EP0650597A1

EP0650597A1 - Diagnostic procedure for evaluating short stature etiology

Info

Publication number: EP0650597A1
Application number: EP93916678A
Authority: EP
Inventors: Barry B. Bercu; Richard F. Walker
Original assignee: University of South Florida
Current assignee: University of South Florida
Priority date: 1992-06-29
Filing date: 1993-06-22
Publication date: 1995-05-03
Also published as: WO1994000759A1; EP0650597A4; CA2139326A1

Abstract

A diagnostic procedure for evaluating the etiology of short stature in children which comprises measuring the effect of a growth hormone releasing compound and the effect of GHRP-6 or a peptide which releases growth hormone by the same cellular mechanism alone or in combination on the growth hormone levels in the blood of the child.

Description

DIAGNOSTIC PROCEDURE FOR EVALUATING SHORT STATURE ETIOLOGY

FIELD OF INVENTION

The present invention provides a method for diagnosing the etiology of short stature in children.

BACKGROUND OF THE INVENTION

Stimulated growth hormone GH secretion using a variety of provocative agents including 1-dopa, clonidine, arginine and insulin have been assessed in slowly or poorly growing children in an attempt to determine the etiology of short stature. However, the reliability of those tests has been the subject of significant debate because differential GH secretion in subsets of short-statured children makes the data difficult to interpret. For example, provocative agents whose action is mediated by growth hormone releasing hormone (GHRH) cannot differentiate hypothalamic defects from pituitary defects in the GH neuroendocrine axis. Thus, the isolation, characterization and synthesis of growth hormone- releasing hormone (GHRH) provided a potentially valuable diagnostic tool for evaluation of growth hormone (GH) deficiency and differentiation between hypothalamic and pituitary decrements (O. Butinandt, Acta Paediatr. Scand [suppl.] 1989; 349. 93-9). However, its diagnostic value has been surprisingly limited because of marked variability in GH secretion following administration of GHRH (A. Pertzelan et al., Hormone Res 1985; .22_., 24-31) . In an attempt to explain this variability, it was proposed that some children with GH deficiency due to hypothalamic etiology were hyporesponsive to a single dose of GHRH because of chronic deprivation of the exposure to the peptide (E.A. Schriock et al., J. Clin. Endocrinol. Metab. 1984, 58.: 1043-1049). This explanation was not completely adequate because subnormal GH responses to a single GHRH injection were also observed in GH sufficient children (P. Chatelain et al., J. Clin. Endocrinol. Metab. 1987: 65: 387-394) .

Another proposal to explain variable responses to provocative GHRH tests was that GH releasing activity of the peptide varied with the time of its administration (P.M. Martha et al., J. Clin. Endocrinol. Metab. 1988; 67, 449-454). Endogenous hypothalamic-somatotroph secretory rhythm affect the GH response to GHRH in humans (J. Deversa et al., Clinical Endocrinology 1989; 30. 367-377) , and rats in vivo (G.H. Albini et al.. Clinical Pharmacol Ther. 1988, .43_., 696-700) and in vitro (G.S. Tannenbaum et al., Endocrinology 1989; 124. 1380-1388) and this rhythm might cause the variation in GH responsivity following exogenous GHRH. Thus, debate has considered whether spontaneous as well as stimulated GH secretion must be characterized to reliably identify children with low GH secretion. Clearly, the greatest information would be derived from both methods, but the high cost and inconvenience of extended hospitalization for long-term analysis of spontaneous GH secretion limits the practical utility of this procedure. On the other hand, the low cost and relatively quick procedure makes provocative testing most attractive, so long as the problem of response variability could be resolved. U.S. patents 5,065,747 issued November 19, 1991, and 4,844,096 issued July 4, 1989 describe methods for reducing the variability in GH levels by administering somatostatin prior to provocative testing or prior to determining the etiology of growth hormone deficiency.

The present invention provides a new and more effective way to use stimulated GH secretion as a reliable and effective tool for diagnosing GH secretory deficits in short- statured children.

SUMMARY OF THE INVENTION The present invention provides a diagnostic procedure for determining whether short stature in a child is due to a deficiency in growth hormone releasing hormone (GHRH) or GHRP-6 or a peptite which causes release of growth hormone (GH) by the same cellular mechanism. The diagnostic procedure of the present invention is carried out by establishing baseline levels of growth hormone in the blood of the child having short stature, then administering sequentially a growth hormone releasing compound, GHRP-6, or a peptide which causes growth hormone release by the same cellular mechanism as GHRP-6, or the combination of GHRH and GHRP-6 and then measuring the changes, if any, in the levels of growth hormone in the blood of the short-statured child resulting from the three different administrations. Children deficient in GHRH would respond best to GHRH. Children deficient in the endogenous analog for GHRP-6 would respond best to GHRP-6. Children deficient in both GH secretogogues would respond best to the combination of GHRH and GHRP-6. The growth hormone releasing compound can be administered either first or second, generally on different days, i.e., before or after the GHRP-6 or peptide which acts similarly at a cellular level. The quantities of each agent to be administered is any quantity known to be effective in causing an increase in growth hormone levels, i.e., an amount which will stimulate release of growth hormone.

