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The AAPS Journal ( # 2012)DOI: 10.1208/s12248-012-9382-1 Theme: Facilitating Oral Product Development and Reducing Regulatory Burden through Novel Approaches to Assess Bioavailability/BioequivalenceGuest Editors: James Polli, Jack Cook, Barbara Davit, and Paul Dickinson Bioequivalence Requirements in the European Union: Critical Discussion Alfredo García-Arieta1,3 and John Gordon2 Received 25 January 2012; accepted 29 May 2012 Abstract. The aim of the present paper is to summarize the revised European Union (EU) Guideline on the Investigation of Bioequivalence and to discuss critically with respect to previous Europeanrequirements and present US Food and Drug Administration guidelines its more relevant noveltiessuch as the following: in order to facilitate the development of generic medicinal products, the EUguideline includes the eligibility for Biopharmaceutics Classification System (BCS)-based biowaivers notonly for BCS class I drugs but also for class III drugs with tighter requirements for dissolution andexcipient composition. The permeability criterion of BCS classification has been substituted with humanabsorbability, as per the Biopharmaceutical Drug Disposition Classification System. The widening of theacceptance range for Cmax is possible only for highly variable reference products with an additionalclinical justification. This scaled widening is carried out with a proportionality constant of 0.760 which ismore conservative than the FDA approach and maintains the consumer risk at a 5% level when the intra-subject CV is close to 30%, due to the smooth transition between the scaled and the constant criteria. Theguideline allows for the possibility of two-stage designs to obtain the necessary information onformulation differences and variability from interim analyses as a part of the pivotal bioequivalencestudy, instead of undertaking pilot studies. The guideline also specifies that the statistical analyses shouldbe performed considering all factors as fixed, which has implications in the case of replicate designs.
KEY WORDS: bioequivalence; generic medicinal products; regulatory requirements.
Union (EU) (Canada ), and South Africa ), hasissued its own corresponding guidelines.
Although bioequivalence (BE) principles have been The first BE guideline of the EU "Investigation of clearly defined since the early 1990s (i.e., 20% acceptance Bioavailability and Bioequivalence" was published in June range (80–125%) for the 90% confidence interval of the ratio 1992 as part of the Rules Governing Medicinal Products in between test and reference least square means after log- the European Communities. This guideline was revised as a transformation of the pharmacokinetic parameters of interest, "Note for Guidance on the Investigation of Bioavailability Cmax and area under curve (AUC)), there is no international and Bioequivalence," released in July 2001 Subsequent- consensus on many of the details regarding the requirements ly, clarification on specific topics has been given through for the design, conduct, and evaluation of bioequivalence Questions and Answers documents ). Beginning in May studies because it has never been a subject of the Interna- 2007, a global update of this guideline was undertaken by the tional Conference of Harmonization. Consequently, each Pharmacokinetic Subgroup of the Efficacy Working Party, regulatory region, e.g., USA ), Japan (European now Pharmacokinetic Working Party, and was adopted by theCommittee for Human Medicinal Products (CHMP) in This manuscript represents the personal opinion of the authors and January 2010 as "Guideline on the Investigation of Bioequi- does not necessarily represent the views or policy of the Spanish valence" (effective 1st August 2010) ). This update was Agency for Medicines and Health Care Products or Health Canada.
necessary, on the one hand, to clarify the requirements in 1 Division of Pharmacology and Clinical Evaluation, General Sub- order to increase the homogeneity within the different directorate for Human Use Medicines, Spanish Agency for Medi- member States of the EU so as to reduce disagreements and cines and Health Care Products, C/Campezo 1, Edificio 8, Planta 2 arbitrations to the Coordination Group for Mutual Recogni- A, 28022 Madrid, Spain.
tion and Decentralised procedures (human) (CMD(h)) and Division of Biopharmaceutics Evaluation, Bureau of PharmaceuticalSciences, Therapeutic Products Directorate, Health Canada, Otta- CHMP, and on the other hand, to take into account the wa, Ontario, Canada.
scientific advances in the field of BE, e.g., requirements for 3To whom correspondence should be addressed. (e–mail: agarciaa@ highly variable drugs and biowaivers based on the Biophar- maceutical Classification System.
1550-7416/12/0000-0001/0 # 2012 American Association of Pharmaceutical Scientists García-Arieta and Gordon In this paper, the revised EU Guideline on the Investi- Specific Products' presented on the FDA web page(which gation of Bioequivalence, whose content is said to be limited simplifies notably the development of generic products for to immediate release dosage forms with systemic action although it contains general principles applicable to BEstudies for any dosage form and defines requirements for WHAT STUDIES SHOULD BE SUBMITTED? several other dosage forms in Appendix II, is summarizedand discussed critically. The specific BE requirements for In contrast to past practice, the revised Guideline modified release products are defined in a different guideline requires the submission of all studies performed with the (that is presently under review.
formulation proposed in the application (i.e., same composi-tion and manufacturing process) with the reference medicinal LEGAL ISSUES: GENERIC MEDICINAL PRODUCTS product marketed in the EU (synopsis only for pilot studies).
VERSUS OTHER TYPES OF APPLICATIONS In addition, synopses of studies conducted during theformulation development should be submitted.
