Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• OVINE INHIBIN The present invention relates to a protein having inhibin activity isolated to substantial homogeneity from material obtained from ovine animals.
• inhibin as a water-soluble substance of gonadal origin which acts specifically at the pituitary level to suppress the secretion of follicle-stimulating hormone (FSH) was postulated by McCullagh more than 50 years ago. Science, 76, 19-20 (1932) . There has been great interest in it, and many laboratories have attempted to isolate and characterize this substance.
• Mason et al. published sequences for the subunits of porcine inhibin derived from studies of cDNA.
• Inhibin may be used to regulate fertility, gonadotropin secretion or sex hormone production in mammalians, both females and particularly males.
• a protein having a molecular weight of about 34,500 daltons (34.5kD) and having inhibin activity has been successfully isolated from ram rete testis flui (RTF).
• the protein has been partially characterized using microsequencing methods.
• the protein was isolated to substantial homogeneity from material obtained from RTF and is hereinafter referred to as ovine inhibin.
• the protein has a molecular weight of about 34.5kD and is composed of two polypeptide chains having molecular weights of about 18,000 and about 16,500 Daltons, respectively, the chains being linked together in the biologically active protein by disulfide bonding.
• amino-terminal residue sequence of the larger 18kD chain of the protein is believed to be Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp- Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu- Pro-Ala-Ala-His-Ala-Asp-Cys.
• the amino-terminal of the 16.5kD chain begins Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn- Ile-Cys-Cys-Lys-Lys-Gln-Phe-Tyr-Val-Ser-Phe-Lys-Asp- ⁇ le- Gly.
• the 34.5kD protein exhibits inhibin activity in that it specifically inhibits the basal secretion of FSH but does not inhibit secretion of luteinizing hormone (LH).
• ovine inhibin Purification of ovine inhibin to substantial homogeneity, i.e., about 90% by weight of total protein in the fraction, was achieved through a combination of protein separation procedures including gel filtration and reverse-phase, high-performance liquid chromatography (RP-HPLC) .
• RP-HPLC reverse-phase, high-performance liquid chromatography
• FIGURE 1 is a chromatogram of the final RP-HPLC purification of an inhibin protein active fractions which was applied directly onto a 0.46 x 25 cm Vydac Cg column with a 5 ⁇ particle size and a 300A pore size, and eluted at 40°C with a gradient of TFA/CH 3 CN buffers from 25% Buffer B to 95% Buffer B in 45 minutes, at a flow rate of 0.7 ml/min. with a back pressure of about 890 psi.
• the 34.5kD peptide was isolated to substantial homogeneity from ram rete testis f uid (RTF) .
• the protein is composed of two chains of 18 D and 16.5kD, and the chains of the intact molecule are held together by disulfide bonding, the linkage between the chains being necessary for biological activity.
• a ino acid analysis of the total protein has been performed, and a partial amino acid residue sequence of each chain has also been determined, beginning at the amino-terminus.
• the chains are rich in Cys residues, and it is believed that internal disulfide bonding is also present.
• the amino-terminal sequence of the 18kD chain is Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp- Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu- Pro-Ala-Ala-His-Ala-Asp-Cys.
• the 18kD chain is estimated to be about 135 residues in length, is likely glycosolated and is linked by one or more disulfide bridges to the 16.5kD chain.
• the 16.5kD chain has between about 115 and about 130 residues and begins at the N-terminus with the following sequence: Gly-Leu-Glu- Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-(Tyr or Phe) .
• the next residues following these are believed to be: Val-Ser-Phe-Lys-Asp-Ile-Gly.
• the C-terminus of either chain may be amidated or free acid.
• the sharp elution peak of the protein which was obtained in the final chromatographic purification step is evidence that the protein has been purified to at least about 90% by weight of total protein.
• the 34.5kD protein is water-soluble, and one of the subunits of the native protein is likely glycosylated.
• a second isolated molecule appears to have an N-shortened version of the 18kD chain, that is shorter by 15 residues, but is linked to an identical 16.5kD chain.
• the 34.5kD protein exhibits inhibin activity in that it specifically inhibits basal secretion of FSH but not LH in a rat anterior pituitary monolayer culture system and exhibits a half-maximal effective dose (EC 5Q ) of about 0.3 ng/ml (10 pM.), based upon the assay described in detail in Endocrinology, 113, 1121-31 (1983).
• the isolated 34.5kD protein, as well as partially purified inhibin preparations blocks the secretion of both LH and FSH _in vitro when cells are stimulated by gonadotropin releasing hormone. Iri vivo, partially purified inhibin preparations are highly selective to decrease plasma FSH and not LH levels. The effects of inhibin on basal gonadotropin secretion ju vitro appears to best reflect the ii vivo situation.
