Arterial polysaccharide complex, a method for its preparation and composition comprising same

An arterial polysaccharide complex of anionic character consisting of hexosamine glycanmonosulphates (FAPA) derived from the aorta of young mammals and a process for its preparation.

This invention relates to a new polysaccharide complex produced by 
extraction from the aorta of young mammals, and its use in the treatment 
of arteriopathies and conditions associated therewith. 
BACKGROUND 
In recent years, numerous drugs have been proposed for aiding the return to 
normal concentrations of the various lipid components of the serum, in 
consideration of the particular seriousness of hyperlipidemias and 
dislipidemias in the etiopathogenesis of numerious illnesses, including 
illnesses of social interest. These drugs can be distinguished 
schematically in the following manner by their action mechanism: 
(1) those which inhibit the intestinal absorption of cholesterol; 
(2) those which act on the secretion of bile acids; 
(3) those which inhibit the synthesis of cholesterol and the mobility of 
the fatty acids of the adipose tissue; 
(4) those which activate fibronolysis and thrombolysis; and 
(5) those which favor increase in the removal of lipoproteins. 
Among these latter drugs, heparin and analogous substances generally known 
as heparinoids have assumed special importance in the treatment of 
hyperlipidemias and dislipidemias. 
Among the various pharmacological actions of heparin, one of particular 
importance is the so-called clarifying action which consists essentially 
of inducing the appearance in the bloodstream of lipoproteinlipase, a 
physiological enzyme which separates the ester bonds of triglycerides to 
transform them into monoglycerides and free fatty acids. This action is 
entirely independent of the anticoagulating action, which represents the 
negative side and determines the limit of use of heparin in this type of 
therapy. 
Again, within the field of antiarteriosclerotic therapy, it has been stated 
in published works (Comi and Coll., Boll, Chim. Farm. 106/5/309/21 (1967); 
French Pat. No. M 4,871filed Nov. 24, 1965) that by administering total 
aorta extracts to rabbits which have been made hypercholesterolemic by 
means of chloresterol-base diets, a strong reduction in atheromatosis 
formations have been obtained. On the other hand, it has been shown 
(Dall'Occhi and Coll. Adv. Exi. Med. Biol. 1967 1,468/83) that the total 
aorta extract leads to reactions of immunity with harmful effects on the 
pulmonary arteries. 
THE INVENTION 
We have now discovered a new and specific polysaccharide complex of anionic 
character which has demonstrated suprising antiarteriosclerotic properties 
in addition to a complete lack of side-effects. 
Also, this invention relates to a means for extracting said polysaccharide 
complex from the aorta of young mammals. 
This new complex, which for simplicity will be indicated hereinafter by the 
letters FAPA (Arterial Antiarteriosclerotic Polysaccharide Factor), is 
characterized unambiguously by the following chemical-physical properties: 
(a) On hydrolysis FAPA liberates hexosamine, uronic acids and sulphates in 
the molar ratios of approximately 1/1/1 with variable quantities of 
nucleosides. 
After acid hydrolysis of the FAPA, analysis shows the following average 
composition of the polysaccharide components. 
(1) Hexosamine (glucosamine): specific colorimetric reaction of the amine 
in hexoses with p-dimethylaminobenzaldehyde: 30.+-.2.25% 
(2) Uronic acid: specific colorimetric reaction with carbazole: 30.5.+-.% 
(3) Organic Sulphate (SO.sub.4): in accordance with FU 8th Ed. Vol. I, page 
106, paragraph B: 16.5.+-.3% 
(4) Sodium (NA): determined by atomic absorption (not routine): 10.+-.2% 
(5) Ash: 15.+-.3% 
(b) Electrophoretically, FAPA separates into three anionic bands 
distinguishable with Alcian Blau and Toluidine Blue. 
The following table shows the relative electrophoretic mobility of the 
three anionic bands. 
