Clinical pharmacology and pharmacokinetics: questions and answers

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The questions and answers (Q&As) on this page provide an overview of the European Medicines Agency’s (EMA) position on specific issues related to clinical pharmacology and pharmacokinetics.


Users should read the Q&As in conjunction with the relevant scientific guidelines.


The Committee for Medicinal Products for Human Use (CHMP) may seek the input of the Pharmacokinetics Working Party (PKWP) to address specific questions in relation to clinical pharmacology. This input may contain general guidance or clarify specific aspects of scientific guidelines. EMA publishes the PKWP's input on this page.

The date refers to when the Q&A was first published.

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Questions about clinical pharmacology, activities of the PKWP and other general enquiries: Send a question to the European Medicines Agency

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Table of contents

1. Pharmacokinetics

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1.1 What does the Agency recommend on the determination of absolute and relative bioavailability? January 2011

Absolute bioavailability

Information on absolute bioavailability is important in the overall evaluation of the pharmacokinetics of the drug substance. For some new chemical entities information on absolute bioavailability facilitates the evaluation of the mass balance study, and enables conclusions regarding the contribution of different elimination routes to drug clearance. 

This information is important when determining the need for studies in subjects with renal and hepatic impairment as well as the need for drug-drug interaction studies at biliary excretion level. The information is also useful when predicting the consequences of pre-systemic drug-drug interactions, both at absorption and metabolism level. 

Therefore, for new active substances intended for systemic action, the absolute bioavailability should, if possible, be determined by comparing the bioavailability of the intended pharmaceutical form for an extra-vascular route of administration with an intravenous administration. For substances with non-linear pharmacokinetics, consideration should be given to the dose(s) used for evaluation of absolute bioavailability. Furthermore, data on absolute bioavailability is valuable in the evaluation of BCS based biowaivers (see Guideline on the investigation of bioequivalence, CPMP/EWP/QWP/1401/98 Rev. 1).

Relative bioavailability

It is recommended to obtain information on the relative bioavailability of different dosage forms (or formulations) used during drug development. By definition relative bioavailability is the comparison of different dosage forms (or different formulations thereof) administered by the same or a different non-intravenous route (e.g. tablets vs. oral solution).

Regarding formulation changes during drug development, unless BCS based biowaiver is applicable bioequivalence studies are needed if there has been a change between the formulation used in phase III and the final marketing formulation which may affect rate or extent of absorption. Relative bioavailability studies (or comparative bioavailability studies) are recommended between different formulations used during phase I, II and III. There is no requirement for demonstration of bioequivalence between phase II and phase III formulations. It is assumed that any difference in rate or extent of absorption between these formulations is taken into account in the design of the phase III studies. 

The clinical relevance of any differences in exposure between formulations used in phase I, II and III studies should be discussed in applications for NCEs in Module 2.5 and 2.7.1 and taken into account in the assessment of pharmacokinetic data in Module 2.7.2.

1.2 Are there any particular recommendations on the determination of absolute and relative bioavailability for suprabioavailable products? January 2011

A suprabioavailable product displays appreciably larger extent of absorption than an approved reference medicinal product.

If suprabioavailability is found, the development of a lower dosage strength should be considered. In this case, the biopharmaceutical development should be reported and a final comparative bioavailability study comparing the reformulated new product with the approved reference medicinal product should be submitted. The potential for a difference in food effect on the rate and/or extent of absorption or a difference in absorption interactions between the reformulated new product and the approved reference product should be discussed and when relevant evaluated in vivo.

In the case where a lower dosage strength has not been developed the dosage recommendations for the suprabioavailable product will have to be supported by clinical studies.

1.3 Regarding the requirement to perform incurred sample reanalysis (ISR), how should the absence of ISR be handled? Is it possible to identify other factors which could be assessed in the absence of ISR to support the validity of the analytical method? December 2012

ISR is considered an element of the validation of the analytical method during study sample analysis. It has been discussed for many years in the scientific community and recently been introduced as regulatory requirement in the European guideline. Like for any deviation from a guideline requirement, the lack of ISR requires a scientific justification by the applicant. Such justification could be considered for validations which have been performed before the new guideline came into force. Its scientific validity will need to be reviewed on a case-by-case basis in the light of the overall validation data, the study outcome, as well as the reliance of the application on these data.

Introduction of ISR as a regulatory requirement

The principles for the implementation of a guideline are outlined in the Procedure for European Union guidelines and related documents within the pharmaceutical legislative framework (EMEA/P/24143/2004 Rev.1). While applicants may, with the agreement of the competent authority concerned, choose to apply a guideline in advance of the date for coming into operation of a guideline, competent authorities should await this date before requiring a guideline to be taken into account for assessments. The Guideline on bioanalytical method validation came into force on 1 February 2012 meaning that as of this date this document sets the applicable requirements for the regulatory review of applications.

It is acknowledged in the above-mentioned principles that in some circumstances it may not be possible for applicants to fully comply with new guidelines within this timeframe (e.g. data generated from trials started before the implementation of the new guideline). In such cases, the applicant should consider whether departure from the new guideline could be justified. The applicant's justification will then be considered on a case-by-case basis by the relevant competent regulatory authorities.

In compliance with this framework, the regulatory assessment requires the review of the bioanalytical method validation in any application against the current regulatory standards as set out in the guideline, including the requirement to address incurred sample reanalysis. If an element of the validation is missing, e.g. lack of incurred sample reanalysis, then this would need to be scientifically justified by the applicant. Such justification can be considered in the framework of the above exception that a particular validation has been performed before the bioanalytical guideline came into force, i.e. February 2012. Any justification will need to be reviewed on a case-by-case basis considering the overall validation data, the study results, as well as the reliance of the application on these data.

Considerations regarding a potential justification for the lack of ISR data

The attempt to scientifically justify the lack of ISR is considered only appropriate for the very practical reason that a study was performed before the Guideline on Bioanalytical Method Validation came into force.

For the scientific justification of the lack of ISR the applicant should take all the following points into consideration:

  • metabolite back conversion:

The applicant should support that back conversion is not an issue for the drug compound or that the risk of back conversion on the outcome of the study results is low as for instance it is known that the drug compound is (almost) not metabolised. For drug compounds for which it is known that back conversion is an issue, i.e. clopidogrel, atorvastatin, ramipril, lack of ISR is considered not acceptable.

  • other ISR data obtained in the same laboratory:

ISR data obtained for the same analyte from other studies carried out in the same laboratory and with the same analytical method may be used as supportive data to justify the lack of ISR.

  • data from repeat analysis:

In most studies repeat analysis of study samples has to be carried out for different reasons. Repeat analysis can be considered as ISR in certain situations, however due to the nature of the reanalysis (for instance run acceptance criteria failure) those data are considered not reliable. The applicant should report the data of these reanalysis and take into account and discuss the reason for the reanalysis in the justification for supportive data.

In case of a multi analyte analysis, if the repeat analysis was due to run acceptance criteria failure for one of the analytes, but the other has passed, the results of the analyte(s) which passed can be used to infer ISR, if analysed.

  • the obtained pharmacokinetic data in the study:

The applicant should compare the obtained pharmacokinetic data with data obtained previously or with reported data and should show that these are comparable

  • 90% confidence interval:

As one element of such justification, if applicable, the applicant could also take into consideration the width of the 90% confidence interval and the ratio to possibly justify that a false positive outcome due to ISR problems has a low probability.

The last two bullet points need to be thoroughly discussed specifically for bioequivalence studies.

The applicant should also consider the overall reliance of the application on the data generated with the bioanalytical method in question.For new molecular entities the pivotal basis of the application normally rests on clinical efficacy and safety studies, nevertheless pharmacokinetic studies in such an application provide significant information (e.g. general pharmacokinetic profile, interactions), which is also reflected in the labelling, hence the validity of such data needs to be sufficiently ensured. Abridged applications may exclusively rely on pharmacokinetic data, e.g. bioequivalence studies, making overall validity of these data paramount. Therefore, the validity of the data needs to be considered for the assessment of the application and the specific study considering whether the data are pivotal or supportive.


The requirement to perform incurred sample reanalysis (ISR) has been introduced with the Guideline on bioanalytical method validation (EMEA/CHMP/EWP/192217/2009). Incurred sample reanalysis (ISR) is applied to assess the reliability of bioanalytical methods used in pre-clinical toxicokinetic studies and for a variety of clinical pharmacology studies including bioavailability, bioequivalence, pharmacokinetic, interaction and comparability studies. The need for incurred sample reanalysis is discussed already since 20061 and regulators supported the need for incurred sample reanalysis also considering significant bioanalytical deficiencies observed in studies. Therefore, although incurred sample reanalysis is a requirement introduced in Europe for the first time with the new EMA Guideline on bioanalytical method validation (EMEA/CHMP/EWP/192217/2009), which came into force in February 2012, it should be noted that the scientific need to perform ISR as an element of bioanalytical method validation was already identified much earlier. ISR should therefore be considered as part of the validation of the analytical method during study sample analysis.

Different sources can be identified which might contribute to the failure of ISR. Some sources may be more likely to occur than other depending on the method, active substance, and analyst, however they cannot be excluded. Sources of ISR failure may be:

  • Execution, i.e. switched samples, instrument issues, scientist performance of method
  • Method, i.e. metabolite interferences, back conversion of metabolites, poor ruggedness, internal standard response
  • Samples, i.e. matrix effects,mislabelling, handling

It is recognized that some of these sources are also likely to occur during validation, like switching samples and mislabelling.

ISR failure and thus lack of the reliability of the study outcome can happen in each study and as such it is difficult to generalise it. Especially with pivotal studies it should be ensured that the results are reliable. However it is also understood that ISR is an additional confirmation of results next to a complete validation.


1. Viswanathan CT, Bansal S, Booth B et al. (2007) Workshop/Conference Report: Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. AAPS J. 9(1), E30–E42

2. Drug interactions

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2.1 Are there alternatives to the recommended 72 hours incubation time for enzyme induction or down-regulation in vitro studies? October 2014

The Guideline on the investigation of drug interactions states that: “the incubation duration of enzyme induction or down-regulation– in vitro studies should generally be 72 hrs. Shorter durations should be well justified.” Most pharmaceutical companies currently use a 48 hrs incubation period with media replenishment every 24 hrs. The mRNA responses are very quick (often <24h). Longer incubation periods bear the risk of study outcome limiting cytotoxicity.

2.1.1 What is the rationale behind recommending a 72 hours incubation time for enzyme induction or down-regulation in vitro studies? Is it acceptable to use shorter incubation times such as 8 to 12 hours measuring mRNA when obtaining EC50 and Emax? This situation could be most relevant for cytotoxic medicines such as used in oncology. 

When drafting the guideline limited experience with induction studies measuring mRNA was available. Based on studies measuring enzyme activity, an incubation duration of 3 days appeared suitable. However, in accordance with the guideline, shorter incubation times can be sufficient if well justified that adequate sensitivity is maintained. The sensitivity of the specific study is verified by the response of the positive control inducer (see the Guideline on the Investigation of Drug Interactions for details).

We have no experience with very short incubations (8-12 hrs) and we are not aware of any literature reference evaluating this. If adequate sensitivity cannot be supported it is recommended to investigate induction in vivo instead, for example by performing a cocktail study.

