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Purpose The purpose of this study was to construct and validate an in vitro three-dimensional blood–brain barrier (3DBBB) model system equipped with brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs). Methods The 3D-BBB system was constructed by seeding hiPS-BMECs onto the capillary lane of a MIMETAS OrganoPlate® 3-lane coated with fibronectin/collagen IV. hiPS-BMECs were incubated under continuous switchback flow with an OrganoFlow® for 2 days. The 3D capillary structure and expression of tight-junction proteins and transporters were confirmed by immunocytochemistry. The mRNA expression of transporters in the 3D environment was determined using qRT-PCR, and the permeability of endogenous substances and drugs was evaluated under various conditions. Results and Discussion The expression of tight-junction proteins, including claudin-5 and ZO-1, was confirmed by immunohistochemistry. The permeability rate constant of lucifer yellow through hiPS-BMECs was undetectably low, indicating that paracellular transport is highly restricted by tight junctions in the 3D-BBB system. The mRNA expression levels of transporters and receptors in the 3D-BBB system differed from those in the 2D-culture system by 0.2- to 5.8-fold. The 3D-cultured hiPS-BMECs showed asymmetric transport of substrates of BCRP, CAT1 and LAT1 between the luminal (blood) and abluminal (brain) sides. Proton-coupled symport function of MCT1 was also confirmed. Conclusion The 3D-BBB system constructed in this study mimics several important characteristics of the human BBB, and is expected to be a useful high-throughput evaluation tool in the development of CNS drugs.

This study investigated the use of HWY hairless rats to predict human plasma concentrations of drugs following dermal application.Utilizing a deconvolution method, pharmacokinetic parameters (e.g. ...

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.

The unbound fractions in plasma (fup) in two mouse models of humanized liver mice, PXB and humanized TK-NOG mice, were compared with human fup values using equilibrium dialysis method. A good relationship between fup values obtained from PXB mice and humans was observed; the fup of 34/39 compounds (87.2%) in PXB mice were within 3-fold of human fup. In contrast, a weak correlation was observed between human and humanized TK-NOG mouse fup values; the fup of 15/24 compounds (62.5%) in humanized TK-NOG mice were within 3-fold of human fup.As different profiles of plasma protein binding (PPB) profiles were observed between PXB and humanized TK-NOG mice, fup 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 fup in PXB mice (38/39 compounds within a 3-fold). Thus, this artificial PXB mouse plasma could be used to evaluate PPB. The unbound fractions in plasma (fup) in two mouse models of humanized liver mice, PXB and humanized TK-NOG mice, were compared with human fup values using equilibrium dialysis method. A good relationship between fup values obtained from PXB mice and humans was observed; the fup of 34/39 compounds (87.2%) in PXB mice were within 3-fold of human fup. In contrast, a weak correlation was observed between human and humanized TK-NOG mouse fup values; the fup of 15/24 compounds (62.5%) in humanized TK-NOG mice were within 3-fold of human fup. As different profiles of plasma protein binding (PPB) profiles were observed between PXB and humanized TK-NOG mice, fup 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 fup in PXB mice (38/39 compounds within a 3-fold). Thus, this artificial PXB mouse plasma could be used to evaluate PPB.

Continuous administration of a 14-amino acid peptide YY (PYY) analog, Ac-[d-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys30]PYY(23-36) (4), which has a high binding affinity and agonist activity for the neuropeptide Y2 receptor (Y2R), has previously shown an antiobesity effect in a 2-week diet-induced obesity (DIO) study in mice. However, there remained a possibility to obtain more potent analogs by further improving its pharmacokinetic profile. A combination of the N-terminal 4-imidazolecarbonyl moiety and three amino acid substitutions, trans-4-hydroxy-d-proline (d-Hyp)24, isovaline (Iva)25, and γ-methylleucine (γMeLeu)28, not only improved the binding affinity of the peptide for Y2R but also increased its anorectic activity in lean mice. In a 2-week DIO study in mice, continuous administration of 4-imidazolecarbonyl-[d-Hyp24,Iva25,Pya(4)26,Cha27,36,γMeLeu28,Lys30,Aib31]PYY(23-36) (31, PYY-1119) at a dose of 0.03mg/kg/day showed a highly potent antiobesity effect, with more than 10% body weight reduction.

1. A physiologically based pharmacokinetic (PBPK) model that includes inhibition constant evaluated in cryopreserved hepatocytes was used to predict drug–drug interactions (DDIs) between orally administered nifedipine, a CYP substrate, and fluconazole or ketoconazole, CYP inhibitors, in rats. 2. The Kp,uu, ratio of unbound inhibitor concentration in liver ([I]liver,u) to that in plasma ([I]sys,u), of fluconazole and ketoconazole was 1.0 and 13.0, indicating that ketoconazole accumulates in liver. The ratios of inhibition constants in rat liver microsomes (Ki,mic,u) to that in rat cryopreserved hepatocytes (Ki,hep,u) for fluconazole and ketoconazole were 1.5 and 25.5, which were similar to the Kp,uu and suggested that cryopreserved hepatocytes could mimic the hepatic accumulation of inhibitors. 3. The increases in AUC of nifedipine predicted by the minimal PBPK model using [I]liver,u/Ki,mic,u and [I]sys,u/Ki,hep,u were within 1.5-fold of the observed values for both inhibitors, whereas the model using [I]sys,u/Ki,mic,u underestimated the AUC increase caused by ketoconazole 21-fold. 4. These results indicated that hepatic accumulation factor of an inhibitor is required for a precise DDI projection and that cryopreserved hepatocytes would be useful to obtain the Ki including hepatic accumulation factor. It was demonstrated that PBPK model using Ki,hep,u could be a valuable approach for quantitative DDI projection.

