Drug stability is crucial in drug development since it affects the safety and efficiency of the drug product. Stability testing and identification of significant degradation products of the active pharmaceutical ingredient (API) are required during drug development. Long-term and accelerated stability testing is performed to provide an in-depth understanding of the stability of the active substance. Accelerated testing lasts 6 months and long-term testing lasts 12 months, which significantly prolongs the development of the drug product. Therefore, early prediction and identification of possible impurities in drug product is important in pharmaceutical development. Stress tests of the active substance can help in the early identification of possible degradation products, which helps to determine degradation pathways and intrinsic stability of the molecule. Stress tests also help to develop stability-indicating analytical methods. Before performing stress tests, we usually try to predict degradation products, using literature data and chemical knowledge. Increasingly, in silico tools that predict degradation products are used, which give us an initial picture of the stability of the molecule under different conditions. Such tools can also be helpful in identifying degradation products obtained in stress testing.
Venetoclax is a selective inhibitor of the anti-apoptotic B-cell lymphoma (Bcl)-2 protein, a first of its kind. It was approved for the treatment of patients with chronic lymphocytic leukemia in the United States in 2016. Because venetoclax is a relatively new active substance, not much is known about its stability in the literature and no stability-indicating analytical method for venetoclax could be found. As the only Bcl-2 protein inhibitor on the market, it has a structure that differs from other known active substances. Thus, the stability of the molecule is also more difficult to predict at first glance.
The purpose of our work was to determine the degradation products of venetoclax using a combined approach with in silico tools and stress tests, as well as to develop an analytical method for monitoring venetoclax and its degradation products. Additionally, we wanted to determine the degradation pathways and degradation chemistry of venetoclax based on the structures of degradation products. The aim was also to include the principles of Analytical Quality by Design (AQbD) in analytical method development, thus improve the robustness of the analytical method, and better understand the effects of critical method parameters on the critical method attributes.
In the introductory chapter, we presented venetoclax and its potentially broad therapeutic applicability in the form of a scientific review article covering the review of the scientific and patent literature. The chapter was expanded with a theoretical background of stability testing and AQbD.
The first chapter of the research work presents the development of a reverse-phase liquid chromatography stability-indicating analytical method for venetoclax using the AQbD principles. Stress samples of venetoclax were utilized for the development process. The development process of the analytical method was composed of initial one-factor-at-a-time (OFAT) experiments, definition of the analytical target profile (ATP), selection of critical method attributes (CMAs), risk assessment, identification of critical method parameters (CMPs), initial screening experiments, optimization, robustness testing, method optimal design region (MODR) determination, and a proposition of a control strategy. The method was qualified in the working point, where we confirmed the analytical target profile: the ability to determine venetoclax in the presence of its degradation products over a range of 80% to 120% of the target concentration with an accuracy of 100% ± 2% and repeatability ⡤ 2% RSD. The method we have developed is suitable for the determination of venetoclax in the presence of major degradation products obtained by stress tests and can be used to determine the stability of venetoclax.
In the second chapter of the research work, we described the implementation of stress tests, the isolation of major degradation products and the determination of the structures of the major degradation products. We hypothesized the main chemistry pathways of venetoclax degradation. We identified four major degradation products of venetoclax with the addition of a strong acid, three major degradation products of venetoclax with the addition of a strong base, and one degradation product of venetoclax formed by oxidation. In addition to the predicted hydrolysis of the N-acylsulfonamide bond, we determined decarboxylation of the acid fragment resulting from said hydrolysis, dimerization of venetoclax via a methylene bridge in the presence of dimethyl sulfoxide, cyclization of the o-nitroaniline moiety, substitution of the amine with a hydroxy group, and oxidation of nitrogen present in the piperazine ring moiety.
The third chapter covers in silico predictions of venetoclax degradation products. Degradation products were predicted under different experimental conditions using a software tool. We compared the results of experimentally obtained degradation products with the predicted ones. The in silico tool successfully predicted three out of the seven experimentally determined degradation products of venetoclax.
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