2.1.2 Would reporter gene data and/or PXR and CAR TR-Fret competitive binding assays be acceptable?

If an induction signal for a PXR inducible enzyme is detected and EC50 and Emax for your investigational drug can be determined, the RIS correlation method (or possibly the mechanistic static model) as described in the Guideline on the investigation of drug interactions could be used with short incubation periods if sensitivity is ensured during the validation.

Receptor binding assays can be used as supportive data only. If using these assays, the applicant needs to provide data supporting the performance of the method, including sensitivity.

2.2 Does the Agency have examples demonstrating in vitro data on down-regulation being confirmed by in vivo findings? Could this be an in vitro artefact? October 2014

The Agency has experience with down-regulation observed in human hepatocytes confirmed in vivo.

2.3 What is the rationale behind the use of 50-fold Cmaxu in the in vitro studies? Can this value be adjusted based on Vd estimates and/or liver-blood partitioning, e.g. for a compound with low human Vd where it is unlikely that liver partitioning is 50-fold? October 2014

The 50-fold safety margin on Cmaxu is experience based and has been applied for more than a decade in the enzyme inhibition assessment in the EU. The safety margin includes factors such as an at least 10-fold inter-study variability in Ki, the possibility of markedly higher concentrations in the hepatocyte than in plasma and higher portal vein concentration than Cmax in plasma during absorption.

The safety factor used for inhibition is also applied in the induction assessment. However, additional issues add to the uncertainty of the IVIVC for induction, such as the possible metabolic and/or chemical degradation during the incubations (37°C for 24 hours) and the lack of control of transporter expression in the cells. Reducing the safety-factor based on Vd cannot be recommended until there is scientific data to support this.

2.4 The mRNA cut-off of 2-fold induction may be stringent given the variability between and within donors. Would the use of modelling approaches be better suitable than fold induction to assess the need for a clinical induction-based drug-drug interaction studies? October 2014

The 2-fold cut-off is used in the basic model. This relates to the first investigation of whether the drug could be an inducer and therefore it is suitable to have a simple approach. For PXR mediated induction the applicant may use alternative methods such as the RIS correlation method and the mechanistic static model as stated in the guideline. At present the use of PBPK is not recommended for this purpose.

2.5 What is the scientific rationale behind recommending CITCO as the positive control for the in vitro assessment for CYP2B6 induction? Is the Agency willing to consider alternative compounds such as Efavirenz which is known to cause CYP2B6 induction-based DDIs in the clinic and is known to be a CAR transactivator? October 2014

If the CAR activator also activates PXR to a significant extent, presence of CAR regulatory pathways cannot be verified. CITCO at the proposed concentration <100 nM is the only substance we are aware of that activates CAR exclusively. Efavirenz is a PXR and CAR agonist (Sharma et al, Biochem Pharmacol 2013). If confirmed that the PXR activation of efavirenz, or another substance, is negligible as compared to the effect on CAR at a certain concentration, the use of that substance as a positive control for CAR could be supported.


CITCO has poor properties which results in variable inductive responses between studies. In addition, CITCO is not an approved drug which limits the applicability to put in vitro data into clinical context.

2.6 What are the expectations with respect to co-regulated enzymes including transporters if a compound induces CYP1A2, CYP2B6 or CYP3A4? Rather than assessing induction of CYP2C in the clinic, can in vitro data or a paper argument be used to avoid additional targeted clinical DDI studies knowing that PXR is involved in the regulation of CYP3A4 and CYP2B6? October 2014

A mechanistic approach to induction is applied. If induction is observed for one of these enzymes, co-regulated enzymes and transporters will be assumed to be also induced. The effect on these enzymes/transporters should preferably be quantified in vivo. Based on present knowledge, lack of CYP2C induction is concluded if the drug does not increase CYP3A4 or CYP2B6 mRNA expression.

2.7 What should the duration of in vivo induction studies be? October 2014

Please note that when the aim of an in vivo induction study is to quantify an induction effect, the duration of the treatment of the inducer should be well thought and justified to the agency based on a conservative enzyme degradation constant (kdeg) and time to reach steady state for the inducer (please see the Guideline on the investigation of drug interactions). 

At present, to evaluate the full induction effect on a CYP3A4 substrate, a duration of 10-14 days is recommended for a perpetrator that does not accumulate during multiple-dose conditions.

3. Bioequivalence (general)

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3.1 Which statistical method for the analysis of a bioequivalence study does the Agency recommend? January 2011

The Guideline on the investigation of bioequivalence (CPMP/QWP/EWP/1401/98 Rev. 1) recommends analysing bioequivalence studies using ANOVA and specifying all factors, including subjects, as fixed rather than random. The analysis presented above show that this approach (Method A) is feasible even for unbalanced replicate design studies. The advantage of this approach is that it is straightforward and that it appears to be software and software option independent. A simple linear mixed model, which assumes identical within-subject variability (Method B), may be acceptable as long as results obtained with the two methods do not lead to different regulatory decisions. However, in borderline cases and when there are many included subjects who only provide data for a subset of the treatment periods, additional analysis using method A might be required.

For highly-variable drugs it is recommended to estimate the within subject variance using data from the reference formulation only.


The Guideline on the investigation of bioequivalence  (CPMP/QWP/EWP/1401/98 Rev. 1) recommends analysing bioequivalence studies using ANOVA and specifying all factors, including subject, as fixed rather than random.

For a 2×2 crossover trial the confidence intervals for the formulation effect will be the same regardless of whether fixed or random effects are used for subject.

For replicate designs the results from the two approaches will differ if there are subjects included in the analysis who do not provide data for all treatment periods. Either approach is considered scientifically acceptable, but for regulatory consistency it is considered desirable to see the same type of analysis across all applications.

For multi-period studies other, more complex statistical models are possible. One of the possibilities is to include a subject by formulation interaction term. Analysis of data currently available shows that the subject by formulation interaction is negligible and therefore models without the interaction effect adequately control the type I error. Thus the same statistical models can be used regardless of the design.


The following text on the general analysis of bioequivalence studies is included in the guidance document. The bold text is the main sentence of interest for this discussion.


Statistical analysis

The assessment of bioequivalence is based upon 90% confidence intervals for the ratio of the population geometric means (test/reference) for the parameters under consideration. This method is equivalent to two one-sided tests with the null hypothesis of bioinequivalence at the 5% significance level. 

The pharmacokinetic parameters under consideration should be analysed using ANOVA. The data should be transformed prior to analysis using a logarithmic transformation. A confidence interval for the difference between formulations on the log-transformed scale is obtained from the ANOVA model. This confidence interval is then back-transformed to obtain the desired confidence interval for the ratio on the original scale. A non-parametric analysis is not acceptable. 

The precise model to be used for the analysis should be pre-specified in the protocol. The statistical analysis should take into account sources of variation that can be reasonably assumed to have an effect on the response variable. The terms to be used in the ANOVA model are usually sequence, subject within sequence, period and formulation. Fixed effects, rather than random effects, should be used for all terms.

Following the publication of revised version of the Guideline on the investigation of bioequivalence  (CPMP/QWP/EWP/1401/98 Rev.1) this paragraph raised several questions from interested parties. The reason for this interest was twofold. Firstly, the new guideline gives more emphasis to replicate design trials and evaluation of such trials is a more complex task compared to a conventional two-period two sequence crossover trial. Secondly, the current standard for the analysis of replicate design trials is a likelihood-based linear mixed model with random subject effects.

The question of whether to use fixed or random effects is not important for the standard two period, two sequence (2×2) crossover trial. In section 4.1.8 of the guideline it is stated that “subjects in a crossover trial who do not provide evaluable data for both of the test and reference products should not be included.” Provided this is followed the confidence intervals for the information effect will be the same regardless of whether fixed or random effects are used. 

Therefore all that remains to be discussed is the analysis method for replicate designs. In section 2 three models for analysing data from replicate bioequivalence trials are considered. To illustrate these approaches, in section 3 data from a four-period unbalanced study (see data set I, Annex I) and data from a three-period balanced study (data set II, Annex I) were analysed using different statistical models and computer programs (Annex II).

3.2 What is the minimum number of subjects that should be included in the second stage of a two-stage bioequivalence study design? February 2013

1. The expected analysis for the combined data in a two-stage design is ANOVA with terms for stage, sequence, sequence*stage, subject (sequence*stage), period (stage), formulation.

2. This model can be fitted provided that in each stage, there is at least one subject randomised to each sequence. This does not supersede the requirement for at least 12 subjects overall.

3. A term for a formulation*stage interaction should not be fitted.


From the perspective of type I error control it is considered that there is no minimal number of subjects to be included in the second stage of a two-stage design, so long as it can be demonstrated that the type I error of the study is controlled. However, the analysis model for analysing the combined data also needs to be considered.

The CHMP Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1/ Corr) states: “When analysing the combined data from the two stages, a term for stage should be included in the ANOVA model.” In addition, to account for the fact that the periods in the first stage are different from the periods in the second stage, a term for period within stage is required. Therefore, the expected ANOVA model for analysis of the combined data from a two-stage design would have the following terms: stage, sequence, sequence*stage, subject (sequence*stage), period (stage), formulation. To fit this model it is necessary to have in each stage at least one patient in each sequence – so a minimum of two patients in each stage of the study, but more if both happen to be randomised to the same sequence.

A model which also includes a term for a formulation*stage interaction would give equal weight to the two stages, even if the number of subjects in each stage is very different. The results can be very misleading hence such a model is not considered acceptable. Furthermore, this model assumes that the formulation effect is truly different in each stage. If such an assumption were true there is no single formulation effect that can be applied to the general population, and the estimate from the study has no real meaning.


According to the Guideline on the investigation of bioequivalence (CPMP/QWP/EWP/1401/98 Rev.1), it is acceptable to use a two-stage approach when attempting to demonstrate bioequivalence. The question was raised whether there were a minimum number of subjects that should be included in the second stage of such a design.

3.3 Regarding the evaluation of orally inhaled medicinal products, to what extent do plasma levels reflect bio-availability in the lung? January 2015

In the European Union (EU), PK bioequivalence studies are considered an acceptable methodology to compare the lung deposition of two inhalation products containing the same active substance. In cases where the oral bioavailability of swallowed drug is negligible, or in case it is made negligible by active charcoal blockade, the plasma concentration time curve reflects both the extent of and the pattern of deposition within the lungs.

To conclude equivalent efficacy, both the amount of drug reaching the lungs and the deposition pattern of drug particles within the lung needs to be equivalent.

The area under the plasma concentration-time curve (or AUC) reflects the amount of drug that has reached the lungs. As the rate of absorption from the inhaled particles is different at different areas of the lung, the deposition pattern within the lung is mirrored by the shape of the plasma concentration-time curve during the absorption phase, i.e. Cmax and tmax.

In the case where intestinal absorption is not prevented, i.e. in a study without charcoal blockade, and thus absorption is the sum of the absorption via the lungs and intestinal absorption, as for other modes of administration, equivalent systemic safety can be concluded if two products give rise to equivalent systemic exposure (AUC and Cmax).

Pharmacokinetic endpoints may be more discriminative than PD or clinical endpoints, in particular the efficacy endpoints available for inhaled corticosteroids.

