AU685789B2 - Cloned human alphaic adrenergic receptor - Google Patents

Description

II WO 94/21660 PCT/US94/02609 1 TITLE OF THE INVENTION CLONED HUMAN ALPHA1C ADRENERGIC RECEPTOR CROSS REFERENCES TO RELATED APPLICATION This is a co tii nf.s. T Application serial 32,849, filed on March 15, 1993, pending.

BACKGROUND OF THE INVENTION i. Field of the Invention: This invention relates to a method for defining the potency and selectivity of compounds for use as human alphalC adrenergic receptor antagonists using cloned human alpha 1 receptors. The invention also relates to the cloned receptors themselves, to compounds identified according to the method of this invention, and to methods of use of such compounds, alone or in combination with other agents.

Particularly preferred are combinations of compounds identified according to this invention and testosterone 5-alpha reductase inhibitors, to alleviate pathologic conditions, particularly benign prostatic 2 hyperplasia (also known as benign prostatic hypertrophy, BPH).

ii. Background: Human adrenergic receptors are integral membrane proteins which have been classified into two broad classes, the alpha and the beta adrenergic receptors. Both types mediate the action of the peripheral sympathetic nervous system upon binding of catecholamines, norepinephrine and epinephrine.

Norepinephrine is produced by adrenergic nerve endings, while epinephrine is produced by the adrenal medulla. The binding affinity of adrenergic receptors for these compounds forms one basis of the classification: alpha receptors bind norepinephrine more strongly than epinephrine and much more strongly than the synthetic compound isoproterenol. The binding affinity of these hormones is reversed for the beta receptors. In many tissues, the functional responses, such as IIRIIC C -r L D IUU WO 94/21660 PCT/US94/02609 2 smooth muscle contraction, induced by alpha receptor activation are opposed to responses induced by beta receptor binding.

Subsequently, the functional distinction between alpha and beta receptors was further highlighted and refined by the pharmacological characterization of these receptors from various animal and tissue sources. As a result, alpha and beta adrenergic receptors were further subdivided into al, c2, 11, and 12 subtypes.

Functional differences between al and a2 receptors have been recognized, and compounds which exhibit selective binding between these two subtypes have been developed. Thus, in WO 92/0073, the selective ability of the enantiomer of terazosin to selectively bind to adrenergic receptors of the alpha 1 subtype was reported. The acl/c2 selectivity of this compound was disclosed as being significant because agonist stimulation of the a2 receptors was said to inhibit secretion of epinephrine and norepinephrine, while antagonism of the a2 receptor was said to increase secretion of these hormones. Thus, the use of nonselective alpha-adrenergic blockers, such as phenoxybenzamine and phentolamine, is limited by their aX2 adrenergic receptor mediated induction of increased plasma catecholamine concentration and the attendant physiological sequelae (increased heart rate and smooth muscle contraction).

For a general background on the a-adrenergic receptors, the reader's attention is directed to Robert R. Ruffolo, Jr., X- Adrenoreceptors: Molecular Biology. Biochemistry and Pharmacology, (Progress in Basic and Clinical Pharmacology series, Karger, 1991), wherein the basis of al/a2 subclassification, the molecular biology, signal transduction (G-protein interaction and location of the significant site for this and ligand binding activity away from the 3'-terminus of alpha adrenergic receptors), agonist structure-activity relationships, receptor functions, and therapeutic applications for compounds exhibiting a-adrenergic receptor affinity was explored.

The cloning, sequencing and expression of alpha receptor subtypes from animal tissues has led to the subclassification of the al receptors into alA, (Lomasney, et al., J. Biol. Chem., 266:6365-6369 1_ WO 94/21660 PCT/US94/02609 3 (1991), rat caA; Bruno et al., BBRC, 179:1485-1490 (1991), human alA), cxlB (Cotecchia, et al., PNAS, 85;7159-7163 (1988), hamster clB; Libert, et al., Science, (1989), dog alB; Ramarao, et al., J. Biol.

Chem., 267:21936-21945 (1992), human alB), and most recently, in a study using bovine brain, a new alC subtype was proposed (Schwinn, et al., J. Biol. Chem., 265:8183-8189, 1990; Hirasawa et al., BBRC 195:902-909 (1993), described the cloning, functional expression and tissue distribution of a human alC adrenergic receptor; Hoehe et al., Human Mol. Genetics i(5):349 (8/92) noted the existence of a two-allele Pstl restriction fragment polymorphism in the clC adrenergic receptor gene; another study suggests that there may even be an alpha-1D receptor subtype, see Perez et al., Mol. Pharm.., 40:876-883, 1992).

Each al receptor subtype exhibits its own pharmacologic and tissue specificities. Schwinn and coworkers noted that the cloned bovine alC receptor exhibited pharmacological properties proposed for the alA subtype. Nonetheless, based on its non-expression in tissues where the alA subtype is expressed, and its sensitivity to chloroethylclonidine, the receptor was given a new designation.

The differences in the a-adrenergic receptor subtypes have 2 relevance in pathophysiologic conditions. Benign prostatic hypertrophy, BPH, is an illness typically affecting men over fifty years of age, increasing in severity with increasing age. The symptoms of the condition include, but are not limted to, increased difficulty in urination and sexual dysfunction. These symptoms are induced by enlargement, or hypertrophy, of the prostate gland. As the prostate increases in size, it impinges on free-flow of fluids through the male urethra.

Concommitantly, the increased noradrenergic innervation of the enlarged prostate leads to an increased adrenergic tone of the bladder neck and urethra, further restricting the flow of urine through the urethra.

The mechanism of prostatic hypertrophy is well understood. The male hormone, 5a-dihydrotestosterone has been identified as the principal culprit. The continual production of dihydrotestosterone by the male testes induces incremental growth of -91- WO 94/21660 PCT/US4102609 4 the prostate gland throughout the life of the male. Beyond the age of aboum fifty years, in many men, this enlarged gland begins to obstruct the urethra with the pathologic symptoms noted above.

The elucidation of the mechanism summarized above has resulted in the recent development of effective agents to control, and in many cases reverse, the pernicious advance of BPH. In the forefront of these agents is Merck Co., Inc.s' product PROSCAR® (finasteride).

The effect of this compound is to inhibit the enzyme testosterone alpha reductase, which converts testosterone into resulting in a reduced rate of prostatic enlargement, and often reduction in prostatic mass.

The development of such agents as PROSCAR® bodes well for the long-term control of BPH. However, as may be appreciated from the lengthy development of the syndrome, its reversal also is not immediate. In the interim, those males suffering with BPH continue to suffer, and may in fact lose hope that the agents are working sufficiently rapidly.

In response to this problem, one solution is to identify pharmaceutically active compounds which complement slower-acting therapeutics by providing acute relief. Agents which induce relaxation of the urethral smooth muscle, by binding to alpha-1 adrenergic receptors, thus reducing the increased adrenergic tone due to the disease, would be good candidates for this activity. Thus, one such agent is alfuzosin, which is reported in EP 0 204597 to induce urination in cases of prostatic hypertrophy. Likewise, in WO 92/0073, the selective ability of the enantiomer of terazosin to bind to adrenergic receptors of the al subtype was reported. In addition, in WO 92/161213, hereby incorporated by reference, combinations of alpha-reductase inhibitory compounds and alphal-adrenergic receptor blockers (terazosin, doxazosin, prazosin, bunazosin, indoramin, alfuzosin) were disclosed. However, no information as to the alA, al B, or al C subtype specificity of these compounds was provided as these refinements were not yet available. The instant invention changes this situation by providing a cloned human Ol C adrenergic receptor and 114' WO 94/21660 PCT/US94/02609 5 a method for identifying compounds which bind the human ca C receptor.

Typically, identification of active compounds is through use of animal tissues known to be enriched in adrenergic receptors. Thus, rat tissues have been used to screen for potential adrenergic receptor antagonists. However, because of species variability, compounds which appear active in animal tissue may not be active or sufficiently selective in humans. This results in substantial wastage of time and effort, particularly where high volume compound screening programs are 1o employed. There is also the danger that compounds, which might be highly effective in humans, would be missed because of their absence of appreciable affinity for the heterologous animal receptors. In this regard, it has been noted that even single amino acid changes between the sequence of biologically active proteins in one species may give rise to substantial pharmacological differences. Thus, Fong et al., (J Biol.

Chem., 267:25668-25671, 1992) showed that there are 22 divergent amino acid residues between the sequence of the human neurokinin-1 receptor and the homologous rat receptor. They further showed, in studies with mutant receptors, that substitution of only two amino acid residues was both necessary and sufficient to reproduce the rat receptor's antagonist binding affinity in the human receptor. Oksenberg et al., (Nature, 360:161-163, 1992) showed that a single amino-acid difference confers major pharmacological variation between the human and the rodent 5-hydroxytryptamine receptors. Likewise, Kuhse et al., (Neuron, 5:867-873, 1990) showed that a single amino-acid exchange alters the pharmacology of the neonatal rat glycine receptor subunit.

This difficulty and unpredictability has resulted in a need for a compound screen which will identify compounds that will be active in humans.

The instant inventors have solved these problems by cloning a novel human adrenergic receptor of the alC subtype. Their efforts have led to the development of a novel screening assay which enables them to identify compounds which specifically interact with the human a C adrenergic receptor. Marshall et al (Br. J. Pharm., p I~LLI n~sUBI*YI6L~ DIY~ C n~ WO 94/21660 I'CT/US94/02609 6 107:327 (1992)) speculated that compounds which specifically interact with the alC adrenergic receptor may be responsible for contraction of the human prostate. The instant invention provides a method for identifying compounds which bind the human alC receptor. In addition, if the compounds are further tested for binding to other human alpha 1 receptor subtypes, as well as counterscreened against other types of receptors, the specificity of the compounds for the human alC adrenergic receptor may be defined.

Compounds identified according to this invention may be used to reduce the acute symptoms of BPH. New agents identified in this manner, or already known agents showing activity in this assay, may now be employed in a novel way to help BPH sufferers contend with the acute symptoms of the syndrome. Thus, this invention is useful to identify compounds which may be used alone or in conjunction with a more long-term anti-BPH therapeutics, such as PROSCAR®. Other uses for the invention include identification of compounds which induce highly tissue-specific, localized alC adrenergic receptor blockade.

Effects of this blockade include reduction of intra-ocular pressure, control of cardiac arrhythmias, and possibly a host of alpha-1C receptor mediated central nervous system events. In addition, the cloned alC receptor can be used for screening of tissue specific expression of alC adrenergic receptors. Effects such as these, induced by or available to analysis with the alC adrenergic receptor also form part of this invention.

SUMMARY OF THE INVENTION The human adrenergic receptor of the alphalC subtype is cloned and used in an in vitro assay to screen for compounds that bind to the receptor, including compounds which specifically inhibit the 0activity of the receptor. The invention includes the assay, the cloned receptor used in the assay (cDNA), an isolated human alphalC adrenergic receptor, cells expressing the cloned receptor, and compounds identified through the use of this novel, cloned receptor, which selectively bind to the human alphalC adrenergic receptor, WO 94/21660 PCT/US94/02609 7 including specific antagonists of the receptor. One embodiment of this invention is a method of treating benign prostatic hypeplasia (BPH) employing compounds having an affinity for the human alpha I C receptor that is at least 12 fold greater than for either the human alpha 1A or the human alpha 1B receptors.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1: Sequence of cDNA obtained by PCR of human heart mRNA, SSEQ. ID:4:.

Fig. 2: Comparison of the open reading frame obtained from human heart, SEQ ID:5:, and the bovine alpha-1C adrenergic receptor sequence, SEQ. ID:6:.

Fig. 3: Sequence of cDNA obtained by screening a human hippocampus cDNA library using the heart mRNA derived sequence from figure 1, SEQ. ID:7: Fig. 4: Sequence of 3' coding region of human alpha-1C gene, obtained by PCR amplification of a human genomic DNA library with oligonucleotides, SEQ. Fig. 5: Sequence of the ligated portions of human alpha-1C DNA shown in figures 3 and 4, SEQ. ID:11:.

Fig. 6: The amino acid sequence of the human alpha-1C adrenergic receptor, SEQ ID:12:.

Fig. 7: The alignment of the nucleotide and amino acid sequence of the human alpha-1C adrenergic receptor, showing the region, SEQ. ID:11: and SEQ. ID:12:.

-I PCT/S94102609 WO 94/21660 8 Fig. 8: Expression of the human alpha-IC adrenergic receptor in COS cells: Binding data using membranes from cells transfected with the expression vector alone and the expression vector containing the human alpha-1C adrenergic receptor coding sequences.

Fig. 9: Binding curves of compounds using membranes from COS cells transfected with the human alpha-lC adrenergic receptor containing expression vector.

Fig. 10: Nucleotide sequence of the human alphalA receptor, SEQ.

ID:13: Fig. 11: Amino acid sequence of the human alphalA adrenergic receptor, SEQ. ID:14: Fig. 12: Partial sequence of the human alphalB adrenergic receptor, SEQ. ID:17: Fig. 13: Partial sequence of the human alphalB adrenergic receptor, 2 SEQ. Fig. 14: Partial sequence of the human alphalB adrenergic receptor, SEQ. ID:23: Fig. 15: Composite human/rat alphalB adrenoreceptror, SEQ. ID:24: Fig. 16: Amino acid sequence of the composite human/rat alphalB adrenergic receptor, SEQ. Fig. 17: Binding curves of compounds using membranes from COS cells transfected with the human alphalA, 1B, and 1C adrenergic receptor expression vectors.

WO 94/21 j WSCTI$94/02609 9 Fig. 18: Sequence of truncated human alphal C adrenergic receptor, SEQ. ID:26:.

Fig. 19: Nucleotide sequence of the human alC adrenergic receptor having a Pstl site, SEQ.ID:27:.

Fig. 20: Amino acid sequence of the human alC adrenergic receptor encoded by the Pstl site encoding allele, SEQ.ID:28:.

Fig. 21: Alignment of the nucleotide and amino acid sequences of figures 19 and 20, SEQ.ID:27: and SEQ.ID:28:.

FIG. 22: Nucleotide sequence of the human alA adrenergic receptor, Seq.ID:29:.

Fig. 23: Amino acid sequence of the human qlA adrenergic receptor, Fig. 24: Alignment of the nucleotide and amino acid sequences of figures 22 and 23, SEQ.ID:29: and DETAILED DESCRIPTION OF THE INVENTION The human alpha adrenergic receptor of the 1-C subtype was identified, cloned and expressed by the instant inventors. A partial coding region for this receptor was generated by reverse transcriptasepolymerase chain reaction technology, RT-PCR. Accordingly, degenerate oligonucleotides encoding amino acids conserved in the fifth and sixth transmembrane domains of all three al receptor subtypes (A, B, C) were used to prime RT-PCR reactions using human heart mRNA as template. The predicted sized products were cloned and sequenced.

Translation of the amplified cDNA yielded an open reading frame encoding a protein 95% homologous to the bovine alC receptor (Fig.2, SEQ. ID:5: and SEQ. This partial sequence was used to obtain a larger cDNA clone from a human hippocampus library (Fig. 3, SEQ.

