PURPOSE To conduct a mechanistic investigation of the interaction between aliskiren and grapefruit juice in healthy subjects. METHODS Twenty-eight subjects received an oral dose of aliskiren 300 mg (highest recommended clinical dose) with 300 mL of either water or grapefruit juice in a two-way crossover design. Safety and pharmacokinetic analyses were performed. In vitro studies were performed in HEK293 cells to investigate the role of organic anion transporting polypeptide (OATP) transporter-mediated uptake of aliskiren. RESULTS Co-administration of a single dose of aliskiren with grapefruit juice decreased the plasma concentration of aliskiren, with mean decreases in the AUC(inf), AUC(last), and C(max) of 38, 37, and 61%, respectively. The uptake of [¹⁴C]aliskiren into OATP2B1-expressing cells was essentially the same as that into control cells, and the inhibitor combination atorvastatin and rifamycin had no effect on [¹⁴C]aliskiren accumulation in either cell type. The uptake of [¹⁴C]aliskiren and [³H]fexofenadine was linear in OATP1A2-expressing cells and was reduced by naringin, with IC₅₀ values of 75.5 ± 11.6 and 24.2 ± 2.0 μM, respectively. CONCLUSIONS Grapefruit juice decreases exposure of aliskiren partially via inhibition of intestinal OATP1A2.

OBJECTIVES AND METHODS--To gain insight into possible mechanisms of and predisposing factors for torsades de pointes during terfenadine therapy, spontaneous reports in the US Food and Drug Administration's Spontaneous Reporting System database were examined. Based on the characteristics of the cases, in vitro cardiac electrophysiologic studies were conducted to test the hypothesis that terfenadine, and not its major metabolite, has actions similar to those of quinidine and is responsible for this form of cardiac toxicity. DESIGN--Spontaneous reports from the general medical community. RESULTS--As of April 1, 1992, 25 cases of torsades de pointes had been reported to the Food and Drug Administration's Spontaneous Reporting System. Predisposing factors in these cases indicated that the parent drug, but not its metabolite, may have actions similar those of quinidine that are responsible for inducing arrhythmia. In vitro studies found that terfenadine is equipotent to quinidine as a blocker of the delayed rectifier potassium current in isolated feline myocytes. The metabolite, terfenadine carboxylate, did not inhibit this potassium current even at concentrations 30 times higher than the concentration of terfenadine producing a half-maximal effect. CONCLUSIONS--Since blockade of the potassium channel did not occur with the major metabolite of terfenadine, episodes of torsades de pointes are most likely the result of a quinidinelike action of the parent drug and of factors that impair the normally rapid metabolism of terfenadine. Dosage restriction and awareness of the clinical conditions and drug interactions capable of inhibiting the metabolism of terfenadine are essential for prevention of this serious reaction.

Fexofenadine, a nonsedating antihistamine, does not undergo significant metabolic biotransformation. Accordingly, it was hypothesized that uptake and efflux transporters could be importantly involved in the drug's disposition. Utilizing a recombinant vaccinia expression system, members of the organic anion transporting polypeptide family, such as the human organic anion transporting polypeptide (OATP) and rat organic anion transporting polypeptides 1 and 2 (Oatp1 and Oatp2), were found to mediate [(14)C]fexofenadine cellular uptake. On the other hand, the bile acid transporter human sodium taurocholate cotransporting polypeptide (NTCP) and the rat organic cation transporter rOCT1 did not exhibit such activity. P-glycoprotein (P-gp) was identified as a fexofenadine efflux transporter, using the LLC-PK1 cell, a polarized epithelial cell line lacking P-gp, and the derivative cell line (L-MDR1), which overexpresses P-gp. In addition, oral and i.v. administration of [(14)C]fexofenadine to mice lacking mdr1a-encoded P-gp resulted in 5- and 9-fold increases in the drug's plasma and brain levels, respectively, compared with wild-type mice. Also, a number of drug inhibitors of P-gp were found to be effective inhibitors of OATP. Because OATP transporters and P-gp colocalize in organs of importance to drug disposition such as the liver, their activity provides an explanation for the heretofore unknown mechanism(s) responsible for fexofenadine's disposition and suggests potentially similar roles in the disposition of other xenobiotics.

