EP0871392A1 - Method of diagnosing pituitary dependent growth hormone deficiency - Google Patents

Abstract

A diagnostic protocol and kit for evaluating pituitary growth hormone (GH) secretory capability as a means to identify the etiology of GH deficiency in children and adults is disclosed. The inventive protocol includes measuring GH secretion, as determined by changes in serum, plasma, or whole blood GH concentrations following administration of the xenobiotic GH releasing hexapeptide (GHRP-6) or any of its peptidyl or non-peptidyl synthetic analogues that release GH by the same cellular mechanism as GHRP-6. The naturally occurring GH releasing compound (GHRC) or its analogues is also administered to children and adults afflicted with disorders related to GH deficiency and GH secretion is measured. After evaluating the effects of independent administration of the GH secretagogues upon changes in blood GH concentrations, the effect of the co-administered GH secretagogues on blood GH concentrations in the child or adult subject is then measured.

Description

METHOD OF DIAGNOSING PITUITARY DEPENDENT GROWTH HORMONE DEFICIENCY

BACKGROUND OF THE INVENTION

TECHNICAL FIELD

The present invention provides a method for directly testing pituitary GH secretory capability in children and adults with clinical symptoms of growth hormone deficiency.

BACKGROUND ART

Stimulated growth hormone (GH) secretion by a variety of provocative agents including L-dopa, clonidine, arginine and insulin has been used to assess GH secretory capability in children with short stature and in adults with a variety of disorders associated with GH deficiency, such as osteoporosis, muscle atrophy, reduced LDL cholesterol metabolism, poor immune function, etc. However, the reliability of those provocative agents has been the subject of significant debate because differential GH secretion in subsets of patients makes the data difficult to interpret. Significant variability presumably occurs because the GH stimulating mechanisms of these non-specific, provocative agents are not well defined and probably affect various different levels of the GH regulatory cascade, ranging from hypothalamic to pituitary sites.

For example, the non-specific provocative agents whose action may be mediated, at least in part, by release of growth hormone releasing hormone (GHRH) from the brain, cannot differentiate between deficient hypothalamic GHRH stores or release capabilities from unresponsive pituitary-GHRH transduction mechanisms. Thus, the isolation, characterization and synthesis of naturally occurring GHRH and its structurally altered analogues that act directly upon the pituitary by the same mechanism as GHRH, seemed to provide a potentially valuable diagnostic tool for differentiating GH deficiency resulting from hypothalamic versus pituitary deficits (Butenandt, 1989) . However, its diagnostic value has been surprisingly limited because of marked and unexpected variability in GH secretion following administration of GHRH, i.e., in some cases GHRH showed extremely low potency even though pituitary GH secretory mechanisms were intact (Pertzelan, 1985) .

In an attempt to explain this variability, it was proposed that some children and adults with GH deficiency due to inadequate hypothalamic GHRH release were hyporesponsive to a single dose of GHRH because of chronic deprivation of the exposure to the peptide (Schriock et al. , 1984) . This explanation was not completely adequate because subnormal GH responses to a single GHRH injection were also observed in GH sufficient children and adults (Chatelain et al . , 1987; Pavlov et al. , 1986) .

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 (Martha et al . , 1988) . Endogenous hypothalamic- somatotroph secretory rhythms affect the GH response to GHRH in humans (Devessa et al . , 1989) , and rats in vivo (Albini et al . , 1988) and in vitro (Tannenbaum et al., 1989) 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 and adults 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 rapid, provocative procedure makes this type of 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 response levels by administering somatostatin prior to provocative testing for the purpose of determining the etiology of growth hormone deficiency. Variability is responses still occur with this protocol suggesting that the responses are modulated by other factors. In addition, there is a reciprocal relationship between endogenous growth hormone (GH) and degenerative changes in form and function associated with aging. For example, the correlation between low serum GH concentrations and somatic involution/physiologic dysfunction was recognized decades ago (Root and Oski,

1969; Rudman et al . , 1981) . However, limited availability of cadaver-derived GH prior to development of recombinant biotechnological methods prevented valid testing of the hypothesis that GH deficiencies and aging decrements were functionally related. When GH became available for experimentation, it was possible to show that administration of the hormone to old men significantly increased insulin-like growth factor-1 (IGF-1) , urinary nitrogen retention, body weight gain, lean body mass, bone density, renal function and improved psychological attitude (Rudman et al . , 1990; Kaiser et al. , 1991; Marcus et al. , 1990) . Thus, while patterns of spontaneous GH secretion deteriorate and serum GH concentration decline during aging (Finkelstein et al . , 1972; Rudman et al . , 1981; Ho et al. , 1987), somatic responsiveness to GH seems to remain intact, since insulin-like growth factor-1 (IGF- 1; somatomedin-C) decrements and certain physical/physiological deficits are readily reversed by administration of exogenous GH (Pavlov et al. , 1986) .

There is progressive resistance to GH secretion during aging that results from either inadequate stimulation of the pituitary gland or degeneration of pituitary-based mechanisms for GH production/secretion. Except for two reports (Wehrenberg and Ling, 1983; Pavlov et al . , 1986) , there is a consensus that GH secretion in response to GHRH administration in vivo declines with advancing age (Ceda et al . , 1986; Ghigo et al . , 1990; Iovino et al . , 1989; Lang et al. , 1987, 1988; Shibasaki et al . , 1984; Sonntag and Gough, 1988; Sonntag et al. , 1980, 1983) . It is possible that changes in the relationship of GH regulatory hormones of hypothalamic origin are the primary etiological factors in age-related decline in GH secretion. Since several hormones may be involved, provocative tests with GHRH alone to determine GH secretory capability in older adults with clinical symptoms of GH deficiency may not be adequate.

It is, therefore, an objective of the present invention to provide a diagnostic protocol that can differentiate between various etiologies of GH deficiency in both children and adults.

