WO2005094808A1

WO2005094808A1 - Regulation of human glycine transporter 2

Info

Publication number: WO2005094808A1
Application number: PCT/EP2005/001926
Authority: WO
Inventors: Tsukimi Yasuhiro; Taijimi Masaomi
Original assignee: Bayer Healthcare AG
Current assignee: Bayer AG
Priority date: 2004-03-02
Filing date: 2005-02-24
Publication date: 2005-10-13
Anticipated expiration: 2006-09-02

Abstract

Reagents which regulate human glycine transporter 2 and reagents which bind to human glycine transporter 2 gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, urological disorder or disease such as detrusor overactivity (overactive bladder), urinary incontinence, neurogenic detrusor oeractivity (detrusor hyperflexia), idiopathic detrusor overactivity (detrusor instability), benign prostatic hyperplasia, and lower urinary tract symptoms.

Description

REGULATION OF HUMAN GLYCINE TRANSPORTER 2

TECHNICAL FIELD OF THE INVENTION

The present invention relates to modulators of human glycine transporter 2. More particularly, the present invention relates to modulators of human glycine transporter 2 and use of such modulators for the treatment or prevention of urological disorder or disease such as detrusor overactivity (overactive bladder), urinary incontinence, neurogenic detrusor overactivity (detrusor hyperflexia), idiopathic detrusor overactivity (detrusor instability), benign prostatic hyperplasia, and lower urinary tract symptoms.

BACKGROUND OF THE INVENTION

The termination of synaptic transmission in the central nervous system (CNS) involves either the enzymatic inactivation of neurotransmitters, or their uptake into pre-synaptic terminals or surrounding glial cells. High-affinity, membrane-associated transporters typically mediate the rapid removal of neurotransmitters from the synaptic cleft, with uptake across a concentration gradient being thermodynamically coupled to transmembrane ion gradients. In the central nervous system, some amino acids are known to be important neurotransmitters. Among them, glycine is one of the major neurotransmitter in the spinal cord, brainstem and retina, where it exerts its effects on the strychnine-sensitive glycine receptors (Betz, H., et al., Ann. N. Y. Acad. Sci., 207:109-115 (1993); Betz, EL, et al., Q. Rev. Biophys. 25:381-394 (1992)). In addition, glycine acts as a co-agonist with glutamate at the NMDA receptor (Kemp, J.A. and Leeson, P.D., Trends. Pharmacol. Sci. 14:20-25 (1993)). Synaptic glycine concentrations are controlled by Na⁺/G^"- dependent high affinity transporters found at nerve-terminals and glial cells (Fedele, E. and Foster, A.C., Brain Res. 572:154-163 (1992)). Two distinct glycine transporters, GlyTl (Smith, K.E., et al., Neuron 8:927-935 (1992)) and GlyT2 (Liu, Q.R., et al., J. Biol. Chem. 268:22802-22806 (1993)), have been isolated, and share approximately 50% identity at both the nucleotide and amino acid levels. Localization of GlyTl and GlyT2 by in situ hybridization techniques reveals distinct patterns of expression in the CNS (Liu, Q.R., et al, J.Biol. Chem. 268:22802-22806 (1993); Zafra, F., et al., J. Neurosci. 15:3952-3969 (1995)). . GlyTl is expressed in the hippocampal and cortical regions of the brain, as well as in the spinal cord and braistem regions. In contrast, GlyT2 is expressed primarily in the spinal cord and cerebellum, and is absent in the hippocampal and cortical regions. Based on their patterns of expression, GlyTl is thought to co-. localize with NMDA receptors, while GlyT2 expression mimics that of strychnine-sensitive glycine receptors (Jursky F and Nelson F, J. Neurochem. 67:336-344 (1996); Liu, Q.R., et al. J. Biol. Chem. 268:22802-22806 (1993). The precise regulation of synaptic glycine concentrations in the CNS is a very important process because glycine is involved in both excitatory and inhibitory neurotransmission (Betz, H., et al., Ann. N. Y. Acad. Sci. 207:109-115 (1993); Benveniste, M.. et al., J. Physiol. (London) 428:333- 357 (1990)). Glycine transporters are likely to be critical to this process. Compounds able to modulate glycine transporter function (i.e., that inhibit GlyT2 or activate GlyTl) would be expected to provide a wide variety of therapeutic benefits. For example, glycine receptor inhibition is known to result in pain transmission (Yaksh TL, Pain, 111-123 (1989)). Compounds that inhibit GlyT2 transporter activity increase the activity of neurons having strychnine-sensitive glycine receptors via increasing synaptic levels of glycine, thus diminishing the transmission of pain-related (i.e., nociceptive) information in the spinal cord, which has been shown to be mediated by these receptors.

