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  Pharmaceutical Patents  

 

Title:  Methods for modulating a drug-related effect or behavior
United States Patent: 
7,365,050
Issued: 
April 29, 2008

Inventors:
 Messing; Robert O. (Foster City, CA), Newton; Philip M. (San Francisco, CA)
Assignee: 
The Regents of the University of California (Oakland, CA)
Appl. No.: 
10/913,697
Filed:
 August 5, 2004


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

The present invention provides a method of reducing or preventing a drug-related effect or behavior in a subject by inhibiting N-type calcium channels. In addition, the invention provides a variety of prescreening and screening methods aimed at identifying agents that modulate a drug-related effect or behavior. These methods involve assaying test agent binding to N-type calcium channels or channel subunits. Alternatively, test agents can be screened for their ability to alter the level of N-type calcium channels, channel subunit polypeptide or RNA, or the depolarization-induced inward calcium current mediated by these channels. Finally, the invention also provides a diagnostic method that entails measuring one or more of these levels and determining risk for a drug-related effect or behavior based on comparison to the corresponding level for a control population.

Description of the Invention

The present invention relates to the discovery that N-type calcium channels modulate certain drug-related effects and behaviors. The role of N-type calcium channels in these effects and behaviors has been demonstrated, for the first time, in vivo using knockout mice that are null for the Ca.sub.v2.2 subunit, which is unique to the N-type channel. Thus, these knockout mice cannot produce functional N-type calcium channels. The results obtained from the studies of these mice described herein indicate that certain drug-related effects and behaviors can be reduced or prevented by inhibiting these channels. In addition, these studies have given rise to various screening methods based on assaying test agents for their ability to bind to N-type calcium channels or channel subunits or to alter an N-type calcium channel-related parameter. Furthermore, the Ca.sub.v2.2 null knockout studies have lead to the development of a diagnostic method for assessing a subject's risk for drug-related effects or behaviors. These methods are of particular interest with respect to drugs of abuse, such as ethanol, cannabinoids, and opioids.

Method of Reducing a Drug-Related Effect or Behavior

A. In General

The invention provides a method of reducing or preventing a drug-related effect or behavior. The method entails inhibiting an N-type calcium channel in a subject, whereby the drug-related effect or behavior is reduced or prevented. Generally, the method is carried out by administering an N-type calcium channel inhibitor to a subject. The method is useful for addressing undesirable effects or behaviors associated with a variety of drugs, particularly sedative-hypnotic and analgesic drugs. In particular embodiments, the method is used to reduce or prevent effects or behaviors associated with drugs such as ethanol, cannabinioids, opioids, and the like. Exemplary cannabinioids include Tetrahydrocannabinol (THC), dronabinol, arachidonylethanolamide (anandamide, AEA). Exemplary opioids include morphine, codeine, heroin, butorphanol, hydrocodone, hydromorphone, levorphanol, meperidine, nalbuphine, oxycodone, fentanyl, methadone, propoxyphene, remifentanil, sufentanil, and pentazocine.

Examples of undesirable drug-related effects or behaviors that can be reduced or prevented according to the method of the invention include sedative and hypnotic effects; drug reward; and drug consumption.

The subject of the method can be any individual that has N-type calcium channels. Examples of suitable subjects include research animals, such as Drosophila melanogaster, mice, rats, guinea pigs, rabbits, cats, dogs, as well as monkeys and other primates, and humans. The subject can be an individual who is regularly, or intermittently, using one or more of the above drugs or an individual who is at risk for such use.

The method of the invention entails inhibiting the N-type calcium channel to a degree sufficient to reduce or prevent the drug-related effect(s) and/or behavior(s) of interest. In various embodiments, the N-type calcium channel is inhibited by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, and 95 percent, as determined by any suitable measure of channel inhibition (such as, for example, any of the assays described herein).

Any kind of N-type calcium channel inhibitor that is tolerated by the subject can be employed in the method of the invention. Thus, the inhibitor can be a polypeptide (such as, e.g., an anti-N-type calcium channel antibody), a polynucleotide (e.g., one that encodes an inhibitory polypeptide), or a small molecule. In particular embodiments, when the inhibitor is a polynucleotide, the polynucleotide is introduced into the subject's cells, where the encoded polypeptide is expressed in an amount sufficient to inhibit N-type calcium channels.

Inhibition N-type channels can be achieved by any available means, e.g., inhibition of: (1) the expression, mRNA stability, protein trafficking, modification (e.g., phosphorylation), or degradation of an N-type calcium channel or one or more of its subunits (e.g., N-type Ca.sub.v2.2), or (2) one or more of the normal functions of an N-type calcium channel, such the depolarization-induced inward calcium current.

As phosphorylation of N-type calcium channels by protein kinase C (PKC) enhances N-type channel function, channel function can be inhibited by inhibiting this phosphorylation. PKC.epsilon. has been implicated as the PKC isozyme that mediates channel phosphorylation. Therefore, N-type channels can be inhibited using a general PKC inhibitor or a selective PKC.epsilon. inhibitor. PKC inhibitors are well-known. For instance, U.S. Pat. No. 5,783,405 describes a large number of peptides that inhibit PKC isozymes. Of these, the .epsilon.V1-1, .epsilon.V1-2, .epsilon.V1-3, .epsilon.V1-4, .epsilon.V1-5 and .epsilon.V1-6 peptides are selective for PKC.epsilon. and are preferred peptide inhibitors. Peptide .epsilon.V1-2 is a particularly preferred inhibitory peptide. Small-molecule inhibitors of PKC are described in U.S. Pat. Nos. 5,141,957, 5,204,370, 5,216,014, 5,270,310, 5,292,737, 5,344,841, 5,360,818, and 5,432,198. These molecules belong to the following classes: N,N'-Bis-(sulfonamido)-2-amino-4-iminonaphthalen-1-ones; N,N'-Bis-(amido)-2-amino-4-iminonaphthalen-1-ones; vicinal-substituted carbocyclics; 1,3-dioxane derivatives; 1,4-Bis-(amino-hydroxyalkylamino)-anthraquinones; furo-coumarinsulfonamides; Bis-(hydroxyalkylamino)-anthraquinones; and N-aminoalkyl amides. Due to their relative ease of administration (for instance, via transdermal delivery or ingestion), small molecule inhibitors of PKC.epsilon. are, in some instances, preferred over peptide inhibitors.

