Advanced translational PBPK model for transferrin receptor-mediated drug delivery to the brain.

Transferrin receptor (TfR)-mediated transcytosis is an attractive pathway for delivering large-molecule therapeutics to the central nervous system across the blood-brain barrier. Despite the clinical success of some drugs conjugated with TfR-binder, the desired drug profile for efficient TfR-mediated delivery to the targeted compartment within the brain, especially considering the species-related differences, has not been fully elucidated. To provide a prospective direction in the TfR-mediated drug delivery system, we developed an advanced physiologically based pharmacokinetic (PBPK) model. The model addresses TfR-mediated trans- and intracellular disposition of anti-TfR antibodies from brain capillary blood, endothelial cells, extracellular fluid (ECF), and eventually to brain parenchymal cells (BPCs), which correspond to pharmacological target sites of interest. The PBPK model is applicable in rats, monkeys, and human TfR knock-in (hTfR-KI) mice with satisfactory prediction accuracy through model calibration using the brain and plasma PK data of anti-TfR monoclonal antibodies, including their fused protein, with diverse binding affinity to TfR (TfR-Kd). The sensitivity analysis to determine drug properties required for the optimal brain delivery revealed 1) a bell-shaped relationship between TfR-Kd and brain exposure; 2) a minimum species difference between monkeys and hTfR-KI mice in the optimal TfR-Kd range, but not with rats; 3) a low TfR-Kd range to be preferably targeted for BPCs compared with ECF; and 4) an increase in brain exposure when using the pH-sensitive antibody. This may advance model-informed drug development, improve molecular design optimization, and provide precise human dose projection of drugs leveraging TfR-mediated shuttle technology into the brain.

Leveraging a physiologically-based quantitative translational modeling platform for designing B cell maturation antigen-targeting bispecific T cell engagers for treatment of multiple myeloma.

Bispecific T cell engagers (TCEs) are an emerging anti-cancer modality that redirects cytotoxic T cells to tumor cells expressing tumor-associated antigens (TAAs), thereby forming immune synapses to exert anti-tumor effects. Designing pharmacokinetically acceptable TCEs and optimizing their size presents a considerable protein engineering challenge, particularly given the complexity of intercellular bridging between T cells and tumor cells. Therefore, a physiologically-relevant and clinically-verified computational modeling framework is of crucial importance to understand the protein engineering trade-offs. In this study, we developed a quantitative, physiologically-based computational framework to predict immune synapse formation for a variety of molecular formats of TCEs in tumor tissues. Our model incorporates a molecular size-dependent biodistribution using the two-pore theory, extravasation of T cells and hematologic cancer cells, mechanistic bispecific intercellular binding of TCEs, and competitive inhibitory interactions by shed targets. The biodistribution of TCEs was verified by positron emission tomography imaging of [89Zr]AMG211 (a carcinoembryonic antigen-targeting TCE) in patients. Parameter sensitivity analyses indicated that immune synapse formation was highly sensitive to TAA expression, degree of target shedding, and binding selectivity to tumor cell surface TAAs over shed targets. Notably, the model suggested a "sweet spot" for TCEs' CD3 binding affinity, which balanced the trapping of TCEs in T-cell-rich organs. The final model simulations indicated that the number of immune synapses is similar (~55/tumor cell) between two distinct clinical stage B cell maturation antigen (BCMA)-targeting TCEs, PF-06863135 in an IgG format and AMG420 in a BiTE format, at their respective efficacious doses in multiple myeloma patients. This result demonstrates the applicability of the developed computational modeling framework to molecular design optimization and clinical benchmarking for TCEs, thus suggesting that this framework can be applied to other targets to provide a quantitative means to facilitate model-informed best-in-class TCE discovery and development.

Sinusoidal Uptake Determines the Hepatic Clearance of Pevonedistat (TAK-924) as Explained by Extended Clearance Model.