Although Gottingen minipigs have been widely used for the evaluation of skin absorption, the correlation of minipig skin permeability with human skin absorption remains unclear. This study was designed to investigate the prediction of human plasma concentrations after dermal application of drug products using skin permeability data obtained from minipigs. First, in vitro skin permeabilities of seven marketed transdermal drug products were evaluated in minipigs, and compared with in vitro human skin permeability data. Next, plasma concentration-time profiles in humans after dermal applications were simulated using the in vitro minipig skin permeability data. Finally, the in vitro-in vivo correlation of minipig skin permeability was assessed. The in vitro skin permeabilities in minipigs were correlated strongly with in vitro human skin permeability data for the same drug products, indicating the utility of minipig skin as an alternative to human skin for in vitro studies. The steady-state plasma concentration or the maximum concentration of drugs was within 2-fold of the clinical data. Bioavailability was approximately 3-fold lower than in vitro permeated fraction. Predictions using in vitro skin permeability data in Gottingen minipig skin can reproduce the human pharmacokinetic profile, although the prediction of in vivo skin absorption underestimates human absorption.

Micronized cocrystal powders and amorphous spray-dried formulations were prepared and evaluated in vivo and in vitro as pulmonary absorption enhancement formulations of poorly soluble itraconazole (ITZ). ITZ cocrystals with succinic acid (SA) or l-tartaric acid (TA) with a particle size diameter of

Although the mechanism of action for peroxisome proliferator-activated receptor gamma (PPARγ) agonists has been extensively explored, the impact of the pharmacokinetic (PK) profile on the pharmacodynamic (PD) effects of PPARγ agonists has not been elucidated in detail. The importance of the PK profile of PPARγ agonist was evaluated for its PD effect based on population PK/PD analysis. Pioglitazone hydrochloride, the PPARγ agonist, was administered orally to Wistar fatty rats once a day (q.d.) or once every other day (q.2d.) as double the amount for the q.d. TREATMENT The plasma glucose lowering effect was selected as a surrogate PD effect for an anti-diabetic effect. The model fitting was conducted using the non-linear mixed effect modeling (NONMEM) method. The indirect response model described well the plasma glucose concentration-time profile. The q.d. treatment showed a stronger impact on the plasma glucose lowering effect than did the q.2d. TREATMENT The results of PK/PD modeling suggested that the sensitivity (i.e. EC50 ) between each group was comparable. On the other hand, the time above the effective concentration in the q.d. treatment group was longer than that in the q.2d. treatment group. The simulation of various dose regimens suggested that the much longer exposure duration within the effective level showed a stronger plasma glucose lowering effect, even with identical exposure to pioglitazone in the plasma. The PK/PD analysis clarified that the PK profile affected the pharmacological response and that continuous exposure at an appropriate effective level would be efficient for the anti-diabetic effect of the PPARγ agonist.

Peripheral administration of PYY3–36, a fragment of peptide YY (PYY), has been reported to reduce food intake by activating the neuropeptide Y2 receptor (Y2R). An N-terminally truncated PYY analogue, benzoyl-[Ala26,Ile28,31]PYY(25–36) (1), showed a relatively potent agonist activity for Y2R but a weak anorectic activity by intraperitoneal administration (2000 nmol/kg) in lean mice because of its markedly poor biological stability in the mouse serum. Notably, two cyclohexylalanine (Cha) substitutions for Tyr residues at positions 27 and 36 (4) improved the stability in the mouse serum concomitant with enhanced anorectic activity. Further optimization at positions 27, 28, 30, and 31 revealed that 21, containing Cha28 and Aib31 residues, showed a more potent anorectic activity than PYY3–36 at a low dose of 300 nmol/kg. The minimum effective dose by intraperitoneal administration of 21 was 30 nmol/kg (ca. 52 μg/kg) in mice, suggesting the biologic potential of short-length PYY3–36 analogues with a potent anorectic effect.

Gastrointestinal peptides such as peptide YY (PYY) can regulate appetite, which is relevant to the study of obesity. The intraperitoneal bolus administration of PYY3–36 and a 12-amino acid PYY analogue, benzoyl-[Cha27,28,36,Aib31]PYY25–36 (1), showed similar anorectic activity by activating the Y2 receptor (Y2R). However, food intake inhibition and body weight loss were not observed upon continuous subcutaneous administration of 1 with osmotic pumps in diet-induced obese (DIO) mice. N-Terminal elongation of 1, together with amino acid substitution at position 24, led to a hydrophilic 14-amino acid peptide, Ac-[d-Hyp24,Cha27,28,36,Aib31]PYY23–36 (18), that showed higher affinity and more potent agonist activity for Y2R and a robust anorectic activity with potency similar to that of PYY3–36. In addition, the continuous subcutaneous administration of 18 at 0.3 mg/(kg·day) induced significant body weight loss in DIO mice. These results suggest that a short-length PYY analogue can be a lead compound for antiob...

Bombesin receptor subtype 3 (BRS-3) is an orphan G protein-coupled receptor. Based on the obese phenotype of male BRS-3-deficient mice, BRS-3 has been considered an attractive target for obesity treatment. Here, we developed a selective BRS-3 agonist (compound-A) and evaluated its antiobesity effects. Compound-A showed anorectic effects and enhanced energy expenditure in diet-induced-obese (DIO)-F344 rats. Moreover, repeated oral administration of compound-A for 7 days resulted in a significant body weight reduction in DIO-F344 rats. We also evaluated compound-A for cardiovascular side effects using telemeterized Sprague-Dawley (SD) rats. Oral administration of compound-A resulted in transient blood pressure increases in SD rats. To investigate the underlying mechanisms of BRS-3 agonist effects, we focused on the suprachiasmatic nucleus (SCN), the main control center of circadian rhythms in the hypothalamus, also regulating sympathetic nervous system. Compound-A significantly increased the messenger RNA expression of Brs-3, c-fos, and circadian rhythm genes in SCN of DIO-F344 rats. Because SCN also controls the hypothalamic-pituitary-adrenal (HPA) axis, we evaluated the relationship between BRS-3 and the HPA axis. Oral administration of compound-A caused a significant increase of plasma corticosterone levels in DIO-F344 rats. On this basis, energy expenditure enhancement by compound-A may be due to a circadian rhythm change in central and peripheral tissues, enhancement of peripheral lipid metabolism, and stimulation of the sympathetic nervous system. Furthermore, the blood pressure increase by compound-A could be associated with sympathetic nervous system stimulation via SCN and elevation of plasma corticosterone levels through activation of the HPA axis.

