delpire et al.. Nat Genet. 1999, 22:192-195.

A transgenic animal in accordance with the present invention may also be prepared using anti-sense or hbozyme CAT constructs, driven by a universal or tissue-specific promoter, to reduce levels of CAT gene expression in somatic cells, thus achieving a "knock-down" phenotype. The present invention also provides the generation of transgenic animals having conditional or inducible inactivation of CAT. Such strains may also comprise additional synthetic or naturally occurring mutations, for example a mutation in upstream or downstream signaling components of CAT-regulated NO production. The present invention also provides transgenic animals with specific "knocked-in" modifications in the disclosed CAT gene, for example to create an over-expression or dominant negative phenotype. Thus, "knocked-in" modifications include the expression of both wild type and mutated forms of a nucleic acid encoding a CAT protein. Knock-in transgenic organisms may be made in any relevant species.

Techniques for the preparation of transgenic animals are known in the art. Exemplary techniques are described in U.S. Patent No. 5,489,742 (transgenic rats); U.S. Patent Nos. 4,736,866, 5,550,316, 5,614,396, 5,625,125 and 5,648,061 (transgenic mice); U.S. Patent No. 5,573,933 (transgenic pigs); 5,162,215 (transgenic avian species) and U.S. Patent No. 5,741 ,957 (transgenic bovine species), the entire contents of each of which are herein incorporated by reference.

CAT modulators can be identified by assessing pain responses in wild type and transgenic animals following administration of a test agent. An altered pain response includes any change in pain sensation that can be objectively measured (or observed). Behavioral indices of changes in pain sensation include ataxia, rapid limb movement, eye movement, breathing, motor activity, emotional behaviors, social behaviors, hyperactivity, hypersensitivity, anxiety, impaired learning, abnormal reward behavior, and abnormal social interaction, such as aggression. Relevant behavioral indices are described in Examples 1-5.

ID. Conformational Assays

The present invention also provides methods for identifying CAT binding partners and modulators that rely on a conformational change of a CAT protein when bound by or otherwise interacting with a test agent. For example, application of circular dichroism to solutions of macromolecules reveals the conformational states of these macromolecules. The technique may distinguish random coil, alpha helix, and beta chain conformational states.

To identify modulators of a CAT protein, circular dichroism analysis may be performed using a recombinantly expressed CAT protein. A CAT protein is purified, for example by ion exchange and size exclusion chromatography, and mixed with a test agent. The mixture is subjected to circular dichroism. The conformation of a CAT protein in the presence of a test agent is compared to a conformation of a CAT protein in the absence of the test agent. A change in conformational state of a CAT protein in the presence of a test agent identifies a CAT binding partner or modulator. Representative methods are described in U.S. Patent Nos. 5,776,859 and 5,780,242. Agonistic or antagonistic activity of the modulator may be assessed using functional assays, such as, NO, citrulline, nitrite or nitrate synthesis.

I.E. Rational Design

The knowledge of the structure of a native CAT protein provides an approach for rational design of CAT inhibitors. In brief, the structure of a CAT protein may be determined by X-ray crystallography and/or by computational algorithms that generate three-dimensional representations. See Saqi et al., Bioinformatics, 1999, 15:521 -522; Huang et al., Pac. Svmp. Biocomput, 2000, 230-241 ; and PCT International Publication No. WO 99/26966. Alternatively, a working model of a CAT protein structure may be derived by homology modeling (Maalouf et al., 1998). Computer models may further predict binding of a protein structure to various substrate molecules that may be synthesized and tested using the assays described herein above. Additional compound design techniques are described in U.S. Patent Nos. 5,834,228 and 5,872,01 1.

