Mechanistic Framework to Predict Maternal-Placental-Fetal Pharmacokinetics of Nifedipine Employing Physiologically Based Pharmacokinetic Modeling Approach.

Nifedipine is used for treating mild to severe hypertension and preventing preterm labor in pregnant women. Nevertheless, concerns about nifedipine fetal exposure and safety are always raised. The aim of this study was to develop and validate a maternal-placental-fetal nifedipine physiologically based pharmacokinetic (PBPK) model and apply the model to predict maternal, placental, and fetal exposure to nifedipine at different pregnancy stages. A nifedipine PBPK model was verified with nonpregnant data and extended to the pregnant population after the inclusion of the fetoplacental multicompartment model that accounts for the placental tissue and different fetal organs within the Simcyp Simulator version 22. Model parametrization involved scaling nifedipine transplacental clearance based on Caco-2 permeability, and fetal hepatic clearance was obtained from in vitro to in vivo extrapolation encompassing cytochrome P450 3A7 and 3A4 activities. Predicted concentration profiles were compared with in vivo observations and the transplacental transfer results were evaluated using 2-fold criteria. The PBPK model predicted a mean cord-to-maternal plasma ratio of 0.98 (range, 0.86-1.06) at term, which agrees with experimental observations of 0.78 (range, 0.59-0.93). Predicted nifedipine exposure was 1.4-, 2.0-, and 3.0-fold lower at 15, 27, and 39 weeks of gestation when compared with nonpregnant exposure, respectively. This innovative PBPK model can be applied to support maternal and fetal safety assessment for nifedipine at various stages of pregnancy.

Total, Unbound, Renal, and Hepatic Clearances of Raltegravir and the Formation and Elimination Clearances of Raltegravir Glucuronide in Pregnant Women.

This work aimed to evaluate the total, unbound, renal, and hepatic clearances of raltegravir (RAL) and the formation and elimination clearances of raltegravir glucuronide (RAL GLU) in pregnant women living with HIV. The participants received RAL 400 mg twice daily during the third trimester (n = 15) of gestation, delivery (n = 15), and the postpartum period (n = 8). Pharmacokinetic parameter values were calculated on the basis of plasma and urine data using noncompartmental methods. RAL clearances for the third trimester of gestation were as follows: total clearance: geometric mean, 63.63 L/h (95% CI, 47.5-85.25); renal clearance: geometric mean, 2.56 L/h (95% CI, 1.96-3.34); hepatic clearance: geometric mean, 60.52 L/h (95% CI, 44.65-82.04); and unbound clearance: geometric mean, 281.14 L/h (95% CI, 203.68-388.05). RAL GLU formation and elimination clearances for the third trimester of gestation were 7.57 L/h (95% CI, 4.94-11.6) and 8.71 L/h (95% CI, 6.71-11.32), respectively. No differences were observed in RAL GLU pharmacokinetic parameters between the third trimester of gestation and the postpartum period, except for higher formation (7.57 vs 4.03 L/h) and elimination (8.71 vs 4.92 L/h) clearances during the third trimester. The findings based on plasma and urine data are consistent with an increase in the hepatic uridine 5' diphospho-glucuronosyltransferase isoenzymes activities involved in RAL metabolism during pregnancy, and the formation of RAL GLU is a minor route of RAL elimination. Compared to the postpartum period, in the third trimester of gestation, the similar RAL plasma exposure in pregnant women reinforces the maintenance of an RAL regimen including a 400-mg oral dose twice daily during pregnancy.

P-Glycoprotein and Organic Anion Transporter Polypeptide 1B/Breast Cancer Resistance Protein Drug Transporter Activity in Pregnant Women Living With HIV.

