Unbound Drug Concentrations Research Articles

Prediction Models for Brain Distribution of Drugs Based on Biomimetic Chromatographic Data.

The development of high-throughput approaches for the valid estimation of brain disposition is of great importance in the early drug screening of drug candidates. However, the complexity of brain tissue, which is protected by a unique vasculature formation called the blood–brain barrier (BBB), complicates the development of robust in silico models. In addition, most computational approaches focus only on brain permeability data without considering the crucial factors of plasma and tissue binding. In the present study, we combined experimental data obtained by HPLC using three biomimetic columns, i.e., immobilized artificial membranes, human serum albumin, and α1-acid glycoprotein, with molecular descriptors to model brain disposition of drugs. Kp,uu,brain, as the ratio between the unbound drug concentration in the brain interstitial fluid to the corresponding plasma concentration, brain permeability, the unbound fraction in the brain, and the brain unbound volume of distribution, was collected from literature. Given the complexity of the investigated biological processes, the extracted models displayed high statistical quality (R2 > 0.6), while in the case of the brain fraction unbound, the models showed excellent performance (R2 > 0.9). All models were thoroughly validated, and their applicability domain was estimated. Our approach highlighted the importance of phospholipid, as well as tissue and protein, binding in balance with BBB permeability in brain disposition and suggests biomimetic chromatography as a rapid and simple technique to construct models with experimental evidence for the early evaluation of CNS drug candidates.

Is the Protein-Mediated Uptake of Drugs by Organic Anion Transporting Polypeptides a Real Phenomenon or an Artifact?

Plasma proteins or human serum albumin (HSA) have been reported to increase the in vitro intrinsic uptake clearance (CLint,uptake) of drugs by hepatocytes or organic anion transporting polypeptide (OATP)-transfected cell lines. This so-called protein-mediated uptake effect (PMUE) is thought to be due to an interaction between the drug-protein complex and the cell membrane causing an increase in the unbound drug concentration at the cell surface, resulting in an increase in the apparent CLint,uptake of the drug. To determine if the PMUE on OATP-mediated drug uptake is an artifact or a real phenomenon, we determined the effect of 1%, 2%, and 5% HSA on OATP1B1-mediated [human embryonic kidney (HEK)293 transfected cells] and passive CLint,uptake (mock HEK293 cells) on a cocktail of five statins. In addition, we determined the non-specific binding (NSB) of the statin-HSA complex to the cells/labware. The increase in uptake of atorvastatin, fluvastatin, and rosuvastatin in the presence of HSA was completely explained by the extent of NSB of the statin-HSA complex, indicating that the PMUE for these statins is an artifact. In contrast, this was not the case for OATP1B1-mediated uptake of pitavastatin and passive uptake of cerivastatin, suggesting that the PMUE is a real phenomenon for these drugs. Additionally, the PMUE on OATP1B1-mediated uptake of pitavastatin was confirmed by a decrease in its unbound IC50 in the presence of 5% HSA versus Hank's balanced salt solution buffer (HBSS). These data question the utility of routinely including plasma proteins or HSA in uptake experiments and the previous findings on PMUE on OATP-mediated drug uptake. SIGNIFICANCE STATEMENT: Here we report, for the first time, that the protein-mediated uptake effect (PMUE) on organic anion transporting polypeptide (OATP)-transported drugs could be an artifact of the non-specific binding (NSB) of the drug-albumin complex to cells/labware. Future experiments on PMUE must take into consideration such NSB. In addition, mechanisms other than PMUE need to be explored to explain the underprediction of in vivo OATP-mediated hepatic drug clearance from in vitro uptake studies.

Population Pharmacokinetics and Dosing Simulations of Ceftriaxone in Critically Ill Patients Receiving Extracorporeal Membrane Oxygenation (An ASAP ECMO Study).

BackgroundDespite the surge in use of extracorporeal membrane oxygenation (ECMO) in the adult intensive care unit, little guidance is available on the appropriate dosing of antimicrobials in this setting. Ceftriaxone is an antimicrobial with a high affinity to plasma protein, a property identified in the literature as susceptible to sequestration into extracorporeal circuits and hypothesised to require dosage adjustments in this setting.ObjectiveThe aim of this study was to describe the pharmacokinetics of ceftriaxone and identify the best dosing regimen for critically ill adult patients receiving ECMO.MethodsSerial blood samples were taken from patients receiving both ECMO and ceftriaxone. Total and unbound drug concentrations were measured in plasma by chromatographic assay and analysed using a population pharmacokinetic approach with Pmetrics®. Dosing simulations were performed to identify the optimal dosing strategy: 60 and 100% of time with free (unbound) drug concentration exceeding the minimum inhibitory concentration (fT>MIC).ResultsIn total, 14 patients were enrolled, of which three were receiving renal replacement therapy (RRT). Total and unbound ceftriaxone was best described in a two-compartment model with total body weight, serum albumin concentrations, creatinine clearance (CrCL), and the presence of RRT included as significant predictors of pharmacokinetics. Patients not on RRT generated a mean renal clearance of 0.90 L/h, non-renal clearance of 0.33 L/h, and central volume of distribution of 7.94 L. Patients on RRT exhibited a mean total clearance of 1.18 L/h. ECMO variables were not significant predictors of ceftriaxone pharmacokinetics. Steady-state dosing simulations found that dosages of 1 g every 12 h and 2 g every 24 h achieved >90% probabilities of target attainment in patients with CrCL of 0 mL/min with RRT and 30 and 100 mL/min and various serum albumin concentrations (17 and 26 g/L).ConclusionsDosing recommendations for critically ill adult patients not on ECMO appear to be sufficient for patients on ECMO. Patients exhibiting augmented renal clearance (> 130 mL/min) or treatment of less susceptible pathogens may require higher doses, which requires further investigation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40262-021-01106-x.