Micturition is mediated by the spinobulbospinal reflex pathway that passes through the pontine micturition center (Blok, B.F., et al, J. Comp. Neurol. 359:300-309 (1995);), and some amino acids are known to be important neurotransmitters for reflex micturition. For example, glutamate is a major excitatory neurotransmitter in both the upper and the lower central nervous system, and it facilitates the micturition reflex. On the other hand, γ-aminobutyric acid (BAG A) is a major inhibitory neurotransmitter in the central nervous system, and it inhibits the micturition reflex at the level of the lumbosacral cord (Igawa, Y., et al., J. Urol. 150:537-542 (1993). Glycine is another important inhibitory neurotransmitter, and higher concentrations of glycine are found in the spinal cord than in supraspinal regions (Aprison, M.H., et al., Comp. Biochem. Physiol. 28:1345-1355 (1969). Recently, it was reported that glycinergic neurons have an important inhibitory effect on the spinobulbospinal and spinal micturition reflexes at the level of the lumbosacral cord (Miyazato, M., et al., Exp. Neurol. 183:232-240 (2003).

Urinary Incontinence

Urinary incontinence (UI) is the involuntary loss of urine. Urge urinary incontinence (UUI) is one of the most common types of UI together with stress urinary incontinence (SUI) which is usually caused by a defect in the urethral closure mechanism. UUI is often associated with neurological disorders or diseases causing neuronal damages such as dementia, Parkinson's disease, multiple sclerosis, stroke and diabetes, although it also occurs in individuals with no such disorders. One of the usual causes of UUI is overactive bladder (OAB) which is a medical condition referring to the symptoms of frequency and urgency derived from abnormal contractions and instability of the detrusor muscle.

There are several medications for urinary incontinence on the market today mainly to help treating UUI. Therapy for OAB is focused on drugs that affect peripheral neural control mechanisms or those that act directly on bladder detrusor smooth muscle contraction, with a major emphasis on development of anticholinergic agents. These agents can inhibit the parasympathetic nerves which control bladder voiding or can exert a direct spasmolytic effect on the detrusor muscle of the bladder. This results in a decrease in intravesicular pressure, an increase in capacity and a reduction in the frequency of bladder contraction. Orally active anticholinergic drugs such as propantheline (ProBanthine), tolterodine tartrate (Detrol) and oxybutynin (Ditropan) are the most commonly prescribed drugs. However, their most serious drawbacks are unacceptable side effects such as dry mouth, abnormal visions, constipation, and central nervous system disturbances. These side effects lead to poor compliance. Dry mouth symptoms alone are responsible for a 70% non- compliance rate with oxybutynin. The inadequacies of present therapies highlight the need for novel, efficacious, safe, orally available drugs that have fewer side effects.

