A phase 1 study to evaluate the safety and LDL cholesterol-lowering effects of RG7652, a fully human monoclonal antibody against proprotein convertase subtilisin/kexin type 9.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) downregulates low-density lipoprotein (LDL) receptors, thereby leading to a rise in circulating LDL cholesterol (LDL-C). RG7652 is a fully human monoclonal antibody against PCSK9. This placebo-controlled, phase 1 ascending-dose study in healthy subjects evaluated the safety of RG7652 and its efficacy as a potential LDL-C-lowering drug. HYPOTHESIS: Anti-PCSK9 antibody therapy safely and effectively reduces LDL-C. METHODS: Subjects (N = 80) were randomized into 10 cohorts. Six sequential single-dose cohorts received 10, 40, 150, 300, 600, or 800 mg of RG7652 via subcutaneous injection. Four multiple-dose cohorts received 40 or 150 mg of RG7652 once weekly for 4 weeks, either with or without statin therapy (atorvastatin). RESULTS: Adverse events (AEs) were generally mild; the most common AEs were temporary injection-site reactions. No serious AEs, severe AEs, AEs leading to study-drug discontinuation, or dose-limiting toxicities were reported. RG7652 monotherapy reduced mean LDL-C levels by up to 64% and as much as 100 mg/dL at week 2; the effect magnitude and duration increased with dose (≥57 days following a single RG7652 dose ≥300 mg). Exploratory analyses showed reduced oxidized LDL, lipoprotein(a), and lipoprotein-associated phospholipase A2 with RG7652. Antidrug antibody against RG7652 tested positive in 2 of 60 (3.3%) RG7652-treated and in 4 of 20 (20.0%) placebo-treated subjects. Simultaneous atorvastatin administration did not appear to impact the pharmacokinetic profile or lipid-lowering effects of RG7652. CONCLUSIONS: Overall, RG7652 elicited substantial and sustained dose-related LDL-C reductions with an acceptable safety profile and minimal immunogenicity.

Evaluation of Cytochrome P450 3A4-Mediated Drug-Drug Interaction Potential for Cobimetinib Using Physiologically Based Pharmacokinetic Modeling and Simulation.

BACKGROUND AND OBJECTIVES: Cobimetinib is eliminated mainly through cytochrome P450 (CYP) 3A4-mediated hepatic metabolism in humans. A clinical drug-drug interaction (DDI) study with the potent CYP3A4 inhibitor itraconazole resulted in an approximately sevenfold increase in cobimetinib exposure. The DDI risk for cobimetinib with other CYP3A4 inhibitors and inducers needs to be assessed in order to provide dosing instructions. METHODS: A physiologically based pharmacokinetic (PBPK) model was developed for cobimetinib using in vitro data. It was then optimized and verified using clinical pharmacokinetic data and itraconazole-cobimetinib DDI data. The contribution of CYP3A4 to the clearance of cobimetinib in humans was confirmed using sensitivity analysis in a retrospective simulation of itraconazole-cobimetinib DDI data. The verified PBPK model was then used to predict the effect of other CYP3A4 inhibitors and inducers on cobimetinib pharmacokinetics. RESULTS: The PBPK model described cobimetinib pharmacokinetic profiles after both intravenous and oral administration of cobimetinib well and accurately simulated the itraconazole-cobimetinib DDI. Sensitivity analysis suggested that CYP3A4 contributes ~78 % of the total clearance of cobimetinib. The PBPK model predicted no change in cobimetinib exposure (area under the plasma concentration-time curve, AUC) with the weak CYP3A inhibitor fluvoxamine and a three to fourfold increase with the moderate CYP3A inhibitors, erythromycin and diltiazem. Similarly, cobimetinib exposure in the presence of strong (rifampicin) and moderate (efavirenz) CYP3A inducers was predicted to decrease by 83 and 72 %, respectively. CONCLUSION: This study demonstrates the value of using PBPK simulation to assess the clinical DDI risk inorder to provide dosing instructions with other CYP3A4 perpetrators.

Population pharmacokinetics and dosing implications for cobimetinib in patients with solid tumors.

