Physiologic Changes During Pregnancy and Impact on Small-Molecule Drugs, Biologic (Monoclonal Antibody) Disposition, and Response.

Pregnancy is a unique physiological state that results in many changes in bodily function, including cellular, metabolic, and hormonal changes. These changes can have a significant impact on the way small-molecule drugs and monoclonal antibodies (biologics) function and are metabolized, including efficacy, safety, potency, and adverse effects. In this article, we review the various physiologic changes that occur during pregnancy and their effects on drug and biologic metabolism, including changes in the coagulation, gastrointestinal, renal, endocrine, hepatic, respiratory, and cardiovascular systems. Additionally, we discuss how these changes can affect the processes of drug and biologic absorption, distribution, metabolism, and elimination (pharmacokinetics), and how drugs and biologics interact with biological systems, including mechanisms of drug action and effect (pharmacodynamics) during pregnancy, as well as the potential for drug-induced toxicity and adverse effects in the mother and developing fetus. The article also examines the implications of these changes for the use of drugs and biologics during pregnancy, including consequences of suboptimal plasma drug concentrations, effect of pregnancy on the pharmacokinetics and pharmacodynamics of biologics, and the need for careful monitoring and individualized drug dosing. Overall, this article aims to provide a comprehensive understanding of the physiologic changes during pregnancy and their effects on drug and biologic metabolism to improve the safe and effective use of drugs.

Pharmacokinetic Evaluation in Pregnancy-Current Status and Future Considerations: Workshop Summary.

As pregnant individuals have traditionally been excluded from clinical trials, there is a gap in knowledge at the time of drug approval regarding safety, efficacy, and appropriate dosing for most prescription medications used during pregnancy. Physiologic changes in pregnancy can result in changes in pharmacokinetics that can impact safety or efficacy. This highlights the need to foster further research and collection of pharmacokinetic data in pregnancy to ensure appropriate drug dosing in pregnant individuals. Therefore, the US Food and Drug Administration and the University of Maryland Center of Excellence in Regulatory Science and Innovation hosted a workshop on May 16 and 17, 2022, titled "Pharmacokinetic Evaluation in Pregnancy." This is a summary of the workshop proceedings.

A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

PURPOSE: Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentration during the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach. METHODS: Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2 µM), estradiol (0.3 µM), estriol (0.8 µM), estrone (0.2 µM), 17α-hydroxyprogesterone (0.1 µM), and human growth hormone (0.005 µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC-MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively. RESULTS: Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used. CONCLUSION: The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.

Prediction of maternal and fetal pharmacokinetics of indomethacin in pregnancy.

AIMS: Indomethacin is used for the treatment of preterm labour, short cervices and idiopathic polyhydramnios during pregnancy. Few studies have described the pharmacokinetics (PK) of indomethacin during pregnancy. This study aimed to determine maternal and fetal PK of indomethacin during different trimesters of pregnancy using physiologically based PK (PBPK) modelling and simulations. METHODS: Full PBPK simulations were performed in nonpregnant subjects and pregnant subjects from each trimester of pregnancy at steady state using Simcyp's healthy volunteers and pregnancy PBPK model, respectively. The fetal exposures were predicted using a fetoplacental pregnancy PBPK model. The models were verified by comparing PBPK-based predictions with observed PK profiles. RESULTS: Predicted exposure (AUC0-6h ) and clearance of indomethacin in nonpregnant women and pregnant women are similar to the clinical observations. AUC0-6h of indomethacin is approximately 14, 24 and 32% lower, consistent with 18, 34 and 52% higher clearance in the first, second and third trimesters of pregnancy, respectively, compared to nonpregnant women. Predicted fetal plasma exposures increased by approximately 30% from the second trimester to the third trimester of pregnancy. CONCLUSION: A mechanistic PBPK model adequately described the maternal and the fetal PK of indomethacin during pregnancy. As the pregnancy progresses, a modest decrease (≤32%) in systemic exposures in pregnant women and a 33% increase in fetal exposures to indomethacin were predicted. Higher fetal exposures in the third trimester of pregnancy may pose safety risks to the fetus. Additional studies are warranted to understand the exposure-response relationship and provide appropriate dosing recommendations during pregnancy that consider both safety and efficacy.

