https://orcid.org/0000-0002-5838-5607
ABSTRACT
Background
SB16 is a biosimilar to reference denosumab (DEN). This study assessed pharmacokinetic (PK) equivalence and evaluated pharmacodynamic (PD), safety, tolerability, and immunogenicity between SB16, European Union-sourced DEN (EU-DEN), and United States-sourced DEN (US-DEN).
Research design and methods
In this double-blind, parallel group, and single-dose study, healthy male subjects were randomized 1:1:1 to receive a single 60 mg dose of either SB16, EU-DEN, or US-DEN subcutaneously. PK, PD, safety, and immunogenicity were evaluated for 197 days. Primary PK endpoints were area under the concentration-time curve (AUC) from time zero to infinity, AUC from time zero to the last quantifiable concentration, and maximum serum concentration (Cmax). Equivalence was determined if 90% confidence intervals (CIs) for the ratio of geometric least squares means (LS Means) were within the equivalence margin of 0.80 to 1.25.
Results
A total of 168 subjects (56 per treatment group) were randomized. All of the corresponding 90% CI of geometric LS Means ratio of primary PK parameters were within the pre-defined equivalence margin. PD, safety, and immunogenicity profiles were also comparable between the treatment groups.
Conclusion
This study demonstrated PK bioequivalence between SB16, EU-DEN, and US-DEN in healthy male subjects.
TrialRegistration
CT.gov identifier: NCT04621318
1. Introduction
Denosumab (Prolia, Amgen, United States [US]) is a human Immunoglobulin G2 (IgG2) monoclonal antibody with affinity and specificity for human receptor activator of nuclear factor kappa-B ligand (RANKL). By preventing RANKL from activating human receptor activator of nuclear factor kappa-B (RANK), its receptor on the osteoclast precursors and osteoclasts, it inhibits osteoclast formation, function, and survival. It has an approximate molecular weight of 147 kilodalton (kDa) and is produced using genetically engineered mammalian Chinese Hamster Ovary (CHO) cells.
Reference denosumab (DEN) contains 60 mg of denosumab per mL of solution and is administered as a single-use pre-filled syringe (PFS) for subcutaneous (SC) administration. It is currently indicated for osteoporosis in postmenopausal women and in men at increased risk of fractures, bone loss associated with hormone ablation in men with prostate cancer at increased risk of fracture, and bone loss associated with long-term systemic glucocorticoid therapy in adult patients at increased risk of fracture by both European Medicines Agency (EMA) and US Food and Drug Administration (FDA). In addition, US FDA approved it for the treatment of bone loss associated with adjuvant aromatase inhibitor therapy for breast cancer.
A biosimilar is a biological product that is highly similar to an approved biologic (reference product), having no clinically meaningful differences in terms of quality, safety, and efficacy [Citation1]. Regulatory approval of biosimilars is based on the totality of evidence, and this includes a stepwise approach of comparative analytical, non-clinical, and clinical studies [Citation1,Citation2]. The use of biosimilars may facilitate patient access to biological medicine at a lower cost and reduce the burden on many healthcare systems [Citation3].
SB16 (Samsung Bioepis, Republic of Korea) was developed as a biosimilar to reference DEN. A quality and a series of non-clinical assays were performed in terms of the structural, physicochemical, and biological characteristics of SB16, demonstrating the high similarity when compared to the reference DEN. Subsequently, a Phase I study was conducted and reported here aimed to compare pharmacokinetic (PK), pharmacodynamic (PD), safety, and immunogenicity between SB16 and reference products (European Union [EU]-sourced denosumab [EU-DEN] and US-sourced denosumab [US-DEN]) in healthy male subjects.
2. Participants and methods
2.1. Study population
The study population comprised healthy male subjects. Key inclusion criteria included an age of 28–55 years, body weight of 60.0–95.0 kg, and a body mass index (BMI) of 20.0–29.9 kg/m2. The absence of clinically significant abnormalities was confirmed by the Investigator, from results of 12-lead electrocardiogram (ECG), vital sign, and physical examination. Key exclusion criteria included osteonecrosis of the jaw (ONJ), risk factors for ONJ such as invasive dental procedures or active periodontal disease, or a history of bone disease or any medical condition that could have affected bone metabolism. Subjects were also excluded if they had abnormal albumin-adjusted serum calcium level or known intolerance to calcium or vitamin D supplements.
