METHODS FOR IDENTIFYING MODULATORS OF CBl AND CB2 CANNABINOID RECEPTORS AND THEIR USE IN WOUND HEALING

Field of the Invention

This invention relates to methods and compositions for the identification of compounds useful for enhancing wound healing and treating wound healing disorders, compounds identified by such screening methods, and the use of such compounds in compositions and methods for enhancing wound healing, treating or preventing scar formation, and treating wound healing disorders.

Background of the Invention

Wounds normally heal without therapeutic intervention, however there are numerous disorders involving impaired wound healing, and circumstances in which enhanced wound healing is necessary or desirable. An acceleration of the natural wound healing process is often desirable, for example, to minimize the danger of bacterial infections or the rest periods of injured or surgical patients. Even following successful wound closure, abnormal or excessive scar formation can result, causing serious physiological and cosmetic concerns. In the case of patients with large-scale burn wounds or extensive lacerations, the quality of life can be dramatically adversely affected. Moreover, wound healing disorders can lead to delayed healing of wounds or to chronic wounds. Such disorders can be caused by the nature of the wounding (e.g., large-area wounds, deep or extensive surgical wounds, and burns), certain medications, as well as the underlying disorder or disease. For example, a significant percentage of patients with Type II diabetes suffer from chronic ulcers, also known as "diabetic foot," of which approximately half require expensive and often unsatisfactory treatments. Until now only a few satisfactory therapies have been developed for enhancing wound healing, treating or preventing scar formation, and treating wound healing disorders. Thus, a need exists for improved wound healing agents and therapeutics.

Cannabinoids, the biologically active constituents of marijuana, have been used for millennia for their medicinal and recreational properties. Cannabinoids exert their effects by binding to specific G protein-coupled receptors ("GPCRs") (see Matsuda et al., Nature (1990)

346:561-564; and Munro et al., Nature (1993) 365:61-65). To date, two cannabinoid receptors have been cloned, cannabinoid receptor 1 ("CBl receptor") and canninoid receptor 2 ("CB2 receptor"). The CBl receptor is abundantly expressed in the brain, especially in the hippocampus, cortex, cerebellum and basal ganglia. The CB2 receptor appears to be absent from the brain but is enriched in immune tissues. Δ ⁹ -Tetrahydrocannabinol (Δ ⁹ -THC), the primary active ingredient in Cannabis sativa, activates both. The endogenous ligands are lipids like arachidonoylethanolamide (anandamide), 2-arachidonoyl-glycerol (2-AG). In addition to their neurological properties, cannabinoids are known for their analgesic, sedative, immunomodulatory and cardiovascular effects. Antagonists and inverse agonists of CB receptors have also been suggested for treatment of reproductive and endocrine disorders, and diseases related to the respiratory and gastrointestinal systems.

A genetically modified mouse is available that lacks both CBl and CB2 receptors (Jarai, Z. et al. (1999) PNAS 96(24):14136-14141). The CB1/CB2 double knockout mice are healthy, are of size and weight similar to their wild-type littermates, and have no gross defects. When the present inventors investigated the wound healing ability of CB 1/CB2 knockout ("KO") mice, they found that the mice had a higher re-epithelialization rate and reduced scarring as compared to control animals. Based on these and other findings, the present inventors have discovered that the use of compounds that modulate CBl and CB2 receptor expression or activity can be of therapeutic value in enhancing wound healing and treating wound healing disorders.

Summary of the Invention

The present invention provides newly identified targets (genes, or the corresponding RNA or protein products) for enhancing wound healing, preventing or reducing scar formation, and for therapeutic intervention in diseases or disorders characterized by an impaired or excessive wound healing response.

In one aspect, the invention provides methods of identifying pharmacologically active agents for enhancing wound healing or treating wound healing disorders using CBl and CB2 receptor proteins or genes. In one embodiment, the methods comprise contacting the CB1/CB2 proteins or nucleic acids encoding the CB1/CB2 proteins with at least one candidate substance, and determining whether the candidate substance(s) binds to or interacts with the target proteins or genes. In another embodiment, the methods comprise contacting a cell exhibiting pathologically disturbed expression of a CBl and/or CB2 polypeptide or nucleic acids encoding

the CB1/CB2 polypeptides with at least one candidate substance, and determining whether the CB1/CB2 polypeptides or nucleic acids return to normal expression. In yet another embodiment, the methods comprise contacting a sample comprising test polypeptides with CBl and CB2 binding substances, and detecting the presence of polypeptides that bind to the CB1/CB2 binding substances.

In another aspect, the invention provides methods of screening candidate substances for the ability to enhance wound healing or treat wound healing disorders. In one embodiment, the methods involve contacting a test model of wound healing with at least one candidate substance, wherein the test model includes cells that express the CB1/CB2 polypeptides or nucleic acids encoding the CB1/CB2 polypeptides, and measuring the effect of the candidate substance on a biological property associated with wound healing in the test model. In another embodiment, the methods comprise providing a non-human wild-type animal and a non-human transgenic animal, wherein the transgenic animal comprises a homozygous disruption in the CBl and CB2 receptor genes and thus lacks production of functional CB1/CB2 receptor protein and exhibits, relative to the wild-type animal, enhanced wound healing. The method further involves administering at least one candidate substance to the wild-type animal, and determining whether the substance(s) enhances the wound healing properties of the wild-type animal to resemble the wound healing properties of the transgenic animal, thereby identifying a pharmacologically active agent capable of enhancing wound healing. In other aspects, the invention relates to tests or arrays for identification of pharmacologically active agents. The tests and arrays contain a test component, which can be a CB 1 and CB2 polypeptide, nucleic acids encoding the CB 1 and CB2 polypeptides, antibodies or antibody fragments directed against the CBl and CB2 polypeptides, a cell expressing the CBl and CB2 polypeptides or nucleic acids encoding the CBl and CB2 polypeptides, or fusion proteins containing the CB 1 and CB2 polypeptides. The components in the tests can optionally be combined or together with suitable additives or auxiliaries, and/or bound to a solid phase. The invention includes methods of using the tests and arrays for the identification of candidate pharmacologically active agents for enhancing wound healing or treating wound healing disorders. In other embodiments, the invention involves the use of the CB1/CB2 target polypeptides and nucleic acids, antibodies or antibody fragments directed against the polypeptide targets, cell containing the CB1/CB2 polypeptide or nucleic acids targets, or fusion proteins

containing the CB1/CB2 polypeptides for identification of wound-healing agents or for the production of tests or arrays to identify such agents.

The invention also provides the use of modulators (antagonists, inverse agonists, and agonists) of the CB1/CB2 targets for enhancing wound healing and treating disorders or conditions characterized by aberrant wound healing. It will be appreciated that antagonists, inverse agonists or inhibitors can be compounds that reduce the activity of the target, antibodies specific for the target, as well as antisense oligonucleotides or ribozymes or transcription inhibitors, that have the effect of reducing the amount of the target produced. The invention also provides the use of agonists or activators of these targets for the treatment of wounds or disorders characterized by excessive wound healing. It will also be appreciated that agonists or activators of these targets can comprise compounds that increase the activity of the target itself, as well as compounds that increase the expression of the target. Increased activity of the target could also be achieved by delivering the target itself, either as protein or its coding region in an appropriate vector as gene therapy. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Brief Description of the Figures FIG.l shows hematoxylin-eosin stained slides of skin samples from CB1/CB2 double knockout mice (kol, ko2) and control animals (wtl, wt2) 5 days after lesioning. Arrows indicate the epidermis. Double knockout animals had an improved wound healing in comparison to wild- type animals.

FIG. 2 shows RT-PCR on different skin tissues from mice (epidermis, dermis, and primary keratinocytes). The CBl receptor mRNA was expressed in all three samples, whereas the CB2 receptor was only expressed in the dermis and in primary keratinocytes. GAPDH was used as a house keeping control gene.

FIG. 3 shows the full thickness eccisional wounds, 5mm each, were made on the either side of the dorsal midline by excising skin and panniculus carnosus. Wounds were left uncovered and harvested 5 days after injury. Mice were housed individually during the healing period. For histological analysis the complete wounds were isolated, bisected, fixed overnight in 4% paraformaldehyde in phophate buffered saline (PFA) and embedded in parafin. Sections (7μm)

from the middle of the wound were stained with hematoxylin-eosin. Morphometric measurements of area and length were performed on slices using OpenLab software (Improvision Ltd., Basel, Switzerland) and are presented in a) for total area and in b) for epithelial length.

Detailed Description of the Invention

The present invention relates, inter alia, to methods of screening candidate substances for the ability to enhance wound healing. The candidate substances can be pharmacological agents already known in the art or can be substances previously unknown to have any pharmacological activity. The substances can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. Members of combinatorial libraries can also be screened for wound healing activity.

In one aspect of the invention, the cannabinoid 1 receptor ("CBl receptor") and the cannabinoid 2 receptor ("CB2 receptor") are contacted with a candidate substance. In one embodiment, the method comprises the steps of contacting the CBl and CB2 receptors with a compound and detecting compound/CBl receptor and compound/CB2 receptor complexes. The methods comprise both cell-free and cell-based assays, including any of the in vitro or in vivo assays described below. The effect of the candidate substance on a biological property associated with wound healing can be measured in a variety of functional assays. These biological properties include, but are not limited to, enhanced wound healing, enhanced tissue regeneration, cell growth, cell replication, cell movement, cell adhesion, DNA synthesis, protein synthesis, mRNA synthesis, and mRNA stability. A candidate substance that modulates one or more of these biological properties is identified as a potential drug candidate for enhancing wound healing or treating wound healing disorders. Optionally, the effect of a candidate substance on a biological property of a wild-type animal can be compared with the same or a similar biological property of a CB1/CB2 receptor double knock-out ("double KO") animal. A candidate substance that alters the biological property of the wild-type animal such that it resembles the biological property of the CB1/CB2 receptor double KO animal is identified as a potential drug candidate for enhancing wound healing.

