Antiatherosclerotic effect of an antibody that binds to extracellular matrix glycosaminoglycans.

OBJECTIVE: Subendothelial retention of proatherogenic lipoproteins by proteoglycans is critical in atherosclerosis. The aim of this study was to characterize the recognition and antiatherogenic properties of a chimeric monoclonal antibody (mAb) that reacts with sulfated molecules. METHODS AND RESULTS: chP3R99 mAb recognized sulfated glycosaminoglycans, mainly chondroitin sulfate (CS), by ELISA. This mAb blocked ≈70% of low-density lipoprotein (LDL)-CS association and ≈80% of LDL oxidation in vitro, and when intravenously injected to Sprague-Dawley rats (n=6, 1 mg/animal), it inhibited LDL (4 mg/kg intraperitoneally, 1 hour later) retention and oxidation in the artery wall. Moreover, subcutaneous immunization of New Zealand White rabbits (n=19) with chP3R99 mAb (100 µg, 3 doses at weekly intervals) prevented Lipofundin-induced atherosclerosis (2 mL/kg, 8 days) with a 22-fold reduction in the intima-media ratio (P<0.01). Histopathologic and ultrastructural studies showed no intimal alterations or slight thickening, with preserved junctions between endothelial cells and scarce collagen fibers and glycosaminoglycans. In addition, immunization with chP3R99 mAb suppressed macrophage infiltration in aorta and preserved redox status. The atheroprotective effect was associated with the induction of anti-CS antibodies in chP3R99-immunized rabbits, capable of blocking CS-LDL binding and LDL oxidation. CONCLUSION: These results support the use of anti-sulfated glycosaminoglycan antibody-based immunotherapy as a potential tool to prevent atherosclerosis.

Effect of D-003, a Mixture of High Molecular Weight Aliphatic Acids, on Glucocorticoid- and Lipopolysaccharides (LPS)-Induced Osteonecrosis.

Osteonecrosis (ON) is characterized through the impairment of osseous blood flow that leads to the collapse of femur head. Corticoid-induced ON in rats and lipopolysaccharide (LPS)-induced in rabbits are useful models to assess the efficacy of potential treatments on this disease. D-003 inhibits the mevalonate pathway, lipid peroxidation and prevents osteoporosis in rats through increasing the osteoclast apoptosis. This study investigated the effects of D-003 on corticoid- and LPS-induced ON in rats and rabbits. Corticoid-induced ON: Rats were randomized into five groups. A negative control and four groups treated with prednisolone 6 mg/Kg: a positive control and three treated with D-003 (5, 25 and 200 mg/Kg) for 80 days. All positive controls presented ON areas. D-003 significantly reduced the numbers and proportions of ON lesions, as compared to the positive control group. LPS-induced ON in rabbits: Rabbits were randomized into five groups: a negative control and four injected with a single intra-venous injection of LPS (10 µg/Kg) including a positive control and three with D-003 (5, 25 and 200 mg/Kg) for 30 days. ON was seen in all positive controls. The incidence of ON and the number of ON lesions in the groups treated with D-003 (25 and 200 mg/Kg) was significantly lower compared to the positive controls. LPS injection significantly increased the size of bone marrow fat cells in positive controls and such increase was significantly decreased by D-003. In conclusion, D-003 reduced ON lesions in corticoid-and LPS-induced ON and also the size of bone marrow fat cells in rabbits with LPS.

Effects of in utero exposure to D-004, a lipid extract from Roystonea regia fruits, in the male rat: a comparison with finasteride.

D-004 is a lipid extract obtained from Cuban royal palm fruits, consisting of a mixture of free fatty acids, that prevents prostate hyperplasia induced with testosterone in rodents. This study investigated the possible alterations due to D-004 of androgen-dependent development after exposure in utero and compared them with those due to finasteride. Rats were randomized into five experimental groups: a control group, three groups treated with D-004 at 500, 750, or 1,000 mg/kg/day, respectively, and a group treated with finasteride (10 mg/kg/day). Male rats were treated 10 weeks before and during mating. Female rats were treated for 15 days prior mating, during mating, during pregnancy, and until lactation (day 21) except for those treated with finasteride, which were only administered the drug on gestational days 12-21. All male offspring were monitored individually until necropsy after postnatal day 90. The results of the present study indicate that D-004 induced no alterations in androgen-dependent development after the exposure in utero. Also, the current study demonstrated a permanent reduction in anogenital distance and retention of nipples in adult male rats exposed to finasteride during late gestation. Significant alterations induced by exposure to finasteride were mainly in tissues dependent on dihydrotestosterone during development.