In addition, interaction of N-type calcium channels with the .beta.-.gamma. subunits of G proteins has been shown to inhibit N-type channel function. Therefore, channel function can be inhibited by any means of enhancing G.beta.-.gamma. interaction with N-type channels. For example, activation of any of a number of G protein-coupled receptors leads to G.beta.-.gamma. inhibition of N-type calcium channels. Ruiz-Velasco and Ikeda, J. Neuroscience 20:2183-2191 (2000). Accordingly, an agonist of such a receptor can be used in the present method to inhibit N-type calcium channels.

In one embodiment, N-type calcium channel inhibition is achieved by reducing the level of N-type calcium channels in a tissue having such channels. N-type calcium channels are expressed in neurons of the central and peripheral nervous systems. Thus, the method of the invention can target N-type calcium channels in brain, dorsal root ganglion neurons, and sympathetic ganglion neurons. In a variation of this embodiment, N-type channel level is reduced by reducing the level of N-type Ca.sub.v2.2 subunits in the tissue. This can be achieved using, e.g., antisense or RNA interference (RNAI) techniques to reduce the level of N-type Ca.sub.v2.2 RNA available for translation.

The N-type calcium channel inhibitor can be non-selective or selective for N-type calcium channels. Examples of non-selective inhibitors suitable for use in the invention include omega conotoxin MVIIC, omega grammotoxin SIA, and omega agatoxin IIIA. Preferred embodiments employ a selective inhibitor, such as, for example, omega-conotoxin MVIIA, omega conotoxin GVIA, omega conotoxin CNVIIA (Favreau et al., 2001), omega conotoxin CVID (AM336; Lewis et al., 2000), Ptul (a toxin from the assassin bug Peirates turpis; Bernard et al. 2001), NMED-126, and NMED-160 (both of the latter two compounds are produced by NeuroMed Technologies, Inc., Vancouver, British Columbia, Calif.). Additional N-type calcium channel inhibitors useful in the invention are described in U.S. Pat. Nos. 6,617,322, 6,492,375; 6,387,897; 6,310,059; 6,267,945; 6,011,035 and in published U.S. application Ser. No. 10/409,868 (published Mar. 4, 2004; Publication No. 20040044004) and Ser. No. 10/409,763 (published Feb. 19, 2004; Publication No. 20040034035).

B. Compositions

For research and therapeutic applications, an N-type calcium channel inhibitor is generally formulated to deliver inhibitor to a target site in an amount sufficient to inhibit N-type calcium channels at that site.

Inhibitor compositions of the invention optionally contain other components, including, for example, a storage solution, such as a suitable buffer, e.g., a physiological buffer. In a preferred embodiment, the composition is a pharmaceutical composition and the other component is a pharmaceutically acceptable carrier, such as are described in Remington's Pharmaceutical Sciences (1980) 16th editions, Osol, ed., 1980.

A pharmaceutically acceptable carrier suitable for use in the invention is non-toxic to cells, tissues, or subjects at the dosages employed, and can include a buffer (such as a phosphate buffer, citrate buffer, and buffers made from other organic acids), an antioxidant (e.g., ascorbic acid), a low-molecular weight (less than about 10 residues) peptide, a polypeptide (such as serum albumin, gelatin, and an immunoglobulin), a hydrophilic polymer (such as polyvinylpyrrolidone), an amino acid (such as glycine, glutamine, asparagine, arginine, and/or lysine), a monosaccharide, a disaccharide, and/or other carbohydrates (including glucose, mannose, and dextrins), a chelating agent (e.g., ethylenediaminetetratacetic acid [EDTA]), a sugar alcohol (such as mannitol and sorbitol), a salt-forming counterion (e.g., sodium), and/or an anionic surfactant (such as Tween.TM., Pluronics.TM., and PEG). In one embodiment, the pharmaceutically acceptable carrier is an aqueous pH-buffered solution.

Preferred embodiments include sustained-release pharmaceutical compositions. An exemplary sustained-release composition has a semipermeable matrix of a solid hydrophobic polymer to which the inhibitor is attached or in which the inhibitor is encapsulated. Examples of suitable polymers include a polyester, a hydrogel, a polylactide, a copolymer of L-glutamic acid and T-ethyl-L-glutamase, non-degradable ethylene-vinylacetate, a degradable lactic acid-glycolic acid copolymer, and poly-D-(-)-3-hydroxybutyric acid. Such matrices are in the form of shaped articles, such as films, or microcapsules.

Where the inhibitor is a polypeptide, exemplary sustained release compositions include the polypeptide attached, typically via .epsilon.-amino groups, to a polyalkylene glycol (e.g., polyethylene glycol [PEG]). Attachment of PEG to proteins is a well-known means of reducing immunogenicity and extending in vivo half-life (see, e.g., Abuchowski, J., et al. (1977) J. Biol. Chem. 252:3582-86. Any conventional "pegylation" method can be employed, provided the "pegylated" variant retains the desired function(s).

