Introduction

Osteoporosis, a systemic skeletal disease, causes low bone mass and microarchitectural breakdown of bone tissue predisposing a person to increased bone fragility and fracture risk [1]. The World Health Organization (WHO) practically defines osteoporosis when bone mineral density (BMD) is 2.5 standard deviation (SD) or more below the average value for young healthy women (a T-score of <  − 2.5 SD) [2].

Osteoporosis is a highly prevalent disease, affecting approximately 30% of postmenopausal women in the USA and the European Union (EU), and over 40% of them will experience fragility fractures in their lifetime [3]. Globally, an estimated 9 million osteoporotic fractures occurred in the year 2000, from which 1.6 million were hip fractures, 1.7 million were forearm fractures, and 1.4 million were clinical vertebral fractures. Women account for 70% of hip fractures. By region, Europe had the most fractures (34.8%), followed by the Western Pacific Area (28.5%), Southeast Asia (17.4%), and the Americas (15.7%) [4].

The management of osteoporosis includes lifestyle and dietary measures (e.g., adequate intake of calcium and vitamin D, regular exercises, smoking cessation, and avoiding excessive alcohol consumption) and pharmacological interventions [5]. At present, two main categories of pharmacological agents are available for the treatment of osteoporosis: antiresorptive agents and anabolic agents [6]. Most pharmacological treatments are currently antiresorptive agents; their primary mechanism of action is to reduce bone resorption by inhibiting osteoclast activity. This class of drugs includes bisphosphonates, estrogens, selective estrogen receptor modulators, strontium ranelate, and denosumab [7].

Teriparatide, a recombinant human parathyroid hormone (rhPTH), is a biologically active N-terminal fragment (1–34) of the 84-amino acid human PTH [8]. It is an approved anabolic agent for osteoporosis treatment that stimulates bone formation to improve bone density and strength [9]. The Fracture Prevention Trial [10] was the basis for the approval of teriparatide by the US Food and Drug Administration (FDA) and the European Medicine Agency (EMA) as the first anabolic agent to treat postmenopausal women with severe osteoporosis. It was later approved for the treatment of osteoporosis in men, as well as for osteoporosis associated with glucocorticoid therapy in men and women at risk of fracture [11]. By improving bone microarchitecture as well as increasing bone mass, teriparatide significantly reduces fracture risk [12]. Teriparatide has an identical sequence to the 34 N-terminal amino acids of human PTH, and it is manufactured by using a strain of Escherichia coli modified by recombinant DNA technology [13].

Intas Pharmaceutical Ltd., India, has developed INTG8 as a potential biosimilar of teriparatide to both EU- and US-referenced products (Forsteo®, Eli Lilly, The Netherlands (EU-teriparatide), and Forteo®, Lilly USA (US-teriparatide)). Comprehensive analytical similarity tests were performed to compare physicochemical and structural properties and in vitro biological activity of INTG8 to EU- and US-teriparatide reference products. The biosimilarity of INTG8 to the reference products was demonstrated in this comparability exercise (data on file). Here, we present the results of a phase 1 trial that compared the pharmacokinetics (PK), pharmacodynamics (PD), safety, tolerability, and immunogenicity of teriparatide biosimilar (INTG8) with the EU- and US-teriparatide reference products in healthy subjects.

Methods

Study design

This was an assessor-blind, randomized, three-treatment, three-period, single-dose, crossover, bioequivalence study in healthy men and postmenopausal women after subcutaneous (SC) administration under fasting conditions (ctri.nic.in/ #CTRI/2020/10/028627). The study conducted was at a single center between 07 December 2020 and 12 February 2021 in compliance with Independent Ethics Committee (IEC) that approved the study protocol, good clinical practice from the International Conference on Harmonization (ICH-GCP, E6 (R2), 2016), the Declaration of Helsinki (Brazil, October 2013), applicable principles of good laboratory practice (GLP), and applicable national or international regulatory requirements. A written informed consent was obtained from each subject before the start of the study. This study was conducted at Lambda Therapeutic Research Ltd., in Ahmedabad, India, which was involved in study conduct, PK, PD, and immunogenicity analyses, as well as statistical analysis.