An evaluation of the safety and preliminary efficacy of peri- and post-operative treprostinil in preventing ischemia and reperfusion injury in adult orthotopic liver transplant recipients.

BACKGROUND: Orthotopic liver transplantation (OLT) is the only treatment option for various end-stage liver diseases. Ischemia and reperfusion (I/R) injury is one of the unavoidable complications/conditions in OLT. In 2019, a total of 8896 livers were transplanted of which >94% organs were procured from deceased donors. An increase in the use of extended criteria donor (ECD) livers for transplantation further unraveled the role of hepatic I/R injury on short-term and long-term graft outcomes. Despite promising outcomes with the use of antioxidants, free radical scavengers, and vasodilators; I/R-mediated liver injury persists and significantly influences the overall clinical outcomes. Treprostinil, a synthetic prostacyclin I2  (PGI2 ) analog, due to its vasodilatory property, antiplatelet activity, and its ability to downregulate pro-inflammatory cytokines can potentially minimize I/R injury. AIM: We investigated the safety and preliminary efficacy of continuous intravenous infusion of treprostinil in liver transplant recipients in a prospective, single-center, non-randomized, interventional study. MATERIAL AND METHODS: This was a dose escalation (3 + 3 design) phase 1/2 study. Deceased donor liver transplant recipients received 5 ng/kg/min for two days, or 2.5, 5, and 7.5 ng/min/kg for 5 days as a continuous infusion. Multiple blood samples were collected for biochemical parameter assessment and for measuring treprostinil levels. Indocyanine green plasma disappearance rate was used as a measure of hepatic functional capacity. RESULTS: Subjects tolerated continuous infusion of treprostinil up to 5 ng/kg/min for 120 h with no occurrence of primary graft non-function (PNF), minimized need for ventilation support, reduced hospitalization time, 100% graft and patient survival, and improved hepatobiliary excretory function comparable to normal healthy adults. DISCUSSION: Treprostinil can be administered to liver transplant patients safely during the perioperative period. CONCLUSION: Based on this phase 1/2 study, further efficacy studies of treprostinil in preventing I/R injury of liver should be conducted to potentially increase the number of livers available for transplantation.

Translational Quantitative Systems Pharmacology in Drug Development: from Current Landscape to Good Practices.

Systems pharmacology approaches have the capability of quantitatively linking the key biological molecules relevant to a drug candidate's mechanism of action (drug-induced signaling pathways) to the clinical biomarkers associated with the proposed target disease, thereby quantitatively facilitating its development and life cycle management. In this review, the model attributes of published quantitative systems pharmacology (QSP) modeling for lowering cholesterol, treating salt-sensitive hypertension, and treating rare diseases as well as describing bone homeostasis and related pharmacological effects are critically reviewed with respect to model quality, calibration, validation, and performance. We further reviewed the common practices in optimizing QSP modeling and prediction. Notably, leveraging genetics and genomic studies for model calibration and validation is common. Statistical and quantitative assessment of QSP prediction and handling of model uncertainty are, however, mostly lacking as are the quantitative and statistical criteria for assessing QSP predictions and the covariance matrix of coefficients between the parameters in a validated virtual population. To accelerate advances and application of QSP with consistent quality, a list of key questions is proposed to be addressed when assessing the quality of a QSP model in hopes of stimulating the scientific community to set common expectations. The common expectations as to what constitutes the best QSP modeling practices, which the scientific community supports, will advance QSP modeling in the realm of informed drug development. In the long run, good practices will extend the life cycles of QSP models beyond the life cycles of individual drugs.

Correction to: FDA Approval of Angiotensin II for the Treatment of Hypotension in Adults with Distributive Shock.

The author name that previously read.

FDA Approval of Angiotensin II for the Treatment of Hypotension in Adults with Distributive Shock.

