0021 -972X/90/7004-975$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society

Vol. 70, No. 4 Printed in U.S.A.

Growth Hormone (GH)-Releasing Peptide Stimulates GH Release in Normal Men and Acts Synergistically with GH-Releasing Hormone* C. Y. BOWERS, G. A. REYNOLDS, D. DURHAM, C. M. BARRERA, S. S. PEZZOLI, AND M. 0. THORNER Section of Endocrinology and Metabolism, Department of Medicine, Tulane University Medical School (C.Y.B., G.A.R., D.D., C.M.B.), New Orleans, Louisiana 70112; and the Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health Sciences Center (S.S.P., M.O.T.), Charlottesville, Virginia 22908

ABSTRACT. The acute GH release stimulated by the synthetic hexapeptide, His-DTrp-Ala-Trp-DPhe-Lys-NH2 [GH releasing peptide (GHRP)], was determined in 18 normal men and compared with the effects of GH-releasing hormone, GHRH-(144)-NH2. Specificity of effect was assessed by measurement of serum PRL, LH, TSH, and cortisol. GHRP was administered at doses of 0.1, 0.3, and 1.0 /xg/kg by iv bolus. GHRH at a dose of 1.0 Mg/kg was administered alone and together with various does of GHRP. No adverse clinical effects or laboratory abnormalities were observed in response to GHRP. A side-effect of mild facial flushing of 1- to 3-min duration occurred in 16 of the 18 subjects who received GHRH-(l-44)-NH2. Mean (±SEM) peak serum GH levels after injection of placebo and 0.1,0.3, and 1.0 Mg/kg GHRP

were 1.2 ± 0.3, 7.6 ± 2.5, 16.5 ± 4.1, and 68.7 ± 15.5 respectively. The submaximal dosages of 0.1 and 0.3 Mg/kg GHRP plus 1 Mg/kg GHRH stimulated GH release synergistically. Serum PRL and cortisol levels rose about 2-fold above basal levels only at the 1 ^g/kg dose of GHRP, and there were no changes in serum LH and TSH over the first hour after administration of the peptide(s). GHRP is a potent secretagogue of GH in normal men. Since GHRP and GHRH together stimulate GH release synergistically, these results suggest that GHRP and GHRH act independently. This supports our hypothesis that the GH-releasing activity of GHRP reflects a new physiological system in need of further characterization in animals and man. (J Clin Endocrinol Metab 70: 975-982, 1990)

stimulates GHRH release from the hypothalamus without influencing somatostatin (SRIF) release (9). Both GHRP and GHRH act directly on pituitary somatotrophs; however, several observations suggest that these effects are mediated via different receptors and that different intracellular mechanisms are involved (10-14). Included in these studies was the development of a GHRP antagonist, His-DTrp-DLys-Trp-DPhe-Lys-NH2, by Bowers, Momany, and Hong which inhibits the in vitro GH response to GHRP, but not to GHRH. These results were recently confirmed by Cheng et al. (15). Additionally, GHRP and GHRH administered together synergistically stimulate GH release in rats (9), rhesus monkeys (3), and cows (our unpublished observations). We now report the effects of varying doses of GHRP alone and in combination with GHRH (1.0 Mg/kg) on serum levels of GH, PRL, LH, TSH, and cortisol in normal men.

ONSIDERABLE evidence exists to suggest that the novel synthetic hexapeptide GH-releasing peptide (GHRP) acts through unidentified pathways to regulate GH secretion. GHRP (His-DTrp-Ala-Trp-DPhe-LysNH2) was developed using a closely integrated theoretical, biological, and synthetic approach (1, 2). It stimulates GH release in rats as well as lambs (2), monkeys (2, 3), chimpanzees (3), steers (4), pigs (5), and cows (6). Its specificity of action has been demonstrated in rats (2). Chronic administration in rats enhanced body weight gain (2, 7) and in cows enhanced milk production (6) (our unpublished observations). In vitro studies indicate that GHRP acts at both the pituitary and hypothalamus (8). Although the endocrine factors involved in the GHRP-induced release of GH are still not understood, evidence exists that GHRP probably Received August 14, 1989. Address requests for reprints to: Dr. C. Y. Bowers, Section of Endocrinology and Metabolism, Department of Medicine, Tulane University Medical School, New Orleans, Louisiana 70112. * This work was supported in part by Grant DK-32632 and GCRC Grant RR-0847. Presented at the 71st Annual Meeting of The Endocrine Society, Seattle, WA, June 1989.

