Effects of policosanol on platelet aggregation in rats

THROMBOSIS RESEARCH 69; 321-327, 1993 0049-3848/93 $6.00 + .OOPrinted in the USA. Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.

EFFECTS OF POLICOSANOL ON PLATELET AGGREGATION IN RATS

M.L.ARRUZAZABALA, D.CARBAJAL, R. MAS, M. GARCIA, AND V. FRAGA From Department of Pharmacology and Toxicology, National Center of Scientific Research Ave 25 y 158, Cubanacan, La Habana, CUBA (Received 28.4.1992; accepted in revised form 4.12.1992 by Editor R. Malmgren) ABSTRACT Policosanol is the trivial name of a mixture of high molecular weight alcohols isolated from sugar cane, wherein octacosanol is the main The effects of Policosanol treatment on rat platelet component aggregation were studied. Depending on the dose, Policosanol (5-20 mg/kg, perorally) inhibited the decrease in circulating platelet counts and collagen-induced malondialdehyde concentration in plasma. In rat clotted whole blood thromboxane Bz formation was inhibited by Policosanol (25 mg/kgl. Policosanol (50-200 mg/kg, single doses) inhibited ADP-induced platelet aggregation in platelet-rich plasma, while lower doses (25 mg/kgI did not change responses to ADP significantly. However, rats treated with this dose (25 mg/kg) for 4 weeks showed a significant inhibition of platelet aggregation in PRP when a submaximal ADP concentrations was administered.

It has been shown that treatment with some lipid-lowering drugs reduces the tendency to platelet hyperaggregation frequently observed in hyperlipidemic patients (Kruger and Soergel, 1983; Overturf el al, 1987). Similarly, anti-aggregatory effects mediated by these compounds have been shown in experimental animals (Wanless, 1984; MC Gregor et al 1980; Renaud et al, 19701. Policosanol is the trivial name of a mixture of high molecular (MWI aliphatic primary alcohols, isolated from sugar cane (Saccharum officinarum, LI, whose main component is octacosanol (CH3(CH212,CH20,41(MW 410.7 pm) followed by triacontanol (MW 438.5 pm) and hexacosanol (MW 382.41 while the other alcohols dotriacontanol (tetracosanol, heptacosanol, nonacosanol, and tetratriacontanoll are minor components.

KEY WORDS:

platelet aggregation, ADP, policosanol, thromboxane A2.

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Taking into account that oral administration of octacosanol in mice subjected to physical training produces a significant decrease in the values of serum triglycerides and phospholipids, as well as in the hepatic content of cholesterol (Shimura et al, 19871, the putative cholesterol-lowering effects of policosanol were investigated in different experimental models. Thus, oral treatment with policosanol reduces serum cholesterol levels in different experimental models (Arruzazabala et al, 19911, (Cruz-Bustillo et al, 1991) (Arruzazabala et al, 1992) healthy human volunteers (Hernandez et al, 19921 and patients with type II hyperlipoproteinemia (Castafioet al, 1991). On the other hand, toxicological preclinical studies showed that policosanol is a very safe treatment when administered to rodents and monkeys (Aleman et al, 1991; Rodriguez et al, 1991) and mutagenicity data indicated that policosanol does not cause genetic changes neither in prokaryotes nor in eukaryotes cells (Fernandez et al, 19911. Moreover, clinical trial demonstrate that policosanol is safe and very well tolerated drug (Castafioet al, 19911. Consequently, taking into account the effects of policosanol on serum lipids profile as well as its safety data, the possible effects of treatment on platelet aggregation were studied. For this purpose, several experimental series using "in vivo" and "ex vivo" models were evaluated. MATERIAL AND METHODS Animals Sprague Dawley male rats weighing 250 to 300 g were used. These Animals were adapted to laboratory conditions for 7 days with free access to food and water and were randomly distributed among several experimental groups (10 animals/group). Administration a~& dosage Policosanol (Batch L-C-11) (Ateromixol, trade name) was supplied by Dalmer S.A. Laboratories (Cuba) and for administration it was prepared as a suspension in Acacia gum/water (10 mg/ml). Animals received no food for 16 hours prior to oral administration of the drug. Policosanol administration was made by oral route (1 ml/100 g body weight) 2, 4 and 24 hours before the experiments, while control animals received equivalent volume of the vehicle. Intravascular platelet aggregation Four experimental groups were used: 1) controls 2) Policosanol 5 mg/kg 31 Policosanol lOmg/kg 4) Policosanol 20 mg/kg. Two hours later animals were anesthetized intraperitoneally with pentobarbital sodium (30-40 mg/kgI. A cannula was inserted into a carotid for blood sampling before and 90 s after 30 Mg/kg collagen intravenous injection into the dorsal vein of the penis. Blood (900 Ml) was collected in plastic tubes containing a 100 /.11 mixture with 0.7 mg/ml indomethacin and EDTA (0.19 %, w/v). One aliquot was used to determine the platelet count on each sample through optic microscopic counting. Each value represents the mean of five cell counts. Inter-assay and intra-assay coeficients of variation were 4.5 % and 3.1 %, respectively. Blood was then centrifuged at 1200 x g for 15 min and plasma malondialdehyde (MDA) concentration was quantified through the thiobarbituric acid method (Satoh 1978). Platelet counts and plasma MDA concentration variations after injecting collagen are expressed as a percentage of the basal value.

