Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 8 to 10 amino acid residues in defined sequence
Reexamination Certificate
1998-06-03
2001-10-09
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
8 to 10 amino acid residues in defined sequence
C514S015800, C530S399000
Reexamination Certificate
active
06300471
ABSTRACT:
This invention pertains to compositions and methods for selectively stimulating or inhibiting the release of follicle-stimulating hormone from the anterior lobe of the pituitary gland.
The brain controls the release of gonadotropin hormones from the anterior pituitary gland. Two important gonadotropins are follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH is critical for spermatogenesis and for ovarian follicle development, while LH is critical to androgen secretion in males, and estrogen secretion, ovulation, and formation of the corpus luteum ain females. A hormone with specific activity for releasing FSH but not LH could be used to increase fertility in humans or other animals, or to correct fertility problems caused by defective hypothalamic control of FSH secretion. Conversely, antisera or other antagonists to an FSH-specific releasing factor will inhibit FSH secretion, thereby inhibiting spermatogenesis in males, or inhibiting development of follicles and ovarian development in females, providing a new antifertility drug. It is also possible that very high doses of an FSH-specific releasing factor will inhibit FSH secretion, rather than stimulate it.
Prior indirect evidence has suggested that separate factors could be responsible for triggering the release of FSH and for triggering the release of LH in mammals. However, this hypothesis could not previously be confirmed, because no previous work successfully isolated a potent factor that selectively induces the release of FSH, but not LH. See McCann, S. et al., Control of follicle-stimulating hormone and luteinizing hormone release by hypothalamic peptides, Annals New York Academy of Sciences, 687:55-59, 1993; Dees, W. et al., Ethanol and the pulsatile release of luteinizing hormone, follicle stimulating hormone and prolactin in ovariectomized rats, Alcohol, 2:641-646, 1985; Dhariwal, A. et al., Separation of follicle-stimulating hormone-releasing factor from luteinizing hormone-releasing factor, Endocrinology, 76:290-294, 1965; Dhariwal, A. et al., Chromatographic behavior of follicle stimulating hormone-releasing factor on Sephadex and carboxy methyl cellulose, Neuroendocrinology, 2:294-303, 1967; Igarashi, M. et al., A hypothalamic follicle stimulating hormone-releasing factor, Endocrinology, 74:446-452, 1964; Lumpkin, M. et al., Effect of destruction of the dorsal anterior hypothalamus on follicle-stimulating hormone secretion in the rat, Endocrinology, 115:2473-2480, 1984; Samson, W. et al., Chromatographic and biologic analysis of ME and OVLT LHRH, Peptides, 1:97-102, 1980; Mizunuma, H., et al., Evidence for an FSH-releasing factor in the posterior portion of the rat median eminence, Life Sci., 33:2003-2009, 1983.
Luteinizing hormone releasing hormone (LHRH, also known as gonadotropin-releasing hormone, or GnRH) has both LH-releasing activity and FSH-releasing activity. See Schally, A. et al., Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle-stimulating hormones, Science, 173:1036-1038, 1971; and D. Lincoln, Gonadotropin-releasing hormone (GnRH): basic physiology, pp. 218-229 in L. DeGroot et al., Endocrinology, 1995. The latter states at page 218: “There is no convincing evidence for the existence of a separate and specific FSH-releasing hormone, although some components of the GNRH precursor and some ONRH analogues appear to differ in the degree to which they stimulate the secretion of the two gonadotropins.”
Sower, S. et al., Primary structure and biological activity of a third gonadotropin-releasing hormone from lamprey brain, Endocrinology, 132:1125-1131, 1993 reported the structure of lamprey GnRH-III (referred to as l-LHRH-III in this specification), and reported that it stimulated estradiol and progesterone release from
Petromyzon marinus
(lamprey) ovaries. (Lampreys, jawless fish, are representatives of what is generally considered to be the most primitive of the extant classes of vertebrates.)
