Hitoshi Arioka

Phase I study of CPT-11 and etoposide in patients with refractory solid tumors.

PURPOSE To determine the maximum-tolerated dose (MTD) and acceptable dose level of a cytotoxic regimen of CPT-11, a new camptothecin derivative, in combination with etoposide (VP-16) and to describe the principal toxicities associated with it. PATIENTS AND METHODS Patients with refractory solid tumors received VP-16 and CPT-11 daily for 3 consecutive days (days 1 through 3) every 3 or 4 weeks. Groups entered the trial at escalating CPT-11/VP-16 dose levels of 40/60, 60/60, 60/80, and 80/60 mg/m2. Thirty-four patients entered this study, of whom 33 were assessable for toxicity and 22 for therapeutic efficacy. RESULTS Granulocytopenia was so severe that this regimen required supportive therapy with recombinant human granulocyte colony-stimulating factor (G-CSF). The majority of the patients experienced a 5% weight loss and diarrhea was the dose-limiting toxicity. The MTDs were 60/80 and 80/60 mg/m2 administered on days 1 through 3. Five of seven previously untreated patients with non-small-cell lung cancer (NSCLC) achieved partial responses (PRs) to this therapy, as did two with NSCLC who had received prior chemotherapy, two with head and neck cancer, and one with an adenocarcinoma (primary tumor unknown). CONCLUSION The recommended dose of CPT-11/VP-16 for this regimen with G-CSF is 60/60 mg/m2 on days 1 through 3 every 3 to 4 weeks. We suggest that the combination of topoisomerase I and II inhibitors is likely to be an effective treatment strategy. The activity of this regimen against NSCLC is particularly encouraging and should be evaluated in a phase II trial.

Mechanism of the radiosensitization induced by vinorelbine in human non-small cell lung cancer cells

Vinorelbine (Navelbine, KW-2307), a semisynthetic vinca alkaloid, is a potent inhibitor of mitotic microtubule polymerization. The aims of this study were to demonstrate radiosensitization produced by vinorelbine in human non-small cell lung cancer (NSCLC) PC-9 cells and to elucidate the cellular mechanism of radiosensitization. A clonogenic assay demonstrated that PC-9 cells were sensitized to radiation by vinorelbine with a maximal sensitizer enhancement ratio at a 10% cell survival level of 1.35 after 24-h exposure to vinorelbine at 20 nM. After 24-h exposure to vinorelbine at 20 nM, the approximately 67% of the cells that had accumulated in the G2/M-phase were cultured in the absence of vinorelbine and then irradiated at a dose of 8 Gy. Flow cytometric analyses showed prolonged G2/M accumulation concomitant with continuous polyploidization, and induction of apoptosis was observed in the cells subjected to the combination of vinorelbine-pretreatment and radiation. Polyploidization and induction of apoptosis were confirmed by morphological examination and a DNA fragmentation assay, respectively. We concluded that vinorelbine at a minimally toxic concentration moderately sensitizes human NSCLC cells to radiation by causing accumulation of cells in the G2/M-phase of the cell cycle. Prolonged G2/M accumulation concomitant with continuous polyploidization and increased susceptibility to induction of apoptosis may be associated with the cellular mechanism of radiosensitization produced by vinorelbine.

Enhancement of cisplatin sensitivity in high mobility group 2 cDNA-transfected human lung cancer cells.