Pharmacokinetics of Cefazolin in Patients With Obesity Undergoing Surgery Requiring Cardiopulmonary Bypass

The authors' objective was to determine the adequacy of an institutional standard dosing practice for infection prophylaxis in open cardiac surgery in patients heavier than 120 kg undergoing cardiopulmonary bypass. A prospective, single-center, open-label study was used to determine if cefazolin serum concentrations were maintained above the minimum inhibitory concentration (MIC) throughout surgery. A pharmacokinetic model describing cefazolin disposition was developed for perioperative patients with morbid obesity, based on these values. Probability of target attainment was evaluated across the clinically relevant MIC spectrum. Maine Medical Center is an academic hospital in Portland, Maine, affiliated with Tufts University School of Medicine. Twenty patients scheduled for cardiac surgery requiring cardiopulmonary bypass who weighed at least 120 kg. All patients received 2 g of cefazolin intravenously (IV) within 1 hour before incision, an additional 1 g injected into the cardiopulmonary bypass circuit at the initiation of bypass, and 2 g administered IV every 3 hours after the initial IV dose. Cefazolin serum concentrations were collected after incision, after initiation of bypass, each hour of bypass, at the end of bypass, and at sternal closure. For patients weighing >120 kg undergoing cardiac surgery, the studied dosing regimen met or exceeded targeted cefazolin concentrations for all study patients. The authors conducted probability of target attainment analyses using both 65% and 100% of time with unbound drug concentrations across clinically relevant MICs. The authors found that their current dosing strategy achieved a probability of target attainment >90% throughout surgery for both total and unbound cefazolin concentrations, independent of cardiopulmonary bypass times.

CTNI-48. A PHASE 0 ‘TRIGGER’ TRIAL OF CDK4/6 PLUS ERK1/2 INHIBITORS IN RECURRENT GLIOBLASTOMA

Abstract BACKGROUND This dual-drug Phase 0 study (NCT04391595) evaluates the tumor pharmacokinetics (PK) and tumor pharmacodynamics (PD) of abemaciclib, a selective CDK4/6-inhibitor, plus LY3214996, a selective ERK1/2 inhibitor, in recurrent GBM patients. METHODS Adult recurrent GBM patients (n=10) with intact RB expression, > 30% pERK expression, and CDKN2A/B deletion or CDK4/6 amplification received six days of abemaciclib (150mg BID) plus LY3214996 (200mg QD) prior to a planned resection at 3-5 or 7-9 hour time interval after the final drug dose in a Time-Escalation Arm. Tumor tissue (gadolinium [Gd]-enhancing and nonenhancing regions), cerebrospinal fluid (CSF), and plasma were collected. Total and unbound drug concentrations were measured using validated LC-MS/MS methods. Tumor PD effects, including RB and RSK phosphorylation, were compared to matched archival or pre-treatment biopsied tissue. A PK ‘trigger’ (i.e., unbound concentration > 5x biochemical IC50) was set for each drug. Gd-nonenhancing tumor tissue exhibiting abemaciclib and LY3214996 concentrations in excess of their trigger threshold qualified patients for postoperative dual-drug therapy. RESULTS No dose-limiting toxicities were observed. In Gd-nonenhancing tumor regions, median unbound concentrations of abemaciclib were 31.2 nM (3-5 hour cohort) and 25.1 nM (7-9 hour cohort) while median unbound concentrations of LY3214996 were 52.0 nM (3-5 hour cohort) and 10.2 nM (7-9 hour cohort). Tumor RB and RSK phosphorylation decreased in 6/10 and 2/10 patients, respectively. Tumor proliferation (MIB-1) was decreased in 8/10 patients. 5/10 patients exceeded PK thresholds for both abemaciclib (12 nM) and LY3214996 (25 nM), thereby entering the study’s therapeutic expansion phase. CONCLUSION Abemaciclib and LY3214996 achieve pharmacologically-relevant concentrations in Gd-non-enhancing GBM tissue and are associated with suppression of RB pathway and tumor proliferation. The Optimal Time Interval (OTI) for tissue sampling was 3-5 hours after the final drug dose. Based on this interim analysis, the trial will accrue an additional 25 patients at this OTI.

