GLS4 is a novel inhibitor of the hepatitis B virus (HBV) capsid assembly with inhibitory activities against nucleot(s)ide-resistant HBV strains. This study investigated the pharmacokinetics, safety, and tolerability of GLS4 and the effects of food and ritonavir in healthy adults. GLS4 was administered in a single-ascending-dose study over 1 to 240 mg and multiple-ascending-dose study that ranged from 30 mg once daily to 180 mg three times daily.
KEYWORDS: GLS4, HBV, safety, pharmacokinetics, CYP 3A4, ritonavir
ABSTRACT
GLS4 is a novel inhibitor of the hepatitis B virus (HBV) capsid assembly with inhibitory activities against nucleot(s)ide-resistant HBV strains. This study investigated the pharmacokinetics, safety, and tolerability of GLS4 and the effects of food and ritonavir in healthy adults. GLS4 was administered in a single-ascending-dose study over 1 to 240 mg and multiple-ascending-dose study that ranged from 30 mg once daily to 180 mg three times daily. The drug interaction study included sequential design (day 1 for 120 mg GLS4 alone, day 5 for 100 mg ritonavir alone, followed by 9 days of both drugs) and a placebo control (9 days of both 240 mg GLS4 and 100 mg ritonavir). The results showed that the steady-state trough concentration of multiple dosing of GLS4 alone was significantly lower than the 90% effective concentration of 55.7 ng/ml, even with increasing dosing frequency and dosage. An initial dose of 100 mg ritonavir significantly boosted plasma concentration at 24 h of 120 mg GLS4 from 2.40 to 49.8 ng/ml (geometric mean ratio, 20.7; 90% confidence interval, 17.0 to 25.3), while a milder effect was observed on the area under the curve from 0 to 24 h, with a 7.42-fold increase, and on the maximum concentration, with a 4.82-fold increase. The pharmacokinetics change in GLS4 persisted after 9 days of chronic dosing, with a trough concentration of 182 ng/ml. Both single and multiple doses of GLS4 up to 240 mg with or without ritonavir were well tolerated. These results support the investigation of a novel HBV treatment regimen containing GLS4 with 100 mg ritonavir added solely to enhance GLS4 concentrations in plasma. (This study was registered at the China Platform for Registry and Publicity of Drug Clinical Trials [http://www.chinadrugtrials.org.cn] under numbers CTR20132137 and CTR20150230.)
INTRODUCTION
Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV), which is a major and global health problem. It can cause chronic infection and is a major cause of morbidity and mortality with a disproportionate impact on Asia and Africa (1). According to the World Health Organization (WHO), approximately 2 billion people have been infected with HBV, 350 million of whom suffer from chronic HBV infection (CHB) (2). The current treatment for HBV infection involves two classes of agents: oral nucleoside- or nucleotide-based reverse transcriptase inhibitors (lamivudine, adefovir, telbivudine, entecavir, and tenofovir) and interferons (IFNs) (3–5).
The clinical use of IFN and peg-IFN is limited by low response rates (20 to 30%) among CHB patients and side effects (6). Some nucleot(s)ide inhibitors are prone to drug resistance and virologic breakthrough due to viral mutation (7). To improve the treatment of HBV infection and reduce drug resistance, novel agents with different therapeutic targets besides a viral polymerase are required.
Nucleocapsid formation and pregenomic RNA packaging are critical steps in the viral life cycle that may offer a better drug target against various genotypes of HBV polymerase-resistant mutants (8–11). Bay41-4109 has been reported to disrupt or misdirect the assembly of the HBV capsid, which leads to capsid depletion and then termination of viral replication (12). However, it was also found to be hepatotoxic at high doses in rats, and follow-up studies of this compound were suspended (13). A new heteroaryldihydropyrimidine compound, 6[R,S]-ethyl-6-(2-bromo-4-fluorophenyl)-4-(morpholinomethyl)-2-(thiazol-2-yl)-1,6-dihydropyrimidine-5-carboxylate mesilate (GLS4), was developed from Bay41-4109 (14, 15), and the development progress has been published (16). GLS4 demonstrated potent inhibitory activities in an HBV HepG2.2.15 cell assay with the 50% effective concentration (EC50) value of 1 nM, and it also exhibited high potency against various polymerase drug-resistant mutants, including lamivudine-resistant HBV strains, telbivudine-resistant HBV strains, and entecavir-resistant HBV strains with EC50 values in the range of 10 to 20 nM (16). Preclinical pharmacokinetic profiles of GLS4 showed it was absorbed rapidly, with an oral bioavailability of approximately 5 to 15%, depending on species and dose-proportional plasma concentrations (14). An in vitro recombinant enzyme test showed that CYP2C19, CYP3A4, and CYP3A5 are involved in GLS4 metabolism, and the main metabolic pathways are dealkylation, hydrolyzation, dehydrogenation, and oxidation. The types of metabolites in dog liver microsomes were the most similar to those in humans (17). All the metabolites show no activities against HBV in vitro assay. GLS4 is predominantly metabolized via the liver and primarily excreted as an unchanged drug, along with its major metabolites M2 and M4 via feces and M2 and M6 via mouse urine. The cumulative dose of radioactive excretion in urine and feces accounts for 21.5 and 62.1% of the dose, respectively. Safety evaluations, including acute toxicity and repeated toxicity studies, indicate that GLS4 is safe enough to support clinical experiments in humans (16).
A first-in-human trial was performed to evaluate the safety, tolerability, and pharmacokinetics profile of single GLS4 (including food effect) and multiple GLS4 administration once or three times daily in healthy adult volunteers. The results showed that the anticipated concentration required for effective antiviral activity could not be achieved using GLS4 alone. An extra ritonavir-boosting study was therefore conducted to evaluate the effect of ritonavir on the pharmacokinetics and safety of GLS4 in healthy adult subjects.
RESULTS
Demographic characters.
Of 170 enrolled subjects, 167 completed the study. One subject in part 1B was lost to follow-up before period 2. Another subject in part 2B withdrew due to an adverse event (AE). The third subject in part 3A discontinued due to a personal matter. All subjects with evaluable data were included in the safety analysis. Data from 126 subjects were included in the pharmacokinetic analyzes (35 subjects who received a placebo and 3 subjects who discontinued the study were not included; 6 subjects on the 1-mg-dose group in part 1A were not included due to low blood concentrations). Baseline demographic characteristics were generally comparable with age, weight, and body mass index (BMI) across dose groups and substudies (Table 1).
TABLE 1.
