Fulvestrant-3-Boronic Acid (ZB716) Demonstrates Oral Bioavailability and Favorable Pharmacokinetic Profile in Preclinical ADME Studies

Fulvestrant-3-boronic acid (ZB716), an oral selective estrogen receptor degrader (SERD) under clinical development, has been investigated in ADME studies to characterize its absorption, metabolism, and pharmacokinetics. ZB716 was found to have high plasma protein binding in human and animal plasma, and low intestinal mucosal permeability. ZB716 had high clearance in hepatocytes of all species tested. ZB716 was metabolized primarily by CYP2D6 and CYP3A. In human liver microsomes, ZB716 demonstrated relatively low inhibition of CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 (when testosterone was used as the substrate), and no inhibition of CYP2B6 and 3A4 (when midazolam was used as the substrate). In assays for enzyme activity, ZB716 induced CYP1A2, 2B6, and 3A4 in a concentration-dependent manner. Single-dose and repeated-dose pharmacokinetic studies in rats and dogs showed oral bioavailability, dose-proportional drug exposure, and drug accumulation as measured by maximum concentration and area under the concentration–time curve (AUC).


Introduction
Fulvestrant was approved for second-line endocrine treatment in 2002 [1], and recently as a first-line therapy for advanced or metastatic estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer [2,3]. Fulvestrant acts as a full estrogen receptor (ER) antagonist that degrades the receptor protein, conferring clinical efficacy in treating patients with progressive disease while on tamoxifen or aromatase inhibitors. However, fulvestrant is not orally bioavailable, and its poor pharmacokinetic properties and insufficient drug exposure are believed to limit clinical response, which could be further improved. Efforts to increase fulvestrant exposure through multiple clinical trials between 2005 and 2009 led to subsequent approval of a higher dosage regimen in 2010 [4]. Still, clinical studies indicate that at the maximum injectable limit of 500 mg, the therapeutic effect has not reached its optimal level [5]. This unmet clinical need has driven the continued search for an ER-degrading antiestrogen that could achieve greater drug exposure through high potency and oral bioavailability.
In the past 10 years, over 13 oral SERDs have entered clinical trials to test their safety and efficacy in ER+ breast cancer patients [6]. These oral SERDs are nonsteroidal small In the past 10 years, over 13 oral SERDs have entered clinical trials to test their safety and efficacy in ER+ breast cancer patients [6]. These oral SERDs are nonsteroidal small molecules characterized by an ER-binding motif and a side chain featuring either an acrylic acid or an amino base group that confer antiestrogenic and ER-degrading activities [7]. In preclinical studies evaluating efficacy and mode of action, the oral SERD candidates are compared with the benchmark fulvestrant-the only clinically approved SERD. In most cases, the oral SERDs are found to be comparable, but not superior, to fulvestrant in ER binding, ER degradation, tissue selectivity, and inhibiting xenograft tumor growth. It is hoped that the oral SERDs, when given to patients, would derive greater efficacy via higher drug exposure than fulvestrant.
The main challenge that oral SERDs seek to overcome, therefore, is the pharmacokinetic limitations of fulvestrant, but not its potency as a pure antiestrogen and ER degrader. If fulvestrant were orally bioavailable and the level of drug exposure could be increased by oral dosage, its clinical efficacy could be conceivably enhanced significantly. In reality, glucuronidation of fulvestrant by UDP-glucuronosyltransferase (UGT) enzymes expressed in both the intestine and liver may effectively inactivate and eliminate the drug before it reaches systemic circulation [8][9][10]. To circumvent the first pass clearance that limits fulvestrant's oral absorption, we implemented a solution where the 3-OH group of fulvestrant is replaced by a boronic acid group to obtain fulvestrant-3-boronic acid (ZB716) (Figure 1) [11]. The rationale for this chemical modification was based on our research findings [12][13][14][15], where boronic bioisosteres can significantly reduce the first-pass metabolism of phenolic compounds. Our studies confirmed that ZB716 retains full binding affinity of the steroidal moiety of fulvestrant, while minimizing glucuronidation and sulfation [11][12][13]. We found that ZB716 acted as a pure antiestrogen and ER degrader in ER+ breast cancer cells, and potently inhibited tumor growth in clinically relevant xenograft breast cancer models [12]. We report preclinical studies in support of the investigational new drug (IND) application for a phase 1 clinical trial of ZB716. These studies include in vitro ADME investigations, and pharmacokinetic (PK) and toxicokinetic (TK) studies in rodents and dogs. The species and strains used in the present studies reflected those employed in the toxicological testing of ZB716, in order to enable meaningful assessment of the exposure levels in the toxicity studies, and provided confidence in the conclusions drawn regarding the safety of ZB716 in humans. Analysis in support of the toxicokinetic (TK) evaluations in pivotal repeat-dose toxicity studies was performed in compliance with the US FDA GLP Regulations for Nonclinical Laboratory Studies (21 CFR Part 58).

