This was a randomized, double-blinded, placebo-controlled, single ascending dose study in 32 healthy Chinese adults at phase I department, Peking University People’s Hospital (China). The study was conducted in compliance with the Declaration of Helsinki, Guideline for Good Clinical Practice, and was approved by the Ethics Committee of Peking University People’s Hospital (No. 2017PHA-090-01). Written informed consent was obtained from each subject before the initiation of the investigation. The study was registered at the Chinese Clinical Trials Registry (registration number: CTR 20171459).

Pharmaceutical and pre-clinical studies showed that the no observed adverse effect level (NOAEL) of LY06006 in the toxicological study of multiple administration in cynomolgus monkeys is 10 mg/kg. In the light of the guidelines issued by FDA, the calculated initial dose should not exceed 19.2 mg based on the mean weight of 60 kg of healthy adults (FDA, 2005). Therefore, we set the initial dose as 18 mg. According to the clinical recommendation dosage and previous clinical studies of Prolia^® in healthy adults, three groups were set with the dose of 18 mg, 60 mg, and 120 mg, respectively (Mcclung et al., 2006; Bekker et al., 2004; Kumagai et al., 2011; Chen et al., 2018).

Subjects were randomly assigned (3:1) to LY06006 or placebo in each group. Only after tolerance evaluation on day 56 of the lower dose group was completed and safety and tolerance were confirmed, the next dose group could be carried out. All subjects were not allowed to be given calcium and Vitamin D supplements throughout the study period. Such preparations could be used as appropriate only after the occurrence of serum calcium reduction (<2.0 mmol/L) with relevant clinical symptoms.

Screening occurred 30 days before dosing. Eligible subjects received a single abdominal subcutaneous injection of the investigational product or placebo on day 0 (D0) and a sequent 4-days hospitalization for observation. Subjects were discharged on day 4. After that, a follow-up period continued during which subjects returned to the hospital for safety evaluations and blood sampling for pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity assessments. Subjects in 18, 60, and 120 mg groups were followed for 4 months (D140), 6 months (D196), and 9 months (D252), respectively.

Healthy adults aged 18–65 years with a body mass index of 19.0–24.0 kg/m² were eligible. Exclusion criteria contain: 1) suffered or ongoing osteomyelitis, jaw necrosis, odontopathy, or maxillary disease in an active stage needing oral surgery; 2) administration of any medications that might affect bone turnover (e.g., denosumab, bisphosphonates or fluoride within 12 months, estrogens, selective estrogen receptor modulators, parathyroid hormone, systemic glucocorticosteroids, Vitamin D supplements (>1000 IU/day), anabolic steroids, calcitriol or available analogs, or diuretics within 6 months, and inhaled or topical glucocorticoid within 2 weeks); 3)recent bone fracture (within 6 months); 4) had acute or chronic infections; 5) hypocalcemia, hypercalcemia, or abnormal serum albumin-adjusted blood calcium levels; 6) history of drug abuse, smoking or alcohol; 7) pregnant or breastfeeding women.

Safety assessment was based on the monitoring of adverse events (AEs) and serious AEs (SAEs) along with routine clinical and laboratory assessment, including physical examination, vital signs, blood tests, urinalysis, and 12-lead electrocardiograms. Safety evaluations were conducted at baseline and regularly throughout the study.

The frequency, severity, and characterization of AEs were continuously recorded using Medical Dictionary for Regulatory Activities (MedDRA version 22.0). AE intensity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 4.03.

Blood samples for PK evaluation were collected at 1 h preceding the injection, and at 6 h (D1), 24 h (D2), 72 h (D4), 120 h (D6), 168 h (D8), 216 h (D10), 264 h (D12), 312 h (D14), 480 h (D21), 648 h (D28), 984 h (D42), 1320 h (D56), 1656 h (D70), 1992 h (D84), 2328 h (D98), 2664 h (D112), 3336 h (D140) after dosing in 18 mg groups. Blood samples collected at 4008 h (D168), 4680 h (D196) were added in 60 mg group and 4008 h (D168), 4680 h (D196), 5352 h (D224), 6024 h (D252) were added in 120 mg group.

15 min of centrifugation was needed to separate the supernatant serum, and these serum samples were stored at −80°C before analysis. Serum levels of LY06006 were detected by Electrochemiluminescence assay (ECLA). The lower limit of quantification (LLOQ) was 39.06 ng/ml and the quantitative range was 39.06–5000 ng/ml. Values determined lower than the LLOQ were set to zero.