2.2. Study design
This was a randomized, double-blind, three-arm, multi-center, parallel group, and single-dose study (CT.gov identifier: NCT04621318, EudraCT number: 2020–002592–35). It was conducted in two centers in US and one center in France. A total of 168 subjects were randomized in a 1:1:1 ratio to receive a single 60 mg dose of either SB16, EU-DEN, or US-DEN subcutaneously in the abdomen on day 1. Subjects were assigned to one of the 3 treatment groups according to a randomization list generated by an independent statistician using SAS software. The subject, the Investigator, the site staff, the Sponsor, and other study personnel who were involved in the treatment or clinical evaluation of subjects were blinded to the treatment group assignments. The investigational product (IP) PFS was packaged and labeled identically to ensure the blinding of the treatment group assignment. All IP administrations were completed by the Investigator or designated staff to ensure compliance. To minimize the risk of hypocalcemia, all subjects received calcium (at least 1000 mg) and vitamin D (at least 400 IU) daily during treatment period. Subjects were observed for 197 days (4704 h) post-dose and PK, PD, safety, and immunogenicity were evaluated throughout the study period.
This study was conducted in accordance with the Declaration of Helsinki (1996) and consistent with the International Council for Harmonization (ICH) E6 (R2) Good Clinical Practice guidelines (GCP), applicable local regulatory requirements and laws. The final study protocol was reviewed and approved by the French Independent Ethics Committee (IEC) and/or the US Institutional Review Board (IRB) for each study center. All subjects signed the agreements on the informed consent form (ICF) prior to any study-related procedures. The consent form was reviewed and approved by the appropriate IEC or IRB.
2.3. Study endpoints
The primary PK endpoints were area under the concentration–time curve from time zero to infinity (AUCinf), area under the concentration–time curve from time zero to the last quantifiable concentration (AUClast), and the maximum serum concentration (Cmax). The secondary PK endpoints included the time to reach Cmax (Tmax), apparent volume of distribution during the terminal phase (Vz/F), terminal rate constant (λz), terminal half-life (t1/2), apparent clearance (CL/F), and percentage of AUCinf due to extrapolation from time of last measurable concentration to infinity (%AUCextrap). The PD endpoint was area under the effect curve from time zero to Day 197 (AUEC0-D197) for serum C-telopeptide of type I collagen (CTX) percent inhibition. The safety endpoints were adverse events (AEs) and serious AEs (SAEs), clinical laboratory values, 12-lead ECG, vital signs, physical examination, and injection site assessment. Immunogenicity endpoints were the incidence of anti-drug antibodies (ADAs) and neutralizing antibodies (NAbs) to denosumab.
2.4. Study assessments
Blood samples for PK analysis were collected at pre-dose, 12, 24, 48, 96, 144, 192, 240, 288, 336, 504, 672, 1008, 1344, 2016, 2688, 3360, 4032, and 4704 h post-dose. Subjects were discharged on Day 3 with the rest of the study period consisting of outpatient visits. Serum denosumab concentrations were measured using a validated electrochemiluminescence immunoassay (ECLIA) specific for the detection and quantification of denosumab. The lower limit of quantification (LLOQ) was 20 ng/mL. For the calculation of PK parameters, below limit of quantification (BLQ) before IP administration was considered as zero and all other BLQ values were set to missing.
Blood samples for PD analysis were collected at pre-dose, 24, 48, 96, 144, 192, 336, 672, 1344, 2016, 3360, 4032, and 4704 h post-dose. Serum CTX samples were analyzed using a validated electrochemiluminescence immunoassay (ECLIA). For the analysis of CTX in human serum, the LLOQ and the upper limit of quantification were 0.043 ng/mL and 1.21 ng/mL, respectively.