This study evaluates the influence of pregnancy and HIV infection in conjunction with the use of raltegravir, lamivudine, and tenofovir disoproxil fumarate (combined antiretroviral therapy [cART]) on intestinal P-glycoprotein (P-gp) and hepatic organic anion transporter polypeptide (OATP) 1B1/1B3 and/or breast cancer resistance protein (BCRP) drug transporter activity using rosuvastatin (OATP1B/BCRP) and fexofenadine (P-gp) probes. Single oral doses of 5-mg rosuvastatin and 60-mg fexofenadine were administered to women living with HIV under cART in the third trimester of gestation (n = 15) and postpartum period (n = 10). A control group of 12 healthy nonpregnant women also was investigated. Pharmacokinetic parameters were estimated by using a noncompartmental method and evaluated by t test (P < .05). The rosuvastatin area under the plasma concentration-time curve from time 0 to the last quantifiable concentration (AUC0-last ) value was higher in the third trimester of pregnancy (19.5 [95%CI, 16.8-22.3] ng • h/mL] when compared to postpartum (13.3 [95%CI, 9.3-17.5] ng • h/mL), while the fexofenadine AUC0-last values did not differ between the third trimester of pregnancy (738.0 [95%CI, 611.4-864.6] ng • h/mL) and postpartum period (874.9 [95%CI, 408.2-1342.0] ng• h/mL). The rosuvastatin AUC0-last values did not differ between healthy nonpregnant women (13.8 [95%CI, 10.0-17.6] ng • h/mL) and women living with HIV in the postpartum period (13.3 [95%CI, 9.3-17.5] ng • h/mL), and the fexofenadine AUC0-last values did not differ between the 2 investigated groups (603.6 [95%CI, 467.5-739.7] ng • h/mL vs 874.9 [95%CI, 408.2-1342.0] ng • h/mL). It is suggested that gestation inhibits the hepatic OATP1B1/1B3 and/or BCRP activity but does not alter intestinal P-gp activity. The influence of HIV infection in conjunction with use of cART on OATP1B/BCRP and intestinal P-gp activity was not observed.

Optimizing Therapeutic Drug Monitoring in Pregnant Women: A Critical Literature Review.

BACKGROUND: More than 90% of pregnant women take at least one drug during pregnancy. Drug dose adjustments during pregnancy are sometimes necessary due to various pregnancy-induced physiological alterations frequently associated with lower plasma concentrations. However, the clinical relevance or benefits of therapeutic drug monitoring (TDM) in pregnant women have not been specifically studied. Clinical pharmacokinetic studies in pregnant women are incredibly challenging for many reasons. Despite this, regulatory agencies have made efforts to encourage the inclusion of this population in clinical trials to achieve more information on the pharmacotherapy of pregnant women. This review aims to provide support for TDM recommendations and dose adjustments in pregnant women. METHODS: The search was conducted after a predetermined strategy on PubMed and Scopus databases using the MeSH term "pregnancy" alongside other terms such as "Pregnancy and dose adjustment," "Pregnancy and therapeutic drug monitoring," "Pregnancy and PBPK," "Pregnancy and pharmacokinetics," and "Pregnancy and physiological changes." RESULTS: The main information on TDM in pregnant women is available for antiepileptics, antipsychotics, antidepressants, antibiotics, antimalarials, and oncologic and immunosuppressive drugs. CONCLUSIONS: More data are needed to support informed benefit-risk decision making for the administration of drugs to pregnant women. TDM and/or pharmacokinetic studies could ensure that pregnant women receive an adequate dosage of an active drug. Mechanistic modeling approaches potentially could increase our knowledge about the pharmacotherapy of this special population, and they could be used to better design dosage regimens.

In vitro metabolism of the new antifungal dapaconazole using liver microsomes.

Dapaconazole is a new antifungal imidazole that has been shown a high efficacy against several pathogenic fungi. This study aimed to investigate the interspecies variation in the in vitro metabolic profiles and in vivo hepatic clearance (CLH,in vivo) prediction of dapaconazole using liver microsomes from male Sprague Dawley rat, male Beagle dog and mixed gender human using a liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method. In addition, the produced metabolites were identified by ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS/MS). The microsomal protein concentration of 0.1 mg/mL and the incubation time of 10 min were employed for the kinetics determination, resulting in a sigmoidal kinetic profile for all species evaluated. The predicted CLH,in vivo was 6.5, 11.6 and 7.5 mL/min/kg for human, rat and dog, respectively. Furthermore, five metabolized products were identified. These findings provide preliminary information for understanding dapaconazole metabolism and the interspecies differences in catalytic behaviours, supporting the choice of a suitable laboratory animal for future pharmacokinetics and metabolism studies.