In another embodiment, a sustained-release composition includes a liposomally entrapped inhibitor. Liposomes are small vesicles composed of various types of lipids, phospholipids, and/or surfactants. These components are typically arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing N-type calcium channel inhibitors are prepared by known methods, such as, for example, those described in Epstein, et al. (1985) PNAS USA 82:3688-92, and Hwang, et al., (1980) PNAS USA, 77:4030-34. Ordinarily the liposomes in such preparations are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the specific percentage being adjusted to provide the optimal therapy. Useful liposomes can be generated by the reverse-phase evaporation method, using a lipid composition including, for example, phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). If desired, liposomes are extruded through filters of defined pore size to yield liposomes of a particular diameter.

Pharmaceutical compositions can also include an inhibitor adsorbed onto a membrane, such as a silastic membrane, which can be implanted, as described in International Publication No. WO 91/04014.

Pharmaceutical compositions of the invention can be stored in any standard form, including, e.g., an aqueous solution or a lyophilized cake. Such compositions are typically sterile when administered to subjects. Sterilization of an aqueous solution is readily accomplished by filtration through a sterile filtration membrane. If the composition is stored in lyophilized form, the composition can be filtered before or after lyophilization and reconstitution.

In particular embodiments, the methods of the invention employ pharmaceutical compositions containing a polynucleotide encoding a polypeptide inhibitor of N-type calcium channels. Such compositions optionally include other components, as for example, a storage solution, such as a suitable buffer, e.g., a physiological buffer. In a preferred embodiment, the composition is a pharmaceutical composition and the other component is a pharmaceutically acceptable carrier as described above.

Preferably, compositions containing polynucleotides useful in the invention also include a component that facilitates entry of the polynucleotide into a cell. Components that facilitate intracellular delivery of polynucleotides are well-known and include, for example, lipids, liposomes, water-oil emulsions, polyethylene imines and dendrimers, any of which can be used in compositions according to the invention. Lipids are among the most widely used components of this type, and any of the available lipids or lipid formulations can be employed with polynucleotides useful in the invention. Typically, cationic lipids are preferred. Preferred cationic lipids include N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), dioleoyl phosphotidylethanolamine (DOPE), and/or dioleoyl phosphatidylcholine (DOPC). Polynucleotides can also be entrapped in liposomes, as described above.

In another embodiment, polynucleotides are complexed to dendrimers, which can be used to introduce polynucleotides into cells. Dendrimer polycations are three-dimensional, highly ordered oligomeric and/or polymeric compounds typically formed on a core molecule or designated initiator by reiterative reaction sequences adding the oligomers and/or polymers and providing an outer surface that is positively changed. Suitable dendrimers include, but are not limited to, "starburst" dendrimers and various dendrimer polycations. Methods for the preparation and use of dendrimers to introduce polynucleotides into cells in vivo are well known to those of skill in the art and described in detail, for example, in PCT/US83/02052 and U.S. Pat. Nos. 4,507,466; 4,558,120; 4,568,737; 4,587,329; 4,631,337; 4,694,064; 4,713,975; 4,737,550; 4,871,779; 4,857,599; and 5,661,025.

For therapeutic use, polynucleotides useful in the invention are formulated in a manner appropriate for the particular indication. U.S. Pat. No. 6,001,651 to Bennett et al. describes a number of pharmaceutical compositions and formulations suitable for use with an oligonucleotide therapeutic as well as methods of administering such oligonucleotides.

C. Administration

Methods for in vivo administration do not differ from known methods for administering small-molecule drugs or therapeutic polypeptides, peptides, or polynucleotides encoding them. Suitable routes of administration include, for example, topical, intravenous, intraperitoneal, intracerebral, intraventricular, intramuscular, intraocular, intraarterial, or intralesional routes. Pharmaceutical compositions of the invention can be administered continuously by infusion, by bolus injection, or, where the compositions are sustained-release preparations, by methods appropriate for the particular preparation.

D. Dose

The dose of inhibitor is sufficient to inhibit N-type calcium channels, preferably without significant toxicity. For therapeutic applications, the dose of inhibitor depends, for example, upon the therapeutic objectives, the route of administration, and the condition of the subject. Accordingly, it is necessary for the clinician to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Generally, the clinician begins with a low dose and increases the dosage until the desired therapeutic effect is achieved. Starting doses for a given inhibitor can be extrapolated from in vitro data.

Methods of Screening for Agents that Modulate a Drug-Related Effect or Behavior

The role of N-type calcium channels in mediating drug-related effects and behaviors makes the N-type channel an attractive target for agents that modulate these effects and behaviors. Accordingly, the invention provides prescreening and screening methods aimed at identifying such agents. The prescreening/screening methods of the invention are generally, although not necessarily, carried out in vitro. Accordingly, screening assays are generally carried out, for example, using purified or partially purified components in cell lysates or fractions thereof, in cultured cells, or in a biological sample, such as a tissue or a fraction thereof.

A. Prescreening Based on Binding to N-Type Calcium Channels or a Subunit Thereof

The invention provides a prescreening method based on assaying test agents for specific binding to an N-type calcium channel or a subunit thereof. Agents that specifically bind to N-type channels, or a subunit thereof, have the potential to modulate channel function, and thereby modulate one or more drug-related effects and/or behaviors.

In one embodiment, therefore, a prescreening method of the invention entails contacting a test agent with an N-type calcium channel or a subunit thereof, such as the N-type Ca.sub.v2.2 subunit. Specific binding of the test agent to the N-type channel or subunit is then determined. If specific binding is detected, the test agent is selected as a potential modulator of a drug-related effect or behavior.