______________________________________ 
Average 
Densitometric 
Ratios of Positive 
Band Anodic Mobility Toluidine Blue Bands 
______________________________________ 
1 U: 2.2 .+-. 0.5 .multidot. 10.sup.-4 cm.sup.2 V.sup.-1 sec..sup.1 
14 .+-. 3% 
2 U: 2.09 .+-. 0.5 .multidot. 10.sup.-4 cm.sup.2 V.sup.-1 sec..sup.1 
9 .+-. 2% 
3 U: 1.93 .+-. 0.05 .multidot. 10.sup.-4 cm.sup.2 V.sup.-1 sec..sup.1 
77 .+-. 8% 
______________________________________ 
(c) FAPA reacts with alkaline and alkaline earth metals to form salts which 
are very soluble in water, whereas the salts which it forms with the 
aliphatic ammonium ion are insoluble. 
(d) The sodium salt is 2% soluble in 50% ethyl alcohol, whereas, the acid 
form is 2% soluble in 70% ethyl alcohol. 
(e) FAPA reacts with Toluidine Blue to give a minimum metachromatic 
reaction (approximately 1/10 of that given by heparin). 
(f) Spectrophotometrically, it shows a maximum of 260.+-.1 nm. 
(g) FAPA shows a rotatory power on polarised light of -11.degree. to 
-22.degree.. 
(h) The infrared spectrum of the substance determined in KBr, to which the 
graph of FIG. 1 refers, shows the following functions: 
EQU --COOH 
EQU --NHR 
EQU --COCH.sub.3 
EQU --OSO.sub.3 H 
EQU --CH.sub.2 OH 
From the total analytical data it can be established that FAPA has the 
following structure: 
##STR1## 
wherein R.sub.1 and R.sub.2 are --H or SO.sub.3 H, R.sub.3 is --SO.sub.3 
H or --CO--CH.sub.3, and m, n, p are whole numbers which can vary over a 
wide range and define a molecular weight of 10,000 to 30,000 daltons for 
the polysaccharide chains. 
These characteristics, and in particular the molecular ratios of the 
components obtained after hydrolysis, indicate that FAPA may be defined 
chemically as a molecular complex consisting of 
hexosaminglycanmonosulphates (chondroitin sulphates B and C and heparitin 
sulphates of the arterial wall). 
The process for producing FAPA according to the present invention is 
characterized essentially by the following operations: 
(1) removing and immediately grinding the aortas of young mammals which 
have just been slaughtered; 
(2) homogenising and suspending said tissues in water or suitable saline 
solutions; 
(3) attack by proteolytic enzymes such as trypsin, chymotrypsin, 
pancreatin, papain, ficin or bromelin, under the respective optimum pH, 
temperature and time conditions; 
(4) separating the digested liquid by filtration and collecting same; 
(5) precipitating FAPA from the digested liquid by solvents miscible with 
water and/or by forming insoluble salts with the aliphatic ammonium ion; 
and 
(6) purifying by salification with alkaline or alkaline earth metal bases, 
decolouration with permanganate, treatment with alkali at 70.degree. C. 
for eliminating the terminal parts of the residual peptide chains, 
separation of the desired salt form with solvents (sodium, potassium, 
calcium salts, etc.), filtration, sterilization and lyophilisation. 
The pharmacological properties of FAPA have been examined in comparison 
with certain antiarteriosclerotic preparations of the aforesaid type; in 
particular it has been examined in comparison with heparin, the clarifying 
drug for amutonomasia, and with the total aorta extract. 
The following basic characteristics were determined by in vivo 
experimentation: 
(1) Action of experimental arteriosclerosis 
This study was carried out on rabbits divided into batches of five animales 
each fed with heterogenous diet rich in cholesterol. 
The blood of the animals was tested for determining the esterified fatty 
acids (EFA), the total cholesterol, the betalipoproteins and the 
coagulation time. The cholesterol content of the aortic tissue was 
determined, together with the incidence of atheromas. Table I summarises 
the results obtained using FAPA, heparin and total aorta extract, after 40 
days of treatment. 
