Evaluation of Pharmacokinetics and Safety of the Biosimilar (B01711) and Insulin Degludec/Insulin Aspart (IDegAsp, Ryzodeg) in Healthy Chinese Adults in a Randomized, Open-Label, Single-Dose, Crossover, Phase I Study

Introduction

The persistent global rise in diabetes prevalence underscores diabetes mellitus as a formidable global health challenge.¹ Poor glycemic management can lead to adverse neurological and cardiac complications, resulting in suboptimal outcomes.² Typically, patients with type 2 diabetes are initially prescribed basal insulin as the first class of insulin therapy. If monotherapy with basal insulin fails to achieve glycemic targets, prandial insulin will be added or therapy will be switched to premixed insulin in routine clinical practices.³

Insulin degludec/insulin aspart (IDegAsp, 70/30) represents the third generation of insulin, characterized by a faster onset time and a more prolonged action period, closely mimicking physiological insulin secretion.⁴ IDegAsp is a fixed-ratio formulation, combining 70% of IDeg and 30% of IAsp. Following subcutaneous injection of IDegAsp, the IAsp ingredient rapidly dissociates into its active monomeric form to induce a peak of insulin;⁵ concurrently, the IDeg ingredient’s stable dihexameric state and reversible albumin binding confer a long-lasting, peakless insulin effect.⁶ The two processes occur independently without mutual interference. Owing to the longer half-life of degludec, IDegAsp may offer superior efficacy compared to the therapy of premixed insulin. Ryzodeg (Novo Nordisk A/S, Denmark) received approval from the European Medicines Agency (EMA) in 2013, the US Food and Drug Administration (FDA) in 2015, and China in 2019.

Affordable prescription medications are essential for managing diabetes effectively. Regrettably, even in developed countries like the United States, approximately 25%~30% of Americans with diabetes report rationing or skipping insulin due to its high cost.^7,8 Improving access to more affordable medications can be achieved through the introduction of generic drugs and biosimilars, extending the market exclusivity period for brand-name drugs, and eliminating anticompetitive pay-for-delay agreements.⁹

B01711, an insulin biosimilar to Ryzodeg, was developed by Hui Sheng Bio-pharmaceutical Co., Ltd., following guidelines from the EMA,¹⁰ FDA,¹¹ and China,^12,13 sharing identical components with Ryzodeg. Preclinical studies in beagles demonstrated that B01711 had similar pharmacokinetic and pharmacodynamic profiles to Ryzodeg at a dose of 0.7 U/kg. At doses of 0.6 U/kg and 1.2 U/kg, no systemic toxic reactions of B01711, aside from expected insulin-related responses, were observed, aligning with findings for Ryzodeg. This was a first-in-human study designed to evaluate the pharmacokinetics (PK) and safety of B01711 compared to Ryzodeg in healthy Chinese volunteers.

Subjects and Methods

Subjects

Written consents from subjects were obtained before the commencement of any activities. Individuals aged 18 to 45 years, with a body mass index (BMI) ranging from 19.0 to 25.0 kg/m², and maintaining euglycemia [fasting glucose <6.1 mmol/L, glycated hemoglobin (HbA1c) <6.5%] were deemed eligible. During the screening, the results of physical examinations, laboratory tests (including complete blood count, liver and renal function tests, anti-insulin antibody assay, urinalysis, and coagulation tests), and a 12-lead electrocardiogram needed to be within normal limits or deemed clinically insignificant by the investigator. Effective contraceptive measures were required from the signing of the informed consent form until 30 days following the trial drug administration.

The primary exclusion criteria included: positive test results for hepatitis B surface antigen, hepatitis C virus antibody, human immunodeficiency virus antibody, or Treponema pallidum antibody; a history of hypoglycemia; a clinically significant history of drug allergy or known hypersensitivity to the study drug or any of its components or related excipients; vaccination or any drug use within four weeks before screening; blood donation or significant blood loss exceeding 400 mL within the past three months; heavy smoking (more than five cigarettes per day) or alcohol abuse (more than 25 g of alcohol per day).

