Analysis of Non-linear Pharmacokinetics of P-Glycoprotein Substrates in a Microfluidic Device Using a Mathematical Model that Includes an Unstirred Water Layer (UWL) Compartment.

PURPOSE: The purpose of this research is to analyze non-linear pharmacokinetics of P-glycoprotein (P-gp) substrates in a cell based assay of a microfluidic device, which might be affected by hydrodynamic barrier (unstirred water layer, UWL). RESULTS: Apparent permeability (Papp) were obtained using non-P-gp substrates (propranolol, metoprolol, and atenolol) and P-gp substrates (quinidine and talinolol) in a commercially available microfluidic device, organoplate ® of Caco-2 cell based assay. The previous UWL resistance model was well fitted to Papp of static and flow condition by assuming UWL including and negligible condition, while P-gp substrates of higher passive permeability (quinidine) was apart from the fitting curve. The concentration dependent non-linear kinetics of P-gp substrates, quinidine and talinolol, was more analyzed in detail, and apparent Vmax discrepancy between static and flow assay condition in the quinidine assay was observed, while that was not observed in talinolol, the lower permeable substrate. Based on the experimental results, a mathematical model for P-gp substrates including UWL compartment on the previous 3-compartment model was developed, and it indicated that the apparent Vmax was variable along with the ratio between passive permeability and UWL permeability. CONCLUSIONS: The mathematical model adding UWL compartment well explained non-linear pharmacokinetics of apparent permeability of P-gp substrate in the microfluidic device. The model also has a potential to be applied to P-gp substrate permeability analysis in vivo.

Main contribution of UGT1A1 and CYP2C9 in the metabolism of UR-1102, a novel agent for the treatment of gout.

UR-1102, a novel uricosuric agent for treating gout, has been confirmed to exhibit a pharmacological effect in patients. We clarified its metabolic pathway, estimated the contribution of each metabolic enzyme, and assessed the impact of genetic polymorphisms using human in vitro materials. Glucuronide, sulfate and oxidative metabolites of UR-1102 were detected in human hepatocytes. The intrinsic clearance by glucuronidation or oxidation in human liver microsomes was comparable, but sulfation in the cytosol was much lower, indicating that the rank order of contribution was glucuronidation ≥ oxidation > sulfation. Recombinant UGT1A1 and UGT1A3 showed high glucuronidation of UR-1102. We took advantage of a difference in the inhibitory sensitivity of atazanavir to the UGT isoforms and estimated the fraction metabolised (fm) with UGT1A1 to be 70%. Studies using recombinant CYPs and CYP isoform-specific inhibitors showed that oxidation was mediated exclusively by CYP2C9. The effect of UGT1A1 and CYP2C9 inhibitors on UR-1102 metabolism in hepatocytes did not differ markedly between the wild type and variants.

A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development under the "Metabolites in Safety Testing" Regulatory Guidance.

Since the introduction of metabolites in safety testing (MIST) guidance by the Food and Drug Administration in 2008, major changes have occurred in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and nonclinical studies to generate this information. In this cross-industry review, we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radio chromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated, which has led to more frequent determination of human metabolite profiles from multiple ascending-dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. Together with technological advances, these events have led to a general shift of focus toward earlier human metabolism studies using high-resolution mass spectrometry and to a reduction in animal radiolabel absorption/distribution/metabolism/excretion studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industry.

A highly specific and sensitive quantification analysis of the sterols in silkworm larvae by high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

The biochemical quantification of sterols in insects has been difficult because only small amounts of tissues can be obtained from insect bodies and because sterol metabolites are structurally related. We have developed a highly specific and sensitive quantitative method for determining of the concentrations of seven sterols-7-dehydrocholesterol, desmosterol, cholesterol, ergosterol, campesterol, stigmasterol, and ß-sitosterol-using a high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC/APCI-MS/MS). The sterols were extracted from silkworm larval tissues using the Bligh and Dyer method and were analyzed using HPLC/APCI-MS/MS with selected reaction monitoring, using cholesterol-3,4-(13)C(2) as an internal standard. The detection limits of the method were between 12.1 and 259 fmol. The major sterol in most silkworm larval tissues was cholesterol, whereas only small quantities of the dietary sterols were detected. Thus, a simple, sensitive, and specific method was successfully developed for the quantification of the sterol concentrations in each tissue of an individual silkworm larva. This method will be a useful tool for investigating to molecular basis of sterol physiology in insects, facilitating the quantification of femtomole quantities of sterols in biological samples.

Cholesterol internalization and metabolism in insect prothoracic gland, a steroidogenic organ, via lipoproteins.

