Development of a PBPK model to quantitatively understand absorption and disposition mechanism and support future clinical trials for PB‐201

Abstract PB‐201 is the second glucokinase activator in the world to enter the phase III clinical trials for the treatment of type 2 diabetes mellitus (T2DM). Combined with the efficacy advantages and the friendly absorption, distribution, metabolism, and excretion characteristics, the indication population of PB‐201 will be broad. Because the liver is the primary organ for PB‐201 elimination, and the elderly account for 20% of patients with T2DM, it is essential to estimate PB‐201 exposure in specific populations to understand the pharmacokinetic characteristics and avoid hypoglycemia. Despite the limited contribution of CYP3A4 to PB‐201 metabolism in vivo, the dual effects of nonspecific inhibitors/inducers on PB‐201 (substrate for CYP3A4 and CYP2C9 isoenzymes) exposure under fasted and fed states also need to be evaluated to understand potential risks of combination therapy. To grasp the unknown information, the physiologically‐based pharmacokinetic (PBPK) model was first developed and the influence of internal and external factors on PB‐201 exposure was evaluated. Results are shown that the predictive performance of the mechanistic PBPK model meets the predefined criteria, and can accurately capture the absorption and disposition characteristics. Impaired liver function and age‐induced changes in physiological factors may significantly increase the exposure under fasted state by 36%–158% and 48%–82%, respectively. The nonspecific inhibitor (fluconazole) and inducer (rifampicin) may separately increase/decrease PB‐201 systemic exposure by 44% and 58% under fasted state, and by 78% and 47% under fed state. Therefore, the influence of internal and external factors on PB‐201 exposure deserves attention, and the precision dose can be informed in future clinical studies based on the predicted results.


INTRODUCTION
Glucokinase (GK), as a glucose sensor in the βcells of the pancreas and a pacemaker in the hepatic conversion of glucose to glycogen, 1 is considered to be the major ratelimiting enzyme for glycolysis. 2 Small-molecule GK activators (GKAs) have overcome physical limitations to effectively control the levels of glycosylated hemoglobin in patients with type 2 diabetes mellitus (T2DM) 3,4 and have the potential to reverse diabetes. 5 Currently, a similar drug has successfully reduced glucose 6 and improved the early insulin secretion index in clinical trials, 7 indicating that GKA will play a unique role in hypoglycemic drugs. As the second GKA in the world to be developed in the phase III clinical trials, 8 PB-201 (imported from Pfizer [PF-04937319] by Suzhou PegBio Co., Ltd.) has demonstrated safe [9][10][11] and effective hypoglycemic effects 11 in various early clinical trials. [9][10][11] Currently, a series of pharmacokinetic (PK) studies of PB-201 tablets in vivo have been completed. The main PK characteristics of PB-201 in vivo indicate that PB-201 has the moderate oral bioavailability (about 68%, the detailed data unpublished) in monkeys. 12 PB-201 is slowly absorbed in humans, with the time (median) to reach maximum plasma concentration after multiple doses (NCT01272804) about 2-3 h. The binding rate of PB-201 to plasma proteins is about 70%, 13 suggesting that PB-201 is widely distributed in vivo (volume of distribution based on the terminal phase ~254.4 L at 100 mg, NCT01044537). Cytochrome P450-mediated oxidative metabolism plays the major role in the elimination of PB-201, which was catalyzed through CYP3A and CYP2C9 isoforms. 13 Moreover, PB-201 is not a specific substrate of OATP1B1, OATP1B3, P-gp, and BCRP (unpublished). The kidney contributes a little to PB-201 elimination in vivo, as unchanged drug in the urine is less than ~1% (NCT01272804). Biliary excretion has a tiny (about 2.3%) contribution to the elimination of PB-201 in the bile duct-cannulated rats. 12 Therefore, the liver plays an important role in the clearance of PB-201, and evaluation of the systemic exposure of PB-201 in the liver impairment population is necessary and pressing in the development of new drugs. 14

WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?
CYP3A4 (metabolism fraction [f m ]-36.0%) and CYP2C9 (f m -32.7%) are the major isoenzymes of PB-201 metabolism in vitro, and food can significantly increase PB-201 exposure in vivo. Clinical drug-drug interaction studies have shown that CYP3A4 has the limited (~26.4%) contribution to PB-201 elimination in vivo, indicating that the f m of CYP2C9 in vivo may be less than 25%.

