Safety, Tolerability, and Pharmacokinetics of the Novel Hepatitis B Virus Capsid Assembly Modulator GST-HG141 in Healthy Chinese Subjects: a First-in-Human Single- and Multiple-Dose Escalation Trial

ABSTRACT

Hepatitis B virus capsid assembly modulators (HBV CAMs) are promising, clinically validated therapeutic agents for the treatment of chronic hepatitis B (CHB). The safety, tolerability, and pharmacokinetic (PK) profiles of GST-HG141, a novel HBV CAM, were evaluated in healthy Chinese volunteers. This phase Ia study included two parts: a double-blinded, randomized, placebo-controlled single-ascending-dose (SAD) (50, 100, 200, 300, 400, or 500 mg) study comprising a food-effect investigation (300 mg) and a multiple-ascending-dose (MAD) (100 or 200 mg twice daily) study. GST-HG141 reached the maximum plasma concentration (C_max) at 1.25 to 3.00 h (median T_max). The exposure exhibited a linear increase, while the mean half-life (t_1/2) ranged from 13.096 h to 22.121 h. The exposure of GST-HG141 (300 mg) was higher after food intake by about 2.4-fold. In the MAD study, steady state was reached at around day 5, and the mean trough steady-state concentrations were 423 and 588 ng/ml for 50- and 100-mg cohorts, respectively. The ratios of GST-HG141 accumulation were <1.5. GST-HG141 was well tolerated in healthy Chinese subjects. The rates of adverse events in the GST-HG141 cohort did not differ from those of the placebo cohort. GST-HG141 was tolerated in healthy Chinese subjects. The safety and PK profiles of GST-HG141 support the further evaluation of its efficacy in individuals with CHB. (This study has been registered in ClinicalTrials.gov under identifier NCT04536337.)

TEXT

The hepatitis B virus (HBV) genome consists of a partially double-stranded DNA molecule that replicates by reverse transcription (1, 2). Despite widespread prophylactic vaccination, approximately 250 million people are chronically infected with HBV worldwide (3). Chronic hepatitis B (CHB) is one of the main causes of liver cirrhosis and hepatocellular carcinoma, which induces considerable liver-related morbidity and mortality, with an estimated 0.6 to 1 million deaths per year (4–6). China has the world’s largest burden of HBV infection, and more than 90 million people harbor persistently high serum HBsAg levels. The World Health Organization has estimated that approximately 6% of pregnant women are HBV carriers in China (https://www.who.int/hepatitis/news-events/hbv-mtct-tenofovir/en/).

Currently, treatment for CHB is mainly based on the third-generation nucleos(t)ide analogue (NUC), which shows viral resistance to some extent. However, lifelong NUC administration is required to avoid viral relapse, such as HBsAg loss, which is observed in less than 10% of patients after 5 years of therapy (7). Another therapeutic option is interferon alpha, which is limited by low response rates (10 to 20%) in CHB patients and a poor tolerability (8). Therefore, safe and novel treatments for CHB are needed to enhance the effectiveness of the current antiviral strategies or to shorten the duration of NUC treatment (9).

Blocking the HBV life cycle at different steps is a potential strategy for drug development, including entry inhibitors, capsid assembly modulators (CAMs), covalently closed circular DNA (cccDNA) disrupters/silencers, translation inhibitors, and secretion inhibitors (10, 11). It has been documented that CAMs can destabilize the HBV core protein assembly and also inhibit the recycling of relaxed circular DNA in the nucleus, leading to reductions in the cccDNA pool (12). Therefore, CAMs may be effective against various HBV genotypes. Several CAMs are in advanced stages of clinical development, including JNJ-379, ABI-H0731, RG-7907, and GLS-4.

