WO2003099333A1

WO2003099333A1 - Methods of controlling fertility, cancers and reproductive tract diseases

Info

Publication number: WO2003099333A1
Application number: PCT/GB2003/002267
Authority: WO
Inventors: Robert E. Garfield
Original assignee: REPRODUCTIVE HEALTH TECHNOLOGIES Inc
Current assignee: REPRODUCTIVE HEALTH TECHNOLOGIES Inc
Priority date: 2002-05-23
Filing date: 2003-05-23
Publication date: 2003-12-04
Anticipated expiration: 2004-11-23
Also published as: AU2003242828A1; AU2003242828A8

Abstract

Fertility, cancers such as breast cancer, endometrial cancer and leiomyoma, and reproductive tract diseases such as endometriosis, may be controlled by antiprogestins in combination with inducible nitric oxide inhibitors (iNOS), Aromatase Inhibitors (AI) and/or nitric oxide (NO).

Description

METHODS OF CONTROLLING FERTILITY, CANCERS AND REPRODUCTIVE R_AC_{T DISEAS}E_S

The invention relates to fertility control, and to the treatment of reproductive tract diseases.

Fertility regulation is a major area of research. Better and more affordable fertility control, whether contraception or methods to improve fertility, is sought by many people throughout the world.

The need for contraceptive options which are safe, effective, reversible, acceptable and affordable to most population groups is presently not being met. There are approximately six hundred million women of reproductive age in the world today, of whom around 228 million are at risk of unwanted pregnancy. It has been reported that up to 64% of all pregnancies world-wide are unintended, and that over 45 million termination of pregnancys occur annually world-wide. Further, it is estimated that at least 78,000 women die each year of termination of pregnancy-related deaths. Even in countries where contraception is easily available, the problem is still rife. In the United States around 57% of all pregnancies (around 2.5 million annually) are unintended, and around 1.3 million end in termination of pregnancy. About half of the pregnancies result from failed contraception.

On the other hand, another major problem is infertility in couples wanting to have children. One of the most basic human desires is to procreate, and the frustration of this desire is very distressing. It is estimated that infertility affects over 37 million married couples around the world. A recent study gave a conservative estimate that there are around 2.3 million infertile couples in the USA at present. This is approximately 9% of the domestic married couples, in which the wife is of childbearing age (15-44). The remaining couples have varying degrees of fertility. Another study found that about 4.9 million US women of childbearing age have an impaired ability to have children. At least 30-50% of infertility is attributable to male infertility. The pathophysiology of male infertility is poorly understood, and has a very poor proginosis in many cases. Male infertility can be treated in only 10-20% of cases.

What is required are new methods for fertility control for both improved contraception and for increasing fertility.

Diseases of the reproductive tract, such as endometriosis and cancer, affect many women. Some estimates suggest that up to 30% of women of reproductive age have endometriosis. Whilst some are asymptomatic, others suffer with severe pain associated with menstruation and intercourse. Tissue, similar to that lining the uterus (the endometrium) develops outside the uterus, throughout the pelvis. This can lead to scar tissue. Importantly endometriosis can result in infertility. The growth and spread of endometriosis is dependent on the female reproductive hormone cycle. Presently, there is no cure for endometriosis.

Endometrial cancer is another important disease of the female reproductive tract. It appears most frequently between the ages of 55 and 65. Many of the risk factors point to increased estrogen stimulation as being a cause of endometrial carcinoma. Further endometrial carcinoma frequently occurs on background of endometrial hyperplasia. Similarly, breast cancer occurs more frequently in women with endometrial cancer. As with most cancers, the survival rate for endometrial cancer is good if it is found early in development. However, any improved treatment for endometrial cancer would be beneficial.

A further complication of the reproductive tract is uterine fibroids. Properly termed leiomyomas, fibroids are benign tumours arising from the smooth muscle cells in the myometrium. Estrogens stimulate the growth of these tumours. They are found in 30%-50% of women during reproductive life. Whilst they can be arymptornatic, fibroids may cause disturbances in menstrual function and may be painful.

Similar regulatory and control pathways to those invoked in fertility are implicated in endometriosis and cancers such as leiomyomas, and breast cancer. Klijn J G M, et al. (Steroids, 2000, Vol. 65, pages 825-830) reviews the treatment of breast cancers using progesterone antagonists or modulators, such as mifepristone. Mifepristone has also been used in combination with tamoxifen to treat breast cancers (El Etreby M F, et al., Breast Cancer Research and Treatment (1998), Vol. 51, pages 149-168).

