The present invention relates to peptides which possess biological activity in respect to the inhibition of growth hormone, insulin secretion and glucagon secretion are provided. The peptides have fewer amino acid components than somatostatin and some of the peptides have dissociated activity.
|
1. A peptide selected from those of the formulae: ##STR3## wherein R1 is selected from the group consisting of Asn and desR1 ; R2 is selected from the group consisting of Trp and D-Trp; R3 is selected from the group consisting of Phe and Thr; R4 is selected from group consisting of Thr and desR4 ; R5 is selected from the group consisting of Ser, Phe and desR5, provided that at least one of R1, R4 and R5 is deleted; X is selected from the group consisting of H, and an alpha-amino protecting group; X1 and X6 are selected from the group consisting of H and a protecting group for Cys selected from S-p-methoxybenzyl, S-acetamidomethyl, S-trityl and S-benzyl; X2 is selected from the group consisting of H and a side chain amino protecting group; X3, X4 and X5 are selected from the group consisting of H and a hydroxyl protecting group selected from the group consisting of acetyl, benzoyl, tert-butyl, trityl, benzyl and benzyloxycarbonyl; with the proviso that at least one of X, X1, X2, X3, X4, X5, and X6 is other than hydrogen; and R6 is selected from the group consisting of hydroxy, methoxy, and an anchoring bond used in solid phase synthesis linked to a solid resin support selected from the group consisting of --O--CH2 -polystyrene resin support and O--CH2 -benzylpolystyrene resin support; and Cys is either L-Cys or D-Cys.
2. A peptide in accordance with
3. A peptide in accordance with
4. A peptide in accordance with
5. A peptide in accordance with
6. A peptide in accordance with
7. A peptide in accordance with
|
|||||||||||||||||
The invention described herein was made in the course of work under a grant or award from the Department of Health and Human Services (formerly DHEW).
The present invention relates generally to peptides having biological activity in respect to the inhibition of growth hormone, insulin and glucagon secretion. More particularly, the present invention is directed to peptides having fewer amino acid moieties than somatostatin which are effective to inhibit the release of growth hormone by the pituitary gland or the release of glucagon or insulin by the pancreas. Various peptides of the invention have dissociated biological activity in respect to the inhibition of growth hormone, insulin and glucagon secretion.
A peptide having inhibitory effect on the secretion of growth hormone has been characterized and is described in U.S. Pat. No. 3,904,594 to Guillemin et al. This peptide has been named "somatostatin". Somatostatin (also known as somatotropin release inhibiting factor) is the tetradecapeptide: ##STR1##
Somatostatin, the linear form of somatostatin (dihydrosomatostatin) and various acylated derivatives of somatostatin and dihydrosomatostatin are described in the aforementioned U.S. patent.
Somatostatin and many analogs of somatostatin exhibit activity in respect to the inhibition of growth hormone (GH) secretion from cultured, dispersed, rat anterior pituitary cells in vitro and inhibition of insulin and glucagon secretion in vivo in the rat. It has been considered highly desirable in the use of somatostatin to selectively inhibit only the secretion of GH, insulin or glucagon. Efforts have been made to develop analogs of somatostatin which possess dissociated biological activity and which inhibit only GH, insulin or glucagon secretion. Although there have been reports citing differences in the amounts of somatostatin required for inhibition of insulin compared to glucagon in the human and the perfused rat pancreas in vitro, somatostatin and some somatostatin analogs exhibit similar potencies on the inhibition of these two hormones in vivo.
The present invention relates to the discovery that certain amino acids can be removed and/or rearranged in somatostatin and dihydrosomatostatin peptides to provide novel peptides having fewer amino acid components and which possess biological activity in respect to the inhibition of GH, insulin or glucagon secretion. Some of the novel peptides of the invention have dissociated activity. The novel peptides of the invention having fewer amino acid components than somatostatin or dihydrosomatostatin are considered to be of great value because of the relative simplicity with which these peptides can be manufactured.