SUMMARY OF THE INVENTION AND ADVANTAGES

According to the present invention, a diagnostic protocol and kit for evaluating pituitary growth hormone (GH) secretory capability as a means to identify the etiology of GH deficiency in children and adults is disclosed. The inventive protocol includes measuring GH secretion, as determined by changes in serum, plasma, or whole blood GH concentrations following administration of the xenobiotic GH releasing hexapeptide (GHRP-6) or any of its peptidyl or non-peptidyl synthetic analogues that release GH by the same cellular mechanism as GHRP-6 (the group/family of compounds are referred to as GHRX) . The naturally occurring GH releasing compound (GHRC) or its analogues is administered to children and adults afflicted with disorders related to GH deficiency and GH secretion is measured. In the preferred embodiment, the changes in serum, plasma or whole blood concentrations of GH following individual administration of these two different GH secretagogues is then expressed as a ratio of peak GH concentrations for GHRX divided by peak GH concentrations for GHRH. This ratio is then used to compare the results for the subject being tested against values derived from normal subjects for reference purposes. After evaluating the effects of independent administration of the GH secretagogues upon changes in blood GH concentrations, the effect of the co-administered GH secretagogues on blood GH concentrations in the child or adult subject is then measured.

The ratio provides a relative measure of the deficiency for each endogenous secretagogue in reference to the co-secretagogue. This is useful in selecting the therapeutic dose for treatment since the more the ratio deviates from the normal range the more aggressive the treatment must be. The present invention provides a new and more effective way to use stimulated GH secretion as a reliable and effective tool for directly evaluating pituitary GH secretory capability and for differentiating specific extra-pituitary factors from intra-pituitary factors that contribute to GH deficiency in children and adults afflicted with medical disorders related to such a hormone deficit. BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

FIGURE 1 is a diagram of the three step provocative testing of the present invention. FIGURE 2 is a dose response curve for GHRP-6 in rats pretreated with vehicle (triangles) , αr-methyl-p- tyrosine (circles) , or GHRH antiserum (squares) . Values represent means ± SEM (8 rats per dose and treatment) in plasma collected 15 minutes after administration of GHRP- 6.

FIGURE 3 is a dose response curve showing the effect of [N-Ac-Tyr¹, D-Arg²] -GRF 1-29 amide, a GH receptor antagonist, on GH release in response to GHRP-6 (30 μg/kg, closed symbols, panel A) or morphine (1.5 mg/kg, open symbols, panel B) . The GHRH receptor antagonist was administered in doses of 0 (circle) , 5 (square) , 50 (triangle) , or 150 (diamond) μg/kg. Values represent mean ± SEM 8 rats per group.

FIGURE 4A-D is a series of dose response curves showing the GHRP-6/GHRH ratio over time after challenge in (A) a control subject, (B) a hypopituitary subject, (C) a constitutional delay subject, and (D) an irradiated subject.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides a diagnostic procedure and kit for determining whether growth hormone deficiency in children and adults is due to a deficiency in endogenous growth hormone releasing hormone, to a deficiency of the endogenous analogue of a new family of xenobiotic GH secretagogues (GHRX) , the prototype of which is GHRP-6, to a deficiency of both endogenous GH secretagogues, or due to intrinsic defects in the pituitary gland not involving either GH secretagogue, that result in GH deficiency.

GHRH has been identified as a naturally occurring GH secretagogue, an endogenous analogue of a group referred to as GH releasing compound (GHRC) . GHRP-6 represents a synthetic analogue of another, yet unidentified, endogenous GH secretagogue. The relationship between GHRP-6 and its endogenous counterpart is presumably analogous to that of morphine and the endorphins.

Since GHRH and GHRP-6 are functional complements, the response of one is amplified in the presence or under the influence of the other. Therefore, robust GH secretion in response to administration of GHRH or GHRP-6 could be interpreted as representing adequate endogenous GHRP-6 or GHRH, respectively. Alternatively, assuming that all pituitary cellular and molecular elements for GHRH- and GHRP-6-mediated GH secretion are functional, a poor response to either GH secretagogue administered individually could represent inadequacy of its endogenous complement. The paradox in this hypothesis is that a normal response to a provocative challenge by a given GH secretagogue will be observed for the peptide that is lacking or deficient in the patient.

Utilizing this differential diagnostic procedure, the integrity of functional pituitary elements can be differentiated from inadequate concentrations of both endogenous complements by administering the GH secretagogues simultaneously. GH secretion in response to co-administered GHRH and GHRP-6 would indicate inadequate endogenous GH secretagogues, whereas lack of GH secretion in response to the co-administered secretagogues would indicate intrinsic defect (s) in pituitary functional capacity.

The basis of the diagnostic test is a comparison of responses to provocative challenges of exogenous GH secretagogues administered sequentially and in combination. In the preferred embodiment, GHRH and GHRP-6 are used as the GH secretagogues. For clarity of discussion, the invention will be discussed mainly in terms of GHRH and GHRP-6. The invention, however, may be applied in an analogous fashion with the analogues of these two GH secretagogues.

The analogues for xenobiotic GHRP-6 include peptidyl and non-peptidyl forms of GHRP-6-like xenobiotic GH secretagogues that release growth hormone from the pituitary gland by the same cellular mechanism as GHRP-6. The group/family of these related molecules will be designated as GHRX for convenience. The analogues of GHRH are members of a group of GH secretagogue referred to as growth hormone releasing compound (GHRC) such as GH releasing hormone (GHRH) , GH releasing factor or any of its synthetic analogues that release GH from the pituitary gland by the same cellular and molecular mechanism as GHRH.