Such prescreening is generally most conveniently accomplished with a simple in vitro binding assay. Means of assaying for specific binding of a test agent to a polypeptide are well known to those of skill in the art. In preferred binding assays, the polypeptide is immobilized and exposed to a test agent (which can be labeled), or alternatively, the test agent(s) are immobilized and exposed to the polypeptide (which can be labeled). The immobilized species is then washed to remove any unbound material and the bound material is detected. To prescreen large numbers of test agents, high throughput assays are generally preferred. Various screening formats are discussed in greater detail below.

B. Screening Based on Binding to Polynucleotides Encoding N-type Calcium Channel Subunits

The invention also provides a prescreening method based on screening test agents for specific binding to a polynucleotide encoding an N-type calcium channel subunit. Agents that specifically bind to such polynucleotides have the potential to modulate the expression of the encoded N-type calcium channel subunit, and thereby modulate one or more drug-related effects and/or behaviors.

In one embodiment, therefore, a prescreening method of the invention entails contacting a test agent with a polynucleotide encoding an N-type calcium channel subunit, such as the N-type Ca.sub.v2.2 subunit. Specific binding of the test agent to the polynucleotide is then determined. If specific binding is detected, the test agent is selected as a potential modulator of a drug-related effect or behavior.

Such prescreening is generally most conveniently accomplished with a simple in vitro binding assay. Means of assaying for specific binding of a test agent to a polynucleotide are well known to those of skill in the art. In preferred binding assays, the polynucleotide is immobilized and exposed to a test agent (which can be labeled), or alternatively, the test agent(s) are immobilized and exposed to the polynucleotide (which can be labeled). The immobilized species is then washed to remove any unbound material and the bound material is detected. To prescreen large numbers of test agents, high throughput assays are generally preferred. Various screening formats are discussed in greater detail below.

C. Screening Based on Levels of N-type Calcium Channels or Channel Subunit Polypeptides or RNA

Test agents, including, for example, those identified in a prescreening assay of the invention can also be screened to determine whether the test agent affects the levels of N-type calcium channels or channel subunit polypeptides or RNA. Agents that reduce these levels can potentially reduce one or more drug-related effects and/or behaviors. Conversely, agents that increase these levels can potentially enhance such drug-related effects and/or behaviors.

Accordingly, the invention provides a method of screening for an agent that inhibits or enhances a drug-related effect or behavior in which a test agent is contacted with a cell that expresses an N-type calcium channel in the absence of test agent. Preferably, the method is carried out using an in vitro assay. In such assays, the test agent can be contacted with a cell in culture or present in a tissue. Alternatively, the test agent can be contacted with a cell lysate or fraction thereof (e.g., a membrane fraction for detection of N-type calcium channels or channel subunit polypeptides). The level of (i) N-type calcium channels; (ii) channel subunit polypeptide; or (iii) channel subunit RNA is determined in the presence and absence (or presence of a lower amount) of test agent to identify any test agents that alter the level. Where channel subunit polypeptide or RNA is determined, the channel subunit is preferably N-type Ca.sub.v2.2. If the level assayed is altered, the test agent is selected as a potential modulator of a drug-related effect or behavior. In a preferred embodiment, an agent that reduces the level assayed is selected as a potential inhibitor of one or more drug-related effects and/or behaviors.

Cells useful in this screening method include those from any of the species described above in connection with the method of reducing a drug-related effect or behavior. Cells that naturally express an N-type calcium channel are typically, although not necessarily, employed in this screening method. Examples include PC12 cells, SH-SY5y cells, NG108-15 cells, IMR-32 cells, SK-N-SH cells, RINm5F cells, and NMB cells. Alternatively, cells that have been engineered to express an N-type calcium channel can be used in the method.

In one embodiment, the test agent is contacted with the cell in the presence of the drug. The drug is generally one that produces one or more undesirable effects or behaviors, such as, for example, sedative-hypnotic and analgesic drugs. In particular embodiments, the drug is ethanol, a cannabinioid, or an opioid.

1. Sample

As noted above, screening assays are generally carried out in vitro, for example, in cultured cells, in a biological sample (e.g., brain, dorsal root ganglion neurons, and sympathetic ganglion neurons), or fractions thereof. For ease of description, cell cultures, biological samples, and fractions are referred to as "samples" below. The sample is generally derived from an animal (e.g., any of the research animals mentioned above), preferably a mammal, and more preferably from a human.

The sample may be pretreated as necessary by dilution in an appropriate buffer solution or concentrated, if desired. Any of a number of standard aqueous buffer solutions, employing one or more of a variety of buffers, such as phosphate, Tris, or the like, at physiological pH can be used.

2. Polypeptide-Based Assays

N-type calcium channels and/or channel subunit polypeptides can be detected and quantified by any of a number of methods well known to those of skill in the art. Examples of analytic biochemical methods suitable for detecting N-type calcium channel subunit or, in some cases, entire channels, include electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunohistochemistry, affinity chromatography, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, and the like.

