Shuichi Kishimoto

Preparation and evaluation of o/w type emulsions containing antitumor prostaglandin

Antitumor prostaglandins(PGs) such as Delta12-PGJ2 and Delta7-PGA1 possess a cyclopentenone or cross-conjugated dienone structures. Antitumor PGs are actively incorporated through cell membrane and control gene expression. Very recent studies clarified that P53 independent expression of p21 and gadd 45, activation of PPARgamma are involved in antitumor mechanism of these PGs. At the low concentration, these PGs exhibit physiological or pathological activity such as osteoblast calcification, promotion of colon cancer cell proliferation. COMPARE PROGRAM using human 38 tumor cell lines suggested that antitumor mechanism of Delta7-PGA1 and 13, 14-dihydro-15-deoxy-Delta7-PGA1 methyl ester (TEI-9826) are quite different from other anticancer agents which are clinically used. Lipid microspheres and Lipiodol formulation were examined as dosage form of the PGs and lipid microspheres were selected for further study. At first lipid microspheres integrated TEI-9038 (Lipo TEI-9038) was chosen as a candidate for clinical trial. However Lipo TEI-9038 failed to exhibit substantial antitumor effect because of its enzymatic instability and toxicity in vivo. Lipo TEI-9826 was then selected as promising candidate for clinical trial because of its stability in serum. Lipo TEI-9826 exhibited marked antitumor effect in several animal models including CDDP resistant nude mice model. Pharmacokinetic and toxicological studies using rats suggested that continuous infusion is the most suitable administration method for Lipo TEI-9826. New type emulsifier, Controlled High Pressure Process Homogenizer (De-BEE 2000 and mini De-BEE) was developed during the preclinical studies on manufacturing process of Lipo TEI-9826. These results warrant the clinical trial for Lipo TEI-9826 in CDDP resistant cancer.

Role of Na+, K+-ATPase α1 subunit in the intracellular accumulation of cisplatin

The present study was undertaken to identify what regulates intracellular cisplatin (CDDP) accumulation and what changes in membrane fraction of CDDDP-resistant cell line. The CDDP-resistant rat hepatoma cell line, H4-II-E/CDDP, shows a significant decrease in intracellular platinum accumulation compared with parental H4-II-E cells, although there was no difference in the efflux of CDDP between these two cell lines. In this study, we examined the contribution of functional change in active transport to the CDDP resistance of H4-II-E/CDDP cells. Compared with the resistant cells, platinum accumulation in the parental cells was clearly decreased by low temperature or ATP depletion. In addition, the Na+, K+-ATPase inhibitor ouabain and the K+ channel inhibitor tetraethylammonium decreased platinum accumulation in parental cells but did not change the accumulation in resistant cells. Amphotericin B, an antifungal agent, increased the intracellular platinum accumulation in resistant cells to the same level as in parent cells. Western blot analysis demonstrated that the Na+, K+-ATPase α1 subunit was reduced in resistant cells compared with the parental cells, although there was no difference in the expression of the β1 subunit between the two cell lines. Furthermore, the Na+, K+-ATPase α1 subunit of H4-II-E was decreased following a 24-h exposure to CDDP. These results suggest that Na+, K+-ATPase-dependent active transport of CDDP does not occur in resistant cells, and, furthermore, our findings provide the first evidence that the Na+, K+-ATPase α1 subunit plays an important role in the transport of CDDP.

The pharmacokinetics of morphine and its glucuronide conjugate in a rat model of streptozotocin‐induced diabetes and the expression of MRP2, MRP3 and UGT2B1 in the liver

Objectives The aim was to investigate the pharmacokinetics of morphine and its metabolite, morphine‐3‐glucuronide (M3G), in a rat model of streptozotocin (STZ)‐induced diabetes.

Effect of absorption rate on pharmacokinetics of ibuprofen in relation to chiral inversion in humans

