DE2037942A1 - Medicines used to prevent atheroclerosis and the recurrence of cardiocascular seizures in atherosclerotic mammals - Google Patents

Description

HESTER M. MOERISON 7012 LaPresa Drive, Hollywood, California, V.St.v.A.

Medicines to prevent atherosclerosis and the recurrence of cardiocascular seizures in atherosclerotic vision Mammals

The invention relates to a medicament for prevention the occurrence of atherosclerotic lesions in fieren of mammal species, including humans, by promoting the formation of the collateral circulation in heart regions, which is supplied by the branches of the coronary arteries as well as to prevent the occurrence of heart attacks, including myocardial infarction, acute coronary insufficiency and acute myocardial ischemia in people with ischemic or coronary heart failure.

Chondroitin sulfates A and C are widespread in human and animal tissues, especially in cartilage and connective tissue. Although conjugated to protein or peptide residues, the pure, acidic mucopolysaccharides can be isolated without significant degradation. Chondroitin sulfates A and "C both contain D-glucuronic acid _r 2-amino-2-deoxy-D-galetose and acetyl and sulfate residues in eq.uiniolaren

109846/1812

Amounts. Structural studies have shown that chondroitin sulfate A and C differ only in the position of the sulfate ester group in the hexosamine residue. Chondroitin sulfate A contains the repeating unit (1 - * - 4) »0-ß ~ D-glucopyranosyluronic acid- (1 ^ 3) -2-acetamido-2-I) eEoxy-" 4-O-Sulpho-ß-B-galactopyranose, chondroitin sulfate C die repeating unit (1 - * - 4-) ~ 0-ß-D-glucopyranosyluronic acid- (l - * - 3) ~ 2- ~ acetaraido-2-oesoxy-6-0-sulpho-J3 ~ D-galactopyra ~ nose. It has now been shown that purified preparations of chondroitin sulfate A of apparently similar, chemical Composition ,, assessed according to the usual analytical methods, differ significantly in their pharmacological and physiological Be able to differentiate between activity. Similar findings were made with purified preparations of chondroitin sulfate C. obtain.

The invention is based on the finding that chondroitin sulfate A and chondroitin sulfate C (hereinafter abbreviated to CSA and CSC) can be obtained in a form which is "active" in the prevention of atherosclerosis and the recurrence of cardiovascular attacks in atherosclerotic Mammals when administered regularly over an extended period. This "active" material must be derived from the in of U.S. Patent No. 3,405,120 be distinguished, which has been found to be inactive, the Plasnia il'hrombus formation time 6 to 12 hours after administration to be extended in rabbits according to the Chandler-Cien method, and that is inactive in preventing atherosclerosis in rats fed the atherogenic diet as described below. So far was from the relevant publications such as the US patent 1 950 100 a difference in the anti-atherosclerotic Activity * of various preparations of CSA and CSC with obviously similar chemical composition not known.

10984B / 1812

The invention relates to a medicament which the Development of atherosclerotic lesions in mammals ver * prevents the formation of the collateral circulation in heart regions that are supplied by branches of the coronary arteries, promotes and the occurrence of heart attacks such as myocardial infarction, acute coronary insufficiency and acute myocardial ischemia in people with ischemic or coronary heart failure prevented.

The medicament of the present invention for preventing atherosclerotic lesions from occurring by promoting education of the collateral circulation in heart regions that are supplied by the branches of the coronary arteries, and for prevention the occurrence of heart attacks, including myocardial infarction, acute coronary insufficiency and acute myocardial ischemia in people with ischemic heart failure is characterized by containing an effective amount of "active" chondroitin sulfate A, "active" chondroitin sulfate C, or mixtures thereof, said activity being due to the prolongation of the plasma thrombus formation time 6 to 12 hours after application found in rabbits by the Chandler-Ösen method.

Treatment with the CSA and CSC according to the invention containing Medicinal product consists of the oral application of the compound in doses of 0.5 g to 10 g per day, whereby normally one third of the daily dose before or after each meal is taken. The treatment time ranges from a short period (about 3 months) to lifelong treatment of the patient. Occasional interruptions in this application program are irrelevant, provided that it is carried out regularly as a whole. The skilled person will immediately recognize the difference recognize between an essentially, regular application over a longer period of time and the error-prone, occasional one or short-term application.

• - 4 -1 0 9 8 4 S / 1 8 1 2

It has been found that there are significant differences in the plasma thrombus formation time 6 to 12 hours after intra- ■ ·. venous application of different batches of chondroitin sulfate A gives, tots the fact that they are based on the following Standard tests are indistinguishable.

test

nitrogen
Schveiel
Optical rotation

pH of 1- $ -
Solution

Combustion residue

Hexosamine
Hexuronic acid

Specific
Viscosity 1%

Parbe

(20 # solution)

Pyrogen

sterility

Heavy metals

method

Kjeldahl Schoninger

U.S.P. XYII, page 911

common ·.

