Biologically active peptides structurally related to regions within growth hormones

Disclosed are novel synthetic peptides having primary structural homology to a continuous sequence of amino acid residues of human growth hormone in a region spanning positions thirty-two to forty-six ("hGH.sub.32-46 ", or "deletion peptide"). In preferred forms, peptides of the invention comprehend: duplicate portions (i.e., sequence fragments) of hGH.sub.32-46 ; stereochemical analogs and fragment analogs of hGH.sub.32-46 including one or more amino acid residues in D-isomeric configuration; and, "interspecies" analogs and fragment analogs of hGH.sub.32-46 including one or more non-homologous amino acid residues duplicating variant residues present in corresponding positions in corresponding regions of heterologous species growth hormones. Peptides of the invention are administered to mammals contemporaneously with exogenous insulin to generate hypoglycemic effects greater than available through administration of insulin alone. A presently preferred heptapeptide has the sequence, NH.sub.2 -Glu-Glu-Ala-Tyr-Ile-Pro-Lys-COOH, and has insulin-potentiating activity greater than hGH.sub.32-46.

BACKGROUND 
The present invention relates generally to novel, biologically active 
synthetic peptides which are structurally related to a region within human 
growth hormone and which are active, inter alia, in potentiating the 
effects of insulin on glucose metabolism in mammals, including humans. 
The diabetes mellitus disease state is a chronic disorder affecting 
carbohydrate, fat and protein metabolism. A characteristic feature of 
idiopathic diabetes mellitus is a defective or deficient insulin secretory 
response giving rise to impaired carbohydrate (glucose) use and resultant 
hyperglycemia. Two major variants of the disease state exist. One variant, 
seen in about ten percent of all idiopathic diabetics is referred to as 
insulin-dependent diabetes mellitus ("IDDM") or juvenile onset diabetes. 
This variant, frequently manifested for the first time in youth and is 
characterized by a progressive loss of insulin secretory function by beta 
cells of the pancreas and hence a progressive "dependence" on exogenous 
insulin for maintenance of carbohydrate metabolism. (This characteristic 
is shared by those non-idiopathic, or "secondary", diabetics whose 
disorders have their origins in pancreatic disease.) The second variant of 
idiopathic diabetes mellitus is referred to as non-insulindependent 
diabetes mellitus ("NIDDM") or adult onset diabetes and accounts for the 
remainder of the idiopathic diabetic population. 
All diabetics, regardless of their genetic and environmental backgrounds or 
the age of onset of the disease, have in common an apparent lack of 
insulin or inadequate insulin function. Because transfer of glucose from 
the blood into muscle and fatty tissue is insulin dependent, diabetics 
lack the ability to utilize glucose adequately. Further, because 
glycogenolysis is ordinarily inhibited by insulin, the rate of 
glycogenolysis is elevated in the diabetic. Both these "derangements" from 
normal metabolic events lead to accumulation of glucose in the blood 
(hyperglycemia) to the point where renal glucose reabsorption capacity is 
exceeded and glycosuria occurs. The major source of energy for the 
diabetic thus becomes fatty acids derived from triglycerides stored in 
fatty tissue. In the liver, fatty acids are oxidized to ketone bodies 
which are circulated and used as an energy source by tissues. In the IDDM 
patient, and sometimes the NIDDM patient, the rate of formation of ketone 
bodies may exceed the rate of their utilization and ketosis along with 
metabolic acidosis may occur. Since tissues appear to be starving for 
glucose, dietary and tissue sources of protein are used in 
gluconeogenesis. Anabolic processes such as synthesis of glycogen, 
triglycerides and proteins are "sacrificed" to catabolic activities 
including glycogenolysis, gluconeogenesis and mobilization of fats. Thus, 
the diabetic state which has its origins as a "simple" insulin defect, 
results in widespread metabolic disturbances having long-term pathologic 
effects on nearly all organs and tissues of the body. Indeed, the diabetic 
state is one of the prime contributors to deaths caused by myocardial 
infarction, renal failure, cerebrovascular disease, atherosclerotic heart 
disease and systemic infections. 
Diabetic therapy for IDDM patients and advanced NIDDM patients has 
consistently focused on administration of exogenous insulin derived from 
bovine and porcine sources. It is frequently the case that use of such 
heterologous species material gives rise to formation of anti-insulin 
antibodies which have activity-limiting effects and result in progressive 
requirements for larger doses in order to achieve desired hypoglycemic 
effects. This, combined with the generally progressive need of the IDDM 
patient for more exogenous insulin as beta-cell function is lost, tends to 
accelerate the pathologic effects of the diabetic state. 
Use of the most common (and convenient) administrative route for exogenous 
insulin may itself exacerbate pathology resulting from insulin therapy. 