The novel peptides of the invention are defined by the formulae: ##STR2## where R1 is selected from Asn and des R1, R2 is selected from Trp and D-Trp, R3 is selected from Phe and Thr, R4 is selected from Thr and des R4, and R5 is selected from Ser, Phe, and des R3 provided that at least one of R1, R4 and R5 is deleted.
The nomenclature used to describe the peptides of the present invention is in accordance with the conventional practice of using the first three letters of the trivial name. Also, in accordance with such practice, it is the L form of the amino acid that is intended, unless otherwise expressly indicated. In this connection, it should be understood that either of the Cys amino acid moieties can be either D-Cys or L-Cys.
Pharmaceutically acceptable acid addition salts of the peptides are also within the scope of the present invention. Such acid addition salts include but are not limited to hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.
Also considered to be within the scope of the present invention are intermediates of the formula:
X-Cys(X1)-R1 -Phe-Phe-R2 -Lys(X2)-R3 (X3)-Phe-R4 (X4)-R5 (X5)- Cys(X6)-R6. III
wherein: X is either hydrogen or an α-amino protecting group. The α-amino protecting groups contemplated by X are those known to be useful in the art in the step-wise synthesis of polypeptides. Among the classes of α-amino protecting groups covered by X are (1) acyl type protecting groups such as formyl, trifluoroacetyl, phthalyl, toluenesulfonyl (tosyl), benzensulfonyl, nitrophenylsulfenyl, tritylsulfenyl, o-nitrophenoxyacetyl, chloroacetyl, acetyl, y-chlorobutyrul, etc.; (2) aromatic urethan type protecting groups such as benzyloxycarbonyl and substituted benzyloxycarbonyl such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups such as α-t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkyl urethan type protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl; (5) thiourethan type protecting groups such as phenylthiocarbonyl; (6) alkyl type protecting groups such as triphenylmethyl (trityl), benzyl; (7) trialkylsilane groups such as trimethylsilane. The preferred α-amino protecting group defined by R is tertbutyloxycarbonyl.
X1 and X6 are each a protecting group for Cys selected from the group consisting of S-p-methoxybenzyl, S-p-methylbenzyl, S-acetamidomethyl, S-trityl, S-benzyl, and the like. The preferred protecting group is S-p-methoxybenzyl. X1 and/or H-Cys-Phe-Phe-D-Trp-Lys-Phe-Phe-Cys-OH
H-Cys-Phe-Phe-Trp-Lys-Thr-Phe-Cys-OH
was synthesized by the following solid phase methodology. Other peptides, described hereinafter were synthesized by a similar technique.
The tertiobutyloxycarbonyl-S-paramethoxybenzyl (Boc-SpOMe-Bzl) derivative of Cys was linked to the resin by any of three known methods; (1) reflux in ethanol in presence of triethyl amine, (2) Cesium salt of the Boc protected amino acid is kept at 50°C in dimethylformamide (DMF) overnight, (3) the potassium salt of the Boc-protected amino acid is kept at 80°C in dimethyl sulfoxide (DMSO) for 2 hours. Only one milliequivalent of the protected Cys per milliequivalent of Cl on the resin is used.
Method (3) is described hereinbelow in more detail. To a slurry of the resin and the dissolved protected Cys in DMSO is added 0.9 mEq of potassium tertiobutoxide (KOtBut) per mEq of amino acid. The reaction mixture is exposed to air as little as possible so that no amber coloration is observed. Reaction at 80°C for 2 hours yields a suitable substituted resin for synthesis of the peptides (approx. 0.2 mEq of amino acid derivative per g of resin). After deprotection and neutralization, the peptide chain is built on resin. Deprotection, neutralization and addition of each amino acid is performed in accordance with schedule I. N.alpha. -t-butyloxycarbonyl (Boc) derivative of each amino acid is used. After deprotection of the first residue (i.e., SpOMe.Bzl.Cys) according to schedule I (steps 3 to 8 included), the N Boc derivative of Phe is added along with a coupling agent. Thereafter, the N Boc derivative of Thr is next added along with a coupling agent which is dicyclohexylcarbodiimide (DCC) (step 9 of schedule I). The side chain of Thr is protected with O-benzyl ether (OBzl). Benzyloxycarbonyl (Z) or benzyloxycarbonyl-2Cl [Z(2-Cl)] was used as the protecting group for the Lys side chain.