The primary objective of this trial was to demonstrate PK bioequivalence of INTG8 to both EU- and US-licensed reference products (Forsteo®, Eli Lilly, The Netherlands (EU-teriparatide) and Forteo®, Lilly USA (US-teriparatide)) following 20-μg single subcutaneous injection in healthy men and postmenopausal women. Secondary objectives included comparing pharmacodynamics (PD), safety, and tolerability. Test and reference products’ immunogenicity was assessed as an exploratory objective.

Subjects were housed in the clinical facility for at least 11 h before the administration of the investigational medicinal products (IMPs) in period I and remained in the clinical facility till the end of period III (for 24 h after the IMP administration of period III). All study periods (periods I to III) were completed continuously. End of study assessments were conducted 28 days after the administration of period III IMPs. For logistic reasons, the study was conducted into three groups (group I, group II, and group III).

After an overnight fast of at least 10 h, eligible subjects were randomized (1:1:1) to receive a single SC dose of teriparatide 20 μg/80 μL of either teriparatide biosimilar, EU-teriparatide, or US-teriparatide. A washout period of 24 h separated three periods of the study; therefore, subjects received a single dose of each treatment on 3 consecutive days in a crossover manner. The sequence of administration of treatments, i.e., “TR1R2” or “R2TR1” or “R1R2T” to the subjects, were determined according to the randomization schedule (R1 = EU reference product, R2 = US-referenced product, T = test product or biosimilar). Equal allocation of subjects in each sequence was ensured. Study drugs were administered into the abdomen of each subject while in the supine position in each period. It was an assessor-blinded study; therefore, coded treatment blinding was not needed. The study staff ensuring the safety of the subjects and laboratory personnel analyzing the samples for PK, PD, and immunogenicity data were blinded.

Study subjects

A healthy subject population was chosen to minimize variability and detect differences between teriparatide biosimilar, EU-teriparatide, and US-teriparatide. Subjects were screened within 28 days prior to the IMP administration in period I. Healthy adult men (18 to 45 years) and postmenopausal women (45 to 65 years), with a body mass index (BMI) of 18.5–30 kg/m2 were eligible to volunteer in this study. Postmenopausal status was defined as serum follicle-stimulating hormone (FSH) levels > 40mIU/mL and 6 months of spontaneous amenorrhea or 6-week post-surgical bilateral oophorectomy with or without hysterectomy prior to the start of the study. The ability to comply with study procedures and to provide voluntary written informed consent, as well as a negative serum pregnancy test at screening, were other inclusion criteria. The main exclusion criteria included orthostatic or systemic hypotension at screening, history or presence of any clinical disease or disorder, history or presence metabolic bone disease, abnormal PTH level at screening, i.e., PTH level (< 15 pg/mL or > 65 pg/mL), and previous treatment, including for investigational purposes, with human PTH or any products derived from PTH.

Pharmacokinetics, pharmacodynamics, and immunogenicity measurements

Pharmacokinetic properties of the test and reference formulations were assessed by measuring serum teriparatide concentration. The primary PK endpoints were maximum serum concentration (Cmax), area under the curve (AUC) from time zero to t (AUC0-t), and AUC from time zero extrapolated to infinity (AUC0-∞). Secondary PK endpoints were time to achieve Cmax (Tmax), half-life (t1/2), terminal rate constant (λz), residual AUC (AUC_%Extrap_obs), volume of distribution (Vd) based on terminal phase, and total body clearance (Cl). Pharmacodynamic properties of the test and reference formulations were compared by measuring corrected total serum calcium levels (baseline-adjusted and non-adjusted). The PD was assessed as secondary endpoints, which included maximum observed effect (Emax), area under the effect curve (AUE) from time zero to the last measurable concentration (AUE0–t), and time to maximum observed effect (Tmax) for total serum calcium levels.

