Building an adaptive dose simulation framework to aid dose and schedule selection.

Establishing a dosing regimen that maximizes clinical benefit and minimizes adverse effects for novel therapeutics is a key objective for drug developers. Finding an optimal dose and schedule can be particularly challenging for compounds with a narrow therapeutic window such as in oncology. Modeling and simulation tools can be valuable to conduct in silico evaluations of various dosing scenarios with the goal to identify those that could minimize toxicities, avoid unscheduled dose interruptions, or minimize premature discontinuations, which all could limit the potential for therapeutic benefit. In this tutorial, we present a stepwise development of an adaptive dose simulation framework that can be used for dose optimization simulations. The tutorial first describes the general workflow, followed by a technical description with basic to advanced practical examples of its implementation in mrgsolve and is concluded with examples on how to use this in decision-making around dose and schedule optimization. The adaptive simulation framework is built with pharmacokinetic, pharmacodynamic (i.e., biomarkers, activity markers, target engagement markers, efficacy markers), and safety models that include evaluations of unexplained interindividual and intraindividual variability and covariate impact, which can be replaced and expanded (e.g., combination setting, comparator setting) with user-defined models. Subsequent adaptive simulations allow investigation of the impact of starting dose, dosing intervals, and event-driven (exposure or effect) dose modifications on any end point. The resulting simulation-derived insights can be used in quantitatively proposing dose and regimens that better balance benefit and adverse effects for further evaluation, aiding dose selection discussions, and designing dose modification recommendations, among others.

Front-loaded linezolid regimens result in increased killing and suppression of the accessory gene regulator system of Staphylococcus aureus.

Front loading is a strategy used to optimize the pharmacodynamic profile of an antibiotic through the administration of high doses early in therapy for a short duration. Our aims were to evaluate the impact of front loading of linezolid regimens on bacterial killing and suppression of resistance and on RNAIII, the effector molecule of the accessory gene regulator system (encoded by agr) in methicillin-resistant Staphylococcus aureus (MRSA). Time-killing experiments over 48 h were utilized for linezolid against four strains of MRSA: USA100, USA300, USA400, and ATCC 29213. A hollow-fiber infection model simulated traditional and front-loaded human therapeutic regimens of linezolid versus USA300 at 10(6) CFU/ml over 240 h. Over 48 h in time-kill experiments, linezolid displayed bacteriostatic activity, with reductions of >1 log(10) CFU/ml for all strains. Front-loaded regimens that were administered over 5 days, 1,200 mg every 12 h (q12h) (total, 10 doses) and 2,400 mg q12h (total, 10 doses) followed by 300 mg q12h thereafter, resulted in sustained bactericidal activity, with reductions of the area under the CFU curve of -6.15 and -6.03, respectively, reaching undetectable limits at the 10-day study endpoint. All regimens displayed a reduction in RNAIII relative expression at 24 h and 240 h compared with that of the growth control. Monte Carlo simulations predicted a <1.27× increase in the fractional decreases in platelets for all front-loaded regimens versus the 600 mg q12h regimen, except for the highest-dose front-loaded regimen. Front-loading strategies for linezolid are promising and may be of utility in severe MRSA infections, where early aggressive therapy is necessary.

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.

Development of Dolutegravir Single-entity and Fixed-dose Combination Formulations for Children.

BACKGROUND: The World Health Organization (WHO) 2019 antiretroviral treatment guidelines recommend use of optimal treatment regimens in all populations. Dolutegravir-based regimens are the preferred first-line and second-line treatment in infants and children with HIV 4 weeks of age and above. There is an urgent need for optimal pediatric formulations of dolutegravir as single-entity (SE) and fixed-dose combination (FDC) to ensure correct dosing and adherence for swallowing and palatability. This article outlines the chronology of dolutegravir pediatric formulation development as granules and conventional and dispersible tablets in a total of 5 pharmacokinetic studies evaluating the relative bioavailability of dolutegravir SE and FDC formulations in healthy adults. METHODS: The relative bioavailability studies were 2-part, Phase I, open-label, randomized studies in healthy adults. Dolutegravir SE study compared conventional dolutegravir 50 and 25 mg with equivalent conventional 10-mg and dispersible 5-mg tablets, respectively. Subsequently, dolutegravir FDC study compared adult FDC of abacavir/dolutegravir/lamivudine and adult FDC of dolutegravir/lamivudine with their respective pediatric FDC formulations, taken as dispersion immediately or swallowed whole. RESULTS: As observed in previous studies, dolutegravir administered as dispersion (granules/dispersible tablets) showed relatively higher bioavailability compared with conventional tablets. The bioavailability of dolutegravir dispersible tablets (both SE and FDC) was approximately 1.6-fold higher when compared with conventional tablets. In addition, the bioavailability of abacavir/lamivudine was not impacted by dispersible formulation. CONCLUSIONS: These studies demonstrate the successful development of pediatric dolutegravir-containing formulations as SE and FDC that permit pediatric dosing in line with WHO recommendations.

