Akihiro Hisaka

Assessment of intestinal availability (FG) of substrate drugs of cytochrome p450s by analyzing changes in pharmacokinetic properties caused by drug-drug interactions.

In this study, we developed the drug–drug interaction (DDI) method as a new assessment technique of intestinal availability (FG, the fraction of drug transferred from the intestinal enterocytes into the liver, escaping from intestinal metabolism) based on the clearance theory. This method evaluates FG from changes caused by DDIs in the area under the blood concentration-time curve and in the elimination half-life of victim drugs. Application of the DDI method to data from the literature revealed that the mean and S.D. of FG values for 20 substrate drugs of CYP3A was 0.56 ± 0.29, whereas that for 8 substrate drugs of CYP2C9, CYP2C19, and CYP2D6 was 0.86 ± 0.11. These results were consistent with the fact that intestinal metabolism is mediated predominantly by CYP3A. The DDI method showed reasonable correlations with the conventional i.v./p.o. method and the grape fruit juice (GFJ) method (coefficients of determination of 0.41 and 0.81, respectively). The i.v./p.o. method was more susceptible to fluctuations in the hepatic blood flow rate compared with the DDI and GFJ methods. The DDI method evaluates FG separating from the absorption ratio (FA) although it requires approximation of FA. Since preciseness of approximation of FA does not greatly affect the evaluation of FG by the DDI method, we proposed a reasonable approximation method of FA for the evaluation of FG in the DDI method. The DDI method would be applicable to a broad range of situations in which various DDI data are utilizable.

Prediction of Drug Transfer into Milk Considering Breast Cancer Resistance Protein (BCRP)-Mediated Transport

ABSTRACTPurposeDrug transfer into milk is of concern due to the unnecessary exposure of infants to drugs. Proposed prediction methods for such transfer assume only passive drug diffusion across the mammary epithelium. This study reorganized data from the literature to assess the contribution of carrier-mediated transport to drug transfer into milk, and to improve the predictability thereof.MethodsMilk-to-plasma drug concentration ratios (M/Ps) in humans were exhaustively collected from the literature and converted into observed unbound concentration ratios (M/Punbound,obs). The ratios were also predicted based on passive diffusion across the mammary epithelium (M/Punbound,pred). An in vitro transport assay was performed for selected drugs in breast cancer resistance protein (BCRP)-expressing cell monolayers.ResultsM/Punbound,obs and M/Punbound,pred values were compared for 166 drugs. M/Punbound,obs values were 1.5 times or more higher than M/Punbound,pred values for as many as 13 out of 16 known BCRP substrates, reconfirming BCRP as the predominant transporter contributing to secretory transfer of drugs into milk. Predictability of M/P values for selected BCRP substrates and non-substrates was improved by considering in vitro-evaluated BCRP-mediated transport relative to passive diffusion alone.ConclusionsThe current analysis improved the predictability of drug transfer into milk, particularly for BCRP substrates, based on an exhaustive data overhaul followed by focused in vitro transport experimentation.

Connexin 43 enhances Bax activation via JNK activation in sunitinib-induced apoptosis in mesothelioma cells

The constituent protein of gap junctions, connexin (Cx), interacts with various proteins via its C-terminus region, including kinases, cell-adhesion proteins, and a pro-apoptotic protein, Bax. This molecular interaction may affect expression and functioning of the interacting proteins and modulate the cellular physiology. In our previous work, Cx43 was found to interact directly with Bax and in the presence of sunitinib, lead to the Bax-mediated apoptosis in mesothelioma cells. In this study, we investigated the mechanism of how Cx43 promotes Bax-mediated apoptosis using the same cell line. Treatment with sunitinib increased the expression of the active conformation of the Bax protein, which was predominantly localized at the mitochondria, only in Cx43-transfected cells. Bax oligomerization and decrease in the mitochondrial membrane potential were also observed. The involvement of c-Jun N-terminal kinase (JNK) in the interaction of Cx43 and Bax was further examined. Treatment with sunitinib increased the expression of phosphorylated (active) form of JNK only in the Cx43-transfected cells. Phosphorylated JNK and active Bax were co-localized, and the co-localization was suppressed by the knockdown of Cx43. Moreover, JNK inhibition clearly suppressed Bax activation. In conclusion, we identified a novel Cx43-JNK-Bax axis regulating the process of apoptosis for the first time.