Dietary sterols including cholesterol and phytosterols are essential substrates for insect steroid hormone (ecdysteroid) synthesis in the prothoracic glands (PGs). In the silkworm Bombyx mori, one of the model species of insects, the steroidogenesis has been well demonstrated that cholesterol biotransformation into ecdysone in the PG cells. Because insects lack the ability to synthesize cellular sterol de novo, lipoprotein, lipophorin (Lp), has been thought to be the major cholesterol supply source; however, details of cholesterol behavior from Lp to the PG cells has not been analyzed till date. In this report, we developed Lp incorporation method using labeled cholesterols such as 22-NBD-cholesterol and cholesterol-25,26,26,26,27,27,27-d7 (cholesterol-d7), and analyzed the internalization and metabolism of cholesterol in PGs in vitro using the silkworm Bombyx mori. The internalization of cholesterol was visualized using 22-NBD-cholesterol. PGs showed an enriched cellular 22-NBD-cholesterol signal, which dissociated from the Lp localizing at the close area of cell membrane. The distribution pattern observed in the PGs was different from other tissues such as the brain, fat body, and Malpighian tubules, suggesting that the internalization of cholesterol in the PGs was distinct from other tissues. The metabolism of cholesterol was traced using LC-MS/MS methods to detect cholesterol-d7, 7-dehydrocholesterol-d7 (an expected intermediate metabolite), and the final product ecdysone-d6. 7-Dehydrocholesterol-d7 and ecdysone-d6 were detected in the PG culture incubated with labeled Lp, showing that the cholesterol of Lp was utilized for ecdysone synthesis in the PGs. Our results reveal the distinct behavior of cholesterol in the PGs, with the first direct evidence of biochemical fate of lipoprotein cholesterol in insect steroidogenic organ. This will aid in the understanding of the involvement of lipoprotein cholesterol in steroid hormone synthesis in insects.

The silkworm glutathione S-transferase gene noppera-bo is required for ecdysteroid biosynthesis and larval development.

Insect molting and metamorphosis are tightly controlled by ecdysteroids, which are important steroid hormones that are synthesized from dietary sterols in the prothoracic gland. One of the ecdysteroidogenic genes in the fruit fly Drosophila melanogaster is noppera-bo (nobo), also known as GSTe14, which encodes a member of the epsilon class of glutathione S-transferases. In D. melanogaster, nobo plays a crucial role in utilizing cholesterol via regulating its transport and/or metabolism in the prothoracic gland. However, it is still not known whether the orthologs of nobo from other insects are also involved in ecdysteroid biosynthesis via cholesterol transport and/or metabolism in the prothoracic gland. Here we report genetic evidence showing that the silkworm Bombyx mori ortholog of nobo (nobo-Bm; GSTe7) is essential for silkworm development. nobo-Bm is predominantly expressed in the prothoracic gland. To assess the functional importance of nobo-Bm, we generated a B. mori genetic mutant of nobo-Bm using TALEN-mediated genome editing. We show that loss of nobo-Bm function causes larval arrest and a glossy cuticle phenotype, which are rescued by the application of 20-hydroxyecdysone. Moreover, the prothoracic gland cells isolated from the nobo-Bm mutant exhibit an abnormal accumulation of 7-dehydrocholesterol, a cholesterol metabolite. These results suggest that the nobo family of glutathione S-transferases is essential for development and for the regulation of sterol utilization in the prothoracic gland in not only the Diptera but also the Lepidoptera. On the other hand, loss of nobo function mutants of D. melanogaster and B. mori abnormally accumulates different sterols, implying that the sterol utilization in the PG is somewhat different between these two insect species.

A Halloween gene noppera-bo encodes a glutathione S-transferase essential for ecdysteroid biosynthesis via regulating the behaviour of cholesterol in Drosophila.

In insects, the precise timing of moulting and metamorphosis is strictly guided by ecdysteroids that are synthesised from dietary cholesterol in the prothoracic gland (PG). In the past decade, several ecdysteroidogenic enzymes, some of which are encoded by the Halloween genes, have been identified and characterised. Here, we report a novel Halloween gene, noppera-bo (nobo), that encodes a member of the glutathione S-transferase family. nobo was identified as a gene that is predominantly expressed in the PG of the fruit fly Drosophila melanogaster. We generated a nobo knock-out mutant, which displayed embryonic lethality and a naked cuticle structure. These phenotypes are typical for Halloween mutants showing embryonic ecdysteroid deficiency. In addition, the PG-specific nobo knock-down larvae displayed an arrested phenotype and reduced 20-hydroxyecdysone (20E) titres. Importantly, both embryonic and larval phenotypes were rescued by the administration of 20E or cholesterol. We also confirm that PG cells in nobo loss-of-function larvae abnormally accumulate cholesterol. Considering that cholesterol is the most upstream material for ecdysteroid biosynthesis in the PG, our results raise the possibility that nobo plays a crucial role in regulating the behaviour of cholesterol in steroid biosynthesis in insects.