Quantitative assessment of hepatic clearance (CLH) of drugs is critical to accurately predict human dose and drug-drug interaction (DDI) liabilities. This is challenging for drugs that involve complex transporter-enzyme interplay. In this study, we demonstrate this interplay in the CLH and DDI effect in the presence of CYP3A4 perpetrator for pevonedistat using both the conventional clearance model (CCM) and the extended clearance model (ECM). In vitro metabolism and hepatocyte uptake data showed that pevonedistat is actively transported into the liver via multiple uptake transporters and metabolized predominantly by CYP3A4 (88%). The active uptake clearance (CLact,inf) and passive diffusion clearance (CLdiff,inf) were 21 and 8.7 ml/min/kg, respectively. The CLact,inf was underpredicted as Empirical Scaling Factor of 13 was needed to recover the in vivo plasma clearance (CLplasma). Both CCM and ECM predicted CLplasma of pevonedistat reasonably well (predicted CLplasma of 30.8 (CCM) and 32.1 (ECM) versus observed CLplasma of 32.2 ml/min/kg). However, both systemic and liver exposures in the presence of itraconazole were well predicted by ECM but not by CCM (predicted pevonedistat plasma area under the concentration-time curve ratio (AUCR) 2.73 (CCM) and 1.23 (ECM))., The ECM prediction is in accordance with the observed clinical DDI data (observed plasma AUCR of 1.14) that showed CYP3A4 inhibition did not alter pevonedistat exposure systemically, although ECM predicted liver AUCR of 2.85. Collectively, these data indicated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat and are consistent with the lack of systemic clinical DDI with itraconazole. SIGNIFICANCE STATEMENT: In this study, we successfully demonstrated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat. Both the conventional and extended clearance models predict CLplasma of pevonedistat well however, only the ECM accurately predicted DDI effect in the presence of itraconazole, thus providing further evidence for the lack of DDI with CYP3A4 perpetrators for drugs that involve complex transporter-enzyme interplay as there are currently not many examples in the literature except prototypical OATP substrate drugs.

Quantitative Systems Pharmacology Approaches for Immuno-Oncology: Adding Virtual Patients to the Development Paradigm.

Drug development in oncology commonly exploits the tools of molecular biology to gain therapeutic benefit through reprograming of cellular responses. In immuno-oncology (IO) the aim is to direct the patient's own immune system to fight cancer. After remarkable successes of antibodies targeting PD1/PD-L1 and CTLA4 receptors in targeted patient populations, the focus of further development has shifted toward combination therapies. However, the current drug-development approach of exploiting a vast number of possible combination targets and dosing regimens has proven to be challenging and is arguably inefficient. In particular, the unprecedented number of clinical trials testing different combinations may no longer be sustainable by the population of available patients. Further development in IO requires a step change in selection and validation of candidate therapies to decrease development attrition rate and limit the number of clinical trials. Quantitative systems pharmacology (QSP) proposes to tackle this challenge through mechanistic modeling and simulation. Compounds' pharmacokinetics, target binding, and mechanisms of action as well as existing knowledge on the underlying tumor and immune system biology are described by quantitative, dynamic models aiming to predict clinical results for novel combinations. Here, we review the current QSP approaches, the legacy of mathematical models available to quantitative clinical pharmacologists describing interaction between tumor and immune system, and the recent development of IO QSP platform models. We argue that QSP and virtual patients can be integrated as a new tool in existing IO drug development approaches to increase the efficiency and effectiveness of the search for novel combination therapies.

Mechanistic Multilayer Quantitative Model for Nonlinear Pharmacokinetics, Target Occupancy and Pharmacodynamics (PK/TO/PD) Relationship of D-Amino Acid Oxidase Inhibitor, TAK-831 in Mice.