For those outside of the EU, it is important to first understand the concept of a generic medicinal product as defined in Directive 2001/83 (), which is different than theUS Food and Drug Administration (US-FDA) concept of The only study design change in the revised version generic. In the EU, those products that show equivalence by relative to the previous guideline is that an additional means of pharmacodynamic or therapeutic equivalence trials multiple dose study is not required for immediate release with clinical endpoints, i.e., locally acting and locally applied products with non-linear pharmacokinetics (PK; dose- or products like inhalation, nasal, cutaneous, gastrointestinal, time-dependent PK). In comparison with the US-FDA ophthalmic products, etc. are not considered to be generics, guideline no major differences seem to exist with regard to but hybrids. Generics are only those whose BE is demon- study design since the standard single-dose 2×2 design is strated by means of bioavailability studies, i.e., pharmacoki- recommended in both regions. Obviously, the parallel design netic studies. In addition, different dosage forms are is acceptable for drugs with very long half-lives if demo- acceptable in the case of immediate release oral dosage graphic characteristics (e.g., age, body weight, sex, ethnic forms, i.e., oral solution and tablet. Furthermore, pharmaceu- origin, smoking status, and metabolic status) that may affect tical alternatives such as different salts, ester, ethers, isomers, the PK of the drug in both treatment groups are comparable.
mixtures of isomers, complexes, or derivatives of an active Therefore, phenotyping and/or genotyping is necessary in substance are considered to be the same active substance, parallel designs. Furthermore, replicate designs are recom- unless they differ significantly with regard to safety and/or mended for highly variable drugs in order to estimate the efficacy. Some, if not all, of these differences are somewhat within-subject variability of the reference product with the difficult to understand when generics are considered to exist aim of widening the acceptance limits for Cmax ().
to be interchangeable with the reference product. The reason Multiple dose studies are only acceptable when single for such criteria is that, in the EU, the pharmaceutical dose studies are not feasible due to the following: (1) legislation only deals with the approvability or prescribability tolerability/safety concerns that require that the study be of medicinal products. A product is approved if the benefit– performed in patients that cannot have a passive wash-out risk relationship is positive, but this does not mean that it is period or (2) in exceptional cases of low analytical sensitivity interchangeable with the reference product. The substitution that precludes the estimation of the plasma concentration- policy is a national issue that is not regulated by the EU.
time profile after a single dose, but that is able to detect the Another issue that may be difficult to understand for higher plasma levels that occur after accumulation in steady those outside the EU is that a product that fails to show BE in state. As Cmax after multiple doses is less sensitive to detect comparative bioavailability (pharmacokinetic) studies can be formulation differences than Cmax after a single dose ), approved based on pharmacodynamic/clinical studies showing the use of a single supra-therapeutic dose is preferred if there equivalence, even though these studies are less sensitive to are neither solubility nor tolerability limitations.
detect differences between products. Again, this is because, inthe EU, the objective is not to interchange these hybrid SELECTION OF THE REFERENCE PRODUCT products but to approve them based on a positive benefit–riskrelationship.
Although the definition of reference medicinal products in Directive 2001/83 ) states clearly that "reference HOW MANY STUDIES ARE REQUIRED? medicinal product" shall mean a medicinal product author-ised under Art. 6, in accordance with the provisions of Art. 8, The number of BE studies required in the EU has to be the Notice to Applicants ) and the Guideline on the deduced based on the physico-chemical characteristics of the Investigation of Bioequivalence () have widened the legal substance (e.g., solubility and chirality), its pharmacokinetic basis that a reference product can have. Now, not only properties (e.g., linearity or dose proportionality, food effect/ products applied based on Art. 8(3), but also those based on food intake recommendations in the Summary of Products article 10a, 10b, or 10c of Directive 2001/83/EC can be Characteristics (SPC)), and proportionality in composition (to considered as an appropriate reference product. As the waive studies for proportional strengths). Presently, the Euro- reference product has to be based on a complete dossier pean Medicines Agency (EMA) does not publish BE recom- (Art. 8(3)), it is understandable that a licence (Art. 10c) of mendations like the ‘Bioequivalence Recommendations for the innovator could be used as reference when the innovator Bioequivalence Requirements in the European Union is not on the market or that a fixed dose combination (Art. 10b) model of healthy volunteers is adequate in most instances to is a complete dossier for the combination. However, an extrapolate the results to other populations, but the rare application based mostly on literature data plus one or only a instances where the extrapolation is not adequate are not few clinical studies, which is considered a mixed dossier, a identified. Therefore, unless these rare instances are identi- type of complete dossier (Art. 8(3)), is a more controversial fied in the literature, it will have to be assumed that the model reference product since generics of the innovator will be of healthy volunteers is always applicable.
confounded with generics of the mixed dossier. From a scientificpoint of view, it is evident that those bibliographical applications STUDY STANDARDIZATION (Art. 10a) of drugs that are considered to be of well-establisheduse simply because they have been marketed in the EU should Standardization of study conditions is in the interest of not be considered appropriate reference products since these the sponsor in order to reduce variability and increase the products are approved based on the literature data obtained with likelihood of demonstrating BE. In the revised guidance, the other products and in most cases no experiments were carried over-night fasting time has been reduced to at least 8 h and out with the Art. 10 a formulation. Therefore, usually no BE or the volume of fluid to be taken with the treatments is comparative bioavailability study has been performed on these identified as at least of 150 ml.
products before reaching the market of the EU to link the When a study is to be conducted in the fed state, the bioavailability of the proposed product to the product described revised guideline indicates that the timing of food adminis- in the literature, the one with a well-established use. In the USA, tration must follow the SPC of the reference product. If this it must be challenging to understand how a marketing author- information is not provided in detail in the SPC, the isation can be granted to a product that lacks pre-clinical and administration of the treatments should follow 30 min after clinical data. In fact, those EU legislators that assume that the the start of the meal, which should be eaten within 30 min.
