WO2008137081A1 - Substituted ( 5, 6 ) -dihydronaphraalenyl compounds as reversible male contraceptives - Google Patents

Abstract

The invention provides a method for inhibiting spermatogenesis in a mammal, as well as a reversible contraceptive for use in a mammal.

Description

SUBSTITUTED (5, 6) -DIHYDRONAPHTHALENYL COMPOUNDS AS REVERSIBLE MALE CONTRACEPTIVES

This application claims priority to U.S. Provisional Application Serial Number 60/927,276, filed May 2, 2007, the contents of which is specifically incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Contraceptive methods for men are considered an essential component of worldwide reproductive health (Mruk, 2008; Anderson & Baird, 2002; Amory et al., 2006). The more common methods, i.e. mechanical devices and chemical intervention, however, can be inconvenient, offer reduced sensation, or have a significant incidence of failure, efficacy issues and undesirable side effects. Therefore, a need exists for a more effective male contraceptive that exhibits few if any side effects and health risks.

An effective male contraceptive would also offer a useful and humane method of population control in domesticated animals, as well as free-ranging wildlife. According to the American Humane Society, about 3 to 4 million dogs and cats are euthanized each year at animal shelters. And feral or wild animal overpopulation such as the overpopulation of feral hogs, deer, and bears, for example, could be managed using a male contraceptive.

Furthermore, a male contraceptive would be useful for reproductive management of animals in captivity so as to maintain genetic diversity and prevent inbreeding.

SUMMARY OF THE INVENTION

The invention involves the discovery of compounds that can inhibit spermatogenesis in a male mammal. The compound can be a pan retinoic acid receptor antagonist/ligand. Alternatively, the compound can be an antagonist/ligand that is selective for a particular retinoic acid receptor subtype. Thus, the invention provides a method of inhibiting or reducing spermatogenesis in a male mammal. The invention also provides an effective male contraceptive that, when properly administered, exhibits few if any side effects, health risks and further complications.

One aspect of the invention is a method of inhibiting spermatogenesis in a male mammal that involves administering to the male mammal for at least about five days an effective amount of a compound having the following formulae:

wherein Ri is selected from the group consisting of hydrogen, an alkyl, a branched lower alkyl, a cyclic alkyl, a heterocylic ring, or an aryl ring.

Compounds that can used in a method of the invention include:

VII),

(VIII) or any combination thereof.

In some embodiments, the compound is a retinoic acid antagonist/ligand, and in some embodiments, the antagonist/ligand is specific for the retinoic acid receptor subtype α, β, or γ. For example, in some embodiments, the antagonist/ligand is specific for the retinoic acid receptor subtype α.

In some embodiment, the retinoic acid antagonist/ligand has the structure:

In some embodiments, the retinoic acid antagonist/ligand is specific for the retinoic acid receptor subtype, α, β, or γ. In some embodiments, the antagonist and/or Iigand is specific for the retinoic acid receptor subtype α.

In general, an effective amount of the present antagonist/ligand is an amount effective for inhibiting at least about 80% of pregnancies in a female with whom the male mammal has mated. In some embodiments, the effective amount is an amount effective for inhibiting at least about 90% of pregnancies in a female with whom the male mammal has mated. In other embodiments, the effective amount is effective for inhibiting at least about 95% of pregnancies in a female with whom the male mammal has mated.

In some embodiments, an effective amount of the present antagoinist/ligand is about 0.01 mg/kg to about 100 mg/kg, for example, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 7 mg/kg, or about 2.5 mg/kg.

In some circumstances the antagonist/ligand is administered daily. In some embodiments, the compound is administered daily for about five to about fourteen days. In other embodiments, the compound is administered daily for at least about fourteen days, for example, for about thirty days; about 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 1 months or more than 1 1 months; or about 1 year, 2 years, 3 years or more than 3 years. In some embodiments, the effective amount of the compound is about 5 mg/kg and the compound is administered for at least 7 days. In other embodiments, the effective amount of the compound is about 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2mg/kg or 2.5 mg/kg and the compound is administered for at least 14 days, for example, for about 30 days; about 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 1 months or more than 1 1 months; or about 1 year, 2 years, 3 years or more than 3 years. .

In some embodiments, the compound is administered as a sustained release dosage form.

The antagonists/ligands have good bioavailability and are generally administered orally or mucosally. However, in some circumstances the compound is administered transdermal Iy.

Another aspect of the invention is a method of inhibiting spermatogenesis in a male mammal that involves administering to the male mammal an effective amount of a retinoic acid antagonist/ligand such that spermatogenesis in the male mammal is inhibited after about 3 weeks and continues to be inhibited for about 5 weeks after administration of the compound.

In another embodiment the method further involves administering the retinoic acid antagonist/ligand to the male mammal for about seven days after a period of about 5 weeks.

In some embodiments, the male mammal is a mouse, rat, bat, dog, cat, rabbit, whale, horse, deer, sheep, goat, buffalo, elk, bear, coyote, mountain lion, raccoon, fox, monkey or human.

In another aspect, the invention provides an article that includes a compound of the invention or any combination thereof and packaging material that includes a label indicating that the compound or compounds can be used to inhibit spermatogenesis in a mammal. In some of embodiments, the compound is formulated as a sustained release dosage form. In some embodiments, the article is a contraceptive that inhibits spermatogenesis in a mammal such as, for example, a mouse, rat, bat, dog, cat, rabbit, whale, horse, deer, sheep, goat, buffalo, elk, bear, coyote, mountain lion, raccoon, fox, monkey or human.

In another aspect, the invention provides for the use of an effective amount of a compound of the invention or any combination thereof in the preparation of a medicament for inhibiting spermatogenesis in a mammal.

Other features and advantages of the invention will be apparent from the following description and from the claims.

DESCRIPTION OF THE FIGURES

Figures IA-F illustrate the results of transactivation assays of the synthetic retinoid BMS-189453 (A-C) and BMS- 195614 (D-F). Increasing concentrations of all-/ra/w-retinoic acid (open circles) stimulated transactivation as indicated. (A-F) indicate dose-response activation of the reporter construct. Closed circles represent the normalized transactivation for each test compound in the same concentration range. Transactivation results for BMS- 189532 were similar to those reported for BMS-195614 (data not shown). Each data point represents the mean of three independent measurements.

Figures 2A-C illustrate the results of transactivation competition assay for assessment of antagonist activity of BMS-189453, BMS-195614 and BMS- 189532. CAT reporter activity was measured in the presence of 10^~7 M all-/r<ms-retinoic acid and increasing concentrations of each synthetic retinoid. (A) BMS-189453 competes the activity of ATRA for RARα (open circle), RARβ (closed circle), RARγ (open square) and is thus considered a pan-antagonist; (B) BMS-195614 selectively competes with ATRA for RARα (open circle) activity with less significant competition seen for RARβ(closed circle) and RARγ (open square) and is thus an RARα selective antagonist; (C) BMS-189532 specifically competes ATRA CAT expression for RARα (open circles) with minimal activity observed for RARβ and RARγ (closed circles and open squares, respectively) and is thus considered an RARα selective/specific antagonist.

Figure 3A-H are histological sections of mice treated with 0 (A), 0.1 (B), 1.0 (C-E) and 5.0 (F-H) mg/kg/day of BMS-189453 and illustrate the acute disruptive effect of BMS- 189453 on spermatid alignment and release in the seminiferous tubules immediately after dosing. A-G: Original magnification, x60. H: Original magnification, x40. pi, Preleptotene spermatocytes; 1, leptotene spermatocytes; p, pachytene spermatocytes; rs, round spermatids; es, elongated spermatids. Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the seminiferous tubules.

Figure 4 is a bar graph illustrating the testicular weight of BMS-189453-treated male mice at different post-dose time points. The bars represent the mean + SD of at least seven mice for each time point. (***) indicates significant differences within the same age group as assessed by the paired student /-test. p<0.0005.

Figures 5A-I are histological sections of the testes of mice, one month after treatment with 0 (A, B), 1.0 (C, D) and 5.0 (E-L) mg/kg/day of BM S- 189453, illustrating the temporal, cell-specific disruption of spermatogenesis. A, C-D, F-G: Original magnification, x40. B, E, H-I: Original magnification, x60. Abbreviations: in, intermediate spermatogonia; ps, pachytene spermatocytes; pl/1, preleptotene or leptotene spermatocytes; z, zygotene spermatocytes; d, diplotene spermatocytes; M I/I I, meiosis I/I I; rs, round spermatids; es, elongated spermatids. Although the asynchronous cell associations complicate staging, an attempt was made to stage the drug-treated tubules using the acrosomal system. Roman numerals followed by asterisks indicate the approximately staged tubules (e.g., stage IX*). Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the seminiferous tubules. Panels h.i show abnormal sperm in the epididymis.

Figures 6A-B are bar graphs illustrating the levels of testosterone in mice at 1 week (A) and 1 month (B) after treatment with the indicated dosages of BMS- 189453.

Figures 7A-D are histological sections of the testes of mice three months after treatment with 1 mg/kg/day (A, C) and 5 mg/kg/day (B, D) of BMS-189453. These histological studies indicate recovery of spermatogenesis. A-B: x40. C-D: x40. sg, spermatogonia; pl/1, preleptotene/leptotene spermatocytes; ps, pachytene spermatocytes; z, zygotene spermatocytes; rs, round spermatids; es, elongated spermatids. Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the seminiferous tubules.

Figures 8A-F are histological sections of mice treated with 0 (A), 0.1 (B), 1 (C, D) and 5 (E, F) mg/kg/day of BMS-189453. These results indicate the recovery of spermatogenesis in testes of mice six months after treatment. A-F: x40. ps, pachytene spermatocytes; z, zygotene spermatocytes; rs, round spermatids; es, elongated spermatids. Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the seminiferous tubules.