CTNI-22. A PHASE 0/1 ‘TRIGGER’ TRIAL OF RIBOCICLIB PLUS EVEROLIMUS IN RECURRENT HIGH-GRADE GLIOMA

Abstract BACKGROUND mTOR activation is a mechanism of resistance in CDK4/6 targeting. We evaluated tumor pharmacokinetics (PK) and tumor pharmacodynamics (PD) of combined CDK4/6 and mTOR inhibition in recurrent high-grade glioma (HGG) patients. METHODS Recurrent HGG patients with (1) intact RB, (2) CDKN2A/B deletion or CDK4/6 amplification, and (3) PTEN loss or PIK3CA mutations receive five days of presurgical ribociclib plus everolimus prior to resection at 2, 8 or 24 hours after the final dose. Beginning at 400mg QD ribociclib plus 2.5mg QD everolimus, six dose-escalations summit at 600mg QD plus 60mg QW. Gadolinium [Gd]-enhancing and nonenhancing tumor regions, CSF, and plasma are collected. Total and unbound drug concentrations are determined using validated LC-MS/MS methods. RB and S6 phosphorylation are compared to matched archival tissue. To select patients for a therapeutic expansion phase of combined drug therapy, the protocol includes a PK ‘trigger’ (i.e., for each drug, unbound concentration in Gd-nonenhancing tumor > 5-fold biochemical IC50) and a PD ‘trigger’ (i.e., for each tumor, > 30% decrease in pRB and pS6). RESULTS 21 patients with WHO Grade III (n=2) and IV (n=19) gliomas were enrolled into the Phase 0 component of the study. No dose-limiting toxicities were observed. In Gd-nonenhancing tumor regions, the median unbound concentration of ribociclib was 719 nM, whereas unbound everolimus tumor concentrations were undetectable. Across all dose-levels, 62% (13/21) and 22% (5/21) of tumors demonstrated decreased tumor RB and S6 phosphorylation, respectively. Tumor proliferation (MIB-1) was decreased in 67% (14/21) of all patients. No patients qualified for the therapeutic expansion phase. CONCLUSION In adult HGG, ribociclib achieves pharmacologically-relevant concentrations in Gd-nonenhancing tumor whereas everolimus exhibits no meaningful tumor penetration. These findings support further clinical development of ribociclib, but not everolimus, for the treatment of high-grade glioma patients.

Effects of membrane transport activity and cell metabolism on the unbound drug concentrations in the skeletal muscle and liver of drugs: A microdialysis study in rats.

The unbound concentrations of 14 commercial drugs, including five non‐efflux/uptake transporter substrates—Class I, five efflux transporter substrates—class II and four influx transporter substrates—Class III, were simultaneously measured in rat liver, muscle, and blood via microanalysis. K puu,liver and K puu,muscle were calculated to evaluate the membrane transport activity and cell metabolism on the unbound drug concentrations in the skeletal muscle and liver. For Class I compounds, represented by antipyrine, unbound concentrations among liver, muscle and blood are symmetrically distributed when compound hepatic clearance is low. And when compound hepatic clearance is high, unbound concentrations among liver, muscle and blood are asymmetrically distributed, such as Propranolol. For Class II and III compounds, overall, the unbound concentrations among liver, muscle, and blood are asymmetrically distributed due to a combination of hepatic metabolism and efflux and/or influx transporter activity.

OS05.8.A A Phase 0/1 ‘Trigger’ Trial of Ribociclib Plus Everolimus in Recurrent High-Grade Glioma

Abstract BACKGROUND The RB-CDK4/6 and mTOR signaling pathways are deregulated in high-grade glioma (HGG) and mTOR activation is a potential mechanism of resistance to CDK4/6 inhibition. This study evaluates the tumor pharmacokinetics (PK) and tumor pharmacodynamics (PD) of combined CDK4/6 and mTOR inhibition in recurrent HGG patients. MATERIAL AND METHODS Eligible patients had recurrent HGG with (1) intact RB expression, (2) CDKN2A/B deletion or CDK4/6 amplification, and (3) PTEN loss or PIK3CA mutations. Six patients received five days of presurgical ribociclib (400mg QD) plus everolimus (2.5mg QD) and then underwent tumor resection at 2, 8 or 24 hours following the last dose. Five subsequent dose-escalation cohorts each enrolled three additional patients, reaching a maximum dose-level of ribociclib (600mg QD) plus everolimus (60mg QW). Tumor tissue (gadolinium [Gd]-enhancing and nonenhancing regions), CSF, and plasma were collected. Total and unbound drug concentrations were determined using validated LC-MS/MS methods. Tumor PD effects, including RB and S6 phosphorylation, were compared to matched archival tissue. A PK ‘trigger’ (i.e., unbound concentration > 5-fold biochemical IC50) and a PD ‘trigger’ (>30% decrease in both pRB and pS6) were set for each drug. Gd-nonenhancing tissue exhibiting both PK and PD effects in excess of these thresholds qualified patients for postoperative combination therapy. RESULTS 21 patients with WHO Grade III (n=2) and WHO Grade IV (n=19) gliomas were enrolled. No dose-limiting toxicities were observed. Following presurgical drug, all patients demonstrated marked decrease in Gd-enhancement on preoperative MRI. In Gd-nonenhancing tumor regions, the median unbound concentration of ribociclib was 719 nM (i.e., > 5-fold biochemical IC50 for CDK4/6 inhibition), whereas the unbound everolimus tumor concentrations in all patients were below the lower limit of quantitation (i.e., < 0.2 nM). The median total concentrations of everolimus in tumors at dose-levels 0 to 5 were 2.9, 8.8, 10.3, 5.0, 15.7, and 13.7 nM, respectively. Across all dose-levels, 62% (13/21) and 22% (5/21) of tumors demonstrated decreased tumor RB and S6 phosphorylation, respectively. Tumor proliferation (MIB-1) was decreased in 67% (14/21) of all patients. CONCLUSION In adult HGG, ribociclib achieves pharmacologically-relevant concentrations in Gd-nonenhancing tumor, consistent with the observed tumor PD effects. Everolimus exhibits very limited penetration into human glioma tissue. Our study supports further development of ribociclib, but not everolimus, for the treatment of glioma patients.