GST-HG141 is a novel CAM developed for CHB patients by Fujian Cosunter Pharmaceutical Co., Ltd., that has been shown to inhibit HBV DNA synthesis in HepG2.2.15 cells, with a 50% effective concentration (EC₅₀) of 8.16 ± 3.65 nM. In addition, GST-HG141 retained potent antiviral activity against both HBV genotypes A to D and NUC-resistant mutants (entecavir and lamivudine). In a biochemical quenching assay, GST-HG141 accelerated HBV core protein assembly with an EC₅₀ of 0.93 ± 0.11 μM, which is lower than that of GLS4 (3.34 ± 0.47 μM). In an adeno-associated virus/HBV model, GST-HG141 (15 to 150 mg/kg of body weight) demonstrated a robust dose-dependent reduction of HBV DNA in the serum and liver following 28 days of treatment (unpublished data). Moreover, GST-HG141 was determined to be well tolerated in rat, mouse, and dog studies (unpublished data).

Here, a first-in-human trial was performed to assess the safety, tolerability, and pharmacokinetic (PK) profile of GST-HG141 in healthy Chinese subjects. Furthermore, the results will contribute to the dosage optimization for subsequent phase Ib and phase II studies in patients with CHB.

RESULTS

Volunteers.

A total of 92 volunteers from 499 screened subjects were enrolled in this study and completed the safety assessment. One volunteer withdrew from the MAD study due to an adverse event (AE), and 91 volunteers ultimately completed the entire study. The demographics of the volunteers were generally comparable in terms of age, weight, and body mass index (BMI) across the dose cohorts, as summarized in Table 1.

TABLE 1.

Demographics of the volunteers in this study^a

Cohort	Age (yr)	Gender		Ht (cm)	Wt (kg)	Body mass index (kg/m2)
		Male	Female
SAD
Placebo (n = 12)	38.8 (10.0)	6 (50.0)	6 (50.0)	161.2 (6.5)	58.5 (8.4)	22.3 (2.2)
50 mg (n = 8)	36.1 (8.6)	4 (50.0)	4 (50.0)	164.2 (8.1)	64.5 (8.4)	24.0 (2.6)
100 mg (n = 8)	34 (9.9)	4 (50.0)	4 (50.0)	166.9 (5.8)	63.5 (7.3)	22.9 (2.1)
200 mg (n = 8)	36.4 (7.7)	4 (50.0)	4 (50.0)	159.2 (6.1)	59.2 (9.4)	23.3 (2.5)
300 mg (A) (n = 8)	39.3 (6.4)	4 (50.0)	4 (50.0)	163.5 (3.7)	63.1 (6.2)	23.6 (2.7)
300 mg (B) (n = 8)	38.3 (6.7)	4 (50.0)	4 (50.0)	160.0 (6.9)	58.0 (6.2)	22.8 (3.0)
400 mg (n = 8)	35.1 (13.4)	4 (50.0)	4 (50.0)	163.7 (5.6)	61.4 (9.7)	22.8 (2.6)
500 mg (n = 8)	37.1 (8.1)	4 (50.0)	4 (50.0)	165.0 (11.3)	64.7 (10.0)	23.9 (2.5)
MAD
Placebo (n = 4)	40.0 (9.66)	2 (50.0%)	2 (50.0%)	163.8 (3.7)	61.9 (10.2)	23.3 (3.0)
100 mg (n = 10)	34.7 (6.83)	5 (50.0%)	5 (50.0%)	162.9 (5.8)	59.7 (7.4)	22.4 (2.9)
200 mg (n = 10)	32.1 (8.74)	5 (50.0%)	5 (50.0%)	163.40 (8.8)	64.6 (7.5)	24.4 (2.5)

^aData are number (%) or mean (SD). Subjects of cohort A in the 300-mg cohort were under a fasting state in session 1 and under a fed state in session 2. Cohort B was the opposite.

Tolerability and safety.