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Thromboxane B2 Assay In this experiment, single doses of Policosanol (25 mg/kgl were used. 200 pl of blood were drawn by cutting the tip of the tail before starting treatment (t=Ol and 2, 4 and 24 hours after Policosanol administration. Blood was incubated at 37'C for one hour, centrifuged at 2000 x g for 5 min and the serum was stored at -20' C. The resulting thromboxane A2 (TxA21 concentration was determined by measuring the stable metabolite thromboxane B2 (TxB21 by means of a radioimmunoassay for which the Amersham TxBz quantification reagent kit was used. The assay was carried out according to instructions supplied by the trading company. Serum TxBz concentration is expressed in pg/L. Platelet aggregation in rats. The rats were distributed in 6 experimental groups. A control group and three single doses of Policosanol (25, 50 and 200 treatment groups that received mg/kgl. The assay was performed 2, 4 and 24 hours after the treatment. The other two groups received Policosanol (25 mg/kgl or vehicle, by oral route for 4 weeks. The assay was performed 24 hours after the last treatment. All animals were anesthetized with ether and blood samples were drawn from vena cava and mixed with 3.8 % w/v sodium citrate (9 volumes of blood per 1 of anticoagulant). Blood was centrifuged at 250 x g for 10 min to obtain platelet-rich plasma (PRP). Once PRP was isolated, the remainder was centrifuged at 1300 x g for 15 min to obtain platelet-poor plasma (PPP). Platelet aggregation was quantified by Born turbidimetric method (Born, 1962). PRP 200 ~1 aliquots were preincubated for 2 min at 37'C on a Elvi 840 aggregometer, at 1000 rpm and stimulated by the addition of submaximal concentration of ADP (Final concentration 2 PM). Platelet aggregation was determined by calibrating the equipmemt at 0% light transmission for PRP and at 100 % for PPP. Aggregation curves were recorded for 5 min. Results are expressed in maximum aggregation percentages (%I. Chemicals Collagen (Type II, bovine achilles tendon), ADP, EDTA, and indomethacin were purchased from Sigma Chemical co. St. Louis, MO and Radioimmunoassay kit for thromboxane B2 was purchased from Amersham International Plc. Statistical Analysis The nonparametric Mann-Whitney U test was used to compare data between groups (* p < 0.05; ** p < 0.011.

RESULTS Intravenous platelet agnregation Depending on the dose, Policosanol (5-20 mg/kgl inhibited the decrease in circulating platelet count and the simultaneous increase in collageninduced MDA concentration in plasma (Table 1). Thromboxane A2 assay In rat clotting whole blood, TxBz formation was inhibited by Policosanol (25 mg/kgl administered 2 and 4 hours before. Nevertheless, when TxB2 assay was performed 24 hours after the policosanol administration the reduction did not achieve significant levels (Table 2).

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TABLE I Effect

of Policosanol

on collagen-induced

intravascular

PLATELETS COUNT(x lOgcells

Control Pollcosanol Pollcosanol Policosanol

5 mg/kg 10 mg/kg 20 mg/kg

n

Before (Basellnel

10 10 10 10

186.16 191.12 197.33 196.51

i f f f

platelet

aggregation.

/ Ll

After 8.5 8.4 8.5 7.4

% of Baseline

collagen (90 sl

149.83 168.71 176.11 194.50

t + f f

78 86 88 99

9.1 7.4 9.0 7.8

?? ?? ??

MALONDIALDEHYDE CONCENTRATION (pM1

Control Pollcosanol Policosanol Policosanol

5 mg/kg 10 mg/kg 20 mg/kg

n

Before (Baseline)

10 10 10 10

4.87 5.21 5.18 4.31

f i k *

After

0.96 1.0 1.12 0.93

collagen (90 sl

6.01 6.08 5.39 4.26

f: f f ?:

% of Baseline

0.98 1.11 1.01 1.11

123 116 104 * 98 ??

