Microsomal Assay Research Articles

Liquid chromatography–tandem mass spectrometry screening method using information‐dependent acquisition of enhanced product ion mass spectra for synthetic cannabinoids including metabolites in urine

The total number of synthetic cannabinoids (SCs) - a group of new psychoactive substances (NPS) - is increasing every year. The rapidly changing market demands the latest analytical methods to detect the consumption of SCs in clinical or forensic toxicology. In addition, SC metabolites must also be included in a screening procedure, if detection in urine is asked for. For that purpose, an easy and fast qualitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) urine screening method for the detection of 75 SCs and their metabolites was developed and validated in terms of matrix effects, recovery, and limits of identification for a selection of analytes. SC metabolites were generated using in vitro human liver microsome assays, identified by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and finally included to the MS/MS spectra in-house library. Sample preparation was performed using a cheap-and-easy salting-out liquid-liquid extraction (SALLE) after enzymatic hydrolysis. Method validation showed good selectivity, limits of identification down to 0.05ng/mL, recoveries above 80%, and matrix effects within ±25% for the selected analytes. Applicability of the method was demonstrated by detection of SC metabolites in authentic urine samples.

Prediction of Transformation Products of Monensin by Electrochemistry Compared to Microsomal Assay and Hydrolysis

The knowledge of transformation pathways and identification of transformation products (TPs) of veterinary drugs is important for animal health, food, and environmental matters. The active agent Monensin (MON) belongs to the ionophore antibiotics and is widely used as a veterinary drug against coccidiosis in broiler farming. However, no electrochemically (EC) generated TPs of MON have been described so far. In this study, the online coupling of EC and mass spectrometry (MS) was used for the generation of oxidative TPs. EC-conditions were optimized with respect to working electrode material, solvent, modifier, and potential polarity. Subsequent LC/HRMS (liquid chromatography/high resolution mass spectrometry) and MS/MS experiments were performed to identify the structures of derived TPs by a suspected target analysis. The obtained EC-results were compared to TPs observed in metabolism tests with microsomes and hydrolysis experiments of MON. Five previously undescribed TPs of MON were identified in our EC/MS based study and one TP, which was already known from literature and found by a microsomal assay, could be confirmed. Two and three further TPs were found as products in microsomal tests and following hydrolysis, respectively. We found decarboxylation, O-demethylation and acid-catalyzed ring-opening reactions to be the major mechanisms of MON transformation.

Detection of the recently emerged synthetic cannabinoid 4F-MDMB-BINACA in "legal high" products and human urine specimens.

Synthetic cannabinoids (SCs) remain one of the largest groups of new psychoactive substances (NPS) on the European drug market. Although the number of new derivatives occurring on the market has dropped in the last two years, newly emerging NPS still represent a challenge for laboratories performing forensic drug analysis in biological matrices. The newly emerged SC 4F-MDMB-BINACA has been reported by several law enforcement agencies in Europe and the USA since November 2018. This work aimed at revealing urinary markers to prove uptake of 4F-MDMB-BINACA and differentiate from the use of structurally similar SCs. Phase-I metabolites detected in human urine specimens were confirmed by phase-I metabolites generated in vitro using a pooled human liver microsomes (pHLM) assay. Seized materials and test-purchased "legal high" products were analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-qToF-MS). Human urine specimens and pHLM assay extracts were measured with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and confirmed by LC-qToF-MS. In January 2019, the Institute of Legal Medicine in Erlangen (Germany) identified 4F-MDMB-BINACA in three herbal blends. During the same time period, the described SC was identified in a research chemical purchased online. Investigation of phase-I metabolism led to the metabolites M10 (ester hydrolysis) and M11 (ester hydrolysis and dehydrogenation) as reliable urinary markers. Widespread distribution on the German drug market was proven by analysis of urine samples from abstinence control programs and by frequent detection of 4F-MDMB-BINACA in "herbal blends" and "'research chemicals" purchased via the Internet.

OATP1B3-1B7 (LST-3TM12) Is a Drug Transporter That Affects Endoplasmic Reticulum Access and the Metabolism of Ezetimibe.

