Blood Esterases Research Articles

Assessing in vitro stability of remdesivir (GS-5734) and conversion to GS-441524 in feline plasma and whole blood

Feline infectious peritonitis (FIP) is a potentially fatal coronavirus-driven disease of cats. Treatment with nucleoside analogue GS-441524 and or prodrug remdesivir (RDV) have produced remission in both experimentally induced and naturally occurring FIP, yet information regarding metabolism of RDV into GS-441524 in cats is scarce. This study assessed possible phase I metabolism of RDV in cats, utilising an in vitro feline microsome model with in vitro t1/2 and in vitro Clint calculated using the substrate depletion method. A previously validated high-performance liquid chromatography (HPLC) fluorescence method was utilised for detection and analysis of RDV and GS-441524. Qualitative yield of RDV and intermediate metabolite GS-441524 were determined following microsome incubation, then compared to whole blood and plasma incubations. In vitro microsome incubation resulted in rapid depletion of RDV, though it did not appear to resemble a conventional phase I-dependent reaction in cats, as it is in humans and dogs. Depletion of RDV into GS-441524 was demonstrated in whole blood in vitro, suggesting cats convert RDV to GS-441524, likely via blood esterases, as observed in mice and rats. RDV metabolism is unlikely to be impacted by impaired liver function in cats. Furthermore, as RDV depletes within minutes, whereas GS-441524 is very stable, whole blood or plasma GS-441524 concentrations, rather than plasma RDV concentrations, are more appropriate for therapeutic drug monitoring (TDM) in cats receiving RDV.

Design of a Supersoft Topical JAK Inhibitor, Which Is Effective in Human Skin but Rapidly Deactivated in Blood.

We describe the discovery and characterization of the supersoft topical JAK inhibitor 3(R), which is potent in biochemical and cellular assays as well as in human skin models. In blood, the neutral ester 3(R) is rapidly hydrolyzed (t1/2 ∼ 6 min) to the corresponding charged carboxylic acid 4 exhibiting >30-fold reduced permeability. Consequently, acid 4 does not reach the intracellular JAK kinases and is inactive in cellular assays and in blood. Thus, hydrolysis by blood esterases leads to the rapid deactivation of topically active ester 3(R) at a rate beyond the maximal hepatic clearance.

Inhibition of Serum Esterases in Juvenile Rats Repeatedly Exposed to Low Levels of Chlorpyrifos.

Chlorpyrifos (CPF) is an organophosphorus insecticide that has gained significant attention cue to the reported toxicity associated with developmental exposure. While the canonical mechanism of toxicity of CPF involves the inhibition of brain acetylcholinesterase (AChE), we have reported that exposure of juvenile rats to levels of CPF that do not yield any inhibition of brain AChE results in neurobehavioral alterations at later ages. However, it is unclear what effect exposure to these low levels of CPF has on blood esterase activities which are frequently used not only as biomarkers of exposure but also to set exposure levels in risk assessment. To determine this, male and female rat pups were exposed orally from postnatal day 10 to 16 to either corn oil (vehicle) or 0.5, 0.75, or 1.0 mg/kg CPF. At 12 h after the final exposure, serum cholinesterase (ChE), butyrylcholinesterase (BChE), and carboxylesterase (CES), and red blood cell (RBC) and brain AChE activities were determined. There were no differences between sexes in either the controls or individual treatments for all enzymes. Only the highest dosage of 1.0 mg/kg CPF yielded significant brain AChE inhibition (22-24%) but all dosages significantly inhibited the blood esterases with inhibition being highest with serum CES (65-85%) followed by serum BChE (57-76%), RBC AChE (35-65%), and then serum ChE (16-32%). Our data verify that blood esterases are inhibited at dosages of CPF that alter neurobehavioral performance in the absence of effects on brain AChE activity.

