WO2010051498A1

WO2010051498A1 - Biotechnology relating to mammalian reproduction

Info

Publication number: WO2010051498A1
Application number: PCT/US2009/062862
Authority: WO
Inventors: Timothy Laurance Vail; Cheryl Ann Dyer; Loretta Powers Mayer
Original assignee: Arizona Board Of Regents On Behalf Of Northern Arizona University
Priority date: 2008-11-03
Filing date: 2009-10-30
Publication date: 2010-05-06

Abstract

Methods and compositions for chemical sterilization of mammals. Active isomers of 4- vinylcyclohexene diepoxide (VCD) are used for application to a non-human mammal. In addition, compositions and methods of use of an epoxide hydrolase inhibitor to potentiate the follicular depletion effect of the VCD isomer(s) are disclosed.

Description

BIOTECHNOLOGY RELATING TO MAMMALIAN REPRODUCTION

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The invention relates generally to improved compositions and methods for limiting mammal reproduction, and, more particularly, to compositions and methods involving 4-vinylcyclohexene diepoxide and an epoxide hydrolase inhibitor in combination with 4-vinylcyclohexene diepoxide.

Description of the Related Art

[0002] As detailed in previous work (see, for example, US Patent Application

Publication 2004/0073962), the occupational chemical VCD, the diepoxide metabolite of 4-vinylcyclohexene (VCH), causes selective loss of small preantral follicles in the ovaries of mice and rats. Compared to vehicle controls, VCD dosing of rats and mice for 12 days is known to cause a significant reduction in the number of primordial and primary follicles.

[0003] However, following 30 days of dosing there was also a significant reduction in the numbers of secondary follicles which was explained as a reduction in the pool of primordial follicles from which secondary follicles could be recruited at that time to develop into large antral follicles that produce 17 β-estradiol. The targeting of primordial and primary follicles by VCD appears to be follicle stage-specific because no direct effects have been observed or measured in larger (secondary to antral) follicles, or other non-ovarian tissues as determined by necropsy, histopathology, plasma lipid profiles, and liver enzyme activity.

[0004] Using a combination of molecular and cellular approaches in our studies in rats, evidence has been collected that VCD-induced follicle loss is by acceleration of the normal rate of atresia. These studies have also demonstrated that alterations in apoptosis-associated intracellular pathways activated by VCD dosing are specific for small preantral follicles, as compared with large preantral follicles or liver. Atresia in the rodent ovary occurs via apoptosis, or programmed cell death, and is a normal process without necrosis-induced responses such as inflammation.

[0005] Evidence of VCD-induced impending follicle loss has been observed as an increase in numbers of unhealthy follicles in the treated group. The unhealthy appearance in VCD-treated ovaries is morphologically and ultrastructurally similar to unhealthy follicles undergoing natural atresia in controls. At the molecular level, VCD- accelerated atresia in small preantral follicles was identified because intracellular events associated with apoptosis were measured selectively in the targeted follicular population. These events include: A) increased Bax/BclxL ratio, B) increased expression of Bad, C) leakage of cytochrome c from mitochondria into the cytosol, D) increased caspase-3 activity, E) activation of the JNK and p38 branch of the MAPK signaling pathway , F) retardation of natural atresia and bax expression (relative to controls) in small follicles following a single dose of VCD, and G) estrogen receptor- mediated protection from VCD-induced follicle loss.

[0006] Collectively, these data provide support at a molecular level that VCD causes follicle loss by enhancing events associated with the normal process of atresia. These molecular events are specific for the small preantral follicles known to be physiologically targeted by VCD.

[0007] Follicle loss resulting from repeated dosing of F344 rats with VCD has been well characterized. The conclusion from characterization performed in preliminary studies is that VCD appears to cause the selective loss of primordial and primary follicles by accelerating the natural process of atresia via apoptosis. In long term studies in B6C3F1 mice and F344 rats that were dosed with the parent compound, 4- vinylcyclohexene (VCH, mice) or the ovotoxic form (VCD, rats), premature ovarian failure occurred within a year. [0008] Previous studies in rodents, dogs and monkeys have indicated that 4- vinylcyclohexene diepoxide (VCD; 160 mg/kg/d delivered for 15 days) is efficacious in eliminating non-regenerating primordial follicles of the ovary resulting in premature ovarian failure or sterilization. Ongoing studies in the laboratory of one of the inventors have indicated that safe dose of VCD with no observable adverse effects can be as high as 750 mg/kg/d delivered orally. However, the volume of VCD required is impractical in many settings.

[0009] The industrial VCD used in previous studies consists of up to 8 stereoisomers in the commercially available mixture. Studies indicate that a single isomer of VCD (lR,2S,4R,7R)-trans-VCD) may be the active pharmaceutical ingredient (API) involved in ovarian atrophy and potentially follicle depletion.

[0010] As mentioned previously, observations of inducing ovarian failure in rats, mice, dogs, and monkeys indicate that 4-vinylcyclohexene diepoxide accelerated the normal process of atresia that eliminates ovarian follicles. Without these follicle populations, which cannot be functionally regenerated to recruit growing follicles, the ovary ultimately runs out of follicles and the individual is sterile. Thus, it has been shown that VCD treatment accelerated follicle depletion leading to chemical sterilization of rodents and canines (preliminary studies).