• the 34.5kD protein is useful for regulating gonadotropin secretion and thus fertility and/or sex hormone production of both male and female mammalians.
• inhibin might have direct gonadal actions on gametogenesis or steroidogenesis is also likely, and some brain actions of inhibin are suggested.
• a purification procedure was used to isolate ovine inhibin from crude RTF which utilized successive purification steps that include Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) with different stationary phases and/or mobile phases and also include gel filtration or permeation Fast Protein Liquid Chromatography(FPLC) .
• RP-HPLC Reverse Phase-High Performance Liquid Chromatography
• FPLC Fast Protein Liquid Chromatography
• the column was washed with the aqueous Buffer A until the UV absorption reached baseline. Flow through the column is maintained at 2.5 ml per minute. The column is loaded at 30% Buffer B, and a gradient for the mobile phase was then begun gradually changing to 95% over 30 minutes.
• the fractions are separated by an Altex 420 gradient liquid chromatography system equipped with a Spectroflow 773 UV detector (Kratos Analytical Instruments, Ramsey, N. J.) and a Servocoder SR 6253 strip chart recorder and are collected and tested for substantial inhibin activity.
• Inhibin protein fractions from the various individual columns were pooled and further purified by a 1 x 30 cm Vydac 5-um-particle-size C. column and a heptafluorobutyric acid (HFBA) buffer system at RT.
• HFBA heptafluorobutyric acid
• Buffer A contains 1 ml of HFBA in 999 ml water
• Buffer B is 400 ml of water, 1 ml of HFBA and 599 ml of acetonitrile.
• Columns were loaded at 30% B followed by a gradient to 58% B in 25 minutes.
• Active zones from reversed phase HPLC were lyophilized and resuspended in column eluant for processing on Pharmacia FPLC system by applying to two 1 x 30 cm Superose(FPLC) 12-B columns, 10 pm (Pharmacia Fine Chemicals, Piscataway, N. J.) linked in series.
• Each column was eluted with 6M guanidine ⁇ Cl, 0.1M ammonium acetate, pH 4.75, and 0.5% DMS in Milli Q H 2 0 at a flow rate of 0.4 ml per minute for about 50 min.
• the column fractions were monitored by UV absorption and by bioassay. Active fractions eluted between
• Inhibin protein fractions from the various individual columns were pooled and further purified by RP-HPLC using 5- ⁇ m-particle-size C. column, 1 x 30 cm, and 0.5% TFA/CH 3 CN buffer system.
• the active fraction was lyophilized and subjected to FPLC cation exchange by being brought up in Buffer A which was 50 mM sodium acetate, 4 M urea, 1 mM CHAPS (3-[ (Cholamidopropyl) dimethylammonio]-1-propanesulfonate) in Milli Q H 2 0, pH 5.3.
• Buffer B was 1 M NaCl in Buffer A.
• a Pharmacia FPLC system equipped with a Mono S HR 5/5 column, V. 1 ml, was used at a flow rate of 1 ml per min. Column was loaded at 0% B followed by a gradient to 30% B in 25 minutes and then to 100% B in 5 minutes.
• the active fraction was applied to a 0.46 x 25 cm Vydac C g Column of reversed phase material with a 5 um particle size and a 300A pore size.
• Buffer A is 0.5% (v/v) TFA in water and Buffer B is 1 ml TFA, 200 ml of water and 799 ml of acetonitrile.
• Flow rate was 0.7 ml/min at 40°C with a back pressure of 900 psi.
• Buffer B was used at 25 volume % for the initial loading, followed by a gradient to 50% in 25 minutes. Two zones of active inhibin protein eluted, and both were separately processed thereafter.
• Each active fraction was applied to a 0.46 x 25 cm Vydac C g Column of reversed phase material with a 5 um particle size and a 300A pore size * Buffer A is 0.1% (v/v) TFA in water, and Buffer B is 1 ml TFA, 200 ml of water and 799 ml of acetonitrile. Buffer B was used at 25 volume % for the initial loading at 40°C and a flow rate of 1.2 ml/min.
• a gradient to 95% is run in 45 minutes at a flow rate of 0.7 ml/min at 40°C with a back pressure of 890 psi, and a detector setting of 215 n , 2.9 AUFS, which was slightly changed to 214 nm. before elution of the peak.
• the purified inhibin protein eluted generally between about 27.0 minutes and about 28.3 minutes after start of the gradient, which is equal to between about 18.9 ml and about 19.8 ml of elutant after start of the gradient.
• FIGURE 1 A chromatogram of the final step, for the later eluting active fraction from the previous step, is depicted in FIGURE 1 and was generated using an Altex 420 System, two Beckman Model 100A pumps, a Datamark, Servocoder SR 6253 strip chart recorder, a Kratos, Spectroflow 773 variable wavelength, UV/visible detector and a Rheodyne 7125 injector with a 2.0 ml loop.
