Modulation of Enzyme-Catalyzed Synthesis of Prostaglandins by Components Contained in Kidney Microsomal Preparations

In the kidney, prostaglandins formed by cyclooxygenase 1 and 2 (COX-1 and COX-2) play an important role in regulating renal blood flow. In the present study, we report our observations regarding a unique modulatory effect of renal microsomal preparation on COX-1/2-mediated formation of major prostaglandin (PG) products in vitro. We found that microsomes prepared from pig and rat kidneys had a dual stimulatory–inhibitory effect on the formation of certain PG products catalyzed by COX-1 and COX-2. At lower concentrations, kidney microsomes stimulated the formation of certain PG products, whereas at higher concentrations, their presence inhibited the formation. Presence of kidney microsomes consistently increased the Km values of the COX-1/2-mediated reactions, while the Vmax might be increased or decreased depending on stimulation or inhibition observed. Experimental evidence was presented to show that a protein component present in the pig kidney microsomes was primarily responsible for the activation of the enzyme-catalyzed arachidonic acid metabolism leading to the formation of certain PG products.

Identification and Quantification of MIDD0301 metabolites

Background: MIDD0301 is an oral asthma drug candidate that binds GABAA receptors on airway smooth muscle and immune cells. Objective: The objective of this study is to identify and quantify MIDD0301 metabolites in vitro and in vivo and determine the pharmacokinetics of oral, IP, and IV administrated MIDD0301. Methods: In vitro conversion of MIDD0301 was performed using liver and kidney microsomes/S9 fractions followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method was developed using synthesized standards to quantify MIDD0301 and its metabolites in urine and feces. Blood, lung, and brain were harvested from animals that received MIDD0301 by oral, IP, and IV administration, followed by LCMS/MS quantification. Imaging mass spectrometry was used to demonstrate the presence of MIDD0301 in the lung after oral administration. Results: MIDD0301 is stable in the presence of liver and kidney microsomes and S9 fractions for at least two hours. MIDD0301 undergoes conversion to the corresponding glucuronide and glucoside in the presence of conjugating cofactors. For IP and IV administration, unconjugated MIDD0301 together with significant amounts of MIDD0301 glucoside and MIDD0301 taurine were found in urine and feces. Less conjugation was observed following oral administration, with MIDD0301 glucuronide being the main metabolite. Pharmacokinetic quantification of MIDD0301 in blood, lung, and brain showed very low levels of MIDD0301 in the brain after oral, IV, or IP administration. The drug half-life in these tissues ranged between 4-6 hours for IP and oral and 1-2 hours for IV administration. Imaging mass spectrometry demonstrated that orally administered MIDD0301 distributes uniformly in the lung parenchyma. Conclusion: MIDD0301 undergoes no phase I and moderate phase II metabolism.

AUTOIMMUNE MARKERS IN MEASLES INFECTION OF VARYING SEVERITY

Measles is an acute viral infectious disease caused by an RNA-containing virus of Morbilliviridae genus. This infectious disease is characterized by the development of catarrhal syndrome, general intoxication syndrome and specific exanthema. The virus is transmitted from person to person by airborne mechanism, when coughing, sneezing and exhaling air droplets during conversation. It is shown that measles is characterized by long-term immunosuppression, which persists for a long period after the patient's clinical recovery and normalization of blood parameters that can lead to an exacerbation of various chronic and autoimmune diseases. Taking into account the impairment of cytokine and immunological reactivity observed in adult patients with measles infection, violations of the dynamics of interferon levels elevation and immunosuppression caused by the virus itself, as well as the lack of studies on autoantibody levels in patients with measles of varying severity, investigating autoimmune markers is of great relevance as can be used to predict and prevent the development and activation of autoimmune responses. The purpose of the study is to determine the involvement of autoimmune reactions in the immunopathogenesis of measles infection in individuals with various degrees of severity of the disease. The study included 65 patients with a diagnosis of measles who were treated at the Kharkiv region clinical infectious hospital in the period for 2017 – 2019. Depending on the severity of the disease and the presence of complications the patients included in the study were divided into 4 groups. The control group consisted of 20 voluntary donors who at the time of blood sampling had no clinical signs of measles, had no contact with infected patients, and had no exclusion criteria. All patients included in the study were tested for serum levels of antibodies to cell membrane phospholipids, IgG to liver and kidney microsomes (anti-LKM-1), and IgG to native DNA (ADNA 2) on the 1st and 10th day of hospital stay. These indicators were assessed by the method of solid-phase enzyme immunoassay (ELISA) using test systems manufactured by “Granum” LLC (Ukraine), EUROIMMUNE (Germany) and BioRad (USA). Statistical processing of the obtained results was carried out using the Statistica 6.0 software. When interpreting the significance of the difference in results, the Student's criterion was used; the critical value of the significance level was considered to be p ˂ 0.05. Results and discussion. It was found that on the day of admission to the hospital there was an increase in the level of autoantibodies to native DNA, liver and kidney microsomes, as well as cell membrane phospholipids in all groups of patients. In dynamics (on the 10th day of hospital stay), the patients of groups 1-4 demonstrated a decrease in the level of all types of autoantibodies; in the patients who had a severe course of the disease and developed complications development, the levels was higher than in the control group. The obtained data indicate that with the development of measles infection against the background of activation of general and specific immune response, there is an activation of autoimmune reactions, whose activity fades as the infectious process goes away. Autoimmune reactions under a favourable clinical course of measles infection, most likely, are not aggressive but protective by their nature and are aimed at eliminating infected and damaged cells. Patients with measles of varying severity have increased levels of autoantibodies to native DNA, liver and kidney microsomes, as well as cell membrane phospholipids. It has been found out that the levels of autoantibodies do not go beyond the reference values, but in severe disease, borderline values of autoantibodies are recorded in a significant number of patients. The study of autoantibody levels can be recommended in cases of severe measles infection, both with and without complications, to predict and prevent the development of autoimmune pathology.