The pharmacokinetic parameters, the area under the plasma drug concentration-time curve (AUC), the time taken to reach the maximum concentration (T_max), peak concentrations of drug in serum (C_max), terminal elimination half-life (t_1/2z), apparent volume of distribution (V_d/F) and the apparent clearance (CL_z/F) were calculated using the plasma concentration-time data collected. The pharmacokinetic properties of LY06006 were assessed based on drug concentrations and basic pharmacokinetic parameters calculated.

Blood samples were collected at pre-dose (within 1 h) and days 14, 28, 42, 56, 84, 140, 196 (only 60 and 120 mg groups), and 252 (only 120 mg groups) after the end of injection to determine anti-drug antibodies (ADAs) and neutralizing antibodies (NAbs).

Validated bridging ECLA was applied for ADA measurement, and negative control response criteria (NRC) was 70.7 electrochemiluminescence unit. The antibody response criteria (ARC) of low concentration positive control and high concentration positive control were 1.59 and 150.80, respectively. The signal-to-noise ratio (S/N) of the screening cut-point was 1.11, and the confirmatory cut point (CCP) of confirmation results was 10.9%.

The plasma concentrations of C-terminal telopeptide of type I collagen (CTX-1), bone-specific alkaline phosphatase (BALP), and parathyroid hormone (PTH) were reported as pharmacodynamic parameters. Pharmacodynamic blood collection points were the same as the pharmacokinetic blood collection time of the corresponding group.

CTX-1, BALP, and PTH were determined by enzyme-linked immunosorbent assays (ELISA). Plasma levels of PTH were detected by ECLA. The detection method was verified by recording its specificity, quantitative limit (LOQ), goodness-of-fit/linearity, accuracy, and precision (intra-assay and inter-assay). The linear range of CTX-1 and BALP are 0.117–2.00 and 7.00–90.0 ng/ml, respectively. All values less than the LLOQ were reported as the value of LLOQ.

Statistical significance was set at p < 0.05 for all tests. Non-compartmental pharmacokinetics analysis for LY06006 was conducted using Phoenix® WinNonlin® 7.0 (Certara, Princeton, NJ, USA) and other statistical analyses were analyzed using SAS (Statistical Analysis System; SAS Institute Inc, Cary, NC, United States, version 9.4).

The full analysis set (FAS) included all subjects who were randomly allocated. The safety set (SS) included all subjects who received randomization and study drug, with safety evaluation data. The PK concentration set (PKCS) included all subjects who received the study drug and had ≥1 quantifiable plasma concentration collected after dosing. The PK parameter set (PKPS) included all subjects who received the study drug dose and had ≥1 valid PK parameter. FAS was used for the analysis of the demographic characteristics. SS was used for safety analysis. PKCS was used for the plasma concentration versus time statistics analysis. And PKPS were used for PK parameters analysis.

The plasma concentration versus time statistics was depicted by semi-log concentration-time curves. The mean, standard deviation, coefficient of variation, quartile, maximum, minimum, and geometric mean of PK parameters of each group were calculated. Differences across treatment groups in PK parameters including C_max, AUC, t_1/2z, V_d/F, and CL_z/F were analyzed using analysis of variance (one-way ANOVA), while T_max was examined by Kruskal–Wallis H test. Meanwhile, all PK parameters except T_max were log-transformed in statistical analysis. A multiple comparisons test with Bonferroni correction was performed when necessary. Dose-exposure proportionality was assessed using a power model. The assumption was that the logarithm of the PK variable (C_max and AUC) is linearly related to the logarithm of dose: ln (PK) = β₀ + β₁ *ln (dose). Dose proportionality was established when the 90% CI for the slope β₁ fell completely within the range 0.882–1.118 (the criterion interval: 1 + [ln (0.80)/ln(r)], 1 + [ln (1.25)/ln(r)], r = the highest dose/the lowest dose) (Smith et al., 2000).

Between 9 January 2018, and 13 May 2019, of 303 individuals assessed for eligibility, 32 were allocated into three groups (Figure 1). 31 participants completed the trial but one participant received 60 mg of study drug did not complete the trial because of pregnancy. Data from the participant who did not complete the study were included in the FAS, SS, PKCS, PKPS, PDOS, PDPS and ADAS. A summary of baseline characteristics is provided in Table 1. Baseline characteristics were comparable between each group.