Sphingomyelin synthase 2 (SMS2) has attracted attention as a drug target for the treatment of various cardiovascular and metabolic diseases. The modification of a high throughput screening hit, 2-quinolone 10, enhanced SMS2 inhibition at nanomolar concentrations with good selectivity against SMS1. To improve the pharmaceutical properties such as passive membrane permeability and aqueous solubility, adjustment of lipophilicity was attempted and 1,8-naphthyridin-2-one 37 was identified as a potent and selective SMS2 inhibitor. A significant reduction in hepatic sphingomyelin levels following repeated treatment in mice suggested that compound 37 could be an effective in vivo tool for clarifying the role of SMS2 enzyme and developing the treatment for SMS2-related diseases.

Accumulation of toxic endogenous and/or exogenous substances can trigger tissue injury. Multidrug and toxin extrusion proteins (MATEs) are transporters at renal proximal tubules involved in the secretion of hydrophilic substances into urine. Multidrug and toxin extrusion protein inhibition can lead to nephrotoxicity via accumulation of toxic substances; however, case studies demonstrating causality are rare, except for drug-drug interaction studies. To explore the involvement of MATE inhibition in nephrotoxicity, MATE1 inhibition, cytotoxicity, and mitochondrial toxicity (MT) of 38 in-house compounds that showed toxicity were assessed in in vivo safety evaluations using rats, dogs, and monkeys and compared considering unbound exposures at minimal steady-state concentration (C24h,u) between nephrotoxicity positive and negative compounds. Logarithmic-corrected means of C24h,u normalized by MATE1 IC50 or cytotoxicity EC50 (C24h,u/IC50 and C24h,u/EC50) were higher for nephrotoxic compounds. An exposure cutoff of C24h,u/IC50 > 0.01 filtered nephrotoxicity with a 54% positive predictive value. Of 7 cases filtered with this cutoff, all the cases showed pathological changes at renal proximal tubules expressing MATE1. Furthermore, all cases with > 0.01 reliable exposure for MATE1 inhibition and cytotoxicity exhibited nephrotoxicity. Although compounds potent for MATE1 inhibition and cytotoxicity without and with MT (potentials of 10, 30, and 40 μM, respectively) were correctly classified as nephrotoxic by evaluation of in vitro potency alone, without considering exposures, these results suggest that MATE1 inhibition potency and cytotoxicity can be used to assess nephrotoxicity, especially at proximal tubules, and could be used for safety assessment in early drug discovery.

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.

1. In order to identify the best inhibitor concentration for the accurate prediction of magnitude of a hepatic cytochrome P450 (CYP)-mediated drug-drug interaction (DDI), the DDI between nifedipine, the CYP substrate probe, and fluconazole, ketoconazole, or ritonavir, the CYP inhibitors, in in situ rat liver perfusion system and rats were investigated. 2. In in situ system, the intrinsic clearance (CLint) of nifedipine was decreased after co-infusion of the CYP inhibitors. The decrease in in situ CLint of nifedipine was most comparable to that in in vitro CLint in rat liver microsomes calculated by using the unbound liver concentrations of inhibitors ([I]liver,u). The ratios of unbound liver concentration to unbound hepatic vein concentration (Kp,uu) of ketoconazole and ritonavir were 4.0-8.0 and 18.4-21.1, suggesting a concentrative uptake of them into liver. 3. In rats, the DDI effects of orally administered nifedipine with constant infusion of the inhibitors were investigated. The most accurate prediction of magnitude of DDI was achieved when [I]liver,u was applied as the inhibitor concentration. 4. These results indicated that [I]liver,u is the most reliable inhibitor concentration for CYP-mediated DDI and it is necessary to consider the concentrative uptake of inhibitors into liver for the quantitative prediction of DDI.

We investigated the gastrointestinal absorption characteristics of oral sustained-release formulations in microminipigs, dogs, and monkeys in order to clarify the similarities in absorption properties between these animals and humans. Time profiles of oral absorption of nifedipine and valproic acid were calculated from the plasma concentration-time profiles of the drugs by a deconvolution method. The curves for both drugs in microminipigs were close to or slightly higher than those in humans, whereas those in monkeys were lower. Furthermore, the plasma concentration-time profiles of the drugs were subjected to non-compartmental analysis. The fractions of a dose absorbed into the portal vein (FaFg) in microminipigs ranged from 50 to 100% of the human values, whereas those in monkeys were less than half the human values. In addition, the other absorption-related parameters for the sustained-release formulation in microminipigs, as well as monkeys, were comparable to those in humans. In conclusion, the oral absorption properties of microminipigs and humans were similar regarding the sustained-release formulations. Therefore, microminipig is a suitable animal model to estimate the oral absorption of sustained-release formulations in humans.

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. 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. 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. 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.