WO 94/21660 VCT/IUS94/02609 10 The remaining coding region was obtained by PCR amplification of human genomic DNA using primers based on the cDNA sequence and the last six amino acids of bovine alC receptor (Fig. 4, SEQ.ID:10:). The complete receptor was then assembled using the partial sequences shown in Fig. 3, SEQ.ID:6: and Fig. 4, SEQ.

to generate the sequence shown in Fig. 5, SEQ. ID:11:. The translation of this sequence is shown in Fig. 6, SEQ. ID:12:, and the alignment of the nucleotide and amino acid sequences, and the untranslated sequences, is shown in Fig. 7, SEQ. ID:11: and SEQ.

ID:12:.

The 3'-terminal six amino acids of the human al C adrenergic receptor were confirmed by screening a human genomic library with the radiolabeled 3'-terminal 512 nucleotides of the SEQ.

clone previously obtained. A complete human exon 2 was generated in this manner and sequenced. The nucleotide sequence of this gene is provided in figure 19, SEQ. ID:27: and the amino acid sequence is provided in figure 20, SEQ. ID:28:. We discovered that this clone was identical to the original 3'-terminal portion of the gene, except that: 1) There are five silent nucleotide changes between the new clone and the previously obtained clone (the last five codons, including the stop codon, each have a silent change in the third nucleotide); and 2) At nucleotide position 1636 (amino acid 347), there is a cytosine to thymine base change resulting in the formation of a Pstl site at that location and a concommitant single amino acid change of Arg to Cys.

Thus, we have confirmed and localized the site of the two-allele Pstl restriction fragment polymorphism (RFLP) noted by Hoehe et al., [Human Mol. Genetics, 1(5):349 Through pharmacological studies using clones of both alleles, we have confirmed that the Arg to Cys change appear to be pharmacologically indistinguishable (see Table II, Example 11, below).

The cloned human alC receptor, when expressed in mammalian cell lines (see Fig. is used to discover ligands that bind to the receptor and alter its function. In addition, the cloned alC c sE WO 94/21660 PCT/US94/02609 11 receptor enables quanititation of mRNA levels in human tissues, including the aorta and prostate, by RNase protection assays. For these purposes, a complete coding sequence of the receptor is provided. However, as long as the ligand binding and signal transduction segments of the receptor (G-protein interaction) are intact, truncation at the 3' end of the sequence does not affect the functioning of the receptor.

Thus, in addition to the sequence provided in SEQ. ID:11:, a sequence, truncated at the 3' end, SEQ. ID:26: is disclosed, which consists entirely of human alphalC sequence.

The specificty of binding of compounds showing affinity for the al C receptor is shown by comparing affinity to membranes obtained from COS cells tranfected with the cloned alC receptor and membranes from tissues known to express other types of alpha or beta adrenergic receptors. In addition, the cloned human alA and a hybrid human/rat alB (with only the cytoplasmic, carboxy terminal region being rat sequence) could be used for this purpose, along with the human alC receptor expressed in COS cells Expression of the cloned human alA, alB, and alC receptors and comparison of their binding properties with known selective antagonists provides a rational way for selection of compounds and discovery of new compounds with predictable pharmacological activities.

Once the human receptor is cloned and expressed in a cell such as COS cells or CHO cells, the receptor is free of other human proteins. The membranes from cells expressing different human alpha adrenergic receptor subtypes are then isolated according to methods well known in the art for membrane associated receptor binding assays.

For example, the method of Schwinn, et al., Biol. Chem., 265:8183- 8189, 1990) may be used. A compound of interest is used to compete with the binding of a known, quantifiable alpha receptor ligand. Thus, radiolabled prazosin, niguldipine, 5-methyl urapidil, terazosin, dozazosin, phenoxybenzamine, WB4101, benoxathian, HEAT hydroxy-3-iodophenyl)ethylaminomethyl]tetralone, or phentolamine may be used for this purpose (see, for example, Robert R. Ruffolo, Jr., a-Adrenoreceptors: Molecular Biology, Biochemistry and s WO) 9it/2 166(i Pf-TION04/0,160P2BO~ 12 Pharmaco:ogy, (Progress in Basic and Clinical Pharmacology series, Karger, 1991), page 29). Because of the ease of 1 2 5 Iodine detection, 125 I-HEAT may be preferred for this purpose. By increasing the.

amount of unlabeled, test compound, the labeled compound is competed off the receptor. From these experiments, IC50 values for each test compound and receptor subtype is determined.

Thus, according to this invention, a method is provided for identifying compounds specific for the human alphal C receptor comprising the following steps: a. Cloning the human alphalC adrenergic receptor; b. Splicing the the cloned alphalC adrenergic receptor into an expression vector to produce a construct such that the alphal C receptor is operably linked to transcription and translation signals sufficient to Sinduce expression of said receptor upon introduction of said construct into a prokaryotic or eukaryotic cell; c. Introducing said construct into a prokaryotic or eukaryotic cell which does not express a human alphalC adrenergic receptor in the absence of said introduced construct; Sd. Incubating cells or membranes isolated from cells produced in step c.

with a quantifiable compound known to bind to human alfha adrenergic receptors, and subsequently adding test compounds at a range of concentrations so as to compete the quantifiable compound from the receptor, such that an IC50 for the test compound is obtained as the concentration of test compound at which 50% of the quanritiable compound becomes displaced from the receptor; e. Incubating cells or membranes of cells which naturally express or have an introduced, cloned human alpha adrenregic receptor of a subtype other than the human alphalC receptor under identical conditions to the incubation conducted in step d, and obtaining the of the test compound for the non-alphalC receptor; and f. Comparing the IC50 for the test compound for the alphalC receptor and for the alpha adrenergic receptor of a subtype other than the IBIIBMsbCdBIBI"~"- ~s~---US~LRPI- WO 94/21660 K'VIIJ89S410209 13 alphalC to identify compounds having a lower IC50 for the alphalC receptor.

In addition to providing a sequence for the human alC adrenergic receptor, the instant inventors have also discovered a different sequence than that reported by Bruno et al., [BBRC 179:1485- 1490 (1991)] for the human alA adrenergic receptor. The new sequence is more homologous to the rat alA adrenergic receptor sequence. Disclosed and claimed herein is the sequence for this new human alA adrenergic receptor (see Example 12 and figures 22, 23, 1 and 24, SEQ. ID:29: and SEQ. ID:30:). While no difference in ligand binding has thus far been observed based on the different amino terminal amino acid sequences between these two receptors, such differences cannot be ruled out except by screening compounds against both clones. Since a new human sequence is provided herein, compounds identified according to the method of this invention using the earlier reported human alA adrenergic receptor sequence can now be confirmed against this clone.

As a result of the cloning, sequencing, expression, and screening efforts described above and further exemplified below, numerous compounds have been tested for their ability to specifically bind to the cloned human alC receptor with high affinity. Compounds specific for the human alpha 1C adrenergic receptor, that is compounds having an affinity for the human alpha 1C receptor that is at least 12 fold greater than for either the human alpha 1A or the human alpha 1B receptors, are identified by this method. Thus, the compounds niguldipine, (S(+)-l,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5pyridinedicarboxylic acid 3-(4,4-diphenyl-l-piperidinyl)-propyl methyl ester hydrochloride), and 5-methyl urapidil (5-methyl-6[[3-[4-(2methoxyphenyl)- 1-piperazinyl]propyl] amino]-1,3-dimethyluracil) have been discovered to selectively bind to the human l C adrenergic receptor. These compounds may be administered in dosages effective to antagonize the alphalC receptor where such treatment is needed, as in

BPH.

Ii RP-- L1 OMI~ WO 94/21660 V)C1 941606US9402 14 Compounds iCentified according to the method of this invention as being selective human alC adrenergic receptor antagonists may further be defined by counterscreening. This is accomplished.

according to methods known in the art using other receptors responsible for mediating diverse biological functions Compounds which are both selective amongst the various human alphal adrenergic receptor subtypes and which have low affinity for other receptors, such as the alpha2 adrenergic receptors, the B-adrenergic receptors, the muscarinic receptors, the serotonin receptors, and others are particularly 0preferred. The absence of these non-specific activities may be confirmed by using cloned and expressed receptors in an analogous fashion to the method disclosed herein for identifying compounds which have high affinity for the various huaman alphal adrenergic receptors.

Furthermore, functional biological tests are used to confirm the effects of identified compounds as alphalC adrenergic receptor antagonists.

Compounds identified according to this patent disclosure may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the human al C adrenergic receptor while minimizing any potential toxicity. In addition, coadministration or sequential administration of other agents which alleviate the effects of BPH is desirable. Thus, in one embodiment, this includes administration of compounds identified according to this disclosure and a human testosterone 5-a reductase inhibitor. Many such compounds are now well known in the art and include such compounds as PROSCAR®, (also known as finasteride, a 4-Aza-steroid; see US Patents 4,377,584 and 4,760,071, for example, hereby incorporated by reference). In addition to PROSCAR®, which is principally active in prostatic tissue due to its selectivity for human 5-a reductase isozyme 2, combinations of compounds which are specifically active in inhibiting isozyme 1 (found particularly in skin) and compounds which act at both of these isozymes, are useful in combination with compounds identified according to this invention.

In the treatment of hyperandrogenic disease conditions, e.g.

benign prostatic hyperplasia (BPH) and/or the prevention and treatment 91~11sB"~W~s~9~""4 rTJUS94O260) WO 94/21660 15 of prostatic cancer, and the treatment of prostatitis, it would be desirable to have one drug entity which is active against both isozymes to significantly inhibit dihydrotesterone production. It would also be desirable to have one drug entity that is active as a dual inhibitor of both sozymes for the treatment of conditions of the skin and scalp, e.g. acne vulgaris, seborrhea, female hirsutism, and androgenic alopecia.

Additionally, such a dual inhibitor of 5a-reductase 1 and 2 could be used in combination with a 5a-reductase 1 inhibitor or with a reductase 2 inhibitor, e.g. finasteride (PROSCAR®), for combination therapy in the treatment of hyperandrogenic conditions, in combination with compounds identified according to this inventionas being selective human alphalC adrenergic receptor antagonists. The dual isozyme inhibitor could also be used in combination with a potassium channel opener, e.g. minoxidil, for the treatment of male pattern baldness, and such combinations in combination with selective human alphalC adrenergic receptor antagonists also form part of the instant invention Compounds that are active as dual 5a-reductase 1 and 2 inhibitors have been described in W093/23420, EP 0572166; WO 93/23050; W093/23038, W093/23048; W093/23041; W093/23040; W093/23039; W093/23376; W093/23419, EP 0572165; W093/23051, each of which is hereby incorporated by reference.

The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds identified 2 according to this invention as the active ingredient for use in the specific antagonism of human alphalC adrenergic receptors can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for systemic administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), PCT[US94/26O 9 WO 94121660 16 intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as an alphalC antagonistic agent.

The daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult human/per day. For oral administration, the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0002 mg./kg to about mg./kg. of body weight per day. The range is more particularly from about 0.001 mg./kg to 7 mg./kg. of body weight per day. The dosages of the alphalC adrenergic receptor and testosterone reductase inhibitors are adjusted when combined to achieve desired effects. As those skilled in the art will appreciate, less reductase inhibitor may be required when the acute symptoms of BPH are alleviated by treatment with alphal C adrenergic receptor inhibitors has been initiated. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.

Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

WO 94/21660 PCT/IUS94102609 17 For the treatment of acne vulgaris, androgenic alopecia including male pattern baldness, seborrhea, female hirsutism, benign prostatic hyperplasia, prostatitis and the prevention and/or treatment of prostatic cancer, compounds exhibiting at least 12 fold selectivity for inhibition of the alphalC adrenergic receptor can be combined with a therapeutically effective amount of a 5x-reductase 2 inhibitor, such as finasteride, in addition to a 5c-reductase 1 inhbitor, such as 4,7B-dimethyl-4-aza-5a-cholestan-3-one, in a single oral, systemic, or parenteral pharmaceutical dosage formulation. Alternatively, a combined therapy can be employed wherein the alphalC adrenergic receptor antagonist and the 5a-reductase 1 or 2 inhibitor are administered in separate oral, systemic, or parenteral dosage formulations. Also, for the skin and scalp related disorders of acne vulgaris, androgenic alopecia including male pattern baldness, Sseborrhea, and female hirsutism, the compounds of the instant invention and dual inhibitors of 5S-reductase 1 and 2 could be formulated for topical administration. For example, niguldipine or urapidil and finasteride can be administered in a single oral or topical dosage formulation, or each active agent can be 2 administered in a separate dosage formulation, in separate oral dosage formulations, or an oral dosage formulation of finasteride in combination with a topical dosage formulation of a compound exhibiting dual inhibiton of both isozymes of 5a-reductase. See, U.S. Patent No.'s 4,377,584 and 4,760,071 which describe dosages and formulations for 5a-reductase inhibitors.

Furthermore, since administration of inhibitors have been found to be useful in combination with a therapeutically effective amount of a potassium channel opener, such as minoxidil, cromakalin, pinacidil, a compound selected from the classes of S-triazine, thiane-1-oxide, benzopyran, and pyridinopyran derivatives or a pharmaceutically acceptable salt thereof, compounds of this invention may also be used in combination therapy for the treatment of androgenic alopecia including male pattern baldness.

The active agents can be administered in a single topical dosage

I

WO 94/21660 PCTIVUS4/02409 18 formulation, or each active agent can be administered in a separate dosage formulation, in separate topical dosage formulations, or an oral dosage formulation of a compound of formula I in combination with a topical dosage formulation of, minoxidil.

See, U.S. Patent No.'s 4,596,812, 4,139,619 and WO 92/02225, published 20 February 1992, for dosages and formulations of calcium channel openers.

For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.

The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentration of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with w WO 94/21660 VCT/IS94/02609 19 an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.

Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.

Disintegrators include, withouit limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

The liquid forms in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents which may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired.

Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.

Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to 2 form, alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations. See, EP 0 285 382.

The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also bex delivered by the use of monoclonal antibodies as individual carriers WO 94/21660 PCTr/US94/02609 20 to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues.

Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, 1 polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

The following examples are provided to further define the invention without, however, limiting the invention to the particulars of these examples.

EXAMELE-1 PCR amplification, cloning and sequencing of phalX: Based on the amino acid homologies of human alA, rat alB and bovine alC receptors, degenerate oligonucleotides were designed to amplify cDNAs encoding all three receptor subtypes. These oligonucleotides are: WL' (SEQ. ID:1) TITTCTAGAT TRTTNARRTA NCCNAGCC 28 MYC (SEQ. ID:2) TTTACTAGTA TCSTNGTNAT GTAYTG 16 WC(SEQ. ID:3) TTTCTAGAG AARAANGGNA RCCARC 26 WC' (SEQ. ID:3) TITTCTAGAG AARAANGGNA RCCARC 26 Il~ auaau~ll-- VCTIrUS94/02609~! WO 94/216(0 21 Oligonucleotides MYC and WL' were used as primers in a reverse transcription PCR amplification of human heart mRNA (Clontech) using the RNA PCR kit from Perkin Elmer Cetus. Briefly, 0.5 ug of mRNA was reverse transcribed in a volume of 20 ul using either random oligonucleotide primers (reaction 1) or oligo dT primer (reaction Reactions 1 and 2 were pooled and served as template for PCR amplification as follows: PCR Reactions: Primary reaction (50 ul) ul 10X buffer from Perkin Elmer Cetus GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP 3ul first strand cDNA 1 ul 25 pMoles oligo MYC 1 ul 25 pMoles oligo WL' 0.25 ul 1.25 units Amplitaq DNA polymersase 31.75 ul water Reaction conditions; 40 cycles of 940C 450C 720C 2' Secondary reaction (100 ul) ul 10X buffer from Perkin Elmer Cetus GeneAmp Kit 16ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP first strand cDNA 2 ul 50 pMoles oligo MYC 2 ul 50 pMoles oligo WYC 2 ul 50 pMoles oligo WC' ul 2.5 units Amplitaq DNA polymersase ul water Reaction conditions; 40 cycles of 940C 450C 720C 2' Prep scale tertiary reaction 3 X 200 ul: i I'MCr/US04/02409 WO 94/21660 22 19.5 ul 10X buffer 32 ul 1.25 mM each stock of dATP, dCTP, dGTP, and dTTP ul secondary PCR reaction 4 ul 100 pMoles oligo MYC 4 ul 100 pMoles oligo WC' 1 ul 5 units Amplitaq DNA polymerase 134.5 ul water Reaction conditions; 30 cycles of 940C 500C 720C 2' The PCR product was purified by Qiagen spin columns and digested with restriction endonucleases Spel and Xbal. The fragment was then ligated into SpeI/Xbal cut pGEM9Zf(-). The ligation mix was used to transform E. coli XL-1 blue. Plasmid DNA was isolated from Swhite transformants and sequenced by the dideoxy chain termination method. The base sequence obtained is shown in Fig. 1, SEQ. ID:4:.