Pharmacokinetics/Toxicokinetics after a Single Dose of ZB716 in Rats
The single-dose PK and relative bioavailability of ZB716 were characterized in male and female Sprague Dawley rats (n = 3) after oral administration of 30, 100, and 400 mg/kg/day as suspension (20% PG, 5% Solutol, and 75% of 40% HP-β-CD in water). The We report preclinical studies in support of the investigational new drug (IND) application for a phase 1 clinical trial of ZB716. These studies include in vitro ADME investigations, and pharmacokinetic (PK) and toxicokinetic (TK) studies in rodents and dogs. The species and strains used in the present studies reflected those employed in the toxicological testing of ZB716, in order to enable meaningful assessment of the exposure levels in the toxicity studies, and provided confidence in the conclusions drawn regarding the safety of ZB716 in humans. Analysis in support of the toxicokinetic (TK) evaluations in pivotal repeatdose toxicity studies was performed in compliance with the US FDA GLP Regulations for Nonclinical Laboratory Studies (21 CFR Part 58).

Pharmacokinetics/Toxicokinetics after a Single Dose of ZB716 in Rats
The single-dose PK and relative bioavailability of ZB716 were characterized in male and female Sprague Dawley rats (n = 3) after oral administration of 30, 100, and 400 mg/kg/day as suspension (20% PG, 5% Solutol, and 75% of 40% HP-β-CD in water). The bioavailability of ZB716 was also investigated in this study after administration of solution (5% DMSO, 20% PEG400, and 75% of 40% HP-β-CD in saline) using dose levels of 2 mg/kg/day (IV) and 6 mg/kg/day (PO). Plasma samples were collected at various times through to 24 h post-dose, and analyzed for ZB716. Time to reach maximum plasma concentration (Tmax) was observed mostly 0.5-2 h post-dose following single oral administration of ZB716. Systemic exposure to ZB716, expressed as the maximum plasma concentration (C max ) and area under the curve up to the last measurable concentration (AUC last ) values, increased with dose in a close to or greater than dose-proportional manner from 6 to 30 mg/kg/day, and increased with dose in a less than dose-proportional manner from 30 to 100 mg/kg/day, and from 100 to 400 mg/kg/day. By comparing exposure in animals given 6 mg/kg/day orally and animals given 2 mg/kg/day IV, bioavailability was 6.36% in males and 5.98% in females. Group mean TK parameters are shown in Table 1. Groups of Sprague Dawley rats (n = 5/sex/group; n = 3/sex/TK group) were orally administered ZB716 (20% PG, 5% Solutol, and 75% of 40% HP-β-CD in DI water) at dose levels of 10, 100, and 400 mg/kg/day for 7 days. At the end of the study, the T max of ZB716 was mostly 0.5-1 h post-dose in animals given 10 and 100 mg/kg/day, and up to 8 h post-dose in animals given 400 mg/kg/day on both day 1 and day 7. Systemic exposure, expressed as C max and AUC last values, increased with dose in a less than dose-proportional manner on days 1 and 7 ( Figure 2). A slight gender difference was observed; generally, systemic exposures were higher in females than in males (male/female AUC last ratio = 0.442:0.883). Accumulation was observed only in male animals given 400 mg/kg/day. Group mean TK parameters are shown in Table 2.
The PK of ZB716 was investigated in a 4-week repeat-dose toxicity study in rats. Groups of Sprague Dawley rats (n = 10/sex/group; n = 3/sex/TK group) were orally administered ZB716 (5% EtOH, 15% PG, 5% Solutol, and 75% of 40% HP-β-CD in DI water) at dose levels of 25, 100, and 400 mg/kg/day for 4 weeks. The plasma concentration of ZB716 was measured at various times through to 24 h post-dose on days 1 and 28. The exposure generally increased with dose in a less than or close to dose-proportional manner on both days 1 and 28. A gender difference was noted with higher exposure at all dose levels for females on day 1, and for females given 25 mg/kg/day on day 28. No accumulation was noted after 28 days of dosing, and a trend for decreased exposure was noted in females given 100 and 400 mg/kg/day after repeat dosing. Systemic exposure data, expressed as C max and AUC last for ZB716, are summarized in Table 3. There was no test-article-related mortality or moribundity during the study. Cmax and AUClast for ZB716, are summarized in Table 3. There was no test-article-related mortality or moribundity during the study. were given ZB716 at 10 mg/kg/day, 100 mg/kg/day, and 400mg/kg/day, respectively.   were given ZB716 at 10 mg/kg/day, 100 mg/kg/day, and 400 mg/kg/day, respectively.  The single-dose PK of ZB716 was characterized in beagle dogs after oral administration as a suspension (20% PG, 5% Solutol, and 75% of 40% HP-β-CD in water), using dose levels of 30 and 100 mg/kg/day. The relative bioavailability of ZB716 was also investigated after IV administration of solution (5% DMSO, 20% PEG400, and 75% of 40% HP-β-CD in saline) using a dose level of 2 mg/kg/day, and oral administration of 6 mg/kg/day in solution (5% DMSO, 20% PEG400, and 75% of 40% HP-β-CD in saline). All groups had one male and one female animal. Plasma samples were collected at various timepoints, through to 24 h post-dose, and analyzed for ZB716. Systemic exposure to ZB716, expressed as C max and AUC last values, increased with dose in a greater than dose-proportional manner in the male animal, and in a less than dose-proportional manner in the female animal, from 6 to 30 mg/kg/day.
The systemic exposure increased with dose in a less than dose-proportional manner from 30 to 100 mg/kg/day for both male and female animals. No obvious gender difference was observed. When comparing exposure in animals given 6 mg/kg/day orally and animals given 2 mg/kg/day IV, bioavailability was 6.18% in males and 6.74% in females. The PK parameters derived from these studies are presented in Table 4.

