Clinical studies with human growth hormone releasing factor in normal adults and patients

Peptides. Vol. 7, Suppl. 1, pp. 281-286, 1986. " Ankho International Inc. Printed in the U.S.A.

I)196-9781/86 $3.00 + .011

Clinical Studies With Human Growth Hormone Releasing Factor in Normal Adults and Patients G. SASSOLAS, S. B I O T - L A P O R T E , R. COHEN, P. C H A T E L A I N , J. P. B O I S S E L , S. F E R R Y , J. A . C H A Y V I A L L E , P. D U P I N , P. G A R R Y , B . C L A U S T R A T , P. G I R A R D , F . B O R S O N , J. T R O U I L L A S AND B. LORAS

C e n t r e de MtSdecine Nucl¢;aire, H ~ p i t a l N e u r o - C a r d i o l o g i q u e , L y o n , F r a n c e

SASSOLAS, G., S. BIOT-LAPORTE, R. COHEN, P. CHATELAIN, J. P. BOISSEL, S. FERRY, J. A. CHAYVIAI,LE, P. DUPIN. P. GARRY, B. CLAUSTRAT, P. GIRARD, F. BORSON, J. TROUILLAS AND B. LORAS. Clinical studies with human growth hormone releasin~,,j~u'tor in m~rmal adults and patients. PEPTIDES 7: Suppl. 1,281-286, 1986.--The recent availability of human growth hormone releasing factor (hGRF) encouraged thorough investigations of human growth hormone secretion. Moreover it is now possible to put forward a therapeutic application for this hormone. Herein, we report the dose-effect relationship obtained between hGRF and GH response in normal young men submitted to IV administration of doses ranging from 2.5 to 600 ~g per subject, in three protocols. In some subjects the 2.5/.tg dose elicited GH secretion as compared with placebo. A highly significant dose-effect was observed (based on GH-AUC and GH-peakt for doses ranging from 5 to 80/tg. Responses were identical above 80/xg. We conclude that the optimal dose required to elicit maximum GH release with minimal unwanted effects is 80/xg in adults. These are related to the dose and observed for doses up to 80--150 #g. Subcutaneous administration also induced GH-release, with relationship to the doses used ( 100, 300 and 600 ttg per subject). The mean response to the highest dose (600 ttg) was comparable in timing and magnitude to that obtained with a 100/,tg intravenous dose. Bioactivity of GH released under hGRF was proven in the Nb2 lymphoma cell multiplication assay and a high correlation was obtained between bioassay and radioimmunoassay. GH was present in blood atier hGRF under 3 molecular forms corresponding to little, big and big-big GH with percentages of 50, 30 and 20, respectively. An early and slight increase in prolactin was found to be related to the hGRF doses above 80/~g. No change was observed for doses less than 80/zg. Pharmacokinetic parameters were calculated from the IR-GRF concentrations obtained in the IV protocols. The half-life of distribution phase was 6.8_+0.4 rain and of the elimination phase was 43+2.98 rain. Comparison of IR-GRF concentrations alter IV and SC administration showed a massive loss of hGRF between SC space and plasma. The time required for IR-hGRF to disappear from the plasma was not greatly different from one route to another. When hGRF was associated with other hypothalamic hormones and the responses to separate and combined administration were compared, FSH, LH, cortisol and GH were comparably stimulated while TSH and prolactin responded significantly more to combined than separate administration. In adults presenting GH deficiency, idiopathic or secondary to hypothalamic tumors, IV administration of hGRF (I/xg/kg) induced GH responses which were always lower than in normal subjects. The older the patient and the longer the post-operative period, the lower the response. Short-term repeated IV or SC administrations of hGRF did not modify the magnitude of the GH responses and, in responders, induced a rise in Sm-C levels. IR-GRF basal concentrations determined in 45 acromegalic patients, were abnormal only in 3 cases with proven GRF-secreting tumors. Responses to IV 100/xg hGRF in acromegalic patients were highly variable without correlation with basal GH levels nor with functional aspects of their GH-secreting pituitary adenomas. Human growth hormone releasing factor (hGRF)