Methodological considerations for ghrelin isoforms assay in clinical evaluation in anorexia nervosa

The growing interest concerning the role of metabolic sensors in various eating disorders requires the implementation of a strict methodology to collect, store and process blood samples in clinical studies. In particular, measurement of isoforms of the appetite-stimulating hormone, ghrelin, has been challenging in clinical settings. Indeed the acyl ghrelin (AG) isoform is rapidly degraded into desacyl ghrelin (DAG) by blood esterases, thus optimal conditions for the conservation of AG and accurate determination of AG/DAG ratio should be used. Here, we compared different protease inhibitors (Aprotinin, PHMB, AEBSF) during blood collection, increasing delays (0–180 min) before centrifugation, plasma supplementation with various HCl concentrations, storage durations of frozen plasma (8 and 447 days) and immunoenzyme-assay procedures (one-step versus sequential) in healthy subjects. Optimal conditions were obtained by collecting blood with aprotinin and supplementation of plasma with 0.1 N HCl with subsequent freezing for at least 8 days and using one-step assay. Under such conditions, different patterns of secretion of ghrelin isoforms were characterized in patients with restrictive-type anorexia nervosa (AN-R) before and after nutritional recovery. We illustrate the pulsatile variations of ghrelin isoforms according to the time around a meal and hunger rates in 3 patients with AN-R. This study offers a comprehensive comparison of various conditions using selective and specific immunoassays for both ghrelin isoforms in order to optimize assay sensitivity and consistency among procedures. These assay conditions could therefore be widely used to elucidate precisely the role of ghrelin isoforms on eating behavior in physiological and pathological situations.

The Current Role of Clevidipine in the Management of Hypertension.

Acute hypertension, which may damage blood vessels, causes irreversible organ damage to the vasculature, central nervous system, kidney, and heart. Clevidipine, the first third-generation calcium channel antagonist approved by the Food and Drug Administration (FDA) in the past 20 years, is an ultra-short-acting calcium channel blocker that inhibits L-type calcium channels with high clearance and low distribution, can be rapidly metabolized into the corresponding inactive acid, and is rapidly hydrolyzed into inactive metabolites by esterase in arterial blood. Clevidipine is the same as nicardipine in that the main physiological effect is vasodilation and the main target is the arterial system, which has a limited effect on capacitor vessels. Unlike nitroglycerin, clevidipine has a limited effect on preload. In contrast to other direct-acting vasodilators, clevidipine has an ultra-short half-life due to metabolism by nonspecific blood and tissue esterases. Clevidipine trials conducted in adult populations have proven that it can rapidly control blood pressure in cardiac surgery situations and that adverse reactions to clevidipine are similar to those withotherantihypertensive agents. In recent years, clinical trials have shown that clevidipine has excellent blood pressure-lowering capability in patients with acute neurological injury (hemorrhage, stroke, and subarachnoid andacute intracerebral hemorrhage), those undergoing coronary artery bypass graft or spinal surgery, and in those with cerebral aneurysm/pheochromocytoma, acute heart failure, acute aortic syndromes, or renal insufficiency with severe hypertension, and it is equivalent to commonly used blood pressure-lowering medicines such as nicardipine or nitroglycerin. However, there is a lack of large-scale clinical trial data on the efficacy and safety of clevidipine in children during the perioperative period.

In-vitro and in-vivo metabolism of different aspirin formulations studied by a validated liquid chromatography tandem mass spectrometry method

Low-dose aspirin (ASA) is used to prevent cardiovascular events. The most commonly used formulation is enteric-coated ASA (EC-ASA) that may be absorbed more slowly and less efficiently in some patients. To uncover these “non-responders” patients, the availability of proper analytical methods is pivotal in order to study the pharmacodynamics, the pharmacokinetics and the metabolic fate of ASA. We validated a high-throughput, isocratic reversed-phase, negative MRM, LC–MS/MS method useful for measuring circulating ASA and salicylic acid (SA) in blood and plasma. ASA-d4 and SA-d4 were used as internal standards. The method was applied to evaluate: (a) the "in vitro" ASA degradation by esterases in whole blood and plasma, as a function of time and concentration; (b) the "in vivo" kinetics of ASA and SA after 7 days of oral administration of EC-ASA or plain-ASA (100 mg) in healthy volunteers (three men and three women, 37–63 years). Parameters of esterases activity were Vmax 6.5 ± 1.9 and Km 147.5 ± 64.4 in plasma, and Vmax 108.1 ± 20.8 and Km 803.2 ± 170.7 in whole blood. After oral administration of the two formulations, tmax varied between 3 and 6 h for EC-ASA and between 0.5 and 1.0 h for plain-ASA. Higher between-subjects variability was seen after EC-ASA, and one subject had a delayed absorption over eight hours. Plasma AUC was 725.5 (89.8–1222) for EC-ASA, and 823.1(624–1196) ng h/mL (median, 25–75% CI) for plain ASA. After the weekly treatment, serum levels of TxB2 were very low (< 10 ng/mL at 24 h from the drug intake) in all the studied subjects, regardless of the formulation or the tmax. This method proved to be suitable for studies on aspirin responsiveness.

