CZ360698A3 - Method of increasing implantation frequency for in-vitro fertilization and pharmaceutical preparation - Google Patents

Description

A method for increasing the frequency of implantation and a pharmaceutical composition for in vitro fertility,

Technical field

The present invention relates to a method for increasing the frequency of implantation after in vitro fertilization (IVF), in the treatment of infertility, and to treat and prevent early pregnancy loss in women using a nitric oxide synthase substrate (L-arginine), a nitric oxide donor or both, separately or in combination with progesterone and / or estrogen, and a pharmaceutical composition. The present invention also relates to novel methods for regulating fertility by inhibiting endometrial receptivity, preventing implantation and inducing menstruation with NOS inhibitors, preferably iNOS inhibitors, in combination with progesterone antagonists and / or progesterone synthase inhibitors.

BACKGROUND OF THE INVENTION

In vitro fertilization in humans is surprisingly successful. The overall fertility rate per IVF treatment cycle in the United States is approximately 14% (Medical Research International Society for Assisted Reproductive Technology [SART], The American Fertility Society [1992]. Fertil Steril 5:15) and 12.5% in the UK (The Human Fertilization and Embryology of Auhority. Annual Report, London 1992).

This success is higher when more than one embryo is transmitted at a time. However, the simultaneous transmission of several embryos increases the incidence of multiple pregnancies and the possibility of miscarriage and premature birth. The reasons for low percent pregnancy after IVF are not yet fully understood. The success rate affects the quality of the embryo and the uterus environment. In general, there is a high percentage of spontaneous early miscarriages among women in fertile cycles. After natural conception, very early loss of perhaps 50-60% of pregnancies occurs (Winston ML, Handyside AH [1993], New challenges in human in vitro fertilization. Science 260: 932-935). This may be the result of both embryonic abnromalities and dyschrony between the embryo and the endometrium at the time of embryo transfer.

Many cases of loss of pregnancy may be due to abnormalities of conceived embryo or still unsuitable culture conditions, as the success of embryo transfer after IVF decreases with increasing time from fertilization (Winston ML, Handyside AH [1993], New challenges in human in vitro fertilization. 260: 932-935.

To avoid possible problems in the culture media, oocyte transfer (gamete intrafallopian transfer - GIFT) or zygote directly into the fallopian tubes (zygote intrafallopian transfer - ZIFT) was performed in women with healthy fallopian tubes. However, these trials only slightly increased the fertility and fertility rates after IVF (Edwards RG [1995] Clinical approaches to increasing uterine receptivity during human implantation. Hum Reprod 10, Suppl 3: 60-67).

The influence of uterine environment on IVF fertility may be equally important. It is well known that successful pregnancy following embryo transfer requires both healthy blastocysts and a receptive uterus. An embryo transferred to a poorly prepared uterus is unlikely to be implanted. In all mammals, the endometrium is receptive to implantation only during a certain period after ovulation. This • 4 »4 · 4 ·

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4 4 4 · ♦ ·· The luteal phase is called the implant window. In women, successful implantation can occur only between days 15 and 20 of the histologically defined 28-day cycle; during periods with the highest levels of progesterone (Navot D, Scott RT, Droesch KD, Veeck LL, Hung-Ching Liu, Rosenwaks Z [1991], The window of embryo transfer and efficiency of human conception in vitro. Fertil Steril 55: 114-118 ). The optimal condition for implantation was estimated to be on days 20-22 of the normal cycle, i.e. 7 days after LH level (Bergh PA, Navot D [1992], The Impact of Embryonic Development and Endometrial Graduation on the Timing of the Implant and He).

Fertil Steril 58: 537-542).

An essential prerequisite for successful implantation is sufficient preparation of the endometrium by progesterone, and the application of antiprogestin during the luteal phase completely prevents implantation (Chwalisz K, Stckermann K, Fuhrmann U,

Fritzemeier KH, Einspanier A, Garfield RE (1995), Mechanism of action of antiprogestins in the pregnant uterus. In Henderson D, Philibert D, Roy AK, and Teutsch G (eds) Steroid Receptors and Antihormones. Ann NY Acad Sci 761: 202-224. In the fertile cycle, progesterone regulates the transport of the fertilized egg by the fallopian tube and induces the secretory changes necessary for implantation in the endometrium. In mammals, implantation is a precisely timed event. Secretory endometrial proteins (Beier HM, Elger W, Hegele-Hartung C, Mootz U, Beier-Hellwig K [1992], Dissociation of the corpus luteum, endometrium and blastocyst in human implantation research. J Reprod Fert 92: 511-523), and perhaps other intracellular and cell-surface proteins, such as integrins, cytokines, and growth factors, produced by endometrial epithelial cells as a result of progesterone stimulation, are necessary for implantation (Edwards RG [1995]

• · ·

Physiological and molecular aspects of human implantation. Hum Reprod 10, Suppl 2: 1-14).

Asynchronia between embryo and endometrial development was previously seen as one of the possible causes of implant failure after IVF (Beier HM, Elger W, Hegele-Hartung C, Mootz U, Beier-Hellwig K [1992], Dissociation of corpus luteum, endometrium and blastocyst in Human implantation research.

J Reprod Fert 92: 511-523. However, to date there are no effective methods available to increase the frequency of implantation.

The most advanced stages of implantation in humans are characterized by the invasion of trophoblastic cells into deciduum and angiogenesis (Loke YW, King A / 1995 / Human Implantation, Cell Biology and Immunology, Cambridge University Press).

These stages are also dependent on progesterone because progesterone antagonists also lead to the termination of early pregnancy (Chwalisz K, Stckermann K, Fuhrmann U, Fritzemeier KH, Einspanier A, Garfield RE / 1995), Mechanism of action of antiprogestins in the pregnant uterus. Henderson D, Philibert D, Roy AK, Teutsch G (eds) Steroid Receptors and Antihormones, Ann NY Acad Sci 761: 202-224).

In early pregnancy, sufficient blood supply to the uterus is essential for embryo development. A disorder in the blood supply to the uterus may endanger pregnancy (Edwards RG [1995] Clinical approaches to increasing uterine receptivity during human implantation. Hum Reprod 10, Suppl 3: 60-67). Patients with impaired blood supply were given aspirin in an effort to improve this supply. Low doses of aspirin are believed to increase the prostacyclin: thromboxane A2 ratio and thereby increase placental perfusion. However, the effect of aspirin on uterine blood supply was only marginal (Goswamy RK, Williams G, Steptoe PC (1988) Decreased uterine perfusion - a cause of infertility. Hum Reprod 3955-3959; Wada I, Hsu CC, Williams G, Macnamee)

MC, Brinsden PR (1994) The benefits of low-dose aspirin therapy in women with impaired uterine perfusion during assisted conception. Hum Reprod 9: 1954-1957).

Contraceptive methods need to be further improved. Particular attention should be paid to further reducing the total monthly dose and the frequency of contraceptive use.