A Phase 0 ‘trigger’ trial of CDK4/6 plus ERK1/2 inhibitors in recurrent glioblastoma.

2005 Background: The RB-CDK4/6 and MAPK signaling pathways are dysregulated in glioblastoma (GBM). Our recent phase 0 study of ribociclib in recurrent GBM patients suggested that CDK4/6 inhibitor monotherapy is not durable. In this ongoing, dual-drug phase 0 study (NCT04391595), we evaluate the tumor pharmacokinetics (PK) and tumor pharmacodynamics (PD) of abemaciclib, a selective CDK4/6-inhibitor, plus LY3214996, a selective ERK1/2 inhibitor, in recurrent GBM patients. Methods: Adult patients eligible for this open-label, multi-center phase 0 protocol had recurrent GBM with (1) intact RB expression, (2) >30% pERK expression, and (3) CDKN2A/B deletion or CDK4/6 amplification. Prior to a planned resection, patients received six days of abemaciclib (150mg BID) plus LY3214996 (200mg QD). In a Time-Escalation Arm, ten patients were assigned to 3-5 hour or 7-9 hour intervals from final drug dose to tumor removal. Tumor tissue (gadolinium [Gd]-enhancing and nonenhancing regions), cerebrospinal fluid (CSF), and plasma were collected. Total and unbound drug concentrations were measured using validated LC-MS/MS methods. Tumor PD effects, including RB and RSK phosphorylation, were compared to matched archival or pre-treatment biopsied tissue. A PK ‘trigger’ ( i.e., unbound concentration > 5x biochemical IC50) was set for each drug. Gd-nonenhancing tumor tissue exhibiting abemaciclib and LY3214996 concentrations in excess of their trigger threshold qualified patients for postoperative dual-drug therapy. Results: In this interim analysis, no dose-limiting toxicities were observed. In Gd-nonenhancing tumor regions, median unbound concentrations of abemaciclib (including its equipotent M2 and M20 metabolites) were 31.2 nM (3-5 hour cohort) and 25.1 nM (7-9 hour cohort). In the same tissue, median unbound concentrations of LY3214996 were 52.0 nM (3-5 hour cohort) and 10.2 nM (7-9 hour cohort). Tumor RB and RSK phosphorylation decreased in 6/10 and 2/10 patients, respectively. Gd-enhancing tumor proliferation (MIB-1) was decreased in 8/10 patients. 5/10 patients exceeded PK thresholds for both abemaciclib (12 nM) and LY3214996 (25 nM), thereby entering the study’s therapeutic expansion phase. Conclusion: Abemaciclib and LY3214996 achieve pharmacologically-relevant concentrations in Gd-non-enhancing GBM tissue and are associated with suppression of the RB pathway and tumor proliferation. Following 6 days of presurgical drug exposure, the Optimal Time Interval (OTI) for tissue sampling was 3-5 hours after the final drug dose. Based on this interim analysis, the trial will accrue an additional 25 patients at this OTI. Clinical trial information: NCT04391595.

Protein binding of clindamycin in vivo by means of intravascular microdialysis in healthy volunteers.