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by SLCO1B3 and SLCO1B7 OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe β-D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7.

Evaluation of the inhibition risk of shikonin on human and rat UDP-glucuronosyltransferases (UGT) through the cocktail approach

Shikonin, a natural red colorant, is widely used for food garnishment and cosmetic ingredient in the world. Shikonin also possesses a variety of pharmacological actions, including anti-inflammation and anti-cancer activities. However, little is known about its effects on the UDP-glucuronosyltransferases (UGT) activity. Therefore, the aim of this study was to evaluate the effect of shikonin on the UGT1A1, UGT1A3, UGT1A6, UGT1A9 and UGT2B7 activities via the human and rat liver microsomal assay and cocktail approach. The results showed shikonin inhibited human and rat liver microsomal UGT activity only in a dose-dependent manner. The further enzyme kinetic studies demonstrated that shikonin was not only a competitive inhibitor of human UGT1A1, UGT1A9, and UGT2B7, but also presented competitive inhibition on rat UGT1A1 and AZTG reactions. In conclusion, shikonin as a reversible inhibitor of UGT enzyme has a high-risk potential to cause the possible toxicity, especially drug-drug or food-drug interactions.

A rapid MS/MS method to assess the deuterium kinetic isotope effect and associated improvement in the metabolic stability of deuterated biological and pharmacological molecules as applied to an imaging agent

The deuterium kinetic isotope effect has been known for a period of 40 years, but it is only relatively recently that new drug entities (NDEs) incorporating deuterium demonstrating beneficial pharmacokinetics, pharmacodynamics, and toxicology have arrived to market. Determination of the precise location to deuterate and subsequently any evaluation for a kinetic isotope effect (KIE) is challenging. Typically, such an evaluation would be performed in an in vitro metabolic assay (e.g. liver microsomes) in separate reaction media for both the deuterated and non-deuterated analogues. Here, we have devised an approach whereby we incubate a 1:1 ratio of both the deuterated and protio-form of an imaging agent together in the same liver microsomal assay and determine the relative rate of consumption of both moieties, based upon specific MS-MS transitions unique to both molecules without the need for liquid chromatography-mass spectrometry (LC-MS) separation and quantification. Any deviation of the ratio of the MS transitions from the initial starting point indicated an observable KIE.A site specific deuteration of PBR111, a neuroinflammation imaging agent, was chosen for a proof-of-concept study. Based upon prior mechanistic knowledge of PBR111, two locations were selected for deuteration; an active and inactive site, to corroborate that there was no significant KIE for the inactive site and confirm the efficacy of the developed methodology.

Metabolic profiling of synthetic cannabinoid 5F-ADB by human liver microsome incubations and urine samples using high-resolution mass spectrometry.

5F-ADB (methyl 2-{[1-(5-fluoropentyl)-1H-indazole-3-carbonyl] amino}-3,3-dimethylbutanoate) is a frequently abused new synthetic cannabinoid that has been sold since at least the end of 2014 on the drug market and has been classified as an illicit drug in most European countries, as well as Turkey, Japan, and the United States. In this study, the in vitro metabolism of 5F-ADB was investigated by using pooled human liver microsomes (HLMs) assay and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). 5F-ADB (5μmol/L) was incubated with HLMs for up to 3hours, and the metabolites were identified using LC-HRMS and software-assisted data mining. The in vivo metabolism was investigated by the analysis of 30 authentic urine samples and was compared to the data received from the in vitro metabolism study. Less than 3.3% of the 5F-ADB parent compound remained after 1hour of incubation, and no parent drug was detected after 3hours. We identified 20 metabolites formed via ester hydrolysis, N-dealkylation, oxidative defluorination, hydroxylation, dehydrogenation, further oxidation to N-pentanoic acid and glucuronidation or a combination of these reactions in vitro. In 12 urine samples (n=30), 5F-ADB was detected as the parent drug. Three of the identified main metabolites 5F-ADB carboxylic acid (M20), monohydroxypentyl-5F-ADB (M17), and carboxypentyl ADB carboxylic acid (M8) were suggested as suitable urinary markers. The screening of 8235 authentic urine samples for identified 5F-ADB metabolites in vitro resulted in 3135 cases of confirmed 5F-ADB consumption (38%).

