Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential—towards a consensus

Abstract

The issue of drug–drug interactions has generated significant concern within the pharmaceutical industry and amongst regulatory authorities in recent years. This has arisen with respect to early termination of clinical development (e.g. furafylline), refusal of approval (e.g. mibefradil in Sweden), severe prescribing restrictions and withdrawal from the market (e.g. sorivudine, terfenadine, mibefradil, astemizole, cisapride), and threatened litigation. This report summarizes the outcomes of a conference held in Basel in November 2000, under the auspices of the European Federation of Pharmaceutical Sciences (EUFEPS), the US Food and Drug Administration (FDA) and the American Association of Pharmaceutical Sciences (AAPS). The meeting followed on from two previous workshops on drug interactions held in Nuremberg (1997) and Arlington (1999) sponsored by the same groups. Whereas previous conferences had identified the main areas of contention, a specific aim of this meeting was to attempt a consensus on the conduct of in vitro and in vivo studies of metabolic and transport interactions. There were five main conference sessions, in which experienced scientists from academia, industry and regulatory bodies were invited to contribute short presentations formulated, where possible, to address specific questions: Session 1. Mechanistic considerations. Moderators: S. E. Clarke, S. Wrighton;Speakers: A. Boobis, J. B. Houston, S. D. Hall, S. Ekins, P. Maurel, H. Lennernaes. Session 2. In vitro assessment;Moderators: J. B. Houston, K. Thummel;Speakers: B. G. Lake, J. Lipscomb, T. Lavé, R. S. Obach, S. Wrighton, S. E. Clarke, P. Langguth, L. Z. Benet, C. Crespi, E. LeCluyse. Session 3: In vitro prediction. Moderators: G. T. Tucker, L. Z. Benet;Speakers: K. Thummel, T. Andersson, B. Clement, H. Suzuki, V. Fischer, M. Dickens, J. Lin, P. Watkins, A. Rostami-Hodjegan. Session 4: In vivo assessment. Moderators: S-M. Huang, U. Fuhr;Speakers: K. S. Reynolds, R. A. Branch, G. Blakey, G. T. Tucker, U. Fuhr, V. Steinijans, P. Milligan, G. Alvan. Session 5: The regulatory view. Moderator: G. Alvan;Speakers: S-M. Huang, Y. Ohno. In addition, the speakers were organized into panels with chairpersons and rapporteurs to develop position statements on the main issues in breakout sessions. These were then presented in open forum at the final session and modified to take account of debate. We have attempted to distil the key features of the discussions with respect to resolved and unresolved issues. Chairperson: J. B. Houston; Rapporteur: K. Thummel In vitro methodology presents a wide spectrum of choices, many of which have unresolved issues. It is convenient to consider the latter under the headings of Experimental systems, Experimental conditions and Effective drug concentrations. Hepatocytes provide cellular integrity with respect to enzyme architecture, phase II metabolism and, potentially, allow for any concentration gradients mediated by transporters that may affect exposure of substrate/inhibitor to enzymes. However, some transporters are rapidly down-regulated after isolation of hepatocytes, and support matrices (sandwich cultures) may introduce artefacts (additional collagen diffusion barrier; loss of enzyme activity). In practice, the evidence that human hepatocytes provide ‘better’ values of intrinsic metabolic clearance and Ki values is limited. Systematic comparisons of Ki values obtained from microsomes and hepatocytes, with appropriate correction for nonspecific binding, are needed. Discrepancies may indicate a role of transporters modulating access of drug to enzyme. While useful for qualitative studies (metabolite identification, comparison of metabolite pattern in animals and humans), it was agreed that the utility of cryopreserved hepatocytes for quantitative estimation of intrinsic clearance or Ki was not well-established. It was felt that, currently, these systems should be used for qualitative estimates of Ki values only (e.g. in high throughput screening). Levels of enzyme expression are variable across systems and, especially with regard to CYP3A, reductase and cytochrome b5 to CYP ratios are often unphysiological. Use of ‘relative activity factors’ (RAF) should take account not only of the relative hepatic abundance of individual CYPs, but also differences in activity per unit enzyme in expression systems relative to liver microsomes. As these factors will rely on the use of specific probe substrates and particular microsomal preparations there are many attendant issues (see later). The qualitative value of in silico predictions of metabolite patterns and active site modelling for identification of inhibitors was recognized. However, although promising, current experience of in silico prediction of CLint and Ki values is limited. Existing pharmacophore models generally do not describe the complete active site space of an enzyme. Good rank order predictions of inhibition potential can be achieved, especially with similar molecules, but there is a need to apply training data sets to much larger series, and to develop more extensive data bases in association with experimental in vitro and, ideally, in vivo data. It was recommended that companies explore the potential of in silico methods with large molecular librairies, developing proof of principle with animal data before moving to prediction in humans with an established and acceptable prediction accuracy. For Ki or IC50 determinations, the calculation of parameters from initial product formation was recommended; use of substrate disappearance is of concern because of the potential for product inhibition. If the former approach is used it is prudent to restrict to < 20% substrate depletion; if the latter, > 