DMPK perspective on quantitative model analysis for chimeric antigen receptor cell therapy: Advances and challenges.

Chimeric antigen receptor (CAR) cells are genetically engineered immune cells that specifically target tumor-associated antigens and have revolutionized cancer treatment, particularly in hematological malignancies, with ongoing investigations into their potential applications in solid tumors. This review provides a comprehensive overview of the current status and challenges in drug metabolism and pharmacokinetics (DMPK) for CAR cell therapy, specifically emphasizing on quantitative modeling and simulation (M&S). Furthermore, the recent advances in quantitative model analysis have been reviewed, ranging from clinical data characterization to mechanism-based modeling that connects in vitro and in vivo nonclinical and clinical study data. Additionally, the future perspectives and areas for improvement in CAR cell therapy translation have been reviewed. This includes using formulation quality considerations, characterization of appropriate animal models, refinement of in vitro models for bottom-up approaches, and enhancement of quantitative bioanalytical methodology. Addressing these challenges within a DMPK framework is pivotal in facilitating the translation of CAR cell therapy, ultimately enhancing the patients' lives through efficient CAR cell therapies.

Evaluation of the drug-drug interaction potential of brigatinib using a physiologically-based pharmacokinetic modeling approach.

Brigatinib is an oral anaplastic lymphoma kinase (ALK) inhibitor approved for the treatment of ALK-positive metastatic non-small cell lung cancer. In vitro studies indicated that brigatinib is primarily metabolized by CYP2C8 and CYP3A4 and inhibits P-gp, BCRP, OCT1, MATE1, and MATE2K. Clinical drug-drug interaction (DDI) studies with the strong CYP3A inhibitor itraconazole or the strong CYP3A inducer rifampin demonstrated that CYP3A-mediated metabolism was the primary contributor to overall brigatinib clearance in humans. A physiologically-based pharmacokinetic (PBPK) model for brigatinib was developed to predict potential DDIs, including the effect of moderate CYP3A inhibitors or inducers on brigatinib pharmacokinetics (PK) and the effect of brigatinib on the PK of transporter substrates. The developed model was able to predict clinical DDIs with itraconazole (area under the plasma concentration-time curve from time 0 to infinity [AUC∞] ratio [with/without itraconazole]: predicted 1.86; observed 2.01) and rifampin (AUC∞ ratio [with/without rifampin]: predicted 0.16; observed 0.20). Simulations using the developed model predicted that moderate CYP3A inhibitors (e.g., verapamil and diltiazem) may increase brigatinib AUC∞ by ~40%, whereas moderate CYP3A inducers (e.g., efavirenz) may decrease brigatinib AUC∞ by ~50%. Simulations of potential transporter-mediated DDIs predicted that brigatinib may increase systemic exposures (AUC∞) of P-gp substrates (e.g., digoxin and dabigatran) by 15%-43% and MATE1 substrates (e.g., metformin) by up to 29%; however, negligible effects were predicted on BCRP-mediated efflux and OCT1-mediated uptake. The PBPK analysis results informed dosing recommendations for patients receiving moderate CYP3A inhibitors (40% brigatinib dose reduction) or inducers (up to 100% increase in brigatinib dose) during treatment, as reflected in the brigatinib prescribing information.

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.

Biodistribution and delivery of oligonucleotide therapeutics to the central nervous system: Advances, challenges, and future perspectives.

Considerable advances have been made in the research and development of oligonucleotide therapeutics (OTs) for treating central nervous system (CNS) diseases, such as psychiatric and neurodegenerative disorders, because of their promising mode of action. However, due to the tight barrier function and complex physiological structure of the CNS, the efficient delivery of OTs to target the brain has been a major challenge, and intensive efforts have been made to overcome this limitation. In this review, we summarize the representative methodologies and current knowledge of biodistribution, along with the pharmacokinetic/pharmacodynamic (PK/PD) relationship of OTs in the CNS, which are critical elements for the successful development of OTs for CNS diseases. First, quantitative bioanalysis methods and imaging-based approaches for the evaluation of OT biodistribution are summarized. Next, information available on the biodistribution profile, distribution pathways, quantitative PK/PD modeling, and simulation of OTs following intrathecal or intracerebroventricular administration are reviewed. Finally, the latest knowledge on the drug delivery systems to the brain via intranasal or systemic administration as noninvasive routes for improved patient quality of life is reviewed. The aim of this review is to enrich research on the successful development of OTs by clarifying OT distribution profiles and pathways to the target brain regions or cells, and by identifying points that need further investigation for a mechanistic approach to generate efficient OTs.