Effects of D-003 on Lipopolysaccharides-induced Osteonecrosis in Rabbits.

D-003, a mixture of high molecular weight acids, inhibits cholesterol synthesis prior to mevalonate and prevents osteoporosis induced by ovariectomy in rats, and both osteoporosis and osteonecrosis induced by corticoids in rats. The aim of this study was to investigate effects of D-003 on lipopolysaccharides-induced osteonecrosis in rabbits. Animals were randomized into 5 groups: a sham and four groups injected with lipopolysaccharides: one treated orally with vehicle and three with D-003 (5, 25 and 200 mg/kg, respectively) during four weeks. We assessed the effects of treatments on the incidence of osteonecrosis (number of animals with osteonecrosis lesions/animals per group), the mean numbers and areas of osteonecrosis per animal and on the mean sizes of the bone marrow fat cells. The incidence of osteonecrosis in the groups of D-003 25 and 200 mg/kg was significantly lower than in the positive controls. The reduction of osteonecrosis increased with the doses, but significant dose-dependence relationship was not achieved. D-003 significantly and dose-dependently decreased the number of osteonecrosis lesions per animal as compared to the positive controls. Likewise, the mean osteonecrosis areas in the proximal femoral and humeral bones were significantly decreased by D-003. The injection of lipopolysaccharides significantly increased the average size of bone marrow fat cells as compared to the negative controls, and such increase was significantly and markedly reduced with D-003. It is concluded that D-003 reduced the incidence, number and percent areas of osteonecrosis lesions, and the size of bone marrow fat cells, a marker of adipogenesis, in rabbits with lipopolysaccharides-induced ostenonecrosis.

Oral subchronic toxicity of a lipid extract from Roystonea regia fruits in mice.

D-004 is a lipid extract of royal palm (Rosytonea regia) fruits that prevents prostate hyperplasia induced with testosterone in rodents. Previous studies have shown no D-004-related toxicity in rats, but no study in mice had been reported. D-004 (500, 1000, and 2000 mg/kg) was evaluated in a subchronic (eight weeks) study in NMRI mice. No evidences of treatment-related toxicity were detected. Thus, body-weight gain, clinical observations, food consumption, blood biochemical, hematology, organ-weight ratios, and histopathological findings were similar in control and treated groups. This study supports that D-004 orally administered up to 2000 mg/kg did not induce treatment-related toxicity.

D-003 does not possess oestrogenic potential in-vivo: findings of the uterotrophic assay.

D-003 is a mixture of long-chain fatty acids purified from sugarcane wax that inhibits both cholesterol synthesis prior to mevalonate formation, and lipid peroxidation. D-003 has been shown to prevent bone loss and bone resorption in ovariectomized rats, and significantly improves bone resorption markers in postmenopausal women with reduced bone mineral density. As hormone-replacement therapy, D-003 displays cholesterol-lowering and anti-resorptive effects. We have studied its potential oestrogenic activity in-vivo using the uterotrophic assay. Rats were randomly distributed into five groups: a sham-operated group and four groups of ovariectomized rats, one treated with vehicle, one with D-003 (50 mg kg(-1)), one with oestradiol benzoate (30 microg kg(-1)) and one with D-003 (50 mg kg(-1)) plus oestradiol benzoate (30 microg kg(-1)). Treatments were administered for 14 days. Ovariectomy decreased the values of relative uterus weight, epithelium cell height and endometrial thickness compared with sham-operated rats, and these effects were all significantly reduced with oestradiol benzoate, but not with D-003. Concurrent administration of D-003 and oestradiol benzoate had statistically similar effects on all variables as oestradiol benzoate alone. In conclusions, D-003 orally given at 50 mg kg(-1), a dose that prevents bone loss and bone resorption in ovariectomized rats, did not display oestrogenic/anti-oestrogenic activity in-vivo, as assessed in the uterotrophic assay.

Protective effect of policosanol on endothelium and intimal thickness induced by forceps in rabbits.