To elucidate the role of high mobility group 2 protein (HMG2) in cis‐diamminedichloroplatinum (II) (cisplatin, CDDP) sensitivity, we constructed a human HMG2‐transfected human non‐small cell lung cancer cell line, PC‐14/HMG2. The HMG2 mRNA expression level was approximately twice those of parental PC‐14 and mock‐transfected PC‐14/CMV. Gel mobility shift assay revealed a CDDP‐treated DNA‐protein complex in the nuclear extract of PC‐14/HMG2, which was not found in the extracts of PC‐14 and PC‐14/CMV. This complex formation was subject to competition by CDDP‐treated non‐specific salmon sperm DNA, indicating that ectopic HMG2 recognizes CDDP‐damaged DNA. PC‐14/HMG2 showed more than 3‐fold higher sensitivity to CDDP than PC‐14 and PC‐14/CMV. The intracellular platinum content of PC‐14/HMG2 after exposure to 300 μM CDDP was 1.1 and 1.5 times that of PC‐14 and PC‐14/CMV, respectively. Cellular glutathione levels were not different in these cell lines. Repair of DNA interstrand cross‐links determined by alkaline elution assay was decreased in PC‐14/HMG2. These results suggest that HMG2 may enhance the CDDP sensitivity of cells by inhibiting repair of the DNA lesion induced by CDDP.

Improvement by eicosanoids in cancer cachexia induced by LLC-IL6 transplantation.

Cachexia frequently occurs in the late stages of cancer, and is difficult to manage. We previously reported that interleukin-6 (IL-6) cDNA transfection into Lewis lung carcinoma (LLC-IL6) induced cachexia-like symptoms in C57BL/6 mice. This was thought to be a useful experimental model of cancer cachexia. We have examined the effects of two eicosanoids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in order to evaluate whether they could relieve cachexia. LLC-IL6-bearing animals were divided into three treatment groups receiving DHA, EPA or water as the control; 80-μl samples of these compounds (purity>95%) were administered orally by catheter daily starting 7 days after tumor transplantation. Tumor growth curves were similar in the three groups. There were no differences in water or food intake in the three groups. However, body weight, a marker of cachexia, was significantly higher in treated mice than in the control group. Sixteen days after tumor transplantation, the mean body weight was 17.45 g (P<0.05), 17.2 g and 16.41 g in the groups receiving DHA, EPA and water respectively. The eicosanoids did not affect serum levels of IL-6. Ubiquitination of muscle protein, a marker of proteolysis coupled to cachexia, was compared in LLC-IL6-and LLC-transplanted mice. The eicosanoids prevented the ubiquitination of approximately 180 kDa protein. These results suggest that eicosanoids may prevent the cachexia mediated by IL-6.

A randomised phase II study of TSU-68 in patients with hepatocellular carcinoma treated by transarterial chemoembolisation.

BACKGROUND TSU-68 is an antitumour drug that acts by inhibiting angiogenesis. We evaluated the efficacy and safety of TSU-68 in combination with transarterial chemoembolisation (TACE) in patients with intermediate-stage hepatocellular carcinoma (HCC). PATIENTS AND METHODS In this multicenter, open-label phase II study, we randomised patients with HCC who had been treated with a single session of TACE to receive either 200mg TSU-68 twice daily or no medication. The primary end-point was progression-free survival (PFS). RESULTS A total of 103 patients were enrolled. Median PFS was 157.0days (95% confidence interval [CI], 124.0-230.0days) in the TSU-68 group and 122.0days (95% CI, 73.0-170.0days) in the control group. The hazard ratio was 0.699 (95% CI, 0.450-1.088). Fatigue, elevated aspartate aminotransferase (AST), elevated alkaline phosphatase, oedema and anorexia were more frequent in the TSU-68 group than in the control group. The most frequent grade 3/4 adverse events were AST elevation (46% of patients in the TSU-68 group and 12% of controls) and alanine aminotransferase elevation (26% of patients in the TSU-68 group and 8% of controls). Two deaths, grade 5 hepatic failure and melena were noted in the TSU-68 group. CONCLUSION This exploratory study shows a trend towards prolonged PFS with TSU-68 treatment after a single session of TACE, but this observation was not statistically significant. The two deaths were related to the study treatment. These results suggest that further examination of the study design is necessary to determine whether TSU-68 has any clinical benefits when combined with TACE.

p16INK4 expression is associated with the increased sensitivity of human non-small cell lung cancer cells to DNA topoisomerase I inhibitors.