Benign Prostatic Hyperplacia

Disease Summary

Benign prostatic hyperplacia (BPH) is the benign nodular hyperplasia of the periurethral prostate gland commonly seen in men over the age of 50. The overgrowth occurs in the central area of the prostate called the transition zone, which wraps around the urethra. BPH causes variable degrees of bladder outlet obstruction resulting in progressive lower urinary tract syndromes (LUTS) characterized by urinary frequency, urgency, and nocturia due to incomplete emptying and rapid refilling of the bladder. Bladder outlet obstruction induces bladder hypertrophy and NGF production in the bladder (J Clin Invest 88:1709, 1991). This NGF sensitizes C-fiber sensory afferent neurons to induce detrusor overactivity (J Urol 165:975, 2001). This might be one of several causes for irritative symptoms in LUTS. The actual cause of BPH is unknown but may involve age-related alterations in balance of steroidal sex hormones. ^'

The selective αl-adrenoceptor antagonists, such as prazosin, indoramin and tamsulosin are used as an adjunct in the symptomatic treatment of urinary obstruction caused by BPH, although they do not affect on the underlying cause of BPH. In BPH, increased sympathetic tone exacerbates the degree of obstruction of the urethra through contraction of prostatic and urethral smooth muscle. These compounds inhibit sympathetic activity, thereby relaxing the smooth muscle of the urinary tract. Uroselective αl -antagonists and l -antagonists with high tissue selectivity for lower urinary tract smooth muscle that do not provoke hypotensive side-effects should be developed for the treatment.

Drugs blocking dihydrotestosterone have been used to reduce the size of the prostate. 5α-reductase inhibitors such as finasteride are prescribed for BPH. These agents selectively inhibit 5α-reductase which mediates conversion of testosterone to dihydrotestosterone, thereby reducing plasma dihydrotestosterone levels and thus prostate growth. The 5α-reductase inhibitors do not bind to androgen receptors and do not affect testosterone levels nor do they possess feminizing side- effects.

Androgen receptor antagonists are used for the treatment of prostatic hyperplasia due to excessive action or production of testosterone. Various antiandrogens are under investigation for BPH including chlormadione derivatives with no estrogenic activity, orally-active aromatase inhibitors, luteinizing hormone-releasing hormone (LHRH) analogues.

Lower Urinary Tract Symptoms

BPH causes variable degrees of bladder outlet obstruction, resulting in progressive lower urinary tract symptoms (LUTS) characterized by urinary frequency, urgency, and nocturia due to incomplete emptying and rapid refilling of the bladder.

It was suggested that one of the maj or causes of LUTS induced by partial outlet obstruction is a markedly enhanced spinal reflex of the bladder neuronal cirquit (J Urol 160:34, 1998; J Urol 162:1890, 1999).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide reagents and methods for regulating a glycine transporter 2. This and other objectives of the invention are provided by one of the embodiments described below.

One embodiment of the invention is a reagent that modulates the activity of a human glycine transporter 2 polypeptide or polynucleotide. Such reagent can be useful to treat urological disorders.

Further embodiment of the invention is a pharmaceutical composition for the treatment of urological disorders. The composition comprises the above-mentioned reagent and a pharmaceutically acceptable carrier.

Another embodiment of the invention is a use of the above-mentioned reagent in the preparation of a medicament for modulating the activity of human glycine transporter 2 in a urological disorder. The urological disorder can be at least one selected from the group consisting of a disorder caused by overactive bladder, urinary incontinence, detrusor hyperflexia, detrusor instability, benign prostatic hyperplasia, and one of lower urinary tract symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows effect of intrathecal administration of GlyT2 inhibitor (A) and intraveous injection of GlyT2 inhibitor (B) on bladder function in conscious rats.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that human glycine transporter 2 increase glycine level in spinal cord and can be regulated to control urological disorders or diseases, such as detrusor overactivity (overactive bladder), urinary incontinence, neurogenic detrusor oeractivity (detrusor hyperflexia), idiopathic detrusor overactivity (detrusor instability), benign prostatic hyperplasia, and lower urinary tract symptoms.

The present invention demonstrated that inhibition of glycine transporter 2 in spinal cord prolonged micturition interval.

