The conduct of in vitro and in vivo drug-drug interaction studies: a PhRMA perspective

Current regulatory guidances do not address specific study designs for in vitro and in vivo drug-drug interaction studies. There is a common desire by regulatory authorities and by industry sponsors to harmonize approaches to allow for a better assessment of the significance of findings across different studies and drugs. There is also a growing consensus for the standardization of cytochrome P450 (CYP) probe substrates, inhibitors, and inducers and for the development of classification systems to improve the communication of risk to health care providers and patients. While existing guidances cover mainly CYP-mediated drug interactions, the importance of other mechanisms, such as transporters, has been recognized more recently and should also be addressed. This paper was prepared by the Pharmaceutical Research and Manufacturers of America (PhRMA) Drug Metabolism and Clinical Pharmacology Technical Working Groups and represents the current industry position. The intent is to define a minimal best practice for in vitro and in vivo pharmacokinetic drug-drug interaction studies targeted to development (not discovery support) and to define a data package that can be expected by regulatory agencies in compound registration dossiers.     

Methodological and conduct principles for pharmacoeconomic research. Pharmaceutical Research and Manufacturers of America

In January 1995, the Pharmaceutical Research and Manufacturers of America (PhRMA) adopted a voluntary set of principles to provide guidance for its member companies on the conduct and evaluation of pharmacoeconomic research. The principles were prepared by a working group of pharmacoeconomic scientists from the PhRMA Task Force on the Economic Evaluation of Pharmaceuticals. The principles were reviewed by a panel of academic experts and outside reviewers at each stage of their development. The PhRMA document consists of a set of broad principles that will foster high quality pharmacoeconomic research without impeding further methodological development of the field. Specific recommendations are offered in those methodological areas for which general agreement exists. However, no attempt was made to force a consensus for those methodological issues which have yet to be resolved. The principles address methodology and reporting of research rather than sponsor-investigator relationships or conflict of interest issues, which have been addressed elsewhere. This approach is based on the belief that the scientific integrity of pharmacoeconomic research is best ensured through the soundness of the research methods used and the full disclosure and transparency of all methods, analyses and results.     

The conduct of drug metabolism studies considered good practice (I): analytical systems and in vivo studies

This review serial outlines practical and scientifically-based approaches to conducting contemporary drug metabolism studies considered good practice for drug development and regulatory filing. The present part addresses analytical methods used in the drug metabolism studies and evaluates advantages and disadvantages of these methods as well as the related sample preparations. The methods described here cover from conventional radioactive labeling of drugs, which includes selection of a proper radioisotope, its labeling position, and modern radio-pharmacokinetics employed in microdosing by using a radionuclide to visualize drug distribution in vivo, to currently widely-used liquid chromatography (LC) in conjunction with mass spectrometry (MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) for quantitative detection of metabolites and characterization of their structures. Although the analytical tools have progressed sufficiently to allow determination of metabolites, proper in vitro models and in vivo studies have to be carefully designed in order to understand drug metabolism. Points for consideration when conducting in vivo drug metabolism studies include interspecies differences in systemic exposure and metabolism pathways, identification of the major metabolites and unique human metabolites that become the regulatory focus, local metabolism in addition to liver metabolism, time points for sampling, and synthesis of the authentic metabolites to confirm their formation. The next part of this serial article will focus on in vitro drug metabolism studies.     

Maraviroc: in vitro assessment of drug-drug interaction potential

AIMS

To characterize the cytochrome P450 enzyme(s) responsible for the N-dealkylation of maraviroc in vitro, and predict the extent of clinical drug–drug interactions (DDIs).

METHODS

Human liver and recombinant CYP microsomes were used to identify the CYP enzyme responsible for maraviroc N-dealkylation. Studies comprised enzyme kinetics and evaluation of the effects of specific CYP inhibitors. In vitro data were then used as inputs for simulation of DDIs with ketoconazole, ritonavir, saquinavir and atazanvir, using the Simcyp™ population-based absorption, distribution, metabolism and elimination (ADME) simulator. Study designs for simulations mirrored those actually used in the clinic.