Figures 9A-D are results of fertility test: A-B illustrate the average numbers of embryos obtained after each assessment time point during the mating period of 14 weeks in mice treated with 5 mg/kg/day/day of BMS- 189453 for 7-day dosing period (B, n=9), as compared to control (A, n=5), and C-D are histological sections of mice treated with 5 mg/kg/day of BMS- 189453. These results show full recovery of spermatogenesis in testes of mice at 3.5 months after treatment and the presence of sperm in the epididymis, c-d: x40. Arabic numerals indicate the step of spermatid differentiation.

Figures 10A-F are results from histological and TUNEL-labeling analysis of adult testes of mice treated with BMS-189453 at 0 mg/kg (A) or 5 mg/kg (B-E) immediately after dosing (A-D) and at 1 month post dose (E). Staining with hematoxylin revealed the appearance of TUNEL-positive spermatids only at the basal lamina in BMS453-treated mice (B-D) and not in the control testis (A). Apoptotic elongated spermatids were detected when they were deeply embedded in the seminiferous epithelium of some of these testes (C). A & E: x20. C & D: x40. Arrows point to the TUNEL-positive elongated spermatids and bracket indicates the TUNEL-positive round spermatids. (F) A bar graph indicating the number of TUNEL-positive germ cells in mice after BMS-189453 treatment (100 tubules scored per testis; 5 to 10 mice per time point). The total number of apoptotic germ cells (excluding elongated spermatids) per 100 seminiferous tubules was counted in testicular sections of both control and BMS-189453-treated males.

Figures 1 IA-F are histological sections of the testes of mice one month after treatment with 0 (A, D), 2 (B, E) and 10 (C, F) mg/kg/day of BMS- 195614 and BMS- 189532, respectively, and illustrate a lack of inhibition of spermatogenesis by low doses of RARα-selective antagonists, BMS-189532 and BMS-195614. A-F: Original magnification, x40. Roman numerals indicate the stage of the seminiferous tubules.

Figure 12 is a bar graph illustrating the testicular weight of male mice at one month after treatment with the indicated dosages of BMS614 or BMS532.

Figures 13A-B are bar graphs summarizing the testosterone levels of male mice at one month after treatment with the indicated dosages of BMS614 or BMS532. Figures 14A-D are histological sections of the testes of mice one month after treatment with 1 mg/kg of BMS309.

Figures 15A-H are histological sections of the testes of mice one month after treatment with 5 mg/kg of BMS309.

Figures 16A-G are results illustrating the testicular morphology of mice in group HA at 4 weeks after the 14-day treatment of 5.0 mg/kg/day of BMS-189453. Testicular integrity was rated on a scale of 1 to 5, in which 1 is the worst and 5 the best.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compounds that inhibit spermatogenesis in a mammal without causing adverse side effects. These compounds can be used as a male contraceptive. The compound can be a pan retinoic acid receptor antagonist/1 igand. Alternatively, the compound can be an antagonist/1 igand that is selective for a particular retinoic acid receptor subtype. Thus, the invention provides a method of inhibiting or reducing spermatogenesis in a male mammal. The invention also provides an effective male contraceptive that, when properly administered, exhibits few if any side effects, health risks and further complications.

As used herein, the term "inhibit" or "inhibiting" means an abolishment of an activity or function such as spermatogenesis or a phenotype such as fertility. The term also includes a reduction in any amount, for example, a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, more than 80% reduction in the activity, function or phenotype.

As used herein, the term "mammal" means any member of the class mammalia, for example, a mouse, rat, bat, dog, cat, rabbit, whale, horse, deer, sheep, goat, buffalo, elk, bear, coyote, mountain lion, raccoon, fox, monkey or human.

As used herein, the term "selective" in reference a compound of the invention such as an antagonist/ligand of a retinoic acid receptor means that the antagonist/I igand shows a preference for a particular retinoic acid receptor subtype and will preferentially bind to the particular receptor subtype over another subtype by about 2 folds or more than 2 folds.

In one aspect, the present invention provides a method for achieving reversible sterility. As used herein, the term "reversible" means that once the methods of the present invention are discontinued, a male subject previously undergoing treatment will be returned to a condition which will permit reproduction under normal conditions. At the very least, the present compounds will not present a continued permanent obstacle to reproduction. However, reversible does not necessarily mean instantaneous. Full potency may not be restored for days or even weeks after treatment is discontinued. For example, in some embodiments, full potency will return within about 4 to about 7 weeks after administration is discontinued.

Compounds

According to the invention, the compounds of the following formulae are useful for inhibiting spermatogenesis and can be used as reversible male contraceptives:

wherein Ri is selected from the group consisting of hydrogen, an alkyl, a branched lower alkyl, a cyclic alkyl, a heterocylic ring or an aryl ring.

Specific examples of compounds that can act as reversible male contraceptives include the following.

(VIII) or any combination thereof.

In general, the present compounds are retinoic acid receptor antagonists/1 igands.

The following definitions are used, unless otherwise described: halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl" embraces only the straight chain radical, a branched chain isomer such as "isopropyl" being specifically referred to. Heterocyclic encompasses a radical attached via a ring carbon of a monocyclic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (Ci-C₄)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a 5- or 6-membered ring with an ethylene, propylene, dimethylene, trimethylene, or tetramethylene di-radical fused thereto. Similarly, Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non- peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (Ci-C₄)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a ethylene, propylene, trimethylene, or tetramethylene di-radical fused thereto.

In particular, (Ci-C₆)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso- butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C₃-C₆)CyClOaIlCyI(C i-C₆)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2- cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl; (C|- C₆)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl, 2- propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1 ,-pentenyl, 2-pentenyl, 3-pentenyl, A- pentenyl, 1 - hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C₂-C₆)alkynyl can be ethynyl, 1 -propynyl, 2-propynyl, 1 -butynyl, 2-butynyl, 3-butynyl, 1 -pentynyl, 2- pentynyl, 3-pentynyl, 4-pentynyl, 1- hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5- hexynyl; (C|-C₆)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C|-C₆)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2- fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; hydroxy(Ci-C₆)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3- hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1 - hydroxyhexyl, or 6-hydroxyhexyl; (C|-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (Ci-C₆)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio; (C₂- C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, or a bicyclic ring as described above; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide). Methods of making the retinoic acid antagonist are known to those of skill in the art. See for example, U.S. Patent Nos. 5,559,248; 5,648,385; 5,945,561 ; and 6,713,515, which are herein incorporated by reference in their entireties.

Compositions

The present compounds, including their salts, are administered to inhibit spermatogenesis and/or act as reversible contraceptives.

To achieve the desired effect(s), the present compounds, and combinations with other agents, may be administered as single or divided dosages. For example, the present compounds can be administered in dosages of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, although other dosages may provide beneficial results. In some embodiments, the amount is about 0.01 mg/kg to about 100 mg/kg, or about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 7 mg/kg, or about 2.5 mg/kg.

The amount administered will vary depending on various factors including, but ^■ not limited to, the compound chosen, the weight, the physical condition, the health, the age of the mammal, and if the compound is chemically modified. In addition, selection of a longer treatment period will enable use of a lower effective dose. For example, to inhibit spermatogenesis in a mammal, a compound of the invention can be administered at 20 mg/kg/day, 10 mg/kg/day or 5 mg/kg/day for 7 days. Alternatively, a compound can be administered at 2.5 mg/kg/day for 30 days. Factors that affect the amount to be administered, as well as the amount to be administered and duration of treatment can be readily determined by the clinician employing animal models or other test systems that are available in the art.

Administration of the compounds in accordance with the present invention may be in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition. In some embodiments, the administration of the compounds of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. For example, when the present compounds are administered for about 5-14 days, spermatogenesis will inhibited starting at about 2 to 3 weeks after administration and continues to be inhibited for about 5 weeks after administration of the compound.

In general, systemic administration is contemplated, for example, through oral administration. However, in some embodiments, the compounds can be locally administered, for example, to the testes. Such local administration can, for example, be through use of a transdermal patch, lotion or through an implanted sustained delivery device.

To prepare the composition, the present compounds are synthesized or otherwise obtained, purified as necessary or desired and then lyophilized and stabilized. The compound(s) can then be adjusted to the appropriate concentration, and optionally combined with other agents. The absolute weight of a given compound included in a unit dose can vary widely. For example, about 0.0001 to about 2 g, or about 0.0005 to about 1 g, of at least one compound of the invention, or a plurality of compounds can be administered. Alternatively, the unit dosage can vary from about 0.001 g to about 5 g, from about 0.001 g to about 1 g, from about 0.005 g to about 5 g, or from about 0.0005 g to about 2 g. In general, the compounds are administered daily at the indicated dosages for at least one week. However, in some instances, these dosages can be administered twice daily. In addition, low dosages can be administered for long periods of time. For example, low dosages can be administered for weeks, e.g. 1 , 2, 3 weeks; months, e.g. 1 month, 2 months, 4 months, 6 months, 8 months, 10 months; or even years, e.g. 1 , 2, 3, 4 or more than 4 years, as needed..

Thus, one or more suitable unit dosage forms comprising the present compounds can be administered by a variety of routes including oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, dermal, transdermal, intrathoracic, intrapulmonary and intranasal (respiratory) routes. In some embodiments, the present compounds are administered orally or transdermally.

The compounds may also be formulated for sustained release (for example, using microencapsulation, see WO 94/ 07529, and U.S. Patent No.4,962,091 ). The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts. Such methods may include the step of mixing the compounds with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

When the compounds of the invention are prepared for oral administration, they are generally combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. For oral administration, the compounds may be present as a powder, a granular formulation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum. The compounds may also be presented as a bolus, electuary or paste. Orally administered compounds of the invention can also be formulated for sustained release, e.g., the compounds can be coated, micro-encapsulated, or otherwise placed within a sustained delivery device. The total active ingredients in such formulations comprise from 0.001 to 99.9% by weight of the formulation.

