Relaxation of smooth vascular muscle

Certain substances are provided which have the ability to relax smooth vascular muscle, having an endothelial lining, when administered in vitro or by intravenous injection. Examples of useful substances which are pharmaceutically-acceptable include phospholipase A.sub.2 (PLA.sub.2) and certain lysolecithins. The concentration required is at least about 3 units of PLA.sub.2 and at least about 10.sup.-5 Molar lysolecithin. The lysolecithins are dissolved in a suitable solvent, such as dimethyl sulfoxide, for injection. Alternatively, phospholipase A.sub.2 is administered directly. The resulting solutions should have a pharmacologically-effective amount of the substance dissolved therein. Importantly, the body does not develop a tolerance to the lysolecithin.

TECHNICAL FIELD 
This invention is concerned with a method for relaxing smooth vascular 
muscle. 
BACKGROUND ART 
There are a number of conditions which cause the muscle to contract, 
resulting in adverse reactions in the body. For instance, contraction of 
coronary arteries can cause severe chest pains, such as those experienced 
with angina pectoris. In addition, contraction of peripheral vasculature 
can result in hypertension. 
In the case of angina pectoris, a common treatment is the placing of 
nitroglycerin under the tongue. This produces nitric oxide (NO) which is 
mediated by cyclic-3',5'-guanosin-monophosphate, resulting in dilation of 
the coronary arteries and relief of the symptoms. However, the body 
eventually develops a tolerance to nitroglycerin, thus rendering it 
ineffective. 
There are many agents for treating hypertension but many problems are 
associated with their use. Some of them cause the patient to develop 
rashes in some cases. Other cause hypotension unless the dosage is 
controlled and monitored with great care. 
Because of these problems with the agents of the prior art, the discovery 
of a new class of agents for relaxing smooth vascular muscle is 
beneficial. 
DISCLOSURE OF INVENTION 
In accordance with the invention, certain substances are provided which 
have the ability to relax smooth vascular muscle when administered by 
intravenous injection. Examples of useful substances which are 
pharmaceutically-acceptable include phospholipase A.sub.2 (PLA.sub.2) and 
certain lysolecithins. The concentration required is at least about 3 
units of phospholipase A.sub.2 and at least about 10.sup.-5 Molar 
lysolecithin. 
The lysolecithins are dissolved in a suitable solvent, such as dimethyl 
sulfoxide, while phospholipase A.sub.2 may be injected directly. The 
solutions thus have a pharmacologically-effective amount of the 
lysolecithin dissolved therein. 
Importantly, in contrast to nitroglycerin, the body does not develop a 
tolerance to the repeated use of the lysolecithin. 
BEST MODES FOR CARRYING OUT THE INVENTION 
When certain substances are administered to a living body suffering from 
vascular constriction due to tension in the smooth vascular muscle, the 
muscle gradually relaxes and normal or improved blood flow is resumed. 
Such constrictions are found in patients suffering from angina pectoris or 
hypertension. Unless these constrictions are removed, the patient can 
suffer serious consequences. 
I have discovered that certain substances, which are 
pharmaceutically-acceptable in phase I experiments at the dosage range 
listed below, have the desired ability to relax smooth vascular muscle. 
Preferred substances are certain of the lysolecithins and phospholipase 
A.sub.2. The lysolecithins that may be advantageously employed in the 
practice of the invention are combinations (esters) of fatty acids, such 
as palmitic, stearic, oleic, etc. with glycerol, phosphates plus, for 
example, choline. Most preferred are the stearoyl, palmitoyl, and oleoyl 
forms of L-.alpha.-lysophosphatidylcholine. The lysolecithin is used in a 
concentration of at least about 10.sup.-7 M and preferably about 10.sup.-5 
M. 
Phospholipase A.sub.2 catalyzes the hydrolysis of the ester bond in 
position 2 of glycerophospholipids to form a free fatty acid and 
lysophospholipid, which in turn may be reacylated by acyl-Co-A in the 
presence of an acyltransferase. Phospholipases split off one fatty acid 
from a lecithin, converting it into a lysolecithin. Phospholipase A.sub.2 
is used in a concentration ranging from at least about 3 units up to about 
5 units. 
The substances utilized in the invention possess a molecular structure 
comprising a hydrophobic portion within the molecule and a hydrophilic 
portion at least at one end of the molecule. 
Solutions of the lysolecithins in a suitable solvent, such as dimethyl 
sulfoxide, are administered to patients having the usual symptoms of 
angina pectoris or hypertension. Phospholipase A.sub.2 may be administered 
directly. 
While the concentration and total dosage in patients will vary, depending 
on the outcome of phase I and II experiments, it appears that the 
concentrations listed above will generally be pharmacologically-effective. 
A single dose of phospholipase A.sub.2 of about 3 units will generally 
produce the desired relaxation in animals in approximately 1 minute. If 
necessary, a second dose may be administered in approximately two to four 
hous. With regard to the lysolecithins, the use of the method of the 
invention does not appear to induce a tolerance in the body, and thus the 
same dose may be employed as needed, without loss of effectiveness. 
Without subscribing to any particular theory, it appears that the effective 
lysolecithins and phospholipase A.sub.2 stimulate cyclic-3', 
5'-guanosin-monophosphate for dilating blood vessels. Specifically, in the 
presence of the endothelial lining of coronary arteries or peripheral 
vasculature, the substances utilized in the practice of the invention 
promote relaxation, and thus dilation, of the associated muscle layer. In 
the body, phospholipase A.sub.2 also causes a marked decrease in 
resistance to blood flow (about 25 to 30%) in coronary arteries and, in 
some instances, an increase on coronary blood flow.