U.S.P. XVII, page 885

Elson-Morgan +

Dische (Carbazole) +

Oswald

own procedure ++

U.S.P. XVII, page 863

U.S.P. XVII, page 829

U.S.P. XVII, page 877

Range 2.5 to 3.5%

5 to 6 fo

(a) £ ⁴ = -23 ° to -25 ° pH 6 to pH less than 3 %

32 to 35 % 36 to 39 y>

10 cps

less than 0.2 optical density unit

pyrogen-free sterile · less than 20 ppm

+ Methods of Enzymology . Volume III, edited by Sidney P. Colcwick and Nathan 0. Kaplan, Academic Press, New York, 1957, pp. 93-101.

-H- The optical density is measured at 420 μ in the Beckmann Jr. Model B spectrophotometer certainly.

1098 ^ 6/1812

None of the tests described differentiate between biologically and physiologically "active" CSA and biologically and physiologically, "active" CSC of inactive material. The biological and physiological activity of CSA and CSC can be determined as follows will. In determining the. biological and physiological activity by prolonging the plasma thrombus formation time according to the Chandler-Ösen method in the modification of Morrison et al CJ · Atheroscler. Res., 8: 319 »1968), preparations of purified chondroitin sulfate resulted A, which were comparable according to the above test series, the following average plasma throrabus formation times in rabbits, 12 hours after injection:

Sample tested Average plasma

Ihrorabus-educational ζ ince 12 hours after the injection Physiological saline solution 12.2 minutes CSA, Lot A-6 49.6 CSA, Lot A-10 46.4 CSA, Lot B-3 14.1 CSA, Lot B-7 16.7 CSA, Lot B-9 31.6 CSA, Lot C-1 52.1 CSA, Lot C-2 13.6 CSA, Lot C-4 18.1 CSA, Lot W-2101 15.2 CSA, batch ¥ -164 22.8

The tests were carried out on white male Ueu-Zealand rabbits weighing from 4 1/2 to 5 1/2 pounds. The plasma thrombus formation time was measured in each animal before Injection and 12 hours after injection. The chondroitin sulfate A was in physiological saline solution dissolved uiid diluted to a concentration of 80 mg / ml. the physiological saline solution controls and the chondro-

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itine sulfate solutions were the. Injected into the ear vein of rabbits at a dose of 1 ml / kg body weight. At least 6 rabbits were used for each group. Injections'. of physiological saline solution did not cause any significant change in the plasma thrombus formation time when blood samples were taken 12 hours after the injection compared to the value "before the injection; Preparations lead to a clearly significant "brightening"; of the plasma thrombus formation time "12 hours after. The" τα $ ection ";; Γ

Activity: showed in this regard * V-It is; from 'I that heparin, soft extremely active, in which, elongation, younger ^ aea? · Ϊ Plasma-Thrombus-Bildurigszeii;: toei blood samples is _a l die; 15 minutes after intravenous injection; no prolongation of the plasma thrombus formation time in the blood pool taken 6 to 12 hours after injection. On the other hand, it was found that samples of chondroitin sulfate lead to a noticeable prolongation of the plasma thrombus - In blood samples taken 12 hours after the injection, there was no "significant effect;

before injection showed when blood samples were taken 15 minutes after, l ::: - ^/ ; taken from the injection. '■ ^: ■. \ - v. ^f "^; r ^v

It has also been shown that in the case of Chdndroitinsul - /]} - ^

"""--.- ■. ■■■" ■ · ■■■ ·.-.-'- / '- ■ - .: fats C purified preparations of this material made on the Iiv' basis of the above tests similarly, they differed significantly in the activity that led to the. Lengthening j of the plasma thrombus formation time in rabbits results when I - ^: the blood samples are taken 12 hours after the injection.