Lamprey l-LHRH-I has been reported to have relatively low activity in releasing either FSH or LH in rats. Yu, W. et al., Selective FSH-releasing activity of [D-Trp
9
] GAP
1-13
: comparison with gonadotropin-releasing abilities of analogs of GAP and natural LHRHs, Brain Res. Bull., 25:867-873, 1990.
Schally, A. et al., Re-examination of porcine and bovine hypothalamic fractions for additional luteinizing hormone and follicle stimulating hormone-releasing activities, Endocrinology, 98:380-391, 1976 reported that in vivo FSH-releasing activity could not be separated from LH-releasing activity from porcine hypothalami by fractionation on Sephadex, and concluded that there was only one gonadotropin-releasing hormone (GnRH).
By contrast, Lumpkin, M. et al., Purification of FSH-releasing factor: Its dissimilarity from LHRH of mammalian, avian, and piscian origin, Brain Res. Bull., 18:175-178, 1987 reported that FSH-releasing activity was separated from the LH-releasing activity in ovine hypothalami on Sephadex G-25, but did not isolate the factor causing FSH release.
Neurons that are immunopositive for l-LHRH-I have been identified in human hypothalami, projecting from the arcuate region to the median eminence. Stopa, E. et al, Polygenic expression of gonadotropin-releasing hormone (GnRH) in human?, Peptides, 9:419-423, 1988.
Lincoln, D., Luteinizing Hormone-Releasing Hormone, pp. 142-151 in DeGroot et al. (ed), Endocrinology, 1989, discloses various agonists and antagonists for mammalian LHRH.
W. Yu et al., “A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat,” Proc. Natl. Acad. Sci. USA, 94:9499-9503, 1997 discloses some of the work reported in the present specification, but is not believed to constitute prior art.
U.S. Pat. No. 4,973,577 discloses a 28,000 dalton protein isolated from porcine follicular fluid that stimulates the release of FSH, but not of LH. This protein has a relatively slow onset of action, and is relatively difficult to synthesize. The protein was said to be a homodimer of two chains of 116 amino acid residues each, or 232 residues total.
U.S. Pat. No. 3,888,836 discloses a method for synthesizing mammalian LHRH. Mammalian LHRH causes increased serum levels of both LH and FSH.
U.S. Pat. No. 4,721,775 discloses certain peptides that non-selectively induce the secretion of both LH and FSH.
Attempts in our laboratory to purify FSH-releasing factor (FSH-RF) by fractionation of lamb hypothalami (discussed in some of the papers cited above) were successful only at certain seasons of the year, and even then we found that activity was lost after samples were stored at −20° C. (unpublished data). Thus our prior work did not successfully isolate or identify the putative FSH-releasing factor.
Other studies in our laboratory confirmed FSH-releasing activity by incubating stalk-median eminence (SME)-extracts in vitro with hemipituitaries from male rats. We confirmed the FSH-releasing activity of sheep and rat SME extracts in this assay, and found that the FSH-releasing activity emerged from columns of Sephadex G-25 just prior to emergence of LHRH, similar to results we had seen in an in vivo assay in ovariectomized, estrogen- and progesterone-blocked female rats. Even where we were able to extract crude or partially purified fractions showing selective FSH-releasing activity, that activity was relatively low compared to the activity of fractions with LH-releasing activity (unpublished data).
Our laboratory also screened known LHRH's from various species for selective FSH-releasing activity; and we also evaluated the activity of 25 analogs of LHRH in in vivo assays. (LHRH's from various species are disclosed in Lumpkin et al., 1987.) One analog was found to have only FSH-releasing activity, but its potency was very low, and the slope of its dose-response curve was flat. Of the known forms of LHRH from other species, we found that only chicken (c) LHRH-II had slightly preferential FSH-releasing activity in vivo (unpublished data).
We have unexpectedly discovered that l-LHRH-III is the long-
McCann Samuel M.
Yu Wen H.
Board of Supervisors of Louisiana State University and Agricultu
Low Christopher S. F.
Lukton David
Runnels John H.
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