• the inhibin protein On SDS-PAGE under non-reducing condition, the inhibin protein showed a single band migrating at about 34.5kD. Under reducing condition, the inhibin protein separated into two bands, one migrating at 18kD and the other at 16.5kD. Electrophoresis showed the protein was more than 90% pure.
• NH_-terminal sequence analyses of the 18kD and 16.5kD chains of the 34.5kD inhibin protein were accomplished by first separating the two chains by SDS-PAGE under reducing conditions. Microsequencing, as described in Spiess, J. et al. Biochemistry, 20, 1982-1988 (1981), of the intact inhibin protein beginning at the NH 2 -terminus consistently revealed two residues of approximately equal concentration at every cycle, indicating that the protein is composed of two chains.
• sequence of the NH 2 ⁇ terminal residues of the 18kD chain of the inhibin protein is Ser-Thr-Pro-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala- Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala- His-Ala-Asp-Cys.
• the first NH 2 -terminal residues of the 16.5kD chain of the inhibin protein are Gly-Leu- Glu-Cys, and it is believed that the next residues are Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-(Tyr or Phe) .
• the next residues following these are believed to be: Val-Ser-Phe-Lys-Asp-Ile-Gly.
• mRNA messenger RNA
• mRNA messenger RNA
• mRNA messenger RNA
• ram testes which produce inhibin
• cDNA is synthesized from the mRNA by reverse transcription.
• the cDNA is inserted into a cloning vector which is used to transform a suitable host to create a cDNA library.
• labelled oligonucleotides are synthesized for detecting cDNA corresponding to each chain. Because of the degeneracy of the genetic code, mixed hybridization probes are prepared and used as probes. These probes are then used to select, from the library, cDNA clones that contain gene sequences encoding the chains. cDNA libraries may also be screened by im unological expression assay with an antibody raised against inhibin or one of the two inhibin chains. Immunological expression assay may also be used to confirm screening with hybridization probes.
• cDNA is excised and inserted into appropriate vectors under the control of suitable promotor sequences, and the vectors are transformed into cell lines for expression of the recombinant inhibin chains.
• vectors containing the genes for both chains could conceivably be transformed into the same cell line, for simplicity, vectors for expression of each chain are preferably transformed separately into cell lines.
• the two inhibin chains can then be isolated from the cellular material and/or the cell culture medium. The two chains are then subjected to oxidizing conditions which promote disulfide bonding between the chains.
• the foregoing molecular biology techniques may also be used to read the gene sequences encoding the separate inhibin chains, and thereby completely characterize the protein chains.
• Substantially pure 34.5kD inhibin or the nontoxic salts thereof, combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition may be administered to mammals, including humans, either intravenously, subcutaneously, percutaneously, intramuscularly or orally for control of fertility, gonadotropin secretion or sex hormone production.
• antibodies raised against synthetic fragments of inhibin e.g. the six N-terminal residues of the 18KD chain, namely Ser-Thr-Pro-Pro-Leu-Pro, have been shown to neutralize the activity of purified inhibin.
• passive (administration of antibodies) or active (administration of immunogenic inhibin as antigen) immunization methods could be employed to block endogenous inhibin and thereby elevate endogenous gonadotropin secretion and exert a profertility effect in sheep (both rams and ewes) , in human beings and in other vertibrate animal species having inhibin of a similar polypeptide structure.
• inhibin induces decreased fertility in female mammals and decreases spermatogenesis in male mammals, and administration of a sufficient amount of inhibin could be employed to induce infertility in sheep, including rams and ewes, and in other mammals. Inhibin is also useful for tests to diagnose infertility.
• Such peptides are often administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron or the like (which are considered as salts for purposes of this application) .
• acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.
• sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.
• Inhibin should be administered under the guidance of a veterinarian or a physician, and pharmaceutical compositions will usually contain an effective amount of the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier.
• the dosage will vary depending upon the specific purpose for which the protein is being administered, and dosage levels in the range of about 0.1 to about 1 milligrams per Kg. of body weight may be used when the protein is administered on a regular basis as a male contraceptive.
• inhibin can be similarly purified from other crude extracts, for example follicular fluid.

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• 1987
  • 1987-06-23 EP EP19870904454 patent/EP0272309A4/fr not_active Withdrawn
  • 1987-06-23 JP JP50410687A patent/JPH01500121A/ja active Pending
  • 1987-06-23 CA CA 540422 patent/CA1305829C/fr not_active Expired - Lifetime
  • 1987-06-23 WO PCT/US1987/001470 patent/WO1988000208A1/fr not_active Application Discontinuation

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