A Phase I Study to Evaluate the Pharmacokinetics and Safety of Cabotegravir in Adults With Severe Renal Impairment and Healthy Matched Control Participants.

This study investigates the impact of severe renal impairment on the pharmacokinetics of cabotegravir, an investigational HIV-1 integrase inhibitor. This was a phase I, open-label, parallel-group, multicenter study conducted in 8 participants with severe renal impairment (creatinine clearance <30 mL/min; no renal replacement therapy) and 8 healthy participants (creatinine clearance >90 mL/min; 2 women/group; 6 men/group) matched for sex, age (±10 years), and body mass index (±25%). Participants received a single 30-mg oral cabotegravir tablet to determine total and unbound plasma cabotegravir concentrations. Arithmetic and geometric least squares means were calculated, and cabotegravir noncompartmental pharmacokinetic parameters were compared using geometric least squares mean ratios with 90% confidence intervals. Safety was assessed throughout the study. Geometric least squares mean ratios (90% confidence intervals) were 0.97 (0.84-1.14) for area under the plasma concentration-time curve extrapolated to infinity, 1.01 (0.87-1.17) for maximum observed plasma concentration, 1.31 (0.84-2.03) for unbound cabotegravir 2 hours after dosing, and 1.51 (1.19-1.92) for unbound cabotegravir 24 hours after dosing. All adverse events were grade 1, except grade 3 lipase elevation in a participant with renal impairment. Severe renal impairment did not impact plasma cabotegravir exposure, and cabotegravir may be administered without dose adjustment in renal impairment among patients not receiving renal replacement.

Stereospecific high-performance liquid chromatographic analysis of tramadol and its O-demethylated (M1) and N,O-demethylated (M5) metabolites in human plasma.

A stereospecific method for simultaneous quantitation of the enantiomers of tramadol (T) and its active metabolites O-demethyl tramadol (M1) and O-demethyl-N-demethyl tramadol (M5) in human plasma is reported. After the addition of penbutolol (IS), plasma (0.5 ml) samples were extracted into methyl tert-butyl ether, followed by back extraction into an acidic solution. The separation was achieved using a Chiralpak AD column with a mobile phase of hexanes:ethanol:diethylamine (94:6:0.2) and a flow rate of 1 ml/min. The fluorescence of analytes was then detected at excitation and emission wavelengths of 275 and 300 nm, respectively. All the six enantiomeric peaks of interest plus three unknown metabolite peaks and IS peak (a total of 10 peaks) eluted within 23 min, free from endogenous interference. The assay was validated in the plasma concentration range of 2.5-250 ng/ml, with a lower limit of quantitation of 2.5 ng/ml, for all the six analytes. The extraction efficiency (n=5) was close to 100% for both T and M1 enantiomers and 85% for M5 and IS enantiomers. The application of the assay was demonstrated by simultaneous measurement of plasma concentrations of T, M1, and M5 enantiomers in a healthy volunteer after the administration of 50 mg oral doses of racemic T.

Divergent effects of nitric oxide donors on the biliary efflux transporters in isolated perfused rat livers: nitric oxide-independent inhibition of ABC transporters by sodium nitroprusside.

Rhodamine 123 (RH-123) and its glucuronidated metabolite (RH-Glu) are excreted into the bile via the ABC efflux transporters P-glycoprotein (P-gp) and multidrug resistance-related protein type 2 (Mrp2), respectively. In this study, we investigated the short-term (2 h) effects of a low or high concentration of nitric oxide (NO) donors sodium nitroprusside (SNP) and isosorbide dinitrate (ISDN) on the hepatobiliary disposition of RH-123 and its metabolite in an isolated perfused rat liver model. Additionally, the effects of ISDN on the hepatobiliary disposition of 5 (and 6)-carboxy-2', 7'- dichlorofluorescein (CDF), a specific marker of Mrp2, were investigated in the same model. Whereas SNP caused a substantial (85-90%) reduction in the P-gp- and Mrp2-mediated transport of RH-123 and RH-Glu, respectively, ISDN did not affect either of these transporters. However, ISDN reduced the biliary recovery of RH-Glu, most likely because of inhibition of the formation of the metabolite. Further studies showed that the effects of SNP on these transporters are due to a substantial (88%) depletion of hepatic ATP levels by this NO donor. Additionally, studies using CDF revealed an almost identical hepatobiliary disposition of this Mrp2 marker in the presence or absence of ISDN. It is concluded that short-term exposure of rat livers to NO does not affect the functions of the efflux transporters P-gp and Mrp2. The observed inhibitory effects of SNP on the functions of both P-gp and Mrp2 are via an NO-independent mechanism.

