Kazumi Sano

Novel camptothecin analogues that circumvent ABCG2-associated drug resistance in human tumor cells

Irinotecan (7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino]‐carbonyloxycamptothecin; CPT‐11) is a widely used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I); however, overexpression of ABCG2 (BCRP/MXR/ABCP) can confer cancer cell resistance to SN‐38, the active form of CPT‐11. We have recently demonstrated that plasma membrane vesicles prepared from ABCG2‐overexpressing PC‐6/SN2‐5H cells transported SN‐38 and its glucuronide conjugate in an ATP‐dependent manner (Nakatomi et al., Biochem Biophys Res Commun 2001;288:827–32). In the present study, we have characterized a total of 14 new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. All of the tested CPT analogues, which have different substitutions at positions 10 and 11, strongly inhibited the Topo I activity in a cell‐free system, as did SN‐38. Their antitumor activities in the SN‐38‐resistant PC‐6/SN2‐5H2 cell line greatly varied, however, being correlated with intracellular accumulation levels. We have examined ATP‐dependent transport of those CPT analogues by using plasma membrane vesicles prepared from both PC‐6/SN2‐5H2 cells and ABCG2‐transfected HEK‐293 cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with high polarity are good substrates for ABCG2 and are therefore effectively extruded from cancer cells. In this context, to circumvent ABCG2‐associated drug resistance, low‐polarity CPT analogues are considered to be potent lead compounds. The present study provides a practical approach to discover new CPT‐based drugs for the chemotherapy of drug‐resistant human cancer.

Pharmacokinetics of gefitinib predicts antitumor activity for advanced non-small cell lung cancer.

Introduction: We assessed the relationship between the plasma concentration of gefitinib and its efficacy in Japanese patients with advanced non-small cell lung cancer (NSCLC). Methods: Plasma trough levels of gefitinib were measured on days 3 (D3) and 8 (D8) by high-performance liquid chromatography in 44 patients with advanced NSCLC treated with 250 mg gefitinib daily. Eligibility criteria included performance status ≤3, age ≤ 80 years, and stages IIIB–IV cancer. Epidermal growth factor receptor mutations in 23 patients were analyzed retrospectively. Results: The median plasma gefitinib values were 662 ng/ml on D3 and 1064 ng/ml on D8, and the D8/D3 ratio was 1.587. The median progression-free survival (PFS) was 71 days, and the median overall survival was 224 days. Adenocarcinoma, never smoking, and high D8/D3 ratio were associated with better PFS. Multivariate analysis showed that PFS was associated with never smoking and high D8/D3 ratio. Never-smokers with a high D8/D3 ratio showed the best PFS. Overall survival was not associated with the D8/D3 ratio. Epidermal growth factor receptor mutation analysis of 23 patients showed that 15 patients had exon 19 deletion and/or exon 21 point mutation. Median PFS was similar between mutation-positive and mutation-negative individuals in the high D8/D3 group, whereas mutation-negative individuals in the low D8/D3 group showed the worst median PFS. Conclusions: A high D8/D3 ratio was independently associated with better PFS in patients with NSCLC treated with gefitinib. Our findings suggest that the pharmacokinetics of gefitinib may be involved in its antitumor activity.

Mesenchymal mode of migration participates in pulmonary metastasis of mouse osteosarcoma LM8

The outcomes of osteosarcoma patients still remain poor because of intractable pulmonary metastasis. We previously established a highly metastatic osteosarcoma cell line, LM8 from Dunn mouse osteosarcoma by in vivo selection. We herein aimed to clarify the characteristic biological features related with high metastatic potential and new target molecules to suppress pulmonary metastasis of osteosarcoma, using this syngeneic spontaneous metastatic model. LM8 cells acquired fibroblastic morphology with striking filopodia on the cell surface. Immunostaining showed faint stress fiber formation and peripherally localized integrin β1, and biochemical analyses showed the activated Cdc42 and autophosphorylation of focal adhesion kinase (FAK) in LM8 cells when compared to Dunn cells. LM8 cells had activated motility in single cell migration mode. LM8 migration was increased by a Rho-associated kinase (ROCK) inhibitor, Y-27632, while decreased by Cdc42 silencing using RNA interference system. We found that a clinically approved camptothecin analog, irinotecan suppressed the migration, Cdc42 activity, and autophosphorylation of FAK, and attenuated integrin β1 distribution selectively in LM8 cells. Daily oral administration of irinotecan significantly reduced the rate and size of pulmonary metastasis in syngeneic C3H mice. The fibroblastic morphology and activated cell migration with the dependency on Cdc42 but not Rho-ROCK signaling pathway argued that LM8 moved in mesenchymal mode of cell migration. This activated mesenchymal migration was a key component of the pulmonary metastasis of LM8 cells. The inhibition of mesenchymal migration by irinotecan, in addition to its cytotoxic effects, might be effective in preventing pulmonary metastasis of osteosarcoma.