Sinusoidal Organic Anion-Transporting Polypeptide 1B1/1B3 and Bile Canalicular Multidrug Resistance-Associated Protein 2 Play an Essential Role in the Hepatobiliary Disposition of a Synthetic Cyclic Dinucleotide (STING Agonist).

The liver is central to the elimination of many drugs from the body involving multiple processes and understanding of these processes is important to quantitively assess hepatic clearance of drugs. The synthetic STING (STimulator of INterferon Genes protein) agonist is a new class of drugs currently being evaluated in clinical trials as a potential anticancer therapy. In this study, we used ML00960317 (synthetic STING agonist) to investigate the hepatobiliary disposition of this novel molecular entity. A bile-duct cannulated (BDC) rat study indicated that biliary excretion is the major route of elimination for ML00960317 (84% of parent dose in bile). The human biliary clearance using in vitro sandwich cultured human hepatocyte model predicted significant biliary excretion of ML00960317 (biliary excretion index (BEI) of 47%). Moreover, the transport studies using transporter expressing cell lines, hepatocytes, and membrane vesicles indicated that ML00960317 is a robust substrate of OATP1B1, OATP1B3, and MRP2. Using relative expression factor approach, the combined contribution of OATP1B1 (fraction transported (ft) = 0.62) and OATP1B3 (ft = 0.31) was found to be 93% of the active uptake clearance of ML00960317 into the liver. Furthermore, OATP1B1 and OATP1B3-mediated uptake of ML00960317 was inhibited by rifampicin with IC50 of 6.5 and 2.3 µM, respectively indicating an in vivo DDI risk (R value of 1.5 and 2.5 for OATP1B1 and OATP1B3, respectively). These results highlighted an important role of OATP1B1, OATP1B3, and MRP2 in the hepatobiliary disposition of ML00960317. These pathways may act as rate-determining steps in the hepatic clearance of ML00960317 thus presenting clinical DDI risk.

Design, synthesis, and structure-activity relationship of TAK-418 and its derivatives as a novel series of LSD1 inhibitors with lowered risk of hematological side effects.

Lysine-specific demethylase 1 (LSD1) is an enzyme that demethylates methylated histone H3 lysine 4 (H3K4). Inhibition of LSD1 enzyme activity could increase H3K4 methylation levels and treat diseases associated with epigenetic dysregulation. However, known LSD1 inhibitors disrupt the interaction between LSD1 and cofactors such as GFI1B, causing the risk of hematological toxicity, including thrombocytopenia. Starting from a known LSD1 inhibitor (±)1 as a lead compound, a novel series of LSD1 inhibitors that do not induce the expression of GFI1 mRNA, an in vitro surrogate marker of LSD1-GFI1B dissociation, has been designed and synthesized. Initial structure-activity relationship (SAR) studies revealed the structural features key to avoiding GFI1 mRNA induction. Such SAR information enables optimization of LSD1 inhibitors with lowered risk of hematological side effects; TAK-418 ((1R,2R)-2n), the clinical candidate compound found through this optimization, has a hematological safety profile in rodents and humans. We further confirmed that oral administration of TAK-418 at 0.3 and 1 mg/kg for 2 weeks ameliorated memory deficits in mice with NMDA receptor hypofunction, suggesting potential of efficacy in neurodevelopmental disorders. TAK-418 warrants further investigation as a novel class of LSD1 inhibitors with a superior safety profile for the treatment of CNS disorders.

Fc-independent functions of anti-CTLA-4 antibodies contribute to anti-tumor efficacy.

