Effects of policosanol chronically administered in male monkeys (Macaca arctoides)

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Pergamon

Fd Chem. Toxic. Vol. 32. No. 6. pp. 565-575. 1994

ElsevierScienceLtd. Printedin Great Bntain 0278-6915(94)E0036-M

EFFECTS OF POLICOSANOL C H R O N I C A L L Y ADMINISTERED IN MALE MONKEYS

(MACACA ARCTOIDES)* C. RODRiGUEZ-ECHENIQUE,R. MESA, R. M~s, M. NOA, R MENENDEZ, R. M. GONZ.I~LEZ, A. M. AMOR, V. FRAGA,V. SOTOLONGO and A. LAGUNA Department of Pharmacology and Toxicology. National Center for Scientific Research. Havana City, Cuba (Accepted220ctober 1993)

Abstract--Policosanol, administered orally, has shown a cholesterol-loweringeffect in differentexperimental models. Because lipid-loweringtherapy is administered chronically, it is necessary to know the effects of these drugs after long-term administration. 18 adult male Macaca arctoides monkeys were used to study the cholesterol-loweringeffects and possible toxicity produced by oral administration of policosanol (0.25. 2.5 and 25 mg/kg) for 54 wk. After 8 wk. a significantreduction of serum total cholesterol and low-density lipoprotein cholesterol was observed in policosanol-treatedanimals when compared with the controls; this effect persisted throughout the study. The animals" behavioural repertoire, physical condition, haematology and blood biochemistry, as well as spermiogram analysis and electrocardiography, were monitored during the study; ophthalmological and pathological anatomy examinations were performed at the end of the administration period. No drug-related toxicity was detected by any examination. The results gave further evidence of the marked and persistent cholesterol-loweringeffects of policosanol that had been observed in different experimental models. There was a significant reduction of spontaneous aortic atherosclerotic lesions in treated animals compared with controls. Policosanol (0.25 25 mg/kg) administered orally for 54 wk brought about a persistent reduction in blood cholesterol levels and was very safe and well tolerated during long-term administration.

INTRODUCTION Hypercholesterolaemia is considered to be a main risk factor for coronary atherosclerosis (Levy, 1982). in this sense, a positive correlation between the cardiovascular morbidity or mortality and elevated levels of serum cholesterol and low-density lipoprotein-cholesterol (LDL-C) has been demonstrated (Glueck, 1983; Levy, 1981). Various effective cholesterol-lowering drugs have been described and marketed, but most have induced adverse reactions and drug-related toxicity (AHFS, 1989; O'Connor et aL, 1990). As lipid-lowering drugs must be administered chronically, long-term safety is a critical aspect for their definitive acceptance. Thus, the search for new effective and safe cholesterol-lowering drugs is justified.

*A brief version of this work was presented at the Sixth Congress of Toxicology, Rome. 28 June-3 July 1992 (Rodriguez-Echenique et al., 1992c). Correspondence should be addressed to Dr R. M. Ferreiro Centro Nacional de lnvestigaciones Cientificas.Ave. 25 y 158, Cubanacan, Playa, Ciudad de La Habana, Cuba Apartado postal 6990. Abbret'iations: ECG -- electrocardiogram; GAGs ---glycosaminoglycans; HDL-C.~ high density lipoproteincholesterol; LDL-C = low density lipoprotein-eholesterol.

at:

Policosanol is a mixture of high molecular weight aliphatic primary alcohols isolated from sugar cane ( S a c c h a r u m officinarum L.) wax, the main component of which is octacosanol [CH3(CH2),6CH_,Ot4 ] (mol. wt 410.7) followed by triacontanol (mol. wt438.5) and hexaeosanol (mol. wt 382.4), while the other alcohols (nonacosanol, dotriacontanol, heptacosanol and tetratriacontanol) are minor components. Policosanol has shown a cholesterollowering effect in rabbits and dogs (Arruzazabala et al., 1991a and 1992), swine (Cruz-Bustillo et al., 1991) and monkeys (Rodriguez-Echcnique et al., 1992a). Previous toxicological studies have shown that acute administration of policosanol to rats and mice of both sexes (Alem:in et al., 1991 and 1992) did not induce drug-related toxicity, the LD~o being higher than 5000 mg/kg in all these species. Similarly, subchronic and chronic studies of policosanol oral toxicity performed in rats (Alem~n et al., 1992) showed that no drug-related toxicity was induced, and the highest dose used (500 mg/kg) did not cause observable toxic effects. Furthermore, chronic administration of policosanol (30--180mg/kg) for l yr to beagle dogs of both sexes also showed that treatment was very safe and no drug-related toxicity was observed. As the anatomy and cardiovascular physiology of monkeys and humans are very similar and the

