Selection of a suitable animal model to evaluate secretion of drugs in the human milk: a systematic approach.

Lack of data on drug secretion in human milk is a concern for safe use of drugs during postpartum.Clinical studies are often difficult to perform; despite substantial improvements in computational methodologies such as physiologically based pharmacokinetic modelling, there is limited clinical data to validate such models for many drugs.Various factors that are likely to impact milk to plasma ratio were identified. A literature search was performed to gather available data on milk composition, total volume of milk produced per day, milk pH, haematocrit, and renal blood flow and glomerular filtration rate in various animal models.BLAST nucleotide and protein tools were used to evaluate the similarities between humans and animals in the expression and predominance of selected drug transporters, metabolic enzymes, and blood proteins.A multistep analysis of all the potential variables affecting drug secretion was considered to identify most appropriate animal model. The practicality of using the animal in a lab setting was also considered.Donkeys and goats were identified as the most suitable animals for studying drug secretion in milk and future studies should be performed in goats and donkeys to validate the preliminary observations.

Intra-amniotic sildenafil administration in rabbits: Safety, pharmacokinetics, organ distribution and histologic evaluation.

BACKGROUND: The effectiveness of sildenafil in the management of pulmonary hypertension in congenital diaphragmatic hernia (CDH) has been reported but has not been systematically evaluated. Our studies have also demonstrated that intra-amniotic (IA) sildenafil administration improves pulmonary hypertension in CDH. METHODS: We evaluated the pharmacokinetics of sildenafil after IA administration in pregnant rabbits. Following maternal laparotomy, fetuses received IA injection of 0.8 mg of sildenafil. Maternal blood, amniotic fluid, and fetal tissues were collected at various time points. The concentrations of sildenafil and its major metabolite in samples were analyzed by liquid chromatography-mass spectrometry. To assess organ toxicity, 7 days after IA sildenafil administration, fetal organs were examined histologically. RESULTS: After IA dosing, sildenafil was absorbed quickly with an absorption half-life of 0.03-0.07 h into the fetal organs. All the organs showed a maximum concentration within 1 h and the disposition half-life ranged from 0.56 to 0.73 h. Most of the sildenafil was eliminated from both mothers and fetuses within 24 h after a single dose. There was no histological evidence of organ toxicity in the fetuses after a single dose of IA administration of sildenafil. CONCLUSION: IA sildenafil is rapidly absorbed into the fetus, distributes into the mother, and is eliminated by the mother without accumulation or fetal organ toxicity. This study confirms the feasibility and the safety of IA administration of sildenafil and enables future applications in the treatment of CDH fetuses.

Assessment of glomerular filtration and tubular secretion in baboons with life-supporting pig kidney grafts.

With pig kidney xenotransplantation nearing clinical reality, it is imperative to measure pig kidney function in the graft recipients. Our aims were (i) to compare inulin clearance after a short intravenous (IV) bolus with steady-state inulin IV infusion, (ii) to use this method to measure the glomerular filtration rate (GFR), and (iii) to determine the tubular secretory function using cefoxitin in a pig-to-baboon renal transplant model. A short IV infusion of inulin and cefoxitin were followed by a maintenance IV infusion of inulin over 5 h in seven healthy baboons, three healthy pigs, and five baboons after bilateral native nephrectomy and intra-abdominal pig renal transplantation. Blood and urine samples were collected. Serum and urinary inulin and serum cefoxitin concentrations measured by validated assays were used to calculate GFR and renal secretion. GFR calculated were similar by both methods. The body weight normalized total body clearance of inulin was similar in pigs and baboons despite differences in absolute clearances. Pig kidney transplanted into baboons provided similar clearance in baboons when normalized to baboon body weight and sustained filtration and secretory functions. The study documented that pig kidneys support the physiologic needs of baboons and are likely to support human recipients as well.

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.

Effect of formulation and route of administration on the distribution of 17-hydroxyprogesterone caproate in rats.

