A first‐in‐human, randomized, double‐blind, single‐ and multiple‐dose, phase I study of recombinant human thymosin β4 in healthy Chinese volunteers

Abstract The study evaluated the safety, tolerability, pharmacokinetics (PK) and anti‐drug antibody (ADA) of the recombinant human thymosin β4 (NL005) for single and multiple intravenous injections in healthy subjects. Seven cohorts, with 54 healthy subjects, were given a single intravenous dose of NL005 or placebo and were observed for 28 days. The cohorts received ascending doses of either 0.05, 0.25, 0.5, 2.0, 5.0, 12.5 or 25.0 μg/kg in the single‐dose trial. A total of 30 healthy subjects were randomly enrolled in the multiple‐dose trial, and 3 cohorts (0.5, 2.0 and 5.0 μg/kg) were administered once human thymosin β4 daily for 10 days and observed for 28 days. The adverse events were mild to moderate in intensity. There were no dose‐limiting toxicities or serious adverse events. The plasma concentration, maximum peak concentration (C max) and AUC of each dose group increased with the increase in the dose. The tendency of terminal clearance in each dose group was consistent, and there was no obvious accumulation after continuous administration. Thus, the drug can be concluded to be well tolerated and safe in healthy people and suitable for use in a clinical study for the treatment of acute myocardial infarction.

and serious adverse events. Also, no tumorigenesis was observed within 6 months. [6][7][8] NL005 (recombinant human thymosin β4,rh-Tβ4) comprised 44 amino acids and was generated via restructuring of products by the Beijing northland biotech co., Ltd, using genetic engineering. It has biological activities identical to that of the natural Tβ4. It has undergone complete preclinical pharmacy, pharmacodynamics, pharmacokinetics and safety evaluation, and the preclinical studies have shown NL005 to be stable, of high-quality, safe and effective for increasing the heart function, improving the heart configuration and increasing collateral circulation. It imparts anti-apoptotic property and cell oxidation resistance with no abnormalities during the synthesis. Further developments are however required for harnessing Tβ4 in treating acute myocardial infarction clinically.
Considering the safety, 0.05 and 0.25 μg/kg cohorts (2 subjects per cohort) were used as the pre-test groups, half consisting of males and half of females, and were administered NL005 once. The other five cohorts of subjects (10 subjects per cohort) were administered one dose of NL005 or placebo in a 4:1 ratio.
In the MAD, 30 healthy subjects were enrolled and three cohorts (0.5, 2, 5 μg/kg) were formed, half consisting of males and half of females. Each cohort (10 subjects per cohort) was given NL005 or placebo at a ratio of 4:1 for 10 consecutive days.

| Subject eligibility
Male (weight ≥50 kg) and female (weight ≥45 kg) volunteers (19 ≤ BMI ≤ 28 kg/m 2 ) between 18 and 50 years and in good health, with no underlying medical conditions that would place them at risk, were evaluated for the study participation. All the subjects provided informed consent. The exclusion criteria included clinically significant abnormalities, smoking history (the previous 3 months > 5 cigarettes per day, or did not stop smoking during the experiment), drinking history (the previous 3 months > 2 units per day (1 unit = 360 mL beer, 150 mL wine or 45 mL liquor with 40% alcohol content) or alcohol test positive) and history of substance abuse or drug screening test positive, pregnancy and lactation plan, ADA positive and allergic to protein preparations and biological products.

| Procedure
The subjects for SAD were admitted to the clinical pharmacology unit on day 1 of the study and were discharged from the unit on day 5. The

| Statistical analysis
All statistical analyses were done with the SAS 9.4 (SAS Institute) in the Statistical Analysis Department, Jiaxing Taimei Medical Technology. The pharmacokinetic parameters and ADA indexes were tested and analysed by Joinn Laboratories (China) Co., Ltd.
The pharmacokinetic parameters were estimated using the Phoenix WinNonlin software (Certara). was used to calculate the pharmacokinetic parameters, and the main parameters obtained were C max , T max, AUC, t 1/2, CL and V z .
All the ADA samples are transported to the testing unit in the cold chain under dry ice (≤−70℃) and were screened and confirmed with the indirect ELISA analysis method, and the positive samples were tested for the antibody titre.

| Demographic and baseline characteristics
Fifty-four subjects were enrolled in SAD (10 placebos, 44 NL005), half of which were males and the other half were females. Thirty subjects were enrolled in MAD (6 placebos, 24 NL005). The study volunteers had fairly consistent characteristics across all cohorts in terms of age, weight and BMI etc (Table 1).

| Safety and tolerability
The adverse events (AEs) were coded by the MedDRA 21.0, and the relationship was assessed by CTCAE4.03, and the clinical safety was evaluated. There were no serious adverse events or dose-limiting toxicities during the SAD. Seven of 10 (70%) placebo subjects demonstrated AEs, 25 of 44 (56.8%) Tβ4 cohorts too. All the AEs were mild and moderate, only one placebo subject experienced remission after medication for upper respiratory tract infection, and the rest experienced spontaneous remission. The incidence and severity of adverse events were equal in the Tβ4 group and the placebo group.
There were no serious adverse events or dose-limiting toxicities during the MAD. Five of six (83.3%) placebo subjects experienced AEs, and the experimental group incidence was 16 of 24 (66.7%).
All the AEs were mild and moderate, untreated and alleviated by themselves. At 5 μg/kg, both placebo and Tβ4 groups had one case of subject shedding due to an abnormal electrocardiogram. The incidence and severity of adverse events were equal in the Tβ4 group and the placebo group (Table 3).

| Pharmacokinetics
In the SAD, the mean plasma concentration increases proportionally with the increase in the dose and drug administration time ranging from 0.25 to 25 μg/kg (Figure 1). The C max , AUC 0-t and AUC 0-∞ demonstrated a trend of linear dynamic characteristics, but the conclusion of linear pharmacokinetic characteristics remains elusive (

| The anti-drug antibody of NL005
A total of 108 ADA samples (D14/D28) were collected from 54 subjects in SAD, and except for one positive sample, all the others were negative, and the positive detection rate was 1/108. Two exfoliated subjects were removed, and out of 28 subjects (56 ADA samples) in MAD, except for two positive samples, all the others were negative, and the positive detection rate was 1/28. All the positive samples became negative after follow-up.

| DISCUSS ION
The pharmacokinetic results of both SAD and MAD showed a proportional increase in the mean plasma concentration, C max and AUC each, with the increasing dose. Among the cohorts, the T max was from 3 to 15 minutes, and t 1/2 was from 0.5 to 2.08 hours; and it was consistent with the reports on chemical Tβ4 products. 4 In the MAD, and no tumour-related adverse events were found in this study. [14][15][16] As far as we know, this study is the first reported investigation of Tβ4, produced using recombinant gene technology in healthy subjects. This study showed that NL005 is well tolerated and safe and should be allowed to develop further drugs to treat acute myocardial infarction.

ACK N OWLED G EM ENTS
Beijing Northland Biotechnology Co., Ltd. is the sponsor of the study.

CO N FLI C T O F I NTE R E S T
The

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.