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monkeys develop atherosclerotic lesions spontaneously (Vesselinovitch, 1988; Malinow et al., 1966), male non-human primates have been considered to be one of the best animal models of atherosclerosis (Vesselinovitch, 1988). Macaca arctoides is one of the species of macaques considered to be useful in these studies, owing to the occurrence of atherosclerotic lesions in the aorta, coronary and cerebral arteries that resemble those of humans (Pick et al., 1974). Previous studies have shown that policosanol also reduces LDL-C and total cholesterol in M. arctoides and that subacute administration of increasing doses of policosanol did not induce toxic symptoms in this species (Rodriguez-Echen:que et al., 1992a). The study described here was undertaken to investigate the effects in M. arctoides of policosanol administered orally for 54wk. As no sex-dependent effect was detected in any previous study, only male monkeys were included. NIATERIALS AND METIIODS

Animals and treatment Experimental animals. 18 adult male M. arctoides (6-8 yr old), weighing 6-10 kg at the beginning of the experiment were selected. Animals had been captured in Vietnam 2 yr earlier and had undergone quarantine in their country of origin and in Cuba. 6 months before starting the experimental phase, at the breeding colony of Centro Nacional de Investigaclones Cientificas (CNIC, Cuba) the animals underwent a prophylactic schedule of oral deparasitization against malaria, filariasis and intestinal parasites, for which the following drugs were administered: chloroquine (25mg/kg for 3 days); primaquine (0.3 mg/kg/day for 14 days); mebendazole (40 mg/kg/ day for 3 days) and metronidazole (30.5 mg/kg/day for l0 days) (all from Industria M6dica Farmac6utica, Ministerio de Salud Pfibliea, Cuba), and diethylcarbamazine (Loxuran, Pharmacochemical Works, Budapest, Hungary) (6 mg/kg/day for 10 days). Animals also received an immersion bath in an Esteladon-300 EC solution (I/1000; Ciba-Geigy, Basel, Switzerland) to remove ectoparasites. 3 months before beginning the study, animals were placed in rooms for acclimatization to experimental conditions. They were kept in individual stainlesssteel holding cages (80 x 60 x 90 cm) with temperature, humidity and light/dark cycles that were optimal for their development. The animals received a balanced diet during the whole experimental period, including root and leaf vegetables, fruit and supplementary chow for monkeys (Table I). In accordance with their dietary requirements (Spector, 1956), the 18 animals daily received a similar low-fat and protein-rich diet between 14.00 and 15.00 hr, with free access to water. Animals were randomly allocated to one control group (six animals) and three experimental groups

(four animals each). The holding cages for the four groups were allocated to three compartments, each having six animals from all groups (two from the control group and four from the three different dose groups), thus avoiding systematic location. (Although animals are referred to in the text by consecutive number in each group, they were identified with arbitrary numbers by the observers. During the experiment, a code number known only by the controllers of the experiment was assigned to each animal.) Drug administration and dosage. The batch of policosanol used for this study had a purity of 97% as assessed by gas chromatography. The relative percentage composition of each alcohol was octacosanol 67, triacontanol 14, hexacosanol 8, dotriacontanol 5 and heptacosanol 3, while the other three alcohols (tetracosanol, tetratriacontanol and nonacosanol) each comprised 1% or more of the total. A mixture of policosanol (26%), lactose (70%), talc ( 3 0 ) and magnesium stearate (1%) was introduced in a piece of banana (Musa paradisiaca sapientum), adjusting the amounts to conform to doses of 0.25, 2.5 and 25 mg/kg, as required, and administered orally once daily from 08.30 to 09.00 hr for 54 wk. Control animals received a placebo mixture comprising only the excipients (talc, magnesium stearate and lactose) and excluding the active ingredient. The observers ensured that animals ate all the piece of fruit with the mixture every day. Body weight was monitored bimonthly and doses were adjusted accordingly. Taking into account the phylogenetic similarities between this species and humans, the highest dose selected for this long-term study represents approximately 86 times the recommended human maximal therapeutic dosage. Table I. Composition of diet received by adult male Macuca urcloides hem

Component

Monkey chow ( 1600 CM-08)*

Fats Proteins Carbohydrates Fibre Phosphorus (P) Total minerals [kcal/kg of chow

Fresh fruit and vcgctablcst

Fats Proteins Carbohydrates Phosphorus (P) Iron (Fc) Calcium (Ca) Ascnrbic acid Other vitamins Vitamin A [kcal/kg

Weight (g/lO0 g dry mass) 7.37 28.63 42.63 5.63 1.65 1409 3.99] (g/ I O0 g) 8 24 68 (mg/100 g) 170 5 155 2 1.28 60 IU 160]

*87.91% dry mass and 12.09% watcr. *¢Dictary requirements recommended for adult male macaques by the Food and Nutrition Board National Research Council. 1953 (Spector, 1956).

