CA1288348C - Product and process for promoting the regression of atherosclerosis in animals - Google Patents

Abstract

PRODUCT AND PROCESS FOR PROMOTING THE REGRESSION OF ATHEROSCLEROSIS
IN ANIMALS
ABSTRACT
Product and process for promoting the regression of atherosclerosis in animals and the diminution of lipid deposits found in the arteries of animals by administration of diethylaminoethyl dextrans.

Description

~l 28~341!3 B~CKGROUND OF TE~E: INVENTION

The long term ingestion by animals including man of diets containinq ~ats or cholesterol containing foods is known to produce lipid arterial deposits or pla~ue and to be a factor in causing high blood pressure and atherosclerosis.

The use of diethylaminoethyl dextran tDEAE-dextran~
for the prevention of atherosclerosis by the reduction of blood lipids and cholesterol has been described in U.S.
Patents 3527872 and 3,851,057. The use of this compound for the purpose of ohtaining antisteatogenic ac~ion and re-duction of body weight has been described in U.S. Patent 4,160,826.

The re~ression of atherosclerot.~c deposits has previously been investigated in animals by means of a low-fat diet or by the use of such hypolipidemic agents as cholestyramine and estrogen,.~lthough such expedients have been found to be somewh~t effective in reducing serum chole~terol levels, their e~ficacy in inducing regression of arterial deposits has not been clearly established.

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SUMMARY OF THE INVENTION
. . _ , ,, It has not heretofor been considered that DEAE-dextran could be effective in the removal of atherosclerotic ar-terial deposits accumulated or formed by the continuation of hyper-cholesterimic diets in animals.

Applicants have now discovered that DEAE-dextran can, by oral administration to animals, be highly effective in causing the regression of lipid arterial deposi-ts.

DEAE-dextran is a product obtained by reacting dextran with diethylaminoethyl chloride hydrochloride, preferably in an aqueous medium in the presence of an alkali metal hydroxide, i.e. sodium hydroxidej ànd precipitation from the reaction mixture either in the form of the free base or the hydrochloride by methods such as axe described in U.S. Patent 3851057. In view o~ its greater stability, the hydrochloride form is pre-ferred.

Dextran, as is well known, is a polysaccharide obtained by the action of backeria growing on a sucrose substrate, con-taining D-glucose units. This invention contemplates the DEAE-dextran product obtained by using dextran or its hydrogenated form as the starting material for its production. The product is khen administered orally in a predetermined dosage to subjects known to have atherosclerosis due to the existence of arterial deposits.

- . -~28~3~3 DEAE-dextran is a polyelectrolyte derivative of dextran, capable of sequestering substances of opposite charge. The interaction oE the counterion with the polyelectrolyte, depends naturally, also on the nature of the ionizable groups of the two substances, the pH, and on the ionic strength of the solvent.
Under conditions of ionic strength in the gut, DEAE-dextran which is a rather strong polycation tends to bind firmly, sub-stances acid in character. The result is a net inhibition of the digestion and absorption of lipids, either by the binding of bile acids which hinders the emulsion of neutral fats and the activation of pancreatic lipases, or by interaction with fatty acids resulting from the hydrolysis of neutral fat; thus their absorption and their action on lipase are blocked, with removal of the sub-strata necessary for triglyceride synthesis.
Compared with insoluble polyelectrolytes such as Cholestyramine DEAE-dextran offers advantages of efficacy and tolerability.
The fact that it is soluble ma~es it more active as a sequestering agent, which permits a dose in humans of only 2 - 6 grams daily (against 16 - 32 grams daily Eor cholestyramine) which at the same time makes it more easily tolerated. Its non-absorption by ~the gastrointestinal mucosa accounts for the absence of direct systemic side effects. In addition, such insoluble poly-electrolytes as cholestyramine are known to produce, or severely worsen pre-existing constipation which strongly contra-indicates use in human therapy especially for the periods which would be required to effectively remove arterial plaque. This problem would not occur with the use oE water solub~e DE~E-dextran.

3~

In order to demonstrate the effecti.veness of DE~E-dextran, a lon~ term investigation was carried out in comparison with cholestyramine upon an animal model known to be susceptible to the formation of atherosclerotic arterial deposits and in whom the progress of this disease could be scientifically re-lated to its progress in other animals including man. This in-vesti~ation is summarized below. The dextran utilized in the investigation was preliminarily hydrogenated to produce a hydro-dextran by the use of sodium borohydride under well known pro-cedures such as described in U.S. Patents 3~22221 or 3234209.
In this procedure, the:~terminai aldehyde group normally present in the dextran molecule is converted to a hydroxide. This results in a dextran derivative which when reacted further to produce other derivatives such as DEAE-dextran, gives a product of greater purity and equal to or superior to derivatives of dextran which has no-t been preliminarily hydrogenated.

