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WO2008137081A1 - Composés de (5,6)-dihydronaphtalényle substitué en tant que contraceptifs masculins réversibles - Google Patents

Composés de (5,6)-dihydronaphtalényle substitué en tant que contraceptifs masculins réversibles Download PDF

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Publication number
WO2008137081A1
WO2008137081A1 PCT/US2008/005695 US2008005695W WO2008137081A1 WO 2008137081 A1 WO2008137081 A1 WO 2008137081A1 US 2008005695 W US2008005695 W US 2008005695W WO 2008137081 A1 WO2008137081 A1 WO 2008137081A1
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WIPO (PCT)
Prior art keywords
compound
months
bms
mice
administered
Prior art date
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PCT/US2008/005695
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English (en)
Inventor
Debra J. Wolgemuth
Peter R. Reczek
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The Trustees Of Columbia University In The City Of New York
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Publication of WO2008137081A1 publication Critical patent/WO2008137081A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/16—Masculine contraceptives

Definitions

  • 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 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.
  • domesticated animals as well as free-ranging wildlife.
  • dogs and cats are euthanized each year at animal shelters.
  • feral or wild animal overpopulation such as the overpopulation of feral hogs, deer, and bears, for example, could be managed using a male contraceptive.
  • a male contraceptive would be useful for reproductive management of animals in captivity so as to maintain genetic diversity and prevent inbreeding.
  • 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.
  • the compound can be an antagonist/ligand that is selective for a particular retinoic acid receptor subtype.
  • 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.
  • 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.
  • the compound is a retinoic acid antagonist/ligand, and in some embodiments, the antagonist/ligand is specific for the retinoic acid receptor subtype ⁇ , ⁇ , or ⁇ . For example, in some embodiments, the antagonist/ligand is specific for the retinoic acid receptor subtype ⁇ .
  • the retinoic acid antagonist/ligand has the structure:
  • the retinoic acid antagonist/ligand is specific for the retinoic acid receptor subtype, ⁇ , ⁇ , or ⁇ . In some embodiments, the antagonist and/or Iigand is specific for the retinoic acid receptor subtype ⁇ .
  • 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.
  • 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.
  • the antagonist/ligand is administered daily.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • the compound is formulated as a sustained release dosage form.
  • 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.
  • 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.
  • 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.
  • BMS-189453 competes the activity of ATRA for RAR ⁇ (open circle), RAR ⁇ (closed circle), RAR ⁇ (open square) and is thus considered a pan-antagonist
  • B BMS-195614 selectively competes with ATRA for RAR ⁇ (open circle) activity with less significant competition seen for RAR ⁇ (closed circle) and RAR ⁇ (open square) and is thus an RAR ⁇ selective antagonist
  • BMS-189532 specifically competes ATRA CAT expression for RAR ⁇ (open circles) with minimal activity observed for RAR ⁇ and RAR ⁇ (closed circles and open squares, respectively) and is thus considered an RAR ⁇ 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.
  • 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 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.
  • FIG. 1 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 RAR ⁇ -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.
  • 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.
  • the compound can be an antagonist/1 igand that is selective for a particular retinoic acid receptor subtype.
  • 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.
  • 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.
  • mammalia 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.
  • 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.
  • the present invention provides a method for achieving reversible sterility.
  • 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.
  • the compounds of the following formulae are useful for inhibiting spermatogenesis and can be used as reversible male contraceptives:
  • 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.
  • the present compounds are retinoic acid receptor antagonists/1 igands.
  • 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-C 4 )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.
  • 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-C 4 )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.
  • (Ci-C 6 )alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso- butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;
  • (C 3 -C 6 )cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;
  • (C 3 -C 6 )CyClOaIlCyI(C i-C 6 )alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2- cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl;
