Note: Descriptions are shown in the official language in which they were submitted.
CA 022l6260 l997-09-23
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METHOD OF DIAGNOSING PITUITARY DEPENDENT
GROWTH HORMONE DEFICIENCY
This application is a Continuation-In- Part
of United States Serial No. 08/405,842, filed March
17, 1995, which is a Continuation-In-Part application
of United States Serial No. 08/171,346, filed December
21, 1993, which was a Continuation application of
United States Serial No. 07/905,760, filed June 29,
1992, now abandoned.
R~CRGROUND OF THE lNv~:NLlON
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
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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 andprobably 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
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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 (Devesa 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
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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 in
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
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biotechnological methods prevented valid testing of
the hypothesis that GH deflciencies 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;
10 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
15 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
25 (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
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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.
Applicants have previously attempted to
provide a protocol which would determine GH secretory
capability, i.e. deficiencies, in adults and children
utilizing combined testing with growth hormone
releasing hormone (GHRH) and xenobiotic GH releasing
hexapeptide (designated GHRP-6 or GHRP) (see United
States patent 5,246,920 and WO 94/00759). However,
unexpected variability in responses and
reproducibility continued utilizing the methods set
forth in the above listed references.
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 and that is
reproducible and with low varlability.
,
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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 provocative challenge to children and adults
afflicted with disorders related to GH deficiency.
The challenge comprises administration first 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). Within two to four hours of the GHRX
administration the naturally occurring GH releasing
hormone (GHRH) or its peptidyl or non-peptidyl
synthetic analogues that release GH by the same
cellular mechanism as GHRH (the group/family of
compounds are referred to as GHRH) is then
administered. GH secretion is measured at regular
intervals for two hours after administration of each
GH secretagogue. After evaluating the effects of
independent, sequential administration, the effect of
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co-administration of the GH secretagogues on blood GH
levels is determined on a different day.
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.
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.
In particular the present invention removes the
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variability and lack of reproducibility of the prior
art methods.
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 lA-B are diagrams of the three step
provocative testing protocol of the present invention.
FIGURE 2 is a dose response curve for GHRP-6
in rats pretreated with vehicle (triangles), ~-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-Tyrl, D-Arg2]-GRF 1-29 amide, a
GHRH receptor antagonist, on GH release in response to
GHRP-6 (30 ~g/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) ~g/kg.
Values represent mean + SEM 8 rats per group.
CA 022l6260 l997-09-23
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FIGURE 4A-D is a series of dose response
curves showing GH concentration over time and the
calculated 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.
DETATT~n DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention provides a diagnostic
procedure and kit for determining whether growth
hormone (GH) deficiency in children and adults is due
to a deficiency in endogenous growth hormone releasing
hormone (GHRH), 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. GHRP-6 represents a
synthetic analogue of another, yet unidentified,
endogenous GH secretagogue. Although the endogenous
substance remains unidentified its receptor has been
defined (Howard et al., 1996). The relationship
between GHRP-6 and its endogenous counterpart is
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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
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indicate the absence of endogenous GH secretagogues or
intrinsic defect(s) in pituitary functional capacity.
The basis of the diagnostic test is a
comparison of responses to provocative challenges of
exogenous/endogenous GH secretagogues administered
sequentially and in combination. However, as shown
herein below Applicants have determined unexpectedly
that the order of the testing and timing of the
injections of the two components is critical to
accurate, reproducible results.
GHRH and GHRP-6 or their peptidyl or non-
peptidyl synthetic analogues that release GH by the
same cellular mechanism as GHRH and GHRP-6
respectively 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 a homologous 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 are designated as GHRX. The
analogues of GHRH are members of a group of GH
secretagogues which can be referred to growth hormone
releasing compound (GHRC) such as GH releasing hormone
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(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.
However, by convention this group/family of related
molecules is referred to 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. Sequential,
independent provocative challenges are administered in
the order GHRX followed by GHRH, and then the
combination of the GH secretagogues is administered.
The timing between the sequential,
independent challenges must be between two and four
hours with the preferred embodiment at two hours (120
minutes after GHRX administration). Blood is drawn at
regular intervals for up to two hours after the
administration of each secretagogue, generally every 5
minutes for the first 20 minutes after administration
and 10-15 minute intervals thereafter for the
remaining two hours. From these blood samples the
peak GH response is measured thereby determining if GH
level changes after each administration has occurred.
On a separate day from the sequential
administration of the two GH secretagogues, a
combination of GHRH and GHRX is administered as a
single intravenous bolus. Blood samples are drawn at
.
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the same time intervals as following administration of
the sequential individual challenges for up to three
hours after administration of the combination.
The diagnostic potential of GHRX and GHRH,
with GHRP and growth hormone releasing hormone
respectively the preferred compounds, for determining
the etiology of GH secretory deficiency is profoundly
affected by the sequence in which the secretagogues
are administered. When GHRH is administered alone,
the GH secretory response it elicits is extremely
variable and is poorly dose related (Table 1).