Objectives Bombesin receptor subtype‐3 (BRS‐3) has been suggested to play a potential role in energy homeostasis. However, the physiological mechanism of BRS‐3 on energy homeostasis remains unknown. Thus, we investigated the BRS‐3‐mediated neuronal pathway involved in food intake and energy expenditure. Materials and Methods Expression of BRS‐3 in the rat brain was histologically examined. The BRS‐3 neurons activated by refeeding‐induced satiety or a BRS‐3 agonist were identified by c‐Fos immunostaining. We also analyzed expression changes in feeding‐relating peptides in the brain of fasted rats administered with the BRS‐3 agonist. Results In the paraventricular hypothalamic nucleus (PVH), dorsomedial hypothalamic nucleus (DMH), and medial preoptic area (MPA), strong c‐Fos induction was observed in the BRS‐3 neurons especially in PVH after refeeding. However, the BRS‐3 neurons in the PVH did not express feeding‐regulating peptides, while the BRS‐3 agonist administration induced c‐Fos expression in the DMH and MPA, which were not refeeding‐sensitive, as well as in the PVH. The BRS‐3 agonist administration changed the Pomc and Cart mRNA level in several brain regions of fasted rats. Conclusion These results suggest that BRS‐3 neurons in the PVH are a novel functional subdivision in the PVH that regulates feeding behavior. As the MPA and DMH are reportedly involved in thermoregulation and energy metabolism, the BRS‐3 neurons in the MPA/DMH might mediate the energy expenditure control. POMC and CART may contribute to BRS‐3 neuron‐mediated energy homeostasis regulation. In summary, BRS‐3‐expressing neurons could regulate energy homeostasis through a novel neuronal pathway.

The discovery of a novel series of β-methyltryptophan (β MeTrp) derivatives as selective and orally active non-peptide somatostatin receptor 2 (SSTR2) agonists for the treatment of Type 2 diabetes is described. In our previous research, Compound A, β-MeTrp derivative with highly potent and selective SSTR2 agonistic activity IC50 (SSTR2/SSTR5)=0.3/>100 (nM), was identified asa drug candidate for treatment of Type 2 diabetes which lowers significantly plasma glucose level in Wistar fatty rats in its oral administrations. However, as serious increase in AUC and phospholipidosis (PLsis) were observed in its toxicological studies in rats, follow-up compounds were searched to avoid risk of PLsis with reference to their in vitro PLsis potentials evaluated on the basis of accumulation of phospholipids in HepG2 cells exposed to the compounds. It has been found that introduction of a carbonyl group onto the piperidine and piperazine or aniline moiety of compounds A and B reduced markedly the in vitro PLsis potentials. And further modification of the compounds and their evaluation led to a discovery of compounds 3k with lower in vitro PLsis potentials exhibiting lowering effect of hypoglycemia-induced glucagon secretion in SD rats (ED50=1.1mg/kg) and glucose excursion in meal tolerance test in Wistar fatty diabetic rats (MED=3.0mg/kg) in oral administrations. Compound 3k was selected asa new drug candidate of selective and orally active non-peptide SSTR2 agonists for treatment of Type 2 diabetes with low in vivo PLsis potential.

Glucagon-like peptide-1 (GLP-1) analogs lower body weight in humans in addition to their potent anti-diabetic effects. Hence, agonistic targeting of the GLP-1 receptor could be a valid approach to target obesity. However, quantitative analyses of the pharmacokinetic/pharmacodynamic (PK/PD) relationship between GLP-1 analogs and their anti-obesity effect have not been reported in either animals or humans. Therefore, the present study was performed to establish a mechanism-based PK/PD model of GLP-1 receptor agonists using the GLP-1 analog, exenatide, for the development of promising new anti-obesity drugs. Exenatide was administered to high-fat diet-induced obese C57BL/6J mice via subcutaneous bolus and continuous infusion. Food intake and body weight reductions were observed, which depended on the plasma concentrations of exenatide. The homeostatic feedback model, in which food intake is assumed to be regulated by appetite control signals, described the relationship among the plasma concentration-time profile of exenatide, food intake, and body weight. The estimated IC50 of exenatide against food intake was 2.05 pM, which is similar to the reported KD value of exenatide in rat brain and the estimated EC50 value for augmentation of insulin secretion in humans. The PK/PD model simulation indicated that subcutaneous infusion would show a stronger effect on body weight reduction than bolus dosing would. This novel, quantitative PK/PD model could be utilized for anti-obesity research and development of GLP-1 analogs, GLP-1 secretagogs, GLP-1 degradation inhibitors, and combinations thereof by allowing the estimation of appropriate pharmacokinetic profiles and dosing regimens.

Using structure-based drug design, we identified and optimized a novel series of pyrimidodiazepinone PLK1 inhibitors resulting in the selection of the development candidate TAK-960. TAK-960 is currently undergoing Phase I evaluation in adult patients with advanced solid malignancies.

To date, the in vitro-in vivo correlation (IVIVC) of P-glycoprotein (P-gp)-mediated drug-drug interaction (DDI) at the blood-brain barrier (BBB) in rats indicated that the cutoff value to significantly affect the brain penetration of digoxin was [I,unbound/Ki] of 1, where I,unbound is the unbound plasma concentration of P-gp inhibitors. On the basis of the IVIVC in rats, we speculated that clinically used P-gp inhibitors do not cause DDI at the human BBB, because none of the compounds studied was [I,unbound/Ki]>1 at therapeutic doses. Recently, positron emission tomography studies with P-gp substrates, such as [(11)C]verapamil, [(11)C]N-desmethyl loperamide, and [(11)C]loperamide, together with potent P-gp inhibitors, have indicated that increases in the influx rate constant for brain entry were observed in humans. Therefore, we aimed to retrospectively analyze the results of P-gp-mediated DDIs with in vitro P-gp inhibition assays and to confirm the appropriate cutoff value. In vitro P-gp inhibition assays using verapamil, N-desmethyl loperamide, and loperamide as P-gp probe substrates were performed in human multidrug resistance protein 1-expressing LLC-PK1 cells. The efflux ratios decreased in the presence of P-gp inhibitors, and the Ki of tariquidar was 10 nmol/L, regardless of probe substrates. Taking the in vitro Ki and unbound plasma concentrations in clinical DDI studies together, the criterion [I,unbound/Ki] of 1 was an appropriate cutoff limit to observe significant P-gp-mediated DDI at the BBB in humans. On the other hand, no significant DDI was observed in cases in which [I,unbound/Ki] was less than 0.1. This criterion was comparable to the previous IVIVC result in rats.