Materials and Methods Peptides GHRP was synthesized at Peninsula Laboratory (Belmont, CA) under Good Manufacturing Practice (GMP) requirements 975

BOWERS ET AL.

976

JCE & M • 1990 Voll26«No4

/xg/kg BW • — A— o— o—

FIG. 1. GHRP dose-response curve. Mean (±SEM) serum GH levels in normal men after iv bolus (60 s) injection of placebo (n = 6) and 0.1 (n = 7), 0.3 (n = 6), and 1.0 (n = 9) ^g/kg GHRP. All doses were not administered in all subjects.

• PLACEBO A .1 GHRP a .3 GHRP o 1 GHRP

X

-60 -40 -20

0

20

t iv bolus

40

60

80 100 120 140 160 180 200 220 240 n=6-9

TIME (min)

90

6000

75

5000

c

o

4000

60

3000

-i

x o

o 30

2000

15

1000

0

Placebo

0.1 0.3 GHRP Dose (/xg/kg)

ESS Peak GH

of the FDA and was formulated at Tulane University. An Investigational New Drug Application for GHRP was obtained after submission of animal toxicological studies. GHRH-(144)-NH2) Somatobiss, was supplied by Drs. David Stroman and Hans Doler of Bissendorf Peptide (Wedemark, West Germany). Both peptides were administered in a volume of 1 mL normal saline. Experimental design Eighteen normal men, aged 23-36 yr (mean, 26 yr), within 15% of ideal BW (range, 55-90 kg; mean 75 kg) were enrolled

EZ3 AUC 1h

jrve

FIG. 2. Response to GHRP as represented by the peak GH, AUC over 1 h, and AUC over 4 h. Duncan's multiple comparison test, based on analysis of variance, showed that the dose of 1.0 fig/ kg was different from the other doses (P < 0.05).

= Area Unde

II

1.0 U3 AUC 4h

in the study after having given written informed consent. The studies were approved by the FDA and the Committee on Use of Human Subjects at Tulane Medical School. Each subject had a normal medical history, physical examination, clinical chemistries (SMAC-20), complete blood count, and urinalysis. Routine chemistries were obtained 1 week before admission to the Clinical Research Center, on the morning of each study, at the end of each study, and 7 days after the final study day. Each subject was admitted to the Clinical Research Center on three to five occasions, separated by at least 7 days, the evening before the study. The subjects fasted from 2400 h until completion of the study at 1300 h the next day, but water was

GHRP STIMULATES GH RELEASE TABLE 1. Dose-response relationship of GH release in response to GHRP Dose (Mg/kg, iv) 1. 2. 3. 4.

Placebo GHRP GHRP GHRP

0.1 0.3 1.0

Peak GH (Mg/L) 1.2 ± 7.6 ± 16.5 ± 68.7 ±

0.3 2.5 4.1 15.5

GHAUC over 1 h 51 ± 250 ± 600 ± 2622 ±

14 89 167 620

Mg/kg GHRP plus 1.0 Mg/kg GHRH (n = 6); and group C, 1.0 fig/kg GHRP, 1.0 Mg/kg GHRH, and 1.0 M g/kg GHRP plus 1.0 Mg/kg GHRH (n = 6). GHRH was administered at the maximally effective dose of 1 Mg/kg in each combination of peptides (16).