Distributive shock is a subset of shock marked by decreased systemic vascular resistance, organ hypoperfusion and altered oxygen extraction. Despite the use of intravenous fluids and either higher dose of catecholamines or other additional exogenous vasopressors to maintain blood pressure in the target range, the rate of mortality remains higher in patients with septic shock. Therefore, there is clearly an unmet need for additional safe and effective treatments. The use of angiotensin II to raise the mean arterial pressure (MAP) could provide additional therapy and the opportunity to evaluate a catecholamine-sparing effect by decreasing the dose of concomitant catecholamines while maintaining a target MAP. ATHOS-3 (Angiotensin II for the Treatment of High-Output Shock phase 3; ClinicalTrials.gov number, NCT02338843) was an adequate and well-controlled trial. The primary endpoint was the rate of MAP response at hour 3 of treatment with study drug, defined as either a 10-mmHg increase from baseline in MAP or a MAP of at least 75 mmHg. The secondary endpoints were changes from baseline in Sequential Organ Failure Assessment (SOFA) scores (total and cardiovascular). Mortality was an exploratory endpoint. The trial provided substantial evidence of the effectiveness of angiotensin II in raising blood pressure over placebo in patients with distributive shock, while keeping catecholamine levels constant. There was no change in the secondary endpoint of total SOFA scores relative to placebo when catecholamine use was reduced in lieu of angiotensin II treatment. There was a slight decrease in the secondary endpoint of cardiovascular SOFA score relative to placebo during the catecholamine-sparing phase, reflecting the catecholamine-sparing effect. There was a consistent trend in decreased mortality relative to placebo over the 28-day study period. Based on the agreements emanating from the special protocol assessment to assess blood pressure effects, the data from this single study supported approval of angiotensin II by the Food and Drug Administration for marketing in the USA.

Human hepatocyte assessment of imatinib drug-drug interactions - complexities in clinical translation.

AIM: Inducers and inhibitors of CYP3A, such as ritonavir and efavirenz, may be used as part of the highly active antiretroviral therapy (HAART) to treat HIV patients. HIV patients with chronic myeloid leukemia or gastrointestinal stromal tumour may need imatinib, a CYP3A4 substrate with known exposure response-relationships. Administration of imatinib to patients on ritonavir or efavirenz may result in altered imatinib exposure leading to increased toxicity or failure of therapy, respectively. We used primary human hepatocyte cultures to evaluate the magnitude of interaction between imatinib and ritonavir/efavirenz. METHODS: Hepatocytes were pre-treated with vehicle, ritonavir, ketoconazole, efavirenz or rifampicin, and the metabolism of imatinib was characterized over time. Concentrations of imatinib and metabolite were quantitated in combined lysate and medium, using LC-MS. RESULTS: The predicted changes in imatinib CLoral (95% CI) with ketoconazole, ritonavir, rifampicin and efavirenz were 4.0-fold (0, 9.2) lower, 2.8-fold (0.04, 5.5) lower, 2.9-fold (2.2, 3.5) higher and 2.0-fold (0.42, 3.5) higher, respectively. These predictions were in good agreement with clinical single dose drug-drug interaction studies, but not with reports of imatinib interactions at steady-state. Alterations in metabolism were similar after acute or chronic imatinib exposure. CONCLUSIONS: In vitro human hepatocytes predicted increased clearance of imatinib with inducers and decreased clearance with inhibitors of CYP enzymes. The impact of HAART on imatinib may depend on whether it is being initiated or has already been dosed chronically in patients. Therapeutic drug monitoring may have a role in optimizing imatinib therapy in this patient population.

Population pharmacokinetics of oseltamivir in non-pregnant and pregnant women.

AIMS: Physiological changes in pregnancy are expected to alter the pharmacokinetics of various drugs. The objective of this study was to evaluate systematically the pharmacokinetics of oseltamivir (OS), a drug used in the treatment of influenza during pregnancy. METHODS: A multicentre steady-state pharmacokinetic study of OS was performed in 35 non-pregnant and 29 pregnant women. Plasma concentration-time profiles were analyzed using both non-compartmental and population pharmacokinetic modelling (pop PK) and simulation approaches. A one compartment population pharmacokinetic model with first order absorption and elimination adequately described the pharmacokinetics of OS. RESULTS: The systemic exposure of oseltamivir carboxylate (OC, active metabolite of OS) was reduced approximately 30 (19-36)% (P < 0.001) in pregnant women. Pregnancy significantly (P < 0.001) influenced the clearance (CL/F) and volume of distribution (V/F) of OC. Both non-compartmental and population pharmacokinetic approaches documented approximately 45 (23-62)% increase in clearance (CL/F) of OC during pregnancy. CONCLUSION: Based on the decrease in exposure of the active metabolite, the currently recommended doses of OS may need to be increased modestly in pregnant women in order to achieve comparable exposure with that of non-pregnant women.