GST-HG141 presented good tolerability in all dose cohorts. No serious AEs were observed in any of the subjects. The safety results are summarized in Table 2. In the SAD study, 24 AEs were reported by 16 subjects (23.5%). Among these, 20 AEs occurred in the GST-HG141 cohort (12 subjects, 21.4%), while the others were observed in the placebo cohort (4 subjects, 33.3%). The most frequent AEs were urinary tract infections, which were observed from urinalysis abnormalities without any symptoms. All of them were recovered in 1 to 5 days without any intervention. In addition, seven AEs were determined to be adverse drug reactions (ADRs). One of the AEs was an exercise-induced creatine kinase (CK) elevation, which was classified as grade 4 and recovered in 10 days without any intervention, and it was not assessed as ADR. The other AEs were classified as grade 1 or 2. The most frequent ADRs included increased serum creatinine level (3.6%), decreased neutrophil count (1.8%), decreased white blood cell count (1.8%), positive red blood cell urine test (1.8%), increased serum bilirubin level (1.8%), and hypertriglyceridemia (1.8%). No ADRs were found to be dose dependent. The subjects with increased serum creatinine level of grade 1 had a baseline near the upper limit of the normal range and recovered in several days. The other five ADRs were recovered in 2 to 9 days without any intervention.

TABLE 2.

Summary of adverse events after the administration of GST-HG141, according to each dose cohort (n = 92)^a

Preferred term	SAD									MAD
	50 mg (N = 8), n (%)	100 mg (N = 8), n (%)	200 mg (N = 8), n (%)	300 mg, fasted (N = 16), n (%)	300 mg, fed (N = 16), n (%)	300 mg, overall (N = 16), n (%)	400 mg (N = 8), n (%)	500 mg (N = 8), n (%)	Placebo (N = 12), n (%)	100 mg BID (N = 10), n (%)	200 mg BID (N = 10), n (%)	Placebo (N = 4), n (%)
Over with TEAE	0	2 (25.0)	0	0	2 (12.5)	2 (12.5)	6 (75.0)	2 (25.0)	4 (33.3)	2 (20.0)	3 (30.0)	2 (50.0)
Serum creatinine increased	0	0	0	0	0	0	2 (25.0)	0	0	0	1 (10.0)	0
ALT increased	0	0	0	0	0	0	1 (12.5)	0	0	0	0	0
AST increased	0	0	0	0	0	0	1 (12.5)	0	0	1 (10.0)	0	0
CK increased	0	0	0	0	0	0	1 (12.5)	0	0	1 (10.0)	1 (10.0)	0
CK-MB increased	0	0	0	0	0	0	0	0	0	1 (10.0)	0	0
Serum bilirubin increased	0	0	0	0	0	0	1 (12.5)	0	0	0	0	1 (25.0)
Hypertriglyceridemia	0	0	0	0	0	0	0	1 (12.5)	0	0	1 (10.0)	0
Hypoalbuminemia	0	0	0	0	0	0	0	0	2 (16.7)	0	0	0
Hyperuricaemia	0	0	0	0	0	0	0	0	0	1 (10.0)	0	0
Neutrophil count increased	0	0	0	0	1 (6.3)	1 (6.3)	0	0	0	0	0	0
Neutrophil count decreased	0	1 (12.5)	0	0	0	0	0	0	0	0	1 (10.0)	0
Lymphocyte count decreased	0	0	0	0	1 (6.3)	1 (6.3)	0	0	0	0	0	0
WBC count increased	0	0	0	0	1 (6.3)	1 (6.3)	0	0	0	0	0	0
WBC count decreased	0	1 (12.5)	0	0	0	0	0	0	0	0	1 (10.0)	0
Urinary RBC positive	0	1 (12.5)	0	0	0	0	0	0	0	0	0	0
Urinary sediments positive	0	0	0	0	0	0	0	0	1 (8.3)	0	0	0
Urinary tract infection	0	0	0	0	0	0	3 (37.5)	1 (12.5)	1 (8.3)	0	0	1 (25.0)
Upper respiratory infection	0	0	0	0	0	0	1 (12.5)	0	0	0	0	0
Cough	0	0	0	0	1 (6.3)	1 (6.3)	0	0	0	0	0	0
Colporrhagia	0	0	0	0	0	0	0	0	0	1 (10.0)	0	0
Myalgia	0	0	0	0	0	0	0	0	0	1 (10.0)	0	0

^aN, number of subjects analyzed; n, number of subjects. TEAE, treatment-emergent AE; WBC, white blood cell; RBC, red blood cell.