| ______________________________________ |
| 1. Schedule for coupling of amino acids in solid phase |
| synthesis (5-10 g resin) |
| Mix times |
| Step Reagents and operations Min. |
| ______________________________________ |
| 1 CH2 Cl2 wash 80 ml (2 times) |
| 3 |
| 2 Methanol (MeOH) wash 30 Ml (2 times) |
| 3 |
| 3 CH2 Cl2 wash 80 ml (3 times) |
| 3 |
| 4 50 percent trifluoroactetic acid (TFA) |
| 10 |
| containing 5 percent 1,2-ethanedithiol |
| in CH2 Cl2 70 ml (2 times) |
| 5 CH2 Cl2 wash 80 ml (2 times) |
| 3 |
| 6 Triethylamine (Et2 N) 12.5 percent in |
| 5 |
| CH2 Cl2 70 ml (2 times) |
| 7 MeOH wash 40 ml (2 times) |
| 2 |
| 8 CH2 Cl2 wash 80 ml (3 times) |
| 3 |
| 9 Boc-amino acid (10 mmoles) in 10 ml |
| DMF (1 times) and 30 ml CH2 Cl2 plus |
| DCC (10 mmoles) in |
| Ch2 Cl2 (2 M) 30 to 120 |
| 10 MeOH wash 40 ml (2 times) |
| 3 |
| 11 Et3 N 12.5 percent in CH2 Cl2 70 ml (2 |
| 3imes) |
| 12 MeOH wash 30 ml (2 times) |
| 3 |
| 13 CH2 Cl2 wash 80 ml (2 times) |
| 3 |
| ______________________________________ |
After step 13, an aliquot is taken for a ninhydrin test:
if the test is negative, go back to step 1 for coupling of the next amino acid; if the test is positive or slightly positive, go back to steps 9 through 13. Schedule I was used for coupling of each of the amino acids of the peptide to Cys.
Cleavage of the peptides from the resin (5 grams) and deprotection of the side chain protecting groups of the peptide was performed in hydrofluoric acid (75 ml) in the presence of anisole (8 ml). After elimination of hydrofluoric acid under high vacuum, the resin-peptide was washed with ether.
The dried resin was immediately extracted with 25% acetic acid (150 ml) and diluted to 3000 ml with degassed H2 O (N2). The pH of the solution was adjusted to 6.6-7.0 with NH4 OH. The solution was titrated dropwise under stirring with potassium ferricyanide solution (1.g/500 ml H2 O) until a permanent yellow color was observed. The solution sat for 10 minutes and pH was adjusted to 5.0 with glacial acetic acid; Bio Rad AG 3-X4A resin (100-200 mesh, chloride form, 10-15 g) was added to the turbid solution and stirred for 15 minutes. The solution was filtered over celite and applied successively onto two columns; (a) Bio Rad AG 3-X4A resin chloride form (10 ml); (b) Bio Rex-70 resin (100 ml) cation form. The celite + resin cake was thoroughly washed with water (500 ml) which was applied onto columns (a) and (b) as a wash. The peptide material was then eluted from the Bio Rex-70 resin column with pyridine; acetic acid:water (30:4:66) or 50% acetic acid. Fractions were collected; only the ones containing peptide (ninhydrin positive) were diluted with water and immediately lyophilized. 950 mg of crude cream colored material was obtained. It was applied onto a Sephadex G-25 F gel column (3×200 cm) equilibrated and eluted with 2 N acetic acid.