PURPOSE: To characterize cobimetinib pharmacokinetics and evaluate impact of clinically relevant covariates on cobimetinib pharmacokinetics. METHODS: Plasma samples (N = 4886) were collected from 487 patients with various solid tumors (mainly melanoma) in three clinical studies (MEK4592g, NO25395, GO28141). Cobimetinib was administered orally, once daily on either a 21-day-on/7-day-off, 14-day-on/14-day-off or 28-day-on schedule in a 28-day dosing cycle as single agent or in combination with vemurafenib. Cobimetinib doses ranged from 2.1 to 125 mg. NONMEM was used for pharmacokinetic analysis. RESULTS: A linear two-compartment model with first-order absorption, lag time and first-order elimination described cobimetinib pharmacokinetics. The typical estimates (inter-individual variability) of apparent clearance (CL/F), central volume of distribution (V2/F) and terminal half-life were 322 L/day (58 %), 511 L (49 %) and 2.2 days, respectively. Inter-occasion variability on relative bioavailability was estimated at 46 %. CL/F decreased with age. V2/F increased with body weight (BWT). However, the impact of age and BWT on cobimetinib steady-state exposure (peak and trough concentrations and AUC following the recommended daily dose of 60 mg 21-day-on/7-day-off) was limited (<25 % changes across the distribution of age and BWT). No significant difference in cobimetinib pharmacokinetics or steady-state exposure was observed between patient subgroups based on sex, renal function, ECOG score, hepatic function tests, race, region, cancer type, and co-administration of moderate and weak CYP3A inducers or inhibitors and vemurafenib. CONCLUSION: A population pharmacokinetic model was developed for cobimetinib in cancer patients. Covariates had minimal impact on steady-state exposure, suggesting no need for dose adjustments and supporting the recommended dose for all patients.

Modeling and Simulation to Support Phase 2 Dose Selection for RG7652, a Fully Human Monoclonal Antibody Against Proprotein Convertase Subtilisin/Kexin Type 9.

RG7652 is a fully humanized monoclonal antibody targeting human PCSK9, a regulator of serum low density lipoprotein cholesterol (LDLc) levels. RG7652 prevents degradation of the hepatic LDLc receptors by blocking PCSK9 binding and thereby resulting in efficient LDLc uptake by hepatocytes. The pharmacokinetics of RG7652 have been evaluated in healthy subjects after single and multiple subcutaneous doses. Pharmacokinetic (PK) and pharmacodynamic (PD) models were developed to explain the antibody PK and LDLc time course data. The PK and PD models based on data from healthy subjects were used to simulate the effects of RG7652 on LDLc levels for a range of potential dose regimens in patients with coronary heart disease. A one-compartment PK model combined with an indirect PD response model was able to adequately describe the PK and LDLc data. Simulations of 400 mg every 4 weeks or 800 mg every 8 weeks regimens show significant LDLc reduction and suggest that dosing RG7652 once every month or once every 2 months is predicted to be optimal for the treatment of hypercholesterolemia. The PK and PD model successfully described the PK and LDLc data from healthy subjects in a Phase 1 study, and the model-based simulations provided useful insights and quantitative understanding for the selection of Phase 2 study doses in patients with coronary heart disease. The approach used in the case study demonstrates the utility of modeling and simulation in designing dose-ranging studies.

Gastric reacidification with betaine HCl in healthy volunteers with rabeprazole-induced hypochlorhydria.

Previous studies have demonstrated that increased gastric pH from the use of acid-reducing agents, such as proton-pump inhibitors or H2-receptor antagonists, can significantly impact the absorption of weakly basic drugs that exhibit pH-dependent solubility. Clinically practical strategies to mitigate this interaction have not been developed. This pilot study evaluated the extent and time course of gastric reacidification after a solid oral dosage form of anhydrous betaine HCl in healthy volunteers with pharmacologically induced hypochlorhydria. Six healthy volunteers with baseline normochlorhydria (fasting gastric pH < 4) were enrolled in this single period study. Hypochlorhydria was induced via 20 mg oral rabeprazole twice daily for four days. On the fifth day, an additional 20 mg dose of oral rabeprazole was given and gastric pH was monitored continuously using the Heidelberg pH capsule. After gastric pH > 4 was confirmed for 15 min, 1500 mg of betaine HCl was given orally with 90 mL of water and gastric pH was continuously monitored for 2 h. Betaine HCl significantly lowered gastric pH by 4.5 (± 0.5) units from 5.2 (± 0.5) to 0.6 (± 0.2) (P < 0.001) during the 30 min interval after administration. The onset of effect of betaine HCl was rapid, with a mean time to pH < 3 of 6.3 (± 4.3) min. The reacidification period was temporary with a gastric pH < 3 and < 4 lasting 73 (± 33) and 77 (± 30) min, respectively. Betaine HCl was well tolerated by all subjects. In healthy volunteers with pharmacologically induced hypochlorhydria, betaine HCl was effective at temporarily lowering gastric pH. The rapid onset and relatively short duration of gastric pH reduction gives betaine HCl the potential to aid the absorption of orally administered weakly basic drugs that exhibit pH-dependent solubility when administered under hypochlorhydric conditions.