Although several mathematical models have been reported for the estimation of human plasma concentration profiles of drug substances after dermal application, the successful cases that can predict human pharmacokinetic profiles are limited. Therefore, the aim of this study is to investigate the prediction of human plasma concentrations after dermal application using in vitro permeation parameters obtained from excised human skin. The in vitro skin permeability of 7 marketed drug products was evaluated. The plasma concentration-time profiles of the drug substances in humans after their dermal application were simulated using compartment models and the clinical pharmacokinetic parameters. The transdermal process was simulated using the in vitro skin permeation rate and lag time assuming a zero-order absorption. These simulated plasma concentration profiles were compared with the clinical data. The result revealed that the steady-state plasma concentration of diclofenac and the maximum concentrations of nicotine, bisoprolol, rivastigmine, and lidocaine after topical application were within 2-fold of the clinical data. Furthermore, the simulated concentration profiles of bisoprolol, nicotine, and rivastigmine reproduced the decrease in absorption due to drug depletion from the formulation. In conclusion, this simple compartment model using in vitro human skin permeation parameters as zero-order absorption predicted the human plasma concentrations accurately.

1. The aim of the present study was to evaluate the usefulness of chimeric mice with humanised liver (PXB mice) for the prediction of clearance (CLt) and volume of distribution at steady state (Vds...

DDI could be caused by the inhibition of OATP-mediated hepatic uptakes. The aim of this study is to set the risk criteria for the compounds that would cause DDI via OATP inhibition at the drug discovery stage. The IC50 values of OATP inhibitors for human OATP-mediated atorvastatin uptake were evaluated in the expression system. In order to set the risk criteria for OATP inhibition, the relationship was clarified between OATP inhibitory effect and severe adverse effects of OATP substrates, rhabdomyolysis, hyperbilirubinemia and jaundice. Rhabdomyolysis would be caused in the atorvastatin AUC more than 9-fold of that at a minimum therapeutic dose. The atorvastatin AUC was 6- to 9-fold increased with the OATP inhibitors of which IC50 values were ≤1 μmol/L. Hyperbilirubinemia and jaundice would be caused with the OATP inhibitors of which IC50 values were ≤6 μmol/L. This investigation showed that the compounds with IC50 of ≤1 μmol/L would have high risk for OATP-mediated DDI that would cause severe side effects. Before the detailed analysis based on the dosage, unbound fraction in blood and effective concentration to evaluate the clinical DDI potency, this criteria enable high throughput screening and optimize lead compounds at the drug discovery stage.

Spiro-pyrazolidinedione derivatives without quaternary chiral center were discovered by structure-based drug design and characterized as potent acetyl-CoA carboxylase (ACC) inhibitors. The high metabolic stability of the spiro-pyrazolo[1,2-a]pyridazine scaffold and enhancement of the activity by incorporation of a 7-methoxy group on the benzothiophene core successfully led to the identification of compound 4c as an orally bioavailable and highly potent ACC inhibitor. Oral administration of 4c significantly decreased the values of the respiratory quotient in rats, indicating the stimulation of fatty acid oxidation.

Spiro-lactone (S)-1 is a potent acetyl-CoA carboxylase (ACC) inhibitor and was found to be metabolically liable in human hepatic microsomes. To remove one of the risk factors in human study by improving the metabolic stability, we focused on modifying the spiro-lactone ring and the benzothiophene portion of the molecule. Spiro-imide derivative 8c containing a 6-methylthieno[2,3-b]pyridine core exhibited potent ACC inhibitory activity and favorable pharmacokinetic profiles in rats.

Highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for measuring digoxin (DGX), a typical P-glycoprotein probe, in human plasma, rat plasma, and rat brain. We extracted DGX and deuterium-labeled DGX (as internal standard) from sample fluids under basic conditions using acetonitrile and sodium chloride-saturated 0.1 mol/L sodium hydroxide. The upper organic layer was diluted with distilled water, and the resulting solution was injected into an LC/MS/MS system in negative ionization mode. Chromatographic separation was achieved on a C(18)-ODS column in the gradient mobile phase, which comprised 0.05% (w/v) ammonium carbonate (pH 9.0) and methanol at a flow rate of 0.7 mL/min. Regardless of the type of biological matrix, intra-day and inter-day validation tests demonstrated good linearity of calibration curves within ranges of 0.1-10 ng/mL for plasma and 0.5-50 ng/g for rat brain and gave excellent accuracy and precision of quality control samples at 4 concentration levels. Unlike existing methods, our approach uses negative ionization to avoid competitive adduct formation of DGX. Our method showed higher sensitivity and wider applicability to various types of biological matrices than existing methods. Our method will support clinical and preclinical investigation of in vivo P-glycoprotein functionality using DGX.