The present invention further provides methods of enhancing wound healing and treating wound healing disorders. In this aspect of the invention, the methods include, without limitation,

treating surgical incisions, cuts, tears, abrasions, burns, scrapes, contusions, bruises, and the like. Methods and compositions of the invention can be used to treat and thus enhance healing of a wound by promoting processes such as reepithelialization, rapid connective tissue proliferation, deposition of organized extracellular matrix, and restoration of normal tissue architecture and function. Surgical adhesions can be prevented by prophylactic treatment of surgical incisions using compositions and methods of the invention. These methods and compositions are useful in any situation in which regeneration or healing of a wound without or with minimal formation of scar tissue is desired, as well as treating chronic wound diseases and disorders.

I. Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below.

As used herein, the terms "CBl and CB2 receptor," "CB1/CB2 receptor," "CBl and CB2 proteins," "CB1/CB2 proteins" and "CB1/CB2" are used interchangeably to refer to the CBl and CB2 receptor proteins. Similarly, "CBl and CB2 polypeptides" and "CB1/CB2 polypeptides" are used interchangeably to refer to the CBl and CB2 proteins and polypeptides having CB 1 and CB2 receptor activity. As will be appreciated by those of skill in the art, the ligand binding assays of the invention are performed using each of the individual CB 1 and CB2 targets (e.g., CBl and CB2 proteins, polypeptides, and nucleic acids). Typically the ligand binding assays will identify distinct active compounds/agents that bind to or interact with the individual CB 1 and CB2 targets. In this case, the functional assays and methods of treatment described herein may comprise the application or administration of both active compounds or agents. However, depending upon the selectivity of the candidate substance or agent, a single compound or agent may bind to or interact with both CBl and CB2 targets. In this case, the functional assays and methods of treatment may comprise the application or administration of a single compound or agent. Accordingly, as used herein, the terms "active agent," "pharmacologically active agent," and "candidate pharmacologically active agent" refer to a compound (substance) or compounds (substances) that interact with or bind to the CB 1 and CB2 targets (e.g., CBl and CB2 polypeptides, nucleic acids encoding the CBl and CB2 polypeptides, or CBl and CB2 binding substances), thereby directly or indirectly modulating CBl and CB2 receptor activity.

As used herein, a "CBl and CB2 receptor activity," "biological activity of CBl and CB2

receptors" or "functional activity of CBl and CB2 receptors," refers to an activity exerted by CBl and CB2 receptor proteins, polypeptides or nucleic acid molecules on, for example, a CB 1 and CB2 receptor-responsive cell or on a CBl and CB2 receptor substrate (e.g., a protein substrate, as determined in vivo or in vitro). Thus, for example, the terms "CBl and CB2 receptor activity" and "CB1/CB2 receptor activity" are used interchangeably to mean the activity exerted by both CBl and CB2 receptor proteins. In one embodiment, a CB1/CB2 receptor activity is a direct activity, such as an association with the CB 1 and CB2 target molecules. A "target molecule" or "binding partner" is a molecule with which the CB 1 and/or CB2 receptor proteins bind or interact in nature, such as a ligand, peptide or chemokine. A CB1/CB2 receptor activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the CBl and CB2 receptor protein with a ligand, peptide, or chemokine. For example, the CB1/CB2 receptor proteins can have one or more of the following activities: (1) the ability to bind a ligand such as a peptide, small molecule, chemokine, etc.; (2) the ability to transduce a signal through a membrane; (3) the ability to interaction with cellular proteins such as G proteins; (4) the ability to modulate second messenger levels such as cAMP and cGMP; and (5) the ability to cause a cell to divide, differentiate and/or migrate.

The CBl and CB2 receptor proteins can modulate the activities of cells in tissues where they are expressed. The present inventors have identified the CBl receptor in mouse epidermis, dermis and primary keratinocytes; the CB2 receptor was detected in dermis and primary keratinocytes and weakly in epidermis, as discussed below. Thus, by antagonizing or inhibiting the CB 1 and CB2 receptor proteins, one can regulate intracellular signaling pathways and cause cells expressing these receptors to proliferate and migrate, thereby enhancing the rate of re- epithelialization, tissue proliferation, and restoration of normal tissue architecture and function. Conversely, by agonizing or activating the CB 1 and CB2 receptor proteins, one can inhibit wound healing activities. Modulators of CB 1 and CB2 receptor proteins therefore may be useful in the treatment of wounds and to enhance or inhibit wound healing.

Accordingly, CBl and CB2 receptors can act as novel targets and therapeutic agents for wound healing and treating wound healing disorders. As used herein, "wound healing" means the healing process of a mechanical wound of the skin, such as for example laceration, skin abrasion or excoriation of the skin (e.g., decubitus or necrotic processes, such as Necrobiosis lipoidica). As used herein, the term "enhanced wound healing" refers to an improvement in one or more of the regenerative processes or phases involved in healing (e.g., recruitment of inflammatory cells,

re-epithelialization, formation of granular tissue, and matrix remodeling) in response to contact with or exposure to a pharmacologically active agent as compared to a untreated test control or animal. As used herein, "wound healing disorders" are diseases or disorders whose pathogenesis is caused by, is related to, or is associated with aberrant CB 1 and CB2 receptor protein function or expression.

"Aberrant expression," as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

As used herein, the term "subject," can refer to a mammal, such as a human, or to an experimental animal or disease model. The subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.

The term "modulates" as used herein refers to the inhibition, reduction, increase or enhancement of CBl and CB2 receptor function, expression, activity, or alternatively a phenotype associated with a disruption in the CB 1 and CB2 receptor genes. The term "modulator" refers to agents that act as agonists, inverse agonists or antagonists of the CB2 and/or CBl receptors. As used herein, the term "treatment" refers to the application or administration of a therapeutic agent to a subject (i.e., patient) having a wound, or to a patient about to undergo, undergoing, or having recently undergone an invasive surgical procedure. The term "treatment" also refers to the application or administration of a therapeutic agent to an isolated tissue or cell line from a patient who has a wound healing disorder or condition, a symptom of disease, or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of disease, or the predisposition toward disease.

As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a therapeutic agent and a pharmaceutically acceptable carrier. As used herein, "pharmacologically effective amount," "therapeutically effective amount" or simply "effective amount" refers to that amount of an agent effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a condition or disorder, a therapeutically effective amount of a drug for the treatment of that condition or disorder is the amount necessary to effect at least a 25% reduction in that parameter.

The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

As used herein, the term "gene" refers to a gene containing at least one of the CBl or CB2 receptor DNA sequences disclosed herein; any DNA sequence that encodes the amino acid sequences encoded by the DNA sequences disclosed herein; and/or any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein. The term includes coding as well as noncoding regions such as sequences necessary for normal gene expression, including promoters, enhancers and other regulatory sequences.

As used herein, the terms "polynucleotide and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes single-, double-stranded and triple helical molecules. "Oligonucleotide" refers to polynucleotides of between 5 and about 100 nucleotides of single- or

double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A "primer" refers to an oligonucleotide, usually single-stranded, that provides a 3'-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis. Polynucleotides include, without limitation, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6- isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1 -methyl guanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.

The term "functional variants" of a polypeptide within the meaning of the present invention includes polypeptides which are regulated, for example, like the polypeptides used according to the invention during disease, or in regenerative processes of the skin, but in particular in wound-healing disorders. Functional variants, for example, also include polypeptides which are encoded by a nucleic acid which is isolated from non-skin-specific tissue, e.g. embryonic tissue, but after expression in a cell involved in wound healing or skin disease have the designated functions.

Functional variants within the meaning of the present invention are also polypeptides that share a sequence homology, in particular a sequence identity, with the CBl and CB2 polypeptides described herein. The terms "homology" and "homologous" as used herein denote a characteristic of a DNA sequence having at least about 70 percent sequence identity as compared to a reference sequence, typically at least about 85 percent sequence identity, preferably at least about 95 percent sequence identity, and more preferably about 98 percent sequence identity, and most preferably about 100 percent sequence identity as compared to a reference sequence. Homology can be determined using a "BLASTN" algorithm. It is understood that homologous sequences can accommodate insertions, deletions and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide

residues do not precisely correspond or align. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome.

A "fragment" of a polynucleotide is a polynucleotide comprised of at least 9 contiguous nucleotides, preferably at least 15 contiguous nucleotides and more preferably at least 45 nucleotides, of coding or non-coding sequences.

The term "gene targeting" refers to a type of homologous recombination that occurs when a fragment of genomic DNA is introduced into a mammalian cell and that fragment locates and recombines with endogenous homologous sequences. The term "homologous recombination" refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of homologous nucleotide sequences.

The terms "target gene," "target gene sequence," "target DNA sequence" and "target sequence" refer to any nucleic acid molecule or polynucleotide of any gene to be modified by homologous recombination. The target sequence includes an intact gene, an exon or intron, a regulatory sequence or any region between genes. The target gene comprises a portion of a particular gene or genetic locus in the individual's genomic DNA. In the context of the present invention, the target genes are the Cnrl and Cnr2 genes encoding CBl and CB2 receptors, respectively. A "CBl receptor gene" and "CB2 receptor gene" as used herein refer to the genes Cnrl and Cnr2, and sequences comprising SEQ ID NOS: 1 and 3, respectively, i.e., comprising the sequences identified in GenBank as GI or NID number: NM_007726; Accession number: NM_009924.

"Disruption" of a CBl or CB2 receptor gene occurs when a fragment of genomic DNA locates and recombines with an endogenous homologous sequence. These sequence disruptions or modifications may include insertions, missense, frameshift, deletion, or substitutions, or replacements of DNA sequence, or any combination thereof. Insertions include the insertion of entire genes, which may be of animal, plant, fungal, insect, prokaryotic, or viral origin. Disruption, for example, can alter or replace a promoter, enhancer, or splice site of a CBl or CB2 receptor gene, and can alter the normal gene product by inhibiting its production partially or completely or by enhancing the normal gene product's activity. As used herein, the term "transgenic cell" refers to a cell containing within its genome the

CB 1 and CB2 receptor genes that have been disrupted, modified, altered, or replaced completely or partially by the method of gene targeting. The term transgenic cell also refers to a cell

comprising heterologous CBl and/or CB2 receptor genes.