bioavailability of the new product will not change significantly The guideline states that although concomitant medica- and the benefit–risk relationship will be similarly positive may be tions should be avoided, contraceptives are permitted as are wrong is some cases, e.g., the use of a small amount of sodium any other medications considered necessary to treat emergent laurylsulphate (SLS) in a product approved based on a issues, however, the use of these medications must be bibliographical application, will increase the bioavailability of reported and it must be demonstrated that they neither alendronate five- to sixfold (unpublished data). If it is question- interfere analytically nor interact pharmacodynamically.
able that a bibliographic product should be marketed, it is easy to For those drugs that are taken always in combination understand that generics of such a product should not be with another drug (e.g., drugs to be boosted with ritonavir), the study may be performed in combination or isolation, Finally, although liposomes are not considered to fulfil the because BE in one of these scenarios indicates BE in the EU definition of generic since clinical and/or preclinical studies other since the extent of the interaction will be the same for may be necessary in addition to bioequivalence pharmacokinet- both products.
ic studies, the EMA has validated as a generic/hybrid medicinalproduct (Doxorubicin Sun) (an application making refer- FASTING OR FED CONDITIONS ence to a product (Caelyx® 2 mg/ml concentrate for solution forinfusion) approved as a hybrid application (formerly Art. 4.8.(a) With respect to the administration of food during BE (iii) of the EEC Directive 65/65) (The reference product studies, the revised EU guideline still differs from the US- containing liposomal doxorubicine was considered a hybrid FDA regulations. The approach of the US-FDA () is that in application that referred to the reference product of conven- order to demonstrate BE a study conducted under fed tional doxorubicine ). Although an abbreviated application conditions is required in addition to a fasted study except cannot refer to another abbreviated application, in this case the for in the following situations: (1) class I drugs when both test generic application refers simultaneously to the liposomal product and Reference Listed Drug (RLD) are rapidly product (hybrid) and the conventional intravenous solution dissolving and have similar dissolution profiles, (2) when the (complete dossier).
SPC of the RLD states that the product should be taken Apart from that, the EU guideline is sound in asking for only on an empty stomach, or (3) when the RLD label does comparisons against the same dosage form of the reference not make any statement about the effect of food on product when available. When the innovator company absorption or administration. In contrast, in general in the develops a line extension, it is recommended that comparison EU (), only a single study conducted in the fasting state is of the new dosage form be made with the one nearest to the required assuming that it is the most sensitive condition to formulation used in phase III trials.
detect formulation differences. Therefore, the food effect may Finally, as per the revised guideline, the applicant should exist but it is not believed that products with conventional justify that the batch of the reference product investigated is pharmaceutical technology will be equivalent in fasting state representative of the reference product in the market and bioinequivalent in fed state as the fasting state is comparing at least two batches from the EU market.
considered more discriminative. Consequently, it is notconsidered necessary to increase the regulatory burden for NUMBER AND SELECTION OF SUBJECTS such products.
Based on this principle, for drugs that are taken only in As seen in other similar guidelines, a minimum number the fasted state or irrespective of food, a BE study with that of 12 subjects has been defined as a requirement to ensure drug must be conducted in fasted state. However, in situations reliable estimates. Interestingly, the guideline stresses that the where it is recommended in its labelling that a reference García-Arieta and Gordon product be taken only in the fed state, a BE study conducted immediate release dosage forms would be Cmax,ss and with that product should generally be conducted in fed state.
AUC(0–τ). If urinary data are employed, Ae(0–t) and Rmax, if This "generally" means that if the fed state is recommended Cmax in plasma is not detectable, should be measured.
in the SPC in order to avoid tolerability problems associated Urinary data are only acceptable if the parent cannot be with chronic use in patients, a fasted state study is acceptable measured in plasma, and it can be justified that urinary as a single dose in healthy volunteers but, if the fed state is excretion reflects plasma exposure. However, the guideline required for pharmacokinetic reasons resulting in a systemic does not indicate a preference between the possible exposure that is notably different, the study should be approaches: a study after steady state for the parent drug in performed in fed state. There is an exception to this approach plasma, a single dose study with a metabolite in plasma, or a for products (test or reference) employing special (not single dose study for the parent drug in urine. This is a case conventional) technology (e.g., microemulsions and solid by case decision. Interestingly, in case of a multiple dose study, dispersions) that can be taken irrespective of food in that Cmax of the parent in plasma does not need to be measured, for these products BE has to be shown in both fasted and fed even if measurable, after the first administration, whereas in state (e.g., cyclosporine microemulsion).
case of a single-dose study for the parent drug in urine the Cmax The advantage of testing the performance of products in in plasma, if measurable, should be used instead of Rmax.
the fasted state and the fed state with a high-fat, high-caloriemeal, such as is required for many conventional products in ANALYTE TO BE MEASURED: PARENT the USA, is that the extremes of the food effect are tested and BE with intermediate meals can be assumed. In the EU,if the SPC of the reference product indicates administration In principle the parent drug has to be measured, even if with food but does not make specific recommendations with inactive, due to the higher sensitivity of its Cmax to detect respect to the composition of the meal, studies should employ formulation differences in release rate ,). However, a high-fat, high-calorie meal and hence, bioequivalence when in the case of pro-drugs or drugs with very low contribution to products are taken with meals with a different more moderate activity, where BE is very difficult to show due to high composition, which might be more realistic, is not investigat- variability associated with low plasma levels that disappear ed. The demonstration of bioequivalence in the fasting state very quickly, it is acceptable to measure only the main active and after a high-fat high-calorie meal would represent a metabolite for practical reasons (e.g., mycophenolate mofetil bracketing approach where all intermediate meal composi- vs. losartan) (). The use of a metabolite as a surrogate of an tions could be assumed. In contrast, demonstration of active parent drug is discouraged. Such a situation would only bioequivalence in the worst-case scenario of a high-fat high- be considered if the state-of-the-art analytical technology is calorie meal could be considered as not representative of all not able to measure the low concentrations of the parent drug possible meal compositions. The high-fat, high-calorie meal after a single dose. In this case, it would be necessary to might be representative of a dinner or a lunch of some justify that a supra-therapeutic dose is not feasible due to European countries but, would not normally be considered a tolerability/safety reasons or solubility limitations, and that typical breakfast.
