Kimio Tohyama

Functional Characterization of Mouse Organic Anion Transporting Peptide 1a4 in the Uptake and Efflux of Drugs Across the Blood-Brain Barrier

This study investigated the role of a multispecific organic anion transporter, Oatp1a4/Slco1a4, in drug transport across the blood-brain barrier. In vitro transport studies using human embryonic kidney 293 cells expressing mouse Oatp1a4 identified the following compounds as Oatp1a4 substrates: pitavastatin (Km = 8.3 μM), rosuvastatin (Km = 12 μM), pravastatin, taurocholate (Km = 40 μM), digoxin, ochratoxin A, and [d-penicillamine2,5]-enkephalin. Double immunohistochemical staining of Oatp1a4 with P-glycoprotein (P-gp) or glial fibrillary acidic protein demonstrated that Oatp1a4 signals colocalized with P-gp signals partly but not with glial fibrillary acidic protein, suggesting that Oatp1a4 is expressed in both the luminal and the abluminal membranes of mouse brain capillary endothelial cells. The brain-to-blood transport of pitavastatin, rosuvastatin, pravastatin, and taurocholate after microinjection into the cerebral cortex was significantly decreased in Oatp1a4(−/−) mice compared with that in wild-type mice. The blood-to-brain transport of pitavastatin, rosuvastatin, taurocholate, and ochratoxin A, determined by in situ brain perfusion, was significantly lower in Oatp1a4(−/−) mice than in wild-type mice, whereas transport of pravastatin and [d-penicillamine2,5]-enkephalin was unchanged. The blood-to-brain transport of digoxin was significantly lower in Oatp1a4(−/−) mice than in wild-type mice only when P-gp was inhibited by N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). Taken together, these results show that Oatp1a4 can mediate the brain-to-blood and blood-to-brain transport of its substrate drugs across the blood-brain barrier. The brain-to-plasma ratio of taurocholate, pitavastatin, and rosuvastatin was close to the capillary volume in wild-type mice, and it was not affected by Oatp1a4 dysfunction. Whether Oatp1a4 can deliver drugs from the blood to the brain remains controversial.

Characterization of binding and inhibitory properties of TAK-063, a novel phosphodiesterase 10A inhibitor.

Phosphodiesterase 10A (PDE10A) inhibition is a novel and promising approach for the treatment of central nervous system disorders such as schizophrenia and Huntington’s disease. A novel PDE10A inhibitor, TAK-063 [1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)-pyridazin-4(1H)-one] has shown high inhibitory activity and selectivity for human recombinant PDE10A2 in vitro; the half-maximal inhibitory concentration was 0.30 nM, and selectivity over other phosphodiesterases (PDEs) was more than 15000-fold. TAK-063 at 10 µM did not show more than 50% inhibition or stimulation of 91 enzymes or receptors except for PDEs. In vitro autoradiography (ARG) studies using rat brain sections revealed that [3H]TAK-063 selectively accumulated in the caudate putamen (CPu), nucleus accumbens (NAc), globus pallidus, substantia nigra, and striatonigral projection, where PDE10A is highly expressed. This [3H]TAK-063 accumulation was almost entirely blocked by an excess amount of MP-10, a PDE10A selective inhibitor, and the accumulation was not observed in brain slices of Pde10a-knockout mice. In rat brain sections, [3H]TAK-063 bound to a single high-affinity site with mean ± SEM dissociation constants of 7.2 ± 1.2 and 2.6 ± 0.5 nM for the CPu and NAc shell, respectively. Orally administered [14C]TAK-063 selectively accumulated in PDE10A expressing brain regions in an in vivo ARG study in rats. Striatal PDE10A occupancy by TAK-063 in vivo was measured using T-773 as a tracer and a dose of 0.88 mg/kg (p.o.) was calculated to produce 50% occupancy in rats. Translational studies with TAK-063 and other PDE10A inhibitors such as those presented here will help us better understand the pharmacological profile of this class of potential central nervous system drugs.