Policosanol is a cholesterol-lowering drug isolated from sugar cane wax with concomitant antiplatelet effects that prevents lipofundin-induced atherosclerotic lesions in rabbits and rats, including foam cell formation, and also reduces foam cell formation in carrageenan-induced granulomas in rats, while it inhibits proliferation of smooth muscle cells induced in rabbit cuffed artery. This study was undertaken to determine whether policosanol prevents endothelium damage and increase in arterial wall thickness in rabbits with arterial walls damaged with a forceps. Artery forceps were placed over the central artery of the right ear of all rabbits, and each artery was injured eight times. Animals were randomly distributed into four groups: a positive control group treated with Tween 20/H2O vehicle, two groups treated with policosanol (5 and 25 mg/kg, respectively), and a group treated with aspirin (8 mg/kg). Treatments were given for 30 days. Damaged arteries were examined by light and electron (transmission and scanning) microscopy. To evaluate intimal thickening, areas of intima were measured, and a significant reduction in policosanol-treated animals was observed. The endothelial surface, studied with scanning electron microscopy, revealed several types of damage. In control group, the endothelial surface was severely damaged. De-endothelialized areas were reduced in policosanol-treated animals. Platelet adhesion to subendothelium was seen in all animals of the control group, whereas policosanol-treated groups exhibited significantly reduced platelet adhesion. Policosanol also reduced, dose-dependently, the platelet sequestration induced in the damaged vessel wall, partially preventing the reduction in platelet count. It is concluded that policosanol prevents endothelium injury and reduces significantly intimal thickness of rabbit arteries damaged with forceps.

Preventive effect of D-002, a mixture of long-chain alcohols from beeswax, on the liver damage induced with CCl4 in rats.

D-002 is a mixture of higher aliphatic primary alcohols purified from beeswax with antioxidant effects. Acute hepatotoxicity induced with CCl4 in rats has been related to increased hepatic lipid peroxidation and prevented with some antioxidants. This study investigated whether D-002 could prevent the acute CCl4-induced hepatotoxicity in rats. Animals were randomly distributed into four groups: a negative control treated orally with the vehicle and three groups injected with CCl4 (1 mL/kg) and treated orally either with the vehicle (positive control) or with D-002 (25 and 100 mg/kg). Eighteen hours after CCl4 dosing, rats were anesthetized, and livers were removed for histopathological studies. Some portions were taken and homogenized for assessing malondialdehyde (MDA) concentrations. Positive, but not negative, controls showed ballooned cells, swollen hepatocytes, and lipid-included hepatocytes, as well as necrotic areas and inflammatory infiltrates. D-002 (25 and 100 mg/kg) dose-dependently and significantly (P < .01) decreased the frequency of all abnormal liver cell types and increased that of normal hepatocytes compared with the positive controls, not showing necrotic areas or inflammatory infiltrates. D-002 dose-dependently decreased hepatic MDA levels, but only in the highest dose group were these levels significantly lower than in the positive control. In conclusion, D-002 effectively prevented the histological liver disturbances and lowered MDA levels, a marker of cellular lipid peroxidation, in rats treated with CCl4. Since increased liver lipid peroxidation has been postulated as a cause of CCl4-induced liver damage in rats, the preventive effects of D-002 could be due to its antioxidant action, but such a proposal still requires further research.

Effect of D-003, a mixture of very-long-chain aliphatic acids purified from sugarcane wax, on cerebral ischemia in Mongolian gerbils.

D-003 is a mixture of very-high-molecular-weight aliphatic acids purified from sugar cane wax (Saccharum officinarum), which inhibits platelet aggregation and lipid peroxidation. The objective of the present study was to evaluate the effect of D-003 on cerebral ischemia induced by ischemia-reperfusion (I-R) in Mongolian gerbils. Two experimental series were conducted. The first series investigated the effects of D-003 on cerebral edema, neurological symptoms, and mortality in Mongolian gerbils with cerebral ischemia induced by I-R, while the second series investigated the effects on histological markers of cerebral injury, such as edema intensity (vacuolization) and cerebral necrosis. Animals were randomly distributed in five experimental groups: a sham-operated group experiencing surgical handling except the clamping and orally treated with Tween/water vehicle and four groups subjected to the I-R surgical procedure. One of these groups was treated with the same vehicle, and the other three groups received D-003 at 25, 100, and 200 mg/kg, respectively. All treatments were administered for 14 days. D-003 (200 mg/kg) significantly reduced the cerebral edema and clinical symptoms provoked by I-R compared with the positive control group, whereas lower doses (25 and 100 mg/kg) were not effective. Positive control animals showed an injury profile characterized by swelling (tissue vacuolization) and necrosis of neurons in all areas of the brain studied (frontal cortex, hippocampus, and striatum). The results of the histological study were consistent with those observed by determining cerebral edema and symptoms observation. Thus, D-003 at 200 mg/kg significantly reduced histological markers of brain injury (swelling and necrosis) compared with the control group. It is concluded that D-003 administered orally at 200 mg/kg for 14 days protected against cerebral damage caused by bilateral cerebral ischemia in Mongolian gerbils.

