United States Patent: 4,049,824


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United States Patent 4,049,824
Diehl September 20, 1977
 
Cetyl myristoleate
 
Abstract
A method is described for immunizing against inflammatory rheumatoid arthritis
in mammals and for immunizing against and relieving at least one of the symptoms
of inflammatory rheumatoid arthritis in mammals.
 
Inventors: Diehl; Harry Weldon (4424 Oak Hill Road, Rockville, MD 20853)
Appl. No.: 682540
Filed: May 3, 1976
Current U.S. Class:514/552; 514/825; 554/223
Intern'l Class: A61K 031/23; C11C 003/02
Field of Search: 424/312 260/410.9 R
 
 
 
References Cited [Referenced By]
 
 
U.S. Patent Documents
3194822Jul., 1965Neiswerder260/410.
3427344Feb., 1969Koshigoe et al.260/410.
 
 
Other References
Chem. Abst. 70 - 69470r (1969).
Primary Examiner: Friedman; Stanley J.
Attorney, Agent or Firm: Smith, Jr.; John C.
 
 
Claims
 

I claim:
1. A method of relieving and inhibiting the symptoms of inflammatory rheumatoid
arthritis in mammals which comprises the parenteral administration to a mammal
of an effective amount of cetyl myristoleate.

2. A method according to claim 1 wherein said cetyl myristoleate is extracted
from the tissues of mice.

3. A method according to claim 1 wherein said cetyl myristoleate is administered
with a compatible pharmaceutical carrier.

4. A method according to claim 3 wherein said compatible pharmaceutical carrier
is mineral oil.

5. A method according to claim 1 wherein about 0.05-0.75 gm of cetyl
myristoleate is administered for each 140-200 gms weight of the animal.

6. A method for preparing a concentrate containing cetyl myristoleate useful for
relieving and inhibiting the symptoms of inflammatory rheumatoid arthritis in
mammals comprising macerating the tissues of mice to form a macerated material,
extracting said macerated material with a solvent to form an extract and
concentrating said extract to form said concentrate containing cetyl
myristoleate.

7. A method according to claim 6 wherein said macerated material is extracted
with methylene chloride.

8. A method according to claim 6 comprising the further step of diluting said
extract with a compatible pharmaceutical carrier.

9. A method according to claim 8 wherein said compatible pharmaceutical carrier
is mineral oil.

10. The compound, cetyl myristoleate, having the structure
CH.sub.3 (CH.sub.2).sub.15 OCO(CH.sub.2).sub.7 CH.dbd.CH(CH.sub.2).sub.3
CH.sub.3.
 

Description
The present invention relates to a method for immunizing against inflammatory
rheumatoid arthritis in mammals and for immunizing against and relieving at
least one of the symptoms of inflammatory rheumatoid arthritis in mammals by
administering parenterally an immunologically or therapeutically effective
amount of cetyl myristoleate

CH.sub.3 (CH.sub.2).sub.15 OCO(CH.sub.2).sub.7 CH.dbd.CH(CH.sub.2).sub.3
CH.sub.3,

to a method of preparing the pharmacologically active cetyl myristoleate by
extracting the compound from the tissues of mice and to the compound itself.
Osteoarthritis is one of the oldest and most common inflammatory diseases in
mammals. It occurs at all ages. Studies show that 97% of all persons over age 60
have an arthritic condition which can be observed by X-ray. The most common
symptoms of arthritis are pain, fever and inflammation, and it is the No. 1
crippling disease in man.

An object of the present invention is to provide a method for immunizing against
inflammatory rheumatoid arthritis in mammals and to immunize against and relieve
the symptoms of inflammatory rheumatoid arthritis in mammals.

Another object of the invention is to inhibit the symptoms, such as pain, fever
and inflammation associated with inflammatory rheumatoid arthritis in mammals by
administering parenterally cetyl myristoleate extracted from the tissues of
mice.