Study Design and Procedures

This was a phase I, single-center, open-label, randomized, single-dose, two-treatment, two-period, two-sequence, crossover study. It comprised a screening visit and two treatment visits with an interval of at least 14 days. The subjects were randomized into one of the two treatment arms (Figure 1). Each participant received both study drugs during the two dosing periods. They arrived at the clinical research unit the day before dosing. A fasting state of at least 10 hours before drug administration was ensured. The trial commenced on the morning of the dosing day. Venipuncture was performed using the median cubital vein for blood sampling, facilitated by heating the area with a warmed blanket. Following pre-dosing blood sample collection, a 0.5-U/kg dose of either B01711 or Ryzodeg was subcutaneously injected into the abdominal wall. Subjects had breakfast post-injection and consumed lunch and dinner as usual. A 4-mL blood sample was collected at −15 (predose), 10, 20, 30, 40, 50, 60, 70, 80, 90, 120, 150, 180, 240, 300, 360, and 480 minutes for IAsp concentration analysis. Another 4-mL blood sample was collected at −0.25 (predose), 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 36, 48, 72, 96, and 120 hours for IDeg level analysis. Additionally, a 0.1-mL blood sample was collected at the aforementioned time points to measure whole blood glucose levels immediately using the Biosen C-line glucose meter. Additional carbohydrates were administered when blood glucose levels fell below 3.6 mmol/L to prevent hypoglycemia. The study protocol was approved by the Ethics Committee of Sichuan University West China Hospital and conducted in accordance with the Declaration of Helsinki, Good Clinical Practice guidelines, and local regulations. This study is registered at https://www.chictr.org.cn (ChiCTR2400083084).

Figure 1 Sketch map of study design and subject flow chart.

PK samples were collected in tubes containing dipotassium ethylenediaminetetraacetic acid (K2EDTA) as a plasma stabilizer. The tubes were centrifuged at 1800 g for 15 minutes to obtain plasma which was then frozen at −60 °C until shipment for analysis. Plasma concentrations of IDeg and IAsp were measured using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method by Labcorp Pharmaceutical Research and Development in Shanghai, China, with lower limits of quantification of 0.25 and 0.20 ng/mL, respectively. The liquid-liquid extraction method was employed to pretreat the PK samples.

Pharmacokinetic Endpoints

The value of PK sample measurements below the lower limit of quantification (BLQ) was regarded as zero. The primary PK endpoints included the maximum concentration of IAsp (C_{IAsp, max}), the area under the curve (AUC) of IAsp from 0 to the time of the last measurable value (AUC_{IAsp, 0-t}), and the AUC of IDeg from 0 to 24 hours (AUC_{IDeg, 0–24 h}). The secondary PK endpoints included half-life (t_1/2), peak time (T_max), and AUC from 0 to infinity (AUC_0-∞) for IAsp; C_max, t_1/2, T_max, AUC_0-∞, AUC from 0 to 12 hours (AUC_0-12 h), AUC from 12 to 24 hours (AUC_12-24 h), AUC from 0 to 120 hours (AUC_0-120 h) for IDeg.

Safety Assessments

Safety and tolerability were investigated through spontaneous reporting and inquiries regarding adverse events (AEs) and treatment-emergent adverse events (TEAEs) throughout the study. Additionally, physical examinations, vital sign measurements, 12-lead electrocardiogram, and laboratory tests, including serum chemistry, urinalysis, and hematology were monitored.

Sample Size and Statistical Methods

No data on the intra-subject coefficient of variation (CV) of IDegAsp was available. However, based on published pieces of literature, the intra-subject CV of primary PK-AUC estimates ranged from 17% to 21.2% for IDeg and 10% to 20% for IAsp, respectively. Assuming the intra-individual CVs were up to 21.2% and B01711 was no more than ±5% different from Ryzodeg, a minimum sample size of 28 subjects in a crossover design was determined to demonstrate bioequivalence with 90% power and a 5% type 1 error. To account for a potential 10% dropout, 32 subjects were planned.