The efficacy of an anti-infective drug is influenced by its protein binding (PB), since only the free fraction is active. We hypothesized that PB may vary in vitro and in vivo, and used clindamycin, a drug with high and concentration-dependent PB to investigate this hypothesis. Six healthy volunteers received a single intravenous infusion of clindamycin 900 mg. Antibiotic plasma concentrations were obtained by blood sampling and unbound drug concentrations were determined by means of in vivo intravascular microdialysis (MD) or in vitro ultrafiltration (UF) for up to 8 h post dosing. Clindamycin was assayed in plasma and MD fluid using a validated HPLC-UV (ultraviolet) method. Non-linear mixed effects modelling in NONMEM® was used to quantify the PB in vivo and in vitro. C max was 14.95, 3.39 and 2.32 mg/L and AUC0-8h was 41.78, 5.80 and 6.14 mg·h/L for plasma, ultrafiltrate and microdialysate, respectively. Calculated ratio of AUCunbound/AUCtotal showed values of 13.9%±1.8% and 14.7%±3.1% for UF and microdialysate, respectively. Modelling confirmed non-linear, saturable PB for clindamycin with slightly different median (95% CI) dissociation constants (Kd) for the alpha-1 acid glycoprotein (AAG)-clindamycin complex of 1.16 mg/L (0.91-1.37) in vitro versus 0.85 mg/L (0.58-1.01) in vivo. Moreover, the estimated number of binding sites per AAG molecule was 2.07 (1.79-2.25) in vitro versus 1.66 in vivo (1.41-1.79). Concentration-dependent PB was observed for both investigated methods with slightly lower levels of unbound drug fractions in vitro as compared with in vivo.

Application of a Physiologically Based Pharmacokinetic Model to Characterize Time-dependent Metabolism of Voriconazole in Children and Support Dose Optimization.

Background: Voriconazole is a potent antifungal drug with complex pharmacokinetics caused by time-dependent inhibition and polymorphisms of metabolizing enzymes. It also exhibits different pharmacokinetic characteristics between adults and children. An understanding of these alterations in pharmacokinetics is essential for pediatric dose optimization. Objective: To determine voriconazole plasma exposure in the pediatric population and further investigate optimal dosage regimens. Methods: An adult and pediatric physiologically based pharmacokinetic (PBPK) model of voriconazole, integrating auto-inhibition of cytochrome P450 3A4 (CYP3A4) and CYP2C19 gene polymorphisms, was developed. The model was evaluated with visual predictive checks and quantitative measures of the predicted/observed ratio of the area under the plasma concentration-time curve (AUC) and maximum concentration (Cmax). The validated pediatric PBPK model was used in simulations to optimize pediatric dosage regimens. The probability of reaching a ratio of free drug (unbound drug concentration) AUC during a 24-h period to minimum inhibitory concentration greater than or equal to 25 (fAUC24h/MIC ≥ 25) was assessed as the pharmacokinetic/pharmacodynamic index. Results: The developed PBPK model well represented voriconazole's pharmacokinetic characteristics in adults; 78% of predicted/observed AUC ratios and 85% of Cmax ratios were within the 1.25-fold range. The model maintained satisfactory prediction performance for intravenous administration in pediatric populations after incorporating developmental changes in anatomy/physiology and metabolic enzymes, with all predicted AUC values within 2-fold and 73% of the predicted Cmax within 1.25-fold of the observed values. The simulation results of the PBPK model suggested that different dosage regimens should be administered to children according to their age, CYP2C19 genotype, and infectious fungal genera. Conclusion: The PBPK model integrating CYP3A4 auto-inhibition and CYP2C19 gene polymorphisms successfully predicted voriconazole pharmacokinetics during intravenous administration in children and could further be used to optimize dose strategies. The infectious fungal genera should be considered in clinical settings, and further research with large sample sizes is required to confirm the current findings.

Pharmacokinetic / pharmacodynamic relationships of liposomal amphotericin B and miltefosine in experimental visceral leishmaniasis.

BackgroundThere is a continued need to develop effective and safe treatments for visceral leishmaniasis (VL). Preclinical studies on pharmacokinetics and pharmacodynamics of anti-infective agents, such as anti-bacterials and anti-fungals, have provided valuable information in the development and dosing of these agents. The aim of this study was to characterise the pharmacokinetic and pharmacodynamic properties of the anti-leishmanial drugs AmBisome and miltefosine in a preclinical disease model of VL.Methodology / Principal findingsBALB/c mice were infected with L. donovani (MHOM/ET/67/HU3) amastigotes. Groups of mice were treated with miltefosine (orally, multi-dose regimen) or AmBisome (intravenously, single dose regimen) or left untreated as control groups. At set time points groups of mice were killed and plasma, livers and spleens harvested. For pharmacodynamics the hepatic parasite burden was determined microscopically from tissue impression smears. For pharmacokinetics drug concentrations were measured in plasma and whole tissue homogenates by LC-MS. Unbound drug concentrations were determined by rapid equilibrium dialysis. Doses exerting maximum anti-leishmanial effects were 40 mg/kg for AmBisome and 150 mg/kg (cumulatively) for miltefosine. AmBisome displayed a wider therapeutic range than miltefosine. Dose fractionation at a total dose of 2.5 mg/kg pointed towards concentration-dependent anti-leishmanial activity of AmBisome, favouring the administration of large doses infrequently. Protein binding was >99% for miltefosine and amphotericin B in plasma and tissue homogenates.Conclusion / SignificanceUsing a PK/PD approach we propose optimal dosing strategies for AmBisome. Additionally, we describe pharmacokinetic and pharmacodynamic properties of miltefosine and compare our findings in a preclinical disease model to available knowledge from studies in humans. This approach also presents a strategy for improved use of animal models in the drug development process for VL.