Weekly intramuscular (250 mg/week) or subcutaneous (275 mg/week) injections of 17-hydroxyprogesterone caproate (17-OHPC) is the only treatment option for the prevention of preterm birth in women with a prior history of preterm delivery.The objective of the current study was to determine the relative distribution of 17-OHPC in selected tissues in adult female SD rats after IM (oily formulation or solution), IV (solution), PO (solution), or intravaginal (suppository) administration.Plasma, uterus, adipose, and liver samples were collected at various times and analysed by LC-MS-MS.The highest concentrations of 17-OHPC were observed in the adipose tissue, after IM (oily formulation), and intravaginal administration.Substantial concentrations of 17-OHPC were also observed in the uterus after IM, intravaginal and IV administration.17-OHPC was not detected in the liver and in any of the tissues tested after PO administration.17-OHPC levels in plasma after intravaginal suppository administration were low despite substantial concentrations in the adipose and the uterus.The distribution of 17-OHPC depends on the formulation, the route of administration, and the sampling time.Low systemic concentrations and substantial distribution in the tissues of interest after intravaginal administration warrants future studies to evaluate the potential of the daily intravaginal route of administration of 17-OHPC.

Treprostinil Supplementation Ameliorates Hepatic Ischemia Reperfusion Injury and Regulates Expression of Hepatic Drug Transporters: An Isolated Perfused Rat Liver (IPRL) Study.

PURPOSE: IR injury is an unavoidable consequence in deceased donor liver transplantation. Cold preservation and warm reperfusion may change the expression and function of drug transporters in the liver due to vasoconstriction, infiltration of neutrophils and release of cytokines. We hypothesize that vasodilation, anti-platelet aggregation and proinflammatory downregulation activities of treprostinil will diminish the IR injury and its associated effects. METHODS: Livers obtained from male SD rats (n = 20) were divided into 1) Control, 2) IR, 3) Treprostinil-1 (preservation only), and 4) Treprostinil-2 (preservation and reperfusion) groups. Control livers were procured and immediately reperfused. Livers in the other groups underwent preservation for 24 h and were reperfused. All the livers were perfused using an Isolated Perfused Rat Liver (IPRL) system. Periodic perfusate, cumulative bile samples and liver tissue at the end of perfusion were collected. Liver injury markers, bile flow rates, m-RNA levels for uptake and efflux transporters (qRT-PCR) were measured. RESULTS: Cold preservation and warm reperfusion significantly increased the release of AST and ALT in untreated livers. Treprostinil supplementation substantially reduced liver injury. Bile flow rate was significantly improved in treprostinil-2 group. m-RNA levels of Slc10a1, Slc22a1, and Slc22a7 in liver were increased and m-RNA levels of Mdr1a were decreased by IR. Treprostinil treatment increased Abcb11 and Abcg2 m-RNA levels and maintained Slc22a1m-RNA similar to control livers. CONCLUSIONS: Treprostinil treatment significantly reduced liver injury. IR injury changed expression of both uptake and efflux transporters in rat livers. Treprostinil significantly altered the IR injury mediated changes in m-RNA expression of transporters.

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.

Pregnancy Alters CYP- and UGT-Mediated Metabolism of Buprenorphine.