Population Pharmacokinetic Model of Plasma and Cellular Mycophenolic Acid in Kidney Transplant Patients from the CIMTRE Study.

Background and ObjectiveMycophenolate mofetil is widely used in kidney transplant recipients. Mycophenolate mofetil is hydrolysed by blood esterases to mycophenolic acid (MPA), the active drug. Although MPA therapeutic drug monitoring has been recommended to optimise the treatment efficacy by the area under the plasma concentration vs time curve, little is known regarding MPA concentrations in peripheral blood mononuclear cells, where MPA inhibits inosine monophosphate dehydrogenase. This study aimed to build a pharmacokinetic model using a population approach to describe MPA total and unbound concentrations in plasma and into peripheral blood mononuclear cells in 78 adult kidney transplant recipients receiving mycophenolate mofetil therapy combined with tacrolimus and prednisone.MethodsTotal and unbound plasma concentrations and peripheral blood mononuclear cell concentrations were assayed. A three-compartment model, two for plasma MPA and one for peripheral blood mononuclear cell MPA, with a zero-order absorption and a first-order elimination was used to describe the data.ResultsMycophenolic acid average concentrations in peripheral blood mononuclear cells were well above half-maximal effective concentration for inosine monophosphate dehydrogenase and no relationship was found with the occurrence of graft rejection. Three covariates affected unbound and intracellular MPA pharmacokinetics: creatinine clearance, which has an effect on unbound MPA clearance, human serum albumin, which influences fraction unbound MPA and the ABCB1 3435 C>T (rs1045642) genetic polymorphism, which has an effect on MPA efflux transport from peripheral blood mononuclear cells.ConclusionThis population pharmacokinetic model demonstrated the intracellular accumulation of MPA, the efflux of MPA out of the cells being dependent on P-glycoprotein transporters. Nevertheless, further studies are warranted to investigate the relevance of MPA concentrations in peripheral blood mononuclear cells to dosing regimen optimisation.Electronic supplementary materialThe online version of this article (10.1007/s40268-020-00319-y) contains supplementary material, which is available to authorized users.

Irinotecan-mediated diarrhea is mainly correlated with intestinal exposure to SN-38: Critical role of gut Ugt

Background and purposeIrinotecan-induced diarrhea (IID) results from intestinal damages by its active metabolite SN-38. Alleviation of these damages has focused on lowering luminal SN-38 concentrations. However, it is unclear if the enteric bioavailability of SN-38 is mostly dependent on luminal SN-38 concentrations. Experimental approachIrinotecan (50 mg/kg, i.p. once daily for 6 days) was administered to female wildtype FVB, Mdr1a (−/−), Mrp2 (−/−) and Bcrp1 (−/−) mice for pharmacokinetic (PK), toxicokinetic (TK) and biodistribution studies. Plasma PK/TK profiles and tissues drug distribution were determined after first or sixth daily doses, along with activities of blood and gut esterases and intestinal Ugts. Caco-2 cells and bile-cannulate mice were used to further investigate intestinal and biliary disposition of irinotecan and its metabolites. Key resultsSignificant differences in IID severity were observed with the susceptible rank of Bcrp1(−/−) > wildtype FVB > Mdr1a(−/−) > Mrp2(−/−). This rank order did not correlate with biliary excretion rates of SN-38/SN-38G. Rather, the severity was best correlated (R = 0.805) with the intestinal ratio of Css SN-38/SN-38G, a measure of gut Ugt activity. On the contrary, IID was poorly correlated with plasma AUC ratio of SN-38/SN-38G (R = 0.227). Increased intestinal esterase activities due to repeated dosing and gut efflux transporter functionality are the other key factors that determine SN-38 enteric exposures. Conclusion and implicationsIntestinal SN-38 exposure is mainly affected by intestinal Ugt activities and blood esterase activities, and strongly correlated with severity of IID. Modulating intestinal SN-38 concentration and gut Ugt expression should be the focus of future studies to alleviate IID.