RESULTS

Maraviroc was metabolized to its N-dealkylated product via a single CYP enzyme characterized by a K_m of 21 µM and V_max of 0.45 pmol pmol⁻¹ min⁻¹ in human liver microsomes and was inhibited by ketoconazole (CYP3A4 inhibitor). In a panel of recombinant CYP enzymes, CYP3A4 was identified as the major CYP responsible for maraviroc metabolism. Using recombinant CYP3A4, N-dealkylation was characterized by a K_m of 13 µM and a V_max of 3 pmol pmol⁻¹ CYP min⁻¹. Simulations therefore focused on the effect of CYP3A4 inhibitors on maraviroc pharmacokinetics. The simulated median AUC ratios were in good agreement with observed clinical changes (within twofold in all cases), although, in general, there was a trend for overprediction in the magnitude of the DDI.

CONCLUSION

Maraviroc is a substrate for CYP3A4, and exposure will therefore be modulated by CYP3A4 inhibitors. Simcyp™ has successfully simulated the extent of clinical interactions with CYP3A4 inhibitors, further validating this software as a good predictor of CYP-based DDIs.

WHAT IS ALREADY KNOWN ABOUT THE SUBJECT

• Maraviroc is known to undergo oxidative metabolism in vivo and is a substrate for cytochrome P450 (CYP).
• Simcyp™ has recently become more widely used for the prediction of CYP-mediated drug–drug interactions (DDIs) using in vitro metabolism data.

WHAT THIS STUDY ADDS

• Maraviroc has been identified as a CYP3A4 substrate and the kinetic constants characterized.
• The predicted DDIs associated with maraviroc as a CYP3A4 substrate using Simcyp™ have been compared against the clinical data.
• This study demonstrates the value of a reliable Simcyp™ model for prediction of DDIs.

     

The conduct of drug metabolism studies considered good practice (II): in vitro experiments

In vitro drug metabolism studies, which are inexpensive and readily carried out, serve as an adequate screening mechanism to characterize drug metabolites, elucidate their pathways, and make suggestions for further in vivo testing. This publication is a sequel to part I in a series and aims at providing a general framework to guide designs and protocols of the in vitro drug metabolism studies considered good practice in an efficient manner such that it would help researchers avoid common pitfalls and misleading results. The in vitro models include hepatic and non-hepatic microsomes, cDNA-expressed recombinant human CYPs expressed in insect cells or human B lymphoblastoid, chemical P450 inhibitors, S9 fraction, hepatocytes and liver slices. Important conditions for conducting the in vitro drug metabolism studies using these models are stated, including relevant concentrations of enzymes, co-factors, inhibitors and test drugs; time of incubation and sampling in order to establish kinetics of reactions; appropriate control settings, buffer selection and method validation. Separate in vitro data should be logically integrated to explain results from animal and human studies and to provide insights into the nature and consequences of in vivo drug metabolism. This article offers technical information and data and addresses scientific rationales and practical skills related to in vitro evaluation of drug metabolism to meet regulatory requirements for drug development.     

Predictive power of an in vitro system to assess drug interactions of an antimuscarinic medication: a comparison of in vitro and in vivo drug-drug interaction studies of trospium chloride with digoxin

The authors studied a potential drug-drug interaction via findings from in vitro and in vivo studies, to assess whether the in vitro system was predictive of in vivo clinical pharmacokinetic outcomes. An in vitro experiment and a clinical study were performed to assess the potential for interaction. The effect of trospium chloride on human P-glycoprotein-mediated transport of [3H]-digoxin was determined in vitro. A randomized, crossover clinical trial in 40 subjects was performed to evaluate the effect of trospium on the pharmacokinetics of digoxin in vivo. The findings from the studies were then compared. The in vitro findings in this study were corroborated by the clinical study via assessment of inhibition and impact on pharmacokinetic parameters. The in vitro system for assessment of a potential interaction of 2 drugs excreted primarily through the kidney was predictive of the pharmacokinetic outcomes obtained from a clinical setting.     

