Mauricio Maia

Systemic pharmacokinetics following intravitreal injections of ranibizumab, bevacizumab or aflibercept in patients with neovascular AMD

Background Data comparing systemic exposure and systemic vascular endothelial growth factor (VEGF) suppression of ranibizumab, bevacizumab and aflibercept following intravitreal injection are lacking. Methods Fifty-six patients with wet age-related macular degeneration received intravitreal ranibizumab (0.5 mg), bevacizumab (1.25 mg), or aflibercept (2.0 mg). Serum pharmacokinetics and plasma free VEGF were evaluated after the first and third injections. Results Following the first dose, systemic exposure to aflibercept was 5-, 37-, and 9-fold higher than ranibizumab, whereas, bevacizumab was 9-, 310-, and 35-fold higher than ranibizumab, based on geometric mean ratio of peak and trough concentrations and area under the curve, respectively. The third dose showed accumulation of bevacizumab and aflibercept but not ranibizumab. Aflibercept substantially suppressed plasma free VEGF, with mean levels below lower limit of quantitation (10 pg/mL) as early as 3 h postdose until ≥7 days postdose. Mean free (unbound) VEGF levels with ranibizumab were largely unchanged, with mean trough level of 14.4 pg/mL compared with baseline of 17 pg/mL. Conclusions There are notable differences in systemic pharmacokinetics and pharmacodynamics among anti-VEGF treatments after intravitreal administration. All three agents rapidly moved into the bloodstream, but ranibizumab very quickly cleared, whereas bevacizumab and aflibercept demonstrated greater systemic exposure and produced a marked reduction in plasma free VEGF. Trial registration number NCT02118831.

Ligand Binding Assays in the 21st Century Laboratory: Recommendations for Characterization and Supply of Critical Reagents

Critical reagents are essential components of ligand binding assays (LBAs) and are utilized throughout the process of drug discovery, development, and post-marketing monitoring. Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. Proactive reagent management assures adequate supply. It also assures that the quality of critical reagents is appropriate and consistent for the intended LBA use throughout all stages of the drug development process. This manuscript summarizes the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in LBAs, with recommendations for antibodies (monoclonal and polyclonal), engineered proteins, peptides, and their conjugates. Recommendations are given for each reagent type on basic and optional characterization profiles, expiration dates and storage temperatures, and investment in a knowledge database system. These recommendations represent a consensus among the authors and should be used to assist bioanalytical laboratories in the implementation of a best practices program for critical reagent life cycle management.

A Therapeutic Anti-VEGF Antibody with Increased Potency Independent of Pharmacokinetic Half-life

Bevacizumab [Avastin; anti-vascular endothelial growth factor (VEGF) antibody] is an antiangiogenic IgG approved for treating patients with certain types of colon, breast, and lung cancer. In these indications, bevacizumab is administered every 2 to 3 weeks, prompting us to study ways to reduce the frequency of administration. Increasing affinity to neonatal Fc receptor (FcRn) may extend the pharmacokinetic half-life of an antibody, but the quantitative effect of FcRn affinity on clearance has not been clearly elucidated. To gain further insight into this relationship, we engineered a series of anti-VEGF antibody variants with minimal amino acid substitutions and showed a range of half-life improvements in primates. These results suggest that, if proven clinically safe and effective, a modified version of bevacizumab could potentially provide clinical benefit to patients on long-term anti-VEGF therapy through less-frequent dosing and improved compliance with drug therapy. Moreover, despite having half-life similar to that of wild-type in mice due to the species-specific FcRn binding effects, the variant T307Q/N434A exhibited superior in vivo potency in slowing the growth of certain human tumor lines in mouse xenograft models. These results further suggest that FcRn variants may achieve increased potency through unidentified mechanisms in addition to increased systemic exposure.

A multiplexed hybrid LC–MS/MS pharmacokinetic assay to measure two co-administered monoclonal antibodies in a clinical study

BACKGROUND Combination therapies with monoclonal antibodies (mAbs) enhance therapeutic activity and may circumvent drug resistance. However, these studies present bioanalytical challenges for ligand-binding assays (LBAs). Recent MS-based protein quantification offers an alternative for bioanalysis. RESULTS A hybrid LC-MS/MS assay was developed to simultaneously measure human serum concentrations of two mAbs. Anti-idiotypic reagents that did not work in LBAs were successfully used for mAb affinity capture enrichment. Stable isotope-labeled peptide internal standards were employed. The mAb quantification involved measuring a signature CDR peptide derived from each mAb as a surrogate. Selected clinical samples were successfully analyzed. CONCLUSION The multiplexed LC-MS/MS method provided a powerful quantitative tool for clinical PK assessment of co-administered mAbs without the requirement for stringent affinity capture reagents.

Comparison of competitive ligand-binding assay and bioassay formats for the measurement of neutralizing antibodies to protein therapeutics.

