Introduction

Abstract Introduction Results Discussion References

Anastrozole (ZD1033), an achiral triazole derivative known as 2,2[5-(1H-1,2,4-triazol-1-ylmethyl)-1,3-phenylene]bis(2-methylproprionitrile) (fig. 1), is a potent inhibitor of aromatase (CYP19), which converts androgens to estrogens. This compound is currently marketed as a treatment for postmenopausal women with advanced breast cancer. Anastrozole exhibits high intrinsic potency demonstrated by the in vitro inhibition of human placental aromatase with an IC₅₀ of 15 nM. Preclinical studies with anastrozole demonstrated its selectivity for aromatase in vivo as compared with inhibition of other enzyme activities responsible for steroid biosynthetic pathways. Cholesterol biosynthesis was minimally inhibited in vitro and plasma cholesterol concentrations were unchanged in preclinical species at doses that were at least 30 times higher than its maximally effective dose (0.1 mg/kg) required for aromatase inhibition (1). Anastrozole, at doses 100-200 times its maximally effective aromatase inhibitory dose, did not interfere with cholesterol side-chain cleavage nor did it affect plasma aldosterone levels, sodium and potassium excretion, or adrenal and prostate gland weights. Anastrozole was a comparatively weak inhibitor of bovine adrenal 11-hydroxylase in vitro (IC₅₀  12 µM), and in vivo changes in circulating 11-deoxycorticosterone or hypokalemia were not observed at 3 and 10 mg/kg doses in monkeys and dogs, respectively. Androgen synthesis was not affected in rats and monkeys at 10 times or, in dogs, at 100 times the effective aromatase inhibitory dose. The selectivity of anastrozole was also established in early clinical studies where doses of 1 to 10 mg in postmenopausal female volunteers produced maximal suppression of estradiol without affecting cortisol or aldosterone secretion (1, 2).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Structure of anastrozole.

Various azole-containing compounds have been shown to be potent inhibitors and inducers of cytochrome P450-mediated drug metabolism both in vitro and in vivo. Lipophilic compounds containing nitrogen heterocycles such as phenylimidazoles and phenylpyridines inhibit rat liver monooxygenase activities with varying microsomal binding affinities depending on the position of heterocycle substitution (3). Many investigators have demonstrated that N- and C4-substituted imidazole drugs such as ketoconazole and cimetidine inhibit CYP¹activity and drug clearance in both animals and man (4-8). The potency of inhibition by these agents varies greatly and is determined by their hydrophobic nature and the effect of steric hindrance on the strength of the bond between their heteroatomic lone pair of electrons and the CYP heme iron. Although many azole-containing compounds are generally thought to be nonspecific inhibitors of monooxygenase activity, the selectivity of CYP inhibition by compounds such as ketoconazole has been shown to be concentration-dependent in human in vitro systems (9).

Information on potential drug:drug interactions is necessary during the development of all compounds intended for therapeutic use. In vitro methodologies are now routinely used to study the potential of a drug candidate to inhibit specific CYP enzymes and classify the drugs most likely to interact during clinical use. This study examined the effects of anastrozole on several of the major drug metabolizing CYP enzymes in human liver using in vitro approaches. Comparisons of the inhibitory effects were made to other known inhibitors.

Materials and Methods

Chemicals. Anastrozole, BMPN-benzoic acid, dehydronifedipine, and dextrorphan were synthesized by Zeneca Pharmaceuticals (Macclesfield, UK or Wilmington, DE). Phenacetin and acetaminophen were obtained from US Pharmacopoeia Convention, Inc. (Rockville, MD). Cimetidine, coumarin, dextromethorphan, ketoconazole, nifedipine, tolbutamide, and umbelliferone were obtained from Sigma Chemical Co. (St. Louis, MO). 4-Hydroxytolbutamide was obtained from Ultrafine Chemicals, Ltd. (Manchester, UK). All other reagents and supplies were obtained from standard commercial sources.