Leiomyomas have been successfully treated with RU486 - see Murphy A A, et ah, Fertility and Sterility (1995), Nol. 64, pages 187-190 and J. Clin. Endocrinol. Metab. (1993), Vol. 76, pages 513-517. The same group have also treated endometriosis using RU486 (Gertility and Sterility (1991), Nol 56, pages 402-407).

There are many publications on the role of nitric oxide (NO) in male and female reproduction. Some papers are contradictory so the picture is not entirely clear. In the male NO appears to be primarily produced by endothelial nitric oxide synthase (elNOS), a system which is required for the control of vascular tone. Attempts to use this system for fertility control would have the side-effect of elevated blood pressure. NO is also produced by inducible nitric oxide synthase (iNOS) in both men and women. NO is thought to be involved in various steps in reproduction, as listed below.

Male a) Erection b) Germ cell development c) Sperm capacitation d) Vascular modulation of testicular vessels e) Sperm output f) Sperm motility and motion characteristics.

Female a) Oocyte development and maturation b) Ovulation c) Egg activation and fertilisation d) Endometrial growth e) Vascular modulation f) Implantation g) Embryo development h) Trophoblast outgrowth i) Placental function j) Inhibition of uterine contractility k) Cervical function.

NO plays a synergistic role with progesterone in regulating the female reproductive tract, in particular in control of uterine contractility, spiral artery functions and cervical ripening. The interaction is particularly important during menstruation, implantation, pregnancy and parturition. NO also modulates many of the vaginal changes brought about by sexual stimuli. NO and steroid hormones (estrogens and progesterone) also have synergistic effects on the extracellular matrix of the vagina and bladder. NO deficiency seems to play a major role in pre-eclampsia.

There is a very finely controlled interaction between the developing embryo and the maternal tissue in pregnancy. The sequence of events is important; the development and location of the blastocyst, the hormone regulation and the uterine receptivity must be synchronised in order for successful implantation of the blastocyst to take place. If one of the criteria required for implantation is removed, the likelihood of a successful pregnancy is largely reduced. The period of pre-implantation is critical. During implantation the inner cell mass of the blastocyst differentiates to produce an epiblast and visceral and parietal endoderm. The trophoblast interacts with the uterus (Enders, et al. (1978), Anat. Rec, Nol. 190, pages 65-77). There are several stages of interaction as the blastocyst implants into the endometrium, the blastocyst traversing the uterine epilthelium and underlying basement membranes before entering the endometrial stroma and approaching the maternal capillaries (Schlafke and Enders (1975), Biol. Reprod., Nol 12, pages 41-65); Blankenship and Given (1992), Anat. Rec. Nol. 233, pages 196-204). The maternal tissue is transformed for implantation, the luteal hormones causing the endometrial stroma to change into metabolically active, enlarged decidual cells (Abrahamsohn (1983), Anat. Embryol., Nol 166, pages 263-274; Parr, et al. (1986), Am. J. Anat., Nol. 176, pages 423-436). The secretions from the uterine glands are also altered. An expanding capsule is formed around the implanting blastocyst by the decidualized cells (Schlafke and Enders (1975), Biol. Reprod., Nol. 12, pages 41-65). These cells and the uterine glands are also responsible for producing the cytokines and growth factors that help nurture the developing embryo. All of the events that take place during implantation are critical; if anyone is altered or removed implantation is much less likely to succeed. In the same respect, if this chain of events can be controlled better by pharmacological intervention, implantation may be more likely to succeed. This period is therefore a good candidate for fertility regulation.

The interaction between antiprogestins and iΝOS inhibitors had not been studied in detail until the inventors' studies. The inventors surprisingly found that antiprogestins and NO from iNOS had synergistic effects on implantation, embryonic development and endometrial function, at doses which individually would have had little or no effect.

Aromatase inhibitors are substances which inhibit the enzyme aromatase (estrogen synthase). This enzyme converts androgens to estrogens. Inhibition of aromatase results in a severe decline in the synthesis of estrogens with resultant changes in the other steroids in the synthetic pathway. The use of aromatase inhibitors as a contraceptive method to stop implantation is known from US 5,583,128. However the inventors have surprisingly found that the contraceptive effects of aromatase inhibitors are vastly increased when used in combination with antiprogestins.