A still further object of the present invention is a method for preparing a
compound useful in immunizing against inflammatory rheumatoid arthritis and in
inhibiting the appearance of the symptoms thereof by extraction from the tissues
of mice.

These and other objects will become apparent in the following detailed
description of the invention.

It is well known that Freund's adjuvant will induce poly-arthritis in rats but
not in mice. It has been common practice to test various compounds and
compositions in laboratories to determine their effectiveness in relieving the
symptoms of inflammatory rheumatoid arthritis by administering test compounds or
compositions to rats having poly-arthritis induced previously by administering
Freund's adjuvant. It was hypothesized that mice must contain some protective
factor or mechanism which prevented the inducement of poly-arthritis in mice.
In accordance with the present invention a substance has been isolated from mice
which, when administered to rats, essentially prevents the formation of
poly-arthritis and the resultant symptoms when the rats are subsequently
injected with Freund's adjuvant. The substance was isolated by extracting
homogenized whole mice with methylene chloride. Upon purification of this
extracted substance it was identified as cetyl myristoleate. The following
example describes in greater detail the isolation of the effective substance
from mice.

EXAMPLE I
Seventy-nine mice totalling 2300 grams were macerated in an electric blender in
batches of about eight mice, each batch being macerated in 400 ml methylene
chloride. The final blend was poured into a 4 liter beaker. The blend was
stirred until the methylene chloride separated, then the mixture was filtered
under light suction through a large Buchner funnel containing filter paper
covered by a thin layer of Filter-Cel. The resultant precipitate was washed with
two 100 ml portions of methylene chloride. The combined filtrate and washings
were separated from a top layer of water after which the methylene chloride was
filtered again. This filtrate was concentrated in vacuo to a thin syrup of 167
grams which was treated with four parts of acetone and then left at -5.degree.
C. for 3 days with brief stirring each day.

The mixture was filtered using Buchner filter paper with a layer of Filter-Cel
and light suction. The precipitate was washed with four 25 ml portions of
-5.degree. C. acetone. The combined filtrate and washings were concentrated in
vacuo to a thin syrup of 121 grams. This material was dissolved in 500 ml of
20:1 legroin (20.degree.-40.degree. C.), dry ether and chromatographed on 2500
ml 40-325 mesh ASTM silica gel in an appropriate column using the 20:1
legroin-ether mixture as eluent. One 1500 ml "blank" and then eleven 100 ml and
six 200 ml fractions were collected.

Fraction Nos. 3-15 inclusive were combined and filtered in vacuo through 120
grams of Darco-X. The filtrate was concentrated in vacuo to a syrup of 0.15
gram. The Darco-X was further washed with several portions of methylene
chloride. The 0.15 gram of syrup was dissolved in these washings which were
concentrated in vacuo to a syrup of 0.8 gram of crude material. This was
chromatographed using 350 ml of silica gel, 70-325 mesh ASTM using 40:1 carbon
tetrachloride-ether as eluent.

Fraction Nos. 67-79 of 7 ml each gave 0.4 gram of material which was
rechromatographed with 125 ml of silica gel using 60:1 carbon
tetrachloride-ether in 2.5 ml fractions.

Fraction Nos. 117-127 gave 0.15 gram of purified syrup which proved to be
principally the desired "immunity factor", cetyl myristoleate
(v.sub.max.sup.neat 1782 cm.sup.-1). Extensive thin-layer chromatography and
frequent bioassays were used throughout to monitor the isolation and
purification of the compound.

The following example describes the steps taken to identify the cetyl
myristoleate product of Example I.

EXAMPLE II
A mixture of 0.15 gram of the material obtained in Example I was heated with 2
ml of acetone and 3 ml of 10% sodium hydroxide solution under reflux with
stirring for fourteen hours and then treated with 0.7 ml of 12 M HCl.

Extractions were made with four 5 ml portions of methylene chloride followed by
drying with Na.sub.2 SO.sub.4 and then evaporated with methylene chloride in
vacuo. The 0.14 gram of material recovered was chromatographed on 140 ml of
silica gel with methylene chloride as eluent. A 400 ml and one hundred ten 6 ml
fractions were collected.