A CAT protein may be isolated using standard biochemical techniques, and the resulting CAT protein is of sufficient purity and concentration for crystallization. The purified CAT protein may be crystallized under varying conditions of at least one of the following: pH, buffer type, buffer concentration, salt type, polymer type, polymer concentration, other precipitating ligands, and concentration of purified CAT. Methods for generating a crystalline protein are known in the art and may be reasonably adapted for determination of a CAT protein as disclosed herein. See e.g., Deisenhofer et al. J^ MoI. Biol.. 1984, 180:385-398; Weiss et al., FEBS Lett. 1990 267:268-272; or the methods provided in a commercial kit, such as the CRYSTAL SCREEN™ kit (available from Hampton Research of Riverside, California, United States of America).

A crystallized CAT protein may be tested for functional activity and differently sized and shaped crystals are further tested for suitability in X-ray diffraction. Generally, larger crystals provide better crystallography than smaller crystals, and thicker crystals provide better crystallography than thinner crystals. Preferably, CAT crystals range in size from 0.1-1.5 mm. These crystals diffract X-rays to at least 10 A resolution, such as 1.5-10.0 A or any range of value therein, such as 1 .5, 1.6, 1.7, 1.8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5 or 3, with 3.5 A or less being preferred for the highest resolution.

|L, Therapeutic Applications

CAT binding partners and CAT modulators identified by the methods disclosed herein are useful for applications specifically related to pain inhibition and for applications related to enhancement of pain sensation as needed to treat congenital insensitivity to pain. Nociceptive sensory neurons that may be subject modulators of CAT and CAT-regulated NO production include injured neurons, neurons at risk of dying, or any other sensory neuron for which pain modulation is desired, including neurons of the central nervous system or the peripheral nervous system. For in vivo applications employing CAT inhibitors, the subject may have suffered a condition associated with pain or heightened sensitivity to pain, including but not limited to tissue trauma, toothache, muscle ache, spondylolysis, neuropathic pain, inflammatory pain, musculoskeletal pain, bony pain, lumbosacral pain, neck or upper back pain, visceral pain, somatic pain, neuropathic pain, cancer pain, pain caused by injury or surgery such as burn pain or dental pain, headaches such as migraines, tension headaches, or combinations of these conditions. One skilled in the art will recognize that these pain conditions may overlap one another. For example, a pain condition caused by inflammation may also be visceral or musculoskeletal in nature. A pain modulator identified by the methods disclosed herein can be administered to a subject at a therapeutically effective dose. A therapeutically effective dose refers to an amount of compound sufficient to result in amelioration of symptoms of the disease or condition for which treatment is sought.

Toxicity and therapeutic efficacy of pain modulators can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Pain modulators that exhibit large therapeutic indices are preferred. While pain modulators which exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell-based assays and animal studies, usually in rodents, rabbits, dogs, pigs, and/or primates, can be used in formulating a range of dosage for use in humans. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans. Typically a minimal dose is administered, and the dose is escalated in the absence of dose-limiting cytotoxicity. Determination and adjustment of an effective amount or dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art of medicine.

The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any pain modulator used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ {i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compositions and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, topical, subcutaneous, intraperitoneal, intraveneous, intrapleural, intraoccular, intraarterial, rectal administration, or within/on implants, e.g., matrices such as collagen fibers or protein polymers, via cell bombardment, in osmotic pumps, grafts comprising appropriately transformed cells, etc. It is also contemplated that pharmaceutical compositions may be administered with other products that potentiate the activity of a pain modulator and optionally, may include other therapeutic ingredients.

A variety of techniques are available for promoting transfer of therapeutic CAT modulators across the blood brain barrier including disruption by surgery or injection, drugs which transiently open adhesion contact between CNS vasculature endothelial cells, and compounds which facilitate translocation through such cells.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Pharmaceutical compositions may also include various buffers (e.g., Tris, acetate, phosphate), solubilizers (e.g., TWEEN®, Polysorbate), carriers such as human serum albumin, preservatives (thimerosol, benzyl alcohol) and anti-oxidants such as ascorbic acid in order to stabilize pharmaceutical activity. The stabilizing agent may be a detergent, such as TWEENO-20, TWEENΘ-80, NP-40 or TRITON-XΘ-100. EBP may also be incorporated into particulate preparations of polymeric compounds for controlled delivery to a patient over an extended period of time. A more extensive survey of components in pharmaceutical compositions is found in Remington's Pharmaceutical Sciences, 1990, 18th ed., A. R. Gennaro, ed., Mack Publishing, Easton, Pa.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

CAT modulators of the invention may be administered as an initial treatment or for treatment of conditions that are unresponsive to conventional pain therapies. In addition, CAT modulators as identified herein may be used in combination with other therapies to thereby elicit additive or potentiated therapeutic effects. CAT modulators may be co-formulated with additional agents, or formulated for consecutive administration prior to or after administration of one or more additional agents.