EXAMPLE 2 Isolation of partial alphalC cDNA Clone: A cDNA library prepared from mRNA isolated from human hippocampus (Stratagene) was screened by plaque hybridization using phalX as a probe. Hybridization conditions were as follows: 1XSSC is 0.15M sodium chloride, 0.015M sodium citrate, Formamide Denhardt's Solution 1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin) 0.15 mg/ml salmon sperm DNA hybridize overnight at 420 C.

WO 94/21660 I'CT/US94/0209 23 Filters were washed 3 times in 2XSSC, 0.1% SDS at room temperature for then 1 time in 1XSSC, 0.1% SDS at 50C for Positive clones were identified by autoradiography. Phagemid DNA was rescued from the positive plaques and sequenced by the dideoxy chain termination method. The base sequence obtained is shown in Fig.

3, SEQ. ID:7:.

EXAMPLE

PCR amplification, cloning and sequencing of 3'CG of alphalC: The 3' end of the coding region of human alphalC adrenergic receptor was amplified from human genomic DNA using two oligonucleotides: S3C (SEQ ID:8:) TTTGAATTCT GATTTCAAGC CCTCTG 3' and 3'C (SEQ ID:9:) TTTGAATTCT TANACYTCYT CNCCRTTYTC 3' as follows: ul 10X buffer from Perkin Elmer Cetus GeneAmp Kit 16 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP 6 ul 1 ug human genomic DNA (Promega) 2 ul 50 pMoles oligo S3C 2 ul 50 pMoles oligo 3'C ul 2.5 units Amplitaq DNA polymersase 63.5 ul water 4 a, VCTIVS94/0240960 WO 94/21660 24 Reaction conditions; 40 cycles of 940C 500C 2'; 720C 2' The PCR product was purified by Qiagen spin columns and digested with restriction endonuclease EcoRI. The fragment was then ligated into EcoRI cut pGEM3Zf(-). The ligation mix was used to transform E. coli XL-1 blue. Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method.

The base sequence is shown in Fig. 4, SEQ. Assembly of complete coding region of human alphalc adrenergic Sreceptor: The complete coding region of human alphalc adrenergic receptor was assembled by ligating the cDNA clone (see Example 2, figure 3, SEQ ID:7:) and 3'CG (see Example 3, figure 4, SEQ ID:10: at their 2 common PvuII site 1552-1557 of figure 3, SEQ ID:7: and 59-64 of figure 4, SEQ ID:10:). The complete nucleotide sequence is shown in figure 5, SEQ ID:11:. The amino acid sequence is shown in figure 6, SEQ. ID:12:. Figure 7 shows the structure of the cDNA, including the sequences. The very 3' twenty seven nucleotides (6 amino acids) shown is the sequence of the PCR primer used to generate the sequence. However, the function of the receptor, both for ligand binding and signal transduction depends on sequences far removed from the carboxy terminus of the receptor. A completely human sequence is shown in figure 18, SEQ. ID:26: which is truncated at the 3' terimuns.

Expression of the cloned alvhalC adrenermic recentor: ~r LLB~ I WO 94/21660 WCTU/94/02609 25 The complete sequence (SEQ ID:11:) of the human alphalC adrenergic receptor was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS-7 cells by electroporation. Cells were harvested after 72 hours and the membranes containing the expressed receptor protein were prepared as described in Schwinn, et al., J. Biol. Chem., 265:8183- 8189, 1990. Membranes (5-25 ug, see figure 8) prepared from the COS-7 cells transfected with the vector containing the alphalC receptor gene specifically bound the alpha 1 antagonist [125 I]-HEAT; membranes prepared from the COS-7 cells transfected with the vector alone did not bind the alpha 1 antagonist [125 I]-HEAT (figure 8), proving the expression of the alphalC adrenergic receptor. Binding reactions (total volume 200 ul) contained 50 mM Tris-HC1 pH. 7.4, mM EDTA, 150 mM NaCI, 100 pM [125 I]-HEAT, and membranes prepared from COS-7 cells transfected with expression plasmids.

Reactions were incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Non specific binding was determined in the presence of 10 uM prazosin.

IICIIUS94/02609~ WO 94/21660 26

EXAMPLE__

Screening assay: Alpha 1 C Adrenergic Receptor Binding Membranes prepared from the transfected COS-7 cells may also be used to identify compounds that bind to the human alphal C adrenergic receptor. These competition binding reactions (total volume 200 ul) contain 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCI, 100 pM [125 I]-HEAT, membranes prepared from COS-7 cells transfected with the SalphalC expression plasmid and increasing amounts of unlabeled ligand.

Reactions are incubated at room temperature for one hour with shaking.

Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Binding data were analyzed and determined by an iterative curve fitting program. Results are shown in Figure 9.

EXAMELE.K

Expression of human alphalA adrenergic receptor: The complete coding region for the human alphalA adrenergic receptor (Buno, et al., BBRC., 179:1485-1490, (1991); see figure SEQ. ID:13: and figure 11, SEQ. ID:14: herein) was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS-7 cells by electroporation. Cells were harvested after 72 hours and the membranes containing the expressed receptor protein were prepared as described in Schwinn, et al., J. Biol.

Chem., 265:8183-8189, 1990. Membranes prepared from the COS-7 cells transfected with the vector containing the alphalA receptor gene specifically bound the alpha 1 antagonist [125 I]-HEAT; membranes prepared from the COS-7 cells transfected with the vector alone did not bind the alpha 1 antagonist [125 I]-HEAT.

WO 94/21660 PCT/US94102609 27 Binding reactions (total volume 200 ul) contained 50 mM Tris- HCI pH. 7.4, 5 mM EDTA, 150 mM NaCI, 100 pM [125 I]-HEAT, and membranes prepared from COS-7 cells transfected with expression.

plasmids. Reactions are incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Nonspecific binding was determined in the presence of 10 uM prazosin.

EXAMPL E..

Expression of human alphalB adrenergic receptor: 1. PCR amplification of partial cDNA for human alphalB adrenergic receptor: Amplification of 5XB clones SEQ. ID:15: 5' TCT AGA CCA TGA AYC CNG AYC TGG 3' A1B, SEQ. ID:16: 5' TTT GAA TTC ACA TWC CGA CYA CAA TGC CC 3' Oligonucleotides 5XB and A1B were used as primers in a reverse transcription PCR amplification of human heart mRNA (Clontech) using the Invitrogen Copy Kit. Briefly, 1.0 ug of mRNA was reverse transcribed in a volume of 20 ul using oligonuleotide WC' as primer.

Primary reaction (50 ul) ul 10X buffer from Perkin Elmer Cetus GeneAmp Kit WO 94/21660 PCT/MS94/02609 28 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP ul first strand cDNA 1 ul 25 pMoles oligo 1 ul 25 pMoles oligo A1B 0.25 ul 1.25 units Amplitaq DNA polymersase 32.75 ul water Reaction conditions; 40 cycles of 940C 580C 720C 2' The PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INVoaF' (Invitrogen). Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 12, SEQ.

ID:17: 2. Amplification of EFK clones EFK, SEQ. ID:18: 5' GAAGGCGCGCTTGAACTC 3' 5B1, SEQ. ID:19: 5' AGAGAACCACCAAGAACC 3' Oligonucleotides EFK and 5B1 were used as primers in a reverse transcription PCR amplification of human aorta mRNA (Clontech) using the Invitrogen Copy Kit. Briefly, 1.0 ug of mRNA was reverse transcribed in a volume of 20 ul using oligo dT as primer.

Primary reaction (50 ul) 5 ul 10X buffer from Perkin Elmer Cetus GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP ul first strand cDNA WO 94/21660 PCT/US94/02609 29 1 ul 25 pMoles oligo EFK 1 ul 25 pMoles oligo 5B1 0.25 ul 1.25 units Amplitaq DNA polymersase 33.25 ul water Reaction conditions; 40 cycles of 940C 580C 72 0 C 2' The PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INVaF' (Invitrogen). Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 13, SEQ.

3. Assembly of partial cDNA for human alphalB adrenergic receptor A partial cDNA clone encoding the human alphalB adrenergic receptor was assembled by joining the 5XB sequence (SEQ. ID:17:) and the EFK sequence (SEQ. ID:20:) at their common BamHI site.

4. Amplification of the 3' end of rat alphalB adrenergic receptor S4B, SEQ. ID:21: 5' TTT GAA TTC ATG TTC AAG GTG GTG TTC 3' 3'B2, SEQ. ID:22: 5' TTT GAA TTC TAA AASTGN CCN GGN SCC AGN GGC AT 3' Oligonucleotides S4B and 3'B2 were used as primers in a reverse transcription PCR amplification of rat heart mRNA using the Invitrogen Copy Kit. Briefly, 0.6 ug of mRNA was reverse transcribed in a volume of 20 ul using oligo dT as primer.

:1_ aerirlUS3r111)21;6!, WO 94/21660 30 Primary reaction (50 ul) ul 10X buffer from Perkin Elmer Cetus GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP ul first strand cDNA 1 ul 25 pMoles oligo EFK 1 ul 25 pMoles oligo 5B1 0.25 ul 1.25 units Amplitaq DNA polymersase 33.25 ul water Reaction conditions; 40 cycles of 94 0 C 580C 72 0 C 2' The PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INVctF' (Invitrogen). Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 14, SEQ.

ID:23:.

Assembly and expression of a functional human/rat hybrid alphalB adrenergic receptor The partial human alphalB adrenergic receptor cDNA was joined to the 3' end of the rat alphalB adrenergic receptor cDNA at their common BssHII restriction endonuclease site. This composite sequence is shown in figure 15, SEQ. ID:24:, and the amino acid sequence is shown in Fig. 16, SEQ. The complete coding region for the human/rat alphalB adrenergic receptor was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS-7 cells by electroporation. Cells were harvested after 72 hours and the membranes containing the expressed receptor protein were prepared as described in Schwinn, et al., J. Biol. Chem., 265:8183- WO 94/21660 PCT/US94/02609 31 8189, 1990. Membranes prepared from the COS-7 cells transfected with the vector containing the alphalB receptor gene specifically bound the alpha 1 antagonist [125 I]-HEAT; membranes prepared from the COS-7 cells transfected with the vector alone did not bind the alpha 1 antagonist [125 I]-HEAT. Binding reactions (total volume 200 ul) S contained 50 mM Tris-HCI pH. 7.4, 5 mM EDTA, 150 mM NaC1, 100 pM [125 I]-HEAT, and membranes prepared from COS-7 cells transfected with expression plasmids. Reactions are incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Non specific binding was determined in the presence of 10 uM prazosin.

EXAMPLE 9 Selective Binding assays Membranes prepared from COS-7 cells cransfected with the human alpha 1 receptor subtype expression vectors may also be used to identify compounds that selectively bind to the human alphalC adrenergic receptor.

These competition binding reactions (total volume 200 ul) contain mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaC1, 100 pM [125 I]- HEAT, membranes prepared from COS-7 cells transfected with the respective alpha 1 subtype expression plasmid and increasing amounts of unlabeled ligand. Reactions are incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Binding data were analyzed and IC50s determined by an iterative curve fitting program.

the results from such an analysis.

Table I shows the results from such an analysis.

I~lllbn~Eurm~ r~lara~-~aurrru~i~sP~---- PCTUS94/02609 WO 94/21660 32 IC0 0nM) xlA aIB lC Compound prazosin terazosin doxazosin phenoxybenzamine WB4101 benoxathian phentolamine urapidil niguldipine 2.4 2 4.3 16 68 650 270 670 42 130 EXAMPLE IDENTIFICATION AND CLONING OF A NEW ALLELE FOR THE HUMAN ALPHA1-C ADRENERGIC

RECEPTOR

Probes: 3'CG: A 525 bps fragment, specific to complete exon.2 of human alphalc AR, was PCR amplified from human genomic DNA using a sense primer based on the isolated cDNA clone and an antisense primer based on the last six amino acids of bovine alphalc cDNA. This PCR product was subcloned and confirmed by sequencing (see Example 3, i I WO 94/21660 PCT/US9/02609 33 ei c ,_rLibrar_ Screeniin Human W138 Fibroblast genomic library synthesized in the Lambda Fix II vector (2 x 10 6 recombinants; Stratagene,La Jolla, CA) was screened with This probe was labelled with 3 2 P-dCTP (Amersham) by random-primed labelling kit (Boehringer Mannheim,Indianapolis,IN). A Total 800,000 plaques were screened, using duplicate Hybond-N.nylone filters (Amersham,UK). Prior to hybridization, filters were denatured (1.5M NaCl+0.5M NaOH), neutralized (1.5M NaCl+ 1M Tris.Cl,pH 8.0) and washed (0.2M Tris.Cl pH 7.5 2 SSC), 5' for each. DNA was cross-linked with UV crosslinker (Stratagene,La Jolla,CA). The filters were, then, hybridized in fcrmamide, 5 x SSC(lxSSC= 0.15M NaCI, 0.015M Na citrate, 0.02% polyvinylpyrophosphate, 0.2% Ficoll, 0.2% bovine serum albumin, 150pg of sheared boiled Salmon sperm DNA, 106 Scpm of 32 p-labelled probe at 42 0 C for 40hrs. Filters were washed in 0.1x SSC SDS solution at 600C for 20'. Two more rounds of screening for 20 "positive" plaques/clones with 3'CG probe confirmed two clones for the alphalC adrenergic receptor, which were named 48.1C and 53.1C. Clone 53.1C was subjected to further analysis/investigation.

Sub-clonnine of Exon.2 53.1C lambda DNA was amplified by plate lysis method and purified with Qiagen midi-lambda kit (Qiagen,Chatsworth,CA). A 2.6Kb band excised with EcoRI restriction enzyme was identified by Southern analysis using 3'CG probe. This fragment was then subcloned into pGEM3Zf(+) vector.

DNA sequencing: Nucleotide sequence analysis of DNA in both direction was performed by Sanger chain termination method.

