Miriam Noa

A 12-month study of policosanol oral toxicity in Sprague Dawley rats.

Policosanol is a natural mixture of higher aliphatic primary alcohols. Oral toxicity of policosanol was evaluated in a 12-month study in which doses from 0.5 to 500 mg/kg were given orally to Sprague Dawley (SD) rats (20/sex/group) daily. There was no treatment-related toxicity. Thus, effects on body weight gain, food consumption, clinical observations, blood biochemistry, hematology, organ weight ratios and histopathological findings were similar in control and treated groups. This study supports the wide safety margin of policosanol when administered chronically.

Toxicity of policosanol in beagle dogs: one-year study

Policosanol is a new chemical entity composed of 8 higher aliphatic alcohols obtained from sugar cane (Saccharum officinarum), L. wax, whose cholesterol-lowering effects have been demonstrated in experimental models, healthy volunteers and patients with type II hypercholesterolemia. This study investigated the oral toxicity of policosanol administered for 52 weeks to beagle dogs. Twenty-four beagle dogs (12 males and 12 females) were distributed randomly in 3 experimental groups (4 animals/group): a control and 2 treated groups receiving policosanol at 30 and 180 mg/kg daily (7 days/week) by gavage. No mortality was observed in any group. Overall, policosanol was well tolerated throughout the study and no toxic symptoms were observed. All groups showed similar weight gain and food consumption. Lipid profile determinations showed that policosanol decreased total cholesterol by 20% approximately from 8 to 52 weeks. Cholesterol-lowering effects did not wear off during the study, thus demonstrating the persistence of the effectiveness. Triglycerides and high density lipoprotein-cholesterol (HDL-C) were not changed significantly. No blood biochemistry or histopathological disturbances attributable to treatment were observed. This study has shown that no drug-related toxicity was induced by policosanol administered up to 180 mg/kg/day for 52 weeks to beagle dogs. Since this dose is approximately 620 times higher than the maximal recommended therapeutic dose (20 mg/day) it indicates a good safety margin of this product.

Effect of D-004, a Lipid Extract from the Cuban Royal Palm Fruit, on Atypical Prostate Hyperplasia Induced by Phenylephrine in Rats

AbstractBackground and objective: Benign prostatic hyperplasia (BPH) is a non-malignant enlargement of the prostate that results in obstructive lower urinary tract symptoms. Saw palmetto (Serenoa repens), the dwarf American palm (Arecaceae family), is commonly used to treat BPH. The Cuban royal palm (Roystonea regia) also belongs to the Arecaceae family, and 200–400mg of D-004, a lipid extract from its fruits, administered orally for 14 days has been shown to prevent testosterone- but not dihydrotestosterone-induced prostatic hyperplasia in rats. D-004 (125–250 μ/mL) added to preparations of rat vas deferens caused a marked, dose-dependent and significant inhibition of noradrenaline-induced smooth muscle contraction, a response mediated through α1-adrenoceptors, and was more effective in these respects than Saw palmetto. However, the in vivo effects of D-004 and Saw palmetto on the hypertensive response induced by noradrenaline were modest (albeit significant), and neither treatment affected resting blood pressure or heart rate in rats. The differential effects of D-004 in in vitro and in vivo models could be related to a differential affinity for adrenoceptor subtypes or to different bioavailabilities in vascular and urogenital targets. Phenylephrine injected into rodents induces prostatic hyperplasia with all the characteristic morphological changes of the condition but does not result in enlargement of the prostate. Therefore, this phenylephrine-induced change in rat prostate tissue is called atypical prostatic hyperplasia. It serves as an in vivo model of prostatic hyperplasia induced by stimulation of α1-adrenoceptors. The objective of this study was to determine whether D-004 can inhibit induction of atypical prostatic hyperplasia by phenylephrine in rats. Methods: Rats were randomly distributed into five groups (ten rats/group). One group was a negative control and received oral vehicle only. The other four groups were injected subcutaneously with phenylephrine (2 mg/kg): of these groups, one was a positive control receiving the vehicle, and the other three groups were treated with D-004 or Saw palmetto (both 400 mg/kg) or tamsulosin 0.4 mg/kg. All active treatments were given orally for 28 days. After completion of treatment, rats were placed unrestrained in metabolic cages and micturition studies were performed. The rats were later killed and their prostates removed and weighed. Prostate samples were processed for histological study, with histological changes being assessed according to a scoring system. Bodyweight was measured at baseline and at weekly intervals. Results: Histological examination of positive control rats revealed features of atypical prostatic hyperplasia, with piling-up, papillary and cribiform patterns and budding-out of epithelial cells. Micturition assessment revealed that phenylephrine significantly lowered both the total volume of urine in 1 hour and the volume per micturition; the latter was considered the main efficacy variable. D-004 and Saw palmetto extracts significantly prevented this reduction in volume per micturition by 70.5% and 68.6%, respectively, while tamsulosin totally abolished the reduction in micturition induced by phenylephrine (100% inhibition). Tamsulosin, D-004 and Saw palmetto significantly reduced the histological changes of atypical prostatic hyperplasia induced by phenylephrine by 73.1%, 61.2% and 50.0%, respectively. Conclusions: Administration of D-004 resulted in marked and significant prevention of phenylephrine-induced impairment of micturition and histological changes in rat prostate. These findings indicate that, in vivo, D-004 effectively opposes these responses to phenylephrine, which are mediated through urogenital α1-adrenoceptors. In this respect, D-004 was moderately more effective than Saw palmetto, a phytotherapeutic standard used to treat BPH, but less effective than tamsulosin, a selective α1A-adrenoceptor antagonist.