TABLE 1
GHRH (,ug/kg) Peak Serum GH (ng/ml)
x + SEM
01.09 + 1.02
0.19.46 + 3.71
0.3316.45 + 8.11
1.013.54 + 6.51
3.321.80 + 6.78
10.017.41 + 4.67
Current knowledge of GHRH stimulated GH
secretion attributes the variability of GHRH to the
physiological state of the GH neuroendocrine axis at
the time that the stimulus is applied (Devessa et al.,
1989; Albini et al., 1988; Tannenbaum et al., 1989).
In other words, the sensitivity or readiness of the GH
.
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neurosecretory system to stimulation is dynamic,
sometimes being greater than other times. Since the
physiological factors affecting sensitivity to GHRH
stimulation are not easily predicted immediately prior
to a GHRH challenge, it is essentially impossible to
eliminate variability if GHRH is administered by
itself or before GHRP.
On the other hand, the response to GHRP
administered alone is not variable (Table 2). The
minimal variability of the GHRP response is presumed
to result from its partial action as a somatostatin
agonist.
TABLE 2
GHRP (~g/kg)Peak Serum GH (ng/ml)
x + SEM
0 1.11 + 1.01
0.1 17.51 + 1.91
0.5 41.31 + 3.97
1.0 67.88 + 6.1
1.5 81.60 + 5.88
Relevant to using GHRH and GHRP as
diagnostic agents is the fact that GHRP eliminates the
endogenous factors causing variability in the GHRH
response. Administration before GHRH creates an
episode of GH secretion that "resets" the sensitivity
of the GH neurosecretory axis to stimulation by GHRH,
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so that at the time of GHRH administration, uniform
baseline conditions in the subject have been achieved.
This then puts the GH neurosecretory axis in a uniform
state of readiness for GHRH so that a subsequent
relatively stable and constant episode of GHRH-
stimulated GH secretion results (Table 3). This GHRH
stimulated GH secretory episode can then be compared
with the stable episode of GHRP- stimulated GH
secretion that preceded it as a basis for the
diagnostic test of the present invention. In Table 3
the GH secretory responses in the same individual to
repeated injections of GHRH preceded by GHRP shows
stable response to GHRP and GHRH. The GHRH
variability is significantly reduced.
TABLE 3
GH Secretagogue Peak Serum GH ( ng/ml)
(~g/kg) x + SEM
GHRP 1.0 69.31 + 4.21
GHRH 1.0 32.66 + 3.33
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
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conditions, a different pattern of GH secretion is
anticipated in response to a provocative challenge
with exogenous GHRC and GHRX. (Table 4). 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 Table 4 the first column lists the
stimulus or provocative challenge given to the
patient. Columns 2-5 list the possible status of
endogenous GH secretagogues. The predicted response,
increase in GH concentration, to the challenge is
listed in each column and is dependent upon the
endogenous status of the GH secretagogues.
TABLE 4
INCREASED BLOOD CONCENTRATIONS OF GH
IN RESPONSE TO PROVOCATIVE CHALLENGE
Provocative GHRH + GHRH - GHRH + Missing Pituitary
Challenge/ GHRP + GHRP + GHRP - Both Mechanistic
Stimulus Defect
GHRX Yes No Yes No No
GHRH Yes Yes No No No
Both Yes Yes Yes Yes No
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In the normal condition, GHRH and the in
vivo endogenous analogue of GHRP-6 are present in
normal concentrations (Column 2, Table 4). Therefore,
the injection of GHRX and then GHRH will release
concentrations of GH that are normal and that reflect
the presence of endogenous GHRH and endogenous
analogue of 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 4), the
injection of GHRX will release subnormal amounts of
GH, if at all. However, an injection of GHRH
following the GHRX will release a single normal
quantum of GH.
If the in vivo endogenous analogue of GHRP-6
is insufficient or absent while endogenous GHRH is
present in normal amounts (Column 4, Table 4), then
the injection of GHRX will release a normal quantum of
GH. However, the subsequent injection of GHRH will
release subnormal amounts of GH, if at all.
If both endogenous GH secretagogues are
insufficient or absent (Column 5, Table 4), then GHRX
and the subsequent GHRH administration individually
will release subnormal quanta of GH, if at all. If
the co-administration of GHRH and GHRX release a
normal quanta of GH, then both endogenous
secretagogues are absent or insufficient. If no or
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reduced GH is released (Column 6, Table 4) 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 and 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 2.9 is considered in the
normal range.
Normal values for GH secretion and normal
ranges are determined for each population tested by
sampling from healthy individuals. In the examples
herein the population tested was primarily European,
male and contained both children and adults. Normal
values may be different for other populations
depending on factors such as ethnicity, pre- and post
menapausal women. In addition environmental factors
may be considered such as time of year in establishing
normal ranges.