Administration of biological therapeutic proteins can lead to unwanted immunogenicity in recipients of these products. The assessment and characterization of such immune reactions can be helpful to better understand their clinical relevance and how they relate to patient safety and therefore, have become an integral part of a product development program for biological therapeutics. Testing for anti-drug antibodies (ADA) to biological/biotechnology-derived therapeutic proteins generally follows a tiered approach. Samples are initially screened for binding antibodies; presumptive positives are then confirmed in a confirmatory assay; subsequently, confirmed-positive samples may be further characterized by titration and with a neutralizing antibody (NAb) assay. Regulatory guidances on immunogenicity state that assessing the neutralizing capacity of antibodies should preferably be done using functional bioassays, while recognizing that competitive ligand-binding (CLB) assays may be substituted when neutralizing bioassays are inadequate or not feasible. This manuscript describes case studies from four companies in which CLB assays and functional bioassays were compared for their ability to detect neutralizing ADA against a variety of biotechnology-derived therapeutic proteins. Our findings indicate that CLB assays are comparable to bioassays for the detection of NAbs, in some cases offering better detection sensitivity, lower variability, and less matrix interference.

Preclinical pharmacokinetics of MHAA4549A, a human monoclonal antibody to influenza A virus, and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza A

ABSTRACT MHAA4549A is a human immunoglobulin G1 (IgG1) monoclonal antibody that binds to a highly conserved epitope on the stalk of influenza A hemagglutinin and blocks the hemagglutinin-mediated membrane fusion in the endosome, neutralizing all known human influenza A strains. Pharmacokinetics (PK) of MHAA4549A and its related antibodies were determined in DBA/2J and Balb-c mice at 5 mg/kg and in cynomolgus monkeys at 5 and 100 mg/kg as a single intravenous dose. Serum samples were analyzed for antibody concentrations using an ELISA and the PK was evaluated using WinNonlin software. Human PK profiles were projected based on the PK in monkeys using species-invariant time method. The human efficacious dose projection was based on in vivo nonclinical pharmacological active doses, exposure in mouse infection models and expected human PK. The PK profiles of MHAA4549A and its related antibody showed a linear bi-exponential disposition in mice and cynomolgus monkeys. In mice, clearance and half-life ranged from 5.77 to 9.98 mL/day/kg and 10.2 to 5.76 days, respectively. In cynomolgus monkeys, clearance and half-life ranged from 4.33 to 4.34 mL/day/kg and 11.3 to 11.9 days, respectively. The predicted clearance in humans was ∼2.60 mL/day/kg. A single intravenous dose ranging from 15 to 45 mg/kg was predicted to achieve efficacious exposure in humans. In conclusion, the PK of MHAA4549A was as expected for a human IgG1 monoclonal antibody that lacks known endogenous host targets. The predicted clearance and projected efficacious doses in humans for MHAA4549A have been verified in a Phase 1 study and Phase 2a study, respectively.

Pharmacokinetics of Ranibizumab after Intravitreal Administration in Patients with Retinal Vein Occlusion or Diabetic Macular Edema

OBJECTIVE To describe the systemic pharmacokinetics of ranibizumab after intravitreal administration in patients with retinal vein occlusion (RVO) or diabetic macular edema (DME). DESIGN A population approach of nonlinear mixed-effect pharmacokinetics modeling based on serum concentrations of ranibizumab measured at various times after intravitreal administration. PARTICIPANTS Patients with RVO (n = 441) and DME (n = 435) from 4 large, randomized, phase 3 clinical trials of monthly ranibizumab intravitreal administration. METHODS A 1-compartment pharmacokinetics model with first-order absorption and elimination rate constants previously developed in patients with age-related macular degeneration (AMD) was fitted separately to RVO and DME data. Population pharmacokinetic parameters and interindividual variability were estimated for each model. Baseline covariates were evaluated for potential effects on systemic pharmacokinetics. Model performance was validated using general diagnostic plots and a visual predictive check. MAIN OUTCOME MEASURES Ranibizumab disposition was determined in RVO and DME patients and compared with that previously seen in AMD patients. RESULTS The AMD pharmacokinetics model correctly predicted the measured serum ranibizumab concentration data for RVO and DME patients. Most observed data points were within the simulated 90% confidence interval, indicating that systemic ranibizumab concentrations were comparable among AMD, RVO, and DME patients. No disease-related covariates were identified by the population pharmacokinetics analysis. CONCLUSIONS The systemic pharmacokinetics of ranibizumab were similar among patients with AMD, RVO, or DME. Disease-related differences and patient demographics, measured in this study, did not lead to variability in ocular elimination or in systemic exposure of ranibizumab after intravitreal administration. In all disease processes tested, ranibizumab exits the eye slowly and then is eliminated rapidly from the circulation, thus minimizing systemic exposure.