Development of a simple and rapid method to determine the unbound fraction of dolutegravir, raltegravir and darunavir in human plasma using ultrafiltration and LC–MS/MS

Dolutegravir, raltegravir and darunavir are three antiretroviral drugs widely used in combined antiretroviral therapies. These three drugs are highly bound to plasma proteins. Compared to the total concentration, the concentration of unbound drug which is considered as the only pharmacological active form should be more informative to improve therapeutic drug monitoring in patients to avoid virological failure or toxicity. The aim of the present study was to develop an ultrafiltration protocol and a LC–MS/MS method to simultaneously determine the concentrations of the unbound dolutegravir, raltegravir and darunavir in human plasma. Finally, 150 μL of plasma was ultrafiltrated using Centrifree® ultrafiltration devices with ultracel YM-T membrane (cutoff 30 KDa) during 5 min at 37 °C at 1500 g. Then, 20 μL of the ultrafiltrate were injected into the LC–MS/MS system. The chromatographic separation was carried out on a BEH C18 column using a mobile phase containing deionized water and acetonitrile, both with 0.05 % (v/v) of formic acid, with a gradient elution at a flow rate of 0.5 mL/min. The run time was only 4 min. The calibration curve ranged from 0.5–200 ng/mL for dolutegravir, 1 to 400 ng/mL for raltegravir and 10–4000 ng/mL for darunavir. This method was validated with a good precision (inter- and intra-day CV% lower than 14 %) and a good accuracy (inter- and intra-day bias between −5.6–8.8 %) for all the analytes. This method is simple, reliable and suitable for pharmacokinetic studies.

Population pharmacokinetics of cefazolin before, during and after cardiopulmonary bypass in adult patients undergoing cardiac surgery.

The aims of the present study were to establish a population pharmacokinetic (PPK) model of cefazolin for adult patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and to assess the probability of target attainment (PTA) for the prophylaxis of surgical site infection (SSI) using cefazolin. Adult patients who underwent cardiac surgery with CPB were enrolled in the prospective study. Blood samples for plasma cefazolin assay were collected, and total and unbound drug concentrations were measured and analysed using the nonlinear mixed-effects modelling (NONMEM) software considering saturable plasma protein binding. Using the PPK model, plasma unbound cefazolin concentration-time courses with current prophylaxis protocols were simulated, and the PTA for common SSI pathogens was estimated. A total of 199 blood samples were obtained from 27 patients. A one-compartment model with first-order elimination plus an on/off CPB compartment best described the data. The population mean for systemic drug clearance (CL) was reduced and that for the volume of distribution (V) was increased during CPB compared with the pre-CPB values. CPB-induced hypoalbuminemia was associated with reduced maximum protein binding (Bmax). The simulation studies suggested that the current dosing protocols are insufficient for attaining PTA > 0.9 throughout surgery against pathogens with minimum inhibitory concentrations (MICs) >8mg/L. A new dosing protocol that achieves a PTA > 0.9 for pathogens with a MIC of 16mg/L was proposed. PPK modelling with simulation may be valuable for devising a cefazolin prophylaxis protocol for patients undergoing cardiac surgery with CPB.

Single-dose pharmacokinetics of temocillin in plasma and soft tissues of healthy volunteers after intravenous and subcutaneous administration: a randomized crossover microdialysis trial.

The antibiotic temocillin has recently been rediscovered as a promising therapeutic option against MDR Gram-negative bacteria. However, some aspects of the pharmacokinetic (PK) profile of the drug are still to be elucidated: subcutaneous administration of temocillin might be of interest as an alternative to the intravenous route in selected patients. Similarly, information on the penetration of temocillin into human soft tissues is lacking. To investigate the feasibility and plasma PK of subcutaneous dosing as well as soft tissue PK of temocillin after intravenous administration to healthy volunteers. Eight healthy volunteers received 2 g of temocillin both as intravenous and subcutaneous infusion in a randomized two-period crossover study. Concentration-time profiles of total temocillin in plasma (after both routes) and of unbound temocillin in plasma, muscle and subcutis (only after intravenous dosing) were determined up to 12 h post-dose. Subcutaneous dosing caused some infusion site discomfort but resulted in sustained drug concentrations over time with only slightly decreased overall exposure compared with intravenous dosing. Plasma protein binding of temocillin showed concentration-dependent behaviour and was higher than previously reported. Still, unbound drug concentrations in muscle and subcutis determined by microdialysis markedly exceeded those in plasma, suggesting good tissue penetration of temocillin. The subcutaneous administration of temocillin is a valid and feasible alternative to intravenous dosing. With the description of plasma protein binding and soft tissue PK of temocillin in healthy volunteers, this study provides important information that adds to the ongoing characterization of the PK profile of temocillin and might serve as input for PK/PD considerations.

The Impact of Plasma Protein Binding Characteristics and Unbound Concentration of Voriconazole on Its Adverse Drug Reactions.

