Population Pharmacokinetics and Pharmacodynamics of Benralizumab in Healthy Volunteers and Patients With Asthma.

Benralizumab is a humanized, afucosylated, anti-interleukin-5 receptor α, immunoglobulin G (IgG) 1 κ monoclonal antibody. We developed a population pharmacokinetic (PK)/pharmacodynamic (PD) model for benralizumab by analyzing PK and blood eosinophil count data from two healthy volunteer studies (N = 48) and four studies in patients with asthma (N = 152). Benralizumab PK was dose-proportional and adequately described by a two-compartment model with first-order elimination from the central compartment and first-order absorption from the subcutaneous dosing site. The estimated systemic clearance and volume of distribution were typical for human IgG. Body weight and high-titer antidrug antibodies were identified as relevant covariates influencing the PK of benralizumab. Depletion of blood eosinophil counts was depicted by a modified transit model in which benralizumab induced depletion of eosinophils in each age compartment. Stochastic simulations supported an every-8-week dosing schedule of benralizumab for a phase IIb study in patients with uncontrolled asthma.

Pharmacogenomics and translational simulations to bridge indications for an anti-interferon-α receptor antibody.

A type I interferon (IFN) gene signature shared by systemic lupus erythematous (SLE) and systemic sclerosis (SSc) was used to evaluate an anti-type I IFN-α receptor (IFN-αR) monoclonal antibody, MEDI-546, in a phase I trial in SSc. MEDI-546 suppressed IFN signature in blood and skin of SSc patients in a dose-dependent manner. To bridge clinical indications to SLE, we developed a model incorporating (i) pharmacokinetics (PK) and pharmacodynamics (PD) in SSc patients, (ii) internalization kinetics of MEDI-546/IFN-αR complex, and (iii) the different IFN signatures between SSc and SLE. Simulations predicted that i.v. administration of MEDI-546 at 300- or 1,000-mg monthly doses could suppress IFN signature in blood to levels of healthy subjects in 53 and 68% of SLE patients, respectively. An innovative approach utilizing a novel biomarker characterized the PD of MEDI-546 by modeling and simulation and allowed rapid progression of MEDI-546 from a phase I study in SSc to a randomized, multiple-dose phase II trial.

Protein engineering and preclinical development of a GM-CSF receptor antibody for the treatment of rheumatoid arthritis.

BACKGROUND AND PURPOSE: For antibody therapies against receptor targets, in vivo outcomes can be difficult to predict because of target-mediated clearance or antigen 'sink' effects. The purpose of this work was to engineer an antibody to the GM-CSF receptor α (GM-CSFRα) with pharmacological properties optimized for chronic, s.c. treatment of rheumatoid arthritis (RA) patients. EXPERIMENTAL APPROACH: We used an in silico model of receptor occupancy to guide the target affinity and a combinatorial phage display approach for affinity maturation. Mechanism of action and internalization assays were performed on the optimized antibody in vitro before refining the modelling predictions of the eventual dosing in man. Finally, in vivo pharmacology studies in cynomolgus monkeys were carried out to inform the predictions and support future clinical development. KEY RESULTS: Antibody potency was improved 8600-fold, and the target affinity was reached. The refined model predicted pharmacodynamic effects at doses as low as 1 mg kg(-1) and a study in cynomolgus monkeys confirmed in vivo efficacy at 1 mg kg(-1) dosing. CONCLUSIONS AND IMPLICATIONS: This rational approach to antibody drug discovery enabled the isolation of a potent molecule compatible with chronic, s.c. self-administration by RA patients. We believe this general approach enables the development of optimal biopharmaceuticals.

Nonclinical aspects of biopharmaceutical development: discussion of case studies at a PhRMA-FDA workshop.