BACKGROUND: In the United States, drug addiction has become a nationwide health crisis. Recently, buprenorphine (BUP), a maintenance therapy approved by the Food and Drug Administration, has been increasingly used in pregnant women for the treatment of opioid use disorder. Pregnancy is associated with various anatomic and physiological changes, which may result in altered drug pharmacokinetics (PKs). Previously, we reported that dose-adjusted plasma concentrations of BUP are lower during pregnancy than after pregnancy. The mechanism(s) responsible for this difference has not yet been defined. Our study aimed to evaluate alterations in cytochromes P450 (CYP)- and uridine diphosphate glucunosyltransferases (UGT)-mediated metabolism of BUP during pregnancy to determine the mechanism(s) responsible for this observation. METHODS: Data from 2 clinical studies were included in the current analysis. Study 1 was a prospective, open-labeled, nonrandomized longitudinal BUP PK study in pregnant women with a singleton gestation, stabilized on twice-daily sublingual BUP opioid substitution therapy. Each subject participated in up to 3 studies during and after pregnancy (the second, third trimester, and postpartum). The design of study 2 was similar to study 1, with patients evaluated at different time points during the pregnancy (first, second-half of pregnancy), as well as during the postpartum period. In addition, the dosing frequency of BUP study 2 participants was not restricted to twice-daily dosing. At each study visit, blood samples were collected before a BUP dose, followed by multiple collection times (10-12) after the dose, for up to 12 hours or till the end of the dosing interval. Plasma concentrations of BUP and 3 metabolites were quantified using validated ultraperformance liquid chromatography-tandem mass spectrometric assays. RESULTS: In total, 19, 18, and 14 subjects completed the PK study during 1/2 trimester, third trimester, and postpartum, respectively. The AUC ratios of norbuprenorphine and norbuprenorphine glucuronide to buprenorphine, a measure of CYP3A mediated N-demethylation, were 1.89, 1.84, and 1.33 during the first and second, third trimesters, and postpartum, respectively. The AUC ratios of buprenorphine glucuronide to BUP, indicative of UGT activity, were 0.71, 2.07, and 0.3 at first/second trimesters, third trimester, and postpartum, respectively. Linear mixed-effect modeling analysis indicated that the AUC ratios of CYP- and UGT-mediated metabolism of BUP were significantly higher during pregnancy compared with postpartum. CONCLUSIONS: The CYP and UGT activities were significantly increased as determined by the metabolic ratios of BUP during pregnancy compared with the postpartum period. The increased UGT activity appeared to account for a substantial part of the observed change in metabolic activity during pregnancy. This is in agreement with the need for BUP dose increment in pregnant women to reach similar BUP exposure and therapeutic effect as in nonpregnant subjects.

Route of administration and formulation dependent pharmacokinetics of 17-hydroxyprogesterone caproate in rats.

1. Weekly intramuscular injections of (250 mg/week) of 17-hydroxyprogesterone caproate (17-OHPC) are the only treatment option for prevention of preterm birth in women with a prior history of preterm delivery. 2. The objective of the current study was to evaluate the use of an alternate formulation and the feasibility of an alternate route of administration of this agent. 17-OHPC was administered to adult female SD rats, as marketed oily formulation intramuscularly, or as a solution IV, IM, or PO. 3. Plasma concentrations of 17-OHPC were measured by LC-MS-MS and pharmacokinetic parameters were calculated by non-compartmental analysis, using WinNonLin (Certara, St. Louis, MO). 4. After IV or IM administration as a solution, the mean half-life of 17-OHPC was around 11 h. The bioavailability was nearly 100% after IM administration, but was very low (<3%) after PO administration of a solution dosage form. 5. Intramuscular injection of the oily formulation resulted in low levels of 17-OHPC that were sustained for a prolonged time period with a projected bioavailability close to 100%. 6. The pharmacokinetics of 17-OHPC is dependent on the formulation and the route of administration. 7. The low bioavailability after oral administration indicates that oral administration of 17-OHPC may not be feasible with simple formulations of this drug.

Pharmacokinetics of cefazolin prophylaxis in obese gravidae at time of cesarean delivery.

OBJECTIVE: The objective of the study was to compare the pharmacokinetics of 2 g and 3 g doses of cefazolin when used for perioperative prophylaxis in obese gravidae undergoing cesarean delivery. STUDY DESIGN: We performed a double-blinded, randomized controlled trial from August 2013 to April 2014. Twenty-six obese women were randomized to receive either 2 or 3 g intravenous cefazolin within 30 minutes of a skin incision. Serial maternal plasma samples were obtained at specific time points up to 8 hours after drug administration. Umbilical cord blood was obtained after placental delivery. Maternal adipose samples were obtained prior to fascial entry, after closure of the hysterotomy, and subsequent to fascial closure. Pharmacokinetic parameters were determined via noncompartmental analysis. RESULTS: The median area under the plasma concentration vs time curve was significantly greater in the 3 g group than in the 2 g group (27204 µg/mL per minute vs 14058 µg/mL per minute; P = .001). Maternal plasma concentrations had an impact by body mass index. For every 1 kg/m(2) increase in body mass index at the time of the cesarean delivery, there was an associated 13.77 µg/mL lower plasma concentration of cefazolin across all time points (P = .01). By the completion of cesarean delivery, cefazolin concentrations in maternal adipose were consistently above the minimal inhibitory concentration for both Gram-positive and Gram-negative bacteria with both the 2 g and 3 g doses. The median umbilical cord blood concentrations were significantly higher in the 3 g vs the 2 g group (34.5 µg/mL and 21.4 µg/mL; P = .003). CONCLUSION: Cefazolin concentrations in maternal adipose both at time of hysterotomy closure and fascial closure were above the minimal inhibitory concentration for both Gram-positive and Gram-negative bacteria when either 2 g or 3 g cefazolin was administered as perioperative surgical prophylaxis. Maternal cefazolin concentrations in plasma and maternal adipose tissue are related to both dose and body mass index.

