Radiopotentiation by the oral platinum agent, JM216: role of repair inhibition

PURPOSE: To test for in vitro radiopotentiation by the orally-administered platinum (IV) complex, JM216; to compare these results to cisplatin and carboplatin; and to investigate whether the mechanism of radiopotentiation involves repair inhibition of radiation-induced DNA damage. METHODS AND MATERIALS: H460 human lung carcinoma cells were incubated with the drugs for 1 h at 37 degrees C, irradiated at room temperature, and returned to 37 degrees C for 20 min. Cells were then rinsed and colony forming ability was assessed. Wild-type V79 Chinese hamster cells and radiosensitive, DNA repair-deficient mutant cells (XR-V15B) were also studied along with H460 cells. Ku86 cDNA, which encodes part of a protein involved in DNA repair, was transfected into XR-V15B cells as previously described. The effect of JM216 on sublethal damage repair (SLDR) was also assessed using split-dose recovery. RESULTS: Using equally cytotoxic doses of JM216, cisplatin, and carboplatin, the radiation dose enhancement ratios (DER) were 1.39, 1.31, and 1.20, respectively; the DER with 20 microM JM216 was 1.57. JM216 (20 microM) did not significantly change the final slope of radiation survival curves, but greatly reduced the survival curve shoulder. V79 cells also showed radioenhancement using 20 microM JM216, but no enhancement occurred using XR-V15B cells. Transfection of Ku86 cDNA into XR-V15B cells restored radiopotentiation by JM216 to wild-type V79 levels. In addition, 20 microM JM216 completely inhibited sublethal damage repair in H460 cells. CONCLUSION: Our results show that JM216 can potentiate the effects of radiation in human lung cancer cells, and that the mechanism of this effect is probably inhibition of DNA repair by JM216.     

Biotransformation of the platinum drug JM216 following oral administration to cancer patients

 This study evaluates the metabolic profile of JM216 [bis(acetato)ammine-dichloro(cyclohexylamine) platinum(IV)], the first orally administrable platinum complex, in plasma ultrafiltrates of 12 patients (n=2–4 time points per patient) following different doses of drug (120, 200, 340, 420, 560 mg/m²). The biotransformation profile was evaluated by high-performance liquid chromatography (HPLC) followed by atomic absorption spectrophotometry (AA). The AA profiles were compared with those previously identified by HPLC on line with mass spectrometry (HPLC-MS) in plasma incubated with JM216. A total of six platinum peaks (Rt=5.5, 7.2, 10.6, 12.4, 15.6, and 21.6 min, respectively) were observed in patients’ plasma ultrafiltrate samples, of which only four appeared during the first 6 h post-treatment. Four of these coeluted with those observed and identified previously in plasma incubation medium. No parent JM216 was detected. The major metabolite seen in patients was the Pt II complex JM118 [cis-amminedichloro-(cyclohexylamine)platinum (II)] and was observed in all the patients. Interestingly, the second metabolite was shown to coelute with the Pt IV species JM383 [bis-acetatoammine(cyclohexylamine)dihydroxoplatinum (IV)]. Both JM118 and JM383 were identified by HPLC-MS in a clinical sample. Peak C, which was a minor product (less than 5% of the free platinum), coeluted with JM559 [bis-acetatoammine-chloro(cyclohexylalamine)hydroxoplatinum (IV)]. The cytotoxicity profile of all three metabolites in a panel of cisplatin-sensitive and -resistant human ovarian carcinoma cell lines was very close to that of the parent drug. In addition, the concentrations of JM118 reached in patients’ plasma ultrafiltrate were comparable with the cytotoxic levels of the compound determined in the ovarian carcinoma panel of cell lines. Two metabolites were seen in patients but not in the in vitro incubation medium, suggesting the involvement of a possible enzyatic reaction. Thus, the biotransformation profile following oral administration of JM216 shows a variety of Pt(IV) and Pt(II) metabolites in plasma that differ significantly from other systemically applied platinum drugs. Received: 8 May 1995/Accepted: 6 October 1995     

Potential radiation-sensitizing effect of semisynthetic epothilone B in human lung cancer cells.

