* Drug Safety Evaluation and
Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, 2800 Plymouth Rd., Ann Arbor, Michigan 481061047
Received August 24, 2000; accepted October 13, 2000
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ABSTRACT |
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Key Words: avasimibe; ACAT inhibitor; dog toxicity; adrenal toxicity.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Compound administration.
Avasimibe was prepared in-house and had nuclear magnetic resonance, and infrared and mass spectra consistent with the structure of the compound. The potency of each lot of compound used in these studies was determined by HPLC and elemental analysis to be >97%. The compound was administered orally as bulk drug in gelatin capsules on a mg/kg body weight basis. Control animals received empty gelatin capsules equal in number to those given to the high-dose group for each study.
Experimental Design
In all studies, clinical signs were monitored daily and body weights were taken weekly.
Escalating-dose study.
The 2-week repeated-dose study was the first study conducted with avasimibe in dogs (see below). The doses were selected based on experience with a previous ACAT inhibitor (Wolfgang et al., 1995). However, the study failed to define a dose-limiting toxicity. Therefore, an escalating dose study was conducted in order to establish a maximum tolerated dose. In the study, 2 male dogs were administered 100 mg/kg on Days 19, 1000 mg/kg once a day on Days 1016 and 1000 mg/kg b.i.d. on Days 1723. The b.i.d. doses were administered 8 h apart. Plasma drug concentrations were determined pre-dose, 1.5, 4, 8, 12, and 24 h post-dose on Days 9 and 16, and pre-dose, 1.5, 4, 8, 9.5, 12, 16, 24, and 32 h post-dose (first dose) on Day 23. Hematological and serum chemistry parameters were measured pre-test and on Days 8, 15, and 22. The animals were not euthanized at the end of the study.
Repeated-dose studies.
The experimental designs for the 2-, 13- and 52-week repeated-dose studies are summarized in Table 1. A full battery of standard clinical chemistry, hematology, and urinalysis assays was conducted at the times indicated. In addition to standard tests, serum lipoproteins were measured by an HPLC procedure described by Kieft et al. (1991). In the 13-week and 52-week studies, a selected battery of serum lipid markers and indices of liver toxicity were monitored at more frequent intervals, as indicated in Table 1
. In the 52-week study, starting in Week 40, 1000 mg/kg males were given a 50% increased food ration due to clinical indications of thinness.
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Tissue Biochemistry
For the 2-, 13- and 52-week studies, hepatic microsomal fractions were prepared from all animals at study termination. In the 2-week study, total CYP (spectral), microsomal protein, methoxyresorufin-O-demethylase (MROD), ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD) and erythromycin-N-demethylase (END) activities of microsomal fractions were determined. Additionally, microsomal CYP 1A, 2B, and 3A were evaluated by Western blot. In the 13- and 52-week studies only total CYP, protein, and END activities were determined on hepatic microsomal fractions. Microsomal assays were performed using procedures described previously (Robertson et al., 1995). Adrenal and liver total cholesterol, esterified cholesterol and free cholesterol were measured at termination in the 2-, 13- and 52-week studies using procedures described by Bocan and Guyton (1985).
Toxicokinetics
Plasma avasimibe concentrations were determined by solid phase extraction followed by HPLC/UV detection. The limit of quantitation was 0.25 µg/ml for the dose-escalating and 2-week studies. The limit of quantitation was 0.08 µg/ml for the 13- and 52-week studies.
Maximum concentrations (Cmax) were recorded as observed. Area under concentration-time profile (AUC) values was estimated using the linear trapezoidal rule. AUC(0-tldc) values were calculated from time zero to the time for the last detectable concentration (tldc). Apparent elimination rate constant (z) values were estimated as the absolute value of the slope of a linear regression of natural logarithm (ln) of concentration versus time during the terminal phase of the concentration-time profile. AUC(0-
) values were calculated as the sum of corresponding AUC(0-tldc) and ldc/
z values. AUC(0-Tau) were estimated using linear trapezoidal rule, where Tau is the dosing interval. Pharmacokinetic parameter values were calculated using SAS (SAS Institute, Inc., Cary, NC) except in the 52-week study, where WinNonlin (Pharsight Corporation, Mountain View, CA) was used.