In general, preparations of purified chondroitin sulfate A, purified chondroitin sulfate C or combinations of the two "active" in inhibiting or preventing the occurrence of ' Atherosclerosis, if it leads to an increase in plasma thrombus

109 846/1812

mm I ^ m

COPY ORlGINAkINSPECTED,

.- 7 - ■

bus formation time in rabbits 12 hours after injection under the conditions given above by at least 80 % compared to values in controls injected with physiological saline solution. It is not claimed that the activity of such preparations which prolongs the riasma-chromium formation time as a result of intravenous application, with the anti-atherogenic activity as a pole of the oral application. tion is correlated, JThe thrombus-prolonging test is only \ T- 'used as a search test for the detection of preparations with anti-athero gener · "X' Ή _έ activity. - · • ready by gereinigtem.Chondroitinsulfat A, _C; or E very combination J station from these anti-atherogenic activity-under den.im, ^ _-:;. -Igenden shows "above conditions, even · active in the encryption * ~ ■ - 'the plasma thrombus Bildungezeit _fa.ngerung in rabbits 1.2> ^Γ ί Α \ hours after injection, are, with values -the at least $ 80 higher than the injected with saline controls. There are also findings that preparations, · ^;; ^ which are particularly active "in the prolongation of the plasma thrombus" formation time in rabbits under the conditions listed above. ,:, conditions are equally active in anti-atherogenic activi- / ;; jbäiiäsiiid *; In contrast, were preparations; of purified chondroitin sulfate. A and / or C with little or no anti-atherogenic activity was never able to measure the plasma thrombus formation time in rabbits in comparison with controls * which were injected with physiological saline solution under the conditions mentioned above to lengthen significantly »-, ·." ■ -

Differences in the activity of different preparations from purified ones Chondroitin sulfate A and C can result from differences in the manufacturing process as well as in the starting material. It was observed that samples of purified chondroitin sulfate A from the same starting material significantly im With regard to their effect on the plasma thrombus formation time 12 hours after injection differed, depending on the manufacturing process. It has also been observed that manufacture ;:

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iAD ORIGINARY \ ~: COPY.]> vV

process "which the one output material au highly active material, according to the test listed above., _(process, have led, led with another starting material to inert material, despite the fact that differences between the various preparations · chemical above Tests «could not be proven.

An experimental model for the induction of atherosclerosis in the aorta and coronary arteries was developed by Hatten. It consists in giving young rats a purified diet containing alcohols, supplemented with 1.25 million U, SP units of vitamin D ₂ (viosterol) per kg / body weight, for 6 weeks. Rats fed an appropriate diet in which cholesterol was _{omitted on the one hand and vitamin D 2 on} the other, showed no such lesions. The distribution and microscopic appearance of such lesions are very similar to those observed in humans with atherosclerotic lesions in these tissues. It has been shown that the oral application of purified preparations of. Chondroitin Sulphate A and C and their combinations which are active in prolonging the plasma thrombus formation time in rabbits _} as indicated above, highly active in reducing or preventing the occurrence of atherosclerotic lesions as a result of the atherogenic ones. Diet mentioned above are.

The following is a description of the experimental conditions for the production of atherosclerosis and the results after application of a biological and physiological called "active" preparation of purified chondrotin sulfate A.

The basic diet was a highly purified mixture of sucrose ($ 61), vitamin-free test casein (24 ^c / °) > cottonseed oil ( $ 10>) , Hubbell, Mendel and Wakeman salt mixture ($ 5)>

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to which the following vitamins were added (amounts per kg / diet): thiamine hydrochloride (10 mg), riboflavin (10 mg), pyridoxine- ■>, hydrochloride (10 mg), galcium panthothenate (60 mg), nicotinic acid (100 mg), ascorbic acid ( 200 mg), biotin (1 mg), folic acid (10 mg), 'p-aminobenzoic acid (200 mg), inositol (400 mg), vitamin B ₁₂ (150 microgr.), 2-methyl ~ 1,4-naphthocmon ( 5 mg), choline chloride (2 g), vitamin A (5000 USP units), vitamin D ₂ (500 USP units) and co-tocopherol acetate (100 mg). The vitamins were added in place of an equal amount of sucrose. 54 male rats of the Long-Evans strain, averaging 145 g body weight - range from 136 to

155 S ~ ^U11 d 54 female rats of the Long-Evans stamine., With an average body weight of 146 g - range from 138 to

156 g - was selected for the following experiment:

The animals were divided into 5 groups of comparable weight * Groups I, II and III consisted of 6 animals of both sexes; Groups IV and V consisted of 18 animals of both sexes. Group I was fed the basic diet mentioned above. Group II was fed the basic diet supplemented with 1 % Purified Chondroitin Sulphate A (Lot 9008). These chondroitin sulfate preparations resulted in a plasma thrombus formation time of 48.8 minutes, 12 hours after the injection, in rabbits, compared to an average thrombus formation time of 13.2 minutes in controls that were injected with physiological saline solution (test conditions as above described). Group III was fed the basic diet to which 1.5 % cholesterol and 0.5% cholic acid were added. Group IV was fed the basic diet supplemented with 1.5 % cholesterol, $ 0.5 cholic acid and vitamin D ₂ (1.25 million USP units kg / diet). Group V was fed the same diet as Group IV with the addition of 1 % purified chondroitin sulfate A (Lot 9008). The supplementary components were added to the basic diet instead of a corresponding amount of sucrose. The animals were placed in metal cages

1098 U / 1812

-10-

2O37Ö42

- ίο -

kept with a raised sieve bottom (3 rats in a cage) and received the various diets ^ d ^ J ^ ibJ ^ duni. The ¹ Iiere were fed daily; the food not consumed after 24 hours was' thrown away. The rats were weighed weekly during the experiment.