Potential interactions between HIV drugs, ritonavir and efavirenz and anticancer drug, nilotinib--a study in primary cultures of human hepatocytes that is applicable to HIV patients with cancer.

Nilotinib is used to treat chronic myeloid leukemia (CML), and is metabolized by CYP3A. With a black-box warning for QT prolongation, which is exposure dependent, controlling for drug interactions is clinically relevant. Treatments of HIV patients with CML are with HAART drugs, ritonavir and efavirenz, may cause complex drug interactions through CYP3A inhibition or induction. We evaluated the interactions of ritonavir or efavirenz on nilotinib using human hepatocytes and compared these interactions with those of ketoconazole or rifampin, classical CYP3A inhibitor and inducer, respectively. Hepatocytes were treated with vehicle, ritonavir (10 µM), ketoconazole (10 µM), efavirenz (10 µM), or rifampin (10 µM) for 5 days. On day 5, nilotinib (3 µM) was coincubated for an additional 24-48 hours. The concentrations of nilotinib were quantitated in collected samples (combined lysate and medium) by LC-MS. Apparent intrinsic clearance (CL(int,app)) of nilotinib was lowered 5.8- and 3.1-fold, respectively, by ritonavir and ketoconazole. Efavirenz and rifampin increased the CL(int,app) of nilotinib by 2.1- and 4.1-fold, respectively. The clinically recommended dose (300 mg twice daily) of nilotinib may have to be reduced substantially (150 mg once daily) or increased (400 mg thrice daily), respectively, to achieve desired drug exposure, when ritonavir or efavirenz is co-administered.

A microphysiological system model of therapy for liver micrometastases.

Metastasis accounts for almost 90% of cancer-associated mortality. The effectiveness of cancer therapeutics is limited by the protective microenvironment of the metastatic niche and consequently these disseminated tumors remain incurable. Metastatic disease progression continues to be poorly understood due to the lack of appropriate model systems. To address this gap in understanding, we propose an all-human microphysiological system that facilitates the investigation of cancer behavior in the liver metastatic niche. This existing LiverChip is a 3D-system modeling the hepatic niche; it incorporates a full complement of human parenchymal and non-parenchymal cells and effectively recapitulates micrometastases. Moreover, this system allows real-time monitoring of micrometastasis and assessment of human-specific signaling. It is being utilized to further our understanding of the efficacy of chemotherapeutics by examining the activity of established and novel agents on micrometastases under conditions replicating diurnal variations in hormones, nutrients and mild inflammatory states using programmable microdispensers. These inputs affect the cues that govern tumor cell responses. Three critical signaling groups are targeted: the glucose/insulin responses, the stress hormone cortisol and the gut microbiome in relation to inflammatory cues. Currently, the system sustains functioning hepatocytes for a minimum of 15 days; confirmed by monitoring hepatic function (urea, α-1-antitrypsin, fibrinogen, and cytochrome P450) and injury (AST and ALT). Breast cancer cell lines effectively integrate into the hepatic niche without detectable disruption to tissue, and preliminary evidence suggests growth attenuation amongst a subpopulation of breast cancer cells. xMAP technology combined with systems biology modeling are also employed to evaluate cellular crosstalk and illustrate communication networks in the early microenvironment of micrometastases. This model is anticipated to identify new therapeutic strategies for metastasis by elucidating the paracrine effects between the hepatic and metastatic cells, while concurrently evaluating agent efficacy for metastasis, metabolism and tolerability.