CYP3A is one of the most important cytochrome P450 isoforms responsible for drug metabolism by humans because it is the major such enzyme in critical tissues such as the gastrointestinal tract and liver, and it is involved in the oxidative biotransformation of numerous clinically useful therapeutic agents. Many factors regulate CYP3A expression but these are being increasingly defined so that the disposition characteristics of a drug whose metabolism is importantly mediated by this isoform can be reasonably well predicted a priori. For example, metabolic clearance is distributed within a population in a unimodal fashion but marked (5- to 20-fold) interindividual variability is present as a consequence of both genetic and nongenetic factors. In addition, first-pass metabolism occurs following oral drug administration and this may be extensive so that bioavailability is low. CYP3A activity can also be readily modulated by inducers like rifampicin and several anticonvulsant agents, and many potent inhibitors exist such as azole antifungal agents and macrolide antibiotics. Accordingly, the potential for drug interactions with these drugs as well as other CYP3A substrates, when given concomitantly, is high. Metabolism involving CYP3A is also likely to be affected by liver disease as well as aging, and modest differences may be present between men and women but these are often clinically unimportant. Because of such predictability, knowledge of the role and importance of CYP3A in the metabolism of a putative drug candidate is becoming increasingly desirable at an early stage in the development process. In vitro studies using human liver preparations, including microsomes, cultured hepatocytes and heterologous expressed enzymes, can provide important insights in this regard. This is particularly the case for identifying potential drug interactions whose clinical significance can be subsequently assessed. Data with respect to terfenadine and cyclosporine obtained several years after their approval and marketing, indicate that, if available and applied during their development, the paradigm of using in vitro studies to rationally direct and prioritize clinical studies would have prospectively prevented the serious adverse effects and inefficacy that were only recognized during their empiric clinical use. Such examples, along with those associated with the genetic polymorphism of CYP2D6, provide strong justification for establishing the role and importance of individual CYP isoforms in a candidate drug's metabolism at an early stage.

Testosterone, terfenadine, midazolam, and nifedipine, four commonly used substrates for human cytochrome P-450 3A4 (CYP3A4), were studied in pairs in human liver microsomes and in microsomes from cells containing recombinant human CYP3A4 and P-450 reductase, to investigate in vitro substrate-substrate interaction with CYP3A4. The interaction patterns between compounds with CYP3A4 were found to be substrate-dependent. Mutual inhibition, partial inhibition, and activation were observed in the testosterone-terfenadine, testosterone-midazolam, or terfenadine-midazolam interactions. However, the most unusual result was the interaction between testosterone and nifedipine. Although nifedipine inhibited testosterone 6beta-hydroxylation in a concentration-dependent manner, testosterone did not inhibit nifedipine oxidation. Furthermore, the effect of testosterone and 7,8-benzoflavone on midazolam 1'-hydroxylation and 4-hydroxylation demonstrated different regiospecificities. These results may be explained by a model in which multiple substrates or ligands can bind concurrently to the active site of a single CYP3A4 molecule. However, the contribution of separate allosteric sites and conformational heterogeneity to the atypical kinetics of CYP3A4 can not be ruled out in this model.

Saquinavir is a HIV protease inhibitor used in the treatment of patients with acquired immunodeficiency syndrome, but its use is limited by low oral bioavailability. The potential of human intestinal tissue to metabolize saquinavir was assessed in 17 different human small-intestinal microsomal preparations. Saquinavir was metabolized by human small-intestinal microsomes to numerous mono- and dihydroxylated species with K(M) values of 0.3-0.5 microM. The major metabolites M-2 and M-7 were single hydroxylations on the octahydro-2-(1H)-isoquinolinyl and (1,1-dimethylethyl)amino groups, respectively. Ketoconazole and troleandomycin, selective inhibitors of cytochrome P4503A4 (CYP3A4), were potent inhibitors for all oxidative metabolites of saquinavir. The cytochrome P450-selective inhibitors furafylline, fluvoxamine, sulfaphenazole, mephenytoin, quinidine, and chlorzoxazone had little inhibitory effect. All saquinavir metabolites were highly correlated with testosterone 6beta-hydroxylation and with each other. Human hepatic microsomes and recombinant CYP3A4 oxidized saquinavir to the same metabolic profile observed with human small-intestinal microsomes. Indinavir, a potent HIV protease inhibitor and a substrate for human hepatic CYP3A4, was a comparatively poor substrate for human intestinal microsomes and inhibited the oxidative metabolism of saquinavir to all metabolites with a Ki of 0.2 microM. In addition, saquinavir inhibited the human, small-intestinal, microsomal CYP3A4-dependent detoxication pathway of terfenadine to its alcohol metabolite with a Ki value of 0.7 microM. These data indicate that saquinavir is metabolized by human intestinal CYP3A4, that this metabolism may contribute to its poor oral bioavailability, and that combination therapy with indinavir or other protease inhibitors may attenuate its low relative bioavailability.

Grapefruit juice, a beverage consumed in large quantities by the general population, is an inhibitor of the intestinal cytochrome P-450 3A4 system, which is responsible for the first-pass metabolism of many medications. Through the inhibition of this enzyme system, grapefruit juice interacts with a variety of medications, leading to elevation of their serum concentrations. Most notable are its effects on cyclosporine, some 1,4-dihydropyridine calcium antagonists, and some 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. In the case of some drugs, these increased drug concentrations have been associated with an increased frequency of dose-dependent adverse effects. The P-glycoprotein pump, located in the brush border of the intestinal wall, also transports many cytochrome P-450 3A4 substrates, and this transporter also may be affected by grapefruit juice. This review discusses the proposed mechanisms of action and the medications involved in drug-grapefruit juice interactions and addresses the clinical implications of these interactions.