Assessment of Drug–Drug Interaction between Warfarin and Aprepitant and Its Effects on PT-INR of Patients Receiving Anticancer Chemotherapy

Aprepitant is a known inducer of CYP2C9, the main warfarin-metabolizing enzyme. Consequently, co-administration of these two drugs may result in reduction of the anticoagulation activity of warfarin. However, the nature and degree of time-dependent changes in prothrombin time international normalized ratio (PT-INR) after aprepitant and warfarin co-treatment in patients receiving anticancer chemotherapy has not been elucidated. We retrospectively examined the changes in warfarin dose, PT-INR, and warfarin sensitivity index (WSI; average of PT-INR value/average of daily warfarin dose) during four weeks, i.e., one week before and three weeks after aprepitant administration. The mean and standard deviation values of WSI for one week before and one, two, and three weeks after the beginning of aprepitant administration were 0.51±0.22 (1.00, n=34), 0.74±0.30 (1.53±0.59, n=30), 0.38±0.15 (0.82±0.22, n=28), and 0.46±0.29 (0.87±0.23, n=24), respectively. Values in parentheses represent relative changes versus WSI of one week before and number of subjects. Although the mean value of WSI significantly increased one week after aprepitant administration compared to that at one week before the administration, it in turn significantly decreased two weeks after compared to one week before (paired t-test, p<0.05 after Bonferoni correction). In patients taking warfarin, PT-INR should be carefully monitored for at least two weeks after the beginning of aprepitant administration because it may fluctuate with both aprepitant and chemotherapy during this period.

Physiologically Based Pharmacokinetic Modeling of Bosentan Identifies the Saturable Hepatic Uptake As a Major Contributor to Its Nonlinear Pharmacokinetics

Bosentan is a substrate of hepatic uptake transporter organic anion–transporting polypeptides (OATPs), and undergoes extensive hepatic metabolism by cytochrome P450 (P450), namely, CYP3A4 and CYP2C9. Several clinical investigations have reported a nonlinear relationship between bosentan doses and its systemic exposure, which likely involves the saturation of OATP-mediated uptake, P450-mediated metabolism, or both in the liver. Yet, the underlying causes for the nonlinear bosentan pharmacokinetics are not fully delineated. To address this, we performed physiologically based pharmacokinetic (PBPK) modeling analyses for bosentan after its intravenous administration at different doses. As a bottom-up approach, PBPK modeling analyses were performed using in vitro kinetic parameters, other relevant parameters, and scaling factors. As top-down approaches, three different types of PBPK models that incorporate the saturation of hepatic uptake, metabolism, or both were compared. The prediction from the bottom-up approach (models 1 and 2) yielded blood bosentan concentration-time profiles and their systemic clearance values that were not in good agreement with the clinically observed data. From top-down approaches (models 3, 4, 5-1, and 5-2), the prediction accuracy was best only with the incorporation of the saturable hepatic uptake for bosentan. Taken together, the PBPK models for bosentan were successfully established, and the comparison of different PBPK models identified the saturation of the hepatic uptake process as a major contributing factor for the nonlinear pharmacokinetics of bosentan.

A Novel Method to Estimate Long‐Term Chronological Changes From Fragmented Observations in Disease Progression

Clinical observations of patients with chronic diseases are often restricted in terms of duration. Therefore, obtaining a quantitative and comprehensive understanding of the chronology of chronic diseases is challenging, because of the inability to precisely estimate the patients disease stage at the time point of observation. We developed a novel method to reconstitute long‐term disease progression from temporally fragmented data by extending the nonlinear mixed‐effects model to incorporate the estimation of “disease time” of each subject. Application of this method to sporadic Alzheimers disease successfully depicted disease progression over 20 years. The covariate analysis revealed earlier onset of amyloid‐β accumulation in male and female apolipoprotein E ε4 homozygotes, whereas disease progression was remarkably slower in female ε3 homozygotes compared with female ε4 carriers and males. Simulation of a clinical trial suggests patient recruitment using the information of precise disease time of each patient will decrease the sample size required for clinical trials.