This strongly suggests that glycine transporter 2 are involved in the spinal reflex mechanism of detrusor overactivity. The present invention provide a link between human glycine transporter 2 and treatment of urological disorders using modulator that inhibit human glycine transporter 2 signaling. Glycine transporter 2 can be regulated to control urological disorder or disease such as detrusor overactivity (overactive bladder), urinary incontinence, neurogenic detrusor oeractivity (detrusor hyperflexia), idiopathic detrusor overactivity (detrusor instability), benign prostatic hyperplasia, and lower urinary tract symptoms.

DEFINITION

Urological disorders as used herein can be diseases of the bladder including but not limited to urinary incontinence including overactive/oversensitive bladder, overflow urinary incontinence, stress urinary incontinence caused by dysfunction of the bladder, urethra or central/peripheral nervous system.

As used herein a urological disorder can be a disorder of the prostate including but not limited to "a prostate disorder" which refers to an abnormal condition occurring in the male pelvic region characterized by, e.g., male sexual dysfunction and/or urinary symptoms. This disorder may be manifested in the form of genitourinary inflammation (e.g., inflammation of smooth muscle cells) as in several common diseases of the prostate including prostatitis, benign prostatic hyperplasia and cancer, e.g., adenocarcinoma or carcinoma, of the prostate. Antibodies

Any type of antibody known in the art can be generated to bind specifically to an epitope of a glycine transporter 2 polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable of binding an epitope of a glycine transporter 2 polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, ep> itopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

An antibody which specifically binds to an epitope of a glycine transporter 2 polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.

Typically, an antibody which specifically binds to a glycine transporter 2 polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably., antibodies which specifically bind to glycine transporter 2 polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a glycine transporter 2 polypeptide from solution.

Human glycine transporter 2 polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a glycine transporter 2 polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Gueriri) and Corynebacteήum parvum are especially useful.

Monoclonal antibodies that specifically bind to a glycine transporter 2 polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al., Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al., Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al., Mol. Cell Biol. 52, 109-120, 1984).

In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al., Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al., Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies that specifically bind to a glycine transporter 2 polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to glycine transporter 2 polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. §5, 11120-23, 1991).

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.

A nucleotide , sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al., 1995, Int. J. Cancer 61, 497-501 ; Nicholls et al., 1993, J. Immunol. Meth. 165, 81-91). Antibodies which specifically bind to glycine transporter 2 polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al., Nature 349, 293-299, 1991).

Other types of antibodies can be constructed and used tlierapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from irnmunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.

Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a glycine transporter 2 polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation.

Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12,

15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides Long. Longer sequences also can be used.

Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of glycine transporter 2 gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleoticles, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or ^"by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of -mother nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphor-amidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.

Modifications of glycine transporter 2 gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the glycine transporter 2 gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it c-auses inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been d escribed in the literature (e.g., Gee et al, in Huber & Carr, MOLECULAR AND IMMU OLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation betwe n an antisense oligonucleotide and the complementary sequence of a glycine transporter 2 polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a glycine transporter 2 polynucleotide, each separated by a stretch of contiguous nucleotides which are not co_mplementary to adjacent glycine transporter 2 nucleotides, can provide sufficient targeting specif! city for glycine transporter 2 mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular glycine transporter 2 polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to a glycine transporter 2 polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., -Agrawal et al, Trends Biotechnol 70, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 275, 3539-3542, 1987.

Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target IO A, followed by endo- nucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a glycine transporter 2 polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the glycine transporter 2 polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target IQNLA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).

Specific ribozyme cleavage sites within a glycine transporter 2 RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate glycine transporter 2 RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybri ization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mec-hanical methods, such as micro injection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease glycine transporter 2 expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme- encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al, U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNLA occurs only when both a ribozyme and a target gene are induced in the cells.

Differentially Expressed Genes

Described herein are methods for the identification of genes whose products interact with human glycine transporter 2. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, overactivity of bladder, hyperflexia, benign prostatic hyperplasia, and CNS disorders. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human glycine transporter 2 gene or gene product may itself be tested for differential expression.

The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences ma-y be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, artd Northern analysis.

