A Comparison of Calcium Aggregation and Ultracentrifugation Methods for the Preparation of Rat Brain Microsomes for Drug Metabolism Studies

Preparation of brain microsomes by the calcium chloride aggregation method has been suggested as an alternative to the ultracentrifugation method. However, the effects of the calcium chloride concentration on the quality of the microsomal fractions are not known. Brain microsomes were prepared from the adult rat brains using the high-speed ultracentrifugation and low-speed calcium chloride (10–100 mM) aggregation methods (<i>n</i> = 5–6 per group). The microsomal protein yield (spectrometry), the cytochrome P450 reductase (CPR) activity (spectrometry), and the monooxygenase activities (UPLC-MS/MS) of CYP2D and CYP2E1 were determined in the obtained fractions. Increasing the concentrations of calcium chloride progressively increased the protein yield of the low-speed microsomal fractions. However, the increased yield was associated with a significant decrease in the activities of CPR, CYP2D, and CYP2E1. Additionally, the CYP2D and CYP2E1 activities were significantly correlated with the CPR activities of the fractions. In conclusion, when an ultracentrifuge is available, preparation of brain microsomes by the ultracentrifugation method might be preferable. However, the calcium aggregation method at a calcium chloride concentration of 10 mM is an acceptable alternative to the ultracentrifuge method.

HEPATOPROTECTIVЕ EFFECT OF THE THOROUGHWAX EXTRACT IN EXPERIMENTAL TETRACYCLINE-INDUCED HEPATITIS

В последние годы возросла распространённость лекарственных поражений печени. В профилактике и лечении таких заболеваний актуальным считают применение препаратов растительного происхождения. Перспективным объектом для разработки таких препаратов является растение володушка золотистая. В ФГБНУ ВИЛАР разработан и стандартизирован сухой экстракт травы володушки золотистой. В составе экстракта содержатся флавоноиды, сапонины, полисахариды, дубильные вещества катехиновой природы. Содержание фенольных соединений в пересчете на рутин составляет не менее 6%. Цель - оценка гепатозащитного действия экстракта володушки при экспериментальном тетрациклиновом гепатите. Методика. Исследовано влияние экстракта володушки (Bupleurum aureum Fisch.) на состояние печени крыс в условиях экспериментального тетрациклинового гепатита в сравнении с известным растительным лекарственным препаратом гепатопротекторного действия Силимаром. Гепатопротекторные и детоксицирующие свойства экстракта володушки оценивали по изменению содержания микросомального белка и цитохрома Р450 в микросомальных фракциях печени экспериментальных групп животных, а также по изменению активности ключевых ферментов системы детоксикации микросом печени, катализирующих ферментативные реакции: цитохрома Р450 и глутатионтрансферазы. Результаты. Установлено, что экстракт володушки в экспериментально-терапевтической дозе (100 мг/кг) обладает статистически значимыми гепатопротекторными и детоксицирующими свойствами, оказывает существенный эффект на активность ферментов микросомальной системы детоксикации (цитохром Р450 и глутатионтрансферазу) в печени крыс. Заключение. Экстракт володушки является перспективным объектом для создания новых высокоэффективных лекарственных средств растительного происхождения для профилактики и лечения лекарственных поражений печени. Prevalence of drug-induced hepatic disorders has greatly increased during recent years, and the use of herbal remedies is considered relevant for their prevention and treatment. A promising object for development of such drugs is thoroughwax (Bupleurum aureum). A dry extract of this herb was developed and standardized at the National Research Institute of Therapeutic and Aromatic Plants. The extract contains flavonoids, saponins, polysaccharides, and tanning substances of the catechine group. The content of phenolic substances is not less than 6% in rutin equivalents. Aim. To study hepatoprotective effects of the thoroughwax (Bupleurum aureum Fisch.) extract in rats with experimental tetracycline-induced hepatitis as compared to a well-known herbal hepatoprotective agent, Silimar. Hepatoprotective and detoxifying properties of the thoroughwax extract were assessed by changes in concentrations of microsomal protein and cytochrome P450 in hepatic microsomal fractions and changes in activities of key enzymes of the hepatic microsomal detoxifying system, which catalyzes enzyme reactions of cytochrome P450 and glutathione transferase. Results. The thoroughwax extract at an experimental therapeutic dose of 100 mg/kg provided significant hepatoprotective and detoxifying effects and induced a strong activation of the microsomal detoxifying enzymes, cytochrome P450 and glutathione transferase, in the rat liver. The thoroughwax extract is promising for development of new, effective herbal drugs for prevention and treatment of drug-induced hepatic disorders.

