Inhibition Kinetic Parameters Research Articles

Demethylzeylasteral exhibits dose-dependent inhibitory behaviour towards estradiol glucuronidation

The disturbance of estradiol level might induce the occurence of some diseases, including cancer. Estradiol is mainly metabolized through the conjugation reactions, including the sulfation and glucuronidation reactions. The present study tried to evaluate the inhibition of estradiol glucuronidation by the major ingredients of Tripterygium wilfordii Hook F. demethylzeylasteral. Selective ion monitoring at negative ion mode ([M⁺ H⁻] = 447) was employed to monitor the two glucuronides of estradiol. The reaction rate was determined through comparison of peak area of these two glucuronides. Lineweaver-Burk plot and Dixon plot were utilized to determine the inhibition kinetic type, and the inhibition kinetic parameters (K i) were calculated using the second plot. Competitive inhibition of demethylzeylasteral towards the formation of two glucuronides of estradiol was demonstrated, and the K i values were calculated to be 453.3 and 110.9 μM, respectively. All these results will remind us the risk of elevated serum concentrations of estradiol due to the inhibition of estradiol glucuronidation by demethylzeylasteral.

Comparison of Inhibition Capability of Scutellarein and Scutellarin Towards Important Liver UDP‐Glucuronosyltransferase (UGT) Isoforms

Scutellarin is an important bioactive flavonoid extracted from Erigeron breviscapus (Vant.) Hand-Mazz, and scutellarein is the corresponding aglycone of scutellarin. The present study aims to compare the inhibition potential of scutellarin and scutellarein towards several important UDP-glucuronosyltransferase (UGT) isoforms, including UGT1A1, UGT1A6, UGT1A9 and UGT2B7. It was demonstrated that scutellarein exerted stronger inhibition towards the tested UGT isoforms than scutellarin. Furthermore, the inhibition kinetic type and parameters (Ki ) were determined for the scutellarein's inhibition towards these UGT isoforms. Competitive inhibition of scutellarein towards all these UGT isoforms was demonstrated, and the Ki values were calculated to be 0.02, 5.0, 5.8 and 35.9 μM for UGT1A1, 1A6, 1A9 and 2B7, respectively. Using in vivo maximum plasma concentration of scutellarein in rat, the in vitro-in vivo extrapolation was performed to predict in vivo situation, indicating the most possible in vivo adverse effects due to the inhibition of scutellarein towards UGT1A1. All these results remind us to monitor the utilization of scutellarin and scutellarein, and the herbs containing these two components.

Structure–inhibition relationship of ginsenosides towards UDP-glucuronosyltransferases (UGTs)

The wide utilization of ginseng provides the high risk of herb–drug interaction (HDI) with many clinical drugs. The inhibition of ginsenosides towards drug-metabolizing enzymes (DMEs) has been regarded as an important reason for herb–drug interaction (HDI). Compared with the deep studies on the ginsenosides' inhibition towards cytochrome P450 (CYP), the inhibition of ginsenosides towards the important phase II enzymes UDP-glucuronosyltransferases (UGTs) remains to be unclear. The present study aims to evaluate the inhibition behavior of ginsenosides towards important UGT isoforms located in the liver and intestine using in vitro methods. The recombinant UGT isoform-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employed as in vitro probe reaction. The results showed that structure-dependent inhibition existed for the inhibition of ginsenosides towards UGT isoforms. To clarify the possibility of in vivo herb–drug interaction induced by this kind of inhibition, the ginsenoside Rg3 was selected as an example, and the inhibition kinetic type and parameters (Ki) were determined. Rg3 competitively inhibited UGT1A7, 2B7 and 2B15-catalyzed 4-MU glucuronidation reaction, and exerted noncompetitive inhibition towards UGT1A8-catalyzed 4-MU glucuronidation. The inhibition parameters (Ki values) were calculated to be 22.6, 7.9, 1.9, and 2.0μM for UGT1A7, 1A8, 2B7 and 2B15. Using human maximum plasma concentration of Rg3 (400ng/ml (0.5μM)) after intramuscular injection of 60mg Rg3, the area under the plasma concentration-time curve (AUC) was extrapolated to increase by 2.2%, 6.3%, 26.3%, and 25% for the co-administered drugs completely undergoing the metabolism catalyzed by UGT1A7, 1A8, 2B7 and 2B15, respectively. All these results indicated that the ginsenosides' inhibition towards UGT isoforms might be an important reason for ginseng–drug interaction.