TABLE 2 Effect

of Pollcosanol

Control Pollcosanol Pollcosanol Policosanol

(after (after (after

(25 mg/kgl

2 hl 4 hl 24 h)

Platelet annrenation in rats Policosanol (SO-200 mg/kg, This platelet aggregation. administered 24 hours before not change responses to ADP wlth Policosanol (25 mg/kgl platelet aggregation when a

Platelet

aggregation

in

on serum thromboxane

82 levels

n

TxB2 (Mg/Ll

10 10 10 10

310.4 41.1 140.8 218.7

+ 68.5 2 2.1 2 30.5 * 27.2

In rats

*

?? ??

single doses1 inhibited ADP-induced “ex viva” effect was not observed when the drug was the assay (Table 31. Lower doses (25 mg/kgI did rats treated significantly (Table 31. However, for 4 weeks showed a significant inhibition of submaxlmal ADP concentration was used (Table 41.

rats

Policosanol (50-200 mg/kg, single doses) inhibited ADP-induced "ex vivo" platelet aggregation. This effect was not observed when the drug was administered 24 hours before the assay (Table 3). Lower doses (25 mg/kg) did not change responses to ADP significantly (Table 3). However, rats treated with Policosanol (25 mg/kg) for 4 weeks showed a significant inhibition of platelet aggregation when a submaximal ADP concentrat
TABLE 3 Effect

of

Control Policosanol Policosanol Policosanol Policosanol Policosanol

Policosanol

(25 (50 (200 (200 (200

mg/kg mg/kg mg/kg mg/kg mg/kg

on “ex vlvo”

after after after after after

2 hl 2 hl 2 hl 4 hl 24 hl

platelet

aggregation

induced

n

Aggregatlon

10 10 10 10 10 10

29.6 20.8 1.9 3.4 10.1 20.8

+ f f t t f

by ADP (2 PM1 (Xl

5.0 4.9 3.4 ??* 1.4 ** 4.1 * 5.9

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TABLE4 inducedby Effectof Policosanol 'ex vlvo"plateletaggregation after4 weeksof treatment. n Control Pollcosanol 25 mg/kg

Aggregation

10

38.5+ 5.9

10

24.5

?: 6.4

ADP

(2

pM)

(X)

??

DISCUSSION Platelet interactions with subendothelial collagen leads to platelet activation, the release of granule contents and the metabolism of arachidonic acid to cyclic endoperoxides. These endoperoxides are in turn transformed into TxAz, a vasoconstrictor and potent platelet aggregating agent. The collagen injection used in our experiments caused intravascular aggregation detected by decreasing circulating platelet counts and MDA production, a product of lipid peroxidation and an indirect marker of TxAz formation (Verstraete, 1983). In this study, Policosanol (5-20 mg/kgl inhibited both collagen-induced decreases in platelet counts and the enhanced MDA production, suggesting an "in vivo" anti-aggregatory effect due to the inhibition of arachidonic acid metabolism. This conclusion is supported by the fact that this drug at 25 mg/kg markedly inhibited TxAz formation in rat clotted whole blood. On the other hand, although a single dose of Policosanol (25 mg/kgl had no effect on ADP-induced PHP aggregation, at higher doses (SO-200 mg/kg) Policosanol administered 2 h prior to blood sampling significantly inhibited ADP-induced PHP aggregation. Furthermore, Policosanol (25 mg/kgl administered during 4 weeks inhibited ADP-induced PHP aggregation suggesting acumulative anti-platelet effect. ADP aggregation in titrated platelet-rich plasma is biphasic, the first wave being due to the exogenous ADP and the second (blocked by aspirin), to the release of proaggregatory substances. Therefore, the fact that policosanol almost completely inhibted platelet aggregation suggest that this process is not only directly related with a reduction in TxAz levels. It is well established that ADP-induced activation leads to the exposure of membrane receptors for fibrinogen (glycoproteins GPIIb/IIIal. This is crucial for the first phase aggregation (Mustard et al 1978; Hantgan et al, 1976; Gerrard and White, 1976). This suggest that policosanol could affect the interaction of fibrinogen with the PGIIb/IIIa receptors or ADP membrane receptors. A similar effect has been described for the anti-thrombotic compound ticlopidine (Minno et al, 19851. However, further studies are needed to clarify this hypothesis. This study demonstrate the antiaggregatory effects of policosanol on "in vivo" and llexvivo" experimental models. These results, the previous data of the scarce toxicity of this drug as well as its cholesterol-lowering effects demonstrated in animal models, healthy volunteers and patients with primary hypercholesterolemia type II (Castafioet al, 19911, suggest a promising value of policosanol in the management of hypercholesterolemia and associated complications as platelet hyperaggregability. Further studies however, will be needed to investigate the antiaggregatory effects of policosanol in other experimental models as well as in human volunteers.