A physiologically based pharmacokinetic (PBPK) approach to evaluate pharmacokinetics in patients with cancer.

Potential differences in pharmacokinetics (PK) between healthy subjects and patients with cancer were investigated using a physiologically based pharmacokinetic approach integrating demographic and physiological data from patients with cancer. Demographic data such as age, sex and body weight, and clinical laboratory measurements such as albumin, alpha-1 acid glycoprotein (AAG) and hematocrit were collected in ~2500 patients with cancer. A custom oncology population profile was built using the observed relationships among demographic variables and laboratory measurements in Simcyp® software, a population based ADME simulator. Patients with cancer were older compared with the age distribution in a built-in healthy volunteer profile in Simcyp. Hematocrit and albumin levels were lower and AAG levels were higher in patients with cancer. The custom population profile was used to investigate the disease effect on the pharmacokinetics of two probe substrates, saquinavir and midazolam. Higher saquinavir exposure was predicted in patients relative to healthy subjects, which was explained by the altered drug binding due to elevated AAG levels in patients with cancer. Consistent with historical clinical data, similar midazolam exposure was predicted in patients and healthy subjects, supporting the hypothesis that the CYP3A activity is not altered in patients with cancer. These results suggest that the custom oncology population profile is a promising tool for the prediction of PK in patients with cancer. Further evaluation and extension of this population profile with more compounds and more data will be needed.

Dogs are more sensitive to antagonists of inhibitor of apoptosis proteins than rats and humans: a translational toxicokinetic/toxicodynamic analysis.

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and are overexpressed in a variety of cancers. GDC-0152 is a potent and selective IAP antagonist being developed as an anticancer agent. In preclinical safety studies, dogs were particularly sensitive to GDC-0152 showing adverse signs of a tumor necrosis factor alpha (TNF-α) driven systemic inflammatory response, related to cellular IAP degradation and activation of NFκB signaling, at lower exposures compared with rat. In addition, downstream increases in systemic levels of cytokines and chemokines, such as monocyte chemotactic protein-1 (MCP-1), were observed. A semimechanistic population toxicokinetic/toxicodynamic (TK/TD) model incorporating transit compartments was used to fit MCP-1 plasma concentrations from rats or dogs given iv GDC-0152 doses. Estimated TD parameters inferred that lower GDC-0152 plasma concentrations triggered more severe increases in plasma MCP-1 in dogs compared with rats. Human simulations performed using dog TD parameters and human pharmacokinetics predicted 300-2400% increases of MCP-1 in humans at iv doses from 0.76 to 1.48mg/kg. Similar simulations using rat TD parameters suggest little or no change. Patients given iv doses of GDC-0152 up to 1.48mg/kg iv showed no substantial increases in systemic MCP-1 or signs of a severe TNF-α driven systemic inflammatory response. Emerging clinical data reported for other IAP antagonists are consistent with our observations. Taken together, the data suggest dogs are more sensitive to IAP antagonists compared with humans and rats. This study illustrates how TK/TD analysis can be utilized to quantitatively translate and context an identified preclinical safety risk in dogs to humans.

Comparative performance of cell life span and cell transit models for describing erythropoietic drug effects.