Ritonavir and efavirenz significantly alter the metabolism of erlotinib--an observation in primary cultures of human hepatocytes that is relevant to HIV patients with cancer.

Erlotinib is approved for the treatment of non-small cell lung and pancreatic cancers, and is metabolized by CYP3A4. Inducers and inhibitors of CYP3A enzymes such as ritonavir and efavirenz, respectively, may be used as part of the highly active antiretroviral therapy drugs to treat patients with human immunodeficiency virus (HIV). When HIV patients with a malignancy need treatment with erlotinib, there is a potential of as-yet-undefined drug-drug interaction. We evaluated these interactions using human hepatocytes benchmarked against the interaction of erlotinib with ketoconazole and rifampin, the archetype cytochrome P450 inhibitor and inducer, respectively. Hepatocytes were treated with vehicle [0.1% dimethylsulfoxide, ritonavir (10 µM)], ketoconazole (10 µM), efavirenz (10 µM), or rifampin (10 µM) for 4 days. On day 5, erlotinib (5 µM) was incubated with the above agents for another 24-48 hours. Concentrations of erlotinib and O-desmethyl erlotinib were quantitated in collected samples (combined lysate and medium) using liquid chromatography and tandem mass spectrometry. The half-life (t(½)) of erlotinib increased from 10.6 ± 2.6 to 153 ± 80 and 23.9 ± 4.8 hours, respectively, upon treatment with ritonavir and ketoconazole. The apparent intrinsic clearance (C(Lint, app)) of erlotinib was lowered 16-fold by ritonavir and 1.9-fold by ketoconazole. Efavirenz and rifampin decreased t1/2 of erlotinib from 10.3 ± 1.1 to 5.0 ± 1.5 and 3.4 ± 0.2 hours, respectively. Efavirenz and rifampin increased the C(Lint, app) of erlotinib by 2.2- and 2-fold, respectively. Our results suggest that to achieve desired drug exposure, the clinically used dose (150 mg daily) of erlotinib may have to be significantly reduced (25 mg every other day) or increased (300 mg daily), respectively, when ritonavir or efavirenz is coadministered.

Population pharmacokinetics of cyclosporine in transplant recipients.

A number of classical pharmacokinetic studies have been conducted in transplant patients. However, they suffer from some limitations, for example, (1) the study design was limited to intense blood sampling in small groups of patients during a certain posttransplant period, (2) patient factors were evaluated one at a time to identify their association with the pharmacokinetic parameters, and (3) mean pharmacokinetic parameters often cannot be precisely estimated due to large intraindividual variability. Population pharmacokinetics provides a potential means of addressing these limitations and is a powerful tool to evaluate the magnitude and consistency of drug exposure. Population pharmacokinetic studies of cyclosporine focused solely on developing limited sampling strategies and Bayesian estimators to estimate drug exposure, have been summarized before, and are, therefore, not a subject of this review. The major focus of this review is to describe factors (demographic factors, hepatic and gastrointestinal functions, drug-drug interactions, genetic polymorphisms of drug metabolizing enzymes and transporters) that have been identified to contribute to the large portion of observed variability in the pharmacokinetics of cyclosporine in transplant patients. This review summarizes and interprets the conclusions as well as the nonlinear mixed-effects modeling methodologies used in such studies. A highly diversified collection of structural models, variability models, and covariate submodels have been evaluated and validated using internal or external validation methods. This review also highlights areas where additional research is warranted to improve the models since a portion of model variability still remains unexplained.

A sensitive and specific CYP cocktail assay for the simultaneous assessment of human cytochrome P450 activities in primary cultures of human hepatocytes using LC-MS/MS.