金沢大学医薬保健研究域薬学系 金沢大学医薬保健研究域医学系, Purpose. The oral bioavailability of some therapeutic agents is markedly lower in cynomolgus monkeys than in humans. We investigated small-intestinal absorption of the P-glycoprotein (P-gp) substrates etoposide and digoxin in monkeys to clarify the influence of efflux transport on their intestinal permeability. Methods. The pharmacokinetics of etoposide and digoxin was examined in monkeys and rats after oral and intravenous administration. Intestinal permeability and segmental differences in permeability were investigated with an Ussing-type chamber. Results. The bioavailability of etoposide was 12.9 and 13.9% in monkeys and rats, respectively. Total body clearance of etoposide in monkeys was much less than hepatic blood flow, suggesting that the bioavailability would be limited at intestinal absorption. Marked vectorial transport of etoposide in the secretory direction was observed in rats, especially in the lower small intestine, and segmental differences were consistent with the distribution of P-gp expression. Vectorial transport was minimal in monkey small intestine. Our kinetic analysis indicated that P-gp contributes little to the intestinal permeability of etoposide and digoxin in monkeys, and apical uptake is rate-limiting. Conclusion. Low bioavailability of etoposide in monkeys is due to poor intestinal uptake resulting from low influx from the apical side, rather than secretion via P-gp. © 2008 Springer Science+Business Media, LLC.

Monoacylglycerol O-acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DG), a triacylglycerol precursor and potential peripheral target for novel anti-obesity therapeutics. High-throughput screening identified lead compounds with MGAT2 inhibitory activity. Through structural modification, a potent, selective, and orally bioavailable MGAT2 inhibitor, compound A (compA), was discovered. CompA dose-dependently inhibited postprandial increases in plasma triglyceride (TG) levels. Metabolic flux analysis revealed that compA inhibited triglyceride/diacylglycerol resynthesis in the small intestine and increased free fatty acid and acyl-carnitine with shorter acyl chains than originally labelled fatty acid. CompA decreased high-fat diet (HFD) intake in C57BL/6J mice. MGAT2-null mice showed a similar phenotype as compA-treated mice and compA did not suppress a food intake in MGAT2 KO mice, indicating that the anorectic effects were dependent on MGAT2 inhibition. Chronic administration of compA significantly prevented body weight gain and fat accumulation in mice fed HFD. MGAT2 inhibition by CompA under severe diabetes ameliorated hyperglycemia and fatty liver in HFD-streptozotocin (STZ)-treated mice. Homeostatic model assessments (HOMA-IR) revealed that compA treatment significantly improved insulin sensitivity. The proximal half of the small intestine displayed weight gain following compA treatment. A similar phenomenon has been observed in Roux-en-Y gastric bypass-treated animals and some studies have reported that this intestinal remodeling is essential to the anti-diabetic effects of bariatric surgery. These results clearly demonstrated that MGAT2 inhibition improved dyslipidemia, obesity, and diabetes, suggesting that compA is an effective therapeutic for obesity-related metabolic disorders.

Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has attracted interest as a novel target for the treatment of obesity and metabolic diseases. Starting from N-phenylbenzenesulfonamide derivative 1 with moderate potency for MGAT2 inhibition, we explored an effective location of the hydrophobic group at the 1-position to enhance MGAT2 inhibitory activity. Shifting the hydrophobic group to the adjacent position followed by introduction of a bicyclic central core to restrict the substituent orientation produced N-phenylindoline-5-sulfonamide derivative 10b, which displayed much improved potency, with an IC50 value of 1.0 nM. This compound also exhibited excellent selectivity (greater than 30,000-fold) against related acyltransferases (MGAT3, DGAT1, DGAT2, and ACAT1). Subsequent optimization efforts were directed toward improving pharmacokinetic profiles, which resulted in the identification of 5-[(2,4-difluorophenyl)sulfamoyl]-7-(2-oxopyrrolidin-1-yl)-N-[4-(trifluoromethyl)phenyl]-2,3-dihydro-1H-indole-1-carboxamide (24d) endowed with potent MGAT2 inhibitory activity (IC50 = 3.4 nM) and high oral bioavailability (F = 52%, mouse). In a mouse oral fat tolerance test, oral administration of this compound effectively suppressed the elevation of plasma triacylglycerol levels.

Cultured epithelial autografts (CEA) with highly expanded mesh skin grafts were used for extensive adult burns covering more than 30% of the total body surface area. A prospective study on eight patients assessed subjective and objective findings up to a 12-month follow-up. The results of wound healing for over 1:6 mesh plus CEA, gap 1:6 mesh plus CEA, and 1:3 mesh were compared at 3, 6, and 12 months using extensibility, viscoelasticity, color, and transepidermal water loss by a generalized estimating equation (GEE) or generalized linear mixed model (GLMM). No significant differences were observed among the paired treatments at any time point. At 6 and 12 months, over 1:6 mesh plus CEA achieved significantly better expert evaluation scores by the Vancouver and Manchester Scar Scales (p < 0.01). Extended skin grafting plus CEA minimizes donor resources and the quality of scars is equal or similar to that with conventional low extended mesh slit-thickness skin grafting such as 1:3 mesh. A longitudinal analysis of scars may further clarify the molecular changes of scar formation and pathogenesis.

We attempted to establish animal models to evaluate the effects of drug degradation in the stomach on oral bioavailability. In addition, we assessed the utilization of animal studies in determining the need for enteric-coated formulations. In order to control the gastric pH in rats and dogs, appropriate dosing conditions were investigated using pentagastrin and rabeprazole, which stimulate and inhibit gastric acid secretion. Using animals controlled for gastric acid secretion, the area under curve (AUC) ratios (AUC with rabeprazole/AUC with pentagastrin) of all compounds unstable under acidic conditions were evaluated. The AUC ratios of omeprazole and erythromycin, which are administered orally to humans, as enteric-coated tablets, were greater than 1.9 in the rats and dogs controlled for gastric acid secretion. On the contrary, the AUC ratios of clarithromycin, azithromycin, and etoposide (commercially available as a standard immediate-release form) were less than 1.3 each. In conclusion, in vivo models using rats and dogs were optimized to evaluate the effects of gastric acid on the oral bioavailability of drugs, and demonstrated that in vivo models can lead to a better understanding of the oral bioavailability, with respect to the formulation development.