Isoliquiritigenin showed strong inhibitory effects towards multiple UDP-glucuronosyltransferase (UGT) isoform-catalyzed 4-methylumbelliferone (4-MU) glucuronidation

Isoliquiritigenin, a herbal ingredient with chalcone structure, has been speculated to be able to inhibit one of the most drug-metabolizing enzymes (DMEs) UDP-glucuronosyltransferase (UGT). Therefore, the aim of the present study was to investigate the inhibition of isoliquiritigenin towards important UGT isoforms in the liver and intestine, including UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9 and 1A10. The recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as probe reactions. The results showed that 100μM of isoliquiritigenin inhibited the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 by 95.2%, 76.1%, 78.9%, 87.2%, 67.2%, 94.8%, and 91.7%, respectively. The data fitting using Dixon plot and Lineweaver–Burk plot showed that the inhibition of UGT1A1, UGT1A9 and UGT1A10 by isoliquiritigenin was all best fit to the competitive inhibition, and the second plot using the slopes from the Lineweaver–Burk plot versus isoliquiritigenin concentrations was used to calculate the inhibition kinetic parameter (Ki) to be 0.7μM, 0.3μM, and 18.3μM for UGT1A1, UGT1A9, and UGT1A10, respectively. All these results indicated the risk of clinical application of isoliquiritigenin on the drug–drug interaction and other possible diseases induced by the inhibition of isoliquiritigenin towards these UGT isoforms.

Glycyrrhetinic Acid Exhibits Strong Inhibitory Effects Towards UDP‐Glucuronosyltransferase (UGT) 1A3 and 2B7

The aim of the present study is to evaluate the inhibitory effects of liver UDP-glucuronosyltransferases (UGTs) by glycyrrhizic acid and glycyrrhetinic acid, which are the bioactive ingredients isolated from licorice. The results showed that glycyrrhetinic acid exhibited stronger inhibition towards all the tested UGT isoforms, indicating that the deglycosylation process played an important role in the inhibitory potential towards UGT isoforms. Furthermore, the inhibition kinetic type and parameters were determined for the inhibition of glycyrrhetinic acid towards UGT1A3 and UGT2B7. Data fitting using Dixon and Lineweaver-Burk plots demonstrated that the inhibition of UGT1A3 and UGT2B7 by glycyrrhetinic acid was best fit to competitive and noncompetitive type, respectively. The second plot using the slopes from Lineweaver-Burk plots versus glycyrrhetinic acid concentrations was employed to calculate the inhibition kinetic parameters (K(i)), and the values were calculated to be 0.2 and 1.7 μM for UGT1A3 and UGT2B7, respectively. All these results remind us the possibility of UGT inhibition-based herb-drug interaction. However, the explanation of these in vitro parameters should be paid more caution due to complicated factors, including the probe substrate-dependent UGT inhibition behaviour, environmental factors affecting the abundance of herbs' ingredients, and individual difference of pharmacokinetic factors.