Identification of Differentially Expressed Genes

To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects an_d from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al, ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed usiitg techniques well known to those of skill in the art, such as, for example, the single-step RN-A isolation process of Chomczynski, U.S. Patent 4,843,155.

Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al, Nature 308, 149-53; Lee et al, Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang & Pardee, Science 25^*7, 967-71, 1992; U.S. Patent 5,262,311). The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human glycine transporter 2. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human glycine transporter 2. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human glycine transporter 2 gene or gene product are up-regulated or down-regulated.

Screening Methods

The invention provides assays for screening test compounds that bind to or modulate the activity of a glycine transporter 2 polypeptide or a glycine transporter 2 polynucleotide. A test compound preferably binds to a glycine transporter 2 polypeptide or polynucleotide. More preferably, a test compound decreases or increases functional activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.

Test Compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds 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. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, 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 approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et «/., J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl 33, 2061; Gallop et al, J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Natur 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Oevlin, Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici. J. Mol Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409).

High Throughput Screening

Test compounds can be screened for the ability to bind to glycine transporter 2 polypeptides or polynucleotides or to affect glycine transporter 2 activity or glycine transporter 2 gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.

Another high throughput screening method is described in Beutel et al, U.S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together. Binding Assays

For binding assays, the test compound is preferably a small molecule that binds to the glycine transporter 2 polypeptide such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the glycine transporter 2 polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound that is bound to the glycine transporter 2 polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to a glycine transporter 2 polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a glycine transporter 2 polypeptide. 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 test compound and a glycine transporter 2 polypeptide (McConnell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to a glycine transporter 2 polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, a glycine transporter 2 polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Barrel et al, BioTechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the glycine transporter 2 polypeptide and modulate its activity.

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. For example, in one construct, polynucleotide encoding a glycine transporter 2 polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, 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., LacZ), 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 DNA sequence encoding the protein that interacts with the glycine transporter 2 polypeptide.

It may be desirable to immobilize either the glycine transporter 2 polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the glycine transporter 2 polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non- covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a glycine transporter 2 polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, the glycine transporter 2 polypeptide is a fusion protein comprising a domain that allows the glycine transporter 2 polypeptide to be bound to a solid support. For example, glutathione-S-transferase 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 test compound or the test compound and the non-adsorbed glycine transporter 2 polypeptide; the mixture is then 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. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined. Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a glycine transporter 2 polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated glycine transporter 2 polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well 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). Alternatively, antibodies which specifically bind to a glycine transporter 2 polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site can be derivatized to the wells of the plate. Unbound target or protein can be 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 which specifically bind to the glycine transporter 2 polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the glycine transporter 2 polypeptide, and SDS gel electrophoresis under non-reducing conditions.

Screening for test compounds which bind to a glycine transporter 2 polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a glycine transporter 2 polypeptide or polynucleotide can be used in a cell-based assay system. A glycine transporter 2 polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a glycine transporter 2 polypeptide or polynucleotide is determined as described above.

Functional Assays

Test compounds can be tested for the ability to increase or decrease a biological effect of a human glycine transporter 2. Such biological effects can be determined for example using functional assays such as those described below. Functional assays can be carried out after contacting either a purified glycine transporter 2 polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound which increases or decreases a functional activity of a glycine transporter 2 polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent.

Ion channels can be tested functionally in living cells. Polypeptides comprising amino acid sequences encoded by open reading frames of the invention are either expressed endogeneously in appropriate reporter cells or are introduced recombinantly. Channel activity can be monitored by concentration changes of the permeating ion, by changes in the transmembrane electrical potential gradient, or by measuring a cellular response (e.g., expression of a reporter gene or secretion of a neurotransmitter) triggered or modulated by the polypeptide's activity.