Dose-dependent inhibition of transporter-mediated hepatic uptake and biliary excretion of methotrexate by cyclosporine A in an isolated perfused rat liver model.

Methotrexate (MTX) and cyclosporine (CyA) are coadministered in a number of diseases. In this study, the effects of CyA on the hepatobiliary disposition of MTX were investigated in an isolated perfused rat liver model. A bolus 5-mg dose of MTX was added to a recirculating perfusate in the absence or presence of a relatively low (0.5 mg) or high (2.5 mg) dose of CyA or vehicle pretreatment, and perfusate, bile, and terminal liver samples were collected for analysis by high-performance liquid chromatography (HPLC). In control and vehicle groups, MTX showed a low hepatic extraction ratio (∼0.1) and was almost completely eliminated by excretion into the bile. The low-dose CyA significantly reduced (60%) the hepatic extraction ratio and clearance of MTX, without affecting the bile/liver concentration ratio, suggesting inhibition of sinusoidal uptake of MTX only. In contrast, the high-dose CyA significantly reduced both hepatic uptake and Mrp2-mediated biliary excretion of MTX. Isolated rat hepatocyte uptake studies showed significant inhibition of [(3)H]MTX uptake in the presence of CyA. It is concluded that CyA significantly alters the hepatobiliary disposition of MTX by inhibiting its sinusoidal uptake and/or biliary transport, potentially reducing enterohepatic recirculation of the drug in vivo.

Effects of P-glycoprotein and Mrp2 inhibitors on the hepatobiliary disposition of Rhodamine 123 and its glucuronidated metabolite in isolated perfused rat livers.

The hepatobiliary disposition of rhodamine 123 (RH-123) and its glucuronidated (RH-Glu) and deacylated (RH-110) metabolites were studied in an isolated perfused rat liver (IPRL) model in the presence and absence of P-glycoprotein (P-gp) and Mrp2 inhibitors. A single dose (180 microg) of RH-123 was added to a recirculating perfusate in the absence (Control) or presence of cyclosporine A (CyA) or dibromosulfophthalein (DBSP) in the perfusate. Serial (0-90 min) perfusate and bile and terminal liver samples were collected for analysis by HPLC. In the Control livers, 25.4 +/- 2.2% (mean +/- SD) of the dose was recovered as RH-123 (11.7 +/- 2.0%) and RH-Glu (13.2 +/- 0.9%) in the bile. Whereas CyA substantially (90%) reduced (p < 0.001) the biliary excretion of RH-123 without affecting the excretion of RH-Glu, DBSP reduced the biliary excretion of RH-Glu by >80% (p < 0.001) with no effect on the biliary excretion of RH-123. Mass balance studies showed that DBSP, in addition to reducing the biliary clearance of RH-Glu, also strongly inhibited the glucuronidation of RH-123, an effect that was confirmed in vitro using the glucuronidation marker umbelliferone. It is concluded that the use of RH-123 in an IPRL model may serve as a dual marker for the determination of the altered functions of P-gp and/or Mrp2.

Route-dependent stereoselective pharmacokinetics of tramadol and its active O-demethylated metabolite in rats.

The effects of route of administration on the stereoselective pharmacokinetics of tramadol (T) and its active metabolite (M1) were studied in rats. A single 20 mg/kg dose of racemic T was administered through intravenous, intraperitoneal, or oral route to different groups of rats, and blood and urine samples were collected. Samples were analyzed using chiral chromatography, and pharmacokinetic parameters (mean +/- SD) were estimated by noncompartmental methods. Following intravenous injection, there was no stereoselectivity in the pharmacokinetics of T. Both enantiomers showed clearance values (62.5 +/- 27.2 and 64.4 +/- 39.0 ml/min/kg for (+)- and (-)-T, respectively) that were equal or higher than the reported liver blood flow in rats. Similar to T, the area under the plasma concentration-time curves (AUCs) of M1 did not exhibit stereoselectivity after intravenous administration of the parent drug. However, the systemic availability of (+)-T was significantly (P < 0.05) higher than that of its antipode following intraperitoneal (0.527 +/- 0.240 vs. 0.373 +/- 0.189) and oral (0.307 +/- 0.136 vs. 0.159 +/- 0.115) administrations. The AUC of the M1 enantiomers, on the other hand, remained mostly nonstereoselective regardless of the route of administration. Pharmacokinetic analysis indicated that the stereoselectivity in the pharmacokinetics of oral T is due to stereoselective first pass metabolism in the liver and, possibly, in the gastrointestinal tract. The direction and extent of stereoselectivity in the pharmacokinetics of T and M1 in rats were in agreement with those previously reported in humans, suggesting that the rat may be a suitable model for enantioselective studies of T pharmacokinetics.