Representative agents useful for combination therapy include, for example, opiod receptors agonists, (i.e., opiates, such as codeine, oxycodeine, hydromorphone, diamorphine, methadone, fentayl, sufentanil, buprenorphine, meperidine, DEMEROL®), non-steroidal anti-inflammatory drugs, such as cyclooxygenase inhibitors, N-methyl-D-aspartate antagonists (e.g., ketamine and agmatine), D₂-adrenergic agonists (e.g., clonidine), leukemia inhibitory factor, heme oxygenase antagonists, vallinoid receptor antagonists (e.g., capsacin and lidocaine), anesthetics, etc. Agents with little role in pain treatment may also be used to promote efficacy of CAT modulators, for example, antidepressants and antiepileptics.

For combination therapies, a CAT modulator and one or more additional therapeutic agents are administered within any time frame suitable for performance of the intended therapy. Thus, the single agents may be administered substantially simultaneously (i.e., as a single formulation or within minutes or hours) or consecutively in any order. For example, single agent treatments may be administered within about 1 year of each other, such as within about 10, 8, 6, 4, or 2 months, or within 4, 3, 2 or 1 week(s), or within about 5, 4, 3, 2 or 1 day(s). The administration of the CAT modulator and a second therapeutic agent preferably elicits greater pain reduction than administration of either alone.

For additional guidance regarding formulation, dose, administration regimen, and measurable therapeutic outcomes, see Berkow et al., The Merck Manual of Medical Information, 2000, Merck & Co., Inc., Whitehouse Station, New Jersey; Ebadi, CRC Desk Reference of Clinical Pharmacology, 1998, CRC Press, Boca Raton, Florida; Gennaro, Remington: The Science and Practice of Pharmacy, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pennsylvania; Katzung, Basic & Clinical Pharmacology, 2001 , Lange Medical Books / McGraw-Hill Medical Pub. Div., New York; Hardman et al., Goodman & Gilman's the Pharmacological Basis of Therapeutics, 2001 , The McGraw- Hill Companies, Columbus, Ohio; Speight & Holford, Averv's Drug Treatment: A Guide to the Properties, Choices, Therapeutic Use and Economic Value of Drugs in Disease Management, 1997, Lippincott, Williams, & Wilkins, Philadelphia, Pennsylvania.

Throughout this application, various publications, patents and published patent applications are referred to by an identifying citation. The disclosures of these publications, patents and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

The following examples have been included to illustrate modes of the invention. Certain aspects of the following examples are described in terms of techniques and procedures found or contemplated by the present co- inventors to work well in the practice of the invention. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations may be employed without departing from the scope of the invention.

EXAMPLES

Example 1

CAT-2 Knockout Mice Withstand Acute Visceral Pain

Acute visceral pain was assessed in both wild type and knockout mice using the abdominal stretch (writhing) assay. A significant decrease in the number of abdominal stretches was indicative to be anti-nociceptive.

Male and female CAT-2 knockout mice and age matched C57B6 wild type mice (20-25 g, Charles River) were housed in groups of five on bedding in a climate controlled room on a 12 hour light/dark cycle with food and water available ad libitum.

Mice were injected intraperitoneal^ (i.p.) with 2 mg/kg of parapheylquinone (PPQ) dissolved in 4% ethanol in distilled water. After PPQ administration, the mice were individually placed in a Plexiglass cage. The total number of abdominal stretches was recorded for 1 -minute periods, beginning 5 and 10 minutes after PPQ injection (n = 10/group).