WO 94/21660 pCTIUS94102609 34 Result and Dicussin: Sequencing analysis of this genomic clone confirmed that clone 53.1c contains sequences for complete exon.2 flanked by an intron at 5'-end. It also revels that there is a nucleotide change from cytosine to thymine(T) at nucleotide position 1636, amino acid position 347.

This change creates a PstI site and changes the codon for arginine (Arg) to cystine (Cys). This data differs from the known/published cDNA sequence of the gene. Southern analysis of human genomic DNA confirms the PstI site in the gene/exon.2.

EXAM PL.- COMPARATIVE PHARMACOLOGY OF ALPHA I-C ALLELES We have cloned two genes for the human alpha-lc receptor. The coding regions differ by a single nucleotide. The genes encode either Cys or Arg at amino acid 347 near the C terminus of the receptor. The nucleotide difference lies within a PstI restriction enzyme recognition site thus creating a Restriction Fragment Length Polymorphism (RFLP). The frequency of allele 1 (LRR) is 0.34; allele 2 (LCR) is 0.66 in 83 unrelated individuals (Hoehe et al "A two-allele PstI RFLP for the alpha-1C adrenergic receptor gene" Human Molecular Genetics 1: 349, 1992; Allele I is defined by a 2.1 kb PstI fragment; allele 2 yields two bands of 1.6 and 0.5 kb). Since the amino acid difference occurs within the intracellular tail of the receptor we would not expect any pharmacological differences between the expressed receptors. To investigate the pharmacological profiles of the two allelic forms of the human alpha-1c adrenergic receptor we ligated the genomic exon 2 fragment of allele 2 to a cDNA clone of allele 1 at a common PvuII restriction site. The two allelic forms were transiently expressed in COS-7 cells using pcDNAI/NEO (Invitrogen) expression vector. Competitive inhibition studies performed in the presence of

S

125 I-HEAT with various antagonists showed no significant difference in their pharmacological profiles (Table II): WO 94/21660 PCT/US94/0209 35 Table I COMPARATIVE PHARMACOLOGY OF ALPHA 1-C ALLELES (nM) LRR LCR phentolamine 15 17 niguldipine 0.8 1.8 prazosin 1.0 0.9 urapidi 3.1 4.3 WB4101 0.9 EXAMELE 12 CLONING OF A NOVEL ALPHA 1-A ADRENERGIC RECEPTOR A cosmid library containing FG293 cell line genomic DNA in the double-cos vector sCos-1 was screened as follows: The published human ala receptor cDNA clone (Bruno et al., BBRC, 179:1485-1490 (1991), and see Fig. 10, SEQ.ID:13:) was cloned into the vector pcDNA1 neo to generate the clone pEXala. Filters containing approximately 200,000 clones were screened by colony hybridization ([Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, 1989 using a mixed exon 1 probe generated by PCR corresponding to ala (TMD1-3), alb (TMD1-5) and al- 25 cycles of 95" C 52 "C 30 sec; 72 "C 1.5' using 10 ng of pEX alb, pEX alc or pEX ala and 10 pmoles each of primers 5' MET GAATCCCGACCTGGAC), SEQ.ID:31:, and 3' BAM SEQ.ID:32:, for alb, 5' 597 CCATGGTGTTTCTCTCGGG), SEQ.ID:33: and 3' 1219 GACGCGGCAGTACATGAC SEQ.ID:34: for alc or 5' 76 GTCATGATGGCTGGGTACTTG SEQ.ID:35:, for ala in a 12 il reaction containing 1.5 tPM each unlabelled dNTP and 50 gCi 3000 Ci/mmol a-[ 3 2 P] dCTP. The filters were incubated with 1 x 106 cpm/ml of probe in 5X SSC, 35% Foimamide, 0.02% SDS, 0.1 WO 94/21660 PCTUS94/02609 36 lauroyl sarcosine, 2% blocking buffer (Bohrenger Mannheim), at 42 "C for 18 hours. The filters were washed with 2 liters of 0.5X SSC, 0.1% SDS, 55 "C and exposed to Kodak XAR-5 film. Twelve primary positives were picked from master plates and re-screened using the alaspecific probe. Cosmid DNA was prepared from second round positive clones, digested with endonucleases Eco RI or Hind III and subjected to Southern blot analysis: Fragments were resolved by electrophoresis, and transferred to a nitrocellulose membrane (Bohrenger Mannheim) with 20X SSC (1X SSC 0.15M Sodium chloride, 0.015M Sodium citrate, pH 7.0) according to the method of Southern ([Southern, 1975 The membrane was hybridized, washed and analyzed as described above. Alpha-la, alb, and alc receptor clones were identified by comparison of restriction patterns with genomic southern blots performed with ala, alb, or alc specific probes. A Cosmid containing ala receptor exon 1 DNA was subjected to restriction digestion by endonuclease Pst I and subjected to southern blot analysis as above using the ala -specific probe. Two fragments of 2.3 and 1.6 kb were detected and subcloned into the Pst I site of PGEM 3ZF The presence of the correct 5' terminal sequences in the 2.3 kb fragment was 2 confirmed by sequencing across the junction between inverted repeat and non-repeat sequences. The 5' end of the ala receptor gene was ligated to the cDNA clone at their common PstI site, see figures 22-24, SEQ.ID:29:, and EXEMPLARY COUNTERSCREENS LAssay Title: Dopamine D2,D4 in vitro screen Objective of the Assav: The objective of this assay is to eliminate agents which specifically affect binding of [3H] spiperone to cells expressing human dopamine receptors D2, D3 or D4.

WO 94/21660 PCT/US94/02609 37 Modified from VanTol et al (1991); Nature (Vol 350) Pg 610-613.

Frozen pellets containing specific dopamine receptor subtypes stably expressed'in clonal cell lines are lysed in 2 ml lysing buffer (10mM Tris-HCl/5mM Mg, pH Pellets obtained after centrifuging these membranes (15' at 24,450 rpm) are resuspended in Tris-HC1 pH 7.4 containing EDTA, MgCl[2], KC1, NaCI, CaCl[2] and ascorbate to give a 1 Mg/mL suspension. The assay is initiated by 1 adding 50-75 gig membranes in a total volume of 500 gl containing 0.2 nM [3H]-spiperone. Non-specific binding is defined using 10 gM apomorphine. The assay is terminated after a 2 hour incubation at room temperature by rapid filtration over GF/B filters presoaked in 0.3% PEI, using 50mM Tris-HCI pH 7.4.

2. Amy Titi: Serotonin Objective of the Assay The objective of this assay is to eliminate agents which specifically affect binding to cloned human 5HTla receptor Modified from Schelegel and Peroutka Biochemical Pharmacology 35: 1943-1949 (1986).

Mammalian cells expressing cloned human 5HTla receptors are lysed in ice-cold 5 mM Tris-HCI, 2 mM EDTA (pH 7.4) and homogenized with a polytron homogenizer. The homogenate is centrifuged at 1000Xg for 30', and then the supernatant is centrifuged again at 38,000Xg for 30'. The binding assay contains 0.25 nM [3H]8- OH-DPAT in 50 mM Tris-HC1, 4 mM CaC12 and 1mg/ml ascorbate.

Non-specific binding is defined using 10 |M propranolol. The assay is terminated after a 1 hour incubation at room temperature by rapid filtration over GF/Cfilters WO 94/21660 PCT/US94/02609 38 EXAMPLE 14 EXEMPLARY FUNCTIONAL ASSAYS In order to confirm the specificity of compounds for the human alphalC adrenergic receptor and to define the biological activity of the compounds, the following functional tests may be performed: 1. IN VITRO RAT, DOG AND HUMAN PROSTATE AND DOG URETHRA Taconic Farms Sprague-Dawley male rats, weighing 250- 400 grams are sacrificed by cervical dislocation under anesthesia (methohexital; 50 mg/kg, An incision is made into the lower abdomen to remove the ventral lobes of the prostate. Each prostate removed from a mongrel dog is cut into 6-8 pieces longitudinally along the urethra opening and stored in ice-cold oxygenated Krebs solution overnight before use if necessary. Dog urethra proximal to prostate is cut into approximately 5 mm rings, the rings are then cut open for contractile measurement of circular muscles. Human prostate chips from transurethral surgery of benign prostate hyperplasia are also stored overnight in ice-cold Krebs solution if needed.

The tissue is placed in a Petri dish containing oxygenated Krebs solution [NaCI, 118 mM; KCI, 4.7 mM; CaCl2, 2.5 mM; KH2PO4, 1.2 mM; MgSO4, 1.2 mM; NaHCC03, 2.0 mM; dextrose, 11 mM] warmed to 37 0 C. Excess lipid material and connective tissue are carefully removed. Tissue segments are attached to glass tissue holders with 4-0 surgical silk and placed in a 5 ml jacketed tissue bath containing Krebs buffer at 37 0 C, bubbled with 5% C02/95% 02. The tissues are connected to a Statham-Gould force transducer; 1 gram (rat, human) or 1.5 gram (dog) of tension is applied and the tissues are allowed to equilibrate for one hour. Contractions are recorded on a Hewlett-Packard 7700 series strip chart recorder.

After a single priming dose of 3 gM (for rat), 10 gM (for dog) and 20 gM (for human) of phenylephrine, a cumulative WO 94/21660 PCT/US94/02609 39 concentration response curve to an agonist is generated; the tissues are washed every 10 minutes for one hour. Vehicle or antagonist is added to the bath and allowed to incubate for one hour, then another cumulative concentration response curve to the agonist is generated.

values are calculated for each group using GraphPad Inplot software. pA2 (-log Kb) values were obtained from Schild plot when three or more concentrations were tested. When less than three concentrations of antagonist are tested, Kb values are calculated according to the following formula Kb =.LB1, x-1 where x is the ratio of EC50 of agonist in the presence and absence of antagonist and is the antagonist concentration.

2. MEASUREMENT OF INTRA-URTHALE PRESSURE N ANESTHETIZED

DOGS

PURPOSE: Benign prostatic hyperplasia causes decreased urine flow rate that may be produced by both passive physical obstruction of the prostatic urethra from increased prostate mass as well as active 2 obstruction due to prostatic contraction. Alpha adrenergic receptor antagonists such as prazosin and terazosin prevent active prostatic contraction, thus improve urine flow rate and provide symptomatic relief in man. However, these are non-selective alpha-i receptor antagonists which also have pronounced vascular effects. Because we have identified the alpha-1C receptor subtype as the predominent subtype in the human prostate, it is now possible to specifically target this receptor to inhibit prostatic contraction without concomitant changes in the vasculature. The following model is used to measure adrenergically mediated changes in intra-urethral pressure and arterial 3 pressure in anesthetized dogs in order to evaluate the efficacy and potency of selective alpha adrenergic receptor antagonists. The goals are to: 1) identify the alpha-1 receptor subtypes responsible for prostatic/urethral contraction and vascular responses, and 2) use this model to evaluate novel selective alpha adrenergic antagonists. Novel WO 94/21660 PCT/US94/02609 40 and standard alpha adrenergic antagonists may be evaluated in this manner.

METHODS: Male mongrel dogs (7-12 kg) are used in this study.

The dogs are anesthetized with pentobarbital sodium (35 mg/kg, i.v.

plus 4 mg/kg/hr iv infusion). An endotracheal tube is inserted and the ventilated with room air using a Harvard instruments positive cx:sph~~ment large animal ventilator. Catheters (PE 240 or 260) are placed in the aorta via the femoral artery and vena cava via the femoral veins (2 catheters, one in each vein) for the measurement of arterial pressure and the administration of drugs, respectively. A supra-pubic incision ~1/2 inch lateral to the penis is made to expose the urethers, bladder and urethra. The urethers are ligated and cannulated so that urine flows freely into beakers. The dome of the bladder is retracted to 1s facilitate dissection of the proximal and distal urethra. Umbilical tape is passed beneath the urethra at the bladder neck and another piece of umbilical tape is placed under the distal urethra approximately 1-2 cm distal to the prostate. The bladder is incised and a Millar micro-tip pressure transducer is advanced into the urethra. The bladder incision is sutured with 2-0 or 3-0 silk (purse-string suture) to hold the transducer. The tip of the transducer is placed in the prostatic urethra and the position of the Millar catheter is verified by gently squeezing the prostate and noting the large change in urethral pressure.

Phenylephrine, an alpha-1 adrenergic agonist, is administered (0.1-100 ug/kg, iv; 0.05 ml/kg volume) in order to construct dose response curves for changes in intra-urethral and arterial pressure. Following administration of increasing doses of an alpha adrenergic antagonist (or vehicle), the effects of phenylephrine on arterial pressure and intra-urethral pressure are re-evaluated. Four or five phenylephrine dose-response curves are generated in each animal (one control, three or four doses of antagonist or vehicle). The relative antagonist potency on phenylephrine induced changes in arterial and intra-urethral pressure are determined by Schild analysis. The family of averaged curves are fit simultaneously (using ALLFIT software WO 94/21660 (9 6CIYVS94/02400) 41 package) with a four paramenter logistic equation constraining the slope, minimum response, and maximum response to be constant among curves. The dose ratios for the antagonist doses (rightward shift in. the dose-response curves from control) are calculated as the ratio of the for the respective curves. These dose-ratios are then used to construct a Schild plot and the Kb (expressed as ug/kg, iv) determined.

The Kb (dose of antagonist causing a 2-fold rightward shift of the phenylephrine dose-response curve) is used to compare the relative potency of the antagonists on inhibiting phenylephrine responses for intra-urethral and arterial pressure. The relative selectivity is calculated as the ratio of arterial pressure and intra-urethral pressure Kb's Effects of the alpha-1 antagonists on baseline arterial pressure are also monitored. Comparison of the relative antagonist potency on changes in arterial pressure and intra-urethral pressure provide insight as to whether the alpha receptor subtype responsible for increasing intra-urethral pressure is also present in the systemic vasculature.

According to this method, one is able to confirm the selectivity of alphal C adrenergic receptor antagonists that prevent the increase in intra-urethral pressure to phenylephrine without any activity at the vasculature.

At the end of the experiment, the dogs are killed via an overdose of intravenously administered pentobarbital or saturated KC1.

WO 94/21660 PCT/US94/02609 42 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Bayne, Marvin L Clineschmidt, Bradley V Strader, Catherine D (ii) TITLE OF INVENTION: CLONED HUMAN ALPHA1C ADRENERGIC RECEPTOR (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Merck Co., Inc.

'TREET: 126 Lincoln Avenue CITY: Rahway STATE: New Jersey COUNTRY: United States of America ZIP: 07065 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/032,849 FILING DATE: 15-MAR-1993 (viii) ATTORNEY/AGENT INFORMATION: NAME: Bencen, Gerard H REGISTRATION NUMBER: 35,746 REFERENCE/DOCKET NUMBER: 18943IA (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (908)594-3901 TELEFAX: (908)594-4720 TELEX: 138825 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 94/21660 PCT/US94/02609 43 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TTTTCTAGAT TRTTNARRTA NCCNAGCC 28 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TTTACTAGTA TCSTNGTNAT GTAYTG 26 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TTTTCTAGAG AARAANGGNA RCCARC 26 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 235 base pairs TYPE: nucleic acid.

STRANDEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 94/21660 PCTIUS94/0.2609 -44 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GCCGCGTCTA CGTGGTGGCC AAGAGGGAGA GCCGGGGCCT CAAGTCTGGC CTCAAC %CCG ACAAGTCGGA CTCGGAGCAA GTGACGCTCC GCATCCATCG GAAAAACGCC CCGGCAGGAG 120 GCAGCGGGAT GGCCAGCGCC AAGACCAAGA CGCACTTCTC AGTGAGGCTC CTCAAGI1TCT 180 CCCGGGAGAA GAAAGCGGCC AAAACGCTGG GCATCGTGGT CGGCTGCTTC GTCCT 235 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 78 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE; NO FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Arg Val Tyr Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly 1 5 10 Leu Lys Thr Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg Ile His 25 Arg Lys Asn Ala Pro Ala Gly Gly Ser Gly Met Ala Ser Ala Lys Thr 40 Lys Thr His Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys 55 Ala Ala Lys Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu 70 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 93 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 3 0 (ii) MOLECULE TYPE: peptide (iii) fPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal WO 94/21660 I'MrUS9410260') 45 SEQUENCE DESCR27TION: SEQ ID NO:6: Val Met Tyr Cys Arg Val Tyr Val Val Ala Lys Arg Giu Ser Arg 10 Leu Lys Ser Giv Leu Lys Thr Asp Lys Ser Asp Ser Giu Gin Val Glv 25 Thr Leu Arg Ile His Arg Lys Asn Aia Gin Vai Giy Giy 40 Ser Gly Val Leu Lys Phe Val Gly Cys Thr Ser Ala Lys Asn Lys Thr His Phe 55 Ser Arg Giu Lys Lys Aia Aia Lys Thr 70 Phe Val Leu Cys Trp Leu Pro Phe Phe Ser Vai Arg Leu Leu Gly Ile Val 75 Leu Val Met Pro INFORMATION FOR SEQ ID NO:7: Wi SEQUENCE CHARACTERISTICS: LENGTH: i601 base pairs TYPE: nucleic acid STRAN'DEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO.7: GAATTCCCTC CTAGAAGCTG GAGAGAGCAG GAGCC TTCGG TGGGGCAGCT CAAAATGTAG

GTAACTGCGG

GCTGATCTTT

AGAATCGTGA

TAATTCTGGA

GGGAGGGAGT

CGCGCGCCCC

CAGGGCTGGC

CTGCCAGGGC

CGCCAGCCCG

GTGTTTCTCT

AACATTTCCA

GCCAGGAGCA

TGGTTTGAGG

ATCTTCCCCC

ATTGCATGTT

CCGGGTCCCG

TGGCCATGTC

CAGGGTTGTC

TCCCTCCAGA

GGAGGTGGCC

CGGGAAATGC

AGGCCATTCT

GCGCCCAGAT GCCATCGGTC' GAGAGACTGG CGCTGGAGTT AGCCAGGACG AATAAGACAG GCAAGGAGTC TCCTGGATCT GCTAGGCCAG CCCGCAGGTG TTTAATGCCC TGCCCCTTCA TCCCACCCGC GCGCGCCGTC AGAGACCTTT TGATTCCCGG CTGGACAGCC GGACCTCGCC TTCCGACAGC TCCAACTGCA GCTCGGGGTG ATCTTGGGGG

CCTGCCTTTG

TITGAATT CCG

CGCGGAAAAG

TCGCACCCAG

GAGAGGGTCC

TGTGGCCTTC

TCACCCCCAG

CTCCCGCGCT

CGGCCCCGGC

CCCAACCGCC

GCCTCATTCT

AGCGTCGACG

AATCATGTGC

CAGATTCTCG

CTTCGGGTAC

CCGGCAGCCC

TGAGGGTTCC

CCAAACCCAC

CCCGCCTCCG

TGGGACCATG

GGCACCGGTG

TTTCGGGGTG

WO 94/21660 IOCT/US94/02009 46 CTGGGTAACA TCCTAGTGAT CCTCTCCGTA GCCTGTCACC GACACCTGCA .CTCAGTCACG CACTACTACA TCGTCAACCT GGCGGTGGCC GACCTCCTGC TTCTCCGCCA TCTTCGAGGT CCTAGGCTAC TGGGCCTTCG TGGGCGGCAG TGGATGTGCT GTGCTGCACC GCGTCCATCA ATCGACCGCT ACATCGGCGT GAGCTACCCG CTGCGCTACC AGGGGTCTCA TGGCTCTGCT CTGCGTCTGG GCACTCTCCC CTCTTCGGCT GGAGGCAGCC GGCCCCCGAG GACGAGACCA CCGGGCTACG TGCTCTTCTC GGCTCTGGGC TCCTTCTACC lo GTCATGTACT GCCGCGTCTA CGTGGTGGCC AAGAGGGAGA CTCAAGACCG ACA.AGTCGGA CTCGGAGCAA GTGACGCTCC CCGGCAGGAG GCAGCGGGAT GGCCAGCGCC AAGACCAAGA CTCAAGTTCT CCCGGGAGAA GAAAGCGGCC AAAACGCTGG GTCCTCTGCT GGCTGCCTTT TTTC TTAGTC ATGCCCATTG AAGCCCTCTG AAACAGTTTT TAAAATAGTA TTTTGGCTCG AACCCCATCA TATACCCATG CTCCAGCCAA GAGGGAATTC INFORMATION FOR SEQ ID NO:8: WI. SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cOMA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUEN4CE DESCRIPTION: SEQ ID NO:B: TTTGAATTCT GATTTCAAGC CCTCTG INFORNATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

TCACCTCCAC

GCAGGGTCTT

TGGGCCTCTG

CAACCATCGT

TGGTCATATC

TCTGCCAGAT

TGCCTCTGGC

GCCGGGGCCT

GCATCCATCG

CGCACTTCTC

GCATCGTGGT

GGTCTTTCTT

GGTGCTGCCC

CTGCAACATC

CATCATCTCC

CACCCAGAGG

CATTGGACCC

CAACGAGGAG

CATCATCCTG

CAAGTCTGGC

GAAAAACGCC

AGTGAGGCTC

CGGCTGCTTC

CCCTGA'FTTC

840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1601 GATATCTAAA CAGCTGCATC

C

WO 94/21660 lfCT/US04/02609 47 (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TTTGAATTCT TANACYTCYT CNCCRTTYTC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 512 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CTGATTTCAA GCCCTCTGAA ACAGTTTTTA AAATAGTATT TTGGCTCGGA GCTGCATCAA CCCCATCATA TACCCATGCT CCAGCCAAGA GTTCAAAAAG ATGTCTTGAG AATCCAGTGT CTCCGCAGAA AGCAGTCTTC CAAACATGCC CCCTGCACCC GCCCAGCCAG GCCGTGGAAG GGCAACACAA GGACATGGTG

TATCTAAACA

GCCTTTCAGA

CTGGGCTACA

CGCATCCCCG

GAATGGAAAT

TGGGATCAAG AGAGACCTTC TACAGGATCT CCAA TTTTCTCTTC CATGCCCCGT GGATCTGCCA GGAT' GTACCACAGC CCGGGTGAGA AGTAAAAGCT TTTT( CAACCCCCAG CCTTGACAAG AACCATCAAG TTCC TCAGTGAGAA CGGCGAAGAG GTTTAAGAAT TC INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 2004 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both GACGGA TGGCGTTTGT TACAGT GTCCAAAGAC CAATCCTCCT GCAGGT CTGCTGCTGT GTAGGGCCCT AACCAT TAAGGTCCAC ACCATCTCCC (ii) (iii) :iv) MOLECULE TYPE: cDNA HYPOTHETICAL: NO ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: P"CTIUJS'4/0260) WO 94/21660 48 GAATTCCCTC CTAGAAGCTG

GTAACTGCGG

GCTGATCTTT

AGAATCOTGA

TAATTCTGGA

OGGAGGGACT

CGCGCGCCCC

CAGGGCTGGC

CTGGCAGGGC

CGCCAGCCCG

GCCAGGAGCA

TGTTTGAGG

ATCTTCCCCC

GAGAGACCAG

GCGCCCAGAT

GAGAGACTOG

AGCCAGGACG

GAGCCPI'CGG

GCCATCGGTC

CGCTGGAGTT

AATAAGACAG

ATTGCATGTT GCAAGGACTC TCCTGGATCT CCGGGTCCCG GCTAGCCCAG CCCGCAGGTG TGGCCATGTC TTTAATGCCC TGCCCCTTCA CAGGGTTGTC TCCCACCCGC GCGCGCCGTC TCCCTCCAGA AGAGACCTTT TGATTCCCGG GGAGGTGGCC CTGGACAGCC GOACCTCOCC TGGGGCAGCT CAAAATGTAG CCTGCCTTTG AGCGTCGACG TTGAJWI'CCG AATCATGTGC CGCGGAAAAG CAGATTCTCG TCGCACCCAG CTTCGGGTAC GAGAGGGTCC CCGGCAGCCC TGTGGCCTTC TGAGGGTITCC TCACCCCCAG CCAAACCCAC CTCCCGCGCT CCCGCCTCCG GTGTTTCTCT CGGGAAATGC TTCCGACAGC AACATTTCCA AGGCCATTCT GCTCGGGGTG CTGGGTAACA TCCTAGTGAT CCTCTCCGTA CACTACTACA TCGTCAACCT GGCGGTGGCC TTCTCCGCCA TCTTCGAGGT CCTAGGCTAC

TCCAACTCCA

ATCTTGGGGG

GCCTGTCACC

CGGCCCCGGC

CCCAACCGCC

GCCTCATTCT

GACACCTGCA

TGGGCGGCAG

ATCGACCGCT

AGGGGTCTCA

CTCTTCGGCT

TGGATGTGCT

ACATCGGCGT

TGGCTCTGCT

GGAGGCAGCC

GTGCTGCACC

GAGCTACCCG

CTGCGTCTGG

GGCCCCCGAG

GACCTCCTGC TCACCTCCAC TGGGCCTTCG CCAGGGTCTT GCGTCCATCA TGGGCCTCTG CTGCGCTACC CAACCATCGT GCACTCTCCC TGGTCATATC GACGAGACCA TCTGCCAGAT

TGGGACCATG

GGCACCGGTG

T1TTCGGGGTG

CTCAGTCACG

GGTGCTGCCC

CTGCAACATC

CATCATCTCC

CACCCAGAGG

CATTGGACCC

CAACGAGGAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 i080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 CCGGGCTACG TGCTCTTCTC GGCTCTGGGC TCCTTCTACC TGCCTCTGGC OTCATGTACT GCCGCGTCTA CTCAAGACCG ACAAGTCGGA CCGGCAGGAG GCAGCGGGAT CTCAAGTTCT CCCGGGAGAA CGTGGTGGCC AAGAGGGAGA GCCGGGGCCT

CATCATCCTG

CAAGTCTGGC

CTCGGAGCAA GTGACGCTCC GCATCCATCG GAAAAACGCC GGCCAGCGCC AAGACCAAGA CGCAC'1rCTC AGTGAGGCTC GAAAGCGGCC AAAACGCTGG GCATCGTGGT CGGCTGCTTC GTCCTCTGCT GGCTGCCTTT TTTC ITAGTC ATGCCCATTG AAGCCCTCTG AAACAGTTTT TAAA.ATAGTA TTTTGGCTCG 3 0 AACCCCATCA TATACCCATG CTCCAG3CCAA GAGTTCAAAA AGAATCCAGT GTCTCCGCAG AAAGCAGTCT TCCAAACATG CCGCCCAGCC AGGCCGTGGA AGGGCAACAC AAGGACA"GG AGAGAGACCT TCTACAGGAT CTCCAAGACG GATGGCGTTT TCCATGCCCC GTGGATCTGC CAGGATTACA GTOTCCAAAG

GGTCTTTCTT

GATATCTAAA

CCCTGATTTC

CAGCTGCATC

AGGCCTITTCA GAATGTCTTG CCCTGGGCTA CACCCTGCAC TGCGCATCCC CGTGGGATCA GTGAATGGAA ATTTTTCTCT ACCAATCCTC CTGTACCACA WO 94/21660 VCVUS940260 -49 GCCCGGGTGA GAAGTAAAAG CTTTTTGCAG GTCTGCTGCT GTGTAGGGCC CTCAACCCCC 1920 AGCCTTGACA AGAACCATCA AGTTCCA.ACC ATTAAGGTCC ACACCATCTC CCTCAGTGAG 1980 AACGGCGAAG AGGTTTAAGA ATTC 2004 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 466 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terainal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Vai Phe Leu Ser Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr Gln 1 5 10 Pro Pro Ala Pro Val Asn Ile Ser Lys Ala Ile Leu Leu Gly Val Ile 25 Leu Giy Gly Leu Ile Leu Phe Gly Val Len Gly Asn Ile Leu Val Ile 40 Len Ser Val Ala Cys His Arg His Len His Ser Val Thr His Tyr Tyr 55 Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu 70 75 Pro Phe Ser Ala Ile Phe Gln Val Len Gly Tyr Trp Ala Phe Giy Arg 90 Val Phe Cys Asn Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala 100 105 110 Ser Ile Met Gly Len Cys Ile Ile Ser Ile Asp Arg Tyr Ile Gly Val 115 120 125 Ser Tyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gin Arg Arg Gly Leu 130 135 140 Met Ala Len Len Cys Val Trp Ala Len Ser Len Val Ile Ser Ile Giy 145 150 155 160 Pro Leu Phe Gly Trp Arg Gin Pro Ala Pro Gin Asp Gln Thr Ile Cys 165 170 175 Gin Ile Asn Gln Gln Pro Gly Tyr Val Len Phe Ser Ala Len Giy Ser 180 185 190 WO 94121660 WO 94/160 C't[US94102609 50 Pro Leu Ala Lys Arg Glu Asp Ser Diii 230 Gly Gly Ser 245 Arg Leu Leu Ile Ile Leu 200 Ser Arg Gly 215 Gin Val Thr Gly Met Ala Lys Phe Ser Tyr Cys Arg Val Tyr 205 Ser dly Leu Lys Thr 220 Ile His Arg Lys Asn 240 Lys Thr Lys Thr His 255 Lys Lys Ala Ala Lys 270 260 265 Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe 275 Phe Leu Val Met Pro Ile Gly 290 295 Ciii Thr 305 Ile Asn Phe Gin Lys His Gly Gin 370 Phe Tyr 385 Ser Ser Ser Ser Cys Cys Val Pro 450 Lys Ile 310 Ile Tyr 325 Leu Arg Gly Tyr Asp Met Ser Lys 390 Arq Gly 405 Thr Aia Val Pro Ile Thr Val 375 Thr Ser Arg 280 Ser Phe Phe Pro Phe Trp Leu Gly 315 Cys Ser Ser Gin 330 Gin Cys Leu Arg 345 Leu His Pro Pro 360 Arg Ile Pro Vai Asp Gly Vai Cys 395 Ala Arg Ile Thr 410 Jal Arg Ser Lys 425 Gly Pro Ser Thr Pro Ser Leu 440 Lys Val His Thr 455 Asp Lys Asn His Gin 445 Ser Giu Asn Gly GlUi 460 Ile Ser Leu Giu Val 465 INFORMATION FOR SEQ ID NO:i3: SEQUENCE CHARACTERISTICS: LENGTH: i621 base pairs TYPE: nucleic acid STRANDEDNESS: doubie TOPOLOGY: both VC1fUS94/0260$) WO 94/21660 51 (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