Use of active charcoal and truncated AUCs

For some inhaled medicinal products, the contribution of intestinal absorption to systemic exposure is negligible (<5%) and a single dose PK study without charcoal can be used for both efficacy and safety comparisons. Reasons for the negligible contribution include poor intestinal absorption (e.g., chromoglycate, nedocromil), or an extensive first-pass metabolism (e.g., beclomethasone, fluticasone, mometasone, ciclesonide). For drugs with significant oral bioavailability (e.g., budesonide, formoterol, salmeterol), a PK study with active charcoal is necessary to assess efficacy, and a study without charcoal is used to assess safety. The charcoal blockade needs to be validated to demonstrate that oral contribution to total bioavailability is negligible. In case the absorption of the drug in the lung is very quick (e.g., tmax ≤ 5 min) and absorption occurs before the contribution of gastrointestinal absorption is significant (e.g., salbutamol/albuterol, salmeterol), AUC0-30 min might be acceptable as a surrogate for efficacy and AUC0-t for safety. Thus, in this case, one study without active charcoal blockade is sufficient.

To be noted, most respiratory medicinal products are now being approved in the EU based on PK studies (e.g., nasal sprays of mometasone in suspension; pMDI in suspension of salbutamol, salmeterol, fluticasone and salmeterol/fluticasone; and DPI of salmeterol/fluticasone).

3.4 Evaluation of orally inhaled medicinal products: can I scale acceptance limits (for Cmax and perhaps AUC) to allow for variability in reference product for fine particle dose? January 2015

In bioequivalence studies, scaling or widening of the acceptance limits is only acceptable for Cmax when it is caused by high intra-subject variability despite similar in vitro characteristics. Scaling is not a suitable solution to the variability in the in vitro characteristics, i.e. the fine particle dose (FPD) of different batches of the reference product.

Widening of the acceptance range

Widening of the conventional 20% acceptance range based on high variability is only possible for Cmax according to the CHMP Guideline on the Investigation of Bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1/Corr) (up to 69.84 – 143.19%) if a replicate design is conducted.

To support safety, it should be demonstrated that the systemic exposure is not higher for the test product than for the reference product, i.e. the upper limit of the 90% confidence interval should not exceed the upper bioequivalence acceptance limit 125.00.

Between-batch variability of the reference product and intra-batch variability over time

Variability in particle-size distribution between batches of the reference product or within a single batch of a reference product through their storage period can be significant. There may even be situations where it may be difficult to demonstrate PK bioequivalence between batches of the same reference product. Therefore, before the in vivo comparison, several batches of both test and reference products could be tested to identify representative batches (within ±15% of the corresponding median fine particle dose (or APSD)) of test and reference, respectively. In case of fixed combinations this may imply, if pre-specified in the protocol, the use of different batches for each component.

The development of an IVIVC may be useful to correct the results of the PK study to justified parts of the APSD of the typical marketed batch of the reference product and the corresponding typical test product batch according to the proposed specifications. The IVIVC could also be used as scientific support of the in vitro specification of the test product.

Another approach that might be acceptable is to show that the side batches (batches in the tails of the distribution) representing the test product specifications are not superior and not inferior to the side batches of the reference product obtained from the market.

3.5 Can I use a 3 period design scheme for the demonstration of within-subject variability for Cmax? June 2015

To demonstrate that the within subject variability for Cmax of the reference product is greater than 30% a replicate design where the reference product is given more than once is required. If a 3 period design is to be used to justify a widening of the limits for Cmax subjects the most efficient study design would randomise subjects to receive treatments in the following order: RRT, RTR or TRR. This design is the most efficient as all subjects receive the reference product twice and hence an estimate of the within subject variability is based on data from all subjects.

The question raised asks if it is possible to use a design where subjects are randomised to receive treatments in the order of TRT or RTR. This design is not considered optimal as explained above. However, it would provide an estimate of the within subject variability for both test and reference products. As this estimate is only based on half of the subjects in the study the uncertainty associated with it is higher than if a RRT/RTR/TRR design is used and therefore there is a greater chance of incorrectly concluding a reference product is highly variable if such a design is used.

The CHMP Guideline on the Investigation of Bioequivalence requires that at least 12 patients are needed to provide data for a bioequivalence study to be considered valid, and to estimate all the key parameters. Therefore, if a 3-period replicate design, where treatments are given in the order TRT or RTR, is to be used to justify widening of a confidence interval for Cmax then it is considered that at least 12 patients would need to provide data from the RTR arm. This implies a study with at least 24 patients in total would be required if equal number of subjects are allocated to the 2 treatment sequences.


The Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev.1), states that: "for the acceptance interval to be widened the bioequivalence study must be of a replicate design where it has been demonstrated that the within-subject variability for Cmax of the reference compound in the study is >30%."

The question was raised whether it is suitable to use a TRT/RTR replicate design to demonstrate that the Cmax of the reference product is highly variable or is it mandatory to use TRTR/RTRT or TRR/RTR/RRT replicate designs?”

3.6 If the SmPC of a reference product allows for the possibility to administer a tablet crushed or disintegrated (and mixed with food), would a specific bioequivalence study with administration of crushed/disintegrated tablets (Test and Reference) always be required for a generic application? November 2016

The bioavailability of an active substance(s) may be altered if products are crushed/disintegrated to assist swallowing and also if a crushed/disintegrated tablet is mixed with food. This change in bioavailability may be formulation/product-specific as well as drug-dependent. Therefore, a test product that is shown to be bioequivalent when administered as a whole tablet in a fasted state, may exhibit significantly different bioavailability compared to the reference product, when both are administered crushed/disintegrated (and dispersed in food). 

Consequently, if the SmPC of the reference product allows for the possibility to administer the tablet crushed/disintegrated (and dispersed in food), bioequivalence should also be demonstrated, in principle, for a test product with this additional mode of administration. 

It is proposed, however, to define conditions which would avoid such an additional bioequivalence study for generic products, if the product cumulatively fulfils the following criteria:

  • the drug substance is highly soluble and stable in the pH-range of the gastrointestinal tract according to BCS-based biowaiver criteria;
  • surface active excipients do not differ qualitatively nor quantitatively, and have no specific formulation characteristics or specific technologies - e.g. an amorphous solid dispersion (ASD) in order to stabilize a polymorphic form;
  • disintegration time is comparable between test and reference;
  • dissolution profiles are similar between test and reference product at pH 1.2, 4.5 and 6.8 at 50 rpm in the paddle apparatus, or 100 rpm in the basket apparatus, and in the QC method if a different medium is used for this.

Therefore, the following data are required to waive additional in-vivo investigations and to allow for the administration of the crushed product:

  • successful bioequivalence with uncrushed products;
  • BCS classification of the drug substance as class 1 or 3;
  • comparative evaluation of excipients, particularly regarding potentially surface active excipients;
  • comparative multimedia in-vitro dissolution;
  • comparative disintegration testing.
3.7 If the test product is an oral solution (or suspension) dosed in single dose sachets and the test product claims the administration with or without water (or makes no claim), how should the test product be administered in the BE study - with or without concomitant fluid intake? If the test product is an oral powder or granulate dosed in a single dose sachet for intake with or without water it is understood that it should be assimilated to an orodispersible tablet, orodispersible film, buccal tablet or film, sublingual tablet or chewable tablet, but how should oral solutions/suspensions be tested? September 2017

The requirements for the investigation of bioequivalence/comparative bioavailability for oral solutions and suspensions regarding fluid intake during administration can be summarised as follows:

  1. In case the SmPC of the reference product states that it should be administered with water and the test product claims the same then during the study both products should be administered with water.
  2. In case the SmPC of the reference product states that the product should be administered without water and the test product claims the same then during the study both products should be administered without water.
  3. In case the SmPC of the reference product states that it should be administered with water and the test product claims that it can be administered both with or without water then during the study both products should be administered with water and additional data for the test product without water (e.g. a third study arm) should be submitted to support this claim.
  4. In case the SmPC of the reference product states that it should be administered with or without water and the test product claims the same then during the study both products should be administered without water as this generally represents “the worst-case scenario”.
  5. In case the SmPC of the reference product states nothing concerning fluid administration and the test product claims the same then during the study both products should be administered without water as this generally represents “the worst-case scenario”.

Changing the volume of fluid intake can affect the gastrointestinal transit time and in certain cases the in vivo solubility of the active substance and/or the concentration of a critical excipient and hence the absorption of the active substance.  Therefore, a possible effect of fluid intake during administration on the bioavailability of an oral solution/suspension cannot be excluded. As the magnitude of this effect is expected to be maximal when administration takes place without water, which generally represents the worst-case scenario, it would be preferable to investigate bioequivalence/comparative bioavailability under these conditions. Deviations from this approach should be scientifically justified and supported by additional study data and/or literature.

3.8 What are the recommendations for demonstration of bioequivalence of gastro-resistant preparations (e.g. omeprazole)? July 2010 and March 2018 (updated April 2018)

According to Guideline on the pharmacokinetic and clinical evaluation of modified release dosage forms (EMA/CPMP/EWP/280/96 Corr1), in gastro-resistant or enteric products bioequivalence should be demonstrated not only in a single dose study in fasted conditions, but also in a single dose study under fed conditions. The fed study should be conducted using a high-fat meal (approximately 50 percent of total caloric content of the meal) and high-calorie (approximately 800 to 1000 kcal) meal. This test meal should derive approximately 150, 250, and 500-600 kcal from protein, carbohydrate, and fat, respectively. The composition of the meal should be described with regard to protein, carbohydrate and fat content (specified in grams, calories and relative caloric content (%)).

Consequently, bioequivalence studies should be performed under both fed and fasting conditions. In general a generic is meant to be bioequivalent with the innovator under fasted and fed conditions. A difference regarding formulation related food interactions indicates product differences thus contradicting the generic by definition. Accordingly, for products where bioequivalence can be shown in the SPC recommended condition but not in the non-recommended state due to a different food effect, the product does not fulfil the requirements of a generic product, but could be eligible for an Article 10(3) application. See also 4.1.1. "Food effect studies with oral modified release forms" for recommendations regarding study design, etc.

Gastric emptying of single unit dosage forms non-disintegrating in the stomach (e.g. enteric coated tablets) is prolonged and highly erratic, most likely due to the effect of the inter-digestive cycle within the Migrating Myoelectric Complex. The consequences of this effect on the enteric coating of delayed release formulations are largely unpredictable: if e.g. the active pharmaceutical ingredient (API) release occurs prior to stomach emptying because of prolonged residence in the stomach either degradation can occur or the release may be considerably delayed. In either case erratic concentration profiles (either non-existing or extremely delayed) can be obtained. Therefore the sampling period should be designed such that measurable concentrations are obtained, taking into consideration not only the half-life of the drug but the possible occurrence of this effect as well. This should reduce the risk of obtaining incomplete concentration-time profiles due to delay to the most possible extent. These effects are highly dependent on individual behaviour. Therefore, but only under the conditions that sampling times are designed to identify very delayed absorption and that the incidence of this outlier behaviour is observed with a comparable frequency in both test and reference products, these incomplete profiles can be excluded from statistical analysis provided that it has been considered in the study protocol.

The general requirements for biowaiver of an additional strength detailed in section 4.1.6 of the Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 rev 1) are applicable also for delayed release tablets and recommendations regarding which strength to study is given in the same section of this guideline and also in section 4.1.1. "Food effect studies with oral modified release forms". When evaluating proportionality in composition, it is recommended to consider the proportionality of gastro-resistant coating with respect to the surface area (not to core weight) to have the same gastro-resistance (mg/cm2).