Several methods of antiprogestin fertility regulation have been proposed in the last decade, based either on inhibition of ovulation or prevention of implantation after intercourse or early luteal phase application (Spit of IM, Bardin CV [1993] Contraception 48: 403-444). Only application of mifepristone after sexual intercourse (Glasier A, Thong KJ, Dewar M, Mackie M, Baird DDT [1992] Mifepristone [RU 486] compared with high-dose estrogen and progestestogen for emergency postcoital conception. N Engl J Med

327: 1041-1044) and its administration in the early luteal phase at LH + 2 (Gemzell-Danielsson K, West lund P, Johannisson E, Swahn ML, Bygdeman M, Seppl [1996] Effect of low weekly doses of mifepristone on ovarian function and endometrial development (Hum Reprod 11: 256-264), however, has been shown to be effective in women. The contraceptive effects of mifepristone using these techniques are probably due to the effect of the substance on the endometrium. Postcoital administration of mifepristone should only be used exceptionally, otherwise it will interfere with ovulation and will lead to induction of amenorrhea. The use of mifepristone at LH + 2 is relatively inappropriate as an increase in LH should be identified at each cycle. Available data on antiprogestins suggest that there is also the possibility of developing a pill to be used once a month for menstruation induction and contraception. This method could be used in emergencies but also regularly. Antiprogestin RU 486 was «· · ·«

administered immediately after sexual intercourse and has been shown to effectively inhibit implantation (Glasier A, Thong KJ, Dewar M, Mackie M, Baird DDT [1992] Mi fepristone [RU 486] compared with high-dose estrogen and progestestogen for emergency postcoital conception. N Engl J Med 327: 1041-1044. However, when administered alone (i.e., without prostaglandin) as a monthly contraceptive, RU 486 does not effectively terminate pregnancy during the late luteal phase (Couzinet B, le Strat N, New Year's E, Schaison G [1990] Latheal administration on the antiprogesterone RU 486 in normal women: Effects on menstrual cycle events and fertility control in a long-term study (Fertil Steril 54: 1039-1044). The efficacy of RU 486 alone at termination of pregnancy, regardless of dose, is approximately 80 to 85% within 10 days after menses have been missed, approximately 65% to 56 days amenrrhoey and less than 40% in later stages of pregnancy (Van Look PFA, Bydgeman M [ 1989] Antiprogestational steroids, A new dimension in human fertility regulation (In: Oxford Reviews of Reproductive Biology 11: 1-60). Administration of RU 486 to pregnant women with amenorrhea lasting 7 weeks or less induced complete abortion in 65 to 85% of women. However, the addition of prostaglandin increases efficacy to 95% or more (Van Look PFA, Bydgeman M [1989] Antiprogestational steroids. A new dimension in human fertility regulation. In: Oxford Reviews of Reproductive Biology 11: 1-60; Aubeny E, Baulieu EE [ 1991] Contragestive activity of RU 486 and oral active prostaglandin combinations Comptes Rendus de 1Academie des Science 312: 539-545). These studies suggest that additional administration of prostaglandin increases the efficacy of antiprogestins at end of advanced pregnancy. However, it remains unclear whether the same synergistic effect can be achieved in very early pregnancy, ie. in the first week of amenorrhey. In addition, prostaglandins are known to induce unwanted gastrointestinal •

Ί effects that may be unacceptable with regular contraceptive use.

One of the most intriguing findings in biology and medicine recently is that nitric oxide is produced by endothelial cells and is involved in vascular tone regulation, platelet aggregation, nerve impulses, and immune system activation. Nitric oxide is an important mediator of vascular smooth muscle relaxation; previously known as EDRF (Endothelin-Derived Relaxing Factor) {Furchgott RF and Zawadzski JV [1980] The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373-376; Moncada S, Palmer RMG and Higgs EA [1991] Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev

43: 109-142. Nitric oxide is synthesized by oxidative deamination of guanidine nitrogen of L-arginine with at least three different isoforms of flavin-containing enzyme, nitric oxide synthase {Moncada S, Palmer RMG and Higgs EA [1991] Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 43: 109-142). Nitric oxide synthesis has been shown to be competitively inhibited by L-arginine analogues, namely, methyl ester of NG-nitro-L-arginine (L-NAME), NG-monoethyl-L-arginine (LMMA), N-iminoethyl-L-arnithine ( L-NIO), L-monomethyl-L-arginine (L-NNMA) and L-NG-methylarginine (LNMA) and Nw-nitro-L-arginine (L-NA).

Nitric oxide increases levels of cGMP (1,4,5-cyclic guanosine monophosphate) in vascular smooth muscle, causing relaxation and reduction of vascular tone {Moncada S, Palmer RMG and Higgs EA [1991] Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 43: 109-142). In these

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ft · nitric oxide was detected in compounds. Nitrogen-based vasodilators are now classified as nitric oxide donors because they are biotransformed or spontaneously release nitric oxide (Moncada S, Palmer RMG and Higgs EA [1991] Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 43: 109-142). Long-term nitrogen-based vasodilators can be considered as substitution treatment for a failing physiological mechanism. Nitric oxide is also produced by macrophages and other cells of the immune system.

So far, three largely mated NOS enzymes have been isolated and identified. These include endothelial NOS (e-NOS, type III), neuronal NOS (n-NOS, type II) and inducible NOS (i-NOS, type I), (Knowles RG, Moncada S [1994] Nitrous oxide synthases in mammals. Biochem J 298: 249-258; Sess WC [1994] The nitric oxide synthase family of proteins. J Vasc Res 31: 131-143; Nathan C [1992] Nitric oxide as a secretory product of mammalian cells. FASEB J 6: 301 -3064).

The constitutive isoform of e-NOS was originally identified in endothelial tissues and the constitutive isoform of b-NOS in neuronal tissue, both rapidly and transiently producing small amounts of NO under basal conditions. The i-NOS isoform is inducible by cytokines or endotoxin (LPS) and produces large amounts of NO for hours or days independent of Ca 2+. Cells expressing i-NOS do not generate NO under basal conditions. The form of e-NOS enzyme is expressed by endothelial cells, in cardiomyocytes, in platelets and in some neurons. NO-derived e-NOS (derived from e-NOS) is the most important vasodilator. It is released in small amounts to maintain constant vasorelaxation and normal blood pressure. It is believed that the n-NOS isoform acts as a neurotransmitter. It is believed to play an important role in mediating functions such as gastrointestinal motility and penile erection.

There is ample evidence from animal experiments that nitric oxide deficiency contributes to the pathogenesis of a number of diseases including hypertension, atherosclerosis and diabetes (Moncada S, Palmer RMG and Higgs EA [1991] Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 43: 109 -142). There are many recent studies showing that inhibition of nitric oxide synthase radically increases blood pressure. The application of nitric oxide synthase inhibitors to pregnant female rats and guinea pigs produces the same symptoms as observed in eclampsia (Chwalisz K and Garfield RE [1994] The role of progesterone during pregnancy: Models of parturition and preeclampsia. From Geburtsh at Perinat 198: 170-180) . Preeclampsia is characterized by increased blood pressure and peripheral vascular resistance, fetal growth retardation, proteinuria and edema. In humans, histopathological and clinical evidence (fetal growth retardation, fetal death) shows that decreased placental perfusion is the earliest and most regularly occurring change observed in preeclampsia (Robert with JM and Redman CWG [1993] pre-eclampsia: more than pregnancy-induced hypertension 341: 1447.-14511; Friedman EA [1988] Preeclampsia: review of the role of prostaglandins. Obstet Gynecol 71: 122-137).