Ipilimumab, a monoclonal antibody that recognizes cytotoxic T-lymphocyte associated protein 4 (CTLA-4), was the first immune checkpoint inhibitor approved by the FDA to treat metastatic melanoma patients. Multiple preclinical studies have proposed that Fc effector functions of anti-CTLA-4 therapy are required for anti-tumor efficacy, in part, through the depletion of intratumoral regulatory T cells (Tregs). However, the contribution of the Fc-independent functions of anti-CTLA-4 antibodies to the observed efficacy is not fully understood. H11, a non-Fc-containing single-domain antibody (VHH) against CTLA-4, has previously been demonstrated to block CTLA-4-ligand interaction. However, in vivo studies demonstrated lack of anti-tumor efficacy with H11 treatment. Here, we show that a half-life extended H11 (H11-HLE), despite the lack of Fc effector functions, induced potent anti-tumor efficacy in mouse syngeneic tumor models. In addition, a non-Fc receptor binding version of ipilimumab (Ipi-LALAPG) also demonstrated anti-tumor activity in the absence of Treg depletion. Thus, we demonstrate that Fc-independent functions of anti-CTLA-4 antibodies contributed to anti-tumor efficacy, which may indicate that non-Treg depleting activity of anti-CTLA-4 therapy could benefit cancer patients in the clinic.

Brain microvascular endothelial cells derived from human induced pluripotent stem cells as in vitro model for assessing blood-brain barrier transferrin receptor-mediated transcytosis.

The blood-brain barrier (BBB), a selective barrier formed by brain microvascular endothelial cells (BMEC), represents a major challenge for the efficient accumulation of pharmaceutical drugs into the brain. The receptor-mediated transcytosis (RMT) has recently gained increasing interest for pharmaceutical industry as it shows a great potential to shuttle large-sized therapeutic cargos across the BBB. Confirming the presence of the RMT pathway by BMEC is therefore important for the screening of peptides or antibody libraries that bind RMT receptors. Herein, a comparative study was performed between a human cell line of BMEC (HBEC) and human induced pluripotent stem cells-derived BMEC-like cells (hiPS-BMEC). The significantly higher gene and protein expressions of transporters and tight junction proteins, excepting CD31 and VE-cadherin were exhibited by hiPS-BMEC than by HBEC, suggesting more biomimetic BBB features of hiPS-BMEC. The presence and functionality of transferrin receptor (TfR), known to use RMT pathway, were confirmed using hiPS-BMEC by competitive binding assays and confocal microscopy observations. Finally, cysteine-modified T7 and cysteine modified-Tfr-T12 peptides, previously reported to be ligands of TfR, were compared regarding their permeability using hiPS-BMEC. The hiPS-BMEC could be useful for the identification of therapeutics that can be transported across the BBB using RMT pathway.

TAK-676: A Novel Stimulator of Interferon Genes (STING) Agonist Promoting Durable IFN-dependent Antitumor Immunity in Preclinical Studies.

Oncology therapies targeting the immune system have improved patient outcomes across a wide range of tumor types, but resistance due to an inadequate T-cell response in a suppressive tumor microenvironment (TME) remains a significant problem. New therapies that activate an innate immune response and relieve this suppression may be beneficial to overcome this hurdle. TAK-676 is a synthetic novel stimulator of interferon genes (STING) agonist designed for intravenous administration. Here we demonstrate that TAK-676 dose-dependently triggers activation of the STING signaling pathway and activation of type I interferons. Furthermore, we show that TAK-676 is a highly potent modulator of both the innate and adaptive immune system and that it promotes the activation of dendritic cells, natural killer cells, and T cells in preclinical models. In syngeneic murine tumor models in vivo, TAK-676 induces dose-dependent cytokine responses and increases the activation and proliferation of immune cells within the TME and tumor-associated lymphoid tissue. We also demonstrate that TAK-676 dosing results in significant STING-dependent antitumor activity, including complete regressions and durable memory T-cell immunity. We show that TAK-676 is well tolerated, exhibits dose-proportional pharmacokinetics in plasma, and exhibits higher exposure in tumor. The intravenous administration of TAK-676 provides potential treatment benefit in a broad range of tumor types. Further study of TAK-676 in first-in-human phase I trials is ongoing. Significance: TAK-676 is a novel systemic STING agonist demonstrating robust activation of innate and adaptive immune activity resulting in durable antitumor responses within multiple syngeneic tumor models. Clinical investigation of TAK-676 is ongoing.

Selective Acetyl-CoA Carboxylase 1 Inhibitor Improves Hepatic Steatosis and Hepatic Fibrosis in a Preclinical Nonalcoholic Steatohepatitis Model.