Lack of protective effect of D-003, a mixture of high-molecular-weight primary acids from sugar cane wax, on liver damage induced by galactosamine in rats.

D-003 is a mixture of very-high-molecular-weight aliphatic primary acids purified from sugar cane wax, wherein octacosanoic acid is the most abundant. Experimental and clinical studies have shown that D-003 lowers cholesterol and prevents plasma lipoprotein peroxidation (LP). D-003 has protected against the histological changes characteristic of CCl4- and paracetamol-induced hepatic injury in rats, in which LP plays a pivotal role for explaining the resulting hepatotoxicity. Galactosamine induces hepatotoxicity associated with depressed RNA and protein synthesis, not with LP. The aim of this study was to evaluate whether D-003 could prevent hepatoxicity induced by mechanisms others than increased LP. We investigated the effects on galactosamine hepatotoxicity in rats distributed into five groups: a negative control group, a positive control group, and three groups treated with galactosamine and D-003 (5, 25, and 100 mg/kg). To induce liver damage, galactosamine (800 mg/kg) was injected intraperitoneally 30 minutes after dosing with vehicle or D-003. Twenty-four hours later, rats were sacrificed, and livers were immediately removed for histopathological studies. Livers from positive controls showed the characteristic pattern of galactosamine-induced damage. Galactosamine significantly reduced the percentage of normal hepatocytes, increasing both necrotic or lipid-rich hepatocytes compared with negative controls. D-003, however, did not increase the percentage of normal hepatocytes compared with positive controls, indicating that treatment was not effective for preventing the hepatic injury induced with galactosamine. Likewise, D-003 failed to change the content of necrotic and lipid-rich hepatocytes relative to positive controls. It is concluded that D-003 did not protect against the histological changes of galactosamine-induced hepatotoxicity.

Effect of D-003 on intimal thickening and circulating endothelial cells in rabbit cuffed carotid artery.

D-003 is a mixture of very-high-molecular-weight aliphatic primary acids purified from sugar-cane (Saccharum officinarum L.) wax, in which octacosanoic acid is the most abundant component. Previous experimental studies have shown that D-003 not only shows cholesterol-lowering and anti-platelet effects, but also reduces thromboxane B2 and increases prostacyclin levels. It acts by inhibiting cholesterol biosynthesis. The positioning of a non-occlusive silicone collar around the rabbit carotid artery results in the formation of a neointima. Collars were placed around the left carotid for 15 days. The contralateral artery was sham-operated. We included three experimental groups: A control group received vehicle, and two others received D-003 at 5 and 25 mg/kg until sacrificed. Samples of arteries were examined by light microscopy. To evaluate intimal thickening the cross-sectional areas of intima and media were measured. Neointima was significantly reduced in D-003-treated animals compared with controls. Furthermore, the circulating endothelial cell has been studied in this experimental model with endothelium damage. The results demonstrate the protective effect of D-003 on vascular endothelium of the studied rabbits. It is concluded that the protective effect of D-003 against neointima formation and circulating endothelial cells in this experimental model could represent potential beneficial pleiotropic effects in the anti-atherogenic profile of this substance, beyond its cholesterol-lowering and anti-platelet effects independently demonstrated.

Protective effect of policosanol on atherosclerotic plaque on aortas in monkeys.