Transport Mechanism-Based Drug Molecular Design: Novel Camptothecin Analogues to Circumvent ABCG2-associated Drug Resistance of Human Tumor Cells

Acquired and intrinsic drug resistance in cancer is the major obstacle to long-term, sustained patient response to chemotherapy. Irinotecan (CPT-11) is a widely-used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I). However, overexpression of ABCG2 (BCRP/MXR/ABCP) reportedly confers cancer cells resistance to SN-38, the active form of CPT-11. To circumvent the ABCG2-associated drug resistance, we have synthesized and characterized a total of fourteen new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. While the lactone E ring is a prerequisite for anticancer activity, modifications of the A or B rings do not significantly affect Topo I inhibition activity. In this context, we have synthesized new CPT analogues with different substitutions at positions 10 or 11 of the A ring. All of the tested CPT analogues strongly inhibited the Topo I activity in a cell-free system. Accordingly, we have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from ABCG2-overexpressing cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with a hydroxyl group at position 10 or 11 of the A ring are good substrates for ABCG2 and therefore effectively extruded from cancer cells. Thus, hydrogen bond formation is considered to be involved in substrate recognition and/or transport processes of ABCG2. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.

Metabolism of sulphobromophthalein. II. Species differences between rats, guinea-pigs and rabbits

Interesting species differences in the metabolism of sulphobromophthalein sodium have been observed between rats, guinea‐pigs and rabbits. The species difference was measured in terms of sulphobromophthalein monoglutathione conjugate (mGSH) positional isomer formation. After an intravenous injection of sulphobromophthalein to rats, 92% of sulphobromophthalein‐mGSH excreted into bile was the α ‐isomer. In contrast, in guinea‐pigs the three isomers α, β and δ were excreted in equivalent amounts. In rabbits, the majority of sulphobromophthalein‐mGSH was excreted as the β‐isomer. The formation ratio of glutathione (GSH) conjugates in‐vitro using cytosolic glutathione S‐transferases (GSTs) prepared from livers generally accounted for the biliary excretion ratio of α‐, β ‐ and δ‐monomercaptide isomers in‐vivo. GSTs from the livers of rat, guinea‐pig, and rabbit were purified and characterized. Although their main GSTs produced different isomers, their 20 amino acid residues showed that they belonged to the same class mu of GSTs. The results suggested differences of the three‐dimensional structure of GSTs that formed sulphobromophthalein‐mGSH isomers between the three animal species.

Pharmacokinetics of Rifabutin in Japanese HIV-Infected Patients with or without Antiretroviral Therapy

Objective Based on drug-drug interaction, dose reduction of rifabutin is recommended when co-administered with HIV protease inhibitors for human immunodeficiency virus (HIV)-associated mycobacterial infection. The aim of this study was to compare the pharmacokinetics of rifabutin administered at 300 mg/day alone to that at 150 mg every other day combined with lopinavir-ritonavir in Japanese patients with HIV/mycobacterium co-infection. Methods Plasma concentrations of rifabutin and its biologically active metabolite, 25-O-desacetyl rifabutin were measured in 16 cases with HIV-mycobacterial coinfection. Nine were treated with 300 mg/day rifabutin and 7 with 150 mg rifabutin every other day combined with lopinavir-ritonavir antiretroviral therapy (ART). Samples were collected at a median of 15 days (range, 5–63) of rifabutin use. Results The mean Cmax and AUC0–24 of rifabutin in patients on rifabutin 150 mg every other day were 36% and 26% lower than on 300 mg/day rifabutin, while the mean Cmax and AUC0–24 of 25–O-desacetyl rifabutin were 186% and 152% higher, respectively. The plasma concentrations of rifabutin plus its metabolite were similar between the groups within the first 24 hours, but it remained low during subsequent 24 to 48 hours under rifabutin 150 mg alternate day dosing. Conclusion Rifabutin dose of 150 mg every other day combined with lopinavir-ritonavir seems to be associated with lower exposure to rifabutin and its metabolite compared with rifabutin 300 mg/day alone in Japanese patients. Further studies are needed to establish the optimal rifabutin dose during ART. The results highlight the importance of monitoring rifabutin plasma concentration during ART. Trial registration UMIN-CTR (https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=search&action=input&language=E) UMIN000001102

High-performance liquid chromatographic determination of sulphobromophthalein and its conjugates.