BACKGROUND AND PURPOSE: To evaluate a semisynthetic epothilone B, BMS-247550, as a potential radiosensitizer in vitro and in vivo. MATERIALS AND METHODS: Human NCI-H460 lung cancer cells were treated with either BMS-247550 or paclitaxel and in combination with radiation at multiple doses for different time periods. Surviving fractions were then analyzed using a clonogenic assay. Cell cycle redistribution by BMS-247550 was measured with propidium iodide and flow cytometry. Percent apoptosis was also measured using 7-amino-actinomycin D staining with flow cytometry. For in vivo studies, the H460 xenograft model was used in athymic nude mice. Tumors were treated with BMS-247550 (5 mg/kg) i.p. injection on days 0, 2, and 4 and/or radiation (2 Gy/day, days 0-4). RESULTS: The in vitro radiation dose enhancement ratios (DER) of 1-h BMS-247550 and paclitaxel were 2.03 and 1.34, respectively. Treatment with BMS-247550 enhanced the G2/M block and induced apoptosis; whereas in combination with radiation, the induction of apoptosis was additive. BMS-247550 in combination with radiation in vivo enhanced the tumor growth delay when compared with either drug or radiation alone. CONCLUSIONS: These results demonstrated that BMS-247550 could enhance the effects of radiation in human lung cancer cells both in vitro and in vivo and that a G2/M block and increased apoptosis might be possible explanations for the enhancement.     

Lack of nephrotoxicity of new oral platinum drug JM216 in lung cancer patients

Purpose: The purpose of this study was to assess renal function in patients treated with the oral platinum drug JM216 [bisacetato-ammine-dichloro-cyclohexylamine-platinum(IV)], since the effects of JM216 on renal function have only partly been investigated using serum parameters or ⁵¹Cr-EDTA clearance. We used a sensitive method that assessed glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and indicators of tubular and glomerular damage. Methods: A group of 24 patients with either non-small-cell lung cancer (NSCLC) stage IIIb/IV or small-cell lung cancer (SCLC), limited disease (LD) or extensive disease (ED), treated with JM216 were studied. All patients had no prior chemotherapy, a performance score <2, a life expectancy of more than 3 months and normal liver, renal and bone marrow functions before treatment. All patients received oral JM216 120 mg/m² per day for 5 consecutive days, repeated every 21 days with a maximum of six cycles. In six SCLC patients the dose was escalated to 140 mg/m² per day after the first cycle. Prior to treatment, after the first cycle and after the end of treatment renal function was assessed by ¹²⁵I-sodium thalamate and ¹³¹I-hippurate clearances to determine acute and cumulative changes in GFR and ERPF, respectively. Furthermore, tubular and glomerular damage were assessed by urinary excretion of β₂-microglobulin, lactic dehydrogenase (LDH), alkaline phosphatase (ALP), gamma-glutamyltransferase (GT) and albumin. Results: In 20 evaluable patients no significant acute impairment of renal function was observed. Median (range) GFR, ERPF and filtration fraction (FF) before treatment were 101 ml/min (53–164 ml/min), 417 ml/min (227–719 ml/min), and 0.25 (0.19–0.33), respectively. After the first cycle values were 117 ml/min (71–189 ml/min), 418 ml/min (228–709 ml/min) and 0.28 (0.21–0.33), respectively. Also, no indications of tubular or glomerular damage were found. In four patients renal function was evaluated at the end of treatment (one after three cycles, one after five cycles and two after six cycles). Median (range) GFR, ERPF and FF were 99 ml/min (74–139 ml/min), 401 ml/min (277–496 ml/min) and 0.26 (0.23–0.30), respectively, revealing no delayed nephrotoxicity. Conclusion: We conclude that oral JM216 shows no nephrotoxicity. Received: 5 May 1999 / Accepted: 9 August 1999     

The farnesyl protein transferase inhibitor SCH66336 synergizes with taxanes in vitro and enhances their antitumor activity in vivo

Purpose: SCH66336 is an orally active, farnesyl protein transferase inhibitor. SCH66336 inhibits ras farnesylation in tumor cells and suppresses tumor growth in human xenograft and transgenic mouse cancer models in vivo. The taxanes, paclitaxel (Taxol) and docetaxel (Taxotere) block cell mitosis by enhancing polymerization of tubulin monomers into stabilized microtubule bundles, resulting in apoptosis. We hypothesized that anticancer combination therapy with SCH66336 and taxanes would be more efficacious than single drug therapy. Methods: We tested the efficacy of SCH66336 and taxanes when used in combination against tumor cell proliferation in vitro, against NCI-H460 human lung tumor xenografts in nude mice, and against mammary tumors in wap-ras transgenic mice. Results: SCH66336 synergized with paclitaxel in 10 out of 11 tumor cells lines originating from breast, colon, lung, ovary, prostate, and pancreas. SCH66336 also synergized with docetaxel in four out of five cell lines tested. In the NCI-H460 lung cancer xenograft model, oral SCH66336 (20 mg/kg twice daily for 14 days) and intraperitoneal paclitaxel (5 mg/kg once daily for 4 days) caused a tumor growth inhibition of 56% by day 7 and 65% by day 14 compared to paclitaxel alone. Male transgenic mice of the wap-ras/F substrain [FVB/N-TgN(WapHRAS)69LlnYSJL] spontaneously develop mammary tumors at 6–9 weeks of age which have been previously shown to be resistant to paclitaxel. Paclitaxel resistance was confirmed in the present study, while SCH66336 inhibited growth of these tumors. Most importantly, SCH66336 was able to sensitize wap-ras/F mammary tumors to paclitaxel chemotherapy. Conclusion: Clinical investigation of combination therapy using SCH66336 and taxanes in cancer patients is warranted. Further, SCH66336 may be useful for sensitizing paclitaxel-resistant tumors to taxane treatment. Received: 30 November 1999 / Accepted: 10 May 2000     