Prolonged time delay in response to drug action is a common feature of hematological responses to pharmacotherapy such as erythropoiesis. The objective of this study was to compare the performance of two competing modeling approaches for delayed drug effects, mechanistic cell life span models, and semi-mechanistic cell transit models. The comparison was performed with an experimental dataset from multiple dose administrations of an erythropoietin mimetic to Cynomolgus monkeys. Comparative performance measures include visual predictive checks, goodness-of-fit plots, model estimation time, estimation status, and estimation error. The analysis revealed that both models resulted in a similarly good description of the erythropoietic drug effect, with precision and bias of the model-based predictions of red blood cell counts of less than 11%. The cell transit model needed slightly longer time to converge compared to the cell life span model. The system and drug effect parameters were similar in both models indicating that the models can be interchangeably used to describe the current data. Thus, model selection would be dependent on the purpose of the modeling exercise, the available data, and the time allocated for model development.

A simple in vitro PK/PD model system to determine time-kill curves of drugs against Mycobacteria.

In vivo tuberculosis is exposed to continually changing drug concentrations for which static minimum inhibitory concentration (MIC) testing may be a poor surrogate. While in vitro approaches to determine time-kill curves for antibiotics have been widely applied in assessing antimicrobial activity against fast growing microorganisms, their availability and application for slow-growing microorganisms including Mycobacterium tuberculosis has so far been scarce. Thus, we developed a novel simple in vitro pharmacokinetic/pharmacodynamic (PK/PD) model for establishing time-kill curves and applied it for evaluating the antimicrobial activity of different dosing regimens of isoniazid (INH) against Mycobacterium bovis BCG as a surrogate for virulent M. tuberculosis. In the in vitro model M. bovis BCG was exposed to INH concentration-time profiles as usually encountered during multiple dose therapy with 25, 100 and 300mg/day in humans who are fast or slow INH metabolizers. Bacterial killing was followed over time by determining viable counts and the resulting time-kill data was analyzed using a semi-mechanistic PK/PD model with an adaptive IC(50) function to describe the emergence of insensitive populations of bacteria over the course of treatment. In agreement with previous studies, the time-kill data suggest that AUC(0-24)/MIC is the PK/PD index that is the most explanatory of the antimicrobial effect of INH. The presented in vitro PK/PD model and associated modeling approach were able to characterize the time-kill kinetics of INH in M. bovis BCG, and may in general serve as a potentially valuable, low cost tool for the assessment of antibacterial activity in slow-growing organisms in drug development and applied pharmacotherapy.

A tandem mass spectrometry assay for the simultaneous determination of acetaminophen, caffeine, phenytoin, ranitidine, and theophylline in small volume pediatric plasma specimens.

BACKGROUND: Acetaminophen, caffeine, phenytoin, ranitidine, and theophylline are widely used in pediatric pharmacotherapy, but only very limited information is available on the pharmacokinetics of these medications in premature neonates. As pharmacokinetic studies in this population are hampered by limitations in the number and volume of plasma samples, we developed an LC-MS/MS assay for the simultaneous determination of these medications in small volume human plasma specimens for pharmacokinetic evaluations in neonates. METHODS: Sample preparation was performed by protein precipitation with methanol after addition of internal standard to 50 microl of plasma specimen. After chromatographic separation on a C18 column using gradient elution, analytes were detected using a triple quadrupole mass spectrometer that was operated in positive ion mode with electrospray ionization. RESULTS: All 5 analytes could be simultaneously quantified in human plasma. The linear quantification range comprised 12.2 to 25,000 ng/ml for acetaminophen, phenytoin, and ranitidine, 24.4 to 25,000 ng/ml for theophylline, and 48.8 to 25,000 ng/ml for caffeine with accuracies ranging from 87.5 to 115.0%. The intra-day and inter-day precision (%CV) was between 2.8 and 11.8% and 4.5 and 13.5%, respectively. CONCLUSIONS: An accurate, sensitive, and reliable LC-MS/MS method was developed and validated to simultaneously quantify 5 drugs frequently used in neonatal pharmacotherapy.

A microbiological assessment of novel nitrofuranylamides as anti-tuberculosis agents.