Policosanol treatment in male monkeys Obserrations. Animals were observed twice a day (from 08.00 to 08.30 hr and from 17.00 to 18.00 hr) for the first 12 wk; subsequently, they were observed once a day (from 08.00 to 08.30 hr) throughout the experiment. The animals" physical condition, their responses to the observer and the characteristics of the faeces and urine (collected on trays that were placed beneath the cages) were recorded. Furthermore, whenever animals were anaesthetized for weighing and blood extraction, they underwent a physical check-up, including evaluation of museulotendinous and palpebral reflexes, palpation of masses and search for signs of toxicity. The behavioural observations were included to determine whether the treatment affected any of the parameters of their behavioural repertoire (Bertrand, 1969; Smith and Byrd, 1983). These observations and the procedures for recording data were similar to those described by the Primates Research Center of San AndrOs Totoltepec of Mexico (Guzm;inFlores et al., 1973; Soils et al., 1985). The behavioural patterns observed were scratching, foetal posture, stereotypes, lipping, vocalizations, startle and grooming. Laboratory assays

Blood and semen samples were drawn under anaesthesia [ketamine hydrochloride (Calypsol, Gedeon Richter Ltd, Budapest, ttungary), 10 mg/kg im]. Blood tests. Blood tests were performed one day before starting treatment (t = 0) and 4, 8, 12, 16, 32, 48 and 54wk thereafter. Blood was drawn after a 12-hr overnight fast by puncture of femoral vessels with a sterile syringe. All laboratory tests were performed on the same day. For haematologicai parameters, haemoglobin values (Hb) were quantified according to the Hainline method (Hainline. 1958) and the percentage haematocrit (Hto) was also determined. Biochemical determinations were performed blind. Blood biochemical parameters were measured by colorimetric enzymatic methods using reagent kits, except for acetylcholinesterase activity, which was estimated according to the method of Voss and Sachsse (1970). The values of serum total cholesterol, blood glucose, glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase were determined at t = 0 (to, baseline) and 4, 8, 16, 32, 48 and 54wk thereafter. At the end of the experiment, triglycerides, high density lipoprotein cholesterol (HDL-C) and LDL-C values were also determined: HDL-C was determined according to the cholesterol content present in the supernatant obtained after fl-lipoprotein precipitation (Seigler and Wu, 1981); LDL-C values were calculated according to the formula LDL-C = Total cholesterol - [(Triglycerides x 0.37) + HDL-C].

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Creatinine, urea and acetylcholinesterase values (AchE) were determined at baseline (t o) and 8, 32, 48 and 54 wk thereafter. Acid phosphatase was determined at to and 32, 48 and 54 wk thereafter; alkaline phosphatase was determined at to and 54 wk thereafter. Urine analysis. After 32 and 54 wk the early m o r n ing urine of all animals was collected and its pH, colour and appearance were noted. In addition, qualitative assays were conducted using reagent strips (L-Combur Test: B6ehringer GmbH, Mannheim, Germany) for detection of proteins, glucose, ketonic bodies, haemoglobin and erythrocytes. The presence of sediment in the urine was assessed by microscopic examination. The Kruskal-Wallis non-parametric statistical test was applied to the results obtained using reagent strips. Spermiogram. Three semen samples were taken (every 3 wk) by means of rectal electroejaculation (Mastroianni and Manson, 1963; Roussel and Austin, 1968). to obtain mean values of the spermiogram parameters before treatment (baseline values). Once treatment had started, samples were taken at wk 8, 12, 24, 32, 48, 51 and 54. The parameters investigated in semen samples were volume, pH, sperm concentration and motility, as well as note of the numbers of living, dead, normal and abnormal spermatozoa, since these are considered to be of most importance in evaluating fertility in primates (Valerio et al., 1970). Ophthahnoh~gieal tests

At wk 54, the ophthalmological tests of refringent means (i.e. cornea, iris, lens, sclera, etc.) and fundoscopy by indirect ophthalmoscopy were performed on all animals. Electrocardiogram (ECG)

At wk 32 and 54 of treatment, unipolar and bipolar limb leads were recorded in all animals. Analysis of the ECG records included the measurements of those parameters commonly used for ECG evaluation in primates (Bcllinger et aL, 1980; Malinow, 1966). Thus, P, QRS complex and T waves amplitude and duration, as well as the PR, ST and RR intervals in the second lead (1I) were analysed. Post mortem stud)'

This study included 14 male monkeys, comprising six controls and the animals treated with the two higher doses of policosanol (2.5 and 25 mg/kg) (four animals/group). All animals were subjected to histopathological examination. Macroscopic examination. After concluding the administration period (54 wk), animals were anaesthetized with 15mg ketamine hydrochloride/kgim and a lethal dose of suxamcthonium chlordihydrate (Asta Werke, Degussa Pharma Gruppe. Germany; 10 mg/kg iv). They subsequently were exsanguinated and subjected to autopsy.