The dissolution profiles should be compared not only in Pharmacopoeial conditions (2 hours at pH 1.2 followed by 45 minutes at pH 6.8), but also at more neutral pHs in the range 2-5, both for single unit non disintegrating and disintegrating dosage forms with multiple units. Hence, at least, two dissolution tests in two steps are required. First a comparison at pharmacopoeial conditions, 2 hours at pH 1.2 followed by 45 minutes in pH 6.8 and then a second separate dissolution test at a higher initial pH mimicking fed state e.g. 2 hours at 4.5 followed by 45 minutes in pH 6.8.

Concluding similarity if dissolution of more than 85% is obtained within 15 minutes is not applicable for gastro-resistant formulations. In case of gastro-resistant formulations the release occurs after gastric emptying (median approx. 13 – 15 min). Therefore, the comparison of dissolution profiles should be performed even if dissolution is more than 85% before 15 min in either products or strengths. Hence, a tight sampling schedule is recommended after the product has been investigated for 2 h in media mimicking the gastric environment (pH 1.2 or 4.5) since profile comparison (e.g. using the f2 calculation) is required.

4. Product-specific bioequivalence

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4.1 Bioequivalence studies for generic products containing clopidogrel. June 2009

The platelet aggregation inhibitor clopidogrel is pre-systemically hydrolysed to the inactive metabolite clopidogrel carboxylic acid. The plasma levels of the unchanged drug are up to 2000 fold lower than those of the carboxylic acid metabolite. Another metabolite, clopidogrel thiol, formed by a parallel pathway, is the pharmacologically active form of clopidogrel and is generated in the intestine and liver primarily by the CYP2C19 enzyme isoform. Due to its chemical instability and low circulating levels, its detection in plasma is problematic. Clopidogrel thiol irreversibly binds to the P2Y12 receptors of ADP on the platelet membranes in portal and systemic circulation, leading to the inhibition of platelet aggregation.


4.1.1 Which substance should be studied in bioequivalence studies: the parent compound clopidogrel or the metabolite(s) of clopidogrel?

The Guideline on the investigation of bioequivalence(CPMP/EWP/QWP/1401/98 Rev 1) states “Also for inactive prodrugs, demonstration of bioequivalence for parent compound is recommended. The active metabolite does not need to be measured.”

At the time of approval of the reference product Plavix, no reliable and validated methodology for the determination of the pharmacokinetics of the parent prodrug clopidogrel or of the active metabolite clopidogrel thiol was available. Thus, at the time, the pharmacokinetic profile of clopidogrel was established based on the pharmacokinetics of clopidogrel carboxylic acid, which is the non-active metabolite. In the meantime, the pharmacokinetic profile characterisation of clopidogrel has improved by development of a sensitive analytical technique (e.g. LC-MS-MS) enabling for a suitable investigation of the parent prodrug, clopidogrel. A more accurate picture of the PK profile of clopidogrel can be obtained.

The demonstration of bioequivalence between the reference and the generic compound should be based on the parent prodrug, clopidogrel.


4.1.2 Is demonstration of bioequivalence under fed conditions necessary in addition to the demonstration under fasting conditions?

The Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev 1) states “In general, a bioequivalence study should be conducted under fasting conditions as this is considered to be the most sensitive condition to detect a potential difference between formulations. For products where the SmPC recommends intake of the reference medicinal product on an empty stomach or irrespective of food intake, the bioequivalence study should hence be conducted under fasting conditions.”

The food effect on the bioavailability(BA) of the unchanged clopidogrel - not recognised in the SPC - was not investigated by the innovator before approval of the originator product since a sensitive analytical method was not available at the time of approval. However, a publication by Nirogi et al. (2006) suggested a significant food effect with a high-fat meal. Similar results have been observed in applications for generic medicinal products. The food effect might be due to a protection from acidic hydrolysis in the stomach in a fasting state, since the BA is enhanced under fed conditions. The EWP-PK subgroup reviewed the solubility properties of clopidogrel salts and these indicate that when administration of clopidogrel occurs under fasting conditions, the dissolution in the gastric media with a subsequent hydrolysis and formation of the inactive carboxy-acid metabolite is maximal. As a consequence, the extent of unchanged drug that still is available for absorption (at the intestine level) is reduced. Conversely, the dissolution of clopidogrel is limited in the gastric media under fed conditions, the acidic hydrolysis in the stomach is reduced and the BA of clopidogrel is improved.

The EWP-PK subgroup acknowledges that as a consequence, the solubility of salts might be important. However, all clopidogrel salts have high solubility at low pH and the risk for acidic hydrolysis may therefore be similar. The food effect could consequently be expected to be similar to the reference product for different salts. Hence, the EWP-PK subgroup considered that there was currently an insufficient scientific rationale to justify a deviation from the revised Guideline on the Investigation of Bioequivalence and bioequivalence should be demonstrated under fasting conditions irrespective of the salt.

Should further information on the food effect of clopidogrel become available, the SPC would be amended accordingly.


At the time the innovative drug-product was developed, no data regarding the effect of food on the bioavailability of clopidogrel parent compound were available. More recently, the investigation of food intake influence on the bioavailability of clopidogrel has been investigated. The results obtained by Nirogi et al.1 indicate that in the fed state the bioavailability of a single oral dose of clopidogrel increases dramatically (500 - 600 %) but the systemic exposure to the major but inactive carboxylic acid metabolite increases only by approximately 10-20 %. The current Summary of Product Characteristics (SPC) for the originator states that clopidogrel should be given as a single daily dose of 75 mg with or without food.


4.1.3 Regarding bioanalytical methods, are there any special requirements to ensure that the risk of back-conversion of the major metabolite to clopidogrel could be excluded?

Within several centralised clopidogrel applications, the CHMP raised concerns about the possible back-conversion of the major metabolite of clopidogrel (clopidogrel carboxylic acid) to clopidogrel during the bio-analytical analysis of the samples. Considering that plasma levels of clopidogrel carboxylic acid observed in patients or healthy volunteers treated with clopidogrel are much higher than that of the parent drug, a minimum back-conversion of the metabolite could potentially lead to a huge over-estimation of clopidogrel plasma levels and would bias the outcome of bioequivalence study.

The EWP-PK subgroup confirmed that back-conversion could potentially occur when methanol is used as (part of) extraction solvent, reconstitution solvent, chromatography mobile phase or for the preparation of calibrators, quality control (QC) solutions and internal standards during bioanalysis. Therefore, testing for the back-conversion of clopidogrel carboxylic acid metabolite should be part of the validation process of analytical methods used for the measurement of clopidogrel plasma levels.

It should be demonstrated that there is no back-conversion of the major metabolite to the parent drug clopidogrel under all conditions for sample handling (including extraction procedures) and storage.


4.1.4 Could the acceptance criteria for Cmax be widened?

According to the Guideline on the investigation of bioequivalence(CPMP/EWP/QWP/1401/98 Rev 1) widening of the acceptance criteria for Cmax is possible for highly variable drug products provided that a wider difference in Cmax is considered clinically irrelevant based on a sound clinical justification. The revised Guideline on the Investigation of Bioequivalence provides detailed advice on how the acceptance criteria can be widened for highly variable drug products with a bioequivalence study of replicate design and using the scaled-average-bioequivalence approach. However, a prerequisite for widening the acceptance criteria is that a wider difference in Cmax is considered clinically irrelevant. This issue was assessed by the EWP-CVS subgroup.

The EWP-CVS subgroup evaluated the request from widening the 90% confidence interval for Cmax from the efficacy and safety perspectives. The EWP-CVS subgroup considered what would be the degree of the impact of the possible variations in the Cmax following the 75 mg dose, since some data suggest the existence of a plateau response in the inhibition of platelets aggregation. However, it is currently not entirely clear what would be the influence of variable clopidogrel concentrations on pharmacodynamics. It is important to note that clopidogrel is approved and recommended for use in acute clinical conditions, for which a high loading dose is advised in order to attain a fast antiplatelet action. Whether in these situations a lower Cmax might be of clinical relevance is unknown, but cannot be completely excluded.

In conclusion, it is not definitely proven that widening Cmax acceptance range for clopidogrel is devoid of clinically relevant implications, both in terms of safety and efficacy, for all situations where the drug is used in clinical practice. Under these circumstances, the widening of 90% confidence intervals for Cmax is not recommended.



1. Nirogi, RV et al. (2006) Effect of food on bioavailability of a single oral dose of clopidogrel in healthy male subjectsArzneimittelforschung56(11); 735-9

4.2 Acceptance criteria for bioequivalence studies for losartan. July 2010

Which analyte, parent and/or metabolite, should be used for the decision of bioequivalence in the case of losartan, and which acceptance criteria should be applied? 

Bioequivalence for losartan should be proven based upon parent data. Regarding what acceptance criteria to apply, the submitted documents do not allow any conclusion to be drawn on this and consequently a conservative approach using 90% CI of 80 – 125% for AUC and Cmax applies.


Losartan is not a pro-drug. It is an angiotensin II antagonist at the AT1-subtype receptor. In humans, losartan competitively binds to the AT1 receptor, while the metabolite E3174 binds non-competitively.

The active metabolite E3174 is not directly formed from losartan, but from an intermediate product, metabolite E3179. Alternatively, the E3179 intermediate can also be hydroxylated to an inactive metabolite. It has been estimated that about 14% of the orally administered losartan dose is converted into E3174. In addition, 5 other minor metabolites exists that exhibit activity but much less than parent.

AUC of the active metabolite is 4 – 8 fold higher than parent, as it is cleared about 10-fold slower than parent.

Plasma free fractions of parent are 1.3% and that of the active metabolite 0.2%. Losartan and its metabolite E3174 shows linear pharmacokinetics.

It has been shown in vitro that the IC50 for binding to the AII receptor in smooth muscle cells is 10-fold more potent for the metabolite than parent and that the in vitro AII concentration dependent contractile response in rabbit aorta is 33-fold higher for the metabolite. In vivo, in normotensive and renal hypertensive rats, the active metabolite has been shown to be 15 – 20-fold more potent compared to the parent.

Based on in vivo studies in rat, in which the potency was 15 – 20-fold higher for the metabolite, and assuming a more or less comparable protein binding as that observed for human plasma (literature indicated for losartan a binding >99% in rat plasma), the metabolite activity is about 76 – 100-fold higher than the parent compound.

Hence, based on total exposure (AUC), the metabolite accounts for the majority of the activity. However, losartan and the active metabolite have different plasma-concentration time course, with considerably higher losartan plasma concentrations during the first hours after administration. Considering the plasma concentration time course, difference in activity and protein binding, losartan may account for a large part of the activity during the first hour after the first drug administration, and at losartan tmax, which occur after about one hour, contribution to activity may be almost equal for losartan and the metabolite. Thereafter, the metabolite’s contribution to activity is much larger.

As the active metabolite E3174 is formed via an intermediate product and not direct from the parent, the pharmacokinetic data for metabolite E3174 may not reflect the rate of absorption of parent.

4.3 What are the requirements for demonstration of bioequivalence for ciclosporine generics? July 2010

The reference product Neoral soft gelatine capsule concerns a specific formulation of ciclosporin which undergoes microemulsification process at administration (in the presence of water). For Neoral, the SmPC indicates a 33% decrease in Cmax and a 13% decrease in AUC, in case the product is taken with a high fat meal.