Subcutaneous injection of insulin gives rise to relatively high insulin 
levels in peripheral tissues and relatively low levels circulating through 
the liver, the primary site of endogenous insulin activity. High levels of 
insulin in peripheral tissue have been associated with blood vessel 
pathology (e.g., blood vessel constriction and permeability changes) and 
pathologic effects on associated peripheral tissues, e.g., diabetic 
retinopathy. The "swamping" effects of subcutaneously administered insulin 
on peripheral circulatory tissues eventually reduces the amount of insulin 
circulating to the liver--again resulting in the need for increased doses 
to achieve desired metabolic effects. 
It will be apparent from the above that substantial long term benefits in 
insulin therapy for diabetics (especially IDDM patients) can be expected 
to attend the development of methods and materials for enhancing the 
hypoglycemic effects of exogenous insulin. If insulin therapy for a given 
patient is expected to continue over a period of decades, it is 
significant that initial doses be as small as possible and that large 
doses of exogenous insulin be avoided for as long as possible. 
The recent past has seen modest advances in the development of chemical 
agents capable of stimulating endogenous insulin secretion and hence 
reducing the need for exogenous insulin in large doses. Further, 
recombinant DNA methods have been brought to bear on the problem of 
securing large scale production of homologous species (human) insulin with 
the hope that use of the "human" material will reduce the progressive need 
for larger doses of insulin resulting from the effects of anti-insulin 
antibodies made against heterologous species materials. As yet, however, 
no significant advances have been reported in research directed toward 
development of compounds which would function to augment hypoglycemic 
effects of any given dose of endogenous insulin and thus guarantee that 
the insulin dose regimen employed can always be set at or near the minimum 
needed for desired metabolic effect and will result in the minimum of 
adverse side effects. There continues to exist, therefore, a need in the 
art for methods and materials for enhancing the hypoglycemic effects of 
exogenous insulin in mammals, including humans. 
Of interest to the background of the invention are the results of certain 
studies on insulin-like activities of human growth hormone ("hGH"). hGH is 
a relatively high molecular weight polypeptide (.about.22,000 Daltons) 
consisting of a continuous sequence of 191 amino acid residues with 
secondary structure provided by two disulfide bonds formed between 
cysteine residues at position numbers 53/165 and 182/189, respectively. 
["Atlas of Protein Sequence and Structure," Vol. 5, Supp. 2, pp. 120-121 
(M. Dayhoff, ed., National Biomedical Resarch Foundation, 1976)]. Early 
studies of the growth promoting effects of hGH revealed, as one of its 
intrinsic properties, the ability to initially raise and then lower blood 
levels of glucose and to lower free fatty acids within one hour of 
administration, followed by later increasing circulating fatty acids. See, 
e.g., Goodman, Metabolism, 19, pp. 849-855 (1970); Goodman, Ann. N.Y. 
Acad. Sci., 148, pp. 419-440 (1968); and Swislocki, et al. Endocrinology, 
76, pp. 665-672 (1965). The hyperglycemic and hypoglycemic effects of 
large doses of hGH are so pronounced in many cases that they constitute a 
substantial adverse side-effect of hGH therapy for growth disorders. 
Determination of the effects of hGH on glycemia prompted a series of 
studies into the in vivo and in vitro actions of peptide fractions and 
synthetic fragments related to amino and carboxy terminal regions of hGH. 
See, e.g., the review by Bornstein appearing at pp.41-44 in "Growth 
Hormones and Related Peptides", A. Pecile, et al., eds. Excerpta Medica, 
Amsterdam-Oxford (1976). A variety of biological effects were noted 
including an insulin potentiating effect on glucose uptake by a fragment 
duplicating the sequence of amino acid residues at hGH positions 1 through 
15 and a hyperglycemic effect for a peptide duplicating residues 176 
through 191. 
The discovery by Lewis, et al. in 1975 [J.Biol.Chem., 253, pp. 2679-2685] 
of a naturally-occurring structural variant of hGH which differed from the 
major form of the hormone by having fewer amino acid residues prompted a 
systematic examination of the variant, 20,000 Dalton polypeptide, and its 
properties. Studies by Frigeri, et al., Biochem. Biophys. Res. Comm., 91, 
pp. 778-782 (1979), Lewis, et al., Biochem. Biophys, Res. Comm., 92, pp. 