No deaths or drug-related serious adverse events (SAEs) occurred. No adverse events (AEs) led to withdrawal from the trial, except for 1 pregnancy. Subsequent post hoc sensitivity analyses showed that the overall incidence of AEs was not affected before and after removing the pregnancy event.

259 treatment-emergent adverse events (TEAEs) were reported in 24 participants receiving LY06006 and 29 TEAEs were reported in seven participants who received a placebo. All were mild or moderate in intensity and resolved. The overall incidence and classification of TEAEs are summarized in Table 2.

The most frequent AEs were elevated serum PTH level (83.3%), hypocalcemia (54.2%), and hypophosphatemia (45.8%). AEs that occurred frequently are listed in Table 3.

The serum concentration-time profile of LY06006 is displayed in Figure 2.

LY06006 was rapidly absorbed with detectable concentrations observed 6 h after the dose in all groups. The T_max was relatively long, with a range of 120–480 h for all dose groups and serum LY06006 concentrations decreased slowly 11–13 days after dosing with a long half-life ranging from 168 to 216 h. Concentration profiles over time showed a biphasic decline that a slow initial phase characterized by an approximately linear decline in serum concentration followed by a more rapid elimination phase.

Pharmacokinetics parameters from the noncompartmental analysis of LY06006 (Table 4) showed that with the dose increased, the exposure to LY06006 increased, which was reflected by both AUC_0–t and C_max. In addition, the median t_1/2z of LY06006 was also observed prolonged with the escalation of dose. The mean CL_z/F decreased from 11.42 to 7.41 from 18 to 120 mg. The mean t_1/2z rose from 343.91 to 470.84 from 18 to 60 mg. MRT_0-t was 692.87 h (18 mg), 945.38 h (60 mg) and 1009.91 h (120 mg).

The dose-proportionality analysis (Table 5) found that values of β of C_max (90% CI: 0.947–1.207), AUC_(0-∞) (90% CI: 1.070–1.374), and AUC_0-day140 (90% CI: 1.063–1.361) were partially within the criterion interval 0.882–1.118.

All participants (32, 100%) had negative ADA test results within 1 h before administration. No participants tested positive for anti-drug antibodies after a single subcutaneous injection of LY06006 injection/placebo, and during follow-up.

After a single subcutaneous injection of 18–120 mg LY06006 injection in healthy subjects, the serum CTX-1 concentration showed a consistent trend over time (Figure 3). A rapid decrease of serum CTX-1 was observed, which was able to be detected as early as 6 h after administration with a decline of 79.05% (4.50), 69.62% (13.70), and 63.55% (12.57) from baseline in 18 mg, 60 mg, and 120 mg group, respectively, while the decline in the placebo group was 53.91% (25.88). The CTX-1 decrease was positively correlated with the dose, based on 18 mg LY06006 could maintain the inhibition level to 84 days, 60 mg to 140 days, 120 mg to 224 days, and then slowly increased. Besides, the serum CTX-1 concentration did not recover to the baseline level during the whole observation period. The mean decline (SD) of the 18 mg (D140), 60 mg (D196) and 120 mg (D252) group was 43.37% (34.25), 58.94% (18.19) and 49.60% (14.69).

Serum BALP concentration showed a consistent trend over time after a single subcutaneous injection of 18–120 mg LY06006 in healthy subjects (Figure 3). The mean (SD) maximum BALP inhibition of the 18 mg group was 39.93% (8.21) on day 98 after administration, and was maintained until day 140. The mean (SD) inhibition of BALP in the 60 mg group reached 41.34% (14.85) on day 84 after administration, and the mean (SD) maximum inhibition reached 43.63% (16.36) on day 140 after administration and remained until day 196. BALP inhibition of the 120 mg dose group reached the maximum inhibition level of 31.30% (17.62) on day 140 after administration and could maintain until day 252. BALP concentration did not return to the baseline level during the observation period. The mean (SD) of the 18 mg group was 34.25% (10.57) on day 140 and the 60 mg group was 41.33% (15.15) on day 196. The 120 mg group had a mean (SD) reduction of 21.72% (12.53) from baseline on day 252.

Decreased levels of serum calcium and phosphorus were observed shortly after injection, with the maximum mean decrease observed on day 7 and day 3, respectively (Figures 4, 5). Serum PTH increased with the maximum mean increase observed on day 7 (Figure 6).