This was a randomized, single-blind, partial-reference-replicated, three-sequence, three-period crossover study in healthy subjects (Fig. 1) confined to a clinical pharmacology unit for dosing and immediate follow-up to assess PK, PD, and safety, including the immunogenicity of pegfilgrastim-cbqv and pegfilgrastim (ClinicalTrials.gov, NCT02650973). The study took place at four clinical sites across the US (Cincinnati, OH; Cypress, CA; West Bend, WI; San Antonio, TX). Each subject was randomly assigned in a 1:1:1 ratio (stratified by site and sex) to a treatment sequence group (A, B, or C) that included one dose of pegfilgrastim-cbqv and two doses of pegfilgrastim, each administered in a separate period with at least 28 days between doses of study drug (Fig. 1).
Compliance with Ethics Guidelines
The protocol and informed consent form were submitted to and approved by the sites’ institutional review boards before initiation of the study. IRB approval was obtained from Schulman Institutional Review Board (Cincinnati, OH), Alpha Independent Review Board (San Clemente, CA), Chesapeake Institutional Review Board (Columbia, MD), and IntegReview Independent Review Board (Austin, TX) in the US. This study was conducted in accordance with all applicable laws and regulations, and it complied with the International Conference for Harmonisation E6 Guideline on Good Clinical Practice and the Declaration of Helsinki. The rationale of the study, procedural details, and investigational goals were explained to each subject, along with potential risks and benefits. Each subject was assured of his/her right to withdraw from the study at any time. Before the initiation of any study procedures, each subject signed and dated an approved informed consent form.
A single dose of pegfilgrastim-cbqv (6 mg/0.6 ml sterile single-use prefilled syringe) or pegfilgrastim (6 mg/0.6 ml 6 ml sterile single-use prefilled syringe) was administered subcutaneously on day 1 of each period, according to the subject’s assigned treatment sequence. Treatment of male subjects was spaced by no fewer than 28 days, whereas female subjects were dosed only at 28-day intervals (± 2 days) to ensure that they were dosed at the same time in their menstrual cycles to decrease intrasubject variability. This study was single-blinded, as the pegfilgrastim-cbqv prefilled syringe is not identical to the pegfilgrastim prefilled syringe. Each syringe was labeled with a unique number, and an unblinded site pharmacist matched the appropriate unique syringe number to the appropriate subject. The subjects, investigational site staff, biostatisticians, and data managers remained blinded to the subject treatment for the duration of the study. The subjects and investigational site staff responsible for assessing adverse events (AEs) and injection site reactions (ISRs) were blinded to study treatments. To strengthen the blinding of the study, access to the randomization scheme was limited, unblinded plasma concentrations were not shared with the study site, and investigational site staff responsible for assessing safety were not the same as the staff performing the injections.
Eligible subjects were healthy male and female participants aged 18–45 years (Table 1). Inclusion criteria were body weight 50–100 kg (110–220 lb), with a body mass index of 18–28 kg/m2. The subjects were required to be medically healthy, with normal hematologic, coagulation, hepatic, and renal function and a fasting lipid profile that did not require medications to control lipid levels. Women of childbearing potential who were not actively breastfeeding must have had a negative pregnancy test and agreed to use an approved method of birth control; alternatively, women could have been > 1 year postmenopausal, and men must have been willing to use barrier contraception and refrain from donating sperm.
Exclusion criteria included previous exposure to pegfilgrastim or filgrastim; presence of hematologic disorder, cancer, diabetes, or any clinically significant condition that might interfere with lymphatic flow or PK of the drug; history of chronic or acute respiratory illness within the past 3 months; use of drugs (prescription, nonprescription, or recreational) or alcohol at screening or during study period; participation in high-impact exercise or activities that could result in blunt trauma to the truncal area; consumption of > 400 mg of caffeine per day; participation in a clinical study within 30 days before screening; blood and/or plasma donation ≥ 500 ml in the previous month; HIV or hepatitis B or C infection; clinically significant food or drug allergies; any condition that would complicate or compromise the study or well-being of the subject. Subjects who did not meet the ANC criteria for dosing (1.7–7. 2 × 109/l) were precluded from subsequent dosing.
The primary PK end points for this study were the peak plasma concentration (Cmax) and the area under the concentration-time curve from time 0 to infinity (AUC0−∞). The primary PD end points were the maximum absolute neutrophil count (ANCmax) and the area under the curve of the ANC from time 0 to the last sample (ANC AUC0−last). Secondary end points included the characterization of the PK profile of pegfilgrastim-cbqv using standard parameters and the characterization of the safety profile (including immunogenicity) and tolerance of pegfilgrastim-cbqv, as assessed by AEs, AEs of special interest (AESIs), laboratory variables, vital signs, incidence of antidrug antibodies/neutralizing antibodies (ADAs/NAbs), and ISRs.