The L-arginine-NO system is present in the uterus (Garfield RE and Yallampalli C [1993] Control of myometrial contractility and labor. In: Basic Mechanisms Controlling Term and Preterm Birth. Ed: K Chwalisz, RE Garfield, Springer-Verlag, New York , pp. 1-29, Chwalisz K and Garfield RE [1994]

Antiprogestins in induction of labor. Ann NY Acad Sci 734: 374-413; Buhimschi I, Yallampalli, C, Dong Y-L and • • 9 • 999 99 • 9 ♦

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Garfield RE [1995] Involvement of nitric oxide cyclic guanosine monophosphate pathway in human uterine contractility control during pregnancy. Am J Obstet Gynecol 172: 1577-1584; Brewer SM, Regenstrin AC, Lykins D et al. [1993] Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy. Am J Obstet Gynecol 169: 1285-1291. This system plays an important role in the regulation of uterine contractility, maintenance of pregnancy, onset of labor, and fetal perfusion. L-arginine and nitric acid led to rapid and substantial relaxation of spontaneous uterine strips taken from rats from mid-gestation to near-gestation (Buhimschi I, Yallampalli, C, Dong YL and Garfield RE [1995] Am J Obstet Gynecol 172: 1577-1584; Garfield RE and Yallampalli C [1993] Control of Myometrial Contractility and Labor In: Basic Mechanisms Controlling Term and Preterm Birth. : K Chwalisz, RE Garfield, Springer-Verlag, New York, pp.1-29, SM Brewer, Regenstrin AC, Lykins D et al [1993] Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy. J Obstet Gynecol 169: 1285-1291, Natuzzi ES, Ursell PC, Harrison M et al [1993] Nitric oxide synthase activity in pregnant uterus decreases in parturition Biochem Biophys Res Commun 194: 108-114, Jennings RW MacGillvray T E and Harrison MR [1995] Nitric oxide inhibits preterm labor in the rhesus monkey. J Mat Fed Med 2: 170-175.

Expression of NOS enzymes in female uterus was studied by immunoblotting with monoclonal antibodies. i-NOS and e-NOS detected in uterus • · «·

• (myometry). I-NOS levels in the uterus decreased during uterine contractions at the time and before the end in the animals induced to give birth before the time. Opposite changes were observed in the cervix (Buhimischi I Ali M, Jain V, Chwalisz K and Garfield RE [1996]

Diffferential regulation of nitric oxide in uterus and cervix during pregnancy and labor. Human Reprod (in print).

NOS is also present in placental tissues and in the uterine arteries. Invasion of uteroplacenary arteries by trophoblasts in relation to the expression of the NO synthase isoform was studied in pregnant guinea pigs using immuno- and histochemical methods and compared with arterial dilation. Significant dilatation of uteroplacental arteries begins in the middle of pregnancy and continues to the term (Nanaev A, Chwalisz K, Frank HG, Kohnen G, Hartung CH and Kaufmann P [1995] Physiological dilatation of uteroplacental arteries in the guinea pig depends on nitric oxide synthesis activity Cell Tissue Res 282: 407-421). This study shows that dilation of uteroplacental arteries can be observed when invading trophoblastic cells coexpressing endothelial nitric oxide synthase (e-NOS) and macrophages (i-NOS) are detected near the blood vessels, i.e. before trophoblastic arterial wall invasion. Conrad et al. (1993) localized NOS to layers of syncythiotrophoblasts in human placenta (Conrad KP, Vili M, McGuire PG, Dail WG, Davis AK [1993] Expression of nitric oxide synthase by syncythiotrophoblast in human placental villi, FASEB J 7: 1269-1276) . Morris et al. (1993) have shown calcium-dependent (calcium-dependent) and calcium-non-dependent (calcium-independent) activity in human placental villi and basal plate (Morris NH, Sooranna SR, Ewaton BM, Steer PJ [1993], NO synthase) • «• φ φ · ♦ ·

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Activity φ »activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity activity Lancet 342: 699-680) and Myatt et al. (1993) have shown that the vascular system of placental villi synthesizes the calcium-dependent isoform of NOS (Myatt L, Brockmann DE, Langdon G, Pollock JS [1993] Constitutive calcium-dependent isoform of nitric oxide synthase in the human placenta villous vascular tree. Myatt L, Brockmann DE, Eis AL, Pollock JS [1993] Immunohistochemical localization of nitric oxide synthase in the human placenta. Placenta 14: 487-495). In addition, Buttery et al. (1994) have shown that endothelial NOS by term is located in the umbilical cord and vein endothelium in the placental syncythiotrophoblast (Buttery LDK, McCarthy A, Springall A et al. [1994] Endothelial nitric oxide synthase in the human placenta: regional distribution and proposed regulators the role of the feto-maternal interface (Placenta 15: 257-267). Moorhead et al. (1995) have also demonstrated that NADPH diaphorase (a non-specific reaction to identify nitric oxide synthase) is present in various parts of the uterus in early pregnancy (Moorhead CS, Lawhun M, Nieder GL [1995] first half of pregnancy and during an artificially-induced decidual cell reaction (J Histochem Cytochem 43: 1053-1060). Finally, Toth et al. (1995) have shown that NOS activity is present in the first trimester of human placental homogenates (Toth M, Kukor Z, Romero R, Hertelendy F [1995] Nitric oxide synthase in the first trimester of human placenta: Characterization and subcellular distribution. Hypertens Pregnancy 14/3 : 283-300).

These studies suggest that nitric oxide is an important factor regulating placental blood supply and myometrial rest during pregnancy. However, no studies have been published to date showing the harmful effects of NOS inhibition on implantation or the beneficial effects of nitric oxide donors or substrates on implantation after IVF or in women who have had early pregnancy loss. In contrast, Haddad et al. (1995) indicated that increased nitric oxide production is associated with early embryo loss in mice and that iNOS inhibitors can be used to induce early miscarriage (Haddad EK, Duclos AJ, Baines MG [1995] J Dec Med 12: 1143-1151).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for increasing the frequency of implantation after IVF, comprising administering a substrate and / or a nitric oxide donor in a mammal.

Another object of the present invention is a method of treating and preventing infertility or early loss of pregnancy, the method comprising administering a substrate and / or a nitric oxide donor in a mammal in the early stages of pregnancy.

Another object of the present invention is to provide a method for increasing the frequency of implantation and treatment and preventing infertility or early pregnancy loss, comprising administering a substrate and / or a nitric oxide donor, wherein the method comprises progesterone or a progestagen in combination with the substrate; or with a nitric oxide donor.

Another object of the present invention is a process using a progestational agent and an estrogenic agent.