511-516 (1980), and Lewis, et al., Endocrine Res. Comm., 8, pp. 155-164 
(1981) established that the 20,000. Dalton variant lacked the hypoglycemic 
and fatty acid lowering effects of hGH but substantially retained its 
growth promotant effects. It was also determined that the "missing" amino 
acid residues were in a region spanning positions thirty-two to forty-six 
of hGH. Following these publications were reports of further studies 
directed toward ascertaining the role of the "missing" residues in the 
growth stimulating and insulin-like activities of hGH. Frigeri, et al., 
[Proc. 64th Ann. Meeting of the Endocrine Society, San Francisco, June 
1982 (Abstract 88), p. 101] reported that, in normal rats, a synthetic 
peptide corresponding to residues 32 to 46 of hGH did not show either the 
late increases in free fatty acids nor the glycemic effects which are 
characteristic of intact hGH. An unspecified degree of improvement in 
glucose tolerance of a GT-impaired strain of mice (YS/Wf Nctr) was 
observed for the peptide, as was an in vitro increase in glucose 
utilization of insulin-stimulated fat cells of older obese rats. Yudaev, 
et al., Biochem. Biophys. Res. Comm., 110, pp. 866-872 (1983) reported 
substantially the same in vitro effect on fat cells for a synthetic 
tetradecapeptide having a sequence of amino acids copying residues at 
positions 31 through 44, and reiterated an earlier report of the absence 
of any in vivo hypoglycemic effect for the tetradecapeptide in rabbits and 
normal rats. In sum, the above-noted studies revealed that while hGH 
displays substantial glycemic effects in vivo which are not shown by the 
20,000 Dalton variant, the "missing" sequence had no glycemic effect in 
vivo unless provided to the test animal as part of the hGH polypeptide. 
Also of interest to the present invention are recent studies revealing 
alteration of biological effects of relatively small synthetic peptides 
resulting from incorporation of amino acids in D-isomeric configuration 
rather than the naturally-occurring L-isomeric forms. See, e.g., Sawyer, 
et al., P.N.A.S. (U.S.A.), 77, pp. 5754-5758 (1980) relating to 
prolongation of effects of alpha-melanotropin through synthesis of the 
tridecapeptide with D-phenylalanine replacing L-phenylalanine in position 
7. Finally, recent advances in recombinant DNA methods for securing large 
scale production of peptides and polypeptides have made possible the 
generation of analogs of naturally occurring substances which differ from 
the natural compounds in terms of the identity or location of one or more 
amino acid residues. Particularly interesting are those new compounds 
wherein variations in the sequence of residues are effected based on the 
identity of residues extant in heterologous species forms of the 
biologically active polypeptide or in differing subtypes of polypeptides 
within a family of related compounds. An example of the latter is the 
disclosure of the construction and use of analogs of human leukocyte 
interferons set out in co-owned, co-pending U.S. patent application Ser. 
No. 483,451, filed Apr. 15, 1983, by Alton, et al. 
BRIEF SUMMARY 
In one of its aspects, the present invention provides novel, biologically 
active synthetic peptides having primary structural homology to a 
continuous sequence of amino acid residues of human growth hormone ("hGH") 
in a region spanning positions thirty-two to forty-six, i.e., 
"hGH.sub.32-46 ", NH.sub.2 
-Glu-Glu-Ala-Tyr-Ile-Pro-Lys-Glu-Gln-Lys-Tyr-Ser-Phe-Leu-Gln-COOH. 
A first class of peptides of the invention consists of peptide "fragments" 
having from three to fourteen amino acid residues in a sequence precisely 
duplicating a continuous portion of the above-noted region. Preferred 
peptides include the sequence of residues at positions 35 through 37 of 
hGH (i.e., have the sequence, RNH-Tyr-Ile-Pro-COR', wherein R is hydrogen 
or an amino acid residue and R' is hydroxyl or an amino acid residue) and 
a presently most preferred group of compounds have the sequence, NH.sub.2 
-Glu-Glu-Ala-Tyr-Ile-Pro-Lys-COR', wherein R' is hydroxyl or an amino acid 
residue. 
A second class of peptides of the invention consists of stereochemical 
analogs of hGH.sub.32-46 or analogs of fragments of hGH.sub.32-46 
including as many as fifteen amino acid residues (analogs) or as few as 
three residues (fragment analogs) in which from one to three of the 
residues exist in a D-isomeric configuration and the remainder are in the 
L-isomeric form. Presently preferred compounds of this class include those 
wherein either a glutamic acid residue corresponding to the residue in 
position thirty-two of hGH.sub.32-46 or an alanine residue corresponding 
to the residue at position thirty-four of hGH.sub.32-46 is in D-isomeric 
form. 
A third class of peptides of the invention consists of "interspecies" 
analogs of hGH.sub.32-46 or analogs of fragments of hGH.sub.32-46 
including a sequence of three to fifteen amino acid residues in which one 
or more (and up to nine) residues present are not duplicative of residues 
present in hGH.sub.32-46 but, rather, duplicate residues present in 
corresponding regions of heterologous species growth hormones (e.g., 
equine, ovine, bovine, murine/rat, and chicken growth hormones). 
Illustrative preferred peptides of this class include the heptapeptide 
having the sequence, NH.sub.2 -Glu-Arg-Thr-Tyr-Ile-Pro-Glu-COOH. 
Also comprehended by the invention are stereochemical, interspecies analogs 
and fragment analogs of hGH.sub.32-46. 