The tissue distribution in BALB/c mice of C-14-labeled JM216, an orally active platinum antitumour compound

 The ammine/amine platinum(IV) dicarboxylates have been developed as orally active platinum antitumor agents, and one of these, [PtCl₂(NH₃)(C₆H₁₁NH₂) (OCOCH₃)₂] (JM216), is undergoing clinical trials at present. A synthesis method was developed to radiolabel JM216 with carbon 14 at the carboxylate carbon. The labeling efficiency was 92%, and the purity as shown by high-performance liquid chromathography (HPLC) was 96% after recrystallisation. The radiolabeled JM216 was given orally to BALB/c mice and detailed tissue-distribution data were obtained (blood plasma, kidney, liver, spleen, brain, lung, muscle and skin) for time points of 2 h and 2, 6 and 10 days. Comparison of these data with previously reported data for distribution of platinum obtained by atomic absorption spectroscopy has shown distinct differences, especially for the liver and the kidney. This clearly indicates a difference in behaviour between the labeled ligand and the platinum centre, suggesting detachment of the ligand in vivo. Received: 26 November 1995 / Accepted: 5 March 1996     

Combination effects of TAS-103, a novel dual topoisomerase I and II inhibitor, with other anticancer agents on human small cell lung cancer cells

Purpose: TAS-103 [6-((2-(dimethylamino) ethyl)amino)-3-hydroxy-7H-indeno(2,1-c)quinolin-7-one dihydrochloride] is a newly synthesized dual inhibitor of topoisomerase I and II. Since anticancer drugs are used in combination with other drugs for effective chemotherapy, we investigated the cytotoxic effect of TAS-103 in combination with other conventional anticancer agents, such as cisplatin, vindesine, doxorubicin, 5-fluorouracil, and the antitopoisomerase inhibitors SN-38 and etoposide in vitro. Methods: Inhibition of the growth of the human small-cell lung cancer cell line SBC-3 was evaluated using the tetrazolium dye (MTT) assay. Drug interactions were evaluated by isobologram analysis and the determination of combination indices supplemented by a three-dimensional model. Results: Simultaneous use of TAS-103 and cisplatin had a supradditive effect, but combinations of TAS-103 with other drugs had an additive or marginally subadditive effect. Three-dimensional model analysis added more information about the synergistic concentration ranges of two drugs (cisplatin 200–400 nM and TAS-103 7–10 nM). Sequential use of TAS-103 and cisplatin had only an additive effect. Conclusion: These results suggest that the concomitant use of TAS-103 and cisplatin has a greater cytotoxic effect on cancer cells than single drug use, and may provide a beneficial effect in the treatment of small-cell lung cancer. Received: 23 March 1998 / Accepted: 15 September 1998     

Combined effects of the oral fluoropyrimidine anticancer agent, S-1 and radiation on human oral cancer cells

We evaluated the orally administered S-1, in combination with ionizing radiation both in vivo and in vitro against human oral cancer cell lines. Human oral cancer cell lines were used as subcutaneous xenografts in nude mice. S-1 (10 mg/kg) was administered orally 1 h before radiation treatments (1.5 Gy), or 1 h after radiation for five consecutive days. Apoptotic cells were detected by TUNEL method. For in vitro analysis, attached cells were treated with S-1 (50 microg/ml) for 1 h and then irradiated (3, 6, 9, 12, 15 Gy), or they were treated with S-1 for 1 h after radiation. Cell survival was determined by clonogenic assay. The combination of S-1 and radiation was more effective than either agent alone. In addition, S-1 administration before radiation was more effective than S-1 administration after radiation. Moreover, the combination of S-1 and radiation could induce apoptosis significantly than either agent alone (P < 0.01). In vitro clonogenic survival experiments demonstrated the dose enhancement ratio of 1.22 (radiation + S-1), 1.45 (S-1 + radiation) in B88 cells, and 1.16 (radiation + S-1), 1.28 (S-1 + radiation) in HSG cells. These data demonstrate that the combination of S-1 and fractionated radiotherapy is more effective against human oral cancer xenografts than either agent alone, and that S-1 administration before radiation is more effective than after radiation, suggesting a potential clinical applicability of combination treatment of S-1 and radiation in oral cancer therapies.     