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 are
CA 022l6260 l997-09-23
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in the normal range. If not, then the patient is
deficient in both endogenous GH secretagogues. The
normal ratio must be achieved with peak GH values for
GHRX stimulation ranging between 40 and 90 ng/ml and
for GHRH stimulation ranging between 20 and 45 ng/ml.
GH values <40 ng/ml or >90 ng/ml from GHRX stimulation
or <20 ng/ml or >45 ng/ml from GHRH stimulation
suggest abnormal changes in concentrations of the
endogenous analogues for both GHRX and GHRH.
Alternatively, the pituitary transduction and GH
release mechanisms may be abnormal. Co-administration
of the GH secretagogues will identify these patients.
Ratios of 3.0 to 3.9 result from relatively
greater response to GHRX than to GHRH. This would be
due to a decline or modest deficit in endogenous GHRX.
Alternatively, an unusually robust response to GHRX
without a concomitant robust response to GHRH (i.e.,
GHRH response remains "normal") can provide a ratio in
this range. This response suggests an unusual
increase in endogenous GHRH. By analyzing both the
ratio and the underlying individual responses these
alternatives can be distinguished and identified.
Ratios less than 1.0 suggest severe
deficiency in endogenous GHRH without deficiency of
endogenous GHRX. Ratios of 1.1 to 1.4 resulting from
relatively greater responses to GHRH than to GHRX
indicate a modest deficit in endogenous GHRH or an
CA 022l6260 l997-09-23
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unusual increase in endogenous GHRX. As discussed
herein above these can be distinguished based on an
analysis of the peak values used to calculate the
ratio.
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 itself, 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
CA 022l6260 l997-09-23
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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 NOCOLUMN
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 l-2
U.S. 4,649,131 1-4
U.S. 4,710,382 1-2
U.S. 4,774,319
The above U.S. patents and, in particular,
the portions indicated above by column number, are
incorporated herein by reference as teaching growth
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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 Illustrated 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-~-
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
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0152.00184 -24-
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
CA 022l6260 l997-09-23
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being exhaustive of the GHRP-6 analogue-compounds
useful in practicing the present invention.
The provocative challenge with GHRH must be
administered after blood concentrations of GH return
to basal levels, i.e. approximately 120 minutes after
administration of GHRX.
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 ~g/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-l (IGF-l) 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
CA 02216260 1997-09-23
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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. ~hildren
or adults deficient in both GH secretagogues, as
indicated by poor responses to sequentially
administered GHRP-6 and GHRH 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 one of a growth hormone releasing
compound (GHRC) described herein above 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
CA 022l6260 l997-09-23
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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
CA 022l6260 l997-09-23
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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 vitro 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.
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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
CA 02216260 1997-09-23
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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
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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
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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.j 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).
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It has been proposed that the activity of
GHRP-6 is greater in vivo than in in vitro 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 (Figures lA-B). 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
CA 02216260 1997-09-23
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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
CA 022l6260 l997-09-23
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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.
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
CA 022l6260 l997-09-23
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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 75~6 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
CA 022l6260 l997-09-23
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(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 ~-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 ~-
methyI-p-tyrosine (L form) which blocks stimulation of
hypothalamic GHRH neurons were used to remove or
reduce concentrations of endogenous GHRH in the young
CA 022l6260 l997-09-23
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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 ~-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
CA 022l6260 l997-09-23
0152.00184 - 39-
administered [N-Ac-Tyrl, D-Arg2]-GRF 1-29, an
antagonist to the pituitary GHRH-6 receptor.
As seen in Figure 3, pre-administration of 5
to 50 ~g/kg of [N-Ac-Tyr1, D-Arg2]-GRF 1-29 amide which
blocks pituitary GHRH receptors also attenuated GHRP
activity. However, 150 ~g/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
(P<0.01) by 150 mg/kg GHRH receptor antagonist (Fig.
3A). Morphine-stimulated GH secretion was
significantly reduced (P<0.05) by 50 and 150 mg/kg
GHRH receptor antagonist (Fig. 3B).
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 complementarity as a useful diagnostic
tool for evaluating pituitary-based, GH deficiency.
CA 022l6260 l997-09-23
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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 4; Figures lA-B). 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 ~g/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 ~g/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
CA 02216260 1997-09-23
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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.
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
CA 022l6260 l997-09-23
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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 GHRP and GHRH nor to the co-
administered peptides.
Radiation of the head for treatment ofcancer 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
CA 022l6260 l997-09-23
0152.00184 -43-
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. 3 8. 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
CA 022l6260 l997-09-23
0152.00184 -44-
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
CA 022l6260 l997-09-23
0152.00184 -45-
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
CA 022l6260 l997-09-23
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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.
CA 02216260 1997-09-23
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