Zona pellucida proteins for contraception

Details Zona pellucida proteins for contraception Zona pellucida proteins for contraception

1999-05-03

2002-02-05

Russel, Jeffrey E. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S015800, C514S021800, C530S300000, C530S326000, C530S327000, C530S328000, C530S350000

Reexamination Certificate

active

06344442

ABSTRACT:

This invention relates to the use of peptides that are derived from zona pellucida proteins for contraception and for pharmaceutical formulations that contain these peptides, without these peptides having an immunizing action.
With the increase in world population, the need for efficient methods of contraception is also growing. In addition to oral contraceptives and spermicides, mechanical contraceptives are also available here, such as, for example, IUDs (intrauterine devices), vaginal rings, and condoms. Another approach is based on preventing fertilization by blocking the egg-sperm interaction. The sperm must penetrate the zona pellucida, an extracellular matrix that consists of glycoproteins and that surrounds the female gametes (the growing oocytes and the ovulated egg). This interaction takes place via a complex interplay of ligands, as well as sperm receptors on the part of the ovocyte or the sperm surface. The zona pellucida of the various mammalian species consists of three to four glycoproteins, which are normally referred to as ZP1, 2, and 3 or ZPA, B and C [Harris, J. et al.: Cloning and Characterization of Zona Pellucida Genes and cDNA's from Variety of Mammalian Species: The ZPA, ZPB and ZPC Gene Families. DNA Sequence: 4, 361-393, (1994)]. In mice, it has been described that the sperm bond first to ZP3 via O-bonded oligosaccharide chains, and the additional bond is probably mediated by ZP2. ZP3 seems to mediate not only the initial bond of the sperm to the zona pellucida, but also another decisive process for fertilization, the acrosome reaction [Dean, J.: Biology of Mammalian Fertilization. J. Clin. Invest.: 89, 1055-1059 (1992); Wassermann, P. M.: Regulation of Fertilization by Zona Pellucida Glycoproteins. J. Reprod. Suppl.: 42, 79-87, (1990)].
In view of the fact that the zona pellucida glycoproteins are unique in the ovary, exhibit antigenic properties, and are accessible to circulating antibodies during the intraovarian growth phase, the research has focused on the development of contraceptive vaccines on the basis of zona pellucida proteins [Naz, R. K. et al.: Development of Contraceptive Vaccines for Humans Using Antigen Derived from Gametes (Spermatozoa and Zona Pellucida) and Hormones (Human Chorionic Gonadotropin): Current Status. Human Reprod. Update: 1, 1-18, (1995); Millar, S. E. et al.: Vaccination with Zona Pellucida Peptides Produces Long-Term Contraception in Female Mice. Science: 246, 935-938, (1989)]. A phenomenon that was noted in almost all animals in the case of immunization with zona pellucida proteins is the induction of an oophoritis. Previously it was not possible to completely explain the reason for the occurrence of an oophoritis. In any case, the formation of an oophoritis makes the longer-term use of zona pellucida proteins or of peptides that are derived from these proteins appear problematical for contraceptive immunization.
There is therefore an urgent need for a new contraceptive that actively prevents fertilization by blocking egg-sperm interaction, without having an immunizing action.
It has now been found that the egg-sperm interaction is prevented by peptides that are derived from zona pellucida proteins.
This invention relates to the use of peptides that are derived from zona pellucida proteins for contraception, without these peptides having an immunizing action.
In another embodiment, this invention relates to the use of peptides that are derived from zona pellucida proteins, for the production of pharmaceutical formulations.
In another preferred embodiment, the peptides are derived from mouse protein or human ZP1, 2, or 3 protein.
In an especially preferred embodiment, peptides are derived from mouse protein or human ZP3 proteins.