A one-way ANOVA was performed to determine statistical significance using a customized SAS-excel application (SAS Institute, Cary, NC). The criterion for significant differences was p<0.05.

As shown in Figures 1A-1 B, both female (Figure 1A) and male (Figure 1 B) CAT-2 knockout mice demonstrated a statistically significant decrease in the number of abdominal stretches observed compared to the CAT-2 wild type mice. These results indicate that the CAT-2 knockout mice are capable of withstanding acute visceral pain greater than wild type mice.

Example 2

CAT-2 Knockout Mice Withstand Acute Inflammatory Pain

Acute inflammatory pain was assessed in both wild type and knockout mice by carageenan injection and measuring latency of response using an infrared photobeam as a radiant heat source (ITTC, Woodland Hills, CA). The heat source was aimed at the plantar surface of the left hind paw (Hargreaves test). An increase in the latency of withdrawal was indicative to be antinociceptive.

Male and female CAT-2 knockout mice and age matched C57B6 wild type mice (20-25 g, Charles River) were housed in groups of five on bedding in a climate controlled room on a 12-hour light/dark cycle with food and water available ad libitum.

Baseline latencies were taken prior to carrageenan administration following a 30 minute habituation period. Twenty-four hours after baseline testing animals were anesthetized with 4% isoflurane/nitrogen anesthesia and received an intraplantar injection of 2% carrageenan (20 μl volume) into the left hind paw. Animals are placed back into test chambers and paw withdrawal latencies were assessed 1 , 3 and 5 hours later (n=10-1 1/group). A cut off latency of 20 seconds was used to avoid tissue damage. The time that elapsed between placement and the first avoidance response of either hind limb, such as licking or rapid shaking, was recorded as the latency of response. A total of three readings were taken for each mouse at each time point and the average of these readings was determined and used for subsequent analyses.

As shown in Figures 2A-2B, both female (Figure 2A) and male (Figure 2B) mice showed a statistically significant increase in the latency response in the hot plate assay 5 hours after caregeenan-induced acute inflammation. The male CAT-2 knockout mice also demonstrated a statistically significant increase in latency of response at the 1 and 3 hour time points relative to their wild type counterparts. While the female CAT-2 knockout mice demonstrated an increase in the latency of response at the 1 and 3 hour time points, the increases observed were not statistically significant (p ≥ 0.05).

Example 3

Identification of Modulators of Acute Pain

Wild type mice are treated with a test agent or a placebo, and latency of response is measured when animals are subjected to a tail-flick assay or PPQ-induced visceral pain assay (see Examples 1-2). Baseline latencies are taken prior to administration of a test agent or placebo. Twenty-four hours after baseline testing, a test agent or placebo is administered, followed by assessment of pain sensation. CAT antagonists which alleviate pain are identified as test agents that alleviate pain sensation, as observed in CAT-2 deficient mice.

Example 4

Identification of Modulators of Neuropathic Pain

To assess the ability of test agents to alleviate neuropathic pain, wild type mice are treated with a test agent or a placebo, and latency of response is measured when animals are subjected to a chronic constriction injury. Baseline latencies are taken prior to administration of a test agent or placebo. Twenty-four hours after baseline testing, a test agent or placebo is administered, followed by assessment of pain sensation. Mice are anesthetized and the common sciatic nerve of the right hind limb is exposed at the level of the middle of the thigh by blunt dissection through biceps femoris. Proximal to the trifurcation, the nerve is freed of the adhering tissue and ligatures are tied loosely around the nerve. The degree of constriction is such that it could retard but not arrest circulation. The incision is then closed in layers. In sham-operated mice, an identical surgery is performed except the sciatic nerve is not ligated. After habituation, a radiant heat source is focused onto the plantar surface of the animal's right hind paw. Latency to withdraw its paw from the heat is recorded. To minimize tissue damage, a cut off latency is used.