Microsome preparations. Transplant quality samples of human liver were obtained from the International Institute for the Advancement of Medicine (Exton, PA). Microsomes were prepared from liver tissue immediately upon receipt by differential centrifugation using previously described methods (10). Microsomal suspensions were stored for subsequent use at 80°C in 10 mM Tris-acetate (pH 7.4) containing 0.1 mM EDTA, 0.1 mM PMSF, and 20% glycerol (v/v). The total protein content of each microsomal sample was determined using the bicinchoninic acid reagent (Pierce Chemical Co., Rockford, IL) using BSA as the standard.

Determination of Human CYP Activities. Phenacetin deethylation to acetaminophen was used to assess CYP1A2 activity in human liver microsomes. Incubation mixtures of 0.5 ml contained 0.1 mg microsomal protein, 50 mM HEPES (pH 7.5), 15 mM MgCl₂, 1 mM NADPH, and phenacetin (50, 100, and 200 µM). Reactions were stopped after 15 min by the addition of 2 ml isopropanol:ethyl acetate (1:9 v/v). The samples were vortexed and then centrifuged to separate aqueous and organic layers. The organic layer was transferred to a clean tube and evaporated under nitrogen to dryness. Samples were reconstituted in HPLC mobile phase (10:90 acetonitrile: 0.1% TFA in water). The concentration of acetaminophen produced in the microsomal incubation was determined by HPLC with UV detection at 248 nm. Separation of acetaminophen from phenacetin and other reaction components was accomplished using a Zorbax SB-C8 column (MacMod Analytical, Inc., Chadds Ford, PA), 4.6 × 75 mm, with a precolumn 0.5 µm filter (Upchurch Scientific, Oak Harbor, WA). Acetaminophen (rt = 1.6 min) was eluted from the column using acetonitrile: 0.1% TFA in HPLC grade water (10:90 v:v) at a flow of 1.5 ml min^-1. Phenacetin (rt = 4.8 min) was washed from the column following a step gradient to 25% acetonitrile.

Data Analysis. Incubation of the CYP-marker substrates with microsomes from 1 to 3 individual donors in the presence or absence of various concentrations of anastrozole was used to determine IC₅₀ values. IC₅₀ values for the inhibition of the CYP activities were determined by nonlinear regression analysis (Origin, version 3.0, Microcal Software, Inc., Northampton, MA) using the following equation: where I is the initial concentration of inhibitor in a microsomal incubation, E₀ is the per cent enzyme activity that is not inhibited at I =  and, IC₅₀ is the inhibitor concentration that inhibits enzyme activity by 50%.

	Results

Abstract Introduction Results Discussion References

The potential inhibitory effects of the aromatase inhibitor, anastrozole, on drug metabolism were evaluated by determining IC₅₀ and K_i values for metabolic reactions that are selectively catalyzed by five different cytochrome P450 forms in human hepatic microsomes (table 1). Anastrozole inhibited less than 10% of the control coumarin hydroxylase (CYP2A6) and dextromethorphan O-demethylase (CYP2D6) activities in the human microsome samples at concentrations up to 500 µM (fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Inhibition of phenacetin O-deethylase (), coumarin hydroxylase (), tolbutamide hydroxylase (), dextomethorphan O-demethylation (), and nifedipine oxidation () in human liver microsomes by anastrozole. Data for phenacetin, tolbutamide, and nifedipine metabolism represent mean ± SE (N = 3 individual microsome samples). Data for coumarin and dextromethorphan represent the average of duplicate incubations.

The O-deethylation of phenacetin as a marker activity for CYP1A2 was inhibited by anastrozole at concentrations between 10 and 100 µM with an IC₅₀ of 30 µM. Dixon plots of the reciprocal deethylation rates when 50, 100, or 200 µM phenacetin was co-incubated with 0 to 500 µM anastrozole exhibited considerable nonlinearity (fig. 3A).²

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Dixon (A) and Cornish-Bowden (B) plots of the effect of anastrozole co-incubation on the rate of acetominophen formation from phenacetin in human liver microsomes. Phenacetin was incubated at initial concentrations of 50 (), 100 (), and 200 () µM.