Data analysis was performed by SAS^® (version 9.4; SAS Institute Inc). and WinNonlin^® (version 8.3; Certara L.P). Subjects with significant deviations affecting PK profiles, or a baseline value of ≥5% of C_{IDeg, max} or C_{IAsp, max}, or those using other drugs interfering with PK assessment, or reaching peak level immediately following drug administration, were excluded from the PK analysis. The primary PK endpoints were calculated using raw, log-transformed data, and analyzed in a mixed-effects model with treatment (two levels), sequence (two levels), and period (two levels) as fixed effects, and subject-within-sequence as a random effect. The least squares (LS) mean of PK estimates of each investigational drug, and the ratio of B01711 to Ryzodeg, along with their 90% confidence intervals (CIs), were estimated. PK equivalence would be established if the 90% CIs of the estimated ratio of primary PK endpoints lay within the limits of 80.0%~125.0%. The normality of the data was examined with Q-Q plots. A nonparametric approach based on the Wilcoxon signed-rank test was applied to assess the differences in time-related parameters. All subjects who received at least one dose of the treatment were included in the safety analysis. A two-sided P value of <0.05 was considered statistically significant.

Results

Baseline Demographics and Characteristics

The study began in April 2022 and concluded in August 2022. As illustrated in Figure 1, a total of 39 participants were initially recruited, with 32 (22 males and 10 females) ultimately enrolling after screening. All enrolled participants received both drug administrations and completed the trial procedures. The participants had an average age of 27.6 years (range: 21~37 years), an average weight of 60.9 kg (range: 49.6~77.1 kg), an average height of 168.1 cm (range: 155.0~181.0 cm), an average BMI of 21.5 kg/m² (range: 19.1~25.0 kg/m²), an average fasting glucose level of 4.72 mmol/L (range: 3.71~5.76 mmol/L) and an average HbA1c level of 5.3% (range: 4.6%~5.9%) (Table 1).

Table 1 Demographics and Baseline Characteristics of the Participants

Pharmacokinetics

Following drug administration, the IAsp ingredient was absorbed rapidly and reached a peak level at around 50 minutes. Approximately 97% of the total IAsp values at the eighth hour postinjection remained BLQ. The time profiles of the IAsp component for both drugs were similar (Figure 2A). The ratios of LS-means for C_{IAsp, max}, and AUC_{IAsp, 0-t} between B01711 and Ryzodeg were both close to 1, with their respective 90% CIs falling within the range of 0.80 to 1.25. Moreover, the ratio of LS-mean of AUC_{IAsp, 0-∞} for B01711 compared to Ryzodeg was 102.3%, with a 90% CI of 99.6% to 105.0%. No significant differences were detected in T_{IAsp, max}, and t_{1/2, IAsp} (P>0.05) (Table 2).

Table 2 Comparison of Pharmacokinetic Parameters Between B01711 and Ryzodeg

Figure 2 The time profile of mean IAsp levels from 0 to 8 hours (A) and time-concentration of mean IDeg from 0 to 120 hours (B) and 0 to 24 hours (C). The error bars represented the standard deviation.

The IDeg component was gradually absorbed after injection, with its time profiles over the intervals of 0 to 120 hours and 0 to 24 hours depicted in Figure 2B and C, respectively. IDeg peak levels were reached at approximately 10~11 hours. Approximately 72%~75% of the total IDeg values were BLQ at the 120 hours post-injection. The LS- mean ratio of AUC_{IDeg, 0–24 h} for B01711 compared to Ryzodeg was 106.1%, with a 90% CI ranging from 101.9% to 110.5%. Furthermore, the values of T_{IDeg, max}, and t_{1/2, IDeg} for both drugs were comparable (P>0.05). All 90% CIs of the LS-mean ratios for secondary PK estimates (AUC_{IDeg, 0–12 h}, AUC_{IDeg, 12–24 h}, AUC_{IDeg, 0–120 h}, AUC_{IDeg, 0-∞}, and C_{IDeg, max}) between the two formulations were within the range of 0.80 to 1.25 (Table 2).

Safety Evaluation

No serious AEs or discontinuations due to treatment-emergent AEs (TEAEs) were reported in either group. Table 3 shows that two TEAEs occurred in two subjects in the B01711 group, and seven TEAEs were reported in seven subjects in the Ryzodeg group. The monitoring of whole blood glucose levels is shown in Figure 3. A participant in the Ryzodeg group experienced a hypoglycemia event. The AEs (hemoglobin decreased, hemorrhage at the injection site, and vomiting) were considered unrelated to the study drug. However, hypokalemia and hypoglycemia were deemed treatment-related AEs (TRAEs) by the investigator. These TRAEs were mild and resolved. About two hours after subcutaneous administration of B01711 to the abdomen, one subject reported allergic dermatitis near the blood sampling site on the forearm, which had been warmed with a blanket. The blanket was removed immediately, and the condition was resolved within four hours. Consequently, the AE ‘allergic dermatitis’ was considered unrelated to the study drug. All AEs were mild in severity. To address the AE ‘hemoglobin decreased’, two subjects received a treatment regimen including a polysaccharide-iron complex capsule (150 mg once daily orally), a folic acid tablet (5 mg three times daily orally), and a compound vitamin B tablet (one tablet three times daily orally) for seven days, leading to normalized hemoglobin levels post-treatment. Both drugs were well tolerated in this study.