As indicated in the Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev 1.), for products with specific formulation characteristics, like Neoral, bioequivalence studies performed under both fasted and fed conditions are required unless the product must be taken only in the fasted state or only in the fed state. Neoral may be taken with or without food, and in clinical practice, ciclosporin is often recommended to be taken in a standardised way in relation to food. Hence, a generic ciclosporin product must be bioequivalent with the originator product both in fasting and in fed state.

As EWP has defined ciclosporin to be a NTID, for which both AUC and Cmax are important for safety and efficacy, a narrowed (90.00-111.11%) acceptance range should be applied for both AUC and Cmax, under fasting as well as under fed conditions, in line with the Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev 1.).

Although a generic product with a reduced food effect could be considered an improvement, this would not be considered acceptable for a ‘generic application’, but could be considered for a “hybrid” application, article 10(3) with additional data to support an application under this legal basis.

4.4 Bioequivalence studies for generic application of omega 3 fatty acid ethylesters in a soft gelatine capsule. October 2013

What do I need to consider in generic applications referring to an oily ‘liquid composition’ in a soft gelatine capsule?

The capsule filling of both the generic and the innovator product comprised 1000 mg of the liquid active substance, (omega 3 fatty acid ethylesters), without any excipients. The active substance fully complied with the Ph Eur Monograph on Omega-3 fatty acid ethylesters (EE) which describes an active substance including an allowed (although not defined) low amount of preservative.

Hence, the gelatin capsules only included the oily, liquid active substance, (omega 3 fatty acid ethylesters). However, the liquid active substance contains a slightly different amount of preservative alpha-tocopherol (as 70% in vegetable oil). Furthermore the composition of the capsule itself was roughly the same as for the innovator product but with a slight difference in the amount of glycerol.

This particular situation is not addressed in the current Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1/ Corr **), i.e. a generic application referring to an oily ‘liquid composition’ in a soft gelatine capsule.


4.4.1 Would a biowaiver be acceptable in this specific type of medicine formulation if fast and comparable disintegration of the capsules has been demonstrated over the whole physiological range (pH 1 – 6.8)?

Bioequivalence (BE) is a means to detect potential formulation differences between generics and innovators. This implies that formulation differences are expected due to e.g. different excipients (quantitatively and/or qualitatively) and/or different manufacturing processes.

Since the oily content of both capsule products including an allowed amount of preservative is considered the active substance (PhEur monograph), a different formulation effect cannot be assumed. Hence, requesting in vivo BE between test and reference could hardly be justified as both capsules would contain the same amount of actives within accepted limits of variability without excipients potentially causing different formulation effects. The possibility of different amounts of impurities is expected to be controlled via the monograph, i.e. this could not be the reason for a BE study as it refers to the active substance rather than the formulation.

Therefore, simple characterisation of capsule quality by comparative disintegration tests is deemed sufficient. It should however be noted that the disintegration of capsule shells cannot be used as a BE tool as such as it has no relation to any in vivo parameter, but simply describes capsule quality.

In summary, a biowaiver would be acceptable in this specific type of drug formulation if fast and comparable disintegration of the capsules has been demonstrated over the whole physiological range (pH 1 – 6.8). Since the liquid oily active substance of the capsules filled with omega-3 fatty acid EEs will be directly available for absorption after rupture and disintegration and a different formulation effect cannot be expected from the allowed preservative, in vivo BE study could be waived.


4.4.2 If a bioequivalence trial is required, what would be the preferred study design (fed or fasted)? In the case of fasted state conditions, would it be possible to determine bioequivalence between medicines including in the analysis subjects that have presented erratic absorption profiles, for which the extrapolation AUCt-inf could not be estimated or was >20% in more than 50% of the subjects?

Should in vivo BE trial be requested, it should be performed under fed conditions for the following reasons:

  • Plasma concentrations are markedly higher under fed conditions than those quantified in the fasted state,
  • Plasma concentrations in the fasted state are rather low and erratic. Unreasonably low values within the PK profiles render them invalid as they indicate the measurements of physiological processes rather than pharmacokinetics.

The last point was addressed in the paragraph above. However, since this is considered a general question not particularly related to the omega-3 fatty acid ethylesters in a soft gelatine capsule, it is further discussed below.

Subjects for which erratic absorption prevent the calculation of extrapolated AUC and/or for which the residual area is more than 20 % should still be included in the regular calculations and evaluation of AUCt since this is the most relevant pharmacokinetic parameter to compare extent of absorption (see section 4.1.8 in the Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1/ Corr **)). However, the cited guideline clearly states that when this is true “in more than 20 % of the observations then the validity of the study may need to be discussed” (see section 4.1.8 Evaluation; Reasons for exclusion). Hence, only in exceptional cases it could still be possible to accept an extrapolation larger than 20% in a significant number of subjects (>20% of the subject’s concentration - time profiles) if it is justified that AUCt has been calculated reliably and it is representative of the extent of drug absorption from the products under comparison. Of note, this rule and reasoning does not apply if the sampling period is 72h or more and AUC0-72h is used instead of AUCt.

4.5 What do I need to consider in a generic application for quetiapine lambda 200, 300, 400 mg prolonged release tablets? October 2013

The clinical development plan for Quetiapine Lambda 200, 300, 400 mg prolonged release tablets consisted of a single-dose study under fasting and fed conditions with 200 mg strength in healthy volunteers and a multiple-dose study with the highest, 400 mg tablet in schizophrenic patients.

The application for the 300 and 400 mg strength was referred to the CMDh. The PKWP input was sought on the following points:

1. Clinical development plan: the need for single dose bioequivalence studies in all strengths, where single-dose study under fasting and fed conditions with 200 mg strength in healthy volunteers and a multiple-dose study with the highest, 400 mg tablet in schizophrenic patients have shown bioequivalence,

2. The need for inclusion of early time points in the calculation of f2 values for a prolonged release tablet in in-vitro dissolution data supportive of a biowaiver.

The PKWP acknowledged the following limitations:

  • Single dose studies with doses higher than 200 mg are not feasible in healthy volunteers due to unacceptably severe adverse effects,
  • Multiple dose studies with doses equal to or higher than 200 mg are not feasible in healthy volunteers due to unacceptably severe adverse effects,
  • Single dose studies in patients are not feasible due to ethical reasons (interruption of treatment).

Hence, the PKWP’s feedback was based on the assumption that it was not possible to conduct the study with the 300mg dose.


4.5.1. Would a multiple dose study in the highest strength be considered sufficient to demonstrate bioequivalence despite differences in the dissolution profiles, in case where a single-dose study can be waived because of safety reasons?

Overall in vivo and in vitro evidence provided points to a positive answer to this question: a multiple dose study in the highest strength can be considered sufficient to demonstrate bioequivalence despite differences in the dissolution profiles (which can be explained because the dissolution profiles become similar when tested at the same dose level per vessel), in case where a single-dose study can be waived because of safety reasons, taking also into consideration the demonstrated BE in the single dose study with the 200 mg strength and a bracketing approach between the 200 and 400 mg strengths. This conclusion cannot be generalised and a case by case approach will be needed in similar situations.


In the case of Quetiapine Lambda the following statement from the Note for guidance on modified release oral and transdermal dosage forms: Section II (pharmacokinetic and clinical evaluation (CPMP/EWP/280/96) applies:

In case of prolonged release single unit formulations with multiple strengths, a single dose study under fasting conditions is required for each strength. Studies at steady state may be conducted with the highest strength only if the same criteria for extrapolating bioequivalence studies are fulfilled as described in the Note for Guidance for immediate release forms (linear pharmacokinetics, same qualitative composition, etc.).

Therefore, the following is required:

  1. Waive multiple dose studies for the 200 mg and 300 mg strengths based on conditions applicable to IR forms as per the Guideline on the investigation of bioequivalence currently in force. All conditions were fulfilled except for dissolution (see below).
  2. Waive single dose studies for the 300 mg and 400 mg studies based on exceptional circumstances: single dose studies are not feasible both in healthy volunteers and patients (see above). In this case the same rules for waiving different strengths should apply.

As a consequence, the only outstanding issue was the comparison of dissolution profiles.

Overall the dissolution data raised doubts on the extrapolation of the BE results only from the 400 mg and the 200 mg strengths because the comparison of 200 mg vs. 400 mg at pH 4.5 and 6.8 does not meet the f2 criterion. On the contrary, bioequivalent (BE) results could be extrapolated to the 300 mg strength on the basis of dissolution data since respective comparisons complied with the f2 criterion.

It was then investigated whether the differences in the dissolution data were due to an active substance effect (as a result of lack of sink conditions) or a formulation effect. As for the lack of sink conditions, the results of a comparison of equivalent strengths of the test product (TP) (2X200 mg vs. 1X400 mg) at pH 4.5 and 6.8 suggested that the noncompliant results could be explained by an active substance effect, not by a formulation effect. However, the results of a comparison of the 200 mg strength of the reference product (RP) the 400 mg strength of the RP at pH 4.5 and 6.8 did not suggest an active substance effect.

Given the exceptional circumstances that the single dose studies cannot be conducted in patients and that the studies with doses higher than 200 mg cannot be conducted in healthy volunteers, only a multiple 200 mg dose study in patients could have clarified these findings. . However, this study would not be ethically acceptable since there was direct evidence that the lack of comparability between 200 mg and 400 mg in the TP was due to the solubility of the active substance, whereas the formulation effect was based on an indirect observation that this was not the case for the RP.

Moreover, BE results should prevail over dissolution data and the 200 mg strength of the TP was BE to the 200 mg strength of the RP, inasmuch as the 400 mg strength of the TP was BE to the 400 mg strength of the RP.

Finally, a bracketing approach could be applicable in this situation since studies were available at the extreme of the strength interval (200 and 400 mg).


4.5.2 Is it acceptable and/or needed to include early time points of the dissolution profiles in the calculation of f2 values for a prolonged release tablet? Because f2 values are sensitive to the choice of dissolution time points, what recommendations can be made for prolonged release tablets in order to reliably conclude that the dissolution profiles can be considered similar?

In this case the 2 h time point should not be omitted not only because there was no scientific reason to exclude it but because the amount released was considered relevant.

The choice of early time points in a comparative dissolution profile test should be based on the relevance (mainly amount released and release controlling mechanism). On the other hand, the conditions stated in Appendix 1 of the Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1/ Corr) should be complied with, namely

  • A minimum of three time points (zero excluded)
  • The time points should be the same for the two formulations
  • Twelve individual values for every time point for each formulation
  • Not more than one mean value of > 85% dissolved for any of the formulations.
  • The relative standard deviation or coefficient of variation of any product should be less than 20% for the first point and less than 10% from second to last time point.


The design of a study comparing two dissolution profiles should take into account, among other factors, the inclusion of relevant sampling time points. It is perfectly reasonable to use 2 h as a first time point in a dissolution test running over 24 h. In the case at hand at 2 h already a relevant amount (10 to 15 %) of the active has been released. On the other hand, early time points, even in the case of a sustained release dosage form, are important in revealing release differences between the products under comparison, because the mechanism controlling the release of the active substance is present from the start.

Even though the choice of sampling time points could be questioned, there is no scientific reason to exclude valid data in a calculation.