A phase I study of oral uracil/ftorafur (UFT) plus leucovorin and bis-acetato-ammine-dichloro-cyclohexylamine-platinum IV (JM-216) each given over 14 days every 28 days

Purpose: To determine the feasibility, maximal tolerated doses, and response rates for a combined regimen of the platinum and 5-fluorouracil oral analogues bis-acetato-ammine-dichloro-cyclohexyl-amine platinum(IV) (JM-216) and uracil/ftorafur (UFT) coadministered as a 14 consecutive-day every 28-day schedule. Methods: Of 20 patients enrolled in this investigation, 17 on the following dose escalation scheme were evaluable for toxicity and/or response: I UFT 300 mg/day, JM-216 5 mg/day (three patients), II UFT 300 mg/day, JM-216 10 mg/day (four patients), III UFT 300 mg/day, JM-216 20 mg/day (ten patients). Results: All 17 evaluable patients were evaluable for toxicity. At dose level III, dose-limiting nausea and emesis were observed in one patient despite maximal antiemetic support. Importantly, neurotoxicity and nephrotoxicity were not observed at the JM-216 dose levels examined in this study. This observation is consistent with results seen with single agent JM-216. Conclusion: For JM-216 and UFT administered at 20 mg/day and 300 mg/day over 14 days, nausea and emesis were observed as the principal dose-limiting toxicities. These doses are considerably below the maximally tolerated doses of single agent JM-216 and UFT. Shorter administration schedules should be explored in an attempt to increase the dose intensity and minimize the toxicity of this combination oral regimen. Received: 5 March 1998 / Accepted: 3 September 1998     

P53 status plays no role in radiosensitizing effects of SN-38, a camptothecin derivative

Purpose: Topoisomerase inhibitors including camptothecin are being studied as potential radiosensitizers. CPT-11 is a derivative of camptothecin and is clinically available. In this study, we investigated the effects of SN-38 (an active metabolite of CPT-11) on four nonirradiated and irradiated murine fibroblast cell lines with different p53 statuses to clarify the role of p53 in the radiosensitizing activity of SN-38. Materials and methods: Four fibroblast cell lines, MT158, MT158/neo, MT158/wtp53 and MT158/mp53 with the same genetic background but with different p53 statuses, were used. Exponentially growing cells were treated with SN-38 (200 nM) and incubated with the drug for 30 min. Cells were then irradiated (0 to 12 Gy) and further incubated with the drug for 2 h. The cell survival rate was determined using a conventional clonogenic assay. The effects of the treatments on the cell cycle were analyzed with a flow cytometric assay. Apoptosis after these treatments was also detected by an annexin V assay. Results: There were no significant differences in sensitivity to radiation or SN-38 treatment among these cell lines. The combined treatment of irradiation and SN-38 showed supraadditive effects in all four cell lines independent of their p53 status. Transient arrest in G₂ with a decreased percentage of cells in both the S and G₁ phases was observed 8 h after treatment with either SN-38 alone, radiation or their combination, regardless of the p53 status. No significant differences in frequency of apoptosis were observed between treatment and control groups in two cell lines with or without wild-type p53. Conclusion: The combination of irradiation and SN-38 treatment showed supraadditive effects in all four cell lines tested, and the p53 status did not play a role in the combination effect. Received: 19 May 1999 / Accepted: 19 November 1999     

Allosteric effectors of hemoglobin as modulators of chemotherapy and radiation therapy in vitro and in vivo