Effects of hepatic ischemia-reperfusion injury on the P-glycoprotein activity at the liver canalicular membrane and blood-brain barrier determined by in vivo administration of rhodamine 123 in rats.

PURPOSE: To investigate the effects of normothermic hepatic ischemia-reperfusion (IR) injury on the activity of P-glycoprotein (P-gp) in the liver and at the blood-brain barrier (BBB) of rats using rhodamine 123 (RH-123) as an in vivo marker. METHODS: Rats were subjected to 90 min of partial ischemia or sham surgery, followed by 12 or 24 h of reperfusion. Following intravenous injection, the concentrations of RH-123 in blood, bile, brain, and liver were used for pharmacokinetic calculations. The protein levels of P-gp and some other transporters in the liver and brain were also determined by Western blot analysis. RESULTS: P-gp protein levels at the liver canalicular membrane were increased by twofold after 24 h of reperfusion. However, the biliary excretion of RH-123 was reduced in these rats by 26%, presumably due to IR-induced reductions in the liver uptake of the marker and hepatic ATP concentrations. At the BBB, a 24% overexpression of P-gp in the 24-h IR animals was associated with a 30% decrease in the apparent brain uptake clearance of RH-123. The pharmacokinetics or brain distribution of RH-123 was not affected by the 12-h IR injury. CONCLUSIONS: Hepatic IR injury may alter the peripheral pharmacokinetics and brain distribution of drugs that are transported by P-gp and possibly other transporters.

Drugs in Human Milk Part 1: Practical and Analytical Considerations in Measuring Drugs and Metabolites in Human Milk.

Human milk is a remarkable biofluid that provides essential nutrients and immune protection to newborns. Breastfeeding women consuming medications could pass the drug through their milk to neonates. Drugs can be transferred to human milk by passive diffusion or active transport. The physicochemical properties of the drug largely impact the extent of drug transfer into human milk. A comprehensive understanding of the physiology of human milk formation, composition of milk, mechanisms of drug transfer, and factors influencing drug transfer into human milk is critical for appropriate selection and use of medications in lactating women. Quantification of drugs in the milk is essential for assessing the safety of pharmacotherapy during lactation. This can be achieved by developing specific, sensitive, and reproducible analytical methods using techniques such as liquid chromatography coupled with mass spectrometry. The present review briefly discusses the physiology of human milk formation, composition of human milk, mechanisms of drug transfer into human milk, and factors influencing transfer of drugs from blood to milk. We further expand upon and critically evaluate the existing analytical approaches/assays used for the quantification of drugs in human milk.

Simplified processing and rapid quantification of buprenorphine, norbuprenorphine, and their conjugated metabolites in human plasma using UPLC-MS/MS: Assessment of buprenorphine exposure during opioid use disorder treatment.