Deglycosylation of Liquiritin Strongly Enhances its Inhibitory Potential Towards UDP‐Glucuronosyltransferase (UGT) Isoforms

The detailed mechanisms on licorice-drug interaction remain to be unclear. The aim of the present study is to investigate the inhibition of important UGT isoforms by two important ingredients of licorice, liquiritin, and liquiritigenin. The results showed that liquiritigenin exhibited stronger inhibition towards all the tested UGT isoforms than liquiritin. Data fitting using Dixon and Lineweaver-Burk plots demonstrated the competitive inhibition of liquiritigenin towards UGT1A1 and UGT1A9-mediated 4-MU glucuronidation reaction. The inhibition kinetic parameters (Ki ) were calculated to be 9.1 and 3.2 μM for UGT1A1 and UGT1A9, respectively. Substrate-dependent inhibition behaviour was also observed for UGT1A1 in the present study. All these results will be helpful for understanding the deep mechanism of licorice-drug interaction. However, when translating these in vitro parameters into in vivo situations, more complex factors should be considered, such as substrate-dependent inhibition of UGT isoforms, the contribution of UGT1A1 and UGT1A9 towards the metabolism of drugs, and many factors affecting the abundance of ingredients in the licorice.

Strong inhibition of deoxyschizandrin and schisantherin A toward UDP-glucuronosyltransferase (UGT) 1A3 indicating UGT inhibition-based herb–drug interaction

Deoxyschizandrin and schisantherin A are major bioactive lignans isolated from Fructus schisandrae which has been widely used as a tonic in traditional Chinese medicine for many years. Inhibition of UDP-glucuronosyltransferases (UGTs) by herbal components might be an important reason for clinical herb–drug interaction. The aim of the present study is to investigate the inhibitory effect of deoxyschizandrin and schisantherin A on major UGT isoforms. Recombinant UGT isoforms were used as enzyme source, and a nonspecific substrate 4-methylumbelliferone (4-MU) was utilized as substrate. The results showed that 100μM of deoxyschizandrin and schisantherin A exhibited strong inhibition on UGT1A3, and negligible inhibition on other tested UGT isoforms. Furthermore, deoxyschizandrin and schisantherin A were demonstrated to inhibit UGT1A3 in a concentration-dependent manner, with IC50 value of 10.8±0.4μM and 12.5±0.5μM, respectively. Dixon and Lineweaver–Burk plots showed that inhibition of UGT1A3 by deoxyschizandrin was best fit to competitive inhibition type, and inhibition kinetic parameter (Ki) was calculated to be 0.48μM. Inhibition of UGT1A3 by schisantherin A gave the best fit for types of noncompetitive inhibition, and the results showed Ki to be 11.3μM. All these experimental data suggested that herb–drug interaction might occur when deoxyschizandrin or schisantherin A containing herbs were co-administered with drugs which mainly undergo UGT1A3-mediated metabolism. However, given that many in vivo factors could influence the in vitro–in vivo extrapolation (IVIVE), these in vitro inhibitory parameters should be considered with caution.

Demethylzeylasteral Exhibits Strong Inhibition towards UDP-Glucuronosyltransferase (UGT) 1A6 and 2B7

Inhibition of UDP-glucuronosyltransferase (UGT) isoforms can result in severe clinical results, including clinical drug-drug interactions (DDI) and metabolic disorders of endogenous substances. The present study aims to investigate the inhibition of demethylzeylasteral (an important active component isolated from Tripterygium wilfordii Hook F.) towards three important UGT isoforms UGT1A6, UGT1A9 and UGT2B7. The results showed that 100 μM of demethylzeylasteral exhibited strong inhibition towards UGT1A6 and UGT2B7, with negligible influence towards UGT1A9. Furthermore, Dixon and Lineweaver-Burk plots showed the inhibition of UGT1A6 and UGT2B7 by demethylzeylasteral was best fit to competitive inhibition, and the inhibition kinetic parameters (Ki) were calculated to be 0.6 μM and 17.3 μM for UGT1A6 and UGT2B7, respectively. This kind of inhibitory effect need much attention when demethylzeylasteral and demethylzeyasteral-containing herbs (e.g., Tripterygium wilfordii Hook F.) were co-administered with the drugs mainly undergoing UGT1A6, UGT2B7-catalyzed metabolism. However, when extrapolating the in vivo clinical results using our present in vitro data, many complex factors might affect final results, including the contribution of UGT1A6 and UGT2B7 to the metabolism of compounds, and the herbal or patients’ factors affecting the in vivo concentration of demethylzeylasteral.