This study investigated voriconazole (VRC) unbound plasma concentration and its relationship with adverse drug reactions (ADRs) in patients with malignant hematologic disease. Plasma samples were collected from patients or spiked in vitro. A time-saving rapid equilibrium dialysis assay was used for the separation of unbound and bound VRC, following a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis method for drug concentration detection. Liver function and treatment details were collected from the electronic medical records of patients. Protein concentration was determined according to instructions. VRC plasma protein binding rate (PPB) in patient is significantly higher [69.5 ± 6.2%] than that in in-vitro samples, influenced by total drug concentration (Ct), plasma protein concentration, and protein type. The α1-acid glycogen (AAG) has the highest affinity with VRC. Relationship between total PPB of VRC with PPB of individual protein is not a simple addition, but a compressive combination. Unbound drug concentration (Cu) of VRC shows significant relationships with Ct, protein concentration, AST level, metabolism type of CYP2C19 and co-administration of high PPB medicines. Unbound plasma concentration of VRC shows a more sensitive relationship with ADRs than Ct.

A Population Pharmacokinetic Model-Guided Evaluation of Ceftolozane-Tazobactam Dosing in Critically Ill Patients Undergoing Continuous Venovenous Hemodiafiltration.

The aim of this work was to describe optimized dosing regimens of ceftolozane-tazobactam for critically ill patients receiving continuous venovenous hemodiafiltration (CVVHDF). We conducted a prospective observational pharmacokinetic study in adult critically ill patients with clinical indications for ceftolozane-tazobactam and CVVHDF. Unbound drug concentrations were measured from serial prefilter blood, postfilter blood, and ultrafiltrate samples by a chromatographic assay. Population pharmacokinetic modeling and dosing simulations were performed using Pmetrics. A four-compartment pharmacokinetic model adequately described the data from six patients. The mean (± standard deviation [SD]) extraction ratios for ceftolozane and tazobactam were 0.76 ± 0.08 and 0.73 ± 0.1, respectively. The mean ± SD sieving coefficients were 0.94 ± 0.24 and 1.08 ± 0.30, respectively. Model-estimated CVVHDF clearance rates were 2.7 ± 0.8 and 3.0 ± 0.6 liters/h, respectively. Residual non-CVVHDF clearance rates were 0.6 ± 0.5 and 3.3 ± 0.9 liters/h, respectively. In the initial 24 h, doses as low as 0.75 g every 8 h enabled cumulative fractional response of ≥85% for empirical coverage against Pseudomonas aeruginosa, considering a 40% fT>MIC (percentage of time the free drug concentration was above the MIC) target. For 100% fT>MIC, doses of at least 1.5 g every 8 h were required. The median (interquartile range) steady-state trough ceftolozane concentrations for simulated regimens of 1.5 g and 3.0 g every 8 h were 28 (21 to 42) and 56 (42 to 84) mg/liter, respectively. The corresponding tazobactam concentrations were 6.1 (5.5 to 6.7) and 12.1 (11.0 to 13.4) mg/liter, respectively. We suggest a front-loaded regimen with a single 3.0-g loading dose followed by 0.75 g every 8 h for critically ill patients undergoing CVVHDF with study blood and dialysate flow rates.

ACTR-10. PHASE 0 TRIAL OF CERITINIB IN BRAIN METASTASES AND RECURRENT GLIOBLASTOMA

Abstract BACKGROUND Ceritinib is an orally bioavailable, small molecule inhibitor for ALK/IGFR1/FAK, which are highly expressed in glioblastoma and brain metastases. Preclinical and clinical reports suggest ceritinib activity in central nervous system (CNS) malignancies, yet there is no direct evidence in patients. This study assessed the pharmacokinetics (PK) and pharmacodynamics (PD) of ceritinib in brain metastases and recurrent glioblastoma patients. METHODS Ten patients with brain metastases (n=3) or glioblastoma (n=7) exhibiting high expression of pSTAT5b/pFAK/pIGFR1 were treated with 10 days of oral ceritinib (750 mg daily) prior to tumor resection. Plasma, tumor, and cerebrospinal fluid (CSF) samples were collected at ~ 4 and 24 h following the last dose. Total and unbound drug concentrations were determined using LC-MS/MS. PD response was assessed by immunohistochemical analysis of pALK, pFAK, pIGFR1, and pIRS1 staining in treated tumor and matched archival tissues. RESULTS Ceritinib was highly bound to human plasma protein (median fraction unbound (Fu), 1.4%) and to brain tumor tissue (median Fu, 0.073% and 0.14% in enhancing and non-enhancing regions respectively). There was a large interindividual variability in drug CNS penetration, with the median unbound concentrations in enhancing, non-enhancing, and CSF of 0.040, 0.006, and 0.012 µM, respectively. The median unbound tumor-to-plasma ratio was 2.44 and 0.33 in enhancing and non-enhancing areas, respectively. In one brain metastasis patient, drug binding to enhancing tumor was significantly lower (Fu, 1.62%), resulting in a higher unbound drug tumor and CSF concentration as compared to all other patients. In all patients, no pharmacodynamic response was detected in sampled tumor tissue. CONCLUSION Ceritinib is highly bound to plasma proteins and tumor tissues. Unbound drug concentrations in brain metastasis and glioblastoma appear insufficient for target modulation. Despite recent reports of clinical response, our findings suggest no role for ceritinib in treating glioblastoma and an unfavorable profile for brain metastases.