Human studies, males

G R O W T H h o r m o n e is s e c r e t e d in a pulsatile p a t t e r n in a n i m a l s a n d in m a n , d u e to t h e i n t e r a c t i o n o f two hypophysiotropic hypothalamic peptides, somatostatin and g r o w t h h o r m o n e releasing factor. T h e r e c e n t availability o f t h e s e f a c t o r s a n d o f t h e i r specific a n t i b o d i e s p r o v i d e s valuable tools for s t u d y i n g the p h y s i o l o g y o f G H s e c r e t i o n in animals. Human GRF (hGRF) has been characterized from t w o p a n c r e a t i c t u m o r s [6, 8, 10, 17] with a c r o m e g a l y a n d s u b s e q u e n t l y r e c o g n i z e d as b e i n g identical to h y p o t h a l a m i c G R F [7]. A n u m b e r o f g r o u p s h a v e i n v e s t i g a t e d the effects of

Growth hormone (GH)

h G R F on G H s e c r e t i o n in m a n [4, 18, 20]. I n c r e a s i n g insight h a s b e e n gained into G H d e f i c i e n c y i n v e s t i g a t e d by direct s o m a t o t r o p h s t i m u l a t i o n [2, 5, 9, 14]: p r e l i m i n a r y trials suggest p o s s i b l e a p p l i c a t i o n s for p r o m o t i n g g r o w t h [19]. O u r g r o u p has p a r t i c i p a t e d in this surge o f i n t e r e s t [11-13]. O u r aim here is to r e p o r t findings c o l l e c t e d in n o r m a l a d u l t s a n d in p a t i e n t s p r e s e n t i n g a b n o r m a l G H s e c r e t i o n . O u r studies w e r e p e r f o r m e d a f t e r a p p r o v a l b y the Ethical C o m m i t t e e de l ' U n i v e r s i t 6 C l a u d e B e r n a r d - L y o n a n d by the National Ethical Committee. h G R F ( G R F - 4 4 - N H2) s y n t h e s i z e d in the L a b o r a t o r i e s for

281

SASSOLAS ET AL.

282

Place~ GH pg/I

GRF

."k

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GRF

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....

+o,

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FIG. I. Means of GH responses obtained after IV injection of different doses of hGRF. The number of subjects was 10, 5 and 7 respectively.

GH

AUC JJg.min./I 225o. 2000.

•

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PHASE

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ONE

STUDIES

These studies led us to describe the dose-response relationship with 1V and SC routes, to consider the bioactivity and the molecular forms of GH released under hGRF, to define pharmacokinetic parameters after IV and SC administration, and to investigate response to hGRF adminstered in association with the other three releasing hormones.

sc

pg sc

, lOO pg iv

r \! Dose-Response Relationship

<<2__ 0

0 5 1 0 2"0 3~3 40 50 60

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FIG. 3. Mean GH responses after IV 100/xg, SC 100, 300 and 600/~g in six subjects.

Neuro Endocrinology of the Salk Institute (kindly provided in lyophilized form by R. Guillemin) and in the laboratories of SANOFI was used. Our methods have been reported previously [12,13]. The studies were carried out: (1) in a group of young normal adult men, belonging to the same age group (20-29 years of age); (2) in patients presenting GH deficiency of various origins; (3) in acromegalic patients with pituitary adenomas.

Three IV dose ranges were employed: (1) 0, 75, 150, 300, 600/~g per subject (N= 10); (2) 0, 1, 2.5, 5, 10/~g per subject (N=5); (3) 0, 2.5, 5, 10, 20, 40, 80/~g (N=7). The results are illustrated in Fig. I. They show the curves of the means of GH responses obtained with the three ranges of doses. An abrupt rise from base-line was observed after the injection of hGRF, maximum levels occurred within 45 minutes, and plasma GH returned to basal values by 90-180 minutes. A true bimodal curve was never observed. Based on statistical analyses performed on GH peaks and AUCs, the data allowed us to determine some parameters of the dose-response relationship (Fig. 2). A highly significant dose effect was observed for GH-AUC as well as for GH-peak, which was subjectindependent, for doses ranging from 2.5 to 80 /~g (3rd protocol). Plotting averages of GH-AUC against increasing doses showed that as from 5/~g the response increased with dose, and that below this dose mean responses were negligible.