Many adverse drug-drug interactions are attributable to pharmacokinetic problems and can be understood in terms of alterations of P450-catalyzed reactions. Much is now known about the human P450 enzymes and what they do, and it has been possible to apply this information to issues related to practical problems. A relatively small subset of the total number of human P450s appears to be responsible for a large fraction of the oxidation of drugs. The three major reasons for drug-drug interactions involving the P450s are induction, inhibition, and possibly stimulation, with inhibition appearing to be the most important in terms of known clinical problems. With the available knowledge of human P450s and reagents, it is possible to do in vitro experiments with drugs and make useful predictions. The results can be tested in vivo, again using assays based on our knowledge of human P450s. This approach has the capability of not only improving predictions about which drugs might show serious interaction problems, but also decreasing the number of in vivo interaction studies that must be performed. These approaches should improve with further refinement and technical advances.

The antihistaminic drug terfenadine, alpha-[4-(1,1-dimethylethyl)phenyl]-4-(hydroxydiphenylmethyl)-1- piperidinebutanol (Seldane), is of interest because of its lack of sedative properties. Major routes of metabolism include oxidative N-dealkylation to 4-(hydroxydiphenylmethyl)-piperidine (1) and oxidation of a tert-butyl methyl group to a primary alcohol (2), which is subsequently oxidized to a carboxylic acid. Rates of formation of 1 and 2 varied approximately 30-fold in the 17 human liver microsomal samples examined and were highly correlated with each other, suggesting that the same enzyme may be involved in both oxidations. The rates of formation of 1 and 2 were both correlated with rates of nifedipine oxidation (a marker of cytochrome P-450 (P-450) 3A4) but not with markers for other human P-450s. Microsomal oxidation of (both enantiomers of) terfenadine to 1 and 2 was markedly inhibited by gestodene, a selective mechanism-based inactivator of P-450 3A enzymes but not by any of several other P-450 inhibitors. Antibodies raised against P-450 3A4 could inhibit most of the oxidation of (both enantiomers of) terfenadine to 1 and 2 in a microsomal sample having high catalytic activity but antibodies recognizing other P-450s had no effect. The oxidation of terfenadine to 1 and 2 was catalyzed by purified human liver microsomal P-450 3A4 and by partially purified yeast recombinant P-450 3A4. These results provide evidence that P-450 3A4 (and possibly other P-450 3A enzymes) play a major role in the oxidation of (both enantiomers of) terfenadine to both of its major oxidation products.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of the nonsedating antihistamine terfenadine has been associated with altered cardiac repolarization in certain clinical settings. For this reason we examined the effects of terfenadine, and its metabolites, on a rapidly activating delayed rectifier K+ channel (fHK) cloned from human heart. fHK was stably expressed in human embryonic kidney cells, and both whole-cell currents and currents from excised inside-out patches were recorded. Terfenadine (3 microM) blocked whole-cell fHK current by 72 +/- 6%. In inside-out patches, terfenadine applied to the cytoplasmic surface blocked fHK with an IC50 value of 367 nM. The main effect of terfenadine was to enhance the rate of inactivation of fHK current and thereby reduce the current at the end of a prolonged voltage-clamp pulse. The blockade displayed a weak voltage dependence, increasing at more positive potentials. The mechanism of action of terfenadine is therefore consistent with blockade of open channels. In contrast, the metabolites of terfenadine were weakly active on fHK. IC50 values for all of the metabolites tested ranged from 27-fold to 583-fold higher than that obtained for terfenadine. It is concluded that terfenadine, but not its metabolites, blocks at least one type of human cardiac K+ channel at clinically relevant concentrations and that this activity may underlie the cardiac arrhythmias that have been associated with the use of this drug.

Terfenadine, a nonsedating H1-selective antihistamine, is widely used in many countries. We report pharmacokinetic results in a patient who developed a prolonged QT-interval in ECG and symptomatic torsades de pointes ventricular tachycardia as a consequence of the interaction of itraconazole and terfenadine. Both drugs were taken in the recommended doses: terfenadine 60 mg b.d. and itraconazole 100 mg b.d. Terfenadine metabolism was delayed by itraconazole, leading to an increased level of unmetabolised terfenadine. Seven weeks after the cessation of itraconazole treatment, terfenadine was rapidly metabolized to its active metabolite and did not prolong the QT-interval when given as a single provocation dose (120 mg). The findings suggest that intraconazole in therapeutic doses inhibits terfenadine metabolism. It is also possible that unmetabolised terfenadine alone, without an increased level of its active metabolite, may cause torsades de pointes. The concomitant use of terfenadine and itraconazole (and ketoconazole) should be avoided.