WHAT QUESTION DID THIS STUDY ADDRESS?
Dual effects of nonspecific inhibitors/inducers on PB-201 exposure under fasted and fed states were evaluated by the physiologically-based pharmacokinetic model to understand potential risks of combination therapy. Meanwhile, the exposure in specific populations was also evaluated to provide supportive information for corresponding clinical trials.

WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?
The nonspecific inhibitor (fluconazole) and inducer (rifampicin) may separately increase/decrease PB-201 systemic exposure by 44% and 58% under fasted state, and by 78% and 47% under fed state. Physiological changes due to impaired hepatic function and age may significantly increase exposure under fasted state by 36%-158% and 48%-82%, respectively.

HOW MIGHT THIS CHANGE DRUG DISCOVERY, DEVELOPMENT, AND/OR THERAPEUTICS?
Combined with the exposure-response analysis results, the effects of nonspecific inhibitors may lead to hypoglycemia in some patients and the nonspecific inducers can significantly reduce PB-201 exposure under fasted and fed states. But the predicted results still need to be confirmed in future clinical trials. Assuming that the same systemic exposure of PB-201 produces the same pharmacological effects in vivo, the predicted results can be served as indicators for the dose design of corresponding clinical trials.
Globally, there are about 536.6 million people aged 20-79 years with diabetes, of whom more than 90% suffer from T2DM, 15 and the elderly account for 20% of patients with T2DM. 16 Generally, drug safety is closely related to the systemic exposure. 17 Specific populations, including the liver impairment population, geriatric population, and so on, usually have increased 18 or delayed absorption, 19 abnormal enzyme activity, [20][21][22] and changes in other physiological factors, which will cause uncertain changes in systemic exposure and induce adverse effects. Thus, assessment of PB-201 systemic exposure in specific populations is the prerequisite for avoiding hypoglycemic events before expanding the clinical indication population. However, the disposition mechanism of PB-201 cannot be summarized based on the PK characteristics described above, and the lack of understanding of the disposition mechanism will increase the difficulty of reasonably designing the dose of PB-201 in the clinical trials with specific populations. Moreover, the improper dosage may also induce hypoglycemia. Therefore, recommending scientific and reasonable doses in clinical trials for specific populations has become the crucial step to ensure the safe and effective implementation of clinical trials.
Up to now, a drug-drug interaction (DDI) study of PB-201 has only been conducted with ketoconazole (a specific strong CYP3A4 inhibitor), which has minimal effect on the systemic exposure of PB-201 (about 26.4%). Due to the limited contribution of CYP3A4 to systemic exposure of PB-201 and extensive metabolism of PB-201 by multiple isoenzymes, the induction of perpetrators on PB-201 exposure has not been evaluated yet. Although CYP2C9 is the secondary CYP isoform for the clearance of PB-201 in vitro, the activity of CYP2C9 can also be inhibited by fluconazole and fluvoxamine, 23 which are also CYP3A4 inhibitors. Compared with the effect of ketoconazole on PB-201 systemic exposure, whether fluconazole and fluvoxamine have similar or intensive effects is worth discussing. Moreover, the systemic exposure of PB-201 was significantly increased under fed state (NCT01513928) compared with the same dose of PB-201 under fasted state (NCT01272804). In order to avoid the treatment failure in phase III studies, there is an urgent need to fully understand the systemic exposure of PB-201 co-administration with perpetrators both under fasted and fed states, so as to provide supportive information for dose decision in the phase III clinical trials.
Considering that the physiologically-based pharmacokinetic (PBPK) model can bridge the relationship between in vitro experimental results and in vivo systemic exposure, 24,25 evaluation of PB-201 co-administration with perpetrators can be performed through the model method after the disposition mechanism of compound validated by DDI studies with strong index perpetrators. 26 Meanwhile, untested scenarios can be simulated based on the population library to explore the effects of external and internal factors on the PB-201 systemic exposure. 27 Herein, we are aimed (i) to develop a mechanistic PBPK model of PB-201 according to preclinical and clinical data to reliably describe the absorption and disposition characteristics of PB-201 in vivo; (ii) to explore the influences of physiological changes on the systemic exposure of PB-201 to support dose decision in clinical trials of specific populations; and (iii) to evaluate the changes in systemic exposure after PB-201 co-administration with potential perpetrators under both fasted and fed states to provide useful information for ensuring the successful implementation of PB-201 phase III clinical trials.

