In vivo evaluation of combination therapy targeting the isoprenoid biosynthetic pathway.

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthetic pathway (IBP), produces the isoprenoid (geranylgeranyl pyrophosphate, GGPP) used in protein geranylgeranylation reactions. Our prior studies utilizing triazole bisphosphonate-based GGDPS inhibitors (GGSIs) have revealed that these agents represent a novel strategy by which to induce cancer cell death, including multiple myeloma and pancreatic cancer. Statins inhibit the rate-limiting enzyme in the IBP and potentiate the effects of GGSIs in vitro. The in vivo effects of combination therapy with statins and GGSIs have not been determined. Here we evaluated the effects of combining VSW1198, a novel GGSI, with a statin (lovastatin or pravastatin) in CD-1 mice. Twice-weekly dosing with VSW1198 at the previously established maximally tolerated dose in combination with a statin led to hepatotoxicity, while once-weekly VSW1198-based combinations were feasible. No abnormalities in kidney, spleen, brain or skeletal muscle were observed with combination therapy. Combination therapy disrupted protein geranylgeranylation in vivo. Evaluation of hepatic isoprenoid levels revealed decreased GGPP levels in the single drug groups and undetectable GGPP levels in the combination groups. Additional studies with combinations using 50% dose-reductions of either VSW1198 or lovastatin revealed minimal hepatotoxicity with expected on-target effects of diminished GGPP levels and disruption of protein geranylgeranylation. Combination statin/GGSI therapy significantly slowed tumor growth in a myeloma xenograft model. Collectively, these studies are the first to demonstrate that combination IBP inhibitor therapy alters isoprenoid levels and disrupts protein geranylgeranylation in vivo as well as slows tumor growth in a myeloma xenograft model, thus providing the framework for future clinical exploration.

Geranylgeranyl pyrophosphate counteracts the cataractogenic effect of lovastatin on cultured rat lenses.

Statins are commonly prescribed cholesterol-lowering agents which inhibit the rate-limiting enzyme of the cholesterol biosynthetic pathway. In addition to inhibiting cholesterol synthesis, statins also inhibit the synthesis of other sterol and non-sterol compounds produced by the pathway including the isoprenoids, farnesyl (FP) and geranylgeranyl pyrophosphate (GGP). Certain proteins, most notably small GTP-binding proteins of the Ras superfamily, must be post-translationally modified by addition of a farnesyl or geranylgeranyl moiety in order to be properly targeted to membranes and to be active. Statins have been shown to affect cellular processes such as proliferation, signaling and apoptosis and it is likely that these effects are due, at least in part, to decreased isoprenoid synthesis. Certain statins have been shown to produce cataracts in experimental animals. We have previously demonstrated that lenses exposed to lovastatin during organ culture may develop cataracts as well, and we proposed that this resulted from decreased prenylation of small GTP-binding proteins. To test our hypothesis, rat lenses were exposed to lovastatin in organ culture with concomitant supplementation of the medium with GGP and/or FP. The results clearly demonstrated that GGP strongly inhibited lovastatin-induced lens opacification in this system while FP had little effect. GGP also markedly reduced the histological changes and the increased epithelial cell apoptosis induced in the cultured lenses by lovastatin. The data indicate that inhibition of protein prenylation, perhaps of Rho GTPases, is an important factor in the lovastatin-induced cataract in vitro.

Lovastatin effects on human breast carcinoma cells. Differential toxicity of an adriamycin-resistant derivative and influence on selenocysteine tRNAS.

Selenocysteine tRNA[Ser]Sec isoacceptors contain the modified nucleotide i6A immediately 3' to the anticodon. Because synthesis of i6A is expected to be inhibited by lovastatin, the status of tRNA[Ser]Sec isoacceptors was examined in human breast carcinoma cells. As part of the initial characterization of these cells, it was determined that an adriamycin resistant derivative of the MCF-7 cell line exhibited a dramatic increase in the sensitivity to the killing effects of lovastatin relative to the parental MCF-7 cells. When MCF-7Adr cells were incubated with high levels of lovastatin, there was a dramatic perturbation in the distribution of isoacceptors within the selenocysteine tRNA population. Lovastatin may therefore be a useful reagent for both the study of differential killing of drug-resistant tumor cells and selenoprotein biosynthesis.

Biochemical changes and morphological alterations of liver and kidney in hamsters after administration of the HMG-coenzyme A reductase inhibitor, simvastatin: prevention and reversibility by mevalonate.

The present study analyses the effects of simvastatin, a specific inhibitor of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA reductase) in male Syrian hamsters fed a standard diet or a diet supplemented with 0.12% cholesterol and 20% coconut oil. In hamsters fed the standard diet, gastric administration of simvastatin (10 mg/kg/day) during 12 days was found to be lethal and to have hepatotoxic and nephrotoxic effects. This toxicity was exacerbated in hamsters fed a hyperlipidaemic diet and was preceded by a progressive anorexia and loss of body weight. Marked elevations in serum aspartate and alanine aminotransferase activities were associated with the organ lesions. All elevated biochemical changes and morphological alterations were prevented or reversed by coadministration of mevalonate, the product of the HMG-CoA reductase. It is suggested that the dramatic effect of simvastatin could result from depletion of a non-sterol metabolite of mevalonate in spite of a lack of protective effects of farnesol and geranylgeraniol in the following study. The toxicity of simvastatin could indeed result from the low basal activity of HMG-CoA reductase in hamster liver coupled with a prolonged inhibition of mevalonate synthesis.

Effects of ethanol, lovastatin and coenzyme Q10 treatment on antioxidants and TBA reactive material in liver of rats.