All AEs and SAEs were recorded during the course of the study and coded according to the Medical Dictionary for Regulatory Activities (MedDRA) version 23.0. Other safety assessments included laboratory tests (hematology, chemistry, and urinalysis), vital signs, 12-lead ECG, physical examination, and injection site assessment.
Blood samples for immunogenicity analysis were collected at pre-dose, on Day 15, Day 29, Day 85, Day 141, and Day 197 to analyze ADAs and NAbs to denosumab. ADAs were detected using a qualitative and quasi-quantitative ECLIA. ADA-positive samples were further tested for NAbs using ECLIA.
2.5. Statistical methods
The sample size was determined based on an inter-subject coefficient of variation (CV) of 35.14%, which was reported in the previously published data [Citation4]. With the sample size of 50 in each of the treatment arms (and the total sample size of 150), a parallel study design would have had 80% power, assuming a true geometric mean ratio of 1.05 to be able to reject both the null hypotheses that 1) the true geometric mean ratio of the test to the reference was less than 0.80 and 2) the true geometric mean ratio of test to the reference was greater than 1.25, where both of these null hypotheses could be rejected simultaneously if the 90% confidence intervals (CIs) for the true geometric mean ratio lay completely between 0.80 and 1.25. Assuming a 10% drop-out rate, a total of 168 subjects (56 per each arm) were randomized.
The PK parameters were calculated based on actual sampling times and non-compartmental analysis methods using Phoenix WinNonlin version 8.1 (Certara, US). The statistical analysis of the loge-transformed primary endpoints was performed by analysis of variance (ANOVA) model with treatment group as a fixed effect. The difference in least squares means (LSMeans) between SB16 and EU-DEN, between SB16 and US-DEN, or between EU-DEN and US-DEN, and the corresponding 90% CIs were determined. Back transformation provided the ratio of geometric LSMeans and 90% CIs for these ratios. Equivalence for the primary endpoints was determined if the 90% CIs for the ratio of geometric LSMeans of SB16 to EU-DEN, SB16 to US-DEN, and EU-DEN to US-DEN, respectively, were within the equivalence margin of 0.80 to 1.25.
3. Results
3.1. Subject disposition and baseline characteristics
A total of 438 subjects were screened, of whom 168 subjects were randomized. Of all the subjects who were randomized, 160 subjects completed the study and 8 subjects discontinued the study (). Among the discontinued subjects, three subjects withdrew consent for further participation, two subjects discontinued due to protocol deviation, two subjects were lost to follow-up, and one subject died (completed suicide, not related to the IP). Baseline characteristics of the subjects were generally comparable among the 3 treatment groups ().
Figure 1. CONSORT diagram of subjects disposition.
Table 1. Baseline characteristics (randomized Set).
3.2. Pharmacokinetic results
The mean serum concentration–time profiles were superimposable and comparable among the 3 treatment groups (). There was a slow increase in the serum concentrations after study drug administration (median Tmax of 192 h for all treatment groups), followed by a slow decrease during the elimination phase. At 4704 h (end of study [EOS]), all serum concentrations in the SB16 were BLQ. These results could be grounds for long enough sampling duration of a reliable estimate of the extent of exposure. A prolonged elimination with long termination half-life (approximately 21 days) and a more rapid terminal elimination were identified with serum concentrations of all 3 treatment groups.
Figure 2. Mean (±standard deviation) serum denosumab concentration over time by treatment group (PK analysis Set) on linear (top) and Semi-logarithmic (bottom) scale.
The descriptive statistics of the PK parameters are summarized in . The primary endpoints were similar between the SB16, EU-DEN, and US-DEN. The arithmetic means of AUCinf for SB16, EU-DEN, and US-DEN were 6617.3 μg·h/mL, 6570.3 μg·h/mL, and 6730.3 μg·h/mL, respectively. The arithmetic means of AUClast for SB16, EU-DEN, and US-DEN were 6496.8 μg·h/mL, 6401.5 μg·h/mL, and 6537.5 μg·h/mL, respectively. The arithmetic means of Cmax were 5.801 μg/mL, 5.688 μg/mL, and 5.473 μg/mL, respectively. All secondary endpoints, including the median Tmax, mean λz, Vz/F, CL/F, and %AUCextrap values were also comparable across the 3 treatment groups.