Acetyl-CoA carboxylase (ACC) 1 and ACC2 are essential rate-limiting enzymes that synthesize malonyl-CoA (M-CoA) from acetyl-CoA. ACC1 is predominantly expressed in lipogenic tissues and regulates the de novo lipogenesis flux. It is upregulated in the liver of patients with nonalcoholic fatty liver disease (NAFLD), which ultimately leads to the formation of fatty liver. Therefore, selective ACC1 inhibitors may prevent the pathophysiology of NAFLD and nonalcoholic steatohepatitis (NASH) by reducing hepatic fat, inflammation, and fibrosis. Many studies have suggested ACC1/2 dual inhibitors for treating NAFLD/NASH; however, reports on selective ACC1 inhibitors are lacking. In this study, we investigated the effects of compound-1, a selective ACC1 inhibitor for treating NAFLD/NASH, using preclinical in vitro and in vivo models. Compound-1 reduced M-CoA content and inhibited the incorporation of [14C] acetate into fatty acids in HepG2 cells. Additionally, it reduced hepatic M-CoA content and inhibited de novo lipogenesis in C57BL/6J mice after a single dose. Furthermore, compound-1 treatment of 8 weeks in Western diet-fed melanocortin 4 receptor knockout mice-NAFLD/NASH mouse model-improved liver hypertrophy and reduced hepatic triglyceride content. The reduction of hepatic M-CoA by the selective ACC1 inhibitor was highly correlated with the reduction in hepatic steatosis and fibrosis. These findings support further investigations of the use of this ACC1 inhibitor as a new treatment of NFLD/NASH. SIGNIFICANCE STATEMENT: This is the first study to demonstrate that a novel selective inhibitor of acetyl-CoA carboxylase (ACC) 1 has anti-nonalcoholic fatty liver disease (NAFLD) and anti-nonalcoholic steatohepatitis (NASH) effects in preclinical models. Treatment with this compound significantly improved hepatic steatosis and fibrosis in a mouse model. These findings support the use of this ACC1 inhibitor as a new treatment for NAFLD/NASH.

Discovery of a novel series of GPR119 agonists: Design, synthesis, and biological evaluation of N-(Piperidin-4-yl)-N-(trifluoromethyl)pyrimidin-4-amine derivatives.

We undertook an optimization effort involving propan-2-yl 4-({6-[5-(methanesulfonyl)-2,3-dihydro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate 1, which we had previously discovered as a novel G protein-coupled receptor 119 (GPR119) agonist. To occupy a presumed hydrophobic space between the pyrimidine and piperidine rings in interaction with GPR119, we replaced the linker oxygen with nitrogen. Subsequently, the introduction of a substituent at the bridging nitrogen atom was explored. We found that the installation of N-trifluoromethyl group 10 not only enhanced GPR119 agonist activity but also considerably improved the human ether-à-go-go-related gene (hERG) inhibition profile. These improvements were not observed for non-fluorinated substituents, such as ethyl analog 8b. The next optimization effort focused on the exploration of a new surrogate structure for the indoline ring and the isosteric replacements of the piperidine N-Boc group to improve solubility, metabolic stability, and oral bioavailability. As a result, N-{1-[3-(2-fluoropropan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}-6-{[1-(methanesulfonyl)piperidin-4-yl]oxy}-N-(trifluoromethyl)pyrimidin-4-amine (27) was identified as a potent and orally bioavailable GPR119 agonist. This compound augmented insulin secretion and effectively lowered plasma glucose excursion in a diabetic animal model after oral administration. In this study, we discuss the designs, syntheses, and biological activities of a novel series of N-(piperidin-4-yl)-N-(trifluoromethyl)pyrimidin-4-amine derivatives as GPR119 agonists, and to determine the distinctive effect of the N-trifluoromethyl group on hERG inhibition, we also discuss the conformational preference of representative compounds.

An Automated Multicycle Immunoaffinity Enrichment Approach Developed for Sensitive Mouse IgG1 Antibody Drug Analysis in Mouse Plasma Using LC/MS/MS.