After 6 weeks of feeding, the rats were anesthetized with sodium pentobarbital, and blood was drawn from the heart into a heparinized syringe. The livers were removed, swabbed off to decix any excess blood, weighed, and stored in a refrigerator until analysis. Fat was extracted from the livers by the Thompson et al (Brit. J. Nutrition, 3: 50-1949) method; N.ieft and Deuel (J. Biol. Chem., 177: 143, 1949). For necropey, the heart and aorta were fixed with 10% igcm, buffered formalin. The hearts were divided into 3 parts, the apical, middle and basal. The aortas were cut transversely at the level of the arch and mid-thorax. Frozen sections of the above-mentioned preparations were prepared, stained with a thickness of 16 to 20 micrometers and to demonstrate lipids with oil red-0 and counterstained with hematoxylin. Ten sections were made from each part of the heart and the thoracic arch portion of the aorta. The sections were examined under the microscope and classified according to the occurrence ν, ηά, severity of the atherosclerotic lesions *

Occurrence and distribution of atherosclerotic lesions in the coronary arteries and aorta of rats of the various Groups are compiled in Table I.

- 11 109846/1812

i 1 e

Virkurifrcn an "active"Chonäroitinsuli'at A preparation on the occurrence and distribution -.zharcz ^ l erotic lesions in the coronary arteries and dei 'aorta of rats with a by oversupply of vitaniin-D-atiierogenen Uiät geZütierz vrnräen .

non-atherogenic diet

* U: rrnl lohe had n 7iän ± der üJisr "

oa

o group

upg body weight (g) last body weight (g) +)

Coronary erosion EGG incidence

had average number of arteries j3, which file in the following of the heres are injured: basal part middle part .apiliTaler part

Acrtinatneroslrlerose- incidence (fo)

fros = ntual proportion of animals per C-i-unpe, which show injuries in the following parts of the aorta Number per injured diamond): intracardiac

arc

Middle thorax

0.0

0.0 0.0 0.0

0.0

0.0

0.0 0.0 0.0

0.0 0.0

0.0
0.0
0.0

0.0

O, O O , 0 O, O O, O O "O O, O O, O O , 0 . O, O

atherogenic diet

Group I Group II Group III ' group IV 6th
1 group V β
9 β
145.3
362.0 β
145.3
565.7 6th
145
371 , 3
, 5 18th
145,
131, • 18th
145,
158,

100.0

4.2
3.5
2.4

94.4

44.4

(2.0) '++)
88.9

66.7- (1.3) ++)

16.7

0.5 0.1 0.2

0.0

0.0 0.0 0.0

4-) lias experiment ended after 6 weeks of feeding.

++) The severity of the injuries was assessed on a scale ranging from 0 to 4.

Table (port setting)

Femal he Batten "Sah! Of animals per group Original Body weight (g) Last body weight (g) +)

Histological findings Coronary alheroISIerose prekomnias

Average number of arteries per rat divided into parts des Eersens are injured:

basal part middle part apical part

Aortic atherosclerosis incidence

a>

^ Percentage of animals per group, which are in the following parts eo aorta, show injuries - * ■ (2 injured rats): *** intracardiac

arc

middle thorax

0.0

0.0 0.0 0.0

0.0

0.0

0.0 0.0 0.0

0.0

0.0

0.0 0.0 0.0

0.0

non-atherogenic diet I. Group II group i atherogenic diet V Group ODVJl] 6th
146.5
229.3 6th
146,
259 III Group IV group , 5
, 2 6th
146,
240, 5
6th 18th
146.5
110.3 . 18th
'146
138

100.0 +++) 16.7

2.7 2.8 2.7

100.0 +++)

O , 0 O, O O, O 87.5 (2.0) O , 0 O, O O, O 100.0 (2.7) O , 0 P » O O, O 93.8 (1.6)

27.8. (1.2) 27.8 (1.2) 11.1 (1.5)

+) The experiment was ended after 6 weeks of feeding.

++) The severity of the injuries was assessed on a scale ranging from O to 4.