Study strategy
PB-201 PBPK model was developed according to the in vitro-in vivo data, such as the permeability and oxidative metabolism of PB-201 in vitro and the renal clearance of PB-201 in humans. Then, the model was validated by the clinical trial results including PK studies results of multiple ascending doses both in White and Chinese patients (NCT01272804 and NCT03973515), PK study results of PB-201 with a single-dose immediate release formation under fed state (NCT01513928), PK study results of PB-201 in adults with T2MD inadequately controlled on metformin (NCT02206607), and the DDI study results about ketoconazole co-administration with PB-201 (NCT01468714). Finally, the mechanistic PBPK model was used to simulate the PK characteristics of PB-201 in specific populations and to evaluate the effects of CYP3A/ CYP2C9 perpetrators on PB-201 systemic exposure both under fasted and fed states in order to provide supportive information for dose decision making in corresponding clinical trials. The detailed strategy diagram is shown in Figure 1, the simulation scenarios are displayed in Table S1, and the perpetrators' model parameters are listed in Tables S2 of the supplementary files.

Apparent permeability and P-gp substrate assessment in Caco-2 cell lines
The integrated Caco-2 monolayers were cultured for 22 days in a 5% CO 2 incubator at 37°C for the assessment of PB-201 apparent permeability. Monolayers were incubated with PB-201 in apical and basolateral (BL) sides for 2 h at the concentration of 0.1 μM, 1 μM, and 5 μM, respectively. Meanwhile, nadolol (with low permeability compound), propranolol (with high permeability), and taxol (P-gp substrate) were selected as the positive controls to ensure the applicability of the incubation system. Moreover, verapamil, a specific inhibitor of P-glycoprotein (P-gp), was added into another incubation system under the same condition mentioned above to evaluate whether PB-201 was a substrate of P-gp. All experiments were conducted in triplicate and analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The apparent permeability coefficient (P app ) was calculated based on the following equation: where P app was cm/s × 10 −6 , dQ/dt (pmol/second) was the rate at which the compound appears in the receiver side, C 0 (nM) was the initial concentration of the compound in the donor side, and A (cm 2 ) represents the surface area of the cell monolayer.

Metabolic stability of PB-201 in human recombinant CYP isoenzymes
Due to CYP-mediated oxidative metabolism was the main route for the elimination of PB-201 in preclinical animal studies, 12 human recombinant CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoenzymes were selected to evaluate the metabolic stability of each isoenzyme to PB-201. The 100 μM PB-201 with the volume of 2 μL was added into the 100 μL CYPs (100 pmol/ mL) working solution. After gently mixing, the mixture was pre-incubated at 37°C for 10 min. The reaction was initiated by adding 98 μL of cofactor working solution. Samples were collected at 0, 5, 15, 30, and 60 min, respectively, and the reaction was terminated with cold acetonitrile. After vortexing vigorously for about 1 min, samples were centrifuged at 3000 g for 15 min at 4°C. PB-201 in the supernatant was removed and quantified by HPLC-MS/ MS. Moreover, to ensure the robustness of the incubation system, the metabolic stability of positive control compounds of corresponding CYP isoenzymes was evaluated by the same method. The intrinsic clearance (CL int ) of each CYP isoenzymes to the metabolism of PB-201 was calculated according to the remaining percentage of PB-201 and the amount of CYP isoform in the incubation system. The detailed equation is listed in Equation 2. Moreover, according to the published protein abundance of each CYP isoenzymes in the human liver, 28 metabolism fraction (f m ) of each CYP isoenzyme for PB-201 systemic metabolism in vitro was calculated based on Equation 3. 29 where CL int j was jth CYP isoform and the P450 j was the jth P450 isoform tested. where P450 j abundance was the protein abundance of P450 j .