4.6 Requirements for demonstration of bioequivalence for mycophenolate mofetil generics. January 2011

Bioequivalence data for inactive pro-drugs in relation to both parent drug and metabolite

The questions relate to the circumstances under which it is acceptable to base bioequivalence decision solely on metabolite data if a pro-drug plasma level is measurable. The revised guideline states: “Also for inactive pro-drugs, demonstration of bioequivalence for parent compound is recommended”.

4.6.1 Does the exact meaning of the word “recommended” in the context of mycophenolate mofetil (MMF), depends on the feasibility of the technical detection limits, in which the concentrations of the inactive prodrug are approximately 12000- to 6000-fold lower, for AUC and Cmax, respectively, than that of the active metabolite mycophenolic acid?Or should specific PK-parameters be taken into account, low exposure of the parent resulting in a short Tmax, which makes it not relevant to measure the parent drug.

The Guideline on the investigation of bioequivalence states “for inactive prodrugs, demonstration of bioequivalence for parent compound is recommended”. The guideline further clarifies: “However, some pro-drugs may have low plasma concentrations and be quickly eliminated resulting in difficulties in demonstrating bioequivalence for parent compound. In this situation it is acceptable to demonstrate bioequivalence for the main active metabolite without measurement of parent compound.” Hence, although the guideline recommends the use of parent compound also for inactive pro-drugs, exceptions are possible. The acceptability of use of main active metabolite instead of parent compound will be determined based both on the feasibility of measuring parent compound and on the pharmacokinetic characteristics for parent compound and active metabolite. For pro-drugs with a very large difference in exposure between parent and active metabolite and where the pro-drug is quickly eliminated, it is expected that there can be difficulties in demonstrating bioequivalence for parent compound and demonstration of bioequivalence based on active metabolite alone can be accepted.

For mycophenolate mofetil (MPM) specifically, the parent compound undergoes extensive presystemic metabolism to the active metabolite MPA.Moreover, MPM half-life is very short (0.60 to 1.20 h as reported) resulting in approximately 12000- and 6000-fold lower AUC and Cmax respectively, for parent compound compared to metabolite. MPM has a tmax of 0.5 h and a t1/2 of less than 1 h, which limits the characterisation of the early plasma concentrations. As a consequence reliable estimation of Cmax will be difficult. “In this situation it is acceptable to demonstrate bioequivalence for the main active metabolite without measurement of parent compound” as stated in the Guideline on the investigation of bioequivalence.

4.6.2 Is it acceptable not to follow this recommendation and use only metabolite data to demonstrate bioequivalence between two products of the same pro-drug mycophenolate mofetil, even when current analytical assays allow measuring the parent with acceptable sensitivity?

A recommendation leaves room for an exceptional decision on a case by case basis. In this case it is clear that the parent compound is inactive and completely converted into the active metabolite yielding a 12000 fold difference in AUC. Due to this, demonstration of bioequivalence between two products of the same pro-drug can be based on metabolite data only. The argument that current analytical assays allow measuring the parent with acceptable sensitivity cannot be readily taken considering the short tmax and t1/2 of the parent compound which will limit a reliable estimation of Cmax of the parent compound.

4.7 Demonstration of bioequivalence for ebastine. October 2013

Is it acceptable for a generic application for inactive pro-drugs to demonstrate bioequivalence based on either the parent ebastine or on the active metabolite carebastine, provided proper justification in the study protocol has been provided, or can only one of these analytes be used?

In the context of the Guideline on the investigation of bioequivalence (CPMP/QWP/EWP/1401/98 Rev. 1), the parent compound ebastine can be considered to be an inactive pro-drug as it has no or very low contribution to clinical efficacy1-6.

Although demonstration of bioequivalence for parent compound is recommended for inactive pro-drugs, demonstration of bioequivalence with ebastine would only be possible by inclusion of a very high number of subjects. Indeed, ebastine has very low plasma concentrations, is rapidly and extensively metabolised resulting in highly variable plasma concentrations of the parent compound, resulting in a higher variability in pharmacokinetics than carebastine.

Therefore, bioequivalence studies using carebastine for bioequivalence evaluation would be considered acceptable to detect formulation related differences between a test and a reference.

In summary, in accordance with the Guideline on the investigation of bioequivalence, it would be acceptable to demonstrate bioequivalence based on the pharmacokinetics of the active metabolite carebastine. However, in case an application is submitted solely with data on the parent ebastine, it is also acceptable to demonstrate bioequivalence based on the pharmacokinetics of the parent ebastine. In case both ebastine and carebastine are analysed, the analyte to be used for bioequivalence evaluation should be prospectively defined in the protocol.


The Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev.1) states that:

Inactive pro-drugs

Also for inactive pro-drugs, demonstration of bioequivalence for parent compound is recommended. The active metabolite does not need to be measured. However, some pro-drugs may have low plasma concentrations and be quickly eliminated resulting in difficulties in demonstrating bioequivalence for parent compound. In this situation it is acceptable to demonstrate bioequivalence for the main active metabolite without measurement of parent compound. In the context of this guideline, a parent compound can be considered to be an inactive pro-drug if it has no or very low contribution to clinical efficacy.


1. T. Yamaguchi, T. Hashizume, M. Matsuda, M. Sakashita, T. Fujii, Y. Sekine, M. Nakashima & T. Uematsu (1994) Pharmacokinetics of the H1-receptor antagonist ebastine and its active metabolite carebastine in healthy subjects. Arzneim.-Forsch. 44(1):59-64

2. Martinez-Tobed A., Tarrús E., Segura J. & Roberts D.J. (1992) Pharmacokinetic studies of ebastine in rats, dogs and man. Drugs Today28(Suppl. B):57-67

3. Wiseman L.R. & Faulds D. (1996) Ebastine: a review of its pharmacological properties and clinical efficacy in the treatment of allergic disorders. Drugs51(2):260-277

4. Presa I.J. (1999) H1-antihistamines: a review. Alergol. Immunol. Clin.14(5):300-312

5. Rico S., Antonijoan R.M. & Barbanoj M.J. (2009) Ebastine in the light of CONGA recommendations for the development of third-generation antihistamines. J. Asthma Allergy2:73-92

6. Wood-Baker R. & Holgate S.T. Dose-response relationship of the H1-histamine antagonist, ebastine, against histamine and methacholine-induced bronchoconstriction in patients with asthma. Agents and Actions (1990) 30:1-2

4.8 CHMP request to PKWP for clarification on demonstrating bioequivalence of low dose acetylsalicylic acid gastro-resistant formulations in fixed dose combinations with substitution indication. December 2016

PKWP response:

CHMP has received requests for clarifications of the bioequivalence requirements for fixed dose combinations (FDC) containing a 100 mg acetylsalicylic acid (ASA) tablet with monolithic gastro- resistant coating and separate tablet/granules with the other active substance(s) encapsulated in one formulation. A substitution indication was being claimed for these.

According to CHMP guidelines1,2 it is required for a substitution indication to show bioequivalence for all active compounds of the formulation, similarity is not sufficient. Although ASA is a well-known active substance for which various formulations in various strengths have been approved for many years, similarity in combination with a bibliographic justification is not sufficient for a substitution indication. Thus, claiming similarity to multi-source bibliographical data with various different formulations in an application file does not make it possible to extrapolate the published data to the selected product.

Therefore, for these FDC, bioequivalence should be demonstrated for each individual component of the FDC, including ASA, since the products are intended for a substitution indication.

Widening the bioequivalence acceptance limits for mean Cmax  and AUC can be accepted for highly variable drugs like ASA provided that the high variability is properly justified in a replicate design study, in accordance with the bioequivalence guideline. If bioequivalence cannot be demonstrated for the reference product for the two treatment periods, then other studies should be undertaken.

Specifically if the AUC is highly variable then the sample size should be increased, while if the Cmax is variable a replicate design could be used.

The metabolism of ASA and production of the metabolite salicylic acid (SA) occurs both pre- and post- absorption, i.e. by some limited pre-absorptive hydrolysis in the stomach and also via a hepatic first- pass effect. In this second step, the metabolic conversion of salicylate and subsequent formation of conjugates and their renal excretion is dose-dependent. Thus metabolites would not be sensitive to evaluate bioequivalence of ASA products.

Hence, available study results do not justify any deviation from existing guidelines on demonstrating bioequivalence for gastro resistant ASA formulations in FDC claiming substitution indications.

5. Bioequivalence in special populations

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5.1 Clarifications regarding bioequivalence studies in children. June 2009

Treatment of children often requires that new formulations or strengths are developed. If chemical-pharmaceutical data are not considered sufficient to establish bioequivalence should bioequivalence studies be conducted in children or would healthy volunteers suffice?

In vivo bioequivalence is almost always established in healthy volunteers unless the drug carries safety concerns that make this unethical. This model, in vivo healthy volunteers, is regarded adequate in most instances to detect significant formulation differences and the results will allow extrapolation to populations in which the drug is approved (the elderly, patients with renal or liver impairment etc.). The same reasoning applies also to children. Hence, in the vast majority of cases bioequivalence studies in healthy volunteers are adequate for products intended for use in children.

5.2 Clarifications on the guideline on the evaluation of the pharmacokinetics of medicinal products in patients with impaired hepatic function. January 2015

5.2.1 Why does the guideline state in Sections 3.4 and 4.1 that it is the free fraction of the drug and metabolites that is to be determined?

Sections 3.4 and 4.1 of the present guideline clearly state that in the hepatic impairment study groups, the free fraction should be determined if the substance(s) measured are highly bound to plasma proteins.The protein binding may be reduced in hepatic impairment. If using total concentration, an increase in the therapeutically relevant free concentration can be masked or underestimated as both the protein bound fraction and hepatic function are affected. No recommendation can be based on the total concentration in this situation. It has been noted that applicants have not observed this requirement resulting in submission of inconclusive studies.

5.2.2 Why does section 2 of the guideline state that biliary secreted drugs should be studied?

In section 2 of the guideline it is stated that biliary secreted drugs should be studied. Biliary secretion as well as hepatic metabolism can be affected by hepatic impairment. Furthermore,in reviewed NCE applications, very marked increases in exposure have been found for drugs subject to extensive hepatic uptake, when given to patients with hepatic impairment due to hepatitis C. In view of these findings it is particularly important to study the effect of hepatic impairment in drugs subject to hepatic uptake.

5.2.3 How should the subjects to be included in the hepatic impairment (HI) study be selected?

The subjects included in the hepatic impairment study should be representative for the actual class, e.g. if moderate impairment is investigated, the subjects should have Child-Pugh scores covering the range of moderate impairment and being spread over the range.

5.2.4 How should hepatic impairment be classified?

Presently, the Child-Pugh classification is being proposed as the most widely used to categorise hepatic function. Presenting the pharmacokinetic effect as a function of the biochemical Child-Pugh components (e.g. S-albumin, bilirubin, prothrombin time, etc.) is encouraged in the guideline. Research in this area is on-going.

5.2.5 What is the role of physiologically based pharmacokinetics (PBPK) when estimating the effect of hepatic impairment?

In Section 3.6, the guideline makes a short statement on the use of PBPK as a tool. Predicting the effects of hepatic impairment by PBPK is an interesting application of PBPK and there is a great deal of ongoing research in this area. However at the present time due to low confidence in the use of PBPK modelling to predict hepatic impairment, it is considered that there is no need to revise the general information given on PBPK modelling.