By "pharmaceutically acceptable" is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

Pharmaceutical formulations containing the compounds of the invention can be prepared by procedures known in the art using well-known and readily available ingredients. For example, the compounds can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives. Binding agents can also be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone. Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and sodium bicarbonate. Agents for retarding dissolution can also be included such as paraffin. Resorption accelerators such as quaternary ammonium compounds can also be included. Surface active agents such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carriers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added. The compositions of the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They may also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like.

For example, tablets or caplets containing the compounds of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Caplets and tablets can also include inactive ingredients such as cellulose, prege latinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, and the like. Hard or soft gelatin capsules containing at least one compound of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coated caplets or tablets containing one or more compounds of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.

The compounds of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes. The pharmaceutical formulations of the compounds of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve.

Thus, the compounds may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion containers or in multi-dose containers. As noted above, preservatives can be added to help maintain the shelve life of the dosage form. The compounds and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the present compounds and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable carriers, vehicles and adjuvants that are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol," polyglycols and polyethylene glycols, C 1 -C4 alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol," isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.

It is possible to add, if desired, additional ingredients, for example, chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes, flavorings and colorings. Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and α-tocopherol and its derivatives can be added.

Additionally, the present compounds are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the compounds, for example, in a particular part of the intestinal or respiratory tract, possibly over a period of time. Coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, draining devices and the like.

For topical administration, the compounds may be formulated as is known in the art for direct application to a target area. Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap. Other conventional forms for this purpose include ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Thus, the compounds of the invention can be delivered via patches (e.g., transdermal patches) or bandages for dermal administration. Alternatively, the compounds can be formulated to be part of an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it might be desirable to use microporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized. The backing layer can be any appropriate thickness that will provide the desired protective and support functions. A suitable thickness will generally be from about 10 to about 200 microns.

Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The compounds can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4, 140,122; 4,383,529; or 4,051 ,842. The percent by weight of a compound of the invention present in a topical formulation will depend on various factors, but generally will be from 0.001% to 95% of the total weight of the formulation, and typically 0.01 -85% by weight.

The compounds may further be formulated for topical administration, for example, to the skin or mucosa. In other embodiments, the active ingredient may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier.

The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non-limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0. The present invention further pertains to an article such as a packaged pharmaceutical composition and can be in a kit or other container. The kit or container holds an effective amount of a pharmaceutical composition that includes a compound of the invention and instructions for using the pharmaceutical composition as a male contraceptive. The pharmaceutical composition includes a carrier and an effective amount of a compound of the invention or combinations of such compounds.

Retinoic Acid Receptors

All-trans-retinoic acid (ATRA), the biologically active form of vitamin A, is an important regulator of cell growth and differentiation (Kastner et al., 1995; DeLuca and Ross, 1997). The effects of ATRA are mediated through two classes of nuclear retinoid receptors, the RARs, which bind ATRA and 9-cis- retinoic acid (9cRA), and the RXRs, which bind only the 9cRA isomer. The RARs and RXRs each have three subtypes, RARα, β, and γ as well as RXRα, β, and γ, that are encoded by distinct genes (Petkovich et al., 1987; Brand et al., 1988; Mangelsdorf et al., 1990; Mangelsdorf and Evans, 1995; Chambon, 1996; Piedrafita and Pfahl, 1999). They function as ligand-dependent transcription factors that bind to retinoic acid response elements (RAREs) in the promoter region of target genes, leading to the modulation of the transcription of these genes (Green and Chambon, 1988). Interactions among these members of the steroid/thyroid hormone receptor superfamily may contribute to many of the pleiotropic biological activities attributed to retinoids, such as the regulation of cell proliferation, differentiation and morphogenesis. While many potential targets of RAR have been identified from studies screening for ATRA-induced up-regulation or down-regulation of genes or for genes containing retinoic acid response elements (RAREs), those which might be important for spermatogenesis, a key physiological target of vitamin A function, remain unknown.

Mutagenesis of the mouse RARα receptor gene results in disruptions in spermatogenesis that were reported to be similar to those observed in the vitamin A deficient (VAD) rat testis (Lufkin et al., 1993). However, only recently have studies by the inventors begun to provide insight into which cells in the testis are affected by the disruption of RARα function and the molecular mechanisms that underlie the lack of function of retinoid signaling through the RARα transcription factor (Chung et al., 2004; Chung et al., 2005; Chung and Wolgemuth, 2004). These studies became feasible when the inventors discovered that the previously reported perinatal lethality of the RARα- deficient mice could be overcome by maintaining the mice in a pathogen-free environment (Chung et al., 2004).

Specific defects in spermatogenesis are observed in RARα-deficient mice, including a failure of the elongated spermatids to align at the lumen of the tubule, defects in spermiation, and a failure of the elongating spermatids to rotate relative to the Sertoli cells at step 8-9 of spermiogenesis. The inventors also observed a novel onset of apoptosis (as assessed by TLTNEL-staining) in many of the elongating spermatids that fail to become entrenched within Sertoli cells. In addition, the inventors noted abnormalities in the progression of meiotic prophase and a temporary arrest at step 8 spermatids as well. These abnormalities are quite specific to the RARα knockout phenotype and although spermatogenesis was also impaired in the RXRα knockout mice (Kastner et al., 1996), the phenotype was distinct and appeared to affect Sertoli cell function (Kastner et al., 1996).

According to the invention, many of these same physiological changes occur when the present compounds are administered to a male mammal. However, while RARα interaction may occur, it is not certain that the effects of the present compounds are due to solely to RARα interaction. For example, the present compounds may target a variety of RARs (not just RARα) or have other as yet unidentified interactions and effects. What is clear is that the present compounds strongly inhibit spermatogenesis in a reversible manner. Pursuant to the invention, the dosages of the present compounds are adjusted to optimally inhibit spermatogenesis while eliminating any negative side effects. Surprisingly, no negative side effects are observed when the present compounds are administered as described herein.

The following Examples further illustrate certain aspects of the invention and are not intended to limit the invention described in the claims in any manner.

EXAMPLES

The following examples relate to the use of the retinoic acid receptor pan-antagonist BMS-189453 in inhibiting spermatogenesis at low doses and without adverse side effects. Results show that CD-I mice treated with BMS- 189453 by oral administration at 5 mg/kg/day for 7 consecutive days exhibited aberrant spermatogenesis that resembled phenotypes observed in vitamin A-deficient and retinoic acid receptor alpha-deficient testes within one month following cessation of treatment. Sterility as measured by the failure to produce progeny, a stringent assessment of contraceptive efficacy, was concomitantly induced. Fertility was restored with no apparent adverse effects within 14 weeks after treatment. Histological analysis showed that spermatogenesis recovered, with normal- appearing testicular tubules and sperm in the epididymides.

BMS- 189453 has been shown to have good (82-98%) oral bioavailability in rat and monkeys (Schulze et al., 2001 ). It is a synthetic RAR antagonist with low molecular weight that is easy to synthesize. It is also fat-soluble and is mainly metabolized in the liver into water soluble components and excreted. The half-life of BMS- 189453 is approximately 6 hours in rats (Cipollone D et al Cardiovascular Path 15, 194-202, 2006). As reported in rat (Schulze et al., 2001), its lack of overt toxicity and other adverse side effects in low dose regimens are particularly important. The following studies show that low doses of BMS- 189453 proved effective in inducing a rapid onset of male sterility and spontaneously reversibility after a sustained interval (~14 weeks), thus demonstrating the activity of a novel, low molecular weight compound in inhibiting spermatogenesis in a reversible manner, and hence its usefulness as a male contraceptive.

Example 1 - Materials and Methods

Source of animals and tissues

All procedures were performed in accord with guidelines of the Institutional Animal Care and Use Committee of the Columbia University Medical Center. Testes of drug-treated mice (BMS- 189453, BMS- 189532 or BMS- 195614) were dissected from anesthetized animals and fixed with 4% paraformaldehyde in PBS or with Bouin's fixative (15 parts picric acid-aqueous solution, 5 parts formaldehyde and 1 part glacial acidic acid). Tissues were fixed overnight at 4⁰ C before being processed.

Retinoid Transactivation Assays and Transactivation Competition Assays

The RAR pan-antagonist, BMS- 189453, and RARα-selective antagonists, BMS- 189532 and BMS- 195614 synthesized by Bristol-Myers Squibb Co., Princeton, NJ were used in the assays (Yu et al., 1996; Zusi et al., 2002). The transactivation methods used are as reported previously ^26>41. Briefly, HeLa cells were transfected using the calcium phosphate co-precipitation method with cDNA constructs encoding each of the three receptors, RARα, β, and γ along with an RARE-CAT (chloramphenicol acetyl transferase) reporter construct and β-galactosidase for normalization of transfection efficiency under tk promoter (Vasios et al., 1989). All-/rø«s-retinoic acid or the compounds to be tested were incubated with transfected cells for 24 hours in order to induce the expression of the CAT reporter. CAT was measured using a commercially available CAT-ELISA kit (5-Prime-3 Prime, Inc.).

Antagonist activity of the synthetic compounds was determined using a transactivation competition assay. Using the same DNA constructs for the RARs and reporter genes described for transactivation, HeLa cells were co-incubated with a sub-maximal dose of ATRA (10^~7 M) was incubated along with increasing concentrations of the tested compound in the range of 0 to 10^"5 M (BMS 189453 and BMS 195614) or 10^"9 to 10^'5 M (BMS 189532). After 24 hours, CAT concentration was measured. Data points for each compound are normalized for RAR stimulation at 10^'7 M ATRA for BMS 189453 (a) and BMS 195614 (b) or 10^'6 M ATRA for BMS 189532 (c) and are reported as the mean of three independent measurements. A compound was considered an antagonist if it inhibited ATRA- induced CAT reporter expression but did not, itself, induce expression of the CAT reporter. By this criterion, all three compounds are designated as RAR antagonists.