A simple, sensitive and selective high-performance liquid chromatographic method for the determination of sulphobromophthalein and its mercaptide conjugates in rat bile was developed. These pigments, which have an absorption maximum at 580 nm in alkaline solution, were separated isocratically on an alkali-resistant ODS column by paired-ion chromatography. Analysis of bile samples obtained after intravenous administration of sulphobromophthalein to rats showed the presence of at least twenty peaks of metabolites, of which thirteen were identified and seven quantified.

Metabolism of sulphobromophthalein I: positional isomers of sulphobromophthalein monoglutathione conjugate

Three positional isomers of sulphobromophthalein glutathione monoconjugate (BSP‐mGSH) were detected using a paired‐ion HPLC method that employs triethylamine phosphate (TEA‐H3PO4) as a pairing agent. To confirm that these compounds were glutathione (GSH) conjugates, sulphobromophthalein (BSP) was incubated with a four‐fold volume of GSH under alkaline ammonium hydroxide. At least 6 metabolites (3 di‐GSH conjugates and 3 isomers of mono‐GSH conjugates) were produced under these conditions. The three mono‐GSH conjugates were each purified and identified as compounds with a molecular weight of 1020 according to FAB mass spectrometry results. Positional isomers of BSP‐GSH were provisionally distinguished via the addition of the symbols α, β and δ to the end of each abbreviation, to reflect the amount of isomers present. Thus, the isomer present in the largest quantity was termed BSP‐mGSH(α), the second most abundant isomer was termed BSP‐mGSH(β) and the third was termed BSP‐mGSH(δ). Interestingly, a species difference was recognized in that rat cytosol GSH S‐transferase (GST) primarily produced BSP‐mGSH(α), whereas guinea‐pig cytosol generated BSP‐mGSH(δ), BSP‐mGSH(α) and BSP‐mGSH(β) equally and rabbit cytosol mainly produced BSP‐mGSH(β).

Gefitinib Increases Serum Concentrations of Oral Irinotecan and SN-38 without Increasing the Biliary Concentration of SN-38 in Rats

Background: Gefitinib competes with topoisomerase I inhibitors at drug efflux pumps in vitro. We evaluated the effects of oral gefitinib on pharmacokinetic parameters of orally coadministered irinotecan. Methods: We measured the serum pharmacokinetic parameters and biliary excretion of irinotecan, SN-38 and its glucuronide after irinotecan (50 or 100 mg/kg) was orally administered with or without gefitinib 100 mg/kg to rats. We measured the concentrations of irinotecan and SN-38 in the small intestine, liver, lungs and kidneys in each rat. Results: The plasma area under the curve (0–24 h) of irinotecan and SN-38 was increased significantly, while the apparent elimination constant of irinotecan was decreased significantly. Gefitinib significantly increased the biliary cumulative amounts of irinotecan, but not of SN-38, and also influenced the enterohepatic circulation of irinotecan, but not of SN-38. Conclusions: Gefitinib increased the serum concentrations of irinotecan and SN-38 following oral administration of irinotecan without increasing the biliary excretion of SN-38 in vivo.

Gefitinib enhances the antitumor activity of CPT-11 in vitro and in vivo by inhibiting ABCG2 but not ABCB1: a new clue to circumvent gastrointestinal toxicity risk.

Background: Despite the potent antitumor activity of CPT-11, late-onset diarrhea owing to enterohepatic circulation of SN-38 is a critical issue. Methods: We aimed to evaluate the inhibitory potency of gefitinib against the ABCB1- or ABCG2-mediated excretion of CPT-11 and its active metabolite SN-38 in vitro and in vivo. Results: Gefitinib dose-dependently enhanced the antiproliferation activity of SN-38 in vitro by inhibiting ABCG2. The inhibitory effect of gefitinib on ABCB1 was marginal. When both CPT-11 and gefitinib were administered orally to nude mice bearing human lung cancer PC-6 cells, tumor growth was markedly suppressed. By gefitinib coadministration, the lactone forms of both CPT-11 and SN-38 in the tumor tissue increased more than 2-fold. Conclusions: Gefitinib significantly enhances the antitumor efficacy of CPT-11 and its tumor distribution in vivo. Coadministration of gefitinib may provide a new means to reduce the dose of CPT-11 and to circumvent the gastrointestinal toxicity risk.