Demographic characters of subjects
| Part and treatment group | No. of subjects | No. male (%) | Mean ± SD |
||
|---|---|---|---|---|---|
| Age (yr) | wt (kg) | BMI (kg/m2) | |||
| Part 1A | |||||
| GLS4 | 54 | 29 (53.7) | 26.87 ± 5.53 | 58.76 ± 5.93 | 21.84 ± 1.40 |
| Placebo | 18 | 9 (50.0) | 26.22 ± 4.88 | 58.53 ± 6.43 | 21.74 ± 1.28 |
| Part 1B | |||||
| GLS4 | 16 | 16 (100) | 25.63 ± 4.21 | 62.69 ± 6.59 | 22.09 ± 1.35 |
| Part 2A | |||||
| GLS4 | 27 | 15 (55.6) | 25.56 ± 5.44 | 60.63 ± 6.80 | 22.04 ± 1.58 |
| Placebo | 9 | 6 (66.7) | 29.11 ± 6.49 | 61.94 ± 5.26 | 21.98 ± 1.36 |
| Part 2B | |||||
| GLS4 | 24 | 14 (58.3) | 26.4 ± 4.8 | 61.1 ± 7.0 | 22.0 ± 1.7 |
| Placebo | 6 | 3 (50.0) | 23.8 ± 3.2 | 59.3 ± 6.2 | 22.0 ± 1.2 |
| Part 3A | |||||
| GLS4 | 8 | 4 (50.0) | 26.0 ± 5.3 | 59.3 ± 4.2 | 22.0 ± 2.2 |
| Part 3B | |||||
| GLS4 | 6 | 3 (50.0) | 27.3 ± 5.3 | 61.7 ± 7.6 | 22.6 ± 1.3 |
| Placebo | 2 | 2 (100) | 28.5 ± 0.7 | 68.6 ± 7.0 | 21.9 ± 0.4 |
Pharmacokinetics.
(i) Part 1: single-ascending-dose and food effect study. After a single oral dose of GLS4 capsules from 2.5 to 240 mg, GLS4 was absorbed rapidly with a median time to maximum concentration (Tmax) of 0.416 to 1.00 h. The drug was eliminated quickly with plasma concentrations in all dose groups decreasing to <3% of peak concentrations 24 h after administration. Subsequently, a slow and long second-elimination phase was observed in the medium to high dosage cohort. The half life (t1/2) of GLS4 varied from 1.00 to 2.81 h for low doses to 54.1 to 58.7 h for the high-dose group. Mean plasma concentration-time profiles and parameters are presented in Fig. 1 and Table 2, respectively. The maximum concentration (Cmax) and the area under the curve from 0 to 168 h (AUC0–168) increased with dose. However, based on the power model assessment, the increases in the Cmax (slope = 1.18, 90% confidence interval [CI] = 1.08 to 1.28) and the AUC0–168 (slope = 1.41, 90% CI = 1.33 to 1.49) were slightly higher than that of the dose increase over the dose range of 2.5 to 240 mg (Fig. 2).
FIG 1.
Mean plasma concentration-time profile in semi-log scale of GLS4 in SAD study (part 1A).
TABLE 2.
Pharmacokinetic parameters of part 1A (SAD study) and part 1B (food effect study)
| Part (study) and dose (mg) | No. | Mean (%CV)a
|
||||||
|---|---|---|---|---|---|---|---|---|
|
Cmax (ng/ml) |
Tmax (h) |
AUC0–tb
(h*ng/ml) |
AUC0–∞ (h*ng/ml) |
t1/2c (h) | CL/F (L/h) | C24 (ng/ml) | ||
| Part 1A (SAD) | ||||||||
| 2.5 | 6 | 4.35 (49.6) | 0.416 (0.333–0.750) | 4.07 (57.9) | 4.45 (56.1) | 1.00 (46.9) | 562 (80.0) | – |
| 7.5 | 6 | 8.71 (62.2) | 0.500 (0.333–1.00) | 15.9 (30.9) | 18.8 (53.5) | 2.81 (162.9) | 398 (53.8) | – |
| 15 | 6 | 40.5 (44.6) | 0.500 (0.333–0.750) | 59.4 (45.6) | 76.0 (88.7) | 21.3 (170.6) | 198 (66.6) | 0.345 (31.1) |
| 30 | 6 | 64.4 (52.8) | 0.500 (0.333–0.750) | 115 (57.5) | 129 (60.9) | 27.0 (108.7) | 232 (60.8) | 0.550 (46.3) |
| 60 | 6 | 231 (46.7) | 0.750 (0.500–0.750) | 480 (46.1) | 510 (46.6) | 55.6 (15.4) | 117 (42.7) | 2.05 (47.7) |
| 120 | 6 | 396 (34.4) | 0.750 (0.500–1.00) | 1,096 (36.7) | 1,138 (36.6) | 55.5 (18.3) | 106 (38.1) | 4.62 (45.4) |
| 180 | 6 | 603 (27.8) | 1.00 (0.750–1.00) | 1,897 (39.1) | 1,978 (41.9) | 58.7 (35.6) | 91.0 (31.6) | 7.28 (34.9) |
| 240 | 6 | 624 (57.7) | 0.625 (0.333–1.00) | 1,809 (30.5) | 1,881 (29.3) | 54.1 (28.9) | 128 (30.5) | 7.49 (23.2) |
| Part 1B (food effect) | ||||||||
| 120 (fasting) | 15 | 514 (48.2) | 0.750 (0.500–1.00) | 1141 (34.4) | 1,223 (34.9) | 34.2 (35.7) | 98.1 (41.1) | 4.70 (44.2) |
| 120 (fed) | 15 | 219 (45.2) | 3.00 (0.500–6.00) | 1183 (34.4) | 1,301 (34.3) | 33.9 (25.0) | 92.2 (42.4) | 7.25 (40.1) |
Values are expressed as geometric means (between-subject percent coefficient of variance [%CV]), except for Tmax, which is expressed as the median (range). –, more than two-thirds of the data were below the lower limit of quantitation.
AUC0–t, the AUC0–168 for the part 1A study and the AUC0–72 for the part 1B study.
The t1/2 value was calculated using data for 0 to 168 h for part 1A and for 0 to 72 h for part 1B.
FIG 2.
Cmax and AUC0–168 versus dose proportionality for GLS4 in SAD study (part 1A). (A) Cmax; (B) AUC0–168. The dotted line represents a slope of 1.
GLS4 is widely metabolized in the human body and generates many metabolites. The main metabolites of GLS4 in plasma include aromatized GLS4 (M1), ester-hydrolyzed GLS4 (M2), morpholine N,N-didealkylated GLS4 (M3), and morpholine N-dealkylated GLS4 (M4). The metabolites M1, M2, M3, and M4 reached peak plasma concentrations at approximately 1 h, which was similar to the performance of GLS4. The t1/2 were 2.00 to 49.0 h, 0.678 to 4.92 h, 15.8 to 43.0 h, and 2.89 to 42.7 h, respectively. The AUC0–t of M3 was about 1- to 4-fold of that of GLS4. The AUC0–t of M1 was similar to that of GLS4. AUC0–t value for M2 and M4 were approximately 10 and 50% of that of GLS4. The Cmax values for the metabolites are listed from high to low: M1 > M3 > M4 > M2.
The concentrations of GLS4 and its metabolites in urine and feces were very low. The accumulative excretion ratio of prototypes and their metabolites in urine and feces accounted for 0.73 to 1.23% and 0.88 to 1.05% of the dose over 0 to 168 h after administration.
