Hypotensive active peptides

The present invention provides a hypotensive composition comprising a peptide having the structure X-Y, Y-X, or a salt thereof, wherein X is proline or proline-proline and Y is arginine, lysine, arginine-arginine, lysine-lysine, arginine-lysine, or lysine-arginine, together with a pharmaceutically suitable diluent.

BACKGROUND OF THE INVENTION 
The present invention relates to short peptides, two to four amino acids in 
length, to be used as antihypertensive agents. 
Hypertension is a serious health problem of epidemic proportions. It is 
estimated that 20% of the adult population of North America have systemic 
arterial pressures above the accepted normal range. A few direct causes of 
hypertension, such as pheochromocytoma and primary hyperaldosteronism, are 
amenable to direct therapeutic intervention. These instances are not the 
norm. In general, pharmacologic treatment of hypertension involves 
treating the symptom. Therapy is directed only to a correction of the 
abnormal pressure. Yet, it is now clear that such therapy can favorably 
affect prognosis and thus greatly decrease the risk of death due to 
cardiovascular disease in the affected person. 
Essential, or primary, hypertension is a poorly defined condition that is 
diagnosed by exclusion. It is not known whether hypertension is a disease 
per se or simply the upper end of a continuous spectrum of blood pressures 
in the population. In both normotensive and hypertensive individuals, 
vascular tone is controlled by sympathetic nerve fibers. Even in 
normotensive individuals, reduction of the vascular tone can reduce the 
blood pressure. Thus, abnormal sympathetic nerve function is probably not 
responsible for the increased peripheral resistance that characterizes 
primary hypertension. 
Pharmacologic therapy of hypertension has generally been directed at 
promoting salt excretion (diuretics) and decreasing vascular tone by both 
direct (direct vasodilators) and indirect (adrenegic blocking agents) 
means. Diuretics are an effective means of reducing overall blood volume 
and thereby reduce blood pressure, yet their use, particularly in moderate 
to severe hypertension, is generally secondary to other agents. Diuretics 
also tend to deplete serum potassium, which can have serious consequences 
in the cardio-comprised patient. 
The indirect vasodilators are probably the most extensively used agents for 
mild to moderate hypertension. These agents, for the most part, act on 
some part of the sympathetic nervous system. Propranolol, a beta 
adrenergic blocker, is widely used in this country to varying degrees of 
success. Propranolol alone, though, provides poor control of hypertension. 
Such therapy can also lead to various uncomfortable side effects including 
a prolonged reduction in cardiac output accompanied by depression, 
impotence, and a pronounced lethargy. Other indirect acting agents, 
including Guanethidine, Pargyline, Methyldopa, Reserpine, Clonidine, and 
the ganglionic blockers, have also displayed varying degrees of success 
with numerous side effects and untoward reactions. 
Similarly, problems with side effects are common with long term use of 
drugs that act directly on the vascular smooth muscle. Diazoxide is both 
hyperuricemic and hyperglycemic. Nitroprusside is a powerful vasodilator 
but its effects are transient and the drug must be given by continuous 
infusion. Hydralazine is used regularly even though the incidence of 
untoward reactions is very high. 
In all of these agents, the main problems facing the physician are efficacy 
and side effects. In the area of antihypertensives, even minor side 
effects can result in the serious problem of patent non-compliance. In 
terms of efficacy, the direct acting agents appear to offer the best 
control of the hypertensive state yet their toxicities and other 
limitations tend to decrease their usefulness, particularly in mild to 
moderate cases. It is in the interest of medical science, therefore, to 
develop new approaches to the treatment of hypertension. 
SUMMARY OF THE INVENTION 
The present invention provides a hypotensive composition comprising a 
peptide having the structure X-Y, Y-X, or a salt thereof, wherein X is 
proline or proline-proline and Y is arginine, lysine, arginine-arginine, 
lysine-lysine, arginine-lysine, or lysine-arginine, together with a 
pharmaceutically suitable diluent. 
It also provides a method for alleviating hypertension in hypertensive 
mammals which comprises administering to the mammal an antihypertensive 
effective amount of a peptide having the structure X-Y, Y-X, or a salt 
thereof, wherein X is proline or proline-proline and Y is arginine, 
lysine, arginine-arginine, lysine-lysine, arginine-lysine, or 
lysine-arginine. 