In the investigation described herein clextran havin~ an average molecular seight of about 500,000 was utilized although other dextran polymers rangin~ from about 40,000 or higher will also be e~fective.

OBJECTIVE OF T~IE INVESTI~TION

To determine i~ lon~-term trea-tment with l)~AE-dextran will regress established atherosclerotic lesions in Japanese quail in comparison with cholestyramine and negative controls on a basal diet, This al50 leads to the determination of the the.rapeutic appIicability of DEAE-dextran to the reduction of es-tablished atherosclerotic deposits in other.animals 'ncluding man.

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Animal~

One hundred twenty-two young male Japanese quail selectively bred for their propen~ity to develop atherosclerosis when fed diets containing cholesterol were individually housed in wire bottom ca~es. The birds were given free acces~ to food (Purina Game Bird Growena) and clean water for a thrce week quarantine period. The special suitability of Japanese ~uail for this pur-pose has been reported by Dr. Charles C. Day et al, in "Laboratory and Animal Science" Vol. 27, No. 5, PP 817-~2 (1977) Study Protocol The birds were divided into two groups. Group ~1 (14 birds) was fed a hasal 40~ dextrose, 10~ fat, semipurified diet. Group #2 (10~ birds) was fed the basal diet supplemented with 0.5 cholesterol (J.T. ~aker Co., Cat. No. 7-F676). The 2 diets were fed for 8 weeks.
Serum cholesterol levels were determined after 6 and 7 weeks. One hundred-three of the group 2 birds were allocated to one of 13 subgroups on the ba~is of the average o~ their 6 and 7 week cholesterol levels. One subgroup was sacriiced on day 56 for arterial atheroscl~ro~is and chole~terol analysis. The remain-ing 93 birds entered the regression phase of the study and were treated as sllown below:

Group I.D n Treatment Durina ~e~ression Phase , 2 10 Sacrificed on day 56 of induction phase A-16 10 ~therogenic Diet (AD)~ Sacrific~d after 16 weeks A-32 10 .~therogenic Diet ~AD); Sacrificed after ~2 weeks ~-4B 10 ~therogenic Diet (~n); Sacriiced after 48 wee~
* Trade Mark , .

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~L2~ 348 Group I.D. n Treatment Dur_~L ~ ression Phase B-16 7 Basal Diet (BD); ~acrificed after 16 weeks B-32 7 Basal Diet (BD); Sacrificed after 32 weeks B-48 7 Basal Diet (BD); Sacrificed after 48 weeks C-16 7 AD -~ 2~ Cholestyramine*; Sacrificed after 16 weeks C-32 7 AD ~ 2~ Cholestyramine*; Sacrificed after 32 weeks C-48 7 AD + 2~ ~holestyramine*; Sacrificed after 48 weeks n-16 7 AD ~ 2% DEAE-Dextran ; Sacrificed after 16 weeks D-32 7 A~ + 2% DEAE-Dextran ; Sacrificed after 32 weeks D-48 7 ~D + 2~ D~AE-Dextran ; Sacrificed after 48 weeks * ~m~erlite XE-268, Rohm and l~aas The 14 group 1 (basal diet only) served as negative controls and were treated as shown below during the re~ression phase.

Group I.D. n Treatment Durin~ Re~ression Phase 1 3 Sacri~iced on c~ay 56 of induction phase E-16 3 Basal Diet (BD~; sacrificed after 16 weeks E-32 4 Basal Diet (BD), sacrificed after 32 weeks E-48 4 Basa]. Diet (BD); sacrificed after 4~ weeks The birds were blec1 after 2, 4 and ~ weeks and every 4 weeks thereafter during the regression phase for serum cholesterol analysis. Food intake and body w~ights were monitored throughout the regression phase. Livers were weic~hed at sacrifice. Heart~
and proximal 1-2 cm oE ~hor~cic aorta and brachiocephallc arteries were stored at -200 C until anal~æed.

l2~3~33~8 Analvtical Methods Serum obtained rom approximately 400 ul of clotted venous blood was analyzed for cholesterol using an automatic enzymic rocedure (Roto Chem, Pmicon).