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In combination with a substrate and / or a nitric oxide donor in an effort to increase the frequency of implantation and to treat and prevent infertility or early pregnancy loss in female mammals.

Another object of the present invention is to provide a method for controlling fertility by administering an antiprogestin (e.g., mifepristone, ORG 31710, ORG 33 628, J867, CDB 2914, ZK 137316) and / or a progesterone synthase inhibitor (e.g. epostane, trilostane) in combination with nitric oxide synthase inhibitor.

A further object of the present invention is to provide a method of controlling fertility, comprising administering an antiprogestin and / or a progesterone synthase inhibitor in combination with a nitric oxide synthase inhibitor as a monthly contraceptive method.

It is a further object of the present invention to provide a method of re-fertility comprising administering an antiprogestin in combination with a nitric oxide synthase inhibitor as a method of terminating early pregnancy.

It is a further object of the present invention to provide a pharmaceutical composition which can be used to carry out such processes under practical conditions. Further study of the disclosure of the present invention and the appended claims will illustrate other possible applications and advantages of the invention to those skilled in the art.

One aspect of the present invention is to provide certain processes, and in this regard, it is to be understood that among these

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999 procedures include a method of increasing the frequency of implantation after IVF or of resolving early pregnancy loss by applying either a nitric oxide substrate or a nitric oxide donor, or both, alone or in combination, to female mammals exhibiting these symptoms. with prostgesterone alone, or in combination with estrogen, in effective amounts to alleviate symptoms, wherein the amount of nitric oxide synthase substrate and nitric oxide donor, or both, effectively increases implantation frequency and fertility by increasing circulating blood levels of L-arginine in the female treated, at least a level corresponding to 10 to 500 pmoles above the normal levels of circulating L-arginine of 50 to 1000 pmoles or an increase in nitric oxide donor levels to about 10 nM to 100 μΜ (micromole), the amount of progestational agent being bioequivalent to 10 to 300 milligrams injected progesterone, and the amount of estrogen is bioequivalent to approximately 2 milligrams per day of estradiol (Progynova.RTM., Schering).

Further, in this aspect of the various methods of the present invention, the present invention also relates to a method of regulating fertility which comprises administering to a female mammal a nitric oxide inhibitor and an antiprogestin, alone or in combination with a progesterone synthase inhibitor. In one possible application, fertility regulation occurs prior to implantation. A nitric oxide inhibitor, preferably an iNOS inhibitor, can be used in combination with an antiprogestin to inhibit implantation after administration during the perioovulatory period (maximum LH +/- 4 days), as an emergency contraceptive (e.g. within 72 hours after unprotected sexual intercourse), to inhibit endometrial receptivity with continuous daily use, to inhibit endometrial receptivity

once weekly, to induce menstruation (for example, during the first week of amenorrhea in the fertile cycle), or periodically to induce menstruation every 28 +/- 3 days.

In another aspect, the present invention provides a variety of products, wherein the invention relates to a pharmaceutical composition comprising at least one nitric oxide synthase substrate (L-arginine) and a nitric oxide donor (e.g., sodium nitroprusside or glyceryltrinitrate) alone, or in another combination with one or more of the following:

progesterone / progestin and / or estradiol / estrogen, wherein the amount of nitric oxide synthase substrate, nitric oxide donor or both per dose unit is sufficient to increase the circulating blood L-arginine level to a minimum of 10 to 500 μΜ above normal levels 50 to 1000 μΜ of circulating L-arginine in the blood, or to raise the nitric oxide donor level to approximately 10 nM to 100 μΜ, the estrogen level being bioequivalent to about 2 milligrams of estradiol (for example Progynova®, Schering), the progesterone amount bioequivalent to 5-300 milligrams of injected progesterone.

As regards further aspects of the various products of the present invention, the invention also relates to a pharmaceutical composition comprising at least one nitric oxide synthase inhibitor, preferably an iNOS inhibitor (e.g. L-NMMA, L-NIO, L-NIL, aminoguanidine , AMT, 4-methyl-2-aminopyridine,

6,4-dimethyl-2-aminoyridine or 2-aminopyridine) in an effective dose in combination with the antiprogestin alone and / or in another combination with a progesterone synthase inhibitor.

• · fe fe fe fe • fefe fefe fefe

With the methods of the present invention, it is possible to achieve an increased frequency of implantation and fertility after IVF, while at the same time ensuring the prevention of early pregnancy loss in a pregnant woman (female mammal) who exhibits symptoms of these possible problems.

Because low percentages of implantation and birth rates and early pregnancy loss are due to (or worsening) blood supply to the congenital fetus due to insufficient or subnormal nitric oxide synthesis, both substrates of nitric oxide synthase, i.e. L-arginine and donor nitric oxide, for example sodium nitroprusside, nitroglycerin, glyceryl trinitrate, SIN-1, isosorbide mononitrate, isosorbide dinitrate and diethylenetriamine / NO (DETA / NO), effective in ameliorating these symptoms, and in one aspect of the present invention a combination of both these characters.

When the progestagenic and / or estrogenic preparation is applied concomitantly with the nitric oxide substrate and / or nitric oxide donor, the effect is additive. In the case of female mammals, the progestagenic agent may be administered concurrently with the estrogen or instead of the estrogen.

Therefore, both in the aspect of the various processes of the present invention and in the case of the different products of the present invention (pharmaceutical compositions), the invention utilizes both a nitric oxide substrate and a nitric oxide donor, optionally one or more estrogens (e.g. Progyn®, Schering) or progestins. (for example, progesterone or hydroxyprogesterone caproate (Proluton® Depot), etc.).

Φ ·· • φ

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Thus, with respect to an aspect of the various methods of the present invention, a synergistic or additive effect is achieved for fertility control when the progesterone receptor antagonist (antiprogestin) and / or the progesterone synthase inhibitor are co-administered with a nitric oxide synthase inhibitor, preferably an iNOS inhibitor.

The following are examples of dosage ranges for typical NO substrates and NO (oral) donors:

total dose

L-arginine sodium nitroprusside nitroglycerin isosorbide mononitrate isosorbide dinitrate

500 mg - 10 g p.o. 500 - 2000 gg / kg / day

0.5 - 10 mg 10 - 100 mg 10 - 100 mg

Examples of combinations of active agents that can be co-administered with a nitric oxide substrate and / or a nitric oxide donor are the following estrogens and progestins with typical oral dosage ranges of estrogen and progestin active ingredients with the nitric oxide substrate or donor:

Estrogens:

a daily dose bioequivalent to about 1 to 2 mg per day, for example Premarin®, Vyeth-Ayerst, 0.625 mg / day, estradiol 25 to 100 gg / day transdermally or vaginal estradiol

• Gels or creams.

Progestins:

a daily dose bioequivalent to 50 to 300 milligrams of progesterone / day, for example hydroxyprogesterone caproate i.m. to provide a weekly dose of 100 to 1000 mg of the same or tablet or dragee providing an effective oral dose of micronized progesterone, or progesterone vaginal gel at a daily dose of 2-300 mg / day.