Statistics
Treatment comparisons were performed on rank-transformed data using a one-factor analysis of variance (ANOVA). A dose-trend test sequentially applied at the 2-tailed 1% significance level was utilized for quantitative clinical laboratory data. If the high-dose linear trend test was not significant, a test for trend reversal was performed. If the trend reversal was significant, then treated groups were compared individually to the control group using Dunnett's test. For the 300 mg/kg dose group added to the 52-week study, a t-test was used to determine statistical differences between the treated groups and their concurrent control.
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RESULTS |
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In remaining animals, clinical signs including reduced food consumption, changes in fecal consistency (diarrhea, soft stool), emesis, and salivation were dose-related in incidence, primarily affecting the 300- and 1000 mg/kg dose groups. Biologically significant group mean body-weight loss (up to 1.5 kg) was noted in both sexes at 1000 mg/kg, but was reversed in males after their food ration was increased (Table 5).
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Changes in clinical biochemistry parameters consistent with the pharmacology of the compound included dose-related decreases in serum cholesterol (decreased up to 86%) and lipoproteins (decreased up to 100%) relative to pretest (Fig. 3). Changes in clinical biochemistry parameters indicative of an effect on the liver (Fig. 4
) were primarily restricted to doses
300 mg/kg, noted in both sexes, and included moderate to marked increases (up to 24-fold) in serum AP and ALT with lesser increases (less than 5-fold) in serum AST and gamma glutamyltranspeptidase (GGT). Serum protein was decreased up to 22% primarily due to decreased albumin (up to 30%). Changes in clinical biochemistry parameters were generally maximal by Weeks 4 to 8 and either remained the same or declined by Week 52. Hepatic effects, as indicated by elevated ALT, were associated with concurrent hypocholesterolemia (Fig. 5
). Abnormal red blood cell (RBC) morphology, characterized by the presence of burr cells, was noted at
300 mg/kg, was dose-related in incidence and severity, and was found almost exclusively in animals with concurrent hypocholesterolemia (Fig. 6
).
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DISCUSSION |
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Hepatic effects appeared to be associated with concurrent hypocholesterolemia in the 52-week study, which was demonstrated by a striking association between ALT elevations and low serum cholesterol (Fig. 5). This result was more than a simple dose response, since dogs at 300 mg/kg, with normal cholesterol, also had normal ALT values, while those dogs with serum cholesterol below the normal range had elevated ALT. Additionally, three 300 mg/kg (2M, 1F) dogs, with no hepatic pathology at termination, had Week 52 serum cholesterol levels within the normal range while 4 of 5 animals with hepatic pathology had Week 52 serum cholesterol levels at or below the lower limit of the normal range (98 and 99 mg/dl for males and females, respectively). The single animal with normal Week 52 cholesterol and hepatic pathology had only minimal hemosiderin pigment associated with macrophages. In addition to these findings, it was clear that the data did not support any speculation about accumulation of hepatic cholesterol due to inhibition of tissue ACAT.
Cholesterol represents a significant component of the RBC membrane and has pronounced effects on membrane fluidity (Telen, 1993). Hypercholesterolemia causes significant alterations in RBC morphology (Telen, 1993
), and the presence of echinocytes (burr cells) noted in burn patients has been attributed to hypocholesterolemia (Harris et al., 1981
). Therefore, since abnormal RBC morphology was observed almost exclusively in animals with concurrent hypocholesterolemia, it seems plausible that RBC effects noted in this study were due, at least indirectly, to an exaggerated pharmacological effect.
If toxicity was the result of exaggerated pharmacology, the greater effect on serum cholesterol noted in the 52-week study may partially explain the increased severity of effects noted in the 52-week study relative to the 13-week study, at the same doses and within the same timeframe. The differences in severity of toxicity cannot be explained by differences in plasma drug concentrations since the Week 12 Cmax and AUC values at 1000 mg/kg in both studies were similar.