the metabolite formation is not saturated at therapeutic doses Another issue of debate is the composition of the high- so that the metabolite exposure reflects the parent exposure.
fat, high-calorie meal. In the US-FDA, the ingredients of the Active metabolites do not need to be measured if the high-fat, high-calorie meal are defined, i.e., an example test parent drug is measured, even if the PK system is non-linear, meal would be two eggs fried in butter, two strips of bacon, although there is experience with some statins showing two slices of toast with butter, four ounces of hash brown discordant results between parent and metabolite. However, potatoes, and eight ounces of whole milk. Substitutions in this as these are very exceptional cases, the risk is considered to test meal can be made as long as the meal provides a similar amount of calories from protein, carbohydrate, and fat and In contrast, in the case of active metabolites formed as a has comparable meal volume and viscosity. In the EU, result of gut wall or other pre-systemic metabolism, the US- however, only the caloric content of each component of the FDA recommends that the metabolite and the parent drug be meal is defined, which leaves room to employ different types measured, but only the parent drug has to be analyzed using a of food according to the dietary habits of the study site.
confidence interval approach. The metabolite data are used Consequently, the volume, texture, and viscosity of the meal as supportive evidence of comparable therapeutic outcome may vary markedly, which could affect the extent of the food and could highlight the existence of marked differences in metabolite exposure. This approach seems to be moreadequate for the assessment of statins but unnecessary for most drugs. In addition, the absence of formal statisticalanalysis makes the data difficult to interpret.
Non-compartmental methods should be used to estimate conventional PK parameters, e.g., AUC(0–t), AUC(0–∞), resid- ual area, Cmax, tmax, λz, and t1/2 in single-dose studies. Theparameters to be analysed statistically in a single dose study The revised guideline introduces new recommendations are Cmax and AUC(0–t), instead of AUC(0–∞). For the first on the need of chiral bioanalytical methods for enantiomer time, AUC truncated at 72 h (AUC(0–72)) is accepted in BE drugs. Chiral methods are necessary when three conditions studies as a substitute of AUC(0–t) For studies conducted are met (or unknown): (1) the enantiomers exhibit different at steady state, the parameters for statistical analysis for pharmacokinetics, (2) the enantiomers exhibit pronounced Bioequivalence Requirements in the European Union difference in pharmacodynamics, and (3) the exposure STRENGTH TO BE INVESTIGATED (AUC) ratio between enantiomers is modified by a differencein the rate of absorption.
If an application includes multiple strengths and these In contrast to the US-FDA requirements, it is not strengths fulfil certain criteria, it may be sufficient to necessary that the primary efficacy and safety activity resides demonstrate BE at only one or two strengths. The criteria with the minor enantiomer, because even if both eutomer and to waive BE studies for some strengths are as follows: distomer have similar exposure the bias of the achiral methodremains In addition, when the AUC ratio between (a) The pharmaceutical products are obtained by the same enantiomers is modified by a difference in rate of absorption, manufacturing process. It should be noted that it is now at a given rate both enantiomers may exhibit similar PK, but possible to manufacture them in different manufacturing at another rate of absorption the PK will differ ( Therefore, the first requirement is fulfilled if the third is (b) The qualitative composition of the different strengths is fulfilled. Consequently, non-chiral methods are acceptable the identical, although certain excipients like colorants only if it is possible to show that enantiomers have similar pharmacodynamic activity (e.g., omeprazole) or that nonlin- (c) The composition of the different strengths are quantita- ear absorption is not present (as expressed by a change in the tively proportional, i.e., the ratio between the amount of enantiomer concentration ratio with change in the input rate each excipient to the amount of active substance(s) is the of the drug) for both enantiomers. Interestingly, the US-FDA same for all strengths. For immediate release products, compares enantiomer concentration ratio, which changes with coating components, capsule shell, colour agents, andfl time, whereas the EU compares AUC for simplicity, but avours are not required to follow this rule.
seems to be less accurate. Unfortunately, according to the In addition, some deviation from exactly proportional revised EMA guideline, a chiral bioanalytical method would compositions are acceptable when the amount of the not be necessary for etodolac although it was the example active substance(s) is less than 5% of the tablet core that illustrated the non-linear absorption since it only affects weight, or the weight of the capsule content, in the strength used in the BE study and the strength to be Although it is not indicated in the guideline, it can be waived, and one of the following conditions applies: deduced that for drugs that are pure enantiomers whereenantiomer inter-conversion exists and inter-conversion 1. The amounts of the different excipients are the depends on rate of absorption, chiral bioanalytical methods same and only the amount of active substance is would be necessary.
2. The amount of a filler is changed to account for the ENDOGENOUS SUBSTANCES change in amount of active substance and the amountsof other excipients is kept constant.
In BE studies of endogenous substances, factors like dietary intake that may affect the baseline levels should be (d) Appropriate in vitro dissolution data should confirm the standardized and baseline correction should be used to similarity of the dissolution profiles, and estimate pharmacokinetic parameters. Supra-therapeutic (e) BE has been investigated at the strength(s) that are most doses, if well tolerated and without solubility limitations, sensitive to detect a potential difference between prod- facilitate the measurement of the concentrations over base- ucts. The strengths to be tested depend on the pharmaco- line provided by the treatment. The type of baseline kinetic linearity, more specifically on AUC dose correction must be pre-defined case by case depending on proportionality. It is important to note that Cmax is not the characteristics of the substance. In some cases the taken into account due to its higher variability, which approach will involve the subtraction of a constant baseline could make the conclusion of PK linearity/dose propor- level, which can be the mean of several pre-dose concen- tionality more difficult, although Cmax is generally more trations of each subject, or subtraction of the pre-dose AUC sensitive than AUC to detect solubility-limited absorption of each subject, when the endogenous levels are not constant.