Introduction: A series of molecules designed to be allosteric effectors of hemoglobin were examined for their potential as radiation sensitizers in vitro and in vivo and for their potential as chemosensitizers in vivo as well as for their antimetastatic effect. Results: At a concentration of 100 μM for 1 h prior to, during and for 1.5 h after radiation exposure, the allosteric effectors decreased the shoulder of the radiation survival curve of normally oxygenated EMT-6 cells and increased the slope of the radiation survival curves of hypoxic EMT-6 cells resulting in dose-modifying factors of 1.8 to 2.1. In vivo the allosteric effectors had antitumor activity against the Lewis lung carcinoma and produced primarily additive tumor growth delay when administered along with fractionated radiation therapy. When administered on days 4 through 18 after tumor implantation, the allosteric effectors, especially JP-7, RSR-13 and RSR-4, were highly effective antimetastatic agents in animals bearing Lewis lung carcinoma. In cell culture, simultaneous exposure to the allosteric effectors (at 100 μM ) effectively sensitized EMT-6 cells to the effects of 4-hydroperoxycyclophosphamide, thiotepa and carboplatin. The allosteric effectors were not very cytotoxic toward EMT-6 tumor cells from tumors treated in vivo with single doses of each molecule nor were these agents very cytotoxic toward bone marrow CFU-GM taken from the same animals. Conclusions: It is likely that the allosteric effectors have a molecular target in addition to hemoglobin. Other possible targets include hydroxymethyl-glutaryl-CoA reductase or microsomal cytochrome b₅. Received: 27 June 1997 / Accepted: 8 October 1997     

Evaluation of toremifene for reversal of multidrug resistance in renal cell cancer patients treated with vinblastine

Purpose: Expression of P-glycoprotein (Pgp), which confers the multidrug resistance (MDR) phenotype, is thought to contribute to the insensitivity of renal cell cancer (RCC) to chemotherapy. The development of Pgp inhibitors for clinical application has been hampered by unacceptable toxicity at doses required to achieve adequate cellular concentration. Toremifene is able to reverse MDR and sensitise RCC to vinblastine in vitro. However, in vivo toremifene is tightly bound to serum proteins, in particular the acute phase protein α₁-acid glycoprotein (AAG), which may limit tissue availability. In this phase I–II study we assessed the tolerability of short courses of high dose toremifene in combination with vinblastine and evaluated the key determinants of MDR reversal in vivo. Methods: Twenty-seven patients with metastatic RCC received escalating doses of oral toremifene for 3 days every 2 weeks in combination with vinblastine 6 mg/m² i.v. on day 3 of each cycle. The serum concentration of toremifene, its metabolites and AAG were measured and the effect of patients' serum on inhibition of Pgp in vitro was determined. Results: Twenty-six patients were evaluable for response. Eight patients (31%) had stable disease and 18 patients (69%) progressive disease. The mean serum concentration of toremifene at 780 mg daily for 3 days was 7.82 μM [standard deviation (SD) 2.48, range 2.50 to 14.70], which exceeds that known to reverse MDR in vitro. The serum concentration of the major metabolite of toremifene, N-demethyltoremifene, which also reverses MDR, was 5.13 μM (SD 1.78, range 1.80 to 9.00). In 60% of patients the pre-treatment AAG concentration was above that known to block the effects of toremifene in vitro. However, addition of serum from patients on toremifene to MCF-7 adr cells in vitro inhibited Pgp-mediated efflux of rhodamine 123. Conclusions: We have shown that short course, high-dose toremifene in combination with vinblastine is generally well tolerated and that the concentration of toremifene required to reverse MDR in vitro is achievable in vivo. Received: 7 July 1999 / Accepted: 15 November 1999     

Preclinical evaluation of the orally active camptothecin analog, RFS-2000 (9-nitro-20(S)-camptothecin) as a radiation enhancer

PURPOSE: To test for enhancement of radiation effects in vitro and in vivo by the orally administered camptothecin derivative, 9-nitrocamptothecin (RFS-2000); to study whether the mechanism of this enhancement involves inhibition of sublethal damage recovery. METHODS AND MATERIALS: In vitro: H460 human lung carcinoma cells were incubated with RFS-2000 for various times at 37 degrees C, irradiated, immediately rinsed, and assessed for colony-forming ability. Sublethal damage recovery (SLDR) was also assessed using two split doses of radiation. In vivo: H460 cell xenografts were used in nude mice. Tumors were grown subcutaneously on the flank, then treated with RFS-2000 (1 mg/kg) and/or radiation (2 Gy) for 5 consecutive days. Tumor growth delay was then measured for each treatment group. RESULTS: Radiation enhancement was observed in vitro for incubation times between 4 and 24 hr with 10 nM RFS-2000. Using a 24-hr treatment, the radiation dose enhancement ratio values (DER) for 5, 10, and 15 nM were 1.22, 1.54, and 2.0, respectively. Incubation with 10 nM RFS-2000 inhibited SLDR by a factor of 2. The results of three independent in vivo experiments showed that RFS-2000 can enhance the effects of fractionated radiotherapy, with an enhancement factor (EF) of 1.64. CONCLUSION: Our results show that RFS-2000 can enhance the effects of radiation in human lung cancer cells both in vitro and in vivo, and that the mechanism of this effect may involve the inhibition of SLDR.     

