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United States Patent 19 11) Patent Number: 5,643,944 Garfield Et Al US005643944A United States Patent 19 11) Patent Number: 5,643,944 Garfield et al. 45 Date of Patent: Jul. 1, 1997 (54) OVULATION CONTROL BY REGULATING 4,851,385 7/1989 Rueske ...................................... 54/15 NTRC OXDE LEVELS 5,068,221 11/1991 Mathias ... ... 514/5 5,130,137 7/1992 Crowley, Jr. ............................ 424/422 75) Inventors: Robert E. Garfield, Friendswood; HER Chandrasekhar Yallampalli, Houston, OT PUBLICATIONS both of Tex. Bonello et al., "Inhibition of Nitric Oxide: Effects on Interleukin-1B-Enhanced Ovulation Rate, Steriod Hor 73) Assignee: Board of Regents, The University of mones, and Ovarian Leukocyte Distribution at Ovulation in Texas System, Austin, Tex. the Rat, "Biology of Reproduction, 54:436.445, 1996. Mani et al., "Nitric Oxide Mediates Sexual Behavior in 21 Appl. No.: 477,189 Female Rats," Proc. Natl. Acad. Sci., 91:6468-64.72, Jul. 1994. (22 Filed: Jun. 7, 1995 Shukovski and Tsafriri, "The Involvement of Nitric Oxide in M the Ovulatory Process in the Rat," Endocrinology, Van Voorhis et al., "Nitric Oxide: An Autocrine Regulator of 62 Elf Ser. No. 165,309, Dec. 10, 1993, Pat No. Human Granulosa-Luteal Cell Steroidogenesis.” 135(5):1799-1806, 1994. (51) Int. Cl. ....................... A61K 31/34; A61K 31/195; A61K 31/045 Primary Examiner. Theodore J. Criares 52 U.S. Cl. ......................... s14,470. 514/565: 514,742, Attorney, Agent, or Firm-Arnold White & Durkee 514/843 (57) ABSTRACT 58) Field of Search .............................. 514/15, 470,565 514/742,843 The stimulation of ovulation in a female may be achieved by administering a nitric oxide source, optionally in further 56 References Cited combination with one or more of clomiphene, a U.S. PATENT DOCUMENTS gonadotropin, and an LH-RH agonist. 4,339.434 7/1982 Ericsson .................................. 424/05 3 Claims, 1 Drawing Sheet U.S. Patent Jul. 1, 1997 5,643,944 Hypothalamus Anterior Pituitary Gland -- -- Estrogen PrOgesterOne ? / O \ Copa or Ova SSU?),Rise AdUE / --O- -D Graafian follicle Follicular Ovulatory Luteal Phase Phase Phase FIG. 1 5,643,944 1 2 OWULATION CONTROL BY REGULATING progesterone is required to initiate the LH surge. LHRH NTRC OXDE LEWELS synthesis in the hypothalamus stimulates the anterior pitu itary to synthesize and secrete LH and FSH. This is a divisional of application Ser. No. 08/165.309, A side view of the brain with hypothalamus and pituitary filed Dec. 10, 1993, now U.S. Pat. No. 5,470,847. enlarged is shown in FIG. 1. Also shown is the ovary in the follicular, ovulatory and luteal phase which produce estro FIELD OF THE INVENTION gen and progesterone in the follicular cells in response to The present invention relates to ovulation control by stimulation respectively from FSH and LH. High levels of agulating nitric oxide levels. To prevent ovulation, nitric progesterone and estrogen feed back on the hypothalamus oxide levels may be lowered ing a us nitric oxide synthesis O and negatively regulate the secretion of LH-RH or GnRH inhibitor, alone or in combination with at least one of a and to decrease production of LH and FSH. During periods progesterone, an estrogen, an antigonadotropin and a GnRH of low serum levels of estrogen and progesterone LH-RH antagonist or the like. Nitric oxide levels may be increased levels rise to stimulate synthesis of FSH and LH. However, to stimulate ovulation using a nitric oxide source, alone or both estrogen and progesterone also have positive feedback in combination with at least one of a gonadotropin and 5 control on the hypothalamus and some progesterone is clomiphene or the like. required for stimulating LH-RH. It is on the basis of this concept that the modern contraceptive "pill" is designed, BACKGROUND OF THE INVENTION Progestins and estrogens in the "pill" inhibit the synthesis of Ovulation is the process where an ovum or ova are LH-RH thus preventing the LH surge which is required for released from the ovaries. The timing of ovulation within the stimulation of growth, maturation and rupture of the Graa menstrual cycle is of foremost importance for fertilization. fian follicle. Since the life span of both spermatozoa and the unfertilized Female contraception methods are based upon the above ovum is limited, fertilization must take place within hours theory of the control of ovulation. Generally, all contracep after ovulation if conception is to occur during that men tive procedures are based upon the principal that high or strual cycle. 