There are a number of factors that are relevant to the underlying basis of the present invention. Recent studies show that GHRH may not be the only endogenous agent that provides stimulation for GH secretion. A xenobiotic hexapeptide, GHRP-6, which has different binding characteristics from GHRH (E.E. Codd et al., Neuropharmacology 1989; ,28_., 1139-1144 and A.D. Blake, et al., J. Endocrinol. 1991; 129. 11-19) and utilizes a different somatotroph second messenger system (K. Cheng et al., Endocrinol. 1989; 124, 2791-2798) is a GH secretagogue that potentiates GHRH efficacy (C.Y. Bowers et al., J. Clin. Endocrinol. Metab. 1990; 7J, 975-982) . Furthermore, like GHRH, GHRP-6 is effective in some, but not all, short-statured children to whom it is administered (C.Y. Bowers et al. , J. Clin. Endocrinol. Metab. 1991; 1 _, 292-298 and V. Merica et al., 1992: Growth Hormone Responses to a Second Generation Growth Hormone Releasing Peptide and to GH Releasing Hormone [GHRH] in Growth Hormone Deficient Children, Endocrine Society Abstracts, 74th Annual Meeting, San Antonio, TX, June 24-27, No. 477, pg 171). The different mechanisms and synergistic effects of GHRH and GHRP-6 in vitro and in vivo, suggest the existence of an endogenous analog for the synthetic hexapeptide that may be physiologically relevant. Support for this hypothesis derives from two of our recent studies. In one study, passive immunization against endogenous GHRH in rats, reduced GHRP-6 activity approximately 90% (B.B. Bercu et al., Endocrinology 1992; 130. 2579-2586) . These data demonstrated the requirement of endogenous GHRH for expression of GHRP-6 activity. A reciprocal relationship, i.e., dependence of GHRH upon a yet unidentified, endogenous analog of GHRP-6 may also exist. Thus, in individuals deficient in the endogenous GHRP-6 analog, GHRH efficacy would be blunted, whereas in individuals deficient in GHRH, GHRP-6 efficacy would be blunted. Support for this hypothesis derives from our recent finding in which old rats that were hyporesponsive to individually administered GHRH or GHRP-6, were hyperresponsive to co-administered GHRH and GHRP-6 (R.F. Walker et al.. Life Science 1992; 49. 1499-1504 and B.B. Bercu et al. , Endocrinol. Abstracts, Annual Meeting San Antonio, TX, June 24-27, 1992). Hyposensitivity to the individually administered peptides suggested that more than one, interdependent, endogenous, stimulatory factor contributed to GH secretion. GHRP-6 is a hexapeptide having the following structure: His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂. In practicing the present invention, GHRP-6, as well as all other synthetic peptides in the same class of drugs that release GH by the same cellular mechanism as GHRP-6 referred to hereinafter as GHRP-6 or analogs thereof, will be used as diagnostic agents in children for differential diagnosis of growth hormone (GH) deficiency of various etiologies. Responses to GHRP-6 or analogs thereof will be compared with those resulting from administration of the endogenous GH releasing hormone (GHRH) and from co-administration of GHRH and GHRP-6 to better understand the cause of growth deficits in short statured children. Since GHRP-6 acts as a functional analog for a yet undefined GHRH co- factor or co-secretagogue, hyposensitivity to a provocative GHRH challenge could represent deficiency of that endogenous co-secretagogue (represented by GHRP-6) rather than down- regulation of pituitary receptor or deficits in GH synthesis and/or release. Such a child could have normal GHRH concentrations/secretion and a normal pituitary, but display poor GH secretion in the absence of the endogenous GHRP-6 analog. On the other hand, this child would respond normally to a challenge dose of GHRP-6 because his/her complement of GHRH would be available. In contrast, a child with GHRH deficiency, but having normal concentrations of GHRP-6 endogenous analog would respond normally to a provocative dose of GHRH, and poorly to GHRP-6 since the child's GHRH complement would be lacking. The paradox in this hypothesis is that a normal response to a provocative challenge by a given GH secretagogue (GHRH or GHRP-6 or analog thereof) will be observed for the peptide that is lacking or deficient in the slow-growing child. Poor responses to individually administered GHRH and GHRP-6 would suggest a deficiency of both endogenous GH secretagogues, and/or a defect in pituitary GH synthesis/release. Thus, the present invention provides a new clinical application for GHRP-6 and other synthetic GH secretagogues that function by the same mechanism as GHRP-6. The invention is important because it not only provides a reliable method for identifying children with low GH secretory capability, but also helps diagnose the etiology of GH deficiency and perhaps provide a key to appropriate treatment.