The activity of ion channel proteins in cells can be determined, for example, by loading the cells with an ion-sensitive fluorescent indicator. Fluorescent indicators can be loaded into cells in 96- well plates or another container, and the activity of ion channel proteins in the presence or absence of various test compounds can be simply and rapidly determined. See, e.g., U.S. Patent 6,057,114. Ion channel currents result in changes of electrical membrane potential (V_m) which can be monitored directly using potentiometric fluorescent probes. These electrically charged indicators (e.g., the anionic oxonol dye DiBAC (3)) redistribute between extra- and intracellular compartments in response to voltage changes across the membrane in which the ion channel resides. The equilibrium distribution is governed by the Nernst-equation. Thus, changes in membrane potential results in concomitant changes in cellular fluorescence. Again, changes in V_m might be caused directly by the activity of the target ion channel or through amplification and/or prolongation of the signal by channels co-expressed in the same cell.

Another approach to determining the activity of ion channel proteins involves the electrophysiological determination of ionic currents. Cells which endogenously express a glycine transporter 2 can be used to study the effects of various test compounds or glycine transporter 2 polypeptides on endogenous ionic currents attributable to the activity of glycine transporter 2. Alternatively, cells which do not express glycine transporter 2 can be employed as hosts for the expression of glycine transporter 2, whose activity can then be studied by electrophysiological or other means. Cells preferred as host cells for the heterologous expression of glycine transporter 2 are preferably mammalian cells such as COS cells, mouse L cells, CHO cells (e.g., DG44 cells), human embryonic kidney cells (e.g., HEK293 cells), African green monkey cells and the like; amphibian cells, such as Xenopus laevis oocytes; or cells of yeast such as S. cerevisiae or P. pastoris. See, e.g., U.S. Patent 5,876,958.

Electrophysiological procedures for measuring the current across a cell membrane are well known. A preferred method is the use of a voltage clamp as in the whole-cell patch clamp technique. Non-calcium currents can be eliminated by established methods so as to isolate the ionic current flowing through ion channel proteins. In the case of heterologously expressed glycine transporter 2, ionic currents resulting from endogenous ion channel proteins can be suppressed by known pharmacological or electrophysiological techniques. See, e.g., U.S. Patent 5,876,958.

A further activity of the glycine transporter 2 which can be assessed is its ability to bind various ligands, including test compounds. The ability of a test compound to bind glycine transporter 2 or fragments thereof may be determined by any appropriate competitive binding analysis (e.g., Scatchard plots), wherein the binding capacity and/or affinity is determined in the presence and absence of one or more concentrations a compound having known affinity for the glycine transporter 2. Binding assays can be performed using whole cells that express glycine transporter 2 (either endogenously or heterologously), membranes prepared from such cells, or purified glycine transporter 2.

Gene Expression

In another embodiment, test compounds that increase or decrease glycine transporter 2 gene expression are identified. A glycine transporter 2 polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the glycine transporter 2 polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

The level of glycine transporter 2 mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a glycine transporter 2 polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a glycine transporter 2 polypeptide.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell that expresses a glycine transporter 2 polynucleotide can be used in a cell-based assay system. The glycine transporter 2 polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used. Pharmaceutical Compositions

The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, a glycine transporter 2 polypeptide, glycine transporter 2 polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a glycine transporter 2 polypeptide, or mimetics, activators, or inhibitors of a glycine transporter 2 polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration. Therapeutic Indications and Methods

Modulating human glycine transporter 2 provides effective controls of urinary disorders such as urinary incontinence, overactive bladder, benign prostatic hyperplasia and lower urinary tract syndromes.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases glycine transporter 2 activity relative to the glycine transporter 2 activity which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be -used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅0/ED₅₀.

Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation. Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single- chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA.