Enhancement of the antitumor activity of ionising radiation by nimotuzumab, a humanised monoclonal antibody to the epidermal growth factor receptor, in non-small cell lung cancer cell lines of differing epidermal growth factor receptor status

The expression and activity of the epidermal growth factor receptor (EGFR) are determinants of radiosensitivity in several tumour types, including non-small cell lung cancer (NSCLC). However, little is known of whether genetic alterations of EGFR in NSCLC cells affect the therapeutic response to monoclonal antibodies (mAbs) to EGFR in combination with radiation. We examined the effects of nimotuzumab, a humanised mAb to EGFR, in combination with ionising radiation on human NSCLC cell lines of differing EGFR status. Flow cytometry revealed that H292 and Ma-1 cells expressed high and moderate levels of EGFR on the cell surface, respectively, whereas H460, H1299, and H1975 cells showed a low level of surface EGFR expression. Immunoblot analysis revealed that EGFR phosphorylation was inhibited by nimotuzumab in H292 and Ma-1 cells but not in H460, H1299, or H1975 cells. Nimotuzumab augmented the cytotoxic effect of radiation in H292 and Ma-1 cells in a clonogenic assay in vitro, with a dose enhancement factor of 1.5 and 1.3, respectively. It also enhanced the antitumor effect of radiation on H292 and Ma-1 cell xenografts in nude mice, with an enhancement factor of 1.3 and 4.0, respectively. Nimotuzumab did not affect the radioresponse of H460 cells in vitro or in vivo. Nimotuzumab enhanced the antitumor efficacy of radiation in certain human NSCLC cell lines in vitro and in vivo. This effect may be related to the level of EGFR expression on the cell surface rather than to EGFR mutation.     

Modification of the antitumor activity of chemotherapeutic drugs by the hypoxic cytotoxic agent tirapazamine

 Purpose: Preclinical studies were performed to examine the interaction of the hypoxic cell toxin tirapazamine (TPZ), a benzotriazine di-N-oxide, with several chemotherapeutic agents, including carboplatin, cyclophosphamide, doxorubicin, etoposide, 5-fluorouracil (5-FU), taxol, and navelbine. Methods: The modification by TPZ of the antitumor drug activity and the effect of schedule were determined with an in vivo/in vitro clonogenic assay using well-established RIF-1 murine tumors transplanted into C3H mice. Results: Additive, or greater than additive, tumor cell killing was observed when TPZ was combined with carboplatin, cyclophosphamide, doxorubicin, etoposide, 5-FU and taxol. With the exception of 5-FU there were only small, or no, enhancements of the systemic toxicities of the drugs by TPZ. The greatest enhancement of antitumor activity was with carboplatin, with the maximum effectiveness when TPZ was given 2–3 h before the carboplatin. The activity of cyclophosphamide, doxorubicin, etoposide and taxol were most enhanced when TPZ was given 24 h before the drug. Additional investigations with three-drug combination treatments using cisplatin and TPZ with either etoposide or navelbine indicated a substantial therapeutic gain from the addition of TPZ. Conclusions: The data for each of the drugs tested in combination with TPZ, with the exception of 5-FU, indicate that potential clinical benefit may be obtained from therapies combining TPZ with conventional chemotherapy. Received: 6 March 1996 / Accepted: 30 June 1996     

Phase I study of oral JM216 given twice daily

JM216 [bis-acetato-ammine-dichloro-cyclohexylamine-platinum(IV)] is an oral platinum complex that is currently in phase II trials in ovarian cancer and lung cancer on a daily-times-5 schedule. This trial examined an alternative schedule of two doses given 12 h apart, which may be better tolerated by patients. A total of 19 patients were given 50 cycles of treatment at doses ranging from 150 to 350 mg/m² b.i.d. The study was stopped before the MTD was reached due to non-linear pharmacokinetics. Toxicity was similar to that encountered in previous phase I studies, with nausea, vomiting and diarrhoea being seen at all dose levels, although this was generally mild and short-lived, and grade 3 and 4 myelosuppression being seen at dose levels ranging from 250 to 350 mg/m². There was no nephro-, oto-, or neurotoxicity, but one patient had an allergic reaction at 300 mg/m² on the fifth and sixth cycles. No response was seen, but two patients with mesothelioma had stable disease and received six cycles. There was considerable interpatient variability in plasma pharmacokinetics at all dose levels. There was no relationship between dose and AUC (dose 1 and dose 2) or C_max after dose 1. In a limited number of patients the first dose was given in the morning rather than in the evening, apparently resulting in lower AUC, C_max and T _max values at the 250-mg/m² dose level, but this was not seen in one patient at 300 mg/m². This study confirms that the pharmacokinetics of JM216 is non-linear and highly variable due to saturable absorption and that the daily times 5 schedule is the optimal schedule for further phase II trials. Received: 8 August 1997 / Accepted: 5 November 1997     