Example 5

Identification of Modulators of Cold Allodvnia

To assess the ability of test agents to alleviate neuropathic pain, wild type mice are treated with a test agent or a placebo, and latency of response is measured when animals are subjected to a chronic constriction injury. Baseline latencies are taken prior to administration of a test agent or placebo. Twenty-four hours after baseline testing, a test agent or placebo is administered, followed by assessment of pain sensation. Cold allodynia, the detection of pain resulting from cold stimuli, is assessed in both wild type and knockout mice by submerging the paw of the mice into ice cool water (4 ±1 ⁰C). An increase in the latency to withdraw is indicative to be anti-nociceptive.

Example 6

Identification of CAT Antagonists Using si RNA

To assess the pain-inhibitory activity of test agents comprising single- stranded interfering RNAs (siRNA), wild type mice are treated with siRNA or a placebo, and latency of response is measured when animals are subjected to a chronic constriction injury. Baseline latencies are taken prior to administration of a test agent or placebo. Twenty-four hours after baseline testing, an siRNA or placebo is administered, followed by assessment of pain sensation. Test agents comprising siRNAs are designed using criteria set out by Elbashir et al., 2001 , Nature, 41 1 (6836):494-498. Selected sequences are subjected to a BLAST search to ensure no significant homology with other genes. A representative sense siRNA template includes an AA dimer at the 5' end followed by an about 10-20 nucleotide complementary to the target sequence. The 3' end of both the sense and anti-sense templates includes an eight nucleotide sequence corresponding to the complementary sequence of the T7 promoter primer required for efficient transcription of the siRNA. Deprotected and desalted oligonucleotide templates and control scrambled sequences are chemically synthesized. For transfection of siRNAs into cultured cells, LIPOFECTAMINE® is used according to the vendor's instructions. Following siRNA-mediated knockdown, an amount of NO production is measured.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying a pain modulator, the method comprising the steps of:

(a) providing a cationic amino acid transporter (CAT) protein;

(b) providing one or more test agents to the cationic amino acid transporter (CAT) protein under conditions sufficient for binding;

(c) assaying binding of said one or more test agents to the cationic amino acid transporter (CAT) protein; and

(d) selecting a test agent which demonstrates specific binding to the cationic amino acid transporter (CAT) protein, and which modulates pain sensation.

2. The method of claim 1 , wherein the cationic amino acid transporter (CAT) protein is a CAT-2 protein.

3. The method of claim 1 or 2, wherein said one or more test agents is a peptide, a protein, an oligomer, a nucleic acid, a small molecule, or an antibody.

4. A pain modulator identified as a test agent that shows specific binding to a CAT protein according to the method of any one of claims 1 to 3.

5. A method for alleviating pain comprising contacting neurons with a binding partner of a cationic amino acid transporter (CAT) protein identified according to the method of any one of claims 1 to 3.

6. A method of identifying a pain modulator comprising the steps of:

(a) providing a CAT protein under conditions for production of nitric oxide (NO); (b) providing one or more test agents or a control agent to the CAT protein of (a);

(c) assaying NO production; and

(d) selecting a test agent that shows altered NO production in the presence of the agent as compared to said control agent.

7. The method of claim 6, wherein the cationic amino acid transporter (CAT) protein is a CAT-2 protein.

8. The method of claim 6 or 7, wherein the one or more test agents is a peptide, a protein, an oligomer, a nucleic acid, a small molecule, or an antibody.

9. The method of any one of claims 6 to 8, further comprising:

(e) selecting a test agent that results in reduced NO production in the presence of the test agent as compared to said control agent.

10. A pain modulator identified as a test agent that elicits altered NO production according to the method of any one of claims 6 to 9.

1 1 . A method of alleviating pain in a subject comprising administering to the subject an effective amount of a pain modulator identified according to the method of claim 9.

12. The method of any one of claims 6 to 8, further comprising:

(e) selecting a test agent that results in enhanced NO production in the presence of the agent as compared to said control agent.

13. A method of treating congenital pain insensitivity in a subject comprising administering to the subject an effective amount of a pain modulator identified according to the method of claim 12.