CCCOTGCAG

GCCGCCCTTG

GGCCCTACGG

CGCTCGGTCA

CCGGTCCACG GAGATGGTGC GGCGGCCGTC GGOGACTG AGCCTTCATC CTTATGGCCG CCGCCACCTG CAGACCGTCA GCTGAGCGCC ACCGTACTGC TGGCCGCGCC TTCTGCGACG ACACCACCAG GGCTACGACC CAGACCAGOG CCAGGATGGC TGATGGCTGG GTACTTGAGT GAGTGGCGCA CGCCCACGTA AGAGGCTGAG GATGGAGGCC GTGCAGCACA GCACGTCCAC TGGTGAGCGC GCAGGGCGTG GGCGTGGGCG TCTTCCTGGC TGGCAGGTAA CCTGCTTrGTC ATCCTCTCAG TGGCCTGCAA CCAACTATTT CATCGTGAAC CTGGCCGTGG CCGACCTGCT CCTTCTCGGC CACCATGGAG CCTCAGCCTC TGCACCATCT CCCAGCCATC ATGACCGAGC CCTGGTGGTG TCCGTAGGGC

TATGGGCCGC

CCGTGGACCG

GCAAGGCGGC

CCCTGCTGGG

CGTGGACGTG

GTACGTGGGC

CGCCATCCTG

CTGGAAGGAG

CGCTGTCTTC

CTGCCGCGTG

CGACAGOC

CGCCGACGGG

GTTCTGGGCT

CTGTGCTGCA

GTGCGCCACT

GCCCTGCTCT

CCCGTGCCCC

TCCTCCGTGT

TACGTGGTCG

AAGGCCTCCG

GCGCACGGCA

CTTCTGCGGT ATCACCGAGG AGGCGCTA CCTGCCCATG GCGGTCATCG TGGTCATGTA CACGCGCAGC CTCGAGGCAG GCGTCAAGCG GCGCATCCAC TGTCGCGGCG CGGCCACGGG CAAGGGCCAC ACCTTCCGCA GCTCGCTCTC

TCTGGGCCTT

CGGCCTCCAT

CACTCAAGTA

GGGTCGTAGC

CTGACGAGCG

GCTCCTTCTA

CGCGCAGCAC

AGGTGGTGCT

TGCGCAGCGC

CCCGTGAGAA

GGTTCCCTTT

AGGGCGTCTT

TCTACCCCTG

GCCGTCGTCG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 CGTGCGCCTG CTCAAGTTCT GAAAGCGGCC AAGACTCTGG CTTCTTTGTC CTGCCGCTCG CAAGGTCATC TTCTGGCTCG TTCCAGCCGC GAGTTCAAGC CCGGCGCCGC CGCCCTCTCT

CCATCGTCGT

GCTCCTTGTT

GCTACTTCA.A

GCGCCTTCCT

GGCGTGTCTA

CCTGCGCCAG

CACCGCGCTC

CGCCAGCCGT

AGACCCACGA

GGCGCGCAGC

GGGTGTCTTC

CCCGCAGCTG

CAGCTGCGTG

CCGTCTCCTG

CGGCCACCAC

CGACCCCCC

CCCAGGCACG

CGCGAGTGGA

GCCAGCCTGT

CAGCGCTCAG

GACTGCGCCC CGAG?1TCGGG

GTGCTCTGCT

AAGCCATCGG

AACCCGCTCA

CGCTGCCAGT

CCCGACCCCG

CGAAGCCACC

CCCAGCTGCG

GCGCAGAGGC

ACCCCGAACC

CAGCGCCTTC

CGCCAAAGTC

AGCGTGCGCC

TGGCGGGCCT CCACCAGCGG CCCGGAGCGC CGCTGGCCCT CCCGAGATGC AGGCTCCGGT GGCTGCTGGG GCCGTTCCGG CGCACAAGAT CGCCGCCGGG AGGTGGAGGC TGTGTCCCTA WO 94/21660 PCIVUS94/02609 -52 GGCGTCCCAC ACGAGGTGGC CGAGGGCGCC ACCTGCCAGG CCTACGAATT GGCCGACTAC 1620 A 1621 INFORMATION FOR SEQ ID NO:14: W1 SEQUENCE CHARACTERISTICS: LENGTH: 501 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: No FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Met Ala Ala Ala Leu 1 Arg Ser Val Met Met Ala Gly Tyr Leu Ser Glu T rp Met Val Ile Cy s Ala Thr Val Leu 145 Thr Pro Thr Ala Val Gln Ser Ala Gln Met Ala Val Arg His Leu Ala Asp Leu 100 Glu Val Leu 115 Ala Ala Val Thr Ile Ser Arg Ser Thr 25 Ser Thr Ser 40 Val Gly Val 55 Gly Asn Leu Thr Val Thr Leu Ser Ala 105 Phe Trp Ala 120 Val Leu Cys 135 Asp Arg Tyr Glu Met Val Gln Arg Leu Arg Thr Ala Ala Val Gly Gly Leu Gly Val Phe Lau Ala Ala Phe Leu Val Ile Leu Ser Val Ala 75 Asn Tyr Phe Ile Val Asn Leu 90 Thr Val Leu Pro Phe Ser Ala 110 Phe Gly Arg Ala Phe Cys Asp 125 Cys Thr Ala Ser Ile Leu Ser 140 Val Gly Val Arg His Ser Leu 155 160 Lys Ala Ala Ale Ile Leu Ala 170 175 Lys Tyr Pro Ala Ile Met Thr Glu Arg 165 Leu Leu Trp Val Val Ala Leu Val Val Ser Val Gly Pro Leu Leu Gly 180 185 190 Trp Lys Glu Pro Val Pro Pro Asp Glu Arg Phe Cys Gly Ile Thr Glu WO 94/21660 PCTPUS94/02609 53 195 Clu Ala Gly 210 200 Tyr Ala Val Phe Ser Ser Val 205 Cys Ser Phe Tyr Leu PL, 215 220 met Ala Val Ile Val Val Met Tyr Cys Arg Val Tyr Val Val Ala 225 Ser Ala Al a Ser Ala 305 Pro Pro Ser Arg Arg~ 385 Ser Gly I Thr Sc r Asp Ser 290 Lysa Phe Se r Cys A.la 370 Arg

G

1 y *Thr Glu Gly 275 Lau Thr Phe Glu Val 355 Phe Pro Leu Pro 230 *Arg Sar Leu 245 Val Val Leu 260 Ala His Gly Ser Val Arg Leu Ala Ile 310 Phe Val Lau 325 Gly Val Phe 340 Asn Pro Leu Leu Arg Leu Leu Trp Arg 390 Arg Gln Asp 405 Leu Ala Leu 9) 420 235 Lys Arg Arg Gly Lys Cly Ser Arg 300 Phe Val Arg Gly 255 Ala Thr 270 Thr Phe Lys Lys Cys Trp Pro Leu Gly Ser Leu Phe Pro Gln Leu Lys Pro Gly Thr Pro Glu Met Gln Ala Pro Val Ala Ser Arg Arg Ser His 435 440 445 Pro Ala Pro Ser Ala Ser Gly dly Cys Trp Gly Arg Ser Gly Asp Pro 450 455 460 Arg Pro Ser Cys Ala Pro Lys Ser Pro Ala Cys Arg Thr Arg Ser Pro 465 470 475 Pro dly Ala Arg Ser Ala Gln Arg Gln 485 Trp Arg Leu Cys Pro 500 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: Arg Ala Pro Ser Ala Gln Arg 490 495 WO 94/21660 'PCT/US94/02609 54 LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID TCTAGACCAT GAAYCCNGAY CTGG 24 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS; single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TTTGAATTCA CATWCCGACY ACAATGCCC 29 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 921 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: both (ii) MOLECULE TYPE: cbNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TCTAGACCAT GAATCCCGAC CTGGACACCG GCCACAACAC ATCAGCACCT GCCCACTGGG GAGAGTTGAA AAATGCCAAC TTCACTGGCC CCAACCAGAC CTCGAGCAAC TCCACACTGC 120 CCCAGCTGGA CATCACCAGG GCCATCTCTG TGGGCCTGGT GCTGGGCGCC TTCATCCTCT 180 TTGCCATCGT GGGCAACATC CTAGTCATCT TGTCTGTGGC CTGCAACCGG CACCTGCGGA 240

II--

WO 94121660 PCT/US94/02609 55 CGCCCACCAA CTACTTCATT GTCAACCTGG CCATGGCCGA TCCTGCCC I'1 CTCAGCGGCC CTAGAGGTGC TCGGCTACTG GTGACATCTG GGCAGCCGTG GATGTCCTGT GCTOCACAGC CCATCTCCAT CGATCGCTAC ATCGGGGTGC GCTACTCTCT CCCGGAGGAA GGCCATCTTG GCCCTGCTCA GTGTCTGGGT TCGGGCCTCT CCTTGGGTGG AAGGAGCCGG CACCCAACGA CCGAAGAACC CTTCTATGCC CTCTTCTCCT CTCTGGGCTC TCATTCTAGT CATGTACTGC CGTGTCTATA TAGTGGCCAA AGGCAGGAGT CATGAAGGAG ATGTCCAACT CCAAGGAGCT AGAACTTTCA CGAGGACACC CTTAGCAGTA CCAAGGCCAA CCATAGCTGT CAAACTTTTT AAGTTCTCCA GGGAAAAGAA TTGTGGTCGG TATGTGAATT C INFORMATION FOR SEQ ID NO:18: Wi SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTT-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GAAGGCGCGC TTGAACTC INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: lin(!ar n (ii) MOLECULE TYPEt cDONA CCTGCTGTTG AGCTTCACCG GGTGCTGGGG CGGATCTTCT GTCCATTCTG AGCCTGTGCG GCAGTATCCC ACGCTGGTCA CTTGTCCAC1 .,TCATCTCCA TGACAAGGAG TGCGGGGTCA CTTCTACATC CCTCTGGCGG GAGAACCACC AAGAACCTAG GACCCTGAGG ATCCATTCCA GGGCCACAAC CCCAGGAGTT AGCAGCTAAG ACGTTGGGCA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID ND:19: WO 94/21660 PCT/US94/02609 56 AGAGAACCAC CAAGAACC 18 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 389 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID AAGAGAACCA CCAAGAACCT AGAGGCAGGA GTCATGAAGG AGATGTCCAA CTCCAAGGAG CTGACCCTGA GGATCCATTC CAAGAACTTT CACGAGGACA CCCTTAGCAG TACCAAGGCC 120 AAGGGCCACA ACCCCAGGAG TTCCATAGCT GTCAAACTTT TTAAGTTCTC CAGGGAAAAG 180 AAAGCAGCTA AGACGTTGGG CATTGTGGTC GGTATGTTCA TCTTGTGCTG GCTACCCTTC 240 TTCATCGCTC TACCGCTTGG CTCCTTGTTC TCCACCCTGA AGCCCCCCGA CGCCGTGTTC 300 AAGGTGGTGT TCTGGCTGGG CTACTTCAAC AGCTGCCTCA ACCCCATCAT CTACCCATGC 360 TCCAGCAAGG AGTTCAAGCG CGCTTTCGT 389 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOq IETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TTTGAATTCA TGTTCAAGGT GGTGTTC 27 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

I

~sRp~lrm~s~i~a~-rrul~l PCT/US9402609 WO 94/21660 57 (ii) (iii) (iv) MOLECULE TYPE: cDNA HYPOTHETICAL: NO ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TTTGAATTCT AAAASTGNCC NGGNSCCAGN GGCAT INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 582 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

GAATTCATGA

CATCTACCCG

GTGCCGCGGT

TTCAAGGTGG

TGCTCCAGCA

GGCCGCCGCC

CCGGCCGTGG ACCCGCGGCG TGACAGCGGC AGCTGCATGA GGGCTACCTG GGTCGAGGAA ACCCGGGGCG CTGCTCAGCT GCCACTCTTC ACCTTCAAGC CAGCAACGGG GGCTGCGACA CAACATGCCC CTGGGCCCGG TGTTCTGGCT GGGCTACTTC AACAGCTGCC TCAATCCCAT AGGAGTTCAA GCGCGCCTTC ATGCGTATCC TTGGGTGCCA GCCGCCGCCG TCGCCGTCTA GGCGCGTGCG CTTACACCTA GCTCGCTGGA GAGATCACAG TCGCGGAAGG ACTCTCTGGA GCGGCCAGAA GAGGACCCTG CCCTCGGCGT CGCCCAGCCC CGCAGCCACC CGTGGAGCTG TGCGCCTTCC CCGAGTGGAA TGCCAGAGCC TCCTGGCCGC CGCGGCCGTC TCGACTCTGG TCCTGGGCGA TCCTGAGAGC CCGGGAACCG AAGCGACAGC CCACGACCCA CCTGGCCAAC GGGCAGCCCG GCTTCAAGAG GCCACTTTTA AAAGCCGAAT TC INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 1567 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii! HYPOTHETICAL: NO WO 94/21660 I)CT/US940260) 58 (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: TCTAGACCAT GAATCCCGAC CTGGACACCG GCCACAACAC ATCAGCACCT GCCCACTGGG GAGAGTTOAA AAATGCCAAC TTCACTGGCC CCAACCAGAC CTCGAGCAAC TCCACACTGC CCCAGCTGGA CATCACCAGG GCCATCTCTG TGGGCCTGGT GCTGCGCC

TTGCCATCGT

CGCCCACCAA

TCCTGCCCTT

GTGACATCTG

GGGCAACATC CTAGTCATCT CTACTTCATT GTCAACCTGG CTCAGCGGCC CTAGAGGTGC GGCAGCCGTG GATGTCCTGT

TGTCTGTGGC

CCATGGCCGA

TCGGCTACTG

GCTGCACAGC

GCTACTCTCT

GTGTCTGGGT

CACCCAACGA

CTCTGGGCTC

CTGCAACCGC

CCTGCTGTTG

GGTGCTGGGG

GTCCATTCTG

GCAGTATCCC

CTTGTCCACC

TGACAAGGAG

CTTCTACATC

'ITCATCCTCT

CACCTGCGGA

AGCTTCACCG

CGGATCTTCT

AGCCTGTGCG

ACOCTGGTCA

GTCATCTCCA

TGCGGGGTCA

CCTCTGGCGG

CCATCTCCAT CGATCGCTAC CCCGGAGGAA GGCCATCTTG TCGGGCCTCT CCTTGGGTGG CCGAAGAACC CTTCTATGCC

ATCGGGGTGC

GCCCTGCTCA

AAGGAGCCGG

CTCTTCTCCT

TCATTCTAGT

AGGCAGGAGT

CATGTACTGC

CATGAAGGAG

CGTGTCTATA TAGTGGCCAA GAGAACCACC ATGTCCAACT CCAAGGAGCT GACCCTGAGG AGAAkCTTTCA CGAGGACACC CTTAGCAGTA CCAAGGCCAA GGGCCACAAC GGGAAAAGAA ACCAGCTAAG

CCATAGCTGT

TTGTGGTCGG

CAAACTTTTT AAGTTCTCCA TATGTTCATC TTGTCCTGGC CCTTlGTTCTC CACCCTGAAG ACTTCAACAG CTGCCTCAAC CCTTCATGCG TATCCTTGGG CTCTAGGCGC GTGCGCTTAC CACAGTCGCG GAAGGACTCT CCCTGCCCTC GGCGTCGCCC AGCTGTGCGC CTTCCCCGAG GCCGCCGCGG CCGTCTCGAC AGAGCCCGGG AACCGAAGCG