The nonlinearity in the results was also evident in the Cornish-Bowden plot (fig. 3B) when the data generated at 500 µM anastrozole was excluded. The Dixon plot could be divided into two distinct linear phases. Therefore, when the data corresponding to the two phases were subjected to linear regression analysis, two apparent K_i values of 8 and 80 µM were determined for the inhibition of phenacetin metabolism to acetaminophen. The high affinity K_i of 8 µM estimated in this in vitro investigation was used to estimate conservatively the in vivo interaction potential towards CYP1A2 substrates. Parallel plots in the Cornish-Bowden transformation of the data indicate that the high affinity inhibition is competitive in nature.

Tolbutamide methyl-hydroxylase activity, a marker of CYP2C9 activity in human liver microsomes, was inhibited by anastrozole with an IC₅₀ of 48 µM when tolbutamide was co-incubated at 1000 µM (fig. 2). At this concentration of tolbutamide, CYP2C8 most likely contributes to the methylhydroxylase activity (16, 17) whereas at lower concentrations, the contribution by this CYP2C form would be negligible. An apparent K_i of 10 µM was determined based on plots of the reciprocal hydroxylation rates when 100, 200, and 1000 µM tolbutamide was co-incubated with 0 to 100 µM anastrozole (fig. 4A). The inhibition of tolbutamide hydroxylase activity by anastrozole exhibited linear behavior on Dixon plots and Cornish-Bowden plots demonstrated parallel best-fit lines indicating competitive-type inhibition (fig. 5B).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Dixon (A) and Cornish-Bowden (B) plots of the effect of anastrozole co-incubation on the rate of 4-hydroxytolbutamide formation from tolbutamide in human liver microsomes. Tolbutamide was incubated at initial concentrations of 100 (), 200 (), and 1000 () µM.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Dixon (A) and Cornish-Bowden (B) plots of the effect of anastrozole co-incubation on the rate of dehydronifedipine formation from nifedipine in human liver microsomes. Nifedipine was incubated at initial concentrations of 10 (), 25 (), and 50 ( 224 ) µM.

Anastrozole inhibited nifedipine oxidation in human liver microsomes with an IC₅₀ of 27 µM. By comparison, ketoconazole was a much more potent inhibitor of this CYP3A-catalyzed reaction with an IC₅₀ of 0.02 µM, while cimetidine was a weaker inhibitor (IC₅₀ = 650 µM) when co-incubated with nifedipine. Dixon plots of the reciprocal oxidation rates exhibited similar biphasicity to that observed for the inhibition of CYP1A2 by anastrozole (fig. 5A). Two distinct intercepts were obtained by linear regression analysis of the data and thus, two apparent K_i values of 10 and 55 µM were determined for the inhibition of CYP3A-catalyzed nifedipine oxidation by anastrozole. Cornish-Bowden plots suggested that the CYP3A inhibition was mixed (competitive/noncompetitive) in nature (fig. 5B).

Because CYP3A4 appears to be the most abundant cytochrome P450 enzyme in both human liver and intestine and this enzyme is involved in the metabolism of many drug substrates, triazole and BMPN-benzoic acid were tested for their ability to inhibit nifedipine oxidation. These two compounds are major metabolites of anastrozole that are produced by N-dealkylation (cleavage of the methylene bridge). When co-incubated with nifedipine at concentrations up to 250 µM, these metabolites of anastrozole did not inhibit nifedipine oxidation.