In the immuno-oncology field, surrogate mouse monoclonal antibodies are often preferred in establishing proper PK/PD/efficacy correlations as well as supporting anticipated mouse to human translation. Thus, a highly sensitive and specific bioanalytical method is needed in quantifying those surrogate mouse antibodies after dosing in mice. Unfortunately, when specific reagents, such as recombinant target antigen and anti-idiotypic antibody, are not available, measuring mouse surrogate antibody drugs in mice is very challenging for ligand binding assay (LBA) due to the severe cross reactivity potential. Different from LBA, if at least one unique surrogate peptide can be identified from the surrogate antibody sequence, the immunoaffinity enrichment based LC/MS/MS assay may be able to differentiate the analyte response from the high endogenous immunoglobulin background and provide adequate sensitivity. Herein, a new automated multicycle immunoaffinity enrichment method was recently developed to extract a surrogate mouse IgG1 (mIgG1) antibody drug from mouse plasma using a commercially available antimouse IgG1 secondary antibody. In the assay, reuse of the capture antibody up to six times mostly resolved the binding capacity issue caused by the abundant endogenous mIgG1 and made the immunoaffinity enrichment step more cost-effective. Combined with a unique surrogate peptide identified from the antibody, the LC/MS/MS assay achieved a limit of quantitation of 5 ng/mL with satisfactory assay precision, accuracy, and dynamic range. The successful implementation of this novel approach in discovery pharmacokinetic (PK) studies eliminates the dependence on specially generated immunoaffinity capturing reagents.

Design, Synthesis, and Evaluation of (2-Aminocyclopropyl)phenyl Derivatives as Novel Positron Emission Tomography Imaging Agents for Lysine-Specific Demethylase 1 in the Brain.

Dysregulation of histone H3 lysine 4 (H3K4) methylation is implicated in the pathogenesis of neurodevelopmental disorders. Lysine-specific demethylase 1 (LSD1) determines the methylation status of H3K4 through flavin adenine dinucleotide (FAD)-mediated histone demethylation. Therefore, LSD1 inhibition in the brain can be a novel therapeutic option for treating these disorders. Positron emission tomography (PET) imaging of LSD1 allows for investigating LSD1 expression levels under normal and disease conditions and validating target engagement of therapeutic LSD1 inhibitors. This study designed and synthesized (2-aminocyclopropyl)phenyl derivatives with irreversible binding to LSD1 as PET imaging agents for LSD1 in the brain. We optimized lipophilicity of the lead compound to minimize the risk of nonspecific binding and identified 1e with high selectivity over monoamine oxidase A and B, which are a family of FAD-dependent enzymes homologous to LSD1. PET imaging in a monkey showed a high uptake of [18F]1e to regions enriched with LSD1, indicating its specific binding to LSD1.

Discovery of a novel series of indolinylpyrimidine-based GPR119 agonists: Elimination of ether-a-go-go-related gene liability using a hydrogen bond acceptor-focused approach.

We previously identified a novel series of indolinylpyrimidine derivatives exemplified by 2 in Figure 1, which is an indoline based derivative, as potent GPR119 agonists. Despite the attractive potency of 2, this compound inhibited the human ether-a-go-go-related gene (hERG) K+ channel. We elucidated crucial roles of the methylsulfonyl group of 2 in its interaction with the hERG channel and the GPR119 receptor, presumably as a hydrogen bond acceptor (HBA). To remove the undesirable hERG inhibitory activity, a strategy was implemented to arrange an HBA on a less conformationally flexible framework at the indoline 5-position instead of the methylsulfonyl group. This successfully led to the discovery of a piperidinone ring as a desirable motif at the indoline 5-position, which could minimize hERG liability as shown by 24b. Further optimization focused on the reduction of lipophilicity in terms of more favorable drug-like properties. Consequently, the introduction of a hydroxy group at the 3-position of the piperidinone ring effectively reduced lipophilicity without compromising GPR119 potency, resulting in the identification of (3S)-3-hydroxy-1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}oxy)pyrimidin-4-yl]- 2,3-dihydro-1H-indol-5-yl}piperidin-2-one ((S)-29) as a novel, potent, and orally bioavailable GPR119 agonist with a well-balanced profile. The pharmacological effects of this compound were also confirmed after single and chronic oral administration in diabetic animal models.

Characterization of plasma protein binding in two mouse models of humanized liver, PXB mouse and humanized TK-NOG mouse.