MNGI-01. A PHASE 0 TRIAL OF RIBOCICLIB IN AGGRESSIVE MENINGIOMA PATIENTS INCORPORATING A TUMOR PHARMACODYNAMIC- AND PHARMACOKINETIC-GUIDED EXPANSION COHORT

Abstract BACKGROUND New approaches are urgently needed for aggressive meningiomas, which remain largely incurable. Forkhead Box M1 (FOXM1) has been identified as a master transcription factor in aggressive meningiomas and Cyclin D-dependent Kinases (CDK) are positive regulators of cell-cycle entry, promoting tumorigenesis through FOXM1 activation. We evaluated the tumor pharmacokinetics (PK), tumor pharmacodynamics (PD), and preliminary clinical response of ribociclib, a selective CDK4/6-inhibitor, in aggressive meningioma patients. METHODS Eight aggressive WHO Grade II/III meningioma patients with intact RB expression were enrolled and administered oral ribociclib daily for 5 days prior to tumor resection. Plasma, tumor, and cerebrospinal fluid (CSF) samples were collected at 2, 8, or 24 h after the last dose. Total and unbound drug concentrations were determined using a validated LC-MS/MS method. PD effects, including RB and FoxM1 phosphorylation, were compared to matched archival tissue. Patients with PK and PD responses in tumor tissue, defined as unbound ribociclib concentration > 5-fold in vitro IC50 (0.04µM) and >20% decrease in pRB levels, respectively, were enrolled into an expansion cohort for preliminary assessment of progression-free survival. RESULTS The median CSF concentration of ribociclib was 0.25 µM. In tumor tissue, the median unbound ribociclib concentration was 1.36 µM and the median unbound tumor-to-plasma ratio was 5.34. Suppression of G1-to-S phase was inferred in tumors with declining FoxM1 phosphorylation (50%), RB phosphorylation (38%), and cellular proliferation (75%). Four patients demonstrated concurrent PK and PD responses and were graduated to continuous ribociclib therapy. At 14 months, two of these patients (one Grade II and one Grade III) demonstrate partial responses per RANO criteria. CONCLUSION Ribociclib achieves pharmacologically-active concentrations in aggressive meningioma tissue. Target modulation was demonstrated by a decrease in FOXM1-mediated tumor proliferation. Further investigation of ribociclib as a therapeutic strategy for aggressive meningiomas is warranted.

392O - A phase 0/II clinical trial of a CDK4/6 inhibitor in aggressive meningioma patients

BackgroundNew approaches are urgently needed for aggressive meningiomas, which remain largely incurable. Forkhead Box M1 (FOXM1) has been identified as a master transcription factor in aggressive meningiomas and Cyclin D-dependent Kinases (CDK) are positive regulators of cell-cycle entry, promoting tumorigenesis through FOXM1 activation. We evaluated the tumor pharmacokinetics (PK), tumor pharmacodynamics (PD), and preliminary clinical response of ribociclib, a selective CDK4/6-inhibitor, in aggressive meningioma patients. MethodsEight aggressive WHO Grade II/III meningioma patients with intact RB expression were enrolled and administered oral ribociclib daily (900mg) for 5 days prior to tumor resection. Plasma, tumor, and cerebrospinal fluid (CSF) samples were collected at 2, 8, or 24h after the last dose. Total and unbound drug concentrations were determined using a validated LC-MS/MS method. PD effects, including RB and FoxM1 phosphorylation, were compared to matched archival tissue. Patients with PK and PD responses in tumor tissue, defined as unbound ribociclib concentration > 5-fold in vitro IC50 (0.04 µM) and >20% decrease in pRB levels, respectively, were enrolled into an exploratory phase 2 cohort. ResultsThe median CSF concentration of ribociclib was 0.25 µM. In tumor tissue, the median unbound ribociclib concentration was 1.36 µM and the median unbound tumor-to-plasma ratio was 5.34. Suppression of G1-to-S phase was inferred in tumors with declining FoxM1 phosphorylation (50%), RB phosphorylation (38%), and cellular proliferation (75%). Four patients demonstrated concurrent PK and PD responses and were graduated to continuous ribociclib therapy. At one year, two of these patients (one Grade II and one Grade III) demonstrate partial responses per RANO criteria. ConclusionsRibociclib achieves pharmacologically-active concentrations in aggressive meningioma tissue. Target modulation was demonstrated by a decrease in FOXM1-mediated tumor proliferation. Further investigation of ribociclib as a therapeutic strategy for aggressive meningiomas is warranted. Clinical trial identificationNCT02933736. Legal entity responsible for the studyThe authors. FundingThe Ben and Catherine Ivy Foundation. DisclosureAll authors have declared no conflicts of interest.