In the context of the present invention, the term "transgenic animal" refers to an animal that contains within its genome a CBl and/or CB2 receptor gene that has been inserted or disrupted using recombinant techniques. The transgenic animal includes both the heterozygote animal (i.e., one defective allele and one wild-type allele) and the homozygous animal (i.e., two defective alleles).

As used herein, the terms "selectable marker" and "positive selection marker" refer to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neo ¹ ) gene are resistant to the compound G418. Cells that do not carry the Neo ^r gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art.

A "host cell" includes an individual cell or cell culture that can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected with the constructs described herein. II. Screening for CB1/CB2 Modulatory Activity

The present invention relates, inter alia, to screening. It includes methods of screening for moieties useful as therapeutic agents or useful in the development of therapeutic agents via drug screening programs. More specifically, the nucleic acid molecules, proteins, and protein homologs described herein can be used to screen for agents for enhancing wound healing and treating wound healing disorders.

In one embodiment, the invention provides methods of evaluating candidate substances for their ability to interact with (e.g., bind) a CBl and/or CB2 polypeptide. The methods include contacting the substance with the subject CBl and/or CB2 polypeptide, and evaluating ability of the substance to interact with (e.g., to bind or form a complex with) the CBl and/or CB2 receptor polypeptides. This method can be performed in vitro, such as in a cell free system or in a two- hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with CBl and CB2 receptors, as well as to find natural or synthetic inhibitors of the CBl and CB2 receptor proteins. Screening methods are discussed in more detail below.

In another embodiment, the invention provides methods of evaluating substances for their

effects on wound healing activities. The methods include the use of in vitro systems (e.g., systems that model different components of the wound healing process) and in vivo models for wound healing, including the CB1/CB2 double knock-out mice described herein, (a) Ligand Binding Assays The invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate substance or test compounds (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) that (1) bind to CBl and CB2 receptor proteins; (2) have a stimulatory or inhibitory effect on, for example, CB1/CB2 protein expression or activity; or (3) have a stimulatory or inhibitory effect on, for example, the expression or activity of a CB1/CB2 protein substrate. Substances thus identified can be used to modulate the activity of target gene products (e.g., CB1/CB2 genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify substances that disrupt normal target gene interactions.

In one embodiment, the invention provides assays for screening candidate substances or compounds that are substrates of CB1/CB2 receptor proteins or polypeptides or biologically active portions thereof. In another embodiment, the invention provides assays for screening candidate substances that bind to or modulate the activity of CB1/CB2 receptor proteins or polypeptides or biologically active portions thereof.

The candidate substances of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non- peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909-13; Erb et al. (1994) Proa Natl. Acad. Sci. U.S.A 91:11422-426; Zuckermann et al. (1994/ J. Med. Chem. 37:2678-85; Cho

et al. (1993) Science 261 :1303; Carrll et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engi. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233-51.

Libraries of compounds can be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555- 556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sd U.S.A 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. MoI. Biol.222:301-310; Ladner supra.). Candidate substances suitable for use in the methods and compositions of the present invention can be agents already known in the art to have a pharmacological activity and/or already known to have CB 1 and/or CB2 modulatory activity.

Numerous compounds have been reported to have CBl and/or CB2 modulatory activity, including, without limitation, SR141716A (N-[piperidin-l-yl]-5-[4-chlorophenyl]-l-[l,2- dichlorophenyl]-4-methyl-lHpyrazole-3-carboxamide HCl); WIN 55212-2(R]-[+]-[2,3-dihydro- 5-methyl-3-{[4-morpholinyl]methyl}pyrrolol[l,2,3-de]l,4-benz oxazin-6-yl]- 1 [naphthalenyl)methanone mesylate); cannabidiol; R(+)-methanandamide, THC (Δ ⁹ - tetrahydrocannabinol); anandamide (arachidonyl ethanolamide); HU-210 ([-]-l 1-OH-Δ ⁹ -THC); 7- (2-chlorophenyl)-8-(4-chlorophenyl)-2-methyl-4-(4-methyl piperazin-l-yl)-pyrazolo[l,5- a] [ 1 ,3,5]triazine; 7-(2-chlorophenyl)-8-(4-chlorophenyl)-2-methyl-4-(4-pyrimidi n-2-ylpiperaz- in- l-yl)pyrazolo[l,5-a][l,3,5]triazine; 7-(2-chlorophenyl)-8-(4-chlorophenyl)-4-[(lS,4S)-5- methanesulfonyl^.S-diazabicyclo^^.ljhept^-ylj^-methylpyrazol otl.S-altl^jSJtriazine; 7-(2- chlorophenyl)-8-(4-chlorophenyl)-2-m-ethyl-4-[4-(propane-2-s ulfonyl)-piperazin-l-yl]- pyrazolo[ 1 ,5-a] [ 1 ,3,5]triazine; 7-(2-chlorophenyl)-8-(4-chlorophenyl)-2-methyl-4-(4- ethanesulfonyl)-piperazin-l-yl)-pyrazolo[l,5-a][l,3,5]triazi ne; 7-(2-chlorophenyl)-8-(4-ch- lorophenyl)-2-methyl-4-piperazin-l-yl-pyrazolo[l,5-a][l,3,5] triazine; 7-(2-chlorophenyl)-8-(4- chlorophenyl)-2-methyl-4-(4-methanesulfonyl)-pipe- razin- 1 -yl)-pyrazolo[ 1 ,5-a] [ 1 ,3,5]triazine; (lS,4S)-5-[7-(2-chlorophenyl)~ 8-(4-chlorophenyl)-2-methylpyrazolo[l,5-a][l,3,5]triazin-4-y l]- 2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester; 7-(2-chlorophenyl)-8-(4- chlorophenyl)-4-[(lS,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl]-2 -methylpyrazolo[l,5- a][l,3,5]triazine; l-{(lS,4S)-5-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2-methyl pyrazolo[l,5- a][l,3,5]triazin-4-yl]-2,-5-diazabicyclo[2.2.1]hept-2-yl}-et hanone; l-{(lS,4S)-5-[7-(2-

chlorophenyl)- -8-(4-chlorophenyl)-2-methylpyrazolo[ 1 ,5-a] [ 1 ,3,5]triazin-4-yl]-2,5- diazabicyclo[2.2.1]hept-2-yl}-2-methylpropan-l-one; l-{(lS,4S)-5-[7-(2-chlorophenyl)-8-(4- chlorophenyl)-2-methylpyrazolo[l,5-a][l,3,5]triazin-4-yl]-2, 5-diazabicyclo[2.2.1]hept-2-yl}- phenylmethanone; 7-(2-chlorophenyl)-8-(4-chlorophenyl)-4-[(lS,4S)-5-ethanesul fonyl-2,5- diazabicyclo[2.2.1]hept-2-yl]-2-m- ethylpyrazolo[l,5-a][l,3,5]triazine; 7-(2-chlorophenyl)-8-(4- chlorophenyl)- -2-methyl-4-[(l S,4S)-5-(propane-2-sulfonyl)-2,5-diazabicyclo[2.2. l]hept-2- yl]- pyrazolo[ 1 ,5-a] [ 1 ,3,5]triazine; and ( 1 S,4S)-5-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2- methylpyrazolo[l,5-a][l,3,5]triazin-4-yl]-2,5-diazabicycl- o[2.2.1]heptane-2-sulfonic acid dimethylamide; l-[7-(2-chlorophenyl)-8-(2,4-dichlorophenyl)-2-methylpyrazol o[ 1 ,5- a][l,3,5]triazin-4-yl]-3-ethylaminoazetidine-3- -carboxylic acid amide; l-[7,8-bis-(2- chlorophenyl)-2-methylpyrazolo[ 1 ,5-a] [ 1 ,3,5]triazin-4-yl]-3-ethylaminoazetidine-3-carboxylic acid amide; 1 -[7-(2-chlorophenyl)-8-(4-cyanophenyl)-2-methylpyrazolo[ 1 ,5-a] [ 1 ,3,5]triazin-4- yl]-3-ethylaminoazetidine-3-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-methylphenyl)-2- methylpyrazolo[l,5-a][l,3,5]triazin-4-yl]-3-ethylaminoazetid ine-3-carboxylic acid amide; l-[7- (2-chlorophenyl)-8-(4-ethylphenyl)-2-methylpyrazolo[ 1 ,5-a] [ 1 ,3,5]triazin-4-yl]-3- ethylaminoazetidine-3-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-methoxyphenyl)-2- methylpyrazolo[l,5-a][l,3,5]triazin-4-yl]-3-ethylaminoazetid ine-3-carboxylic acid amide; l-[7- (2-chlorophenyl)-8-(3-chlorophenyl)-2-methylpyrazolo[l,5-a][ l,3,5]triazin-4-yl]-3- ethylaminoazetidine-3-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2- methylpyrazolo[l,5-a][l,3,5]triazin-4-yl]-4-methylaminopiper idine-4-carboxylic acid amide; 1- [7-(2-chlorophenyl)-8-(4-fluorophenyl)-2-methylpyrazolo[ 1 ,5-a] [ 1 ,3,5]triazin-4-yl]-4- ethylaminopiperidine-4-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2- methylpyrazolotl^-ajfl^^jtriazin^-ylj^-ethylaminopiperidine^ -carboxylic acid amide; l-[7- (2-chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[ 1 ,5-a] [ 1 ,3 ,5]triazin-4-yl] -4- isopropylaminopiperidine-4-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2- methylpyrazolo[l,5-a][l,3,5]triazin-4-yl]-3-ethylaminoazetid ine-3-carboxylic acid amide; l-[7- (2-chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[l,5-a][ l,3,5]triazin-4-yl]-3- isopropylaminoazetidine-3-carboxylic acid amide; 3-amino-l-[7-(2-chlorophenyl)-8-(4- chlorophenyl)-2-methylpyrazolo[l,5-a][- l,3,5]triazin-4-yl]-azetidine-3-carboxylic acid amide; 1- [7-(2-chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[l,5- a][l,3,5]triazin-4-yl]-3- methylaminoazetidine-3-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2- methylpyrazolotljS-aJtljS.SJtriazin^-ylJ-S-dimethylaminoazet idine-S-carboxylic acid amide; 1-