Assessing the contribution of UGT isoforms on raltegravir drug disposition through PBPK modeling.

This work aimed to develop a physiologically based pharmacokinetic (PBPK) model for raltegravir accounting for UDP-glucuronosyltransferase (UGT) metabolism to assess the effect of UGT gene polymorphisms. Raltegravir elimination was evaluated using Km and Vmax values from human recombinant systems and UGT tissue scalar considering liver, kidney, and intestine. The predicted/observed ratios for raltegravir PK parameters were within a 2-fold error range in UGT1A1 poor and normal metabolizers, except in Asian UGT1A1 poor metabolizers. This PBPK modeling approach suggests that UGT1A3 is the main contributor to raltegravir's metabolism. UGT1A3 and UGT1A1 gene polymorphisms might have an additive effect on raltegravir's drug disposition and response. The final model accounting for hepatic, renal, and intestinal UGT metabolism, biliary clearance, and renal excretion improved model predictions compared with the previously published models. This PBPK model with the quantitative characterization of raltegravir elimination pathways can support dose adjustments in different clinical scenarios.

Prospective Prediction of Dapaconazole Clinical Drug-Drug Interactions Using an In Vitro to In Vivo Extrapolation Equation and PBPK Modeling.

This study predicted dapaconazole clinical drug−drug interactions (DDIs) over the main Cytochrome P450 (CYP) isoenzymes using static (in vitro to in vivo extrapolation equation, IVIVE) and dynamic (PBPK model) approaches. The in vitro inhibition of main CYP450 isoenzymes by dapaconazole in a human liver microsome incubation medium was evaluated. A dapaconazole PBPK model (Simcyp version 20) in dogs was developed and qualified using observed data and was scaled up for humans. Static and dynamic models to predict DDIs following current FDA guidelines were applied. The in vitro dapaconazole inhibition was observed for all isoforms investigated, including CYP1A2 (IC50 of 3.68 µM), CYP2A6 (20.7 µM), 2C8 (104.1 µM), 2C9 (0.22 µM), 2C19 (0.05 µM), 2D6 (0.87 µM), and 3A4 (0.008−0.03 µM). The dynamic (PBPK) and static DDI mechanistic model-based analyses suggest that dapaconazole is a weak inhibitor (AUCR > 1.25 and <2) of CYP1A2 and CYP2C9, a moderate inhibitor (AUCR > 2 and <5) of CYP2C8 and CYP2D6, and a strong inhibitor (AUCR ≥ 5) of CYP2C19 and CYP3A, considering a clinical scenario. The results presented may be a useful guide for future in vivo and clinical dapaconazole studies.

Pharmacokinetics of Benznidazole in Experimental Chronic Chagas Disease Using the Swiss Mouse-Berenice-78 Trypanosoma cruzi Strain Model.

Chronic Chagas disease might have an impact on benznidazole pharmacokinetics with potential alterations in the therapeutic dosing regimen. This study aims to investigate the influence of chronic Trypanosoma cruzi infection on the pharmacokinetics and biodistribution of benznidazole in mice. Healthy (n = 40) and chronically T. cruzi (Berenice-78 strain)-infected (n = 40) Swiss female 10-month-old mice received a single oral dose of 100 mg/kg of body weight of benznidazole. Serial blood, heart, colon, and brain samples were collected up to 12 h after benznidazole administration. The serum and tissue samples were analyzed using a high-performance liquid chromatography instrument coupled to a diode array detector. Chronic infection by T. cruzi increased the values of the pharmacokinetic parameters absorption rate constant (Ka ) (3.92 versus 1.82 h-1), apparent volume of distribution (V/F) (0.089 versus 0.036 liters), and apparent clearance (CL/F) (0.030 versus 0.011 liters/h) and reduced the values of the time to the maximum concentration of drug in serum (Tmax) (0.67 versus 1.17 h) and absorption half-life (t1/2a ) (0.18 versus 0.38 h). Tissue exposure (area under the concentration-versus-time curve from 0 h to time t for tissue [AUC0-t,tissue]) was longer and higher in the colon (8.15 versus 21.21 µg · h/g) and heart (5.72 versus 13.58 µg · h/g) of chronically infected mice. Chronic infection also increased the benznidazole tissue penetration ratios (AUC0-t,tissue/AUC0-t,serum ratios) of brain, colon, and heart by 1.6-, 3.25-, and 3-fold, respectively. The experimental chronic Chagas disease inflammation-mediated changes in the regulation of membrane transporters probably influence the benznidazole pharmacokinetics and the extent of benznidazole exposure in tissues. These results advise for potential alterations in benznidazole pharmacokinetics in chronic Chagas disease patients with possibilities of changes in the standard dosing regimen.