HUMAN GROWTH RELEASING FACTOR

Placebo - -

Prolachn (}Jg/I)

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12J= 1113 i1: 10. ~ 9.

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FIG. 5B. Serum IR-GRF levels at various times after IV injection of 600, 300, 160, 80, 40, 20/xg bGRF (mean±SEM, No. of subjects).

However, in some subjects doses as low as 2.5 ~g could seemingly elicit GH secretion. In our 2nd protocol, responses (GH-peak and AUC) were significantly different between 2.5 /xg and placebo. It was thus concluded that the minimum effective dose is low, between 2.5 and 5 txg in adult man. From the data provided by the 1st and 3rd trials, we concluded that the optimal dose required to elicit maximum release in the healthy adult, with minimal unwanted effects, is approximately 80 ~xg. Indeed, this dose, in most of the subjects, elicits higher G H - A U C responses than smaller doses do, despite the fact that the mean responses did not differ signficantly, owing to high intersubject variability. This phenomenon of interindividual variability makes reliable estimates difficult in small scale studies. It has been consistently observed in all reports even with supra maximal doses. In our 1st trial, the significance of these variations was evaluated (p<0.01 for A U C and p<0.007 for GH peak) and we noted the consistency of the responses of individual subjects. Three subcutaneous doses were investigated in six subjects. They received 100,300 and 600/.g subcutaneously at 7

day intervals. Statistical analyses of GH curves showed a significant difference in A U C s between doses of 600 and I00 txg (p<0.01) and between doses 600 and 300/xg (/)<0.05). No difference was found between 100 and 300 txg. Moreover, the response curve with 600 txg SC is comparable in timing and magnitude of GH peak to that obtained in the same subjects with 100 txg IV, a dose of maximal effect (Fig. 3). We concluded that hGRF given subcutaneously at doses of 5-7 #g/kg can be as effective in promoting GH release as IV plateau doses [11]. BIOACTIVITY AND CIRCULATINGFORMSOF GH RELEASED UNDER hGRF At 30 and 60 min after adminstration of hGRF, in protocol 1, a marked increase in plasma GH bioactivity was detected in the Nb2 lymphoma cell multiplication assay. When regression analysis was performed between GH values measured by the Nb2 bioassay and IRA at times zero, 30 and 60 min, a significant correlation was found (r=0.713, p<0.001). Gel filtration chromatography of plasma drawn 60 min after hGRF injection showed that immunoreactive GH eluted in three major regions, corresponding to dissociation

284

SASSOLAS ET AL. Comparison Between IV and SC Administration

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constant values of 0.47-0.50, 0.31-0.34 and 0.15-0.18. These regions corresponded to molecular forms previously described as little, big, and big-big GH. The mol wts at the peaks averaged 22,000, 45,000 and 88,600 respectively. The percentage of little, big, big-big GH was approximately 5WA 30% and 20% respectively. These results have been previously reported by ourselves [12] and by Stolar 116]. PROLACTIN

In two out of the 3 protocols, we studied plasma prolactin levels in particular detail. The results are shown in Fig. 4. When the change in PRL after hGRF was compared with the change after placebo, a significant increase in plasma PRL 09<0.001) was found in the 1st protocol and a dosedependent relationship between hGRF dose and PRL response is shown in the left-hand side of the figure. No change was observed with doses ranging from 2.5 to 80/zg (righthand side of the figure). The release of prolactin under hGRF has been observed in the primate for large doses of GRF [16] and it has been reported in some in vitro trials [1]. This could indicate a direct effect. However, mediation by an extrapituitary factor cannot be disregarded. Bearing this in mind, we determined plasma levels of VIP in our dose-response protocols and found no significant change (unpublished). PHARMACOKINET1C

STUDIES

They were undertaken in 2 protocols involving 1V route ( 1 and 3) and in the protocol of subcutaneous administration. IV hGRF Our data confirmed that the means of the experimental plasma GRF-AUC were strongly related to the dose (Figs. 5a and 5b). The experimental data were fitted to a biexponential equation. The half-time of each phase was 6.80 min (SEM=0.40) and 43.07 (SEM=2.98) for distribution and elimination phases respectively. The MCR was 200 i/day (SEM=23). These results are comparable with those obtained with GRF 1-40 [15].