The diagnostic procedure of the present invention is carried out by establishing baseline levels of growth hormone (GH) in the blood of the child or adult being evaluated. Sequentially provocative challenges are then administered of a GHRC and GHRX, and then the combination of the GH secretagogues is administered. The levels of growth hormone in the blood of the patient is measured thereby determining if GH level changes after each of the three different administrations has occurred. The order of the provocative challenges can be changed as long as enough time between injections is provided for clearance of the previous challenge. In summary, five conditions are anticipated: normal, an insufficiency of GHRH, an insufficiency of the in vivo endogenous analogue of GHRP-6, an insufficiency of both or a defective pituitary GH secretory mechanism. The last four conditions will present with a GH insufficiency. Obviously, the cause in each case is different. In each of these conditions, a different pattern of GH secretion is anticipated in response to a provocative challenge with exogenous GHRC and/or GHRX. (Table 1, Figure 1) It is these different responses that are used to differentially diagnose the etiology of GH insufficiency, i.e. to determine whether the GH insufficiency is based upon GHRH and/or in vivo endogenous analogue of GHRP-6 insufficiency, or upon an inherent, functional pituitary defect.

In the normal condition, GHRH and the in vivo endogenous analogue of GHRP-6 are present in normal concentrations (Column 2, Table 1) . Therefore, the injection of either GHRC or GHRX will release concentrations of GH that are normal and that reflect the presence of the endogenous and exogenous GHRH and GHRP.

If endogenous GHRH is insufficient or absent while the in vivo endogenous analogue of GHRP-6 is present in normal amounts (column 3, Table 1) , then an injection of GHRC will release a single normal quantum of GH. The injection of GHRX will release subnormal amounts of GH, if at all.

If the in vivo endogenous analogue of GHRP-6 is insufficient or absent while endogenous GHRH is present in normal amounts (Column 4, Table 1), then an injection of

GHRC will release subnormal amounts of GH, if at all. The injection of GHRX will release a normal quantum of GH.

If both endogenous GH secretagogues are insufficient or absent (Column 5, Table 1) , then GHRC or GHRX administered individually will release subnormal quanta of GH, if at all. If the co-administration of GHRC and GHRX release a normal quanta of GH, then both endogenous secretagogues are absent or insufficient. If no or reduced GH is released (Column 6, Table 1) than an intrinsic defect in the pituitary gland not involving either GH secretagogue has been shown.

The responses are evaluated as representing optimal concentrations of both endogenous GH secretagogues or deficiencies of one or the other endogenous complementary secretagogue by calculating a ratio of peak GH concentrations for GHRX divided by peak GH concentrations for GHRH. The normal range of ratios is calculated and the response of the patient compared with the normal values. A ratio in the range of 1.4 to 2.5 is considered in the normal range while a ratio of less than 1.4 indicates a deficiency in endogenous GHRH and a ratio greater than 2.5 indicates a deficiency in endogenous GHRP. A deficiency in both endogenous peptides would produce a ratio in the normal range. Therefore, whenever a normal ratio is observed the peak values must be checked to determine if the peak responses were in the normal range (Table 1) . If not, then the patient is deficient in both endogenous GH secretagogues.

The ratio provides a relative measure of the deficiency for each endogenous secretagogue in reference to the co-secretagogue. This is useful in selecting the therapeutic dose for treatment since the more the ratio deviates from the normal range the more aggressive the treatment must be.

The growth hormone levels in the blood of the child or adult can be measured by any well known procedures, e.g., using an immunoradiometric assay as described by Bowers et al . (1990) or any other contemporary, scientifically accepted method.

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 analogues 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 analogue 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:

COUNTRY PATENT NO COLUMN

U.S. RE 33,699 1-4

U.S. 4,517,181 2

U.S. 4,518,586 1-4

U.S. 4,528,190 1-2

U.S. 4,529,595 1-4

U.S. 4,562,175 1-2

U.S. 4,563,352 1-4

U.S. 4,585,756 1-2

U.S. 4,595,676 1-2

U.S. 4,605,643 1-2

U.S. 4,610,976 1-2

U.S. 4,617,149 1-4

U.S. 4,622,312 1-4

U.S. 4,626,523 1-2

U.S. 4,649,131 1-4

U.S. 4,710,382 1-2

U.S. 4,774,319 1 The above U.S. patents and, in particular, the portions indicated above by column 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 analogues thereof (GHRX) means GHRP-6 and any peptide or nonpeptide compound that releases GH by the same cellular mechanism. The term analogue (Dorland ' s Illustra ted Medical Dictionary, 25th Edition, W.B. Saunders, Philadelphia, PA., p. 78) in the present invention is used to refer to functional and metabolic analogues that are peptides or nonpeptides that cause the release of GH by the same cellular mechanism as GHRP-6, i.e., they are compounds of similar activity. GHRP-6 is the hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂ which is believed to act directly on the pituitary to release GH. In addition to GHRP-6, the pentapeptide Tyr- D-Trp-Gly-Phe-Met-NH₂ (Cheng et al . , 1989) is a useful analogue in the release of GH.

Other compounds considered analogues of GHRP-6 for purposes of the present invention have been reported. For example, C.Y. Bowers et al . (1980) teaches that, 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-jS-Nal-Ala-Trp-D-Phe-Lys-NH₂ is also useful in the release of growth hormone. Also, Momany et al . (1981) 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 . , (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. Further, United States Patents 4,839,344 and 4,880,778 to Bowers et al. disclose many of the above peptides. All of these compounds are useful in the diagnostic procedure of the present invention.

A nonpeptide functional or metabolic analogue of GHRP-6 has been disclosed by Smith et al . (1993) that acts through the same site as GHRP-6. The compound, L-692,429, is antagonized by the same agents as is GHRP-6 and interacted with GRF. This and related compounds activate the same cellular receptors and second messengers as GHRP- 6 in the course of initiating its relevant action (e.g. growth hormone release) . All of these analogues and compounds of similar activity are useful in practicing the present invention as described herein; however, these peptides and nonpeptides should not be considered as being exhaustive of the GHRP-6 analogue-compounds useful in practicing the present invention.

The provocative challenges can be administered on the same day provided adequate time for clearance of the previous challenge occurs between. Clearance is determined by monitoring the blood levels of GH and when they return to the base line established prior to the first provocative challenge, clearance has occurred. 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, and are adjusted to take into account age, sex and body weight as are known in the medical arts. In general, 1 μg/kg body weight has been found to be effective.