OBJECTIVES: Nitrofuranylamides (NFAs) are nitroaromatic compounds that have recently been discovered and have potent anti-tuberculosis (TB) activity. A foundational study was performed to evaluate whether this class of agents possesses microbiological properties suitable for future antimycobacterial therapy. METHODS: Five representative compounds of the NFA series were evaluated by standard microbiological assays to determine MICs, MBCs, activity against anaerobic non-replicating persistent Mycobacterium tuberculosis, post-antibiotic effects (PAEs), antibiotic synergy and the basis for resistance. RESULTS: The antimicrobial activity of these compounds was restricted to bacteria of the M. tuberculosis complex, and all compounds were highly active against drug-susceptible and -resistant strains of M. tuberculosis, with MICs 0.0004-0.05 mg/L. Moreover, no antagonism was observed with front-line anti-TB drugs. Activity was also retained against dormant bacilli in two in vitro low-oxygen models for M. tuberculosis persistence. A long PAE was observed, which was comparable to that of rifampicin, but superior to isoniazid and ethambutol. Spontaneous NFA-resistant mutants arose at a frequency of 10(-5)-10(-7), comparable to that for isoniazid (10(-5)-10(-6)). Some of these mutants exhibited cross-resistance to one or both of the nitroimidazoles PA-824 and OPC-67683. Cross-resistance was associated with inactivation of the reduced F(420)-deazaflavin cofactor pathway and not with inactivation of the Rv3547, the nitroreductase for PA-824 and OPC-67683. CONCLUSIONS: Based on these studies, NFAs have many useful antimycobacterial properties applicable to TB chemotherapy and probably possess a unique mode of action that results in good activity against active and dormant M. tuberculosis. Therefore, the further development of lead compounds in this series is warranted.

Pharmacokinetically-guided lead optimization of nitrofuranylamide anti-tuberculosis agents.

In an effort to develop novel and more potent therapies to treat tuberculosis, a new class of chemical agents, nitrofuranylamides, is being developed. The present study examines biopharmaceutic properties and preclinical pharmacokinetics of nitrofuranylamides at early stages of drug discovery to accelerate the optimization of leads into development candidates. The first tested compound, Lee 562, had high anti-tuberculosis activity in vitro, but exhibited poor metabolic stability resulting in a high systemic clearance, a short elimination half-life and low oral bioavailability in vivo in rats. Thus, two follow-up compounds were designed and tested that included structural modifications for increased metabolic stability. Both compounds showed improved metabolic stability compared to Lee 562, with Lee 878 being much more stable than Lee 952. As a consequence, the oral bioavailability of Lee 878 reached approximately 27% compared to 16% for the other two compounds. This observation prompted us to select compounds based on metabolic stability screening and a new set of nine compounds with high in vitro activity were tested for metabolic stability. The most stable compound in the assay, Lee 1106 was selected for further pharmacokinetic evaluation in rats. Surprisingly, Lee 1106 exhibited poor oral bioavailability, 4.6%. Biopharmaceutic evaluation of the compound showed that the compound has poor aqueous solubility and a high clogP. Based on these results, a screening paradigm was developed for optimization of the nitrofuranylamide lead compounds in a timely and cost-effective manner that might also be applicable to other classes of anti-infective drugs.

Biopharmaceutics, pharmacokinetics and pharmacodynamics of antituberculosis drugs.

Tuberculosis (TB) is the leading cause of mortality due to a single infectious agent. The currently used combination drug regimens produce cure rates that exceed 95%, given good patient adherence during the multiple months treatment period. However the recent surge in HIV infections and the synergy between HIV and TB as well as the emergence of resistance resulted in an unforeseen increase in the number of TB cases, including multi-drug resistant (MDR) and extensively-drug resistant (XDR) forms of TB. Consequently, there is an urgent need to develop novel, fast acting antituberculosis drugs with high potency that can provide treatment options for all forms of TB. It is well known that the current TB drugs exhibit differences in their in vivo activity profile and these differences are largely determined by their pharmacodynamics (PD), i.e. intrinsic antibacterial activity, biopharmaceutical properties such as solubility and permeability, and pharmacokinetic (PK) properties such as drug exposure, tissue distribution, and protein binding. An understanding of the relationships among these properties is considered key for a rational use of antituberculosis therapeutics. The current review provides a comprehensive summary of physicochemical/biopharmaceutical, PK, and PD properties of currently used antituberculosis drugs and novel agents under development. Also, a brief review of PK/PD parameters of current TB drugs is given and properties of a desirable TB drug target and drug molecule are outlined. The information provided herewith may be useful in the optimization of biopharmaceutical and PK/PD characteristics in the development of novel TB therapeutics and in the design of optimal treatment regimens.