Examples of progestins are as follows:

Product Ingredients Dose ProlutonR Depot (Schering) i.m. hydroxyprogesterone- kaproát 250-1000 mg / week Progesterone-Depot (J enapharm) i.m. hydroxyprogesterone- kaproát 250-1000 mg / week

Examples of estrogens are as follows: Product Ingredients Dose (mg / day) Climaval (Sandoz). estradiol valerate 0.5-4 mg Progynova (Schering) estradiol valerate 0.5-4 mg

· · · · · · · · · · · · · · · · · · · · · · ·

Examples of antiprogestins are as follows:

11β- [4-N, N- (dimethylamino) phenyl] -17α-hydroxy-17β (3-hydroxypropyl) -13α-methyl-4,9 (10) -gonadien-3-one,

11β- (4-acetylphenyl) -17β-hydroxy-17α- (3-hydroxyprop 1 (Z) -enyl) -4,9 (10) -estradien-3-one (EP-A 0 190 759),

11β, 19- [4- (cyanophenyl) -o-phenylene] -17b-hydroxy17a- (3-hydroxyprop-1 (Z) -enyl) -4-androsten-3-one (WO-A 93/23020),

11β, 19- [4- (3-pyridinyl) -o-phenylene] -17β-hydroxy-17α- (3-hydroxyprop-1 (Z) -enyl) -4-androsten-3-one (WO-A 93/23020) .

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Examples of iNOS inhibitors are as follows:

Dose L-NMMA (Νθ-monomethyl-L-arginine) 1 to 1000 mg / day L-NIO (L-N 1 - (1-iminoethyl) ornithine) 1 to 1000 mg / day L-NIL (L-N 4 - (1-iminoethyl) lysine) 1 to 1000 mg / day aminoguanidine 1 to 1000 mg / day AMT (2-amino, 5,6-dihydro-6-methyl-4H-1,3-thiazine) (Abbott) 0.1 to 100 mg / day 4-methyl-2-aminopyridine 0.1 to 100 mg / day 6,4-dimethyl-2-aminopyridine 0.1 to 100 mg / day 2-aminopyridine 0.1 to 100 mg / day

The pharmacologically active compounds used in the present invention can be administered as a mixture of conventional excipients, i. pharmaceutically acceptable liquids, semi-liquid or solid organic or inorganic carrier substances suitable, for example, for parenteral or enteral administration, and substances which do not react undesirably with the active ingredient in the composition. Suitable pharmaceutically-acceptable carriers include, but are not limited to, the scope of the present invention. Water, salt solutions, alcohols, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, monoglycerides and diglycerides of fatty acids, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinypyrrolidone, and the like.

The pharmaceutical preparations may be sterilized and, if necessary, mixed with adjuvants such as lubricants, preservatives, stabilizers, humectants, emulsifiers, salts for affecting the osmotic pressure, buffers, colorants, flavors and / or flavorings and the like other substances which do not react undesirably with the active substances.

For parenteral administration, solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories, transdermal patches and vaginal gels, creams and foams, are particularly suitable. Ampoules represent suitable dosage units.

Preferably, a combination of substances used in the present invention that is adapted for digestion is used.

Particularly suitable for enteral administration are unit dosage forms, for example tablets, dragees or capsules containing talc and / or a carbohydrate carrier or binder or the like, the carrier material being preferably lactose and / or corn starch and / or potato starch, solid particulate matter, for example granules and liquids or semi-liquid, for example syrups and elixirs, and the like, using sweetened vehicle. It is also possible to formulate the compositions in a sustained release form, including those in which the active compound is protected by a coating with a different degradation rate, for example in the form of microcapsulation, multilayer coatings, and the like.

Tablets, coated tablets, capsules, pills, granules, suspensions and solutions are suitable, for example, for oral administration. Each dosage unit, for example each teaspoon of liquid or each tablet or dragee, contains, for example, 5 to 5000 milligrams of each active ingredient.

For transdermal administration, transdermal patches and gels are suitable, inter alia.

Solutions for parenteral administration also contain, for example, 0.01 to 1% of each active ingredient in an aqueous or alcoholic solution.

The nitric oxide substrate and / or donor may be administered as a mixture with an estrogen and / or progestational agent and any other optional active ingredient or as a separate unit dosage form, either simultaneously or each at different times of the day.

The active agent combination is ideally applied at least once a day (unless administered in a dosage form that releases the active ingredients continuously) and more preferably several times a day, for example in 2 to 6 divided doses. A typical dose is about 0.5 to 1000 milligrams of each active ingredient, although some less active ingredients, such as L-arginine, need much higher oral doses, such as 500 to 10,000 milligrams, and others, such as sodium nitroprusside, require lower doses, for example 500 to 2000 g / kg / day. Doses of nitroglycerin are typically administered orally at 2.6 milligrams twice daily, sublingually at 0.8 milligrams 1 to 4 times per day, and transdermally at 0.2 to 0.5 milligrams / hour. The dosage form used in the preferred embodiment is transdermal administration. Since the LD q hodnoty values of most of these active substances are known from the prior art, administration can be initiated with a lower dosage regimen and increased until a positive effect is achieved, or a higher dosage regimen can be initially used, e.g. a condition where symptoms disappear. The substance combinations can be used either continuously or sequentially.

In humans, both L-arginine and progesterone (or a bioequivalent to another progestin) should be administered in a ratio that provides levels of 50-5000 gmol of L-arginine in the plasma, 10-100 nmol of estradiol and 100-1000 nmol of progesterone.

The nitric oxide synthase inhibitor, preferably an iNOS inhibitor, may be administered in admixture with an antiprogestin and / or a progesterone synthase inhibitor (e.g. epostane, trilostane) and any optional active ingredient or as a separate dosage form, either simultaneously or each in a different daily time.

Description of the attached figures

Various other possible applications, characteristics and other advantages of the present invention will be more appreciated in the following with reference to the accompanying drawings in which like references (like reference symbols) designate like or similar parts in all instances of their occurrence. pictures as follows:

Giant. 1 shows the effects of L-NAME (NOS inhibitor) after implantation in rats. Animals were administered L-NAME at doses of 25 and 50 mg / animal / day in a continuous s.c. infusion from day 1 postcoital (p.c.) until day 7 p.c. An autopsy was performed on day 12 p.c.

Giant. 2 shows the effects of L-NAME (NOS inhibitor) after implantation in rats. Animals were administered L-NAME at doses of 25 and 50 mg / animal / day in a continuous s.c. infusion 5.-7. day (p.c.) An autopsy was performed on either day 9 p.c. or on day 12 p.c.

Giant. 3 shows the effects of L-NAME (NOS inhibitor, 50 mg / animal, day; continuous sc infusion) alone, onapristone at low dose (0.3 mg / animal, sc, oil injection) alone or in combination with L-NAME (50 mg / animal, day, continuous sc infusion) plus onapristone at low dose (0.3 mg / animal, sc injection in oil) after implantation in rats. Administration to animals was performed 5-8. den p.c., ie. in the early phase after implantation. An autopsy was performed on day 9 p.c. In this case, note the significant (p <0.05) increase in the number of pathological implant sites (small and blood stained implant sites) and the reduction in size and weight of the implant sites after L-NAME plus application. and onapristone.