Combined treatment of Dunning R3327 rat prostatic tumor with the 5α-reductase inhibitor PNU 157706 and the antiandrogen bicalutamide

 PNU 157706 [N-(1,1,1,3,3,3-hexafluorophenyl- propyl)-3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide], a novel, potent and selective dual 5α-reductase inhibitor, was reported to be effective in inhibiting the growth of established tumors in the Dunning R3327 rat prostatic carcinoma model. Purpose: We investigated the efficacy of treatment with PNU 157706 in combination with the antiandrogen bicalutamide in this prostatic tumor model. Methods: Rats with tumor diameters of about 1 cm were treated orally 6 days a week for 9 weeks with PNU 157706 (10 mg/kg per day) alone or in combination with bicalutamide (0.2 and 1 mg/kg per day). Animals were killed 24 h after the last treatment, and ventral prostates were removed for testosterone (T) and dihydrotestosterone (DHT) determination. Results: PNU 157706 reduced the growth of established tumors by 39%; bicalutamide proved ineffective at 0.2 mg/kg per day, but reduced tumor growth by 45% at a dose of 1 mg/kg per day. The combination of PNU 157706 with both doses of bicalutamide caused an additive tumor growth inhibition (50% and 64%). Castration resulted in marked tumor growth inhibition (72%). Ventral prostate weight was markedly reduced by PNU 157706 (78%) treatment and by bicalutamide (59% and 77%); combined treatment was as effective as castration. Prostatic DHT content was markedly reduced by PNU 157706 (88%), whereas prostatic T increased slightly (60%). Concomitant treatment with bicalutamide antagonized the T increase induced by PNU 157706 and did not modify the already remarkable suppression of DHT. Conclusions: These data show that the inhibitory effect of PNU 157706 and bicalutamide on Dunning prostatic tumor growth is additive, thus suggesting a possible role of PNU 157706 in the therapy of advanced prostate cancer, in combination with antiandrogens, to provide an effective peripheral androgen ablation therapy with minimal side effects. Received: 1 February 1999 / Accepted: 8 June 1999     

F 11782, a dual inhibitor of topoisomerases I and II with an original mechanism of action in vitro, and markedly superior in vivo antitumour activity, relative to three other dual topoisomerase inhibitors, intoplicin, aclarubicin and TAS-103

Purpose: F 11782 (2′′,3′′-bis pentafluorophenoxyacetyl-4′′,6′′-ethylidene-β-d-glucoside of 4′-phosphate-4′-dimethylepipodophyllotoxin, di-N-methyl glucamine salt) is a newly synthesized dual catalytic inhibitor of topoisomerases I and II with major in vivo antitumour activity. In this study, we compared and contrasted F 11782 with three other known inhibitors of both these nuclear enzymes, namely aclarubicin, intoplicin and TAS-103, and established its novel mechanism of action. Methods: In vitro growth-inhibitory effects against a panel of murine and tumour cell lines were measured by cell counting, clonogenicity or tetrazolium metabolic dye (MTT) assays. In vivo antitumour activities were evaluated against two murine tumour models (i.v. P388 leukaemia and s.c. B16 melanoma). Finally, interactions with either DNA or DNA-topoisomerases were determined using various methodologies: DNA-intercalator displacement, pBR322 DNA relaxation, kDNA decatenation, topoisomerase II extractability measurements, stabilization of topoisomerase-induced cleavable complexes (CC) in vitro and in cells, and gel retardation assays. Results: F 11782 had a different profile of sensitivities and proved generally less cytotoxic than the other dual inhibitors tested in vitro, while showing significantly superior antitumour activity in vivo. F 11782, which did not stabilize CC either in vitro or in cells, was the only compound of this series capable of inhibiting the catalytic activity of both DNA-topoisomerases without interacting with DNA, and of completely impairing the binding of these nuclear proteins to DNA. Moreover, only cotreatment of cells in vitro with F 11782 enhanced the cytotoxic activity of etoposide. Conclusion: These results emphasize the novel mechanism of action of F 11782 vis-à-vis the other dual inhibitors of topoisomerases I and II and so augur well for its future clinical development. Received: 7 January 2000 / Accepted: 10 April 2000     