During the study, 51 blood samples (3 pre-dose samples of 8 mL each and 48 post-dose samples of 3 mL each) for PK, 34 blood samples (3 pre-dose samples and 31 post-dose samples, each of 2 mL) for PD, and 2 blood samples (each of 10 mL) for immunogenicity were collected from each subject to evaluate the PK, PD, and immunogenicity profiles of teriparatide biosimilar and EU- and US-teriparatide. Blood samples for PK evaluation were collected at pre-dose (0.000 h, within 5 min before dosing) and at 0.083, 0.167, 0.250, 0.333, 0.500, 0.750, 1.000, 1.250, 1.500, 1.750, 2.000, 2.500, 3.000, 3.500, 4.000, and 4.500 h following IMP administration in each period. Blood samples for PD measurement (corrected total serum calcium) were obtained at pre-dose (0.000 h, within 5 min before dosing) and at 0.500, 1.000, 2.000, 3.000, 4.000, 5.000, 6.000, 8.000, 12.000, 16.000, and 24.000 h following IMP administration in each period. Blood samples for immunogenicity analysis were obtained before the first dose (0.000 h) and at the end of the study (28 days after dosing of period III). After collection, samples were separated in an ice-cold water bath and stored at − 65 °C ± 10 °C until analysis.

Teriparatide was determined in serum samples using a validated enzyme-linked immunosorbent assay (ELISA) method. Corrected calcium was estimated by using a validated spectrophotometry method based on Vitros 5–1 FS chemistry analyzer. Potential immunogenicity of the study drugs was determined by measuring anti-drug antibodies (ADA) against teriparatide in a screening assay using a validated indirect ELISA-based method. The screening-positive samples were subjected to the confirmatory assay, and the confirmed ADAs positive samples were checked for their specificity, titer, and neutralizing capacity using the validated cell-based method.

Safety and tolerability assessments

Safety was assessed from the screening period to the end of the study. It was assessed through clinical examination, vital sign assessment, 12-lead ECG, chest X-ray (posterior–anterior view) recording, clinical laboratory parameters (e.g., biochemistry, hematology, immunology, and urine analysis), serum pregnancy test (for female subjects), abdominal pelvis ultrasonography (for female subjects), FSH, estradiol measurement and gynecological examination (for female subjects), measurement of orthostatic hypotension, injection site assessment, subjective symptomatology, and monitoring of adverse events (AEs). All AEs are coded and summarized by a system organ class (SOC) and preferred term (PT) using the Medical Dictionary for Regulatory Activities (MedDRA) version 22.0 or higher. The severity of AEs was rated by the principal investigator or the clinical research physician as mild, moderate, or severe. Additionally, the severity of AEs was also graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), version 4 or higher.

Sample size calculation and statistical analysis

To achieve at least 80% power based on the expected test to reference ratio of 90.0–111.1% and previously observed intra-subject coefficient of variation (CV) of ~ 29% for AUC0-t (teriparatide), with 5% significance level (α) and bioequivalence window of 80.00% to 125.00%, 73 subjects were required to complete the study. Considering ~ 30% dropouts or withdrawals (wherein ~ 10% subjects were assumed for dropout due to failure of device), 105 subjects (in comparable proportions, i.e., the male to female ratio should be between 40 and 60%) were enrolled in this study.

Descriptive statistics are provided for all of the PK and PD parameters. As the study was conducted in groups, log (ln)-transformed PK and PD parameters were analyzed using analysis of variance (ANOVA). The ANOVA; power and ratio analysis for the ln-transformed PK parameters Cmax, AUC0-t, and AUC0-∞ for teriparatide; and ln-transformed PD parameters Emax and AUEC0-t for baseline-adjusted and non-adjusted corrected total serum calcium levels were done. Using two one-sided tests for bioequivalence, 90% CI for the ratio of geometric least squares means (LSMs) were calculated for the ln-transformed PK parameters Cmax, AUC0-t, and AUC0-∞ of teriparatide. For the PD parameters, 90% CI and 95% CI for the ratio of geometric LSMs were calculated for the ln-transformed Emax and AUEC0-t for baseline-adjusted and non-adjusted corrected total serum calcium levels.