20% substrate depletion is desirable to offset analytical constraints that will severely limit the range of inhibition that can be measured. Also, there may be different criteria for high throughput assays and optimal parameter determination. The turnover numbers of CYPs in microsomal preparations are influenced by accessory factors such as pH and ionic strength. Should, for example, incubations be carried out at pH 7.4 or would a lower pH be more reflective of intracellular conditions? The addition of magnesium to microsomal incubations may create artefacts in kinetic parameter estimation. It remains an open question as to whether it should be omitted from the incubation matrix or, at least, added in a physiological concentration. To obtain valid data on enzyme kinetics the substrate concentration should be in > 10 – fold excess. It is common practice to work with a mid-range nM CYP concentration in studies with recombinant enzymes. However, for human tissue incubations the mid- µm range of total CYP is used. While there is minor concern for the low abundance CYPs, for CYPs 3A4 and 2C9 the optimal ratio will not be established. For inhibition studies it is usual to cover a concentration range which includes the Km. In this case, the ratio will probably approach unity. Optimum conditions for Michaelis-Menten kinetics are probably also violated in vivo as total CYP concentration is approximately 20 nmol g−1 liver. Furthermore, because CYPs are membrane bound, it cannot necessarily be assumed that enzyme kinetics in vivo will be the same as those in a more spatially homogenous microsomal suspension in vitro. It is prudent to correct enzyme kinetic parameters for any significant nonspecific binding to microsomes and to the incubation container. The use of low protein concentrations will minimize the former, but may exacerbate the latter, particularly when using high throughput multiwell plate materials. Binding to the apparatus should be considered when carrying out all in vitro studies, as exemplified by artefacts introduced by such binding in recent transporter studies using the Caco2 system. Albumin has been noted to promote microsomal metabolism of some drug substrates, primarily CYP2C9 reactions. Accordingly, this observation has been used to refute the ‘free drug hypothesis’ with regard to effective substrate concentration. However, the addition of albumin to microsomal incubations is un-physiological. Possible explanations of the effect include a protein–protein interaction modifying the access of substrate to the active site, a carrier effect whereby the albumin surface helps to project the substrate towards an access channel, a surrogate effect whereby albumin mimics a constitutive intracellular protein that also binds acidic drugs (ligandin?) and promotes metabolism, or a fatty-acid clean-up effect whereby addition of albumin sequesters fatty acids that may inhibit CYP2C9. Further investigation is indicated to resolve this issue. Clearly, the use of pooled microsomes ignores the important issue of interindividual variability in metabolism. It was recommended that in vitro–in vivo extrapolations should always encompass a range rather than an average. Also, there was concern over the of liver tissue in that the range of variability in CYP expression may be that in The of CYP and (e.g. to of is Systematic studies of liver are indicated to any in activity across different CYPs that with a to at the of The of a site of enzyme kinetics in vitro is especially with of is models of the data are by issues of parameter and the in vivo of the is As a for in vitro inhibition studies it was recommended that IC50 values be using at two low concentrations of at two substrates of which kinetics should be when in vitro CLint and appropriate parameters used for in vivo it be that would be more to than the liver because of factors and a range of concentrations. However, the issue may be by of substrates and Further of with respect to is the pharmaceutical there is an concern over developing that or inhibition of drug metabolism, not only because of drug–drug interactions but also because of to binding of metabolic to and of a modified to for It was considered that inhibition should be in in vitro because the cannot be with complete from of potential was with the addition of to loss of initial product formation should be and, in the of formation can be followed of inhibition kinetics in vitro should be followed with in vivo studies in only a minor metabolic is the would be in vivo as a in the clearance on is a of that is the in a and be but and interaction studies may then be indicated in the early of human This will the concentration of substrate/inhibitor that the enzyme and, the value of enzyme kinetic the active of for example, can be considered as or or a of is there is evidence to support the of models of hepatic drug clearance that the intracellular drug concentration the metabolic the hepatic of substrate and the of substrate should be However, estimates of drug concentrations are useful for estimates of the of an Chairperson: S. Rapporteur: S. E. It was agreed that the desirable of would be the same qualitative across similar quantitative or and the same and to the same and regulatory of for an established probe substrate or Ki for a should be within of although it was that the latter often to values are in especially when nonspecific binding is into and can often be by experimental of 20% for and Ki values for the same of was considered to be a consensus was on appropriate for of the human were to the to provide in vitro parameters and not for in vivo or for with respect to The were into and of and not used or than optimal with regard to some This is in 1. of > inhibition of a with the was considered should be for to appropriate of data. was primarily on