+ -H-) Two rats in this group died in the course of the experiment. The information in this group ° ^ are based on observations in the surviving 16 rats. . CD

The findings show that the oral application of a biologically "active" preparation of chondroitin sulfate A in the diet of 1 $> resulted in a significant reduction in the incidence and severity of atherosclerotic lesions in the coronary arteries and aorta of rats with a . were fed by an oversupply of vitainin-D atherogenic diet. Further investigation with a biologically "active" preparation of chondroitin sulfate C indicated that this material was similarly active in this regard. In contrast, preparations of purified chondroitin sulfate A and C, which were similar in chemical composition to the preparations used above, according to the tests described earlier, but with regard to the prolongation of the plasma thrombus formation time in rabbits (determined according to the Chandler Ösen method in blood samples taken 12 hours after injection) were "inactive", little or no activity in reducing the incidence and severity of atherogenic lesions under the test conditions given above.

An additional finding, unique in the medication of anti-atherogenic activities, is that the antiatherogenic activity of "active" preparations of chondroitin sulfate A and C does not lower plasma and liver cholesterol or total liver lipid levels to any detectable extent . The increase in the plasma and liver cholesterol and liver total lipids in rats an atherogenic D by oversupply of vitamins "diet, which supplemented an" active "preparation of chondroitin sulfate A or C with 1 $> "were fed " was the same size in rats fed with the non-atherogenic basic diet as in rats fed with the atherogenic diet with continued chondroitin sulfate A or C.

The effectiveness of biologically "active" preparations of chondroitin sulfate A and C in promoting the formation of the collagen

-14-109846 / 1812

-H-

teralkreislauies in heart regions, which from branches of the coronary arteries is shown by the following results.

Rats were anesthetized with atherogenic diet (Group IV) as stated above, lined, that is, the basal diet was 1.5 i ° cholesterol, 0.5 fo ^v cholic acid and vitamin D ₂ (1.25 million U.S.P. units kg / diet) supplemented. These rats showed a significant decrease in the number of coronary artery acts, as determined by the actual number of coronary artery branches in cross-sections of all three parts of the heart, that is, the basal, middle and apical parts, compared to rats fed the non-atherogenic diet (Groups I, II and III). This effect only affected the coronary arteries, not the main coronary arteries. If rats were fed an atherogenic diet, as indicated above, which additionally contained an "active" preparation of chondroitin sulfate A with a content of 1 % in the diet (group V), the number of coronary artery branches was almost the same as in Rats fed the non-atherogenic diet (groups I, II and III). Similar findings were obtained in both male and female rats. The results are summarized in Table II.

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Table II

Effects of an "Active" Ohondroitin Sulphate A Preparation on Coronary Artery Branches

in had who were fed a diet that was atherogenic to vitamin D because of an oversupply of vitamin D. +)

Diä-cgruppe

Number of animals per group

Average number of coronary arteries per cross-section of the following parts of the heart:
Basal section Middle section Apical section Main.-A. Branches main.-A. Branches main.-A. Branches

Male rats 6th
6th
6th 4.7
5.3
3.5 25.3
28.2
23.0 6.0
6.5
5.0 31.6
33.0
27.5 3.8
4.5
3.8 26.0
28.0
23.0 σ
co Non-atherogenic diet:
Group I.
Group II
Group III 13th
18th 3.7
4.8 14.3
20.9 5.5
5.7 20.0
27.3 3.3
3.6 15.6
22.8 co
σ> Atherogenic diet:
Group IV
Group v VOVOVO 5.2
4.0
4.3 20.0
25.3
27.7 5.2
5.7-
5.0 28.3
32.5
30.0 4.2
3.3
4.0 1812 Female rats 18th
18th 3.2
4.0 11.7
21.4 .4.8
5.6 18.0
29.1 V, 1 25.3
26.2
25.5 light-atherogenic diet
Group I.
Group II
Group III 14.5
22.1 Atherogenic diet
Group IV.
Group v

■ f) The experiment was ended 6 weeks after feeding.

It can be seen that under the conditions of the above-mentioned experiment, in which a reduction in the number of coronary? ₍ If there were any serum branches in rats fed an atherogenic diet, the competitive application of an "active" preparation of chondroitin sulfate A in a content of 1 % of the diet led to a highly significant increase in the number of coronary arteries. To the extent that one If an increase in the coronary artery branches in a particular region of the heart is associated with an increase in the circulation in that region, "active" preparations of chondroitin sulfate A are active in promoting the development of the collateral circulation in the regions of the heart supplied by the coronary artery branches. Additional studies show that "Active" preparations of chondroitin sulfate C are equally active in this regard, whereas, preparations of purified chondroitin sulfate A and C which were "inactive" in prolonging the plasma thrombus formation time in rabbits under the test conditions stated earlier were inactive in terms of activity on the prevention of decrease the number of coronary arteries in rats fed a diet that was atherogenic by an excess of vitamin D.