[0011] The use of 4-vinylcyclohexene diepoxide is described in US Patent Application Publication 2004/0073962. This publication describes the use of a commercial mixture of isomers, 4-vinylcyclohexene diepoxide, which may be used to induce ovarian failure in non-human mammals. Administration of this mixture is not practical due to the dosing protocol described in the application.

[0012] In summary, the present compositions and methods involving VCD are stressful for research animals. Undue stress introduced in research models can compromise data collection. Further, performance of the VCD technology with multiple injections is costly thereby increasing the cost of research. The chemical nature of VCD is not conducive to current "off-the-shelf products designed for routine drug delivery.

[0013] Accordingly, it would be desirable to provide improved compositions and methods for effecting chemical sterilization of mammals involving VCD.

SUMMARY OF THE INVENTION

[0014] The invention generally involves methods and compositions for chemical sterilization of mammals. Active isomers of 4-vinylcyclohexene diepoxide (VCD) are isolated and prepared for application to a non-human mammal. In addition, compositions and methods including an epoxide hydrolase inhibitor hi combination with VCD to potentiate the follicular depletion effect of the isomer(s) are disclosed.

[0015] Current VCD protocols used for mammals require repeated exposure via daily injections of a commercially available mixture of VCD isomers. This disclosure involves the development and use of a purified isomer preparation with an epoxide hydrolase inhibition preconditioning drug delivery protocol that will result in a commercializable chemical sterilant that is practical.

[0016] The novelty of a purified single or multi-isomer VCD delivery system is that it utilizes the chemical properties of VCD in an advanced formulation to deliver an efficacious and commercializable dose of VCD for chemical sterilization.

[0017] Various other purposes and advantages of the invention will become clear from its description in the specification that follows. Therefore, to the accomplishment of the objectives described above, this invention includes the features hereinafter fully described in the detailed description of the preferred embodiments, and particularly pointed out in the claims. However, such description discloses only some of the various ways in which the invention may be practiced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] A particular isomer of VCD may be used or a mixture of isomers. For example, the 4-vinylcyclohexene diepoxide in compositions of the invention may comprise (lR,2S,4R,7R)-trøn.s-4-vinylcyclohexene diepoxide. Furthermore, the 4- vinylcyclohexene diepoxide compositions of the invention may consist of (lR,2S,4R,7R)-trøfts-4-vinylcyclohexene diepoxide.

[0019] In terms of the epoxide hydrolase inhibitors used in combination with VCD, one or more may be used to stabilize and/or be administered with VCD to potentiate greater activity and reduce the dosage and/or frequency of administration. For example, known inhibitors of microsomal expoxide hydrolase include trichloropropene oxide, Zn²⁺, and elaidamide (Morisseau and Hammock, Annu. Rev. Pharmacol. Toxicol. 2005. 45:311-33).

[0020] Thus, the composition combining VCD of one or more isomers with an epoxide hydrolase inhibitor may be used for a method for chemical sterilization of a non-human mammal, with the method including the step of administering a composition comprising an epoxide hydrolase inhibitor in combination with an amount of 4-vinylcyclohexene diepoxide effective to cause follicular depletion in the non- human mammal. Dosing amounts and protocols such as those described in US Patent Application Publication 2004/0073962 may be practiced.

[0021] While VCD can be given orally and through injections, the use of a chemical spray product (against, for example, feral and stray dogs in rural and international developing country settings where canine overpopulations compete for resources and are vectors for disease) is contemplated.

[0022] In one embodiment, the method may include spraying the combination composition on the non-human mammal. [0023] Various changes in the details and components that have been described may be made by those skilled in the art within the principles and scope of the invention herein described in the specification and defined in the appended claims. Therefore, while the present invention has been shown and described herein in what is believed to be the most practical and preferred embodiments, it is recognized that departures can be made therefrom within the scope of the invention, which is not to be limited to the details disclosed herein but is to be accorded the full scope of the claims so as to embrace any and all equivalent processes and products. AU references cited in this application are hereby incorporated by reference herein.

Claims

What is claimed is:

1. A method for chemical sterilization of a non-human mammal, comprising the step of administering a composition comprising an epoxide hydrolase inhibitor in combination with an amount of 4-vinylcyclohexene diepoxide effective to cause follicular depletion in said non-human mammal.

2. The method of claim 1, wherein said 4-vinylcyclohexene diepoxide comprises (lR,2S,4R,7R)-£rans-4-vinylcyclohexene diepoxide.

3. The method of claim 1 , wherein said 4-vinylcyclohexene diepoxide consists of ( 1 R,2S,4R,7R)-trαn5-4-vinylcyclohexene diepoxide.

4. The method of claim 1, wherein said step of administering comprises spraying said composition on said non-human mammal.

5. A composition comprising an epoxide hydrolase inhibitor in combination with 4-vinylcyclohexene diepoxide.

6. The composition of claim 5, wherein said 4-vinylcyclohexene diepoxide comprises (lR,2S,4R,7R)-trøns-4- vinyl cyclohexene diepoxide.

7. The composition of claim 5, wherein said 4-vinylcyclohexene diepoxide consists of (lR,2S,4R,7R)-trans-4-vinylcyclohexene diepoxide.

8. The composition of claim 5, wherein said inhibitor comprises elaidamide.

9. The composition of claim 5, wherein said inhibitor comprises trichloropropene oxide.

10. The composition of claim 5, wherein said inhibitor comprises Zn²⁺.