Mean plasma concentration-time profiles and pharmacokinetic parameters after a single oral dose of 120 mg GLS4 under fasting and fed conditions are presented in Fig. 3 and Table 2, respectively. The AUC0–72 of GLS4 was similar in fed versus fasting conditions (geometric mean ratio [GMR] = 103, 90% CI = 84.4 to 125%). The Cmax decreased by 58.2% (GMR = 41.8%, 90% CI = 31.72 to 55.15%). Similar to the parent drug, the AUC0–72 values of M1, M3, and M4 did not change significantly after meal administration. The Cmax decreased by 58.5% (GMR = 41.5%, 90% CI = 34.0 to 50.7%), 31.7% (GMR = 68.3%, 90% CI = 60.7 to 76.8%), and 54.2% (GMR = 45.8%, 90% CI = 39.5 to 53.1%), respectively. The AUC0–72 of M2 decreased by 47.9% (GMR = 52.1%, 90% CI = 47.4 to 57.3%), and the Cmax decreased by 75.0% (GMR = 25.0%, 90% CI = 20.6 to 30.4%). The Tmax was significantly prolonged for the parent drug and its four metabolites.
FIG 3.
Mean plasma concentration-time profile of GLS4 in semi-log scale in food effect study (part 1B).
(ii) Part 2: multiple-ascending-dose study, including once daily and three times daily. The pharmacokinetic parameters of part 2 are shown in Table 3. The plasma concentration-time profiles are shown in Fig. 4. In part 2A, based on visual inspection of the mean trough concentrations, GLS4 and its metabolites reached steady state on day 6. The accumulation ratios (Racc) of AUC0–24 were 1.05, 0.903, and 0.813 for 30, 60, and 120 mg, respectively, indicating no drug accumulation for once daily multiple dosing. No significant change was observed in Tmax at a steady state compared to day 1. Over the dose range of 30 to 120 mg, both the AUC at steady state (AUCτ) (slope = 0.924, 90% CI = 0.660 to 1.19) and the Cmax at steady state (Cmax,ss) (slope = 0.918, 90% CI = 0.603 to 1.23) of GLS4 increased less proportionally than doses increased (Fig. 5).
TABLE 3.
Pharmacokinetic parameters of part 2A (once daily) and part 2B (three times daily)
| Study and parametera | Mean (%CV)b
|
|||||
|---|---|---|---|---|---|---|
| Part 2A (once daily) |
Part 2B (three times daily) |
|||||
| 30 mg | 60 mg | 120 mg | 60 mg | 120 mg | 180 mg | |
| Initial dosing | ||||||
| No. | 9 | 9 | 9 | 8 | 8 | 7 |
| Cmax (ng/ml) | 90.8 (65.0) | 122 (47.4) | 401 (51.3) | 186 (56.4) | 276 (48.0) | 523 (37.5) |
| Tmax (h) | 0.500 (0.333–0.750) | 0.750 (0.500–1.00) | 0.750 (0.333–1.00) | 0.625 (0.500–0.750) | 0.750 (0.500–2.00) | 0.750 (0.500–1.00) |
| AUC0–24 or AUC0–8 (h ⋅ ng/ml) | 135 (68.6) | 229 (41.2) | 632 (34.3) | 298 (53.3) | 454 (40.8) | 1,023 (50.3) |
| t1/2 (h) | 12.5 (22.6) | 12.2 (24.9) | 12.0 (45.5) | 10.6 (37.8) | 11.5 (15.8) | 9.17 (31.2) |
| C24 or C8 (ng/ml) | 0.604 (78.2) | 1.03 (44.9) | 2.68 (34.6) | 4.66 (70.2) | 6.30 (38.3) | 12.5 (57.8) |
| Steady state | ||||||
| No. | 9 | 9 | 9 | 8 | 8 | 7 |
| Cmax,ss (ng/ml) | 71.4 (55.8) | 94.5 (52.8) | 255 (57.0) | 87.5 (68.0) | 86.5 (46.4) | 217 (46.0) |
| Cmin,ss (ng/ml) | 1.21 (71.3) | 2.03 (59.2) | 4.45 (36.4) | 6.45 (52.7) | 8.08 (46.7) | 14.4 (41.7) |
| Cav,ss (ng/ml) | 5.93 (52.6) | 8.62 (58.2) | 21.3 (40.5) | 22.7 (48.7) | 24.7 (43.3) | 60.5 (38.2) |
| Tmax,ss (h) | 0.500 (0.333–1.00) | 0.750 (0.333–1.00) | 0.750 (0.500–0.750) | 0.500 (0.500–0.750) | 0.750 (0.500–1.00) | 0.500 (0.500–1.00) |
| AUCτ (h ⋅ ng/ml) | 142 (52.5) | 207 (58.2) | 512 (40.5) | 182 (48.7) | 197 (43.3) | 484 (38.2) |
| t1/2 (h) | 53.7 (21.3) | 62.2 (22.9) | 74.1 (18.1) | 79.9 (35.7) | 68.0 (20.9) | 61.6 (45.8) |
| Racc(AUC0–24) or Racc(AUC0–8) | 1.05 (30.5) | 0.903 (31.5) | 0.813 (27.4) | 0.612 (23.2) | 0.435 (42.4) | 0.474 (18.1) |
| Racc(Cmax) | 0.787 (53.4) | 0.777 (31.9) | 0.636 (25.8) | 0.471 (36.5) | 0.314 (40.2) | 0.415 (32.0) |
The t1/2 values were calculated using data for 0 to 24 h for initial dosing, from 0 to 168 h for the steady state of part 2A and from 0 to 120 h for the steady state of part 2B.
Values are expressed as geometric means (%CV) except for Tmax, which is expressed as the median (range).
FIG 4.
Mean plasma concentration-time profile of GLS4 in semi-log scale in MAD study (part 2). (A) MAD once daily (part 2A). Frequent blood samples at steady state were taken on day 7 for 30 mg and on day 14 for 60 and 120 mg. (B) MAD three times daily (part 2B). The dotted lines represent EC90.
FIG 5.
Cmax,ss and AUCτ versus dose proportionality for GLS4 in part 2. (A) Cmax,ss; (B) AUCτ. The dotted line represents a slope of 1.
After three-times-daily oral administration of GLS4, the Racc values calculated by using the AUC0–8 for GLS4 on day 9 were 0.612, 0.435, and 0.474 for 60, 120, and 180 mg, respectively. Values for Cmax,ss (slope = 0.707, 90% CI = 0.211 to 1.20) and AUCτ (slope = 0.780, 90% CI = 0.361 to 1.20) increased in a less-than-dose-proportional manner from 60 to 180 mg (Fig. 5). The AUCτ values of M1 and M4 were similar to that of GLS4. The AUCτ values of M2 and M3 were approximately 20% and 3-fold that of GLS4 at steady state. The Racc of M1 decreased from 0.886 to 0.746 with the increase in once-daily dosage and also declined to 0.480 to 0.641 with the increased dosing frequencies. A similar trend could also be found in that of M2. In contrast to M1 and M2, the Racc of M3 increased from 1.69 to 1.74 for once daily to 2.46 to 3.72 for three times daily due to its long half-life. The Racc values of M4 were all near 1.