The development of PB-201 PBPK model
The , which was obtained through sensitivity analysis and fitting the PK data in clinical trials. The full-PBPK model with the predicted steady-state distribution volume and tissue to plasma partition coefficient (K p ) was used to describe the distribution characteristics of PB-201 in vivo. Moreover, K p scalar was estimated to be 1.50 to match the observed concentration-time profiles. Because the metabolic profile in human hepatocytes revealed that PB-201 was metabolized through oxidative (major) and hydrolytic pathways (minor), 13 the oxidative pathway of PB-201 in vivo was characterized by the CL int of the corresponding CYP isoenzyme according to the metabolic stability studies in human recombinant incubation systems and the hydrolytic pathway of PB-201 in vivo was characterized by CL int_Hep of hepatocytes. Meanwhile, renal clearance (CL R ) obtained from a clinical trial was used to represent the unchanged PB-201 in urine. Because the intrinsic clearance of PB-201 hydrolysis in hepatocytes was absent and the contribution percentage of CYP3A4 to the PB-201metabolism in vivo had been confirmed in the DDI study (NCT01468714), CL int_Hep and inter-system extrapolation factors (ISEFs) scaling of recombinant CYP isoforms in vitro kinetic data were fitted according to the contribution of CYP isoforms in vitro and in vivo (NCT01468714). Last but not least, physicochemical parameters including molecular weight, protein binding, B/P ratio, Log P, and pK a were obtained from experimental outcomes.

Apparent permeability and the P-gp specific substrate assessment
The intact Caco-2 cell monolayers were utilized for PB-201 apparent permeability and specific substrate evaluation. The P app values of PB-201 at the concentration of 0.1, 1, and 5 μM were 9.57, 9.33, and 7.75 × 10 −6 cm/s, respectively, indicating that PB-201 was a compound with medium permeability. Although the efflux ratio (ER) of PB-201 was decreased by 49.99% when incubated with verapamil (P-gp specific inhibitor) in BL, the ER was less than 1 in the absence of verapamil. Therefore, PB-201 is not a specific substrate of P-gp. The detailed results are shown in Table 1.

The metabolic stability of PB-201 in human recombinant CYP isoenzymes
The oxidative metabolic stability of PB-201 in the liver was evaluated in human recombinant CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoenzymes. All isoenzymes were involved into the metabolism of PB-201, and the remaining percentage of PB-201 in the different incubation systems over time is presented in Figure S1 of the supplementary file. The CL int of each CYP isoenzyme to PB-201 metabolism was calculated according to Equation 2, as shown in Table 2