Figure 6 shows mean plasma IR-hGRF obtained with the 3 doses administered subcutaneously and with 3 doses administered intravenously. The delay in reaching the peak obviously differs between the two routes, since it occurs at 5 minutes with IV and at 20 minutes with SC injections. A comparable maximal value is observed with SC 600/zg and IV 20 /xg. This suggests a massive loss of the peptide between the SC space and the plasma, which is in keeping with the striking difference observed after IV and SC injection of the same 600/xg dose (207/~g/! versus 6.5/~g/l respectively). The time required for IR-hGRF to disappear from the plasma is not greatly different from one route to another (between 90 and 120 minutes for SC 600/zg and between 60 and 90 minutes for IV 20/xg). This suggests that hGRF has no prolonged and delayed delivery from the SC space into the plasma. ASSOCIATED ADMINISTRATION OF 4 HYPOTHALAMIC RELEASING HORMONES: PITUITARY TESTS

Ten normal young men were given successively and intravenously each of the 4 releasing hormones (TRH, GnRH, oCRF, and hGRF) separately and then an association of all four hormones sequentially. TSH, PRL, FSH, LH, Cortisol and GH were assayed by conventional radioimmunoassay performed on samples removed from 20 minutes before to 120 minutes after injection of separate or associated releasing hormones. The results showed no significant difference in response (peak-AUC) between separate or associated tests for the following hormones: FSH-LH, Cortisol, GH. On the other hand, a higher response was found for TSH and PRL after administration of the four hormones in association. This trial enabled us to describe the normal ranges of response to pituitary test valid in one age and sex group. The test is very well tolerated and can readily be applied to test pituitary function, for example, in patients having undergone surgical removal of pituitary tumors. CLINICAL

INVESTIGATION

IN ADULTS

WITH GH DEFICIENCY

IV Administration o f hGRF IV administration of hGRF (1/xg/kg) was performed in 10 adult subjects presenting GH deficiency proved by classical tests (insulin hypoglycemia, glucagon-propranolol) and by the lack of sleep related-GH peak. None of the subjects had been treated by growth hormone. They received treatment substituting other hormonal deficits. In six cases, GH deficiency was related to suprasellar tumor, formerly treated by surgery alone or combined with radiotherapy, or recently investigated, untreated, in one case. In the three patients having undergone treatment the longest time ago, presenting global pituitary deficiency unresponsive to hypophysiotropic agents, hGRF did not induce a rise in plasma GH. In contrast, in the three most recent cases, hGRF induced a significant response. The course of time of disorders appears to be a determining factor in GH response to hGRF. This suggests that authentic pituitary somatotroph deficits are constituted gradually after treatment of suprasellar tumors. In four cases of idiopathic growth hormone deficiency, the disorder was included in hypopituitarism (cases 1-4). The hypothalamic origin was suggested by the normal or

HUMAN GROWTH RELEASING FACTOR

285

slightly e l e v a t e d levels of prolactinemia stimulated by T R H , and by an important and delayed r e s p o n s e o f T S H to T R H . h G R F induced a minimal r e s p o n s e in 3 cases and a m o r e marked one in one case. M a x i m a l values under G R F w e r e always higher than those obtained with classical tests and l o w e r than those of normal subjects. T h e s e findings suggest a c o m b i n e d hypothalamic and pituitary disorder. GRF IN ADULTS WITH GH DEFICIENCY

No.

Age

1 2 3 4 5

46 19 21 37 18

6

33

7 8

9

10

hormone Diagnosis idiopathic idiopathic idiopathic idiopathic craniopharyngioma

craniopharyngioma 20 years post surgery 23 craniopharyngioma 10 years post XRT 21 Craniopharyngioma 7 year post surgery and XRT 28 craniopharyngioma 1 year post surgery 18 suprasellar T. 2 year post surgery and XRT