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Pathological anatomy studies were performed in blind fashion in all the controls and all the animals treated with the two higher doses. During the autopsy, the contents of the thorax, cranium and abdomen were analysed in situ before and after evisceration. The weight of the heart, lungs, thyroid, brain, liver, spleen, kidneys, adrenal glands, testes and seminal vesicles was determined. The following organs and tissues were assessed macroscopically: brain (three levels), spinal cord, hypophysis, eyes, turbinates, bone marrow (costal), lymph nodes (mandibular, axillar, thoracic and mesenteric), larynx, thyroid and parathyroid glands, lungs (each Iobule), bronchi, heart, aorta and coronary arteries, salivary glands, oesophagus, stomach, duodenum, jejunum (three levels), caecum, colon (two levels), rectum, pancreas, liver (one segment per Iobule), spleen, kidneys, ureters, suprarenal glands, bladder, sciatic nerve, skeletal muscles and skin. Samples of these organs were fixed in 10% formalin buffered with sodium dibasic phosphate plus potassium monobasic phosphate (pH -- 7.2). Samples of the testes, epididymis, prostate and seminal vesicles were tixed in Bouin's fluid (25 ml formaldehyde, 5 ml acetic acid and 75 ml picric acid). Mh'roscopic observations. Organs and tissues fixed with buffered formalin and Bouin's fluid were stained with haematoxylin and eosin and histopathological examination was performed on paraffin-embedded sections, while tissues tixed with 10% calciumformalin (aorta and liver) were stained with Oil Red O and frozen to be sectioned for microscopic examination. Special anatomopathological obsen,ations. Aortas were removed and opened lengthwise from the aortic vessel up to the bifurcation into the lilac arteries. After observation, a scale of O-3 was established to classify lesions according to their apparent thickness: [0 = no lesion; I = mild lesion (plaque diameter less than 2 mm); 2 = moderate lesion (plaque diameter 2-3 mm); 3 = severe lesion (plaque diameter more than 3 mm)]. Immediately after assessment, aortas were fixed in 10% formaldehyde. Two samples were taken from three standard aortic zones: the zone immediately after the outlet of the aortic arch, the zone at the level of the inferior thoracic artery (seventh intercostal artery) and the abdominal region (below the outlet of the renal arteries). The proximal ends of the three areas selected were sectioned in a cryostat (IEC) and stained with Oil Red O and Sudan IV for lipids; the distal ends were embedded in paraffin and stained with haematoxylin and eosin Alcian blue, pH 2.5 and Stevenel blue (Noa et al., 1985) for glycosaminoglycans (GAGs). Coronary samples were studied taking four equidistant (2 mm intervals) heart segments and processed by means of paraffin embedding. Segments were stained with haematoxylin and eosin, Alcian blue and Stevenel blue. Areas of maximum thickening

of the intima and the media of every animal were measured (Atkinson et al., 1989), using an ocular graticule and an Olympus micrometric slide, with the same magnification of x 100. Four arterial segments per animal were always evaluated. Measurement of the intima was always performed from the elastica and its values were expressed in ram. Mean values and standard deviations for the intima and media of each animal were obtained and afterwards averaged for each group. Animal care and handling, and each of the procedures performed in this work, followed the ethical principles concerning animal experimentation recommended by the Canadian Council on Animal Care (1984).

Statistical analysis Comparisons between groups, of behavioural observations, were performed using the non-parametric Mann-Whitney U-test. The a priori level of significance selected was P < 0.05. Comparisons of percentage changes in cholesterol and of ECG data used the Mann-Whitney U-test. Statistical comparisons of body and organ weight values, haematological and blood biochemical parameters, as well as the parameters relating to semen samples, were carried out by CSS statistical package using the analysis of variance. Fisher's exact probability test was used for comparison between groups of the frequency of animals showing ophthalmological and/or pathological anatomical lesions. RF.~IUI,TS AND DISCUSSION

General ph)'sical condition, behaviour and survival No animal died during the study. No signs of toxicity were noted in any of the animals. Only one animal from the control group (no. 6) and another from the group treated with 0.25 mg policosanol/kg (no. 4) exhibited totally self-depilated regions in both arms and legs 47 wk after treatment started, without presenting any sign of ectoparasitism. Both also presented abraded skin regions produced by selfinflicted bites and scratches, slightly more marked in the monkey treated with policosanol at 0.25 mg/kg. This type of behaviour has been frequently described for primates undergoing long periods of isolation (Sackett, 1972). From 377 daily observations performed, only on 26 occasions (6.9%) did some animals show pasty or semi-liquid faeces without any other alteration, probably owing to something eaten the previous day. This was not related to policosanol administration, since this discrete alteration in the faeces was found in both control and treated animals. Urine colour seldom varied; it depended on the type of food intake (for example of highly pigmented items such as beet, Beta vulgaris), which was observed in both control and treated animals. Beharioural obserrations. The behavioural patterns studied did not show significant statistical differences

Policosanol treatment in male monkeys

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Fig. I. Body weight (mean values) of adult male Macaca arctoides in control (11) group (n = 6) and in groups (each n = 4) receiving 0.25 mg (g~), 2.5 mg (I--1) or 25 mg (1~) policosanol/kg for 54 wk. No significant differences were found among groups (Mann-Whitney U-test). (Mann-Whitney U-test) when groups were compared (scratching, 139.8 __+82.6; foetal posture, 88.7 + 54.9; stereotype, 71. I + 82.7; lipping, 23.9 + 43.8; vocalization, 106.2 + 115.2; flips, 15.6 + 10.9 and grooming. 187.2 +96.5). Some of these patterns varied markedly within the same group. Thus, whereas one of the animals behaved in a given manner repeatedly, another from the same group hardly followed the same behavioural pattern at all. Such variations in behavioural patterns are reflected by the standard deviations. Self-aggressive behaviour found in one of the treated animals (no. 4, 0.25 mg/kg) as well as in one from the control group (no. 6), can be attributed to the long period during which animals remained in individual holding cages. Body weight. Body weight gain was similar in all the groups during the entire study (Fig. I).

