(M1530-08-43) Photo-Oxidation Mechanisms in Liquid Formulations: Thinking beyond Peroxide and Free Radical Induced Degradation

Purpose: Oxidation is one of the most common degradation pathways for active pharmaceutical ingredients (APIs), involving 1-electron processes via peroxy radicals and 2-electron processes. In liquid formulations, several factors can impact oxidative instabilities including pH, excipient impurities, headspace oxygen and photo-oxidation. Photo-oxidation mechanisms can occur via peroxy radical and peroxide reactions (including those initiated by superoxide which is formed by transfer of energy to molecularly dissolved oxygen from a photosensitizer) as well as degradation by singlet oxygen, an electronically excited state of molecular oxygen generated by photosensitizers in the presence of light. The mechanisms of oxidation due to light exposure are more variable than the traditional peroxide and peroxy radical mechanisms, which have been well established. In this work, the mechanism of a particular oxidative degradation product of MK-1454 in a sterile solution formulation was evaluated to investigate the role of singlet oxygen in its degradation when exposed to UV light.

Methods: High performance liquid chromatography (HPLC) was used to assay MK-1454 and its oxidative degradation products as well as photo-oxidative degradate. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) was used for proposed structures of MK-1454 oxidative as well as photo-oxidative degradation products.

Results: MK-1454 is highly sensitive to hydrogen peroxide oxidation and is not significantly prone to free radical oxidation via 2,2’-Azobis(2-methylpropionitrile) [AIBN]. Under photo-stress conditions, a new photo-degradation product was observed in the MK-1454 formulation (Figure 1). To gain a fundamental understanding of singlet oxygen generation and its role in photo-degradation of MK-1454 in an injectable formulation, Rose Bengal (a well-known singlet oxygen generator) was used to interrogate this potential mechanism observed during photo-stress studies. To demonstrate production of singlet oxygen from Rose Bengal, furfuryl alcohol (FFA) was used as a model compound. The MK-1454 photo-degradation product (RRT 0.88) was observed under cool white light stress (32 kLux·hour) in the presence of Rose Bengal (Table 1). Dissolved oxygen measurements demonstrated a correlation between the decrease in dissolved oxygen after light exposure and the loss of MK-1454. A similar technique was used to evaluate the role of superoxide formed by UV stress of L-Tryptophan in the presence of MK-1454 and showed no significant growth of its photo-degradation product (Table 2).

Conclusion: These studies demonstrate the importance of considering alternative routes of photo-oxidation, such as singlet oxygen or superoxide, during formulation development and understanding photochemical degradation of APIs in liquid formulations. Although the photo-induced oxidation of MK-1454 is minimal without the presence of a photosensitizer, these studies highlight enhanced predictive tools and innovative analytical methods that can be used to elucidate the mechanism of degradation induced by exposure to light.


Figure 1. (A) Representative degradation of MK-1454 under AIBN and peroxide stress conditions and (B) Representative degradation of MK-1454 under 1.1x ICH Q1B light stress conditions.


Table 1. Summary of the production of singlet oxygen from irradiated Rose Bengal as measured by furfuryl alcohol (FFA) degradation and the degradation of MK-1454 by Rose Bengal irradiated by cool white light for 4 hours (32 kLux·hour). All values are represented as µM and are shown as Mean ± SD (N=5). Statistical comparisons between conditions were performed using a Two Sample T-Test.


Table 2. Summary of MK-1454 loss, individual and total degradation growth, and dissolved oxygen under UV light stress (720 Watt·hour/m2). All values are represented as µM and are shown as Mean ± SD (N=5). Statistical comparisons between conditions were performed using a Two Sample T-Test.