Opioid use disorder (OUD) is a chronic neurobehavioral ailment and is prevalent in pregnancy. OUD is commonly treated with methadone or buprenorphine (BUP). Pregnancy is known to alter the pharmacokinetics of drugs and may lead to changes in drug exposure and response. A simple, specific, and sensitive analytical method for measuring the parent drug and its metabolites is valuable for assessing the impact of pregnancy on drug exposure. A new liquid chromatography-tandem mass spectrometric method that utilized a simple protein precipitation procedure for sample preparation and four deuterated internal standards for quantification was developed and validated for BUP and its major metabolites (norbuprenorphine [NBUP], buprenorphine-glucuronide [BUP-G], and norbuprenorphine-glucuronide [NBUP-G]) in human plasma. The standard curve was linear over the concentration range of 0.05-100 ng/mL for BUP and NBUP, and 0.1-200 ng/mL for BUP-G and NBUP-G. Intra- and inter-day bias and precision were within ±15% of nominal values for all the analytes. Quality controls assessed at four levels showed high recovery consistently for all the analytes with minimal matrix effect. Adequate analyte stability was observed at various laboratory conditions tested. Overall, the developed method is simple, sensitive, accurate and reproducible, and was successfully applied for the quantification of BUP and its metabolites in plasma samples collected from pregnant women in a clinical study assessing BUP exposure during OUD treatment.

Inhaled PLGA particles of prostaglandin E1 ameliorate symptoms and progression of pulmonary hypertension at a reduced dosing frequency.

This study sought to investigate the efficacy of a noninvasive and long acting polymeric particle based formulation of prostaglandin E1 (PGE1), a potent pulmonary vasodilator, in alleviating the signs of pulmonary hypertension (PH) and reversing the biochemical changes that occur in the diseased lungs. PH rats, developed by a single subcutaneous injection of monocrotaline (MCT), were treated with two types of polymeric particles of PGE1, porous and nonporous, and intratracheal or intravenous plain PGE1. For chronic studies, rats received either intratracheal porous poly(lactic-co-glycolic acid) (PLGA) particles, once- or thrice-a-day, or plain PGE1 thrice-a-day for 10 days administered intratracheally or intravenously. The influence of formulations on disease progression was studied by measuring the mean pulmonary arterial pressure (MPAP), evaluating right ventricular hypertrophy and assessing various molecular and cellular makers including the degree of muscularization, platelet aggregation, matrix metalloproteinase-2 (MMP-2), and proliferating cell nuclear antigen (PCNA). Both plain PGE1 and large porous particles of PGE1 reduced MPAP and right ventricular hypertrophy (RVH) in rats that received the treatments for 10 days. Polymeric porous particles of PGE1 produced the same effects at a reduced dosing frequency compared to plain PGE1 and caused minimal off-target effects on systemic hemodynamics. Microscopic and immunohistochemical studies revealed that porous particles of PGE1 also reduced the degree of muscularization, von Willebrand factor (vWF), and PCNA expression in the lungs of PH rats. Overall, our study suggests that PGE1 loaded inhalable particulate formulations improve PH symptoms and arrest the progression of disease at a reduced dosing frequency compared to plain PGE1.

Simultaneous quantitation of ketamine, norketamine and dehydronorketamine in human milk using a novel ultra high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) assay.

There is a paucity of data on the transfer of ketamine from maternal blood into human milk. Quantification of ketamine in human milk provides information about the potential exposure of the infant to ketamine and its metabolites from the mother during lactation. A highly specific, reproducible, and sensitive UPLC-MS/MS based analytical method was developed and validated for the quantitation of ketamine and its metabolites (norketamine and dehydronorketamine) in human milk. Samples were subjected to a simple protein precipitation and ketamine-d4 and norketamine-d4 were used as internal standards. Separation of the analytes was achieved using an Acquity UPLC equipped with BEH RP18 1.7 µm, 2.1 × 100 mm column. Mass spectrometric analysis of the analyte ions was carried out using electrospray with positive ionization and multiple reaction monitoring mode. The assay was linear over a concentration range of 1-100 ng/mL for ketamine and norketamine, and 0.1-10 ng/mL for dehydronorketamine. Acceptable intra-day and inter-day accuracy and precision were observed for all the analytes. High recovery of the analytes and minimal matrix effect were observed. Stability of analytes was confirmed at the tested conditions. This assay was successfully used to measure analytes in human milk samples collected from lactating women enrolled in a clinical research study. This is the first validated method that simultaneously quantified ketamine and its metabolites in human milk.