[T^-chlorophenyO-S^-chlorophenyOpyrazolofljS-aJtl.S^Jtriazin -^y-lj-S- isopropylaminoazetidine-3-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)- pyrazolo[l,5-a][l,3,5]triazin-4-yl]-4-ethylaminopiperidine-4 -carboxylic acid amide; l-[7-(2- chlorophenyl)-8-(4-chlorophenyl)-pyrazolo[l,5-a][l,3,5]triaz in-4— yl]-3-ethylaminoazetidine-3- carboxylic acid amide; and l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)pyrazolo[l,5- a][l,3,5]triazin-4-y- l]-3-methylaminoazetidine-3-carboxylic acid amide; l-[7-(2-chlorophenyl)- 8-(4-chloro- phenyl)-2-methylpyrazolo[ 1 ,5-a] [ 1 ,3,5]triazin-4-yl]-4-ethylaminopiperidine- -4- carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[l, 5- a][l,3,5]triazin-4-yl]-3-ethylaminoazetidine-3-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4- chlorophenyO-l-methylpyrazolotljS-altljSjSJtriazin^-ylJ-S-is opropylaminoazetidine-S- carboxylic acid amide; 3-amino-l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2-methylpyr azolo[l,5- a][- l,3,5]triazin-4-yl]-azetidine-3-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4- chlorophenyO-l-methylpyrazolotljS^tl.SjSjtriazin^-yπ-S-meth ylaminoazetidine-S-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)pyrazolo[l,5-a][l,3, 5]triazin-4-y- l]-3- isopropylaminoazetidine-3-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)- pyrazolo[l,5-a][l,3,5]triazin-4- yl]-4-ethylaminopiperidine-4-carboxylic acid amide; and l-[7- (2-chlorophenyl)-8-(4-chlorophenyl)-pyrazolo[l,5-a][l,3,5]tr iazin-4~ yl]-3-ethylaminoazetidine- 3-carboxylic acid amide; l-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[l, 5- a][l,3,5]triazin-4-yl]-3-ethylaminoazetidine-3-carboxylic acid amide; Preferred compounds include: 2-[7-(2-chlorophenyl)-8-(4-chloropheny- l)-2-methylpyrazolo[l,5-a][l,3,5]triazin-4-yl]- 5-methyl-2,5,7-triazaspiro[- 3.4]octan-8-one; 2-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2- methylpyrazolo- [ 1 ,5-a][ 1 ,3,5]triazin-4-yl]-2,5,7-triazaspiro[3.4]octan-8-one; 8-[7-(2- chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[ 1 ,5-a][ 1 ,3,5]triazin-4-yl]- 1 -isopropyl- 1 ,3,8- triazaspiro[4.5]decan-4-one; and 2-[7-(2-chlorophenyl)-8-(4-chlorophenyl)-2- methylpyrazolo[l,5-a][l,3,5]triazin-4-yl]-6,6-dimethyl-2,5,7 -triazaspiro[3.4]octan-8-one; 8-[7-(2- chlorophenyl)-8-(4-chlorophenyl)-2 -methyl- pyrazolo[l,5-a][l,3,5]triazin-4-yl]-l -isopropyl- 1,3, 8- triazaspiro[4.5]decan-4-one; -[5-(4-chloro-phenyl)- 1 - -(2-chloro-phenyl)-4-methyl- lH-pyrazol-3- yl]-2-piperidin-l -yl-ethanone; 1 -[5-(4-chloro-phenyl)- 1 -(2-chloro-phenyl)-4-methyl- 1 H-pyrazol- 3-yl]-2-moφholin-4-yl-ethanone; 1 -[5-(4-chloro-phenyI)- 1 -(2-chloro-phenyl)-4-methyl- - 1 H- pyrazol-3-yl]-2-[4-(l-methyl-lH-pyrrole-2-carbonyl)-piperazi n-l-yl]-ethanone; l-[5-(4-chloro- phenyl)- 1 -(2-chloro-phenyl)-4-methyl- 1 H-pyrazol-3-y- l]-2-[4-( 1 -methyl -cyclopropanecarbonyl)- piperazin-l-yl]-ethanone; N-(l-{2-[5-(4-chloro-phenyl)-l-(2-chloro-phenyl)-4-methyl-lH -

pyrazol-3-yl- ]-2-oxo-ethyl}-piperidin-4-yl)-2,2,2-trifluoro-acetamide; l-[5-(4-chloro-phenyl)-l- (2-fluoro-phenyl)-4-methyl-lH-pyrazol-3-yl]-2-moφholin-4-yl -ethanone; l-[5-(4-chloro-phenyl)- l-(2-fluoro-phenyl)-4-methyl- -lH-pyrazol-3-yl]-2-piperidin-l-yl-ethanone; l-[5-(4-chloro- phenyl)- 1 -(2-fluoro-phenyl)-4-methyl- 1 H-pyrazol-3-yl]-2-(4-trifluoroacetyl-piperazin- 1 -y- I)- ethanone; l-[l-(2-chloro-phenyl)-5-(4-chloro- phenyl)-4-methyl-lH-pyrazol-3-yl]-2-pyrrolidin-l- yl-ethanone; 1 -[ 1 -(2-chloro- phenyl)-5-(4-chloro-phenyl)-4-methyl- lH-pyrazol-3-yl]-2- [l,4]oxazepan-4-yl- ethanone; and l-[5-(4-chloro-phenyl)-l-(2-chloro-phenyl)-4-methyl-lH- pyrazol-3-yl]-2-( 1 -oxa-8-aza-spiro[4.5]dec-8-yl)-ethanone; 2-(benzyl-isopropyl-amino)- 1 -[ 1 -(2- chloro-phenyl)-5-(4-chloro-phenyl)-4-methyl-lH-pyrazol-3-yl] -ethanol; l-[5-(4-chloro-phenyl)-l- (2-chloro-phenyI )-4-methyl-lH-pyrazol-3-y- l]-2-(3,5-dimethyl-piperidin-l-yl)-ethanol; l-{2-[l- (2-chloro-phenyl)-5-(4— chloro-phenyl)-4-methyl-lH-pyrazol-3-yl]-2-hydroxy-ethyl}-4- isopropylamin-o-piperidine-4-carboxylic acid amide; l-[5-(4-chloro-phenyl)-l-(2-chloro-phenyl)- 4-methyl-lH-pyrazol-3-yl]-2-(3,3-dimethyl-piperidin-l-yl)-et hanol; l-[5-(4-chloro-phenyl)-l-(2- chloro-phenyl)-4-methyl-lH-pyrazol-3-yl]-2-pi- peridin-1-yl-ethanol; and l-[5-(4-chloro-phenyl)- l-(2-chloro-phenyl)-4-methyl-lH-pyrazol-3-yl]-2-moφholin-4- yl-ethanol; 2-[5-(4-chloro- phenyl)-l-(2-chloro-phenyl)-4-methyl-lH-pyrazol-3-yl]-4-cycl ohexyl-moφholine; 2-[5-(4- chloro-phenyl)- 1 -(2-chloro-phenyl)-4-methyl- 1 -H-pyrazol-3-yl]-4-(propane-2-sulfonyl)- moφholine; 2-[5-(4-chloro-phenyl)— l-(2-chloro-phenyl)-4-methyl-lH-pyrazol-3-yl]-4-(toluene- 4-sulfonyl)-moφholine; l-{2-[l-(2-chloro-phenyl)-5-(4-chloro-phenyl)-4-methyl-lH-py razol-3- - yl]-moφholin-4-yl}-2-methyl-propan-l-one; and 2-[l-(2-chloro-phenyl)-5-(- 4-chloro-phenyl)-4- methyl-lH- pyrazol-3-yl]-4-(4-trifluoromethyl-benzyl)-moφholine; l-[l-(4-chloro-phenyl)-2- (2,4-dichloro-phenyl)-5-methyl-lH-imidazol-4-yl]- -2-piperidin-l-yl-ethanone and l-[l-(4- chloro-phenyl)-2-(2,4-dichloro-phenyl)-5-methyl-lH-imidazol- 4-yl]-2-moφholin-4-yl-ethanone; 3-(4-chlorophenyl)-2-(2-chlorophenyl)— 5-methyl-7-(4-methylpiperazin-l-yl)-pyrazolo[l,5- a]pyrimidine; 3-(4-chlorophenyl)-2-(2-chlorophenyl)-5-methyl-7-(4-pyrimidi n-2-yl-pipera- zin- 1 - yl)-pyrazolo[l,5-a]pyrimidine; 3-(4-chloro-phenyl)-2-(2-chlorophenyl- )-7-[(lS,4S)-5- methanesulfonyl^jS-diazabicycloP^.lJhept^-yll-S-methylpyrazo lofljS-aJpyrimidine; 3-(4- chlorophenyl)-2-(2-chlorophenyl)-5-methyl-7- -[4-(propane-2-sulfonyl)-piperazin- 1 -yl]- pyrazolo[ 1 ,5-a]pyrimidine; 3-(4-chlorophenyl)-2-(2-chlorophenyl)-7-(4-ethanesulfonyl-pi perazin- 1-yl)- -5-methylpyrazolo[l,5-a]pyrimidine; 3-(4-chlorophenyl)-2-(2-chlorophenyl)— 7-(4- methanesulfonylpiperazin- 1 -yl)-5-methylpyrazolo[ 1 ,5-a]pyrimidine; 1 -{4-[3-(4-chlorophenyl)-2- (2-chlorophenyl)-5-methylpyrazolo[l,5-a]pyrimidin-7-yl]-pipe razin-l-yl}-ethanone; 4-[3-(4-

