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Introduction to BCS and its application
The oral bioavailability of a small molecule drug depends on the rate and extent of its absorption. These, in turn, are determined by drug properties such as solubility, permeability and formulation (i.e., presence of excipients or nano formulation). Determination of in vitro solubility and permeability is a good basis for predicting and understanding in vivo absorption, by the classification of a drug into 4 different classes of the Biopharmaceutics Classification System (BCS) :
Class 1: High Solubility – High Permeability
Class 2: Low Solubility – High Permeability
Class 3: High Solubility – Low Permeability
Class 4: Low Solubility – Low Permeability
Potential practical applications of the BCS have been studied extensively over the past decades, and it was adopted by regulatory agencies globally as a guiding principle for developing specific test requirements for specific drug classes. The BCS system currently has two major applications in the regulatory environment:
- can be used to justify inclusion or omission of certain assays (e.g., efflux transporter substrate assessment assays) as part of drug approval processes , or
- as reference for granting a waiver of clinical bioequivalence studies (biowaiver) .
The most common application of the BCS classification is for demonstrating bioequivalence, meaning a new drug is proven to have the same characteristics as a previously registered compound. This determination can lead to waiving the need for clinical studies and hence is a cost and time saving approach in drug development. Beyond the BCS class determination approach, other methods also exist for demonstrating bioequivalence, however, these are more costly, and time-consuming, thus less widespread.
The assay setup for BCS class determination should, however, strictly adhere to the latest guidance and be run on a characterized Caco-2 assay in order to be used in biowaiver applications. The ICH M9 harmonized international guidance issued in May 2021  describes globally accepted requirements, and has now replaced the previously independently existing FDA, EMA and WHO guidance documents for the same purpose.
Did you know?
Since the early 2000s, various regulatory bodies were developing their own guidance recommendations for biowaivers that also applied the BCS, including the FDA, the EMA and the WHO [4-6]. In order to facilitate international applications and consolidate the different biowaiver criteria, the ICH (International Council for Harmonization of technical requirements for pharmaceuticals for human use), started working in 2018 on the ICH M9 umbrella guidance for biowaiver submission, and released their updated final guidance in May 2021.
Since then, this harmonized ICH M9 guidance for “Biopharmaceutics classification system-based biowaivers” has been adopted by the major drug regulators such as the FDA and EMA and has replaced their previous, territory-based requirements. ICH M9-compliant submissions can therefore be internationally submitted and accepted without additional adjustments.
Setting up and results of SOLVO’s BCS calibrated Caco-2 assay
SOLVO’s fully validated Caco-2 assay is suitable for the determination of the permeability class of a compound in line with the BCS classification system in full agreement with the ICH M9 criteria . While in vitro assays are often used to predict in vivo permeability and bioavailability (fraction absorbed) of a compound, most systems, such as a PAMPA assay or non-validated Caco-2 systems only provide system-specific permeability values that are not reproducible across systems or correlated to in vivo, thus cannot be used for regulatory purposes.
To overcome this, in vitro permeability of a set of internal permeability standard drugs has been characterized in our Caco-2 system and plotted against their in vivo fraction absorbed resulting in a reliable regulatory compliant correlation of in vitro permeability to predict in vivo absorption. The 28 internal standard compounds have been selected based upon the guidance document and latest literature in the field. At least 5 compounds were chosen for low-, moderate- and high permeability ranges exceeding minimal regulatory requirements. As part of system characterization and validation, the observed in vitro permeability of these internal standards was plotted against the known human in vivo fraction absorbed to separate high- and low permeability groups.
Minoxidil was identified as the class boundary reference (Figure 1). In the permeability class determination assay, a test compound is tested in parallel with a selection of the previously characterized internal permeability standards, for which the results from each experiment needs to be within acceptance criteria to ensure data reliability. This experiment allows classification of the test compound along BCS criteria as either high- or low permeable based on direct comparison to the high-low permeability boundary cut-off compound, Minoxidil.
Figure 1 – Characterization of SOLVO’s Caco-2 assay with 28 reference compounds. Calculated Papp (A to B direction) at apical pH 6.5 and basolateral pH 7.4, or at apical pH 7.4 and basolateral pH 7.4 were plotted against human oral absorption data (Fa). BCS classification criterion for high permeability is shown as a dotted blue line. Orange triangle and red circle indicates minoxidil, which was chosen as a high permeability internal standard based on rank-order correlation.
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A recurring reason highlighted by regulatory agencies for rejecting submissions containing BCS data was the lack of bi-directional permeability data provided for the selected internal permeability standards . In contrast to previous versions of regulatory guidance documents, the updated ICH M9 guidance document now explicitly includes this criterion for assay characterization. In line with this, SOLVO’s permeability assay characterization report includes bi-directional permeability information on all internal standard compounds tested. This rigor is maintained in the study assay setups as well, where the selected permeability standards are also applied bidirectionally to prove high system reliability and data reproducibility.
