III IIII USO05721278A United States Patent (19) 11) Patent Number: 5,721,278 Garfield et al. 45) Date of Patent: Feb. 24, 1998 54 OWULATON CONTROL BY REGULATING 58) Field of Search ............................ 514/15, 121, 651, NTRC OXDE LEVELS 514/652,561, 841, 843, 648 Inventors: Robert E. Garfield, Friendswood; 56 References Cited Chandrasekhar Yallampalli, Houston, U.S. PATENT DOCUMENTS both of Tex. 4,338,305 7/1982 Corbin ................................... 424/177 73 Assignee: Board of Regents, The University of 4,851,385 7/1989 Rueske ..................................... 514/15 Texas System, Austin,Tex. 5,470,847 11/1995 Garfield et al. ......................... 514/171. Primary Examiner. Theodore J. Criares 21 Appl. No.: 477,187 Attorney, Agent, or Firm-Arnold, White & Durkee 22 Filed: Jun. 7, 1995 57 ABSTRACT Inhibition of ovulation in a female may be achieved by Related U.S. Application Data administering a nitric oxide synthase inhibitor, alone or in 62 Division of Ser. No. 165,309, Dec. 10, 1993, Pat. No. combination with one or more of a progestin, an estrogen, 5,470,847. and an LH-RH antagonist, thereby preventing conception. The stimulation of ovulation in a female may be achieved by 51) Int. Cl. ......... A61K 31/195; A61K 31/135: administering a nitric oxide source, optionally in further A61K 31/56 combination with one or more of clomiphene, a 52 U.S.C. ....................... 514/652; 514/171; 514/561; gonadotropin, and an LH-RH agonist. 514/563; 514/651; 514/565; 514/841; 514/843; 514/648 4 Claims, 1 Drawing Sheet U.S. Patent Feb. 24, 1998 5,721,278 HypothalamuS Anterior Pituitary Gland ProgesterOne Estrogen / \ CorpOra OVa lutea \(es j \s 63 O Graafian follicle Follicular Ovulatory Luteal PaSe Phase Phase FIG. 1 5,721,278 1. 2 OWULATION CONTROL BY REGULATING amounts of LH-RH will be produced. However, some NITRC OXDE LEWELS progesterone is required to initiate the LH Surge. LHRH synthesis in the hypothalamus stimulates the anterior pitu This application is a divisional application of Ser. No. itary to synthesize and secrete LH and FSH. 08/165.309, filed Dec. 10, 1993, now U.S. Pat. No. 5,470, 5 Aside view of the brain with hypothalamus and pituitary 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 regulating nitric oxide levels. To prevent ovulation, nitric progesterone and estrogen feedback on the hypothalamus oxide levels may be lowered using a nitric oxide synthesis 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 15 both estrogen and progesterone also have positive feedback in combination with at least one of a gonadotropin and 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 20 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 25 tive procedures are based upon the principal that high or strual cycle. 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 30 control pills to control ovulation and prevent pregnancy. The can be functionally divided into three phases; the follicular, methods of hormonal regulation offertility 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 35 a) Cyclic combined estrogen-progesterone method. gonadotropin) and a concomitant initiation of ovarian foli b) Sequential estrogen and progestogen method. cular growth. Luteinizing hormone (LH, the other c) Continuous (noncyclic) low-dose, progesterone only gonadotropin) blood levels also rise but start one or two days treatment. 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 (E1) 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 45 6. LHRH antagonists or agonists both of which interfere Graafian follicle, follicular rupture and discharge of the 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 50 alerted to the fact that both hormone components may be ovulatory ovarian follicle cells are luteinized to form a 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 55 hypercholesterolemia, hypertension, diabetes, heavy estrogens increase, LH and FSH decline throughout most of 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 65 liver function, pregnancy, undiagnosed vaginal bleeding, amounts of LH-RH are produced. Inversely, when proges pregnancy-associated jaundice, and hyperlipidemia. In terone and estrogen levels are low in the blood, high many other disorders, a relative contraindication should be 5,721,278 3 4 individually evaluated and use of oral contraceptives cau embodiment, an inhibitor of the enzyme activity is a com 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. O herein. These include N-aminoarginine, N-nitroarginine, Agents which stimulate ovulation also function by acting 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 anovulation is Many substituents, for example, on the guanidino group of clomiphene (MER 41). Clomiphene is a nonsteroidal anti arginine or analogous citruline or ornithine functional estrogen that competes for estrogens at their binding sites.