Ethics statement
The Phase I study was conducted in the Third Xiangya Hospital of Central South University and in accordance with the Declaration of Helsinki and the Good Clinical Practice of the International Conference on Harmonization (ICH-GCP). The study protocol and informed consent documents were approved by the Ethics Committee of the Third Xiangya Hospital of Central South University, Changsha, Hunan, P. R. China (approval No.13072). Written informed consents were provided by all subjects prior to participating in any study-related activities in the study (Registered on China, No. ChiCTR2000041577 and ChiCTR2100041802).
Study drugs
The test recombinant humanized monoclonal antibody against HER2 for injection was manufactured by Qilu Pharmaceutical Co. Ltd (specification:150mg/vail; batch number:20130401), the reference product Herceptin was manufactured by Roche Pharma (Schweiz) Ltd. (specification:20mL:440mg; batch number:SH0027).
Volunteers
Eligible subjects were healthy males aged 18 to 45 years, weight ≥ 50 kg with a body mass index between 19 and 25 kg/m2 and a baseline left ventricle ejection fraction (LVEF) ༞60%. Physical examination, blood pressure, electrocardiography, echocardiography and routine laboratory tests were performed for all subjects at screening period. Exclusion criteria included hemoptysis, thrombotic or hemorrhagic event or cerebral vascular accident, transient ischemic attack, or subarachnoid hemorrhage within 6 months before administration of study drug; undergone surgery within 4 weeks before the trial; allergic or known to be allergic to this product or any excipients; with a history of clinical serious illness or any other diseases or physiological conditions that can interfere with the results of the study.
Study design
This phase I study consists of two parts. Part I was a first-in-human dose-escalating study, which adopted an open-label, sequential-cohort design to define the maximum dose of QLHER2 (range from 0.2 to 6 mg/kg) in 16 healthy volunteers. Part II was a randomized, double-blind, parallel study to evaluate the bioequivalence of QLHER2 and Herceptin in 60 healthy volunteers. Volunteers received either drug at a single dose on day 1 and follow up to day 70. The flow chart of the two-part phase I study is shown in Fig. 1.
In part I, QLHER2 0.2 to 6 mg/kg (0.2, 1, 2, 4 and 6 mg/kg) and Herceptin 4 mg/kg was intravenous infusion at a single dose escalated in cohorts of one or three subjects. Drugs were diluted with 250 mL of 0.9% NaCl and infused intravenously for 90 min. One subject infused QLHER2 0.2 mg/kg without collecting any blood samples but only for observation, to evaluate the overall safety of QLHER2 in healthy adults during the infusion period and one week thereafter. If no severe allergic reactions or other conditions observed, we started the next sequential dosing in 3 volunteers from 1 to 6 mg/kg. Among the same dosing cohort, one out of every three volunteers administrated the drug first, with a 1-3 days’ observation period proved its safety, then the other two subjects received the same dose and observed for another week. The only exception is that in the 4 mg/kg dose cohort, 6 volunteers were randomized equally to receive QLHER2 4 mg/kg or Herceptin 4 mg/kg. Blood samples were collected at determined time points. If any serious allergic reactions or other conditions observed in any of the dose groups, the dose-escalating study will be terminated.
In part II, 60 subjects were enrolled and randomized in a 1:1 ratio to either receiving 4-mg/kg QLHER2 or 4-mg/kg Herceptin. All volunteers underwent intensive monitoring for one week after drug administration. Blood samples were collected at determined time points.
Safety evaluation
Safety was assessed based on adverse event (AE) monitoring and follow-up interviews. All subjects received physical examinations, vital signs (blood pressure, heart rate, respiratory rate and body temperature), 12-lead electrocardiography, echocardiography, laboratory tests (routine blood, routine urine, liver and kidney function, blood glucose, blood lipid, plasma electrolytes, myocardial enzymes, coagulation function, four pre-transfusion tests) during the study period and at the 70-day follow-up visit period. The clinical significance of abnormal laboratory test values was determined by the physicians. Treatment-related AEs were graded based on the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.0). Subjects with AEs were monitored until the conditions were resolved or stabilized(Yang et al., 2020).
Sample collection and analysis
Venous blood samples (5 mL) were collected prior to dosing, at 0.5, 1, 1.5 h during infusion and 0.5, 1,1.5, 2, 3, 4, 6, 8, 12h on day 1and day 2, 3, 4, 5, 6, 7, 14 ± 1, 21 ± 1, 28 ± 1, 35 ± 1, 42 ± 1, 49 ± 1, 56 ± 1, 63 ± 1, 70 after infusion. Blood samples were centrifuged at 3000 rpm/min for 15 min at 4°C. Plasma was separated and kept at -70°C until further bioanalysis. Serum QLHER2 and Herceptin were determined using enzyme-linked chemiluminescent assays (ELISA). The lower limit of quantitation (LLOQ) was 3.125 ng/mL (calibration range, 3125 to 200 ng/mL). Raw data were input and processed by SoftMax Pro Software (version 1.6.3). The range of precision deviation between batches of precision range (CV) was below 15%. These values were within the criteria shown in the guidelines for the ligand-binding assay(Committee, 2015), suggesting that plasma concentrations of QLHER2 and Herceptin measured were reliable.
Data Analysis
Major pharmacokinetic parameters include Cmax (peak concentration), AUC0−∞ (area under the indacaterol concentration in plasma vs time curve from dosing to infinity), AUC0−t (area under the indacaterol concentration in plasma vs time curve from dosing to any of time-point), Tmax (time to reach peak concentration) and t1/2 (half elimination time) were calculated using the noncompartmental model (NCA module), Phoenix WinNonlin 6.2 (Pharsight Corporation, Mountain View, CA, USA). Linear up-log down method was employed in the estimation of AUC. Cmax, Tmax, AUC0−t were compared to assess the bioequivalence of QLHER2 and Herceptin. The 90% confidence intervals (CIs) were estimated from the ANOVA model. 90% CIs of Cmax, AUC0−t, AUC0−∞ for the QLHER2/Herceptin ratio between 80.00-125.00% considered to be equivalence(Zou et al., 2020).