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Publication numberUS3914412 A
Publication typeGrant
Publication dateOct 21, 1975
Filing dateOct 11, 1973
Priority dateOct 11, 1973
Publication numberUS 3914412 A, US 3914412A, US-A-3914412, US3914412 A, US3914412A
InventorsRonald Lee Gendrich, Riemond Henry Rippel, John Hunter Seely
Original AssigneeAbbott Lab
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
{8 Des{13 Gly{9 {0 10 -Gn{13 RH nonapeptide amide analogs in position 6 having ovulation-inducing activity
US 3914412 A
Abstract
An unnatural nonapeptide with improved biological activities surpassing those of the natural follicle-stimulating luteinizing hormone-releasing hormone (Gn-RH) is described. The new peptide induces ovulation in warm-blooded animals at an oral dose of 5 - 100 mu g./kg.
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United States Patent [1 1 Gendrich et a1.

[ Oct. 21, 1975 1 [DES-GLY] 10 -GN-RH NONAPEPTIDE AMIDE ANALOGS IN POSITION 6 HAVING OVULATION-INDUCING ACTIVITY [75] Inventors: Ronald Lee Gendrich, Waukegan;

Riemond Henry Rippel, Gumee; John Hunter Seely, Lake Bluff, all of [73] Assignee: Abbott Laboratories, North Chicago, Ill.

[22] Filed: Oct. 11, 1973 21 Appl. No.: 405,334

[52] US. Cl 424/177; 260/112.5 [51] Int. Cl. A61K 37/00; C07C 103/52 [58] Field of Search 260/1'125; 424/177 [56] References Cited UNITED STATES PATENTS 3,784,535 1/1974 Flouret 260/112.5

OTHER PUBLICATIONS Fujino et al.: Biochem. Biophys. Res. Comm., 49, 863-869 (1972).

Primary Exan'tir'iF-Lewis Gotts Assistant Examiner-Reginald J. Suyat Attorney, Agent, or Firm-Paul D. Burgauer; Robert L. Niblack 57 ABSTRACT v 3'Claims, No Drawings DESGLY] l -GNRH NONAPEPTIDE AMIDE ANALOGS IN POSITION 6 HAVING OVULATION-INDUCING ACTIVITY DETAILED DESCRIPTION OF THE INVENTION GnRI-I which consists of the sequence of pGlu-H- isTrpSerTyrGly-Leu ArgPro- GlyNH in which all the optically active aminoacids are in the L-configuration, has been known for several years. It has found utility particularly in animal husbandry where it has been used to induce ovulation. The method and systems used have been described in Proc. Soc. Exper. Biol. and Med. 143, 55 (1973) by Rippel, et al. Unfortunately, this natural hormone has the disadvantage of having very little oral activity.

It is therefore an object of the present invention to provide a peptide with oral activity. It is another object of this invention to provide a synthetic peptide which shows superior hormonal activity over natural occurring peptides. It is a further object of this invention to provide a synthetic peptide that can be readily assembled from easily accessible aminoacids.

These and other objects are accomplished by providing a compound of the formula:

pGluHisTrp-Ser-Tyr-XLeuArg- Pro-NHCJ-l; I

wherein all animoacids are in the L-configuration with the exception of X, which represents an aminoacid in the D-configuration and having the formula:

in which R represents a hydrocarbon chain of between I and 4 carbon atoms in linear or branched configuration.

In a general embodiment, the compounds of the above formula can easily be made by using the method described by Merrifield in J. Am. Chem. Soc., 85, 2149 (1963). More particularly, N-blocked proline is esterified to a chloromethylated divinylbenzene-styrene copolymer. After deblocking, N -blocked arginine carrying a labile protective group on the imino-N is coupled to the now free imino group of the proline ester and after deblocking, this sequence of coupling and de blocking steps are repeated with other aminoacids in the sequence of the formula shown under formula I. All of the aminoacids are used in their L-form except for the aminoacid identified as X in said formula.

After all of these aminoacids are linked in the above sequence with the arginine, tyrosine, serine and optionally the histidine carrying protective groups, the nonapeptide is removed from the resin support in known fashion; the polyprotected peptide is then transesterified/ammonolyzed whereby the resin link is replaced by the ethylamide terminal. Subsequent treatment in known fashion removes all the protective groups mentioned above, producing the compound of the structure shown in formula I in substantially pure form 'and acceptable yield.