Advanced Yeast Models of Familial Alzheimer Disease Expressing FAD-Linked Presenilin to Screen Mutations and γ-Secretase Modulators.

γ-Secretase is a multisubunit membrane protein complex containing catalytic presenilin (PS1 or PS2) and cofactors such as nicastrin, Aph-1, and Pen2. γ-Secretase hydrolyzes the transmembrane domains of type-I membrane proteins, which include the amyloid precursor protein (APP). APP is cleaved by γ-secretase to produce amyloid β peptide (Aβ), which is deposited in the brains of Alzheimer disease patients. However, the mechanism of this unusual proteolytic process within the lipid bilayer remains unknown. We have established a yeast transcriptional activator Gal4p system with artificial γ-secretase substrates containing APP or Notch fragments to examine the enzymatic properties of γ-secretase. The γ-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4 release from the membrane bound substrates as assessed by growth of yeast or β-galactosidase assay. We also established an in vitro yeast microsome assay system which identified different Aβ species produced by trimming. The yeast system allows for the screening of mutations and chemicals that inhibit or modulate γ-secretase activity. Herein we describe the genetic and biochemical methods used to analyze γ-secretase activity using the yeast reconstitution system. By studying the loss-of-function properties of PS1 mutants, it is possible to successfully screen FAD suppressor mutations and identify γ-secretase modulators (GSMs), which are promising Alzheimer disease therapeutic agents.

A Novel RAD51 Inhibitor, CYT01B, Shows Anti-Cancer Activity in Preclinical Models of Aid Expressing Lymphomas

Activation Induced Cytidine Deaminase (AID) is a DNA directed cytidine deaminase that is normally only expressed in activated B-cells to promote somatic hypermutations and immunoglobulin class switching. Unlike site-specific recombinases (e.g. RAG1/2), AID lacks target site specificity and can generate DNA damage at widespread locations throughout the genome. In cancer cells, AID expression promotes high levels of DNA replication stress, and results in dependency upon the homologous recombination factor RAD51. We have shown previously that a novel RAD51 inhibitor, CYT01B, functions by disrupting RAD51 focus formation which reduces the nuclear concentration of RAD51 and promotes RAD51 protein degradation. Here we present the in vitro and in vivo pharmacological characterization of CYT01B. We first analyzed the permeability and stability of the compound using Caco-2 and liver microsome assays. CYT01B shows low efflux and is stable with low intrinsic clearance rates (< 30 ml/min/mg protein) in mouse, rat, dog, and human, but not in cynomolgus monkey liver microsomes. In vivo, CYT01B showed oral bioavailability that correlated well with Caco-2 permeability (36.9% in monkeys up to 86.5% in rats). Additionally, CYT01B exhibited a minimum half-life of 4 hours in all species tested (mouse, rat, dog, and monkey). We then examined off target liabilities by performing kinome inhibition and Panlabs safety panel screens. CYT01B showed negligible (<50%) inhibition across 371 kinases tested, while only two targets showed greater than 50% inhibition in the Panlabs panel. CYT01B displayed activity in several human-to-mouse cell line xenograft models. In a systemic model of chronic lymphoblastic leukemia (CLL), the tumor burden in the bone marrow of CYT01B-treated mice was reduced by approximately 30%. In two different subcutaneous engraftment models of either a Burkitt's lymphoma cell line (Daudi) or an acute lymphoblastic leukemia cell line (CCRF-SB), mice treated with CYT01B showed tumor growth inhibition of 49% and 88%, respectively. Lastly, CYT01B was well tolerated in rat 7-day dose range finding studies. Rats were treated with 20, 80, and 240 mg/kg once per day by oral gavage. There were no observed changes in hematology or clinical chemistry, and no observed histopathological toxicities. Taken together, these data demonstrate that CYT01B is a cell permeable, metabolically stable, and orally bioavailable small molecule, with anti-cancer activity in multiple preclinical lymphoid cancer models. Overall, this provides the basis for continued preclinical development of an AID/RAD51 synthetic lethal therapeutic paradigm that may be applicable to various hematologic malignancies. DisclosuresPatrick:Cyteir Therapeutics: Consultancy. Bedard:Cyteir Therapeutics: Consultancy. Vacca:Cyteir Therapeutics: Consultancy. McComas:Cyteir Therapeutics: Consultancy. Castro:Cyteir Therapeutics: Consultancy. Day:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Design, synthesis, and evaluation of novel diphenyl ether derivatives against drug-susceptible and drug-resistant strains ofMycobacterium tuberculosis.