Table 2. Summary statistics for pharmacokinetic parameters (PK analysis Set).
The 90% CIs of the geometric LSMeans ratio for AUCinf, AUClast, and Cmax between the treatment groups were completely contained within the pre-defined equivalence margin of 0.80 to 1.25 ().
Table 3. Statistical comparison of the primary pharmacokinetic parameters (PK analysis Set).
3.3. Pharmacodynamic results
The serum CTX percent change from baseline profiles was comparable among the 3 treatment groups (). There was a rapid decrease in the serum CTX concentration immediately after IP administration and this inhibition continued over the course of the study. The mean maximum CTX percent inhibition (Imax) values were comparable across the 3 treatment groups (84.48%inhibition, 84.91%inhibition, and 86.24%inhibition for SB16, EU-DEN, and US-DEN, respectively). The median time to Imax values were 1344.00 h, 1321.40 h, and 1344.00 h for SB16, EU-DEN, and US-DEN, respectively. Also, the mean AUEC0-D197 for CTX percent inhibition for SB16, EU-DEN, and US-DEN was comparable (352205.95% inhibition·h 348,254.45% inhibition·h, and 374,823.13% inhibition·h, respectively).
Figure 3. Percent change from Baseline in median (IQR) serum CTX over time by treatment group (PD analysis set).
3.4. Safety and immunogenicity results
A total of 170 treatment emergent adverse events (TEAEs) were reported in 90 (53.6%) subjects (). The majority of TEAEs were mild to moderate in severity. The incidence of TEAEs in subjects with SB16, EU-DEN, and US-DEN were 62.5%, 44.6%, and 53.6%, respectively (). The most frequent TEAEs were COVID-19 in the SB16, constipation in the EU-DEN, and blood creatine phosphokinase increased in the US-DEN. In the SB16, 6 (10.7%) subjects had 6 events of COVID-19; in the EU-DEN, 5 (8.9%) subjects had 5 events of constipation; in the US-DEN, 5 (8.9%) subjects had 5 events of blood creatine phosphokinase increased. Three SAEs were reported for 3 (5.4%) subjects in the SB16 treatment group (i.e. suicide, facial bones fracture, and depression). All the SAEs were considered not related to the IP by the Investigator.
Table 4. Summary of all adverse events (safety Set).
Laboratory data, vital signs, and ECG parameters did not show any relevant significant changes over time which might be considered related to the IP.
In addition, the overall incidence of subjects with post-dose ADA positive to denosumab was 2 (3.6%), 0 (0.0%), and 4 (7.1%) subjects in the SB16, EU-DEN, and US-DEN, respectively. None of the subjects with post-dose ADA positive to denosumab had a positive result for NAb.