Investigation of drug-drug interaction potential of bortezomib in vivo in female Sprague-Dawley rats and in vitro in human liver microsomes.

Bortezomib (Velcade, PS-341), a dipeptidyl boronic acid, is a first-in-class proteasome inhibitor approved in 2003 for the treatment of multiple myeloma. In a preclinical toxicology study, bortezomib-treated rats resulted in liver enlargement (35%). Ex vivo analyses of the liver samples showed an 18% decrease in cytochrome P450 (P450) content, a 60% increase in palmitoyl coenzyme A beta-oxidation activity, and a 41 and 23% decrease in CYP3A protein expression and activity, respectively. Furthermore, liver samples of bortezomib-treated rats had little change in CYP2B and CYP4A protein levels and activities. To address the likelihood of clinical drug-drug interactions, the P450 inhibition potential of bortezomib and its major deboronated metabolites M1 and M2 and their dealkylated metabolites M3 and M4 was evaluated in human liver microsomes for the major P450 isoforms 1A2, 2C9, 2C19, 2D6, and 3A4/5. Bortezomib, M1, and M2 were found to be mild inhibitors of CYP2C19 (IC(50) approximately 18.0, 10.0, and 13.2 microM, respectively), and M1 was also a mild inhibitor of CYP2C9 (IC(50) approximately 11.5 microM). However, bortezomib, M1, M2, M3, and M4 did not inhibit other P450s (IC(50) values > 30 microM). There also was no time-dependent inhibition of CYP3A4/5 by bortezomib or its major metabolites. Based on these results, no major P450-mediated clinical drug-drug interactions are anticipated for bortezomib or its major metabolites. To our knowledge, this is the first report on P450-mediated drug-drug interaction potential of proteasome inhibitors or boronic acid containing therapeutics.     

The effect of methylbromophenvinfos (Polfos) on some enzymes in vivo and in vitro. I. In vivo studies

The experiment was carried out on Wistar rats receiving orally either oil or oily solution of methylbromophenvinfos (Polfos) either in a single dose of 0.5 LD50, or doses of 0.1 LD50 once daily for a period of 2, 4 or 6 weeks. The activities of cholinesterase (ChE), beta-glucuronidase (beta-glu), lipase and amylase were assayed in the blood serum, the activity of acetylcholinesterase (AChE)-in brain homogenates, and the activities of lipase and amylase-in homogenates of the pancreas. Cholinesterases were inhibited in the course of both acute and chronic poisoning with Polfos. During the acute poisoning a sharp increase in the activity of beta-glu in the blood serum, 1 and 2 h after the pesticide administration, was observed. Polfos inhibited lipase and amylase both after acute and chronic treatment.     

The covalent interaction of 1,4-dibromobenzene with rat and mouse nucleic acids: in vivo and in vitro studies.

1,4-Dibromobenzene (1,4-DBB) was covalently bound to DNA from liver, kidney, lung and stomach of mice after intraperitoneal administration. The covalent binding index (CBI) value (23 in mouse liver) was typical of weak initiators. On the contrary, no interaction with DNA from rat organs was observed (CBI detection limit: 1.3-2.6). The in vitro interaction of 1,4-DBB with calf thymus DNA was mediated mainly by microsomes, especially those from liver of both species and from mouse lung. Mouse subcellular fractions were more active then rat subcellular fractions. Unlike liver cytosol, subcellular cytosolic fractions from lung, kidney and stomach were capable of bioactivating 1,4-DBB, although to a lesser extent than liver microsomes. Both cytochrome P-450 and GSH-transferases are involved in 1,4-DBB bioactivation.     