The patients are selected for evaluation by the inventive protocol according to the following criteria. Slow growth in children or disorders in children and adults associated with growth hormone deficiency, with or without low blood concentrations of insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IBP-3) , such as obesity, osteoporosis, skeletal muscle atrophy, reduced lean body mass, frailty, increased serum LDL cholesterol, reduced immune function, etc., are candidates for the differential diagnostic procedure.

Upon testing with the present inventive protocol, patients deficient in GHRH, as indicated by a poor response to GHRP-6 challenge, would be best treated by GHRH replacement therapy. Children or adults deficient in the endogenous analogue for GHRP-6, as indicated by a poor response to GHRH, would be best treated by GHRP-6 replacement therapy. Children or adults deficient in both GH secretagogues, as indicated by poor responses to sequentially administered GHRH and GHRP-6 but with a robust response to co-administered GHRH and GHRP-6, would be best treated by GHRH and GHRP-6 replacement therapy. Children and adults with intrinsic defects in pituitary cellular and/or molecular mechanisms such as GH secretion involving signal transduction (receptors and second messengers) , gene expression (transcription or translation) , or hormone release, as indicated by a poor response to all challenges, would not respond to therapy with GH secretagogues. Instead, these patients would best benefit from recombinant GH replacement therapy.

The present invention also provides for a kit for the differential diagnosis of pituitary dependent growth hormone deficiency. The kit includes an amount of growth hormone releasing compound (GHRC) known to be effective to cause an increase in growth hormone levels in the blood to be administered to the patient for a provocative challenge. The kit also includes an amount of a molecule known to be effective to cause an increase in growth hormone levels in the blood, the molecule being selected from GHRP-6 or an analogue of GHRP-6 which causes release of growth hormone by the same cellular mechanism as GHRP-6 (GHRX) . Enough material of each agent is included in order to administer each agent individually for a provocative challenge as well as to co-administer the two agents. The kit can optionally also include the reagents for measuring GH levels in the blood of patients following the provocative challenge. The kit can optionally be configured for testing for multiple patients or for a single patient.

There are a number of factors that are relevant to understanding the underlying basis of the hypothesis which lead to the present invention both in determining the etiology of short stature in children and in understanding the etiology of GH deficiencies in adults and children as discussed herein below. The interpretation of these factors is not to be construed as limiting the present invention to this one mode of action. 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 (Codd et al . , 1989; Blake, et al . , 1991) and utilizes a different somatotroph second messenger system (Cheng et al . , 1989) is a GH secretagogue that potentiates GHRH efficacy

(Bowers et al . , 1990) . Furthermore, like GHRH, GHRP-6 is effective in some, but not all, short-statured children or adults to whom it is administered (Bowers et al . , 1991; Merica et al . , 1992) . The different mechanisms and synergistic effects of GHRH and GHRP-6, in vi tro and in vivo, suggested to applicants the existence of an endogenous analogue for the synthetic hexapeptide that may be physiologically relevant. Initial support for this hypothesis derives from one study in which passive immunization against endogenous GHRH in rats, reduced GHRP-6 activity approximately 90% (Bercu et al . , 1992a) . These data demonstrated the requirement of endogenous GHRH for expression of GHRP-6 activity.

This unexpected reciprocal relationship, i.e., dependence of GHRH upon an endogenous analogue of GHRP-6, is the basis of the present invention. Thus, in individuals deficient in the endogenous GHRP-6 analogue, GHRH efficacy would be blunted, whereas in individuals deficient in GHRH, GHRP-6 efficacy would be blunted. However, Robinson, et al . (1992) taught that GHRP-6 efficacy was not dependent upon endogenous GHRH. It was therefore unexpected for applicants to find the interdependence of the GH secretagogues.

Further support for the present invention derives from applicants' 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 (Walker et al . , 1992; Bercu et al . , 1992b) . Hyposensitivity to the individually administered peptides suggested that more than one, interdependent, endogenous, stimulatory factor contributed to GH secretion. The progressive decrement of GH or age-related loss of GHRH efficacy, as seen in aging, could result from several factors. One of these factors could be that chronic reduction in pituitary stimulation by GHRH causes desensitization to the GH secretagogue because hormones often induce their own receptors. Support for reduced stimulation of the pituitary by GHRH derives from the fact that available and/or appropriate GH secretagogues seem to decline during aging (De Gennaro Colonna et al., 1989; Morimoto et al. , 1988; Ono et al. , 1986) . However, an alternative hypothesis has been suggested by applicants that age-related, reduced efficacy of GHRH may be due to the absence or reduced concentrations of other, yet undefined, endogenous co-secretagogue (s) (Goth et al., 1992) .

Deficits in pituitary GH concentrations observed in animals may also contribute to age-related insensitivity to GHRH and subsequent GH deficiency (Bercu et al, 1992b; Walker et al, 1994a) . However, depletion of pituitary GH may not occur in humans because the only published report states that pituitary GH concentrations did not vary significantly with age when measured at autopsy (Gershberg, 1957) . However, this report has not been corroborated by other investigators and applicants have shown that in old laboratory animals, pituitary GH concentrations are significantly lower than in young animals of the same species. The low concentrations of pituitary GH in old animals are probably not due to inherent defects in pituitary function, but more likely to reduced stimulation of the gland. Support for this view derives from the fact that pituitary GH concentrations, as well as pituitary GH mRNA, were significantly increased in old rats chronically administered GHRH and GHRP-6 (Walker et al. , 1994a) . As discussed herein above, it has been hypothesized that a factor having the potential to reduce GHRH efficacy during aging is deterioration of GHRH- pituitary-binding-sites. GHRH binding sites with high and low affinities have been identified on rat pituitary membranes (Abribat et al. , 1990) . Since decreased capacity and loss of the high affinity site correlated with reduced GH secretion during aging, it was suggested that the high affinity binding site mediates GHRH action in somatotrophs (Abribat et al. , 1991) . Prior to this GHRH binding study, applicants (Codd et al. , 1989) reported that GHRP-6 has two binding sites on pituitary and hypothalamic membranes. GHRH did not compete for these GHRP-6 binding sites, suggesting that the two peptides bind different entities. These differential binding sites may be functionally related, since somatotrophs lacking GHRH receptors are unresponsive to GHRP administered alone (Jansson et al. , 1986), or in combination with GHRH (Bercu et al. , 1992a).