CCCCCCGACG

CCCATCATCT

TGCCAGTGCC

ACCTACCGOC

CTGGATGACA

AGCCCGGGCT

TGGAAACCCG

TCTGGGCCAC

TACCCTTCTT

CCGTGTTCAA

ACCCATGCTC

GCGGTGGCCG

CGTGGACCCG

GCGGCAGCTG

ACCTGGGTCG

GGGCGCTGCT

CATCGCTCTA

GOTGGTGTTC

CAGCAAGGAG

CCGCCGCCGC

CGGCGGCTCG

CATGAGCGGC

AGGAACGCAG

CAGCTTGCCA

AAGAACCTAG

ATCCATTCCA

CCCAGGAGTT

ACGTTOGGCA

CCGCTTGGCT

TGGCTGGGCT

TTCAAGCGCG

CGCCGTCGCC

CTGGAGAGAT

CAGAAGAGGA

CCACCCGTGG

GAGCCTCCTG

GGCGATCCTG

ACCGACCTG

720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1567 TCTTCACCTT CAAGCTCCTG ACAGCCAGCA ACGGGGGCTG CGACACCACG CCAACGGGCA OCCCGGCTTC AAGAGCAACA TGCCCCTGGG CCCGGGCCAC TTTTAAAAGC

CGAATTC

f2 INFORMATION FOR SEQ ID WO 94/21660 C/S4120 PCTIIJS94/02609 59 SEQUENCE CHARACTERISTICS: LENGTH: 515 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID Met Asn Pro Asp Leu Asp Thr Gly His Asn Thr Ser Ala Pro Ala His 1 5 10 Trp Gly Glu Leu Lys Asn Ala Asn Phe Thr Gly Pro Asn Gin Thr Ser Ser Asn Ser Thr Leu Pro Gin Leu Gly Leu Val Leu Val Ile Asn Tyr Phe Thr Val Leu Leu Gly Arg 115 Cys Thr Ala 130 Ile Gly Val 145 Lys Ala Ile Ser Ile Gly Lys Glu Cys 195 Phe 55 Ala Leu Ala Asp Se r 135 Leu Leu Ile 25 Asp Ile Thr Arg Ala Ile Ser Val Leu Phe Ala Ile Val Gly Asn Ile Cys Asn Arg His Leu Ala Met Ala Asp Leu Ala Leu Glu Val Leu 105 Ile Trp Ala Ala Val 120 Leu Cys Ala Ile Ser 140 Gin Tyr Pro Thr Leu 155 Ser Val Trp Val Leu 170 Trp Lys Glu Pro Ala 185 Glu Pro Phe Tyr Ala 200 Arg Thr Pro Leu Leu Ser Gly Tyr Trp 110 Asp Val Leu 125 Ile Asp Arg Val Thr Arg Ser Thr Val 175 Pro Asn Asp 190 Leu Phe Ser 205 LeU Gly Gly Val Thr Glu Leu Gly Ser Phe Tyr Ile Pro Leu Ala Val le Leu Val Met Tyr Cys 210 220 Arg Val Tyr Ile Val Ala Lys Arg Thr Thr Lys Asn Leu Glu Ala Gly WO 94121660 I'MUS94/0260901 60 230 235 240 Val Met Lys Giu Met Ser Asn Ser Lys Clu Leu Thr Leu Arg Ile His Ser His Glu Leu 305 Ser Gly Lys Gly Thr 385 Arg Arg' Thr I Ala Ser( 465 Gly 1 Leu A Lys Asn Lys 290 Cys Thr Tyr Glu Gly 370 Tyr Lys Thr Gln Leu 450 Gly :hr vla 245 250 Asn Phe His Glu Asp Thr Leu Ser 260 265 Pro Arg Ser Ser Ile Ala Val Lys 275 280 Lys Ala Ala Lys T-r Leu Gly Ile 295 Trp Leu Pro Phe Phe Ile Ala Leu 310 Leu Lys Pro Pro Asp Ala Val Phe 325 330 Phe Asn Ser Cys Leu Asn Pro Ile 340 345 Phe Lys Arg Ala Phe Met Arg Ile 355 360 Arg Arg Arg Arg Arg Arg Arg Arg 375 Arg Pro Trp Thr Arg Gly Cly Ser 390 Asp Ser Leu Asp Asp Sar Gly Ser I 405 410 Leu Pro Ser Ala Ser Pro Ser Pro 420 425 Pro Pro Val Giu Leu Cys Ala Phe I 435 440 Leu Ser Leu Pro Glu Pro Pro Gly 455 Pro Leu Phe Thr Phe Lys Leu Leu C 470 4 Glu Ala Thr Ala Ser Asn Gly Gly C 485 490 Asn Gly Gin Pro Gly Phe Lys Ser A 500 505 Ser Thr Lys Leu Phe Lys 285 Val Val Gly 300 Pro Leu Gly 315 Lys Val Val Ile Tyr Pro Leu Gly Cys 365 Leu Gly Ala 380 Leu Glu Arg 395 Cys Met Ser G1y Tyr Leu Pro Giu Trp I 445 krg Arg Gly 460 liy Asp Pro C '75 Ala 270 Phe Met Ser Phe Cya 350 Gin s Ser ly Gly 430 Lys krg lu 255 Lys Gly Ser Arg Phe Ile Leu Phe 320 Trp Leu 335 Ser Ser Cys Arg Ala Tyr Gin Ser 400 Gin Lys 415 Arg Gly Pro Gly Leu Asp Ser Pro 480 :ys Asp Thr Thr Thr Asp 495 ~sn Met Pro Leu Giy Pro 510 Gly His Phe 515 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 1987 base pairs TYPE: nucleic acid STRANDEONESS: both i, r, PCTUS94102609) WO 94121660 61 TOPOLOGY: both (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GAATTCCCTC CTAGAAGCTG GAGAGAGCAG GAGCCTTCGG TGGGOCAGCT CAAAATGTAG OTAACTGCGG CCCAGGAGCA GCTGATCT T

AGAATCGTGA

TAATTCTGGA

TGGTX'TGAGG

ATCTTCCCCC

ATTGCATGTT

GCGCCCAGAT

GAGAGACTOG

AGCCAGGACG

GCA.AGGAGTC

GCTAGGCCAG

TTTAATGCCC

GCCATCGGTC CCTGCCTTTG AGCGTCGACG CGCTGGAGTT 'rTGAATTCCG AATCATGTGC AATAAGACAG CGCGGAAAAG CAGATTCTCG TCCTGGATCT TCGCACCCAG CTTCGGGTAC CCCGCAGGTG GAGAGGGTCC CCGGCAGCCC TGCCCCTTCA TGTGGCCTTC TGAGGGITCC GGGAGGGAGT CCGGGTCCCG CGCGCGCCCC TGGCCATGTC CAGGGCTGGC CAGGGTTOTC CTGGCAGGGC TCCCTCCAGA CGCCAGCCCG GGAGGTGGCC TCCCACCCGC GCGCGCCGTC TCACCCCCAG CCAAACCCAC AGAGACCTTT TGATTCCCGG CTCCCGCGCT CCCGCCTCCG CTGGACAGCC GGACCTCGCC CGGCCCCGGC GTGTTTCTCT CGGGAA.ATGC AACATTTCCA AGGCCATTCT TTCCGACAGC TCCAACTGCA GCTCGGGGTG ATCTTGGGG

CCCAACCCCC

GCCTCATTCT

GACACCTGCA

TGGGACCATG

GGCACCOOTG

TTCGGGGTG

CTCAGTCACG CTGGGTAACA TCCTAGTGAT CCTCTCCGTA GCCTGTCACC 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 CACTACTACA TCGTCAACCG CTAGTGGCGG TGGCCGACCT CCTGCTCACC TCCACGGTGC

TGCCCTTCTC

ACATCTGGGC

TCTCCATCGA

AGAGGAGGOG

GACCCCTCTT

CGCCATCTTC GAGGTCCTAG GGCAGTGGAT GTGCTGTGCT

CCGCTACATC

TCTCATGGCT

CGGCTGGAGG

AGGAGCCGGG CTACGTGCTC TCCTGGTCAT GTACTGCCGC CTGGCCTCAA GACCGACAAG ACGCCCCGGC AGGAGGCAGC GGCTCCTCAA GTTCTCCCGG GCTTCGTCCT CTGCTGGCTG

GGCGTGAGCT

CTGCTCTGCG

CAGCCGGCCC

TTCTCGGCTC

GTCTACGTGG

TCGGACTCGG

GGGATGGCCA

GAGAAGAAAG

CCTTTTTTCT

GCTACTGGGC

GCACCGCGTC

ACCCGCTGCG

TCTGGGCACT

CCGAGGACGA

TGGGCTCCTT

TGGCCAAGAG

AGCAAGTGAC

GCGCCAAGAC

CGGCCAAAAC

TAGTCATGCC

CTTCGGCAGG

CATCATGGGC

CTACCCA.ACC

CTCCCTGGTC

GACCATCTGC

CTACCTGCCT

GGAGAGCCGG

GCTCCGCATC

GTCTTCTGCA

CTCTGCATCA

ATCGTCACCC

ATATCCATTG

CAGATCAACG

CTGGCCATCA

GGCCTCAAGT

CATCGGAAAA

CAAGACGCAC T1TCTCAGTGA GCTGGGCATC GTGGTCGGCT CATTGGGTCT TTCTTCCCTG WO 94121660 PCT/US94/02609 62 ATTTCAAGCC CTCTGAAACA GTTTTTAAAA TAGTATTTTG GCTCGGATAT CTAAACAGCT GCATCAACCC CATCATATAC CCATGCTCCA GCCAAGAGTT CAAAAAGGCC TTTCAGAATG TCTTGAGAAT CCAGTGTCTC CGCAGAAAGC AGTCGCTAGT TCCAAACATG CCCTGGGCTA CACCCTGCAC CCGCCCAGCC AGGCCGTGGA AGGGCAACAC AAGGACATGG TGCGCATCCC CGTGGGATCA AGAGAGACCT TCTACAGGAT CTCCAAGACG GATGGCGTTT GGAGA ATTTTTCTCT TCCATGCCCC GTGC-ATCTGC CAGGATTACA GTGTCCAAAG ACCAATCCTC CTGTACCACA GCCCGGGTGA GAAGTAAAAG CTTTTTGCAG GTCTGCTGCT GTGTAGGGCC CTCAACCCCC AGCCTTGACA AGMACCATCA AGTTCCAACC ATTAAGGTCC ACACCATCTC

CCTCAGT

INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 1997 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: AATTCCCTCC TAGAAGCTGG_ AGAGAGCAGG AGCCTTCGGT GGGGCAGCTC AAAATGTAGG TAACTGCGGG CCAGGAGCAG CGCCCAGATG CCATCGGTCC CTGCCTTTGA GCGTCGACGG CTGATCTTTT GGTTTGAGGG AGAGACTGGC GCTGGAGTTT TGAATTCCGA ATCATGTGCA 1560 1626 1680 1740 1800 1860 1920 1980 1987 120 180

GAATCGTGAA

ATCGA

GGAGGGAGTC

GCGCGCCCCT

AGGGCTGGCC

TGGCAGGGCT

GCCAGCCCGG

TGTTTCTCTC

ACATTTCCAA

TGGGTAACAT

TCTTCCCCCA GCCAGGACGA ATAAGACAGC GCGGAAAAGC TTGCATGTTG CAAGGAGTCT CCTGGATCTT CGCACCCAGC CGGGTCCCGG CTAGCCCAGC CCGCAGGTGG AGAGGGTCCC GGCCATGTCT TTAATGCCCT GCCCCTTCAT GTGGCCTTCT AGGGTTGTCT CCCACCCGCG CGCGCCGTCT CACCCCCAGC CCCTCCAGAA GAGACCTTTT GATTCCCGGC TCCCGCGCTC GAGGTGGCCC TGGACAGCCG GACCTCGCCC GGCCCCGGCT GGGAAATGCT TCCGACAGCT CCAACTGCAC CCAACCGCCG GGCCATTCTG CTCGGGGTGA TCTTGGGGGG CCTCATTCTT CCTAGTGATC CTCTCCGTAG CCTUTCACCG ACACCTGCAC

AGATTCTCGT

TTCGGGTACG

CGGCAGCCCC

GAGGGTTCCC

CAAACCCACC

CCGCCTCCGC

GGGACCATGG

GCACCGGTGA

TTCGGGGTGC

TCAGTCACGC

WO 94/21660 PICTUS0'4102609) 63

ACTACTACAT

TCTCCGCCAT

GGGCGGCAGT

TCGACCGCTA

GGGGTCTCAT

TCTT1CGGCTG

CGGGCTACGT

TCATGTACTG

CGTCAACCTG

CTTCGAGGTC

GGATGTGCTG

CATCGGCGTG

GGCTCTGCTC

GAG GCAGCCG

GCTCTTCTCG

CCGCGTCTAC

GCGGTGGCCG ACCTCCTGCT CACCTCCACG GTGCTGCCCT CTAGGCTACT GGGCCTTCGG CAGCfGTCTTC TGCAACATCT TGCTGCACCG CGTCCATCAT GGGCCTCTGC ATCATCTCCA AGCTACCCGC TGCGCTACCC AACCATCOTC ACCCAGAGGA TGCGTCTGGG CACTCTCCCT GGTCATATCC GCCCCCGAGG ACGAGACCAT CTGCCAGATC GCTCTGGGCT CCTTCTACCT GCCTCTGGCC GTGGTGGCCA AGAGGGAGAG CCGGGGCCTC

ATTGGACCCC

AACGAGGAGC

ATCATCCTGG

AAGTCTGGCC

840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 TCAAGACCGA CAAGTCGGAC TCGGAGCAAG TGACGCTCCG CATCCATCGG AAAAACGCCC

CGGCAGGAGG

TCA.AGTTCTC

TCCTCTGCTG

CAGCGGGATG

CCGGGAGA.AG

GCTGCCTTTT

GCCAGCGCCA AGACCAAGAC GCACTTCTCA GTGAGGCTCC AA.AGCGGCCA AAACGCTGGG CATCGTGGTC GGCTGCTTCG TTCTTAGTCA TGCCCATTGG GTCTTTCTTC CCTGATTT1CA AGCCCTCTGA AACAGTTTTT AAAATAGTAT TTTGGCTCGG ATATCTAAAC ACCCCATCAT ATACCCATGC TCCAGCCAAG AGTTCAAAAA GGCCTITCAG GAATCCAGTG TCTCTGCAGA AAGCAGTCTT CCAAACATGC CCTGGGCTAC CGCCCAGCCA GGCCGTGGAA GGGCAACACA AGGACATGGT GCGCATCCCC GAGAGACCTT CTACAGGATC TCCAAGACGG ATGGCGTTTG TGAATGGAAA CCATGCCCCG TGGATCTGCC AGGATTACAG TGTCCAAAGA CCAATCCTCC CCCGGGTGAG AAGTAAAAGC TTTTTGCAGG TCTGCTGCTG TGTAGGGCCC GCCTTGACMA GAACCATCAA GTTCCA.ACCA TTAAGGTCCA CACCATCTCC ACGGGGAGGA AGTCTAG