The elution pattern as observed at 280 nm showed one major symmetrical peak. After lyophylization the center cut yielded 550 mg which were submitted to counter current distribution (solvent system n-butanol:acetic acid:water, 4:1:5) 10 ml lower phase per tube. 100 transfers were performed and the major peak was found in tubes 57-68. The compound (250 mg) appeared homogeneous on tlc.
The specific optical rotation was [α]23 =-67.8±2: (c=1 in 1% acetic acid). Amino acid analysis of this material showed the expected ratio for the different amino acids.
Active esters can be used in solid phase synthesis and the classical method of synthesis can also be used to prepare the peptides of the invention.
In vitro Bioassay: The effects of the various peptides of the invention were tested in vitro on the secretion of growth hormone by primary cultures of enzymatically dissociated rat anterior pituitary cells by the method of Vale et al., Endocrinology 91: p. 562-571 (1972). The assay is made by treating pituitary glands removed from rats to separate cells therefrom. The cells are placed in culture dishes in Dulbecco's Modified Eagle Medium (Dulbecco et al., Virology, Vol. 8, p. 396, 1949). Carbon dioxide gas and oxygen are supplied to the cell cultures which are maintained at 37°C for 4-5 days prior to use in the assay. Following media changes, cell cultures are incubated for a period of 4 hours and particular somatostatin peptides are added thereto. Radioimmunoassay analysis is used to determine the rate of growth hormone secretion which is expressed in nanograms per hour.
An investigation of the effect of somatostatin, dihydrosomatostatin, (as controls) and the peptides of the invention to inhibit the release of glucagon and insulin was made as follows:
In vivo Bioassay: Male Sprague-Dawley-CD rats weighing 180-200 g housed in temperature and humidity controlled quarters with 14h light and 10h dark (light 0700-21100) were used in all experiments. Animals were fed a standard ration and tap water ad libitum. Experiments were carried out at least 5 days after arrival of rats from the supplier between the hours 1400 to 1600. After ether anesthesia, peptides or saline were administered in a volume of 0.2 ml. via the external jugular vein. Animals remained anesthetized until the time of blood collection from the portal vein. The blood samples were placed into chilled tubes containing 10 mg EDTA and 50 μl of M Benzamidine per ml of blood.
Plasma was stored at -20°C for insulin and glucagon determinations. Insulin levels were determined by the method of Herbert et al, J. Chem. Endocr. Metab. 25:1375, 1956 1965, utilizing porcine insulin antisera and (125I) iodinated insulin tracer. Human insulin standard was obtained from Schwarz-Mann, Orangeburg, New York. Glucagon was determined by the method of Faloona and Unger, in Jaffe et al ed., Methods of Hormone Radioimmunoassay, Academic Press, New York, 1974, p. 317, utilizing glucagon antisera 30K. Cellulose was determined by the glucose oxidase method, utilizing a Beckman Glucose Analyzer.
GH determinations were performed on tissue culture media utilizing the following reagents: NIAMDD rat GH standard (GH-RP-1), NIAMDD monkey anti-rat GH (GH-Serum-3), and highly purified rat GH for iodination.
All experiments were carried on in a randomized block design. Following analysis of variance difference between treatments were determined by the multiple range tests of Dunnett and Duncan. Potency values were calculated from four or six point bioassays.
Various peptides in accordance with the invention were prepared in accordance with the solid phase methodology described above. The composition of the peptides is reported hereinbelow in Table I. Table I also sets forth the percent effectiveness of the peptide for inhibiting secretion of growth hormone (GH), insulin and glucagon, with somatostatin taken as the base.