Pituitary under GRF deficiency global deficiency GH + FSH + LH GH + FSH + LH global deficiency GH + ACTH + TSH + FSH + LH + AVP global deficiency

global deficiency global deficiency

GH + FSH + LH

Peak GH (p.g/I) 4.4 8 3.3 3.3 14.2

<1

1.3 <1

R I A in 45 acromegalic patients. High levels (3-10-50 ~g/l) w e r e only found in 3 patients presenting a c r o m e g a l y and with previously detected associated tumors, later p r o v e n to be G R F secreting. In all o t h e r cases, I R - h G R F levels were negligible (below our detection limit value: 0.15/zg/1. Secondly, we administered h G R F to 15 patients before surgery. R e s p o n s e s were highly variable (mean A G H = 6 9 . 5 ~g/l ranging from 5 to 280). N o correlation was found bet w e e n h G R F - i n d u c e d - G H rise and basal G H values. N o relationship was found b e t w e e n the magnitude of the r e s p o n s e s to h G R F and the cytofunctional characteristics of the a d e n o m a s , particularly as c o n c e r n s the secretory granules. N i n e of the tumors r e m o v e d in these patients w e r e studied in vitro with short-term administration of h G R F . The same variability b e t w e e n the responses was found. In contrast with our in v i v o studies, we noted a relationship bet w e e n high r e s p o n s e s to h G R F and the d e m s e l y granulated c h a r a c t e r of the tumors (submitted). In another group of patients previously unsuccessfully treated by surgery, with m e a n G H basal values of 27.7___9.2 /~g/1 (range 7-98), response to h G R F was on average higher than in the first group (mean A GH: 151 ~g/l, ranging from 4-620). The highest responses were o b s e r v e d in patients presenting large and evolutive r e m n a n t tumors. Nine patients cured by surgery p e r f o r m e d from 12 years to 1 year before h G R F test had a mean response of 8.6-+3.2 p.g/1. Six of these patients fulfilled all the criteria of cure, i.e., no G H rise under T R H , undectable G H values under O G T T and r e s p o n s i v e n e s s to G R F .

16.4 CONCLUSION

global deficiency

4.9

Repeated Administration o f h G R F

IV injections o f h G R F (1 /xg/kg) were repeated twice a day during 5 days in 3 patients of e a c h group. N o significant change in the magnitude of the responses was o b s e r v e d . Plasma s o m a t o m e d i n - C levels increased only in the patients with G H response to G R F . S u b c u t a n e o u s injections (3.5/~g/kg per pulse, six pulses per day during 5 days) induced a rise in s o m a t o m e d i n - C (0.11 to 0.67 U/ml at the sixth day) in one patient. CLINICAL INVESTIGATION IN ACROMEGALIC PATIENTS Firstly, basal h G R F plasma levels were d e t e r m i n e d by

Clincial investigations confirm that human G R F is a reliable and potent stimulator of h G H release. T h e y indicate that this peptide can be directly and safely used to explore G H secretion. They suggest its applicability for therapy in G H deficient children responding to G R F . F u r t h e r therapeutic d e v e l o p m e n t s will depend on the availability of galenic form suitable for long-term administration, inducing pulsatile G H secretion and subsequent body growth.

ACKNOWLEDGEMENTS The authors are grateful to the technical staff of the laboratoire de Radio-Immunologie, to the staff nurses of the Centre de M6decine Nuclraire and to Miss Shaw and Mrs. Metral for the preparation of this manuscript. These studies were supported by Fondation pour la Recherch Mrdicale Franqaise, by INSERM (Contrat No. 83-4015) and by SANOFI.

REFERENCES 1. Almeida, O. F. X., H. M. Schulte, R. S. Rittmaster, G. P. Chrousos, D. L. Loriaux and G. R. Merriam. Potency and specificity of a growth hormone-releasing factor in a primate and in vitro. J Clin Endocrinol Metab 58: 309-312, 1984. 2. Borges, J. L. C., M. C. Gelato, A. D. Rogol, M. L. Vance, R. M. Mac Leod, D. Lynn-Loriaux, J. Rivier, R. M. Blizzard, R. Furlanetto, W. S. Evans, D. L. Kaiser, G. R. Merriam, J. Spiess, W. Vale and M. O. Thorner. Effects of human pancreatic tumour growth hormone releasing factor on growth hormone and somatomedin C levels in patients with idiopathic growth hormone deficiency. Lancet 2: 119-123, 1983.

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