According to the invention there is provided a pharmaceutical composition comprising antiprogestins and inducible nitric oxide synthase inhibitors (iNOS). The invention further provides the use of a antiprogestins in combination with an inducible nitric oxide synthase (iNOS) inhibitor to prevent pregnancy.

Preferred iNOS inhibitors for use in the inventions include: NG-nifro-L-arginine methyl ester (L-NAME), NG-monoethyl-L-arginine (LMMA), N-j^:mmoemyl-L-amithine (L-NIO), L-monome yl-L-arginine (L-NNMA), L-NG-methylarginine (LNMA), Nw-nifro-L-arginine (L-NA) and Aminaguanidine.

Preferred examples of antiprogestins for use in the inventions include:

onapristone,

(l l.beta.-[4(Dimemylamino)phenyl]-17.alpha.-hydroxy-17.beta.-(3-hydroxypropyl)-13. alpha.-estra-4,9-diene-3-one), RU 486,

(ll.beta.-[4-(Dimemylan ino)phenyl]-17.beta.-hydroxy-17.alpha.-(l-propinyl)estra-4,9- diene-3-one),

(Z)- 11.beta.-[4-(Dimethylamino)phenyl]- 17.beta.-hydroxy- 17.alpha.-(3-hydroxy- 1 - ρropenyl)estr-4-ene-3-one (EP-A 0 404283), l l.beta.-(4-Acetylphenyl)-17.beta.-hydroxy-17.alpha.-(l-propinyl)estra-4,9-diene-3-one

(EP-A 0 190 759),

4^5'-Dihydro-ll.beta.-[4-(dimemylamino)phenyl]-6.beta.-methylspiro[estra-4,9-diene-17. beta.,2 3Η)-furan]-3-one,

4', 5 '-Dihydro- 11.beta.- [4-(dimethylamino)phenyl]-7.beta, -methylspiro [estra-4,9-diene- 17. beta.,2'(3Η)-furan]-3-one, l l.beta.-(4-Acetylphenyl)-19,24-dinor-17,23-epoxy-17.alpha.-chola-4,9,20-triene-3-one,

(E)- 11.beta.-[4-[[(Acetyloxy)imino]methyl]phenyl]- 17.beta.-methoxy- 17.alpha.-

(methoxymethyl)estra-4,9-diene-3-one,

(E)-l l.beta.-[4-[[[(E oxycarbonyl)oxy]immo]methyl]phenyl]-17.beta.-methoxy-17.alpha.-

(methoxymethyl)estra-4,9-diene-3-one.

A preferred dosage of antiprogestin is 0.01 to 10 mg/kg. daily.

Preferred aromatase inhibitors include anήnoglutethimide.

Pregnancy is preferably prevented by prevention the implantation of the blastocyst into the uterus lining.

Also provided is a method of preventing pregnancy comprising the step of administering an antiprogestin and an iNOS inhibitor to a female mammal. Preferably the mammal is a primate, more preferably a primate, more preferably a human.

Preferably the antiprogestin and iNOS may be administered either pre- or post-coitally.

When administered post-coitus, the antiprogestin and iNOS inhibitor are preferably administered during the pre-implantation period.

Preferably the antiprogestin and iNOS are administered immediately pre-implantation or immediately post-implantation. Oral treatment for two days may be used.

Further provided is the use of an antiprogestin to prevent pregnancy, m combination with treatment with an iNOS inhibitor.

The invention also provides the use of an iNOS inhibitor to prevent pregnancy, in combination with treatment with an antiprogestin.

A second aspect of the invention provides the use of an antiprogestin in combination with an iNOS inhibitor to induce termination of pregnancy or labour.

Also provided is a method of inducing termination or labour comprising the step of administering an antiprogestin and an iNOS inhibitor to a pregnant female mammal.

Further provided is the use of an antiprogestin to induce termination or labour in combination with treatment with an iNOS inhibitor.

The invention also provides the use of an iNOS inhibitor to induce termination or labour in combination with treatment with an antiprogestin.