Drug Treatment

The RAR pan-antagonist, BMS-189453, and RARα-selective antagonists, BMS- 189532 and BMS- 195614 (gifts from Bristol-Myers Squibb Co., Princeton, NJ) were used. For oral administration, these drugs were suspended in a vehicle of aqueous 1.5% microcrystalline cellulose and carboxymethylcellulose sodium (Avicel® CL-61 1 , FMC BioPolymer, Philadelphia, PA) to achieve the desired concentrations.

CD-I male mice, 7 weeks of age with body weight <30g, were purchased from Charles River. BMS-189453 was administered orally via gavage (10 mice/group) at daily doses of 0, 0.1 , 1 and 5 mg/kg for 7 days. Control groups (5 mice/group) received 0 mg/kg of BMS- 189453 in aqueous 1.5% Avicel® for 7 days. Four time points were selected— I week (end of dosing period) and 1 , 3 and 6 months post-dose. During the dosing period, the animals were observed at least once daily for changes in condition and behavior. Body weight was recorded and physical examinations were performed weekly.

Analysis of Effects on Spermatogenesis

At the specified time points, testes were dissected from euthanized animals and weighed. One testis was fixed in Bouin's fixative for subsequent morphological analysis and the second testis was fixed in 4% paraformaldehyde for future studies involving in situ hybridization or immunohistochemical analyses. One member of the pair of epididymides was fixed for morphological evaluation as above. The second member of the pair was used to collect cauda epididymal sperm (if present), which were assessed for motility according to The Jackson Labs' protocols for male infertility evaluation

(http://reprogenomics.iax.org/maleprotocol.htmn. Serum testosterone levels were determined by standard RIA (DELFIA; Wallac, Inc., Turku, Finland) to assess the effects, if any, on the function of Leydig cells when spermatogenesis is disrupted. The specimens were examined grossly for any sign of aberrant growths (tumor-like lesions). Testicular weight within the same age group was assessed by statistical analysis using Student's Mest using the GraphPad Statistical Analysis-Software.

Histology

Fixed tissues were embedded in paraffin, sectioned at 5 μm, and mounted on Superfrost slides (Fisher). Histological sections were deparaffinized in histoclear, hydrated through an alcohol series, and washed with H₂O. Slides were stained with hematoxylin according to standard procedures. They were viewed on a Nikon Eclipse 800 photomicroscope under bright-field optics. Photomicrographs were taken using a digital camera. For staging of the mouse seminiferous epithelium according to Oakberg et al, (1956) and Russell et al. (1990), the testis sections were stained for the Periodic Acid Schiff (PAS) reaction before hematoxylin counterstaining (Leblond & Clermont, 1952).

Sperm Count

Cauda epididymal sperm were collected from CDl mice (Charles River, Wilmington, MA) The two caudae were placed them into a tube containing 1 ml of modified Whitten's medium (Mod W) (22 mM HEPES, 1.2 mM MgCl₂, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, 4.8 mM lactic acid hemi-calcium salt, pH 7.3) at 37 °C as previously described (Travis et al., 2001). The caudae were minced by a pair of small scissors to release the sperm. After mincing, a round forcep was used to gently roll the Cauda pieces to squeeze the sperm from the tubules. 1 ml of cells and tissue fragments were layered onto 300 μl of 3% BSA in PBS. After the heavy tissue fragments fall through the BSA to the bottom of the tube, the upper layer was removed and filtered through a 100 μM filter to remove any remaining debris. A 1 : 10 dilution was made and the sperm were counted by a hematocytometer.

TUNEL Staining ofApoptotic Cells

Histological sections from animals treated with 0.1 , 1.0, and 5.0 mg/kg doses of compound were deparaffinized in histoclear, hydrated through a graded alcohol series, and washed with H₂O. In situ labeling of apoptotic cells was performed on tissue sections from BMS453-treated testes using the in situ cell death detection kit, horseradish peroxidase (POD) (Roche, Indianapolis, IN), according to the manufacturer's instructions. In brief, sections were treated with 20 mg/ml proteinase K in 1 OmM Tris/HCI (pH 8.0) and endogenous peroxidase was blocked in 0.3% H₂Ch for 30 min. Single- and double- strand breaks were 3' labeled with fluorescein-labeled nucleotides, using terminal deoxynucleotidyl transferase. Incorporated fluorescein was detected by anti-fluorescein antibody Fab fragments from sheep, conjugated with POD. Bound antibodies were covered with 0.4 mg/ml 3,3' diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, MO) in 0.1 M Tris (pH 7.2). Only clearly stained cells were considered to be positive and only round-shaped tubules were evaluated. At least 100 tubular cross- sections of each testis from ten different animals were counted per experimental time point (Beumer et al., 1997; Rodriguez et al., 1997). The results of individual counts were compared to counts within the same age group with different drug treatment and time points. Significant differences were assessed by statistical analysis using Student's t-test.

Assessment of Fertility

The fertility assessment was based on a well established protocol (Elbetieha & Da'as, 2003; Al-Thani et al., 2003). In brief, CD-I mice (Charles River) were treated by gavage with 5 mg/kg of BMS453. A seven-day dosing period and six months post-dose time point was selected for assessing both the disruption of fertility and the restoration of fertility. During the dosing period, the animals were observed at least once daily for changes in overall condition of health and behavior. Body weights were measured and physical examinations were conducted weekly. After the dosing period, the 5 mg males (n=9) were immediately placed in an individual cage with two untreated virgin females of the same strain. The animals were caged together for 14 days during which approximately three estrus cycles should have elapsed. At the end of the fourteen day period, the mated females were sacrificed by cervical dislocation under light ether anesthesia and the following measurements were recorded: number of pregnant females, number of implantation sites, number of viable fetuses, number of resorptions, and number of females with resorptions. Another two females were replaced continuously at 2-week intervals until either fertility was restored or the animals reached the 6-month post-dose time point. At that time, the experimental and control males were removed and sacrificed for further evaluations as described.

Example 2 - Retinoic Acid Receptor Antagonists

This Example describes the initial characterization of retinoic acid receptor antagonists that may be useful as reversible male contraceptives.

The effects of all /rørø-retinoic acid (ATRA), the active metabolite of retinol, are mediated through two classes of nuclear retinoid acid receptors, the RARs, which bind ATRA and 9-cis- retinoic acid (9cRA), and the RXRs, which bind only the 9cRA isomer. The RARs and RXRs each have three subtypes, α, β, and γ, that are encoded by distinct genes (Piedrafita et al., 1999; Petkovich & Pfahl, 1987; Mangelsdorf et al. 1990; Mangelsdorf et al., 1995; Chambon, 1996; Brand et al., 1988). These receptors function as ligand-dependent transcription factors that bind to retinoic acid response elements (RAREs) in the promoter region of target genes, leading to the modulation of transcription (Piedrafita & Pfahl, 1999; Chambon, 1996).

Ligands directed specifically at individual nuclear retinoid receptors were synthesized and identified through the combined use of binding affinity and transactivation analysis (Chen et al., 1995a; Chen et al., 1995b; Chen et al., 1996a; Chen et al., 1996b; Starrett, 1997; Yu et al., 1996; Loeliger, 1980). As a result, novel receptor- selective transcriptional antagonists for RARα, RARβ and RARγ were identified and characterized (Chen et al., 1996b; Zusi et al., 2002; P. Reczek, unpublished observations). These compounds, which include BMS 189453, BMS-189532 and BMS- 195614, fit the three criteria for antagonism: (1) they bound to the RARs with a K_d for binding comparable to that of ATRA; (2) there was no detectable activity of the compounds in a transactivation assay; and (3) the compounds were able to compete with the activity of ATRA in a transactivation assay. The structures of BMS-189453, BMS- 189532 and BMS- 195614 are shown below.

BMS-1894653 BMS-189532

BMS-195614

Chemically, BMS-189453 is 4-[[(E)-[5,6-Dihydro-5,5-dimethyl-8-phenyl]-2- naphthalenyl]benzoic acid. BM 189453 has good oral bioavailability (82-98%) in rats and monkeys (Schulze et al., 2001). The bioavailability of BMS-189532 and BMS-195614 is less well-characterized, but is predicted to be similar to BMS- 189453. These compounds were the first antagonists of the RARs to be described (Chen et al., 1995a).

Activity of the compounds were assessed using direct binding to recombinant receptor made in bacteria and a transactivation assay involving the co-transfection of receptor cDNA and the chloramphenicol acetyl transferase (CAT) reporter gene linked to the RARE from the laminin B gene. As shown in FIGs. IA-C, in transactivation assays for the ability to stimulate transcription of a chloramphenicol acetyl transferase (CAT) reporter gene linked to the RARE from the laminin B gene (Vasios et al., 1989), BMS- 189453 showed minimal transactivation activity for RARα (FIG. I A) and RARγ (FIG. 1C), and a small but reproducible transactivation for RARβ (FIG. I B). Given the minimal transactivation activity, BMS-189453 was subsequently tested in a transcription assay for its ability to compete with ATRA, i.e. to act as an antagonist for the RARs. Results in FlG. 2A show that BMS-189453 exhibited potent antagonist activity for all three RAR's. The compound binds well to recombinant receptor protein of all three subtypes with a Kj for binding in the sub-nanomolar range: apparent Ka of 0.4 nM, 0.2 nlM, and 0.8 nM for RARα, β, and γ, respectively (see also Chen et al., 1995a; P. Reczek unpublished observations).

The transactivation properties of BMS-189532 and 195614, as well as their ability to act as antagonists were evaluated as described above for BMS-189453. In brief, the effects of the compounds were assessed using direct binding to recombinant receptor (DBA assay) made in bacteria and a transactivation assay involving the co-transfection of receptor cDNA and the chloramphenicol acetyl transferase (CAT) reporter gene linked to the RARE from the laminin B gene (RTCA). Results are summarized in the following table and in FIGs. I D-F (Note: Very similar data were obtained from transactivation by BMS-189532) and FIGs. 2B- C.