Functional linkage has been demonstrated many times in the ability of GHRP-6 to enhance GHRH efficacy

(Cheng et al., 1989; Bowers et al. , 1990), and applicants showed that passive immunization against GHRH reduced GHRP activity approximately 90% (Bercu et al., 1992a) . The ability of GHRP-6 to increase GHRH activity in aging rats (Walker et al., 1991) may result from facilitation of GHRH binding through cooperativity/allosteric interactions. Changes in intracellular transduction of GHRH has also been associated with aging. GHRH-activated adenylate cyclase activity and cAMP concentrations were lower in pituitaries from old rats than from young ones (Ceda et al., 1986; Parenti et al . , 1987; Parenti et al . , 1991) . However, this apparent deficit was immediately reversed by co-administration of GHRH and GHRP-6 in vivo (Walker et al . , 1991), again suggesting cooperativity between the peptides and deficiency or loss of a co-secretagogue (s) for GHRH during aging. Similarly, gene defects involving transcription of GH mRNA could account for reduced synthesis and secretion of GH in the elderly, and applicants showed that GHRH and GHRP-6 administered together positively affect the molecular biology of GH endocrinology in old rodents (Walker et al., 1994b) . Finally, age-related loss of GHRH efficacy could result from increased exposure of the pituitary gland to somatostatin (SRIF; Shibasaki et al. , 1984; Sonntag and Gough, 1988; Sonntag et al . , 1980) , a peptide that inhibits GH secretion. Passive immunization with SRIF antiserum increased GH release more in old rats than in young rats (Sonntag et al. , 1981; Sonntag and Gough, 1988) , and reduced pulsatile GH secretion in aging female rats (Sonntag et al . , 1980) ; correlated with SRIF hypersecretion (Takahashi et al . , 1987) . In a clinical study, administration of the acetylcholinesterase inhibitor pyridostigmine to block endogenous SRIF release partially restored GH responsiveness to GHRH in elderly subjects (Ghigo et al . , 1990) . Direct measurement of hormones released from perfused hypothalamic explants confirmed increased SRIF secretion from tissues of old rats (Ge et al . , 1989) . It has been proposed that the activity of GHRP-6 is greater in vivo than in in vi tro because the hexapeptide is a functional antagonist of SRIF, perhaps acting at the pituitary to prevent SRIF hyperpolarization of the somatotroph (DeBell et al . , 1991; Goth et al, 1992; Jaffe et al. , 1992) . These possibilities are consistent with applicants finding that co-administration of the complementary GH secretagogues, GHRH and GHRP-6 immediately restore youthful patterns of GH secretion in old animals (Walker et al . , 1991, Walker et al . , 1994 a,b) .

Therefore, age-related pituitary GH hyposecretion probably reflects decrements in the combined influence of several peptides, including SRIF on the pituitary, requiring analysis of responses to co- administered substances as provocative, diagnostic challenges. This hypothesis is in agreement with a report in which GH regulation was shown to be complex, involving several secretagogues acting in concert with GHRH to provide tightly controlled, temporal and quantitative secretion of GH (Goth et al . , 1992) . Accordingly, applicants devised a model for direct testing of pituitary, growth hormone secretory capability (Fig. 1) . The model assumes that a "normal" response to administration of exogenous GHRP-6 or GHRH requires the presence of its endogenous analogue, i.e., GHRH or GHRP, respectively. Blunted responses to either exogenous GH secretagogue is interpreted as indicating a deficiency of its endogenous complement. Blunted responses to both exogenous GH secretagogues, administered sequentially, implies deficiencies of both endogenous complements. This condition can be differentiated from inherent pituitary problems such as those involving receptor or second messenger deficits by a "normal" response to GHRH and GHRP co-administration. A blunted response following co- administration of both GH secretagogues would indicate inherent pituitary dysfunction rather than inadequate endogenous stimuli.

Thus, the present invention provides a new clinical application for the co-secretagogues GHRP-6 and GHRH and other synthetic GH secretagogues that function by the same mechanism as GHRP-6 or GHRH. The invention is important because it not only provides a reliable method for identifying children and adults with low GH secretory capability, but also helps diagnose the etiology of GH deficiency and provides a key to appropriate treatment . Once a child or adult is diagnosed as having either a deficiency in either or both endogenous GHRH and GHRP, the appropriate replacement therapy can ensue whereby either a growth hormone releasing compound, a GHRP-6 or analogue thereof or a combination of both GH secretagogues is administered to the child or adult. Robinson et al . (1992) has stated that endogenous GHRH does not contribute to the effect of a provocative dose of GHRP. If this were correct, then a poor response to GHRP would suggest a defect in the mechanism for GHRP-6 signal transduction/processing, when in fact it could simply result from deficient complement (endogenous GHRH) . As the result of the conclusion of Robinson et al. (1992) that endogenous GHRH does not contribute to endogenous GHRP-6-like activity, the therapy resulting from a poor GHRX provocative test would be GHRP- 6 replacement in an attempt to "prime" the pituitary to respond or, failing that, to use recombinant GH therapy.

Therefore, by the conclusion of the Robinson et al . (1992) diagnostic procedure and those of his colleagues, especially Bowers, GHRH replacement would not be indicated in a patient who responded poorly to GHRP-6, even though GHRH deficiency could be the possible cause of such poor response to a GHRP-6 provocative challenge. Therefore, in practicing the present invention, patients who present with clinical signs of GH deficiency, such as obesity, osteoporosis, reduced lean body mass, skeletal muscle atrophy, frailty and the like, as well as children with short stature, are identified as potentially requiring growth hormone or some agent that would stimulate growth hormone production and would be monitored for the purpose of establishing a baseline level of growth hormone in the blood. Once baseline levels are established, the patient is administered GHRC at a dose ranging from 100 ng/kg body weight to 100 mg/kg body weight. In a preferred embodiment, the GHRC is administered and blood samples are monitored over a period of 120 minutes to determine whether the exogenous GHRC increased growth hormone levels. After the effects of GHRC administration are determined, the patient is administered GHRP-6 or an analogue thereof at a dose of 100 ng/kg body weight to 1 mg/kg body weight. Blood samples are measured for any change in growth hormone levels for 120 minutes as for GHRC administration.