A sensitive and specific CYP cocktail assay for simultaneous measurement of the activities of major human cytochrome P450 enzymes (CYP1A2 (phenacetin), CYP3A4/5 (midazolam), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin) and CYP2D6 (dextromethorphan)) in primary cultures of human hepatocytes, was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hepatocyte incubation medium was processed by a solid phase extraction (SPE) using Oasis SPE extraction cartridges prior to chromatography. The metabolites derived from each of the substrates were simultaneously quantitated using the corresponding stable isotope-labeled internal standards by a positive electrospray ionization mode using multiple reactions monitoring with a single eight minute run. The mean accuracy was in the range of 98-114%. The interday and intraday precision over the concentration ranges evaluated for all the analytes were lower than 15%, and 14%, respectively. All the generated metabolites were stable under the conditions used for sample analysis. Additionally, the interaction of a cocktail substrate on other CYP substrates was also analyzed. Due to substantial inter-substrate interaction, chlorzoxazone (CYP2E1) and bupropion (CYP2B6) were removed from the initial seven probes CYP cocktail assay. Therefore, the final CYP cocktail assay consisting of five probes provides a robust method to simultaneously measure activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5 in primary cultures of human hepatocytes.

All-human microphysical model of metastasis therapy.

The vast majority of cancer mortalities result from distant metastases. The metastatic microenvironment provides unique protection to ectopic tumors as the primary tumors often respond to specific agents. Although significant interventional progress has been made on primary tumors, the lack of relevant accessible model in vitro systems in which to study metastases has plagued metastatic therapeutic development--particularly among micrometastases. A real-time, all-human model of metastatic seeding and cancer cells that recapitulate metastatic growth and can be probed in real time by a variety of measures and challenges would provide a critical window into the pathophysiology of metastasis and pharmacology of metastatic tumor resistance. To achieve this we are advancing our microscale bioreactor that incorporates human hepatocytes, human nonparenchymal liver cells, and human breast cancer cells to mimic the hepatic niche in three dimensions with functional tissue. This bioreactor is instrumented with oxygen sensors, micropumps capable of generating diurnally varying profiles of nutrients and hormones, while enabling real-time sampling. Since the liver is a major metastatic site for a wide variety of carcinomas and other tumors, this bioreactor uniquely allows us to more accurately recreate the human metastatic microenvironment and probe the paracrine effects between the liver parenchyma and metastatic cells. Further, as the liver is the principal site of xenobiotic metabolism, this reactor will help us investigate the chemotherapeutic response within a metabolically challenged liver microenvironment. This model is anticipated to yield markers of metastatic behavior and pharmacologic metabolism that will enable better clinical monitoring, and will guide the design of clinical studies to understand drug efficacy and safety in cancer therapeutics. This highly instrumented bioreactor format, hosting a growing tumor within a microenvironment and monitoring its responses, is readily transferable to other organs, giving this work impact beyond the liver.

Effect of voriconazole and other azole antifungal agents on CYP3A activity and metabolism of tacrolimus in human liver microsomes.

Azole antifungal agents are known to inhibit cytochrome P450 3A (CYP3A) enzymes. Limited information is available regarding the effect of voriconazole on CYP3A activity. We examined the effect of voriconazole on CYP3A activity in human liver microsomes as measured by the formation of 6ß-hydroxytestosterone from testosterone. We also evaluated the interaction between voriconazole and tacrolimus, an immunosuppressive drug, using human liver microsomes. The effect of voriconazole on CYP3A activity and tacrolimus metabolism was compared to that of other azole antifungal agents. CYP3A4 activity and the metabolism of tacrolimus were measured in the absence and in the presence of various concentrations of voriconazole (0-1.43 mM), fluconazole (0-1.63 mM), itraconazole (0-14 µM) and ketoconazole (0-0.19 µM). At a concentration of 21.2 ± 15.4 µM and 29.8 ± 12.3 µM, voriconazole inhibited the formation of 6ß-hydroxytestosterone from testosterone and the metabolism of tacrolimus by 50%, respectively. The rank order of inhibition of 6ß-hydroxytestosterone formation from testosterone and the metabolism of tacrolimus, is ketoconazole > itraconazole > voriconazole > fluconazole. Our observations suggest that voriconazole at clinically relevant concentrations will inhibit the hepatic metabolism of tacrolimus and increase the concentration of tacrolimus more than two-fold. Close monitoring of the blood concentrations and adjustment in the dose of tacrolimus are warranted when transplant patients receiving tacrolimus are treated with voriconazole.

Effects of transient overexpression or knockdown of cytochrome P450 reductase on reactive oxygen species generation and hypoxia reoxygenation injury in liver cells.