The effectiveness of "active" preparations of chondroitin sulfate A and C in preventing the occurrence of heart attacks (including myocardial infarction, acute coronary insufficiency and acute myocardial ischemia) in people with ischemic or coronary heart failure is supported by the following results.

The term "ischemic heart failure" as used herein describes an insufficient blood supply to the myocardium. This does not just mean the presence of coronary artery calcification ("Silent" atherosclerosis is in the coronary arteries given to the majority of North American adults), but the port stepping of the atherosclerotic process to the Stage of a ^ clinically and / or electrocardiographically detectable Abnormality. 120 patients with ischemic heart failure, determined by electrocardiograms and patient

109846/1812 - 17 -

history, including angina and / or condition after myocardial infarction lasting between 6 months., and 20 years' were treated in 2 groups divided by 60 patients each, who were classified according to age, gender, clinical and laboratory findings were as comparable as possible. A Terglelch of both groups is shown in Table III given.

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Table III

Conditions of 60 patients with ischemic heart failure treated with CSA and 60 control patients Gate of the Study.

Average age

(Years)

Angina (cases)

Previous myocarial infarction (cases)

Under antihigh pressure coagulant

r ^? Ω ^^ _Ä ^ Therapy
(Trap) ( _Pä iie)

Total number

from

Patient

Men;

CSA

Controls

66 66

15 19

12th

6th
8th

6th
7th

16 25

Women:

CSA

Controls

64 67

25 20

6. 3

20th
19th

44 35

Together;

CSA

Controls

65 66

40 39

13 15

9 10

60 60

- 19 - · ■

Both groups of patients are chosen at random as they appear in clinical practice. All comprehensive, thera-. _The therapeutic measures taken before the test were continued or extended. These included diets with low sodium, low cholesterol, limited sugar intake where appropriate, applications of vasodilators to coronary arteries, cerebral arteries or peripheral arteries, sedatives, oral anti-coagulants, vitamins and / or supplementary nutrients (including highly unsaturated oils and fats), Thyroid extracts and female sex hormones. The two groups differed in only one way. Group II persons were treated daily with an "active" preparation of 'Chondro- ™ itin sulfate A, those in group I were not. During the first year of the study, the persons in group II were treated daily with 1.5 to 10 g of an "active" chondroitin sulfate A preparation, initially in powder form, later in the form of tablets, each with 0.5 g of purified chondroitin sulfate per tablet. During the second and third years of the study, the dose of "active" Ghondroitin Sulphate A applied daily was either 1.5 or 3 g in the form of tablets.

The chondroitin sulfate A was made from bovine tracheal cartilage. Each batch of Chondroitin Sulphate A was prepared for its J Activity tested in increasing plasma thrombus formation time as noted above. Only those preparations were made used for the test that were found to be "active" after the "Bio-Test".

Laboratory tests of the hemopoietic, hepatic, renal and other systems were performed in both patient groups. In group II, these tests were usually all one performed for up to two months during the first two-year period, then during the third year of the treatment study every four months. The tests were: complete blood count, sedimentation rate, urinalysis, protein-bound iodine,

109846/1812 ~ ²⁰ "

Test with J * -labeled triiodothyronine, sodium, potassium, Thyroid turbidity test, serum glutamate oxaloacetate transaminase, '■ Serum glutamate pyruvate transaminase, serum bilirubin (direct and indirect), calcium, phosphorus, orreatinine, glucose, Total protein, albumin, globulin, urea nitrogen, uric acid, alkaline phosphatase, cephalin flocculation, Cholesterol and lipoprotein.

In addition, chest x-rays and resting electrocardiograms were taken of each patient. Exercise electrocardiograms After the master’s degree, ophthalmological examinations were carried out by qualified personnel in most of the patients Ophthalmologists were performed in all cases; Photographs microcirculation in the conjunctival vessels made in 27 patients.

Coronary risk factors such as obesity, hypercholesterolemia, Hyperlipemia, hyperbetalipoproteinemia, hypertension, smoking, diabetes were about the same in both groups Frequency given.

Harmful effects in connection with chondroitin sulfate A application - clinically or on the basis of laboratory findings - were not observed in any person during the three-year period Observation time established.

Table IV gives details of the mortality and morbidity in the 120 patients returned during the three-year trial period.

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- 21 -

Tabel

I V

Acute heart attacks occurred in 60 treated with CSA
Patients and 60 control patients with ischemic heart failure during a three-year trial period.