Effects of in vivo hepatic ischemia-reperfusion injury on the hepatobiliary disposition of rhodamine 123 and its metabolites in isolated perfused rat livers.

PURPOSE: A few studies have shown that normothermic hepatic ischemia-reperfusion (IR) injury may affect the mRNA and/or protein levels of canalicular transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2). However, the effects of the injury on the functions of these canalicular transporters with respect to the biliary excretion of drugs remain largely unknown. Therefore, the purpose of this study was to investigate the effects of warm hepatic IR on the hepatobiliary disposition of rhodamine 123 (RH-123), a P-gp substrate, and its glucuronidated metabolite (RH-Glu), an Mrp2 substrate, in rats. METHODS: Twenty four or 72 h following a 60-min partial ischemia or sham operation in rats, livers were isolated and perfused ex vivo with a constant concentration (~100 ng/mL) of RH-123. The concentration of RH-123 and its glucuronidated (RH-Glu) and deacylated (RH-110) metabolites were determined in the outlet perfusate, bile, and the liver tissue using HPLC, and relevant pharmacokinetic parameters were estimated. RESULTS: Twenty-four-h IR caused a significant reduction in the hepatic extraction ratio of RH-123 (IR: 0.857 ± 0.078; Sham: 0.980 ± 0.017) and the biliary recovery of the parent drug and RH-Glu by 43% and 44%, respectively. The reductions in the biliary recovery were associated with significant reductions in the apparent biliary clearance of RH-123 and RH-Glu. Mass balance data showed that the formation of the glucuronidated or deacylated metabolite was not significantly affected by the 24-h IR injury. In contrast to the 24-h IR, the injury did not have any effect on the hepatobiliary disposition of RH-123 or its metabolites following 72 h of reperfusion. CONCLUSIONS: It is concluded that the pharmacokinetics of drugs that are subject to biliary excretion by the canalicular P-gp and Mrp2 transporters may be altered shortly after hepatic IR injury.

Two Innovative Approaches to Optimize Vancomycin Dosing Using Estimated AUC after First Dose: Validation Using Data Generated from Population PK Model Coupled with Monte-Carlo Simulation and Comparison with the First-Order PK Equation Approach.

The revised consensus guidelines for optimizing vancomycin doses suggest that maintaining the area under the concentration-time curve to minimal inhibitory concentration ratio (AUC/MIC) of 400-600 mg·h/L is the target pharmacokinetic/pharmacodynamic (PK/PD) index for efficacy. AUC-guided dosing approach uses a first-order pharmacokinetics (PK) equation to estimate AUC using two samples obtained at steady state and one-compartment model, which can cause inaccurate AUC estimation and fail to achieve the effective PK/PD target early in therapy (days 1 and 2). To achieve an efficacy target from the third or fourth dose, two innovative approaches (Method 1 and Method 2) to estimate vancomycin AUC at steady state (AUCSS) using two-compartment model and three or four levels after the first dose are proposed. The feasibility of the proposed methods was evaluated and compared with another published dosing algorithm (Method 3), which uses two samples and a one-compartment approach. Monte Carlo simulation was performed using a well-established population PK model, and concentration-time profiles for virtual patients with various degrees of renal function were generated, with 1000 subjects per group. AUC extrapolated to infinity (AUC0-∞) after the first dose was estimated using the three methods, whereas reference AUC (AUCref) was calculated using the linear-trapezoidal method at steady state after repeated doses. The ratio of AUC0-∞: AUCref and % bias were selected as the indicators to evaluate the accuracy of three methods. Sensitivity analysis was performed to examine the influence of change in each sampling time on the estimated AUC0-∞ using the two proposed approaches. For simulated patients with various creatinine clearance, the mean of AUC0-∞: AUCref obtained from Method 1, Method 2 and Method 3 ranged between 0.98 to 1, 0.96 to 0.99, and 0.44 to 0.69, respectively. The mean bias observed with the three methods was -0.10% to -2.09%, -1.30% to -3.59% and -30.75% to -55.53%, respectively. The largest mean bias observed by changing sampling time while using Method 1 and Method 2 were -4.30% and -10.50%, respectively. Three user-friendly and easy-to-use excel calculators were built based on the two proposed methods. The results showed that our approaches ensured sufficient accuracy and achieved target PK/PD index early and were superior to the published methodologies. Our methodology has the potential to be used for vancomycin dose optimization and can be easily implemented in clinical practice.