1. Literature data suggest that the electron-donating enzyme, cytochrome P450 reductase (CPR), might act as a source of reactive oxygen species (ROS). However, the role of CPR in pathophysiological conditions associated with oxidative stress is unknown. The aim of the present study was to study the role of CPR in the generation of ROS and cellular injury under basal conditions, and after simulated in vitro ischaemia-reperfusion (IR). 2. Plasmid DNA or siRNA approaches were used to transiently overexpress or knockdown the human CPR gene in rat liver epithelial (WB-F344) or human hepatoblastoma (HepG2) cells, respectively. The generation of ROS and/or cellular injury was then studied under the basal conditions and after simulated IR (4 h of ischaemia plus 30 min of reoxygenation). 3. Under the basal conditions, transient overexpression of CPR protein in WB-F344 cells caused a 90% increase in the CPR activity, which was associated with a 100% increase in the ROS production. In contrast, after simulated IR, a 2.5-fold higher CPR activity did not significantly affect the magnitude of ROS generation or cell death. Similarly, although the knockdown of CPR protein resulted in a significant reduction (∼30%) in the CPR activity, the ROS production was not substantially altered after simulated IR in HepG2 cells. 4. Our data suggest that CPR plays a major role in the ROS generation by liver cells under the basal conditions. However, the role of CPR in the ROS generation during simulated in vitro IR injury in these cells is minimal, if any.

Inhibition of NADPH-cytochrome P450 reductase by tannic acid in rat liver microsomes and primary hepatocytes: methodological artifacts and application to ischemia-reperfusion injury.

Tannic acid (TA) inhibits nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) activity, which is measured by reduction of cytochrome c, in rat liver microsomes (RLMs). In the current study, we noticed that TA directly reduces cytochrome c in the absence of microsomes, thus confounding the CPR activity assay. A method is presented that measures CPR activity in the presence of TA by subtracting the cytochrome c reduction in the absence of NADPH (TA effect) from that in the presence of NADPH (TA plus CPR effect). The method was used to determine the inhibitory effect of TA in RLMs, recombinant CPR enzyme, and primary hepatocytes. Additionally, application of TA in a study of role of CPR in a primary rat hepatocyte model of ischemia-reperfusion (IR) was investigated. TA showed concentration-dependent, complete inhibition of CPR with half maximal inhibitory concentration (IC(50) ) values of 58.2 µM in RLMs and 54.6 and 275 µM in primary rat hepatocytes in the absence and presence of serum in the medium, respectively. Additionally, inhibition of CPR by TA was associated with a significant reduction in reactive oxygen species and cell death after IR injury. These data may be useful in future studies using TA as an inhibitor of CPR in microsomes and primary hepatocytes.

Delivery of NADPH-cytochrome P450 reductase antisense oligos using avidin-biotin approach.

Although avidin-mediated intracellular delivery of oligonucleotides or proteins has been shown before, the efficacy studies are lacking. Here, we tested the effectiveness of avidin for delivery of a cytochrome P450 reductase (CPR) antisense oligo in rat liver epithelial cells. A phosphorodiamidate morpholino oligo (PMO) against CPR was biotinylated using four reagents with short, cleavable, or long linkers, followed by conjugation with avidin. The dose-inhibitory response of the unmodified PMO in the presence of a transfection reagent (Endoporter, EP) and the effectiveness of the EP-assisted and avidin-assisted delivery of biotinylated PMOs were tested by Western blot analysis. Additionally, in a preliminary study, the avidin-biotin PMO with a long linker was also tested in vivo in rats. The biotinylated oligos were at least as effective as the unmodified oligo. Whereas the avidin conjugate of biotinylated PMO with the short linker was ineffective, those with the long linkers showed significant reductions in CPR protein expression. Finally, the in vivo study showed modest, but significant, reductions in CPR activity. In conclusion, these studies show for the first time that avidin-mediated intracellular delivery of biotinylated oligos can effectively knock down target genes in vitro, depending on the length of the linker. Additionally, the avidin-biotin approach may be of potential value for in vivo gene knockdown.