机理性灭活体外细胞色素P450及其对体内药物相互作用定量预测的研究进展

近10年来，以体外细胞色素P450抑制数据预测潜在临床药物相互作用的方法得到极大发展。药物相互作用（drug-drug interaction）研究一直是临床实践和新药开发中的重要环节。本文对近年来引起广泛重视的机理性灭活作用（mechanismbased inactivation）进行总结分析，综合传统和经简化的鉴定机理性灭活作用方法，并介绍预测体内由机理性灭活引起药物相互作用的模型和影响参数。     

Lack of interaction between cefdinir and calcium polycarbophil: in vitro and in vivo studies

The effect of calcium polycarbophil on the absorption of cefdinir, cephalosporin derivative, was evaluated in both in vitro and in vivo studies. In the in vitro study, the release of cefdinir from a cellulose membrane in the presence or absence of metal cations was measured using the dissolution test procedure. In the in vivo study, volunteers and a randomized crossover design with two phases were used. In the first phase, the volunteers received 200 mg of cefdinir alone (Study 1); in the other phase, they received 200 mg of cefdinir and 1200 mg of fine calcium polycarbophil granules concomitantly (Study 2). The cefdinir concentrations in the samples or serum were measured by an UV-VIS spectrophotometer or high-performance liquid chromatography. Release in the presence of iron ions was slower than that in the absence of metal ions, however no difference was observed between release in the presence of calcium ions and that in the absence of metal ions. No difference was observed in AUC(0-10), C(max) and t(max) between Study 1 and Study 2. The absorption of cefdinir was not affected by co-administration of calcium polycarbophil. Moreover, the in vitro study on the release of drugs from a cellulose membrane may predict the absorption of a drug caused by the formation of chelate complexes between the drug and metal ions.     

Human cytochrome P-450 3A4: in vitro drug-drug interaction patterns are substrate-dependent.

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.     

Cytarabine release from comatrices of albumin microspheres in a poly(lactide-co-glycolide) film: in vitro and in vivo studies.

Cytarabine (ara-C) was included in albumin microspheres and these microspheres were immersed in a poly(lactide-co-glycolide) (PLGA) film to constitute a comatrix system to develop a prolonged form of release. Cytarabine-loaded albumin microspheres were synthesized by emulsion, and 25 or 50 mg of drug were included in the disperse phase. Thus, microspheres with 46+/-4 microg drug/mg microspheres and 50+/-5 microg drug/mg microspheres were obtained, which means a percentage of incorporation efficiency of 42+/-4% and 25+/-2%, respectively. These cytarabine-loaded microspheres were used to prepare PLGA-comatrices. Kinetic release studies indicated that total cytarabine release only takes place in the presence of protease, probably due to the fact that glutaraldehyde establishes covalent links with the amine side group of the drug and cross-links it with the protein matrix. A slower kinetic release of the drug was obtained from PLGA-comatrices, although only 80% of the included cytarabine was released on day 7. The comatrices were subcutaneously implanted in the back of rats and in both cases the ara-C administered dose was 36 mg of ara-C per kg of body weight. The drug was detected in plasma 10 days. The mean residence time (MRT) of the drug administered by these comatrices was 87-91 times larger when compared to the value obtained when the drug was administered in solution by intraperitoneal injection. The histological studies show that a degradative process of the comatrices takes place. The comatrices do not damage surrounding tissue; a normal regeneration of the implanted zone was observed.     

Mirtazapine and paroxetine: a drug-drug interaction study in healthy subjects

Paroxetine inhibits cytochrome P(450) 2D6, which is involved in the metabolism of mirtazapine. The possible drug-drug interaction between two pharmacologically distinct antidepressants, mirtazapine and paroxetine, has been investigated in a randomized, three-way crossover study in 24 healthy male and female subjects. After a titration phase of 3 days, each subject received single daily doses of 30 mg mirtazapine, 40 mg paroxetine or the combination for 6 days. Assessments included serial blood sampling for pharmacokinetics at steady state, cognitive testing using the test battery of CDR Ltd, a visual analogue mood rating scale (Bond and Lader) and the Leeds Sleep Evaluation Questionnaire. Paroxetine inhibits the metabolism of mirtazapine, as shown by increases of approximately 17% and 25% of the 24 h AUC's of mirtazapine and its demethyl metabolite, respectively. Mirtazapine did not alter the pharmacokinetics of paroxetine. The combined administration of mirtazapine and paroxetine probably does not alter cognitive functioning or result in major changes on the visual analogue mood rating scale and Sleep Evaluation Questionnaire, compared with the administration of either drug alone. The incidence of adverse events was lower during combined administration of mirtazapine and paroxetine than during administration of either drug alone. Fatigue, dizziness, headache, nausea, anxiety and somnolence were the most common adverse events during combined administration. These data suggest that the combination of mirtazapine and paroxetine is unlikely to lead to clinically relevant drug-drug interactions and can be used without dose adjustment of either drug. The combination may even be better tolerated than either drug alone. Copyright 2001 John Wiley & Sons, Ltd.     