  • - C 6 )alkoxy can be methoxy, ethoxy, propoxy,
  • the present compounds are administered to inhibit spermatogenesis and/or act as reversible contraceptives.
  • the present compounds, and combinations with other agents may be administered as single or divided dosages.
  • 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.
  • 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.
  • 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.
  • 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.
  • systemic administration is contemplated, for example, through oral administration.
  • 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.
  • 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.
  • the present compounds are administered orally or transdermally.
  • 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.
  • a pharmaceutically acceptable carrier diluent or excipient
  • 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.
  • 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.
  • compositions containing the compounds of the invention can be prepared by procedures known in the art using well-known and readily available ingredients.
  • the compounds can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like.
  • 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.
  • 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.
  • inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like
  • liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,” polyg
  • antioxidants 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 ⁇ -tocopherol and its derivatives can be added.
  • 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.
  • 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.
  • aerosol formulations e.g., sprays or foams
  • soaps e.g., sprays or foams
  • soaps e.g., detergents, lotions or cakes of soap.
  • Other conventional forms for this purpose include ointments, creams, lotions, pastes, jellies, sprays, and aerosols.
  • the compounds of the invention can be delivered via patches (e.g., transdermal patches) or bandages for dermal administration.
  • the compounds can be formulated to be part of an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer.
  • an adhesive polymer such as polyacrylate or acrylate/vinyl acetate copolymer.
  • 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.
  • 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.
  • pharmaceutically acceptable carriers 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.
  • ATRA All-trans-retinoic acid
  • RARs nuclear retinoid receptors
  • 9cRA 9-cis- retinoic acid
  • the RARs and RXRs each have three subtypes, RAR ⁇ , ⁇ , and ⁇ as well as RXR ⁇ , ⁇ , and ⁇ , 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).
  • RAREs retinoic acid response elements
  • RAR retinoic acid response elements
  • spermatogenesis Specific defects in spermatogenesis are observed in RAR ⁇ -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.
  • the inventors noted abnormalities in the progression of meiotic prophase and a temporary arrest at step 8 spermatids as well.
  • the present compounds may target a variety of RARs (not just RAR ⁇ ) 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.
  • 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.
  • mice 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 4 0 C before being processed.
  • the RAR pan-antagonist, BMS- 189453, and RAR ⁇ -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 .
  • HeLa cells were transfected using the calcium phosphate co-precipitation method with cDNA constructs encoding each of the three receptors, RAR ⁇ , ⁇ , and ⁇ along with an RARE-CAT (chloramphenicol acetyl transferase) reporter construct and ⁇ -galactosidase for normalization of transfection efficiency under tk promoter (Vasios et al., 1989). All-/r ⁇ «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.
  • the RAR pan-antagonist, BMS-189453, and RAR ⁇ -selective antagonists, BMS- 189532 and BMS- 195614 giftss from Bristol-Myers Squibb Co., Princeton, NJ
  • BMS- 189532 and BMS- 195614 giftss from Bristol-Myers Squibb Co., Princeton, NJ
  • 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.
  • 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
  • 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.
  • 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 ⁇ l 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 ⁇ M filter to remove any remaining debris.
  • a 1 : 10 dilution was made and the sperm were counted by a hematocytometer.
  • 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 H 2 O.
  • 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.
  • 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% H 2 Ch 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.
  • DAB 3,3' diaminobenzidine tetrahydrochloride
  • the fertility assessment was based on a well established protocol (Elbetieha & Da'as, 2003; Al-Thani et al., 2003).
  • 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.
  • the animals were caged together for 14 days during which approximately three estrus cycles should have elapsed.
  • 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.
  • This Example describes the initial characterization of retinoic acid receptor antagonists that may be useful as reversible male contraceptives.
  • RAREs retinoic acid response elements
