Antioxidants, such as vitamin E, are being investigated for efficacy in cancer prevention. There are indications that it may have efficacy against bladder (Liang et al, 2008
) and mammary cancer (Shet et al
, 2008; Sharhar et al, 2008
; Suh et al, 2007
). The studies presented in this manuscript were carried out to determine the toxicity profile of PMCol in rats and dogs. In general, toxicity in rats was greater than that seen in dogs in the studies performed, which was apparently due to the lower dose in dogs on mg/m2
basis in addition to apparent lower bioavailability in dogs. Mild hepatotoxicity at higher dose levels was observed in rats. Hepatotoxicity of PMCol was exhibited in both male (TBILI, ALB, and ALT levels were significantly elevated at 2000 mg/kg/day) and female (TBILI, ALB, and TP levels were significantly elevated at 500 and 2000 mg/kg/day) rats. However, the apparent hepatoxicity observed in the clinical pathology parameters evaluated did not cause concommittant changes in liver morphology. In studies conducted by other investigators on vitamin E, rats had increased lipid content in the liver (Wheldon et al, 1983
). Body weight gains were significantly decreased on day 8 in the 2000 mg/kg/day dose group males, which was due to significantly decreased food consumption during the first week of dosing in rats receiving 2000 mg/kg/day. These findings are likely due to the gastroirritation observed following histopathological evaluation of the rats receiving 2000 mg/kg/day, and may not be related to the mild hepatoxicity observed. In male rats, administration of PMCol caused a dose-dependent decrease in prothrombin time (PT), which was significant in the 500 and 2000 mg/kg/day dose groups. Fibrinogen concentrations were also dose-dependently increased, significantly in the 500 and 2000 mg/kg/day dose group males. In females, PT was dose-dependently decreased, significantly in the 100 mg/kg/day dose group. Thus PMCol appears to influence glycogen and protein (e.g., albumin and fibrinogen) synthesis metabolic pathways in the liver, likely a result of the mild hepatoxicity.
Nephrotoxicity was also observed at high dose administration of PMCol in rats. In the 2000 mg/kg/day dose group, significant increases in BUN levels (males and females) and in BUN/CREA ratio (females) were seen. Significant decreases in IP concentrations were observed in the 500 and 2000 mg/kg/day males, likely due to decreased reabsorption in the renal tubules. These evident changes in kidney function were confirmed by histopathology. Microscopic evaluation of the kidneys revealed apoptosis and tubular regeneration.
Significant hematological changes were noted in the rats at doses above 500 mg/kg/day, including statistically significant decreases in RBC counts, as well as HBG and HCT levels. These decreases appear to be the result of RBC destruction (i.e., hemolytic anemia), however this cannot be confirmed with these data obtained in this study. There was no evidence of any effect on hematopoiesis in the bone marrow sections evaluated or evidence of extramedullary hematopoiesis in the spleen sections evaluated histopathologically. Abdo et al, reported that following 2000 mg/kg/day dietary administration rats showed hemorrhagic diatheses, which resulted in similar decreases in hematological values.
PMCol treatment at 800 mg/kg/day resulted in statistically significant decreases in body weights and body weight gains in dogs, likely due to decreases in food consumption. At 800 mg/kg, clinical chemistry changes including decreased levels of total protein (due to hypoalbuminemia) were seen. Biologically relevant changes consistent with slight toxic injury of the liver were seen in week 4, as increased levels of ALP and ALT. Statistically significant decreases in triiodothyronine (T3) and thyroxin (T4) were seen in dogs dosed at 800 mg/kg/day, which were likely due to liver clearance and decreased albumin concentrations, rather than pituitary function since the thyroid-stimulating hormone levels were not increased. The free forms of the T4 were below the limits of detection, but still may be of sufficient quantity to sustain thyroid-pituitary balance and not stimulate a response by the pituitary. If there were any toxic effects on the pituitary, they were masked by the hepatotoxicity and decreased kidney function in the dogs, as well as the decreased nutritional status of the dogs as seen in the decreased food consumption. Lack of histopathological findings in the pituitary also reduces the potential of direct toxicity. This is an advantage over vitamin E administration, in which significant increases in TSH were observed at doses as low as 125 mg/kg/day following 90 days of administration (Abdo et al, 1986
). It should however be noted that if the animals were continued on PMCol treatment for a longer period of time, effects on TSH may eventually develop as a result of the effects on T4. Treatment-related histopathological findings correlated to clinical chemistry changes were limited to the dogs dosed at 800 mg/kg/day. Histopathology lesions included a microvesicular vacuolation of midzonal to periportal hepatocytes and atrophy of the thymus, which was considered normal due to the age of these dogs.
There were significant differences noted in the toxicity profiles between dogs and rats. For example, plasma hormone levels (T4 and TSH) were increased in rats whereas in dogs T4 and T3 were significantly decreased in the 800 mg/kg dose group (). Histopathological changes in dogs and rats also differed in that there were no lesions in the liver of rats, but dogs had histopathological changes in the liver consistent with mild toxicity related to administration of PMCol at 800 mg/kg/day.
Analysis of plasma drug levels indicated there was a large difference in peak plasma levels between dogs and rats at the doses administered in the studies (, Panel B). At 2 hours post-dosing in week 4, plasma levels in rats were approximately 400 ng/mL (500 mg/kg/day) or greater, where dogs were mostly below 10 ng/mL at a dose of 800 mg/kg/day at this same time point. This is probably due to a lower bioavailability in dogs versus rats or possible differences in metabolism; however, differences in the preparation of the dosage formulation between the rats and dogs could play a role. The majority of PMCol in dogs appeared to be protein bound, which likely played a role in the lower toxicity observed in dogs. Results of this analysis indicate that the free fraction of PMCol was determined to be at less than 15% of the total circulating PMCol. However, this was based on a limited number of samples. PMCol is rapidly metabolized, as multiple peaks were observed in the LC/MS/MS chromatograms in addition to the parent compound during the analyses of both rat and dog plasma samples. When comparing dose administered to body weight (mg/kg), dogs were more sensitive than rats. This can be seen by the results of the 14-day toxicity study in dogs in which a dose of 2000 mg/kg resulted in moribund sacrifice of this dog. This was following a reduction in the dose to 1000 mg/kg after four days of administration at 2000 mg/kg. However, when an adjustment is made for relative body surface area to the dose levels (see and ), it appears that rats were more sensitive than dogs. This is likely due to the increased levels of PMCol parent compound in circulation exhibited by rats over dogs; however, differences in absorption, metabolism, and distribution may also have a role in the differences observed in toxicity between dogs and rats.
The results of these studies indicated that administration of PMCol at higher dose levels resulted in severe toxicity in dogs and moderate toxicity in rats, however, administration at lower levels is considered to be less likely to result in toxicity following 28 days of exposure. Due to the low levels of drug detected in the plasma of dogs, an unequivocal determination of toxicity as it relates to exposure cannot be made. However, even at these low exposure levels toxicity is evident in dogs at the higher dose level. In addition, obvious metabolite formation observed during plasma analysis confirmed exposure. It was not possible to identify and measure the metabolites within the scope of this study. Sex-related differences were seen in rats. Male rats appeared to have greater sensitivity to nephrotoxicity, while female animals had a greater incidence of hepatoxicity and changes in hematological parameters evaluated, especially at a dose of 500 mg/kg/day, which was likely due to the higher plasma drug levels in female rats. It appeared that dogs were generally more sensitive than rats to oral administration of PMCol. Further examination of the potential toxic effects of PMCol in longer term studies is required prior to understanding the full risks of PMCol administration as a chemopreventative agent.