Overall, the incidence rates of AEs for the GST-HG141 and placebo cohorts in the MAD study were 25.0% and 50%, respectively. One event of an increased level of CK reported by one subject on day 6 in the 100-mg MAD cohort was classified as grade 4 and considered to be related to the drug. The same subject also reported an increased level of creatine kinase isoenzymes (CK-MB) and increased levels of AST, myalgia, and hyperuricemia. This subject discontinued study and recovered in 4 days without any intervention. Another event of an increased level of CK (grade 2) was reported by one subject in the 200-mg MAD cohort who recovered in 6 days without any intervention. All of the other AEs were reported by only one subject. No clinically significant changes were observed in vital signs or electrocardiography.

PK of GST-HG141. (i) SAD and food-effect study.

The mean GST-HG141 plasma concentration–time profiles and PK parameters after a single dose of GST-HG141 treatment are shown in Fig. 1 (Prism 5 software; GraphPad Software, San Diego, CA, USA) and Table 3. GST-HG141 was absorbed rapidly after a single administration of GST-HG14 in the dose range of 50 to 500 mg, with a median T_max between 1.25 h and 3.00 h. The mean terminal phase elimination half-life (t_1/2) of GST-HG141 ranged from 13.096 h to 21.927 h across all dose cohorts. The C_max of GST-HG141 increased from 124 ng/ml to 990 ng/ml in the dose range of 50 to 500 mg. The C_max and area under the concentration-time curve from 0 h to infinity (AUC_0–∞) values increased in a dose-dependent manner. However, the estimated values of the power model exponent β and 95% confidence intervals (CIs) were 0.37 (0.28, 0.46) for C_max and 0.52 (0.38, 0.66) for AUC_0–∞. The increases in the C_max and the AUC_0–∞ values were not consistent with the dose increase, suggesting that GST-HG141 saturation was reached. The PK parameters were not significantly different between males and females, except for the volume of distribution (V_Z/F) (P = 0.0073) in the 400-mg cohort and C_max (P = 0.0260) in the 500-mg cohort.

FIG 1.

Pharmacokinetic profiles of GST-HG141 in healthy subjects in the single-ascending-dose (SAD) study.

TABLE 3.

Plasma pharmacokinetic parameters in the single-ascending-dose study^a

Dose (mg)	N	AUC0–t (h·ng/ml)	AUC0–∞ (h·ng/ml)	Cmax (ng/ml)	Tmax (h)	t1/2 (h)	CL/F (ml/h)	VZ/F (ml)
50	8	1,620 ± 540	1,710 ± 557	124.4258 ± 27.8143	3.00 (1.5–6)	13.096 ± 9.6354	32,000 ± 10,100	534,000 ± 217,000
100	8b	2,400 ± 1,080	2,310 ± 1,050	146.8452 ± 54.1367	1.375 (0.50–3.00)	16.247 ± 10.3146	46,200 ± 18,600	1,230,000 ± 722,000
200	8	4,160 ± 1,590	4,370 ± 1,700	190.4728 ± 38.8466	2.00 (1–4)	17.146 ± 10.7704	52,800 ± 21,600	1,160,000 ± 501,000
300 (fasted)	16	4,570 ± 1,380	4,720 ± 1,560	244.4718 ± 52.2741	1.75 (1.00, 6.00)	21.927 ± 18.2576	69,200 ± 28,700	2,160,000 ± 1,430,000
300 (fed)	16	10,400 ± 2,020	10,600 ± 2,280	968.2026 ± 143.8727	3.00 (1.00, 6.00)	14.461 ± 13.4291	28,900 ± 6,050	790,000 ± 979,000
400	8	4,620 ± 1,870	4,780 ± 2,000	257.4554 ± 61.3238	1.25 (0.75–3)	19.399 ± 12.0606	96,300 ± 35,200	2,540,000 ± 1,610,000
500	8	10,700 ± 3,160	10,800 ± 3,070	990.4379 ± 186.5262	2.50 (2–4)	18.246 ± 14.6804	49,400 ± 12,300	1,340,000 ± 1,290,000

^aData are expressed as mean ± SD unless otherwise specified. T_max is expressed as median (range).