Robust assessments of the nonclinical safety profile of biopharmaceuticals are best developed on a scientifically justified, case-by-case basis, with consideration of the therapeutic molecule, molecular target, and differences/similarities between nonclinical species and humans (ICH S6). Significant experience has been gained in the 10 years ensuing since publication of the ICH S6 guidance. In a PhRMA-FDA-sponsored workshop, "Nonclinical Aspects of Biopharmaceutical Development," industry and US regulatory representatives engaged in exploration of current scientific and regulatory issues relating to the nonclinical development of biopharmaceuticals in order to share scientific learning and experience and to work towards establishing consistency in application of general principles and approaches. The proceedings and discussions of this workshop confirm general alignment of strategy and tactics in development of biopharmaceuticals with regard to such areas as species selection, selection of high doses in toxicology studies, selection of clinical doses, the conduct of developmental and reproductive toxicity (DART) studies, and assessment of carcinogenic potential. However, several important aspects, including, for example, appropriate use of homologues, nonhuman primates, and/or in vitro models in the assessment of risk for potential developmental and carcinogenic effects, were identified as requiring further scientific exploration and discussion.

Pegfilgrastim, a sustained-duration form of filgrastim, significantly improves neutrophil recovery after autologous marrow transplantation in rhesus macaques.

Daily administration of filgrastim decreases the duration of severe neutropenia in the clinical setting. A sustained-duration form of filgrastim, pegfilgrastim, significantly reduces scheduling protocols to a single injection per chemotherapy cycle while maintaining therapeutic efficiency. We examined the ability of a single injection of pegfilgrastim to significantly improve neutrophil recovery following autologous bone marrow transplantation (AuBMT) in rhesus macaques. On day 1, postmyeloablation (920 cGy x-irradiation) and AuBMT, animals received either 0.1% autologous serum for 18 consecutive days (n=13), or single doses of pegfilgrastim via the subcutaneous (s.c.) or intravenous (i.v.) route (300 or 100 micro g/kg), or a single dose of filgrastim at 300 micro g/kg via the s.c. or i.v. route, or filgrastim at 10 micro g/kg via the s.c. route (n=4) on a daily basis (range=days 12-17). Pharmacokinetic parameters and neutrophil recovery were assessed. A single dose of pegfilgrastim via the i.v. or s.c. route was as effective as daily filgrastim administration, resulting in significant improvement of neutrophil recovery after myeloablation and ABuMT. Effective pegfilgrastim plasma concentrations were maintained in neutropenic animals until after the onset of hematopoietic recovery. Enhanced pharmacokinetics in AuBMT cohorts are consistent with self-regulating, neutrophil-mediated clearance.

Topiramate and phenytoin pharmacokinetics during repetitive monotherapy and combination therapy to epileptic patients.

PURPOSE: To evaluate the potential pharmacokinetic interactions between topiramate (TPM) and phenytoin (PHT) in patients with epilepsy by studying their pharmacokinetics (PK) after monotherapy and concomitant TPM/PHT treatment. METHODS: Twelve patients with epilepsy stabilized on PHT monotherapy were enrolled in this study, with 10 and seven patients completing the phases with 400 and 800 mg TPM daily doses, respectively. TPM was added at escalating doses, and after stabilization at the highest tolerated TPM dose, PHT doses were tapered. Serial blood and urine samples were collected for PK analysis during the monotherapy phase or the lowest PHT dose after taper and the concomitant TPM/PHT phase. Potential metabolic interaction between PHT and TPM also was studied in vitro in human liver microsomal preparations. RESULTS: In nine of the 12 patients, PHT plasma concentrations remained stable, with a mean (+/-SD) area under the curve (AUC) ratio (combination therapy/monotherapy) of 1.13 +/- 0.17 (range, 0.89-1.23). Three patients had AUC ratios of 1.25, 1.39, and 1.55, respectively, and with the addition of TPM (800, 400, and 400 mg daily, respectively), their peak PHT plasma concentrations increased from 15 to 21 mg/L, 28 to 36 mg/L, and 27 to 41 mg/L, respectively. Human liver microsomal studies with S-mephenytoin showed that TPM partially inhibited CYP2C19 at very high concentrations of 300 microM (11% inhibition) and 900 microM (29% inhibition). Such high plasma concentrations would correspond to doses in humans that are 5 to 15 times higher than the recommended dose (200-400 mg). TPM clearance was approximately twofold higher during concomitant TPM/PHT therapy CONCLUSIONS: This study provides evidence that the addition of TPM to PHT generally does not cause clinically significant PK interaction. PHT induces the metabolism of TPM, causing increased TPM clearance, which may require TPM dose adjustments when PHT therapy is added or is discontinued. TPM may affect PHT concentrations in a few patients because of inhibition by TPM of the CYP2C19-mediated minor metabolic pathway of PHT.