chlorophenyl^-^-chloropheny- l)-5-methylpyrazolo[l,5-a]pyrimidin-7-yl]-piperazine-l- carboxylic acid tert-butyl ester; 3-(4-chlorophenyl)-2-(2-chlorophenyl)-5-methyl-7-[(lS,4- S)-5- (propane-2-sulfonyl)-2,5-diazabicyclo[2.2.1]hept-2-yl]-pyraz olo[l,5-a- ]pyrimidine; 1-{(1S,4S)- 5-[3-(4-chlorophenyl)-2-(2-chlorophenyl)-5-methylpyrazolo[l, 5-a]pyrimidin-7-yl]-2,5- diazabicyclo[2.2.1]hept-2-yl}-ethanone; and (lS,4S)-5-[3-(4-chlorophenyl)-2-(2-chlorophenyl)-5- methylpyrazolo[l,5- -a]pyrimidin-7-yl]-2,5-diazabicyclo[2.2.1]heptane-2-carboxyl ic acid tert- butyl ester. Someone of skill in the art can determine without undue burden, whether any of the indicated substances is a agonist, antagonist or reverse agonist of the CB 1 receptor and/or CB2 receptor. Based on this determination the above indicated compounds might be more suitable to be used in a method of treating excessive wound healing as observed, e.g. in keloid formation (agonists) or in the treatment of diseases with a reduced wound healing, e.g. as in Diabetes mellitus (antagonists and reverse agonists)

Several of these compounds have already been determined to be agonists of the CBl receptor and/or the CB2 receptor. Particularly preferred agonists usable in the methods of the present invention are endocannabinoids, such as anandamide, 2-arachidonoyl-glycerol, palmitoyl- ethanolamide, docosatetraenylethanolamide, Homo-g-linoenylethanolamide and oleamide. Also natural cannabinoids, such as cannabinol, cannabidiol, delta 8-THC, and Δ ⁹ -tetrahydrocannbinol (THC), are known as agonists for CBl and/or CB2 receptors. Moreover, alkylamides from Echinacea extracts may act as cannabinoids. Further preferred compounds which may act as agonists include R(+)-methanandamide, arachidonyl-2-chloroethylamide (ACEA), (R)-(+)-[2,3- dihydro-5-methyl-3-{[4-moφholinyl]methyl}pyrrolol[l,2,3-de] l,4-benzoxazin-6-yl]- 1 [naphthalenyl)methanone mesylate (WIN 55212-2), l-propyl-2-methyl-3-(l-naphthoyl)indole (JWH-015), 1 -(2,3-dichlorobenzoyl)-2-methyl-3-(2-[ 1 -morpho-lino]ethyl)-5-methoxyindole

(L768242), the l-methoxy-Delta(8)-THC derivative l-methoxy-Delta(8)-THC-DMH (L759633), l-methoxy-Delta(9(l l))-THC-DMH (L759656), l-methoxy-3-(l',l ^l -dimethylhexyl)-Delta(8)- THC (JWH-229), l-deoxy-3-(l',l ^I -dimethylbutyl)-Delta(8)-THC (JWH- 133), (-)-7'- isothiocyanato-11 -hydroxy- l',l'-dimethylheptylhexahydrocannabinol (AM841), (lR,3R,4R)-3-[2- hydroxy-4-(l,l-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyc lohexan-l-ol (CP55940), 11- hydroxy- T, 1 '-dimethylheptylhexahydrocannabinol (AM4056), (-)-7'-bromo- 11 -hydroxy- 1 ', 1 '- dimethylheptylhexahydrocannabinol (AM4043), [-]-l l-OH-Δ ⁹ -THC (HU-210), HU-308, HU- 320, (2-iodo-5-nitro-phenyl)-[l-(l-methyl-piperidin-2-ylmethyl)-l H-indol-3-yl]-methanone

(AM1241), l-(2,3-Dichlorobenzoyl)-5-methoxy-2-methyl-(2-(moφholin-4-y l)ethyl)-lH-indole

(GW405833).

Several synthetic compounds are known which function as antagonists or reverse agonists for the CBl receptor and/or the CB2 receptor. Among these antagonists/reverse agonists are 6'- azidohex-cis-2'-ene-Delta8-tetrahydrocannabinol (0-1238), 6'-azidohex-2'-yne-Delta8- tetrahydrocannabinol (0-1184), methyl arachidonyl fluorophosphonate (MAFP), [N-(piperidin-l- yO-S^-chloropheny^-l^^-dichlorophenyO^-methyl-lH-pyrazole-S- carboxamide HCl]

(SR141716A), N-[(lS)-endo-l,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl]-5-(4-chloro-3- methylphenyl)-l-(4-methylbenzyl)-pyrazo le-3-carboxamide] (SR144528), N-(Piperidin-l-yl)-5- (4-iodophenyl)-l-(2,4-dichlorophenyl)-4-methyl-lH-pyrazole-3 -carboxamide (AM251), l-(2,4- Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-moφholinyl-lH -pyrazole-3-carboxamide

(AM281), [6-methoxy-2-(4-methoxyphenyl)benzo[b]-thien-3-yl][4-cyanoph enyl] methanone (LY320135), and 6-iodo-pravadoline (AM630). SR141716 is known as a CBl receptor antagonist and SR 144528 and JTE-907 are known as a CB2 receptor antagonist.

In one embodiment, an assay is a cell-based assay in which a cell which expresses CB1/CB2 receptor proteins or biologically active portions thereof is contacted with at least one candidate substance, and the ability of the candidate substance(s) to modulate CB1/CB1 receptor protein activity is determined. Determining the ability of the candidate substance(s) to modulate CB1/CB1 receptor protein activity can be accomplished by monitoring a variety of properties, for example, the ability of CBl and CB2 proteins to (1) bind their natural ligands such as chemokines; (2) interact with a G protein; (3) transduce a signal through a membrane; (4) affect second messenger levels in the cells; and (5) stimulate the cell to divide or migrate. The cell, for example, can be of mammalian origin, e.g., human.

The ability of the candidate substance(s) to modulate CBl and CBl protein binding to a compound, e.g., a substrate, or to bind to CBl and CB2 proteins can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to CB1/CB2 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, CB1/CB2 could be coupled with a radioisotope or enzymatic label to monitor the ability of a candidate substance to modulate CB1/CB2 binding to a substrate in a complex. For example, compounds (e.g., CB1/CB2 substrates) can be labeled with ¹²⁵ I, ¹⁴ C, ³⁵ S or ³ H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish

peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

The ability of a compound (e.g., a CB1/CB2 substrate) to interact with CB1/CB2 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with CB1/CB2 without the labeling of either the compound or the CB1/CB2 protein. (McConnell et al. (1992) Science 257:1906-1912.) As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light- addressable potentiometric sensor ("LAPS"). Changes in this acidification rate can be used as an indicator of the interaction between a compound and CB1/CB2 protein.

In yet another embodiment, a cell-free assay is provided in which CB1/CB2 proteins or biologically active portions thereof are contacted with a candidate substance and the ability of the candidate substance to bind to the CB1/CB2 protein or biologically active portions thereof is evaluated. Soluble and/or membrane-bound forms of isolated proteins (e.g., CB1/CB2 proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n- dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N- methylglucamide, Triton® X-IOO, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether) _n , 3-[(3-cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-l-propane sulfonate (CHAPSO), or N- dodecyl=N,N-dimethyl-3-ammonio- 1 -propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target gene protein and the candidate substance 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.

The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer ("FET") (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; and Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, "donor" molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, "acceptor" molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the "donor" protein molecule can simply utilize the natural fluorescent

energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the "acceptor" molecule label can be differentiated from that of the "donor." Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the "acceptor" molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determining the ability of the CB1/CB2 proteins to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis ("BIA") (see, e.g., Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance ("SPR")), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

In one embodiment, the target gene product or the candidate substance is anchored onto a solid phase. The target gene product/candidate substance complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the candidate substance (which is not anchored) can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize either CBl /CBl proteins, anti-CBl and anti-CB2 antibodies or their target molecules to facilitate separation of complexed from uncomplexed forms of the proteins, as well as to accommodate automation of the assay. Binding of a candidate substance to CB1/CB2 proteins, or interaction of CB1/CB1 proteins with a target molecule(s) in the presence and absence of a candidate compound(s), can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione- S-transferase/target fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the candidate substance or the

candidate substance and either the non-adsorbed target protein or CB1/CB2 proteins, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of CB1/CB2 binding or activity determined using standard techniques.

Other techniques for immobilizing either CB1/CB2 proteins or target molecules on matrices include using conjugation of biotin and streptavidin. Biotinylated CB1/CB2 proteins or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

In order to conduct the assay, 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 or selective for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactive with CB1/CB2 proteins or target molecules but which do not interfere with binding of the CB 1/CB2 proteins to their target molecules. Such antibodies can be derivatized to the wells of the plate, and unbound targets or CB1/CB2 proteins trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the CB1/CB2 proteins or target molecules, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the CB 1/CB2 proteins or target molecules.

Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard

techniques, including but not limited to differential centrifugation (see, for example, Rivas and Minton (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley, New York.); and immunoprecipitation (see, for example, Ausubel et al., eds. (1999J Current Protocols in Molecular Biology, J. Wiley, New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard ( 1998) J MoI Recognit 11:141-8; Hage and Tweed ( 1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer can also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution. In one embodiment, the assay includes contacting the CB1/CB2 proteins or biologically active portions thereof with a known compound(s) that binds CB1/CB2 to form an assay mixture, contacting the assay mixture with a candidate substance, and determining the ability of the candidate substance(s) to interact with CB1/CB2 proteins, wherein determining the ability of the candidate substance(s) to interact with CB1/CB2 proteins includes determining the ability of the candidate substance(s) to preferentially bind to CB1/CB2 or biologically active portions thereof, or to modulate the activity of a target molecule, as compared to the known compound(s). Exemplary compounds known to interact with or bind to CBl and CB2 proteins are described elsewhere herein.