the use of human hepatocytes in the drug development rather than the use of binding assays and methods which were considered to be to for activity was considered to be the with and useful primarily for are to allow for inhibition by were agreed the conduct of with of in should be followed by with the (e.g. should be used. to five preparations are generally for evidence of although may be with a if is by regard to appropriate it was agreed that or for and 10 for should significant for and However, the of a Chairperson: L. Z. Rapporteur: H. of the expression of transporters and activity in different human is at an early more than were in the It was agreed that there was a need for much more in vivo data to in vitro methods of drug In the drug metabolism and transport remains a significant in data. in vitro evidence for the with respect to drug is support for an in vivo transport and intracellular metabolism was considered to be only It remains for example, is in the when it is not a for transport that would exposure to concentrations. It was that the of and active transport across different membrane need to be Further these issues are to the under by regulatory It was the consensus of the that there are significant drug–drug interactions for it was that clearance of drugs in humans can be with from animal data. of transport clearance and interactions from animal models is more in vitro models to the transport across the were considered to be in because of variability in transporter expression as a of the of and across need to develop and in vitro for hepatic transport systems (e.g. the use of was with regard to in vivo transporter binding data obtained in vitro using methods on was considered to be useful in the of a drug–drug or it was felt that such is to in vivo membrane have binding to transporter protein and transport in It is important to consider that binding may and to that some binding transport was recommended as an in vivo probe for interactions in the it metabolism. were and However, because of high it was felt that may only be a probe for the of and not for inhibition. The need for the development of specific probe and inhibitors for individual transporters was In order to the to drug–drug or interactions at the transporter it was agreed that there is a need the in vitro methodology to and inhibitors of the of transporters of of the of the in drug the interactions and of all the drug interaction for and in more concentrations transporters of the of transporter systems and tissue specific the of in vitro preparations in vivo data. and more specific inhibitors of individual transport the of in transport Chairperson: V. Rapporteur: J. in these discussions were to the value of in vitro data in the of drug development rather than in the of of CYP inhibition and were identified as Also, to in vitro to and in vivo kinetics and the quantitative of a drug–drug interaction and variability was as a issue. The use of hepatocytes and liver was for identification of and, the use of for the relative of phase and II metabolism. The of the of and in incubations was The need to have an of drug clearance in humans was also in order to the of metabolism to clearance in humans is often from animal investigation of in It was generally agreed that more than approach should be to the in the metabolism of a The methods were the use of with inhibitors or and of enzymes. of was considered to be a prediction of the of specific to metabolic clearance can be on the of relative abundance in the liver and values from data on Session The use of or was considered to be the similar if low protein concentrations are used. In it was that use of hepatocytes may be a useful but with attendant of where a with hepatocytes would be is where inhibition by by in is by by phase II It was considered important to Ki rather than IC50 values in order to the of inhibition. prediction of the of drug–drug interaction enzyme an of the concentration of to which the are and on the of on metabolic of In with the of the in Session it was that enzyme activity should be used as the Hepatocytes were as the experimental useful only for by systems and binding assays were felt only to be appropriate for initial value is to be established. of the of drug–drug interactions in vivo from in vitro have been only on data. The that there is a of with using such data in that the to is not when in vitro data there is a need to on the and in individual approach to this is to develop whereby in vitro data on drug metabolism are into and all such can be used to different in vitro–in vivo and may in the optimal of in vivo Chairperson: G. T. Rapporteur: U. It was generally agreed that the with respect to in vivo interaction studies should not be on in vitro if a high of inhibition. on values and the metabolism the of were but it was felt that it was to for on the of on the in of substrate when is is in but the is In practice, be and also take account of as as of drug–drug of on the ratio of the of substrate < in the and of a The and of using the probe substrates or the were and, on the former were by the for initial of the potential for drug interaction to in vitro interaction studies would be using initial for the CYPs, with are indicated in 2. The need for in vivo probe of and transporter systems was also These were to and in the where some the value of that and clinical in order to minimize the of studies The value of using more than substrate of was to be with the in vitro of this enzyme. The consensus of the was that from the use of of CYP probe substrates in vivo to in vitro predictions of interactions was to more specific The of this approach the of a was to to the of the However, it the that of the approach was not as by some an and an vivo The former that a is it is of inhibition from in vitro and that