In another especially preferred embodiment, the peptides are
-RYPRQGNVSS-  (SEQ ID NO:1)
-TPSPLPDPNSSPY-  (SEQ ID NO:2)
-SRNRRHVTDEADVT-  (SEQ ID NO:3)
-CSNSSSSQFQIHGPR-  (SEQ ID NO:4)
-TRKCHSSSYLVSLPQ-  (SEQ ID NO:5)
-SQFQIHGPRQ-  (SEQ ID NO:6)
-TPT..PDQNASPY-  (SEQ ID NO:7)
-CGTPSHSRRQPHVMS-  (SEQ ID NO:8)
-SRNRRHVTEEADVT-  (SEQ ID NO:9)
-TRRCRTASHPVSASE-  (SEQ ID NO:10)
 -SRRQPHVMSQ-  (SEQ ID NO:11)
In another especially preferred embodiment, the peptides are
-RYPRQGNVSS-  (SEQ ID NO:1)
-TPT..PDQNASPY-  (SEQ ID NO:7)
-SRNRRHVTDEADVT-  (SEQ ID NO:3)
-TRRCRTASHPVSASE-  (SEQ ID NO:10)
-SQFQIHGPRQ-  (SEQ ID NO:6)
The synthesis of peptides was carried out according to Fmoc strategy [Carpino, L. A. and Han, G. Y. (1970) J. Amer. Chem. Soc. 92:5748-5749; Carpino, L. A. and Han, G. Y. (1972) J. Org. Chem. 37:3404-3409] on solid vehicles [Merrifield, R. B., (1963) J. Am. Chem. Soc. 85, 2149] on an automatic peptide synthesizer. Should the peptides carry a free C-terminus (COOH), HMP-resin [Wang, S. -W. (1973) J. Am. Chem. Soc. 95, 1328] would be used for the synthesis. Should the C-terminus be amidated (CONH
2
), Rink amide resin [Rink, H., (1987) Tetrahedron Lett. 28, 3787] would be used. Should the peptides N-terminally carry a free amino group (H
2
N-), the Fmoc protective group would be cleaved in the last synthesis cycle. Should the N-terminus be acetylated (Ac-), however, the amino group of the N-terminal amino acid could be reacted with acetic anhydride after Fmoc cleavage.
The cleavage of the protective groups was carried out for 0.1-1.5 g of peptide resin with:
0.75 g of phenol,
0.25 ml of ethanedithiol,
0.5 thioanisole
0.5 ml of H
2
O
10 ml of trifluoroacetic acid
and three hours of incubation while being stirred at 37° C. in a water bath.
Peptide isolation was carried out either by precipitation with tert-butylmethyl ether cold (ice bath) and subsequent centrifuging, or after spinning-in in a vacuum by precipitation with ether and subsequent filtering. After drying, the peptides were purified via HPLC as needed.
Execution of the Tests
I. In Vitro Fertilization
In vitro fertilization represents a test system that makes possible a first review of a contraceptive effect of substances. In this case, the possibility exists of studying whether the contraceptive effect can be attributed primarily to an inhibition of the sperm function(s) or to an inhibiting effect on the oocytes, or to the two above-mentioned effects.
I.1. Animal Material
Fertile female mice (CB
6
F
1
strain) about 12 weeks of age; fertile male mice (CB
6
F
1
strain) at least 14 weeks of age.
I.2. Preparation and Execution
Pyrex and bulb bottles are autoclaved.
The medium according to Brinster, Whitten and Wittingham
Substance
mg/100 ml dd H
2
O
NaCl
599.5
KCl
35.6
CaCl
2
25.1
KH
2
PO
4
16.2
MgSO
4
29.3
NaHCO
3
208.4
Glucose
100
Na-pyruvate
11.2
Na-lactate
0.44 ml
Penicillin
8.0
Streptomycin Sulfate
5.0
Phenol Red
0.5
BSA
a) 0.3%
b) 2.0%
The medium is then set at pH 7.4 with 0.5 M NaOH.
Sterile bulb bottles are exposed to gas with carbogen for 5 minutes, and the medium is then filtered into the latter (with a shelf life of no more than one week at 4° C.). To equilibrate the oil, 100 ml of paraffin oil (uvasol) is heated for 2 hours at 100° C. and finally cooled. After cooling, 20 ml of BWW/0.3% BSA is added and exposed to gas for 20 minutes with carbogen.
All instruments that are to be used are sterilized overnight at 140° C. The incubator is set at 37° C., 5% CO
2
, and 95% O
2
.
The small Petri dishes:
a) are filled with paraffin oil that is exposed to carbon gas and kept warm at 37° C. to produce oocytes.
b) are pipetted onto the bottom of the small dish with 0.5 ml of BWW/2% BSA for sperm capacitation (prepared before the removal of the tubes), covered with equilibrated oil, and kept at 37° C.
c) 0.1 ml of BWW/0.3% BSA, corresponding to the concentrations that are to be tested, is mixed with or without substance (control) for the fertilization (prepared after removal of the tubes), covered with equilibrated oil, and kept at 37° C.
I.2.1. Induction of Superovulation
10 injection units o

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