No significant difference in the main pharmacokinetic parameters of GLS4 or its metabolites was seen between males and females in part 2.
(iii) Part 3: Ritonavir-boosting study. Part 3 is a trial involving GLS4 administered with ritonavir. The pharmacokinetic parameters of GLS4 with its metabolites and ritonavir are listed in Tables 4 and 5, respectively. A plasma concentration-time file for GLS4 is shown in Fig. 6. Compared to the administration of GLS4 alone, the initial coadministration with ritonavir significantly increased concentrations at 24 h (C24) (GMR = 20.7, 90% CI = 17.0 to 25.3), while a milder effect was observed on AUC0–24, with a 7.42-fold increase (GMR = 8.42, 90% CI = 7.28 to 9.73), and Cmax, with a 4.81-fold increase (GMR = 5.81, 90% CI = 4.53 to 7.45). The Tmax significantly prolonged from 2 to 3 h (P = 0.025). At the same time, the AUC0–24 of M1, M3, and M4 decreased by 35, 90, and 81%, respectively, and the Cmax decreased by 75, 91, and 88%, respectively, whereas the AUC0–24 and Cmax of M2 increased by 2.82- and 2.19-fold.
TABLE 4.
Pharmacokinetic parameters of GLS4 in part 3 (ritonavir boost study)
| Study and parametera | Mean (%CV)b
|
||||
|---|---|---|---|---|---|
| GLS4 120 mg alone (n = 7) | GLS4 120 mg + ritonavir 100 mg |
GLS4 240 mg + ritonavir 100 mg |
|||
| Initial dosing (n = 7) | Chronic dosing (n = 7) | Initial dosing (n = 6) | Chronic dosing (n = 6) | ||
| GLS4 | |||||
| Cmax or Cmax,ss (ng/ml) | 135 (31.0) | 783 (36.0) | 931 (20.5) | 1,800 (20.6) | 1,620 (31.3) |
| C24 or Cmin,ss (ng/ml) | 2.40 (23.2) | 49.8 (33.1) | 182 (28.2) | 106 (30.7) | 282 (21.0) |
| Tmax or Tmax,ss (h) | 2.00 (2.00–3.00) | 3.00 (2.00–4.00) | 3.00 (1.00–3.00) | 3.00 (2.00–4.00) | 3.00 (2.00–3.00) |
| AUC0–24 or AUCτ (h ⋅ ng/ml) | 429 (17.6) | 3,610 (27.3) | 7,400 (23.3) | 8,230 (21.3) | 13,800 (22.4) |
| t1/2 (h) | 35.2 (25.2) | 16.0 (51.9) | 40.3 (6.5) | 12.1(26.7) | 62.5 (35.3) |
| Racc(AUC0–24) | 2.05 (18.7) | 1.68 (25.9) | |||
| Racc(Cmax) | 1.19 (26.1) | 0.896 (21.8) | |||
| M1 | |||||
| Cmax or Cmax,ss (ng/ml) | 118 (16.1) | 29.5 (73.8) | 22.5 (49.7) | 104 (69.3) | 139 (37.6) |
| AUC0–24 or AUCτ (h ⋅ ng/ml) | 497 (14.7) | 325 (55.6) | 294 (57.2) | 901 (45.9) | 1,160 (38.9) |
| t1/2 (h) | 7.14 (149.5) | 16.5 (81.1) | 41.7 (12.2) | 12.7 (59.4) | 63.4 (30.8) |
| Racc(AUC0–24) | 0.904 (84.3) | 1.29 (95.0) | |||
| M2 | |||||
| Cmax or Cmax,ss (ng/ml) | 11.5 (24.8) | 36.9 (31.2) | 38.5 (20.5) | 115 (32.2) | 65.5 (46.8) |
| AUC0–24 or AUCτ (h ⋅ ng/ml) | 41.1 (36.8) | 160 (22.2) | 254 (24.8) | 474 (25.2) | 427 (38.8) |
| t1/2 (h) | 2.18 (36.8) | 7.86 (22.5) | 44.9 (13.8) | 9.23 (17.1) | 71.7 (31.2) |
| Racc(AUC0–24) | 1.59 (11.4) | 0.902 (14.1) | |||
| M3 | |||||
| Cmax or Cmax,ss (ng/ml) | 52.7 (24.3) | 4.67 (93.8) | 5.68 (21.0) | 20.4 (77.5) | 36.4 (95.1) |
| AUC0–24 or AUCτ (h ⋅ ng/ml) | 505 (12.5) | 49.4 (73.2) | 104 (10.7) | 162 (64.7) | 564 (104.8) |
| t1/2 (h) | 30.7 (23.1) | 30.2 (48.4) | 155 (48.5) | 24.4 (37.5) | 134 (49.1) |
| Racc(AUC0–24) | 2.11 (68.7) | 3.49 (97.1) | |||
| M4 | |||||
| Cmax or Cmax,ss (ng/ml) | 51.0 (17.4) | 5.97 (89.2) | 10.1 (26.3) | 31.3 (66.5) | 58.6 (78.7) |
| AUC0–24 or AUCτ (h ⋅ ng/ml) | 286 (16.2) | 53.5 (61.1) | 155 (23.2) | 236 (48.3) | 782 (73.7) |
| t1/2 (h) | 24.2 (17.5) | 23.1 (73.2) | 52.1 (18.8) | 20.1 (17.8) | 50.9 (17.3) |
| Racc(AUC0–24) | 2.90 (74.8) | 3.32 (56.8) | |||
The t1/2 was calculated using data for 0 to 72 h for GLS4 dosing alone, for 0 to 24 h for initial dosing, and for 0 to 120 h for chronic dosing.
Values are expressed as geometric means (%CV) except for Tmax, which is expressed as the median (range).
TABLE 5.
Pharmacokinetics parameters of ritonavir in part 3 (ritonavir boost study)
| Parameter | Mean (%CV)a
|
||||
|---|---|---|---|---|---|
| Ritonavir alone (n = 7) | GLS4 120 mg + ritonavir 100 mg |
GLS4 240 mg + ritonavir 100 mg |
|||
| Initial dosing (n = 7) | Chronic dosing (n = 7) | Initial dosing (n = 6) | Chronic dosing (n = 7) | ||
| Cmax or Cmax,ss (ng/ml) | 458 (31.3) | 658 (21.3) | 677 (28.6) | 587 (61.4) | 415 (73.8) |
| Cmin,ss (ng/ml) | 17.1 (33.5) | 6.24 (42.9) | |||
| Tmax,ss (h) | 4.00 (3.00–6.00) | 4.00 (4.00–6.00) | 4.00 (3.00–6.00) | 4.00 (3.00–4.00) | 4.00 (3.00–4.00) |
| AUC0–24 or AUCτ (h ⋅ ng/ml) | 3,890 (27.8) | 5,160 (28.3) | 4,710 (16.0) | 4,300 (41.2) | 2,760 (52.2) |
| t1/2 (h) | 6.07 (15.1) | 4.71 (19.0) | 4.99 (13.1) | 4.08 (14.1) | 4.72 (9.8) |
| Racc(AUC0–24) | 0.914 (21.7) | 0.643 (46.6) | |||
| Racc(Cmax) | 1.03 (19.7) | 0.707 (44.8) | |||
Values are expressed as the geometric mean (%CV) except for Tmax, which is expressed as the median (range).