DETAILED DESCRIPTION OF THE INVENTION 
This invention provides a series of 24 small peptides, two to four amino 
acids in length, which represents a new class of therapeutically useful 
antihypertensive agents. The general structure of the peptides included in 
the present invention can be defined as XY or YX, where X is proline (Pro) 
or proline-proline; and Y is arginine (Arg), lysine (Lys), 
arginine-arginine, lysine-lysine, arginine-lysine, or lysine-arginine. 
This yields the following 24 possible combinations: 
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XY YX 
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Pro-Arg Arg-Pro 
Pro-Lys Arg-Pro-Pro 
Pro-Arg-Arg Lys-Pro 
Pro-Lys-Lys Lys-Pro-Pro 
Pro-Arg-Lys Arg-Arg-Pro 
Pro-Lys-Arg Arg-Arg-Pro-Pro 
Pro-Pro-Lys Lys-Lys-Pro 
Pro-Pro-Arg-Arg Lys-Lys-Pro-Pro 
Pro-Pro-Lys-Lys Arg-Lys-Pro 
Pro-Pro-Arg-Lys Lys-Arg-Pro 
Pro-Pro-Lys-Arg Lys-Arg-Pro-Pro 
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The compositions of the present invention may be synthesized by known 
polypeptide synthesis methods. These peptides are then administered to the 
hypertensive mammal in a suitable diluent, such as normal saline, the 
peptide in an amount effective to reduce the mammal's blood pressure. 
The present invention may be better understood by reference to the 
following examples.

EXAMPLE 1 
Corticotropin Inhibiting Peptide (CIP) contains the following amino acid 
sequence, the underlined sequences of which are the sequences which are 
the subject of the present invention: 
Human CIP (ACTH 7-38): 
Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-As 
n-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu [M.W. 3659.68] 
This CIP fragment was obtained from the Peninsula Laboratories, San Carlos, 
CA. When administered to dogs, this peptide resulted in dose-related 
hypotension. 
The dog blood pressure assay was conducted according to the following 
method: 
Mongrel dogs of either sex weighing between 6-15 kg were anesthetized by 
intravenous injection of sodium pentobarbital (30 mg/kg). The femoral 
artery and vein were cannulated for the measurement of blood pressure and 
the injection of drugs, respectively. Arterial blood pressure was 
determined with a Statham pressure transducer and was recorded on a Grass 
polygraph. Mean arterial pressure (MAP) were calculated for the periods 
before and after each peptide injection. A tracheotomy was performed to 
insure a patient airway for spontaneous respiration. 
Hypotensive screening studies involved injecting each dog with 0.1; 0.5; 
1.0; and 2.0 ug/kg. The CIP as supplied by Peninsula Laboratories was in 
the lyophilyzed form. Prior to injection the CIP was dissolved in saline. 
Table 1 summarizes the results obtained. 
TABLE 1 
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Dose-dependent hypotensive activity of CIP 
Dose (ug/kg) 
Blood Pressure Decrease (mmHg) 
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0.1 7 
0.5 10 
1.0 19 
2.0 21 
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EXAMPLE 2 
The above-described dog blood pressure assay was also used for a 
determination of the antihypertensive effect of the following peptides, 
all of which were procured from Peninsula Laboratories. For each peptide, 
the amino acid sequences described by the present invention are 
underlined: 
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Peptide Amino acid sequence 
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Bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe- 
Arg [M.W. 1060.24] 
Xenopsin pGlu-Gly-Lys-Arg-Pro-Trp-Ile-Leu 
[M.W. 980.32] 
Neurotensin pGlu-Leu-Tyr-Glu-Asn-Lys-Pro- 
Arg-Arg-Pro-Tyr-Ile-Leu [M.W. 
1673.15] 
Substance P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe- 
Gly-Leu-Met-NH.sub.2 [M.W. 1347.80] 
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The maximum decrease in blood pressure of the hypotensive effects of these 
peptides in dogs by the assay described above in Example 1 is shown in 
Table 2. Each datapoint represents the mean of 4 replications. 