For each period, subjects were admitted to a clinical pharmacology unit for the first 4 nights of the treatment period and discharged the morning of day 5. Subjects returned for daily blood samples on days 6–13 and day 21 of each period as well as day 28 of period 3. The on-treatment blood sampling schedule for the plasma drug levels and ANC took place before dose administration and at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, 60, 72, 84, and 96 h after dose and on days 6, 7, 8, 9, 10, 11, and 13. On days 21 and 28 (period 3 only), ANC was assessed, including white blood cell and differential counts. Plasma samples were analyzed for pegfilgrastim concentrations using a validated enzyme-linked immunosorbent assay. For the immunogenicity assessment, plasma samples for ADAs and NAbs were collected before dose administration on days 1 and 11 in each period and on day 28 following the last dose of the study drug. The presence of ADAs was assessed using a validated electrochemiluminescent bridging assay. Confirmed ADA-positive samples were characterized for binding specificity to polyethylene glycol (PEG) or G-CSF and semiquantitation (titer) and tested in a validated cell-based assay to determine if they were neutralizing. NAb-positive samples were further tested to assess the neutralization of endogenous G-CSF.
Safety assessments took place at the intervals specified in the protocol and included serum chemistry measurements, hematologic measurements, urinalysis, vital signs, physical examination (including an abdominal examination), clinical AE reports, ISRs, immunogenicity assessment, electrocardiograph, and collection of concomitant medications. AESIs included those related to the spleen, acute respiratory distress syndrome, anaphylaxis, sickle cell crisis, leukocytosis, glomerulonephritis, potential for tumor growth stimulatory effects on malignant cells, and cytokine release/capillary leak syndromes.
A sample size of 120 subjects was determined based on statistical methods provided by Golkowski et al. and through simulations to assess intrasubject variation and power . This study was designed to demonstrate PK bioequivalence (PK-BE) with approximately 90% power with 78 evaluable subjects based on a two-sided 90% CI to evaluate the GMR of pegfilgrastim-cbqv/pegfilgrastim for Cmax and AUC0−∞. The planned sample size assumed an intrasubject coefficient of variation (CV) ≤ 44% for Cmax and AUC0−∞ and an expected true GMR of pegfilgrastim-cbqv/pegfilgrastim of 1.05 for Cmax and AUC0−∞. Additionally, 78 evaluable subjects would provide > 95% power to demonstrate PD bioequivalence based on a two-sided 90% CI to evaluate GMR of pegfilgrastim-cbqv/pegfilgrastim for ANCmax and ANC AUC0−last. This planned sample size assumed an intrasubject CV ≤ 25% for ANCmax and ANC AUC0−last and an expected true GMR of pegfilgrastim-cbqv/pegfilgrastim of 1.0 for ANCmax and ANC AUC0−last.
A planned interim analysis of intrasubject CV for pegfilgrastim 1/pegfilgrastim 2 for AUC0-∞ was conducted when the period 3 PK samples from the initial cohort were available to allow for readjustment of the sample size, if necessary. A predefined cutoff of ≤ 43% was prospectively determined to maintain adequate study power. When PK samples from period 3 were available, the intrasubject CV of AUC0−∞ for pegfilgrastim 1/pegfilgrastim 2 was calculated by an unblinded independent statistician in subjects who had received at least two doses of pegfilgrastim and had sufficient plasma concentration–time data to calculate at least one of the primary PK end points . Because the intrasubject CV was ≤ 43%, it was determined that no additional subjects were required.
Pegfilgrastim plasma concentration data were summarized for each time point using descriptive statistics from subjects who received at least two doses of study drug—one of pegfilgrastim-cbqv and at least one of pegfilgrastim—and had sufficient plasma-concentration time data to calculate at least one of the primary PK end points (PK-BE evaluable population). Standard PK variables were calculated using standard noncompartmental methods using plasma concentration data from the collected blood samples and summarized by treatment group and study period using the PK bioequivalence evaluable population. For AUC and Cmax, the pegfilgrastim average was the geometric mean of the two reference pegfilgrastim doses of each subject if both were available; otherwise, the single available measurement was used. ANC data were summarized for each time point using descriptive statistics for subjects who received at least two doses of study drug (one of pegfilgrastim-cbqv and at least one of pegfilgrastim) and had sufficient data to calculate at least one of the key PD end points (PD evaluable population). The safety population consisted of all randomly assigned subjects who received ≥ 1 dose of either study drug. Descriptive analysis was conducted in PK, PD, or safety profile based on sex or ethnicity. Summary statistics of clinical laboratory data and vital signs were generated by treatment group and time point. The presence of ADAs and NAbs was listed by subject and summarized descriptively by treatment and time point. The ADA/NAb impacts on PK, PD, and safety were evaluated.
PK and PD bioequivalence analysis used a two one-sided tests procedure for an unscaled average bioequivalence approach for the partial reference-replicated three-treatment sequence, three-period design . Bioequivalence was demonstrated if the 90% CIs for the GMR of pegfilgrastim-cbqv:pegfilgrastim for the primary PK and PD end points were entirely within the range of 80–125%.