Fraction Nos. 20-55 gave 40 mg of cetyl alcohol having a melting point of
49.degree.-50.degree. C. after recrystallization from ethanol. The analysis
calculated for C.sub.16 H.sub.34 O is

C -- 79.3%

h -- 14.1%

the analysis found was

C -- 79.2%

h -- 14.3%

fraction Nos. 89-110 produced 60 mg of a syrup which was further purified by
chromotography. The resultant acid gave (v.sub.max.sup.neat 1712, 1722 (sh
cm.sup.-1). The analysis calculated for C.sub.14 H.sub.26 O.sub.2 is

C -- 74.4%

h -- 11.6%

neut. equiv. -- 226.3

What was found is as follows:

C -- 74.1%

h -- 11.9%

neut. equiv. -- 225.5

Both the cetyl myristoleate extracted from the tissues of mice as described in
Example I and cetyl myristoleate prepared synthetically as described in Example
III have been found to be effective in immunizing against rheumatoid arthritis
and the symptoms thereof.

EXAMPLE III
A charge of 150 mg of cetyl alcohol, 150 mg of myristoleic acid, 50 mg of
p-toluenesulfonic acid monohydrate of 20 ml of benzene were heated together
under reflux conditions for four hours and then washed with a -10% sodium
hydroxide solution. The benzene layer was recovered, dried and evaporated in
vacuo.

This procedure produced 300 mg of a mobile oil which was identified as cetyl
myristoleate (v.sub.max.sup.neat 1782 cm.sup.-1) (nuclear-magnetic-resonance and
infrared spectroscopy).

EXAMPLE IV
Following the procedure set out in Example II, 150 mg of cetyl alcohol obtained
in Example III and 150 mg of the acid obtained in Example II produced 300 mg of
an ester (v.sub.max.sup.neat 1782 cm.sup.-1) which was identified by similar
means with the products in Examples I and III.

EXAMPLE V
The cetyl myristoleate or "immunity factor" produced both synthetically and by
isolation from whole mice, respectively, were administered parenterally in
mineral oil as a compatible carrier to male rats (Sprague Dawley Strain) ranging
in weight between 140 and 200 grams. It is not necessary, however, that a
carrier be used since cetyl myristoleate is itself an oil.

One set of rats was innoculated subcutaneously with 1.0 ml each of a mixture of
mineral oil and 0.05 gm of the immunity factor. Twenty-four hours later the rats
were innoculated with Bacto m. Butyricum (Disco 0640-33). A control group
received only the Butyricum.

Another set of rats were given 1.0 ml each of a mixture of mineral oil
containing 0.075 gm of the synthetically produced cetyl myristoleate. Two days
later the rats were innoculated with Bacto m. Butyricum (Disco 0640-33). Another
control group received only the Butyricum.

The rats in both control groups developed severe poly-arthritis during the
following 10 to 18 day period which persisted through 32 days. All of these rats
gradually lost weight.

About 70% of the first group (those treated with "immunity factor" plus
Butyricum) were completely protected from the poly-arthritis. They showed no
swelling or other symptoms. The other 30% were partially protected during the
32-day period.

All of the second group (those treated with synthetically produced cetyl
myristoleate plus Butyricum) were protected from the poly-arthritis and showed a
steady gain in weight.

It was found that the purer the cetyl myristoleate the more dramatic were the
results in protecting the rats from poly-arthritis. Also, an effective dosage of
cetyl myristoleate or "immunity factor" preferably ranges between 0.05 and 0.75
gm for each 140-200 gms weight of the animal. However, doses smaller than and
greater than this range will effectively immunize mammals against inflammatory
rheumatoid arthritis and immunize against and relieve the symptoms thereof such
as pain, fever and inflammation.

Thus havig described the invention in detail, it will be understood by those
skilled in the art that certain modifications and variations may be made without
departing from the spirit and scope of the invention as described herein and
defined in the appended claims.
 
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