Chiral Discrimination of P-glycoprotein in Parturient Women: Effect of Fluoxetine on Maternal-Fetal Fexofenadine Pharmacokinetics.

BACKGROUND AND OBJECTIVE: Fluoxetine, antidepressant widely-used during pregnancy, is a selective inhibitor for P-glycoprotein (P-gp). Fexofenadine, an in vivo P-gp probe, is an antihistamine drug for seasonal allergic rhinitis and chronic urticaria treatment during pregnancy and it is available as a racemic mixture. This study evaluated the chiral discrimination of P-gp investigating the effect of fluoxetine on maternal-fetal pharmacokinetics of fexofenadine. METHODS: Healthy parturient women received either a single oral dose of 60 mg racemic fexofenadine (Control group; n = 8) or a single oral dose of 40 mg racemic fluoxetine 3 h before a single oral dose of 60 mg racemic fexofenadine (Interaction group; n = 8). Maternal blood and urine samples were collected up to 48 h after fexofenadine administration. At delivery, maternal-placental-fetal blood samples were collected. RESULTS: The maternal pharmacokinetics of fexofenadine was enantioselective (AUC0-∞R-(+)/S-(-) ~ 1.5) in both control and interaction groups. Fluoxetine increased AUC0-∞ (267.7 vs 376.1 ng.h/mL) and decreased oral total clearance (105.1 vs 74.4 L/h) only of S-(-)-fexofenadine, whereas the renal clearance were reduced for both enantiomers, suggesting that the intestinal P-gp-mediated transport of S-(-)-fexofenadine is influenced by fluoxetine to a greater extent that the R-(+)-fexofenadine. However, the transplacental transfer of fexofenadine is low (~16%), non-enantioselective and non-influenced by fluoxetine. CONCLUSIONS: A single oral dose of 40 mg fluoxetine inhibited the intestinal P-gp mediated transport of S-(-)-fexofenadine to a greater extent than R-(+)-fexofenadine in parturient women. However, the placental P-gp did not discriminate fexofenadine enantiomers and was not inhibited by fluoxetine.

A Pilot Study of the Maternal-Fetal Pharmacokinetics of Furosemide in Plasma, Urine, and Amniotic Fluid of Hypertensive Parturient Women Under Cesarean Section.

The third trimester of pregnancy is related to physiological changes that can modify the process of absorption, distribution, metabolism, and excretion and, consequently, the efficacy and toxicity of drugs. However, little is known about furosemide pharmacokinetics and placental transfer in pregnancy. This study evaluated the maternal-fetal pharmacokinetics and distribution to amniotic fluid of furosemide in hypertensive parturient women under cesarean section. Twelve hypertensive parturient women under methyldopa (250 mg/8 h) and/or pindolol (10 mg/12 h) treatment received a 40-mg single oral dose of furosemide 1 to 10 hours before delivery by cesarean section. Blood and urine samples were collected for 12 hours after furosemide administration. At delivery, samples were obtained from maternal and umbilical cord blood (n = 8) to assess the transplacental transfer. Amniotic fluid (n = 4) was collected at the time of delivery. The following furosemide pharmacokinetic parameters were obtained as median (interquartile range): Cmax , 403 ng/mL (229 to 715 ng/mL); Tmax , 2.00 hours (1.50 to 4.83 hours); elimination half-life (t1/2 ), 2.50 hours (1.77 to 2.97 hours); AUC0-12 h , 1366 ng⋅h/mL (927 to 2531 ng⋅h/mL); AUC0-∞ , 1580 ng⋅h/mL (1270 to 2881 ng⋅h/mL); CL/F 25.3 L/h (13.8 to 31.4 L/h); CLR, 2.50 L/h (1.77 to 2.97 L/h); CLNR, 22.7 L/h (12.1 to 25.6 L/h); and Vd /F 82.8 L (34.4 to 173 L). The transplacental transfer of furosemide was 0.43 (0.10 to 0.73), and the amniotic fluid concentration was 11.0 ng/mL (5.51 to 14.6 ng/mL). From a clinical point of view, these results suggest that substrates of uridine diphosphate-glucuronosyltransferase isoenzymes such as furosemide may have increased clearance during pregnancy and could require dose adjustment in this population.