SOLVO’s BCS permeability package for clients: approach and important considerations
SOLVO’s BCS Permeability classification package applies a stepwise approach, where test compound permeability is assessed in two separate experiments alongside an extensive selection of control experiments, as illustrated in Table 1. Prior to this, a set of supporting assays is also conducted, such as solubility testing and compound tolerability assessment in the Caco-2 system, to assure the right conditions are selected for the definite permeability determination.
|BCS Biowaiver Caco-2 package||Bioanalytical method development||Development of fully validated bioanalytical method.|
|Assay buffer solubility testing||Solubility testing in assay buffer at pH 6.5 and pH 7.4, in absence and presence of reference standard(s).|
|Caco-2 assay tolerability test||Assessing the tolerability of the Caco-2 cells to the selected assay concentrations of TA and identification of suitable conditions for BCS classification testing.|
|Preliminary unidirectional permeability test||Assessment of apical to basolateral permeability of test article in presence of controls as preliminary assessment.|
|Definite bi-directional permeability test||Assessment of apical to basolateral and basolateral to apical permeability of test article in presence of controls, including required selection of permeability reference standards and transporter functionality controls as definite assessment.|
|Reporting||Preparation of full report according to regulatory standards.|
|QC activity||Throughout the study, quality is monitored by a quality scientist and checklists reported.|
Table 1. – Short description of elements of SOLVO’s assay package for BCS Permeability class determination package.
Compound properties beyond permeability may also affect further study outcome, therefore our set of preliminary assays included with the package aims to ascertain the permeability assays are run at optimal conditions, tailored to best fit each individual compound. Selected characteristics, such as limited aqueous solubility, non-specific binding to plastic or cells or cytotoxic effects at certain concentrations or incubation times, may not necessarily make a compound un-classifiable, however, if not characterized and considered early on, they can affect assay outcomes and potentially lead to inconclusive results. For example, in case a test article has toxic effects to the Caco-2 cells in the assay system, the monolayer integrity may be compromised and overall higher permeability for all markers would be detected, in which case, the data cannot be used for classification. Addressing this in an early stage allows for a simple fix by adjusting the concentration range for further experiments. Such effects are tested at both apical pH levels applied in the experiments (pH 6.5 and pH 7.4) as well as in the presence and absence of the co-tested reference compounds. This data, taken together with any system-specific solubility limitations, is used for selecting the most optimal assay parameters for subsequent measurements. This ensures that the data obtained from permeability tests is high-quality and strongly reliable and can also help avoid unnecessary-repeat experiments, and the associated elevated costs and delays in study timelines.
Information provided on transporter activity and functionality in the in vitro systems used is crucial and has been consistently highlighted in regulatory feedback to biowaiver submissions. Insufficient data is often cited as reason for rejection. Therefore, as part of our BCS Permeability classification study, a set of controls are applied to confirm efflux transporters are functional in the assay system: efflux of control substrates for BCRP, MDR1 and MRP2 are tested in parallel with the test compound assessment, in the presence and absence of a reference inhibitor.
To be suitable for BCS classification, both the test compound and the set of reference permeability standards used need to be mainly passively permeable and not substrates to active (efflux) transport (Efflux ratio (ER) < 2). This also further highlights the importance of bi-directional permeability assessment for all compounds and references in order to obtain an ER value. Compounds that do not fulfil this criterion are likely not classifiable by the standard BCS approach. SOLVO’s stepwise study construction allows for early confirmation of the test compound as non-substrate for active transport, and subsequent, more complex permeability tests can be confidently conducted, while in case efflux is observed, it is possible to re-consider the next steps before the core assays are conducted.
All activities are also conducted with our Regulatory level quality control applied (including archiving) as default to ascertain all regulatory requirements are met.
 J. R. C. Gordon L. Amidon, Hans Lennernas, Vinod P. Shah, “A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability,” Pharm. Res., vol. 12, no. 3, pp. 413–420, 1995
 FDA, In Vitro Drug Interaction Studies — Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions Guidance for Industry, January 2020
 ICH, “M9 Biopharmaceutics Classification System- Based Biowaivers Guidance for Industry M9 Biopharmaceutics Classification System- Based Biowaivers Guidance for Industry,” no. May, 2021.
 FDA, Center for Drug Evaluation and Research. Guidance for Industry: Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System, Office of Training and Communications, Division of Communications Management, Drug Information Branch, HFD-210, Rockville, Maryland 20857. Vol. 2000 Food and Drug Administration; Rockville, MD: Aug, 2000.
 EMA, GUIDELINE ON THE INVESTIGATION OF BIOEQUIVALENCE, London, 20 January 2010
 WHO, Technical Report Series, No. 937, 2006 Annex 8 - Proposal to waive in vivo bioequivalence requirements for WHO Model List of Essential Medicines immediate-release, solid oral dosage forms, 2006
 A.K. Nair et al., „Statistics on BCS Classification of Generic Drug Products Approved Between 2000 and 2011 in the USA” The AAPS Journal, Vol. 14, No. 4, December 2012