^bFor subjects with AUC_{_%Extrap} of >20%, AUC_0–∞, t_1/2, and AUC_{_%Extrap} were not descriptively analyzed. PK parameters, including AUC_0–∞, t_1/2, and AUC_{_%Extrap}, were descriptively analyzed for 7 subjects in the 100-mg group.

The mean GST-HG141 plasma concentration-time profiles in the food-effect study are presented in Fig. 2. T_max was delayed and the C_max and AUC_0–∞ values of GST-HG141 increased, with geometric least-squares method ratios (90% CI) of 401% (351 to 457%) and 233% (199 to 273%), respectively, under the fed condition. The bioavailability was quite high in the fed state compared to the fasted state, indicating that postprandial dosing could improve the bioavailability of GST-HG141. GST-HG141 excretion was 2.772% and 16.728% in the urine and fecal samples from 0 h to 120 h postdose, respectively.

FIG 2.

Pharmacokinetic profiles of GST-HG141 (300 mg) in healthy subjects in the food-effect study.

(ii) MAD study.

The mean GST-HG141 concentration-time profiles after multiple doses are presented in Fig. 3. The plasma PK parameters of GST-HG141 on days 1 and 7 are shown in Table 4. The median T_max values were 2.5 to 3 h and 2 to 2.5 h on days 1 and 7, respectively. The mean steady-state half-life (t_1/2,ss) of GST-HG141 ranged from 11.820 h to 18.406 h, while the accumulation ratio (R_acc) values were <1.5, indicating that there was no apparent plasma accumulation of GST-HG141 on day 7. Steady-state conditions were reached on day 5. The mean steady-state trough concentrations of GST-HG141 were 135.5 and 200.2 ng/ml in the 50-mg and 100-mg cohorts, respectively. The trough concentrations collected before the first administration on days 4 to 7 in 100-mg and 200-mg twice daily (BID) cohorts were 193.3 to 229.5 and 287.3 to 414.0 ng/ml, respectively. However, the trough concentrations collected before the second administration on day 6 were 145.5 and 159.0 ng/ml, which were lower than the above-described trough concentrations.

FIG 3.

Pharmacokinetic profiles of GST-HG141 in healthy subjects in the multiple-ascending-dose (MAD) study.

TABLE 4.

Plasma pharmacokinetic parameters in the multiple-ascending-dose study

Treatment	Day 1						Day 7
	t1/2 (h)	Tmax (h)	Cmax (ng/ml)	AUC0–t (h·ng/ml)	CL/F (ml/h)	VZ/F (ml)	t1/2,ss (h)	Tmax,ss (h)	Cmax,ss (ng/ml)	AUC0–t,ss (h·ng/ml)	AUC0–∞,ss (h·ng/ml)	CL/Fss (ml/h)	Vss (ml)	DF (100%)	RAUC (100%)
100 mg BID (n = 10)	3.798 ± 0.6532	2.5 (1.25–6)	470.5102 ± 111.3529	2850 ± 684	29900 ± 9400	181000 ± 35800	11.82 ± 5.8231	2 (1–4)	600.2593 ± 128.8865	6040 ± 1460	6100 ± 1510	26500 ± 5020	461000 ± 249000	143.318 ± 28.0843	145.62 ± 32.803
200 mg BID (n = 10)	3.8 ± 0.2064	3 (1.5–4)	572.9516 ± 125.5634	3820 ± 1040	43100 ± 12500	300000 ± 104000	18.406 ± 10.2862	2.5 (1.5–3.02)	713.7377 ± 152.9935	7580 ± 2100	7740 ± 2220	42800 ± 9450	1070000 ± 504000	128.060 ± 33.3666	132.95 ± 32.712