Pharmacokinetics/Toxicokinetics after Repeated Doses of ZB716 in Dogs
The TK of ZB716 was investigated following once-daily administration via oral gavage for 7 consecutive days. Beagle dogs (n = 1/sex) were orally administered ZB716 at dose levels of 100 and 200 mg/kg/day. Following 7-day repeat oral administration, the T max of ZB716 was observed mostly at 0.5-2 h post-dose.
The TK of ZB716 was investigated following once-daily administration via oral gavage for 7 consecutive days. The C max and AUC last values of ZB716 increased with dose in a greater than dose-proportional manner. However, the exposure in the female given 100 mg/kg/day was higher than that in the female given 200 mg/kg/day on days 1 and 7 ( Figure 3). During the study, females had higher exposure at 100 mg/kg/day, and males had higher exposure at 200 mg/kg/day. Accumulation was observed at all doses after 7 days of dosing. The PK parameters derived from this study are presented in Table 5.
The TK of ZB716 was investigated in a 4-week repeat-dose toxicity study in dogs. Groups of dogs (n = 3/sex/group) were orally administered ZB716 (5% EtOH, 15% PG, 5% Solutol, and 75% of 40% HP-β-CD in DI water) at dose levels of 25, 100, and 200 mg/kg/day for 4 weeks. The plasma concentration of ZB716 was measured at various times up through to 24 h post-dose on days 1 and 28. Following oral administration of ZB716 at 25, 100, and 200 mg/kg/day, the exposure generally increased with dose in a less than doseproportional manner on both days 1 and 28, except that the values did not increase with dose from 100 to 200 mg/kg/day in females on day 1, or in males on day 28. The lower exposure in females given 200 mg/kg/day on day 1 was likely due to emesis noted in all females in this group. Higher exposure was noted in females given 25 mg/kg/day on days 1 and 28, and lower exposure was noted in females given 200 mg/kg/day on day 1. Accumulation was noted at all dose levels after 28 days of dosing, with a day 28/day 1 AUC ratio of 2.06:5.49, except for males given 400 mg/kg/day. The PK parameters derived from this study are presented in Table 6. There was no test-article-related mortality or moribundity during the study.  The TK of ZB716 was investigated in a 4-week repeat-dose toxicity study in dogs. Groups of dogs (n = 3/sex/group) were orally administered ZB716 (5% EtOH, 15% PG, 5% Solutol, and 75% of 40% HP-β-CD in DI water) at dose levels of 25, 100, and 200 mg/kg/day for 4 weeks. The plasma concentration of ZB716 was measured at various times up through to 24 h post-dose on days 1 and 28. Following oral administration of ZB716 at 25, 100, and 200 mg/kg/day, the exposure generally increased with dose in a less than doseproportional manner on both days 1 and 28, except that the values did not increase with dose from 100 to 200 mg/kg/day in females on day 1, or in males on day 28. The lower exposure in females given 200 mg/kg/day on day 1 was likely due to emesis noted in all females in this group. Higher exposure was noted in females given 25 mg/kg/day on days 1 and 28, and lower exposure was noted in females given 200 mg/kg/day on day 1. Accumulation was noted at all dose levels after 28 days of dosing, with a day 28/day 1 AUC ratio of 2.06:5.49, except for males given 400 mg/kg/day. The PK parameters derived from this study are presented in Table 6. There was no test-article-related mortality or moribundity during the study.