Deep understanding of the interaction between thienorphine and UDP-glucuronosyltransferase (UGT) isoforms

Thienorphine has been demonstrated to be a potent, long-acting partial opioid agonist. It is being developed as a good candidate to treat opioid dependence.The thienorphine’s glucuronide was detected after thienorphine was incubated with human liver microsomes (HLMs). Recombinant UGT isoforms screening experiment and enzyme kinetic study showed that UGT1A1 completely contributed to the glucuronidation of thienorphine.Among the tested UGT isoforms, UGT1A3 and UGT2B7 were inhibited by thienorphine, with other UGT isoforms negligibly influenced. The inhibition type is competitive, and inhibition kinetic parameters (Ki) were 1.65 and 5.27 μM for UGT1A3 and UGT2B7, respectively. However, due to low plasma concentration of thienorphine, in vivo drug–drug interaction might not occur.

The Inhibitory Effect of 20(S)‐Protopanaxatriol (ppt) Towards UGT1A1 and UGT2B7

Ginseng, a commonly used natural product, has been frequently reported to induce herb-drug interaction with many clinical drugs. The intestinal bacterial metabolites of ginsenosides have been widely regarded as the substance basis for ginseng-drug interactions. To date, little is known about the inhibitory effect of intestinal bacterial metabolites of ginsenosides towards UDP-glucuronosyltransferases (UGTs). In vitro investigation of the inhibition of 20(S)-protopanaxatriol (ppt) towards UGT1A1 and UGT2B7 was carried out. The results showed that ppt exhibited strong noncompetitive inhibition towards UGT1A1 and competitive inhibition towards UGT2B7. The inhibition kinetic parameters (Ki ) were calculated to be 8.8 and 2.2 μM for UGT1A1 and UGT2B7, respectively. Using the maximum plasma concentration of ppt, the alteration of area under the concentration-time curve was calculated to be 20% and 70% respectively for UGT1A1-mediated and UGT2B7-mediated metabolism. However, given that the varied contribution of these two UGT isoforms towards drug metabolism and the influence of herb complexity and individual difference, the explanation of these results should be paid more caution.

Strong Inhibition of Celastrol Towards UDP-Glucuronosyl Transferase (UGT) 1A6 and 2B7 Indicating Potential Risk of UGT-Based Herb-Drug Interaction

Celastrol, a quinone methide triterpene isolated from Tripterygium wilfordii Hook F., has various biochemical and pharmacological activities, and is now being developed as a promising anti-tumor agent. Inhibitory activity of compounds towards UDP-glucuronosyltransferase (UGT) is an important cause of clinical drug-drug interactions and herb-drug interactions. The aim of the present study is to investigate the inhibition of celastrol towards two important UDP-glucuronosyltransferase (UGT) isoforms UGT1A6 and UGT2B7. Recombinant UGT isoforms and non-specific substrate 4-methylumbelliferone (4-MU) were used. The results showed that celastrol strongly inhibited the UGT1A6 and 2B7-mediated 4-MU glucuronidation reaction, with 0.9 ± 0.1% and 1.8 ± 0.2% residual 4-MU glucuronidation activity at 100 μM of celastrol, respectively. Furthermore, inhibition kinetic study (Dixon plot and Lineweaver-Burk plot) demonstrated that celastrol noncompetitively inhibited the UGT1A1-mediated 4-MU glucuronidation, and competitively inhibited UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameters (Ki) were calculated to be 0.49 μM and 0.045 μM for UGT1A6 and UGT2B7, respectively. At the therapeutic concentration of celastrol for anti-tumor utilization, the possibility of celastrol-drug interaction and celastrol-containing herbs-drug interaction were strongly indicated. However, given the complicated nature of herbs, these results should be viewed with more caution.