GHAUC over 4 h 406 ± 588 ± 959 ± 4589 ±

977

93 101 256 1101

Hormone assays

Values are the mean ± SEM of six or seven subjects. Analysis of variance for dose factor significant at P — 0.0001. Duncan's multiple comparison test showed the dose of 1.0 Mg/kg w a s different from other doses, P < 0.05. given freely throughout the study. At 0700 h an indwelling cannula (Butterfly, Abbott Hospitals, Inc., Chicago, IL) was placed in the antecubital vein, and patency was maintained by injection of 1-2 mL heparin (10 U/mL) after each blood sample was drawn. At 0900 h, placebo or peptide(s) was administered iv in the antecubital vein of the opposite arm over 60 s. The subjects remained recumbent throughout the study. Blood pressure, pulse, and temperature were measured every 30 min. Fivemilliliter blood samples were collected from 0800-1300 h at varying (3-, 10-, and 30-min) intervals. The serum was frozen and stored at - 2 0 C until assayed. Placebo and each dose of 0.1, 0.3, and 1.0 Mg/kg GHRP were administered in six or seven subjects; however, all doses were not administered in the same subject. GHRP (1 Mg/kg) a n d GHRH (1 fig/kg) were administered to the same nine subjects on separate occasions. Additionally, to determine whether GHRP and GHRH act in a synergistic manner when administered together, three groups of subjects received different combinations of peptides. These were: group A, 0.1 Mg/kg GHRP, 1.0 fig/kg GHRH, and 0.1 Mg/kg GHRP plus 1.0 M g/kg GHRH (n = 7); group B, 0.3 Mg/kg GHRP, 1.0 Mg/kg GHRH, and 0.3

Serum GH levels from 0800-1300 h were measured by immunoradiometric assay (IRMA) against equine serum standards (Nichols Institute Diagnostics, San Juan Capistrano, CA). The sensitivity of the assay was 0.5 Mg/L; the intra- and interassay coefficients of variation were 6.92% and 7.64%, respectively. All samples were analyzed in duplicate. Serum PRL, LH, TSH, and cortisol were measured for 1 h after administration of peptide(s) or placebo, from 0900-1000 h. Serum PRL was measured by IRMA (Hybritech, Inc., San Diego, CA), cortisol by RIA (Becton Dickinson, Orangeburg, NY), and LH and TSH by IRMA (Nichols Institute Diagnostics). The sensitivity of the assays and the intra- and interassay coefficients of variation were as follows: PRL, 0.3 Mg/L, 3.2%, 4.4%; cortisol, 0.04 nmol/L, 4.5%, 6.4%; LH, 0.1 IU/L, 2.6%, 5.4%; and TSH, 1.0 mU/L, 5.58%, 4.13%. Methods of statistical analysis All values are expressed as the mean ± SEM. The GH response was measured in terms of changes in three response variables: 1) GH peak, the maximum peak observed within 90 min after bolus injection; 2 ) area under the curve (AUC) over 1 h, total GH observed in the first hour after the bolus; and 3) AUC over 4 h, total GH observed in the 4-h period after the bolus. AUC was calculated using the trapezoid rule. Linear regression was used to test for a significant dose-response relationship using the GHRP dose and GH response variables.

BW o — o 1 GHRP A — A 1 GHRH

FIG. 3. Effect of GHRP vs. GHRH. Comparison of mean (±SEM) serum GH levels in nine normal men after iv bolus (60 s) injection of 1 Mg/kg GHRP vs. 1 Mg/kg GHRH. The same subject received both peptides.

X

-60 -40 -20

0 t

20

iv bolus

40

60

80 100 120 140 160 180 200 220 240 n=9

TIME (min)

BOWERS ET AL.

978

JCE & M • 1990 Vol 126 • No 4

TABLE 2. GHRP plus GHRH synergism Dose (Mg/kg, iv) Group A (n = 7) 1. GHRP (0.1) 2. GHRH (1.0) 3. GHRP + GHRH (0.1 + 1.0) 4. Synergy" t test of synergy = 0 (P) Group B (n = 6) 1. GHRP (0.3) 2. GHRH (1.0) 3. GHRP + GHRH (0.3 + 1.0) 4. Synergy" t test of synergy = 0 (P) Group C (n = 6) 1. GHRP (1.0) 2. GHRH (1.0) 3. GHRP + GHRH (1.0 + 1.0) 4. Synergy" t test of synergy = 0 (P)

Peak GH (Mg/L)

GHAUC over 1 h

(Mg/L)

7.6 ± 2.5 36.0 ± 3.9 80.0 ± 14.6

250 ± 89 1454 ± 117 3242 ± 558

588 ± 101 2836 ± 238 5889 ± 1020

36.4 ± 11.8

1538 ± 458

2465 ± 932

Growth hormone (GH)-releasing peptide stimulates GH release in normal men and acts synergistically with GH-releasing hormone.

The acute GH release stimulated by the synthetic hexapeptide, His-DTrp-Ala-Trp-DPhe-Lys-NH2 [GH releasing peptide (GHRP)], was determined in 18 normal...
775KB Sizes 0 Downloads 0 Views