Co-administration of both GH secretagogues then follows with the same dosages levels. Additional administration of peptides may be made, but a day of rest, i.e., a day during which neither GH secretagogue is administered, may intervene administration days and, in one embodiment, each provocative challenge is spaced twenty-four hours apart. The dosage amount can be varied to determine optimal dosages. The changes, if any, in growth hormone levels to either or both of GHRC or GHRX thereof will provide an insight to the etiology of the GH deficient condition of the patient. The above discussion provides a factual basis for the method of differential diagnostic protocol and kit for evaluating pituitary growth hormone (GH) secretory capability as a means to identify the etiology of GH deficiency. The methods used with and the utility of the present invention can be shown by the following examples.

EXAMPLES

Animals Fischer 344 rats (Charles River) , both male and female, were used. Young animals are defined as sexually mature between 2 and 7 months of age. Old animals are defined as those over 16 months of age.

EXAMPLE 1

GHRH-efficacy has been reported to be reduced 50 to 75% in old rats (Sonntag et al . , 1983) ; therefore, applicants tested the effect of GHRP-6 alone and in combination with GHRH on GH release in old rats. Peak plasma GH concentrations resulting from GHRP-6 administration in old rats were approximately 60% less than in young rats. In contrast, peak plasma GH concentrations were greater in old rats than in young rats administered GHRP-6 and GHRH. Since target organs sometimes become hyperresponsive when tonic and/or phasic stimulation decreases, than one would expect exaggerated responses to provocative exogenous stimuli under experimental conditions. GH hypersecretion observed in naive, old rats administered a single bolus of GHRH and GHRP (Walker et al. , 1991) provides support for the hypothesis that deficits in stimulated GH secretion in aged rats were due to insufficient signals or inappropriately transduced GH releasing stimuli.

The first possible cause of age-related decrements in GH secretion that applicants considered was that a progressive reduction in pituitary stimulation occurs during aging. This reduction could be attributed to GHRH and/or to an endogenous ligand for GHRP-6. Although the search has begun, a purported endogenous ligand for GHRP-6 has not yet been identified (Bercu et al . , 1992a) . Nonetheless, applicants were able to demonstrate the functional dependence of GHRP-6 upon endogenous GHRH by causing deficits in GHRH that, in turn, attenuated responses to GHRP-6 (Bercu et al. , 1992a) . Young rats were passively immunized against endogenous GHRH or administered α-methyl-p-tyrosine. If GHRH and the endogenous ligand of GHRP-6 are physiological co-agonists of GH secretion in the young rat, then removal of one or the other should be expressed as attenuated activity of its co-agonist when administered alone. Passive immunization which inactivates GHRH with neutralizing antibodies, or cx-methyl-p-tyrosine (L form) which blocks stimulation of hypothalamic GHRH neurons were used to remove or reduce concentrations of endogenous GHRH in the young experimental animals . These treatments were intended to simulate the aged condition in which concentrations of GHRH and GHRH mRNA are low (De Gennaro Collona et al. , 1989; Morimoto et al. , 1988) . Afterwards, GHRP-6 was administered to test and compare its efficacy with that in old rats (Walker et al . , 1991) .

As seen in Figure 2, GHRP-6 activity was significantly attenuated in young female rats administered GHRH antiserum or α-methyl-p-tyrosine to reduce endogenous GHRH concentrations. Presumably, naturally occurring decrements in endogenous GHRH during aging contributed to the blunted response to GHRP-6 that applicants observed (Walker et al . , 1991) . If GHRH activity is also dependent upon a yet unidentified endogenous co-secretagogue whose concentrations decline during aging, then applicants' data support the hypothesis that extrinsic pituitary deficits contribute, at least in part, to attenuated GH secretory responses to administered GHRH.

The second possible cause of age-related decrements in GH secretion applicants considered was that deterioration in GH secretagogue signal transduction at the pituitary level contributes to the progressive insensitivity to provocative stimuli. In a test of this hypothesis, applicants examined GH secretory responses to GHRP-6 in young rats administered [N-Ac-Tyr¹, D-Arg²] -GRF 1-29, an antagonist to the pituitary GHRH-6 receptor. As seen in Figure 3, pre-administration of 5 to

50 μg/kg of [N-Ac-Tyr¹, D-Arg²] -GRF 1-29 amide which blocks pituitary GHRH receptors also attenuated GHRP activity. However, 150 μg/kg of [N-Ac-Tyr¹, D-Arg²] -GRF 1-29 amide potentiated GHRP-6 activity, presumably due to partial agonist activity of the GHRH receptor agonist at the higher dose (Bercu et al . , 1992a) . GHRP-6 stimulated GH secretion as a function of time was significantly reduced (P<0.05) by 50 mg/kg GHRH receptor antagonist and significantly increased (PcO.01) by 150 mg/kg GHRH receptor antagonist. Morphine-stimulated GH secretion was significantly reduced (P<0.05) by 50 and 150 mg/kg GHRH receptor antagonist .

These data suggest that activation of pituitary GHRH receptors contributes to full expression GHRP activity in vivo. .

EXAMPLE 2 A study in children has been performed. The results of this study support the basic concept of GH secretagogue complementarily as a useful diagnostic tool for evaluating pituitary-based, GH deficiency. The protocol used in the pediatric study from which data presented below were collected involved the sequential administration of exogenous GHRP and exogenous GHRH as set forth in the present invention (Table 1, Figure 1) . Briefly, each subject began testing at approximately 0900 h, one half hour after placement of an intravenous catheter. Two blood samples for basal concentrations of serum GH were drawn, and GHRP (1 μg/kg) was administered as a bolus . Blood samples were then drawn at five minute intervals for 20 minutes during which the GH secretory response occurred, and at longer intervals for about two hours thereafter.