PBPK model validation
The PB-201 PBPK model was validated by several clinical data, and the predictive performance was satisfactory.
The predicted systemic exposure of PB-201 in the absence or presence of ketoconazole was in good agreement with the observed results (Figure 2a,b), suggesting that the metabolic mechanism of PB-201 was well-captured by the model. Meanwhile, the absorption phase of PB-201 under fed ( Figure 2c) and fasted states (other picture in Figure 2) matched well, indicating that the model had the ability to capture absorption characteristics. Moreover, the 90% CI of the predicted plasma concentration-time curve included the observed concentration-time points (Figure 2), and the major PK parameters were within the predefined boundaries ( Figure S2). Accordingly, the PBPK model was able to balance the absorption and disposition characteristics of PB-201 in vivo. On the basis of the validated CYP3A4 metabolic pathway, the amount of unchanged PB-201 in urine and the systemic metabolism of PB-201 in the human recombinant CYP isoform incubation system, the contribution percentage of each organ/isoenzyme to the elimination of PB-201 in Chinese patients under regimen A was calculated in SimCYP and presented in Figure 3. The final PB-201 PBPK model parameters and data sources are summarized in Table S3 of the supplementary file. states, respectively. Although erythromycin is a moderate CYP3A inhibitor, the time-dependent inhibition of erythromycin has a powerful effect on CYP3A isoenzyme. 31 Compared with CYP3A4 strong inhibitor itraconazole, erythromycin had a similar impact on PB-201 systemic exposure (AUC 0-120 h increased by 31% and 61% under fasted and fed states, respectively). All predicted results are presented in Figure 5.