In our efforts to develop druggable diphenyl ethers as potential antitubercular agents, a series of novel diphenyl ether derivatives (5a-f, 6a-f) were designed and synthesized. The representative compounds showed promising in vitro activity against drug-susceptible, isoniazid-resistant, and multidrug-resistant strains of Mycobacterium tuberculosis with MIC values of 1.56μg/ml (6b), 6.25μg/ml (6a-d), and 3.125μg/ml (6b-c), respectively. All the synthesized compounds exhibited satisfactory safety profile (CC50 >300μg/ml) against Vero and HepG2 cells. Reverse phase HPLC method was used to probe the physicochemical properties of the synthesized compounds. This series of compounds demonstrated comparatively low logP values. pKa values of representative compounds indicated that they were weak acids. Additionally, in vitro human liver microsomal stability assay confirmed that the synthesized compounds possessed acceptable stability under study conditions. The present study thus establishes compound 6b as the most promising antitubercular agent with acceptable drug-likeness.

Optimization of Experimental Conditions of Automated Glucuronidation Assays in Human Liver Microsomes Using a Cocktail Approach and Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

UDP-glucuronosyltransferase (UGT)-mediated metabolism is possibly the most important conjugation reaction for marketed drugs. However, there are currently no generally accepted standard incubation conditions for UGT microsomal assays, and substantial differences in experimental design and methodology between laboratories hinder cross-study comparison of in vitro activities. This study aimed to define optimal experimental conditions to determine glucuronidation activity of multiple UGT isoforms simultaneously using human liver microsomes. Hepatic glucuronidation activities of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17 were determined using cocktail incubations of 10 UGT probe substrates. Buffer components and cosubstrates were assessed over a range of concentrations including magnesium chloride (MgCl2; 0-10 mM) and uridine 5'-diphosphoglucuronic acid (UDPGA; 1-25 mM) with either Tris-HCl or potassium phosphate buffer (100 mM, pH 7.4). Greater microsomal glucuronidation activity by different hepatic UGT isoforms was obtained using 10 mM MgCl2 and 5 mM UDPGA with 100 mM Tris-HCl buffer. The influence of bovine serum albumin (BSA; 0.1%-2% w/v) on glucuronidation activity was also assessed. Enzyme- and substrate-dependent effects of BSA were observed, resulting in decreased total activity of UGT1A1, UGT1A3, and UGT2B17 and increased total UGT1A9 and UGT2B7 activity. The inclusion of BSA did not significantly reduce the between-subject variability of UGT activity. Future in vitro UGT profiling studies under the proposed optimized experimental conditions would allow high-quality positive control data to be generated across laboratories, with effective control of a high degree of between-donor variability for UGT activity and for chemical optimization toward lower-clearance drug molecules in a pharmaceutical drug discovery setting.

Structure-Based Drug Design and Characterization of Sulfonyl-Piperazine Benzothiazinone Inhibitors of DprE1 from Mycobacterium tuberculosis.