In one embodiment, N-type calcium channels are detected/quantified using a ligand binding assay, such as, for example, a radioligand binding assay. Briefly, a sample from a tissue expressing N-type calcium channels is incubated with a suitable ligand under conditions designed to provide a saturating concentration of ligand over the incubation period. After ligand treatment, the sample is assayed for radioligand binding. Any ligand that binds to N-type calcium channels can be employed in the assay, although N-type-selective calcium channel ligands are preferred. Any of the N-type calcium channel inhibitors discussed above can, for example, be labeled and used in this assay. An exemplary, preferred ligand for this purpose is .sup.125I-.omega.-conotoxin GVIA. Binding of this ligand to cells can be assayed as described, for example, in Solem et al. (1997) J. Pharmacol. Exp. Ther. 282:1487-95. Binding to membranes (e.g., brain membranes) can be assayed according to the method of Wagner et al. (1995) J. Neurosci. 8:3354-3359 (see also, the modifications of this method described in McMahon et al. (2000) Mol. Pharm. 57:53-58).

In another embodiment, channel subunit polypeptide(s) are detected/quantified in an electrophoretic polypeptide separation (e.g. a 1- or 2-dimensional electrophoresis). Means of detecting polypeptides using electrophoretic techniques are well known to those of skill in the art (see generally, R. Scopes (1982) Polypeptide Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to Polypeptide Purification, Academic Press, Inc., N.Y.).

A variation of this embodiment utilizes a Western blot (immunoblot) analysis to detect and quantify the presence channel subunit polypeptide(s) in the sample. This technique generally comprises separating sample polypeptides by gel electrophoresis on the basis of molecular weight, transferring the separated polypeptides to a suitable solid support (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the support with antibodies that specifically bind the target polypeptide(s). Antibodies that specifically bind to the target polypeptide(s) may be directly labeled or alternatively may be detected subsequently using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to a domain of the primary antibody.

In a preferred embodiment, channel subunit polypeptide(s) are detected and/or quantified in the biological sample using any of a number of well-known immunoassays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a general review of immunoassays, see also Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds. (1991).

Conventional immunoassays often utilize a "capture agent" to specifically bind to and often immobilize the analyte (in this case a channel subunit polypeptide). In preferred embodiments, the capture agent is an antibody.

Immunoassays also typically utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the target polypeptide. The labeling agent may itself be one of the moieties making up the antibody/target polypeptide complex. Thus, the labeling agent may be a labeled polypeptide or a labeled antibody that specifically recognizes the already bound target polypeptide. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the capture agent/target polypeptide complex. Other polypeptides capable of specifically binding immunoglobulin constant regions, such as polypeptide A or polypeptide G may also be used as the labeling agent. These polypeptides are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, and Akerstrom (1985) J. Immunol., 135: 2589-2542).

Preferred immunoassays for detecting the target polypeptide(s) are either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured target polypeptide is directly measured. In competitive assays, the amount of target polypeptide in the sample is measured indirectly by measuring the amount of an added (exogenous) polypeptide displaced (or competed away) from a capture agent by the target polypeptide present in the sample. In one competitive assay, a known amount of, in this case, labeled channel subunit polypeptide is added to the sample, and the sample is then contacted with a capture agent. The amount of labeled channel subunit polypeptide bound to the antibody is inversely proportional to the concentration of channel subunit polypeptide present in the sample.

Detectable labels suitable for use in the present invention include any moiety or composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Examples include biotin for staining with a labeled streptavidin conjugate, magnetic beads (e.g., DYNABEADS magnetic beads), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, coumarin, oxazine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, Oregon, USA), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold (e.g., gold particles in the 40 -80 nm diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

The assays of this invention are scored (as positive or negative or quantity of target polypeptide) according to standard methods well known to those of skill in the art. The particular method of scoring will depend on the assay format and choice of label. For example, a Western Blot assay can be scored by visualizing the colored product produced by the enzymatic label. A clearly visible colored band or spot at the correct molecular weight is scored as a positive result, while the absence of a clearly visible spot or band is scored as a negative. The intensity of the band or spot can provide a quantitative measure of target polypeptide concentration.

In preferred embodiments, immunoassays according to the invention are carried out using a MicroElectroMechanical System (MEMS). MEMS are microscopic structures integrated onto silicon that combine mechanical, optical, and fluidic elements with electronics, allowing convenient detection of an analyte of interest. An exemplary MEMS device suitable for use in the invention is the Protiveris' multicantilever array. This array is based on chemo-mechanical actuation of specially designed silicon microcantilevers and subsequent optical detection of the microcantilever deflections. When coated on one side with a protein, antibody, antigen or DNA fragment, a microcantilever will bend when it is exposed to a solution containing the complementary molecule. This bending is caused by the change in the surface energy due to the binding event. Optical detection of the degree of bending (deflection) allows measurement of the amount of complementary molecule bound to the microcantilever.

Antibodies useful in these immunoassays include polyclonal and monoclonal antibodies.

3. Polynucleotide-Based Assays

Changes in N-type calcium channel subunit expression level can be detected by measuring changes in levels of mRNA and/or a polynucleotide derived from the mRNA (e.g., reverse-transcribed cDNA, etc.).

Polynucleotides can be prepared from a sample according to any of a number of methods well known to those of skill in the art. General methods for isolation and purification of polynucleotides are described in detail in by Tijssen ed., (1993) Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Elsevier, N.Y. and Tijssen ed.

i. Amplification-Based Assays

In one embodiment, amplification-based assays can be used to detect, and optionally quantify, a polynucleotide encoding a channel subunit of interest. In such amplification-based assays, the channel subunit mRNA in the sample act as template(s) in an amplification reaction carried out with a nucleic acid primer that contains a detectable label or component of a labeling system. Suitable amplification methods include, but are not limited to, polymerase chain reaction (PCR); reverse-transcription PCR (RT-PCR); ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren et al. (1988) Science 241: 1077, and Barringer et al. (1990) Gene 89: 117; transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874); dot PCR, and linker adapter PCR, etc.