TABLE I 
__________________________________________________________________________ 
EFA Cholesterol 
B-lipoproteins 
coag. time 
aort. cholesterol 
mg/100 ml of 
mg/100 ml of 
mg/100 ml of 
log. of 
mg/100 g of dry 
serum serum serum minutes 
substance 
atheromas 
__________________________________________________________________________ 
controls 456 .+-. 14 
215 .+-. 19 
78 .+-. 4 
0.78 .+-. 0.06 
2.85 .+-. 0.12 
++++ 
FAPA 1 mg/kg 
180 .+-. 5 
90 .+-. 15 
40 .+-. 2.5 
0.81 .+-. 0.04 
1.98 .+-. 0.08 
++ 
FAPA 10 mg/kg 
162 .+-. 8 
76 .+-. 13 
35 .+-. 13 
0.9 .+-. 0.04 
1.7 .+-. 0.04 
+ 
Heparin 1 mg/kg 
171 .+-. 11 
78 .+-. 13 
38 .+-. 1 
1.9 .+-. 0.4 
2 .+-. 0.05 
+ 
Heparin 10 mg/kg 
138 .+-. 8 
76 .+-. 15 
30 .+-. 2 
2.3 .+-. 0.8 
1.8 .+-. 0.03 
+ 
Total aorta 
430 .+-. 14 
190 .+-. 14 
79 .+-. 3 
0.75 .+-. 0.1 
2.9 .+-. 0.2 
++++ 
extract 1 mg/kg 
Total aorta 
218 .+-. 4 
97 .+-. 1 
36 .+-. 4 
0.78 .+-. 0.1 
1.6 .+-. 0.04 
++ 
extract 10 mg/kg 
__________________________________________________________________________ 
4+ = marked occurrence of atheromas in terms of number and extension 
2++ = about 50% of occurrence in controls 
1+ = occurrence approximately equal to that in normal rabbits 
(2) Biological activity 
The aspects of biological activity determined were the anticoagulating 
activity (in accordance with USP) and the clarification activity on rats 
(Morton Grosman Method, J. Lab. and Clin. Med., March 1954, page 445). 
With regard to this latter determination, modifications were introduced in 
the present case to enable the effect to be expressed in international 
lipasemic units (ULI), in accordance with the UIB definition 
(International Biochemical Union), where one international unit (ULI) is 
equal to the activity which transforms the substrate at a speed of 1 
micromole per minute. 
The results of the biological activity aspects of FAPA are shown in Table 
II, in comparison with heparin and aorta extract. 
TABLE II 
______________________________________ 
Heparin Aorta Extract 
FAPA 
______________________________________ 
Anticoagulating 
135-148 About 0.5 5-15 
Activity (USP/mg) 
Clarification activity 
480 .+-. 100 
15 .+-. 7 270 .+-. 80 
(UL/l of plasma) at 
a dose of 0.5 mg/kg 
______________________________________ 
(3) Product Tolerance 
(a) Toxicity: FAPA proved to be well tolerated by rats, rabbits and guinea 
pigs treated either by parenteral administration or by oral administration 
with per diem doses of 200 mg/kg. 
(b) Immunological tests: In guinea pigs treated with FAPA, immunological 
tests on antibody and anaphylaxis production gave no antibody or 
anaphylactogenic response. 
(c) Vasokinetic investigations: no effect, either of histamine or 
adrenaline type, was noted even with doses of 50 mg/kg. 
All of the aforesaid tests show that FAPA can be chosen as a drug for 
antiarteriosclerotic use as its effectiveness is of the order to magnitude 
of heparin, but without giving rise to the risks of haemorrhage typical of 
this latter substance. 
Furthermore, the biological activity of FAPA is approximately ten times 
greater than that of total aorta extract, but without the immunoreactive 
side-effects, and thus with considerable advantage with regard to posology 
and the risk of sensitisations during therapy.

Some practical examples of preparation of the new arterial polysaccharide 
complex according to the invention are given hereinafter, in order to 
enable the process to be easily reproduced, but without in any way 
limiting it. 
EXAMPLE 1 
Calf aorta (50 kg.) were finely ground and homogenised in water (66 l.) 
After slowly heating the mass to 50.degree. C., papain (0.6 kg.) were 
added, previously suspended in water (9 l.) containing sodium 
metabisulphite (10 g.). 