Macozinone (MCZ) is a tuberculosis (TB) drug candidate that specifically targets the essential flavoenzyme DprE1, thereby blocking synthesis of the cell wall precursor decaprenyl phosphoarabinose (DPA) and provoking lysis of Mycobacterium tuberculosis As part of the MCZ backup program, we exploited structure-guided drug design to produce a new series of sulfone-containing derivatives, 2-sulfonylpiperazin 8-nitro 6-trifluoromethyl 1,3-benzothiazin-4-one, or sPBTZ. These compounds are less active than MCZ but have a better solubility profile, and some derivatives display enhanced stability in microsomal assays. DprE1 was efficiently inhibited by sPBTZ, and covalent adducts with the active-site cysteine residue (C387) were formed. However, despite the H-bonding potential of the sulfone group, no additional bonds were seen in the crystal structure of the sPBTZ-DprE1 complex with compound 11326127 compared to MCZ. Compound 11626091, the most advanced sPBTZ, displayed good antitubercular activity in the murine model of chronic TB but was less effective than MCZ. Nonetheless, further testing of this MCZ backup compound is warranted as part of combination treatment with other TB drugs.

Synthesis and Pharmacological Evaluation of Novel Silodosin-Based Arylsulfonamide Derivatives as α1A/α1D-Adrenergic Receptor Antagonist with Potential Uroselective Profile.

Benign prostatic hyperplasia (BPH) is the most common male clinical problem impacting the quality of life of older men. Clinical studies have indicated that the inhibition of α1A-/α1D adrenoceptors might offer effective therapy in lower urinary tract symptoms. Herein, a limited series of arylsulfonamide derivatives of (aryloxy)ethyl alicyclic amines was designed, synthesized, and biologically evaluated as potent α1-adrenoceptor antagonists with uroselective profile. Among them, compound 9 (3-chloro-2-fluoro-N-([1-(2-(2-(2,2,2-trifluoroethoxy)phenoxy]ethyl)piperidin-4-yl)methyl)benzenesulfonamide) behaved as an α1A-/α1D-adrenoceptor antagonist (Ki(α1) = 50 nM, EC50(α1A) = 0.8 nM, EC50(α1D) = 1.1 nM), displayed selectivity over α2-adrenoceptors (Ki(α2) = 858 nM), and a 5-fold functional preference over the α1B subtype. Compound 9 showed adequate metabolic stability in rat-liver microsome assay similar to the reference drug tamsulosin (Clint = 67 and 41 µL/min/mg, respectively). Compound 9 did not decrease systolic and diastolic blood pressure in normotensive anesthetized rats in the dose of 2 mg/kg, i.v. These data support development of uroselective agents in the group of arylsulfonamides of alicyclic amines with potential efficacy in the treatment of lower urinary tract symptoms associated to benign prostatic hyperplasia.

Development of Novel Monoamine Oxidase-B (MAO-B) Inhibitors with Reduced Blood-Brain Barrier Permeability for the Potential Management of Noncentral Nervous System (CNS) Diseases.

Studies indicate that MAO-B is induced in peripheral inflammatory diseases. To target peripheral tissues using MAO-B inhibitors that do not permeate the blood-brain barrier (BBB) the MAO-B-selective inhibitor deprenyl was remodeled by replacing the terminal acetylene with a CO2H function, and incorporating a para-OCH2Ar motif (compounds 10a-s). Further, in compound 32 the C-2 side chain corresponded to CH2CN. In vitro, 10c, 10j, 10k, and 32 were identified as potent reversible MAO-B inhibitors, and all four compounds were more stable than deprenyl in plasma, liver microsomal, and hepatocyte stability assays. In vivo, they demonstrated greater plasma bioavailability. Assessment of in vitro BBB permeability showed that compound 10k is a P-glycoprotein (P-gp) substrate and 10j displayed mild interaction. Importantly, compounds 10c, 10j, 10k, and 32 displayed significantly reduced BBB permeability after intravenous, subcutaneous, and oral administration. These polar MAO-B inhibitors are pertinent leads for evaluation of efficacy in noncentral nervous system (CNS) inflammatory disease models.

A novel calcimimetic agent, evocalcet (MT-4580/KHK7580), suppresses the parathyroid cell function with little effect on the gastrointestinal tract or CYP isozymes in vivo and in vitro.

