Pharmaceutical Forced Degradation (Stress Testing) Endpoints: A Scientific Rationale and Industry Perspective.

Forced degradation (i.e., stress testing) of small molecule drug substances and products is a critical part of the drug development process, providing insight into the intrinsic stability of a drug that is foundational to the development and validation of stability-indicating analytical methods. There is a lack of clarity in the scientific literature and regulatory guidance as to what constitutes an "appropriate" endpoint to a set of stress experiments. That is, there is no clear agreement regarding how to determine if a sample has been sufficiently stressed. Notably, it is unclear what represents a suitable justification for declaring a drug substance (DS) or drug product (DP) "stable" to a specific forced degradation condition. To address these concerns and to ensure all pharmaceutically-relevant, potential degradation pathways have been suitably evaluated, we introduce a two-endpoint classification designation supported by experimental data. These two endpoints are 1) a % total degradation target outcome (e.g., for "reactive" drugs) or, 2) a specified amount of stress, even in the absence of any degradation (e.g., for "stable" drugs). These recommended endpoints are based on a review of the scientific literature, regulatory guidance, and a forced degradation data set from ten global pharmaceutical companies. The experimental data set, derived from the Campbell et al. (2022) benchmarking study,1 provides justification for the recommendations. Herein we provide a single source reference for small molecule DS and DP forced degradation stress conditions and endpoint best practices to support regulatory submissions (e.g., marketing applications). Application of these forced degradation conditions and endpoints, as part of a well-designed, comprehensive and a sufficiently rigorous study plan that includes both the DS and DP, provides comprehensive coverage of pharmaceutically-relevant degradation and avoids unreasonably extreme stress conditions and drastic endpoint recommendations sometimes found in the literature.

Assessing the Relevance of Solution Phase Stress Testing of Solid Dosage Form Drug Products: A Cross-Industry Benchmarking Study.

Stress testing (also known as forced degradation) of pharmaceutical products has long been recognized as a critical part of the drug development process, providing foundational information related to intrinsic stability characteristics and to the development of stability-indicating analytical methods. A benchmarking study was undertaken by nine pharmaceutical companies and the Brazilian Health Regulatory Agency (Agência Nacional de Vigilância Sanitária, or ANVISA) with a goal of understanding the utility of various stress testing conditions for producing pharmaceutically-relevant chemical degradation of drugs. Special consideration was given to determining whether solution phase stress testing of solid drug products produced degradation products that were both unique when compared to other stress conditions and relevant to the formal drug product stability data. The results from studies of 62 solid dosage form drug products were compiled.  A total of 387 degradation products were reported as being observed in stress testing studies, along with 173 degradation products observed in accelerated and/or long-term stability studies for the 62 drug products.  Among these, 25 of the stress testing degradation products were unique to the solution phase stress testing of the drug products; however, none of these unique degradation products were relevant to the formal stability data. The relevant degradation products were sufficiently accounted for by stress testing studies that included only drug substance stressing (in solution and in the solid state) and drug product stressing (in the solid state). Based on these results, it is the opinion of the authors that for solid dosage form drug products, well-designed stress testing studies need not include solution phase stress testing of the drug product in order to be comprehensive.

"Dark" Singlet Oxygen and Electron Paramagnetic Resonance Spin Trapping as Convenient Tools to Assess Photolytic Drug Degradation.

Forced degradation studies are an important tool for a systematic assessment of decomposition pathways and identification of reactive sites in active pharmaceutical ingredients (APIs). Two methodologies have been combined in order to provide a deeper understanding of singlet oxygen-related degradation pathways of APIs under light irradiation. First, we report that a "dark" singlet oxygen test enables the investigation of drug reactivity toward singlet oxygen independently of photolytic irradiation processes. Second, the photosensitizing properties of the API producing the singlet oxygen was proven and quantified by spin trapping and electron paramagnetic resonance analysis. A combination of these techniques is an interesting addition to the forced degradation portfolio as it can be used for (1) revealing unexpected degradation pathways of APIs due to singlet oxygen, (2) clarifying photolytic drug-drug interactions in fixed-dose combinations, and (3) synthesizing larger quantities of hardly accessible oxidative drug degradants.

"Base effect" in the auto-tandem palladium-catalyzed synthesis of amino-substituted 1-methyl-1H-α-carbolines.

An auto-tandem Pd-catalyzed process consisting of an intramolecular direct arylation and an intermolecular Buchwald-Hartwig reaction for C-ring amino-substituted 1-methyl-1H-α-carboline synthesis has been developed. A mechanistic study of the direct arylation reaction revealed a rate effect of the inorganic base on the C-H activation step ("base effect"). The amines, reagents in the tandem protocol, appear to have a similar effect on the direct arylation.

Photochemical stability of 4'-azido-2'-deoxy-2'-methylcytidine hydrochloride: structural elucidation of major degradation products by LC-MS and NMR analysis.