In practicing the present invention, children who are shorter than other children of a comparable age who have been identified as in need potentially of growth hormone or some agent that would stimulate growth hormone production would be monitored for the purpose of establishing a baseline level of growth hormone in the blood. Once baseline levels are established, the child is administered GHRH at a dose ranging from 100 ng/kg body weight to 100 mg/kg body weight. The GHRH peptide is administered and blood samples are monitored over a period of 120 minutes to determine whether the exogenous GHRH increased growth hormone levels. After the effects of GHRH administration are determined, the child is administered GHRP-6 or an analog thereof at a dose of 100 ng/kg body weight to 1 mg/kg body weight for one day. Blood samples are measured for any change in growth hormone levels. Additional administration of peptides may be made but a day of rest, i.e., a day during which neither peptide type is administered should intervene administration days. The dosage amount can be varied to determine optimal dosages. The changes, if any, in growth hormone levels to either or both of GHRH or GHRP-6 or analog thereof will provide an insight to the etiology of the short stature condition of the child.

In addition to GHRH, any growth hormone releasing compound can be utilized in the diagnostic procedure of the present invention. However, GHRH is the preferred growth hormone releasing compound for use in the claimed invention. The growth hormone releasing compounds which may be used in practicing the present invention are any such compounds known to induce growth hormone (GH) secretion and include growth hormone releasing hormone (GHRH) (1-44) and analogs GHRH (1-40) and GHRH (1-29) thereof. There are numerous growth hormone releasing compounds known in the art and any of these known compounds will be useful in practicing the present invention. U.S. Patent 4,622,312 provides an excellent description of GHRH and analog thereof, which can be used in the presently claimed invention. Reissue patent RE33,699 provides a summary of patents which teach growth hormone releasing compounds. The growth hormone releasing compounds taught in each of the following U.S. patents are suitable for invention. Reissue Patent No. RE 33,699 provides a summary of patents which teach growth hormone releasing compounds taught in each of the following U.S. patents are suitable for use in the method of the present invention: COU PATENT NO COLUMN LINE

The above U.S. patents, and in particular the portions indicated above by column and line number, are incorporated herein by reference as teaching growth hormone releasing compounds that are useful in the practice of the presently claimed invention. The term GHRP-6 and analogs thereof means GHRP-6 and any peptide compound that releases GH by the same cellular mechanism. In addition to GHRP-6, the pentapeptide Tyr-D-Trp- Gly-Phe-Met-NH₂ (K. Cheng et al. , Endocrinol. 1989; 124. 2791-2798) is a useful analog in the release of GH. Other compounds considered analogs of GHRP-6 for purposes of the present invention have been reported. For example, C.Y. Bowers et al., Endocrinol. 1980; 106. 663 teaches in addition to Tyr-D-Trp-Gly-Phe-Met-NH₂, compounds Tyr-D-Phe-Gly-Phe-Met-NH₂ and Trp-D- Phe-Pro-Phe-Met-COOH as being useful in the release of growth hormone, and GHRP-1 having the formula Ala-His-D-β-Nal-Ala-Trp-D-Phe-Lys-NH₂ disclosed in the 74th Annual Meeting of the Endocrine Society, June 24-27, 1992, p. 182, San Antonio, Texas, is also useful in the release of growth hormone. Also, F.A. Mo any et al., Endocrinol. 1981; 108. 31 teaches the following compounds as being useful in the release of growth hormone: Try-Ala-D-Trp-Phe-Met-NH₂; Tyr-D- Trp-D-Trp-Phe-Met-NH₂; Tyr-D-Trp-D-Trp-Phe-NH₂; Tyr-D-Trp-D-Trp-Phe-COOH; and D-Trp-D-Trp-Phe- NH₂. Additionally, F.A. Momany et al., Endocrinol. 114 , 1531 (1984) teaches the following compounds as being useful in the release of growth hormone: His-D-Trp-Ala-Trp-D- Phe-NH₂; His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂; Tyr-D- Trp-Ala-Trp-D-Phe-NH₂; His-D-Trp-Ala-Trp-D-Phe- Arg-NH₂; and His-D-Trp-Ala-Trp-D-Phe-Lys-COOH. All of these compounds are useful in the diagnostic procedure of the present invention.