Cinacalcet hydrochloride (cinacalcet), an oral calcimimetic agent has been widely used for the management of secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD). In sharp contrast to vitamin D receptor activators, cinacalcet suppresses SHPT without inducing hypercalcemia or hyperphosphatemia. Nevertheless, some patients remain refractory to SHPT with this agent, as the dose cannot be sufficiently increased due to gastrointestinal symptoms. In order to resolve this issue, we have developed a newly synthesized calcimimetic agent, evocalcet (MT-4580/KHK7580). In a rat model of CKD induced by 5/6 nephrectomy, oral administration of evocalcet efficiently suppressed the secretion of parathyroid hormone (PTH). With regard to the gastro-intestinal effects, cinacalcet induced a significant delay in gastric emptying in rats, while evocalcet did no marked effects on it. Evocalcet also demonstrated the less induction of emesis compared to cinacalcet in common marmosets. The pharmacological effects of evocalcet were observed at lower doses because of its higher bioavailability than cinacalcet, which may have contributed to the reduced GI tract symptoms. In addition, evocalcet showed no substantial direct inhibition of any CYP isozymes in in vitro liver microsome assay, suggesting a better profile in drug interactions than cinacalcet that inhibits cytochrome P450 (CYP) 2D6. These findings suggest that evocalcet can be a better alternative to cinacalcet, an oral calcimimetic agent, with a wider safety margin.

Investigation of hepatic warfarin metabolism activity in rodenticide-resistant black rats (Rattus rattus) in Tokyo by in situ liver perfusion

Anti-blood coagulation rodenticides, such as warfarin, have been used all over the world. They inhibit vitamin K epoxide reductase (VKOR), which is necessary for producing several blood clotting factors. This inhibition by rodenticides results in lethal hemorrhage in rodents. However, heavy usage of these agents has led to the appearance of rodenticide-resistant rats. There are two major mechanisms underlying this resistance, i.e., mutation of the target enzyme of warfarin, VKOR, and enhanced metabolism of warfarin. However, there have been few studies regarding the hepatic metabolism of warfarin, which should be related to resistance. To investigate warfarin metabolism in resistant rats, in situ liver perfusion of warfarin was performed with resistant black rats (Rattus rattus) from Tokyo, Japan. Liver perfusion is an in situ methodology that can reveal hepatic function specifically with natural composition of the liver. The results indicated enhanced hepatic warfarin hydroxylation activity compared with sensitive black rats. On the other hand, in an in vitro microsomal warfarin metabolism assay to investigate kinetic parameters of cytochrome P450, which plays a major role in warfarin hydroxylation, the Vmax of resistant rats was slightly but significantly higher compared to the results obtained in the in situ study. These results indicated that another factor like electron donators may also contribute to the enhanced metabolism in addition to high expression of cytochrome P450.

Structure-Dependent in Vitro Metabolism of Alkyl-Substituted Analogues of Triphenyl Phosphate in East Greenland Polar Bears and Ringed Seals

Organophosphate esters (OPEs), used as plasticizers and flame retardants, are major emerging environmental contaminants in the Arctic. OPEs of environmental interest include triphenyl phosphate (TPHP) and a growing array of alkyl-substituted TPHP analogues. Using a microsomal assay of the liver tissue of polar bears and their ringed seal prey, the comparative in vitro metabolism of TPHP was investigated relative to the analogues of isodecyl diphenyl phosphate (IDDPP), (p-tert-butylphenyl) diphenyl phosphate (TBPDPP), tris(p-tert-butylphenyl) phosphate (TTBPP), and two tris(isopropylphenyl) phosphate isomers (T2IPPP and T4IPPP). Polar bear metabolism of the p-tert-butylphenyl-substituted OPEs, TBPDPP and TTBPP, had a substantially slower rate and percent metabolic depletion compared to those with TPHP. Isodecyl- and isopropyl-substituted OPEs, IDDPP, T2IPPP, and T4IPPP, were also more slowly depleted by polar bears than TPHP was. TPHP, IDDPP, T2IPPP, TBPDPP, TTBPP, and T4IPPP were all slowly metabolized by...

Characterized in Vitro Metabolism Kinetics of Alkyl Organophosphate Esters in Fish Liver and Intestinal Microsomes.