A further aspect of the invention provides a pharmaceutical composition comprising an antiprogestin and an AL

The invention also provides the use of an antiprogestin in combination with an aromatase inhibitor (Al) to prevent pregnancy.

Pregnancy is preferably prevented by prevention the implantation of the blastocyst into the uterus lining. Also provided is a method of preventing pregnancy comprising the step of administering an antiprogestins and an Al to a female mammal.

Preferably the mammal is a primate, such as a human.

Preferably the antiprogestin and Al may be administered pre- or post-coitally. When administered post-coitus, the antiprogestin and Al are preferably adniinistered in the pre-implantation period.

Preferably oral treatment with antiprogestin and an iNOS inhibitor or aromatase inhibitor is carried out for two days.

Further provided is the use of an antiprogestin to prevent pregnancy, in combination with treatment with an Al.

The invention also provides the use of an Al to prevent pregnancy, in combination with treatment with an antiprogestin.

Another aspect of the invention provides the use of an antiprogestin in combination with an Al to induce termination of pregnancy or labour.

Also provided is a method of inducing termination or labour comprising the step of administering an antiprogestins and an Al to a pregnant female mammal.

Further provided is the use of an antiprogestin to induce termination or labour in combination with treatment with an Al.

The invention also provides the use of an Al to induce termination or labour in combination with treatment with an antiprogestin.

Another aspect of the invention provides the use of an antiprogestins in combination with a nitric oxide synthase (iNOS) inhibitor to treat endometriosis.

Also provided is the use of an antiprogestin to treat endometriosis, in combination with an iNOS inhibitor.

The invention further provides the use of an iNOS inhibitor to treat endometriosis, in combination with treatment with an antiprogestin. Also provided is a method of treating endometriosis comprising the step of aclministering an antiprogestin and an iNOS inhibitor to a female mammal.

Another aspect of the invention provides the use of an antiprogestin in combination with an aromatase inhibitor to treat endometriosis.

Also provided is the use of an antiprogestin to treat endometriosis, in combination with an aromatase inhibitor.

The invention further provides the use of an aromatase inhibitor to treat endometriosis, in combination with treatment with an antiprogestin.

Also provided is a method of treating endometriosis comprising the step of administering an antiprogestin and an aromatase inhibitor to a female mammal.

Another aspect of the invention provides the use of an antiprogestin in combination with an iNOS inhibitor to treat cancer.

Also provided is the use of an antiprogestin to treat cancer, in combination with an iNOS inhibitor.

The invention further provides the use of an iNOS inhibitor to treat cancer, in combination with treatment with an antiprogestin.

Also provided is a method of treating cancer comprising the step of administering an antiprogestin and an iNOS inhibitor to a female mammal.

Another aspect of the invention provides the use of an antiprogestin in combination with an aromatase inhibitor to treat cancer.

Also provided is the use of an antiprogestin to treat cancer, in combination, with an aromatase inhibitor.

The invention further provides the use of an aromatase inhibitor to treat cancer, in combination with treatment with an antiprogestin. Also provided is a method of treating cancer comprising the step of administering an antiprogestin and an aromatase inhibitor to a female mammal.

Preferably the cancer is breast cancer, endometrial cancer or leiomyoma.

A yet further aspect of the invention provides a pharmaceutical composition comprising antiprogestins and NO in combination with one or more pharmaceutically acceptable carriers.

The invention further provides the use of progesterone in combination with nitric oxide in the preparation of a medicament to treat infertility.

Infertility is defined as being a reduced ability, compared with the average for a population, to conceive carrying offspring to term.

Preferably the medicament treats infertility by improving the likelihood of successful blastocyst implantation, and/or by reducing the likelihood of pre-term labour.

Preferred dosage regimes are a daily dose bioequivalent to 50-300 mg of progesterone/day, e.g., an injectable suspension of medroxyprogesterone acetate to provide a weekly dose thereof of 100-1000 mg or tablets or dragees providing an oral dose thereof of 5-100 mg/day, an injectable solution of hydroxyprogesterone caproate which provides a weekly dose of 250-500 mg; tablets, capsules or dragees of norethindrone acetate which provide a daily dose of 5-20 mg.

Examples of dosage ranges of typical NO-substrates and NO-donors (per os or transdermally) are:

total dose:

L-Arginine 500 mg- 10 g p.o.