Model-based meta-analysis to evaluate optimal doses of direct oral factor Xa inhibitors in atrial fibrillation patients

The noninferiority of direct oral factor Xa (FXa) inhibitors (rivaroxaban, apixaban, and edoxaban) in treatment of atrial fibrillation were demonstrated compared with warfarin by several large clinical trials; however, subsequent meta-analyses reported a higher risk of major bleeding with rivaroxaban than with the other FXa inhibitors. In the present study, we first estimated the changes of prothrombin time (PT) in 5 randomized trials based on reported population pharmacokinetic and pharmacodynamic models and then carried out a model-based meta-analysis to obtain models describing the relationship between PT changes and the event rates of ischemic stroke/systemic embolism (SE) and of major bleeding. By using the models, we simulated the optimal therapeutic doses for each FXa inhibitor. It was suggested that dose reduction of rivaroxaban from the current 20 mg/d to 10 mg/d would decrease patient deaths from major bleeding (hazard ratio [HR], 0.69; 95% confidence interval [CI], 0.64-0.74) with little increase in those for ischemic stroke/SE (HR, 1.11; 95% CI, 1.07-1.20). The overall decrease in the mortality caused by both events was estimated as 5.81 per 10 000 patient-years (95% CI, 3.92-8.16), with an HR of 0.87 (95% CI, 0.83-0.91). For apixaban and edoxaban, no distinct change in the overall mortality was simulated by dose modification. This study suggested that the current dose of rivaroxaban might be excessive and would need to be reduced to decrease the excess risk of major bleeding.

Investigation of Metabolomic Changes in Sunitinib-Resistant Human Renal Carcinoma 786-O Cells by Capillary Electrophoresis-Time of Flight Mass Spectrometry

Acquired resistance to sunitinib is a challenge in the treatment of renal cell carcinoma (RCC). The dysregulation of cellular metabolism is prevalent during resistance acquisition. It is known that in sunitinib-resistant RCC 786-O (786-O Res) cells sunitinib is mainly sequestered in the intracellular lysosomes. However, the relevance between sunitinib resistance and cellular metabolism has not been examined. In this study, we examined the metabolic changes in 786-O Res by using capillary electrophoresis-time of flight mass spectrometry. The cell line 786-O Res was established via persistent treatment with sunitinib, where increase in intracellular sunitinib, and sizes of lysosomes and nuclei were enhanced as compared with those in the parental 786-O (786-O Par) cells. Metabolic analyses revealed that out of the 110 metabolites examined, 13 were up-regulated and 4 were down-regulated in the 786-O Res cells. The glycolysis, tricarboxylic acid cycle and pentose phosphate pathway (PPP) were identified as being altered in the sunitinib-resistant cells, which resulted in the enhanced metabolisms of energy, nucleic acids, and glutathione redox cycle. As sunitinib was sequestered in the enlarged lysosomes in 786-O Res, the enriched energy metabolism might contribute to the maintenance of luminal pH in lysosomes via the H+ ATPase. The changes in the PPP could contribute to nuclei enlargement through up-regulation of nucleic acid biosynthesis and protect 786-O Res from cytotoxicity induced by sunitinib through up-regulation of reduced glutathione. Though the direct link between sunitinib resistance and metabolic alternation remains to be elucidated, this metabolomics study provides fundamental insights into acquisition of sunitinib resistance.