Laboratory assays Policosanol orally administered at 0.25, 2.5 and 25 mg/kg to male M. arctoides for 54 wk caused no changes in the blood biochemical and haematological

parameters analysed, apart from the serum lipid profile. Lipid prt~ile. At baseline (to), values were statistically similar in all groups. 8 wk after treatment, the percentage changes of serum total cholesterol in the group treated with the highest dose differed significantly from that of the control. Comparisons between all treated groups and controls were significant from wk 16 until the end of the experiment (wk 54), when all treated groups showed the same significance level (P < 0.01) compared with the control (Table 2 and Fig. 2). No significant differences between groups, for triglyceride and HDL-C values, were found at the end of the experiment. On the other hand, LDL-C levels were significantly lower in monkeys treated with any of the three doses of policosanol (0.25, 2.5 and 25 mg/kg) than in control group animals (Fig. 3). These results demonstrate that policosanol, administered orally for 54wk, has a cholesterol-lowering effect in M. arctoides and that the reduction in serum cholesterol levels is associated with a decrease in

Tahle 2. Effect o f policosanol administered orally for 54 wk on serum cholesterol levels (mmol;litre) in male Macaca arctoides Policosanol (mg/kg)

Treatment (wk)

Control group (n = 6)

0,25 (n = 4)

2.5 (n = 4)

25 (n = 4)

0 4 8 16 32 40 54

3.50 + 0.58 2.92 + 0.36 3 . 8 3 + 1.46 3.48+0.45 3.37+0.38 3.47+0.24 3.27 + 0.45

3.85 + 1.07 2.95 + 0.34 3.08±0.33 343_+ 0,61 ° 2.90 + 0.37* 2 . 7 5 + 0 . 3 1 *° 2.58 4- 0,42 "o

3,53 + 0.95 2.55 + 0.31 2.90+0.18 2.78+0.40* 2.30 + 0.47"* 2.7g ± 0.54* 2.35 + 0.49 *°

3.53 + 0.45 2,70 ± 0.32 2 , 8 5 + 0 . 1 0 °* 2.70+0.35** 2,98 + 0.10 °° 2.93 + 0.15"* 2.75 + 0.24**

Values arc means + SD. Asterisks indicate significant differences from controls ( * P < 0.05. , , . p < 0.01; M a n n - W h i t n e y U test).

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t (weeks) Fig. 2. Effect of policosanol (11, 0 mg; L~, 0.25 rag: f"'l, 2.5 mg; I~1, 25 mg/kg) on percentage changes in serum cholesterol in male Macaca arctoides. Significant differences from control: "P < 0.05: bP < 0.01 (Mann-Whitney U-test).

LDL-C values. Thus, these data are in agreement with previous results obtained when policosanol was administered orally in other experimental models (Alem:in et al., 1991; Arruzazabala et al., 1991a and 1992; Cruz-Bustillo et al., 1991; Rodriguez-Echenique et al., 1992a, b). Moreover, as the cholesterol-lowering effect of policosanol persisted during the study, we can conclude that no tachyphylaxis to treatment is developed. The slight upward shift of total cholesterol values observed in the control group during the study can be

associated with the dietary conditions. For 54 wk these animals received a diet that was relatively rich in protein and carbohydrate and low in fat. This type of diet favours a moderate increase of serum cholesterol values, owing to increased cholesterol endogenous synthesis, and has been discussed in studies on the development of models of atberosclerosis in nonhuman primates (Howard, 1976; Middleton et al., 1967). The haematological and blood biochemical parameters studied are shown in Table 3. This table

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Fig. 3. Effect of poficosanol (El. 0 rag; ~J, 0.25 mg; I"I,2.5 mg; I~. 25 mg/kg) on serum triglyceride(TG). high density lipoprotein-cholesterol(HDL) and low density lipoprotein-cholesterol (LDL-C) levelsin male Macaca arctoides. Significant differences from control: "P < 0.05; bp < 0.01 (Mann-Whitney U-test),

Policosanol treatment in male monkeys Table 3. Haematological and blood biochemical values in male .$tacaca arctoide~

Parameters

Means + SD

Haemoglobin (g, %) Haematocrlt (%) Glycaemia ImmolAitre) Glutamic-oxaloacetic transaminas¢ {U:litre) Glutamic-pyruvic transaminase (U/litre) Creatine (#mol/litre) Urea (mmol, litre) Acetylcfiolinesterase ( .u tool thiocholine, I 0 .u I blood, 10 min) Acid phosphatase (U, litre) Alkaline phosphatase (U litre)