Lipolytic activity of cis-beta-carboxyacrylamidine: studies in vivo and in vitro.

cis-β-Carboxyacrylamidine, a product isolated from the fermentation broth of the actinomycete, Streptomyces fur longus var.fur longus nov. sp., stimulated lipolysis in rat adipose tissue and isolated fat cells. Lipid-mobilizing activity in vivo was indicated by the increase in plasma FFA concentrations that occurred after the compound was administered orally. Daily doses reduced body weight gain but after approximately 5 days the rats became resistant to this effect of the compound. Resistant animals still responded to the lipid-mobilizing activity of the agent with an increase in plasma FFA levels. cis-β-Carboxyacrylamidine did not increase cyclic AMP concentrations in incubated adipose tissue. Also it neither stimulated adenylate cyclase or cyclic AMP-dependent protein kinase nor inhibited cyclic AMP phosphodiesterase or phosphoprotein phosphatase. The lipolytic mechanism was found to differ from the mechanism of N-ethylmaleimide. Also cis-β-carboxyacrylamidine inhibited soluble hormone-sensitive lipase, suggesting that soluble hormone sensitive lipase may not be the lipase mainly responsible for the response of adipose tissue and isolated fat cells to lipolytic hormones.     

FDA evaluations using in vitro metabolism to predict and interpret in vivo metabolic drug-drug interactions: impact on labeling.

Recent advances in in vitro metabolism methods have led to an improved ability to predict clinically relevant metabolic drug-drug interactions. To address the relationships of in vitro metabolism data and in vivo metabolism outcomes, the Office of Clinical Pharmacology and Biopharmaceutics in the Center for Drug Evaluation and Research, Food and Drug Administration, evaluated a number of recently approved new drug applications. The goal of these evaluations was to determine the contribution of in vitro metabolism data in (1) predicting in vivo drug-drug interactions, (2) determining the need to conduct an in vivo drug-drug interaction study, and (3) incorporating findings into drug product labeling. Ten cases are presented in this article. They fall into two major groups: (1) in vitro data were predictive of in vivo results, and (2) in vitro data were not predictive of in vivo results. Discussion of these cases highlights factors limiting predictability of in vivo metabolic interactions from in vitro metabolism data. The integration of these findings into drug product labeling is also discussed.     

In vitro and in vivo CYP3A64 induction and inhibition studies in rhesus monkeys: a preclinical approach for CYP3A-mediated drug interaction studies.