The entire mass was heated to 65.degree. C. and kept under these conditions 
for three hours, taking care that the pH did not descend below 6 by 
suitably adding alkali. After this time had passed, the mass was heated to 
95.degree. C. for 15 minutes. 
After standing for 30 minutes, the mass was filtered through cloth with a 
precoat of filter earth. 
Cetyltrimethylammonium chloride (0.6 kg.) dissolved in water (35 l.) was 
added to the filtrate. 
After leaving overnight, the overlying liquid was syphoned off. The 
precipitate was collected by filtration and dissolved in water (25 l.) 
containing sodium chloride (3 kg.) by heating to 70.degree. C. This 
solution was clarified by filtration. The filtrate was treated with an 
equal volume of acetone. The precipitate, namely the crude sodium salt of 
FAPA, was washed several times with 70% acetone and then dried. Crude 
sodium salt of FAPA (85 g.) was obtained in this manner. 
This precipitate was washed in water (1500 ml), the pH of the mixture was 
adjusted to 8 and it was then treated with potassium permanganate (1.5 g.) 
and gradually heated to 90.degree. C. This mixture was filtered to 
eliminate the precipitate formed and the pH of the filtrate was adjusted 
to 11 with sodium hydrate and kept at 70.degree. C. for 10 hours. 
After 10 hours, the liquid was cooled, its pH adjusted to 7 with 
hydrochloric acid, and filtered through a 0.8 micron membrane. The liquid 
obtained was treated with 2 volumes of ethyl alcohol. 
After 2 hours of standing, the precipitate formed was collected by 
filtration and washed with 75% alcohol (500 ml). The precipitate was 
dissolved in water (800 ml) and concentrated until the residual alcohol 
was eliminated. The concentrate was filtered through a 0.2 micron 
membrane, and the clear filtrate was lyophilised. 
In this manner 42 g of the sodium salt of FAPA was obtained, having the 
following characteristics: 
uronic acid: 35%, 
hexosamine: 30.5%, 
--SO.sub.3 Na: 18.1%, 
--nucleosides: 5.2%, 
inorganic salts: 8% (mainly chlorides), 
clarification activity: 210.+-.20 ULI/l, 
anticoagulation activity: 6 UL/mg 
EXAMPLE 2 
Pig aorta (50 kg.) was processed as in Example 1. Sodium salt of FAPA (36 
g) was obtained with chemical-physical characteristics closely analogous 
to those given Example 1, including a clarification activity of 
180.+-.ULI/l. 
EXAMPLE 3 
Calf aorta (50 kg.) was lysised with ficin instead of papain, and the 
procedure of Example 1 was then followed. There was thus obtained 35 g of 
the sodium salt of FAPA with chemical-physical characteristics closely 
analogous to those given in Example 1, including a clarification activity 
of 280+40 ULI/l. 
EXAMPLE 4 
Calf aorta (50 kg.) was homogenized in water (150.1) and lysised with 
pancreatin (2 kg.) at pH 8 and 40.degree. C. for 24 hours, in the presence 
of toluene as the bacteriostatic. 
After filtering, the clear liquid was treated with 3 volumes of acetone. 
The precipitate obtained was hydrolysed with alkali at pH 11 and 
70.degree. C. for 15 hours. After filtering, the liquid was treated with 
cetylpiridinochloride (400 g) to afford the corresponding insoluble 
quaternary ammonium salt of FAPA. The precipitate was then dissolved in 
water (1500 ml.) containing calcium chloride (90 g). After filtering, the 
liquid was treated at pH 8 with potassium permanganate (1 g), heated to 
90.degree. C. and filtered. The filtrate was dialysed for 15 hours against 
a stream of water to eliminate the excess of salts, and then filtered 
through a 0.45 micron membrane and lyophilized. In this manner 35.5 of the 
calcium salt of FAPA was obtained, with chemical-physical characteristics 
closely analogous to those of the product of Example 1, including a 
clarification induction activity of 315.+-.30 ULI/l.