Distribution
A range of in vitro studies have been performed to investigate the binding of ZB716 to serum and plasma proteins, and to investigate ZB716's interactions with a range of cellular transporters.

Plasma Protein Binding
The in vitro binding of ZB716 to plasma proteins was determined by equilibrium dialysis technique in mouse, rat, dog, monkey, and human plasma (EDTA-K2 as anticoagulant), over the concentration range of 0.1-10 µM. The free concentration of ZB716 was below the lower limit of quantitation at 0.1, 1, and 10 µM in human, monkey, dog, rat, and mouse plasma. The results showed that ZB716 had a high protein binding in human, monkey, dog, rat, and mouse plasma ( Table 7).

Intestinal Mucosal Permeation
The bidirectional permeability and absorption mechanism of ZB716 was evaluated across Caco-2 cell monolayers. As shown in Table 8, the efflux ratio of ZB716 at concentrations of 1, 5, and 15 µM was >0.935, >4.55, and 2.87, respectively. The apparent permeability P app (A to B) was <0.778 cm/s × 10 −6 , <0.159 cm/s × 10 −6 , and 0.188 cm/s × 10 −6 , respectively. The concentration of the receiver was below the lower limit of quantitation at 1 and 5 µM, and the exact value of P app and the efflux ratio could not be calculated. According to the results at 15 µM, ZB716 is a compound with low permeability, and may be a substrate of efflux transporters. To determine which CYP enzymes are responsible for the metabolism of ZB716, heterologously expressed recombinant human CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5 isoforms, along with human liver microsomes, were treated with ZB716 in the presence and absence of CYP-specific chemical inhibitors.
The phenotyping results (Table 9) indicated that CYP2D6 and 3A may be the main CYP isoforms involved in the metabolism of ZB716. The metabolism of ZB716 was observed in incubations with heterologously expressed CYP2D6, 3A4, and 3A5 enzymes, with Cl int values of 0.773, 2.35, and 6.86 µL/min/pmol, respectively. In human liver microsome incubations, with and without specific inhibitors for 2D6 and CYP3A, the remaining percentage after incubation with the CYP2D6 and 3A inhibitors quinidine and ketoconazole was 65.6% and 74.4%, respectively ( Table 10). The inhibition ratios (%) were 74.7% and 88.2%. Based on the data on other isoforms, CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, and 2E1 may be also involved-to a much lesser extent-in ZB716 metabolism.

Enzyme Inhibition
The potential for ZB716 to inhibit human CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 was investigated using human liver microsomes and specific probe substrates (Table 11). Over the test concentration range of 0.05-15 µM, the inhibition of CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 (when testosterone was used as the substrate) activity by ZB716 was observed with the mean IC 50 values of >15.0, 6.27, 4.77, 2.20, 12.5, and 11.2 µM, respectively (Table 11). The inhibition of CYP2B6 and 3A4 (when midazolam was used as the substrate) by ZB716 was not observed. The inhibitory potential of ZB716 on human CYP enzyme activities appears relatively low. Furthermore, ZB716 showed some inhibitory potential for CYP2C8, 2C9, and 2C19.