If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

Preferably, a reagent reduces expression of a glycine transporter 2 gene or the activity of a glycine transporter 2 polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a glycine transporter 2 gene or the activity of a glycine transporter 2 polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to glycine transporter 2pecific mRNA, quantitative RT-PCR, immunologic detection of a glycine transporter 2 polypeptide, or measurement of glycine transporter 2 activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic Methods

Human glycine transporter 2 also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the polypeptide. For example, differences can be determined between the cDNA or genomic sequence encoding glycine transporter 2 in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl Acad. Sci. USA 85, 4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Altered levels of glycine transporter 2 also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLE 1

Establishment of stable transfectants expressing recombinant glycine transporter polypeptides

Human glycine transporter cDNA (accession no AF085412) is inserted in pcDNA3.1 expression vector (Invitrogen). A stable HEK293 transformant expressing mitchondrial aequorin is transfected with this construct by Lipofectamine method (Invitrogen). Human GlyT2-expressing HEK293 clones are selected in the presence of G418 (lmg/ml; GIBCO). Cells are cultured in 45 % Dulbecco's modified Eagle Medium, 45 % F12 (DMEM/F12) and 10 % fetal calf serum at 37 °C in 5% CO₂/95% air and 100 % humidified condition.

EXAMPLE 2

Identification of test compounds that bind to glycine transporter 2 polypeptides

Stable transformants expressing recombinant glycine transporter 2 polypeptides are put in wells of 96-well microtiter plates (2x10⁵ cells/80 μl/well) in a binding assay buffer, such as 20 mM Tris- HC1 (pH 8.0), 5 mM MgCl₂, 1 mM EDTA, 0.1% BSA and lx protease inhibitor cocktail (Roche Applied Bioscience). The test compounds dissolved in dimethylsulfoxide are diluted by the assay buffer and aliquots are added to each well (10 μl/well). After shaking for 5 min, the plates are incubated at room temperature for 1 hr. Mixture is transferred to Multiscreen™-FB filters (Millipore) precoated with 0.5% polyethylenimine and washed 3 times with cold assay buffer. After complete drying, Microscinti-PS (Packard, Downers Grove, IL) is added (30 μl/well) and remaining radioactivity is measured by Topcount (Packard). A test compound that decrease the remaining radio activity in a well by at least 15% relative to radio activity in a well in which a test compound is not incubated is identified as a compound which binds to a glycine transporter 2 polypeptide.

EXAMPLE 3

Identification of a test compound which inhibits glycine transporter 2

A stable transfectant of HEK293 expressing the GlyT2 (HEK293/GlyT2) is prepared as described above. On day 0, 40,000 per well of the cells are plated on wells of poly-D lysine-coated 96-well plate with 0.1 ml per well of DMEM containing 10 % FBS supplemented with 0.3 mg/ml of G418. On day 2, the culture medium is replaced with 75 μl/well of the Assay Buffer [10 mM HEPES (pH7.5), 140 mM NaCl, 5.5 mM KC1, 0.8 mM MgSO₄, and 1.8 mM CaCl₂] containing a test sample. Then, 25 μl/well of the Glycine Solution Mix (2 μCi/ml of ³H-glycine and 200 μM of cold glycine in the Assay Buffer) was added to each well. After incubating at room temperature for 30 minutes, the cells are washed once with the Assay Buffer using Wallac Plate Washer. The amounts of ³H-glycine up-taken by the cells are determined by Microbeta counter after dissolving the cells by dispensing 30 μl of Ultima Gold scintillant (Packard) to each well. Compounds that decreases the uptake of ³H-glycine into the HEK293/GlyT2 cells are identified as inhibitors.

EXAMPLE 4

Identification of a test compound which decreases glycine transporter 2 gene expression

Human HEK293 cells transfected with a glycine transporter 2 expression construct are treated with the test compound at 37°C for 10 to 45 minutes. The same cells that have not been transfected are incubated for the same time without the test compound as a negative control.

Total RNA samples are isolated from the two cultures as described in Chirgwin et al. (Biochem. 18, 5294-99, 1979). Twenty μg each of the total RNA samples were subjected to an agarose gel electrophoresis and blotted to a nylon membrane. Levels of GlyT2 transcripts in the samples were determined by hybridizing with a ³²P-labeled glycine transporter 2-pecifιc probe at 65 ° C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement strand of the nucleotide sequence of human glycine transporter 2. A test compound that decreases the glycine transporter 2 specific signal relative to the signal obtained in the absence of the test compound is identified as an modulator that inhibits glycine transporter 2 gene expression.