Giant. 4 shows concentrations in different groups with L-NAME (NOS inhibitor, 50 mg / animal / day; continuous sc infusion) alone, onapristone at low dose (0.3 mg / animal, sc injection in oil) alone and a combination of L- NAME (50 mg / animal / day, continuous sc infusion) plus onapristone at low dose (0.3 mg / animal, sc injection in oil) as described in Figure 3.

Giant. 5 shows the effects of L-NAME (NOS inhibitor, 50 mg / animal / day; continuous sc infusion) alone, onapristone at a low dose (0.3 mg / animal, sc injection in oil) alone and a combination of L-NAME (50 mg / animal) animal / day, continuous sc infusion) plus onapristone at low dose (0.3 mg / animal, sc injection in oil) after implantation in rats. Administration to the animals was performed on day 1-4 of p.c., i.e. during the pre-receptive phase. An autopsy was performed on day 9 p.c. In this case, note the significant (p <0.05) inhibition of endometrial receptivity (no receptivity sites in 6/7 animals) and a radical reduction in the size and weight of implant sites (in 1/7 animals) after L-NAME plus onapristone administration. .

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in more detail with reference to the following non-limiting examples. A description of the pharmacological experiments performed is also given, the results of which are shown in the accompanying drawings.

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Experiment 1

Effect of L-NAME on endometrial receptivity on early implantation in rats after application of post coitum days 1-7 (p.c.).

In the experiments whose results are shown in Fig. 1, pregnant rats (n = 6 / group) were administered L-NAME at doses of 25 and 50 mg / animal / day from day 1 p.c. until day 7 p.c., i.e. during the pre-implantation and early implantation periods (in rats, implantation takes place on day 5 p.c.). Control animals received vehicle. At necropsy (day 9 (study part 1) and day 12 pc (study part 2); n = 6 / group in each part), implantation sites were evaluated microscopically, their diameter was measured and the weight of isolated implantation sites (embroynal) was recorded. and placental tissue).

Experiment 2

Effect of L-NAME on Frequent Implantation in Rats after Application on Days 5-7 p.c.

Giant. 2 shows the results of a second study in pregnant rats receiving L-NAME (25 and 50 mg / animal / day) on days 5-7, i.e. shortly after nidation. An autopsy was performed on day 12 p.c. Implants showing haemorrhagic changes were defined as pathological implantation sites. Based on these results, it can be concluded that the nitric oxide synthase inhibitor, L-NAME, has a significant effect on implantation. Dose-dependent inhibition of the size and weight of implantation sites was observed. In addition, L-NAME induced pathological changes at the implant site (mainly bleeding).

99 · · · · · · · · · · · · · · · ·

8 9

Experiment 3

Effect of L-NAME in combination with low dose antiprogestin (onapristone) on early implantation after application on days 5 to 8 p.c. rats.

The aim of the study was to evaluate whether nitric oxide interacts with progesterone during early implantation in rats. Rats (n = 8 / group) were days 5 to 8 p.c. the following substances were administered: L-NAME (50 mg / day, sc infusion)), onapristone (progesterone antagonist) at subabortif patient dose (0.3 mg / day, sc), L-NAME plus onapristone at equivalent doses and vehicle (controls) . The uterus was removed for morphometric and histological examination of implantation sites on day 9 p.c.

Results: Both L-NAME and minidose of onapristone significantly (a = 0.05) significantly reduce the mean and weight of implant sites when administered at an early stage of pregnancy (Fig. 3). Decidual tissue size decreased after application of L-NAME and onapristone. However, normal mesometrial and antimesometrial decidualization was maintained. The combination of L-NAME and onapristone radically reduced the diameter and weight of implant sites, accompanied by a radical reduction in the extent of decidualization, decidial cell necrosis and embryos. Neither application showed any significant effect on serum progesterone concentrations (Fig. 4), indicating that the effect of L-NAME plus onapristone on implantation was a direct effect.

Conclusions: NOS inhibition does not alter endometrial receptivity. However, when administered early in pregnancy, both NOS inhibition and the application of an ultra-low dose of onapristone alone attenuated the decidualization process and delayed the development of implant sites. The combination application resulted in a synergistic effect and a significantly impaired implantation.

9 9 9 9 9 999 999

9 9 99 9 9 9 9

9 1 · · 9 9 9 1 and 9 1 • 9 9 9

Experiment 4

Effect of L-NAME in combination with a low dose of antiprogestin (onapristone) on endometrial receptivity after application on Days 1-4 p.c. in rats.

In this study, pregnant rats (n = 7 / group) received L-NAME (50 mg / animal / day, sc infusion) with a low dose of onapristone (0.3 mg / animal / day) during pre-receptive phase 1.- 4. den p.c. An autopsy was performed on day 9 p.c.

The results of the study are presented in Fig. 5. Control animals (7/7) showed normal implantation sites. Administration of L-NAME alone had minimal effect on endometrial receptivity for embryo implantation and macroscopically normal implantation sites were found in all animals (7/7) during autopsy. Administration of onapristone alone led to a reduction in the number of implant sites and there were no implant sites at all in most animals (5/7). In contrast, administration of L-NAME plus onapristone completely prevented implantation in 6/7 animals. Of the remaining two animals (2/7), one had morphologically normal implantation sites, but was very small (Fig. 5). The other animal had morphologically abnormal (hemorrhagic), extremely small implantation sites. From this study, it can be concluded that combined administration of L-NAME plus onapristone, when imaplication occurred during the pre-receptive phase, prevented implantation (inhibited uterine receptivity to embryo implantation).

Experiment 5

Spatial-temporal distribution of nitric oxide synthase (NOS) isoforms at implantation sites in mice.

The aim of this study was to detect the presence of or fefe fefe ‘i β · ···· <

• 1 absence of the three major NOS isoforms (iNOS, ecNOS and bcNOS) during nidation in mice. Implant sites from mid-4th day to mid-7th day of pregnancy were examined for the presence of NOS isoforms. Mice were anesthetized and implantation sites were removed, fixed in 4% paraformaldehyde, embedded in paraffin, sliced and stained using standard immunohistochemical techniques. Polyclonal antibodies (Transduction Laboratories) against iNOS, ecNOS and bcNOS were used. INOS staining was most pronounced in macrophages and monocytes between the inner and outer muscle layers of the myometrium in all time intervals examined. Decidua also showed positive staining scattered throughout the tissue. An increase in iNOS positive cells relative to implantation sites was found. ecNOS showed very pronounced staining on days 6 and 7 in the primary decidual zone adjacent to the anti-mesometrial pole of the embryo. Staining was also present in the vessels of the myometrium at all times, as well as in the basal portion of the luminal epithelial cells prior to implantation. Specific staining of bcNOS was observed predominantly in the outer layer of myometrium and within the mesometrium without major differences between time periods. Negative and positive controls showed sufficient staining. In conclusion, these data show that both iNOS and ecNOS show increased expression in mice during the peri-implantation period.