Two Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single-Ascending-Dose Studies To Investigate the Safety, Tolerability, and Pharmacokinetics of an Anti-Influenza A Virus Monoclonal Antibody, MHAA4549A, in Healthy Volunteers

ABSTRACT Hospitalized patients with severe influenza are at significant risk for morbidity and mortality. MHAA4549A is a human monoclonal immunoglobulin (Ig) G1 antibody that binds to a highly conserved stalk region of the influenza A virus hemagglutinin protein and neutralizes all tested seasonal human influenza A virus strains. Two phase 1 trials examined the safety, tolerability, and pharmacokinetics of MHAA4549A in healthy volunteers. Both single ascending-dose trials were randomized, double blinded, and placebo controlled. Trial 1 randomized 21 healthy adults into four cohorts receiving a single intravenous dose of 1.5, 5, 15, or 45 mg/kg MHAA4549A or placebo. Trial 2 randomized 14 healthy adults into two cohorts receiving a single intravenous fixed dose of 8,400 mg or 10,800 mg of MHAA4549A or placebo. Subjects were followed for 120 days after dosing. No subject was discontinued in either trial, and no serious adverse events were reported. The most common adverse event in both studies was mild headache (trial 1, 4/16 subjects receiving MHAA4549A and 1/5 receiving placebo; trial 2, 4/8 subjects receiving MHAA4549A and 2/6 receiving placebo). MHAA4549A produced no relevant time- or dose-related changes in laboratory values or vital signs compared to those with placebo. No subjects developed an antitherapeutic antibody response following MHAA4549A administration. MHAA4549A showed linear serum pharmacokinetics, with a mean half-life of 22.5 to 23.7 days. MHAA4549A is safe and well tolerated in healthy volunteers up to a single intravenous dose of 10,800 mg and demonstrates linear serum pharmacokinetics consistent with those of a human IgG1 antibody lacking known endogenous targets in humans. (These trials have been registered at ClinicalTrials.gov under registration no. NCT01877785 and NCT02284607).

Phase 1 Randomized, Double-Blind, Placebo-Controlled Study of RG7667, an Anticytomegalovirus Combination Monoclonal Antibody Therapy, in Healthy Adults

ABSTRACT Cytomegalovirus can cause debilitating and life-threatening disease in newborns infected in utero and immunocompromised individuals, including transplant recipients. RG7667 is a unique combination of two monoclonal antibodies that binds glycoprotein complexes on the surface of cytomegalovirus and inhibits its entry into host cells. A phase 1 first-in-human, randomized, double-blind, placebo-controlled, dose-escalation study of RG7667 given intravenously was conducted in 181 healthy adults. The study involved a single ascending dose stage (1, 3, 5, and 10 mg/kg each antibody; n = 21), a multiple ascending dose stage (5 and 10 mg/kg each antibody monthly for 3 doses; n = 10), and a multiple dose expansion stage (10 mg/kg each antibody monthly for 3 doses; n = 150). Subjects were followed for 85 to 141 days to evaluate safety, tolerability, pharmacokinetics, and immunogenicity. Most adverse events were mild, and the incidence of adverse events was similar among the RG7667 and placebo groups. RG7667 had dose-proportional pharmacokinetics in all three dosing stages, a mean terminal half-life of 20 to 30 days, and an overall pharmacokinetic profile consistent with that of a human monoclonal antibody that lacks endogenous host targets. The proportion of subjects developing an antitherapeutic antibody response was not higher in the RG7667 group than in the placebo group. In summary, single and multiple doses of RG7667 were found to be safe and well-tolerated in healthy adults and had a favorable pharmacokinetic and immunogenicity profile. This study supports further development of RG7667 as a therapy for the prevention and treatment of cytomegalovirus infection in susceptible populations. (This study has been registered at ClinicalTrials.gov under registration no. NCT01496755.)

Phase 2 Randomized Trial of the Safety and Efficacy of MHAA4549A, a Broadly Neutralizing Monoclonal Antibody, in a Human Influenza A Virus Challenge Model

ABSTRACT MHAA4549A, a human monoclonal antibody targeting the hemagglutinin stalk region of influenza A virus (IAV), is being developed as a therapeutic for patients hospitalized with severe IAV infection. The safety and efficacy of MHAA4549A were assessed in a randomized, double-blind, placebo-controlled, dose-ranging study in a human IAV challenge model. One hundred healthy volunteers were inoculated with A/Wisconsin/67/2005 (H3N2) IAV and, 24 to 36 h later, administered a single intravenous dose of either placebo, MHAA4549A (400, 1,200, or 3,600 mg), or a standard oral dose of oseltamivir. Subjects were assessed for safety, pharmacokinetics (PK), and immunogenicity. The intent-to-treat-infected (ITTI) population was assessed for changes in viral load, influenza symptoms, and inflammatory biomarkers. MHAA4549A was well tolerated in all IAV challenge subjects. The 3,600-mg dose of MHAA4549A significantly reduced the viral burden relative to that of the placebo as determined by the area under the curve (AUC) of nasopharyngeal virus infection, quantified using quantitative PCR (98%) and 50% tissue culture infective dose (TCID50) (100%) assays. Peak viral load, duration of viral shedding, influenza symptom scores, mucus weight, and inflammatory biomarkers were also reduced. Serum PK was linear with a half-life of ∼23 days. No MHAA4549A-treated subjects developed anti-drug antibodies. In conclusion, MHAA4549A was well tolerated and demonstrated statistically significant and substantial antiviral activity in an IAV challenge model. (This study has been registered at ClinicalTrials.gov under identifier NCT01980966.)