Bioequivalence of the test product with that of the reference products was concluded, if the 90% CI for the ratio of geometric LSMs for the ln-transformed PK parameters Cmax, AUC0-t, and AUC0-∞ fell within the acceptance range of 80.00% to 125.00%. Bioequivalence is based on the comparisons of teriparatide biosimilar (test) vs. EU- and US-teriparatide (reference products).

The PK and PD parameters were calculated by non-compartmental model using Phoenix® WinNonlin® version 8.1 (Certara L.P.). Statistical comparison of the PK and PD parameters was carried out using SAS version 9.4.

Results

Subject disposition

A total of 105 subjects were enrolled and dosed in the study, and all these 105 subjects completed the clinical phase of the study successfully. Subject disposition is outlined in Fig. 1.

Fig. 1
figure 1

Subject disposition. A washout period of 24 h was maintained between the dosing days of any two consecutive periods

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All 105 subjects included in the PK set and PD set for baseline non-adjusted corrected total serum calcium levels. However, one subject had pre-dose sample as non-reportable (NR) in period I and III due to that post-dose sample could not be baseline-adjusted, and hence, this subject was excluded from statistical analysis. Hence, 104 subjects were included in the PD set for baseline-adjusted corrected total serum calcium levels.

Demographics and baseline characteristics

Baseline demographic characteristics of 105 subjects enrolled and dosed in the study were similar across the three-treatment sequence groups. Of 105 subjects, 62 (59.05%) were male, and 43 (40.95%) were female. All subjects were Asian and of Indian ethnicity with a mean age of 41.1 years (range 19–63 years), mean weight 61.5 kg (range 45.1–89.3 kg), mean height 160.8 cm (range 143.0–179.0), and mean BMI 23.81 kg/m2 (range 18.66–29.85 kg/m2). The detailed demographic characteristics are provided in Table 1.

Table 1 Baseline demographics and clinical characteristics
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Pharmacokinetics

The combined data from the three treatment periods showed that the mean serum teriparatide concentrations were similar following a single SC dose of teriparatide biosimilar, EU-teriparatide, or US-teriparatide (Fig. 2). The peak mean concentration was reached at similar time following a single SC dose of study drugs. Summary of serum teriparatide PK parameters following a single dose of teriparatide biosimilar, EU-teriparatide, and US-teriparatide is presented in Table 2.

Fig. 2
figure 2

Mean serum concentration: time profiles of teriparatide following administration of teriparatide biosimilar, EU-teriparatide, and US-teriparatide (linear scale)

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Table 2 Summary of serum teriparatide pharmacokinetic parameters
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The bioequivalence analysis (i.e., geometric LSMs, ratio, 90% CIs, intra-subject CV, and power) of teriparatide biosimilar vs. EU-teriparatide and teriparatide biosimilar vs. US-teriparatide is summarized in the Table 3. The 90% CIs of the geometric LSM ratios, derived from the analysis on the ln-transformed Cmax, AUC0-t, and AUC0-∞ of teriparatide biosimilar relative to EU-teriparatide and US-teriparatide were entirely contained within the predefined acceptance range of 80.00% to 125.00%, thereby demonstrating the bioequivalence of teriparatide biosimilar to both EU-and US-teriparatide reference biologic products. Additionally, the corresponding 90% CI for US-teriparatide vs. EU-teriparatide and EU-teriparatide vs. US-teriparatide was also within the range of 80.00% to 125.00%.

Table 3 Statistical comparison of primary pharmacokinetic endpoints between teriparatide biosimilar and reference products (N = 105)
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Pharmacodynamics

The descriptive statistics of PD parameters of corrected total serum calcium levels (baseline-adjusted and non-adjusted) for teriparatide biosimilar, EU-teriparatide, and US-teriparatide are summarized in the Table 4.