To determine the level of the channel subunit mRNA, any of a number of well known "quantitative" amplification methods can be employed. Quantitative PCR generally involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in PCR Protocols, A Guide to Methods and Applications, Innis et al., Academic Press, Inc. N.Y., (1990).

ii. Hybridization-Based Assays

Nucleic acid hybridization simply involves contacting a nucleic acid probe with sample polynucleotides under conditions where the probe and its complementary target nucleotide sequence can form stable hybrid duplexes through complementary base pairing. The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label or component of a labeling system. Methods of detecting and/or quantifying polynucleotides using nucleic acid hybridization techniques are known to those of skill in the art (see Sambrook et al. supra). Hybridization techniques are generally described in Hames and Higgins (1985) Nucleic Acid Hybridization, A Practical Approach, IRL Press; Gall and Pardue (1969) Proc. Natl. Acad. Sci. USA 63: 378-383; and John et al. (1969) Nature 223: 582-587. Methods of optimizing hybridization conditions are described, e.g., in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, Elsevier, N.Y.).

The nucleic acid probes used herein for detection of channel subunit mRNA can be full-length or less than the full-length of these polynucleotides. Shorter probes are generally empirically tested for specificity. Preferably, nucleic acid probes are at least about 15, and more preferably about 20 bases or longer, in length. (See Sambrook et al. for methods of selecting nucleic acid probe sequences for use in nucleic acid hybridization.) Visualization of the hybridized probes allows the qualitative determination of the presence or absence of the channel subunit mRNA of interest, and standard methods (such as, e.g., densitometry where the nucleic acid probe is radioactively labeled) can be used to quantify the level of the channel subunit polynucleotide.)

A variety of additional nucleic acid hybridization formats are known to those skilled in the art. Standard formats include sandwich assays and competition or displacement assays. Sandwich assays are commercially useful hybridization assays for detecting or isolating polynucleotides. Such assays utilize a "capture" nucleic acid covalently immobilized to a solid support and a labeled "signal" nucleic acid in solution. The sample provides the target polynucleotide. The capture nucleic acid and signal nucleic acid each hybridize with the target polynucleotide to form a "sandwich" hybridization complex.

In one embodiment, the methods of the invention can be utilized in array-based hybridization formats. In an array format, a large number of different hybridization reactions can be run essentially "in parallel." This provides rapid, essentially simultaneous, evaluation of a number of hybridizations in a single experiment. Methods of performing hybridization reactions in array based formats are well known to those of skill in the art (see, e.g., Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) Nature Biotechnology 14:1685; Chee (1995) Science 274: 610; WO 96/17958, Pinkel et al. (1998) Nature Genetics 20: 207-211).

Arrays, particularly nucleic acid arrays, can be produced according to a wide variety of methods well known to those of skill in the art. For example, in a simple embodiment, "low-density" arrays can simply be produced by spotting (e.g. by hand using a pipette) different nucleic acids at different locations on a solid support (e.g. a glass surface, a membrane, etc.). This simple spotting approach has been automated to produce high-density spotted microarrays. For example, U.S. Pat. No. 5,807,522 describes the use of an automated system that taps a microcapillary against a surface to deposit a small volume of a biological sample. The process is repeated to generate high-density arrays. Arrays can also be produced using oligonucleotide synthesis technology. Thus, for example, U.S. Pat. No. 5,143,854 and PCT Patent Publication Nos. WO 90/15070 and 92/10092 teach the use of light-directed combinatorial synthesis of high-density oligonucleotide microarrays. Synthesis of high-density arrays is also described in U.S. Pat. Nos. 5,744,305; 5,800,992; and 5,445,934.

In a preferred embodiment, the arrays used in this invention contain "probe" nucleic acids. These probes are then hybridized respectively with their "target" nucleotide sequence(s) present in polynucleotides derived from a biological sample. Alternatively, the format can be reversed, such that polynucleotides from different samples are arrayed and this array is then probed with one or more probes, which can be differentially labeled.

Many methods for immobilizing nucleic acids on a variety of solid surfaces are known in the art. A wide variety of organic and inorganic polymers, as well as other materials, both natural and synthetic, can be employed as the material for the solid surface. Illustrative solid surfaces include, e.g., nitrocellulose, nylon, glass, quartz, diazotized membranes (paper or nylon), silicones, polyformaldehyde, cellulose, and cellulose acetate. In addition, plastics such as polyethylene, polypropylene, polystyrene, and the like can be used. Other materials that can be employed include paper, ceramics, metals, metalloids, semiconductive materials, and the like. In addition, substances that form gels can be used. Such materials include, e.g., proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.

In preparing the surface, a plurality of different materials may be employed, particularly as laminates, to obtain various properties. For example, proteins (e.g., bovine serum albumin) or mixtures of macromolecules (e.g., Denhardt's solution) can be employed to avoid non-specific binding, simplify covalent conjugation, and/or enhance signal detection. If covalent bonding between a compound and the surface is desired, the surface will usually be polyfunctional or be capable of being polyfunctionalized. Functional groups that may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like. The manner of linking a wide variety of compounds to various surfaces is well known and is amply illustrated in the literature.

Arrays can be made up of target elements of various sizes, ranging from about 1 mm diameter down to about 1 .mu.m. Relatively simple approaches capable of quantitative fluorescent imaging of 1 cm.sup.2 areas have been described that permit acquisition of data from a large number of target elements in a single image (see, e.g., Wittrup (1994) Cytometry 16:206-213, Pinkel et al. (1998) Nature Genetics 20: 207-211).