Direct chiral LC-MS/MS analysis of fexofenadine enantiomers in plasma and urine with application in a maternal-fetal pharmacokinetic study.

This study shows the development and validation of two enantioselective LC-MS/MS methods for the determination of fexofenadine in biological matrices including the elution order determination. Plasma (200 µL) or urine (50 µL) aliquots were added to the internal standard solution [(S)-(-)-metoprolol] and extracted in the acid medium with chloroform. Resolution of the (R)-(+)- and (S)-(-)-fexofenadine enantiomers was performed in a Chirobiotic V column. The methods showed linearity at the range of 0.025-100 ng/mL plasma and 0.02-10 µg/mL urine for each fexofenadine enantiomer. These methods were applied to the maternal-fetal pharmacokinetics of fexofenadine enantiomers in plasma and urine of parturient women (n = 8) treated with a single oral 60 mg dose of racemic fexofenadine. Enantiomeric ratio in plasma (AUC0-∞(R)-(+)/(S)-(-)) was close to 1.5, nevertheless in urine was closed to unity. The transplacental transfer was approximately 18% for both fexofenadine enantiomers. The enantioselective methods can also be useful in future clinical studies of chiral discrimination of drug transporters.

A background subtraction approach for determination of endogenous cortisol and 6ß-hydroxycortisol in urine by UPLC-MS/MS with application in a within-day variability study in HIV-infected pregnant women.

Different methods have been used for CYP3A phenotyping, such as probe drugs or the urinary index 6ß-hydroxycortisol/cortisol ratio (6ß-OHF:C). This work describes a simple and affordable method for the simultaneous determination of the endogenous compounds cortisol and 6ß-hydroxycortisol in urine using a background subtraction approach. The method was applied to investigate the CYP3A activity in HIV-infected pregnant women (n = 9) in the third trimester and postpartum periods. Also, the within-day variability in the 6ß-OHF:C index was also evaluated. The sample preparation consists of a pre-cleanup with acetonitrile followed by liquid-liquid extraction with ethyl acetate. The analytes were resolved by employing an Acquity UPLC®BEH C18 column with a mobile phase that consisted of a mixture of acetonitrile containing 0.1% formic acid and 0.1% formic acid in gradient mode. The method presented linearities of 1-1.000 ng/mL and 2-1.000 ng/mL for C and 6ß-OHF, respectively, and presented acceptable precision and accuracy. Qualitative and quantitative matrix effects tests were also performed. A high 6ß-OHF:C within-day variability was observed in both phases. In the third trimester period, the 6ß-OHF:C ranged from 2.57 to 51.69, with a mean ± standard deviation (SD) of 15.12 ± 5.41 (n = 9). Similar values were obtained in the postpartum period, with 6ß-OHF:C ranging from 3.48 to 44.54 with a mean ± SD of 14.37 ± 5.73 (n = 7). Even though the 6ß-OHF:C is a non-invasive index for CYP3A phenotyping, its use is susceptible to high within-day variability.

Determination of raltegravir and raltegravir glucuronide in human plasma and urine by LC-MS/MS with application in a maternal-fetal pharmacokinetic study.