Pharmacokinetics of metformin in the rat: assessment of the effect of hyperlipidemia and evidence for its metabolism to guanylurea

Metformin pharmacokinetics are highly dependent upon organic cationic transporters. There is evidence of a change in its renal clearance in hyperlipidemic obese patients, and no information on its metabolic fate. To study some of these aspects, the influence of poloxamer 407 (P407)-induced hyperlipidemia on metformin pharmacokinetics was assessed. Control and P407-treated adult male rats were administered 30 mg/kg metformin intravenously (i.v.). The pharmacokinetic assessments were performed at 2 time points, 36 and 108 h, following the intraperitoneal dose of P407 (1 g/kg). mRNA and protein expressions of cationic drug transporters were also measured. There was no evidence of a change in metformin pharmacokinetics after i.v. doses as a consequence of short-term hyperlipidemia, and a change in transporter mRNA but not protein expression was observed in the P407- treated rats 108 h after P407 injection. Urinary recovery of unchanged drug was high (>90%) but incomplete. Presumed metabolite peaks were detected in chromatograms of hepatocytes and microsomal protein spiked with metformin. Comparative chromatographic elution times and mass spectra suggested that one of the predominant metabolites was guanylurea. Hyperlipidemia by itself did not affect the pharmacokinetics of metformin. Guanylurea is a putative metabolite of metformin in rats.

Equine 5α-reductase activity and expression in epididymis

The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies.

Ghrelin Octanoylation Is Completely Stabilized in Biological Samples by Alkyl Fluorophosphonates

Ghrelin is a peptide hormone involved in multiple physiological processes related to energy homeostasis. This hormone features a unique posttranslational serine octanoylation modification catalyzed by the enzyme ghrelin O-acyltransferase, with serine octanoylation essential for ghrelin to bind and activate its cognate receptor. Ghrelin deacylation rapidly occurs in circulation, with both ghrelin and desacyl ghrelin playing important roles in biological signaling. Understanding the regulation and physiological impact of ghrelin signaling requires the ability to rapidly protect ghrelin from deacylation in biological samples such as blood serum or cell lysates to preserve the relative concentrations of ghrelin and desacyl ghrelin. In in vitro ghrelin O-acyltransferase activity assays using insect microsomal protein fractions and mammalian cell lysate and blood serum, we demonstrate that alkyl fluorophosphonate treatment provides rapid, complete, and long-lasting protection of ghrelin acylation against serine ester hydrolysis without interference in enzyme assay or ELISA analysis. Our results support alkyl fluorophosphonate treatment as a general tool for stabilizing ghrelin and improving measurement of ghrelin and desacyl ghrelin concentrations in biochemical and clinical investigations and suggest current estimates for active ghrelin concentration and the ghrelin to desacyl ghrelin ratio in circulation may underestimate in vivo conditions.

Identification of a novel putative interaction partner of the nucleoporin ALADIN

It has been shown that the nucleoporin ALADIN employs a significant role in the redox homeostasis of the cell but the function in steroidogenesis contributing to adrenal atrophy in triple A syndrome remains largely unknown. In an attempt to identify new interaction partners of ALADIN, co-immunoprecipitation followed by proteome analysis was conducted in different expression models using the human adrenocortical tumour cell line NCI-H295R. Our results suggest an interaction of ALADIN with the microsomal protein PGRMC2. PGRMC2 is shown to be activity regulator of CYP P450 enzymes and therefore, to be a possible target for adrenal dysregulation in triple A syndrome. We show that there is a sexual dimorphism regarding the expression of Pgrmc2 in adrenals and gonads of WT and Aaas KO mice. Female Aaas KO mice are sterile due to delayed oocyte maturation and meiotic spindle assembly. A participation in meiotic spindle assembly confirms the recently investigated involvement of ALADIN in mitosis and emphasises an interaction with PGRMC2 which is a regulator of cell cycle. By identification of a novel interaction partner of ALADIN we provide novel aspects for future research of the function of ALADIN during cell cycle and for new insights into the pathogenesis of triple A syndrome.

MAPPING IN VITRO AND IN VIVO DERIVED CYP2C9 AND CYP2C19 ONTOGENY FUNCTIONS: A CRITICAL COMPARISON BETWEEN VARIOUS ONTOGENY MODELS

In vivo derived ontogeny profiles for CYP1A2 and CYP3A4, show improved clearance (CL) predictions within a paediatric physiologically based pharmacokinetic (p-PBPK) model1. The aim of this study is to derive ontogeny functions (OF) for CYP2C9 and CYP2C19 based on age related CL data on ibuprofen and pantoprazole & lansoprazole, respectively.A literature review was undertaken to collect age related CL data for these probes, the values were deconvoluted back to intrinsic CL values (per mg of liver microsomal protein) as described previously. The 'best-fit' algorithm for ratio of paediatric to mean adult intrinsic CL with age was determined in Graphpad Prism5 to obtain in vivo OF for CYP2C9 and CYP2C19. These were compared for performance with previously established ‘in vitro' OF in Simcyp Paediatric simulator (v14) using validation datasets.CYP2C9 and CYP2C19 enzyme activities showed an increase with age to values higher than adults by ages 2 and 1 month respectively, maximum values were reached at 1.5 years and 6 months, respectively before declining to typical adult levels by around 25 years.The CYP2C9 in vivo derived OF led to improved predictions of diclofenac and S-Warfarin CL compared to in vitro derived OF across the age range. For CYP2C19 there is a dearth of suitable validation compounds due to lack of clinical data with a possibility of using omeprazole or voriconazole. The reasons for discrepancy between in vitro and in vivo derived OF require further investigation.