Feeding a diet with excess cystine to rats resulted in hypercholesterolemia. To understand the mechanism of the hypercholesterolemia, cholesterol synthesis and degradation, bile acid content of bile, and fecal steroids were determined. The in vivo incorporation of tritiated water into hepatic cholesterol, and activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in rats fed a high-cystine diet were significantly higher than those in rats fed a control diet. The activity of hepatic cholesterol 7 alpha-hydroxylase was similar between two groups. Little effect of cystine supplementation was found on fecal sterol excretion although there were some changes in biliary excretion of cholic acid derivatives. These results indicate that hypercholesterolemia caused by feeding of a high-cystine diet may be due to the stimulation of hepatic cholesterol synthesis.

It has been reported that in vivo biliary clearance can be predicted using sandwich-cultured rat and human hepatocytes. The predicted apparent biliary clearance (CL(bile, app)) from sandwich- cultured rat hepatocytes (SCRH) based on medium concentrations correlates to in vivo CL(bile, app) based on plasma concentrations of angiotensin II receptor blockers (ARBs), HMG-CoA reductase inhibitors (statins), β-lactam antibiotics, and topotecan. However, the predicted biliary clearance from SCRH was 7- to 300-fold lower than in vivo biliary clearance. We speculated that the process of biliary excretion might not have been evaluated using sandwich-cultured hepatocytes. To evaluate this issue, intrinsic biliary clearance (CL(bile, int)) based on intracellular compound concentrations was evaluated to investigate the in vitro-in vivo correlation of this process among ARBs, statins, β-lactam antibiotics, and topotecan. Intrinsic biliary clearance in SCRH correlated to in vivo values obtained by constant intravenous infusion of six compounds, but not rosuvastatin and cefmetazole, to rats. Moreover, differences between SCRH and in vivo CL(bile, int) (0.7-6-fold) were much smaller than those of CL(bile, app) (7-300-fold). Therefore, in vivo CL(bile, int) is more accurately reflected using SCRH than CL(bile, app). In conclusion, to predict in vivo biliary clearance more accurately, CL(bile, int) should be evaluated instead of CL(bile, app) between SCRH and in vivo.

The decision tree to determine whether the P-glycoprotein (P-gp)/multidrug resistance protein 1 (MDR1)-mediated drug–drug interaction (DDI) study is recommended has been proposed by the International Transporter Consortium. We, therefore, designed an in vitro P-gp inhibition assay and determined the appropriate risk criteria for P-gp-mediated DDI at the drug discovery stage. Effects of P-gp inhibitors on digoxin transport across a monolayer of MDR1-expressing cells were examined. The IC50 (half-maximal inhibitory concentration) values generated from the efflux ratio (ER) were smaller than those generated from basolateral-to-apical directional apparent permeability. The difference in IC50 values was kinetically described in a compartment model analysis. This analysis indicated that ER is a highly sensitive parameter that can be used for the degree of P-gp inhibition. Considering IC50 values and the increase in digoxin exposure in clinical DDI studies, the risk criteria of [I2]/IC50 = 30 ([I2], theoretically maximal gastrointestinal concentration) was the optimal cutoff value to predict a clinically relevant DDI. We also investigated whether the IC50 value itself is applicable to assess the DDI risk. In conclusion, compounds with IC50 values less than 2 μM exhibit high risk for P-gp-mediated DDIs. However, compounds with IC50 values greater than or equal to 2 μM are inconclusive because clinical doses should be considered for the precise DDI risk assessment. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4013–4023, 2011

The magnitude of P-glycoprotein [(P-gp)/multidrug resistance protein 1 (MDR1)]-mediated drug-drug interaction (DDI) at the blood-brain barrier (BBB) in rats was estimated by in vitro-in vivo correlation (IVIVC). In in vitro studies, rat Mdr1a-expressing LLC-PK1 cells were examined for the evaluation of P-gp inhibitory activity using digoxin as a P-gp probe substrate. The in vitro K(i) value was calculated using a modified corrected flux ratio that reflects the P-gp function. In in vivo studies, digoxin with or without P-gp inhibitors was administered to rats by constant intravenous infusion to evaluate the effect of P-gp inhibition on digoxin transport to the brain under steady-state conditions. In the presence of elacridar, the brain-to-plasma concentration ratio (K(p,brain)) of digoxin was approximately 14 times the control value. However, no significant change in the K(p,brain) was observed in the presence of clinically used P-gp inhibitors, with the exception of cyclosporine A. A positive correlation was found between the in vivo K(p,brain) of digoxin and [I(,unbound)/K(i)] (where I(,unbound) is the unbound plasma concentration of P-gp inhibitors). Compounds with [I(,unbound)/K(i)] values of >1 increased K(p,brain) of digoxin in rats. In summary, we used a quantitative approach to evaluate the impact of P-gp-mediated DDI at the rat BBB. We successfully established the IVIVC, which indicated the potential DDI in the presence of potent P-gp inhibitors. On the basis of the IVIVC in rats and K(i) values in human MDR1, we speculated that clinically used P-gp inhibitors do not cause DDI at the human BBB, because none of the compounds studied showed [I(,unbound)/K(i)] values of >1 at therapeutic doses.

Feeding a diet high in excess histidine (5% L-histidine) resulted in hypercholesterolemia and enlargement of the liver in rats. To clarify the mechanism of the hypercholesterolemia, cholesterol synthesis and degradation were followed. We found that hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in histidine-excess diet rats was significantly higher than in rats fed a basal diet. Incorporation of [3H] water into cholesterol of liver slices from rats fed the histidine-excess diet was higher than incorporation into liver slices from rats fed the basal diet (expressed per liver per 100 g body weight). In vivo incorporation of [3H] water into hepatic cholesterol was also higher, but the incorporation into cholesterol of the small intestine was lower in histidine-fed rats than in rats fed the basal diet (expressed per liver per 100 g body weight). Hepatic cholesterol 7 alpha-hydroxylase activity was similar in both groups. The data suggest that the hypercholesterolemia caused by histidine-excess diet appears to be due to the stimulation of cholesterol synthesis in the liver.