Determinants of Intestinal Availability for P-glycoprotein Substrate Drugs Estimated by Extensive Simulation With Mathematical Absorption Models

In this study, intestinal drug-drug interactions (DDIs) for substrate drugs of P-glycoprotein were simulated extensively using the extended QGut model and translocation model to explore the determinants of DDI. The results of analyses using both models suggested that permeability and active efflux clearance were the major factors that influenced the fraction absorbed (FA). The results of simulation for 100 virtual drugs in which parameters were generated considering the actual values of commercially available drugs suggested that the ratio of the pH-corrected passive permeability to the intrinsic efflux clearance (Pu/CLeff) relative to that of digoxin would be a useful and quantitative index of P-glycoprotein (P-gp)-mediated DDI risk at lower doses. At higher doses, such as 100 mg, the risk of P-gp-mediated DDI would be significantly reduced because of saturation of P-gp efflux. The simulation suggested that although drugs with lower permeability were more susceptible, even drugs with higher permeability than metoprolol, a representative highly permeable drug, such as BCS class 1 and 2, may experience DDIs owing to P-gp inhibition. Overall, this study demonstrated the usefulness of mathematical intestinal models when only limited observational data are available.

Abstract 5375: Combined treatment of trichostatin A enhances cytotoxic effects of sunitinib on renal cell carcinoma cells

Introduction: Sunitinib (SU) is one of the multi-targeted tyrosine kinase inhibitors (TKIs) and currently approved for use in advanced renal cell carcinoma (RCC) by inhibition of angiogenic signaling. Although it is a really attractive drug for patients who cannot have surgical treatment, SU treatment hasn9t been always successful because of frequent resistance and severe side effects. Combination therapy is one of the strategies to solve these problems. The combinations of SU and IFN-α or mTOR inhibitors in the treatment of RCC have been already explored. However, both of which resulted in failure due to dose-limiting toxicities. Therefore, it is important to establish a novel combination strategy for RCC treatment. In the context of seeking for a prominent combination agent for SU, we paid attention to epigenetic agents. There has been several reports where epigenetic agents were proven to induce apoptotic cells in combination with chemotherapeutic agents, such as gemcitabine and sorafenib. To investigate whether epigenetic agents could be a sensitizer of SU as well in RCC cells, we used trichostain A (TSA), a well-known and strong inhibitor of histone deacetylase (HDAC), and also examined what the mechanism is. Methods: We used 786-O, ACHN and Caki-1 RCC cell lines. The cells were treated with SU alone, TSA alone or the combination, then cytotoxicity was measured by the MTT cytotoxicity method under normoxic or hypoxic conditions. To explore the effect on cell cycle after the combination treatment, flowcytometry was performed. The expression of RTK signaling proteins and p21, which is one of the major targets of TSA, were detected by western blotting. Results: TSA significantly enhanced SU cytotoxicity in all of RCC cells, especially in 786-O. VEGF protein expression, a major angiogenic factor showed increasing trend in 786-O after 8 h of hypoxic exposure, which was decreased by the combined treatment. Flowctometry has revealed that suggested apoptotic cell population (sub-G1) was significantly higher in 1 μM SU and 0.5 μM TSA combination group compared with control or single SU treatment group. On the other hand, ACHN cells didn9t show significant increase of sub-G1 population although cell cycle-arrest at S and G2/M phase was observed in combined treatment group (2.5 μM SU and 0.25 μM TSA). Additionally, cell cycle regulator protein, p21 was significantly increased in both 786-O and ACHN cells in TSA concentration dependent manner, which was accompanied with increase of acetylated histone H3. Conclusion: These findings suggest that combined treatment of SU and TSA is effective against RCC cells. The combination possibly enhances apoptosis or growth inhibition, where increase of p21 by TSA might be involved. Citation Format: Hiromi Sato, Tatsuro Kashiba, Miaki Uzu, Takuya Fujiwara, Yukihiro Shibata, Rina Suzuki, Katsunori Yamaura, Akihiro Hisaka. Combined treatment of trichostatin A enhances cytotoxic effects of sunitinib on renal cell carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5375. doi:10.1158/1538-7445.AM2015-5375