The unbound fractions in plasma (f up) in two mouse models of humanized liver mice, PXB and humanized TK-NOG mice, were compared with human f up values using equilibrium dialysis method. A good relationship between f up values obtained from PXB mice and humans was observed; the f up of 34/39 compounds (87.2%) in PXB mice were within 3-fold of human f up. In contrast, a weak correlation was observed between human and humanized TK-NOG mouse f up values; the f up of 15/24 compounds (62.5%) in humanized TK-NOG mice were within 3-fold of human f up. As different profiles of plasma protein binding (PPB) profiles were observed between PXB and humanized TK-NOG mice, f up evaluation is necessary in each mouse model to utilize these humanized liver mice for pharmacological, drug-drug interaction (DDI), and toxicity studies. The unbound fraction in the mixed plasma of human and SCID mouse plasma (85:15) was well correlated with f up in PXB mice (38/39 compounds within a 3-fold). Thus, this artificial PXB mouse plasma could be used to evaluate PPB.

A Translational Physiologically Based Pharmacokinetics/Pharmacodynamics Framework of Target-Mediated Disposition, Target Inhibition and Drug-Drug Interactions of Bortezomib.

Bortezomib is a potent 20S proteasome inhibitor approved for the treatment of multiple myeloma and mantle cell lymphoma. Despite the extensive clinical use of bortezomib, the mechanism of the complex time-dependent pharmacokinetics of bortezomib has not been fully investigated in context of its pharmacodynamics (PD) and drug-drug interaction (DDI) profiles. Here, we aimed to develop a mechanistic physiologically based (PB) PK/PD model to project PK, blood target inhibition and DDI of bortezomib in patients. A minimal PBPK/PD model consisting of six compartments was constructed using a bottom-up approach with pre-clinical data and human physiological parameters. Specifically, the target-mediated drug disposition (TMDD) of bortezomib in red blood cells (RBC), which determines target inhibition in blood, was characterized by incorporating the proteasome binding affinity of bortezomib and the proteasome concentration in RBC. The hepatic clearance and fraction metabolized by different CYP isoforms were estimated from in vitro metabolism and phenotyping experiments. The established model adequately characterized the multi-exponential and time-dependent plasma pharmacokinetics, target binding and blood proteasome inhibition of bortezomib. Further, the model was able to accurately predict the impact of a strong CYP3A inducer (rifampicin) and inhibitor (ketoconazole) on bortezomib exposure. In conclusion, the mechanistic PBPK/PD model successfully described the complex pharmacokinetics, target inhibition and DDIs of bortezomib in patients. This study illustrates the importance of incorporating target biology, drug-target interactions and in vitro clearance parameters into mechanistic PBPK/PD models and the utility of such models for pharmacokinetic, pharmacodynamic and DDI predictions.

Direct Drug Delivery of Low-Permeable Compounds to the Central Nervous System Via Intranasal Administration in Rats and Monkeys.

PURPOSE: Intranasal administration enhances drug delivery to the brain by allowing targeted-drug delivery. Here, we investigated the properties that render a compound suitable for intranasal administration, and the differences between rodents and non-human primates in delivery to the brain. METHODS: The delivery of 10 low-permeable compounds to the brain, including substrates of efflux drug transporters expressed in the blood-brain barrier (didanosine, metformin, zolmitriptan, cimetidine, methotrexate, talinolol, ranitidine, atenolol, furosemide, and sulpiride) and two high-permeable compounds (ropinirole and midazolam) was evaluated following intranasal and intravenous administration in rats. Six of the 12 compounds (metformin, cimetidine, methotrexate, talinolol, sulpiride, and ropinirole) were also evaluated in monkeys, which have a similar nasal cavity anatomical structure to humans. RESULTS: In rats, most of the low-permeable compounds displayed an obvious increase in the brain/plasma concentration ratio (Kp) by intranasal administration (despite their substrate liability for efflux drug transporters); this was not observed with the high-permeable compounds. Similarly, intranasal administration increased Kp for all low-permeable compounds in monkeys. CONCLUSIONS: Compound permeability is a key determinant of Kp increase by intranasal administration. This route of administration is more beneficial for low-permeable compounds and enhances their delivery to the brain in rodents and non-human primates.

Prediction of human pharmacokinetics of long half-life compounds using chimeric mice with humanised liver.