FIG 6.
Mean plasma concentration-time profile of GLS4 in semi-log scale in a ritonavir boost study (part 3). In part 3A, frequent blood samples were taken on day 1 for 120 mg GLS4 alone, on day 9 for initial dosing of 120 mg GLS4 and ritonavir, and on day 17 for steady state. In part 3B, frequent blood samples were taken on day 1 for initial dosing of 240 mg GLS4 and ritonavir and on day 9 for steady state. The dotted line represents EC90.
After continuous coadministration of GLS4 and ritonavir for 9 days in part 3A, chronic dosing of AUCτ and Cmax,ss of GLS4 were 2.05- and 1.19-fold that of the initial dosing. Ritonavir boosted GLS4 to achieve a minimum concentration at steady state (Cmin,ss) as high as 182 ng/ml. The AUCτ values of M1, M2, M3, and M4 were only 3.97, 3.43, 1.41, and 2.09% that of GLS4 at a steady state. The t1/2 was 40.3 h at a steady state, which is similar to that of GLS4 alone, indicating that ritonavir had less effect on the elimination of GLS4.
At a steady state of coadministration of 240 mg of GLS4 and 100 mg of ritonavir, the AUCτ and Cmax,ss increased 1.86- and 1.73-fold compared to 120 mg of GLS4, and the AUCτ of M1, M2, M3, and M4 increased 2.6-, 3.0-, 3.4-, and 4.3-fold, respectively.
The AUC0–24 and Cmax of ritonavir coadministered with the first dose of GLS4 were increased by 33% (GMR = 1.33, 90% CI = 1.19 to 1.47) and 43% (GMR = 1.43, 90% CI = 1.26 to 1.63), respectively, compared to those with the administration of ritonavir alone, indicating that GLS4 had a weak effect on the pharmacokinetics of ritonavir. The cohort of coadministration of 240 mg GLS4 and 100 mg ritonavir had lower ritonavir exposures compared to the GLS4 120-mg cohort. This suggested that GLS4 induced the enzyme in a dose-dependent manner. In part 3A and part 3B, the Racc values of the AUC of ritonavir were 0.914 and 0.643, and the Racc values of Cmax were 1.03 and 0.71, respectively. No drug accumulation for ritonavir was seen in the two groups.
Safety and tolerability.
In part 1, a total of 22 of 70 subjects (31.4%) who received GLS4 reported 27 AEs, and 5 of 18 subjects (27.8%) who received placebo reported 9 AEs (Table 6). No discontinuation for AEs was reported. Fifteen AEs in 13 subjects with GLS4 and six AEs in 4 subjects with placebo were considered to be related to the study drug. Among drug-related AEs, the most frequently reported AEs were decreasing white blood cell (WBC) count and decreasing potassium in serum. One subject receiving 240 mg experienced a WBC change from 4.19 × 109/liter to 2.89 × 109/liter before recovering after 3 days, and another underwent a WBC count decrease from 4.06 × 109/liter to 2.83 × 109/liter before recovering after 13 days. The WBC count of one subject in the placebo group changed from 3.50 × 109/liter on screening to 2.57 × 109/liter on day 2 and from 4.85 × 109/liter on day 5 to 2.92 × 109/liter on day 8 before this subject recovered on day 17. Of the subjects reporting decreased in serum potassium, one subject in the 30-mg group changed from 3.91 to 3.21 mmol/liter and recovered after 3 days, one subject in the 240-mg group changed from 3.96 to 3.17 mmol/liter and recovered after 4 days, and one subject receiving the placebo changed from 4.17 to 3.21 mmol/liter and recovered after 3 days. Several subjects in part 1 experienced dizziness, intercostal pain, gastrointestinal AEs, and laboratory- or electrocardiogram (ECG)-related AEs that were related to study drug, but no other individual AEs were reported more than three times.
TABLE 6.
Summary of AEs in part 1A (SAD) and part 1B (food effect study)
| Parameter or AEa | No. (%) of AEsb
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Part 1A (at various GLS4 doses in mg) |
Part 1B (GLS4 120 mg) |
|||||||||||
| 1 | 2.5 | 7.5 | 15 | 30 | 60 | 120 | 180 | 240 | Placebo | Fast | Fed | |
| No. of subjects | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 18 | 15 | 16 |
| No. of subjects with AEs | 2 | 1 | 2 | 1 | 3 | 2 | 1 | 1 | 3 | 5 | 4 | 3 |
| Investigations | ||||||||||||
| TSH, decreased | 1 (16.7)* | – | – | – | – | – | – | – | – | – | – | – |
| TSH, increased | – | – | – | – | – | – | – | – | – | – | 1 (6.7)* | 1 (6.3)* |
| Potassium, low | – | – | – | – | 1 (16.7)* | – | – | – | 1 (16.7)* | 1 (5.56)* | – | – |
| WBC, decreased | – | – | – | – | – | – | – | – | 2 (33.3)* | 2 (11.1)* | – | – |
| Total bilirubin elevation | – | – | 1 (16.7)* | – | – | – | – | – | – | – | – | – |
| Elevated triglycerides | 1 (16.7) | – | – | – | – | – | 1 (16.7) | – | – | 1 (16.7) | 3 (20.0) | 2 (12.5) |
| Elevated creatine kinase | – | – | – | 1 (16.7) | – | – | – | – | – | – | – | – |
| Decreased hemoglobin | – | – | – | – | – | – | – | 1 (16.7) | – | – | – | – |
| T wave flat | – | – | – | – | 1 (16.7)* | – | – | – | – | 1 (5.56)* | – | – |
| PR gap prolongation | – | – | – | – | – | – | – | 1 (16.7)* | – | – | – | – |
| Ectopic atrial rhythm | – | 1 (16.7) | – | – | – | – | – | – | – | – | – | – |
| Nervous system disorders | ||||||||||||
| Dizziness | – | – | 1 (16.7)* | – | – | – | – | – | 1 (16.7) | – | – | – |
| Gastrointestinal disorders | ||||||||||||
| Acid regurgitation | – | – | – | – | – | – | – | – | – | 1 (5.56)* | – | – |
| Stomach burning | – | – | – | – | – | 1 (16.7)* | – | – | – | – | – | – |
| Diarrhea | – | – | – | – | – | 1 (16.7)* | – | – | – | 1 (5.56)* | – | – |
| Abdominal pain | – | – | – | – | – | 2 (33.3)* | – | – | – | 1 (5.56)* | – | – |
| Musculoskeletal and connective tissue disorders | ||||||||||||
| Intercostal pain | – | – | – | – | 1 (16.7)* | – | – | – | – | – | – | – |
| Respiratory, thoracic, and mediastinal disorders | ||||||||||||
| Upper respiratory tract infection | – | – | – | – | – | – | – | – | – | 1 (16.7) | – | – |
TSH, thyroid-stimulating hormone.