Population pharmacokinetic-pharmacodynamic modeling of filgrastim (r-metHuG-CSF) in healthy volunteers.

The pharmacokinetic-pharmacodynamic (PK-PD) relationship of the granulopoietic effects of Filgrastim in healthy volunteers was characterized via a population approach. Healthy male volunteers were enrolled into a four-way crossover clinical trial. Subjects received four single doses of Filgrastim (375 and 750 micrograms i.v. and s.c.) with an intervening washout period of 7 days. Serum concentrations of Filgrastim were determined using an enzyme-linked immunosorbent assay. Absolute neutrophil count (ANC) was determined. Data analysis was performed using mixed-effects modeling as implemented in the NONMEM software package. The final PKPD model incorporates a two-compartment PK model with bisegmental absorption from the s.c. site, first-order and saturable elimination pathways, and an indirect PD model. A sigmoidal Emax model for the stimulation of ANC input rate (kin) was superior to the conventional Emax model (mean +/- SE: Emax = 12.7 +/- 1.7; EC50 = 4.72 +/- 0.72 ng/ml; Hill = 1.34 +/- 0.19). In addition, a time-variant scaling factor for ANC observations was introduced to account for the early transient depression of ANC after Filgrastim administration. The absolute bioavailability of subcutaneously administered Filgrastim was estimated to be 0.619 +/- 0.058 and 0.717 +/- 0.028 for 375 micrograms and 750 micrograms s.c. doses, respectively. The time profiles of concentration and ANC, as well as the concentration approximately ANC relationship of Filgrastim in healthy volunteers were well described by the developed population PK-PD model.

Treatment of steroid-refractory acute graft-versus-host disease with anti-CD147 monoclonal antibody ABX-CBL.

ABX-CBL, an immunoglobulin M murine monoclonal antibody, recognizes CD147 and initiates cell killing through complement-mediated lysis. In a dose-finding trial, 27 patients with steroid-refractory acute graft-versus-host disease (GVHD) received ABX-CBL at 0.01 (presumed no effect dose), 0.1, 0.2, or 0.3 mg/kg per day, and an additional 32 patients were given ABX-CBL at 0.2 or 0.15 mg/kg per day. All patients had undergone allogeneic transplantation for malignant or nonmalignant disorders and received GVHD prophylaxis, generally with methotrexate- and cyclosporine-containing regimens. None responded to methylprednisolone, given for a minimum of 3 days. ABX-CBL was started 20 to 236 (median, 47) days after transplantation; it was given for 7 consecutive days and was followed by 2 infusions per week for 2 more weeks. Among 51 patients evaluable for efficacy, 26 (51%) responded, including 13 with complete responses (CR) and 13 with partial responses (PR). CR lasting 14 days or longer or PR lasting 7 days or longer occurred in 21 (41%; 8 CR, 13 PR) patients, including 19 of 43 (44%) patients who received 0.1 to 0.3 mg/kg ABX-CBL and 2 of 8 (25%) patients given 0.01 mg/kg per day. Myalgias at doses 0.2 mg/kg or greater were dose limiting and resolved without sequelae. Causes of death included organ failure, progressive GVHD, and infection. No death was attributed to ABX-CBL. At 6 months after the initiation of ABX-CBL therapy, 26 (44%) patients were surviving. These results are encouraging. Further studies on the use of ABX-CBL in the management of GVHD are warranted.

Cyclic immune thrombocytopenia responsive to thrombopoietic growth factor therapy.