After thawing, the ascending and thoracic portions of the aorta and brachiocephalic arteries were removed from the heart, cleaned of adventitial lipid, opened lon~it~ldinally and flattened.
The exposed endothelial surface was photographed using a Polaroid*
~P=3 Land Camera and Type sa film. The rinht brachiocephalic artery was removed and placed in 10% buffered formalin fox sub-sequent histological examination. The r~maining left brachio-cephalic artery and aorta were weighed and transferred to a stop-pered lfi x lO0 mm glass test tube containing 3 ml isopropyl alcohol.
The arteries were homogenized using a rolytron tis~ue homogenizer (Brinkmann Instruments, ~estbury, t~l.Y.). mwenty ul aliquots of alcoholic extract were taken for cholesterol analysis by an auto-m~ted enzymic procedure similar to that used for serum cholesterol.

Frozen sections of the right brachiocephalic artery were st~ined wlth oil red 0 and photographed using the Polaroid ~IP-3 camera.

With the present study we determined the effects of resin treatment for 16, 32 and 48 weeks respectively on arterial cholesterol concentrations.

The body wei~Jhts of birds during th& induction and regression phases are shown in the Table below. There was no significant effect on growth of feedin~ the atheroqenic diet ~0.5~ cholesterol) alone or containin~ 2~ cholestyramine or DEAE-dextran, * Trade Mark 2~383~3 Ten birds were fed the atherogenic diet and three birds fed the basal diet were sacrificed at the encl of the induction period. In a~reement with previous findin~s liver wei~Jhts were increased by 75~ and arterial cholesterol concentrations were 5 times higher in the birds fed the athero~enic diet compared to normal controls (Table lA).

Food intake during the regression phase of the experiment was slightly higher in resin treated groups compared to diet without resin. Similar amounts of cholestyramine and DEAE-dextran were ingested by treated ~roups.

The effects of diet and drug treatment on serum cholesterol levels for representative groups are shown in Table 2. After 7 weeks the 103 birds fed the athero~enic diet (Group 2) were selected into 13 subgroups on the basis of the avera~e of their week 6 and week 7 cholesterol level. Serum cholesterol levels at the end of the induction study (Day 56) ran~ed between 890 - 1086 mg/100 ml in the 13 subgroups receivin~ the atherogenic diet.

After two weeks treatment with cholestyramine, DE~E-clextran, or switching to the basal diet without cholesterol, there was a marked reduction in serum cholesterol levels. After four weeks, serum cholesterol levels in the 3 treated groups had returned to normal. Ilowever, as shown in Table 2, after treatment for 36,40, 49 and ~8 weeks serum cho:Lesterol levels were reduced substantially after 36 weeks but remained at a fairly stable level thereafter by all three treatments.
Tables l-B, l-C,and l-D desc~ibe results showing regression of arterial deposits after 16, 32,and 48 weeks treatment respectively.

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TABLE 1 Body Wei~hts, Liver Weights, and Arterial Cholesterols in Jap'anese Quail Sacrlficed at Various Sta~es A - Day_56 of Induction Phase (8 weeks) Arterial - Bod~ Weight (~) L ~ Cholesterol Treat nt (mg/g) ~ Day 56 Day 56 Day 56 1 Basal Diet 109 114 1.88 4.7 2 Atherogenic 104 112 3.30 23.9 Diet ~ , j . .. .... _ _ . . . .
All aata expresse~ as mean.
B - After 16 Weeks Treatment Durin~ .~e~ression Phase -.
Arterial Grou~ Regression Body Wei~ht Iq) Liver Wei~ht (~ Cholesterol Treatment Week 8 Week 29 Week 24 Week 24 A-16 Atherogenic Diet (AD) 10~ 10~ 4.68 50.6 ~-16 Basal Diet 106 116 1.77 23~5 C-16 AD+2% choles-tyramine 10~ 110 2.25 12.8 D-16 AD+2~ DE~E-dextran 108 111 2.14 14.5 ~-16 Basal Diet 112 121 1.69 4.4 _ _ . .. . . .. . .. . ... .. . ....
C - After 32 P1ee~s ~reatment Durinq Regression Phase Group ~egresslon Body Wei~hts ~ iver Wei~hts(~) Arterial Treatment l~eek a t^1eek 40 Week 40 Cholesterol.
~-32 ~thero~enic l~eek 40 Diet ~AD~ 109 113 3.94 ~14.6 B-32 Basal Diet 106 111 1.33 14.1 C-32 ~D-~2~ choles-tyramine 107 113 2.19 14 . 3 D-32 AD~2~ DEAE-dextran 110 115 2.34 .,-' 10.7 E-32 ` Basal Ne~ative Control 104 114 1.89 2.7' , 834~3 D After 48 ~eeks ~reatment Durincl ~.egression Phase Group Regression B~d,~ ~ Liver l~eights(g) Arterial Treatment Cholesterol .. . .
~eek 8 Week 56 ~eek 56 Week 56 A-48 Atherogenic Diet (~D) 109123 2.61 50.1 B-48 Basal Diet 106127 2.36 11.4 C-48 AD~2~ Choles-tyr~mine 107122 2.03 12.0 D-48 AD-~2% DEAF,-Dextran 110105 1.62 7.8 E-48 Basal Negative Control 104112 1.47 5.3 2 112 3.30 23.9 ~wk-8) .. . .... . __ .. _ _ ~ . _.... . .. _ . _ TABLE 2 Effects of Basal Diet, Cholestyramine and DEAE-Dextran on Serum Cholesterol Levels (mg/100 ml) in Japanese ~ . _ . . .
Quail Fed on ~thero~enic Diet for Eight Weeks Pxior to Resin Trea-tment ~eqression Incluction Group Regression Treatment Wk 8 Wk36(44~7k40(~8)Wk44~52)Wk48(56) . _ _ . . .. . _ _ . _ _ .. _ A-48 Atherogenic Diet890 727 928 672 489 B-48 Basal Diet 1001 250 263 271 321 C 48 2% Choles-tyramine995 250 271 2$1 295 D-48 2~ DE~E-Dextran 1082 248 279 309 244 E-~8 Basal MecJative Control 256 232 253 270 275 _, . , ~ . _ ~ _ . __.~ .. . ... _ 38~48 As the above Table 1 clearly shows, DEAE-Dextran is substantially m~re effective than cholestyramine in long term therapy. Arterial cho]esterol decreased from 23.9 mg/g before treatment to 7.8 mg~g after 48 weeks with DEAE-dextran, ancl from 23.9 mg!g to 12.0 mg/~ with cholestyramine. It is note-worthy that this degree of improvement did not become apparent until the 40th week. Similarly, serum cholesterol levels were lower with DEAE-dextran after 48 weeks after showlng a lesser degree of reduction than cholestyramine throughout the preceding weeks of the test. r'hese results are illustrated in Table 2.