EXAMPLES 
Example 1 
A total of 20 rabbits were used; seven aortic strips were obtained from 
each animal for the study of the effect of lysolecithin and its 
inhibitors. The effect of lysolecithin was tested by bioassay of rabbit 
aortic strips suspended in oxygenated Krebs-Henseleit solution at a 
resting tension of 1.5 g. Male white New Zealand rabbits weighing 2.4 to 
3.1 kg were anesthetized with pentobarbital (30 mg/kg) and heparinized 
with 500 IU/kg I.V. Tracheostomy was performed and the animals were 
ventilated with a respirator (Bird Mark 10, Space Technology, Palm 
Springs, CA) to assure sufficient oxygen supply. Median sternotomy was 
performed and the thoracic aorta was removed and immersed in ice cold 
Krebs-Henseleit solution. After removal of adjacent superficial connective 
and adipose tissue, the aorta was cut in rings of about 3 mm in width. 
These rings were cut into transverse strips. Endothelium was removed by 
gently rubbing the intimal surface with moistened filter paper wrapped 
around a wooden stick. 
Strips were mounted in an organ chamber of 20 ml capacity with both ends 
fastened. One end was tied to the bottom of the chamber, while the other 
end was attached to an isometic pressure transducer (UL-20-Gr, Shinkoh, 
Minebea Company, Ltd., Tokyo, Japan). The chambers were carefully 
oxygenated with 95% O.sub.2 and 5% CO.sub.2 by slow bubbling to prevent 
foaming. Strips were allowed to equilibrate for 60 minutes, and basal 
tension of the strips was adjusted to 1.5 g. 
Tension development was induced by addition of histamine (10.sup.-5 M) to 
the organ chamber. After a steady state was reached, acetylcoline 
(10.sup.-6 M) was added. Lysolecithin was dissolved in dimethyl sulfoxide 
(DMSO) prepared by placing lysolecithin powder (5 mg) in a Watch glass, 
adding 100 .mu.l DMSO plus 0.5 ml Krebs-Henseleit gradually, and stirring 
continuously with a glass rod until all solid particles had disappeared. 
Lysolecithin (10.sup.-7, 10.sup.-6, and 10.sup.-5 M) in DMSO was then 
added to the muscle bath. Hemoglobin (10.sup.-6 and 10.sup.-5 M) or 
methylene blue (10.sup.-5 M) were added during lysolecithin induced 
relaxation, while indomethacin (10.sup.-5 M) and nordihydroguiaretic acid 
(3.times.10.sup.-5 M) were added prior to precontraction with histamine. 
Superoxide dismutase (150 U/ml) was administered after the addition of 
lysolecithin. 
Lysolecithin was prepared by phospholipase A.sub.2 from egg 
L-.alpha.-phosphatidylcholine, and contained primarily palmitic and 
stearic acids. 
Regarding the effect of lysolecithin on the precontracted unrubbed strip, 
10.sup.-6 M lysolecithin resulted in a slight decrease in tension, while 
10.sup.-5 M lysolecithin caused a marked decline in tension, comparable to 
the relaxation induced by 10.sup.-6 M acetylcholine. As compared to the 
relaxation following acetylcholine, the fall in tension with lysolecithin 
was more gradual. Both hemoglobin (10.sup.-6 and 10.sup.-5 M) and 
methylene blue (10.sup.-5 M) completely inhibited relaxation. Indomethacin 
(10.sup.-5 M) had no effect on relaxation induced by lysolecithin, while 
nordihydroguiaretic acid partially inhibited relaxation. Relaxation was 
slightly potentiated by superoxide dismutase. The addition of DMSO alone 
to the bath had no effect on tension. 
In the rubbed strips, the effect of lysolecithin was markedly reduced. The 
decline in tension was very slight and extended over several minutes. 
Acetylcholine had no effect on developed tension in the rubbed strip. 
Hemoglobin did not alter tension, while methylene blue caused a gradual 
increase. 
Example 2 
Rabbit aortic strips (4 mm wide), prepared as in Example 1, were subjected 
to 1.5 g tension, with histamine added to increase tension, as in Example 
1. Various lysolecithins were added, to compare the effect of the fatty 
acid moity. Relaxation of the lysolecithin was measured as a decline in 
tension. Hemoglobin (10.sup.-5 M) was added 20 minutes prior to histamine 
addition to block relaxation. 
The Table below provides a comparison of the effect of the various 
lysolecithins on relaxation. 
TABLE 
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Effect of Lysolecithin on Relaxation 
of Vascular Smooth Muscle 
Hemoglobin add'n, 
Compound % Relaxation 
% Relaxation 
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L-.alpha.-lysophosphatidyl 
stearoyl 51.0 92.5 
L-.alpha.-lysophosphatidyl 
oleoyl 58.8 104.9 
L-.alpha.-lysophosphatidyl 
palmitoyl 59.5 93.2 
L-.alpha.-lysophosphatidyl 
caproyl 88.6 96.5 
L-.alpha.-lysophosphatidyl 
myristoyl 100.0 112.2 
L-.alpha.-lysophosphatidyl 
decanoyl 95.0 108.1 
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In the Table, the addition of histamine is expressed as 100%. Accordingly, 
the lower the % relaxapalmitoyl lysolecithins are seen to be the preferred 
compounds. 
Thus, there has been provided a method for relaxing smooth vascular muscle. 
It will be appreciated by those skilled in the art that various changes 
and modifications of an obvious nature may be made without departing from 
the spirit and scope of the invention, as defined by the appended claims.