Characterizations of Human UDP-Glucuronosyltransferase Enzymes in the Conjugation of p-Cresol

p-Cresol is a uremic toxin that is formed by intestinal microbiota and extensively conjugated by first-pass metabolism. p-Cresol glucuronide exerts various forms of cellular toxicity in vitro and is accumulated in the plasma of subjects with kidney disease, where associations with adverse cardiovascular and renal outcomes are evident. The objective of this study was to determine the contributions of human UDP-glucuronosyltransferase (UGT) enzymes in the formation of p-cresol glucuronide. Utilizing commonly expressed hepatic or renal human recombinant UGTs (ie, hrUGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17), hrUGT1A6 and hrUGT1A9 exhibited the highest catalytic activities in the generation of p-cresol glucuronide. The kinetics of p-cresol glucuronide formation in hrUGT1A6 and pooled human liver microsomes were best described by the Hill equation and in hrUGT1A9 and pooled human kidney microsomes by substrate inhibition. Using inhibitory and selective UGT inhibitors (ie, acetaminophen or amentoflavone for UGT1A6 and niflumic acid for UGT1A9), UGT1A6 was identified the predominant enzyme responsible for p-cresol glucuronide production in pooled human liver (78.4%–81.3% contribution) and kidney (54.3%–62.9%) microsomes, whereas UGT1A9 provided minor contributions (2.8% and 35.5%, respectively). The relative contributions of UGT1A6 (72.6 ± 11.3%, mean ± SD) and UGT1A9 (5.7 ± 4.1%) in individual human liver microsomes from 12 adult donors were highly variable, where an inverse association (R = −.784, p = .003) between UGT1A6 contribution and UGT1A9 probe substrate activity (ie, mycophenolic acid) was evident. Our novel findings provide valuable tools for conducting further mechanistic studies and for designing clinical interventions to mitigate the toxicities associated with p-cresol glucuronide.

Urinary Elimination of Bile Acid Glucuronides under Severe Cholestatic Situations: Contribution of Hepatic and Renal Glucuronidation Reactions

Biliary obstruction, a severe cholestatic complication, causes accumulation of toxic bile acids (BAs) in liver cells. Glucuronidation, catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, detoxifies cholestatic BAs. Using liquid chromatography coupled to tandem mass spectrometry, 11 BA glucuronide (-G) species were quantified in prebiliary and postbiliary stenting serum and urine samples from 17 patients with biliary obstruction. Stenting caused glucuronide- and fluid-specific changes in BA-G levels and BA-G/BA metabolic ratios. In vitro glucuronidation assays with human liver and kidney microsomes revealed that even if renal enzymes generally displayed lower KM values, the two tissues shared similar glucuronidation capacities for BAs. By contrast, major differences between the two tissues were observed when four human BA-conjugating UGTs 1A3, 1A4, 2B4, and 2B7 were analyzed for mRNA and protein levels. Notably, the BA-24G producing UGT1A3 enzyme, abundant in the liver, was not detected in kidney microsomes. In conclusion, the circulating and urinary BA-G profiles are hugely impacted under severe cholestasis. The similar BA-glucuronidating abilities of hepatic and renal extracts suggest that both the liver and kidney may contribute to the urine BA-G pool.