Alcohol metabolism may result in oxidant stress and free radical injury through a variety of mechanisms. Lovastatin may also produce oxidant stress by reducing levels of an endogenous antioxidant, coenzyme Q (CoQ). The separate and combined effects of ethanol, 20 EN% in a total liquid diet, and lovastatin, 67 mg/kg diet, on alpha-tocopherol, retinol palmitate, CoQ9 and thiobarbituric acid reactive (TBAR) material in liver from rats were determined. The effect of exogenous CoQ10 on these treatment groups was also determined. Food consumption, weight gain, liver lipid and TBAR material were similar between treatment groups. Compared to control animals, ethanol reduced retinol palmitate significantly, from 143 to 90 micrograms/g wet weight. Lovastatin had no effect on retinal palmitate nor did it act additively with ethanol. Ethanol decreased liver alpha-tocopherol from 28 to 12 micrograms/g wet weight and lovastatin diminished it to 12 micrograms; no additive effect was evident. Ethanol had no effect, but lovastatin decreased CoQ9 from 83 to 55 micrograms/g wet weight. Supplementation with CoQ10 did not modulate the effect of ethanol on retinal palmitate, but it did reverse the effect of lovastatin on CoQ9. Supplementary CoQ10 did not alter control levels of alpha-tocopherol, but it appeared to reverse most of the decrease in alpha-tocopherol attributable to ethanol or lovastatin separately. It only partially reversed the effect of ethanol and lovastatin combined on alpha-tocopherol, however. As expected, lovastatin had no effect on CoQ10 levels in supplemented animals. Ethanol, either separately or in combination with lovastatin, diminished liver stores of CoQ10 by almost 40%. We conclude that 20 EN% ethanol given in a liquid diet for 5 weeks is sufficient to lower retinol palmitate and that lovastatin reduces CoQ9. Both diminish alpha-tocopherol, an effect largely overcome by CoQ10 supplementation with either drug alone, but not with the combination. Since many individuals chronically consume the levels of ethanol represented by this experiment, and since a certain number of those also take lovastatin, further research into the possible clinical significance of these observations is warranted.

Biochemical changes and morphological alterations of the liver in guinea-pigs after administration of simvastatin (HMG CoA reductase-inhibitor).

Simvastatin is a potent competitive inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) which is the rate-limiting enzyme of cholesterol synthesis. In guinea-pigs, administration of a high oral dose of simvastatin (125 mg/kg/day at the beginning of the study) during 18 days had a major hepatotoxic effect whereas a lower oral dose (30 mg/kg/day) did not seem to cause any liver damage. A significant reduction in microsomal Cyt P 450 content was only observed on a high dose of simvastatin whereas HMG CoA reductase activity was reduced in the group with the low simvastatin dose. The hepatic microsomal aminopyrine N-demethylase activity remained unchanged in all groups. The liver lesion was hepatocellular necrosis accompanied in some animals by a biliary duct proliferation. It was associated with a 10-fold elevation in serum aspartate and alanine aminotransferase activities, as well as a great reduction in daily food intake and body weight (28%). The hepatotoxicity of simvastatin could result from the low basal content of HMG-CoA reductase in guinea-pig liver, the prolonged inhibition of mevalonate synthesis and probably, from the absence of HMG-CoA reductase enzyme de novo synthesis.

Toxicity of the HMG-coenzyme A reductase inhibitor, lovastatin, to rabbits.

Lovastatin, a specific inhibitor of the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, has been shown to be highly effective in lowering serum cholesterol in animals and humans and thus represents a promising approach to the treatment and prevention of cardiovascular disease. During the preclinical safety assessment of lovastatin, oral doses that were tolerated by dogs, rats and mice were found to be lethal to rabbits in subacute studies. Postmortem findings in rabbits consisted of centrilobular hepatic necrosis, frequently accompanied by renal tubular necrosis and occasionally gallbladder necrosis. The liver lesions were associated with up to 300-fold elevations in serum aspartate and alanine aminotransferase activities, whereas the kidney lesions resulted in accumulations of serum urea nitrogen and creatinine. The organ damage was preceded by a progressive decline in food consumption and loss of body weight. All histopathological and serum biochemical changes induced by lovastatin were completely prevented by coadministration of mevalonate, the product of the inhibited HMG-CoA reductase enzyme. In addition, administration of mevalonate after the onset of lovastatin-induced hepatotoxicity effectively reversed the toxicity despite continued drug treatment. These findings indicated that the toxicity of high doses of lovastatin to rabbits is a consequence of a highly exaggerated pharmacologic action in blocking mevalonate synthesis. However, supplementation of lovastatin-treated rabbits with oral doses of the major product of mevalonate metabolism, cholesterol, paradoxically enhanced the liver and kidney damage, which suggested that the toxicity of lovastatin stemmed from depletion of a nonsterol metabolite(s) of mevalonate critical for cell viability.

Preclinical evaluation of lovastatin.

Administration of lovastatin to animals at high dosage levels produces a broad spectrum of toxicity. This toxicity is expected based on the critical nature of the target enzyme (HMG CoA reductase) and the magnitude of the dosage levels used. The information reviewed in this paper demonstrates that these adverse findings in animals do not predict significant risk in humans. The reason for this derives from the fact that all the available evidence suggests that the adverse effects observed are produced by an exaggeration of the desired biochemical effect of the drug at high dosage levels. The presence of clear and high no-effect doses for these toxic effects along with the fact that most of the changes observed are clearly mechanism-based (directly attributable to inhibition of mevalonate synthesis) indicate that it is unlikely that similar changes will be observed at the therapeutic dosage levels in humans. This hypothesis is supported by the extensive human safety experience described by Tobert in the following report.