Metabolic Soft Spot and Pharmacokinetics: Functionalization of C-3 Position of an Eph–Ephrin Antagonist Featuring a Bile Acid Core as an Effective Strategy to Obtain Oral Bioavailability in Mice

UniPR129, an L-β-homotryptophan conjugate of the secondary bile acid lithocholic acid (LCA), acts as an effective protein-protein interaction (PPI) inhibitor of the Eph–ephrin system but suffers from a poor oral bioavailability in mice. To improve UniPR129 bioavailability, a metabolic soft spot, i.e., the 3α-hydroxyl group on the LCA steroidal ring, was functionalized to 3-hydroxyimine. In vitro metabolism of UniPR129 and 3-hydroxyimine derivative UniPR500 was compared in mouse liver subcellular fractions, and main metabolites were profiled by high resolution (HR-MS) and tandem (MS/MS) mass spectrometry. In mouse liver microsomes (MLM), UniPR129 was converted into several metabolites: M1 derived from the oxidation of the 3-hydroxy group to 3-oxo, M2–M7, mono-hydroxylated metabolites, M8–M10, di-hydroxylated metabolites, and M11, a mono-hydroxylated metabolite of M1. Phase II reactions were only minor routes of in vitro biotransformation. UniPR500 shared several metabolic pathways with parent UniPR129, but it showed higher stability in MLM, with a half-life (t1/2) of 60.4 min, if compared to a t1/2 = 16.8 min for UniPR129. When orally administered to mice at the same dose, UniPR500 showed an increased systemic exposure, maintaining an in vitro valuable pharmacological profile as an EphA2 receptor antagonist and an overall improvement in its physico-chemical profile (solubility, lipophilicity), if compared to UniPR129. The present work highlights an effective strategy for the pharmacokinetic optimization of aminoacid conjugates of bile acids as small molecule Eph–ephrin antagonists.

The Functions of Cytochrome P450 ω-hydroxylases and the Associated Eicosanoids in Inflammation-Related Diseases

The cytochrome P450 (CYP) ω-hydroxylases are a subfamily of CYP enzymes. While CYPs are the main metabolic enzymes that mediate the oxidation reactions of many endogenous and exogenous compounds in the human body, CYP ω-hydroxylases mediate the metabolism of multiple fatty acids and their metabolites via the addition of a hydroxyl group to the ω- or (ω-1)-C atom of the substrates. The substrates of CYP ω-hydroxylases include but not limited to arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid, epoxyeicosatrienoic acids, leukotrienes, and prostaglandins. The CYP ω-hydroxylases-mediated metabolites, such as 20-hyroxyleicosatrienoic acid (20-HETE), 19-HETE, 20-hydroxyl leukotriene B4 (20-OH-LTB4), and many ω-hydroxylated prostaglandins, have pleiotropic effects in inflammation and many inflammation-associated diseases. Here we reviewed the classification, tissue distribution of CYP ω-hydroxylases and the role of their hydroxylated metabolites in inflammation-associated diseases. We described up-regulation of CYP ω-hydroxylases may be a pathogenic mechanism of many inflammation-associated diseases and thus CYP ω-hydroxylases may be a therapeutic target for these diseases. CYP ω-hydroxylases-mediated eicosanods play important roles in inflammation as pro-inflammatory or anti-inflammatory mediators, participating in the process stimulated by cytokines and/or the process stimulating the production of multiple cytokines. However, most previous studies focused on 20-HETE,and further studies are needed for the function and mechanisms of other CYP ω-hydroxylases-mediated eicosanoids. We believe that our studies of CYP ω-hydroxylases and their associated eicosanoids will advance the translational and clinal use of CYP ω-hydroxylases inhibitors and activators in many diseases.

Human and rat microsomal metabolites of N-tert-butoxycarbonylmethamphetamine and its urinary metabolites in rat

Purpose N-tert-Butoxycarbonylmethamphetamine (BocMA), a masked derivative of methamphetamine (MA), converts into MA under acidic condition and potentially acts as a precursor to MA following ingestion. To investigate the metabolism and excretion of BocMA, metabolism tests were conducted using human liver microsomes (HLM), rat liver microsomes (RLM) and rat. Methods BocMA metabolites were analyzed after 1000-ng/mL BocMA incubation with microsomes for 3, 8, 13, 20, 30, and 60 min. Rats were administered intraperitoneal injections (20 mg/kg) of BocMA and their urine was collected in intervals for 72 h. Metabolites were detected by liquid chromatography–tandem mass spectrometry with five authentic standards. Results Several metabolites including 4-hydroxy-BocMA, N-tert-butoxycarbonylephedrine and N-tert-butoxycarbonyl-cathinone were detected for HLM and RLM. In the administration test, three glucuronides of hydroxylated metabolites were detected. The total recovery values of BocMA and the metabolites during the first 72 h accounted for only 0.3% of the administered dose. Throughout the microsomal and administration experiments, MAs were not detected. Conclusion Hydroxylation, carbonylation and N-demethylation were proposed as metabolic pathways. However, BocMA and phase I metabolites were hardly detected in urine. This study provides useful information to interpret the possibility of BocMA intake as the cause of MA detection in biological sample.

Corticosteroid Biosynthesis Revisited: No Direct Hydroxylation of Pregnenolone by Steroid 21-Hydroxylase

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism.

Evaluation of fireground exposures using urinary PAH metabolites

Background Firefighters have increased cancer incidence and mortality rates compared to the general population, and are exposed to multiple products of combustion including known and suspected carcinogens. Objective The study objective was to quantify fire response exposures by role and self-reported exposure risks. Methods Urinary hydroxylated metabolites of polycyclic aromatic hydrocarbons (PAH-OHs) were measured at baseline and 2–4 h after structural fires and post-fire surveys were collected. Results Baseline urine samples were collected from 242 firefighters. Of these, 141 responded to at least one of 15 structural fires and provided a post-fire urine. Compared with baseline measurements, the mean fold change of post-fire urinary PAH-OHs increased similarly across roles, including captains (2.05 (95% CI 1.59–2.65)), engineers (2.10 (95% CI 1.47–3.05)), firefighters (2.83 (95% CI 2.14–3.71)), and paramedics (1.84 (95% CI 1.33–2.60)). Interior responses, smoke odor on skin, and lack of recent laundering or changing of hoods were significantly associated with increased post-fire urinary PAH-OHs. Significance Ambient smoke from the fire represents an exposure hazard for all individuals on the fireground; engineers and paramedics in particular may not be aware of the extent of their exposure. Post-fire surveys identified specific risks associated with increased exposure.