Binding and Transactivation Properties

DBA (K_n in nM): RARα RARβ RARγ

BMS-195614 3 12.5 350

BMS-189532 13 12 1 15

RTCA IC50 in M):

BMS-195614 4 x 10^"7 4 x 10^"6 < 50%

BMS-189532 7x 10^"7 NA NA

DBA: Direct binding assay

RTCA: Retinoid transactivation competition assay (antagonism)

BMS-189532 and 195614 were found to be selective for RARα. Thus, while BMS453 functions as a pan-RAR antagonist, BMS532 and BMS614 exhibit specificity for RARα. The receptor specificities of BMS532 and BMS614 are thought to be due to the amide bonds in place of the double carbon bond found in the pan-antagonist BMS453. This in turn is believed to interact with serine 232 of the RARα protein. Serine is replaced by alanine in RARβ and RARγ (Ostrowski et al., 1995; Ostrowski et al., 1998). In addition, BMS-189453, BMS-195614 and BMS-189532 had been shown previously to exhibit a KQ for binding comparable to that of ATRA in the sub-nanomolar range (Chen et al., 1995a).

Toxicity of BMS-189453 has been evaluated by Schulze et al. (2001). In particular, Schulze et al. investigated the potential target-organ toxicity of BMS453 in rats following a month of oral administration (Schulze et al., 2001). In addition, subsequent 1-week and 1 -, 3-, or 7-day oral toxicity studies were conducted in rats and a 1-week oral toxicity study was conducted in rabbits. As described in Schulze et al. (2001), Sprague Dawley rats were given daily oral doses of 15, 60, or 240 mg/kg body weight BMS-189453 for 1 month. Increases in leukocyte counts, alkaline phosphatase and alanine aminotransferase levels, and striking testicular degeneration were observed at these doses. Overt signs of toxicity death occurred at 240 mg/kg, whereas body weight and food consumption decreased at the 60 and 240 mg/kg doses. Importantly, in studies in which rats were administered doses of BMS-189453 daily for only one week and at doses ranging from 12.5 to 100 mg/kg, only minimal changes in testicular morphology and no other side effects were observed immediately after the dosing period. However, one month after the dosing ceased, marked testicular degeneration was observed at all doses. Additional studies involved dosing at 2, 10, or 50 mg/kg for 1 , 3, or 7 consecutive days. Although no changes were observed at the time of cessation of dosing, testicular atrophy was observed one month later, and the degeneration was still obvious if not worse after 4 months. Testicular atrophy was also observed in the rabbits reported in the published study (Schulze et al., 2001 ) and also in unpublished studies with mice (P. Reczek, unpublished observations).

The significance of these observations in contraceptive approaches lies in the specific and apparently irreversible testicular toxicity following low and few doses of BMS453 that are otherwise tolerated. Example 3 - Inhibition of Spermatogenesis by BMS-189453: Cellular and Temporal Specificity

The following study was conducted to determine whether spermatogenesis can be disrupted using concentrations of BMS-189453 lower than previously reported (specifically 0.1 , 1.0, and 5.0 mg/kg/day), but at the same length of dosing period (7 days). Because mice can still produce progeny even with substantially lowered sperm counts, histological evaluation of the testes at sequential time points post-dosing was used.

At a dose of 0.1 mg/kg/day of BMS- 189453, no changes in testicular morphology was observed at any of the time points examined, either immediately following the dosing period (FIG. 3B) or at 1 month (data not shown), 3 months (data not shown), and 6 months post-dosing (FlG. 8B). However, there were distinct changes in testicular morphology at both 1.0 mg/kg/day (designated 1 mg mice) and 5.0 mg/kg/day (designated 5 mg mice) as described below. No other side effects were observed during or immediately after the dosing period.

Immediately after the dosing period, a failure of spermatid alignment and sperm release was found in the testes of mice given 1 mg (FIG. 3C, D) and 5 mg (FIG. 3F) of BMS- 189453, in striking contrast to the normally appearing stage VIII and IX tubules in testes of mice treated with vehicle alone (FIG. 3A). (The developmental progression of the cells within the tubules is assessed using the acrosomal system of Russell et al., 1990. The approximately staged tubules are referred to using a roman numeral followed by an asterisk.) Interestingly, a failure of spermatids to align at stage VIII has been a consistent observation in the RARd'^' testes (Chung et al., 2005). Round spermatids exhibited crescent-like condensation of the chromatin with a clear space alongside the nuclei at both doses (FIGs. 3E & G) and vacuolar-like spaces were particularly prominent in the 5 mg mice (FIG. 3H). No abnormalities in epididymal spermatozoa were observed in all mice examined at this time point (data not shown).

At 1 month after treatment, the testicular weight of the mice given 5 mg (but not 1 mg) of BMS-189453 was slightly, but significantly reduced (FIG. 4), and there was an overall depletion in the number of sperms in the epididymal sections of the 5 mg mice (n=7; data not shown). Abnormally-shaped round spermatids with crescent-like condensation of the chromatin with a clear space alongside the nuclei were consistently observed in both 1 and 5 mg mice (FIG. 5C & F). Nine of the ten mice in the 1 mg group had predominantly normal-appearing tubules, whereas seven often of the 5 mg mice had quite striking abnormalities in their tubules. Vacuolar-like spaces were now more prominent in the 1 mg mice (FIG. 5D); however, there was still a large proportion (-70%) of normal-appearing tubules (FIG. 5D). In contrast, in the 5 mg mice, spermatogenesis was severely disrupted. The majority (-62%) of tubules that contained round spermatids exhibited sloughing of these cells into the lumen and the diameter of tubules was reduced by -35%. Of those spermatids that did develop to the elongated stage, they were consistently retained within the tubules rather than being released at spermiation (FIG. 5E) (tubule at stage IX*).

Also of interest was the observation of the presence of two to three different steps of spermatids within the same plane of a section within a single tubule (FIG. 5E). In some severely defective tubules, entire layers of given cell types were missing (FIG. 5G). For example, at stage XII*, MI/MII spermatocytes with chromosomes aligned at the metaphase plate, and zygotene and diplotene spermatocytes were readily seen (FIG. 5G, right bottom tubule). However, step 1 or 12 spermatids were missing. In another example, zygotene spermatocytes were missing in the stage XII* tubule depicted in FIG. 5G. Finally, abnormally shaped round spermatids were detected in the caput (head) of the epididymis, and abnormal spermatozoa with massive cellular debris were found in the cauda epididymis (FIG. 5H & I). There was also a considerable amount of cellular debris along with giant multi-nucleated germ cells. This drastic reduction or disappearance of spermatozoa in the epididymis observed in the 5 mg mice by 1 month treatment was likely responsible for the infertility in these mice (see below).

Caudal epididymal sperm were also examined for morphological and motility abnormalities. No epididymal sperm morphological changes were noted in all the mice treated with 0.1 to 5 mg/kg immediately after dosing, as well as 0.1 to 1 mg/kg at the 1 month post-dose. Abnormal epididymal sperm morphology was observed only in mice (n=4) given 5 mg/kg at the 1 month post-dose. Few mice (n = 2) showed highly reduced numbers of sperm in their epididymis; instead, considerable amount of cellular debris, along with giant multi-nucleated germ cells were observed. The motility of at least one hundred spermatozoa was determined for all mice. A spermatozoon is considered motile when forward sperm progression or regular movement of the flagellum is observed. All sperm were normal in all doses at these two points except mice given 5 mg/kg at 1 month post-dose. Sperm motility slowed down tremendously as compared to control and some of the sperm were observed to swim in a circular motion.

The specimens that were examined did not show any sign of aberrant growths or tumor-like lesions. In addition, testosterone levels in mice were not affected by administration with BMS-189453 (FIGs. 6A-B). Because testosterone levels varied tremendously, it was difficult to interpret the high levels of testosterone in some of the control males. The analytical methods employed were not the source of the variability as duplicates were used for each sample and the readings were very consistent and reproducible. The standard graph and the positive control were perfect; the intra-assay coefficient of variation (CV) was within the range (3.1 -1 1.9 %).

At three months after treatment with this compound, an apparent recovery of spermatogenesis was evidenced at the histological level by the presence of normal- appearing tubules in the testes of mice given 1 mg (n=5, FlG. 7A) and 5 mg (n=6, FIG. 7B) of BMS- 189453. There was some variability in the recovery as assessed at the morphological level, however. That is, two animals from the 1 mg group and one animal from the 5 mg group still exhibited -18.2 % and -21.6 % tubules with severe germ cell depletion, respectively (FIG. 7C & D, respectively), although the significance of this disruption on fertility remained to be determined (see below). Neither abnormal spermiation nor defective alignment of spermatids was found in any the samples (stage IX in FIG. 7C), suggesting that the effect of BMS- 189453 on spermiation and spermatid alignment was an acute response and did not re-occur when the treatment stopped. That is, the effect of the antagonist on these two biological properties was acute, but reversible. There were, however, some abnormally shaped round spermatids consistently observed in those few still aberrant tubules in both groups (data not shown). This observation indicates that the dramatic effects of BMS-189453 on spermatogenesis, even at low doses, were reversible.

At six months after treatment, neither abnormal spermiation nor mis-alignment of spermatids was found in either group (stage IX in FIG. 8C-E), again supporting the observation that these abnormalities are an acute response. There was an apparent full recovery of spermatogenesis in the testes of all but two of the mice given 1 mg of BMS- 189453 (n=5, FIG. 8C), as evidenced by the presence of three to four layers of germ cells with correct cellular associations, cell types, and cell numbers in virtually all of the tubules (FIG. 8C). The testes of one male from this group still had around 25% of the tubules with some level of germ cell depletion (usually missing 1 layer of cells) while another one exhibited more severe germ cells depletion (-35%, n=l) and a mixture of cell types were found (tubules in the middle and left-hand side in FIG. 8D. In the mice given 5 mg of drug, full recovery of spermatogenesis was found in all but one mouse (n=6, FIG. 8E), and in that mouse, all but 10% of the tubules (n=150) appeared normal (FIG. 8F). Occasional recovering-stage I tubules (labeled with asterisk, FIG. 8F) with correct cellular association, but fewer numbers of cells and some vacuoles were detected (FlG. 8F) as compared to normal stage I tubule (tubule on left-hand side, FIG. 8F).