Table 4 Total serum calcium levels after administration of teriparatide biosimilar, EU-teriparatide, and US-teriparatide
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The values of PD parameters AUEC0-t, Emax, and Tmax for serum-corrected total serum calcium levels (baseline-adjusted and non-adjusted) were comparable for the three study drugs. The geometric LSM ratios for baseline-adjusted Emax and AUEC0-t were 102.4% and 98.1% for teriparatide biosimilar vs. EU-teriparatide and 106.8% and 121.7% for teriparatide biosimilar vs. US-teriparatide. The geometric LSM ratios for baseline non-adjusted Emax and AUEC0-t were 99.9% and 100.1% for teriparatide biosimilar vs. EU-teriparatide and 99.9% and 101.6% for teriparatide biosimilar vs. US-teriparatide. According to these results, teriparatide biosimilar and EU-teriparatide, as well as teriparatide biosimilar and US-teriparatide, both showed similar PD responses.

Immunogenicity

All 105 subjects were tested for immunogenicity. Of the 105 tested subjects, 13 and 14 subjects were found to be screening-positive at pre-dose and at the end of the study, respectively. Of the 13 screening-positive subjects at pre-dose, 5 subjects were confirmed positive for ADA at pre-dose, and 2 subjects out of the 5 confirmed positive at pre-dose remained positive at the end of the study. Amongst these subjects, none of the subject was found positive for neutralizing antibodies at pre-dose or at the end of the study.

Safety and tolerability

A safety analysis was conducted on all 105 subjects enrolled in the study. A total of 19 AEs were reported by 13 (12.38%) of 105 subjects during the study: 11 AEs in 8 (7.62%) subjects after administration of teriparatide biosimilar, 5 AEs in 4 (3.81%) subjects after administration of EU-teriparatide, and 3 AEs in 3 (2.86%) subjects after administration of US-teriparatide. The summary of AEs by system organ class and preferred term is outlined in Table 5. All the AEs were mild. The majority (12 AEs) of the AEs were considered as related to study drug by the investigator. Except for four subjects, all of the subjects with AEs recovered. The outcome of 7 AEs in these 4 subjects was unknown since they were considered lost to follow-up. Nausea was the most commonly reported adverse event (≥ 5% subjects), reported in 5 (4.76%) of the 105 subjects. As none of the subjects discontinued the study due to AEs, study drugs were well tolerated. No deaths or serious AEs were reported during the study. A single injection site reaction was observed in one subject (EU-teriparatide) indicating acceptable local tolerance.

Table 5 Summary of adverse events by system organ class and preferred term
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Discussion

This phase 1 assessor-blind, randomized, three-treatment, three-period, single-dose, crossover study was conducted with the primary objective of demonstrating the PK equivalence of teriparatide biosimilar (INTG8, Intas Pharmaceuticals Limited, India) to US-teriparatide (Forteo®, Lilly USA, LLC) and EU-teriparatide (Forsteo®, Eli Lilly, the Netherlands) in healthy men and postmenopausal women after SC administration. As secondary objectives, PD profile, safety, and tolerability of the teriparatide biosimilar were compared to the reference biologics. Exploratory objective included comparing the immunogenicity of test and reference products.

According to the European Medicines Agency (EMA), a biosimilar is a biological medicinal product containing the active ingredient of an already authorized original biological medicinal product (reference medicinal product), which needs to be similar in terms of quality, biological activity, safety, and efficacy to the licensed reference product based on the extensive comparison exercise [14]. For demonstrating biosimilarity, stepwise development is generally recommended. This may include comparing the proposed biosimilar with the reference product in terms of structure, function, animal toxicity, and clinical assessments (PK, PD, immunogenicity, safety, and efficacy), and a totality-of-the-evidence approach is used to assess biosimilarity [15]. This study aimed to provide clinical PK similarity of teriparatide biosimilar and reference products, along with providing supportive PD data to show the biosimilar properties of INTG8 compared to EU- and US-referenced product.