The simplest member of the class represented by X in formula I is alanine or the compound wherein R represents the methyl group. Other aminoacids that 'can take the place of X are the leucine, isoleucine, valine and the unnatural a-aminobutyric acid. The nonapeptide of this sequence show a resemblance to the aminoacid sequence in GnRH but distinguishes therefrom in several major respects: GnRH is a decapeptide, ending with an unsubstituted amido group while in the above nonapeptide, the terminal glycinamide group of GnRH is replaced by the ethylamide group which is directly linked to the now terminal proline. An even more striking difference over GnRH is the use of a dextro-acid in the center of the aminoacid sequence. With this change, it was totally unexpected that the new aminoacid chain had biological activity in general; it was even more surprising to find that the biological activity of this compound containing a D- aminoacid had biological activity similar to GnRI-l but at a much higher and much improved level, particularly in view of the fact that most of the biologically active peptides uncovered in recent years are composed of aminoacids exclusively in the L-configuration.

The compounds of formula I act much in the same way as shown in the article referred to above by Rippel, et al; however, the compounds of the present invention induce ovulation in warm-blooded animals at intravenous, intramuscular or subcutaneous single doses of between 0.02 and 1.0 ugjkg. and with a single oral dose of between 2 and ug./kg. More specifically, a single injection to a proestrus rat at a level of 0.05 to 0.3 ,ugjkg. or an oral dose of 30 eg/kg. produces almost certain ovulation. This effect can be easily and beneficially employed in animal husbandry.

In order to show the preparation and use of the compounds of the present invention, reference is made to the following examples which, however, are not intended to limit the invention in any respect.

EXAMPLE 1 Proline carrying as a blocking group the tbutyloxycarbonyl substituent (elsewhere herein referred to as Boc) on the amino group is esterified by combining it with a chloromethylated divinylbenzenestyrene copolymer (marketed by Schwarz-Mann as Merrifield resin) containing 2% of cross linking, using the method described by Stewart, et al. in SOLID PHASE PEPTIDE SYNTHESIS, (published in 1969 by Freeman & Company), San Francisco, (page 1). In this manner, a resin is produced which by hydrolysis and aminoacid analysis shows to contain 0.47 millimoles of proline/g. of resin. In an automatic synthesizer developed according to the previously cited Merrifield apparatus, 4.6 g. of this resin/aminoacid material is used for the synthesis of the desired nonapeptide. Each N-blocked aminocacid is added in a three-fold access and allowed to couple to the existing aminoacid-resin ester in the usual coupling cycle. The coupling reaction is carried out for 4.5 hours with continuous shaking and the reaction is subsequently washed six times with methanolchloroform 1:2for 1.5 minutes each and 4 times with ethanol for 1.5 minutes each. In each instance, a total volume of 48 milliliters is used and the drain time after shaking usually is about 1.5 minutes.

After coupling, the mixture is washed four times for 1.5 minutes each with dioxane, twice with 4N hydrochloric acid/dioxane for 5 minutes and 25 minutes, respectively, five times with dioxane for 1.5 minutes each, three times with ethanol for 1.5 minutes each, three times with chloroform for 1.5 minutes each, three times with 10% triethylamine/chloroform for 1.5 minutes each, four times with chloroform for 1.5 minutes each and six times with dichloromethane for 1.5 minutes each. Ordinarily the solvent used for the coupling reaction is dichloromethane or, when the solubility of the blocked aminoacid is low, a mixture of dichloromethane and dimethylformamide. Coupling is effected by the addition of a solution of dicyclohexylcarbodiimide in dichloromethane at a 2.9 fold excess.

The sequence used for deprotection, neutralization and coupling of the next aminoacid is done in a fully automatic system as described above. In this manner, the peptide is assembled using in turn BocArg(Tos), Boc-Leu, BocDLeu, Boc-Tyr(Cl Bzl), Boo-- Ser(Bzl), Boc-Trp, BocI-Iis(DNP), and pGlu wherein all aminoacids are in the L-form except in the leucine so designated.

The resin is removed from the vessel and suspended in 200 ml. of 5% triethylamine/methanol and 100 ml. of distilled ethylamine is added thereto. After 24 hours, the resin is removed by filtration and the solution evaporated to yield a solid. The solid is taken up in glacial acetic acid and applied to a 3 X 50 cm. column of silica gel equilibrated with 5% methanol/chloroform.

The column is eluted with 5% methanol in chloroform until all traces of N-ethyl dinitroaniline, the yellow by-product of the histidine protecting group DNP is removed. The eluant is then changed to 33% methanol/chloroform and fractions of about 30 ml. each are collected. The compound is located by thin-layer chromatography of aliquots of the fractions (Silica gel G. 33% MeOH/CHCI Cl /tolidine spray). The fractions containing the product are pooled and evaporated to give a solid which is precipitated from methanol with ether. This triprotected nonapeptide (protective groups at Ser, Tyr and Arg) is thus obtained in an amount of 1.69 g., representing an overall yield of 43% of theory.