Raltegravir (RAL) is a HIV-integrase inhibitor recommended for treatment of HIV type 1 infection during pregnancy. The elimination of RAL to RAL glucuronide (RAL GLU) is mediated primarily by UDP glucuronosyltransferase 1A1 (UGT1A1). The present study shows the development and validation of 4 different methods for the analysis of RAL and RAL GLU in plasma and in urine samples. The methods were applied to evaluate the maternal-fetal pharmacokinetics of RAL and RAL GLU in a HIV-infected pregnant woman receiving RAL 400 mg twice daily. The sample preparation for RAL and RAL GLU analysis in 25 µL plasma and 100 µL diluted urine (10-fold with water containing 0.1% formic acid) were carried out by protein precipitation procedure. RAL and RAL GLU generate similar product mass fragments and require separation in the chromatographic system, so a suitable resolution was achieved for unchanged RAL and RAL GLU employing Ascentis Express C18 (75 × 4.6 mm, 2.7 µm) for both plasma and urine samples. The methods showed linearities at the ranges of 0.1-13.5 µg/mL RAL and 0.15-19.5 µg/mL RAL GLU in urine and 10-2000 ng/mL RAL and 2.5-800 RAL GLU in plasma. Precise and accurate evaluation showed coefficients of variation and relative errors ≤ 15%. The methods have been successfully applied in a maternal-fetal pharmacokinetic study.

Lopinavir/ritonavir treatment increases the placental transfer of bupivacaine enantiomers in human immunodeficiency virus-infected pregnant women.

AIMS: The present study evaluated the placental transfer and amniotic fluid distribution of bupivacaine enantiomers in health pregnant women and in human immunodeficiency virus (HIV)-infected pregnant women receiving epidural anaesthesia for caesarean section. METHODS: Twelve HIV-infected pregnant women (HIV group) were treated long-term (at least 8 weeks) with lopinavir/ritonavir (400/100 mg twice daily), and 12 healthy pregnant women (Control group) who submitted to epidural anaesthesia with racemic bupivacaine (75 mg) during caesarean section were investigated. At delivery, samples of maternal and fetal blood and amniotic fluid were collected (10-20 min after drug administration). RESULTS: The placental transfer ratio of bupivacaine enantiomers was significantly higher among the pregnant women from the HIV group when compared with those from the Control group (Mann-Whitney test, P ≤ 0.05). Placental transfer ratios (median and 25th - 75th percentiles) for (+)-(R)-bupivacaine were 0.58 (0.38-0.82) in the HIV group vs. 0.25 (0.18-0.33) in the Control group, and for (-)-(S)-bupivacaine, they were 0.54 (0.34-0.69) in the HIV group vs. 0.25 (0.19-0.29) in the Control group. The transplacental distribution of bupivacaine was stereoselective only in the HIV group. The umbilical artery/umbilical vein ratio and amniotic fluid/maternal vein ratio were low and nonstereoselective, and no statistically significant differences were observed between the groups. CONCLUSIONS: This study supports that the placental transfer of both bupivacaine enantiomers was 100% higher in HIV-pregnant women treated with lopinavir/ritonavir when compared with that in healthy pregnant women receiving epidural anaesthesia for caesarean section.

Leishmanicidal Effects of Piperlongumine (Piplartine) and Its Putative Metabolites.

Piperlongumine is an amide alkaloid found in Piperaceae species that shows a broad spectrum of biological properties, including antitumor and antiparasitic activities. Herein, the leishmanicidal effect of piperlongumine and its derivatives produced by a biomimetic model using metalloporphyrins was investigated. The results showed that IC50 values of piperlongumine in promastigote forms of Leishmania infantum and Leishmania amazonensis were 7.9 and 3.3 µM, respectively. The IC50 value of piperlongumine in the intracellular amastigote form of L. amazonensis was 0.4 µM, with a selectivity index of 25. The piperlongumine biomimetic derivatives, Ma and Mb, also showed leishmanicidal effects. We also carried out an in vitro metabolic degradation study showing that Ma is the most stable piperlongumine derivative in rat liver microsome incubations. The results presented here indicate that piperlongumine is a potential leishmanicidal candidate and support the biomimetic approach for development of new antileishmanial derivatives.

Analytical Methods for the Determination of Rosuvastatin in Pharmaceutical Formulations and Biological Fluids: A Critical Review.