We report a patient with cyclic thrombocytopenia and antiplatelet antibodies, a variant of chronic immune thrombocytopenic purpura (ITP), with a several year history of periodic fluctuation of the platelet count, megakaryocytic hyperplasia and high-titer anti-GPIb-specific antiplatelet antibodies. The patient was resistant to multiple forms of therapy but has responded to the thrombopoietic growth factor, pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF). This case suggests that some patients with classic ITP may respond to thrombopoietic growth factors.

Randomized, dose-escalation study of SD/01 compared with daily filgrastim in patients receiving chemotherapy.

PURPOSE: To explore the use of SD/01 (a polyethylene glycol-conjugated filgrastim shown in preclinical studies to have a prolonged half-life) in patients with chemotherapy-induced neutropenia. PATIENTS AND METHODS: Thirteen patients with non-small-cell lung cancer were randomized to receive daily filgrastim (5 microg/kg/d) or a single injection of SD/01 (30, 100, or 300 microg/kg) 2 weeks before chemotherapy and again 24 hours after administration of carboplatin and paclitaxel. Pharmacodynamic, pharmacokinetic, and safety analyses were performed. RESULTS: Peak serum concentrations of SD/01 and the duration of increased serum concentrations were dependent on the SD/01 dose. SD/01 concentrations remained increased longer in patients with chemotherapy-induced neutropenia. Prechemotherapy median absolute neutrophil counts (ANCs) in patients receiving SD/01 were increased in a dose-dependent fashion, with the duration of this effect also being dose dependent. After chemotherapy, median ANC nadirs were similar in the filgrastim cohort and the cohort receiving SD/01 30 microg/kg, with higher nadirs seen in the cohorts receiving SD/01 100 or 300 microg/kg. Dose-limiting toxicities were not noted. CD34(+) cells were mobilized in all cohorts. CONCLUSION: A single dose of SD/01 increases the serum concentration of SD/01 for several days in a dose-dependent fashion and is not associated with significant toxicity. The effects of SD/01 on ANC and CD34(+) cell mobilization are comparable or greater than those achieved with daily filgrastim. The self-regulation of this molecule provides a potential therapeutic advantage in a variety of clinical settings associated with neutropenia.

Pharmacokinetic analysis of pegylated megakaryocyte growth and development factor in humans.

Phase I studies with pegylated megakaryocyte growth and development factor (PEG-rHuMGDF), a c-Mpl ligand that stimulates megakaryopoiesis, have demonstrated that PEG-rHuMGDF is biologically active alone and causes a dose-related enhancement of platelet recovery when administered after chemotherapy. Here we report the dose-ranging pharmacokinetics of PEG-rHuMGDF. Pre-injection blood samples were drawn daily for pharmacokinetic studies on 43 patients. An ELISA, established using PEG-rHuMGDF as the standard, was able to quantitate Mpl ligand at concentrations > 0.02 ng/mL. Over the dose range 0.03 to 5.0 microg/kg/day, subcutaneous administration produced linear increases in steady-state serum levels. Maximum levels of PEG-rHuMGDF attained after 5.0 microg/kg/day were 5.88 to 10.9 ng/mL. After discontinuation of PEG-rHuMGDF, concentrations of Mpl ligand returned to baseline within 5 days. The pharmacokinetics were best described by a one-compartment model with first-order absorption, an absorption delay, and non linear clearance over the first 48 hours. The mean terminal half-life was 33.3 + 16.7 hours, and the average apparent at steady state was 27.7 + 14.0 mL/h/kg; both were independent of administered dose. The apparent clearance of PEG-rHuMGDF was not predicted by platelet count. Administration of chemotherapy and Filgrastim did not alter the pharmacokinetics of PEG-rHuMGDF.

A population approach to enzyme characterization and identification: application to phenacetin O-deethylation.