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.

Acute and oral subchronic toxicity of D-003 in rats

D-003 is a mixture of higher aliphatic primary acids purified from sugar cane wax (Saccharum officinarum) with cholesterol-lowering and antiplatelet effects experimentally proven. The present work reports the results of two studies investigating the acute and subchronic oral toxicity of D-003 in rats. Oral acute toxicity of D-003 (2000 mg/kg) was investigated according to the Acute Toxic Class (ATC) method (an alternative for the classical LD(50) test), which was performed in Wistar rats. The results obtained in this study defined D-003 oral acute toxicity as unclassified. In the subchronic study, rats of both sexes were orally treated with D-003 at 50, 200 and 1250 mg/kg for 90 days. At this time, animals were sacrificed. No evidence of treatment-related toxicity was detected during the study. Thus, data analysis of body weight gain, food consumption, clinical observations, blood biochemical, haematology, organ weight ratios and histopathological findings did not show significant differences between control and treated groups. It is concluded that D-003 orally administered to rats was safe and that no drug-related toxicity was detected even at the highest doses investigated in both acute (2000 mg/kg) and subchronic (1250 mg/kg) studies.

Comparison of the effects of D-003 and policosanol on lipid profile and endothelial cells in normocholesterolemic rabbits

Abstract Background: Policosanol is a cholesterol-lowering drug purified from sugar cane wax that consists of a mixture of higher aliphatic primary alcohols. D-003 is a mixture of long-chain aliphatic primary acids isolated from the same source with experimentally proven cholesterol-lowering effects. Objective: The present study was undertaken to compare the cholesterol-lowering effects of D-003 and policosanol in normocholesterolemic New Zealand rabbits. In addition, the effects of the 2 drugs on levels of plasma circulating endothelial cells (PCEC) before and after citrate-induced endothelial damage were compared. Methods: Animals were randomly distributed to 3 experimental groups: 1 control and 2 treatment groups. Rabbits in the treatment groups received orally administered policosanol or D-003 daily at 5 mg/kg, the lowest effective dose of policosanol in this model, for 30 days. Results: After 15 days of treatment, both D-003 and policosanol significantly ( P P P P P P P P P Conclusions: D-003 administered orally at 5 mg/kg for 30 days to normocholesterolemic rabbits induced more beneficial changes in serum LDL-C levels than policosanol at the same dose. In addition, a slightly greater beneficial effect on HDL-C levels and PCEC count was noted with D-003 versus policosanol. Although D-003 was more effective than policosanol in these experimental conditions, additional comparative studies that include a wide dose range of both drugs must be conducted before definitive conclusions can be made about the comparative cholesterol-lowering effects of D-003.

Effect of D-003, a Mixture of Very High Molecular Weight Aliphatic Acids, on Prednisolone-Induced Osteoporosis in Sprague-Dawley Rats

AbstractBackground: 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.

Effect of Policosanol on Lipofundin-induced Atherosclerotic Lesions in Rats

Policosanol is a mixture of higher aliphatic alcohols isolated from sugar cane wax, showing cholesterol‐lowering effects and preventing the development of lipofundin‐induced lesions in New Zealand rabbits. This study was conducted to determine whether policosanol orally administered to rats also protects against the development of lipofundin‐induced atherosclerotic lesions.

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 &mgr;g, 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.

Six-Month Toxicity Study of Oral Administration of D-003 in Sprague Dawley Rats

AbstractBackground: 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 (1000 mg/kg /day) represented a non-toxic dose level in the present chronic toxicity study in rats.