DISCUSSION

This is the first report on the safety, tolerability, and PK characteristics of GST-HG141 in healthy Chinese subjects. The results of the safety assessment indicated that single or multiple doses of GST-HG141 (≤500 mg/day) were well tolerated in healthy subjects under both the fed and fasted conditions. No serious AEs or deaths were observed. The incidence or severity of all AEs was found to be uncorrelated with the dose. The rates of AEs in both the SAD and MAD studies did not differ between the GST-HG141 and placebo cohorts. Among the ADRs, increased serum creatinine levels were observed in three subjects, and increased levels of CK were found in two subjects from the GST-HG141 cohort. Among these, only one subject experiencing a creatinine increase in the 200-mg BID cohort had higher exposures than the other subjects in the same cohort. The above-described ADRs were not GST-HG141 exposure related and not observed in preclinical toxicology studies. All of the subjects recovered within several days. These two AEs should also be monitored in further clinical trials.

The PK results of the SAD study confirmed that GST-HG141 was rapidly absorbed, showing a half-life fit for once daily or BID. Increasing the GST-HG141 dose administered to subjects did not result in the corresponding dose-proportional exposure increase, indicating exposure saturation. Single doses of GST-HG141 had moderate variability for AUC_0–∞ and C_max.

In the food-effect study, the absorption of GST-HG141 was delayed, and the t_1/2 values were decreased with a reduced elimination under the fed state. Drug absorption can be altered by food intake due to various changes in gastrointestinal physiology, such as the gastric emptying time, pH value, and bile salt concentration (13). Delayed gastric emptying could improve the absorption of GST-HG141, which may be due to its low solubility and permeability. In addition, the improved bioavailability might result from the increased solubility of GST-HG141 in the intestine and inhibition of efflux transporters (14). The C_max and AUC_0–∞ values increased by 4- and 2.33-fold, respectively, after a high-fat meal. Consequently, the remaining cohorts were studied under postprandial conditions (a moderate-fat meal) to elevate the exposures. The C_max and AUC_0–∞ values in the MAD study increased by 3- and 2-fold, respectively, after a moderate-fat meal from the robust calculation based on the results of the SAD study. The exposures might also be affected by the fat content in the meals.

Steady state was considered to be reached on day 5 in the MAD study, which was consistent with a t_1/2 value ranging from 13.096 h to 21.927 h. BID was chosen to elevate the exposure and minimum plasma concentration (C_min), because we found that the C_12h value decreased by about 50% to 70% relative to C_max in the SAD study. Systemic exposure to GST-HG141 was slightly higher on day 7 than day 1, indicating no significant accumulation over the 7-day dosing period. In addition, GST-HG141 might exhibit chronopharmacokinetics, as the trough concentrations at steady state were higher in the morning than in the evening on day 6 or 7. Circadian oscillations affect drug absorption, distribution, metabolism, and excretion (15). As mentioned above, GST-HG141 absorption was strongly affected by the gastric emptying time, which is influenced by the circadian rhythm. The gastric emptying in the waking state is much slower in the evening (20:00) than in the morning (08:00), which may affect the speed of drug absorption (16). Circadian variations cause the estimated hepatic blood flow in humans to be the highest at 08:00, which may also affect metabolism and hepatobiliary excretion (17). The specific chronopharmacokinetic characteristics of GST-HG141 and its mechanism should be investigated in future trials.

The main route of GST-HG141 elimination was through the bile, which is excreted more during the daytime. However, the metabolites of GST-HG141 require further investigation.