In another of its aspects, the present invention provides improvements in 
insulin therapy methods for securing reduction in circulating glucose in 
mammals, including humans, which involve periodic parenteral 
administration of exogenous insulin. The improved methods comprise 
augmenting the effectiveness of insulin as a hypoglycemic agent by means 
of contemporaneous (e.g., simultaneous) administration of an effective 
amount of one or more of the above-noted novel peptides of the invention. 
Also comprehended by the present invention are novel pharmaceutical 
compositions including insulin and one or more peptides of the invention 
(in ratios of from about 1 mU insulin to 100 .mu.g peptide to about 100 mU 
insulin to 1 .mu.g peptide, and preferably about 1 mU insulin to 1 .mu.g 
peptide along with a pharmaceutically acceptable diluent, adjuvant or 
carrier. 
In another of its aspects, the present invention is seen to comprise a 
novel process for the formulation of (homologous or heterologous species) 
exogenous insulin-containing compositions for use in controlling the 
levels of circulating glucose in mammals wherein a selected desired 
hypoglycemic effect is determined to require the use of a predetermined 
quantity of insulin. According to the improved process, less than the 
predetermined quantity of insulin is incorporated but there is 
incorporated for contemporaneous administration an effective quantity of 
one or more peptides of the invention. 
Other aspects and advantages of the present invention will be apparent upon 
consideration of the following detailed description of the invention 
including illustrative examples of the practice thereof. As employed 
therein and in the claims, the terms, "hGH.sub.32-46 ", "deletion 
peptide", and "DP" shall be used synonymously to designate a peptide of 
the sequence: NH.sub.2 
-Glu-Glu-Ala-Tyr-Ile-Pro-Lys-Glu-Gln-Lys-Tyr-Ser-Phe-Leu-Gln-COOH. 
DETAILED DESCRIPTION 
Incorporated by reference herein are the disclosures and detailed 
descriptions of co-owned, co-pending, contemporaneously-filed U.S. patent 
application Ser. No. 501,023, by co-inventor Christopher G. Rudman, 
entitled, "Potentiation of the Effects of Insulin by Peptides". Briefly 
put, the patent application relates to the discovery of unexpected 
physiological activity for a synthetic pentadecapeptide having an amino 
acid residue sequence duplicative of a region of residues spanning 
positions thirty-two through forty-six of human growth hormone. More 
specifically, it was discovered that while "deletion peptide" ("DP" or 
"hGH.sub.32-46 ") lacked hypoglycemic effects in various model animal test 
systems the substance would, when contemporaneously administered with 
exogenous insulin, potentiate insulin activity and augment the 
hypoglycemic effectiveness of even quite small parenteral doses of 
insulin. 
According to the present invention, three classes of novel peptides have 
been synthesized which have primary structural homology to deletion 
peptide. One class of peptides of the invention may be characterized as 
comprehending deletion peptide fragments, i.e., sequences of from three to 
fourteen amino acids which duplicate a continuous portion of the sequence 
of amino acid residues in deletion peptide. A second class of peptides of 
the invention comprehends stereochemical analogs and fragment analogs of 
hGH.sub.32-46 which duplicate the amino acid residue sequence of deletion 
peptide or fragments thereof, but wherein from one to three amino acids 
are present in a D-isomeric configuration. Still a third class of peptides 
of the invention comprehends analogs and fragment analogs of DP which 
include one or more amino acid residues which are not homologous to human 
growth hormone. Rather, these residues are duplicative of residues extant 
at corresponding positions in corresponding regions of heterologous 
species growth hormones including equine, ovine, bovine, rat/murine and 
chicken species. Peptides of this class are herein referred to from time 
to time as "interspecies analogs". 
Preliminary screenings of the biological activities of representative 
peptides of the invention has revealed, inter alia, a number of compounds 
which, at the doses tested, possess insulin potentiating activity. In at 
least one instance the activity displayed is substantially greater than 
that of deletion peptide, demonstrating utility of the compounds as a 
substitute for deletion peptide. Thus, in processes for formulating 
insulin-containing pharmaceutical compositions for use in controlling the 
levels of circulating glucose in a mammal wherein a selected desired 
reduction in circulating glucose is determined to require the use of a 
predetermined quantity of insulin, the present invention comprehends 
incorporating less than the predetermined quantity and incorporating for 
contemporaneous administration an effective amount of a peptide of the 
invention.

The following illustrative examples therefore relate to: (1) the synthesis 
of representative members of each of the three related classes of peptides 
of the invention; and, (2) tests for glycemic effects of peptides of the 
invention including, specifically, tests of the insulin potentiating 
activity of the compounds in various animal model systems. 