PURPOSE: TAK-831 is a highly selective and potent inhibitor of D-amino acid oxidase (DAAO) currently under clinical development for schizophrenia. In this study, a mechanistic multilayer quantitative model that parsimoniously connects pharmacokinetics (PK), target occupancy (TO) and D-serine concentrations as a pharmacodynamic (PD) readout was established in mice. METHODS: PK, TO and PD time-profiles were obtained in mice and analyzed by mechanistic binding kinetics model connected with an indirect response model in a step wise fashion. Brain distribution was investigated to elucidate a possible mechanism driving the hysteresis between PK and TO. RESULTS: The observed nonlinear PK/TO/PD relationship was well captured by mechanistic modeling framework within a wide dose range of TAK-831 in mice. Remarkably different brain distribution was observed between target and reference regions, suggesting that the target-mediated slow binding kinetics rather than slow penetration through the blood brain barrier caused the observed distinct kinetics between PK and TO. CONCLUSION: A quantitative mechanistic model for concentration- and time-dependent nonlinear PK/TO/PD relationship was established for TAK-831 in mice with accounting for possible rate-determining process. The established mechanistic modeling framework will provide a quantitative means for multilayer biomarker-assisted clinical development in multiple central nervous system indications.

A Citrulline-Based Translational Population System Toxicology Model for Gastrointestinal-Related Adverse Events Associated With Anticancer Treatments.

Gastrointestinal (GI)-related adverse events (AEs) are commonly observed in the clinic during cancer treatments. Citrulline is a potentially translatable biomarker of GI AEs. In this study, irinotecan-induced citrulline changes were studied for a range of doses and schedules in rats. A translational system toxicology model for GI AEs using citrulline was then developed based on new experimental data and parameters from a literature intestinal cell dynamic model. With the addition of feedback-development and tolerance-development mechanisms, the model well captured the plasma citrulline profiles after irinotecan treatment in rats. Subsequently, the model was translated to humans and predicted the observed GI AE dynamics in humans including dose-scheduling effect using the cytotoxic and feedback parameters estimated in rats with slight calibrations. This translational toxicology model could be used for other antineoplastic drugs to simulate various clinical dosing scenarios before human studies and mitigate potential GI AEs.

A validated simultaneous quantification method for vonoprazan (TAK-438F) and its 4 metabolites in human plasma by the liquid chromatography-tandem mass spectrometry.

Vonoprazan fumarate (TAK-438) is a potassium-competitive acid blocker which was approved in Japan for a treatment of acid-related diseases. In this study a simple and validated bioanalytical method, which can simultaneously determine vonoprazan (TAK-438F) and its four metabolites (M-I, M-II, M-III and M-IV-Sul) in human plasma, was developed. The method is based on protein precipitation and subsequent ultra-high performance liquid chromatography separation followed by tandem mass spectrometry detection. The mass spectrometric parameters for detection of TAK-438F, M-I, M-III and M-IV-Sul were modified from their optimum values in order to achieve a simultaneous quantification while retaining enough sensitivity and wide dynamic ranges for all the target analytes. The validity and robustness of the method was verified through a validation study as per the regulatory guidance on bioanalytical method validation. The calibration ranges are 0.1-100 ng/mL for TAK-438F and M-III, and 1-1000 ng/mL for M-I, M-II and M-IV-Sul using the 100 µL of human plasma. The total run time per sample is 5 min. The working solution for M-III was recommended to be prepared separately, especially for the long-term use, in order to avoid the instability of M-III in the mixed working solutions, which could cause the high consumption of reference standards. The established method was applied to clinical pharmacokinetic studies and concentrations of all the analytes in human plasma were successfully determined with high reproducibility ensured by incurred sample reanalysis, indicating the suitableness of the established method.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Single Rising TAK-438 (Vonoprazan) Doses in Healthy Male Japanese/non-Japanese Subjects.