TABLE 2 
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Dose Maximum Decrease in 
Hypotensive agent 
(moles/kg) 
Blood Pressure (mmHg) 
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Substance P 5 .times. 10.sup.-10 
50 
Bradykinin 3 .times. 10.sup.-9 
44 
Neurotensin 7 .times. 10.sup.-10 
40 
Xenopsin 3 .times. 10.sup.-9 
32 
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EXAMPLE 3 
The following peptides include amino acid sequences (underlined) described 
by the present invention: 
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Peptide Amino acid sequence 
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Tuftsin Thr-Lys-Pro-Arg 
Neurotensin (8-13) 
Arg-Arg-Pro-Tyr-Ile-Leu 
CIP fragment Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys 
Contraceptive Thy-Pro-Arg-Lys 
tetrapeptide 
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When administered to a rat, each peptide exerted dose-related hypotensive 
effects. These results are shown in Table 3. Each datapoint represents the 
mean of 4 replications. 
The rat blood pressure assay was conducted as follows: 
Sprague-Dawley rats of both sexes, weighing between 100 and 200 grams, were 
anesthetized with sodium pentobarbital and the right carotid artery and 
jugular vein were cannulated with polyethylene tubing PE50. The 
hypotensive peptide was administered and blood pressure was recorded in 
the anesthetized rats as described above for dogs. 
TABLE 3 
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Dose-Dependent Hypotensive 
Activity of Selected Peptides 
Blood Pressure Decrease (mmHg) 
Contra- 
ceptive 
Neurotensin 
CIP tetra 
Dose (ug/kg) 
Tuftsin (8-13) fragment peptide 
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0.1 0 
1.0 13 
3.0 48 
10.0 63 1 
50.0 18 
100.0 35 2 
200.0 3 
300.0 38 
500.0 9 
1000.0 1 13 
2000.0 15 
3000.0 12 
5000.0 30 
9000.0 46 
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EXAMPLE 4 
The dipeptides proline-lysine and arginine-proline were also tested for 
their hypotensive action in rats. These dipeptides were synthesized by 
Vega Biochemicals, Tucson, AZ, and tested in the rat blood pressure assay 
according to the same method described above in Example 3. The results 
were as follows: 
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Blood Pressure Change in mm Hg at dose of 
Dipeptide 
1 mg/kg 2 mg/kg 3 mg/kg 
______________________________________ 
L Arg-L Pro.sup.A 
-0.8 .+-. 0.8 
-1.3 .+-. 0.8 
-13.8 .+-. 4.4 
L Pro-L Lys.sup.B 
+0.2 .+-. 2.6 
-2.6 .+-. 1.9 
-7.0 .+-. 3.4 
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.sup.A All values are average of four replications. 
.sup.B All values are average of five replications. 
EXAMPLE 5 
These dipeptides, L Arg-L Pro and L Pro-L Lys, were also tested in the rat 
tail artery helical strip in vitro assay. This assay is conducted 
according to the following method: 
Male and female Sprague-Dawley rats were anesthetized with pentobarbital 
(50 mg/kg) before removal of the tail artery. Upon isolation the artery 
was placed in ice cold Krebs-Hensleit solution (KHS) which was oxygenated 
with 95% O.sub.2, 5% CO.sub.2. The vessels were cut helically and strips 
of approximately 1.5 cm were secured in a Sawyer-Bartleston chamber 
containing KHS. The development of force of the helical strips was 
measured with a Grass FT.03 force displacement transducer and recorded on 
a Grass Model 79D polygraph. Isolated tail artery helical strips were 
equilibrated for one hour prior to addition of any peptide. In all cases, 
the strips were first contracted by adding arginine vasopressin (AVP), to 
the bath. The peptide being studied was then added to the bath and the 
degree of relaxation was determined. 
The pure amino acids L-proline, L-arginine, and D-arginine were included in 
this assay as controls. 
The results were as follows: 
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Tension change in gm at dose of 
Dipeptide 100 mg/ml 200 mg/ml 500 mg/ml 
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L Arg-L Pro 
-0.07 -0.18 -0.34 
L Pro-L Lys 
-0.09 -0.16 -0.25 
L Pro 0 0 0 
L Arg 0 0 0 
L Arg 0 0 0 
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EXAMPLE 6 
Addition of lysine to the bradykinin molecule above results in the 
following amino acid sequence: 
Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg. 
When tested in the rat blood pressure assay this analog was even more 
hypotensive than bradykinin itself. 
The above examples are provided by way of illustration only, and are not to 
be construed as a limitation on the scope of the invention defined by the 
following claims.