	Discussion

Abstract Introduction Results Discussion References

Many, if not most, clinically relevant drug interactions are caused by inhibition of drug metabolizing enzymes leading to a decreased metabolic clearance and increased exposure to the inhibited drug. Cytochrome P450 enzymes mediate the rate limiting step of the primary Phase I metabolic reactions for the vast majority of drug compounds as well as the biosynthesis or catabolism of a number of endogenous substances with potent physiological and pathophysiological functions. Specific cytochrome P450 enzymes in the CYP1A, 2A, 2C, 2D, and 3A subfamilies are responsible for the oxidative metabolism of most drugs in humans (18, 19). Toxicities and other adverse events may therefore develop during clinical therapy with drugs that have narrow therapeutic margins when the concomitant administration of CYP inhibitors causes potent metabolic inhibition of drug clearance. Probably the most prominent example of this type of potentially harmful interaction is the cardiotoxicity that can be produced following co-administration of CYP3A4 inhibitors such as ketoconazole and erythromycin with terfenadine, a prodrug metabolized exclusively by CYP3A enzyme(s) (20). In vitro studies are now being used routinely to investigate the interaction potential for drugs resulting from inhibition of CYP-mediated metabolism (21, 22). The experiments reported here examined the ability of anastrozole to inhibit the metabolism of substrates for CYP1A2, CYP2A6, CYP2C9, CYP2D6, and CYP3A. The results were used 1) to compare the inhibition of drug metabolizing CYPs by anastrozole to its in vitro ability to inhibit estrogen synthesis mediated by human aromatase and 2) to predict the potential for anastrozole to cause metabolic drug interactions.

Anastrozole inhibited specific reactions catalyzed by CYP1A2, CYP2C9, and CYP3A but did not inhibit CYP2A6 or CYP2D6-mediated pathways. The inhibition of both CYP1A2 and CYP3A activities were biphasic in nature although the basis of the nonlinear results has not been established. Two apparent K_i values were exhibited by anastrozole for inhibition of CYP1A2 and CYP3A metabolism. The low K_i values for these two enzymes was similar to the single apparent K_i determined for inhibition of CYP2C9 activity by anastrozole (K_i = 8 -10 µM corresponds to 2.3 to 2.9 µg ml¹). The low K_i values were used to estimate conservatively the potential for inhibitory drug interactions with anastrozole during clinical use of the drug. Average steady-state C_max concentrations in patients chronically administered the 1-mg marketed dose of Arimidex were approximately 0.08 µg ml¹ (0.3 µM). These plasma concentrations are 30-fold lower than the apparent K_i values determined in these in vitro studies, suggesting that anastrozole would cause little or no inhibition in vivo of the metabolism of drugs that are substrates for CYP1A2, CYP2C9, and CYP3A enzymes (per cent inhibition in vivo predicted to be approximately 3%). Although a lack of inhibition potential can be predicted from these in vitro results, any effect of intracellular binding or accumulation of anastrozole in hepatocytes on the in vivo inhibition have not been accounted for in these investigations.³

Nifedipine oxidation in human liver microsomes was not decreased by two major metabolites (triazole and BMPN-benzoic acid) of anastrozole at concentrations up to 250 µM. The lack of CYP inhibition by these metabolites is consistent with requirement for both hydrophobic character for active site access and the heteratomic lone pair of electrons in triazole for Type II binding to the heme iron (23).

The results herein also show that the ability of anastrozole to inhibit drug metabolizing CYP enzymes in liver microsomes is much weaker than the inhibition of aromatase. When comparing the IC₅₀ for inhibition of human placental aromatase activity (15 nM) with the inhibition of the hepatic microsomal CYP produced by anastrozole in vitro, a margin of selectivity of at least 500-fold is obtained.

In conclusion, the results of the in vitro experiments performed in human liver microsomes indicate that, although anastrozole can inhibit CYP1A2, 2C9, and 3A-mediated catalytic activities, the level of inhibition in vivo during clinical therapy with Arimidex (Zeneca, Ltd., Macclesfield, UK) 1-mg would not be expected to cause clinically significant interactions with other CYP-metabolized drugs.