(e.g., glimepiride). A simple criterion to conclude AUC However, these two scenarios do not address the possible dose-proportionality has been included in this guideline feedback mechanisms that may occur after the exogenous for this purpose only: the difference in dose-adjusted administration of the endogenous substance. Therefore, the mean AUCs should be no more than 25% between the sponsor is expected to justify the adequacy of a proposed investigated strength and the to be waived strength (i.e., a baseline correction strategy. In rare cases where the endog- ratio within 0.75 and 1.33) enous levels are negligible with respect to the exogenousones, baseline correction is not necessary (e.g., supra-thera- In the case of drugs with linear PK, it is sufficient to peutic doses or patients without or with very low endogenous establish BE with only one strength, usually the highest but, if the drug is highly soluble, any lower strength is acceptable. In Interestingly, the guideline clarifies that it is essential to any case, for reasons of tolerability or safety, studies with a ensure the sensitivity of the study by demonstrating separa- lower strength will be accepted. On the contrary, a supra- tion in exposure following administration of different doses, therapeutic dose using preferably multiple units of the highest either in a pilot study or as part of the BE study using strength may be acceptable for analytical reasons, if there is different doses of the reference formulation, if this has not neither tolerability problems nor absorption/solubility limita- been established previously.
tions at that dose.
García-Arieta and Gordon In the case of non-linear PK with greater than propor- with respect to the batch of the test product. This strategy tional increases in AUC with increasing dose, the BE study should be clearly pre-defined in the study protocol according should be conducted at the strength in the curve part of the to the certificate of analysis of both products.
AUC vs. dose curve, which is generally the highest strength.
The guideline stipulates that subjects that do not provide In the case of non-linear PK with less than proportional data for both test and reference product in a cross-over trial increase in AUC with increasing the dose, it is essential to (or one period in a parallel study) should not be included in identify the cause of the non-linearity. In the case of the statistical analysis. Therefore, the use of statistical saturation of transporters (e.g., gabapentin), the lowest methods that impute the missing observations based on the strength or any strength in the linear part of the AUC vs.
observations of the other subjects are not acceptable.
dose curve should be tested since at the highest strength the Data from treatments that are not relevant for the curve is flat and insensitive. In contrast, in the case of non- comparison of interest should be excluded, e.g., data from linearity caused by solubility/dissolution limitations, the references outside of the EU or fed/fasted arms in a 4 period lowest and the highest strengths should be studied. The study when investigating alternatively BE in the fasted and lowest strength (or any strength in the linear part of the AUC the fed state. Otherwise, all subjects receiving treatment vs. dose curve) would be the most sensitive if both formula- should be included in the statistical analysis. In fact, "spare tions exhibit a similar non-linearity but, if the new formula- subjects," who are treated but whose samples are analysed tion were able to avoid the solubility/dissolution limitations only if other subjects withdraw, are not acceptable and all or, in the extreme case exhibit dose-proportionality, the treated subjects should be analysed even if there are no drop- highest strength would be the most sensitive.
For reasons of safety/tolerability or low bioanalytical The guideline stresses that the decision to withdraw a sensitivity, the dose can be modified as described above for subject must be made before the analysis of his/her samples.
drugs with linear PK.
Reasons for withdrawal are acceptable if pre-defined in the In addition, the guideline now includes the bracketing protocol (e.g., vomiting, diarrohea, need to administer approach to investigate only two strengths (extreme cases) concomitant medication) but, removal on the basis of the when the formulations do not fulfil the criteria to waive BE statistical analysis (i.e., outliers) or for pharmacokinetic studies at some strengths (e.g., formulations are not quanti- reasons (e.g., implausible values, extrapolation of AUC larger tatively proportional in composition and dissolution profiles than 20%) is not accepted. However, as described also in the are not similar). Interestingly, when two strengths have to be US-FDA guideline, those subjects with significant pre-dose investigated and fed and fasting studies are required, it may levels (>5% of Cmax) should be excluded since such a carry- be sufficient to assess only one strength in both fasting and over effect might be unequal between sequences and bias the fed state. Waiver of either the fasting or the fed study at the BE point estimate. Interestingly, an additional reason to other strength has to be based on previous knowledge or the exclude "a" subject has been included in the guideline, but it information obtained with the strength tested in both fasted is not clear if it refers to only one exceptional case or if more and fed state to select the most sensitive condition (fasted or cases (e.g., two or three) are acceptable, and how many cases fed). This is controversial since the Applicant should justify are necessary to conclude that the study validity is question- which study can be waived and it may be difficult to agree able. According to the guideline, if a subject exhibits no levels during assessment (e.g., sirolimus immediate release products or insignificant levels (<5% of the geometric mean of the since the different strengths of the reference product are other subjects) and this erratic behaviour is observed with the not bioequivalent when they are tested at the same dose reference product, the test product should not be penalised and, consequently, that subject could be removed from the In the case of fixed combinations the proportional statistical analysis. However, this might question the reliabil- composition requirement should be fulfilled for all active ity of the study, similar to cases when AUC extrapolation is substances taking into account that when considering the more than 20% in more than 20% of the subjects.
amount of each active substance, the other active substance The statistical analysis recommended in the guideline is (s) can be considered as excipients. In the case of bilayer based on the conventional 90% confidence interval for the tablets, each layer may be considered independently.
ratio of the population least square means test/reference of It is noteworthy that the guideline refers only to strength the pharmacokinetic parameters of interest after log-trans- and does not address the possible need of testing the formation (geometric means). Interestingly, the revised administered single dose when the single dose is higher than guideline does not require a non-parametric 90% confidence the maximum strength, e.g., in case of low solubility drugs the interval for tmax but, simply a visual inspection of medians and differences might be detected only at the highest adminis- variability if the onset of action is relevant for efficacy or tered dose since the low solubility might not be critical at the maximum strength.