Total
cases 2
14th Myocardium j
fatal Infarcts
/ not deadly or outpatient Acute
coronary
insuffis
enz +) Myocardial
i-ischemia
++) ·. Men: CSA
Controls 2
15th 2
4th 0
6th 0
"■ 4 0
0 Women: CSA
Controls 4th
29 1
2 0
4th 1 +++)
4th 0
5 Together: +) stationary
++) stationary
+++) fatal • CSA
Controls 3
6th 0
10 1 +++)
8th 0
• 5

In the 60 patients in Group I who did not receive CSA, "the following acute heart attacks occurred:

1. Myocardial infarction (fatal) - -6 cases

2. Myocardial infarction (non-fatal) = 10 cases

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2037842

3. Acute coronary insufficiency or acute myocardisemia

• (not fatal) = 8 cases

4. Myocardisemia (non-fatal) = 5 cases

All of the patients listed above were hospitalized with the exception of two fatal cases of myocardial infarction, who have been diagnosed as such on the death certificate by a doctor who is not treating them. Patient in hospital Diagnosed as "acute coronary insufficiency" or "impending myocardial infarction" were initially in an intensive care unit treated for acute heart disease in a hospital. Among the 6 fatal cases with myocardial infarction were 4 men aged 76, 71, 65 and 56 years and 2 women aged 65 and 79 years. Three patients died during the first year, the others during the following 2 years of the examination, while the above described, general therapeutic measures were in progress. Of the 6 cases with myocardial infarction (fatal), 4 patients had a medical history with chronic angina pectoris,

8 patients survived the myocardial infarction, 2 of them had 2 attacks each, so that a total of 10 attacks of non-fatal myocardial infarction were present. Among these 8 patients, 5 were men and 3 were women. Their ages were between 49 and 83 years, with an average age of 68 years. Neither of them received anti-coagulant therapy prior to the attack. »5 patients had previously had myocardial infarction.

There were 8 cases (7 patients, 1 P. twice) of acute coronary insufficiency. All had been admitted to the hospital's acute hera disease intensive care unit, some with the diagnosis of an "impending" or "imminent" myocardial infarct, a "protracted angina pectoris" or "status anginosus ". All 7 patients recovered from the acute co-

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ronarinsuffizienz, but in 2 patients suddenly appeared mas sive Myokaidinfarkte on which a 12-month period (not ■> fatal), the other 21 months later (fatal). The first of the two patients had been hospitalized twice for acute coronary insufficiency.

5 patients with myocardial disorders were due to states of oppression in inpatient treatment due to their cardiovascular system. The cases were, however not that hard, and the EKG and blood enzyme changes weren't so critical that an admission to the intensive care unit would have been necessary for acute heart disease; these patients were consequently subject to the general rules Care of the hospital. All 5 patients were women; their ages ranged from 55 "to 75 years old, with an average age from 65 years

The following occurred in the 60 patients who received active CSA acute heart attacks on:

1. Myocardial infarction (fatal) = 3 cases

2. Myocardial Infarction (non-fatal) = 0 cases

3. Acute coronary insufficiency.
(terminal) = 1 case

4. Myocardial ischemia = 0 cases

Two of the fatal myocardial infarctions occurred in men (68 and 77 years old) and the third in a woman (65 years old). ·

In the first patient with fatal myocardial infarction in which An autopsy was performed, the CSA was discontinued two months before the iodine. The second patient in this category had taken CSA intermittently for a year, then discontinued it a year before death. The third pa-

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patient with fatal myocardial infarction suffered from persistent High pressure that is often difficult or impossible to control could be. The patient suffering from terminal coronary insufficiency and who died from decompensated heart failure, this had terminal. Complications as a consequence of massive cerebrovascular Bleeding in chronic fibrillation and fluttering of the heart. The 3 patients had a medical history with Angina pectoris.

Some patients in the CSA-treated group had to accept frequent, brief interruptions in the CSA medication take, be it because of delivery difficulties of the drug, because of occasional "influenza" infections in the respiratory system and Digestive tract, or indigestion caused by food. The interruptions were brief ' Duration, between a few days to a maximum of a few weeks.

So far, there are no new heart attacks in the 60 patients treated with CSA, except for the 4 patients mentioned above occurred. Strictly speaking, 2 of these 4 represent patients ten no cases of failure of CSA in preventing an acute heart attack, since one attack (fatal Myocardial infarction in the 77-year-old man) the patient enters the CSA At the second pail the patient had actually developed a massive cerebrovascular disease year before his death Bleeding died in which the heart failure appeared as a terminal complication. .

Another patient in this group died. The autopsy at the. 62 year old revealed a vicious, cerebral Astrocytoma. The patient was already showing cerebral symptoms, when therapy with CSA began, there were 2 admissions beforehand to the hospital for cerebral and more general neurological purposes Investigations are carried out. These did not reveal the presence of cerebral lesions, they became sudden detected in an operation before death and at autopsy.