Example 4 - Inhibition of Spermatogenesis and Fertility by BMS-189453 is Reversible

This Example illustrates that RAR Antagonists reduce the fertility of male mice in a reversible manner. A series of mating studies using mice administered 5 mg of BMS-189453 was conducted to evaluate the kinetics of the drop in fertility and its restoration. Morphological assessment of spermatogenesis was performed as described above. The decrease in fertility and its recovery in B M S453 -treated males were followed for 3.5 months following treatment.

1 st round of mating (between 1-2 weeks post-dose): Immediately after dosing and in the period of 1 -2 weeks post BMS453 drug treatment, when the BMS453-treated males (n=9) were mated with 2-3 young fertile CD l females for two weeks, some (n=3) of the males failed to yield pregnancies. One male produced 2/7 and 4/6 resorptions in two females (compared to 1/14 or 1/16 in control). As anticipated, the control males generated normal litter sizes and very few resorptions were found (data not shown).

2nd round of mating (between 3-4 weeks post-dose): In the second round of mating of BMS453-treated males (n=9) with 2-3 young fertile females, only one male (#8) was able to produce pregnant females. That is, 8 out of 9 males were infertile in the time period of 3-4 weeks post-dose. In contrast, all our control males generated normal embryos in the young females. 3rd round of mating (between 5-6 weeks post-dose): The results of the 3rd round of mating of BMS453-treated males (n=9) with 2-3 young fertile females (8-week-old) fluctuated a bit. Five out of nine males remained infertile in the time period of 5-6 weeks post-dose). Another two males (n=2) became fertile again, but generated smaller embryonic litter sizes (6 and 4 embryos, respectively). The single male (n=l) that was able to produce pregnant females in the 2nd round of mating remained fertile. Finally, one male, which was infertile in our previous round of mating, produced an embryonic litter size of 12 and 13, suggesting a fast recovery of spermatogenesis. As usual, all our control males generated normal embryos in the young females.

4th round of mating (between 7-8 weeks post-dose): All the B M S453 -treated males became fertile (n=9) and could generate embryonic litter sizes that were comparable to controls (10.4 + 1.64). This result was unexpected as no resumption of spermatogenesis was reported by Schultz et al. (2001). However, the dose used by Schultz et al., i.e. 12.5 mg/kg, was a little more than twice the dose administered in these studies.

5th round of mating (between 9-10 weeks post-dose): All the BMS453-treated males were consistently fertile (n=9) and most of them (5/9) produced litters of embryos of comparable size to the controls. However 2/9 only produced pregnancy in one of the test females and in another 2, the sizes of the litters were reduced, being able to generate 4 and 2 embryos, respectively. This indicated that these mice remained in the process of recovering spermatogenesis. Consistently, everything was normal for control mice.

6th round of mating (between 1 1 -12 weeks post-dose): All the BMS453-treated males were back to normal and remained fertile (n=9) at 1 1-12 weeks post-dose and most of them produced normal embryonic litter sizes. However, one produced litters that were smaller in size (4 embryos only). In general, the males in this round of mating were more fertile than in the 5th test mating. Again, everything was normal for control mice.

7th round of mating (between 13-14 weeks post-dose): All BMS453-treated males were fertile (n=9) at 13-14 weeks post-dose and produced normal litter sizes. The mating regimen was stopped because the full recovery of fertility in the males treated with 5 mg/kg of BMS453 had been documented. Again, everything was normal for control mice. The number of embryos per litter per round of mating/2 -week period of individual BMS-treated and control males was plotted, illustrating the disruption and restoration of fertility phases (FIG. 9A & B, respectively).

After fertility testing was terminated, the BMS453-treated mice were sacrificed. There were no obvious variations in testicular weight and sperm number in both wild- type and drug-treated males (data not shown). The tissues were collected and the morphology of the testes was examined, as shown in FIG. 9C-D. All of the slides of histological sections from testes of the BMS453-treated males at 3.5-months time point were examined. Results indicate full recovery of spermatogenesis (e.g. FlG. 9C) and spermatozoa were detected in the epididymides in the corresponding males accordingly (e.g. FIG. 9D). There were few tubules (2-5 tubules per mouse) with germ cell sloughing which had not yet been recovered (data not shown). However, these minor abnormalities did not affect the sperm numbers, as reflected from the sperm counts and morphological examination (data not shown).

In sum, control males generated normal litter sizes, and very few resorptions were found throughout the assessment period (FIG. 9A). The results of fertility tests found in mice treated with 5mg/kg/day of BMS453 are consistent with the morphological results - (FIG. 9B). By 4 weeks after treatment, all but one mouse (n=8) became infertile. Immediately after dosing and in the period of 1 -2 weeks post-treatment, three males failed to yield pregnancies, indicating that BMS- 189453 has immediate effects on sperm function in some males. By 14 weeks, fertility resumed completely in all males (FIG. 9B) indicating that the anti-fertility effects of this RAR pan-antagonist are reversible. That is, all the BMS- 189453-treated males became fertile (n=9) and could generate litters of embryos of comparable sizes to controls (12.59 + 0.75 experimental [n=9] vs 1 1.90 + 2.16 controls [n=5]). Further, there were no obvious differences in testicular weight (FIG. 4) and sperm number (data not shown) in drug-treated males versus controls after regaining fertility. Full recovery of spermatogenesis was observed, with tubules of comparable size to control (FIG. 9C, n=9) and the presence of spermatozoa in the corresponding epididymides (FIG. 9D, n=9), as compared to the control testes (data not shown; n=5). This finding was unexpected in view of the results of Schultz et al. (2001), that is, at little more than twice the dose of the present studies, 12.5 mg/kg/day, it was reported that there was no resumption of spermatogenesis. Example 5 - TUNEL Staining of Λpoptotic Cells in Degenerating Testicular Tubules

To determine whether the cells in the degenerating testicular tubules and the abnormally aligned elongating spermatids in BMS453-treated testes were undergoing apoptosis, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) staining was used in combination with morphological analysis as described above. In situ labeling of apoptotic cells was performed on tissue sections from BMS453-treated testes using the in situ cell death detection kit, horseradish peroxidase (POD) (Roche, Indianapolis, IN).

Results indicate that in normal adult testis, the most frequent TUNEL-positive cells were spermatogonia and early and meiotically dividing spermatocytes (references in Chung et al., 2005). Some tubules with TUNEL-positive germ cells in B M S453 -treated testes looked very similar to those seen in control testes (FIG. 1 OA versus B & D, respectively). However, the presence of TUNEL-positive spermatids at the periphery of some of the tubules was noted in B M S453 -treated testes (FlGs. 1 OB, C & D) but never in control testes (FIG. 10A). These TUNEL-positive spermatids often lay close to the basal lamina (arrows in FIG. 1 OB & D). However, similar to R A Ra-/- testes, TUNEL-positive spermatids were also found in the seminiferous tubules when they were embedded in the seminiferous tubules (arrows in FIG. 10C).

Tubules exhibiting TUNEL-positive elongated spermatids, as well as occasional TUNEL-positive spermatogonia and spermatocytes were consistently found in some of the B M S453 -treated testes (FIG. 1 OB & D). Most interestingly, TUNEL-positive round spermatids were only detected in the B M S453 -treated testes (bracket in FIG. 10D), but not in RARα /- testes. Though more severe sloughing of germ cells were observed in the BMS453-treated tubules at 1 month post-dose (FIG. 10E), the number of TUNEL- positive germ cells peaked at 5 mg/kg dose of BMS453-treated mouse testes at 1 month post-dose (FIG. 1 OE & F), indicating more cells undergo programmed cell death. Further, it also explains the drastic drop in the fertility observed in the mice treated with 5 mg/kg of BMS453 at 1 month post-dose. Example 6 - Lack of Inhibition of Spermatogenesis by Low Doses of RARa-specific Antagonists, BMS-189532 and BMS-195614

The following experiment was conducted to examine the in vivo effects of two RARα-selective antagonists, BMS-189532 and BMS-195614. No toxicology or other in vivo studies examining the effect of these compounds on male fertility or testicular histology have been published, but preliminary assessments suggested a profile similar to BMS- 189453 (C. Zusi, personnal communication).

CD-I mice (Charles River) were treated with BMS-195614 and BMS-189532 at doses of 2.0 and 10 mg/kg/day over a seven-day dosing period. These doses were chosen to account for differences in bioavailability due to possibly greater hydrophobicity compared to BMS-189453. A one-month post-dose time point was selected for assessing the effects of compounds on testicular morphology and sperm function. During the dosing period, the animals were observed at least once daily for changes in overall condition of health and behavior. Body weights were measured and physical examinations were conducted weekly. Morphological examination was conducted for all the testes and epididymides from mice treated with BMS614 and BMS532 at both 2 mg/kg (n =5) and 10 mg/kg (n =5), as well as those from untreated control (n =10), respectively.