The statistical model should be pre-defined in the protocol. Traditionally in the EU, the factors of the ANOVA in a 2×2 cross-over design are sequence, period, subjectnested in the sequence and formulation. It is not common to Potency correction is only acceptable when the differ- consider the phase within the period when all subjects cannot ence in potency between the tested products is larger than be dosed on the same day. Importantly, the model has to be 5%. Deviations of greater than 5% are only acceptable when analysed as if all factors were fixed. Therefore, subjects it is not possible to find in the European market a batch of the should not be considered as random. This has no implication reference product with a potency difference lower than 5% in 2×2 designs since subjects with missing data are excluded Bioequivalence Requirements in the European Union as imputation like the one performed by SAS® Proc Mixed is NARROW THERAPEUTIC INDEX DRUGS not acceptable. Therefore, SAS® General Lineal Model(GLM) and SAS® Proc Mixed give the same results when In contrast to US-FDA, NTI drugs have a tighter there are not missing data, however, the results will be slightly acceptance range in the EU. This revised guideline has different in case of replicate designs ().
defined a 90.00–111.11% acceptance range for AUC of all The guideline also clarifies that the observation of a NTI drugs. However, the classification of drugs as NTI drugs significant sequence effect (or period effect) is inconsequen- depends on the CHMP and they are not listed in the tial since the existence of a (unequal) carry over effect can be guideline. Cmax acceptance range has to be tightened to addressed directly with pre-dose samples. However, this is not 90.00–111.11 if it is of particular importance for efficacy or applicable to endogenous substances.
safety of drug monitoring, which is again a decision of the For the first time, this guideline acknowledges the CHMP. For example, requirements for AUC and Cmax of possibility of a two-stage design to show BE. In this instance, immediate release cyclosporine formulations have to be the following should be noted: tightened both in fasted and fed state studies while only theAUC requirement for immediate release tacrolimus formula- (a) The first stage is an interim analysis and the second stage tions needs to be tightened is the analysis of the full data set. The second data setcannot be analysed separately.
HIGHLY VARIABLE DRUG PRODUCTS (b) In order to preserve the overall type I error, the significance level needs to be adjusted to obtain a In order to confirm that a product is highly variable (CV, coverage probability higher than 90%. Therefore, it is >30%) for a given pharmacokinetic parameter, it is necessary not acceptable to perform a 90% CI at the interim to perform a replicate design to estimate its intra-subject analysis and a 95% confidence interval in the final analysis with the full data set.
In contrast to the US-FDA, the EU guideline only accepts (c) The plan to spend alpha must be pre-defined in the widening of the acceptance range of Cmax, not for AUC, and it is protocol. The same or a different amount of alpha can be necessary to demonstrate that a larger difference in Cmax is spent in each analysis. If the same alpha is spent in both clinically irrelevant. Previously, such justification was required stages, the Bonferroni rule (95% confidence interval in to widen the acceptance range to 75–133%. Now, this decision both analyses) is too conservative and 94.12% confidence depends on the intra-subject variability of the reference product, interval can be used. It is also possible to distribute the the one in the market whose large variability generally has no alpha differently, and as an extreme case, it is acceptable clinical relevance, and it can vary from 80.00 to 125.00 when to plan no alpha expenditure in the interim analysis when variability is 30% to 69.84–143.19 when it is 50%, the maximum it is designed to obtain information on formulation that is accepted. Intra-subject variabilities larger than 50% are differences and intra-subject variability and 90% CI are not frequent. Although the proper statistical methodology is to not estimated at the interim stage.
scale the average BE, in the guideline, the limits have been (d) A term for the stage should be included in the ANOVA scaled for simplicity.
model. However, the guideline does not clarify what the The guideline gives a table as example with the acceptance consequence should be if it is statistically significant. In range that corresponds to different intra-subject variabilities principle, the data sets of both stages could not be but, the values for other intra-subject variabilities can be obtained with the following formula: (U, L)=exp (±k·sWR),where U and L are the widened limits, sWR is the intra-subject Although the guideline is not explicit, even if the final variability of the reference product and k is the regulatory sample size is going to be decided based on the intra-subject constant that has been defined as 0.760 to be consistent with the variability estimated in the interim analysis, a proposal for a variability where scaling starts (CV=30%). This has been done final sample size must be included in the protocol so that a in order to have a smooth transition between scaling and no significant number of subjects (e.g., 12) is added to the interim scaling, and to avoid an excessive consumer risk at intra-subject sample size to avoid looking twice at almost identical samples.
variability slightly higher than 30%, which are very frequent This proposed final sample size should be recruited even if (). In contrast, the US-FDA employs a proportionality the estimation obtained from the interim analysis is lower constant that is more permissive (wider limits) and there is a than the one pre-defined in the protocol in order to maintain lack of consistency between the CV that corresponds to that the consumer risk.
constant and the CV where scaling starts to be acceptable (CV= In the revised guideline, the acceptance range has 30%), which increases the consumer risk.
now been defined with two decimal units (80.00–125.00%, It is worth noting that the guideline clarifies that the except for narrow therapeutic index drugs), like in the estimation of the intra-subject variability has to be reliable and not the result of outliers, the point estimate has to be When several studies have been performed the complete constrained within 80.00–125.00, and any replicate design is body of evidence must be considered. It is not acceptable to ignore failed studies simply because another one has passed.