Sodium Nitroprusside range 500-2000 ,mu.g/kg/day p.o.

Nitroglycerin 0.5-10 mg p.o.

Nitroglycerin 0.1-10 mg/24 hours transdermal

Isosorbid mononitrate 10-100 mg/day p.o.

Isosorbid dinitrate 10-100 mg/mg p.o. The nitric oxide donors (e.g., nitroglycerin) can be administered preferentially by a transdermal patch, orally, etc.

Also provided is a method of treating infertility comprising the steps of ao-ministering a progesterone and nitric oxide to a woman suffering from reduced fertility.

Preferably the progesterone and NO may be administered separately or simultaneously.

Further provided is the use of progesterone in the treatment of infertility, in combination with treatment with NO .

The invention also provides the use of NO in the treatment of infertility in combination with treatment with progesterone.

Two or more antiprogestins may be used in combination with any of the above aspects of the invention. Aromatase inhibitors may also be used in combination with iNOS.

The invention will now be described in detail with reference to the figures in which:

Figure 1 shows termination of pregnancy induced with L-NAME (50 mg/day), onapristone (3 mg/kg) or combination given on day 17 of pregnancy.

Figure 2 shows synergistic effects of RU 486 (RU) and aminoglutethimide (AG) on implantation in rats treated for 2 days (days 4 and 5 of pregnancy).

Figure 3 shows synergistic effects of RU 486 and aminoglutethimide (AG) to terminate pregnancy when given on day 17 of gestation (animals sacrificed on day 19 of gestation).

Figure 4 shows synergistic effects of RU 486 + letrozole to terminate pregnancy when given at day 17 of pregnancy and animals examined on day 19.

Implantation studies

Studies were carried out on rodents, with treatments being made during either days 2-6 (pre-implantation treatment) or days 5-8 (peri-implantation treatment). The animals were sacrificed at either day 9 or 19 of gestation. Interruption of pregnancy studies

These studies were also carried out in rodents. In rats treatment was made on day 17 with antiprogestins and then on day 17 with either iNOS inhibitors or aromatase inhibitors.

Effects of antiprogestin (onapristone) and NO inhibition with L-NAME (LN) or aminoguanidine (A) on implantation in rats.

Our studies in guinea pigs (Chwalisz, unpublished), demonstrate that INOS inhibitors (L-Name and aminoguanidine) and antiprogestins (onapristone) act synergistically at the endometrial level. Pre-implantation treatment was very effective in preventing the establishment of pregnancy in both species, there were no changes in blood steroids following these treatments. This effect was accompanied by a dramatic vasoconstriction of endometrial blood vessels prior to and during early placentation. In addition, the combined treatment during the pre-implantation phase completely blocked early embryonic development and most likely fertilisation. The available data also suggests that an iNOS inhibitor substantially enhances endometrial effects of antiprogestins.

Effects of iNOS inhibition on ability of antiprogestins to terminate pregnancy late in pregnancy-

Treatment of rats with RU486 (1 mg/day, orally) at day 17 and 18 of gestation did not influence pregnancy. However, when RU486 was combined with the iNOS inhibitor >95% of the pups were delivered prematurely. We conclude that an antiprogestin acts synergistically with an iNOS inhibitor to terminate pregnancy. When animals were treated with onapristone + L-NAME a similar synergistic effect occurred (Figure 1).

Studies of pregnant rodents showed that L-NAME by itself dose-dependently induced pre-term birth when given at day 50 of pregnancy. In addition, treatment of rodents at day 41 of gestation, with either low dose RU486 or L-NAME, terminated pregnancy in only 1 of 5 animals. However, a combination of low dose RU486 and L-NAME terminated pregnancy effectively. Similarly, rodents treated late in gestation, starting on day 61 of gestation, with the iNOS inhibitor aminoguanidine alone delivered prior to control animals.

Effects of aromatase inhibitors.

When we treated rats with RU486 with aminoglutethimide (a P450, aromatase inhibitor) we obtained the same effects as seen with antiprogestin in combination with iNOS inhibitors (i.e. almost 100% inhibition of implantation (Figure 2) or termination of pregnancy (Figure 3) with little effect of the mesoprogestin, antiprogestin or an moglutetlumide alone). Similar effects were seen when Letrozole was used (Figure 4).