Photographs and microphotographs of the stained frozen sections of the arteries at the various stages of treatment, when compared with corresponding photo~raphs of the controls, and untreated quail from the atherogenlc diet, clearly illustrated that plaque lipid was removed as the above data indicate.

In the investigation descrihed above, the DE~-dextran was introduced as a powder into the diet of the animals. However, for pharmaceutical use for oral administration, the compound may be admixed with conventional edible carriers used by those skilled in the art in preparing oral dosage form products. Generally, these are compounded in a "unitary dosage form" containing a pre-determined quantity of the product per unit, whether in pills, capsules or solution or suspension Althouyh th~ animal dosa~e described herein represents approximatel~ 2~ oE the dail~ foocl r~tion, it is known that much higher dosages based upon wei~ht of the animal, and food consump-tion are required than for humans. Accordingl~ the dosage for various animal patients includinq humans wlll vary and must be individually determined.

_12 ~28834~3 In view of the solubility of the acid salts of DEAE-dextran it may be formulated as a liquid or solid as clesired.
In solid form it may be tabletted and since it is somewhat hygroscopic the tablets may be packed in plastic or foil "bubble-pack" packages in which the tablets are individually covered, or the tablets may be individually coated with a soluble or digestible plastic coatiny. The powder may also be formulated into capsules. In lic~uid form, a stablized aqueous solution may be incorporated in capsules or dispensed as a liquid in containers. This adaptability to formulation is in contrast to the insoluble polyelectrolytes such as cholestyramine which must be administered in water suspension and are difficult and inconvenient to take especially in the large quantities required.
In view of the demonstrated relationship of the animal model utilized in the above described tests to the screening for effective anti-atherosclerotic agents in man, it can be assumed that the results obtained can also be attributed to similar ap-plication to human therapy. It appears clear from the data ob-tained that long term therapy with suitable dosages of DEAE-dextran can reduce established atherosclerotic coronary deposits in ani-mals, incluc1ing man, and that such therapy should be carried out for periods of 6-12 months or longer. Pre~erred daily dosages appear to be in the range of 2-6 grams per day.

Claims (5)

1. An orally administered product for the regression of established atherogenic lipid deposits in animal arteries which comprises diethylaminoethyl dextran or its pharmaceutically acceptable acid salt either alone or in combination with a pharmaceutical carrier.

2. A product according to claim 1 wherein the diethylaminoethyl dextran is derived from hydrogenated dextran.

3. A product according to claim 1 wherein the diethylaminoethyl dextran is in the form of an aqueous solution of its acid salt.

4. The product according to claim 1 in which the dextran component of the diethylaminoethyl dextran has a molecular weight of approximately 500,000.

5. The product according to claim 2 wherein the hydrogenated dextran has a molecular weight of approximately 500,000.