The effect of absorption rate on the pharmacokinetics of ibuprofen enantiomers was investigated in 12 healthy Han Chinese male volunteers following oral administration of immediate‐release (IR) and sustained‐release (SR) preparations containing racemic ibuprofen (rac‐ibuprofen). The area under the curve of the plasma concentration‐time curve (AUC; (mean ± s.d.) values for rac‐ibuprofen were 192.90 ± 43.47 for the SR preparation and 195.90 ± 31.69 μg h mL−1 for the IR preparation. AUC values for the enantiomers after administration of the SR formulation were 55.38 ± 17.79 and 92.51 ± 30.68 μg h mL−1 for R‐ and S‐ibuprofen, respectively, and were 65.94 ± 20.06 and 100.81 ± 32.28 μg h mL−1 for R‐ and S‐ibuprofen after administration of the IR preparation. These values did not differ significantly. Cmax values were significantly decreased with the SR preparation: 25.11 ± 5.71, 12.24 ± 3.79 and 12.38 ± 3.55 μg mL−1 for rac‐, R‐, and S‐ibuprofen, respectively, after administration of the SR preparation, vs 46.21 ± 8.20, 20.82 ± 5.90 and 23.46 ± 7.30 μg mL−1 for rac‐, R‐, and S‐ibuprofen, respectively, after administration of the IR preparation. Mean residence time was significantly increased: 7.01 ± 1.29, 5.52 ± 1.25 and 7.04 ± 1.30 h for rac‐, R‐, and S‐ibuprofen, respectively, after administration of the SR preparation vs 4.34 ± 0.89, 3.43 ± 0.64 and 4.51 ± 0.79 h for rac‐, R‐, and S‐ibuprofen, respectively, after administration of the IR preparation. AUC values for S‐ibuprofen were significantly larger than those for R‐ibuprofen in both preparations, indicating unidirectional chiral inversion. The S/R ratio of serum concentrations of enantiomers was 1.78‐fold higher at 6 h after administration of the SR preparation compared with the IR preparation (P < 0.01).

Effects of insulin on CYP3A activity and nicardipine disposition in streptozotocin-induced diabetic rats.

Objectives  The aim of the study was to clarify the effect of insulin treatment on drug metabolism and disposition.

Altered expression of MRP2, MRP3 and UGT2B1 in the liver affects the disposition of morphine and its glucuronide conjugate in a rat model of cholestasis

Objectives The aim was to investigate the disposition of morphine and morphine‐3‐glucuronide (M3G) in a rat model of cholestasis induced by bile duct ligation (BDL).

Population Pharmacokinetic–Toxicodynamic Modeling and Simulation of Cisplatin-Induced Acute Renal Injury in Rats: Effect of Dosing Rate on Nephrotoxicity

Nephrotoxicity is the major dose-limiting toxicity of cisplatin (CDDP). The aim of this study was to develop a pharmacokinetic (PK)/toxicodynamic (TD) model of CDDP-induced acute renal injury in rats and to simulate nephrotoxicity at various dosing rates. CDDP was administered to rats by a 30-s bolus or a 2-h infusion (1.0, 2.5, 5.0, and 7.5 mg/kg). Unbound CDDP concentrations in plasma and urine were determined up to 2 h after administration in the PK study, and plasma creatinine (Cr) levels were monitored for up to 7 days as an index of nephrotoxicity in the TD study. The PK was linear and was fitted with a traditional 2-compartment model. The TD was nonlinear and differed between dosing rates. The creatinine concentration profiles were fitted with a signal transduction-indirect response model. Population analysis using a nonlinear mixed-effect model was adapted to the developed PK/TD model and was well-validated. Dosing simulations from the developed population PK/TD model indicated that CDDP-induced nephrotoxicity was due to not only Cmax but also the time above the toxic concentration of CDDP. Prolongation of infusion time will not necessarily attenuate acute nephrotoxicity. This study demonstrated the potential utility of PK/TD modeling for preventing nephrotoxicity.

Intracellular uptake of an antitumor-active azole-bridged dinuclear platinum(II) complex in cisplatin-resistant tumor cells.

A cationic azolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH3)2}2(μ-OH)(μ-methyl-pyrazolate)]2+ (4M-PzPt), was developed to overcome resistance to cisplatin (CDDP). This study aimed to assess the cytotoxicity of 4M-PzPt against a CDDP-resistant cell line, H4-II-E/CDDP, and compare the intracellular accumulation of CDDP and 4M-PzPt. H4-II-E and H4-II-E/CDDP displayed similar sensitivity to 4M-PzPt; however, the sensitivity of H4-II-E/CDDP to CDDP was approximately 19-fold lower than that of H4-II-E. The difference in the sensitivity to both platinum complexes corresponded with the difference in the amount of intracellular platinum accumulation after exposure to CDDP or 4M-PzPt in both cell lines. In H4-II-E, HepG2, and HuH-7 cells, the intracellular uptake of CDDP and 4M-PzPt occurred via active transport and passive transport. Results of co-exposure with the transport inhibitors ouabain, tetraethylammonium, and cimetidine indicated that the intracellular uptake of CDDP was dependent on Na+/K+-ATPase and that of 4M-PzPt was dependent on organic cation transporters (OCTs), probably OCT1. This study suggested that 4M-PzPt could inhibit the growth of a CDDP-resistant tumor via an intracellular uptake mechanism different from that of CDDP.