High-dose toremifene as a cisplatin modulator in metastatic non-small cell lung cancer: targeted plasma levels are achievable clinically

Purpose: The triphenylethylenes tamoxifen and toremifene have been reported to enhance the cytotoxicity of cisplatin by inhibition of protein kinase C (PKC) signal transduction pathways. However, the concentrations of tamoxifen and toremifene required for chemosensitization in preclinical models are generally ≥5 μM, at least tenfold higher than plasma levels observed in patients receiving these agents as antiestrogenic therapy. As part of a translational phase II trial investigating the efficacy and potential molecular mechanism of high-dose toremifene as a cisplatin modulator in metastatic non-small-cell lung cancer, plasma concentrations of toremifene and its active metabolite N-desmethyltoremifene were measured to determine whether targeted levels could be achieved clinically. Methods: Treatment consisted of toremifene, 600 mg orally on days 1–7, and cisplatin, 50 mg/m² intravenously on days 4 and 11, repeated every 28 days. Toremifene and N-desmethyltoremifene were measured by reverse-phase HPLC assay on days 4 and 11 prior to cisplatin infusion. Results: In the initial 14 patients, the mean total plasma concentrations of toremifene plus its N-desmethyl metabolite on days 4 and 11 were 14.04 (±8.6) μM and 9.8 (±4.4) μM, respectively. Variability in concentrations achieved did not correlate with renal or hepatic function, gender, or body surface area. Levels of N-desmethyltoremifene were higher on day 11 relative to toremifene concentrations. Conclusions: We conclude that plasma levels achieved compare favorably with the levels required for cisplatin chemosensitization and PKC modulation in vitro. Targeted toremifene levels can be achieved clinically with 600 mg orally daily in combination with cisplatin and are well tolerated. Received: 25 November 1997 / Accepted: 9 March 1998     

Antiangiogenic potential of camptothecin and topotecan

Purpose: To determine the inhibitory nature of sublethal doses of camptothecin (CPT) and topotecan (TPT) treatments on normal human endothelial cells in vitro, as well as the in vivo antiangiogenic activity as compared to another antiangiogenic compound, TNP-470 and to a nonspecific cytotoxic agent, cisplatin. Methods: Growth inhibition was determined by the crystal violet assay to measure relative cell numbers. ³H-thymidine uptake was used to determine the inhibitory effect of CPT and TPT on DNA synthesis in vitro. Cell viability was determined using trypan blue exclusion assays. Cell cycle response to CPT was determined by flow cytometric analysis of propidium iodide-stained nuclei. In vivo inhibition of angiogenesis was determined by the disc angiogenesis system (DAS), where surgical sponge discs were placed subcutaneously in the rat dorsum and the ability of systemic treatment with liposomal CPT (LCPT), TPT, TNP-470 or cisplatin to inhibit vascular growth into the discs was evaluated. Quantitation of vascular growth was determined using toluidine blue staining of sectioned discs followed by digital image analysis. Results: Treatment with 50 nM CPT or TPT inhibited human umbilical venular endothelial cell (HUVEC) growth as shown by crystal violet staining, but was not cytotoxic to the cells. This was evidenced by the fact that cell numbers did not increase or decrease with treatment, but remained static while cells were viable for over 96 h posttreatment. ³H-thymidine uptake in HUVEC was inhibited as early as 5 min, reached a maximum inhibition at 24 h and lasted over 96 h posttreatment. Cell cycle analysis of CPT-treated HUVEC showed arrest in S-phase at 12 h with a concurrent decrease in population of cells in G₁. Accumulation of cells at the G₂/M-phase was discernible at 24 h along with the S-phase inhibition. Treatment of rats with 1 mg/kg LCPT or TPT every other day for 14 days resulted in approximately 30% inhibition of vascular growth into the discs. This inhibition was similar to the inhibition seen with TNP-470, an established and potent angiogenic inhibitor. In contrast, cisplatin was not as effective in inhibiting vascular growth into the discs. Conclusions: In this work we showed that CPT and TPT inhibit human endothelial cell growth in vitro in a non-cytotoxic manner and that this inhibition lasts more than 96 h after drug removal. We also showed that LCPT and TPT, unlike a nonspecific cytotoxic agent, cisplatin, are as effective as TNP-470 in inhibiting angiogenic growth in the in vivo disc angiogenesis model. From this observation we propose that in addition to their proven tumoricidal activities, camptothecins may have an indirect in vivo antitumor effect mediated through the inhibition of angiogenesis. Received: 1 October 1998 / Accepted: 8 March 1999