DISCUSSION
Absorption is a complex process that can be affected by formulation factors, physiological parameters, food composition, and so on. As the weakly basic insoluble oral immediate release solid formulation with moderate permeability, dissolution in the gastrointestinal tract is the first and pivotal step for PB-201 to enter into the systemic circulation. values, such as the log K m:w, neutral/ion . Moreover, the K p scalar for the volume of distribution was adjusted to match the PK profile, but the value in this paper was the optimal solution, which was obtained by performing a sensitivity analysis both S o and K p . Furthermore, there were no significant species differences among rats, dogs, and monkeys because the allometric exponent (b) in rats, dogs, and monkeys was about 0.74, which was calculated based on the clearance ( Generally, there exist a positive bidirectional association between T2DM and nonalcoholic fatty liver disease, 32,33 and the elderly account for about 20% of the total number of patients with diabetes. 16 The impaired liver function and physiological changes in patients with T2DM may be the key factors leading to the interindividual variation of PB-201 in vivo. Hence, simulation of PB-201 in liver impairment and geriatric populations is the crucial link to understand the potential effect of internal factors on systemic exposure and the necessary step to broaden the application groups. 14 However, the PBPK model may have a minor drawback in predicting the hepatic metabolic mechanism. Because the hydrolysis pathway in the hepatocytes was compensated by the "fit-for-purpose" method, and the value of CL int_Hep was obtained by fitting the PK profiles of PB-201 in the absence/presence of ketoconazole under the condition of fixing the CL int of each isoform (NCT01468714). Moreover, according to the percentage of remaining PB-201 in the incubation system and the protein abundance in human liver, 28 the f m calculated in this paper was significantly different from previously published results calculated based on the generation of metabolites (M1). 13 Excluding the contribution of nonoxidative pathways (about 35.3%) to PB-201 elimination in vivo, the f m of CYP3A4 in vitro calibrated according to the overall contribution percentage of oxidative metabolism (about 64.7%) in vivo was about 23.3%, which was close to the f m of CYP3A4 mediated PB-201 elimination in vivo (26.4%). Therefore, the metabolic stability of PB-201 in human recombinant CYP isoenzymes obtained in this paper was used to characterize the oxidative metabolic characteristics of PB-201 in vivo. Meanwhile, according to the calibrated f m in vitro, the ISEF was used to adjust the f m of each CYP isoform to the elimination of PB-201 in vivo. Although just the CYP3A4 pathway was confirmed by a DDI study in vivo, the predicted systemic clearance was consistent with the observed one and the liver metabolism was the main pathway to the elimination of PB-201 in the model. Therefore, the predicted systemic exposure of PB-201 in the specific populations can serve as an indicator for dose decision making in corresponding clinical trial designs.
PB-201 was eliminated through various cytochrome oxidases, among which CYP3A4 and CYP2C9 were the main isoenzymes of PB-201 metabolism, because the f m of CYP3A4 and CYP2C9 was 36.0% and 32.7%, respectively, among the isoenzymes investigated in this paper. Due to the PB-201 systemic exposure was slightly increased (about 26.4%) after co-administration with the strong CYP3A inhibitor in the DDI clinical study (NCT01468714), the f m of CYP2C9 to PB-201 metabolism in vivo might be less than 25%. Therefore, the DDI study in vivo did not further evaluate the effect of CYP2C9 perpetrators on PB-201 systemic exposure. Although ketoconazole could simultaneously inhibit the activities of CYP3A and CYP2C9 in SimCYP, the K i value (10 μM; Table S2) of ketoconazole against CYP2C9 was greater than the maximum concentration of ketoconazole in the liver (8.63 μM). Therefore, ketoconazole has no positive potential to inhibit CYP2C9-mediated metabolic pathways during simulation. However, other potential DDI scenarios need to be simulated by PBPK model, as some CYP3A4 inhibitors/inducers can simultaneously impact the activity of CYP3A and CYP2C9 in vivo. Under the preset scenarios, fluconazole (moderate CYP3A and CYP2C9 inhibitors) increased PB-201 systemic exposure in adults aged 20-50 years by 44% under fasted state and 78% under fed state, which were the greatest influence of the specific inhibitors mentioned in this paper both under fasted and fed stated, respectively. But the predicted systemic exposure (AUC 0-120 h and C max ) was still within the range of exposure-response analysis (unpublished). With the assumption that the same systemic exposure of PB-201 will produce the same pharmacological effect in vivo, PB-201 co-administration with specific inhibitors mentioned in this paper under both fasted and fed states may lead to hypoglycemia in some patients according to the exposure-response analysis results (unpublished). Considering that the CYP2C9 pathway has not been validated, it is better to evaluate the potential effect of fluconazole on PB-201 in clinical study. Additionally, rifampin (CYP3A strong inducer and CYP2C9 moderate inducer) and efavirenz (moderate CYP3A inducer) could significantly reduce PB-201 systemic exposure both under fasted and fed states. Considering that PB-201 is not a specific substrate of P-gp and the bioavailability of PB-201 in monkeys is moderate, 12 rifampin will not produce complex induction during absorption and metabolism. 27 Hence, the predicted results about the rifampin and efavirenz induction can be used as the reference, but still need to be confirmed in future clinical trials.
Chronic kidney disease is a common complication of T2DM, affecting 50% of the worldwide patients with T2DM. 34 Renal failure can reduce CL R , which in turn affects the activities of transporters and metabolic enzymes in the liver and gastrointestinal tract. 22,35 Therefore, the dosage of compounds eliminated through non-renal transport and metabolism should also be adjusted in patients with the nephropathy. 35 However, simulation in such a population was not performed in this study, as the parent drug of PB-201 is scarcely secreted (<1%) by the kidneys. Furthermore, the renal impairment population in the SimCYP population library has not established the relationship between the changes of liver and gastrointestinal function and renal impairment. Therefore, the simulation results of PB-201 in the renal impairment population cannot represent the systemic exposure in the patients with T2DM with chronic kidney disease, even if the simulation is performed. Accordingly, it is desirable to estimate PB-201 systemic exposure in patients with T2DM complications of chronic kidney disease to manage unknown risks clinically 36 and to expand the indication population in the future.
In retrospect, the PBPK model has become the favorable tool for applicants to understand untested scenarios in advance and then guide the clinical trial designs. Similarly, the mechanistic PB-201 PBPK model allows a conservative evaluation of the possible scenarios. Due to the finite clinical data, simulations of PB-201 in specific populations and co-administration with an inducer have not been validated. Therefore, such predicted results can only serve as a reference for investigators/regulators to inform precision doses in a scientific and well-founded manner. Additionally, because the liver impairment and geriatric populations in the SimCYP default population database are developed based on White patients, 37 and there is no racial difference in PB-201 exposure (unpublished data of population PKs), the predicted results in specific populations can also be used as indicators for future clinical trials in China.