EXAMPLE 5

In order to clarify whether or not GlyT2 inhibitor could improve the symptom of overactive bladder, effect of GlyT2 inhibitor on acetic acid-induced detrusor hyperreflexia in conscious rats. For this purpose, effect of ORG25543 was examined in 2 different routes, ie, intrathecal or intravenous administration (Caufield, W.L., et al., J. Med. Chem. 44:2679-2682 (2001)).

Catheterization for drug administration

Female Sprague-Dawley rats was anesthetized by ether. In the case of intrathecal catheterization, the atlanto-occipital membrane was exposed through a dorsal incision and a small hole was made in the dura. A polyethylene catheter (PE10) filled with saline was inserted into the subarachnoid space and advanced caudally until the tip reached the level of L6-S1 spinal cord. A suture in the superficial dorsal muscle layer fixed the catheter. In the case of intravenous catheterization, a polyethylene catheter (PE10) was implanted into right jugular vein. The other end of catheter was reached to back through subcutanous tunnel. A suture in the superficial dorsal muscle layer fixed the catheter.

Bladder catheterization and cystometry

A polyethylene catheter (PE50) was implanted into the bladder dome through a midline incision in the abdomen under ether anesthesia. The other end of catheter was reached to the back through subcutaneous tunnel and fixed to the skin.

After recovery from anesthesia, the animals were placed into strain cage (Ballman's cage) and left for over 1 hr. The bladder catheter was connected via T-tube to a pressure transducer and to a microsyringe pump. Room temperature saline was infused into the bladder continuously at a flow rate of 3.6ml/hr. Intravesical pressure was recorded continuously by means of electric recorder. After stabilizing cystometric parameters, the infused solution was changed from saline to 0.2% acetic acid solution which is diluted with saline. Thirty min later, test compound was administered. Micturition interval (min) was determined and expressed as % change of that just before compound administration.

In the case of intrathecal adminsitration, vehicle or test compound was adminsitered at the volume of 10 micro litter. While in the case of intravevous administration, vehicle or test compound was administered at the volume of 50 micro litter / kg of body weight.

Intrathecal administration of saline (vehicle for glycine) or 100 nmol of hydrochloride had little effect on micturition interval. Glycine (lOmicro mol) and ORG25543 (100 nmol) prolonged micturition interval (Fig.l (A)). Significant effect was observed in the case of ORG25543 administration. Their combination showed a tendency of further prolongation of micturition interval.

Intravenous administration of vehicle (DMSO:Tween 80:saline=l :l :8) had little effect on micturition interval (Fig. 1 (B)). ORG25543 significantly prolonged micturition interval in a dose dependent manner.

Claims

1. A method of reducing the activity of human glycine transporter 2, comprising the step of: contacting a cell with a reagent which specifically binds to human glycine transporter 2 polynucleotide or a human glycine transporter 2 polypeptide, whereby the activity of human glycine transporter 2 is reduced and a urological disorder is ameliolated.

2. A reagent that modulates the activity of a human glycine transporter 2 polypeptide or polynucleotide, wherein said reagent is useful to treat urological disorders.

3. A pharmaceutical composition for the treatment of urological disorders, comprising: the reagent of claim 1 or 2, and a pharmaceutically acceptable carrier.

4. Use of the reagent of claim 1 or 2 in the preparation of a medicament for modulating the activity of human glycine transporter 2 in a urological disorder.

5. Use of claim 4, wherein the urological disorder is at least one selected from the group consisting of detrusor overactivity (overactive bladder), urinary incontinence, neurogenic detrusor oeractivity (detrusor hyperflexia), idiopathic detrusor overactivity (detrusor instability), benign prostatic hyperplasia, and lower urinary tract symptoms.