OBJECTIVES: To evaluate safety, tolerability, pharmacokinetics, and pharmacodynamics of TAK-438 (vonoprazan, a potassium-competitive acid blocker) in healthy male subjects. METHODS: In two phase I, randomized, double-blind, placebo-controlled, single rising-dose studies, healthy male subjects (Japan N=84; UK N=63) received a single TAK-438 dose (1-120 mg in Japan and 1-40 mg in the UK). Assessments included safety, tolerability, pharmacokinetics, and pharmacodynamics (intragastric pH). RESULTS: Plasma concentration-time profiles of TAK-438 at all dose levels showed rapid absorption (median Tmax up to 2 h). Estimated mean elimination half-life was up to 9 h. Exposure was slightly greater than dose proportional. No clear difference in TAK-438 pharmacokinetics was observed between Japanese and non-Japanese subjects. Acid suppression was dose dependent and similar in both studies. The 24-h intragastric pH ≥4 holding time ratio with 40 mg TAK-438 was 92% in Japan and 87% in the UK. TAK-438 was well tolerated, with no adverse events reported in Japanese subjects; 10 of 63 UK subjects experienced 12 treatment-emergent adverse events (non-serious). Increases in serum gastrin and pepsinogen I and II concentrations were observed at doses ≥10 mg, but there were no changes in alanine aminotransferase concentrations. CONCLUSIONS: Single oral doses of TAK-438 20-120 mg caused rapid, profound, and 24-h suppression of gastric acid secretion in healthy male subjects, regardless of geographical region, and TAK-438 was well tolerated at all doses studied, making it a potential alternative to proton pump inhibitors for the treatment of acid-related disorders.

Retrospective data analysis and proposal of a practical acceptance criterion for inter-laboratory cross-validation of bioanalytical methods using liquid chromatography/tandem mass spectrometry.

The purpose of this study is to conduct a retrospective data analysis for inter-laboratory cross-validation studies to set a reasonable and practical acceptance criterion based on a number of cross-validation results. From the results of cross-validation studies for 16 compounds and their metabolites, analytical bias and variation were evaluated. The accuracy of cross-validation samples was compared with that of quality control (QC) samples with statistical comparison of the analytical variation. An acceptance criterion was derived with a confidential interval approach. As the results, while a larger bias was observed for the cross-validation samples, the bias was not fully caused by analytical variation or bias attributable to the analytical methods. The direction of the deviation between the cross-validation samples and QC samples was random and not concentration-dependent, suggesting that inter-laboratory variability such as preparation errors could be a source of bias. A derived acceptance criterion corresponds to one prescribed in the Guideline on bioanalytical method validation from the Ministry of Health, Labour and Welfare in Japan and is a little wider than one in the European Medical Agency. In conclusion, thorough retrospective data analysis revealed potential causes of larger analytical bias in inter-laboratory cross-validation studies. A derived acceptance criterion would be practical and reasonable for the inter-laboratory cross-validation study.

Chemical reactivity of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) in vitro.

Ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) was metabolized to cyclohexene and phenyl ring moieties in non-clinical pharmacokinetic studies and it was suggested that the cyclohexene ring moiety of TAK-242 is tightly bound to endogenous macromolecules. After incubation of TAK-242 and glutathione (GSH) in phosphate buffer (pH 7.4) at 37 °C, TAK-242 reacted with GSH to produce a glutathione conjugate of the cyclohexene ring moiety of TAK-242, which had been observed as a metabolite (M-SG) in non-clinical pharmacokinetic studies. Formation of M-SG was time dependent with a first order reaction and M-I, a metabolite from the phenyl ring moiety of TAK-242, was also produced in parallel. The formation of M-SG was accelerated with increasing pH, therefore it was indicated that TAK-242 reacted with GSH by a nucleophilic substitution reaction. Because glutathione transferase (GST) enhanced M-SG formation in vitro, it is expected that the conjugation of TAK-242 with GSH is also facilitated by GST in vivo in addition to a spontaneous chemical reaction. When radio-labeled TAK-242 ([cyclohexene ring-U-¹4C]TAK-242) was incubated with rat serum albumin (RSA) or human serum albumin (HSA) in vitro, the radioactive material was covalently bound to RSA and HSA, and M-I was generated simultaneously in the reaction mixture. The chemical structure of the TAK-242 adduct covalently bound to HSA was characterized by the accurate mass spectra that cyclohexene ring moiety of TAK-242 was covalently bound to the lysine residue in HSA. The adduct was also detected in the plasma of rats and humans after single i.v. dosing of TAK-242 (in vivo).