4. Discussion
SB16 is being developed as a proposed biosimilar of denosumab and has been shown to have identical structure, similar biochemical properties, and biologic functions as the reference DEN. The primary objective of this phase I study was to assess PK similarity between SB16, and reference DEN to support a demonstration of biosimilarity. The primary endpoints for this study were selected in alignment with the EMA and FDA guidelines for SC administration. The results for primary endpoints (i.e. AUCinf, AUClast, and Cmax) demonstrated bioequivalence of SB16 to reference products, EU-DEN and US-DEN. The 90% CIs for the test-to-reference ratios of the calculated primary PK parameters were within the pre-defined bioequivalence criteria of 0.80 to 1.25 for comparison of SB16 to EU-DEN, SB16 to US-DEN, and EU-DEN to US-DEN, demonstrating PK similarity among the 3 treatment groups. Healthy subjects were selected as the study population in accordance with EMA and FDA guidelines to effectively assess the study objectives while avoiding potential interference associated with concomitant treatments or medical conditions [Citation1,Citation2]. Healthy volunteers were judged to be adequately sensitive to detect any difference of PK characteristics between treatments, as well as differences in common AEs and immunogenicity. Especially, healthy male subjects were only participated as the study population to avoid any risk of reproductive toxicity and formation of ADA to denosumab in females who were more likely to require denosumab for the treatment of osteoporosis. A single SC injection of 60 mg denosumab in healthy male subjects was considered suitable to demonstrate PK similarity between SB16 and reference DEN. Serum concentration profiles at the elimination phase showed prolonged elimination and dropped more sharply when serum concentration was below 100 ng/mL. At the last sampling point (i.e. 4704 h), all serum concentrations in the SB16 were BLQ, and the mean serum concentrations in the EU-DEN and US-DEN were almost BLQ (i.e. 20.2 ng/mL and 65.1 ng/mL, respectively). The last sampling points of each treatment group were not appeared in the semi-logarithmic plot (), but the serum concentration from 4032 h to 4704 h rapidly decreased. These profiles were similar to those observed references from the regulatory agencies and academic literatures, and this phenomenon could be explained by non-linear PK characterized by target-mediated drug disposition (TMDD) [Citation5–9]. The binding of denosumab to RANKL and subsequent degradation of the drug-target complex constitutes an additional elimination pathway affect denosumab PK and contributes to its non-linear PK behavior. Slower clearance observed at the higher concentration (over 100 ng/mL) would be due to the saturation of target-mediated clearance.
Denosumab inhibits a resorptive effect on bone and results in a rapid decline in bone resorption and subsequently bone-formation markers. These inhibition effects have been reported for the bone-resorption markers (i.e. CTX and N-telopeptide of type I collagen [NTX]), and bone-formation markers (i.e. serum procollagen N-propeptide of type I collagen [PINP] and bone alkaline phosphatase (BALP) [Citation10–13]. Especially, notable inhibition of bone resorption after denosumab administration was assessed by CTX levels [Citation14]. In previous studies, CTX level was reduced by approximately 85% by 3 days, with maximal reductions occurring by 1 month after 60 mg denosumab administration [Citation15]. In our study, the CTX levels in all treatment groups were reduced by approximately 70% by 3 days and 85% by 7 days. In addition, PD profiles in terms of AUEC0-D197 for CTX percent inhibition were comparable following administration of SB16, EU-DEN, and US-DEN.
Treatment with a single dose of SB16 was generally safe and well-tolerated in healthy male subjects, and a safety profile in terms of incidence of TEAEs was comparable with EU-DEN and US-DEN. The development of post-dose ADAs to denosumab was also comparable between all treatment groups and no NAbs were detected.
Limitations of this Phase I study include factors related to the study population. Only healthy male subjects were included in current phase I study because they were considered to be a more homogeneous and represented sensitive population to compare PK characteristics than patients with various disease-related factors and females with risk of reproductive toxicity and immunogenicity.
Biosimilars, which have been clinically studied and have demonstrated to be bioequivalent in PK, and comparability in safety, efficacy, and immunogenicity, may be more cost-effective product of biologics which may help to increase patient access. Our results support the demonstration of PK similarity between SB16 and reference DEN (EU-DEN and US-DEN). In addition, the comparability of PD, safety, and immunogenicity was also verified.
5. Conclusions
This randomized, double-blind phase I study of denosumab biosimilar candidate SB16 and reference DEN in healthy male subjects showed comparable PK, PD, safety, and immunogenicity between the treatment groups, confirming the bioequivalence of 60 mg single SC dose between SB16 and EU-DEN as well as US-DEN. Overall, the results further support to conduct a phase III study of SB16.
Declaration of interests
All the authors are employees of Samsung Bioepis Co., Ltd. for the present study. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.
Author contributions
H. Lee and S. Kim contributed to manuscript preparation, study design, data analysis, and/or interpretation. H. Lee, S. Kim, H. Seo, and S. Kim reviewed and approved the final version.
Acknowledgments
The authors thank the participants who volunteered in the study and all the investigators who contributed.
Data availability statement
The authors confirm that the data supporting the findings of this study are available within the article.
Additional information
Funding
References
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