Gossypol Exhibits a Strong Influence Towards UDP-Glucuronosyltransferase (UGT) 1A1, 1A9 and 2B7-Mediated Metabolism of Xenobiotics and Endogenous Substances

Gossypol, the polyphenolic constituent isolated from cottonseeds, has been used as a male antifertility drug for a long time, and has been demonstrated to exhibit excellent anti-tumor activity towards multiple cancer types. The toxic effects of gossypol limit its clinical utilization, and enzyme inhibition is an important facet of this. In the present study, in vitro human liver microsomal incubation system supplemented with UDPGA was used to investigate the inhibition of gossypol towards UGT1A1, 1A9 and 2B7-mediated metabolism of xenobiotics and endogenous substances. Estradiol, the probe substrate of UGT1A1, was selected as representative endogenous substance. Propofol (a probe substrate of UGT1A9) and 3'-azido-3'-deoxythimidine (AZT, a probe substrate of UGT2B7) were employed as representative xenobiotics. The results showed that gossypol noncompetitively inhibits UGT-mediated estradiol-3-glucuronidation and propofol O-glucuronidation, and the inhibition kinetic parameters (Ki) were calculated to be 34.2 and 16.4 μM, respectively. Gossypol was demonstrated to exhibit competitive inhibition towards UGT-mediated AZT glucuronidation, and the inhibition kinetic parameter (Ki) was determined to be 14.0 μM. All these results indicated that gossypol might induce metabolic disorders of endogenous substances and alteration of metabolic behaviour of co-administered xenobiotics through inhibition of UGTs’ activity.

Foot and mouth disease leader protease (Lbpro): Investigation of prime side specificity allows the synthesis of a potent inhibitor

Foot and mouth disease virus expresses its genetic information as a single polyprotein that is translated from the single-stranded RNA genome. Proteinases contained within the polyprotein then generate the mature viral proteins. The leader protease (Lbpro) performs the initial cleavage by freeing itself from the growing polypeptide chain; subsequently, Lbpro cleaves the two homologues of the host cell protein eukaryotic initiation factor 4G (eIF4G). We showed that Lbpro possesses specific binding sites at the non prime side from S1 down to S7 [Santos et al. (2009) Biochemistry, 48, 7948–7958]. Here, we demonstrate that Lbpro has high prime side specificity at least down to the S′5 site. Lbpro is thus not only one of the smallest papain-like cysteine peptidases but also one of the most specific. It can still however cleave between both K↓G and G↓R pairs. We further determined the two-step irreversible inhibition (E + I ↔ EI→ E − I) kinetic parameters of two known irreversible epoxide-based inhibitors of cysteine proteinases, E64 and CA074 on Lbpro that show for the reversible step (E + I ↔ EI) Ki = 3.4 μM and 11.6 μM, and for the irreversible step (EI→E−I) k4 = 0.16 and 0.06 min−1, respectively. Knowledge of the Lbpro specificity led us to extend E64 by addition of the dipeptide R–P. This compound, termed E64-R-P-NH2, irreversibly inhibited Lbpro with a Ki = 30 nM and k4 = 0.01 min−1 and can serve as the basis for design of specific inhibitors of FMDV replication.

Kinetic Studies for the Inhibition Effect of Pentachlorophenol on Alkaline Phosphatase Activity <i>In Vitro</i>

The inhibition kinetic parameters of pentachlorophenol (PCP) on bovine intestinal mucosa alkaline phosphatase (ALP) activity were studied by spectrophotometry. The results showed that PCP inhibited ALP activity in a concentration dependent manner, and the 50% inhibitory concentration (IC50) was estimated to be 4.20 mM. The apparent Michaelis–Menten constant (Km) and apparent maximum reaction rate (Vmax) was found to be decreased in the presence of PCP. Lineweaver–Burk plots indicated that the nature of the inhibition was of an uncompetitive type. Kinetic analysis indicated that the value of the inhibition constant (Ki) was 5.67 mM.