Furthermore, the efficacy of GST-HG141 in patients with CHB requires further clinical trials. The phase Ib study of GST-HG141 in patients is currently being planned. Targeted concentration for efficacy is about 85 ng/ml, which is about 5-fold above the EC₅₀. The mean steady-state trough concentration for the 100-mg BID cohort (139 ng/ml) reached the target concentration. As the variability of trough concentrations was moderate, we just compared mean values to the target efficacy concentration. It was estimated that trough concentrations for 50 mg BID could reach the target, as exposure saturation existed. Taking safety issues into account, 25 mg BID should be tested first in patients. Therefore, considering the safety and efficacy issues of GST-HG141, postprandial doses of 25 mg BID, 50 mg BID, and 100 mg BID will be chosen in the phase Ib study.

Conclusions.

GST-HG141 is safe and well tolerated in healthy Chinese subjects. The preclinical pharmacodynamics, safety profiles, and PK characteristics of GST-HG141 support the further evaluation of its safety and efficacy in CHB patients. Based on the data described here, postprandial doses of 25 mg BID, 50 mg BID, and 100 mg BID will be used in a future clinical trial.

MATERIALS AND METHODS

Healthy subjects.

Healthy Chinese subjects aged 18 to 55 years old, with a body mass index of 18 to 28 kg/m², were recruited for this study. The qualification of the volunteers was determined after a review of the following items: medical history, physical examination, 12-lead electrocardiography, X-ray, color Doppler ultrasound of the abdomen and heart, blood chemistry, hematology, urinalysis, coagulation function, serum pregnancy test, hepatitis B and C tests, and HIV diagnostic profiles. Volunteers were excluded from this study if they showed significantly abnormal signs or symptoms, had a history of alcohol or drug abuse, or were smokers. Other exclusion criteria included the administration of any drug within the previous 14 days, a positive drug screening, or ingestion of any food or beverage containing alcohol, caffeine, or xanthine within 24 h of the initial dosing. All subjects were required to practice contraception and have no plan to conceive during the next 6 months.

GST-HG141 was approved by the Research Ethics Board of the First Hospital of Jilin University, Jilin, China. This phase Ia study was performed in healthy Chinese subjects between May 2020 and December 2020 at the Phase I Clinical Trial Unit of the First Hospital of Jilin University, Changchun, Jilin, China. This study was conducted in accordance with the Declaration of Helsinki and followed the principles of Good Clinical Practice. All recruited volunteers provided written informed consent.

Study design and drug administration.

This double-blinded, randomized, and placebo-controlled phase Ia trial was comprised of two parts. Part A was a single-ascending-dose (SAD) study with six cohorts of GST-HG141 administration (50, 100, 200, 300, 400, and 500 mg; n = 10 per dose cohort). In addition, eight extra subjects were enrolled in the 300-mg cohort to assess the food effect of GST-HG141 by a two-period crossover food-effect study with a 10-day washout period. Subjects in the 300-mg cohort were dosed 30 min after a high-fat meal in one period. Subjects in the 500-mg cohort were dosed 30 min after a standardized medium-fat meal, while the other cohorts in the SAD study were dosed under the fasted state. The subjects in the SAD study were randomly assigned to receive GST-HG141 or the placebo, with an allocation ratio of 4:1. Randomization lists for this study were created using SAS V9.4 software (SAS Institute, Cary, NC, USA). GST-HG141 treatment was initiated with the lowest dose group. A maximum recommended starting dose of 50 mg was calculated based on the standard 10-fold safety margin from the no-observed-adverse-effect-level exposure study in rats and the lowest effective dose observed in mice, according to each regulatory guideline (18). Tolerance was assessed based on the medical review of safety results at days 2 and 4. The next dose level was determined only when the principal investigator and the sponsor had reviewed and guaranteed the safety data.

Part B was a multiple-ascending-dose (MAD) study (100 and 200 mg; n = 12 per cohort; twice-daily dosing [BID] for 7 days). Subjects were dosed 30 min after a moderate-fat meal and randomly assigned to receive GST-HG141 or the placebo, with an allocation ratio of 5:1. GST-HG141 and the placebo were provided in identical packaging and appearance by the sponsor (Fujian Cosunter Pharmaceutical Co., Ltd.), ensuring that the group allocation was hidden to both the participants and investigators. The doses were chosen according to the PK and safety results from the SAD study.