I 1.85 + 0.73

38.93 4- 2.25 3.55 _ 0.68 17.58 _ 4.02 28.59 _+ 8.72 125.74 _+.+11.71 5.05 + 1.20 0.46 _+ 0.05 10.98 _.+2.57 81.81 + 29,87

*Values for control (n = 6) animals only are shown because all these parameters were unaffected by treatment and no significant differences (ANOVA. P < 0.05) were found throughout the whole study.

shows mean and standard deviations of these values in control animals only, because no statistically significant differences were found between groups. Urine analysis. After 32 and 54 wk of treatment with policosanol, qualitative analysis of the urine collected did not reveal alterations in any of the 18 animals. In no case did the reagent strips show traces of proteins, glucose, ketone bodies or blood. No pathological sediments were found on the microscopic analysis.

Ophthalmological examination Through indirect ophthalmoscopy, some alterations in the cornea, lens and retina of some animals were observed; however, these were not related to policosanol administration, since they were present in control and treated animals alike. Most of these changes involved congenital alterations or trauma previous to this research and can be summarized as follows: I. Cornea with nebecula of possible traumatic origin: two animals from control group (nos 2 and 4) and from the 2.Smg/kg dose (nos I and 2). 2. Retinal atrophic plate, probably resulting from a previous swelling process (more than 2 yr): only one animal from the 25 mg/kg dose (no. i). 3. Circumpapillary atrophy, probably congenital: only one animal from the 25 mg/kg dose (no. 2). 4. Retinal and papillary angiomatosis (congenital): only one animal from the 0.25mg/kg dose (no. 4). Comparison between groups of such findings was not significant.

Electrocardiograms ECGs did not reveal pathological alterations in any of the animals. At wk 32, leads II and VL of animal no. I from the 2.5 mg policosanol/kg group had a small notch in the descending branch of wave R (QRS complex), but the duration of the complex (0.4 and 0.45 min, respectively) remained within normal

571

limits. Thus, the existence of blocking signs in this branch was ruled out. In addition, the ECG of the same animal at wk 54 did not reveal any alteration. On the other hand, at wk 54, animal no. I from the 25 mg policosanol/kg group had a slight ST segment shift (less than 0.2 mV) on lead I derivation and a slightly marked Q wave (0.15 mV) on the VL lead, but this was so moderate that it could not be considered pathological. The remainder of the tracings did not reveal any other alteration. Means and standard deviations of the parameters measured in all animals were as follows. P wave duration (msec) and amplitude (mV): 0.10+0.008 and 0.29+0.09; QRS wave: 0.09+0.017 and 1.00+0.44; T wave: 0.19+0.033 and 0.35+0.06; PR interval duration (msec): 0.11 + 0.016; ST interval: 0.18 + 0.020; RR interval: 0.76 + 0.120. Statistical analysis did not reveal any significant differences.

Spermiogram Statistical analysis revealcd that in this experiment there were no major differences in the parameters of the spermiogram, indicating that treatment with poEcosanol for 54 wk has no adverse effect on spermiogenesis. Nevertheless, between May and July a period of very low sperm concentration was noted in all groups, in which several animals presented azoospermia; from December to the following February, the sperm concentration was highest in almost all animals, indicating a seasonal effect on spermiogenesis.

Pathological anatomy 14 animals were killed, comprising six control animals and eight animals treated with the two higher doses (2.5 mg/kg and 25 mg/kg, four animals each). No alterations were observed in the cranial, thoracic or abdominal cavities in any of the animals. Between groups, comparison of organ weight did not reveal significant differences. Arterial pathology o~',he blood ressels. Macroscopic examination post mortem revealed that some monkeys studied presented atherosclerotic lesions in the aorta located at the outlet of the arch. These lesions were classified according to the scale previously described. Table 4 lists these data, showing that policosanol reduced the proportion of treated animals presenting severe lesions (level 3) compared with the controls (P < 0.05, Fisher's test). The comparison of mild lesions between groups (level I) did not reveal significant differences. In three control animals, very severe striated, offwhite lesions, with a diameter of more than 3 mm (level 3) were observed. However. such severe lesions were found in any of the animals treated with policosanol (two different doses). In treated groups, aortic lesions at the outlet of the aortic arch were also observed, although these were less severe than those observed in the control group. In this case, lesions were very restricted plaques (less than 2mm in

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Table 4. Effect of policosanol administered orally for 54 wk on the emergence of aortic lesions in Macaca arctoides male monkeys Percentage of animals presenting lesions scored* as Treatment Control animals Policosanol Policosanol

Dose (mg. kg)

0

I

2

3

0 2.5 25.0

50 50 50

0 50 50

0 0 0

50 0* 0*

° P < 0.05 (Fisher's exact probability test) tO = no lesion; I = mild lesion; 2 = moderate lesion; 3 = severe lesion. For further information, see Materials and Methods.