No change in testicular morphology was observed in mice treated with 2 or 10 mg/kg of BMS614 (FIG. 1 I A-C) or BMS-532 (FIG. 1 I D-F) as assessed at one month post-dose when compared to control. Further, no other side effects were observed during the dosing period or during the one-month interval after the dosing period. In control testes, tubules were observed in which mature step 16 spermatids were aligned along the tubular lumen of the seminiferous tubule in preparation for spermiation (FIG. 1 I A). At one month after the dosing period, no failure of spermatid alignment or sperm release was found in the testes of mice treated with 2 mg/kg or 10 mg/kg of BMS614 (FIG. 1 I B & C, respectively) or mice treated with BMS-453 (FIG. 1 I E & F). After spermiation, three layers of spermatogenic cells were readily observed at stage IX tubule in mice treated with BMS-614 (FIG. 1 I B, C) and BMS-453 (FIG. 1 I E, F). No abnormal - looking nuclei of round spermatids were found in the testes of mice treated with either 2 mg/kg or 10 mg/kg of BMS-614 (FIG. 1 1 B & C, respectively) or BMS-453 (FIG. 1 1 E & F, respectively). Finally, no vacuolar-like spaces were found in the tubules of the testes of mice even when treated with 10 mg/kg of BMS-614 (FlG. 12C) or BMS-453 (FIG. 1 I F). Caudal epididymal sperm was also examined for morphological and motility abnormalities. No morphological changes were noted in epididymal sperm in all the mice treated with either 2 or 10 mg/kg of BMS614 or BMS-453. The motility of at least one hundred spermatozoa was determined for all mice. A spermatozoon is considered motile when forward sperm progression or regular movement of the flagellum is observed. No impairment of motility was noted.

The testicular weight of mice treated with BMS532 showed no significant difference (FIG. 12). Like mice treated with BMS-189453, testosterone levels varied tremendously for the BMS614-treated mice. Results in FIG. 13 show that there is no effect on testosterone levels of mice administrated with BMS614 and BMS532. In addition, the specimens examined did not show any sign of aberrant growths or tumor- like lesions.

In sum, results indicate no abnormalities in the mice administered either of the doses of these two antagonists after one month post-dose (FlG. 1 I A-F). Testicular histology indicated no effect on spermatogenesis even in the mice treated at 10 mg/kg/day of BMS-195614 or BMS- 189532. In addition, no morphological change or aberrant motility was noted in epididymal sperm. Although BMS-189532 and BMS- 195614 are potent antagonists in vitro, they are relatively poor testicular toxins in vivo, as doses in the range of 75 mg/kg/day are required affect spermatogenesis in Wistar rats (C. Zusi, personnal communication).

Example 7- Evaluation of BMS213309

The following study was conducted evaluate the RAR antagonist BMS213309 to determine a minimum dose sufficient to induce testicular change and inhibit spermatogenesis. The structure of BMS213309 is shown below:

Experimental groups of mice were as described in studies involving BMS- 189453. CD-I mice (Charles River) were treated by gavage with 1 mg/kg or 5 mg/kg of BMS213309. A five-day dosing period and a one month post-dose time point were selected for assessing the effects of the compounds on testicular morphology and sperm function. During the dosing period, the animals were observed at least once daily for changes in overall condition of health and behavior. Body weights were measured and physical examinations were conducted weekly. The effect on spermatogenesis was evaluated as described above using specimens for the one month time point.

The morphology of all the testes and epididymides from mice treated with BMS213309 at both 1 mg/kg (n =10) plus those from untreated control (n =5), respectively, were examined.

At the one month post-dose time point, 9 of 10 mice administered 1 mg/kg of BMS213309 exhibited no morphological abnormalities in the testes and epididymides (FIGs. 14A & B, respectively). Further, no other side effects were observed during the dosing period or during the one-month interval after the dosing period. Tubules were observed in which mature step 16 spermatids were aligned along the tubular lumen of the seminiferous tubule in preparation for spermiation (right upper corner tubule in FlG. 14A). Unlike the striking phenotype observed following treatment with BMS 189453, no failure of spermatid alignment or sperm release was found in the testes of mice treated with 1 mg/kg of BMS213309 one month after the dosing period (right upper corner tubule in FIG. 14A). After spermiation, three layers of spermatogenic cells were readily observed at stage X tubule (left bottom corner tubule in FIG. 14A). No abnormal-looking nuclei of round spermatids were found in the testes with treatment at 1 mg/kg (FIGs. 14A & C). Finally, no vacuolar-like spaces were found in the tubules of testes of the mice treated with 1 mg/kg of BMS213309 (n=9, FIG. 14A). Cauda epididymal sperm for morphological and motility abnormalities were also examined. No morphological changes were noted in epididymal sperm in all the mice treated with 1 mg/kg of BMS213309 at 1 month post-dose. The motility of at least one hundred spermatozoa was determined for all mice. A spermatozoon is considered motile when forward sperm progression or regular movement of the flagellum is observed. No impairment of motility was noted. Only one of ten BMS213309-treated males had tubules with germ cell sloughing (-20%) (FIG. 14C). Normal spermatozoa, however, were found in the epididymis (FIG. 14D). In sum, results indicate that in BMS213309- treated mice, there was: (1) no failure of alignment of spermatids; (2) no distorted orientation; (3) no asynchronization of cellular association; (4) no vacuoles found; and (5) no significant variation in testicular weight or sperm morphology and number. These effects were not a result of compound degradation metabolism as [¹H]-NMR analysis of BMS213309 indicated that the compound was not degraded or metabolized.

At the one month post-dose time point, mice administered 5 mg/kg of BMS2 I 3309 exhibited variation in the extent of morphological abnormalities. There was no change in testicular morphology in some males (n=4) given 5 mg/kg for consecutive 5 days compared to control (FIG. 15A). However, vacuolar-like spaces in the seminiferous tubules of testes became very prominent in one of the mice resulting in very few spermatogenic cells in one-third of the tubules (FIG. 15B). Interestingly, the remaining tubules of the testes looked completely normal, with full development of spermatogenesis, a finding consistent with non-uniform bioavailability of the compound. For example, the tubules on the left corner showed the normal stage XI spermatogenic cycle (FIG. 15B). Three layers of spermatogenic cells were found in this tubule after spermiation. In some severely defective tubules, -61 % of the tubules were missing the entire layers of given cell types (n=l , FIG. 15C). Consequently, there were no spermatozoa found in the caput (head) of the epididymis (FIG. 15D) and fewer numbers of spermatozoa in the corpus (middle) of the epididymis (FIG. 15E). Counting the spermatozoa in the two caudae epididymides of this mouse revealed a low total sperm count (5.5 x 10⁶) compared to ~2x 10⁷ sperm total from the two caudae of normal mice (it varied from mouse to mouse in the range of 8 x 10⁶ to 5 x 10⁷). Finally, severe sloughing of round spermatids was also found in some of the mice (n=3) and round spermatids were detected in the caput of the epididymis, while abnormally shaped round spermatids were found in the corpus of the epididymis (FIGs. 15G & H, respectively). In addition, there was a considerable amount of cellular debris, along with giant multi-nucleated germ cells (FIG. 15H). The testicular weight of mice treated with 5mg/kg at 1 month post-dose was significantly reduced in some of the mice (data not shown).

The cauda epididymal sperm were also examined in these drug-treated animals in order to check for any morphological abnormalities, and cauda epididymal counts were obtained as well. No morphological changes in epididymal sperm were noted in all the mice treated with 5 mg/kg at the 1 month post-dose. Few mice (n = 3) showed highly reduced numbers of sperm in their epididymis (data not shown); instead, considerable amount of cellular debris, along with giant multi-nucleated germ cells were observed. As anticipated, no aberrant growth or tumor-like lesions were observed in treated animals.

Therefore, although 1 mg/kg of the RARγ antagonist BMS213309 failed to exhibit testicular toxicity, higher doses of BMS213309 did result in germ cell sloughing in some mice (n=4).

Example 8 - Kinetics of Inhibition of Spermatogenesis and its Reversibility

As determined above, administration of 5.0 mg/kg/day of BMS- 189453 for 7 days results in transient disruption of spermatogenesis in mice. Dose optimization, however, involves weighing the benefits of low dosages with efficacy of the compound, e.g. duration of inhibition of spermatogenesis, efficacy of the treatment (100 % sterility during the treatment period, i.e. the dosing period and the duration of infertility), and reversibility. Thus, to understand the kinetics of the inhibition of spermatogenesis and its reversibility, as well as to extend the period of infertility, while still permitting reversibility, the following experiments were conducted.

The first set of experiments involves examining the effects of the 5.0 mg/kg/day for 7-days protocol but for 3 sequential monthly doses (with 21-day no-dose rest periods) on the length of inhibition of spermatogenesis and the reversibility of this effect. Thus, Group I animals were given a seven-day course of BMS- 189453 (5.0 mg/kg/day) followed by a three week rest period for three consecutive months. This protocol assesses the effect of intermittent dosing such as is typical for female birth control pills. The second set of experiments is a comparative study at two doses of BMS- 189453 (5.0 mg/kg/day and 2.5 mg/kg/day) on adult mice but for a longer period of treatment. Group II animals were given 5.0 mg/kg/day for 2 weeks or 2.5 mg/kg/day for four weeks. This protocol assesses the ability of the testis to recover fertility after a longer, chronic, treatment regime. This protocol also assesses whether a lower dose (2.5 mg/kg/day) of compound given for a longer period of time (4 weeks) could bring about reversible infertility, while maintaining the same compound burden on the animal.

Group I: Intermittent Schedule of 5.0 mg/kg/day of Compound for a Seven-day

Dosing Followed by a Three Week No-dose for Three Months

All experimental groups received a treatment of 5.0 mg/kg/day of BMS- 189453 by oral gavage for 7 days. The animals were then placed into three sub-groups that received different treatment regimens, Group IA, Group IB, and Group 1C. All animals were assessed for fertility at 2-week intervals as long as they were part of the experimental protocol (i.e. have not been sacrificed). Mice were then examined at various timepoints as follows.

Group IA: At 4 weeks after the 7-day treatment, one-half (n= 10) of Group IA were sacrificed and the remaining animals (n=10) are assessed for recovery by mating. Group IB received two 7-day doses at 4-week intervals. Then half (n=10) of the starting group were sacrificed at the end of the second 4-week period post-initial dose, and the remaining animals (n=10) are assessed for fertility for a period up to 9 months. Group IC received the full three 7-day treatment at monthly intervals. Then half (n=10) of the animals were sacrificed at the end of the third 4-week period, while the remaining animals (n=10) were assessed for fertility for a period up to 12 months, at which time they are sacrificed if fertility has not been restored by that time. For Groups IA and IB, the animals are sacrificed for histological evaluation when they have successfully sired two consecutive, normal-sized litters. The experimental endpoints evaluated at the time of sacrifice for both Group I and Group II animals are outlined below.