The photochemical stability of (1'R,2'S,3'S,4'R)-4'-azido-2'-deoxy-2'-methylcytidine hydrochloride, a new anti-HCV agent, was investigated. Aqueous solutions and bulk drug powder of the drug candidate were exposed to UV-visible light, complying with ICH requirements. The nucleoside analog decomposed via loss of nitrogen to yield products derived from a highly reactive azide intermediate. Major photolysis products were identified by LC-MS and NMR analysis, revealing three main photodegradation pathways. The first one led to the formation of a ring-expanded imidate ester. The other degradation pathways involved exocyclic or endocyclic bond cleavage with imine or imino lactone formation. The latter were prone to rapid hydrolysis, eventually resulting in the release of cytosine, 2-methyl malonaldehyde and (E)-cytosyl-2-methylpropenal.

Structure-activity relationship of antiparasitic and cytotoxic indoloquinoline alkaloids, and their tricyclic and bicyclic analogues.

Based on the indoloquinoline alkaloids cryptolepine (1), neocryptolepine (2), isocryptolepine (3) and isoneocryptolepine (4), used as lead compounds for new antimalarial agents, a series of tricyclic and bicyclic analogues, including carbolines, azaindoles, pyrroloquinolines and pyrroloisoquinolines was synthesized and biologically evaluated. None of the bicyclic compounds was significantly active against the chloroquine-resistant strain Plasmodium falciparum K1, in contrast to the tricyclic derivatives. The tricyclic compound 2-methyl-2H-pyrido[3,4-b]indole (9), or 2-methyl-beta-carboline, showed the best in vitro activity, with an IC(50) value of 0.45 microM against P. falciparum K1, without apparent cytotoxicity against L6 cells (SI>1000). However, this compound was not active in the Plasmodium berghei mouse model. Structure-activity relationships are discussed and compared with related naturally occurring compounds.

Synthesis and antiplasmodial activity of aminoalkylamino-substituted neocryptolepine derivatives.

A series of chloro- and aminoalkylamino-substituted neocryptolepine (5-methyl-5H-indolo[2,3-b]quinoline) derivatives were synthesized and evaluated as antiplasmodial agents. The evaluation also included cytotoxicity (MRC5 cells), inhibition of beta-hematin formation, and DNA interactions (DNA-methyl green assay). Introduction of aminoalkylamino chains increased the antiplasmodial activity of the neocryptolepine core substantially. The most efficient compounds showed antiplasmodial activities in the nanomolar range. N(1),N(1)-Diethyl-N(4)-(5-methyl-5H-indolo[2,3-b]quinolin-8-yl)pentane-1,4-diamine 11c showed an IC(50) of 0.01 microM and a selectivity index of 1800.

Structural and solvent effects on the 13C and 15N NMR chemical shifts of indoloquinoline alkaloids: experimental and DFT study.

Indoloquinoline alkaloids represent an important class of antimalarial, antibacterial and antiviral compounds. They have been shown to bind to DNA via intercalation preferentially at GC-rich sequences containing nonalternating CC sites. The stability of complexes formed with biological macromolecules depends on noncovalent binding. In the present study, the ability of indoloquinolines to form intermolecular interactions with solvents was investigated by using NMR spectroscopy and density functional theory (DFT) (B3LYP/6-31G**) calculations. NMR data measured for indoloquinoline bases and the corresponding hydrochlorides are discussed in relation to the structure. DFT calculations of shielding constants in vacuo and in solution allowed the investigation of the influence of the environment on the NMR parameters. Calculations incorporating solvent effects indicated significant changes in the anisotropy of the electron distribution, reflected in the span of the chemical shielding tensor (Omega = sigma11 - sigma33). Solvent effects on the span of the 13C and 15N shielding tensor depended on the type of atom and the data indicated a significant influence of solute-solvent interactions.

Isoneocryptolepine, a synthetic indoloquinoline alkaloid, as an antiplasmodial lead compound.

The antiprotozoal activities of three naturally occurring isomeric indoloquinoline alkaloids, i.e., cryptolepine (1), neocryptolepine (2), and isocryptolepine (3), and two dimeric indoloquinoline alkaloids, cryptoquindoline (6) and biscryptolepine (7), originally obtained from the plant Cryptolepis sanguinolenta, were compared with those of a new synthetic indoloquinoline isomer, isoneocryptolepine (4), and a quaternary derivative, N-methyl-isocryptolepinium iodide (5). The latter compounds showed a high antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum strain K1 (IC50 of 0.23 +/- 0.04 and 0.017 +/- 0.004 microM, respectively), while the cytotoxicity (L6 cells) was 4.32 +/- 0.04 and 12.7 +/- 2.0 microM, respectively. Isoneocryptolepine (4) was found to act as an inhibitor of beta-hematin formation and as a DNA-intercalating agent.