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REFERENCES l- KRUEGER B.F and SOERGEL A. Influencia de1 etofibrato sobre la agregacion plaquetaria. Teraniecwoche 33, 3297-99, 1983. 2- OVERTURF M; SYBERS H; SCHAPER I and FAEGTMEYER H. Hypertension and atherosclerosis in cholesterol fed rabbits. Atherosclerosis 66, 63-76, 1987. 3- WANLESS I.R. The effzct of dietarycho&terol on platelet survival in the rabbit: A study using C-serotonin and chromiun double-labeled platelets. Thromb.Haemostasis 52, 85-89, 1984. 4- MCGREGOR L; MORAi!AINR and RENAUD S. Effects of dietary linoleic acid on platelet function in the rat. Thromb.Res. 20, 499, 1980. 5- RENAUD S; KINLOUGH R.L and MUSTARD J.F. Relationship between platelet aggregation and the thrombotic tendency in rats fed hyperlipemic diets. LA Invest. 22, 339, 1970. 6- SHIMURA S; HASEGAWA T; TAKINO S and SUZUKI T. Studies on the effect of octacosanol on motor endurance in mice. Nutrition Renorts & 36, 1029-1038, 1987. 7- ARRU2AZABALA M:L; CARBAJAL D; MA'S R; CASTANO G; SOTOLONGO R and MESA R. Efecto de1 Ateromixol (PPG) sobre 10s niveles de colesterol en perros Beagle. Rev 4-9 CENIC 22, 60-61, 1991. 8- CRUZ-BUSTILLO D; MEDEROS C:M; MAS R; ARUZAZABALA M:L; BARRETO B and MARTINEZ 0. Efecto hipocolesterolemico de1 Ateromixol (PPG) en el cerdo de ceba. Rev. CENIC, 22, 62-64, 1991. 9- ARRUZAZABALA M:L: CARBAJAL D; MAS R; ILLNAIT J; LAGUNA A and CASTANO G.. conejos Efecto de1 Ateromixol sobre el perfil lipidic0 de Venezolanos de Farmacologia y Terapeutica.11, normoco1esterolemicos. Arch A 1992. lo- HERNANDEZ F; ILLNAIT J; MAS R; CASTANO G; FERNANDEZ L; GONZALEZ M; CORDOVI N and FERNANDEZ J.C. Effect of Ateromixol (policosanol) on serum lipids and lipoproteins in healthy volunteers. Current Ther. Research 51, 1-8, 1992. ll- CASTAfiOG; ZARDOYA R; ILLNAIT J; MA'SR; FERNANDEZ L; SURRIBAS E; NODARSE M; and FERNANDEZ J:C. Efectos de1 tratamiento con Ateromixol (PPG) (5 mg) en Ciencias Pronresos II. con hiperlipoproteinemia tip0 Paciente en Medicas.(Venezuela) 5, 21-30, 1991.12-ALEMAM C.; MA'S R.; RODEIRO I.; NOA M.; HERNANDEZ C.; CAPOTE A.; MENENDEZ R.; GONZALEZ R.; AMOR A.M. and JIMENEZ S. Toxicologia aguda de1 Ateromixol (PPG) en roedores. Rev, CENIC, 22, 102-105, 1991. 13- RODRIGUEZ-ECHENIQUE C.; MESA R.; MA'SR.; AMOR A:M: and CASTANO G. Estudio de1 efecto sobre lipidod y lipoproteinas sericos y de la tolerancia al tratamiento oral con dosis crecientes de Ateromixol (PPG) en monos Macaca arctoides. Arch venezolanos de Farmacologia y Terapeutica. 11, 1992 A 14- FERNANDEZ S.I.; RENDON A.; DE LAS CAJIGAS and LOPEZ M. Estudio genotbxico de1 Ateromixol (PPG) un nuevo medicamento hipolipemiante. Rev. CENIC, 22,

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98-101, 1991. 15-SATOH K; Serum lipid peroxide in cerebrovascular disorders determined by a new calorimetric method. Clin Chem.Acta 90, 34-43, 1978. A 16- BORN C. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature (Land) 194, 927-929, 1962. 17- VERSTRAETE M. Introduction: Thromboxane in biological systems and the possible impact of its inhibition. Br. J. Clin. Pharmac.. 15,75-115, 1983. 18- MUSTARD J; PACKHAM M; KINLOUGH-RATHBONE R; PERRY D and REGOEZI E. Fibrinogen and ADP-induced platelet aggregation. Blood 52, 453-466, 1978. 19- HANTGAN R; TAYLOR R and LEWIS J. activation. Blood 65, 1299-1310, 1976.

Platelet interact with fibrin after

20-GERRARD J. and WHITE J. Mechanism of ADP induced platelet aggregation. Am. J. Pathol. 82, 513-516, 1976. 21- DI MINNO G; CARBONE A and MATTOLI P. Functionally thrombasthenic state in normal platelets following the administration of ticlopidine. J. Clin. Invest. 75, 328-338, 1985.