Insights into the Chemical Discovery of Remifentanil.

Design, Synthesis, and Pharmacological Evaluation of Ultrashort- to Long-acting Opioid Analgetics. By Feldman PL, James MK, Brackeen MF, Bilotta JM, Schuster SV, Lahey AP, Lutz MW, Johnson MR, Leighton HJ. J Med Chem 1991; 34:2202-8. Copyright 1991 American Chemical Society. Reprinted with permission.In an effort to discover a potent ultrashort-acting µ-opioid analgetic that is capable of metabolizing to an inactive species independent of hepatic function, several classes of 4-anilidopiperidine analgetics were synthesized and evaluated. One series of compounds displayed potent µ-opioid agonist activity with a high degree of analgesic efficacy and an ultrashort to long duration of action. These analgetics, 4-(methoxycarbonyl)-4-[1-oxopropyl)phenylamino]-1-piperidinepropanoic acid alkyl esters, were evaluated in vitro in the guinea pig ileum for µ-opioid activity, in vivo in the rat tail withdrawal assay for analgesic efficacy and duration of action, and in vitro in human whole blood for their ability to be metabolized in blood. Compounds in this series were all shown to be potent µ agonists in vitro, but depending upon the alkyl ester substitution, the potency and duration of action in vivo varied substantially. The discrepancies between the in vitro and in vivo activities and variations in duration of action are probably due to different rates of ester hydrolysis by blood esterase(s). The [structure-activity relationships] with respect to analgesic activity and duration of action as a function of the various esters synthesized is discussed. It was also demonstrated that the duration of action for the ultrashort-acting analgetic, 8, does not change upon prolonged infusion or administration of multiple bolus injections.

SUN-227 Monitoring Testosterone (T) Levels in Men Receiving Oral Testosterone Undecanoate (TU): Challenges Due to Post-Collection Conversion of TU-to-T and a Path Forward

Introduction: Measuring T levels, in addition to monitoring symptoms, during T replacement therapy is critical to guide dosing decisions. T levels in blood samples from men receiving oral TU can be elevated substantially above actual circulating T levels due to post-collection conversion of TU to T by esterases in blood. Because erroneous T values can result in inappropriate dose-titration decisions, we identified a method for monitoring T that addresses this problem. Methods: Post-collection conversion of TU to T was evaluated in blood drawn from men who had received oral TU (either JATENZO®, Clarus’s oral TU, or Andriol®) and in blood spiked with TU after collection. Blood was collected in Plain, EDTA or NaF-EDTA tubes and then incubated for selected times (up to 3 hours) at room temperature (RT) or on ice. After incubation, blood was centrifuged and the matrix (serum or plasma) was used for measurement of T and TU concentrations by LC/MS-MS. Regression analysis of rate of change of T concentration during incubation versus TU concentration was used to develop algorithms to correct for T overestimation. Algorithm accuracy was tested using results from the Phase 3 inTUne Trial of JATENZO. Results: T concentrations increase in blood containing TU as the blood sits pre-centrifugation, regardless of whether the TU is in the blood when collected or spiked post-collection. The rate of TU conversion depends on the TU concentration, incubation temperature, and presence of NaF, an esterase inhibitor. Incubation temperature had the greatest impact on conversion - rate at RT >5-fold faster than on ice; NaF had a smaller effect. Most clinic T levels are routinely measured in serum from Plain tubes; however, titration in the inTUne Trial of oral TU was based on T in NaF-EDTA plasma. Based on regression analysis of the TU to T conversion rates measured in serum and NaF-EDTA plasma and the NaF effect on measured T levels, a conversion factor of 1.214 was derived to convert a NaF-EDTA plasma T value to an equivalent serum T value for samples collected 6 hours post-dose (the optimal dose-titration sample point for JATENZO). This conversion factor was tested against T data collected during the final PK Visit of the inTUne Trial (87% of subjects attained eugonadal range based on NaF-EDTA plasma T levels) where serum T levels were also measured. Using the conversion factor to compare the measured serum T value with its matched plasma value, we observed a mean error of only 3.1% (n=155 sample pairs; 95% CI 0.4%, 5.8%). Conclusion: Post-collection conversion of TU to T can cause overestimation of circulating T levels in men dosed with oral TU. By accounting for the conversion with different tube types / handling conditions, a conversion factor was derived to allow monitoring of T concentration in JATENZO patients using serum. This conversion factor was validated for JATENZO against the Phase 3 inTUne data.