diameter) and were present in two animals of each treated group. In segments stained with haematoxylin and eosin the above-mentioned lesions were characterized by a n increase of intimal thickness due to the presence of smooth muscle cells, elastin and collagen fibres, as well as an abundance of GAGs, the presence was revealed by Alcian blue and Stevenel blue. In treated animals the arterial lesions were much smaller than those in the control group and no lesions were observed in 50% of animals. The reduction in intimal thickening in policosanol-treated animals compared with controls was highly significant for the groups treated with policosanol at 2.5 mg/kg (P < 0.0001) and at 25 mg/kg (P < 0.001) (Table 5). Nevertheless, comparison between values of mean thickening of the aortic media did not reveal any statistically significant differences. in the study of coronary arteries, thickening of the coronary intima of two monkeys in the control group was observed. The histological characteristics of these lesions were similar to those in the aortas. It is important to point out that these lesions were not observed in coronary arteries of the treated animals. The lesions involved intimal thickening with cell proliferation in the neointima, fibroplasia and GAGs; no foam cells were found. These lesions could be classified as advanced atherosclerotic lesions (fibrous plaques) according to the Haust classification (Haust, 1983). They have been described in various animal species including monkeys (Kramsch et al., 1973; Vesselinovitch et al., 1980), rabbits (Kritchevsky, 1972) and guinea pigs (Noa and lllnait, 1987), as well as in humans (Haust, 1980 and 1983). The existence of high concentrations of G A G s in atherosclerotic lesions of experimental animals, including non-human primates, has been described previously (Wagner and Salisbury, 1978). In studies evaluating the regression of the atherosclerotic lesion,

variations in G A G s were found, indicating a higher G A G content in arteries with intimal thickening. This characteristic has also been found in atherosclerotic lesions of human aortas (Kumar et al., 1967). These results indicate a protective effect of policosanol (2.5 and 25 mg/kg) on the development of atherosclerotic lesions in M. arctoides. This effect became more obvious when comparing the presence of aortic and coronary lesions, since they were the most frequent, which agrees with data obtained in previous studies performed in this and other species of macaques (Middleton et al., 1967; Pick el al., 1974). The development of atherosclerotic lesions is a multifactorial process that is highly dependent on factors such as hypercholesterolaemia (Levy, 1983; Schettler and Habcnitch, 1989), elevated serum thromboxane A: (TxA:) levels (Ellis et al., 1976; Jacobsen, 1983), platelet hyperaggregability (McGregor et al., 1980; Renaud el al., 1970; Wanless, 1984) and endothelial cells circulating in the plasma (Hladovec et al., 1978), among others. Thus, since policosanol lowers total cholesterol and LDL-C levels (Alem:in el al., 1991; Arruzazabala et al., 1991a and 1992; Castafio et al., 1991; Cruz-Bustillo et al., 1991; Herniindez et al., 1992; lllnait et al., 1991; Rodriguez-Echenique et al., 1992a,b), significantly increases prostacyclin levels and reduces serum thromboxane Bz (a stable metabolite of TxA:) levels in rodents (Arruzazabala et al., 1991b and 1993), decreases plasma endothelial cells in rats with endothelial damage induced by sodium citrate (Noa et al., 1991), and produces antiplatelet effects (Arruzazabala et al., 1993), it would be logical to expect that it could prevent the development of atherosclerotic lesions. Macroscopic obserration o f other tissues. Adhesions between the pleura and the diaphragm (probably attributable to previous pleurisy) was found in animal

Table 5. Values of maximum intimal and medial aortic thickening in male Macaca arctaides treated with policosanol for 54 wk Thickness (pm) Trcatmcnt ConLrol animals Policosanol Policosanol

Dose {mg, kg)

Intima

Media

(0) 2.5 25.0

229.38 4- 64.26 75.63 4- 67.72"'" 100.31 4- 88.27 °*

493.75 _+ 64.79 462.50 4- 22.36 478.13 4- 25.62

Values are m e a n s 4- SD: * * p < 0.001; * * * p < 0.0001 (Mann-Whitney

U-test).