6. Biowaivers

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6.1 What is the effect of sorbitol on the pharmacokinetics of highly permeable drug substances? September 2012

The CMDh asked for a view on the extent to which the results reported by Chen et al (1) regarding the effect of sorbitol on bioavailability of metoprolol, taken together with relevant regulatory experience regarding the influence of sorbitol on the oral bioavailability of drug substances, are applicable to other highly permeable drug substances (BCS class 1 and 2).

There is scarce information in the literature1-5 regarding the effect of sorbitol on the absorption of BCS class I and II (highly permeable drug substances). The article by Chen et al1 (showing no effect on metoprolol absorption) and another one by Fassihi2 (showing no effect on Cmax or AUC but an effect on Tmax of theophylline upon 10 g of sorbitol) are worth mentioning.

In Chen et al’s article1, the effect of sorbitol on the absorption of metoprolol (BCS class I) and ranitidine (BCS class III) has been studied. No significant effect of sorbitol (5 g) on the extent (AUC) and a 23% reduction in rate (Cmax) of absorption of a single dose of metoprolol has been recorded, whereas a significant effect has been observed on both AUC and Cmax (44% and 51% reduction, respectively) when sorbitol (5 g) and ranitidine (BCS class III) were administered concomitantly. From these data, the best estimate of a single dose threshold for the sorbitol effect on drug bioavailability is probably around 1 g, affecting all drug BCS classes but mainly low permeability drug substances.

Therefore there is no straightforward answer to this question until more data is collected to determine the actual threshold by exploring sorbitol doses lower than 1.25 g.

The putative effect of sorbitol on GI physiology affecting drug absorption is generally accepted to derive from its osmotic effect, accelerating intestinal transit and increasing intestinal water content. The first effect suggests a higher impact on the absorption of low permeability drugs. The latter can lower the diffusion driving force due to dilution, affecting all drug BCS classes.

Therefore any correlation of sorbitol absorption effect with solubility or permeability is in principle difficult to establish.

It also needs to be recognized that sorbitol intolerance is largely described in the literature6,7. This means that a dose effect relationship cannot be established universally due to individual susceptibility. Even minute amounts of sorbitol can elicit a GI effect in a sub-population.

Consistently with these results, the Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1) states in Appendix II, Oral solutions:

“If the test product is an aqueous oral solution at time of administration and contains an active substance in the same concentration as an approved oral solution, bioequivalence studies may be waived. However if the excipients may affect gastrointestinal transit (e.g. sorbitol, mannitol, etc.), […], a bioequivalence study should be conducted, unless the differences in the amounts of these excipients can be adequately justified by reference to other data. The same requirements for similarity in excipients apply for oral solutions as for Biowaivers (see Appendix III, Section IV.2 Excipients).”

Further recommendations in Appendix III, section IV.2 on excipients state: “As a general rule, for both BCS-class I and III drug substances […] Excipients that might affect bioavailability should be qualitatively and quantitatively the same in the test product and the reference product.”

Therefore, strict compliance with the Guideline on the investigation of bioequivalence is recommended to be followed in the development and assessment of generic applications.

Sorbitol intolerance should be taken into consideration in the labeling of sorbitol containing drug products.


  1. Chen, M., Straughn, A., Sadrieh, N. et al (2007). A Modern View of Excipient Effects on Bioequivalence: Case Study of Sorbitol. Pharm. Res.24, 73-80.
  2. R. Fassihi, R. Dowse & S. S. D. Robertson (1991). Influence of Sorbitol Solution on the Bioavailability of Theophylline. Int. J. Pharm.72:175-178
  3. D. A. Adkin, S. S. Davis, R. A. Sparrow, P. D. Huckle, A. J. Philips & I. R. Wilding (1995). The Effects of Pharmaceutical Excipients on Small Intestinal Transit. Br. J.Clin. Pharmacol.39:381-387
  4. D. A. Adkin, S. S. Davis, R. A. Sparrow, P. D. Huckle & I. R. Wilding (1995). The Effect of Mannitol on the Oral Bioavailability of Cimetidine. J. Pharm. Sci.84:1405-1409
  5. S. van Os, M. Relleke & P.M. Piniella (2007) Lack of Bioequivalence between Generic Risperidone Oral Solution and Originator Risperidone Tablets.Int. J. Clin. Pharmacol., 45: 293-299
  6. Born P. (2011) The clinical impact of carbohydrate malabsorption. Arab J Gastroenterol. 12(1):1-4
  7. Fernández-Bañares F, Esteve M & Viver J.M. (2009)Fructose-sorbitol malabsorption.Curr Gastroenterol Rep.11(5):368-74.
6.2 Is it possible to accept an “additional strengths biowaiver” when bioequivalence to the reference product has been established with a BCS-based biowaiver? October 2013

Biowaiver of additional strength should be applied only when the test product have shown bioequivalence to the reference product by means of an in vivo bioequivalence study.


Bioequivalence is in principle demonstrated by means of in vivo bioavailability studies. These in vivo studies can be waived if the product fulfils the requirements defined in surrogate tests like the BCS biowaiver approach.

This is in accordance with the Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1/ Corr) which states in this respect that: “The BCS (Biopharmaceutics Classification System)-based biowaiver approach is meant to reduce in vivo bioequivalence studies, i.e., it may represent a surrogate for in vivo bioequivalence. In vivo bioequivalence studies may be exempted if an assumption of equivalence in in vivo performance can be justified by satisfactory in vitro data”.

An additional strength biowaiver is a waiver designed to avoid repeating the same in vivo study at the other strength level. Hence, when the Guideline on the Investigation of Bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1/ Corr) states that: “If bioequivalence has been demonstrated at the strength(s) that are most sensitive to detect a potential difference between products, in vivo bioequivalence studies for the other strength(s) can be waived”, this implies that when bioequivalence has been demonstrated in vivo for the test product, in vivo bioequivalence studies for the other strength can be waived.

Indeed, the reference in the sentence above to the sensitivity to detect differences between test and reference products only makes sense in the case of in vivo comparisons. This sensitivity varies depending on the solubility and the pharmacokinetic linearity. In the case of highly soluble drugs, the only drugs for which a BCS biowaiver is acceptable, the sensitivity to detect differences in vitro is the same at all strengths. Thus, the reference to higher sensitivity at the highest strength refers to in vivo studies. Further, the different sensitivities arising from non-linear pharmacokinetics only apply to in vivo studies. Therefore, the intent of this text was to refer to in vivo studies as evidence of bioequivalence.


The Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1/ Corr) states that: “If bioequivalence has been demonstrated at the strength(s) that are most sensitive to detect a potential difference between products, in vivo bioequivalence studies for the other strength(s) can be waived.”

The PKWP was asked to comment on the acceptability of this approach when the bioequivalence of the “reference” strength to the reference product has been investigated using the BCS (Biopharmaceutics Classification System)-based biowaiver approach i.e., without an in vivo bioequivalence study.

6.3 Clarification on how to apply the reference made in Appendix II of the Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev.1/Corr**), when waiving in vivo studies for oral solutions. December 2016

Conceptually, bioequivalence investigates whether two products exhibit comparable in vivo release from the formulation and therefore they exhibit similar bioavailability. Consequently, oral solutions may be considered less critical particularly in the case of aqueous solutions containing completely solubilized active substances, because neither the manufacturing process nor the formulation affects drug release and the formulation impact on absorption should be minimal. However, excipients might impact the bioavailability in different, not necessarily foreseeable ways, since systematic investigations of specific excipients are rarely available and in general, in vivo susceptibility of active substances towards excipient effects seems to be different.

The guideline paragraph under discussion specifically indicates certain ‘critical excipients’ known to potentially affect in vivo bioavailability, by means of in vitro and/or in vivo interactions. In addition, the last sentence including the reference to Appendix III defines that similarity of excipients should be handled according to requirements specified for BCS-based biowaivers. Consequently, differences in excipients could be handled more flexible with BCS class 1 drug substances, but qualitative similarity and very close quantitative similarity of excipients is expected in the case of BCS class 3 drug substances.

This is considered justified because the scientific rationale regarding the potential interaction between highly soluble drug substances (BCS class 1 or 3) and excipients, likewise applies to drug substances already in solution and immediate release formulations (rapid or very rapid in-vitro dissolution).

For clarification, the evaluation and comparison of excipients of solutions containing BCS class 2 and BCS class 4 drug substances, should be handled according to BCS 3 requirements. Of note, non-relevant excipients, e.g. colorants, flavours, buffers, are not considered relevant in this context.

In conclusion, the current guideline indicates the relevance of section IV.2 in Appendix III for oral solutions (Appendix II) and the drug substance BCS class should be considered accordingly, unless deviating from this general requirement can be adequately justified by reference to other data.

7. Biosimilars

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7.1 What are the key pharmacokinetic considerations in the assessment of biosimilarity? February 2018

Pharmacokinetics (PK) is an integral part of biosimilarity assessment. A biosimilar product contains a version of the active substance of an already authorised original product (see EMEA/CHMP/BMWP/42832/2005 Rev. 1). This is in contrast to small molecule generics where the active substances are chemically identical and as such the disposition of the active substance is the same once the compound is absorbed. For a biologically derived medical product the comparability assessment is more complex, as apart from the absorption also the disposition (distribution and elimination) can differ for a biosimilar compared to the reference product. Therefore, in the assessment of a biosimilar the PK comparison should also reflect the distribution and elimination processes (e.g. plasma clearance, volume of distribution, half-life) in addition to the absorption.

Biotechnology-derived proteins can be very different in complexity. For example, evaluating a well-characterisable polypeptide biosimilar without any post-translational modifications having the same structure as the reference product, the probability of differences in disposition of the active substance would be considered to be low. On the other hand, differences in glycan structures of complex biologicals or in pegylation may be more likely to affect distribution or elimination characteristics.

In summary, the comparability PK study for a biosimilar product is a test of formulations and active substance, in contrast to small molecule generics where the comparison of formulations is sufficient. The role of the comparative PK study in the assessment of biosimilarity is to exclude any relevant PK differences that could indicate presence of structural and/or functional differences that could impact the efficacy, safety or immunogenicity of the product. Following are issues that need to be considered in assessment of biosimilarity.


Linear clearance. The simplest case is single IV administration of a compound with concentration independent clearance. In this case clearance (CL) is directly reflected by the primary PK parameter AUC(0-inf) when the same dose of the biosimilar and reference product is given. The extrapolation to AUC(0-inf) from AUC(0-t) for substances having linear elimination is straightforward.

Linear clearance + Nonlinear clearance.For therapeutic proteins the total clearance often consists of a linear (nonspecific) clearance and a nonlinear (target mediated) clearance. Clearance may then be dependent on concentration of the compound itself as well as on the concentration of target and thus target dynamics. Target-mediated clearance may not be evident in the therapeutic concentration range due to saturation of the target, but can be observed at low plasma concentrations when the target is not saturated. AUC(0-inf) includes both nonspecific and target-mediated elimination, but non-compartmental analysis (NCA) cannot differentiate between the two elimination pathways. If pharmacokinetics of the biological medical product is characterised by a nonspecific and target mediated clearance, partial AUCs reflecting the different elimination pathways or PK modelling may support the assessment of PK similarity between the biosimilar and innovator product. Knowledge regarding any nonlinearity should in general be known from the reference product. However, it may be useful to plot individual PK curves for each treatment group to reveal any patterns or trends that could be masked in the mean plasma concentration time curves.