PURPOSE: To determine the enzyme kinetics (EK) and identify the human cytochrome(s) P450 (CYP) involved in the deethylation of phenacetin to acetaminophen using a population-based method. METHODS: A sparse data set was generated from incubations containing human liver microsomes (n = 19) with phenacetin. Estimates of the EK parameters were obtained by fitting the concentration-velocity data to Michaelis-Menten models by using nonlinear mixed effects modeling. Relationships between the EK parameters and the CYP activities determined for these liver microsomes were examined. RESULTS: A two-enzyme kinetic model with a saturated, low KM enzyme and an unsaturated, high KM enzyme capable of forming acetaminophen best fit the data. The population estimates of the EK parameters were Vmax1, 911 pmol/min/mg protein; KM1, 11.3 microM; and Cl(int2), 0.4 microl/min/mg. The coefficients of variation for interliver variability in Vmax1 and residual error of the model were 39% and 15%, respectively. When the selective catalytic activities were examined as potential covariates, 7-ethoxyresorufin O-deethylation (CYP1A2) activity was found to be associated with the low KM enzyme, however, the high KM enzyme(s) could not be identified. CONCLUSIONS: The population approach characterized the EK parameters and identified the low KM enzyme responsible for phenacetin O-deethylation as CYP1A2. Population modeling of EK provides valuable information on inter- and intraliver variability in CYP dependent activities.

Effects of megakaryocyte growth and development factor on platelet production, platelet life span, and platelet function in healthy human volunteers.

The effects of thrombopoietic stimulation on megakaryocytopoiesis, platelet production, and platelet viability and function were examined in normal volunteers randomized to receive single bolus subcutaneous injections of 3 microg/kg pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo in a 3:1 ratio. PEG-rHuMGDF transiently doubled circulating platelet counts, from 237 +/- 41 x 10(3)/microL to 522 +/- 90 x 10(3)/microL (P <.0001), peaking on day 12. Baseline and day-12 samples showed no differences in responsiveness of platelets to adenosine diphosphate or thrombin receptor agonist peptide (P >.4 in all cases); expression of platelet ligand-induced binding sites or annexin V binding sites (P >.6 in both cases); or density of platelet TPO-receptors (P >.5). Platelet counts normalized by day 28. The life span of autologous (111)In-labeled platelets increased from 205 +/- 18 hours (baseline) to 226 +/- 22 hours (P <.01) on day 8. Platelet life span decreased from 226 +/- 22 hours (day 8) to 178 +/- 53 hours (P <.05) on day 18. The theoretical basis for senescent changes in mean platelet life span was illustrated by biomathematical modeling. Platelet turnover increased from 43.9 +/- 11.9 x 10(3) platelets/microL/d (baseline) to 101 +/- 27.6 x 10(3) platelets/microL/d (P =.0009), and marrow megakaryocyte mass expanded from 37.4 +/- 18.5 fL/kg to 62 +/- 17 x 10(10) fL/kg (P =. 015). Although PEG-rHuMGDF initially increased megakaryocyte volume and ploidy, subsequently ploidy showed a transient reciprocal decrease when the platelet counts exceeded placebo values. In healthy human volunteers PEG-rHuMGDF transiently increases megakaryocytopoiesis 2-fold. Additionally, peripheral platelets expand correspondingly and exhibit normal function and viability during the ensuing 10 days. The induced perturbation in steady state thrombopoiesis resolves by 4 weeks. (Blood. 2000;95:2514-2522)

The prolonged hematologic effects of a single injection of PEG-rHuMGDF in normal and thrombocytopenic mice.

A single injection of > or =10 microg/kg PEG-rHuMGDF in mice causes a dose-dependent increase in circulating platelets beginning on day 3 and peaking on days 5-6. The mean platelet volume and platelet distribution width at doses > or =100 microg/kg initially increase in a dose-dependent fashion and later decrease. However, the mean platelet volume does not change when platelets are incubated with PEG-rHuMGDF in vitro. The number of marrow megakaryocytes increases in a dose-dependent fashion as early as day 1 and peaks on day 3. Marrow megakaryocyte colony-forming units (CFU-Meg) do not increase on days 1-3 at a dose of 100 microg/kg (a dose that increases platelet numbers two- to threefold and may be clinically relevant), but the relative frequency of high ploidy megakaryocytes and the proportion of large marrow megakaryocytes (29-50 microm in diameter) increases. After a dose of 1,000 microg/kg the percentage of megakaryocytes in mitosis peaks at 24-48 hours and the percentage of megakaryocytes incorporating BrdU is maximal at 48 hours, the relatively delayed peak of BrdU incorporation most likely representing endomitosis. The relative frequency of type II and III megakaryocytes peaks on days 3 and 4, respectively. Pharmacokinetic analysis of PEG-rHuMGDF shows peak serum concentrations at 2-4 hours and a terminal half-life of 11.4+/-2.5 hours. A single injection of PEG-rHuMGDF ameliorates carboplatin-induced megakaryocytopenia and thrombocytopenia in a dose-response dependent fashion. In conclusion, a single injection of PEG-rHuMGDF increases megakaryocyte and platelet production in normal and myelo-suppressed mice.