Abstract 4742: Comparison of enhancement of the antitumor effect of CDDP by PXR antagonists

We have already found that a nuclear receptor PXR can modulate the cytotoxicity of CDDP for cancer cells, and ketoconazole, a PXT antagonist, can be an agent enhancing the antitumor activity of CDDP due to increase of the intracellular platinum content as the mechanism. However, nuclear receptor ligands have a variety of functions, and there is a possibility that PXR antagonist may enhance the cytotoxicity of CDDP through the mechanism other than the inhibition of transporter-mediated efflux of CDDP. The goal of this study was to compare the enhancing ability of three PXR antagonists, ketoconazole (KTZ), phenethyl isothiocyanate (PEITC) and metformin (MFM), for the antitumor effect of CDDP and clarify the contribution of inhibition of the efflux transporter as the mechanism of PXR antagonist. Cell lines used in this study were human hepatoma cell line HepG2. Changes in mRNA expression were assessed by real-time RT-PCR, and the ability of apoptosis induction was assessed as caspase-3 activity. Total intracellular platinum contents were determined by ICP-AES. When HepG2 cells were treated for 24 hr with 10 μM of CDDP, no change of caspase-3 activity was observed when compared to control cells. Also, 30 μM of KTZ, 30 μM of PEITC or 5 mM of MFM had no affect on the caspase-3 activity in treated cells compared with control cells. When KTZ, PEITC or MFM was exposed to HepG2 cells from 24 hr before treatment and during 24-hr treatment with 10 μM of CDDP, the caspase-3 activity was significantly increased to 461%, 355% and 326% of the control, respectively, in comparison with that in CDDP alone. When HepG2 cells were treated for 8 hr with 25 μM of CDDP, the intracellular platinum content was 110 ng/mg protein. On the other hand when KTZ, PEITC or MFM was exposed to HepG2 cells from 24 hr before treatment and during 8-hr treatment with 25 μM of CDDP, the intracellular platinum content was apparently increased to 227%, 491% and 161% of CDDP alone, respectively. Three PXR antagonists resulted in an increase in the intracellular platinum content that seems to be due to inhibition of the platinum efflux transporter, but there is no correlation between increase in intracellular platinum content and enhancement of antitumor activity by PXR antagonists. These results concluded that PXR antagonist can be an agent enhancing the antitumor activity of CDDP, but the ability of PXR antagonist for enhancing antitumor activity of CDDP can not be determined from only the increase in the intracellular platinum content. Citation Format: Shuichi Kishimoto, Megumi Yasuda, Megumi Tomita, Yuki Yamane, Ryosuke Suzuki, Shoji Fukushima. Comparison of enhancement of the antitumor effect of CDDP by PXR antagonists. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4742.

Abstract 840: Enhancement of antitumor effect of platinum complexes by PXR antagonist

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The goal of this study was to evaluate the enhancing ability of PXR antagonists for the antitumor effect of platinum complex. Cell lines used in this study were human hepatoma cell line HepG2, human ovarian cancer cell line 2008 and human colon cancer cell line HCT116. Changes in mRNA expression were assessed by real-time RT-PCR, and the ability of apoptosis induction was assessed as caspase-3 activity. Cytotoxicity of platinum complexes was assessed by SRB assay, and IC75 and IC90 values of them were determined. They did not induce apoptosis at IC75 but did it markedly at IC90. Previously, we reported that though apoptosis in HepG2 was induced by IC90 dose of CDDP, addition of a PXR agonist, rifampicin, from 24-hr before treatment and during treatment with CDDP was almost suppressed. Moreover, we found that apoptosis in 2008 and HCT116 induced by IC90 dose of CDDP or oxaliplatin was suppressed by rifampicin. That suggests a nuclear receptor PXR affects on the cytotoxicity of platinum complexes for cancer cells other than HepG2. Ketoconazole (KTZ) and phenethyl isothiocyanate (PEITC) were selected as PXR antagonists that were added with platinum complex. Both drugs at 30 μM induced cytostatic cell growth inhibition in HepG2, 2008 and HCT116 but did not affect on caspase-3 activity compared with control in those cells. When these PXR antagonists at 30 μM were added to cancer cell lines from 24-hr before treatment and during treatment with CDDP at IC75, caspase-3 activity was significantly increased compared with that in CDDP alone. Also, when cells were exposed to CDDP and a PXR antagonist without PXR antagonist pretreatment, caspase-3 activity was increased but the level was reduced compared with that in the combination with pretreatment. Similarly, oxaliplatin in combination with KTZ or PEITC markedly induced apoptosis in HCT116. In HepG2, KTZ reduced the level of MRP2 mRNA. That suggests that pretreatment with KTZ might increase the intracellular platinum content and enhance the cytotoxicity of platinum complex. We concluded that PXR antagonist can be an agent enhancing the antitumor activity of platinum complex, and one of the mechanism is to suppress the expression of the platinum efflux transporter. Citation Format: Shuichi Kishimoto, Erika Bou, Kaho Higashi, Ryosuke Suzuki, Shoji Fukushima. Enhancement of antitumor effect of platinum complexes by PXR antagonist. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 840. doi:10.1158/1538-7445.AM2014-840