Teriparatide is rapidly absorbed after SC injections of 20, 40, and 80 μg, reaching peak serum concentrations at approximately 30 min after SC injection and being rapidly eliminated with a half-life of approximately 1 h. The absolute bioavailability of teriparatide is 95% following SC administration, and its pharmacokinetics are not affected by age (range 31 to 85 years). The recommended dose of teriparatide is 20 μg administered once a day [16, 17].

Given the once daily dose and short elimination half-life of teriparatide, a single-dose, three-period crossover study was conducted after single SC 20 μg injection of teriparatide biosimilar and EU- and US-teriparatide on 3 consecutive days with a washout of 24 h. The healthy subject population and crossover design were selected in this study to minimize variability and detect differences between the biosimilar and reference products, which is consistent with the guideline for investigating bioequivalence (CPMP/EWP/QWP/1401/98 Rev.1/Corr**). The standard 80.00% to 125.00% bioequivalence criteria was used for the determination of PK equivalence of primary PK parameters Cmax, AUC0-t, and AUC0-∞ [18]. The serum calcium concentration increases transiently after teriparatide 20 μg is administered once a day, starting between 2 and 4 h after the dose and reaches peak level between 4 and 6 h (median increase, 0.4 mg/dl), and it returns to the baseline by 16 to 24 h after each dose [16]. Therefore, corrected total serum calcium levels (baseline-adjusted and non-adjusted) are measured as PD markers to compare the PD profile of teriparatide biosimilar with the reference products, and these data are provided as the supportive information.

The PK parameters for three study drugs (i.e., teriparatide biosimilar, EU-teriparatide, and US-teriparatide) were similar following a single SC 20 μg dose of teriparatide. The 90% CIs of the geometric LSM ratios of Cmax, AUC0-t, and AUC0-∞ of teriparatide biosimilar relative to EU-teriparatide and US-teriparatide were within the acceptance range of 80.00% to 125.00%, thus demonstrating the PK equivalence of teriparatide biosimilar to the EU-teriparatide and US-teriparatide reference products. The values of PD parameters AUEC0-t, Emax, and Tmax for serum-corrected total serum calcium levels (baseline-adjusted and non-adjusted) after single SC dose of the three study drugs were comparable indicating similar PD responses of teriparatide biosimilar to both the reference products. Based on the safety analysis, the biosimilar and reference products were generally well tolerated in healthy volunteers, with similar incidence of AEs. There were no subject discontinuations, serious adverse events, or deaths observed in the study due to AEs. Overall, no meaningful differences in the safety profile were observed between teriparatide biosimilar, EU-teriparatide, and US-teriparatide reference products. None of the 105 subjects tested for immunogenicity had either pre-dose or post-dose neutralizing antibodies.

As with many phase 1 biosimilarity studies, one of the key limitations is the ability to assess immunogenicity. Although immunogenicity was assessed as a secondary outcome, the nature of the requirements for assessing bioequivalence, e.g., duration of the study, crossover design, and low sample size as per the guideline on the investigation of bioequivalence [18], is not conducive to a full assessment of immunogenicity. The microbial origin of INTG-8 and hence the absence of any post-translational modification, the low molecular weight, and the fully human sequence of the molecule would support a low immunogenic potential. The originator molecule has indeed shown a low level of immunogenicity [17].

This phase 1 study in healthy volunteers (men and postmenopausal women) demonstrated PK equivalence of teriparatide biosimilar (INTG8) to both EU- and US-teriparatide reference products with similar PD, safety, tolerability, and immunogenicity profiles. Further, teriparatide biosimilar has also shown the high degree of similarity to the reference products in the comprehensive analytical comparability exercise, which includes comparison of physicochemical and structural properties and in vitro biological activity (data on file). Overall, the totality of evidence from the analytical comparability exercise and this phase 1 study supports the similarity of teriparatide biosimilar candidate to both EU- and US-licensed reference products.