The results of these studies (both functional and histochemical) show that nitric oxide plays a crucial role during implantation. It appears to be important for placental perfusion throughout the implantation process. Nitric oxide deficiency in the uterus can lead to either resorption of implant sites or spontaneous abortion. Therefore, nitric oxide donors or substrates, alone or in combination with steroid hormones (progesterone estradiol), effectively increase frequency.

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 In addition, combinations of nitric oxide inhibitors with antiprogestins and / or progesterone synthase inhibitors will effectively prevent or prevent pregnancy.

Example 1

Increased frequency of implantation with nitric oxide substrate after in vitro fertilization.

A woman (50-90 kg) who has received IVF is treated with L-arginine at a dose of 0.5 to 20 grams orally daily in three divided doses for the first 2 to 6 weeks of pregnancy or longer.

Example 2

Increased frequency of implantation by nitric oxide donor after in vitro fertilization.

Nitroglycerin (5-15 milligrams / day) is administered transdermally to a woman (50-90 kilograms) in IVF for the first 2 to 6 weeks of pregnancy or longer.

Example 3

Treatment of infertility with nitric oxide substrate.

An infertile woman (weighing 50 to 90 kilograms) is administered L-arginine at a dose of 0.5 to 20 grams orally daily in three divided doses.

Example 4

Treatment of infertility by a nitric oxide donor.

Nitroglycerin (5 to 15 milligrams / day) is transdermally administered to a infertile woman (50-90 kilograms).

Example 5

Increased frequency of nitric oxide substrate implantation in combination with progestron after in vitro fertilization.

A woman (50-90 kg) who has undergone IVF is transdermally injected with nitroglycerin (5-15 milligrams / day) in combination with progesterone (Proluton® Depot / Schering / 250-1000 milligrams / week im) for the first 2 to 6 weeks of pregnancy or longer.

Example 6

Infertility treatment with nitric oxide substrate in combination with nitric oxide substrate after in vitro fertilization.

A infertile woman (50-90 kilograms) is transdermally administered nitroglycerin (5-15 milligrams / day) in combination with L-arginine at a dose of 0.5 to 20 grams orally per day in three divided doses.

Example 7

Increased frequency of nitric oxide substrate implantation in combination with progesterone and estradiol after in vitro fertilization.

Nitroglycerin (5 to 15 milligrams / day) in combination with progesterone (Proluton® Depot (Schering) 250 to 1,000 milligrams / week) is transdermally administered to a woman (50-90 kg) who has received IVF. 99 99 9 9 9

9 • 9 99

i.m.) and estradiol valerate at a dose of 0.5 to 4 milligrams / day for the first 2 to 6 weeks of pregnancy or longer.

Example 8

Postcoital contraception with nitric oxide synthase inhibitor in combination with antiprogestin.

0.5 to 400 milligrams of antiprogestin / day (e.g., mifepristone) are administered in combination with an inhibitor of nitric oxide synthase, preferably an iNOS inhibitor, at an effective dose within 72 hours of sexual intercourse.

Example 9

Endometrial contraception by a nitric oxide synthase inhibitor in combination with an antiprogestin for daily administration.

Antiprogestin is administered at a daily dose that does not prevent ovulation (e.g., 0.1 to 1 milligram of mifepristone / day) in combination with a nitric oxide synthase inhibitor, preferably an iNOS inhibitor at an effective dose.

Example 10

Endometrial contraception with a nitric oxide synthase inhibitor in combination with an antiprogestin given once weekly.

Once a week, antiprogestin is administered at a dose that does not prevent ovulation (e.g., 5 to 20 milligrams of mifepristone / week) in combination with a nitric oxide synthase inhibitor, preferably an iNOS inhibitor at an effective dose.

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φ * φ φ · · ΦΦΦ ♦ φ φ φ

Example 11

Induction of menstruation by a nitric oxide synthase inhibitor in combination with an antiprogestin.

Antiprogestin (eg, mifepristone) is administered at a dose of 0.5 to 400 milligrams / day in combination with a nitric oxide synthase inhibitor, preferably an effective iNOS inhibitor, on day 28 +/- 3 days of the menstrual cycle.

Example 12

Induction of menstruation by a nitric oxide synthase inhibitor in combination with a progesterone synthase inhibitor.

A progesterone synthase inhibitor (eg, epostane) is administered at a dose of 1 to 600 milligrams / day in combination with a nitric oxide synthase inhibitor, preferably an iNOS inhibitor at an effective dose, on day 28 +/- 3 days of the menstrual cycle.

Example 13

Induction of menstruation with a nitric oxide synthase inhibitor in combination with an antiprogestin and in another combination with a progesterone synthase inhibitor.

Antiprogestin (e.g., mifepristone) is administered at a dose of 0.5 to 400 milligrams / day in combination with a progesterone synthase inhibitor (e.g., epostane) at a dose of 1 to 600 milligrams, and a nitric oxide synthase inhibitor, preferably an iNOS inhibitor at an effective dose, 28. day +/- 3 days of the menstrual cycle.

The foregoing examples can be readily repeated by one of ordinary skill in the art with similar success by replacing the generic or specifically described substances and / or application materials. · 4

44 of the present invention in the previous examples.

From the foregoing description, one of ordinary skill in the art will readily recognize the essential characteristics of the invention and, without departing from its spirit and scope, can make various changes and modifications to the invention and adapt it to different uses and conditions.

Claims (50)

