Virtual Screening Directly Identifies New Fragment-Sized Inhibitors of Carboxylesterase Notum with Nanomolar Activity.

Notum is a negative regulator of Wnt signaling acting through the hydrolysis of a palmitoleoylate ester, which is required for Wnt activity. Inhibitors of Notum could be of use in diseases where dysfunctional Notum activity is an underlying cause. A docking-based virtual screen (VS) of a large commercial library was used to shortlist 952 compounds for experimental validation as inhibitors of Notum. The VS was successful with 31 compounds having an IC50 < 500 nM. A critical selection process was then applied with two clusters and two singletons (1-4d) selected for hit validation. Optimization of 4d guided by structural biology identified potent inhibitors of Notum activity that restored Wnt/ß-catenin signaling in cell-based models. The [1,2,4]triazolo[4,3-b]pyradizin-3(2H)-one series 4 represent a new chemical class of Notum inhibitors and the first to be discovered by a VS campaign. These results demonstrate the value of VS with well-designed docking models based on X-ray structures.

Determination of camostat and its metabolites in human plasma - Preservation of samples and quantification by a validated UHPLC-MS/MS method.

OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at -20 °C and -80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1-800 ng/mL. CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.

Structural Insights into Notum Covalent Inhibition.

The carboxylesterase Notum hydrolyzes a palmitoleate moiety from Wingless/Integrated(Wnt) ligands and deactivates Wnt signaling. Notum inhibitors can restore Wnt signaling which may be of therapeutic benefit for pathologies such as osteoporosis and Alzheimer's disease. We report the identification of a novel class of covalent Notum inhibitors, 4-(indolin-1-yl)-4-oxobutanoate esters. High-resolution crystal structures of the Notum inhibitor complexes reveal a common covalent adduct formed between the nucleophile serine-232 and hydrolyzed butyric esters. The covalent interaction in solution was confirmed by mass spectrometry analysis. Inhibitory potencies vary depending on the warheads used. Mechanistically, the resulting acyl-enzyme intermediate carbonyl atom is positioned at an unfavorable angle for the approach of the active site water, which, combined with strong hydrophobic interactions with the enzyme pocket residues, hinders the intermediate from being further processed and results in covalent inhibition. These insights into Notum catalytic inhibition may guide development of more potent Notum inhibitors.

NOTUM from Apc-mutant cells biases clonal competition to initiate cancer.

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.

Larvicidal and ovicidal activities of phenyl acetic acid isolated from Streptomyces collinus against Culex quinquefasciatus Say and Aedes aegypti L. (Diptera: Culicidae).

The bio-efficacy of crude ethyl acetate extract, fractions and a compound phenyl acetic acid from the ethyl acetate extract of Streptomyces collinus was evaluated on Culex quinquefasciatus Say and Aedes aegypti L. mosquitoes (Diptera: Culicidae). The larvae were exposed to concentrations of 2.5, 5.0, 7.5 and 10.0 ppm for fractions and 0.5, 1.0, 1.5 and 2.0 ppm for compound. After 24 h, the larval mortality was assessed and the LC50 and LC90 values were calculated. Similarly, per cent ovicidal activity was calculated for eggs after 120 h post treatment for phenyl acetic acid. Among the eleven fractions screened, fraction 7 from the ethyl acetate extract of Streptomyces collinus exhibited good larvicidal activity against both mosquito species. The LC50 and LC90 values of fraction 7 were 4.42, 6.23 ppm against Cx. quinquefasciatus larvae and 5.13, 14.51 ppm against Ae. aegypti larvae, respectively. Further, the isolated compound, phenyl acetic acid from fraction 7 recorded 100% larvicidal activity at 2 ppm concentration with LC50 and LC90 values of 2.07, 4.87 ppm on Cx. quinquefasciatus larvae and 3.81, 9.87 ppm on Ae. aegypti larvae, respectively. Phenyl acetic acid presented 50.3% and 42.0% ovicidal activity against Cx. quinquefasciatus and Ae. aegypti eggs at 2 ppm concentration after 120 h post treatment. The compound, phenyl acetic acid could be used in mosquito control programme.

Small-molecule inhibitors of carboxylesterase Notum.