BACKGROUND: Policosanol is a cholesterol-lowering drug isolated from sugar cane wax with concomitant antiplatelet effects. Previous studies have shown that policosanol prevents lipofundin-induced atherosclerotic lesions in rabbits and rats, including foam cell formation, as well as the development of foam cells in carrageenan-induced granulomas in rats. Policosanol also inhibits smooth muscle cells proliferation induced on rabbit cuffed artery and on forceps-induced arterial wall damage. Furthermore, policosanol administered long term lowered serum cholesterol and prevented the development of atherosclerotic lesions in Macaca arctoides monkeys. The present study was undertaken to determine whether policosanol could change some characteristic features of atherosclerotic lesions, such as macrophage number and immunohistochemical localization of apoA-1 and apoB in aortas of M. arctoides monkeys. METHODS: Fourteen adult male monkeys weighing 6-10 kg and receiving a low fat, protein-rich diet were randomly distributed in three groups: control group (six monkeys) and two other groups (four monkeys/group) treated with policosanol (2.5 and 25 mg/kg) for 54 weeks. Samples of arteries were examined by light microscopy. Monoclonal antibodies were used to evaluate the presence of macrophage, apoA-1 and apoB. RESULTS: Policosanol reduced the presence of macrophages and the occurrence of apoB, whereas increased apoA-1 localization in aortic atherosclerotic lesions compared with control monkeys. CONCLUSIONS: These results suggest the policosanol potential benefit on plaque composition and stability and could explain the protective effects of policosanol on atherosclerosis development.

Effect of D-003, a mixture of very high molecular weight aliphatic acids, on prednisolone-induced osteoporosis in Sprague-Dawley rats.

BACKGROUND: Drugs inhibiting cholesterol biosynthesis may affect bone metabolism through inhibition of the mevalonate pathway resulting in the inhibition of protein prenylation required for osteoclast activity. D-003 is a mixture of high molecular weight aliphatic primary acids purified from sugar-cane (Saccharum officinarum) wax, with cholesterol-lowering effects demonstrated in experimental and clinical studies. D-003 inhibits cholesterol biosynthesis through indirect regulation of HMG-CoA reductase activity. A previous study demonstrated that D-003 prevented bone loss and bone resorption on ovariectomy-induced osteoporosis in rats. Corticosteroid-induced osteoporosis is the result of changes affecting calcium homeostasis, but the hallmark of corticosteroid-induced bone loss is the direct effects on bone cells, such as inhibition of osteoblastogenesis, promotion of apoptosis of osteoblasts and osteocytes, and decrease in bone formation. OBJECTIVE: To determine whether D-003 could prevent the bone loss induced with prednisolone in Sprague-Dawley rats. METHODS: Rats were randomly distributed in five groups (ten rats per group): a sham-operated control and four groups orally treated with prednisolone 6 mg/kg for 80 days; a positive control orally treated with vehicle; and three groups orally treated with D-003 at 5, 25 and 200 mg/kg, respectively. Rats were killed, bones removed and histological variables of bone resorption and formation studied for histomorphometry. RESULTS: Compared with the sham group, prednisolone significantly (p < 0.01) reduced trabecular bone volume (TBV), while D-003 significantly (p < 0.001) and dose-dependently prevented the prednisolone-induced reduction of TBV. Treatment with prednisolone lowered (p < 0.001) trabecular thickness (TbTh) and number (TbN), while increasing (p < 0.001) the gap between trabeculae. D-003 (5, 25 and 200 mg/kg/day) significantly (p < 0.001) and dose-dependently prevented the reduction of TbTh and TbN and the increase of trabecular gap induced with prednisolone. Treatment with prednisolone increased both the surface and number of osteoclasts compared with sham (p < 0.001). D-003 (5-200 mg/day), however, prevented this effect (p < 0.001 for all comparisons). D-003 also prevented (p < 0.001) the reduction of osteoblast surface (ObS/BS) induced by prednisolone. Osteonecrotic areas were observed in all positive controls, but in none of the sham animals. Positive controls showed hypertrophy of bone marrow adipocytes and lipid-laden pluripotential stromal cells in bones. A significant and dose-dependent reduction of the frequency of animals showing prednisolone-induced osteo-necrosis was observed across the doses of D-003 (5, 25 and 200 mg/kg) investigated here. CONCLUSIONS: D-003 (5, 25 and 200 mg/kg) prevented trabecular bone loss and femoral neck osteonecrosis induced with prednisolone in Sprague Dawley rats, also increasing osteoblast surface and reducing bone resorption parameters. These results suggest that D-003 could be useful for managing corticosteroid-induced osteoporosis.

Comparison of the cholesterol-lowering effects and toxicity of D-003 and lovastatin in normocholesterolaemic rabbits.