Some complexity is added for subcutaneously (SC) or intramuscularly (IM) administrated products since AUC(0-inf) then reflects CL/F. For SC and IM administrated products Cmax, in addition to AUC(0-inf), is a primary PK parameter for statistical comparison between the biosimilar and reference product.

Anti-drug antibodies

Large variations in PK and nonlinear elimination kinetics can also be caused by an immune response and formation of anti-drug antibodies (ADA) that could increase clearance. If there is a high incidence of ADA formation, it may be useful to visualise the individual PK profiles in subjects with and without ADA. A subgroup analysis of ADA negative and ADA positive subjects comparing PK parameters between treatment groups could be performed if feasible. This analysis should if possible be included in the statistical analysis plan.

Batch selection and protein content correction

Representative batches of the biosimilar and innovator product should be used in the comparative PK study and it should be documented how the used batches have been selected. Protein content of the batch and also delivered volume, when pre-filled syringes, injection pens, etc. are being used, should be considered in selection of the batches. The protein content of the selected biosimilar and reference product batches should be determined beforehand and analysed using the same analytical method. Pre-specified and well justified protein content adjustment can be acceptable, provided that the difference in delivered dose is not reflecting a consistent difference between the biosimilar and reference product (see EMEA/CHMP/BMWP/42832/2005 Rev1). Further, proportional adjustment for protein content in case of non-linear pharmacokinetics should be thoroughly discussed. Alternative methods to ensure delivery of the same protein dose could be considered. For example, the same content of the biosimilar and reference product in prefilled syringes could be transferred into identical syringes, thus avoiding any dose correction due to the device or protein content. Such a solution requires further discussion on potential effects of the devices on the delivered doses, where needed, supported with additional data e.g. looking for systematic differences in delivered volume, effects of needle size etc., to support that there is no difference in local delivery of the product.

Study Design 

Parallel design is often utilized due to long half-life or the risk of an immunogenic response. It is important to scrutinise the pre-specified exclusion criteria for individual PK data and for which PK parameters the criteria are valid. After exclusion of subjects for pre-specified reasons, it should be examined whether the groups are balanced and if not, whether this could affect the results (i.e. discuss the robustness of the results in case the fraction of excluded subjects was large e.g. by PK modelling). If a cross-over design is being used, the length of wash out need to be well supported taking active substance and any PD marker that is measured as study endpoint into account. For comparability reasons, a cross-over study design is preferred as the subject is its own control, however, in case of relevant ADA incidence, appropriateness of cross-over design is generally questioned. Even if ADAs are washed out, an increased sensibility for ADA development is carried over to the second period. Besides ADAs, potential effects on target density should be considered as well. When target-mediated clearance is relevant, any change in target density as a result of treatment could affect clearance and thus contribute to a carry-over effect.

Study population. The selected study population should be justified and the sensitivity for detecting PK differences should be considered when assessing the PK study. The variability is in general less in healthy volunteers and therefore, they are generally acceptable as study population in the PK study. However, it should be considered that the target density may be different in healthy volunteers vs. patients and patients in different therapeutic areas or severity of disease. In case both target mediated and non-target mediated elimination need to be considered, comparable PK should preferably be supported for the predominant (or both) mechanism(s). This could in some cases be handled by dose selection (see below). If not, other supportive data could be collected. This could include partial AUCs under the target mediated elimination phase in the healthy volunteers as well as concentrations measured during the efficacy, safety and/or PD studies. For example Ctrough at different time points during the treatment can be used as a supportive PK parameter.

Dose level. If possible, the most sensitive dose should be selected to investigate any target mediated clearance. The dose selection is of particular importance when target mediated clearance is expected to be larger in the patient population compared to healthy subjects. In such case a low dose (i.e. assuming target not saturated) or the lowest recommended therapeutic dose and a high dose, usually the highest recommended therapeutic dose (assuming target is saturated and nonspecific CL is dominating), should be considered for PK comparison (see EMA/CHMP/BMWP/403543/2010).

Sampling times. It is important to ensure long enough plasma sampling also considering subjects with the longest half-life to avoid large extrapolations when estimating AUC(0-inf). This is particularly important when clearance is nonlinear, which can slightly overestimate AUC(0-inf), as the extrapolation to AUC(0-inf) using non-compartmental analysis assumes linear elimination. An extrapolated AUC of ≤20% is considered to be acceptable. In line with the requirements for chemical entities, if more than 20% of the observations are excluded then the validity of the study may need to be discussed.

Statistical Comparison 

Acceptance criteria. The acceptance criteria for the main PK parameters should be defined and justified prior to conducting the study based on obtaining a precise quantification of relative exposure, and including consideration of differences in those parameters that are unlikely to have any clinically relevant impact on pharmacodynamic response.  In the absence of a rigorous clinical justification, acceptance criteria of 80–125% might be used but this might have consequences for interpretation of trial results (see below).

Analysis. Typically, the comparability of the pharmacokinetic parameters is analysed using ANOVA, which is adequate for the analysis of a cross-over trial. However, for a parallel group study it may be desirable to adjust for baseline characteristics that could affect the PK results (e.g. body weight) and which may be imbalanced between the two treatment arms. Therefore, analysis of comparability of the pharmacokinetic parameters by ANCOVA is acceptable in parallel group studies, provided that the choice of covariates has been justified and this is pre-specified in the statistical analysis plan. It should be noted that this model cannot be used to adjust for any covariates measured after randomisation, such as ADA formation.

Statistically significant difference. As stated in the introduction, the active substance in a biosimilar is not exactly the same as the innovator product and the purpose of the clinical comparability studies is not to show the products are identical but to exclude an important impact arising from any functional or structural differences compared to the reference product.Therefore a biosimilar product should be considered to be acceptably similar to the reference product in terms of PK if the 90%-CIs for the primary PK parameters are contained within the pre-specified and justified acceptance limits. In the absence of rigorous and acceptable justification for the pre-specified acceptance criteria, and in particular where 80-125% is chosen rather arbitrarily, a point estimate or substantive part of the confidence interval lying towards the extremes of the acceptance criteria will require further discussion.This includes instances where unity is excluded from the 90% CI.Specifically, results should be explained and justified in the context of evidence for similarity coming from other comparative studies/assays within the development programme.

If the 90%-CIs for the primary PK parameters are not contained completely within the pre-specified acceptance limits, a root cause analysis should be initiated. Findings from the root cause analysis should be reflected in the planning and conduct of any subsequent PK study, but any changes to the design should not compromise the sensitivity of the study to detect differences if they exist. Even in the presence of identified root causes, it is very important for assessment to understand why the evidence of failed PK comparability can be disregarded with plausibility.

In case there remain uncertainties concerning root causes, or if no causes could be identified, assessment of PK similarity should be based on results from both the original and subsequent PK studies. The existence of a study which demonstrates biosimilarity does not mean that those which do not can be ignored. It should be thoroughly discussed and justified that the biosimilarity claim has been demonstrated overall, and that the evidence from the positive study or studies is sufficient to dominate the results from those that were not. Where relevant, a combined analysis of all studies can be provided in addition to the individual study analyses as part of this justification. It is not acceptable to pool together several negative studies to justify PK biosimilarity in the absence of a study that is positive alone.

Limitations of the PK study

It is important to be aware of that the comparative PK study cannot be used to outweigh substantial differences in quality, nonclinical or efficacy and safety studies. The results of the PK investigations should always be interpreted and weighed in the context of all other data. The extent to which potential differences in disposition between biosimilar and reference product could occur depends on the nature of the molecular differences between both products. It is therefore important to consider also the quality characteristics and data on binding properties when judging the likelihood of potential pharmacokinetic differences.

8. Modified release products

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8.1 PKWP is requested to provide clarification on the requirements for sensitisation and irritation tests for transdermal products in Appendix I of the Guideline on the pharmacokinetic and clinical evaluation of modified release dosage forms (EMA/CPMP/EWP/280/96 Corr1). June 2018

Appendix I of the Guideline on the pharmacokinetic and clinical evaluation of modified release dosage forms (EMA/CPMP/EWP/280/96 Corr1) defines the situations where skin irritation and sensitisation should be assessed for transdermal products and recommends study designs and scoring systems that can be used accordingly.

These general requirements of Appendix I remain valid, however, this Q & A has been developed to clarify aspects of study designs and scoring systems to reflect the evolving scientific knowledge on the topic as follows:

1. The Appendix suggests one overall study design and it is now emphasised that this design is likely to need adoption to the particular question(s) to be addressed regarding e.g. specific formulation characteristics, differences in composition as compared to the innovator and intended dosing intervals.
2. Due to scientific and ethical reasons sensitisation testing by means of the human repeat insult patch test as described should be conducted in exceptional cases only as subjects might unnecessarily be sensitised and the sample size may not include sensitive subjects (i.e. individuals that would actually react).
3. As examinations of skin reactions is highly subjective this should be done by trained and experienced persons blinded to the treatment.
4. Other numerical scores than the dermal response (Table 1) and other effect (Table 2) scores that are currently presented in the Appendix have been found to be more appropriate (e.g. for Table 1, ‘definite oedema’ does not happen in isolation to ‘erythema’ and ‘papules’. For Table 2, the use of the term ‘glazing’ is deemed more likely with cosmetics rather than transdermal patches). The following scores may therefore be considered as possible alternate examples, but it is acknowledged that still others are available in the literature and the choice of score should be appropriately justified:

Table 1: Seven-point dermal response scale*

0no reaction
1minimal (barely perceptible) erythema
2mild but well defined erythema only
3moderate erythema only or mild erythema plus edema and/or papules
4severe erythema only or moderate erythema plus edema and/or papules
5severe erythema plus edema and/papules or any vesicular reaction
6bullous reaction or any grade 3 – 5 skin reactions that spread beyond the patch site

*Robinson, M. K. (2001), Intra‐individual variations in acute and cumulative skin irritation responses. Contact Dermatitis, 45: 75-83. doi:10.1034/j.1600-0536.2001.045002075.x

Table 2: Sensitisation score to assess allergic reactions (in line with the German Contact Dermatitis Group)*

0no reaction
0.5erythema no infiltration
1erythema, infiltration, discrete papules
2erythema, infiltration, papules, vesicles
3erythema, infiltration, confluent vesicles

*German Contact Dermatitis Group (Schnuch A, Aberer W, Agathos M, Becker D, Brasch J, Elsner P, Frosch PJ, Fuchs T, Geier J, Hillen U,Loeffler H, Mahler V, Richter G, Szliska C, fuer die Deutsche Kontaktallergie‐Gruppe (2007) LEITLINIEN DER DEUTSCHEN DERMATOLOGISCHEN GESELLSCHAFT (DDG) UND DER DEUTSCHEN GESELLSCHAFT FUER ALLERGIE‐ UND KLINISCHE IMMUNOLOGIE (DGAKI) ZUR DURCHFUEHRUNG DES EPIKUTANTESTS MIT KONTAKTALLERGENEN; issued 14.11.1998, updated 4.5.2007)

In conclusion, applicants are advised to use Appendix I and this Q&A to address sensitisation and irritation testing of transdermal patches carefully, taking account of product characteristics and based on current knowledge.

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