AGCTGCATCA

AATGTCTTGA

ACCCTGCACC

GTGGGATCAA

TTTTTCTCTT

TGTACCACAG

TCAACCCCCA

CTCAGTGAGA

1560 1620 1680 1740 1800 1860 1920 1980 1997 INFORMATION FOR SEQ ID NO:28: Wi SEQUENCE CHARACTERISTICS: LENGTH: 466 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Met Val Phe Leu Ser Gly Asn Ala Sec Asp Ser Sec Asn Cys Thr Gln 1 5 10 WO 94/21660 PCT1US94102609 64 Pro Pro Ala Pro Val Asn Ile Ser Lys Ala Ile Leu Leo Gly Val Ile 25 Leu Gly Gly Leo Ile Leu Phe Gly Val Leo Gly Asn Ile Leo Val Ile Leo Ser Ile Val Pro Phe Val Phe Ser Ile Ser Tyr 130 Met Ala 145 Pro Leo Gin Ilie Phe Tyr Val Val 210 Asp Lys 225 Ala Pro Phe Ser Thr Leo C 2 Phe Leo V 290 Glu Thr V 305 Ile Asn P Va Asi Sei Cys Met 115 Prc LetA Phe Asn Leo 195 Alia Ser lia tal fly 75 'ai ai ro 1 Ala Cys iLeo Ala rAla Ile Asn Ile 100 Gly Leo Leo Arg Leo Cys Gly Trp 165 Gbu Giu Pro Leo Lys Arg Asp Ser Gly Gly 245 Arg Leo 260 Ile Val M~et Pro Phe Lys Ile Ile 90 Trp Ala Ala Val Asp Val Leo Cys 105 Ile Ile Ser Ile Asp 120 Tyr Pro Thr 135 Val Trp Ala 150 Arg Gin Pro Pro Giy Tyr Ala Ile Ile 200 Glu Ser Arg 215 Glu Gin Val 230 Ser Gly Met Leo Lys Phe Vai Gly Cys 1 .280 Ile Gly Ser 295 Ile Val Phe 310 Tyr Pro Cys S Ile Val Thr Arg *Gin 140 Val Asp Ser Tyr Se r 220 Ile Lys Lys Cys' Asp 300 TyrI Tyr 125 Arg Ile Gbu Ala Cys 205 Gly His rhr Lys Trp 285 The Leu Cys Thr Ala 110 Ile Gly Val Arg Gly Leo Ser Ile Giy 160 Thr Ile Cys 175 Leo Gly Ser 190 Arg Val Tyr Leo Lv r; Thr Arg Lys Asn 240 Lys Thr His 255 Ala Ala Lys 270 Leo Pro Phe Lys Pro Ser Asn Ser Cys 320 The Phe Pro rrp Leo Gly 315 ~er Ser Gin Glu Phe Lys Lys Ala 335 325 330 Phe Gin Asn Val Leo Arg Ile Gin Cys Leo Cys Arg Lys Gin C, r Ser PCT/US94/02609 WO 94/21660 65 340 Lys His Ala Leu dly Tyr 355 Gly Gin His Lys Asp Met 370 Phe Tyr Arg Ile Ser Lys 385 390 Ser Ser Met Pro Arg Gly 405 Ser Ser Cys Thr Thr Ala 420 Cys Cys Cys Val Gly Pro 435 Val Pro I'hr Ilie Lys Val 450 345 Thr Leu His 360 Vai Arg Ile 375 Thr Asp Giy 350 Pro Pro Ser Gin Ala Val Giu 365 Pro Val Giy Ser Arg Giu Thr 380 Vai Cys Giu 395 Ser Ala Arg Ile 410 Thr Val Trp Lys Phe Phe 400 Ser Lys Asp Gin 415 Phe Leu Gin Val Arg Val Arg Ser Lys Ser 425 Ser Thr Pro Ser Leu Asp 440 His Thr Ile Ser Leu Ser 455 460 430 Lys Asn His Gin 445 Giu Asn G1y Giu Giu Val 465 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 1776 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ii) MOLECULE TYPE: cDMA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DE~SCRIPTION: SEQ ID NO:29: CTCCCTGCCG GCCGCTCGTT CTGTGCCCCG GCCCGGCCAC CGACGGCCGG ACTTTCCGCG ATCTCCTGAG CGTCAGTTTC GAGGGACCCC GCCCGGACAG GGCTCCAGCG CGGGCGGCGG CGGGGGCGGC GCGGGCGGCG CGGCCCCCTC GCGGTGGGCG GCGTGCCGGG GGGCGCGGGC GGCGGCGGCG GCGTGGTGGG GOCCAGGACA ACCGGAGCTC CGCGGGGGAG CCGGGGAGCG CGGGCGCGGG AATGGCACGG CGGCCGTCGG GGGACTGGTG GTGAGCGCGC AGGGCGTGGG TTCCTGGCAG CCTTCATCCT TATGGCCGTG GCAGGTAACC TGCTTGTCAT GCCTGCAACC GCCACCTGCA GACCGTCACC AACTATTTCA TCGTGAACCT GACCTGCTGC TGAGCGCCAC CGTACTGCCC TTCTCGGCCA CCATGGAGGT

CGTTGAGATG

CAGCGCAGGG

GGAGGGCCCG

CGCAGGCAGC

CGGCGACGTG

CGTGGGCGTC

CCTCTCAGTG

GGCCGTGGCC

TCTGGGCTTC

PIUS94/02609 WO 94/21660 66 TGGGCCTTTG GCCGCGCCTT CTGCGACGTA TGGGCCGCCG

GCCTCCATCC

CTCAAGTACC

LTCGTACCCC

GACGAGCGCT

TCCTTCTACC

CGCAGCACCA

GTGGTGCTGC

CGCACGCCA

TCAGCCTCTG

CAGCCATCAT

TGGTGGTGTC

TCTGCGGTAT

CACCATCTCC

GACCGAGCGC

CGTAGGGCCC

CACCGAGGAG

GTGGACCGGT

AAGGCGGCCG

CTGCTGGGCT

GCGGGCTACG

TGGACGTGCT

ACGTGGGCGT

CCATCCTGGC

GGA.AGGAGCC

CTGTCTTCTC

GCCGCGTGTA

AGCGAGGCAA

CCGACGGGGC

TGCGCCTGCT

TGCCCATGGC GGTCATCGTG GTCATGTACT CGCGCAGCCT CGAGGCGGGC GTCAAGCGCG GCATCCACTG TCGCGGCGCG GCCACGGGCG AGGGCCACAC CTTCCGCAGC TCGCTCTCCG

GTGCTGCACG

GCGCCACTCA

CCTGCTCTGG

CGTGCCCCCT

CTCCGTGTGC

CGTGGTCGCG

GGCCTCCGAG

GCACGGCATG

CAAGTTCTCC

GCTCTGCTGG

GCCATCGGAG

CCCGCTCATC

CTGCCAGTGC

GCGGGCCTCC

CGGAGCGCCG

CGAGATGCAG

GCTGCTGGGG

GCACAAGATC

CGTGAGAAGA AAGCGGCCAA GACTCTGGCC ATCGTCGTGG GTGTCTTCGT TTCCC'TTTCT TCT FTGTCCT GGCGTCTTCA AGGTCATCTT GCCGCTCGGC TCCTTGTTCC CTGGCTCGGC TACTTCAACA GTTCAAGCGC GCCTTCCTCC TACCCCTGTT

CGTCGTCGCC

ACCAGCGGCC

CTGGCCCTCA

GCTCCGGTCG

CCGTTCCGGA

CCAGCCGCGA

GGCGCCGCCG

TGCGCCAGGA

CCGCGCTCCC

CCAGCCGTCG

GACCCACGAC

CCCTCTCTGG

CTGCGCCCCG

CGACCCCGAC

AAAGCCACCC

CCAGCTGCGC

CGCAGAGGCA

CGAGGTGGCC

CGTGTCTACG

AGTTCGGGCG

CCCGAACCCC

AGCGCCTTCC

GCCAAAGTCT

CGCA GCTGAA

GCTGCGTGAA

GTCTCCTGCG

GCCACCACTG

ACGCGCCCCC

CAGGCACGCC

GCqAGTGGAG

CCAGCCTGTC

6( 0l 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1776 CGCGCCGGGG GCGCGCAGCG GTGTCCCTAG GCGTCCCACA GCCGACTACA GCAACCTACG GCGTGCGCCC AGCGCTCAGA GGTGGAGGCT GAGGGCGCCA CCTGCCAGGC CTACGAATTG GGAGACCGAT ATTTAA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 572 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID WO 94/21660 11CIUS94OZ609 67 Val Ser Phe Met Thr Phe Arg Asp Leu Leu Ser 1 5 Glu Gly Pro Arg Pro 10 Asp Ser Ser Ala Gly Gly Ser Ser Ala Gly Gl'y Gly Gly Gly Gly Ala C ly G ly Asn Val1 Val1 Gly Tb r Lau 145 Phe Val1 Asp Thr Leu 225 Pro Phe Gly Ala A rg Asn Gly Asn Val 130 Se r Trp, Leu Arg clu 210 Val Asp Se r Ala Ala Pro Gly Gly Gly Ser Ser Ala Gly Thr Ala Val Gly Val 100 Leu Leu Val 115 Thr Asn Tyr Ala Thr Val Ala Phe Gly 165 Cys Cys Thr 180 Tyr Val Gly 195 Arg Lys Ala Val Ser Val Glu Arg Phe 245 Ser Val Cys 260 7 Glu dly 40 Gly Val 55 Glu Pro Val Gly Leu Ala Leu Ser 120 Ile Val 135 Pro Phe Ala Phe Ser Ile Arg His 200 Ala Ile 215 Pro Leu Gly Ile Phe Tyr Val Val 280 Glu Arg 295 Ala Ala His Thr 25 Pro Ala Val Gly Gly Val Pro Gly Val Gly Ala dly Ser Gly Glu Asp dly Ser Ala Gly Ala Gly Gly Asp 75 Gly Leu Val Val Ser Ala Gln Gly 90 Ala Phe Ile Lau Met Ala Val Ala 105 110 Val Ala Cys Asn Arg His Leu dln 125 Asn Lau Ala Val Ala Asp Leu Lau 140 Ser Ala Thr Met Glu Val Leu dly 155 160 Cys Asp Val Trp Ala Ala Val Asp 170 175 Leu Ser tLeu Cys Thr Ile Ser Val 185 190 Ser Leu Lys Tyr Pro Ala Ile Net 205 Leu Ala Leu Leu Trp Val Val Ala 220 Lau Gly Trp Lys Glu Pro Val Pro 235 240 Thr Glu Glu Ala Gly Tyr Ala Val 250 255 Leu Pro Met Ala Val Ile Val Val 265 270 Ala Arg Ser Thr Thr Arg Ser Leu 285 Gly Lys Ala Ser Glu Val Val Leu 300 T1hr Gly Ala Asp Gly Ala His Gly 315 320 Phe Arg Ser Ser Leu Ser Val Arg 330 335 Met Tyr Cys Arg Val Tyr 275 Val Lys Arg Cys Arg Gly 310 Ala Lys Gly 325 WO 94/21660 WO 94/1660 CTIIUS941026c09 68 Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys Th- Leu Ala Ile 340 Val Val Gay Val Phe 355 Pro Leu Gly Ser Leu 370 Lys Val Ile Phe Trp 385 Ile Tyr Pro Cys Ser 405 Leu Arg Cys Gin Cys 420 Val Tyr Gly His His 435 Cys Ala Pro Ser Ser 450 Thr Ala Leu Pro Asp 465 Gin Ala Pro Val Ala 485 345 350 Trp Phe Pro Phe Phe Phe Val Leu Val Leu Cys 360 Phe Pro Gin 375 Leu Gly Tyr 390 Ser Arg Giu Arg Arg Arg Trp, Arg Ala 440 Gly Asp Ala 455 Pr-o Asp Pro 470 Ser Arg Arg Trp Arg Leu Leu Gly Pro Phe Arg Arg Pro Thr Thr Gin Leu Arg Ala 500 505 510 Lys Val Ser Ser Leu Ser His Ly 515 52 Ala Glu Ala Ala Cys Ala Gin Ar 530 535 Gly Val Pro His Glu Vl Ala GlI 545 550 Leu Ala Asp Ty:, Ser Asn Leu Ari 565 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH; 16 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:. NO Arg Ala Glu Val Ala Thr 555 Thr Asp 570 Gly Ala Gin Arg 525 Ala Val Ser Leu Gin Ala Tyr Glu 560 WO 94/21660 PCT/US94/02609 69 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GAATCCCGAC CTGGAC 16 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GGATCCTCAG GGTC 14 INFORMATION FOR SEQ ID 1,0:33: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CCATGGTGTT TCTCTCGGG 19 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: WO 94/21660 PCT/US94102609 GACGCGGCAG TACATGAC 18 INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID GTCATGATGG CTGGGTACTT G 21

I

Claims (10)

1. A method of alleviating the effects of benign prostatic hyperplasia in a subject, which method comprises administering to the subject a therapeutically effective amount of a compound which specifically binds to the human alpha 1C adrenergic receptor in conjunction with a compound effective to inhibit human testosterone 5-alpha reductase.

2. The method of claim 1 wherein the compound which specifically binds to the human alpha 1C adrenergic receptor has at least twelve-fold specificity for the human alpha 1C adrenergic receptor as compared with the human alphalA and alphalB receptor and an inhibitorily effective amount of a human testosterone 5-alpha reductase inhibitor.

3. The method of claim 1 or claim 2 wherein the compound which specifically binds to the human alpha 1C adrenergic receptor is S(+)-niguldipine, (S(+)-l,4-Dihydro-2,6-dimethyl-4-(3- acid 3-(4,4-diphenyl-1-piperidinyl)-propyl methyl ester hydrochloride), or 5-methyl urapidil, 5-methyl-6[[3-[4-(2-methoxyphenyl)-l-piperazinyl]propyl]amino]- 1,3-dimethyluracil.

4. The method of any one of the preceding claims wherein the compound effective to inhibit human 5-alpha reductase is a 5-alpha reductase type 1 selective inhibitor, a type 2 selective inhibitor, or a type 1 and a type 2 selective inhibitor.

The method of any one of claims 1 to 3 wherein the compound effective to inhibit human testosterone 5-alpha reductase is a dual 5-alpha reductase isozyme 1 and an isozyme 2 inhibitor.

6. The method of any one of claims 1 to 3 wherein the compound effective to inhibit human testosterone 5-alpha reductase is a 5-alpha reductase isozyme 1 inhibitor.

7. The method of any one of claims 2 to 6 wherein the 5-alpha reductase inhibitor is finasteride.

8. A method of alleviating the effects of benign prostatic hyperplasia in a subject, which method comprises administering to the subject a pharmaceutically effective amount of finasteride and S(+)-niguldipine or 5-methyl urapidil.

9. A method of alleviating the effects of benign prostatic hyperplasia in a subject, which method comprises administering to the subject a therapeutically effective amount of a compound which specifically binds to the human alpha IC adrenergic receptor in conjunction with a compound 30 effective to inhibit human testosterone 5-alpha reductase, substantially as hereinbefore described with reference to any one of the Examples. d.

10. The method of any one of the preceding claims wherein the compound which specifically S' binds to the humal alpha 1C adrenergic receptor and the compound effective to inhibit human testosterone 5-alpha reducase are administered together with a suitable excipient, diluent and/or adjuvant in the form of a composition. Dated 23 October, 1997 Merck Co., Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON IN:\LIBFF100406:JVR il~'l