BACKGROUND: D-003 is a mixture of long-chain aliphatic primary acids isolated from sugar-cane wax and having cholesterol-lowering effects and a safety profile that have been proven in animals and in previous clinical studies in healthy volunteers. Lovastatin, the first member of the statin class, is an effective and well tolerated cholesterol-lowering drug. Some lovastatin-related adverse effects have been reported, and preclinical assessment has shown that the rabbit is the most sensitive species to lovastatin toxicity. OBJECTIVE: To compare the cholesterol-lowering effects and toxicity pattern of D-003 and lovastatin in normocholesterolaemic rabbits. METHODS: In order to study cholesterol-lowering effects, rabbits were randomly distributed into three groups (eight animals/group): one control group, only receiving the vehicle, and two groups treated with D-003 or lovastatin at 5 and 10 mg/kg/day, respectively. All treatments were orally administered for 30 days. To study toxicity, rabbits were distributed into four groups (six animals/group): one control group and three groups treated with D-003 200 and 400 mg/kg, respectively, or lovastatin 100 mg/kg. RESULTS: After 30 days, D-003 5 mg/kg and lovastatin 10 mg/kg significantly (p < 0.05) and similarly lowered serum total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) levels versus baseline. D-003, but not lovastatin, increased high-density lipoprotein-cholesterol (HDL-C) significantly (p < 0.05), whereas only lovastatin decreased (p < 0.05) triglycerides. Low doses of both drugs did not change safety indicators. D-003 (200 and 400 mg/kg) and lovastatin (100 mg/kg) administered for 10 days reduced TC and LDL-C levels significantly (p < 0.05). HDL-C values increased significantly (p < 0.05) with D-003, but were unchanged with lovastatin. Neither treatment affected triglycerides. No significant changes in lipid profile were observed in the control groups of the two series. Lovastatin 100 mg/kg impaired bodyweight gain and food consumption versus the controls, while D-003 did not. Lovastatin 100 mg/kg increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) values (p < 0.05 versus baseline and controls) and liver weight (p < 0.05 versus controls). D-003 200 or 400 mg/kg did not affect AST, ALT or liver weight. Lovastatin 100 mg/kg, but not D-003 200 or 400 mg/kg, induced typical hepatocellular and renal tubular necrosis in the rabbits. CONCLUSIONS: D-003 5 mg/kg/day administered orally for 30 days to normocholesterolaemic rabbits lowered LDL-C and TC, as did lovastatin 10 mg/kg. D-003 was more effective in increasing HDL-C, while lovastatin was more effective in lowering triglycerides. Administration of higher doses for 10 days did not show D-003-related toxicity, but did demonstrate the typical pattern of lovastatin-induced toxicity in rabbits.

Effect of D-003 on isoproterenol-induced myocardial necrosis in rats.

D-003 is a mixture of high-molecular-weight aliphatic primary acids purified from sugar cane wax with antiplatelet and cholesterol-lowering effects. Cardiac lesions induced by isoproterenol (ISO) are characterized by myocardial necrosis and exudative infiltration. The objective of this study was to determine whether D-003 shows protective effects against ISO-induced myocardial necrosis in rats. Effects of orally administered single doses of D-003 (25-400 mg/kg) and acetylsalicylic acid (ASA, 30 mg/kg), as well as repeated doses of D-003 (5-200 mg/kg), on characteristic markers of ISO-induced myocardial necrosis in rats were investigated. D-003 administered as single doses dose-dependently decreased necrosis area, percent of infarct area, and the presence of polymorphonuclear cells (PMNs) in myocardial tissue, but only the reductions induced by 200 and 400 mg/kg were significant. Oral acute treatment with ASA also decreased necrosis area and percent of infarct area, but the occurrence of PMNs was unchanged. D-003 administered repeatedly for 10 days also decreased all myocardial necrosis indicators in a dose-dependent manner, with results effective from 25 mg/kg to the highest dose tested, indicating that the repeated dose scheme was more effective to prevent the damage. It is concluded that D-003 shows a protective effect on the myocardial necrosis induced by ISO in rats.

Ultrastructural evidences of HCV infection in hepatocytes of chronically HCV-infected patients.

In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied by transmission electron microscopy (EM) and immunoelectron microscopy (IEM). The 13 biopsies showed ultrastructural cell damage typical of acute viral hepatitis. In four of the 13 liver biopsies enveloped virus-like particles (VLPs) inside cytoplasmic vesicles and in the cytoplasm of hepatocytes were observed. We also detected the presence of unenveloped VLPs mainly in the cytoplasm and in the endoplasmic reticulum. IEM using anti-core, E1 and E2 monoclonal antibodies (mAbs) confirmed the specific localization of these proteins, in vivo, inside cytoplasm and endoplasmic reticulum. Thus, this work provided evidence for hepatocellular injury related to HCV infection. It also suggested the presence of HCV-related replicating structures in the cytoplasm of hepatocytes and raised the possibility of hepatitis C virion morphogenesis in intracellular vesicles.