EXAMPLE 1 
Peptides of the present invention are all suitably manufactured according 
to the general method of Stewart, et al., Solid Phase Peptide Synthesis, 
(W. H. Freeman, San Francisco, 1969). Briefly put, peptides are 
constructed by means of a series of amino acid residue additions to an 
initial, column-bound residue selected to form the carboxy terminal 
residue of the peptide. Each selected carboxyl terminal amino acid is 
coupled to the polystyrene resin as a BOC-protected amino acid. All 
subsequent amino acid additions are carried out with 
dicyclohexylcarbodiimide using the appropriate BOC amino acid with side 
chain protecting groups as follows: Glutamic acid as the Y-benzyl ester; 
Tyrosine as O-2,6-dichlorobenzyl tyrosine; Lysine as 
2-chlorobenzylcarbonyl lysine; Glutamine as xanthyl glutamine; and, Serine 
as O-benzyl serine. Finished protected peptides are cleaved from the resin 
with simultaneous deprotection using anhydrous HF. Individual peptides are 
purified by a combination of chromatography on Sephadex G10 and G25 or 
preparative thin layer chromatography. Desired products are isolated as 
stable lyophilized white to pale tan powders. Composition of peptides is 
determined by amino acid analysis after HCl digestion as described in 
"Protein Sequence Determination" page 197 (S. Needleman, ed., 
Springer-Verlag, 1975). Sequence verification is performed by automated 
amino acid analysis. Purified products migrate as a single spot on TLC. Rf 
values are determined with a pH 4-4.5 solvent comprising butanol, acetic 
acid, water and pyridine (15:3:12:10). Purity is further verified by 
reverse phase high pressure liquid chromatagraphy on a C.sub.18 .mu. 
bondapak column with a 0.1% trifluoroacetic acid/acetonitrile gradient. 
For purposes of illustration, thirteen representative peptides according to 
the invention are specified in Table I below, with the tabular 
presentation designed to readily display primary structural homology with 
the amino acid residues of hGH.sub.32-46. Following the Table is a 
discussion of individual peptides and of their relationship to the three 
above-noted classes ("fragments", "stereochemical analogs" and 
"interspecies analogs") of compounds. 
TABLE I 
__________________________________________________________________________ 
hGH Residues (15)Position No. 
##STR1## 
__________________________________________________________________________ 
Synthetic Peptide Residues 
No. 1 (4) TYR--ILE--PRO--LYS 
No. 2 (5) ALA--TYR--ILE--PRO--LYS 
No. 3 (5) PRO--LYS--GLU--GLN--LYS 
No. 4 (6) GLU--ALA--TYR--ILE--PRO--LYS 
No. 5 (6) LYS--TYR--SER--PHE--LEU--GLN 
No. 6 (7) GLU--GLU--ALA--TYR--ILE--PRO--LYS 
No. 7 (9) TYR--ILE--PRO--LYS--GLU--GLN--LYS--TYR--SER 
No. 8 (12) GLU--GLU--ALA--TYR--ILE--PRO--LYS--GLU--GLN--LYS--TYR--SER 
No. 9 (7) 
##STR2## 
No. 10 (7) 
##STR3## 
No. 11 (7) 
##STR4## 
No. 12 (7) 
##STR5## 
No. 13 (7) 
##STR6## 
__________________________________________________________________________ 
.sup.a D-Alanine 
.sup.b D-Glutamic Acid 
.sup.c Residue extant in equine, murine, rat, bovine, ovine, and chicken 
GH 
.sup.d Residue extant in bovine, ovine, and chicken GH 
A. DP Fragments 
Synthetic Peptide Nos. 1 through 8 illustrated in Table I comprise 
representative members of that class of peptides of the invention 
comprehending from 3 to 14 (and, preferably, from 4 to 12) amino acid 
residues which duplicate continuous sequences extant in the region 
spanning residues at positions thirty-two through forty-six of human 
growth hormone. Peptide Nos. 1, 2, 4, 6, 7, and 8 are representative of 
presently preferred compounds within this and all classes, i.e., those 
including the sequence of residues, -Tyr-Ile-Pro-, duplicating residues at 
positions thirty-five through thirty-seven of hGH. As discussed in detail, 
infra, one of the heptapeptides (Peptide No. 6) constitutes the presently 
preferred compound of the invention based on insulin potentiating effects. 
Rf values of Peptide Nos. 1 through 8 are as follows: 
Peptide No. 1, 0.56; 
Peptide No. 2, 0.47; 
Peptide No. 3, 0.44; 
Peptide No. 4, 0.28; 
Peptide No. 5, 0.57; 
Peptide No. 6, 0.38; 
Peptide No. 7, 0.33; and, 
Peptide No. 8, 0.19. 
B. Stereochemical Analogs of DP 
Synthetic Peptide Nos. 9 and 10 illustrated in Table I comprise 
representative members of that class of compounds of the invention 
comprehending sequences of from 3 to 15 (and, preferably, from 4 to 12) 
amino acid residues which duplicate the sequence or a portion of the 
sequence of deletion peptide. Included in the sequence, however, are from 
one to three residues of amino acids in D-isomeric form, with the 
remainder being in L-isomeric form. The class is thus seen to include 
stereochemical analogs of hGH.sub.32-46 as well as stereochemical analogs 
of fragments of hGH.sub.32-46. Rf values for Peptide Nos. 9 and 10 are as 
follows: 
Peptide No. 9, 0.38; and 
Peptide No. 10, 0.38. 