Rosuvastatin calcium (ROS), ( Figure 1 ) belongs to the "statins" group, which is the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. This drug is indicated for dyslipidemias treatment and can help to decrease the level of "bad cholesterol" and can consequently reduce the development of atherosclerosis and the risk of heart diseases. ROS was developed by Astra-Zeneca and it was approved in 2003 by the FDA in the United States. In 2015, under the trade name Crestor®, it was the fourth largest selling drug in the United States with sales above $5 billion. This study presents a literature review of analytical methods for the quantification of ROS in pharmaceutical preparations and biological fluids. The major analytical methods described in this study for ROS were spectrophotometry, high-performance liquid chromatography (HPLC) coupled to ultraviolet (UV) detection, and tandem mass spectrometry (LC-MS/MS).

In Vitro Inhibition of Human CYP450s 1A2, 2C9, 3A4/5, 2D6 and 2E1 by Grandisin.

Grandisin, a lignan isolated from many species of plants, such as Virola surinamensis, is a potential drug candidate due to its biological properties, highlighted by its antitumor and trypanocidal activities. In this study, the inhibitory effects of grandisin on the activities of human cytochrome P450 enzymes were investigated by using human liver microsomes. Results showed that grandisin is a competitive inhibitor of CYP2C9 and a competitive and mechanism-based inhibitor of CYP3A4/5. The apparent Ki value for CYP2C9 was 50.60 µM and those for CYP3A4/5 were 48.71 µM and 31.25 µM using two different probe substrates, nifedipine and midazolam, respectively. The apparent KI, kinact, and kinact/KI ratio for the mechanism-based inhibition of CYP3A4/5 were 6.40 µM, 0.037 min-1, and 5.78 mL ·â€Šmin-1 µmol-1, respectively, by examining nifedipine oxidation, and 31.53 µM, 0.049 min-1, and 1.55 mL ·â€Šmin-1 µmol-1, respectively, by examining midazolam 1'-hydroxylation. These apparent kinact/KI values were comparable to or even higher than those for several therapeutic drugs that act as mechanism-based inhibitors of CYP3A4/5. CYP1A2 and CYP2D6 activities, in turn, were not substantially inhibited by grandisin (IC50 > 200 µM and 100 µM, respectively). In contrast, from a concentration of 4 µM, grandisin significantly stimulated CYP2E1 activity. These results improve the prediction of grandisin-drug interactions, suggesting that the risk of interactions with drugs metabolized by CYP3A4/5 and CYP2E1 cannot be overlooked.

A non-michaelian behavior of the in vitro metabolism of the pentacyclic triterpene alfa and beta amyrins by employing rat liver microsomes.

Pharmacological studies employing alpha and beta amyrin have demonstrated potential application in several biological activities suggesting their application as promising drugs. In the early drug development, metabolism studies may give important parameters regarding the efficacy and safety of the drug candidate. Therefore, the aim of this work was to determine the enzymatic kinetic parameters of these pentacyclic triterpenes. Chromatographic analyzes were performed using a Shimadzu GC-MS system. The resolution of amyrins was achieved with a DB5-MS column of 0.25 µM film thickness, 30.0 cm length and 0.25 mm diameter. At this condition, the retention times of beta- and alpha-amyrin were 21.3 and 20.2 min, respectively. The proposed method showed to be linear over the concentration range of 0.16-42.18 µM for beta amyrin and 0.11-28.12 µM for alpha amyrin. The lowest concentration quantified by the validated method was 0.16 µM for beta and 0.11 µM for alpha amyrin. The stability study showed that amyrins were stable at room temperature for 12h and at 37°C for 1h. The absolute recovery of the amyrin isomers from the rat microsome was 54.3-59.2%. The enzymatic kinetics presented sigmoidal plots. It was observed a Vmax=0.698 ± 0.022 µmol/mg protein/min, S50=4.4 µM and Hill coefficient of 2.7 ± 0.17 for alpha amyrin and a Vmax=0.775 ± 0.034 µmol/mg protein/min, S50=7.0 µM and Hill coefficient of 2.5 ± 0.21 for beta amyrin. The obtained results give the first clues regarding amyrin metabolism and suggests a more detailed study conducted employing isolated CYP isoforms.