Organotypic cornea equivalents are used as in vitro models for permeation studies. Many ophthalmic drugs are applied as ester prodrugs to achieve a higher bioavailability. The esterase activity of three corneal human cell lines (epithelial, stromal, endothelial cells) as well as of excised porcine cornea, human donor cornea and human cornea construct (HCC) was investigated and compared. Esterase activity was determined using p-nitrophenyl acetate and hydrocortisone acetate (HCA) as esterase substrates. Hydrocortisone acetate permeation across porcine cornea, human donor cornea and HCC was studied in vitro using Franz-diffusion cells. Corneal epithelial cells showed the highest esterase activity and only small differences to keratocytes and endothelial cells were detectable. The permeation barrier properties of the different corneal tissues were very similar in the case of HCA permeation whereas HCA metabolism rates were in the ranking order of porcine cornea > HCC > human donor cornea. Permeation and metabolism studies indicate that the in vitro permeation model HCC is able to adequately convert hydrocortisone acetate to hydrocortisone.
Here we have presented a sensitive and selective LC-MS/MS method for the quantification of tyrphostin A9, which is a selective inhibitor for platelet derived growth factor receptor tyrosine kinase and has been investigated in vitro as a potent oxidative phosphorylation uncoupler. The murine analytical method was developed for three biological matrices: cell culture media, 3T3-L1 cell lysate, and murine plasma. For each matrix the limit of detection and the limit of quantification were found to be 0.5 ng/mL and 1.0 ng/mL, respectively. The range of standard curve for each matrix was 1.0–100 ng/mL, linearity was >0.99, and the precision and accuracy were within 20%. 3-(3,5-di-
tert
-butyl-4-hydroxyphenyl) propanoic acid was found to be the most suitable internal standard. The validated LC-MS/MS method was used to investigate stability and in vitro pharmacokinetics of tyrphostin A9. It was found that tyrphostin A9 is susceptible to hydrolysis, and the degradation product was identified as 3,5-di-
tert
-butyl-4-hydroxybenzaldehyde. Tyrphostin A9 was not stable in biological matrices, and the rate of its degradation in murine plasma was faster than that in cell culture media. In vitro pharmacokinetic studies revealed that tyrphostin A9 concentrations in the cell culture media declined in a bi-exponential manner and the concentrations inside the adipocytes remained constant, suggesting tyrphostin A9 has an intracellular binding site and is retained within the cell. The LC-MS/MS method presented here paves the way for further quantitative investigations involving tyrphostin A9.
The specific pathways, timescales, and dynamics driving the progression of fibrosis in NAFLD and NASH are not yet fully understood. Hence, a mechanistic model of the pathogenesis and treatment of fibrosis in NASH will necessarily have significant uncertainties. The rate of fibrosis progression and the heterogeneity of pathogenesis across patients are not thoroughly quantified. To address this problem, we have developed a continuous-time Markov chain model that is able to capture the heterogeneity of fibrosis progression observed in the clinic. We estimated the average time of disease progression through various stages of fibrosis using seven published clinical studies involving paired liver biopsies. Sensitivity analysis revealed therapeutic intervention at stage F1 or stage F2 results in greatest potential improvement in the average fibrosis scores for a typical patient cohort distribution. These results were in good agreement with a retrospective analysis of placebo-controlled pioglitazone clinical trials for the treatment of NAFLD and NASH. This model provides support for determining patient populations, duration, and potential successful endpoints for clinical trial design in the area of NAFLD and NASH.
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