FAQ – About the role of in vitro bioequivalence testing in the development of new drugs

In August 2023, the European Federation of Pharmaceutical Sciences (EUFEPS) and the American Association of Pharmaceutical Scientists (AAPS) successfully held the fifth Global Bioequivalence Harmonization Initiative (GBHI) conference, bringing together professionals from all over the world.  This meeting conducted an in-depth discussion on promoting the harmonization of global bioequivalence assessment standards.  The meeting provided a comprehensive discussion of the appropriateness and potential benefits of in vitro testing as an alternative to clinical efficacy studies, moving toward a unified standard for bioequivalence assessment.  The meeting aimed to find an optimal coordination strategy to increase consistent approval rates across agencies, minimize inconsistencies with past approved products, and reduce consumer risk.

Bioequivalence (BE) testing is of great significance to the development of new drugs.  The BE test is a method of studying bioavailability, using pharmacokinetic parameters as indicators, to compare different dosage forms of the same drug under the same test conditions and to evaluate whether there is a statistical difference in the degree and rate of absorption of its active ingredients in the human body.  The role of in vitro BE testing in the process of new drug development is reflected in the following aspects:

Using pharmacokinetics as an indicator to understand the release kinetics of drugs in the in vitro environment is a key factor in ensuring that the drug reaches a stable blood concentration in the patient’s body.  It helps scientific researchers better understand the release rate of drugs in different preparations, helps to formulate more precise dosing plans, and improves the controllability and effect of drug treatment.

For some drugs, such as phosphate binders or bile acid binders, in vitro studies such as binding equilibrium tests and binding kinetics tests can be used as effectiveness evaluation methods to replace the traditional pharmacokinetics research as the endpoint of human bioequivalence.

In terms of drug design, by simulating the in vitro environment, researchers can observe the release rate, absorption characteristics and possible metabolic pathways of the drug, providing strong support for drug design and optimization.  More efficient and convenient dosage forms can be designed to improve patient compliance.

By conducting in vitro BE testing at an early stage, potential problems can be discovered earlier that could reduce the risk of failure when entering the clinical trial stage, and save time and costs.

During many years of research services, Medicilon’s CMC department has established a “Solid Preparation and Liquid Preparation Evaluation Platform”, “Ophthalmology Evaluation Platform”, “Inhalation Preparation Evaluation Platform”, “Topical Preparation Evaluation Platform” and “In vitro BE Evaluation Platform”.  For drugs that have effects in the gastrointestinal tract and cannot be evaluated for in vivo equivalence, such as sevelamer carbonate tablets, and sucralfate suspension; Medicilon also has extensive experience in in vitro bioequivalence evaluation.  Medicilon’s Cloud Lecture Hall invited Xiaoyu Huang, Project Manager of the Preparation Department, as a guest to share her research experience in in vitro BE projects for gastrointestinal drugs.  The following is a collection of questions related to in vitro BE experiments during the live broadcast.  We hope this could help you to further understand the confusion related to bioequivalence experiments.

Q: Referring to the FDA guidance, how do you understand the binding kinetics study of cholestyramine in the FDA guidance, which uses 0.3 and 3mM bile salts and contains 0.1M sodium chloride?

A: This part is the operating requirements for cholestyramine binding kinetics in the FDA guidelines. Then, we need to follow the steps of the FDA guidelines in the experiment.  My personal understanding of this is this: The combined kinetic experiments are conducted under simulated human physiological conditions.  The concentration of bile salts in the normal human body is in the range of 0.3-3 grams/liter.  The practices in the guideline are based on the lowest concentration and the highest concentration of bile salts.

Because cholestyramine is a polymer quaternary ammonium salt type ion exchange resin, the area of action is in the gastrointestinal tract, and the presence of other anions may competitively affect the results of cholestyramine binding to bile salts.  The purpose of using 0.1M sodium chloride is to examine the binding of cholestyramine with different concentrations of bile salts at different times under simulated human physiological concentrations. (The normal concentration of chloride ion electrolyte in the human body is about 0.1M, so this also corresponds to the concentration of sodium chloride in the guideline being 0.1M.)

So why are chloride ions considered instead of other ions, such as phosphate ions?  Since this method is already required by FDA guidelines, it is speculated that it will only be adopted after some investigation and comparison with the impact of other anions.

Pharmaceutical research mainly includes APIs and preparation development, quality control and stability study, etc. Medicilon has a wide experience in pharmaceutical research services such as green enzyme chemistry, API quality research, insoluble innovative drug technology, and high-quality preparations.

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Q: What are the factors that affect pepsin activity detection?

A: The first thing to consider is the grade of enzyme that are purchasing.  Secondly, the effect of enzyme concentration on enzyme activity needs to be considered.  Regarding the rate of enzyme reaction and enzyme concentration, within a certain range, when the substrate molecules are sufficient, the greater the amount of enzyme, the faster the conversion rate of the substrate.  Other influencing factors are the concentration of the substrate and pH value. In addition, the detection method will also affect the results.

Q: Which module of the pharmaceutical information should the in vitro BE submission information be submitted to?

A: First of all, it should be noted that the in vitro BE information is not submitted under the pharmaceutical information module. The in vitro BE research information is submitted under the clinical 2.7 module, and is ultimately reviewed by the CDE clinical module examiner.  This was confirmed after we communicated with the CDE examiners.

There are currently no clear document requirements for the materials that need to be submitted, but according to my experience, at least the following are required: test method development, development reports and charts, methodology validation reports and charts under various pH conditions, and in vitro BE study protocol reports and charts, in addition, the technical guidelines for bioequivalence studies of lanthanum carbonate chewable tablets published by NMPA require the provision of data for each formulation unit in each trial, as well as the results of statistical analysis.  These are the information that I think are at least necessary.

Q: k1 is not used to calculate the 90%CI confidence interval, so what is the significance of k1?

A: Although the 90% confidence interval is calculated from the mean ratio of k2 and k1 is not involved in the calculation, the resulting value of k1 ultimately needs to be reported according to FDA guidance.  I understand that k1 is an affinity constant, related to the size of the force in the combination, and it is obtained by the ratio of the slope to the intercept of the linear equation.  Although it is not included in the 90% confidence interval calculation formula, as an affinity constant, its result value should fluctuate within a relatively fixed range, and the size and selection of this value may be related to the environmental or experimental parameters of the selected pH value.  There is a great correlation, that is, the result of k1 may have a certain indicative effect on the rationality of the test conditions.

Q: How many dissolved lines do in vitro BE required and does it have to be a distinction?

A: According to the FDA individual drug guidelines, not all in vitro BE studies of this type of drugs require dissolution studies.  In the currently available individual drug guidelines, dissolution studies are required for calcium acetate and lanthanum carbonate chewable tablets, and this dissolution study is in addition to the dissolution method specified in the quality standard (this method is used for stability and quality control determination), and the data also needs to be provided.  The specific parameter requirements for the dissolving method have been given in the FDA Individual Drug Guide.  We only need to carry out the work in accordance with this requirement and do not consider whether there is any distinction.