25 moderate progesterone or estrogen levels inhibit LHRH and Ovulation is under the control of circulating estrogen and the LH surge and thus prevent ovulation. Thus, estrogen progesterone levels from the ovary and gonadotropins from and/or progesterone are typically prescribed to inhibit ovu the pituitary. During the normal menstrual cycle in women lation. In the USA alone, about 75 million women take birth these hormones exhibit cyclic patterns. The menstrual cycle control pills to control ovulation and prevent pregnancy. The can be functionally divided into three phases; the follicular, methods of hormonal regulation of fertility can be outlined the ovulatory and luteal phases. The follicular period begins as follows: in the late luteal phase of the preceding cycle with a rise in 1. Oral contraception. blood levels of follicle stimulating hormone (FSH, a a) Cyclic combined estrogen-progesterone method, gonadotropin) and a concomitant initiation of ovarian folli b) Sequential estrogen and progestogen method. cular growth. Luteinizing hormone (LH, the other 35 c) Continuous (noncyclic) low-dose, progesterone only gonadotropin) blood levels also rise but start one or two days treatent. later than FSH levels. In the second half of the follicular 2. Long acting injectable hormone preparations. stage, ovarian secretion of estradiol (E) and estrone (E) by 3. Hormone-releasing intrauterine systems. the ovary increases slowly at first, then rapidly reaches a 4. Interception-usually a large dose of estrogen in cases maximum on the day before the LH peak. The rise in plasma of unprotected intercourse. estrogen levels is accompanied by a decrease in FSH levels. 5. Antiprogesterones-which block action of progester During the ovulatory phase there is a rapid rise in blood OS LH levels which leads to the final maturation of the ovarian 6. LHRH antagonists or agonists both of which interfere Graafian follicle, follicular rupture and discharge of the 45 with normal processes and inhibit steps in ovulation. ovum some 16 to 24 hours after the LH peak. Just prior to 7. Antigonadotropins-such as Danazol which is thought ovulation blood E levels drop dramatically and plasma to block implantation. progesterone levels begin to rise. Potential users of these hormone contraceptives should be Following ovulation, during the luteal phase, the post alerted to the fact that both hormone components may be ovulatory ovarian follicle cells are luteinized to form a 50 associated with a slightly increased risk of cardiovascular corpus luteum. The most important feature of the luteal disease. In an asymptomatic woman younger than 35 years, phase of the menstrual cycle is the marked increase in the risk is not a deterrent to use but should be considered progesterone secretion by the corpus luteum. There is a additive to other cardiac risk factors. Thus, Smaller increase in estrogen levels. As progesterone and hypercholesterolemia, hypertension, diabetes, heavy estrogens increase, LH and FSH decline throughout most of 55 Smoking, or a family history of early coronary disease may the luteal phase but FSH begins to rise at the end to initiate augment the risk. Discontinuance of oral contraceptives and follicular growth for the next cycle. use of an effective alternative should be considered in the Progesterone and estrogen secretion by the ovary are management of hypertension or major glucose intolerance. controlled respectively by levels of LH and FSH. Negative Use of these agents by women older than 35 years should be and positive feedback inhibition of progesterone and estro avoided by those who smoke and reevaluated for others. gens regulate the hypothalamus to control luteinizing Absolute contraindications to oral contraceptives include hormone-releasing hormone (LHRH also termed gonadot thrombotic disorders, known or suspected cancer of an ropin releasing hormone, GnRH). During periods of high estrogen-dependent organ (e.g., breast or uterus), impaired circulating blood levels of progesterone and estrogen, low liver function, pregnancy, undiagnosed vaginal bleeding, amounts of LH-RH are produced. Inversely, when proges 65 pregnancy-associated jaundice, and hyperlipidemia. In terone and estrogen levels are low in the blood, high many other disorders, a relative contraindication should be amounts of LH-RH will be produced. However, some individually evaluated and use of oral contraceptives cau 5,643,944 3 4 tiously explored. Because the frequency of arterial throm petitive inhibitor of NO synthase such as, for example, an bosis appears to be increased after elective surgery, it is N substituted arginine or arginine ester or an NN disubstituted arginine which is administered to a female recommended that oral contraceptives be discontinued a desiring contraception. The arginine analogues of the month before surgery. present invention are preferably of the L-configuration and The present invention offers many advantages over the include any pharmaceutically acceptable addition salts as normal hormonal regulation of ovulation because the meth commensurate with planned treatments. ods and compositions use either no estrogen and progester Preferred NO synthase inhibitors are N-substituted argi one or lower amounts of these hormones than current nine analogues of the L-configuration for uses as described methods. herein. These include N-aminoarginine, N-nitroarginine. Agents which stimulate ovulation also function by acting 10 and N-alkyl arginines such as N-methylarginine, on the above pathways. The best known agent which stimu N-ethylarginine, N-propylarginine or N-butylarginine. lates ovulation and is used for treatment of an ovulation is Many substituents, for example, on the guanidino group of clomiphene (MER 41).
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