GHRH (1 μg/kg) was then administered as a bolus, when basal GH concentrations were again established. Blood samples were similarly collected after GHRH administration as they were following GHRP administration. GH concentrations in each serum was then determined by radioimmunoassay.

The study population was composed of children with normal GH secretory function (serving as controls and normal baseline values) and those with GH secretory dysfunction of various clinical diagnoses . The purpose of evaluating different etiologies of GH secretory dysfunction was to validate the hypothesis that complementary endogenous GH secretagogues must be present for optimal expression of GHRP or GHRH stimulatory potential. As seen in Fig. 4A, peak concentrations of serum GH were reached at the 15 or 20 minute time point following stimulation with both secretagogues. In all five normal children tested, peak GH concentrations following GHRP-6 administration were greater than those following GHRH administration. The greater response to the xenobiotic GH secretagogue was not due to its being administered first, because reversal of the sequence produced the same differential response. Peak concentrations of serum GH following GHRP-6 administration in normal children ranged between 42 and 66 ng/ml, whereas peak concentrations of serum GH following GHRH administration ranged between 18 and 36 ng/ml. Thus, the ratio of GHRP-6 to GHRH evoked responses in the normal children ranged between 1.8 (66/36) and 2.3 (42/18) , reflecting the greater responses to GHRP-6 in these subjects.

Three primary assumptions about these data were used with the data collected from children with GH secretory dysfunction. These assumptions were that:

1. peak concentrations of serum GH following administration of exogenous GHRP-6 or GHRH reflected not only the dose of GH secretagogue administered, but also the "normal concentration" of endogenous complementary GH secretagogue present in the subject;

2. GHRP-6:GHRH ratios between 1.4 and 2.5 (allowing for standard errors) reflected the "normal concentrations" of endogenous GH secretagogues contributing to the peak concentrations measured in normal individuals; and

3. peak concentrations of serum GH in response to exogenous GHRP-6 or GHRH should occur within 15 or 20 minutes of their administration.

With these considerations in mind, applicants tested children with various causes of GH secretory dysfunction ranging from hypopituitarism to that resulting from radiation therapy. The results of these tests substantiated the basic premises of the test hypothesis.

The data presented in Fig. 4B show that children with inherently dysfunctional pituitary glands did not respond to any combination of exogenous GH secretagogue administration. This representative subject had atrophic, developmentally retarded pituitary gland which responded to neither sequentially administered GHRH and GHRP-6 nor to the co-administered peptides.

Radiation of the head for treatment of cancer in children retards growth, presumably by reducing the secretion of endogenous GHRH from hypothalamic neurosecretory neurons. In this clinical study, children that were presumably deficient in endogenous GHRH responded poorly to GHRP-6, but exuberantly to GHRH, resulting in a GHRP:GHRH ratio significantly <1. As seen in Fig. 4D, a representative subject from this group produced a peak response of 18.9 ng/ml serum GH in response to GHRP-6, whereas the response to GHRH was 49.4 ng/ml, resulting in a GHRP to GHRH ratio of 0.38. The data were interpreted to mean that the poor response to GHRP-6 resulted from a paucity of endogenous GHRH after radiation. The reduced availability of this GHRP complement, therefore, attenuated the response to a provocative dose of exogenous GHRP-6. Conversely, the data also suggest that radiation did not alter the availability of the endogenous analogue GHRP because the response to a provocative dose of GHRH was amplified.

This finding could be explained by enhanced sensitivity of the pituitary to GHRH resulting either from increased exposure to the endogenous analogue of GHRP and/or by a mechanism involving low level exposure to GHRH that, in turn, increased the pituitary response to a substantial bolus of the secretagogue. The actual mechanism of this change in responsivity is unknown, but data collected from other patients suggest that the reciprocal affect also occurs. As seen in Fig. 4C, some children responded poorly to both secretagogues, but much better to GHRP than to GHRH. This pattern of response suggests a greater deficiency in endogenous GHRP than in GHRH giving a GHRP:GHRH ratio >3. A peak response of 13.5 ng/ml serum GH occurred after administration of GHRP-6, whereas a peak response of only 4.4 ng/ml occurred after administration of GHRH. The GHRP to GHRH ratio for this subject was 3.1. Interestingly, in all individuals with GHRP to GHRH ratios greater than 3, the quantitative responses to both exogenous secretagogues were significantly lower than those observed in normal subjects. These data suggest that the endogenous analogue of GHRP-6 deficiency may be more tightly linked to GHRH deficiency than the reciprocal, because as shown above, quantitatively normal and even supranormal responses to GHRH can occur in the presence of poor responses to exogenous GHRP-6. This observation suggests that endogenous GHRH deficiency is not necessarily accompanied by endogenous GHRP deficiency. However, since applicants have not observed quantitatively normal responses to exogenous GHRP-6 in the presence of poor responses to GHRH, a deficit in endogenous GHRP may always be accompanied by a deficit in GHRH.