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2037Ö42

A total of 29 heart attacks occurred among the 60 control patients, during the 3-year observation period, in contrast to only 4 'heart attacks in the comparison patient group, the daily oral doses of an "active" preparation of chondroitin sulfate A received.

The medicament according to the invention is preferably formulated for oral administration, the active substance being the usual pharmaceutical excipients and carriers are added.

Production of "active" CSA

A starting material for the production of "active" chondroitin sulfate A is bovine trachea. The material is obtained from the slaughterhouse, as soon as possible after the animals have been slaughtered. It is frozen until processed. During processing, it is freed from the tissue and finely ground. The ground material is degreased with 5 parts of acetone. In 2 extractions the fat content is reduced to about 1 # or less. The defatted material is dried and ground again. In a content of 5 $> the material in a Oji M calcium acetate buffer is suspended, the 1 i <> papain and containing 0.005 M of cysteine hydrochloride and 0.005 M of disodium lthylendiaminotetraacetat (verses) is. The latter components serve as enzyme activators. The mixture is incubated with gentle stirring for 24 to 30 hours at 62 ° C ⁰ 0 + 3. About 85 to 95% of the trachea is loosened. The supernatant is decanted and twice the volume of acetone is added. The acetone supernatant is separated, the remaining precipitate is dissolved in physiological saline in a concentration of 3 to $ 5>. Saturated potassium permanganate solution is added to this solution with constant stirring in portions of 2 to 5 ml

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CMQiNAL INSPECTED

Addition of complete decolorization is awaited. Dependent on on the nature of the raw material can '' between 50 and 200 ml of potassium permanganate solution per 6 pounds Starting material may be required. As soon as the decolorization time lasts longer than 5 minutes, no further permanganate is produced more admitted. The solution is then left to stand overnight so that the manganese dioxide flocculates can and reactions can run until they are complete. The manganese dioxide is obtained by centrifugation or filtration separated by coarse filter paper. The manganese dioxide cake is washed with physiological saline solution. In some cases the addition of a small amount of formaldehyde or methanol will cause a flocculation of manganese dioxide to lead. The combined supernatants are mixed with a volume of acetone, an oily precipitate forms, which is separated off after decanting. The precipitate is freed from the solvent or dissolved in as little water as possible and obtaining the final product by lyophilizing the solution. Paper chromatography shows that the product is essentially consists of pure chondroitin sulfate A. Infrared spectrophotometry of the product shows the absorption curve typical for chondroitin sulfate A. Determining the optical Rotation gives values of (a) jj = -24 ° »the nitrogen content is 3.3 #.

The production of "active" CSC

A starting material for the manufacture of "active" chondroitin sulfate C are shark cartilage. This material is ground in dry form and degreased with 3 to 5 parts of acetone. One extraction is generally sufficient. The extracted H. shark cartilage will be treated further as it is described above for dried bovine trachea following the defatting step.

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Claims (6)

PATENT CLAIMS '-η 1.) Medicines to prevent the occurrence of atherosclerotic Läeionen by promoting the formation of the KoI lateral circulation in regions of the heart which are supplied by the branches of the coronary arteries, and for prevention the occurrence of heart attacks, including myocardial infarction, acute coronary insufficiency, and acute myocardial ischemia in people with ischemic heart failure, characterized by containing an effective amount of "Active" chondrotin sulphate A, "active" chondroitin sulphate C or mixtures thereof, said activity being in the extension of the plasma thrombus formation time 6 to 12 hours after application in rabbits after the Chandler Eyes Method is noted. 2.) Medicament according to claim 1, characterized in that the effective amount of active chondroitin sulfate between is about 0.5 and 10 g in the daily administered dose. 3.) A method for producing the active ingredient of the medicament according to claim 1 or 2, characterized in that the starting material is crushed and defatted, then in one, | a protease and a SH-group-containing substance-containing buffer is incubated at elevated temperature, the supernatant is mixed with acetone and the precipitate obtained is dissolved in physiological saline solution and treated with permanganate until it remains discolored, MnO _{2 is} separated off and the desired product is removed from the solution with acetone is felled. · 4.) The method according to claim 5, characterized in that bovine trachea or shark cartilage is used as the starting material. - 28 109846/1812 2037S42 5.) The method according to claim 3 or 4 _t, characterized in that an Oji-M calcium acetate buffer which contains 1 $ papain \ and 0.005 M of cysteine and 0.005 M of ethylenediaininotetraacetate is used. 6.) The method according to claim 3 to 5> characterized in that it is incubated ^{at a temperature between 59 and 65 0 C.} 109846/1812