The above studies indicate that antiprogestins might be used in combination with an aromatase inhibitor both to inhibit implantation and to terminate pregnancy.

These studies show that RU486 and iNOS inhibitors, and RU486 and aromatase inhibitors are synergistic in implantation. It is therefore expected that compounds such as RU486 in combination with aromatase inhibitors or iNOS inhibitors are expected to treat cancers, such as leiomyomas and breast cancer. They are also expected to be useful in the treatment of endometriosis.

Claims

1. A pharmaceutical composition comprising an antiprogestin and an inducible nitric oxide synthase inhibitor (iNOS).

2. An antiprogestin in combination with an inducible nitric oxide synthase (iNOS) inhibitor for use to prevent pregnancy.

3. A method of preventing pregnancy comprising the step of administering an antiprogestin and an iNOS inhibitor to a female mammal.

A ^~An antiprogestin in combination with an iNOS inhibitor for use to induce termination of pregnancy or labour.

5. A method of inducing termination of pregnancy or labour comprising the step of administering an antiprogestin and an iNOS inhibitor to a pregnant female mammal.

6. The use of an antiprogestin to induce termination of pregnancy or labour in combination with treatment with an iNOS inhibitor.

7. The use of an iNOS inhibitor to induce termination of pregnancy or labour in combination with treatment with an antiprogestin.

8. A pharmaceutical composition comprising an antiprogestin and an aromatase inhibitor (Al).

9. An antiprogestin in combination with an aromatase inhibitor (Al) for use to prevent pregnancy.

10. A method of preventing pregnancy comprising the step of adrninistering an antiprogestin and an Al to a female mammal.

11. The use of an antiprogestin to prevent pregnancy, in combination with treatment with an Al.

12. The use of an Al to prevent pregnancy, in combination with treatment with an antiprogestin.

13. An antiprogestin in combination with an Al for use to induce termination of pregnancy or labour.

14. A method of inducing termination of pregnancy or labour comprising the step of administering an antiprogestin and an Al to a pregnant female mammal.

15. The use of an antiprogestin to induce termination of pregnancy or labour in combination with treatment with an Al.

16. The use of an Al to induce termination of pregnancy or labour in combination with treatment with an antiprogestin.

17. An antiprogestin in combination with an inducible nitric oxide synthase (iNOS) inhibitor for use to treat endometriosis.

18. The use of an antiprogestin to treat endometriosis, in combination with an iNOS inhibitor.

19. The use of an iNOS mhibitor to treat endometriosis, in combination with treatment with an antiprogestin.

20. A method of treating endometriosis comprising the step of administering an antiprogestin and an iNOS inhibitor to a female mammal.

21. An antiprogestin in combination with an aromatase inhibitor for use to treat endometriosis.

22. The use of an antiprogestin to treat endometriosis, in combination with an aromatase inhibitor.

23. The use of an aromatase inhibitor to treat endometriosis, in combination with treatment with an antiprogestin.

24. A method of treating endometriosis comprising the step of administering an antiprogestin and an aromatase inhibitor to a female mammal.

25. An antiprogestin in combination with an inducible nitric oxide synthase (iNOS) inhibitor for use to treat cancer.

26. The use of an antiprogestin to treat cancer, in combination with an iNOS inhibitor.

27. The use of an iNOS inhibitor to treat cancer, in combmation with treatment with an antiprogestin.

28. A method of treating cancer comprising the step of administering an antiprogestin and an iNOS inhibitor to a female mammal.

29. An antiprogestin in combination with an aromatase inhibitor for use to treat cancer.

30. The use of an antiprogestin to treat cancer, in combination with an aromatase inhibitor.

31. The use of an aromatase inhibitor to treat cancer, in combination with treatment with an antiprogestin.

32. A method of treating cancer comprising the step of administering an antiprogestin and an aromatase inhibitor to a female mammal.

33. A pharmaceutical composition comprising PRM and NO in combination with one or more pharmaceutically acceptable carriers.

34. Progesterone in combination with nitric oxide in the preparation of a medicament to treat infertility.

35. A method of treating infertility comprising the steps of administering a progesterone and nitric oxide to a woman suffering from reduced fertility.

36. The use of progesterone in the treatment of infertility, in combination with treatment with NO.

37. The use of NO in the treatment of infertility in combination with treatment with progesterone.