MP58-17 MONITORING TESTOSTERONE (T) LEVELS IN MEN RECEIVING ORAL TESTOSTERONE UNDECANOATE (TU): DEALING WITH POST-COLLECTION CONVERSION OF TU TO T

INTRODUCTION AND OBJECTIVES:Measuring T levels during T replacement therapy is critical to guide dosing decisions. Post-collection conversion of TU to T by esterases in blood from men receiving ora...

Accurate measurement of androgen after androgen esters: problems created by exvivo esterase effects and LC-MS/MS interference.

BackgroundEx vivo androgen prodrug conversion by blood esterases after oral androgen ester administration may result in an overestimation of the measured blood androgens.ObjectiveWe investigated whether blood collection tubes with esterase inhibitors decreased the conversion of testosterone undecanoate (TU) and dimethandrolone undecanoate (DMAU) to their active metabolites, testosterone (T), and dimethandrolone (DMA), providing a more accurate assessment of circulating T/DMA levels.MethodsBlood was collected in tubes with/without esterase inhibitors from: (i) four healthy and four hypogonadal men receiving no androgens and spiked ex vivo with TU/DMAU; (ii) four men taking oral TU (Andriol®); and (iii) eight hypogonadal men dosed with oral 316 mg TU and 15 healthy men with 200 mg DMAU. T/DMA levels were measured by LC‐MS/MS.ResultsSodium fluoride (NaF, an esterase inhibitor) decreased measured T levels by 14.2% in men not receiving TU. Increasing amounts of TU/DMAU added to blood collected into plain tubes resulted in a concentration‐dependent overestimation of T/DMA that was reduced by collecting blood into NaF tubes (by 30–85%), and keeping samples at 4 °C and minimizing time prior to centrifugation. After oral TU/DMAU administration to men, when TU/DMAU levels were >15/10 ng/mL, respectively, blood collected in NaF tubes yielded lower measured T concentrations by 15–30% and DMA by 22% due to an additional inhibitory effect of NaF on blood esterases.ConclusionNaF directly lowers plasma T/DMA levels measured by LC‐MS/MS and also inhibits blood esterase activity. Overestimation of T/DMA in blood collected in tubes without NaF after oral TU/DMAU administration is important for pharmacokinetics studies in drug development clinical trials but may have limited impact in clinical practice/utilization because the differences between measured and true androgen values are modest and the wide therapeutic androgen efficacy ranges obviate the need for highly accurate androgen measurements during therapy.

Investigation of the Esterase Status as a Complex Biomarker of Exposure to Organophosphorus Compounds

Development of biomarkers of human exposures to organophosphorus compounds OPCs and their quantification is a vital component of a system of prediction and early diagnostics of OPC-induced diseases. Our study was focused on investigation of esterase status as a complex biomarker of exposure to OPCs and an aid in accurate diagnosis. We suggest that this complex biomarker should be more effective and informative than standard assays of plasma butyrylcholinesterase (BChE), erythrocyte acetylcholinesterase (RBC AChE), and lymphocyte neuropathy target esterase (NTE). It will help: 1) to assess an exposure as such and to confirm the nonexposure of individuals suspected to have been exposed; 2) to determine if the exposure was to agents expected to produce acute and/or delayed neurotoxicity; 3) to perform dosimetry of the exposure, which provides valuable information for medical treatment. To confirm this hypothesis, we have examined the changes in activity of blood AChE, NTE, BChE and carboxylesterase (CaE) 1 h after i.p. administration of increasing doses of three OPCs with different esterase profiles: the known neuropathic compound O,O-dipropyl-O-dichlorovinyl phosphate (C3H 7O)2P(O)OCH=CCl2 (diPr-DClVP) as the control compound and two model dialkylphosphates (C2H5O)2P(O)OCH(CF3)2 (diEt-PFP) and (C4H9O)2P(O)OCH(CF3)2 (diBu-PFP). The esterases assay was performed in hemolysed blood by spectrophotometric (AChE, BChE, CaE) and biosensor (NTE) methods. Analysis of the obtained dose-dependences for blood esterases inhibition showed that blood BChE and CaE were the most sensitive biomarkers, allowing detection of low doses. Inhibition of blood NTE and AChE can be used to assess the likelihood that an exposure to OPC would produce cholinergic and/or delayed neuropathic effects.

