Site-directed deuteration of dronedarone preserves cytochrome P4502J2 activity and mitigates its cardiac adverse effects in canine arrhythmic hearts.

Cytochrome P4502J2 (CYP2J2) metabolizes arachidonic acid (AA) to cardioprotective epoxyeicosatrienoic acids (EETs). Dronedarone, an antiarrhythmic drug prescribed for treatment of atrial fibrillation (AF) induces cardiac adverse effects (AEs) with poorly understood mechanisms. We previously demonstrated that dronedarone inactivates CYP2J2 potently and irreversibly, disrupts AA-EET pathway leading to cardiac mitochondrial toxicity rescuable via EET enrichment. In this study, we investigated if mitigation of CYP2J2 inhibition prevents dronedarone-induced cardiac AEs. We first synthesized a deuterated analogue of dronedarone (termed poyendarone) and demonstrated that it neither inactivates CYP2J2, disrupts AA-EETs metabolism nor causes cardiac mitochondrial toxicity in vitro. Our patch-clamp experiments demonstrated that pharmacoelectrophysiology of dronedarone is unaffected by deuteration. Next, we show that dronedarone treatment or CYP2J2 knockdown in spontaneously beating cardiomyocytes indicative of depleted CYP2J2 activity exacerbates beat-to-beat (BTB) variability reflective of proarrhythmic phenotype. In contrast, poyendarone treatment yields significantly lower BTB variability compared to dronedarone in cardiomyocytes indicative of preserved CYP2J2 activity. Importantly, poyendarone and dronedarone display similar antiarrhythmic properties in the canine model of persistent AF, while poyendarone substantially reduces beat-to-beat variability of repolarization duration suggestive of diminished proarrhythmic risk. Our findings prove that deuteration of dronedarone prevents CYP2J2 inactivation and mitigates dronedarone-induced cardiac AEs.

Investigations on detoxification mechanisms of novel para-phenylenediamine analogues through N-acetyltransferase 1 (NAT-1).

Para-phenylenediamine (PPD) is one of the most used chemicals in oxidative hair dyes. However, its use has been associated with adverse effects on health, including contact dermatitis and other systemic toxicities. Novel PPD derivatives have been proposed as a safer replacement for PPD. This can be achieved if these molecules minimally permeate the skin and/or are easily metabolised by enzymes in the skin (e.g., N-acetyltransferase-1 (NAT-1)) into innocuous compounds before gaining systemic entry. This study investigated the detoxification pathway mediated by NAT-1 enzymes on 6 synthesized PPD analogues (namely, P1-P6) with different chemical properties, to study the role of functional groups on detoxification mechanisms in HaCaT skin cells. These compounds were carefully designed with different chemical properties (whereby the ortho position of PPD was substituted by nucleophile and electrophile groups to promote N-acetylation reactions, metabolism and clearance). Compounds P2-P4 N-acetylated at 54-49 nmol/mg/min, which is 1.6 times higher than N-acetylation of PPD, upregulated NAT-1 activity from 8-7% at 50 µM to 22-11% at 100 µM and showed 4 times higher rate of elimination (k equal to 0.141 ± 0.016-0.124 ± 0.01 h-1) and 3 times faster rate of clearance (0.172 ± 0.007-0.158 ± 0.005 h-1mgprotein-1) than PPD (0.0316 ± 0.0019 h-1, 0.0576 ± 0.003 h-1mg protein-1, respectively). The data suggest that nucleophile substituted compounds detoxify at a faster rate than PPD. Our metabolic and detoxification mechanistic studies revealed significantly higher rates of N-acetylation, NAT-1 activity and higher detoxification of P2-P4 in keratinocytes, suggesting the importance of nucleophilic groups at the ortho position in PPD to reduce toxicity of aniline-based dyes on human skin cells.

Preliminary discovery of novel markers for human cell line activation test (h-CLAT).

The human cell line activation test (h-CLAT) is an OECD approved (Test No. 442E) assay to identify novel skin sensitizers. h-CLAT simulates dendritic cell activation in the skin sensitization pathway and is based on the measurement of CD54 and CD86 overexpression on monocytic, leukemic THP-1 cells. However, the current h-CLAT markers show inconsistent results with moderate and weak sensitizers. Moreover, these markers have accessory roles in cell adhesion and signaling rather than a direct role in cellular inflammation. Therefore, we have explored other inflammation-related markers in this study. PBMCs comprises a mixture of cells that resemble the complex immunological milieu in adults and were primarily used to identify markers. PBMCs (n = 10) and THP-1 cells were treated with 1-chloro-2,4-dinitrobenzene (DNCB, strong) and NiCl2 (Ni, moderate) sensitizers or DMSO (control) and incubated for 24 h. The samples were subjected to RNA sequencing to obtain log2fold change in gene expression. DNCB and NiCl2 significantly upregulated 80 genes in both cell types. Of these, CD109, CD181, CD183, CLEC5A, CLEC8A & CD354 were experimentally validated. DNCB and Ni but not isopropyl alcohol (non-sensitizer) significantly induced the expression of all novel markers except CLEC8A. Moreover, the percentage induction of all novel markers except CLEC8A satisfied the OECD acceptance criteria. In summary, we identified five novel markers that may supplement the current repertoire of h-CLAT markers.