Table 3 Summary of Treatment-Emergent Adverse Events Reported in the Study

Figure 3 The monitored whole blood glucose levels in the study (mean with standard deviation).

Discussion

The American Diabetes Association suggests maintaining a reasonable HbA1c target level below 7.0% (53 mmol/mol), and an astringent lower HbA1c level if possible without increasing the risk of hypoglycemia or other negative side effects.¹⁴ However, most diabetic patients eventually fail to achieve the glycemic goal with lifestyle modifications and oral antidiabetic drugs alone.¹⁵ Initiating and intensifying treatment with insulin is often the next step. Premixed insulin delivers both basal and mealtime coverage in a single injection. However, the protamine component can influence the soluble portion, resulting in a ‘shoulder effect’ and increasing the risk of hypoglycemia. Moreover, the protamination of short-acting insulin transforms it into intermediate-acting insulin with an extended glucose-lowering effect lasting approximately six hours, which is inadequate for consistent basal coverage compared to long-acting insulin.¹⁶

The IDegAsp co-formulation, consisting of 70% IDeg and 30% IAsp in a single pen, offers a continuous basal glucose-lowering effect and mealtime insulin coverage in one injection without the need for re-suspension. This study assessed the PK and safety of an IDegAsp biosimilar (B01711), with Ryzodeg as the reference. Hui Sheng Bio-pharmaceutical Co., Ltd. manufactures the IDeg component (B01411) and the IAsp component (B01511). Previous phase I euglycemic clamp studies (http://www.chinadrugtrials.org.cn/, No: CTR20190161 and CTR20192122) and phase III studies (http://www.chinadrugtrials.org.cn/, No: CTR20190121 and CTR20202005) demonstrated the pharmacokinetic and pharmacodynamic equivalence, efficacy, and safety of B01511 and B01411 in comparison to Novo Nordisk’s reference products.¹⁷ These results prompted the National Medical Products Administration of China to waive the pharmacodynamic comparison for the two drugs, thereby omitting the euglycemic clamp technique for pharmacodynamic evaluation.

The study was conducted in healthy subjects using the LC-MS/MS method to determine the concentrations of IAsp and IDeg, ensuring no interference from endogenous insulin.^18–20 Following subcutaneous injection, the IAsp component was rapidly absorbed into the circulation, reaching a peak level of around 2.28~2.50 ng/mL at approximately 50 minutes. The IDeg component gradually dissolved into the circulation and lasted for a long period. Compared with the reference product Ryzodeg, the 90% CIs of the LS-mean ratios for the primary PK endpoints (AUC_{IAsp, 0-t}, C_{IAsp, max}, and AUC_{IDeg, 0–24 h}) were all in the acceptable range of 0.80~1.25. Moreover, the 90 CIs of the LS-mean ratios for the secondary PK parameters also stayed within the equivalent range. Time-related parameters were comparable between the two drugs. These results support the PK equivalence for B01711 compared to Ryzodeg.

The incidence of TEAEs was low, and all reported TEAEs were mild in this study. All AEs were resolved without any lasting effects. Both drugs were well-tolerated by healthy participants when administered as a single dose.

One limitation of this study is the absence of pharmacodynamic evaluation. Additionally, this study applied a single dose, and PK assessments were not conducted under multiple-dose or steady-state conditions in healthy subjects. Consequently, the obtained PK data cannot be directly extrapolated to clinical settings. A multicenter, randomized, open-label, parallel phase III study (http://www.chinadrugtrials.org.cn/, No: CTR20212490) was conducted in patients with type 2 diabetes to evaluate the efficacy and safety of B01711 compared to Ryzodeg.

Conclusion

This phase I study demonstrated the PK similarity of B01711 compared to Ryzodeg without significant safety concerns at a single dose.