Group II: Extended Dosing Period of 2 or 4 Weeks for 5.0 mg/kg/day or 2.5 mg/kg/day

In order to examine the effect of chronic administration of BMS- 189453, as well as the effect of a lower dose administered for a longer period, mice were administered 5.0 mg/kg/day or 2.5 mg/kg/day for the extended dosing period of 2 and 4 weeks as follows. Group HA (n=30) received 5.0 mg/kg/day for 2 weeks. One-third of the animals (n=10) were sacrificed at the end of four weeks post-initial dose, the second one-third of the animals (n=10) are sacrificed during the infertile/recovering period, and the remaining third of the animals (n=10) are assessed for fertility as described for Group I. Group HB animals Cn=OO) received the same absolute amount of BMS-189453 in a different dosing regimen. This group was treated with a dose of 2.5 mg/kg/day for 4 weeks and then are sacrificed and evaluated for fertility as for group HA. Note that the same absolute amount of drug were administered to Group IB, but under a distinct regimen, i.e. two 7- day regimens of 5.0 mg/kg/day separated by an intervening three-week interval of no treatment.

Effects on Fertility and Morphology

After the mice were treated with the different regimens as described above, fertility assessment are performed as described above. In brief, the males were placed in an individual cage with two virgin females of the same strain. The animals were caged together for 14 days during which approximately three estrus cycles elapsed. At the end of the 14-day period, the mated females were sacrificed and number of conceptuses recorded. Another two females were replaced continuously until the end of protocol. When these drug-treated males were sacrificed after the assessment, sperm counts were also made from the caudal epididymides of these drug-treated mice and the caput epididymides were used for morphological assessment. One testis was fixed in Bouin's fixative for better morphological examination; while the second testis was fixed in 4% paraformaldehyde for in situ hybridization or immunohistochemical analyses.

In Group IA, at 4 weeks after the 7-day treatment of 5.0 mg/kg/day of BMS- 189453, almost all mice (n=9/I 0) became infertile. This is consistent with previous finding in which eight out of nine mice became infertile at 4 weeks after treatment. These mice (n=10) were sacrificed immediately, i.e. at 4 weeks after the 7-day treatment. Very low sperm counts, ranging from 0.34 x 10⁶ to 1.41 x 10⁶ were observed in 8 of the 9 infertile males, and zero sperm were observed in the remaining infertile one. This is far below the sperm count of normal males which is around 2 x 10⁷ sperm in total from the two caudal epididymides (sperm count normally varies from mouse to mouse in the range of 8 x 10⁶ to 5 x 10⁷).

In group 1IA, one-third of the animals (n=10) were sacrificed at 2 weeks after the 14-day treatment, the time immediately before the second round of two-week fertility test. Nine of these ten mice showed zero sperm count in the two caudal epididymides, and empty caput epididymides were found as assessed by morphological evaluation. Accordingly, at 4 weeks after the 14-day treatment of 5.0 mg/kg/day of BMS453 (group HA), all the remaining mice (n=20) in the second round of mating failed to yield pregnancies. This indicated the absolute efficacy of the treatment.

To determine the origin of loss of fertility, the morphology of the testes and epididymides from the first cohort of mice that were sacrificed before the second mating (FIGs. 16A-G) were examined. Testicular integrity was rated on a scale of 1-5 in which 5 indicates normal testes characterized by three to four layers of spermatogenic cells in the seminiferous tubules. In group HA (i.e. 2 weeks after treatment), one mouse, given a testicular integrity rating of 1 , exhibited severe sloughing of germ cells (n=l/10; FlG. 16A, noted as scale 1). The majority of the testes in this mouse consisted of near empty tubules with very few Sertoli cells or spermatogenic cells (FIG. 16A). Six mice, given a testicular integrity rating of 2, exhibited various abnormalities. Examples of these abnormalities included tubules with less severe sloughing, resulting in spermatogonia and/or Sertoli cell-only tubules (n=l/10; FIG. 16B, scale 2), and shrunken tubules due to the slough of germ cells (n=l/10, FIG. 16C, scale 2). One male, given a rating of 3, exhibited moderate sloughing of germ cells (n=l/10, scale 3). Interestingly, severe germ cell sloughing was not observed in some of the mice (n=2/10), although caput epididymides were devoid of sperm (FIG. 16F). These mice were given a rating of 4. Detailed examination of the seminiferous tubules revealed that various steps of spermatids were found in the same plane of the section of the seminiferous tubule (FIG. 16E, scale 4) and some tubules were missing whole layers of pachytene spermatocytes (FIG. 16G, scale 4).

In the second one-third cohort of animals (n=10), five mice were sacrificed during the infertile/recovering period, i.e. at 4-weeks after 14-day treatment. Empty caput epididymides were consistently found as shown by morphological assessment, and very low sperm counts ranging from 0.28 x 10⁶ to 0.74 x 10⁶ were observed in 4 males. One male lacked sperm completely. Sperm count in normal males is about 2 x 10⁷ sperm in the two caudal epididymides (normal sperm count typically ranges from about 8 x 10⁶ to 5 x 10⁷). Such a low sperm count explained the infertility of these mice. Since sperm were present, this implies a possible recovery of this group of mice (Group HA) starting at 4 weeks after treatment. Accordingly, at 6-weeks after treatment, 4 of these infertile males (n=15) became fertile and could generate pups, with normal litter size (n=l 1.71 + 2.6). The eleven remaining infertile males in group IIA continuously failed to yield pregnancies. These results indicate that the extended treatment regimen (5.0 mg/kg/day, 14-day dosing, group HA) may be a more powerful dosing regimen for male contraceptive purposes.

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All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.

The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an antibody" includes a plurality (for example, a solution of antibodies or a series of antibody preparations) of such antibodies, and so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting spermatogenesis in a male mammal, comprising: administering to the mammal an effective amount of a compound having either of the following formulae for at least about five days:

wherein R| is selected from the group consisting of hydrogen, an alkyl, a branched lower alkyl, a cyclic alkyl, a heterocylic ring or an aryl ring.

2. The method of claim 1 , wherein the compound is selected from the group consisting of:

(VIII) or any combination thereof.

3. The method of claim 1 , wherein the compound is:

4. The method of claim 1 , wherein the compound is a retinoic acid receptor antagonist/1 igand.

5. The method of claim 4, wherein the antagonist/ligand is specific for the retinoic acid receptor subtype α, β, or γ.

6. The method of claim 5, wherein the antagonist/ligand is specific for the retinoic acid receptor subtype α.

7. The method of claim 1 , wherein the effective amount is an amount effective for inhibiting at least about 80% of pregnancies in a female with whom the male mammal has mated.

8. The method of claim 1 , wherein the effective amount is an amount effective for inhibiting at least about 90% of pregnancies in a female with whom the male mammal has mated.

9. The method of claim 1 , wherein the effective amount is an amount effective for inhibiting at least about 95% of pregnancies in a female with whom the male mammal has mated.

10. The method of claim 1 , wherein the effective amount is about 0.01 mg/kg to about 100 mg/kg.

1 1. The method of claim 1 , wherein the effective amount is about 0.5 mg/kg to about 10 mg/kg.

12. The method of claim 1 , wherein the effective amount is about 1 mg/kg to about 7 mg/kg.

13. The method of claim 1 , wherein the effective amount is about 2.5 mg/kg.

14. The method of claim 1 , wherein the compound is administered daily.

15. The method of claim 1 , wherein the compound is administered daily for about five to about fourteen days.

16. The method of claim 1 , wherein the compound is administered daily for at least about fourteen days.

17. The method of claim 16, wherein the compound is administered daily for about thirty days.

18. The method of claim 16, wherein the compound is administered daily for about 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 1 months or more than 1 1 months.

19. The method of claim 16, wherein the compound is administered daily for about 1 year, 2 years, 3 years or more than 3 years.

20. The method of claim 1, wherein the effective amount is about 5 mg/kg and the compound is administered for at least 7 days.

21. The method of claim 1 , wherein the effective amount is about 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2mg/kg or 2.5 mg/kg and the compound is administered for at least 14 days.

22. The method of claim 21 , wherein the compound is administered daily for about 30 days.

23. The method of claim 21 , wherein the compound is administered daily for about 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 1 months, or more than 1 1 months.

24. The method of claim 21 , wherein the compound is administered daily for about 1 year, 2 years, 3 years or more than 3 years.

25. The method of claim 1 , wherein the compound is administered as a sustained release dosage form.

26. The method of claim 1 , wherein the compound is administered orally or mucosally.

27. The method of claim 1 , wherein the compound is administered transdermally.

28. The method of claim 1 , wherein spermatogenesis is inhibited after about 3 weeks and continues to be inhibited for about 5 weeks after administration of the compound.

29. The method of claim 1 , wherein the method further comprises administering the compound to the male mammal for about seven days after a period of about 5 weeks.

30. The method of claim 1, wherein the mammal is a mouse, rat, bat, dog, cat, rabbit, whale, horse, deer, sheep, goat, buffalo, elk, bear, coyote, mountain lion, raccoon, fox, monkey or human.

31. An article comprising a compound of any one of formulae 1 to VIlI or any combination thereof and packaging material, wherein the packaging material comprises a label indicating that the compound or any combination thereof can be used to inhibit spermatogenesis in a mammal.

32. The article of claim 31 , wherein the compound is in a sustained release dosage form.

33. The article of claim 31 , wherein the mammal is a mouse, rat, bat, dog, cat, rabbit, whale, horse, deer, sheep, goat, buffalo, elk, bear, coyote, mountain lion, raccoon, fox, monkey or human.

34. Use of an effective amount of the compound of any one of formulae 1 to VIII or any combination thereof in the preparation of a medicament for inhibiting spermatogenesis in a mammal.