In vivo relationship between thalamic nicotinic acetylcholine receptor occupancy rates and antiallodynic effects in a rat model of neuropathic pain: persistent agonist binding inhibits the expression of antiallodynic effects.

We have recently clarified that nicotinic acetylcholine receptors (nAChRs) expressed in the thalamus play an important role in antiallodynic effects produced by the nAChR agonist, 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5IA). This study aimed to reveal the in vivo relationship between thalamic nAChR occupancy rates and antiallodynic effects using 5IA and [¹²5I]5IA. We partially ligated the sciatic nerve of a rat to induce neuropathic pain. Antiallodynic effects were evaluated at 15, 30, 60, and 90 min after intracerebroventricular (i.c.v.) administration of multiple doses (1-100 nmol) of 5IA by the von Frey filament test. Receptor occupancy rates were measured by autoradiography at 15 and 90 min after administration. Antiallodynic effects of repetitive treatment of 5IA (5 and 50 nmol) were also examined. A significant and dose-dependent antiallodynic effect was observed 15 min after administration. It showed a good correlation with receptor occupancy rates (r = 0.97), indicating the binding of 5IA to nAChRs expressed in the thalamus involved in the antiallodynic effect. Five, 50, and 100 nmol of 5IA occupied the thalamic nAChRs until 90 min after administration, while the antiallodynic effect diminished. Five nanomoles of 5IA (which occupied 40% of thalamic nAChRs) showed a significant antiallodynic effect (percentage of the maximal possible effect (%MPE): 35 ± 7) after the second administration, while 50 nmol of 5IA (which occupied 80% of thalamic nAChRs) did not (%MPE: 7 ± 1). These findings suggest that not clearance of 5IA but desensitization of nAChRs caused by persistent binding of 5IA is responsible for the disappearance of antiallodynic effects.

Studies on 1-alkylamine adduct formation in electrospray ionization mass spectrometry for quantitative analysis.

We investigated the formation of 1-alkylamine adduct ions using 1,2-, 1,3-, and 1,4-cyclohexanediol (CHD) as model compounds and the relationships of the peak intensity between the protonated molecules ([M+H]+), sodium adduct ions ([M+Na]+), and 1-alkylamine adduct ions ([M+A+H]+) of 16 model compounds using electrospray ionization mass spectrometry. When 1-octylamine was added to 1,2-, 1,3-, and 1,4-CHD solutions, the peak intensity of the 1-octylamine adduct ions ([M+Oct+H]+) was higher than those of [M+H]+ and [M+Na]+. The highest peak intensity of [M+Oct+H]+ was observed in 1,2-CHD, followed by 1,3-CHD and 1,4-CHD and this order was the same as that of [M+Na]+ for CHDs in the solution without 1-octylamine. These results suggest that the mechanism of formation of [M+Oct+H]+ is similar to that of [M+Na]+ and that adduct formation seems to occur between 1-octylamine and two oxygen atoms in CHD in a similar manner to [M+Na]+. Based on these results, 16 model compounds including CHDs were investigated with respect to the relationship between [M+Na]+ and [M+A+H]+. A positive correlation was observed between the peak intensities of [M+Na]+ and [M+A+H]+, supporting that the formation mechanism of [M+A+H]+ is potentially similar to the [M+Na]+ formation mechanism. These data indicate that a sensitivity enhanced quantitative analysis using [M+A+H]+ could be a feasible approach for compounds generating [M+Na]+.