Notum has recently been identified as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group from Wnt proteins. There are emerging reports that Notum plays a role in human disease, with published data suggesting that targeting Notum could represent a new therapeutic approach for treating cancer, osteoporosis and neurodegenerative disorders. Complementary hit-finding strategies have been applied with successful approaches that include high-throughput screening, activity-based protein profiling, screening of fragment libraries and virtual screening campaigns. Structural studies are accelerating the discovery of new inhibitors of Notum. Three fit-for-purpose examples are LP-922056, ABC99 and ARUK3001185. The application of these small-molecule inhibitors is helping to further advance an understanding of the role Notum plays in human disease.

Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C.

Mycobacterium tuberculosis virulence is highly metal-dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu2+ ≥ Zn2+ > Ni2+ > Co2+ ) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for kcat rather than KM . Removal of the artificial N-terminal 6xHis-tag did not change the metal-dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn2+ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal-dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.

Carboxylesterase Notum Is a Druggable Target to Modulate Wnt Signaling.

Regulation of the Wnt signaling pathway is critically important for a number of cellular processes in both development and adult mammalian biology. This Perspective will provide a summary of current and emerging therapeutic opportunities in modulating Wnt signaling, especially through inhibition of Notum carboxylesterase activity. Notum was recently shown to act as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group. Inhibition of Notum activity may represent a new approach to treat disease where aberrant Notum activity has been identified as the underlying cause. Reliable screening technologies are available to identify inhibitors of Notum, and structural studies are accelerating the discovery of new inhibitors. A selection of these hits have been optimized to give fit-for-purpose small molecule inhibitors of Notum. Three noteworthy examples are LP-922056 (26), ABC99 (27), and ARUK3001185 (28), which are complementary chemical tools for exploring the role of Notum in Wnt signaling.

Westalsan: A New Acetylcholine Esterase Inhibitor from the Endophytic Fungus Westerdykella nigra.

A new cytochalasan alkaloid, westalsan (1), along with two known cytochalasan compounds, phomacin B (2) and 19-hydroxy-19,20-dihydrophomacin C (3), were isolated from the solid rice culture of Westerdykella nigra, a marine-derived endophytic fungus, isolated from the roots of mangrove Avicennia marina (Forssk.) Vierh. The structures of compounds 1-3 were established on the basis of extensive 1D and 2D NMR spectroscopic techniques in combination with HR-ESI-MS. The ability of the isolated compounds to inhibit acetylcholine esterase activity was evaluated. Compound 3 showed the highest acetylcholine esterase inhibitory activity (IC50 0.056±0.003 µM), followed by compound 1 (IC50 0.088±0.005 µM) and compound 2 (IC50 0.140±0.007 µM) compared to donepezil (IC50 0.035±0.002 µM). This was further confirmed by molecular docking experiment.

5-Phenyl-1,3,4-oxadiazol-2(3H)-ones Are Potent Inhibitors of Notum Carboxylesterase Activity Identified by the Optimization of a Crystallographic Fragment Screening Hit.

Carboxylesterase Notum is a negative regulator of the Wnt signaling pathway. There is an emerging understanding of the role Notum plays in disease, supporting the need to discover new small-molecule inhibitors. A crystallographic X-ray fragment screen was performed, which identified fragment hit 1,2,3-triazole 7 as an attractive starting point for a structure-based drug design hit-to-lead program. Optimization of 7 identified oxadiazol-2-one 23dd as a preferred example with properties consistent with drug-like chemical space. Screening 23dd in a cell-based TCF/LEF reporter gene assay restored the activation of Wnt signaling in the presence of Notum. Mouse pharmacokinetic studies with oral administration of 23dd demonstrated good plasma exposure and partial blood-brain barrier penetration. Significant progress was made in developing fragment hit 7 into lead 23dd (>600-fold increase in activity), making it suitable as a new chemical tool for exploring the role of Notum-mediated regulation of Wnt signaling.

Caffeine inhibits Notum activity by binding at the catalytic pocket.

Notum inhibits Wnt signalling via enzymatic delipidation of Wnt ligands. Restoration of Wnt signalling by small molecule inhibition of Notum may be of therapeutic benefit in a number of pathologies including Alzheimer's disease. Here we report Notum activity can be inhibited by caffeine (IC50 19 µM), but not by demethylated caffeine metabolites: paraxanthine, theobromine and theophylline. Cellular luciferase assays show Notum-suppressed Wnt3a function can be restored by caffeine with an EC50 of 46 µM. The dissociation constant (Kd) between Notum and caffeine is 85 µM as measured by surface plasmon resonance. High-resolution crystal structures of Notum complexes with caffeine and its minor metabolite theophylline show both compounds bind at the centre of the enzymatic pocket, overlapping the position of the natural substrate palmitoleic lipid, but using different binding modes. The structural information reported here may be of relevance for the design of more potent brain-accessible Notum inhibitors.