In this study, induction and inhibition of rhesus monkey CYP3A64 versus human CYP3A4 were characterized in vitro, and the corresponding pharmacokinetic consequences were evaluated in rhesus monkeys. In monkey hepatocytes, rifampin markedly induced CYP3A64 mRNA (EC50 = 0.5 microM; Emax = 6-fold) and midazolam (MDZ) 1'-hydroxylase activity (EC50 = 0.2 microM; Emax = 2-fold). Compound A (N-[2(R)-hydroxy-1(S)-indanyl-5-[2(S)-(1,1-dimethylethylaminocarbonyl)-4-[(furo[2,3-b]pyridin-5-yl)-methyl]piperazin-1-yl]-4(S)-hydroxy-2(R)-phenylmethylpentanamide), a known potent and mechanism-based inhibitor of CYP3A4, strongly inhibited the formation of 1'-hydroxy MDZ by recombinant CYP3A64 in a concentration- and time-dependent manner (KI = 0.25 microM; k(inact) = 0.4 min(-1)). Similar corresponding results also were obtained with human CYP3A4 in the presence of rifampin or compound A. In rhesus monkeys, MDZ exhibited a relatively high metabolic clearance (primarily via 1'-hydroxylation followed by glucuronidation) and a low hepatic availability (Fh = 16%). Consistent with the induction of hepatic metabolism of a high-clearance compound, pretreatment with rifampin (18 mg/kg p.o. for 5 days) did not significantly affect the i.v. kinetics of MDZ, but caused a pronounced reduction (approximately 10-fold) in the systemic exposure to MDZ and, consequently, its Fh following intrahepatic portal vein administration (i.pv.) of MDZ. A comparable extent of the pharmacokinetic interaction also was obtained after a 1.8 mg/kg rifampin dose. Also consistent with the in vitro CYP3A64 inhibition finding, compound A (6 mg/kg i.v.) markedly increased (10-fold) the i.pv. administered MDZ exposure. At the doses studied, plasma concentrations of rifampin or compound A reached or exceeded their respective in vitro EC50 or KI values. These findings suggest the potential applicability of the in vitro-in vivo relationship approach in rhesus monkeys for studying CYP3A-mediated interactions in humans.     

In vitro and in vivo safety studies of a proprietary whey extract.

A proprietary whey growth factor extract (WGFE) or Lactermin (Lact milk; ermin growth factors) is a whey fraction of milk containing the major proteins lactoperoxidase and lactoferrin, together with a variety of minor proteins and peptides such as the growth factors IGF-I, IGF-II, PDGF, FGF, TGF-ss and betacellulin. This growth factor component of milk has been suggested to possess biological properties such as the promotion of tissue repair and anti-inflammatory activity. In this study the safety of Lactermin has been evaluated using genotoxicity assays (Ames, mouse lymphoma and micronucleus assay) and in a subchronic (13 week) rat oral toxicity study. In vitro Lactermin did not show any mutagenic properties in the Ames or mouse lymphoma assay and in vivo did not show any adverse clinical effects or in the bone marrow of male or female mice. In the subchronic oral toxicity study in which 10 rats per sex were fed Lactermin mixed with rat diet to deliver doses of 300, 1000 and 3000 mg/kg/day for 13 weeks, male and female rats did not show any test article-related clinical observations or effects on body weight, food consumption, ophthalmic effects, functional observational battery, organ weights, locomotor activity, hematology, serum chemistry, urinalysis or macroscopic or microscopic pathology. The results from the genotoxicity studies and the subchronic oral toxicity study suggest Lactermin is safe for consumption with a no-observed-adverse-effect level (NOAEL) of 3000 mg/kg/day.     

某市药品生产企业质量管理中存在的问题及对策

目的:探讨药品生产企业质量管理中存在的问题,并提出相应的对策。方法:采用问卷调查的方式,对某市7家药品生产企业的基本情况、人员资质、检验仪器设备、质量控制等方面进行调查,并对相关数据进行统计汇总,分析其存在的问题。结果与结论:所调查的7家企业共有36条生产线、263个药品批准文号、1 268名职工。7家企业中,质量管理人员大专以上学历者占61.9%,质量检验人员大专以上学历者占36.4%;质量管理和检验人员工龄结构较合理,10年以下和10年以上各占50%左右。部分企业检验仪器设备不能满足现实要求,需要委托检验。各企业基本能按《药品生产质量管理规范》的要求对纯化水、注射用水系统及洁净室的洁净度定期进行监测。但各企业发展不均衡,人员素质有待提高,检验能力参差不齐,监管机制和法规体系不完善。建议企业应强化培训,不断提高质量管理和检验人员专业水平,并及时购置相关检验仪器设备;政府相关部门应完善监管机制,健全法规体系,强化技术监督,提高监管水平,确保药品质量。