Pharmacokinetic and Pharmacodynamic Modeling of Hedgehog Inhibitor TAK-441 for the Inhibition of Gli1 messenger RNA Expression and Antitumor Efficacy in Xenografted Tumor Model Mice

6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-3-(2,2,2-trifluoroethoxy)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (TAK-441) is a potent, selective hedgehog signaling pathway inhibitor that binds to Smo and is being developed for the treatment of cancer. The objectives of these studies were to explore the possibility of establishing of a link between the pharmacokinetics of TAK-441 and the responses of Gli1 mRNA in tumor-associated stromal or skin cells and the antitumor effect of hedgehog inhibition. To this end, we built pharmacokinetic and pharmacodynamic models that describe the relationship of the concentrations of TAK-441 plasma to the responses of Gli1 mRNA in the tumor (target) and skin (surrogate) and to tumor growth inhibition in mice bearing xenografts of human pancreatic tumors (PAN-04). The responses of Gli1 mRNA and tumor growth were described by an indirect response model and an exponential tumor growth model, respectively. The IC50 values for Gli1 mRNA inhibition in the tumor and skin by TAK-441 were estimated to be 0.0457 and 0.113 μg/ml, respectively. The IC90 value for tumor growth inhibition was estimated to be 0.68 μg/ml. These results suggest that a >83% inhibition of Gli1 mRNA expression in the skin or a >94% inhibition of Gli1 mRNA expression in the tumor would be required to sufficiently inhibit (>90%) hedgehog-related tumor growth in the xenografted model mice. We conclude that Gli1 mRNA expression in the tumor and skin could be a useful biomarker for predicting the antitumor effect of hedgehog inhibitors

Disease‐Associated Changes in Drug Transporters May Impact the Pharmacokinetics and/or Toxicity of Drugs: A White Paper From the International Transporter Consortium

Drug transporters are critically important for the absorption, distribution, metabolism, and excretion (ADME) of many drugs and endogenous compounds. Therefore, disruption of these pathways by inhibition, induction, genetic polymorphisms, or disease can have profound effects on overall physiology, drug pharmacokinetics, drug efficacy, and toxicity. This white paper provides a review of changes in transporter function associated with acute and chronic disease states, describes regulatory pathways affecting transporter expression, and identifies opportunities to advance the field.

Mechanism for Covalent Binding of MLN3126, an Oral Chemokine C-C Motif Receptor 9 Antagonist, to Serum Albumins

N-{4-Chloro-2-[(1-oxidopyridin-4-yl)carbonyl]phenyl}-4-(propan-2-yloxy)benzenesulfonamide (MLN3126) is an orally available chemokine C-C motif receptor 9 selective antagonist. In nonclinical pharmacokinetic studies of MLN3126, nonextractable radioactivity was observed in plasma after oral administration of 14C-labeled MLN3126 ([14C]MLN3126) to Sprague-Dawley (SD) rats. In this study, the nonextractable radioactive component was digested with trypsin or a nonspecific protease, pronase, after chemical reduction to obtain drug-peptide adducts or drug-amino acid adducts. The chemical structure of these adducts was characterized by liquid chromatography/mass spectrometry. The results demonstrated that the major part of the nonextractable radioactivity was accounted for by covalent binding via the Schiff base formed specifically between the ε-amino group of lysine residue 199 in rat serum albumin and the carbonyl group of MLN3126. The half-life (t1/2) of the total radioactivity in plasma during and after 21 daily multiple oral administrations of [14C]MLN3126 to SD rats was approximately 5-fold shorter than the reported t1/2 of albumin in rats. The data indicated that the covalent binding was reversible under physiologic conditions. The formation of the covalent binding was also confirmed in in vitro incubations with serum albumins from rats, humans, and dogs in the same manner, indicating that there are no qualitative interspecies differences in the formation of the Schiff base.

Abstract 4508: Nonclinical pharmacokinetics of TAK-441, a hedgehog pathway inhibitor