[Hereditary angioedema due to C1-esterase inhibitor deficiency†: novel approaches]. / Angiœdèmes héréditaires sur ­déficit en C1-inhibiteur†: nouveautés ­thérapeutiques.

Hereditary angioedema type 1 and 2 are due to a deficiency in C1--esterase inhibitor. This molecule inhibits the generation of bradykinin, a potent inflammatory mediator that increases vascular permeability. Upon accumulation of bradykinin, patients affected develop painful subcutaneous or submucosal edemas that last for several days. In case the upper airways are affected, there is risk of suffocation. This type of angioedema does not respond to antihistamines, cortico-steroids or epinephrine. Management of angioedema attacks consists in injecting C1-esterase inhibitor concentrate or icatibant, a bradykinin receptor B2 antagonist. Preventive measures aim at reducing the frequency and the severity of angioedema attacks. Inhibition of -plasma kallikrein by lanadelumab, a monoclonal antibody adminis-tered subcutaneously, is effective and well tolerated.

Les angiœdèmes (AE) héréditaires de types I et II sont dus à un déficit en inhibiteur de la C1 estérase ou C1-inhibiteur (C1-INH). Cette molécule régule la formation de bradykinine, un puissant médiateur vasoactif. En cas d'accumulation de bradykinine, les patients développent des œdèmes sous-cutanés ou sous-­muqueux douloureux qui persistent plusieurs jours. L'atteinte des voies aériennes comporte un risque de suffocation. Ce type d'angiœdème ne répond pas aux antihistaminiques, aux corticoïdes ni à l'adrénaline. Le traitement de la crise comprend ­l'injection de concentré de C1-INH ou d'icatibant, un antagoniste du récepteur B2 de la bradykinine. Le traitement préventif vise à diminuer la fréquence et la sévérité des crises. L'inhibition de la kallicréine plasmatique par lanadélumab, un anticorps mono­clonal administré en sous-cutané, constitue une approche efficace et bien tolérée.

Crystal structure of pathogenic Staphylococcus aureus lipase complex with the anti-obesity drug orlistat.

Staphylococcus aureus lipase (SAL), a triacylglycerol esterase, is an important virulence factor and may be a therapeutic target for infectious diseases. Herein, we determined the 3D structure of native SAL, the mutated S116A inactive form, and the inhibitor complex using the anti-obesity drug orlistat to aid in drug development. The determined crystal structures showed a typical α/ß hydrolase motif with a dimeric form. Fatty acids bound near the active site in native SAL and inactive S116A mutant structures. We found that orlistat potently inhibits SAL activity, and it covalently bound to the catalytic Ser116 residue. This is the first report detailing orlistat-lipase binding. It provides structure-based information on the production of potent anti-SAL drugs and lipase inhibitors. These results also indicated that orlistat can be repositioned to treat bacterial diseases.

Two flavonoid-based compounds from Murraya paniculata as novel human carbonic anhydrase isozyme II inhibitors detected by a resazurin yeast-based assay.

Human carbonic anhydrase isozyme II has been used as protein target for disorder treatment including glaucoma. Current clinically used sulfonamide-based CA inhibitors can induce side effects, and so alternatives are required. This study aimed to investigate a natural CA inhibitor from Murraya paniculata. The previously developed yeast-based assay was used to screen 14 compounds isolated from M. paniculata and identified by NMR analysis for anti-human CA isozyme II (hCAII) activity. Cytotoxicity of the compounds was also tested using the same yeast-based assay but in a different cultivation condition. Two flavonoid candidate compounds, 5, 6, 7, 8, 3', 4', 5'-heptamethoxyflavone (4) and 3 ,5, 7, 8, 3', 4', 5'-heptamethoxyflavone (9), showed potent inhibitory activity against hCAII with a minimal effective concentration of 10.8 and 21.5 µM, respectively, while they both exhibited no cytotoxic effect even at the highest concentration tested (170 µM). The results from an in vitro esterase assay of the two candidates confirmed their hCAII inhibitory activity with IC50 values of 24.0 and 34.3 µM, respectively. To investigate the potential inhibition mechanism of compound 4, in silico molecular docking was performed using the FlexX and Swissdock software. This revealed that compound 4 coordinated with the Zn2+ ion in the hCAII active site through its methoxy oxygen at a distance of 1.60 Å (FlexX) or 2.29 Å (Swissdock). The interaction energy of compound 4 with hCAII was -13.36 kcal/mol. Thus, compound 4 is a potent novel flavonoid-based hCAII inhibitor and may be useful for further anti-CAII design and development.