The growth hormone levels in the blood of the child can be measured by any well known procedures, e.g., using an immunoradiometric assay as described by C.Y. Bowers et al., J. Clin Endocrinol. Metab 1990; 7J), 975-982 or any other contemporary, scientifically accepted method.

Once the short-statured child is diagnosed as having either a deficiency in GHRH or GHRP-6 the appropriate replacement therapy can ensue whereby either a growth hormone releasing compound or a GHRP-6 or analog thereof or a combination of both GH secretagogues is administered to the child.

Claims

1. A method for measuring the effect of a growth hormone releasing compound and GHRP-6 or a peptide which causes release of growth hormone by the same cellular mechanism as

GHRP-6 on the blood levels of growth hormone in a child of short stature which comprises the steps of:

(a) establishing a baseline level of growth hormone in the blood of said child;

(b) administering an amount of growth hormone releasing compound known to be effective to cause an increase in growth hormone levels in the blood; (c) administering an amount of GHRP-6, or a peptide which causes release of growth hormone by the same cellular mechanism as GHRP-6, known to be effective to cause an increase in growth hormone levels in the blood; (d) administering a combination of GHRH and GHRP-6 or a peptide which causes release of growth hormone by the same cellular mechanism as GHRP-6 in amounts known to be effective to cause an increase in growth hormone levels in the blood; and

(e) measuring levels of growth hormone in the blood after each of steps (b) , (c) and (d).

2. The method of claim 1 wherein the growth hormone releasing compound is growth hormone releasing hormone.

3. The method of claim 2 wherein the GHRP-6 or analog thereof is selected from GHRP-6, GHRP-1, or one of the following peptides:

Tyr-D-Trp-Gly-Phe-Met-NH₂; Tyr-D-Phe-Gly-Phe-Met-NH₂;

Trp-D-Phe-Pro-Phe-Met-COOH;

Tyr-Ala-D-Trp-Phe-Met-NH₂;

Tyr-D-Trp-D-Trp-Phe-Met-NH₂;

Tyr-D-Trp-D-Trp-Phe-NH₂; Tyr-D-Trp-D-Trp-Phe-COOH;

D-Trp-D-Trp-Phe-NH₂;

His-D-Trp-Ala-Trp-D-Phe-NH₂;

His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂;

Tyr-D-Trp-Ala-Trp-D-Phe-NH₂; His-D-Trp-Ala-Trp-D-Phe-Arg-NH₂;

His-D-Trp-D-Trp-Ala-Trp-D-Phe-Lys-COOH.

4. The method of claim 1 wherein the amount of growth hormone releasing compound administered is 100 ng/kg-100 mg/kg of body weight once daily.

5. The method of claim 4 wherein the amount of GHRP-6 or a peptide which causes release of growth hormone by the same cellular mechanism administered is 100 ng/kg-l mg/kg of body weight once daily.

6. The method of claim 5 wherein the growth hormone releasing compound is administered first.

7. The method of claim 5 wherein the growth hormone releasing compound is administered second.

8. The method of claim 1 wherein a combination of GHRH and GHRP-6 or a peptide which causes release of growth hormone by the same cellular mechanism is administered.

9. The method of claim 8 wherein the amount of GHRH administered is 100 ng/kg to 100 mg/kg of body weight and the amount of GHRP-6 or a peptide which acts by the same cellular mechanism as GHRP-6 is from 100 ng/kg to 1 mg/kg of body weight, each being administered once daily for one day.

10. The method of claim 1 wherein a day during which no peptide or hormone is administered intervenes each day on which a dosage is administered.

11. A method for diagnosing the etiology of short stature in a child which comprises administering sequentially an agent which is a growth hormone releasing compound and an agent which is GHRP-6 or a peptide which causes growth hormone release by the same cellular mechanism, and a combination of each said agent, each of said agents being administered in a quantity known to be effective in increasing blood levels of growth hormone and measuring the effects each agent has either alone or in combination on the growth hormone levels in the blood of said child.

12. The method of claim 11 wherein a baseline level of growth hormone in the blood is established for the child prior to the diagnostic procedure.

13. The method of claim 12 wherein the growth hormone releasing compound is GHRH.

14. The method of claim 13 wherein the GHRP-6 or peptide which causes growth hormone release by the same cellular mechanism is GHRP-6.

15. The method of claim 14 wherein a day during which neither agent is administered intervenes the days on which dosing occurs.