PATENT CLAIMS ·· ftft ft ft ft A method for increasing the frequency of implantation in pregnant female mammals after in vitro fertilization, comprising administering to said affected female mammal an effective amount of a nitric oxide synthase substrate, a nitric oxide donor, or both, to increase circulating L-arginine levels. in the blood to at least about 50-5000 pmol above normal levels of 50-1000 gmol circulating levels, and optionally progestin or even estrogen and progestin in effective amounts that increase the frequency of pregnancy. The method of claim 1, wherein the mammal is a non-pregnant human being of the female genus suffering from infertility, or a pregnant woman suffering from regular abortion or a pregnant woman exhibiting symptoms of imminent abortion. The method of claim 1, wherein the nitric oxide substrate is L-arginine. 4. The method of claim 1 wherein the mammal is a pregnant human being of the female genus and is administered a nitric oxide donor. The method of claim 4, wherein the nitric oxide donor is sodium nitroprusside, nitroglycerin, glyceryl trinitrate, SIN-1, isosorbide mononitrate or isosorbide dinitrate. The method of claim 4, wherein the nitric oxide donor is administered orally. φ φ φ · · · · ΦΦΦ ΦΦΦ φ φ φ · β β ΦΦ φ φ φ · · · · · • · · · 7. The method of claim 4 wherein the nitric oxide donor is administered transdermally. 8. The method of claim 1, wherein the mammal is a pregnant human female being and the nitric oxide substrate or donor is administered in combination with progesterone. The method of claim 1, wherein the mammal is a pregnant human female being and the nitric oxide substrate or donor is administered in combination with progesterone and / or estradiol. The method of claim 8, wherein the progestin is progesterone or hydroxyprogesterone caproate. The method of claim 9, wherein the estrogen is estradiol valerate. 12. A method of regulating fertility, characterized in that it is carried out using a nitric oxide synthase inhibitor in combination with an antiprogestin. The method of claim 12, wherein the antiprogestin is mifepristone, ORG 31710, ORG 33 628, J867, CDB 2914 or ZK 137316. 14. The method of claim 12, wherein the administration is postcoital. 15. A pharmaceutical composition comprising a mixture of φ · Φ 4 ΦΦΦ Φ (A) a nitric oxide synthase substrate, (b) a nitric oxide donor, or both (c) a progestin; and (d) an estrogen in an effective amount that increases the frequency of pregnancy or alleviates abortion symptoms in a pregnant or infertile female mammal when administered in an effective amount in the form of an estrogen equivalent to 1. up to 2 milligrams of estradiol and progestin with a bioequivalent dose of 50-300 milligrams of injected progesterone and an amount of nitric oxide synthase substrate, nitric oxide donor, or both that effectively raises circulating L-arginine levels to at least 50-5000 pmol above normal levels of 50 to 1000 gmol or increases nitric oxide donor levels to about 10 nM to 100 μιηοΐ. The pharmaceutical composition of claim 15, wherein (a) is a nitric oxide synthase substrate. Pharmaceutical composition according to claim 16, characterized in that the nitric oxide synthase substrate (b) is L-arginine. The pharmaceutical composition of claim 14, wherein (b) is a nitric oxide donor. The pharmaceutical composition of claim 18, wherein the nitric oxide donor is sodium nitroprusside, nitroglycerin, glyceryltrinitride, SIN-1, isosorbide mononitrate or isosorbide dinitrate, etc. 20. The pharmaceutical composition of claim 15. 99 99 φ · ·· · · · · · ··· ··· 99 99 9 9 999 9, wherein the progestin (d) is progesterone or hydroxyprogesterone caproate. 21. The pharmaceutical composition of claim 15 wherein the estrogen (c) is estradiol valerate. The method of claim 12, wherein the NOS inhibitor is L-NMMA, L-NIO, L-NIL, aminoguanidine, AMT, 4-methylaminopyridine, 6,4-dimethyl-2-aminopyridine or 2-aminopyridine . The method of claim 12, wherein the pharmaceutical composition comprises in a mixture of (a) a nitric oxide inhibitor and (b) an antiprogestin for fertility control purposes. 24. The pharmaceutical composition of claim 23 wherein the NOS inhibitor is L-NMMA, L-NIO, L-NIL, aminoguanidine, AMT, 4-methylaminopyridine, 6,4-dimethyl-2-aminopyridine or 2-aminopyridine. 25. A method for increasing the frequency of implantation and / or pregnancy in a female mammal, comprising administering to said female mammal in which pregnancy is desired, an effective amount of (a) a nitric oxide synthase substrate, a nitric oxide donor, or both, optionally in combination with (b) a progestin and (c) optionally in another combination with an estrogen. 26. A method according to claim 25, wherein the mammal is a non-pregnant human being of a female gender suffering from infertility, a pregnant woman suffering from repeated abortion, or a pregnant woman exhibiting symptoms of imminent abortion. . 27. The method of claim 25, wherein the nitric oxide synthase substrate is L-arginine. 28. The method of claim 25, wherein the mammal is a non-pregnant human being of the female genus and is administered a nitric oxide donor. The method of claim 28, wherein the nitric oxide donor is sodium nitroprusside, nitroglycerin, glyceryltrinitride, SIN-1, isosorbide mononitrate or isosorbide dinitrate, etc. 30. The method of claim 28 wherein the nitric oxide donor is administered orally. 31. The method of claim 28 wherein the nitric oxide donor is administered transdermally. 32. The method of claim 25, wherein the mammal is a pregnant human female being and the nitric oxide substrate or donor is administered in combination with a progestin. 33. The method of claim 25, wherein the mammal is a pregnant human female being and the nitric oxide substrate or donor is administered in combination with a progestin and an estrogen. 34. The method of claim 32, wherein the progestin is progesterone or hydroxyprogesterone caproate. The method of claim 33, wherein the estrogen is estradiol valerate. 36. A method of regulating fertility of female mammals, comprising administering to said affected female mammal in which pregnancy is not desired and at risk of developing pregnancy, an effective amount of a nitric oxide synthase inhibitor in combination with an antiprogestin. The method of claim 36, wherein the antiprogestin is mifepristone, ORG 31 10, ORG 33 628, J867, CDB 2914 or ZK 137316. 38. The method of claim 36, wherein the administration is postcoital. 39. The method of claim 25, wherein the mammal is a human human being, and the amount of nitric oxide synthase substrate, nitric oxide donor, or both applied effectively increases circulating levels. L-arginine in a woman's blood to a value of at least 50-5000 pmol. 40. The method of claim 25, wherein the mammal is a human human being, and the amount of nitric oxide synthase substrate, nitric oxide donor, or both applied effectively raises nitric oxide donor levels to about 1-1000 nmol. 41. The method of claim 25, wherein the mammal is a human human being and the amount of progestin administered is bioequivalent to 50 to 300 milligrams of injected progesterone, and the amount of estrogen administered when administered is bioequivalent to 1 to 2 milligrams. estradiol. 42. A pharmaceutical composition comprising a mixture of effective amounts of (a) a nitric oxide synthase substrate, a nitric oxide donor, or both; and (b) a progestin and optionally (c) estrogen in amounts effective to increase or reduce pregnancy rate. wherein the amount of nitric oxide synthase substrate, nitric oxide donor, or both effectively increases circulating levels of L-arginine in said mammalian strain to which the combination is administered at least about 50-5000 gmol above the normal 50-1000 μπιοί circulating levels and / or effectively raises nitric oxide donor levels to about 1-100 nmol; the amount of progestin administered is bioequivalent to the dose of 50 to 300 milligrams of injected progesterone and the amount of estrogen administered, if administered, is bioequivalent to the dose of 1 to 2 milligrams of estradiol. ⁱⁿ 43. The pharmaceutical composition of claim 42 wherein (a) is a nitric oxide synthase substrate. 44. The pharmaceutical composition of claim 43, wherein the nitric oxide synthesis substrate is L-arginine. • «• 4 • 4 • · ·· 44 45. The pharmaceutical composition of claim 42 wherein (a) is a nitric oxide donor. A combination according to claim 45, wherein the nitric oxide donor is sodium nitroprusside, nitroglycerin, glyceryltrinitide, SIN-1, isosorbide mononitrate or isosorbide dinitrate. 47. The pharmaceutical composition of claim 42, wherein (b) is progesterone or hydroxyprogesterone caproate. 48. The pharmaceutical composition of claim 42, wherein (c) is estradiol valerate. 49. A pharmaceutical composition according to claim 25 wherein components (a) and (b) are applied sequentially. 50. The pharmaceutical composition of claim 25 wherein components (a) and (b) are administered simultaneously.