Structural characterization of melatonin as an inhibitor of the Wnt deacylase Notum.

The hormone melatonin, secreted from the pineal gland, mediates multiple physiological effects including modulation of Wnt/ß-catenin signalling. The Wnt palmitoleate lipid modification is essential for its signalling activity, while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it. Notum enzyme inhibition can therefore upregulate Wnt signalling. While searching for Notum inhibitors by crystallographic fragment screening, a hit compound N-[2-(5-fluoro-1H-indol-3-yl)ethyl]acetamide that is structurally similar to melatonin came to our attention. We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high-resolution structures (≤1.5 Å) of their complexes. In each of the structures, two compound molecules bind with Notum: one at the enzyme's catalytic pocket, overlapping the space occupied by the acyl tail of the Wnt palmitoleate lipid, and the other at the edge of the pocket opposite the substrate entrance. Although the inhibitory activity of melatonin shown by in vitro enzyme assays is low (IC50 75 µmol/L), the structural information reported here provides a basis for the design of potent and brain accessible drugs for neurodegenerative diseases such as Alzheimer's disease, in which upregulation of Wnt signalling may be beneficial.

Butyrlycholine esterase inhibitory activity and effects of extracts (fruit, bark and leaf) from Zanthoxylum armatum DC in gut, airways and vascular smooth muscles.

BACKGROUND: Fruit, bark and leaves of Zanthoxylum armatum DC are popular remedies for gastrointestinal, cardiovascular and respiratory disorders in the subcontinent traditional practices. The aim of the study was to individually probe the profile of methanol extracts from three different parts of Zanthoxylum armatum. METHODS: The ex-vivo muscle relaxant effects of extracts were assessed in the isolated intestine, trachea and thoracic aortic rings and were compared with the positive controls and CRC were constructed. The anti-diarrheal effect of extracts was evaluated in mice by inducing diarrhea with castor oil. The extracts were also studied for acute toxicity and butyrylcholine esterase inhibition. RESULTS: The extracts from fruit, bark and leaves of Z. armatum showed inhibitory effect against the butyrylcholine esterase enzyme with percent inhibition of 50.75 ± 1.23, 82.57 ± 1.33, and 37.52 ± 1.11respectively, compared to standard serine (IC50: 0.04 ± 0.001 µmol/L). The fruit and bark extracts provided 75, and 52% diarrheal protection, compared to verapamil (96%). In isolated rabbit jejunum strips, increasing addition of the extracts inhibited the spontaneous and high K+ precontractions with EC50 values of 0.71 and 3 mg/mL for fruit, EC50 values of 0.61 and 0.5 mg/mL for bark, EC50 0.81 and 3.1 mg/mL for leaves, like verapamil. The extracts induced a concentration-dependent relaxation of the carbachol (1 µM) and high K+ (80 mM) precontractions with EC50 values of 2.4 and 0.9 mg/mL for fruit, EC50 values of 1.2 and 3 for leaves. The bark extract was equipotent against both contractions with EC50 3.1 and 0.7 mg/mL, respectively. In the aortic rings, the fruit extract completely relaxed the phenylephrine (1 µM)-induced contractions with (EC50 value = 0.8 mg/ml) and a partial inhibition of high K+ induced contractions. The leaves extract completely relaxed the aortic contractions with (EC50 values = 1.0 and 8.5 mg/ml). The extracts caused no acute toxicity up to 3 g/kg dose. CONCLUSIONS: The experiments revealed that the extracts of aerial parts of Z. armatum have antidiarrheal properties in vivo and showed spasmolytic effect in intestinal and tracheal preparations with possible mechanism involving the blockage of Ca++ channels. These experiments provide enough justification for use of this plant in ethnomedicine in diarrhea, gut and bronchial spasms.

Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium.

A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche1,2. Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor α (PPAR-α)3, and lowered PPAR-α activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.

Allelopathically inhibitory effects of eucalyptus extracts on the growth of Microcystis aeruginosa.

Microcystis aeruginosa (M. aeruginosa), as the dominant algae in eutrophic water bodies, has caused a serious harm to the local eco-environment. A biological tool, employing allelopathic inhibitory of eucalyptus to control M. aeruginosa, has been receiving tremendous attention. This work presents the results of the allelopathic inhibitory effects of eucalyptus (Eucalyptus grandis × E.urophylla 'GLGU9') extracts of roots (ERE), stems (ESE), and leaves (ELE) on culture solutions of M. aeruginosa and its eco-physiological mechanism. The inhibitory effects of the extracts on the growth of M. aeruginosa varied greatly with ELE exhibiting the highest level of potency. Modes of action by which ELE inhibited M. aeruginosa growth were established. They involved reduction in photosynthesis, disruption of the cell membrane integrity, and inhibition of esterase activities of the cyanobacterial cells. However, ELE did not exhibit any gradients of toxicity towards zebrafish nor Washington grass plant. Species abundance and diversity in the systems remained likewise unaffected by ELE. The synergistic interaction between ELE and single-component allelochemicals (e.g., gallic acid and berberine) was ascribed to the increase in efficacy of allelochemicals in the various systems. The results of this study provide an underlying, novel, and attractive approach for controlling the growth of M. aeruginosa in aquatic environments.

Role of esterase mediated hydrolysis of simvastatin in human and rat blood and its impact on pharmacokinetic profiles of simvastatin and its active metabolite in rat.

Simvastatin is known as a pro-drug, which could be hydrolyzed by esterases to its active form, simvastatin acid. Although pharmacokinetics of simvastatin and simvastatin acid have been widely studied, hydrolysis of simvastatin to simvastatin acid during blood sampling and plasma preparation has been overlooked in the previous studies, leading to underestimation of simvastatin concentration and overestimation of simvastatin acid concentration in plasma. Since both efficacy and adverse drug reaction of simvastatin are highly dependent on simvastatin and simvastatin acid concentrations in vivo, accurate assessment of the two compounds are critical in their pharmacokinetic and pharmacodynamic studies. The current study was proposed aiming to investigate the esterase mediated hydrolysis of simvastatin in human and rat blood and its impact on the pharmacokinetic study of simvastatin and simvastatin acid. Using various esterase inhibitors including potassium florid (KF), bis(4-nitrophenyl) phosphate (BNPP), and ethylenediaminetetraacetic acid (EDTA), carboxylesterase was found to be the major esterase that hydrolyzed simvastatin in rat blood, while carboxylesterase and paraoxonase were the major esterases mediating the hydrolysis of simvastatin in human blood. Further studies using human recombinant enzymes identified simvastatin as substrates of PON1, CES1b, PON3 and CES1c with Clint of 8.75, 5.77, 3.93, and 2.45 µL/min/mg protein. Therefore, inhibition treatments with 20 mM BNPP and 50 mM KF/ 10 mM EDTA were developed to efficiently prevent the hydrolysis of simvastatin during blood sampling and plasma preparation in rat/human. The subsequent pharmacokinetics of orally administered simvastatin at 8.66 mg/kg in rats found that the Cmax and AUC0-∞ of simvastatin in absence of such esterase inhibitors in the blood sampling process were only 17.04 ± 6.60% and 15.30 ± 6.76% of those in presence of the inhibitors, whereas the Cmax and AUC0-∞ of simvastatin acid were 1.60 ± 0.30 and 1.80 ± 0.22 times of that obtained in presence of the inhibitors. Nevertheless, T1/2 of simvastatin and simvastatin acid remained the same regardless of the blood sampling method. Our current study for the first time demonstrated the importance for assessment of simvastatin stability during the blood sampling and plasma preparation process, which may be applicable to therapeutic drug monitoring of not only simvastatin but also other pro-drugs/compounds sharing similar metabolic properties.

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

BACKGROUND: Ex vivo androgen prodrug conversion by blood esterases after oral androgen ester administration may result in an overestimation of the measured blood androgens. OBJECTIVE: We 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. METHODS: Blood 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. RESULTS: Sodium 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. CONCLUSION: NaF 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.