The reasons for the failure should be discussed (e.g., lack of IN VITRO DISSOLUTION AND VARIATIONS statistical power). A combined analysis (meta-analysis) of allstudies can be provided if relevant, however, it is not In vitro dissolution tests of the test and reference bio- acceptable to combine failed studies to show BE.
batches at three different buffers (usually 1.2, 4.5 and 6.8) and García-Arieta and Gordon the Quality Control media have to be reported for quality the active substance is not absorbed in the mouth. However, purposes and to define specifications but, in vivo studies as the BCS biowaiver is based on the intake of the tablet with prevail if in vitro data differ. However, the discrepancy should a glass of water (i.e., solubility in 250 ml) and the orodisper- be addressed and justified. Similarly, if in vitro data do not sible tablets are usually taken without water, it would seem reflect the in vivo data or are unable to discriminate between appropriate that the solubility criterion be amended accord- batches with acceptable and non-acceptable in vivo performance, ingly and dissolution should be compared both in the all attempts should be made to develop an alternative method.
conventional vessels and in vessels, for example, resembling The same dissolution test should be carried out to waive the dissolution in the mouth (e.g., 5 ml of volume) that have proportional formulations. However, where sink conditions are not yet been developed.
not achievable at certain pH values, the profiles might differ It is noteworthy that the demonstration of BE without between strengths. To show that this difference is simply due to water is considered the worst case scenario and it is assumed the different dose, the sponsor should perform studies at the that the formulation will be also equivalent with concomitant same dose per vessel (e.g., two tablets of 5 mg vs. one tablet of intake of water. However, such an assumption is questionable 10 mg) or, alternatively, demonstrate the same trend in the when either the test or the reference orodispersible tablet reference product by comparing each strength of the test with contains mannitol since the presence of water might increase the corresponding strength of the reference.
the differences in absorption due to the osmotic effect of The BE guideline is the only guideline in the EU that addresses specific technical requirements for variations since For studies conducted without water, the guideline there is no specific guideline similar to Scale-Up and Post specifies a method of administration to standardize the Approval Changes guidelines in the US-FDA, but only a administration conditions and to ensure the availability of Regulation () and a Directive (about classification. This enough saliva (to wet the mouth with 20 ml of water directly revised guideline stresses that after reformulation or a change before the administration and not to take water within 1 h of in the manufacturing method that may affect bioavailability, administration). The same rules apply for similar dosage an in vivo study is required unless in vitro data are considered forms: orodispersible films, buccal tablets, sublingual tablets, a valid surrogate. This would only be true in instances of an and chewable tablets.
existing level A in vitro in vivo correlation (IVIVC) defined The guideline stresses the importance of excipients in taking into account such a change, or in the case of a oral solutions since in the past, low solubility drugs, in Biopharmaceutics Classification System (BCS) biowaiver solution thanks to the addition of co-solvents in the formu- approach. Therefore, for products containing a low solubility lation, were not required to show BE. However, different co- drug where an IVIVC has not been established, a new BE study solvents might have a different solubilisation capacity and is always required for changes that may impact bioavailability.
precipitation might differ between different formulations, The guideline does not specify what may affect bioavailability which in turn might affect bioavailability. Similarly, excipients and it must be decided according to current knowledge.
affecting gastrointestinal transport, absorption, or in vivo For BE studies required for a variation, the reference stability have to be assessed more carefully since a low product should again be the innovator product in case of amount of sorbitol can affect certain drugs like risperidone generics or hybrids, and the previous formulations in the case or small amounts of surfactants are able to increase the of applications that did not make reference to another bioavailability of low permeability drugs like alendronate, product (i.e., complete dossiers, mixed dossiers, fixed dose which can be increased up to five- to sixfold ().
combinations, and licences). It seems somewhat illogical to For intravenous aqueous solutions, a waiver of BE require a BE study for a change in bibliographical product studies is not possible if there are differences in composition when such a comparative BE study vs. the product described with respect to excipients that interact with the drug (e.g., in the literature was not required for its authorisation.
complex formation). For intravenous aqueous solutions with In those cases where dissolution studies are considered a different concentration compared with the concentration of sufficient to ensure equivalent in vivo performance after a the reference product in a hybrid application, a waiver can be change, the guideline refers to other guidelines of the Quality granted since the drugs are diluted in the plasma, as long as section, but it can be assumed that the new product has to be there are no safety/tolerability issues related to a higher compared with the existing one.
The comparison of dissolution profiles should be per- For other parenteral routes, the importance of similarity formed with the f2-similarity factor, taking into account not in viscosity has been highlighted in the revised guideline when more than one mean value with more than 85% dissolved for different excipients, but comparable ones, are used. This is any of the formulations and other prerequisites.
ensured if the same qualitative and (similar) quantitativecomposition is employed in the test product.
BIOEQUIVALENCE REQUIREMENTS FOR SPECIFIC The guideline also clarifies that demonstration of BE is not required for lipids for intravenous parenteral nutrition.
BE requirements for comparison of intravenous emul- Although the guideline deals only with immediate sions can be waived if the composition is qualitatively and release formulations, its Appendix II provides some guidance quantitatively the same and the physicochemical character- not only for immediate release dosage forms, but also for istics (e.g., size distribution, Zeta potential, and rheology) are other types of formulations.
similar, although the guideline does not indicate how similar According to the guideline, a BCS biowaiver might be these have to be, and the conventional quality character- considered for orodispersible tablets if it is demonstrated that isation does not include a proper comparability exercise.