Hybridization assays according to the invention can also be carried out using a MicroElectroMechanical System (MEMS), such as the Protiveris' multicantilever array.

iii. Polynucleotide Detection

Channel subunit RNA is detected in the above-described polynucleotide-based assays by means of a detectable label. Any of the labels discussed above can be used in the polynucleotide-based assays of the invention. The label may be added to a probe or primer or sample polynucleotides prior to, or after, the hybridization or amplification. So called "direct labels" are detectable labels that are directly attached to or incorporated into the labeled polynucleotide prior to conducting the assay. In contrast, so called "indirect labels" are joined to the hybrid duplex after hybridization. In indirect labeling, one of the polynucleotides in the hybrid duplex carries a component to which the detectable label binds. Thus, for example, a probe or primer can be biotinylated before hybridization. After hybridization, an avidin-conjugated fluorophore can bind the biotin-bearing hybrid duplexes, providing a label that is easily detected. For a detailed review of methods of the labeling and detection of polynucleotides, see Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y., (1993)).

The sensitivity of the hybridization assays can be enhanced through use of a polynucleotide amplification system that multiplies the target polynucleotide being detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBAO, Cangene, Mississauga, Ontario) and Q Beta Replicase systems.

In a preferred embodiment, suitable for use in amplification-based assays of the invention, a primer contains two fluorescent dyes, a "reporter dye" and a "quencher dye." When intact, the primer produces very low levels of fluorescence because of the quencher dye effect. When the primer is cleaved or degraded (e.g., by exonuclease activity of a polymerase, see below), the reporter dye fluoresces and is detected by a suitable fluorescent detection system. Amplification by a number of techniques (PCR, RT-PCR, RCA, or other amplification method) is performed using a suitable DNA polymerase with both polymerase and exonuclease activity (e.g., Taq DNA polymerase). This polymerase synthesizes new DNA strands and, in the process, degrades the labeled primer, resulting in an increase in fluorescence. Commercially available fluorescent detection systems of this type include the ABI Prism.RTM. Systems 7000, 7700, or 7900 (TaqMan.RTM.) from Applied Biosystems or the LightCycler.RTM. System from Roche.

D. Screening Based on Level of Calcium Current

The invention also provides a screening method based on determining the effect, if any, of a test agent on the level of the depolarization-induced inward calcium current mediated by N-type calcium channels. Agents that reduce this current can potentially reduce one or more drug-related effects and/or behaviors. Conversely, agents that increase this current can potentially enhance such drug-related effects and/or behaviors.

Accordingly, the invention provides a method of screening for an agent that inhibits or enhances a drug-related effect or behavior in which a test agent is contacted with a cell that expresses an N-type calcium channel in the absence of test agent. Preferably, the method is carried out using an in vitro assay. In such assays, the test agent can be contacted with a cell in culture or present in a tissue. Alternatively, the test agent can be contacted with channels in in synaptoneurosomes or purified channel proteins reconstitued in lipid bilayers. The level of depolarization-induced inward calcium current is determined in the presence and absence (or presence of a lower amount) of test agent to identify any test agents that alter the level. If the level of the calcium current is altered, the test agent is selected as a potential modulator of a drug-related effect or behavior. In a preferred embodiment, an agent that reduces the calcium current is selected as a potential inhibitor of one or more drug-related effects and/or behaviors.

The calcium current can be measured using any available technique An indirect measurement of calcium current can be carried out described by McMahon et al. (2000) Mol. Pharm. 57:53-58). In this method, cells are loaded with a dye that fluoresces in the presence of calcium (such as fura-2 AM) prior to depolarization. Cells are generally also preincubated in the presence or absence of an N-type calcium channel-specific inhibitor (e.g., 1 .mu.M .omega.-conotoxin GVIA) to determine the extent of the calcium current that is attributable to N-type calcium channels. Cells are subsequently depolarized by incubation in a 50 mM KCl buffer in the continued presence or absence of the inhibitor. The resulting calcium current can then be calculated based on fluorescence, as described by Solem et al. (1997) J. Pharmacol. Exp. Ther. 282:1487-95. Ruiz-Velasco and Ikeda (J. Neuroscience (2000) 20:2183-91 describe the direct measurement of calcium currents using a whole-cell variant of the patch-claim technique, which can also be employed in the present invention.

Cells useful for screening based on calcium current include any of those described above in connection with screening based levels of N-type calcium channels or channel subunit polypeptides or RNA.

In one embodiment, the test agent is contacted with the cell in the presence of the drug. The drug is generally one that produces one or more undesirable effects or behaviors, such as, for example, sedative-hypnotic and analgesic drugs. In particular embodiments, the drug is ethanol, a cannabinioid, or an opioid.

E. Test Agent Databases

In a preferred embodiment, generally involving the screening of a large number of test agents, the screening method includes the recordation of any test agent selected in any of the above-described prescreening or screening methods in a database of agents that may modulate a drug-related effect or behavior.

The term "database" refers to a means for recording and retrieving information. In preferred embodiments, the database also provides means for sorting and/or searching the stored information. The database can employ any convenient medium including, but not limited to, paper systems, card systems, mechanical systems, electronic systems, optical systems, magnetic systems or combinations thereof. Preferred databases include electronic (e.g. computer-based) databases. Computer systems for use in storage and manipulation of databases are well known to those of skill in the art and include, but are not limited to "personal computer systems," mainframe systems, distributed nodes on an inter- or intra-net, data or databases stored in specialized hardware (e.g. in microchips), and the like.