The target gene products (i.e., CB1/CB2 receptor proteins) can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as "binding partners." Compounds that disrupt such interactions can be useful in regulating the activity of the target gene products. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the CB1/CB2 genes described herein. In an alternative embodiment, the invention provides methods for determining the ability of the candidate substance(s) to modulate the activity of CB1/CB2 proteins through modulation of the activity of a downstream effector(s) of the CB1/CB2 target molecules. For example, the activity of the effector molecule(s) on an appropriate target can be determined, or the binding of the effector(s) to an appropriate target can be determined, as previously described.

To identify compounds that interfere with the interaction between the target gene products and their cellular or extracellular binding partner(s), a reaction mixture containing the target gene

products and the binding partner(s) is prepared, under conditions and for a time sufficient, to allow the products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the candidate substance(s). The candidate substance(s) can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target genes and their cellular or extracellular binding partners. Control reaction mixtures are incubated without the candidate substance or with a placebo. The formation of any complexes between the target gene products and the cellular or extracellular binding partner(s) is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the candidate substance, indicates that the candidate substance interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the candidate substance and normal target gene product can also be compared to complex formation within reaction mixtures containing the candidate substance and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify substances that disrupt interactions of mutant but not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product(s) or the binding partner(s) onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the substances being tested. For example, candidate substances that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the candidate substance. Alternatively, candidate substances that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the candidate substance to the reaction mixture after complexes have been formed. The various formats are briefly described below

In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific or selective for the species to be anchored can be used to anchor the species to the solid

surface.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the candidate substance. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific or selective for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, candidate substances that inhibit complex formation or that disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the candidate substance, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific or selective for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific or selective for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, candidate substances that inhibit complex or that disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a candidate substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, candidate substances that disrupt target gene product-binding partner interaction can be identified.

In yet another aspect, the CB1/CB2 proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-

232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300) to identify other proteins that bind to or interact with CB1/CB2 proteins ("CBl/CB2-binding proteins" or "CBl/CB2-bp") and are involved in CB1/CB2 receptor activity. Such CBl/CB2-bps can be

activators or inhibitors of signals by the CB1/CB2 proteins or CB1/CB2 targets as, for example, downstream elements of CBl/CB2-mediated signaling pathways.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a CB protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the CB1/CB2 proteins can be fused to the activator domain.) If the "bait" and the "prey" proteins are able to interact in vivo forming CBl/CB2-dependent complexes, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., IacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein(s) that interacts with the CB1/CB2 receptor proteins.

In another embodiment, modulators of CB1/CB2 expression are identified. For example, a cell or cell free mixture is contacted with a candidate substance and the expression of CB1/CB2 mRNA or protein evaluated relative to the level of expression of CB1/CB26 mRNA or protein in the absence of the candidate substance. When expression of CB1/CB2 mRNA or protein is greater in the presence of the candidate substance than in its absence, the candidate substance is identified as a stimulator of CB1/CB2 mRNA or protein expression. Alternatively, when expression of CB1/CB2 mRNA or protein is less (statistically significantly less) in the presence of the candidate substance than in its absence, the candidate substance is identified as an inhibitor of CB1/CB2 mRNA or protein expression. The level of CB1/CB2 mRNA or protein expression can be determined by methods described herein for detecting CB1/CB1 mRNA or protein.

In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of CB1/CB2 proteins can be confirmed in vivo, e.g., in an animal such as an animal model for aberrant CB1/CB2 receptor function or expression.

This invention further pertains to novel agents for enhancing wound healing and treating

wound healing disorders identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a CBl and CB2 modulating agent, an antisense CB1/CB2 nucleic acid molecule, a CB1/CB2- specific antibody, or a CBl/CB2-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

(a) Functional Assays

The present invention also relates to the use of CB 1 and CB2 polypeptides, nucleic acid molecules encoding the CB1/CB2 polypeptides, cells expressing the CB1/CB2 polypeptides, and antibodies or antibody fragments directed against the CBl and CB2 polypeptides for functional assays (also referred to herein as "physiological assays") of wound healing.

In general, agents that promote a more rapid influx of fibroblasts, endothelial and epithelial cells into wounds should increase the rate at which wounds heal. Substances that are useful in treating wound healing can be identified and tested in a number of in vitro and in vivo models. Substances identified using the ligand binding assays described above may be further assays for wound healing activities using any of a number of well known methods, including those described below and in the Examples hereof.

In vitro systems model different components of the wound healing process, for example the return of cells to a "wounded" confluent monolayer of tissue culture cells, such as fibroblasts

(Verrier et al., 1986), endothelial cells (Miyata et al., 1990) or epithelial cells (Kartha et al., 992).

Other systems permit the measurement of endothelial cell migration and/or proliferation (Muller et al., 1987; Sato et al., 1988).

In vivo models for wound healing are also well-known in the art, including wounded pig epidermis (Ohkawara et al., 1977) or drug-induced oral mucosal lesions in the hamster cheek pouch (Cherrick et al., 1974).

A suitable in vitro system can be produced, for example, by the stable transformation of epidermal or dermal cells with expression vectors which contain selectable marker genes and CB1/CB2 nucleic acids. The expression of the CB1/CB2 nucleic acids can be manipulated (disrupted or overexpressed) such that they correspond to the pathologically disturbed expression in vivo. For wound healing disorders, pathological behavior of the cells in vivo can thus be mimicked, and agents identified which reproduce the non-pathological state. Anti-sense

oligonucleotides corresponding these the CB1/CB2 nucleic acids described herein (or disrupted nucleic acids) can also be employed for this purpose.

A number of cells lines and expression vectors suitable for use in the in vitro test systems described herein are available in the art, including, for example, HaCaT cells and the expression vector pCMV4 (Anderson et al., 1989, J. Biol. Chem. 264: 8222-9). The CB1/CB2 nucleic acids as described above can be integrated into the expression vectors, both in the sense and in the anti- sense orientation, such that the functional concentration of mRNA of the corresponding genes in the cells is either increased, or is decreased by hybridization with the antisense RNA. After the transformation and selection of stable transformants, the cells in culture in general show an altered proliferation, migration and/or differentiation behavior in comparison with control cells. This behavior in vitro often correlates with the function of the corresponding genes in regenerative processes in the body (Yu et al., 1997, Arch. Dermatol. Res. 289: 352-9; Mils et al., 1997, Oncogene 14: 15555-61; Charvat et al., 1998, Exp Dermatol 7: 184-90; Werner, 1998, Cytokine Growth Factor Rev. 9: 153-65; Mythily et al., 1999, J. Gen. Virol. 80: 1707-13;) and can be detected using tests which are simple and rapid to carry out, such that test systems for pharmacologically active substances based thereon can be constructed. Thus, the proliferation behavior of cells can be detected very rapidly by, for example, the incorporation of labeled nucleotides into the DNA of the cells (see, for example, Savino and Dardenne, 1985, J. Immunol. Methods 85: 221-6; Perros and Weightman, 1991, Cell Prolif. 24: 517-23; Fries and Mitsuhashi, 1995, J. Clin. Lab. Anal. 9: 89-95), by staining the cells with specific stains (Schulz et al., 1994, J. Immunol. Methods 167: 1-13) or by means of immunological processes (Frahm et al., 1998, J. Immunol. Methods 211 : 43-50). The migration can be detected simply by the migration index test (Charvat et al., supra) and comparable test systems (Benestad et al., 1987, Cell Tissue Kinet. 20: 109-19, Junger et al., 1993, J. Immunol. Methods 160: 73-9). Suitable differentiation markers are, for example, keratin 6, 10 and 14 and also loricrin and involucrin (Rosenthal et al., 1992, J. Invest. Dermatol. 98: 343-50), whose expression can be easily detected, for example, by means of generally obtainable antibodies.

III. Pharmaceutical Compositions Compounds that modulate expression or activity of CB1/CB2 receptor activities

(antagonists, inverse agonists, inhibitors, activators, and agonists), also referred to herein as "modulators," "active compounds," "active agents," and "pharmacologically active agents"), that

have been identified by the screens and assays described above, can be incorporated into pharmaceutical compositions. Such compositions typically include the active compound(s) and a pharmaceutically acceptable carrier.

The present invention encompasses agents that modulate expression or activity of CB1/CB2 receptor proteins. An agent can, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,. including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of

microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active agent can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the active agents are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such

as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active agents are formulated into ointments, salves, gels, or creams as generally known in the art.

The active agents can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active agents are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅ 0 (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 LD ₅₀ /ED ₅₀ . Active agents that exhibit high therapeutic indices are preferred. While active agents that exhibit toxic side effects can be used, care should

be taken to design a delivery system that targets such agents 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 culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC ₅₀ (i.e., the concentration of the candidate substance 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 can be measured, for example, by high performance liquid chromatography.

Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

Advances in rodent genetics have generated a number of rodent models for the study of various human diseases, including diseases characterized by wound healing disorders (e.g., rat models are available for diabetes (Brown et al., J. Surg. Res. (1994) 56:562-270). Such models are useful for studying the effects of modulators of CB1/CB2 receptor activity on wound healing, and thus are useful for in vivo testing of the active agents of the present invention as well as for

determining a therapeutically effective dose. Mouse repositories can be found, for example, at: The Jackson Laboratory, Charles River Laboratories, Taconic, Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Network and at the European Mouse Mutant Archive.

In other embodiments of the invention, the nucleic acids and polypeptides, fragments thereof, as well as anti-CBl/CB2 antibodies of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecules, proteins, or antibodies and a pharmaceutically acceptable carrier.