Maternal-Fetal Pharmacology of Drugs: A Review of Current Status of the Application of Physiologically Based Pharmacokinetic Models.

Pregnancy and the postpartum period are associated with several physiological changes that can alter the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs. For certain drugs, dosing changes may be required during pregnancy and postpartum to achieve drug exposures comparable to what is observed in non-pregnant subjects. There is very limited data on fetal exposure of drugs during pregnancy, and neonatal exposure through transfer of drugs via human milk during breastfeeding. Very few systematic clinical pharmacology studies have been conducted in pregnant and postpartum women due to ethical issues, concern for the fetus safety as well as potential legal ramifications. Over the past several years, there has been an increase in the application of modeling and simulation approaches such as population PK (PopPK) and physiologically based PK (PBPK) modeling to provide guidance on drug dosing in those special patient populations. Population PK models rely on measured PK data, whereas physiologically based PK models incorporate physiological, preclinical, and clinical data into the model to predict drug exposure during pregnancy. These modeling strategies offer a promising approach to identify the drugs with PK changes during pregnancy to guide dose optimization in pregnancy, when there is lack of clinical data. PBPK modeling is also utilized to predict the fetal exposure of drugs and drug transfer via human milk following maternal exposure. This review focuses on the current status of the application of PBPK modeling to predict maternal and fetal exposure of drugs and thereby guide drug therapy during pregnancy.

A pilot study of oral treprostinil pharmacogenomics and treatment persistence in patients with pulmonary arterial hypertension.

BACKGROUND AND AIMS: Treprostinil is a prostacyclin analog used to treat pulmonary arterial hypertension. Dosing is empiric and based on tolerability. Adverse effects are common and can affect treatment persistence. Pharmacogenomic variants that may affect treprostinil metabolism and transport have not been well-characterized. We aimed to investigate the pharmacogenomic sources of variability in treatment persistence and dosing. METHODS: Patients were prospectively recruited from an IRB approved biobank registry at a single pulmonary hypertension center. A cohort of patients who received oral treprostinil were screened for participation. Pharmacogenomic analysis was for variants in CYP2C8, CYP2C9, and ABCC4. A retrospective review was conducted for demographics, clinical status, dosing, and response. Fisher's exact test was used for categorical data and Kruskal-Wallis test or Wilcoxon rank sum were used for continuous data. RESULTS: A total of 15 patients received oral treprostinil and were consented. Their median age was 53 years, 73% were female, and 93% were White. The median total daily dose was 22.5 mg (13.5, 41) at last clinical observation. 40% of patients discontinued treatment with a majority due to adverse effects. Approximately 27% of patients had a loss-of-function variant in CYP2C8 (*1/*3 or *1/*4), whereas 47% of patients had a loss-of-function variant in CYP2C9 (*1/*2, *1/*3, or *2/*2). Minor allele frequencies for ABCC4 (rs1751034 and rs3742106) were 0.17 and 0.43, respectively. Survival analysis showed that increased CYP2C9 activity score was associated with decreased risk for treatment discontinuation [hazard ratio (HR): 0.13; 95% confidence interval (CI): 0.02, 0.91; p = 0.04]. Genetic variants were not significantly associated with dosing. CONCLUSION: Genetic variants responsible for the metabolism and transport of oral treprostinil were common. Increased CYP2C9 activity score was associated with decreased risk for treatment discontinuation. However, dosing was not associated with genetic variants in metabolizing enzymes for treprostinil. Our findings suggest significant variability in treatment persistence to oral treprostinil, with pharmacogenomics being a potentially important contributor.The reviews of this paper are available via the supplemental material section.

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.