Enzyme Induction
The potential for ZB716 to induce the activity of CYP1A2, 2B6, and 3A4 was investigated in plated cultures of cryopreserved human hepatocytes from three donors at 0.15, 1.5, 5, and 15 µM. As shown in Table 12, based on the enzyme activity, the induction of CYP1A2 was observed in two donors (ZSE and VKB) at concentrations of 1.5-15 µM. The induction fold was 2.09-7.62 compared to the vehicle control, and the induction percentage was 20.3-80.0% of the positive control. The induction of CYP2B6 was observed in all three donors at 1.5-15 µM. The induction fold was 2.98-6.40 compared to the vehicle control, and the induction percentage was 37.4-92.1% of the positive control. However, the induction of CYP3A4 was observed in one donor at the concentrations of 5-15 µM. The induction fold ratio was 3.01-2.92 compared to vehicle control, and the induction percentage was 22.4-21.5% of the positive control. The effect was dose-dependent; the decrease in fold induction at 15 µM may be caused by the slight cytotoxicity, and so ZB716 may have an induction effect on CYP1A2, 2B6, and 3A4.  Metabolic stability of ZB716 was assessed at a single concentration of 1 µM at t = 0 and at t = 120 min. The calculated in vitro Cl int of ZB716 incubated with human, monkey, dog, rat, and mouse hepatocytes was 39.7 µL/min/10 6 cells, 50.3 µL/min/10 6 cells, 51.9 µL/min/10 6 cells, 48.7 µL/min/10 6 cells, and 40.3 µL/min/10 6 cells, respectively, with estimated half-lives of 34.9, 27.6, 26.7, 28.4, and 34.4 min, respectively. The results indicate that ZB716 had high clearance in hepatocytes of all species tested (Table 13).

Discussion
Systemic exposure profiles, based on maximum concentration (C max ) and area under the concentration-time curve (AUC), were evaluated in rats and dogs. In rats, exposure increased with dose in a close to dose-proportional manner in males, and in a greater than dose-proportional manner in females, at lower doses (6 to 30 mg/kg), and in a less than dose-proportional manner in both sexes at higher doses (30 to 100 mg/kg). No other gender differences were noted in single-dose PK in rats. In dogs, exposure increased in a greater than dose-proportional manner in males in one case, and in a less than doseproportional manner in both sexes in another study. No other gender differences were noted in single-dose PK in dogs. Repeat-dose TK was evaluated in Sprague Dawley rats and beagle dogs. In rats, exposure generally increased in a less than dose-proportional manner. Some evidence of a gender difference was observed, where females showed higher exposure than males. Accumulation was noted in males at 400 mg/kg/day for 7 days, which was not observed after 28 days of dosing. In dogs, exposure generally increased with dose in a less than dose-proportional manner. Although some gender differences were noted for area under the concentration-time curve from 0 to the time of the last measurable concentration (AUC last ) ratios on specific days and at specific doses, no clear pattern emerged. Accumulation was evident with repeat dosing. Repeat oral administration of ZB716 once daily in beagle dogs at doses up to 200 mg/kg/day for 28 consecutive days was tolerated. Abnormal clinical signs in animals given 100 mg/kg/day, and lower body weight gains in animals given 200 mg/kg/day, were also observed. Alterations in clinical chemistry parameters and microscopic findings were noted in the liver and thymus. These findings were fully or partially recovered by the end of the 14-day recovery period.
The absorption of ZB716 appears to be relatively rapid, with a C max in general at 0.5-8 h in rats and dogs. At higher doses, the exposure tends to be less than doseproportional. In both rats and dogs, the plasma levels of ZB716 were generally higher in females than in males.
The in vitro metabolic stability of ZB716 in hepatocytes of five different species all indicated extensive metabolism and rapid clearance, predicting high clearance in vivo. Compared to the positive control verapamil-an oral drug-ZB716 showed a longer half-life and a lower degree of hepatic clearance, consistent with the moderate oral bioavailability of ZB716. As a highly lipophilic compound, ZB716 showed high plasma protein binding in mouse, rat, dog, monkey, and human plasma, with >99% bound at 1 µM. The metabolic pattern obtained in liver microsomes is overall similar in rats, dogs, and humans. The hydrophobic and slightly acidic properties of ZB716 contribute to its high binding to plasma proteins, which likely increases its solubility in plasma. A connection with the plasma proteins protects ZB716 from oxidation, potentially increasing its half-life in vivo.
While CYP2D6 and CYP3A were the primary metabolizing enzymes for ZB716, several CYP isozymes-including CYP1A2, CYP2D6, CYP3A, 2A6, 2B6, 2C8, 2C9, 2C19, and 2E1were also involved in the metabolism of ZB716, suggesting that drug interactions by single CYP inhibitors are unlikely to modulate the exposure to ZB716 and its metabolites. In human liver microsomes, ZB716 demonstrated relatively low inhibition of CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 (when testosterone was used as the substrate), and no inhibition of CYP2B6 and 3A4 (when midazolam was used as the substrate). Although ZB716 showed the potential to induce CYP1A2, 2B6, and 3A4, the overall results suggest a relatively low risk for drug interactions involving these enzymes in vivo. At therapeutically effective levels of ZB716 (~100 nM), inhibition of CYP enzymes by ZB716 is unlikely.
In summary, the present results support the nonclinical toxicological program of ZB716, as well as the clinical development of ZB716 as an oral SERD for phase 1/2 clinical study in breast cancer patients.