As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody, unconjugated or conjugated as described herein, can include a single treatment or, preferably, can include a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). Generally, partially human antibodies and fully human antibodies have a longer half- life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration. A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. U.S.A 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can

comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. In a preferred embodiment the present invention is directed at the use of an agonist of the

CBl receptor and/or the CB2 receptor for the preparation of a pharmaceutical composition for modulating wound healing or treating a wound healing disorder characterized by excessive wound healing, e.g. proliferation of kerationzytes and/or connective tissue, which might lead to excessive scarring, keloid formation and tissue necrosis etc. In these embodiments the agonist of the CBl receptor and/or the CB2 receptor may be selected from the group consisting of anandamide, 2-arachidonoyl-glycerol, palmitoyl-ethanolamide, docosatetraenylethanolamide, Homo-g-linoenylethanolamide and oleamide. Also natural cannabinoids, such as cannabinol, cannabidiol, delta 8-THC, and Δ ⁹ -tetrahydrocannbinol (THC), are known as agonists for CBl and/or CB2 receptors and it is similarly preferred to use these agonists for the preparation of a pharmaceutical composition for the treatment of wound healing. Moreover, alkylamides from Echinacea extracts, which act as cannabinoids can be used. Further agonists include R(+)- methanandamide, arachidonyl-2-chloroethylamide (ACEA), (R)-(+)-[2,3-dihydro-5-methyl-3-{[4- moφholinyl]methyl}pyrrolol[l,2,3-de]l,4-benzoxazin-6-yl]-l[ naphthalenyl)methanone mesylate (WIN 55212-2), l-propyl-2-methyl-3-(l-naphthoyl)indole (JWH-015), l-(2,3-dichlorobenzoyl)-2- methyl-3-(2-[l-morpho-lino]ethyl)-5-methoxyindole (L768242), the l-methoxy-Delta(8)-THC derivative l-methoxy-Delta(8)-THC-DMH (L759633), l-methoxy-Delta(9(l l))-THC-DMH (L759656), l-methoxy-3-(l',l'-dimethylhexyl)-Delta(8)-THC (JWH-229), l-deoxy-3-(l',r- dimethylbutyl)-Delta(8)-THC (JWH-133), (-)-7'-isothiocyanato-l 1 -hydroxy- l',l'- dimethylheptylhexahydrocannabinol (AM841), (lR,3R,4R)-3-[2-hydroxy-4-(l, 1- dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-l-ol (CP55940), 11 -hydroxy- 1',T- dimethylheptylhexahydrocannabinol (AM4056), (-)-7'-bromo- 11 -hydroxy- 1',T- dimethylheptylhexahydrocannabinol (AM4043), [-]-l l-OH-Δ ⁹ -THC (HU-210), HU-308, HU- 320, (2-iodo-5-nitro-phenyl)-[l-(l-methyl-piperidin-2-ylmethyl)-l H-indol-3-yl]-methanone

(AM1241), and l-(2,3-Dichlorobenzoyl)-5-methoxy-2-methyl-(2-(moφholin-4-y l)ethyl)-lH- indole (GW405833).

In these embodiments, the wound healing disorder is preferably selected from the group consisting of treatment of excessive scarring, keloide formation, and tissue fibrosis in response to

wound healing.

In other embodiments the present invention is directed at the use of an antagonist of the CBl receptor and/or the CB2 receptor for the preparation of a pharmaceutical composition for enhancing wound healing or treating a wound healing disorder characterized by reduced wound healing in a subject. Preferred antagonist of the CBl receptor and/or the CB2 receptor is selected from the group consisting of 0-1238, 0-1184, MAFP ₅ SR141716, SR144528, AM251, AM281, LY320135, AM630, and JTE-907. In preferred embodiments the wound healing disorder is selected from the group consisting of arterial occlusive diseases, psoriasis, Crohn's disease, epidermolysis bullosa, age-related skin changes, innervation disorders and ulcers caused by diabetes mellitus.

It is preferred that the pharmaceutical compositions for the various treatments indicated above are formulated as topical formulations, e.g. in an ointment or gel.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

IV. Methods of Treatment

Most skin lesions heal rapidly and efficiently within a week or two. However, the result of this process is not perfect; often there remains a connective tissue scar where the collagen matrix has been poorly reconstituted; epidermal appendages that have been lost at the site of damage do not regenerate. In some conditions, wounds become chronic and do not heal at all.

Wound healing involves a wide range of cellular, molecular, physiological and biochemical events. During the healing process, cells migrate to wound sites where they proliferate and synthesize extracellular matrix components in order to reconstitute a tissue closely similar to the uninjured original. This activity is regulated by mediators secreted from the wound border cells such as platelet-derived growth factor ("PDGF"), epidermal growth factor ("EGF"), transforming growth factor ("TGF") beta and other cytokines. Beneficial effects of these agents on cells has been demonstrated both in vitro and in vivo (reviewed by Moulin, Eur. J. Cell Biol. 68; 1-7, 1995), including benefit of administering PDGF in rat models of diabetes (Brown et al, J. Surg. Res. 56:562-570, 1994). The present invention provides for methods of enhancing wound healing, prophylactic and therapeutic methods of treating surgical patients, and patients having a disorder associated with aberrant wound healing, such as chronic wound diseases.

In one aspect, the invention provides a method for enhancing wound healing in a subject (e.g., in conjunction with surgery or as a result of injury) by administering to the subject an agent that antagonizes or inhibits CB1/CB2 protein expression or at least one CB1/CB2 activity. The appropriate agent can be determined based on screening assays described herein. In another aspect, the invention provides a method for preventing in a subject, a wound healing disorder or condition associated with an aberrant or excessive CB1/CB2 expression or activity (e.g., chronic wound diseases) by administering to the subject an agent that antagonizes or inhibits CB1/CB2 protein expression or at least one CB1/CB2 activity. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the CB1/CB2 aberrance, such that the wound healing disorder is prevented or, alternatively, delayed in its progression.

It is possible that some CBl/CB2-associated healing disorders can be caused, at least in part, by an aberrant or deficient level of gene product, or by the presence of a gene product exhibiting abnormal activity (e.g., excessive healing or scarring). As such, an increase in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.

Examples of disorders in which wound healing plays a role, include, for example, diabetes mellitus, arterial occlusive diseases, psoriasis, Crohn's disease, epidermolysis bullosa, age-related skin changes or innervation disorders. Wound healing disorders lead to a delayed healing of wounds or to chronic wounds. These disorders can be caused by the nature of the wound (e.g., large-area wounds, deep and mechanically expanded operation wounds, burns, trauma, decubitus), medicinal treatment of the patients (e.g. with corticoids), as well as by the nature of the disorder itself. For example, approximately 25% of the patients with Type II diabetes suffer from chronic ulcers known as "diabetic foot," of which approximately half heal poorly and necessitate expensive hospitalized treatments. Diabetic foot causes more stays in hospital than any other complication associated with diabetes. The number of these cases in diabetes Type I and II is on the increase and represents over 2% of all hospital admissions.

As discussed above, successful enhancement of wound healing can be brought about by techniques that serve to inhibit the expression or activity of target gene products. In addition, successful treatment of wound healing disorders caused by aberrant or excessive CB1/CB2 expression or activity can be accomplished using techniques that inhibit the expression or activity of CB1/CB2 proteins. For example, compounds, e.g., an agent identified using an assays

described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of CBl/CB2-associated wound healing disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, human, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab').sub.2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules can be utilized in treating or preventing a disease characterized by CB 1/CB2 expression is through the use of aptamer molecules specific for CB1/CB2 proteins. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically or selectively bind to protein ligands (see, e.g., Osborne et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid molecules can in many cases be more conveniently introduced into target cells than therapeutic protein molecules can be, aptamers offer a method by which CB1/CB2 protein activity can be specifically decreased without the introduction of drugs or other molecules which can have pluripotent effects. Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies can, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of wound healing

disorders.

In circumstances wherein injection of an animal or a human subject with CB1/CB2 proteins or epitopes for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against CB1/CB2 through the use of anti-idiotypic antibodies (see, for example, Herlyn (1999) Ann Med 37:66-78; and Bhattacharya-Chatterjee and Foon (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the CB1/CB2 protein. Vaccines directed to a wound healing disorders characterized by CB1/CB2 expression can also be generated in this fashion. In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies can be preferred. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al. (1993) Proc. Natl. Acad. ScL U.S.A 90:7889-7893). The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to enhance healing of a wound (e.g., by promoting processes such as re-epithelialization, connective tissue proliferation, deposition of organized extracellular matrix), and to prevent, treat or ameliorate CBl/CB2-associated healing disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be

formulated in animal models to achieve a circulating plasma concentration range that includes the IC ₅₀ (i.e., the concentration of the candidate substance 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 can be measured, for example, by high performance liquid chromatography.

Another example of determination of effective dose for an individual is the ability to directly assay levels of "free" and "bound" compound in the serum of the test subject. Such assays can utilize antibody mimics and/or "biosensors" that have been created through molecular imprinting techniques. The compound which is able to modulate CB1/CB2 activity is used as a template, or "imprinting molecule," to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix that contains a repeated "negative image" of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea (1994) Trends in Polymer Science 2:166-173. Such "imprinted" affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis et al (1993) Nature 361:645-647. Through the use of isotope- labeling, the "free" concentration of compound which modulates the expression or activity of CB1/CB2 can be readily monitored and used in calculations of IC ₅₀ .

Such "imprinted" affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC ₅₀ . A rudimentary example of such a "biosensor" is discussed in Kriz et al (1995) Analytical Chemistry 67:2142-2144.

Another aspect of the invention pertains to methods of modulating CB1/CB2 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of CB1/CB2 protein activity associated with the cell. An agent that modulates CB1/CB2 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of CB1CB2 protein (e.g., a CB1/CB2 substrate or receptor),

a CB1/CB2 antibody, a CB1/CB2 agonist or antagonist, a peptidomimetic of a CB1/CB2 agonist or antagonist, or other small molecule.