it would allow quantitative prediction of the of interactions. Furthermore, if have agreed to have in in vitro there is need at for a The vivo the value of in in vivo studies of the potential for metabolic out that not all clinical are to data in vivo Furthermore, a specific was where studies with liver microsomes indicated that the was a of all the common CYPs, but was then by a to have in Further in vitro investigation using hepatocytes a of because of of phase by phase II Clearly, there is always to be a of in from in vitro especially in the where an in value is high in vivo should at provide useful with regard to the of in vitro to in vivo The question as to the of a with regard to the of interaction with is a key issue. In at for inhibition with respect to a site Michaelis-Menten kinetics and when the of the of interaction on and is of However, the may not be valid and there is the that, because of the variability with a particular probe and a may provide a much has been to that there are interactions of the drugs there is a need for of the of the The final consensus on was that there was not a consensus and that the issue debate. The issue of a probe in studies with substrates to provide was the felt that there are data in the on to be that would not if a were The of the used to the activity of an enzyme in vivo on it to the intrinsic metabolic clearance of the substrate by that enzyme. For such as or metabolic ratios ratios or a of of metabolite are often used with some probe It was considered that the for such should be established and in order to allow for the of In the of metabolic mediated by different but to the formation of the same metabolite the same should be For example, the ratios of to and of to have been as convenient of and 3A4 However, of the are to a common metabolite by the enzyme. Accordingly, any of ratio may of inhibition of the Also, it should be that metabolic ratios of the drug in the of ratios and of the metabolite in the of If drug or metabolite are in the of and of pH can a significant to the variability of these Clearly, this has for the of probe substrate and the of the to a drug The with respect to for some has been established. For example, the or ratio has been established and by to be the of of the of all is in order to It was generally agreed that the of a drug–drug interaction should be in the same as studies, on the of While it was considered as a position for drugs to if the of the ratio of or in the and of is within the for for it was that the should be and clinical considerations. However, as with the should be for specific drugs is not a of exposure in the of an was agreed be a for The of kinetics to drug–drug interactions was considered to be useful and to in vitro studies and studies in is to the issue of should be as to an interaction rather than the of The of recent of drugs that the of a specific of was by drug–drug interactions in a of it would also be that at would be those where a specific of drug interaction to For example, in the where a is by and the would have a in drug if or were similar presents for a drug that is by and by when the also has the of drug interactions is to the and Accordingly, the important question was in the as to whether current in vitro and in vivo address the in the While it was that studies with were not to it would be to include in studies evidence of would be to to the on variability from in vitro and in vivo and the and of the to to the Chairperson: G. Rapporteur: S-M. The of of drug–drug interactions and the potential for interactions to cannot be over drugs astemizole, and which were from the US market to drug–drug are substrates or inhibitors of cytochrome enzymes. The by provide similar with regard to to drug–drug interactions. the use of a For example, the the use of an approach It is useful when evidence for and a drug–drug interaction is at all of drug and in vitro studies of drug metabolism and drug–drug interactions to which in vivo studies should be early phase in vivo studies to the important potential drug–drug interactions by in vitro and phase studies to the range of potential interactions and to allow of drug–drug interactions. for drug–drug interactions. and November of and in the in vitro of and potential as a of assessment. For example, the has recommended in vitro studies of CYP (e.g. to the conduct of specific clinical of values should be for of the CYP and a low value that in vivo studies for that particular CYP may not be the current to in vivo interactions there are in all as these are only to apply to However, is of in vitro studies, the US is the of has for The indicated that these studies are to be the of which that should take the to the and of data. These for example, of analytical and of and of any in the The recommended are similar the a the For example, the the use of the recommended the and the substrates or in the initial in vivo studies The was on the utility of to the potential of recommended the use of a approach in the of data (e.g. ratios of the substrate with and the To and the use of with as a only In addition, the the use of to agreed that the animal data may a potential interaction when the are However, animal do not in There was on the role of data (see The studies on the that may from of interaction data. on drug interaction in the have been or to to (e.g. with the use of It was agreed that the issues need to be more in of of drug–drug interactions. of in The is for and industry to address the and is a to in in vivo We that this conference a useful to a and of drug development and The in in vitro were and were a of was on probe for in vitro and in vivo studies, and the need to at particular of specific drug–drug interactions was The was identified as a and as were the issues of enzyme the and and the of enzyme in vitro.