Identification of the human cytochromes P450 responsible for the in vitro formation of the major oxidative metabolites of the antipsychotic agent olanzapine.

The formation kinetics of 2-hydroxymethyl olanzapine (2-OH olanzapine), 4'-N-oxide olanzapine (N-O olanzapine) and 4'-N-desmethyl olanzapine (NdM olanzapine) were analyzed in vitro. Biphasic kinetics were observed for formation of 2-OH and NdM olanzapine. The high-affinity enzyme responsible for 2-OH olanzapine formation by two human liver samples exhibited an intrinsic clearance (CLint) of 0.2 microliter/min/mg. NdM olanzapine formation by two human liver samples exhibited a CLint of 1.0 microliter/min/mg for the high affinity enzyme. The formation of N-O olanzapine was linear up to 300 microM olanzapine, yielding a CLint of 0.32 to 1.70 microliters/min/mg. The formation of 7-hydroxy olanzapine (7-OH olanzapine) exhibited an apparent Km of 24.2 microM. The rates of 2-OH olanzapine formation correlated with CYP2D6 levels and activity, and it was formed to the greatest extent by cDNA-expressed CYP2D6. N-O olanzapine formation correlated with human liver flavin-containing monooxygenase (FMO3) levels and activity. NdM olanzapine and 7-OH olanzapine formation correlated with CYP1A2 catalytic activities and they were formed to the greatest extent by expressed CYP1A2. These results suggest that CYP1A2 catalyzes NdM olanzapine and 7-OH olanzapine formation, CYP2D6 catalyzes 2-OH olanzapine formation and FMO3 catalyzes N-O olanzapine formation.

Effect of fluoxetine, norfluoxetine, sertraline and desmethyl sertraline on human CYP3A catalyzed 1'-hydroxy midazolam formation in vitro.

The ability of fluoxetine, norfluoxetine, sertraline and desmethyl sertraline to inhibit the CYP3A subfamily of cytochromes P450 was examined in vitro, using the formation of 1'-hydroxy midazolam as a probe for CYP3A catalytic activity. The inhibition observed with these four compounds was modeled using competitive, noncompetitive, uncompetitive and mixed competitive/noncompetitive relationships by nonlinear regression analysis. The best fit model of the inhibition of CYP3A-mediated 1'-hydroxy midazolam formation by all four compounds examined was determined to be mixed inhibition. The calculated Ki values were 65.7 +/- 12.0 microM for fluoxetine, 19.1 +/- 1.9 microM for norfluoxetine, 64.4 +/- 11.6 microM for sertraline and 48.1 +/- 11.6 microM for desmethyl sertraline. Steady-state plasma levels of fluoxetine and norfluoxetine can approach a concentration of 1 microM (approximately 350 ng/ml of plasma). Assuming an inhibitor concentration of 1 microM and a concentration of the substrate substantially below its Km (at least 10-fold lower), the results reported predict that fluoxetine and norfluoxetine together would inhibit CYP3A catalytic activity by less than 7% (less than 0.7% if the unbound plasma concentration of fluoxetine is considered). By using the same assumptions and concentrations for sertraline and desmethyl sertraline, these agents together would be predicted to inhibit the metabolic clearance of a coadministered CYP3A metabolized drug by less than 4%. The observations reported here suggest that fluoxetine and sertraline would have little effect on CYP3A-mediated clearance of coadministered drugs.