Tris(2-butoxyethyl) phosphate (TBOEP) and tris( n-butyl) phosphate (TNBP) are the most commonly used alkyl organophosphate esters (alkyl-OPEs), and they increasingly accumulate in organisms and create potential health hazards. This study examined the metabolism of TNBP and TBOEP in Carassius carassius liver and intestinal microsomes and the production of their corresponding monohydroxylated and dealkylated metabolites. After 140 min of incubation with fish liver microsomes, the rapid depletion of TNBP and TBOEP were both best fitted to the Michaelis-Menten model (at administrated concentrations ranging from 0.5 to 200 μM), with a CLint (intrinsic clearance) of 3.1 and 3.9 μL·min-1·mg-1 protein, respectively. But no significant ( P > 0.05) biotransformation was observed for these compounds in intestinal microsomes at any administrated concentrations. In fish liver microsomes assay, bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) and bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate (3-OH-TBOEP) were the most abundant metabolites of TBOEP, and dibutyl-3-hydroxybutyl phosphate (3-OH-TNBP) was the predominant metabolite of TNBP. Similarly, the apparent Vmax values (maximum metabolic rate) of BBOEHEP and 3-OH-TNBP were also respectively highest among those of other metabolites. Further inhibition studies were conducted to identify the specific cytochrome P450 (CYP450) isozymes involved in the metabolism of TNBP and TBOEP in liver microsomes. It was confirmed that CYP3A4 and CYP1Awere the significant CYP450 isoforms catalyzing the metabolism of TNBP and TBOEP in fish liver microsomes. Overall, this study emphasized the importance of hydroxylated metabolites as biomarkers for alkyl-OPEs exposure, and further research is needed to validate the in vivo formation and toxicological implications of these metabolites.

Novel aromatic sulfonyl naphthalene-based boronates as 20S proteasome inhibitors

A novel series of non-peptide proteasome inhibitors (PIs) that act on chymotrypsin-like (ChT-L) of the proteasome were developed. These PIs bearing 4-aromatic sulfonyl naphthalene-based scaffold and Leu-boronic moiety as covalent bonding group displayed far better activity than PI-8182 for inhibiting ChT-L in preliminary biological activity test. The results showed that 2a (IC50 = 6.942 μM, MCF-7) and 2c (IC50 = 6.905 μM, MCF-7) displayed higher anti-proliferative activities than Bortezomib (IC50 = 18.37 μM, MCF-7) under our experimental conditions. Furthermore, in the microsomal stability assay, 2a demonstrated excellent metabolic stability profiles with 56% remaining after 40 min, as compared to Bortezomib of which approximately 30% was remaining. The compounds 2a, 2c emerged as promising lead compounds for the development of novel non-peptide boronate PIs.

Phase I metabolism of the recently emerged synthetic cannabinoid CUMYL-PEGACLONE and detection in human urine samples.

Indole-, indazole-, or azaindole-based synthetic cannabinoids (SCs), bearing a cumyl substituent are a widespread, recreationally used subgroup of new psychoactive substances (NPS). The latest cumyl-derivative, CUMYL-PEGACLONE, emerged in December 2016 on the German drug market. The substance features a novel γ-carboline core structure, which is most likely synthesized to bypass generic legislative approaches to control SCs by prohibiting distinct core structures. Using liquid chromatography-tandem mass spectrometry and liquid chromatography-high resolution mass spectrometry techniques, the main in vivo phase I metabolites of this new substance were detected. A pooled human liver microsome assay was applied to generate in vitro reference spectra of CUMYL-PEGACLONE phase I metabolites. Additionally, 30 urine samples were investigated leading to 22 in vivo metabolites. A metabolite mono-hydroxylated at the γ-carbolinone core system and a metabolite with an additional carbonyl group at the pentyl side chain were evaluated as highly specific and sensitive markers to proof CUMYL-PEGACLONE uptake. Moreover, 3 immunochemical assays commonly used for SC screening in urine were tested for their capability of detecting the new drug but failed due to insufficient cross-reactivity.