Animals
Four-to-six-week-old Sprague Dawley rats (Crl: CD (SD)) (SPF/VAF) were purchased from Beijing Vital River Laboratory Animal Technology, Co. Ltd., Beijing, China). Sixmonth-old beagle dogs were purchased from Beijing Marshall Biotechnology Co., Ltd. Animals were quarantined/acclimated for 2-6 weeks prior to dose initiation. Food was provided ad libitum, except during overnight fasting prior to blood collection for clinical chemistry and necropsy. Water was provided ad libitum. The feed was analyzed for concentrations of specified heavy metals and nutrient components. The water was routinely analyzed for specific microbes and contaminants, including total dissolved solids, inorganic matter, total chlorinated organic chemicals, and heavy metals. No contaminants were detected in the feed, bedding, or water at levels that might have interfered with the outcome of the study. Records of all dogs vaccinated against distemper, hepatitis, leptospirosis, parvo, parainfluenza, and rabies, as well as prophylactic treatments for parasites, were provided by the vendor. Pharmaron's Institutional Animal Care and Use Committee (IACUC) reviewed the protocol and approved the animal care and use application. All study activities were in accordance with Pharmaron's IACUC policies and procedures.

Metabolic Stability
Hepatocytes of humans (#X008001), dogs (#M00205), rats (#M00005), and mice (#M005052) were purchased from BioIVT, NY, USA. Monkey hepatocytes were procured from RILD, Shanghai, China. The metabolic stability of ZB716 in human, monkey, dog, rat and mouse hepatocytes was investigated by incubating ZB716 with human, monkey, dog, rat, and mouse hepatocytes (0.5 × 10 6 cells/mL), or with incubation medium only. The incubations were carried out at a test concentration of 1 µM at 37 • C, over a total incubation period of 120 min. Verapamil was used as the positive control substrate for metabolism across species. Timepoints were taken at 0, 15, 30, 60, 90, and 120 min by adding acetonitrile with IS to stop the reaction. The percentage of the parent drug remaining, clearance, and t 1/2 were calculated (the calculation details are available in the Supplementary Materials).

CYP Induction
To investigate the potential of ZB716 to induce the expression and activity of CYP1A2, CYP2B6, and CYP3A4 in plated cultures, cryopreserved human hepatocytes from 3 different donors (QBU, ZSE, and VKB) were purchased from BioIVT, NY, USA. For CYP induction study, all incubations were performed in triplicate; the test concentrations of ZB716 in the incubation medium were 15, 5, 1.5, and 0.15 µM, and 0.1% DMSO was used as a vehicle control. The dosing medium with ZB716 or control articles was renewed every 24 h. On the last day of the incubation, the concentrations of ZB716 in the medium were measured at 0, 1, 2, 4, 6, and 24 h. After 72-h treatment, the plate was incubated with specific substrates of CYP1A2, CYP2B6, and CYP3A4 for 30 min. The UPLC-MS/MS (SHIMADZU, LC-30AD) equipped with waters Xselect ® HSS T3 2.5 µm (2.1 × 50 mm) was used to detect the marker metabolites in the enzyme activity assay. The fold-induction enzyme activity was determined using the ratio calculation (the calculation details are available in the Supplementary Materials).