An interesting and logical extension of these observations is to differentiate the responses of individuals with GHRP to GHRH ratios of <1 from those with ratios >3, because in GH deficient animals which provide a more homogenous population than humans, co-administration of GHRH and GHRP produced supranormal GH secretory responses (Walker et al . , 1991) . In limited studies with GH deficient human subjects, Bowers et al . (personal communication) observed attenuated mean (population) responses to the co-administered GH secretagogues. However, he did not screen the subjects for their individual responses to GHRP and GHRH, so that the variance in his data could result from differences in the endogenous secretagogues, i.e. robust responders may have had GHRP to GHRH ratios >3, while poor responders had ratios <1. Throughout this application various publications are referenced. Full citations for the referenced publications not included herein above are listed below. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

TABLE 1

PRODUCTION OF GH IN RESPONSE TO PROVOCATIVE CHALLENGE

Provocative GHRH + GHRH - GHRH + Missing Pituitary- Challenge GHRX + GHRX + GHRX - Both Def iciency

GHRX Yes No Yes No No

GHRC Yes Yes No No No

Both Yes Yes Yes Yes No

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Claims

CLAIMSWhat is claimed is:

1. A method for evaluating pituitary growth hormone (GH) secretory capability which includes the steps of establishing a baseline level of growth hormone in the blood of a patient; administering an amount of a molecule known to be effective to cause an increase in growth hormone levels in the blood, the molecule being selected from the group (GHRX) consisting of GHRP-6, wherein GHRP-6 is a hexapeptide of amino acid sequence His-D-Trp-Ala-Trp-D-Phe- Lys-NH₂, and an analogue which causes release of growth hormone by the same cellular mechanism as GHRP-6; measuring levels of growth hormone in the blood in response to GHRX wherein a poor response indicates deficient growth hormone releasing hormone (GHRH) ; administering an amount of growth hormone releasing compound (GHRC) known to be effective to cause an increase in growth hormone levels in the blood; measuring levels of growth hormone in the blood in response to GHRC wherein a poor response to GHRC indicates deficient endogenous in vivo analogue of GHRP-6; co-administering a combination of GHRC and GHRX in amounts known to be effective to cause an increase in growth hormone levels in the blood; and measuring levels of growth hormone in the blood in response to the co-administration.

2. The method of claim 1 wherein the response is evaluated by calculating a ratio of peak GH concentrations for GHRX divided by peak GH concentrations for GHRH.

3. The method of claim 1 wherein the growth hormone releasing compound (GHRC) is growth hormone releasing hormone.

4. The method of claim 1 wherein the analogue which causes release of growth hormone by the same cellular mechanism as GHRP-6 is selected from the group consisting of GHRP-1; 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;

Tyr-D-Trp-Ala-Trp-D-Phe-NH₂; His-D-Trp-D-Trp-Ala-Trp-D-Phe-Lys-COOH

His-D-Trp-Ala-Trp-D-Phe-Arg-NH₂; and

L-692,429.

5. The method of claim 1 wherein the amount of GHRC administered is 100 ng/kg to 100 mg/kg of body weight .

6. The method of claim 5 wherein the amount of GHRX administered is 100 ng/kg to 1 mg/kg of body weight.

7. The method of claim 6 wherein the GHRC is administered first.

8. The method of claim 6 wherein the GHRC is administered second.

9. The method of claim 1 wherein a combination of GHRH and GHRP-6 is co-administered.

10. The method of claim 9 wherein the amount being co-administered of GHRC is 100 ng/kg to 100 mg/kg of body weight and the amount of GHRX is from 100 ng/kg to 1 mg/kg of body weight.

11. The method of claim 1 wherein a day during which no GH secretagogue is administered intervenes each day on which a dosage is administered.

12. A method for determining an etiology of short stature in a child, which includes the steps of establishing a baseline level of growth hormone in the blood of the child; administering an amount of growth hormone releasing compound (GHRC) known to be effective to cause an increase in growth hormone levels in the blood; measuring levels of growth hormone in the blood in response to GHRC wherein a poor response to GHRC indicates deficient endogenous in vivo analogue of GHRP-6; administering an amount of a molecule known to be effective to cause an increase in growth hormone levels in the blood, the molecule being selected from the group (GHRX) consisting of GHRP-6, wherein GHRP-6 is a hexapeptide of amino acid sequence His-D-Trp-Ala-Trp-D-Phe- Lys-NH₂, and an analogue which causes release of growth hormone by the same cellular mechanism as GHRP-6; measuring levels of growth hormone in the blood in response to GHRX wherein a poor response indicates deficient growth hormone releasing compound; co-administering a combination of GHRC and GHRX in amounts known to be effective to cause an increase in growth hormone levels in the blood; and measuring levels of growth hormone in the blood in response to the co-administration wherein a poor growth hormone response indicates intrinsic defects in the pituitary gland not due to deficiency of GHRH and endogenous in vivo analogue of GHRP-6.

13. The method of claim 12 wherein the response is evaluated by calculating a ratio of peak GH concentrations for GHRX divided by peak GH concentrations for GHRH.

14. The method of claim 12 wherein the growth hormone releasing compound (GHRC) is growth hormone releasing hormone.

15. The method of claim 12 wherein the analogue which causes release of growth hormone by the same cellular mechanism as GHRP-6 is selected from the group consisting of GHRP-1;

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;

Tyr-D-Trp-Ala-Trp-D-Phe-NH₂;

His-D-Trp-D-Trp-Ala-Trp-D-Phe-Lys-COOH His-D-Trp-Ala-Trp-D-Phe-Arg-NH₂; and

L-692,429.

16. The method of claim 12 wherein the amount of GHRC administered is 100 ng/kg to 100 mg/kg of body weight.

17. The method of claim 16 wherein the amount of GHRX administered is 100 ng/kg to 1 mg/kg of body weight.

18. The method of claim 12 wherein the GHRC is administered first.

19. The method of claim 12 wherein the GHRC is administered second.

20. The method of claim 12 wherein a combination of GHRH and GHRP-6 is co-administered.

21. The method of claim 20 wherein the amount being co-administered of GHRC is 100 ng/kg to 100 mg/kg of body weight and the amount of GHRX is from 100 ng/kg to 1 mg/kg of body weight .

22. The method of claim 12 wherein a day during which no peptide or hormone is administered intervenes each day on which a dosage is administered.

23. A kit for the differential diagnosis of an etiology of pituitary dependent growth hormone deficiency comprising an amount of growth hormone releasing compound (GHRC) known to be effective to cause an increase in growth hormone levels in the blood; and an amount of a molecule known to be effective to cause an increase in growth hormone levels in the blood, the molecule being selected from the group consisting of GHRP-6 and an analogue which causes release of growth hormone by the same cellular mechanism as GHRP-6.