Blood, urine, and feces sampling and drug analysis.

In the SAD study, blood samples were collected in chilled collection tubes containing the anticoagulant K₂-ethylenediaminetetraacetic acid at baseline (within 30 min prior to dosing) and 0.25 h, 0.5 h, 0.75 h, 1 h, 1.25 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h after dosing. For the food-effect cohort, the time points of blood sampling on days 1 and 11 were the same as those of the SAD cohorts. In the MAD study, the blood samples were collected on days 1 and 7 at the time points described above. The trough concentration samples on days 4 and 5 were collected only before the first administration. In addition, the trough concentration sampling on day 6 was conducted before each dosing. On day 7, PK blood samples were also obtained at 24 h, 48 h, 72 h, 96 h, and 120 h postdose. The samples were centrifuged at 2,000 × g and 2 to 8°C for 10 min to isolate the plasma. For the food-effect study, urine samples were collected at 0 h, 0 to 6 h, 6 to 12 h, 12 to 24 h, 24 to 48 h, 48 to 72 h, 72 to 96 h, and 96 to 120 h after dosing. Fecal samples were collected at 0 to 120 h postdose. All of the samples were maintained at −80°C for subsequent analysis.

A validated liquid chromatography-tandem mass spectrometry method was used to quantify the concentrations of GST-HG141 in human plasma, urine, and fecal samples (unpublished data). The linear range was 1 to 2,000 ng/ml. The precision rate was ≤8.8%, with accuracy ranging from −7.6% to 3.7%.

PK analysis.

The PK parameters were calculated with WinNonlin 8.0 (Certara, Princeton, NJ, USA) using noncompartmental analysis. For the SAD study, the PK parameters of GST-HG141, including C_max, T_max, AUC_0–t, AUC_0–∞, t_1/2, CL/F, V_Z/F, and renal clearance (CL_R), were measured. The amount (A_e0–120h) and apparent fraction (F_e0–120h) of GST-HG141 recovered in the urine and fecal samples were calculated. For the MAD study, C_min at steady state (C_min,ss), C_avg,ss, AUC_ss, drug fluctuation coefficient (DF), and accumulation ratio (R_acc) were also calculated. The R_acc at steady state after multiple dosing was calculated as follows (AUC_0–24 or C_max on day 7)/(AUC_0–24 or C_max on day 1). Attainment of the steady state was determined by visual inspection of trough concentrations (Cτ) on days 4, 5, 6, and 7.

Evaluation of tolerability and safety.

The safety and tolerability of GST-HG141 in all treated subjects were assessed. Safety was evaluated by the presence of adverse events (AEs), clinical laboratory tests (hematology, urinalysis, and blood chemistry), vital signs, physical examinations, and 12-lead electrocardiography. All laboratory abnormalities and AEs were described and graded according to National Cancer Institute (NCI) Common Toxicity Criteria for AEs, version 5.0.

Statistical analysis.

Statistical analyses were performed by SAS 9.4 software (SAS Institute, Inc., Cary, NC). Descriptive statistics were applied to the demographic parameters and safety data. Formal comparisons between dose cohorts were not made in this study. The GST-HG141 plasma concentrations along with the PK parameters were summarized and compared among the different dose cohorts using descriptive statistics. To examine dose proportionality for C_max and AUC, a power model was fit to the data (19).

In the food-effect study, C_max and AUC_0–∞ were compared between the fasted and fed states using a mixed-effects model for the log-transformed PK values with a treatment sequence. Period and treatment were set as fixed effects, and subjects nested in a sequence was set as a random effect. The 90% confidence intervals (CIs) of the geometric mean ratios for the variables C_max and AUC_0–∞ were calculated.