A 250 mg. sample of the above is placed in a hydrogen fluoride reaction vessel with 250 mg. of anisole and about 5 ml. of anhydrous hydrogen fluoride is distilled into it. After 1 hour at C., the hydrogen fluoride is removed with a stream of dry nitrogen and the residue is taken up in 1% acetic acid. This solution is extracted with ether, and the aqueous phase applied to a 1 X 30 cm. column of a highly basic ion exchange resin (marketed by Bio-Rad as AGl X 2 resin) in the acetate form. The product is eluted with 0.1 N acetic acid and localized using thin-layer chromatography (CI-ICl /MeOI-I/32%HOAc: 120/90/40, silica gel G. Cl /tolidine). The product bearing solution is lyophilized, rechromatographed on a Sephadex G-25 (marketed by Pharmacia of Uppsala, Sweden) column. The product eluted is collected and lyophilized to yield a fluffy white solid [a],," 3l.7 (c 1, 1 I-IOAc) in a 25% overall yield. An aminoacid analysis shows the expected ratio of all desired aminoacids assembled in the above fashion.

When in the above synthesis, the Boc-D-leucine is replaced by the correspondingly protected a-aminobutyric acid, alanine, isoleucine or valine, the above synthesis proceeds in the same fashion, again in all instances, using the automatic synthesizer described above.

EXAMPLE 2 The nonapeptide made in the previous example and having the structure of formula I wherein X is the D- leucyl link, was dissolved in physiological saline for injection into a group of mature, female rats weighing an average of 200 g. Each animal received only one injection and the number of animals ovulating was counted. For comparison, a group of rats were also injected with GnRH and the results are given in parentheses in the table below:

While the above table shows that the compound of structure I shows ovulatory effect at a much lower dose than the same effect with Gn-Rl-I, the even more surprising fact is that ovulation can be induced in rats at a dose of 0.003 mg./kg. upon oral administration while GnRI-I requires 62 250 ug/kg. orally to produce the same results.

Since the above compounds are easily soluble in physiological saline, the preparation of injectable solutions is simple and solutions containing 1 10% of the above compound can easily be prepared. If desired, a preservative such as benzyl alcohol is added to improve storage stability of solutions that are not intended to be used promptly, although this is not necessary since the peptide chain does not decompose rapidly.

For oral preparations, any number of pharmaceutical forms can be used, e.g., syrups, elixirs, suspensions or the compounds can be processed into wafers, pills, tablets and the like. However, since the dosage producing ovulatory effects is extremely small, the usual tableting methods require the use of fillers and other excipients to prepare tablets of manageable size. In a preferred embodiment, the oral dosage form consists of a tablet containing between 0.1 to 5.0 mg. of the above peptide per tablet. Such tablets can be coated in the usual fashion, preferably using a readily soluble coating material, e.g., sugar, etc. or the above amount can be incorporated into gelatin capsules which promptly dissolve upon introduction into the stomach. In any event, the usual flavoring and coloring agents can be used without effect on the active peptide so incorporated.

In practice and as well known by those skilled in the art of animal husbandry, ovulation can obviously be induced only in animals that have a suitably mature ovarian follicle. Thus, a female in the reproductive age can be given the above dose of the compound of formula I in repeated administrations until ovulation occurs. Once this has been accomplished, no further ovulation can be induced until a mature follicle has been formed again at which time the administration of the new compounds will have the desired effect again.

What is claimed is:

l. The process of inducing ovulation in a warmblooded animal possessing a suitably mature follicle comprising administering to said animal a dose sufficient to induce ovulation of a compound of claim 1 the 3 ,9 l 4,4 1 2 5 6 wherein all aminoacids are present in their optical L- l 4 carbon atoms. configuration and wherein X represents an aminoacid 2 The process f claim 1 wherein Said dose i adminin the Dconfiguration and having the formula: istered orally and contains between 2 and 100 pug/kg.

of said compound.

3. The process of claim 1 wherein said dose is administered parenterally and contains between 0.02 and 1 .0 p.g./kg. of said compound wherein R is a linear or branched hydrocarbon chain of