The effects on liver and serum lipids of rats of excess cystine added to their casein or soy protein isolate diets were studied. When excess cystine was added to the casein diet, liver lipids were not changed. Using soy protein isolate as a protein source, both food consumption and body weight gain were depressed with increasing levels of dietary cystine. Liver total lipids, cholesterol, triacylglycerols, and phospholipids were increased in rats with 2.5% or 3.5% cystine diets. Serum cholesterol of rats on the 3.5% cystine diet was significantly higher than those of the other groups, which yielded similar values. Serum triacylglycerols were increased by the addition of 0.5% or 1.5% cystine, and decreased by the addition of 2.5% or 3.5% cystine to the diet. Serum free fatty acids increased or tended to increase when liver lipid accumulation was observed. The addition of either methionine or choline to the cystine-excess diet containing soy protein isolate prevented the accumulation of triacylglycerols and phospholipids in the liver although serum triacylglycerols did not return to the levels of rats on the diet without cystine, and serum free fatty acid levels were not changed. Thus, it is surmised that this fatty liver might be due to the choline deficiency induced by the excess cystine.

The relationships between mast cell-derived chymase, angiotensin II, and extracellular matrix production in the skin after intense pulsed light (IPL) were clarified in hamsters. Dorsal areas of the hamsters were irradiated once or twice a week by IPL. The index of extracellular matrix production in the skin was defined as the depth stained with Azan-Mallory stain from the epidermis to the dermis at the point of maximum thickness. The index had significantly increased 7 days after IPL irradiation in sections treated once or twice with IPL compared with that of untreated control sections. The numbers of mast cells, chymase-positive cells, and angiotensin II-positive cells had also significantly increased in IPL-irradiated areas. Significant increases in chymase and angiotensin II activities were observed in the extracts obtained from IPL-irradiated skin. Mast cell-derived chymase may be involved via angiotensin II formation in the dermal extracellular matrix production that occurs after IPL irradiation.

Oral bioavailability of some drugs is substantially lower in cynomolgus monkeys than in various other species, including humans. In the present study, midazolam was used as a model drug to investigate the reason for the lower bioavailability in these monkeys. The bioavailability of midazolam after oral administration was minimal in monkeys and rats, being only 2.1 and 1.1%, respectively. In monkeys, this low bioavailability could not be explained simply in terms of a hepatic first-pass effect. To examine the roles of intestinal metabolism and transport, we evaluated apical-to-basal and basal-to-apical transport of midazolam, and the formation of metabolites in small intestinal tissues using an Ussing-type chamber. The values of mucosal extraction ratio were estimated to be 0.97, 0.93, and 0.89 during apical-to-basal transport in the upper, middle, and lower small intestine of monkeys, respectively, whereas the corresponding values for rats were close to zero, indicating that extensive metabolism of midazolam occurs, particularly in the upper region of the small intestine in monkeys, but not rats. Interestingly, formation of the metabolites was much greater during transport in the apical-to-basal direction than in the basal-to-apical direction, and this could be well explained by a mathematical model based on the assumption that extensive metabolism is associated with the uptake process of midazolam from the apical cell surface. Thus, we conclude that an asymmetric distribution of metabolic activity in the small intestine, leading to extensive metabolism during uptake from the apical cell surface, accounts for the minimal oral bioavailability of midazolam in cynomolgus monkeys.

Effects of dietary carbohydrates on triglyceride production and hepatic lipogenic enzyme activities were examined in Wistar fatty rats, an animal model of noninsulin dependent diabetes mellitus, fed fructose or glucose and were compared with those of Wistar lean rats. Carbohydrates were supplied in 10% drinking solutions for 21 days. As compared with lean rats, Wistar fatty rats were characterized by hyperglycemia, hyperinsulinemia and hypertriglyceridemia, the last of which was associated with an increased hepatic activity of fatty acid synthetase and an increased rate of triglyceride secretion from the liver to the circulation. Feeding fructose to genetically obese diabetic rats produced a threefold increase in the hepatic activity of fatty acid synthetase, a twofold increase in NADPH-generating enzymes (malic enzyme and glucose-6-phosphate dehydrogenase) and a 56% increase in the rate of triglyceride secretion, with a resultant 86% increase in plasma triglyceride concentrations. Feeding glucose produced a similar increase in the activity of NADPH-generating enzymes and triglyceride production in the fatty liver but it differed in producing no change in plasma triglyceride concentrations or hepatic fatty acid synthetase activity. Neither dietary fructose nor glucose changed glycemia or insulinemia. These results show that in genetically obese, diabetic rats feeding fructose and glucose is associated with an increase in hepatic lipogenic enzyme activities and triglyceride production, and suggest that fructose stimulates triglyceride production but impairs triglyceride removal, whereas glucose stimulates both of them.

Publisher Summary This chapter proposes that ideal vitamin intake should be based on how different vitamin concentrations affect vitamin function. Detection of nutrient action should be targeted to specific biochemical and molecular action of the nutrient. Vitamin function should be studied in situ (i.e., in position). “ In situ ” refers to organelles, cells, animals, or ideally humans. Because studies on humans are difficult, experiments with normal human tissue may provide useful information. In situ studies are essential, because vitamin function in living tissue may differ from function in isolated reactions or with isolated proteins. The goal is to determine vitamin function in relation to different nutrient concentrations, and, therefore, this approach to optimal vitamin ingestion is termed “ in situ kinetics,” which has biochemical and clinical components. The biochemical component determines how vitamin concentration affects vitamin biochemical or molecular function in situ . The clinical component determines how the effective vitamin concentrations are achieved in people.