Policosanol treatment in male monkeys no. 4 of the control group; reddish areas were noted in the lungs of animals nos I, 2 and 3 from the control group, animal no. I from the 2.5 mg/kg dose group and animal no. 4 from the 25 mg/kg dose group. An enlarged mesenteric lymph node was found in animal no. 3 from the control group. An 8-cm granular lesion, with some hyperaemic areas, was found in the gastric mucosa of the fundic region of control animal no. 6, and a hyperaemic gastric mucosa was found in animal no. 4 of the same group. In four of the monkeys the liver was slightly yellowish; three were control animals (nos I, 5 and 6) and one was from the 2.5 mg/kg policosanol group (no. 3). in animal no. 4 of the 25 mg policosanol/kg group there was a yellowish area in one of the liver Iobules and no. 5 from the control group displayed filiform whitish areas, probably caused by fibrosis resulting from parasitic invasion. In control animal no. 3 a granular area (of the same colour of a nutmeg) was found in one of the liver Iobules, as well as an increase in the size of mescnteric ganglia. A whitish, spheroidal, hypoplastic testicle (left), of 2 cm diameter in the long axis and showing a brown, heartshaped central area when sectioned was found in animal no. 3 of the control group. Several animals (nos 2, 3 and 5 of the control group, nos 2 and 3 of the 2.5 mg/kg group and nos 3 and 4 from the 25 mg/kg group) displayed adhesions of the tunica albuginea (testes and epididymis), probably caused by non-infectious trauma. In six monkeys, one or more whitish, friable and irregular unattached growths were observed in the bladder (three from the control group, one from the 2.5 mg/kg and two from the 25 mg/kg policosanoltreated group). Microscopic observation of other tissues. Some monkeys showed lung congestion with haemorrhage and oedema, which is frequently observed as a result of anaesthetic administration. These areas coincided with the reddish ones observed macroscopically. Emphysema was found in animal no. I of the control group and animal no. I from the group given 2.5 mg policosanol/kg. There were signs of non-specific adenitis with enlarged mesenteric ganglia in control animal no. 3. Hyperplasia of the gastric mucosa, resulting from a stomach lesion, was found in control animal no. 6. Histological sections of the liver, stained with haematoxylin and eosin, had a yellowish tinge, without no structural alteration. No fatty changes were observed in sections stained for lipids. In the liver of animal no. 3 of the control group, a histological section ofan irregular area of the Iobule showed hepatocyte necrosis with haemorrhage and congestion in the sinusoids. Slight hyperplasia of the pancreatic duct was found in two monkeys of the control group (nos 2 and 5). Testicular hypoplasia with some atrophic and residual seminiferous tubules was found in a control

573

animal (no. 3) with reduced testes, the remainder of this organ being fibrotic. Discrete cystic structures in suprarenal adrenal medulla were found in one control animal (no. 6) and in two animals treated with 2.5 mg policosanol/kg (nos I and 3): a cortical microadenoma was found in one control animal (no. 4). A recently infarcted small area of a papillary muscle (involving less than 10% of the total area) was found in animal no. 1 of the 25 mg policosanol/kg group. This was the monkey in which the ECG had shown a slight ST segment shift on bipolar lead II and a slightly marked Q wave on the unipolar VL lead, hut these electrical phenomena should not necessarily be considered a reflection of the small infarcted area. No atherosclerotic lesions were found in the coronary vessels of this animal, suggesting that the infarct was probably attributable to vasospasm of the coronary irrigating the papillary muscle. Thus, myocardial infarctions are relatively frequent in monkeys (Malinow and Blaton, 1984); although coronary atherosclerosis accounts is implicated in 90% of cases, vasospasm is one of the most frequent of the other causes (Cheitlin and Virmani, 1989; Conti, 1985). Fixed and stained sections of the unattached growths found in the bladder revealed irregularlyshaped protein material with scant nuclei in some sections of some of the unattached growths not related to policosanol treatment. The bladders of these animals did not show any histological changes. The lesions observed in some of these monkeys were not apparently drug-related. Moreover, lesions such as gastric mucosal hyperplasia, testicular hypoplasia and hepatic focal necrosis were observed only in control animals. Other lesions, such as adhesions of the testes and epididymis, were observed in most animals, probably as a result of traumatic aseptic inflammation. The unattached growths observed in the bladder were similar in control and policosanol-treated animals. The general conclusion with regard to structural pathology indicates that policosanoi, administered orally at 2.5 and 25 mg/kg for 54 wk to male M. arctoides did not cause any drug-related toxic alteration detectable on macroscopic or microscopic examination, but prevented the development of atherosclerotic lesions in this monkey. Conclusions Policosanol administered orally at 0.25, 2.5 and 25 mg/kg for 54 wk to male M. arctoides monkeys had the following effects: i. A remarkable and maintained reduction in blood cholesterol levels, which was associated with a decrease in LDL-C values. 2. A significant reduction in aortic medial thickening and in the number of animals displaying

574

C. ROlaRiGt;Ez-EcHENIQUE et al. severe atherosclerotic aortic lesions, c o m p a r e d with controls.

At the doses used in this study, policosanol, administered orally, did not induce any o f the following:

I. toxic symptoms; 2. significant changes in behavioural repertoire; 3. significant changes in haematological or blood biochemical parameters: 4. significant changes in spermatogenesis, urine, E C G or results o f o p h t h a l m o l o g i c a l tests; 5. changes in macroscopic or microscopic pathological a n a t o m y , a t t r i b u t a b l e to treatment in the two higher doses studied. These results indicate that policosanol, even at the highest dose administered (25 mg/kg, which is approximately 90 times higher than the maximal h u m a n therapeutic dose o f 20 mg/day), did not have any observable toxic effect. This study has thus d e m o n strated that policosanol, administered orally for 54 wk to male M. arctoules, has a persistent cholesterol-lowering effect a n d a high degree o f safety, a n d is very well tolerated. Acknowh'dgements--Thc authors are grateful to Esteban P6rez, BSc, Professor Jestis A. Nufiez and Mrs Joselina Mederos for their invaluable assistance with the manuscript. REFERENCES

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