C. Interspecies Analogs of DP 
Synthetic Peptide Nos. 11 through 13 illustrated in Table I comprise 
representative members of that class of peptides of the invention 
comprehending sequences of from 3 to 15 (and, preferably, from 4 to 12) 
amino acid residues which duplicate partially (i.e., in terms of from 2 to 
14 residues) the identity and relative position of residues in deletion 
peptide and wherein one or more of the non-homologous residues are 
selected from among residues at a corresponding position in a 
corresponding heterologous species growth hormone. Table II, below, 
illustrates the rationale for synthesis of peptides of this class by 
providing corresponding sequences of various species growth hormones. The 
hGH.sub.32-46 sequence is set out in capital letters and homology to the 
hGH sequence in the heterologous species hormones is indicated by use of 
capital letters. Alignment of corresponding regions (comprehending 
residues at positions 33-47 of ovine and bovine species, and sequence 
residues at positions 32-46 of the remaining hormones) reveals that there 
is a total interspecies homology at six positions within the region. 
Development of interspecies analogs and fragment analogs is carried out in 
the context of consideration of the lack of homology at the remaining nine 
positions. 
TABLE II 
__________________________________________________________________________ 
GH Species 
Position Nos. 
Residues 
__________________________________________________________________________ 
Human 
32-46 
##STR7## 
Equine 
32-46 GLU--Arg--ALA--TYR--ILE--PRO--Glu--Gly--GLN--Arg--TYR--SER-- 
Ile--Gln--Asn 
Murine/rat 
32-46 GLU--Arg--ALA--TYR--ILE--PRO--Glu--Gly--GLN--Arg--TYR--SER-- 
Ile--Gln--Asn 
Bovine 
33-47 GLU--Arg--Thr--TYR--ILE--PRO--Glu--Gly--GLN--Arg--TYR--SER-- 
Ile--Gln--Asn 
Ovine 33-47 GLU--Arg--Thr--TYR--ILE--PRO--Glu--Gly--GLN--Arg--TYR--SER-- 
Ile--Gln--Asn 
Chicken 
32-46 GLU--Arg--Thr--TYR--ILE--PRO--Glu--Asp--GLN--Arg--TYR--Thr-- 
Gln--Lys--Gln 
__________________________________________________________________________ 
*Residue common to all species noted 
As reflected by Table II, an interspecies analog of deletion peptide may 
include one or more and up to nine amino acid residues which are 
non-homologous to hGH.sub.32-46. Interspecies fragment analogs of deletion 
peptide comprehend sequences of 3 to 14 (and preferably 4 to 12) residues 
which may include one or more (but correspondingly fewer than nine, 
depending on the fragment length) amino acid residues which are 
non-homologous to hGH.sub.32-46. Peptide Nos. 11 through 13 of Table II 
are thus seen as representing interspecies fragment analogs of 
hGH.sub.32-46 according to the invention, including one (e.g., Peptide No. 
11) or more residues duplicative of residues extant at corresponding 
positions within a corresponding continuous sequence of residues of an 
heterologous species growth hormone. 
While not specifically exemplified in Table I, peptides of the invention 
also include stereochemical, interspecies analogs and analog fragments 
wherein interspecies analogs and analog fragments as defined above 
additionally include from one to three amino acids in a D-isomeric 
configuration. 
EXAMPLE 2 
A series of experimental studies was conducted to ascertain biological 
effects (specifically the glycemic effects) of compounds of the invention. 
The protocols for these studies and the results obtained are set out 
below. 
A. Insulin Potentiating Effects in Normal Rats 
A study was conducted to determine insulin potentiating effects of Peptide 
Nos. 3, 5, and 6. Also tested was a dipeptide NH.sub.2 -Ile-Pro-COOH, a 
dipeptide outside the scope of the invention. 
Test groups of 5 normal male Sprague-Dawley rats weighing about 200 grams 
were fasted for 18 hours and received (intraperitoneally in rapid 
succession) 1.0 ml of 0.75M glucose, and either 15 mU of insulin with 1% 
bovine serum ablumin in normal saline (pH 7.4) or a mixture of insulin as 
above with 25 .mu.g Peptide Nos. 3, 5, 6 and dipeptide. Controls received 
only bovine serum albumin and saline. Blood was drawn after one hour and 
plasma glucose levels were determined. 
The results of plasma glucose determinations (means .+-. standard error) 
are set out below in Table III and indicate that Peptide No. 6 is an 
exceptionally active insulin potentiator. 
TABLE III 
______________________________________ 
Plasma Glucose 
(mg/ml at 60 min.) 