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL TAK-441 is a potent and selective smoothened (Smo) antagonist that blocks hedgehog (Hh) signaling. TAK-441 is being developed for the treatment of advanced nonhematologic and hematologic malignancies. In vivo pharmacokinetic (PK) studies using rats and dogs and various in vitro studies were conducted to describe the PK of TAK-441 to support its clinical development. After oral administration of TAK-441 and/or [14C]TAK-441 to rats and dogs, plasma, urine, feces and bile were collected at designated time points. The concentrations of TAK-441 and total radioactivity in the biological samples were measured by high performance liquid chromatography / tandem mass spectrometry and liquid scintillation counter, respectively. The PK of TAK-441 was characterized in rats and dogs by low plasma clearance (397.9 and 161.3 mL/hr/kg), moderate plasma volume of distribution at steady state (681.6 and 2181.3 mL/kg), a moderate terminal elimination half-life (t1/2) (1.7 and 9.8 hours), and an oral bioavailability of 31.7% and 90.3%, respectively. A potential food effect was observed in dogs with an approximate 2-fold increase in the extent of absorption when TAK-441 was dosed to fed dogs (compared with fasted dogs), producing both a higher maximal drug concentration (Cmax) and area under the plasma concentration versus time curve from 0 to 24 hours (AUC0-24h). At 1 μg/mL, TAK-441 had high plasma protein binding in mice (99.7%) and rats (96.2%) and had lower plasma protein binding in dogs (79.6%) and humans (87.7%). No metabolites unique to humans were detected after incubation in hepatic microsomes. CYP3A4/5 was the major CYP isozyme contributing to the metabolism of TAK-441, followed by CYP2C19 and 2C9. The rank order of contribution of the five major human CYP isozymes to TAK-441 metabolism (determined using chemical inhibitors) was 3A4/5 (80.3%) >2C19 (14.4%) >2C9 (5.2%) >1A2 and 2D6 (<1%). TAK-441 showed no appreciable induction of CYP1A2 or CYP3A4/5 activity in human primary hepatocytes. TAK-441 was a weak inhibitor of CYP2C8 (IC50 of 25 μM) and had no inhibitory effect on the other CYP isoforms. TAK-441 was not a time-dependent CYP inhibitor in human liver microsomes. Taken together, there is a low potential for TAK-441 to affect the PK of other concomitantly administered CYP substrates. However, the PK of TAK-441 could be affected by concomitantly administered strong CYP3A4/5 inhibitors and inducers. TAK-441 was mainly excreted in the feces as metabolites. Urinary excretion of the parent TAK-441 was quite low (LOQ and <1% of dose in rats and dogs, respectively). TAK-441 has high permeability in Caco-2 cells, is a poor substrate for efflux pump P-glycoprotein (P-gp), and is a weak inhibitor of P-gp (IC50 of 6.59 μM). In conclusion, TAK-441 has a favourable nonclinical absorption, distribution, metabolism, and excretion (ADME) profile for clinical evaluation and development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4508. doi:10.1158/1538-7445.AM2011-4508

Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury

Fibrosis of the lung constitutes a major clinical challenge and novel therapies are required to alleviate the associated morbidity and mortality. Investigating the antifibrotic efficacy of drugs that are already in clinical practice offers an efficient strategy to identify new therapies. The phosphodiesterase 4 (PDE4) inhibitors, approved for the treatment of chronic obstructive pulmonary disease, harbor therapeutic potential for pulmonary fibrosis by augmenting the activity of endogenous antifibrotic mediators that signal through cyclic AMP. In this study, we tested the efficacy of several PDE4 inhibitors including a novel compound (Compound 1) in a murine model of lung fibrosis that results from a targeted type II alveolar epithelial cell injury. We also compared the antifibrotic activity of PDE4 inhibition to the two therapies that are FDA‐approved for idiopathic pulmonary fibrosis (pirfenidone and nintedanib). We found that both preventative (day 0–21) and therapeutic (day 11–21) dosing regimens of the PDE4 inhibitors significantly ameliorated the weight loss and lung collagen accumulation that are the sequelae of targeted epithelial cell damage. In a therapeutic protocol, the reduction in lung fibrosis with PDE4 inhibitor administration was equivalent to pirfenidone and nintedanib. Treatment with this class of drugs also resulted in a decrease in plasma surfactant protein D concentration, a reduction in the plasma levels of several chemokines implicated in lung fibrosis, and an in vitro inhibition of fibroblast profibrotic gene expression. These results motivate further investigation of PDE4 inhibition as a treatment for patients with fibrotic lung disease.