Inactivation of p16INK4, an inhibitor of cyclin‐dependent kinases 4 (CDK4) and 6 (CDK6), may be essential for ontogenesis in non‐small cell lung cancer (NSCLC). We examined the sensitivity of two clones of P16INK4‐transfected NSCLC cell line with homozygous deletion of p16INK4, A549/pl6‐l and 2, to DNA topoisomerase I (topo I) inhibitors. A549/pl6‐l and ‐2 showed 7.7‐ and 9.1‐fold increases in sensitivity to CPT‐11 (11,7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino]carbonyloxycamptothecin), respectively, compared with A549 cells. Ectopic p16INK4‐expressing cells also showed ∼4.0‐fold increase in sensitivity to SN‐38 (7‐ethyl‐10‐hydroxycamptothecin), the active metabolite of CPT‐11, compared to the parent cells. The topo I‐mediated DNA relaxation activities of ectopic p16INK4‐expressing cells were approximately 5 times higher than those of the parent cells. Northern and western blot analyses indicate that these increased topo I activities of ectopic p16INK4‐expressing cells were due to an elevated topo I mRNA level and an increase in topo I protein. The chemosensitivity to topo I inhibitors, topo I mRNA level, protein content and activity of a pl6INK4 revertant, lacking functional p16INK4, tended to be restored toward those of the parental phenotype to some extent. These results suggest that p161NK4 expression is closely associated with the increased sensitivity of ectopic pl6INK4‐expressing NSCLC cells to topo I inhibitors. The up‐regulation of topo I mRNA level, protein content and activity may he responsible for this hypersensitivity.

A Randomized Cross‐over Study of High‐dose Metoclopramide plus Dexamethasone versus Granisetron plus Dexamethasone in Patients Receiving Chemotherapy with High‐dose Cisplatin

We carried out a randomized, single‐blind, cross‐over trial to compare the antiemetic effect, for both acute and delayed emesis, of granisetron plus dexamethasone (GRN+Dx) with that of high‐dose metoclopramide plus dexamethasone (HDMP + Dx). Fifty‐four patients with primary or metastatic lung cancer, given single‐dose cisplatin (> 80 mg/m2) chemotherapy more than twice, were enrolled in this study. They were treated with both HDMP+Dx and GRN+Dx in two consecutive chemotherapy courses. On day 1, patients experienced a mean of 2.5 (SD=4.3) and 0,1 (SD = 0.4) episodes of vomiting in the HDMP+Dx and the GRN + Dx groups, respectively (P=0.0008). Complete response rate on day 1 was 45 and 90% in the HDMP+Dx and the GRN+Dx groups, respectively (P= 0.0001). Patients treated with GRN+Dx had a tendency to suffer more episodes of vomiting than the HDMP+Dx group on days 2–5, but it was not statistically significant. Twenty‐four patients (57%) preferred the GRN+Dx treatment and 14 patients (33%), HDMP + Dx. In the HDMP + Dx group, nine patients (21%) had an extrapyramidal reaction, and 5 patients (12%) had constipation that lasted for at least two days. In contrast, no patients had extrapyramidal reactions, and IS patients (43%) had constipation in the GRN+Dx group (P < 0.01). GRN+Dx was more effective than HDMP+Dx only in preventing the acute emesis induced by cisplatin. An effective treatment for delayed emesis is still needed.