GENDER SPECIFIC CHANGES IN MOUSE BLOOD ESTRERASE PROFILE AT THE ACUTE INTOXICATION BY 2-(О-CRESYL)-4Н-1,3,2-BENZODIOXAPHOSPHORIN-2-OXIDE

A comparative investigation of the esterase profiles of blood of mice of both genders exposed to various doses of 2-(o-crezyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide (CBDP) was made an hour after its percutaneous injection to animals. A lesser amount of esterase in males blood made them more susceptible to CBDP action as compared to females. It was shown that CBDP equally inhibits the activity of carboxyl esterase and butyryl choline esterase in blood serum of both male and female mice. Statistically significant differences in inhibition degree of enzymes between males and females were found out and therefore it is not recommended to use mixed groups of animals when performing testing of serine esterase inhibitors.

Non-Gradient Blue Native Polyacrylamide Gel Electrophoresis.

Gradient blue native polyacrylamide gel electrophoresis (BN-PAGE) is a well established and widely used technique for activity analysis of high-molecular-weight proteins, protein complexes, and protein-protein interactions. Since its inception in the early 1990s, a variety of minor modifications have been made to this gradient gel analytical method. Here we provide a major modification of the method, which we call non-gradient BN-PAGE. The procedure, similar to that of non-gradient SDS-PAGE, is simple because there is no expensive gradient maker involved. The non-gradient BN-PAGE protocols presented herein provide guidelines on the analysis of mitochondrial protein complexes, in particular, dihydrolipoamide dehydrogenase (DLDH) and those in the electron transport chain. Protocols for the analysis of blood esterases or mitochondrial esterases are also presented. The non-gradient BN-PAGE method may be tailored for analysis of specific proteins according to their molecular weight regardless of whether the target proteins are hydrophobic or hydrophilic. © 2017 by John Wiley & Sons, Inc.

Neuropathy target esterase in mouse whole blood as a biomarker of exposure to neuropathic organophosphorus compounds.

The adult hen is the standard animal model for testing organophosphorus (OP) compounds for organophosphorus compound-induced delayed neurotoxicity (OPIDN). Recently, we developed a mouse model for biochemical assessment of the neuropathic potential of OP compounds based on brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE) inhibition. We carried out the present work to further develop the mouse model by testing the hypothesis that whole blood NTE inhibition could be used as a biochemical marker for exposure to neuropathic OP compounds. Because brain NTE and AChE inhibition are biomarkers of OPIDN and acute cholinergic toxicity, respectively, we compared NTE and AChE 20-min IC50 values as well as ED50 values 1 h after single intraperitoneal (i.p.) injections of increasing doses of two neuropathic OP compounds that differed in acute toxicity potency. We found good agreement between the brain and blood for in vitro sensitivity of each enzyme as well for the ratios IC50 (AChE)/IC50 (NTE). Both OP compounds inhibited AChE and NTE in the mouse brain and blood dose-dependently, and brain and blood inhibitions in vivo were well correlated for each enzyme. For both OP compounds, the ratio ED50 (AChE)/ED50 (NTE) in blood corresponded to that in the brain despite the somewhat higher sensitivity of blood enzymes. Thus, our results indicate that mouse blood NTE could serve as a biomarker of exposure to neuropathic OP compounds. Moreover, the data suggest that relative inhibition of blood NTE and AChE provide a way to assess the likelihood that OP compound exposure in a susceptible species would produce cholinergic and/or delayed neuropathic effects. Copyright © 2016 John Wiley & Sons, Ltd.