Determination of Time-Dependent Inactivation of CYP3A4 in Cryopreserved Human Hepatocytes and Assessment of Human Drug-Drug Interactions

Assessment of time-dependent inhibition (TDI), especially CYP3A4, is an important parameter for preclinical and clinical development. The use of human liver microsomes (HLM) is the most common in vitro matrix to assess TDI, but this often leads to an overprediction of an actual effect observed clinically. Recently, the use of human hepatocytes has been hypothesized as a more relevant and possibly predictive matrix for the assessment of CYP3A4 TDI. Our work evaluates and optimizes three different human hepatocyte assays for the assessment of CYP3A4 TDI using pooled cryopreserved human hepatocytes. Using two of the optimized methods, the time-dependent inhibition kinetic parameters (K(I) and k(inact)) for four known CYP3A4 TDI (diltiazem, erythromycin, verapamil, and troleandomycin) were determined. When comparing TDI in HLM, the K(I) values from hepatocytes were in general 4- to 13-fold higher than that in HLM, whereas the k(inact) values in human hepatocytes were similar or slightly higher or lower depending on the inhibitor. The inactivation potency (k(inact)/K(I)) for four tested CYP3A4 inactivators in human hepatocytes was generally lower than that in HLM due to either lower affinity (K(I)) or lower inactivation rate (k(inact)) or both. When drug interactions were simulated with Simcyp using either HLM or human hepatocyte data, the predictions using the kinetic parameters from human hepatocytes resulted in a much better simulated change in pharmacokinetics compared with observed clinical data.

3-[(4-amino-2- methyl-5-pyrimidinyl) methyl]-5- (2-hydroxyethyl)-4-methyl thiazolium chloride hydrochloride as green corrosion inhibitor of copper in HNO3 solution and its adsorption characteristics

The inhibitory effect of 3-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-5-(2-hydroxyethyl)-4-methyl thiazolium chloride hydrochloride (thiamine hydrochloride) or vitamin B1 hydrochloride on acid corrosion of copper in 2.5 M nitric acid (HNO3) solution was investigated using a weight loss method. Thiamine hydrochloride inhibited the corrosion of copper in 2.5 M HNO3 solutions. The inhibition efficiency increased with increasing concentration of thiamine hydrochloride but decreased with increasing exposure. Adsorption of thiamine hydrochloride molecules on copper surface was found to obey Langmuir adsorption isotherm with standard free energy of adsorption (Δ) –30.55 kJ mol−1. The inhibition mechanism was characterized from trends of inhibition efficiency, thermodynamic, and kinetic parameters.

Investigation of UDP‐glucuronosyltransferases (UGTs) Inhibitory Properties of Carvacrol

UDP-glucuronosyltransferases (UGTs), the most important phase II drug metabolizing enzymes (DMEs), could metabolize many drugs and various endogenous substances including bilirubin, steroid hormones, thyroid hormones, bile acids and fat-soluble vitamins. Evaluation of the inhibitory effects of compounds on UGTs is clinically important because inhibition of UGT isoforms could not only result in serious drug-drug interactions (DDIs), but also induce metabolic disorders of endogenous substances. The aim of the present study was to investigate the inhibitory effects of carvacrol on major UGT isoforms. The results showed that carvacrol could inhibit the activity of UGT1A9 with negligible effects on other UGT isoforms. When 4-methylumbelliferone (4-MU) was used as a nonspecific probe substrate and recombinant UGT enzymes were utilized as an enzyme resource, the inhibition of UGT1A9 was best fit to the competitive type and the inhibition kinetic parameter (K(i)) was calculated to be 5.7 µM. Furthermore, another specific probe substrate, propofol, was employed to determine the inhibitory kinetics of UGT1A9, and the results demonstrated that the inhibitory type was noncompetitive. The inhibition kinetic parameter (K(i)) was determined to be 25.0 µM. Because this substrate-dependent inhibition of UGT1A9 might confuse the in vitro-in vivo extrapolation, these in vitro inhibition kinetic parameters should be interpreted with special caution.