Phase I and Pharmacokinetic Study of Paclitaxel by 24‐Hour Intravenous Infusion

Paclitaxel, a new antitubular agent, appears to be one of the most promising single agents for the chemotherapy of various solid tumors. The primary objectives of this phase I study of paclitaxel using 24‐h continuous intravenous infusions were to determine the maximum tolerated dose of paclitaxel administered by this schedule to Japanese patients with solid tumors and to evaluate the pharmaco‐kiiietics of paclitaxel. Eighteen patients received one of five doses of paclitaxel, 49.5, 75, 105, 135 or 180 mg/m2. Prcmedication with diphenhydramine, dexamethasone, and ranitidine was used to prevent acute hypersensitivity reactions. Pharmacokinetic data were obtained from all 18 patients. Dose‐limiting toxicities observed at 180 mg/m2 consisted of grade 4 granulocytopenia associated with grade 3 infection. No severe HSRs or cardiac toxicity were detected. Reversible toxicities observed included liver dysfunction, alopecia, peripheral neuropathy and myalgias. Pharmacokinetic studies performed using high‐performance liquid chromatography demonstrated that plasma concentrations of paclitaxel increased during the 24‐h infusion and declined immediately upon cessation of the infusion with a half life of 13.1‐24.6 h (75‐180 mg/m2). Less than 10% of paclitaxel was excreted in the urine within 72 h. The peak plasma concentrations and the areas under the concentration‐versus‐time curves increased linearly with the dose administered. Antitumor activity was observed in one patient with pulmonary metastasis from pharyngeal cancer. Based on these studies a phase II trial dose of 135 mg/m2 administered over 24 h was chosen.

In vitro and in vivo growth of B16F10 melanoma cells transfected with interleukin-4 cDNA and gene therapy with the transfectant

In an attempt to develop the most effective cytokine gene therapy, we transfected mouse interleukin(IL)-2, mouse IL-4, and human IL-6 cDNAs into mouse melanoma cells, B16F10. Transfection with IL-4 cDNA decreased the tumorigenicity of B16F10 most strongly. We investigated whether gene therapy with IL-4-transfected B16F10 cells was possible. Flowcytometric analysis showed that major histocompatibility complex class I and II expression in B16F10 and IL-4-cDNA-transfected B16F10 (B16F10-IL4) cells did not differ. Doubling times of B16F10 and B16F10-IL4 were 20.1 and 21.1 h respectively. The growth of B16F10 cells was retarded if C57BL/6 mice were inoculated with B16F10-IL4 at the contralateral sides. When 5×105 B16F10 cells were transplanted subcutaneously into the flanks of C57BL/6 mice, they all developed a tumor mass, whereas no tumor masses formed in those transplanted with B16F10-IL4 cells within 60 days. No nude, severe combined immunodeficient or beige mice were able to reject parental B16F10 or B16F10-IL4 cells, although, B16F10-IL4 tumor growth in all these immunodeficient mice was slower than that of B16F10. Therefore, we concluded that T and natural killer cells are necessary for rejection of B16F10-IL4 tumor cells.

A Topoisomerase II Inhibitor, NK109, Induces DNA Single- and Double-strand Breaks and Apoptosis

2,3‐(Methylenedioxy)‐5‐methyl‐7‐hydroxy‐8‐methoxybenzo[c]phenanthridinium hydrogensulfate di‐hydrate, called NK109, is a benzo[c]phenanthridine derivative, which inhibits DNA topoisomerase II activity by stabilizing the DNA‐enzyme‐drug complex, and shows strong growth‐inhibitory effects on several human cancer cells. In the present study, NK109 treatment induced DNA fragmentation and a rise in the level of cytoplasmic nucleosomes, which are markers of apoptosis, in human small‐cell lung carcinoma SBC‐3 cells. These effects were inhibited by zinc ions and enhanced by cycloheximide or actinomycin D. Dose‐dependent single‐ and double‐strand DNA breaks were observed, using alkaline and neutral elution assays, in SBC‐3 cells treated with more than 0.2 μM NK109 for 4 h. Treatment with NK109 caused more DNA single‐ and double‐strand breaks than treatment with an equimolar amount of VP‐16. These results suggest that NK109 induces DNA strand breaks and apoptosis. In addition, it appears that this process does not require protein or RNA synthesis, but involves a specific endonuclease which is inhibited by zinc ions.