1. The prediction of human pharmacokinetic (PK) parameters is an important theme to select drug candidates from preclinical studies. It is essential to improve the prediction accuracy of compound half-life (t1/2) in humans. In this study, the predictability of t1/2 in humans using PXB mice®, chimeric mice with humanised liver, was assessed using 14 compounds showing long t1/2 in humans. 2. After intravenous administration of the compounds to PXB mice, the plasma concentration-time profiles were fitted using one- or two-compartment models and the human clearance (CLt) and distribution volume (Vdss) were predicted from single-species scaling. Using the obtained parameters, the t1/2 in humans was predicted. Using PXB mice, the predicted t1/2 values of 71.4% of the compounds were within two-fold of the actual values. Meanwhile, based on predictions using SCID mice, the host strain of the PXB mice, only 7.1% of tested compounds were within two-fold. 3. In conclusion, we demonstrated the novel utility of PXB mice for human PK predictions of compounds having long t1/2 in humans.

Application of unbound liver-to-plasma concentration ratio to quantitative projection of cytochrome P450-mediated drug-drug interactions using physiologically based pharmacokinetic modelling approach.

1. This study evaluated the prediction accuracy of cytochrome P450 (CYP)-mediated drug-drug interaction (DDI) using minimal physiologically-based pharmacokinetic (PBPK) modelling incorporating the hepatic accumulation factor of an inhibitor (i.e. unbound liver/unbound plasma concentration ratio [Kp,uu,liver]) based on 22 clinical DDI studies. 2. Kp,uu,liver values were estimated using three methods: (1) ratio of cell-to-medium ratio in human cryopreserved hepatocytes (C/Mu) at 37 °C to that on ice (Kp,uu,C/M), (2) multiplication of total liver/unbound plasma concentration ratio (Kp,u,liver) estimated from C/Mu at 37 °C with unbound fraction in human liver homogenate (Kp,uu,cell) and (3) observed Kp,uu,liver in rats after intravenous infusion (Kp,uu,rat). 3. PBPK model using each Kp,uu,liver projected the area under the curve (AUC) increase of substrates more accurately than the model assuming a Kp,uu,liver of 1 for the average fold error and root mean square error did. Particularly, the model with a Kp,uu,liver of 1 underestimated the AUC increase of triazolam following co-administration with CYP3A4 inhibitor itraconazole by five-fold, whereas the AUC increase projected using the model incorporating the Kp,uu,C/M, Kp,uu,cell, or Kp,uu,rat of itraconazole and hydroxyitraconazole was within approximately two-fold of the actual value. 4. The results indicated that incorporating Kp,uu,liver into the PBPK model improved the accuracy of DDI projection.

T-448, a specific inhibitor of LSD1 enzyme activity, improves learning function without causing thrombocytopenia in mice.

Dysregulation of histone H3 lysine 4 (H3K4) methylation has been implicated in the pathogenesis of several neurodevelopmental disorders. Targeting lysine-specific demethylase 1 (LSD1), an H3K4 demethylase, is therefore a promising approach to treat these disorders. However, LSD1 forms complexes with cofactors including growth factor independent 1B (GFI1B), a critical regulator of hematopoietic differentiation. Known tranylcypromine-based irreversible LSD1 inhibitors bind to coenzyme flavin adenine dinucleotide (FAD) and disrupt the LSD1-GFI1B complex, which is associated with hematotoxicity such as thrombocytopenia, representing a major hurdle in the development of LSD1 inhibitors as therapeutic agents. To discover LSD1 inhibitors with potent epigenetic modulation and lower risk of hematotoxicity, we screened small molecules that enhance H3K4 methylation by the inhibition of LSD1 enzyme activity in primary cultured rat neurons but have little impact on LSD1-GFI1B complex in human TF-1a erythroblasts. Here we report the discovery of a specific inhibitor of LSD1 enzyme activity, T-448 (3-((1S,2R)-2-(cyclobutylamino)cyclopropyl)-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzamide fumarate). T-448 has minimal impact on the LSD1-GFI1B complex and a superior hematological safety profile in mice via the generation of a compact formyl-FAD adduct. T-448 increased brain H3K4 methylation and partially restored learning function in mice with NMDA receptor hypofunction. T-448-type LSD1 inhibitors with improved safety profiles may provide unique therapeutic approaches for central nervous system disorders associated with epigenetic dysregulation.