*, adverse events (AEs) that were considered probably or possibly related to the study drug. –, no AEs were detected.
In part 2, a total of 14 of 51 subjects (27.5%) who received GLS4 reported 21 AEs and 3 of 15 subjects (20.0%) who received placebo reported 5 AEs (Table 7). Fourteen AEs in ten subjects with GLS4 and two AEs in two subject receiving placebo were considered to be related to the study drug. Among the drug-related AEs, the most frequently reported AE in part 2 was elevated alanine transaminase (ALT), with one in the 120-mg once-daily group changing from 18 to 90 IU/liter before recovering after 11 days, and one in the 120-mg three-times-daily group changing from 16 to 54 IU/liter with aspartate aminotransferase (AST) changing from 18 to 62 IU/liter concurrently and recovering after 6 days, 1 subject in the placebo group changing from 22 to 79 IU/liter before recovering after 15 days, and another placebo group member changing from 19 to 72 IU/liter before recovering after 7 days. One subject in the 180-mg cohort of part 2B developed nausea 5 min after the first dose on day 2, but the effect resolved within 90 min. The subject experienced a second episode of nausea and vomiting 8 min after the third dose on day 3. The subject was then assessed by an investigator and withdrew from the study. No significant abnormalities were detected in blood routine, blood chemistry, or physical examination during this period, pharmacological intervention was not required, and the subject was stable on discharge. Another subject in this cohort also developed nausea on day 2. No other individual AEs were reported more than three times.
TABLE 7.
Summary of AEs in part 2
| Parameter or AEa | No. (%) of AEsb
|
|||||||
|---|---|---|---|---|---|---|---|---|
| Part 2A: once daily (at various GLS4 doses in mg) |
Part 2B: three times daily (at various GLS4 doses in mg) |
|||||||
| 30 | 60 | 120 | Placebo | 60 | 120 | 180 | Placebo | |
| No. of subjects | 9 | 9 | 9 | 9 | 8 | 8 | 8 | 6 |
| No. of subjects with AEs | 2 | 2 | 2 | 2 | 4 | 2 | 2 | 1 |
| Investigations | ||||||||
| ALT, increased | – | – | 1 (11.1)* | 1 (11.1)* | – | 1 (12.5)* | – | 1 (16.7)* |
| AST, increased | – | – | – | – | – | 1 (12.5)* | – | – |
| TSH, increased | – | – | – | – | 1 (12.5)* | 1 (12.5)* | – | – |
| Potassium, low | 1 (11.1)* | – | – | – | – | – | – | – |
| WBC, decreased | – | 1 (11.1)* | 1 (11.1)* | – | – | – | – | – |
| WBC, increased | – | – | – | – | 1 (12.5) | – | – | – |
| Elevated neutrophils count | – | – | – | – | 1 (12.5) | – | – | – |
| Elevated triglycerides | – | – | 1 (11.1) | 2 (22.2) | – | – | – | – |
| Elevated creatine kinase | – | 1 (11.1) | – | 1 (11.1) | – | – | – | – |
| Urinary occult blood positive | – | – | – | – | 2 (50.0)c | – | – | – |
| Elevated NAG | – | – | – | – | 1 (12.5)* | – | – | – |
| Gastrointestinal disorders | ||||||||
| Nausea | – | – | – | – | – | – | 3 (37.5)* | – |
| Vomiting | – | – | – | – | – | – | 1 (12.5)* | – |
| Respiratory, thoracic, and mediastinal disorders | ||||||||
| Swelling of tonsil | – | – | – | – | 1 (12.5) | – | – | – |
| Cough | 1 (11.1) | – | – | – | – | – | – | – |
NAG, urinary N-acetyl-d-glucosaminidase.
*, adverse events (AEs) that were considered probably or possibly related to the study drug. –, no AEs were detected.
Between two AEs, only one AE with elevated NAG was probably or possibly related to the study drug.
In part 3, a total of 1 of 14 subject (7.14%) who received GLS4 reported 2 AEs, and no AE was reported in subjects who received placebo (Table 8). The AE of increased AST from 16 to 45 IU/liter was found in the same person with increased ALT in the 120- mg group (from 10 to 63 IU/liter). The patient recovered after 12 days.
TABLE 8.
Summary of adverse events in part 3
| Parameter or AE | No. (%) of AEsa
|
||
|---|---|---|---|
| Part 3A (GLS4 dose, 120 mg) | Part 3B (GLS4 dose in mg) |
||
| 240 | Placebo | ||
| No. of subjects | 8 | 6 | 2 |
| No. of subjects with AEs | 1 | 0 | 0 |
| Investigations | |||
| ALT, increased | 1 (12.5)* | – | – |
| AST, increased | 1 (12.5)* | – | – |
*, adverse events (AEs) that were considered probably or possibly related to the study drug. –, no AEs were detected.
GLS4 was well tolerated without serious AEs or dose-limited toxicity. All AEs were mild in severity and resolved spontaneously. The overall incidence of AEs was similar in subjects receiving GLS4 and placebo subjects. As noted above, only one subject in part 2B led to drug discontinuation due to an AE. There was no dose-related trend with regard to the total number of AEs with GLS4.
DISCUSSION
Both single and multiple doses of GLS4 with or without ritonavir were well tolerated in this first-in-human study. No dose correlation between the incidence and severity of all AEs was found. The frequency of AEs of administration of GLS4 up to 240 mg was similar to that of placebo. Side effects associated with ritonavir reported in previous studies(18, 19) were neurological and gastrointestinal disturbances and lipodystrophy, which were dose related (20). Patients with higher ritonavir concentrations were reportedly at a higher risk of experiencing side effects (19), and side effects were much less frequent and less severe at 100- to 200-mg doses for pharmacokinetic enhancement compared to 600 mg twice daily (18). Coadministration with ritonavir was not associated with any additional safety signals.
When GLS4 was supplied alone under fasting conditions, it was absorbed and distributed rapidly. The blood concentration of GLS4, M1, M2, and M4 for all single dose groups was lower than the 1/10 to 1/20 of Cmax at 24 h after administration, and the average t1/2 of drug elimination was 1.00 to 14.7 h, based on concentrations at 0 to 24 h. However, when the sampling time lasted 168 h, a long second elimination phase was observed after a single oral medium to high dose, which contributed to a long apparent t1/2. Similar results have been reported with entecavir (21), which had a terminal t1/2 of 128 to 149 h and an effective t1/2 of approximately 24 h. The variation in t1/2 from 1.00 h for a low dose to ∼55 h for a high dose in part 1A was an artifact of the plasma concentration of the low dose reaching the lower limit of quantitation prior to full determination of the terminal phase. The AUC and Cmax of GLS4 showed more than dose proportional pharmacokinetics over the single dose range of 2.5 to 240 mg, with a less-than-dose-proportional increase over the multiple-dose range of 30 to 120 mg once daily and a dose range of 60 to 180 mg three times daily.