Glucose + 
Peptide Glucose Glucose + Insulin + 
No. Alone Insulin Peptide 
______________________________________ 
No. 3 162.0 .+-. 10 
114.0 .+-. 7 
110.0 .+-. 6 
No. 5 194.0 .+-. 21 
128.0 .+-. 3 
140.0 .+-. 19 
No. 6 150.0 .+-. 11 
121.0 .+-. 28 
52.0 .+-. 28 
NH.sub.2 --Ile-- 
165.0 .+-. 21 
105.0 .+-. 15 
95.0 .+-. 30 
Pro--COOH 
______________________________________ 
B. Insulin Potentiating Effects on Genetically Altered Mice at Differing 
Ages 
A study was conducted to ascertain insulin potentiating effects of Peptide 
Nos. 6, 9 and 10 on db/db mice aged 8 weeks and 14 weeks. 
Test groups of 5 each of db/db mice weighing from 40 to 60 grams each were 
used. Glucose was administered intraperitoneally at a dosage of 0.1 ml/20 
mg of a solution containing 135 mg/ml glucose. Insulin was 
intraperitoneally administered at a dose of 0.001 mU/10 g and Peptide Nos. 
6, 9 and 10 were each simultaneously administered at a dose of 5 .mu.g/10 
g. 
Plasma glucose determination generated by this study are set out in Table 
IV and again indicate significant effectiveness of Peptide No. 6. 
TABLE IV 
______________________________________ 
Plasma Glucose 
(mg/ml at 60 min.) 
Treatment Age: 8 weeks 
Age: 14 weeks 
______________________________________ 
Glucose Alone 189.0 .+-. 69 
318.0 .+-. 25 
Glucose + Insulin 
135.7 .+-. 11 
268.4 .+-. 65 
Glucose + Insulin + 
92.3 .+-. 12 
124.3 .+-. 34 
Peptide No. 6 
Glucose + Insulin + 
90.8 .+-. 16 
228.0 .+-. 38 
Peptide No. 9 
Glucose + Insulin + 
111.0 .+-. 24 
246.0 .+-. 43 
Peptide No. 10 
______________________________________ 
C. Insulin-Potentiating Effects in Mice and Rats of Peptide No. 6 as 
Compared to DP 
A study was conducted to determine the relative insulin potentiating 
effects of Peptide No. 6 as compared to those of deletion peptide 
("hGH.sub.32-46 "). 
Animals in test groups of five each were meployed in these procedures. 
Sprague-Dawley rats weighed approximately 200 grams; homozygous 
genetically abnormal mice (db/db and ob/ob) had weights in the range of 40 
to 60 grams; heterozygous normal mice all weighed approximately 25 grams. 
Glucose was administered intraperitoneally at a dosage of 0.05 ml/0 g of a 
solution containing 135 mg/ml glucose, except for one group of rats which 
were given an oral dose of 1 ml of 270 mg/ml glucose solution. Insulin was 
intraperitoneally administered at a dose of 0.001 mU/10 g and both Peptide 
No. 6 and deletion peptide were administered were simultaneously 
administered at a dose of 5 .mu.g/10 g. 
Plasma glucose levels determined by this study are set out in Table V below 
and indicate that Peptide No. 6 was uniformly more effective in 
potentiating insulin effects than deletion peptide alone. 
TABLE V 
______________________________________ 
Plasma Glucose 
(mg/ml at 60 min.) 
Glucose + 
Glucose + 
Insulin + 
Animal Glucose Glucose + Insulin + 
Polypeptide 
Model Alone Insulin hGH.sub.32-46 
No. 6 
______________________________________ 
Mouse ob/ob 
222.0 .+-. 35 
167.4 .+-. 26 
138.2 .+-. 11 
118.0 .+-. 20 
Mouse ob/+ 
120.0 .+-. 12 
99.5 .+-. 6 
56.0 .+-. 9 
28.5 .+-. 5 
(normal) 
Mouse db/db 
320.0 .+-. 75 
198.2 .+-. 31 
134.0 .+-. 13 
109.2 .+-. 22 
Mouse db/m 
99.0 .+-. 9 
102.0 .+-. 10 
49.0 .+-. 12 
22.0 .+-. 10 
(normal) 
Rat 150.0 .+-. 11 
121.0 .+-. 28 
73.0 .+-. 10 
52.0 .+-. 6 
(normal) 
Rat* 184.0 .+-. 14 
144.0 .+-. 12 
93.3 .+-. 14 
-- 
(normal) 
______________________________________ 
*Glucose administered orally 
D. Insulin Potentiating Effects in Primates of Peptide No. 6 as Compared to 
Deletion Peptide 
In a manner analogous to Study C, above, Peptide No. 6 and deletion peptide 
were studied for insulin potentiating effects in Rhesus monkeys. Blood 
samples were drawn from normal female monkeys (in four experimental groups 
of 3) five minutes before administration of an oral dose of 3.0 ml/kg of 
0.5 g/ml glucose and intramuscular administration of either: (1) 0.5 ml/kg 
phosphate buffered saline (PBS), pH 7.4; (2) deletion peptide 0.1 ml/kg of 
1.0 mg/ml solution in PBS; (3) deletion peptide as above combined with 0.5 
ml/kg of 20 mU/ml insulin in PBS; or (4) insulin alone as above or (5) 
Peptide No. 6 0.1 ml/kg of a 0.5 mg/ml solution in PBS combined with 
insulin as above. Blood samples were then periodically withdrawn over two 
hours and analyzed for plasma glucose levels. Plasma glucose level data is 
set out in Table VI and reveals that insulin potentiating effects of 
Peptide No. 6 are essentially on par with, or superior on a weight basis 
to, those of deletion peptide under the conditions of the procedure. 