| TABLE I |
| __________________________________________________________________________ |
| Somatostatin (control) Growth |
| Peptides of Invention Hormone |
| Insulin |
| Glucagon |
| R1 |
| R2 |
| R3 |
| R4 |
| R5 100 100 100 |
| __________________________________________________________________________ |
| desR 1 |
| D-Trp |
| PheThr |
| desR4 |
| desR5 |
| 6 80 100 |
| desR1 |
| D-Trp |
| PheThr |
| desR4 |
| desR5 (D-Cys)11 |
| 14 100 100 |
| desR1 |
| D-Trp |
| PheThr |
| Thr desR5 |
| 35 10-100 |
| 100 |
| Asn D-Trp |
| PheThr |
| desR4 |
| desR5 |
| <1 100 <1 |
| Asn Trp PheThr |
| Thr desR5 |
| 3 100 <1 |
| Asn D-Trp |
| PheThr |
| Thr desR5 |
| 10 200 10-100 |
| __________________________________________________________________________ |
Rivier, Jean E. F., Vale, Jr., Wylie W., Brown, Marvin R.
| Patent | Priority | Assignee | Title |
| 4428942, | May 17 1982 | The Salk Institute for Biological Studies | Analogs of somatostatin |
| 6123916, | Feb 22 1990 | Novartis AG | Therapeutic use of somatostatin peptides |
| 7572895, | Jun 07 2004 | MacroGenics, Inc | Transferrin receptor antibodies |
| 7572896, | Feb 03 2005 | MacroGenics, Inc | Antibodies to oncostatin M receptor |
| 7674619, | Feb 02 2005 | MacroGenics, Inc | ADAM-9 modulators |
| 7687242, | Jan 12 2005 | MacroGenics, Inc | KID31 and antibodies that bind thereto |
| 7718774, | Nov 08 2006 | MacroGenics, Inc | TES7 and antibodies that bind thereto |
| 7790855, | Sep 18 2003 | MacroGenics, Inc | KID3 and KID3 antibodies that bind thereto |
| 7847070, | Jan 31 2005 | MacroGenics, Inc | LUCA2 and antibodies that bind thereto |
| 8216570, | Nov 08 2006 | MacroGenics, Inc | TES7 and antibodies that bind thereto |
| 8216578, | Feb 03 2005 | MacroGenics, Inc | Antibodies to oncostatin M receptor |
| 8313916, | Jan 12 2005 | MacroGenics, Inc | KID31 and antibodies that bind thereto |
| 8361475, | Feb 02 2005 | MacroGenics, Inc | ADAM-9 modulators |
| 8440197, | Sep 18 2003 | MacroGenics, Inc | KID3 and KID3 antibodies that bind thereto |
| Patent | Priority | Assignee | Title |
| 3842067, | |||
| 3882098, | |||
| 3904594, | |||
| 3917578, | |||
| 3933784, | Jan 27 1975 | American Home Products Corporation | Des-(ser13)-srif and intermediates |
| 4011207, | Dec 27 1974 | American Home Products Corporation | Des-(Ala1, Gly2,Lys4 -Srif, Des-(Ala1, Gly2, Lys4)-D-Trp8 -Srif and intermediates |
| 4105603, | Mar 28 1977 | The Salk Institute for Biological Studies | Peptides which effect release of hormones |
| Executed on | Assignor | Assignee | Conveyance | Frame | Reel | Doc |
| Aug 31 1979 | The Salk Institute for Biological Studies | (assignment on the face of the patent) | / |
| Date | Maintenance Fee Events |
| Date | Maintenance Schedule |
| Mar 17 1984 | 4 years fee payment window open |
| Sep 17 1984 | 6 months grace period start (w surcharge) |
| Mar 17 1985 | patent expiry (for year 4) |
| Mar 17 1987 | 2 years to revive unintentionally abandoned end. (for year 4) |
| Mar 17 1988 | 8 years fee payment window open |
| Sep 17 1988 | 6 months grace period start (w surcharge) |
| Mar 17 1989 | patent expiry (for year 8) |
| Mar 17 1991 | 2 years to revive unintentionally abandoned end. (for year 8) |
| Mar 17 1992 | 12 years fee payment window open |
| Sep 17 1992 | 6 months grace period start (w surcharge) |
| Mar 17 1993 | patent expiry (for year 12) |
| Mar 17 1995 | 2 years to revive unintentionally abandoned end. (for year 12) |