The 90% effective concentration (EC90) of GLS4 in 50% human serum albumin for HBV-DNA in HepG.2.2.15 cells is ∼55.7 ng/ml. Therefore, it is possible that the concentration should be kept above 55.7 ng/ml during the 24 h after taking the drug, so that it can continue to exert an antiviral effect and reduce the rebound and resistance capacity of the virus. When GLS4 was dosed alone, the plasma GLS4 concentrations were low. The Cmin,ss of the highest dose group in part 2A of the study (120 mg) was 4.45 ng/ml, which was significantly lower than the expected concentration. However, the pharmacokinetic profile showed an autoinduction effect with frequency and dose increasing. The expected trough concentration was still not achieved, with the Cmin,ss being 14.4 ng/ml in part 2B of the study.
Preliminary studies of GLS4 in dog and human liver microsomes indicate that GLS4 is a sensitive CYP3A substrate and that first-pass metabolism plays an important role in GLS4 elimination. The major metabolic pathway involved was N-dealkylation at the morpholine ring, which was mainly catalyzed by CYP3A4 (17). However, GLS4 displayed moderate CYP3A4 induction in a concentration-dependent manner in this study. With increasing frequency and dose, the accumulation ratio showed a tendency to decrease.
Ritonavir is an HIV-1 protease inhibitor and a strong CYP3A inhibitor (22). There is clinical evidence that ritonavir can increase plasma levels of substrates of CYP3A, achieve viral suppression throughout the dosing interval, and allow for a reduced dosing frequency and/or a decrease in doses needed to maintain therapeutic drug concentrations (23–25). Because GLS4 is metabolized mainly by CYP3A4 and the first-pass effect is strong, as mentioned above, ritonavir has the potential to improve GLS4 blood concentrations and achieve superior antiviral effects.
Compared to the administration of GLS4 alone, concomitant administration with ritonavir had a profound impact on the pharmacokinetics of GLS4. Both initial and chronic dosing of ritonavir 100 mg once daily significantly increased the Cmin,ss of GLS4, whereas relatively fewer effects on GLS4 AUCτ and Cmax,ss were observed. The t1/2 was slightly prolonged from 35.2 h for dosing alone to 40.3 h for coadministration, with no significant difference. Compared to coadministration of 240 mg GLS4 and ritonavir, low doses of GLS4 can achieve an effective trough concentration, which was >3 times the EC90 against HBV-DNA in the presence of 50% human serum.
The systemic exposure of oral drugs can be increased by increasing oral bioavailability (either by increasing absorption or decreasing first-pass loss or both) and decreasing elimination (by inhibiting metabolism and/or excretion) (25). GLS4 is reported to be distributed preferentially by the liver (16), which could be a useful characteristic since the disease organ of chronic HBV infections is the liver. Reddy et al. suggested that the effect of the inhibitor on clearance was expected to be minor for compounds with high hepatic extraction, while the effect of the inhibitor on hepatic first-pass extraction is expected to be major (24). Moyle and Back (26) indicated that low-dose ritonavir combined with drugs with high first-pass metabolism primarily boosts the Cmax, Cmin, and AUC and modestly prolongs the t1/2. In our study, more significant effects on the Cmin,ss, Cmax,ss, and AUC of GLS4 than on the t1/2 were observed, indicating that the increase in oral bioavailability was mainly due to inhibited presystemic clearance. This was consistent with a study of the effect of ketoconazole on the pharmacokinetics of GLS4 in dogs, suggesting that the interaction between GLS4 and CYP3A modulators occurs in the duodenal wall (17).
When GLS4 was administered with ritonavir, the geometric mean Cmin,ss was still higher than the Cmax associated with GLS4 alone. The production of the main metabolites M1, M3, and M4 was inhibited, and the exposure of M2 was increased. Because M2 is an esterase metabolite, its metabolism is not affected by ritonavir. When the main metabolic pathway of GLS4 was suppressed, there may have been metabolic compensation in the esterase metabolic pathway, resulting in an increase in the amount of M2. A small rebound in the concentration of the metabolites M1, M3, and M4 at 24 h after the final coadministration of GLS4 and ritonavir was observed, due to low concentrations of ritonavir at that point. Ritonavir also reduced the interindividual variability in Cmax,ss, Cmin,ss, and AUC0–t.
A dose of 100 mg of ritonavir has been used as a pharmacokinetic enhancer for various HIV protease inhibitors (26). In contrast, with the administration of 120 mg GLS4, the Cmax of ritonavir was only 677 ng/ml, which accounted for only 6% of the Cmax of the drug given at a therapeutic dosage of 600 mg twice daily (20).
In conclusion, the exposure of GLS4 increased in a greater than dose proportional manner over the single dose range of 2.5 to 240 mg. However, with the frequency and dosage of multiple dosing increasing, GLS4 showed autoinduction effect. The exposure of GLS4, especially in terms of Cmin,ss, at initial and chronic coadministration with ritonavir was significantly increased. These results support the investigation of a novel HBV treatment regimen contains GLS4, with small amounts of ritonavir added to enhance concentrations in plasma. The efficacy of this regimen is under investigation in phase II clinical trials for which the 120-mg once-daily dosage of GLS4 and the 100-mg once-daily dosage of ritonavir have been selected.
MATERIALS AND METHODS
This study was conducted at the Peking University First Hospital phase I unit (Beijing, China). It was in accordance with the Declaration of Helsinki and followed the principles of Good Clinical Practice. The protocol and informed consent documentation were reviewed and approved by the study center ethics committee. Written informed consent was obtained for all subjects prior to participating in any study procedures.
Study design.
Briefly, the study comprised three parts.
(i) Part 1A was a randomized, double-blind, placebo-controlled single-ascending-dose (SAD) study with nine dose cohorts of GLS4 (1, 2.5, 7.5, 15, 30, 60, 120, 180, and 240 mg). Eight subjects in each cohort were randomized to receive a single dose of GLS4 (six subjects) or a matching placebo (two subjects) after an overnight fast. The starting dose of GLS4 at 1 mg was selected based on a no-observed-adverse-effect-level exposure from rat and dog studies according to regulatory guidance. Dose escalation was performed after the safety profile of the preceding dose was determined. Part 1B was a randomized, two-period, two-sequence, open-label, crossover study conducted to evaluate the effect of food on the bioavailability of GLS4. Sixteen subjects were randomized to fast or receive a standard high-fat, high-calorie breakfast of at least 800 kilocalories 30 min prior to the administration of a 120-mg oral dose of GLS4.
(ii) Part 2 was a randomized, double-blind, placebo-controlled multiple-ascending-dose (MAD) study with different doses and dosing frequencies. Part 2A compromised three cohorts, with 12 subjects in each cohort randomized to receive GLS4 (30, 60, or 120 mg) once daily for 7 days (for 30 mg) or 14 days (for 60 and 120 mg) or a matching placebo at 3:1. Part 2B comprised three cohorts, with 10 subjects in each cohort randomized to receive GLS4 (60, 120, or 180 mg) three times daily from days 2 to 8 (eight subjects) or a matching placebo (two subjects).