Bioequivalence Requirements in the European Union Similarly, BE requirements for intravenous micelle forming sublingual and buccal) and orodispersible tablets since formulations can be waived if the micelles disassemble upon satisfactory dissolution methodology is not developed yet dilution in plasma and the composition is qualitatively and and, as explained above, the orodispersible tablets are usually quantitatively the same. Furthermore, such a waiver is also taken without water, therefore, the definition of solubility extended to cases with minor changes in qualitative or based on 250 ml does not apply.
quantitative composition, as long as the surfactant is not In this guideline, the classification as of a drug as highly altered. However, it is not evident that other excipients (co- soluble is based on the maximum single dose and not simply solvents) or differences in their amount do not affect the maximum strength, the pH range of interest varies from 1 bioavailability or the safety/tolerability profile. For example, to 6.8 instead of 7.5 , and the pH characterisation a change in co-solvents may cause a different stability and requirements do not include the pKa±1, but only pKa.
more frequent precipitation in storage, which does not In this document, the concept of permeability has been preclude marketing but, may facilitate misuse since these changed to absorbability and the criterion of highly absorb- products are not interchangeable. Again, the guideline able is based exclusively on "human" absorption suggests some in vitro test (e.g., critical micelle concentration, determined by means of mass balance studies or absolute solubilisation capacity, free, and bound drug and micelle size) bioavailability studies, greater, or equal to 85% of the but it does not define a complete list of tests and their administered dose. Data from animals or culture cells are corresponding acceptance ranges to ensure similarity.
only considered to be supportive. The data from the mass The guideline clarifies that a waiver of clinical studies is balance studies have to be interpreted in the light of the only possible for locally acting and locally applied products Biopharmaceutical Drug Disposition Classification System formulated as solutions with the same qualitative and (), taking into account that oxidative and conjugative quantitative composition, or with minor differences in exci- metabolites are formed only systemically after absorption.
pients, as long as it is justified that the minor differences do Although the guideline indicates that BE between a solid not alter the local availability of the drug and, therefore, oral dosage form and an oral solution is supportive, as it is therapeutic equivalence. Importantly, the guideline stresses indicative that absorption limitations due to the dosage form the need of comparative bioavailability studies with only a are negligible, it does not signal that absorption is complete.
superiority limit of 125.00% for safety reasons when there is a In such situation, dissolution similarity is less relevant for risk of systemic adverse reactions. This highlights that the class III drugs as BE between solid dosage forms and clinical point of view prevails in locally acting, locally applied solutions is generally more easily accomplished for low products as a quality approach would require BE within permeability drugs than for extremely permeable drugs.
80.00–125.00% since a safer product can be a different but As per the guideline, dissolution profiles should be not an interchangeable product. In the EU, the clinical compared at pH 1.2, 4.5, 6.8, and the pH of minimum demonstration of efficacy would prevail over pharmacokinet- solubility in more than one batch of test and reference ic differences, even if clinical endpoints are less sensitive than products. The agitation speed for these studies has been PK, because products are approved to be marketed, not to be defined as usually 50 rpm for the paddle and 100 rpm for the interchangeable. As mentioned earlier, interchangeablility is basket apparatus. There is no guidance on when a different a national policy which can be impaired by the way the speed would be acceptable. A different agitation speed, e.g., medicinal products are assessed and approved.
75 rpm with the paddle apparatus as recommended by WorldHealth Organization , is questionable since it would facilitate the demonstration of similarity.
Dissolution profiles must be similar and rapid (>85% in The main advancement of the EU guideline in the area 30 min) for class I drugs, and similar and very rapid (>85% in of BCS biowaivers is the acceptance of biowaivers not only 15 min) for class III drugs (). Although rapid dissolution is for class I drugs, which was mentioned in the previous less critical for some products containing class III drugs version, but also for class III drugs under strict conditions.
(perhaps not for those with an absorption window), the Although there are several differences in approach compared requirement of a very rapid release is to ensure that a with the US-FDA approach, like the US-FDA, narrow solution is emptied from the stomach and therefore it can be therapeutic index drugs are excluded and the biowaiver considered as similar to oral solutions.
policy only applies to products with the same immediate In the EU guideline, special attention is paid to release solid oral dosage forms (capsule vs. tablets is not excipients as excipients that may affect bioavailability have acceptable, although this is allowed by the definition of to be included in identical amounts in test and reference generic medicinal products in Directive 2001/83). Similarly, products. In contrast, the US-FDA asserts that large amount in spite of the fact that different ester, ethers, isomers, of surfactants or mannitol and sorbitol are necessary to alter mixtures of isomers, complexes, or derivatives of an active bioavailability (). However, experience in the EU has shown substance are considered to be the same active substance for that small amounts of surfactants (e.g., SLS) and sorbitol the EU definition of generic medicinal product, only different affect the bioavailability of drugs (e.g., 4 mg of SLS increases salts of class I drugs are acceptable for biowaivers.
five- to sixfold the bioavailability of alendronate, and 7 mg of Although the guideline states that it only applies to sorbitol decreases the Cmax of risperiodone with 60 mg also products with systemic action, the same scientific principles decreasing the AUC).
could be applicable to gastrointestinal locally acting products For class I drugs, excipients that are not known to affect (e.g., acarbose). In contrast, it is not applicable to systemically bioavailability can be different but, for class III drugs, even these acting products that are not absorbed in the gut (e.g., excipients have to be the same and in very similar amounts.
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Exogenous plant mir168a specifically targets mammalian ldlrap1: evidence of cross-kingdom regulation by microrna

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