F. Test Agents Identified by Screening

When a test agent is found to alter the level of N-type calcium channels, channel subunit polypeptide or RNA, or depolarization-induced inward calcium current, a preferred screening method of the invention further includes combining the test agent with a carrier, preferably pharmaceutically acceptable carrier, such as are described above. Generally, the concentration of test agent is sufficient to alter the level of N-type calcium channels, channel subunit polypeptide or RNA, or depolarization-induced inward calcium current when the composition is contacted with a cell. This concentration will vary, depending on the particular test agent and specific application for which the composition is intended. As one skilled in the art appreciates, the considerations affecting the formulation of a test agent with a carrier are generally the same as described above with respect to methods of reducing a drug-related effect or behavior.

In a preferred embodiment, the test agent is administered to an animal to measure the ability of the selected test agent to modulate a drug-related effect or behavior in a subject, as described in greater detail below.

G. Screening for Modulation of a Drug-Related Effect or Behavior

The invention also provides a method of screening for an agent that that modulates a drug-related effect or behavior in a subject. The method entails selecting an N-type calcium channel modulator as a test agent, and measuring the ability of the selected test agent to modulate a drug-related effect or behavior in a subject. Any agent that modulates N-type calcium channels that can be administered to a subject can be employed in the method. Modulators selected through any of the prescreening or screening methods of the invention can be tested for modulation of drug-related effects or behavior. Alternatively, known N-type channel modulators can be employed. In a preferred embodiment, the selected test agent is an N-type calcium channel inhibitor.

Test agents can be formulated for administration to a subject as described above for N-type calcium channel inhibitors.

The subject of the method can be any individual that has N-type calcium channels and in which drug-related effects or behaviors can be measured. Examples of suitable subjects include research animals, such as Drosophila melanogaster, mice, rats, guinea pigs, rabbits, cats, dogs, as well as monkeys and other primates, and humans. In preferred embodiments, an animal model established for studying particular drug-related effects or behaviors is employed. Exemplary animal models for studying the effects and behaviors associated with ethanol are described in the Examples below.

The test agent is administered to the subject before, during, and/or after administration of the drug of interest, and the subject is tested or observed to determine whether the test agent modulates a particular drug-related effect or behavior. Test agents can be administered by any suitable route, as described above for N-type calcium channel inhibitors. Generally, the concentration of test agent is sufficient to alter the level of N-type calcium channels, channel subunit polypeptide or RNA, or depolarization-induced inward calcium current in vivo.

The drug and drug-related effect or behavior studied can be any of those described above. The drug is administered by any suitable route and in an amount sufficient to produce the drug-related effect or behavior under examination. The drug-related effect or behavior is measured and compared with that observed in the absence of test agent and/or in the presence of a lower amount of test agent.

Method of Assessing Risk of Drug-Related Effects or Behaviors

Another aspect of the invention is a method of assessing a subject's risk for experiencing a drug-related effect or developing a drug-related behavior. The method entails measuring one of several N-type calcium channel-related parameters in a biological sample from the subject. Suitable parameters include the levels of N-type calcium channels, channel subunit polypeptides (e.g., N-type Ca.sub.v2.2 subunit polypeptide), channel subunit RNA (e.g., N-type Ca.sub.v2.2 subunit RNA), and depolarization-induced inward calcium current. The considerations affecting sample preparation and assay are as described above, with the additional consideration that sample collection is preferably minimally invasive to the subject.

The risk for experiencing a drug-related effect or developing a drug-related behavior is directly correlated with each of these levels. To determine whether the subject has a normal, elevated, or reduced risk, the level measured for the selected N-type calcium channel parameter is compared to that of an appropriate matched (e.g., for age, sex, etc.) control population. The control population can be representative of the general population to allow a determination of risk of the individual subject as compared to, for example, the average risk in the general population.

If a subject is determined to have a high risk for experiencing a drug-related effect or developing a drug-related behavior, an N-type calcium channel inhibitor can be administered to the subject to reduce this risk. Preferably, the inhibitor dose is sufficient to reduce the levels N-type calcium channels, channel subunit polypeptides (e.g., N-type Ca.sub.v2.2 subunit polypeptide), channel subunit RNA (e.g., N-type Ca.sub.v2.2 subunit RNA), and/or depolarization-induced inward calcium current to within a normal range (i.e., the range observed in the control population).

Kits

The invention also provides kits useful in practicing the methods of the invention. In one embodiment, a kit of the invention includes an N-type calcium channel inhibitor in a suitable container. In a variation of this embodiment, the N-type calcium channel inhibitor is formulated in a pharmaceutically acceptable carrier. The kit preferably includes instructions for administering the N-type calcium inhibitor to a subject to reduce or prevent a drug-related effect or behavior.

In another embodiment, the kit is a diagnostic kit for use in assessing a subject's risk for experiencing a drug-related effect or developing a drug-related behavior. The kit includes at least one component that specifically binds to N-type calcium channels, N-type Ca.sub.v2.2 subunit polypeptides, or N-type Ca.sub.v2.2 subunit RNA. This binding component can be used to detect the presence of its binding partner in a biological sample from the subject. In a preferred embodiment, the binding component is labeled with a detectable label or, alternatively, the kit includes a labeling component that is capable of binding to, and thereby labeling, the binding component when the diagnostic method of the invention is carried out. The kit preferably includes instructions for carrying out the diagnostic method of the invention.

Instructions included in kits of the invention can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions.
 

Claim 1 of 14 Claims

1. A method of reducing an ethanol-related effect or behavior, the method comprising administering an N-type calcium channel inhibitor to a subject in need of reduction of the ethanol-related effect or behavior, whereby the ethanol-related effect or behavior is reduced, and wherein the ethanol-related effect or behavior comprises an effect or behavior selected from the group consisting of a sedative effect, a hypnotic effect, drug reward, and drug consumption.

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