In one embodiment, the agent stimulates one or more CB1/CB2 activities. In another embodiment, the agent inhibits one or more CB1/CB2 activities. Examples of such inhibitory agents include antisense CB1/CB2 nucleic acid molecules, anti-CBl/CB2 antibodies, and CB1/CB2 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of CB1/CB2 proteins or nucleic acid molecules. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) CB1/CB2 expression or activity. In another embodiment, the method involves administering CB1/CB2 proteins or nucleic acid molecules as therapy to compensate for reduced or aberrant CB1/CB2 expression or activity. Stimulation of CB1/CB2 activity is desirable in situations in which CB1/CB2 proteins are abnormally downregulated and/or in which increased CB1/CB2 activity is likely to have a beneficial effect. For example, stimulation of CB1/CB2 activity is desirable in situations in which a CB1/CB2 is downregulated and/or in which increased CB1/CB2 activity is likely to have a beneficial effect. Likewise, inhibition of CB1/CB2 activity is desirable in situations in which CB1/CB2 is abnormally upregulated and/or in which decreased CB1/CB2 activity is likely to have a beneficial effect.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

Example 1: In Vivo Assay of Wound Healing

Transgenic CB1/CB2 double knock-out mice were prepared as described in Jarai, Z. et al., PNAS (1999) 96(24): 14136-14141. Control (wild-type) mice were obtained from The Jackson

Laboratory.

Full-thickness excisional wounds of 0.5 cm diameter were made on the back of the trangenic and control mice (10-12 weeks old) by excising skin and panniculus carnosus. Wounds were left uncovered without dressing. For histological analysis, the complete wounds with 3 mm of adjacent normal tissues were isolated, bisected, fixed overnight in 4 % PFA in PBS and embedded in paraffin. Sections from the middle of the wound were stained with hematoxylin/eosin. Only littermates of the same sex were used for direct histologic comparison.

Day 5 wounding is characterized by massive granulation tissue in both genotypes. However, the trangenic CB1/CB2 double knock-out mice had a higher re-epithelialization rate than control animals (Figure 1). The CB1/CB2 double knock-out mice exhibited better wound healing than control mice, as evidenced by an increased rate or re-epithelialization and a decrease in scar tissue.

Example 2: Identification of CBl and CB2 Receptor mRNA in Skin Tissue

CBl receptor was identified in mouse epidermis, dermis and primary keratinocytes by RT-PCR analysis. CB2 receptor cDNA was identified in dermis and primary keratinocytes and wealkly in epidermis (Figure 2). Epidermis, dermis and primary keratinocytes were prepared from mouse skin. The RNA was isolated using Trizol® reagent. Two micrograms were reversed transcribed with an oligo-dT Primer and 200 μg were used for each Polymerase chain reaction

(PCR). Primers from the coding region of the CBl and CB2 were used. GAPDH was a house- keeping control gene. PCR was performed with a hot star Taq Polymerase as follows: 1. activation of the enzyme; 15 min 95 °C; 2. 30 sec 95 ⁰ C; 3. 1 min 60 °C; 4. 1 min 72 °C. Steps

2-4 were repeated 34 times.

Example 3: In Vitro Biological Assays

Bioassay systems for determining the CB-I and CB-2 binding properties and pharmacological activity of cannabinoid receptor ligands are described by Roger G. Pertwee in

"Pharmacology of Cannabinoid Receptor Ligands," Current Medicinal Chemistry (1999) 6:635-

664, which is incorporated herein by reference in its entirety.

The following assays, which are being provided for illustration purposes, can be used to detect compounds that inhibit the binding of ^3H SR141716A (selective radiolabeled CB-I ligand) and ^3H 5-(l,l-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)-cyc- lohexyl]-phenol ( ^3H CP- 55940; radiolabeled CB-l/CB-2 ligand) to their respective receptors. Rat CB-I Receptor Binding Protocol

PelFreeze brains (available from Pel Freeze Biologicals, Rogers, Ark.) are cut up and placed in tissue preparation buffer (5 mM Tris HCl, pH =7.4 and 2 mM EDTA), polytroned at high speed and kept on ice for 15 minutes. The homogenate is spun at 1,000 X g for 5 minutes at 4 ⁰ C. The supernatant is recovered and centrifuged at 100,000 X G for 1 hour at 4° C. The pellet is then re-suspended in 25 ml of TME (25 mM Tris, pH=7.4, 5 mM MgCl ₂ , and 1 mM EDTA) per brain. A protein assay is performed and 200 μl of tissue totaling 20 μg is added to the assay.

The candidate substances are diluted in drug buffer (0.5% BSA, 10% DMSO and TME) and then 25 μl is added to a deep well polypropylene plate. ^3H SR141716A is diluted in a ligand buffer (0.5% BSA plus TME) and 25 μl is added to the plate. A BCA protein assay is used to determine the appropriate tissue concentration and then 200 μl of rat brain tissue at the appropriate concentration is added to the plate. The plates are covered and placed in an incubator at 20° C for 60 minutes. At the end of the incubation period 250 μl of stop buffer (5% BSA plus

TME) is added to the reaction plate. The plates are then harvested by Skatron onto GF/B filtermats presoaked in BSA (5 mg/ml) plus TME. Each filter is washed twice and dried overnight. In the morning the filters are counted on a Wallac Betaplate® counter (available from

PerkinElmer Life Sciences.TM., Boston, Mass.).

Human CB-I Receptor Binding Protocol

Human embryonic kidney 293 (HEK 293) cells transfected with the CB-I receptor cDNA are harvested in homogenization buffer (10 mM EDTA, 10 mM EGTA ₅ 10 mM Na Bicarbonate, protease inhibitors; pH=7.4), and homogenized with a Dounce Homogenizer. The homogenate is then spun at 1,000 X g for 5 minutes at 4° C. The supernatant is recovered and centrifuged at

25,000 X G for 20 minutes at 4° C. The pellet is then re-suspended in 10 ml of homogenization buffer and re-spun at 25,000 X G for 20 minutes at 4 C. The final pellet is re-suspended in 1 ml of TME (25 mM Tris buffer (pH=7.4) containing 5 mM MgCl ₂ and 1 mM EDTA). A protein assay is performed and 200 μl of tissue totaling 20 μg is added to the assay.

The candidate substances are diluted in drug buffer (0.5% BSA, 10% DMSO and TME) and then 25 μl are added to a deep well polypropylene plate. ^3H SR141716A is diluted in a ligand

buffer (0.5% BSA plus TME) and 25 μl are added to the plate. The plates are covered and placed in an incubator at 30° C for 60 minutes. At the end of the incubation period 250 μl of stop buffer (5% BSA plus TME) is added to the reaction plate. The plates are then harvested by Skatron onto GF/B filtermats presoaked in BSA (5 mg/ml) plus TME. Each filter is washed twice and dried overnight. In the morning the filters are counted on a Wallac Betaplate®. counter (available from PerkinElmer Life Sciences.TM., Boston, Mass.). CB-2 Receptor Binding Protocol

Chinese hamster ovary-Kl (CHO-Kl) cells transfected with CB-2 cDNA are harvested in tissue preparation buffer (5 mM Tris-HCl buffer (pH=7.4) containing 2 mM EDTA), polytroned at high speed and kept on ice for 15 minutes. The homogenate is then spun at 1,000 X g for 5 minutes at 4° C. The supernatant is recovered and centrifuged at 100,000 X G for 1 hour at 4° C. The pellet is then re-suspended in 25 ml of TME (25 mM Tris buffer (pH=7.4) containing 5 mM MgCl ₂ and 1 mM EDTA) per brain. A protein assay is performed and 200 μl of tissue totaling 10 μg is added to the assay. The candidate substances are diluted in drug buffer (0.5% BSA, 10% DMSO, and 80.5%

TME) and then 25 μl are added to the deep well polypropylene plate. ^3H CP-55940 is diluted a ligand buffer (0.5% BSA and 99.5% TME) and then 25 μl are added to each well at a concentration of 1 nM. A BCA protein assay is used to determine the appropriate tissue concentration and 200 μl of the tissue at the appropriate concentration is added to the plate. The plates are covered and placed in an incubator at 30° C for 60 minutes. At the end of the incubation period 250 μl of stop buffer (5% BSA plus TME) is added to the reaction plate. The plates are then harvested by Skatron format onto GF/B filtermats presoaked in BSA (5 mg/ml) plus TME. Each filter is washed twice and dried overnight. The filters are then counted on the Wallac Betaplate.TM. counter. CB-I GTP λ ^3ss Binding Assay

Membranes are prepared from CHO-Kl cells stably transfected with the human CB-I receptor cDNA. Membranes are prepared from cells as described by Bass et al., in "Identification and characterization of novel somatostatin antagonists," Molecular Pharmacology, 50:709-715 (1996). GTP λ ^35S binding assays are performed in a 96 well FlashPlate® format in duplicate using 100 pM GTP λ ^35S and 10 μg membrane per well in assay buffer composed of 50 mM Tris HCl, pH 7.4, 3 mM MgCl ₂ , pH 7.4,10 mM MgCl ₂ , 20 mM EGTA, 100 mM NaCl, 30 μM GDP, 0.1% bovine serum albumin and the following protease inhibitors: 100 μg/ml bacitracin, 100

μg/ml benzamidine, 5 μg/ml aprotinin, 5 μg/ml leupeptin. The assay mix is then incubated with increasing concentrations of antagonist (10 ^'10 M to 10 ^'5 M) for 10 minutes and challenged with the cannabinoid agonist CP-55940 (10 μM). Assays are performed at 30° C for one hour. The FlashPlates® are then centrifuged at 2000 X g for 10 minutes. Stimulation of GTP λ ³⁵ binding is then quantified using a Wallac Microbeta.ECs ₀ calculations done using Prism® by Graphpad.