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US3784535 *Sep 13, 1971Jan 8, 1974Abbott LabNonapeptide intermediate to gonadotropin releasing hormone
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US4003884 *May 16, 1975Jan 18, 1977Hoechst AktiengesellschaftPeptides having LH-RH/FSH-RH activity
US4005063 *May 15, 1975Jan 25, 1977Abbott Laboratories[Des-gly]10 -GnRH nonapeptide anide analogs in position 6 having ovulation-inducing activity
US4008209 *Jul 11, 1975Feb 15, 1977Takeda Chemical Industries, Ltd.Nonapeptide amide analogs of luteinizing releasing hormone
US4010261 *Nov 25, 1974Mar 1, 1977Abbott LaboratoriesMethod to prevent reproduction with [Des-Gly]10 -GN-RH nonadeptide amide analogs in position
US4018914 *Nov 13, 1975Apr 19, 1977Abbott LaboratoriesParturition inducement
US4024248 *Aug 7, 1975May 17, 1977Hoechst AktiengesellschaftPeptides having LH-RH/FSH-RH activity
US4034082 *Mar 1, 1976Jul 5, 1977Abbott LaboratoriesMethod to prevent reproduction in warm-blooded female animals with nonapeptides
US4086219 *Dec 27, 1976Apr 25, 1978Parke, Davis & CompanyNonapeptides and methods for their production
US4100274 *Apr 22, 1977Jul 11, 1978Imperial Chemical Industries LimitedPolypeptide
US4111923 *Dec 27, 1976Sep 5, 1978Parke, Davis & CompanyOctapeptides and methods for their production
US4338305 *Aug 11, 1975Jul 6, 1982American Home Products CorporationUse of LRH and LRH agonists
US4751215 *Apr 15, 1986Jun 14, 1988Innofinance Altalanos Innovacios PenzintezetProcess for increasing the sexual activity of birds and useful domestic mammals and for preparing spermatozoa suitable to their propagation
US4753928 *Jul 18, 1986Jun 28, 1988Innofinance Altalanos Innovacios PengintezetProcess for obtaining sexual products from mammals suitable for natural or artificial fertilization
US5068221 *May 9, 1989Nov 26, 1991Mathias John RGonadotropin Releasing Hormone, oligopeptides for gastrointestinal disorders
US5434136 *Oct 19, 1992Jul 18, 1995Mathias; John R.Administering an analog of gonadotropin releasing hormone to a patient suffering from systemic lupus erythematosis
US5916582 *Jun 13, 1997Jun 29, 1999Alza CorporationAqueous formulations of peptides
US5932547 *Jun 13, 1997Aug 3, 1999Alza CorporationNon-aqueous polar aprotic peptide formulations
US5981489 *Jun 13, 1997Nov 9, 1999Alza CorporationNonaqueous peptide compound which consists essentially of about 30% to about 50% (w/w) of the lhrh-related compound leuprolilde acetate in polyoxyethylene glcol or mixture
US6066619 *Apr 16, 1999May 23, 2000Alza CorporationNon-aqueous protic peptide formulations
US6068850 *Feb 11, 1999May 30, 2000Alza CorporationA stable aqueous formulation of a peptide related compound comprising atleast one peptide compoun and water, wherein the formulation is stable at 37 degree c. atleast 2 months, useful in an implantable drug delivery device
US6124261 *Apr 19, 1999Sep 26, 2000Alza CorporationNon-aqueous polar aprotic peptide formulations
US6235712Feb 28, 2000May 22, 2001Alza CorporationNon-aqueous polar aprotic peptide formulations
US7700559Apr 5, 2004Apr 20, 2010Medical Research CouncilGonadotropin releasing hormone analogues conjugates with steroid hormones
US8013117Nov 21, 2006Sep 6, 2011Nanokem S.A.Solution-phase synthesis of leuprolide and its intermediates
DE3624422A1 *Jul 18, 1986Feb 5, 1987Innofinance Altalanos InnovaciMittel zur beeinflussung, vor allem foerderung, der geschlechtstaetigkeit von saeugetieren und dabei insbesondere vervielfachung der geschlechtsprodukterzeugung, bezogen auf die fuer die art charakteristische lebensleistung, sowie zur gewinnung von fuer die natuerliche und die kuenstliche befruchtung geeigneten geschlechtsprodukten von saeugetieren im prae- und postnatalen leben, im infantilen alter, der vorpubertaet und im ausgewachsenen zustand (zusammenfassend ausgedrueckt: medien), sowie ihre verwendung
DE3624422C2 *Jul 18, 1986Jul 11, 1991Innofinance Altalanos Innovacios Penzintezet, Budapest, HuTitle not available
EP0000764A1 *Aug 1, 1978Feb 21, 1979Hoechst AktiengesellschaftAnticonceptive agent containing a peptide
EP0037127A1 *Aug 1, 1978Oct 7, 1981Hoechst AktiengesellschaftUse of a luteinising peptide as anti-contraceptive
WO2006077428A1Jan 20, 2006Jul 27, 2006Astex Therapeutics LtdPharmaceutical compounds
WO2007059921A1Nov 21, 2006May 31, 2007Cpc Cellular Process ChemistrySolution-phase synthesis of leuprolide and its intermediates
WO2008044041A1Oct 12, 2007Apr 17, 2008Astex Therapeutics LtdPharmaceutical combinations
WO2008044045A1Oct 12, 2007Apr 17, 2008Astex Therapeutics LtdPharmaceutical combinations
Classifications
U.S. Classification514/10.3, 930/DIG.698, 930/20, 514/800, 930/130, 530/313, 930/21
International ClassificationC07K7/23, A61K38/00
Cooperative ClassificationY10S930/13, C07K7/23, A61K38/00, Y10S514/80
European ClassificationC07K7/23