  • 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-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).
  • BMS-189532 and 195614 were evaluated as described above for BMS-189453.
  • 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).
  • DBA assay direct binding to recombinant receptor
  • CAT chloramphenicol acetyl transferase reporter gene linked to the RARE from the laminin B gene
  • RTCA Retinoid transactivation competition assay (antagonism)
  • Toxicity of BMS-189453 has been evaluated by Schulze et al. (2001).
  • Schulze et al. investigated the potential target-organ toxicity of BMS453 in rats following a month of oral administration (Schulze et al., 2001).
  • 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.
  • Sprague Dawley rats were given daily oral doses of 15, 60, or 240 mg/kg body weight BMS-189453 for 1 month.
  • mice can still produce progeny even with substantially lowered sperm counts, histological evaluation of the testes at sequential time points post-dosing was used.
  • 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.
  • mice did not show any sign of aberrant growths or tumor-like lesions.
  • 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 %).
  • 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).
  • TUNEL-positive elongated spermatids 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 RAR ⁇ /- testes. Though more severe sloughing of germ cells were observed in the BMS453-treated tubules at 1 month post-dose (FIG.
  • 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.
  • 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.
  • 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.
  • no morphological change or aberrant motility was noted in epididymal sperm.
  • 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).
  • 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:
  • 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.
  • 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.
  • 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).
  • 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).
  • the remaining tubules of the testes looked completely normal, with full development of spermatogenesis, a finding consistent with non-uniform bioavailability of the compound.
  • 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.
  • Counting the spermatozoa in the two caudae epididymides of this mouse revealed a low total sperm count (5.5 x 10 6 ) compared to ⁇ 2x 10 7 sperm total from the two caudae of normal mice (it varied from mouse to mouse in the range of 8 x 10 6 to 5 x 10 7 ). 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).
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • group HA i.e. 2 weeks after treatment
  • the majority of the testes in this mouse consisted of near empty tubules with very few Sertoli cells or spermatogenic cells (FIG.
  • 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).
  • 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 10 6 to 0.74 x 10 6 were observed in 4 males.
  • Sperm count in normal males is about 2 x 10 7 sperm in the two caudal epididymides (normal sperm count typically ranges from about 8 x 10 6 to 5 x 10 7 ).
  • Such a low sperm count explained the infertility of these mice.
  • a retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects. Proc Natl Acad Sci USA, 89, 7129- 33.
  • Cyclin-binding motifs are essential for the function of p21 CIP 1. Molecular and Cellular Biology, 16, 4673-82.
  • RAR-specific agonist/antagonists which dissociate transactivation and API transrepression inhibit anchorage- independent cell proliferation. Embo J, 14, 1 187-97.
  • Retinoic acid receptor gamma mediates topical retinoid efficacy and irritation in animal models. J Invest Dermatol, 104, 779-83.
  • Vitamin A functions in the reproductive organs.
  • N-terminal portion of domain E of retinoic acid receptors alpha and beta is essential for the recognition of retinoic acid and various analogs.
  • BMS- 189453 a novel retinoid receptor antagonist, is a potent testicular toxin. Toxicol Sci, 59, 297-308. Starrett, J. E., Yu, K. L., Mansuri, M.M., et al. (1997). Retinoid Like Compounds. US patent 5,618,839. Travis, A.J. et al. Functional relationships between capacitation-dependent cell signaling and compartmentalized metabolic pathways in murine spermatozoa. J Biol Chem 276, 7630-6 (2001).
  • retinoic acid- responsive element is present in the 5' flanking region of the laminin Bl gene. Proc Natl Acad Sci USA 86, 9099-103. (1989).
  • Protein synthesis inhibitors prevent the induction of laminin Bl , collagen IV (alpha 1), and other differentiation-specific mRNAs by retinoic acid in F9 teratocarcinoma cells. J Cell Physiol, 136, 305-1 1.
  • Retinoic acid receptor antagonist BMS453 inhibits the growth of normal and malignant breast cells without activating RAR-dependent gene expression.
  • Retinoic acid receptor beta,gamma-selective ligands synthesis and biological activity of 6-substituted 2-naphthoic acid retinoids. J Med Chem, 39, 241 1-21.

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Abstract

La présente invention concerne un procédé d'inhibition de la spermatogenèse chez un mammifère, ainsi qu'un contraceptif réversible en vue d'une utilisation chez un mammifère.
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