Formulation and evaluation of co-prodrug of flurbiprofen and methocarbamol

The current work envisages synthesis of an ester prodrug of flurbiprofen whereby its carboxylic group was condensed with a skeletal muscle relaxant methocarbamol, with the aim of synergistic activity of two drugs, avoid flurbiprofen mediated gastro-intestinal damage and minimize the ulceration tendency of flurbiprofen. The synthesized prodrug was characterized and confirmed by physicochemical and spectroscopic studies. Solubility and partition coefficient studies indicated an increased lipophilicity and thus better suitability for oral administration than the parent drugs and the protein binding studies revealed a low protein binding capacity of the mutual prodrug. Subsequently, in-vitro hydrolysis was studied in different pH, simulated gastric fluid, simulated intestinal fluid and plasma and quantitative evaluation was performed by high performance liquid chromatography. It was found that the prodrug remained unhydrolyzed in the stomach after absorption however, underwent rapid cleavage by the esterases in blood to give the parent drug. Furthermore, the mutual ester prodrug was evaluated for its anti-inflammatory, analgesic, skeletal muscle relaxation, ulcerogenic and total acid content activity and was found to possess comparable activity with that of the parent drugs. Microscopic structures of the stomach tissues revealed significant reduction in gastric ulcer formation of mice gastric mucosa as compared to parent carboxylic acid drug.

Importance of measuring testosterone in enzyme-inhibited plasma for oral testosterone undecanoate androgen replacement therapy clinical trials.

Aim:Testosterone undecanoate (TU) is metabolized by nonspecific esterases in blood to testosterone (T). Typical clinical practice has been to analyze testosterone in human serum. The degradation of TU to testosterone was evaluated in conditions typically used in clinical studies.Methods & Results:Freshly collected whole blood was fortified with TU at known concentration. Serum was prepared and T concentration was determined by LC–MS/MS. It was observed that TU degrades extensively to T in human blood under conditions typical of harvesting serum causing overestimation of T concentration of up to 243%. These results were confirmed in a clinical study in which serum and plasma samples were compared.Conclusion:It was demonstrated that T must be analyzed in enzyme-inhibited plasma when TU is the administered medication.

Screening of stabilizers for LC-MS/MS analysis of clevidipine and its primary metabolite in dog whole blood.

Clevidipine is an ester-containing antihypertensive agent that undergoes rapid hydrolysis in blood. A reliable stabilizer cocktail containing citric acid and ascorbic acid was established and the LC-MS/MS method was validated for simultaneous determination of clevidipine and its major metabolite in beagle dog whole blood. The stabilizer could nearly completely inhibit the esterase activity. Both analytes were extracted from whole blood by toluene and detected by MS/MS in positive ESI mode. The linearity range was 0.1-100.0 ng/ml for clevidipine and 1.0-1000.0 ng/ml for the primary metabolite. The stabilizer cocktail was able to effectively suppress the activity of esterase in blood. The method was successfully applied to a PK study of clevidipine in beagle dogs.

Comparative intestinal esterases amongst passerine species: Assessing vulnerability to toxic chemicals in a phylogenetically explicit context

Inhibition of blood esterase activities by organophosphate (OP) pesticides has been used as a sensitive biomarker in birds. Furthermore, compared to mammalian vertebrates, less is known about the role of these enzyme activities in the digestive tracts of non-mammalian vertebrates, as well as the environmental and biological stressors that contribute to their natural variation. To fill this gap, we examined butyrylcholinesterase (BChE) and carboxylesterases (CbE) in the digestive tracts of sixteen passerine species from central Chile. Whole intestine enzyme activities were positively and significantly correlated with body mass. After correcting for body mass and phylogenetic effect, we found only a marginal effect of dietary category on BChE activity, but a positive and significant association between the percentage of dietary nitrogen and the mass-corrected lipase activity. Our results suggest that observed differences may be due to the dietary composition in the case of lipases and BChE, and also we predict that all model species belonging to the same order will probably respond differently to pesticide exposure, in light of differences in the activity levels of esterase activities.