Fluorescent Imaging Analysis for Distribution of Fluorescent Dye Labeled- or Encapsulated-Liposome in Monocrotaline-Induced Pulmonary Hypertension Model Rat

Pulmonary hypertension (PH) is a life-threatening lung disease. Despite the availability of several approved drugs, the development of a new treatment method is needed because of poor prognosis. Tissue selective drug delivery systems can avoid the adverse effects of current therapy and enhance efficacy. We evaluated the possibility of delivering drugs to the lungs of a PH rat model using fluorescence dye-labeled nanosized liposomes. To evaluate the tissue distribution following systemic exposure, fluorescent dye-labeled, 40-180 nm liposomes with and without polyethylene glycol (PEG) were intravenously administered to a monocrotaline-induced PH (MCT) rat model and tissue fluorescence was measured. Fluorescent dye-containing liposomes were intratracheally administered to the MCT model to evaluate the distribution of the liposome-encapsulated compound following local administration to reduce systemic exposure. The lung vascular permeability, plasma concentration of surfactant protein (SP)-D, lung reactive oxygen species (ROS) production, and macrophage marker gene cluster of differentiation (CD68) expression were measured. PEG and 80-nm liposome accumulation in the lung was elevated in the MCT model compared to that in normal rats. The intratracheally administered liposomes were delivered selectively to the lungs of the MCT model. The lung vascular permeability, plasma SP-D concentration, and CD68 expression were significantly elevated in the lungs of the MCT model, and were all significantly and positively correlated to liposome lung accumulation. Liposomes can accumulate in the lungs of an MCT model by enhancing vascular permeability by the inflammatory response. Therefore, drug encapsulation in liposomes could be an effective method of drug delivery in patients with PH.

Combination effects of alogliptin and pioglitazone on steatosis and hepatic fibrosis formation in a mouse model of non-alcoholic steatohepatitis

This study aimed to evaluate the effects of combination therapy with a dipeptidyl peptidase-4 inhibitor, alogliptin, and a peroxisome proliferator-activated receptor-γ agonist, pioglitazone, in a preclinical model of nonalcoholic steatohepatitis using low-density lipoprotein receptor-knockout mice fed a modified choline-deficient l-amino acid-defined diet. Monotherapy with either alogliptin (10-200 mg/kg) or pioglitazone (6-20 mg/kg) significantly decreased hepatic triglyceride content and fibrosis. The concomitant treatment of alogliptin (30 mg/kg), pioglitazone (20 mg/kg) also decreased hepatic triglyceride and hepatic collagen-I mRNA at greater extent compared to monotherapy. Hepatic expression of CD11b mRNA and monocyte chemoattractant protein-1 were also reduced by the concomitant treatment. These results suggest that via an anti-inflammatory potential in addition to anti-metabolic effects, the combination therapy of alogliptin and pioglitazone may provide therapeutic benefits to type 2 diabetes patients with nonalcoholic steatohepatitis, which will be proven in controlled clinical trials.

Pre-clinical Characterization of Absorption, Distribution, Metabolism and Excretion Properties of TAK-063

TAK‐063 is currently being developed to treat schizophrenia. In this study, we investigated the absorption, distribution, metabolism and excretion (ADME) properties of TAK‐063 using several paradigms. Following oral administration of TAK‐063 at 0.3 mg/kg, bioavailability of TAK‐063 was 27.4% in rats and 49.5% in dogs with elimination half‐lives of 3.1 hr in rats and 3.7 hr in dogs. TAK‐063 is a highly permeable compound without P‐glycoprotein (P‐gp) or breast cancer resistance protein substrate liability and can be readily absorbed into systemic circulation via the intestine. TAK‐063 can also cross the blood–brain barrier. TAK‐063 was metabolized mainly by CYP2C8 and CYP3A4/5, while incubation with human liver microsomes produced the major human metabolite, M‐I as well as several unknown minor metabolites. Metabolism of TAK‐063 to M‐I occurs through hydroxylation of the mono‐substituted pyrazole moiety. In vitro, TAK‐063 was observed to inhibit CYP2C8, CYP2C19 and P‐gp with IC50 values of 8.4, 12 and 7.13 μM, respectively. TAK‐063 was primarily excreted in the faeces in rats and dogs with M‐I as a predominant component. The pre‐clinical data from these ADME studies demonstrate a favourable pharmacokinetic profile for TAK‐063 with good brain distribution supporting the feasibility of targeting central nervous system regions involved in schizophrenia pathophysiology. TAK‐063 has recently been investigated in a phase 2 clinical trial (NCT02477020).