Kinetics of biological methane oxidation in the presence of non-methane organic compounds in landfill bio-covers

In this experimental program, the effects of non-methane organic compounds (NMOCs) on the biological methane (CH4) oxidation process were examined. The investigation was performed on compost experiments incubated with CH4 and selected NMOCs under different environmental conditions. The selected NMOCs had different concentrations and their effects were tested as single compounds and mixtures of compounds. The results from all experimental sets showed a decrease in CH4 oxidation capacity of the landfill bio-cover with the increase in NMOCs concentrations. For example, in the experiment using compost with 100% moisture content at 35 degrees C without any NMOCs the V(max) value was 35.0 microg CH4 h(-1)gwet wt(-1). This value was reduced to 19.1 microg CH4 h(-1) gwet wt(-1) when mixed NMOCs were present in the batch reactors under the same environmental conditions. The experimental oxidation rates of CH4 in the presence of single and mixed NMOCs were modeled using the uncompetitive inhibition model and kinetic parameters, including the dissociation constants, were obtained. Additionally, the degradation rates of the NMOCs and co-metabolic abilities of methanotrophic bacteria were estimated.

Acetylcholinesterase/Butyrylcholinesterase inhibition activity of some new carbacylamidophosphate deriviatives

Eight newly synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl2 with R = pCl–C6H4 1a, pBr–C6H4 2a, C6H5 3a, and pMe–C6H4 4a and RC(O)NHP(O)(NC4H8O)2 R = pCl–C6H4 1b, pBr–C6H4 2b, C6H5 3b, pMe–C6H4 4b, were selected to compare the inhibition kinetic parameters, IC50, Ki, kp and KD, on human erythrocyte acetylcholinesterase (hAChE) and bovine serum butyrylcholinesterase (BuChE), Also, the in vivo inhibition potency of compound 2a, 2b and 3a, were studied. The data demonstrates that compound 2a and compound 2b are the potent sensitive as AChE and BuChE inhibitors respectively, and the inhibition of hAChE is about 10-fold greater than that of BuChE.

Kinetics of the time-dependent inactivation of CYP2D6 in cryopreserved human hepatocytes by methylenedioxymethamphetamine (MDMA)

Methylenedioxymethamphetamine (MDMA) was investigated in cryopreserved human hepatocytes as a time-dependent inactivator (TDI) of CYP2D6 using dextromethorphan (DEX) as a probe substrate. Inhibition kinetic parameters k inact, the maximal rate of inactivation, and K I, the inhibitor concentration at half the maximal activation rate, were determined. Time- and concentration-dependent inhibition were confirmed, and the influence of different elements of study design (e.g. cell number, stability of hepatocytes, dilution after preincubation) on estimated kinetic parameters were evaluated. Dilution factors (DF) of 1.2, 5 or total removal of inhibitor (by washing cells after preincubation, WR) resulted in k inact and K I (±S.E.) values of 0.02 ± 0.002 min −1 and 0.88 ± 0.31 μM, 0.01 ± 0.001 min −1 and 1.23 ± 0.70 μM, and 0.01 ± 0.001 min −1 and 2.10 ± 1.32 μM, respectively; indicating that insufficient dilution may lead to overestimation of CYP2D6 inactivation. Accounting for MDMA depletion during the preincubation, corrected K I values were significantly lower (0.11 ± 0.05 μM, 0.15 ± 0.09 μM, 0.24 ± 0.16 μM for DF of 1.2, 5, and WR, respectively). Inactivation efficiency in hepatocytes, as measured by k inact/ K I, was 10-fold less than that previously reported in human liver microsomes or recombinantly expressed systems. Possible causes for the observed differences between in vitro systems warrant further investigation. These may include differences in metabolic consumption of MDMA in each system, non-specific binding and presence of active efflux in hepatocytes.