Our previous ACAT inhibitors, PD 132301 and PD 13814215, also induced hepatic toxicity characterized by elevated AP and transaminases. RBC changes were not noted, possibly since the studies were only 2 and 13 weeks in duration, respectively (Dominick et al., 1993b; Wolfgang et al., 1995
). In the case of PD 13814215, moribundity of 2 dogs at 100 mg/kg was associated with extreme hypocholesterolemia (
27 mg/dl). While it is tempting to speculate about a causal association between hypocholesterolemia and hepatic toxicity and/or RBC morphologic changes, it seems unlikely that they are directly related. Chronic treatment of dogs with statin drugs, which reduced serum cholesterol as much as 88%, produced a plethora of effects including cataracts, CNS toxicity, and gall bladder, as well as hepatic, toxicity (Berry, 1988; Gerson, 1989; Hartman, 1996), quite a different profile from the toxicity in dogs treated with avasimibe. Marked serum cholesterol depletion alone cannot explain the divergent effects noted in all these studies. Differences may be due to tissue specificity of cholesterol depletion or other pharmacological effects such as marked depletion of LDL-C or VLDL-C.
The minimal, but statistically significant, changes noted in serum AP activity at doses of 10 and 100 mg/kg (Fig. 3) were similar between the 2 studies. These findings were not associated with hepatic pathology, and AP increases are a consequence of hepatic microsomal induction in both dogs (Robertson et al., 1993
) and humans (Perucca, 1987
). Therefore, these findings were not considered toxicologically relevant.
Adrenal changes noted in these studies were different from adrenal lesions reported for other ACAT inhibitors (Fig. 9). The ACAT inhibitors PD 1323012, PD 13814215, and FR145237 produced significant decreases in adrenal weight, corresponding to marked zonal atrophy/ablation of the zona fasciculata and reticularis at pharmacologic doses in as little as 2 weeks when orally administered to dogs (Dominick et al., 1993b
; Matsuo et al., 1996
; Wolfgang et al., 1995
). Avasimibe did not produce adrenal pathology in the 13-week study and adrenal effects in the 52-week study were restricted to minimal to mild cortical cytoplasmic vacuolization and fibrosis at doses
300 mg/kg. Adrenal weights were unaffected, lesions were microscopic and of limited severity, and as such, were not considered physiologically significant. Although adrenal function was not assessed, our previous experience with ACAT-induced adrenal toxicity in dogs demonstrated that functional effects, as measured by adrenal corticotrophic responsiveness (ACTH challenge), were not evident until frank morphologic lesions were present (Wolfgang et al., 1995
). It has been hypothesized that ACAT inhibitors induce accumulation of free cholesterol, which is responsible for adrenal toxicity (Warner et al., 1995
). This was clearly not the case with avasimibe. The absence of adrenal findings in the 13-week studies and the minimal findings in the 52-week study were evident in spite of marked depletion of adrenal total and esterified cholesterol (decreased up to 68% and 78%, respectively, at Week 52) and little change in free cholesterol. Based on these findings, we conclude that frank adrenal toxicity should not be considered a necessary class effect of ACAT inhibitors.
The decreased plasma Cmax and AUC values at 300 mg/kg, relative to 100 mg/kg in the 52-week study, are unexplained. Although different bulk drug lots were used in the study, the drug lot used for the 300 mg/kg group was the same at each time point as those used for the remaining groups. Therefore, the discrepancy cannot be explained by chemical specification differences in bulk drug lots. Additionally, the toxicity findings were clearly dose-related, supporting the contention that the toxicity noted in the 52-week study was more clearly associated with dose and pharmacodynamics than with plasma drug concentrations. Induction of CYP 3A and associated decreases in plasma drug concentrations subsequent to Day 1 were noted in all repeated dose studies. This finding has been confirmed in an early clinical trial of avasimibe (Vora et al., 1997).
In conclusion, avasimibe is an ACAT inhibitor that has minimal adrenal toxicity in dogs, with dose limiting toxicity defined by readily monitored and reversible changes in hepatic function. The excellent safety profile of avasimibe has been born out in early clinical trials of the compound (Koren et al., 1998).
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NOTES |
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