Abstract
A new chemical entity, which is a chiral indane dimer, PH46A, has been developed by our research group. As a clinical candidate. PH46A has recently completed Phase I clinical studies in man. Previously, during its pre-clinical development, in in vivo pre-clinical studies PH46A showed potent anti-inflammatory properties, which can be targeted at a range of diseases, including inflammatory bowel disease (IBD). To support the pre-clinical development of this drug candidate, we developed a LCMS/MS method for determining PH46 (the acid form of PH46A salt) in both dog and rat plasma using Compound 1 as internal standard (IS). Those species were selected for safety pharmacology and toxicology, as well as pharmacokinetics studies.The method was validated over the range 10−10000 ng/mL for both matrices and the linearity, accuracy, precision and specificity over this range were demonstrated to be acceptable. No significant matrix effects or carryover were observed for both PH46 and IS and recovery was consistent. PH46 was found to be stable in both dog and rat plasma under the test conditions, such as at room temperature for >24 h, through 3 freeze/thaw cycles, and at -20 °C for >1 month. PH46 and IS in dog and rat plasma extracts were also found to be stable in the autosampler against fresh standard extracts on re-injection after 143.5 h and 243.5 h, respectively at 4 °C. 10- and 100-fold dilutions with control matrix were found not to affect the performance of the assay. This method was successfully applied to a pharmacokinetic study in the dog. With the exception of one dog, 003 M, oral administration of PH46A in gelatine capsules was well tolerated at a dose level of 100 mg/kg. The highest Cmax was observed in animal 003 M. The rapid absorption and high plasma concentration observed for animal 003 M compared to the data for animals 001 M and 002 M may account for the sickness observed in this animal; however, the reasons for this have not been investigated.
Highlights
Our research group have developed a series of indane dimers and the biological activities of these molecules have been investigated [1,2,3,4,5,6]
The in vitro comparative metabolism study of PH46A that we recently published showed that PH46A metabolism in rat is most similar to human, and all putative human metabolites are present in rat, dog, mouse and cynomolgus monkey [11]
Quality control (QC) samples were prepared in control matrix from the primary PH46 stock solution in similar manner as calibration standards (CSs), to give QCs at 10 [Lower Limit of Quantification (LLOQ)], 25, 300 and 8000 ng/mL plasma concentrations and aliquots (50 L) of QCs were extracted for analysis
Summary
Our research group have developed a series of indane dimers and the biological activities of these molecules have been investigated [1,2,3,4,5,6]. Rat and dog were selected as appropriate and useful species for use in subsequent mandatory and pivotal GLP safety pharmacology and toxicology studies in order to guide the clinical development of PH46A. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. As part of the preclinical investigations, the characterisation of the pharmacokinetics (PK) of PH46A in the rat and the dog were studied To this end, this manuscript describes the development and validation of sensitive and specific LC–MS analytical methods for determining PH46 (the free acid form of PH46A salt) (Fig. 1) in dog and rat plasma according to the Food and Drug Administration (FDA) [13] and the European Medicines Agency (EMA) [14] guidelines. The method was subsequently applied to a PK study in male Beagle dogs to quantitate levels of PH46 and evaluate its oral bioavailability following oral administration of PH46A in capsule
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