TABLE VI 
______________________________________ 
Plasma Glucose 
(mg/ml) 
Glucose + 
Glucose + 
Insulin + 
Glucose Glucose + Insulin + 
Peptide 
Time Alone Insulin hGH.sub.32-46 
No. 6 
______________________________________ 
-5 min. 
84.6 .+-. 7.5 
71.0 .+-. 3.0 
70.0 .+-. 2.5 
72.0 .+-. 11 
+5 min. 
82.5 .+-. 6.2 
79.0 .+-. 9.0 
64.0 .+-. 4.0 
62.0 .+-. 6.0 
+15 min. 
106.0 .+-. 14 
72.0 .+-. 10 
69.0 .+-. 9.0 
69.5 .+-. 6.0 
+30 min. 
117.0 .+-. 16 
85.0 .+-. 11 
80.0 .+-. 5.3 
68.0 .+-. 9.7 
+45 min. 
132.0 .+-. 13 
88.0 .+-. 13 
80.0 .+-. 5.9 
73.0 .+-. 3.5 
+60 min. 
125.0 .+-. 12 
109.0 .+-. 14 
96.0 .+-. 8.0 
79.0 .+-. 6.5 
+120 126.0 .+-. 12 
108.0 .+-. 14 
97.0 .+-. 6.6 
88.0 .+-. 12 
min. 
______________________________________ 
The foregoing illustrative examples are believed to establish with 
certainty that the hypoglycemic effects of exogenous insulin are 
substantially enhanced or potentiated when accompanied by contemporaneous 
administration with one or more peptides of the invention. While practice 
of the methods of the invention may comprehend contemporaneous parenteral 
administration of peptide prior to or subsequent to insulin 
administration, it is expected that the most highly augmentative effects 
will be observed by simultaneous administration of both. In this regard, 
it is expected that significant beneficial effects will attend parenteral 
(e.g., subcutaneous, intraperitoneal, intramuscular) administration of 
pharmaceutical compositions of the invention comprising admixtures of 
insulin and one or more peptides of the invention along with 
pharmaceutically acceptable diluents, adjuvants and carriers such as are 
commonly employed in administration of insulin alone. Suitable 
compositions are expected to result from use of admixtures of insulin and 
peptide in relative weight ratios varying from 1 mU insulin to 100 .mu.g 
peptide to about 100 mU insulin to 1 .mu.g peptide with a preferred ratio, 
based on the procedures of the above examples of about 1 mU insulin to 1 
.mu.g peptide. 
While solid phase synthesis according to the procedures of Example 1 
constitutes the presently preferred method for securing production of 
peptides of the invention in quantity, use of alternative methods such as 
liquid phase synthesis or microbial synthesis by recombinant DNA 
techniques (for all but the stereochemical analogs) is contemplated. 
While the foregoing illustrative examples have necessarily concentrated on 
insulin potentiating biological effects of peptides, it will be understood 
that the absence of such effects in the experimental procedures practiced 
is not necessarily preclusive of potentiating utility at higher doses or 
utility in other physiological contexts, especially those involving 
carbohydrate fat and protein metabolism. It may be noted, for example, 
that ongoing studies of the in vivo biological activities of peptides of 
the invention have revealed preliminary evidence of insulin secretory 
stimulation effects, effects on levels of free fatty acids and effects on 
glucose uptake by hepatic and muscle tissue. The results of these studies 
indicate utility for the peptides used alone, for example, in diseases 
requiring stimulation of insulin secretion or depression of free fatty 
acids. 
Numerous modifications and variations in practice of the present invention 
are expected to occur to those skilled in the art upon consideration of 
the foregoing detailed description of illustrative embodiments thereof. As 
one example, illustrative test procedures were conducted using individual 
peptides of the invention even though it is within the scope of the 
invention to employ such peptides either singly or in combination with 
others to develop desired biological effects. Consequently, only such 
limitations should be placed on the scope of the invention as appear in 
the appended claims.