(iii) Part 3A was an open-label, multiple-dose, sequential-design drug interaction study that consisted of one cohort of eight healthy subjects. Subjects received 120 mg GLS4 alone on day 1, 100 mg ritonavir alone on day 5, and both drugs from day 9 to day 17 (for a total of nine doses). Part 3B was a randomized, double-blind and placebo-controlled study. Eight subjects in each cohort were randomized to receive GLS4 (240 mg once daily) and ritonavir (100 mg once daily) for 9 days (six subjects) or matching placebo (two subjects).
Study populations.
For all three parts of the study, healthy males and females (except for part 1B, with only male), between 18 and 45 years of age, with a BMI between the range of 19 and 24 kg/m2, and weight of at least 50 kg, were eligible to participate in this study. Subjects could only participate in a single cohort and were not allowed to participate in other parts. Subjects were not eligible if clinically significant abnormalities relevant were present during a physical exam, were observed while vital signs were recorded, or if detected by ECG or clinical laboratory evaluations at screening. Subjects were also excluded if they had used or consumed any of the following before study drug administration: known inhibitors or inducers of drug metabolism within the previous 1 month or any medication (prescription, over-the-counter, or herbal products) within 2 weeks; an alcohol consumption history averaging >14 U/week within 6 months, or >1 cigarette/day. Subjects with a history of drug abuse in the previous 12 months were also excluded.
Blood, urine, and feces sampling.
In part 1A, a series of blood samples for measurement of GLS4 plasma levels were collected in heparin lithium tubes at predetermined time points: predose; 10, 20, 30, and 45 min postdose; and 1, 2, 3, 4, 6, 8, 12, 16, 24, 36, 48, 72, 96, 120, 144, and 168 h postdose. The blood samples in part 1B were collected according to the time points of part 1A, but only for 72 h in both two periods. Urine samples were obtained predose and at predetermined collection intervals for the ≥30-mg cohorts. Feces samples voided 0 to 168 h postdose were pooled for the cohort of ≥60 mg in part 1A.
In part 2A, frequent blood samples were taken on days 1 and 14 (on day 7 for 30 mg only). Time points were similar with part 1A. Trough samples were collected before dosing on days 2 to 14 (day 7 for 30 mg). In part 2B, blood samples were collected predose and at 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after the first dose, predose on days 7 to 9 and 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, and 120 h after the last dose on day 9.
In part 3A, on days 1 to 4, blood samples were collected for analysis of the GLS4 concentrations (predose and 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 h postdose). On days 5 to 8, blood samples were taken for analysis of the ritonavir concentrations (predose and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 h postdose). Frequent blood samples were taken on time points similar with day 5 but only for 24 h after initial coadministration on day 9 and lasting for 120 h after dosing on day 17. Predose taken on days 15 to 17 were collected to evaluate trough concentrations. In part 3B, frequent blood samples were taken on time points similar to part 3A for 24 h after the initial dose and for 120 h after the last dose on day 9.
Pharmacokinetic assessment.
Plasma, urine, and feces samples were prepared using protein precipitation prior to analysis. A Shimadzu LC-30AD liquid chromatography-SCIEX Qtrap5500 mass spectrometer was used to detect GLS4 and its four metabolites (M1, M2, M3, and M4) in plasma, and a Shimadzu LC-20AD liquid chromatography-SCIEX API4000 mass spectrometer was used to detect them in urine and feces. An isotope-labeled internal standard was used in both of the analytical methods. The mobile phase A was 0.1% formic acid and 5 mM ammonium formate in water, and the mobile phase B was 40% acetonitrile for plasma, urine, and feces samples. The flow rate was 0.6 ml/min for all samples. The column was maintained at room temperature, and the mass spectrometer was used in a positive scan mode. Quantitation was accomplished by triple-quadrupole mass spectrometry and ion monitoring used the MRM mode. The linear calibration ranges of GLS4 and its four metabolites (M1, M2, M3, and M4) were 0.2 to 150 ng/ml for plasma, 5 to 1,500 ng/ml for urine, and 50 to 5,000 ng/g for feces.
Ritonavir in plasma sample was detected using a Shimadzu LC-20AD liquid chromatography- SCIEX Qtrap5500 mass spectrometer. The mobile phases were acetonitrile and 5 mM ammonium formate, including 0.1% formic acid in water (72:28 [vol/vol]). The linear calibration range was 1.00 to 1,500 ng/ml.
All bioanalytical methods were validated by the Shanghai Institute of Materia Medica and met the acceptance criteria of standard operating procedures and method validation plans. All samples were analyzed within established storage stability periods. The long-term stability of GLS4 and its four metabolites in human plasma was demonstrated for 179 days at –70°C, and ritonavir plasma samples were stable for 50 days at –70°C.
A noncompartmental method using Phoenix WinNonlin 6.1 (Certara, Princeton, NJ) was used to calculate the pharmacokinetic parameters for GLS4, its four metabolites, and ritonavir from the plasma concentration-time data. In part 1, the Cmax and Tmax were obtained from the observed data. The AUC for plasma concentration versus time from 0 to the last measurable time point (AUC0–τ), the AUC from 0 to infinity (AUC0–∞), the t1/2, and the apparent plasma clearance after extravascular administration were calculated. In addition, urine and feces concentration data were used to determine the accumulated amount of excretion and the excretion ratio.
In parts 2 and 3, pharmacokinetics parameters similar to those described for part 1 were determined. In addition, the Cmax,ss, time to Cmax,ss (Tmax,ss), Cmin,ss, AUCτ, accumulation ratio of AUC0–τ, and Cmax,ss at a steady state to the corresponding parameter for the first dose were also determined.
Safety assessment.
Safety assessments included monitoring for AEs, vital signs, ECGs, physical examinations, and clinical laboratory assessments (hematology, urinalysis, blood chemistry, lymphocyte subpopulations, blood coagulation function, and thyroid function).
Statistical methods.
Descriptive statistics were provided for demographic parameters and safety data. Dose proportionality was assessed across doses for the Cmax and AUC values by using a power model (27, 28). The effects of food and coadministration of ritonavir on the pharmacokinetics of GLS4 were assessed in parts 1B and 3 by using analysis of variance. The 90% CI of the GMR for the variables AUC0–τ and Cmax were calculated. The Tmax was evaluated by using a nonparametric test. Statistical analyses were performed using IBM SPSS Statistics 20.0 (IBM, Inc.) and SAS 9.2 software (SAS Institute, Inc., Cary, NC).
ACKNOWLEDGMENTS
We are grateful to Shanghai Institute of Materia Medica for pharmacokinetic analysis support.
This study was supported by National Major Science and Technology Projects of China (project 2016ZX09101065) and The State Key Laboratory of Anti-Infective Drug Development (2015DQ780357).
L.L., Z.H., X.W., Q.R., and Y.Z. are employees of Sunshine Lake Pharma Co., Ltd.
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