Effect of policosanol on carbon tetrachloride-induced acute liver damage in Sprague-Dawley rats.

BACKGROUND: Policosanol is a cholesterol-lowering drug purified from sugarcane (Saccharum officinarum, L.) wax. Beneficial pleiotropic effects of policosanol, such as inhibition of the susceptibility of low density lipoprotein to lipid peroxidation, have been shown. Policosanol has a good safety profile and well tolerated and, to date, no drug-related adverse effects have been demonstrated. Specifically, policosanol has not been shown to affect liver function or to increase liver enzyme levels in experimental or clinical studies. AIM: This study was conducted to determine whether policosanol prevents liver damage induced by carbon tetrachloride (CCl4) in rats, since this model has been associated with an increased rate of lipid peroxidation. METHODS: Male Sprague-Dawley rats were randomised to four experimental groups: negative controls (no CCl4 or policosanol, group 1); positive controls (CCl4 but no policosanol, group 2); policosanol 25 mg/kg (group 3) and policosanol 100 mg/kg (group 4). Acute liver injury was induced in groups 2, 3 and 4 by CCl4 suspended in olive oil and administered at a dose of 1590 mg/kg via intraperitoneal injection. Eighteen hours after CCl4 dosing, the rats were anaesthetised and their livers removed for histopathological studies. RESULTS: Policosanol 25 and 100 mg/kg dose dependently and significantly (p < 0.01) decreased the percentage of ballooned cells and hepatocytes with lipid inclusions and increased the percentage of normal hepatocytes compared with positive controls. The percentage inhibition of the occurrence of ballooned cells and hepatocytes with lipids was marked, reaching 71 and 49%, respectively, with the higher dose (100 mg/kg). The percentage of swollen hepatocytes was unchanged by policosanol compared with positive controls. No histological alterations in liver sections were found in the negative control group. Necrotic areas and inflammatory infiltrates were observed in the liver of seven of eight (87.5%) animals in the positive control group. However, only one of eight (12.5%) animals treated with policosanol 25 mg/kg and none (0%) treated with the higher dose (100 mg/kg) showed such a pattern. CONCLUSIONS: Policosanol protected against the histological changes characteristic of CCl(4)-induced hepatic injury in rats, a model of hepatotoxicity in which the process of lipid peroxidation plays a role. Further studies aimed at demonstrating the connection between such hepatoprotective and antioxidant effects of policosanol must be initiated.

Six-month toxicity study of oral administration of D-003 in Sprague Dawley rats.

BACKGROUND: D-003 is a mixture of high molecular weight aliphatic primary acids purified from sugarcane wax (Saccharum officinarum) having cholesterol-lowering and antiplatelet effects. AIM: This study was undertaken to investigate the toxicity induced by long-term oral administration of D-003 for 6 months to Sprague Dawley rats of both sexes. METHODS: Rats were randomly divided into four groups (20 rats of each sex/group): a control group. which received the vehicle, and three treatment groups, which received oral D-003 at doses of 250, 500 and 1000 mg/kg/day, respectively. Daily clinical observations and control of bodyweight and food consumption were conducted throughout the study period. On completion of active treatment, animals were sacrificed. Pharmacological effects associated with D-003 such as inhibition of platelet aggregation and increase in bleeding time were assessed in two satellite groups (14 animals of each sex/group): a control group and a group treated with the highest dose of D-003. Assessments of platelet aggregation to collagen were performed at baseline and at 6 months, and assessments of bleeding time were done at baseline, after 3 and 6 months of treatment, and after 30 days' washout. RESULTS: As expected, D-003 significantly inhibited platelet aggregation. Bleeding time was increased after 3 months of treatment with D-003; this increase was maintained at 6 months, and was reversible after washout. Coagulation factors such as prothrombin time and kaolin-activated thromboplastin-time, which were determined in eight male animals from each group, were unaffected by D-003. Data analyses of bodyweight gain, food consumption, clinical observations, blood biochemistry, haematology, organ weight ratios and histopathological findings did not show trends related to D-003 dose or significant differences between control and treated groups. CONCLUSION: It was concluded that the highest studied dose of D-003 (1,000 mg/kg/day) represented a non-toxic dose level in the present chronic toxicity study in rats.