In vitro interactions of a new derivative of spicamycin, KRN5500, and other anticancer drugs using a three-dimensional model

Purpose: KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus and is known to have a wide range of antitumor activities against human cancer cell lines. Because of its unique structure, this compound seems to have a different mode of action from other antitumor drugs and nonoverlapping toxicities. Therefore, KRN5500 is expected to be a suitable candidate for combination chemotherapy. Methods: We investigated the effects of combinations of KRN5500 and other anticancer drugs on the growth of a human non-small-cell lung cancer cell line, PC14, using a revised three-dimensional model. Results: Synergism was observed when KRN5500 and cisplatin were combined at concentrations in the ranges 0.005 to 0.25 μg/ml and 0.025 to 0.25 μg/ml, respectively. In combination with carboplatin, an analog of cisplatin, and etoposide, a marked synergistic interaction was also found. Conclusion: These results suggest the usefulness of combinations of KRN5500 with cisplatin, carboplatin or etoposide for chemotherapy for non-small-cell lung cancer. Received: 27 April 1998 / Accepted: 15 September 1998     

Phase II trial of combination chemotherapy with cisplatin, carboplatin and etoposide in stage IIIB and IV small-cell lung cancer

Purpose: A phase II trial combining cisplatin, carboplatin and etoposide was conducted in previously untreated patients with stage IIIB and IV small-cell lung cancer, in an attempt to increase response rates and prolong survival. Methods: Previously untreated patients with small-cell lung cancer, with measurable disease, aged ≤ 72 years, performance status ≤ 2, and adequate hematologic, hepatic and renal function were enrolled in the study. They were treated with 80 mg/m² cisplatin on day 1, 100 mg/m² carboplatin on days 2, 3 and 8, and 50 mg/m² etoposide on days 1, 2, 3 and 8. Results: A total of 46 patients (20 with stage IIIB and 26 with stage IV disease) were enrolled in the study. A total of 186 courses of chemotherapy were given, and the dose was reduced in 27 courses (15%). The chemotherapy was repeated for four or more courses in 30 patients. There were 10 complete responses and 32 partial responses, for a total response rate of 91% (95% confidence interval, 79% to 98%). The median survival time and 2-year survival rates were 18 months and 22% for stage IIIB disease, and 14 months and 15% for stage IV disease. Major side effects were hematologic: leukopenia, anemia, and thrombocytopenia of grade 3 or more occurred in 48%, 46%, and 43% of patients, respectively. Conclusions: The three-drug regimen of cisplatin, carboplatin and etoposide is feasible and active against small-cell lung cancer. Received: 21 May 1997 / Accepted: 11 September 1997     

Cellular sensitization to cisplatin and carboplatin with decreased removal of platinum-DNA adduct by glucose-regulated stress

Purpose: Stress conditions, such as glucose starvation and hypoxia, that induce glucose-regulated proteins (GRPs) in cells, are seen in most solid tumors. These conditions have been shown to cause cellular resistance to multiple anticancer drugs, such as etoposide, doxorubicin, and camptothecin. We examined the effect of the GRP-inducing conditions on cellular sensitivity to cisplatin and carboplatin, which are widely used drugs against solid tumors. Methods: We generated the GRP-inducing culture conditions by exposing cells to 2-deoxyglucose (2DG), calcium ionophore A23187 and tunicamycin, and examined cellular sensitivity to cisplatin and carboplatin under these conditions. We next measured platinum accumulation and DNA-bound platinum in 2DG-stressed cells after cisplatin exposure. Results: The GRP-inducing stress conditions led to cellular sensitization to cisplatin and carboplatin. This sensitization was reversible, as the cellular sensitivity returned to normal levels 12 h after removal of 2DG. Platinum accumulation and DNA-bound platinum that were found immediately after exposure to cisplatin for 1 h were slightly increased in 2DG-stressed cells as compared with nonstressed cells. After a drug-free recovery incubation of 8 h, the DNA-bound platinum in the nonstressed cells was reduced by 33% while the amount in the 2DG-stressed cells was sustained at the initial levels. Conclusions: These results indicated that the decreased removal of platinum-DNA adducts was associated with increased sensitivity to cisplatin and carboplatin in the stressed cells. The sensitization of cancer cells under the GRP-inducing stress conditions would explain, in part, the clinical potency of platinum drugs against solid tumors. Received: 27 May 1998 / Accepted: 20 October 1998     

Activity of fenretinide plus chemotherapeutic agents in small-cell lung cancer cell lines

Purpose: Fenretinide [N-(4-hydroxyphenyl)retinamide, 4HPR], a synthetic retinoid, is a potent inducer of apoptosis in small-cell lung cancer (SCLC) cell lines that may act through the generation of reactive oxygen species, suggesting that it may enhance the activity of other cytotoxic agents. In light of 4HPR's clinical potential and potent activity against SCLC cells, we evaluated the in vitro activity of 4HPR in combination with cisplatin, etoposide or paclitaxel. Methods: The growth-inhibitory activities of single-agent 4HPR, cisplatin, etoposide or paclitaxel, and combinations of 4HPR and individual chemotherapeutic agents, were evaluated using an MTT assay in two SCLC cell lines. Each two-drug combination was studied over a range of concentrations at a fixed ratio corresponding to the ratio of the IC₅₀ values of the individual agents. Data were analyzed by median-effect analysis as previously applied to drug combination studies. Results: All four agents inhibited growth in a dose-dependent manner in the NCI-H82 and NCI-H446 SCLC cell lines. At clinically reported drug concentrations that resulted in over 50% growth inhibition, the activities of the combinations 4HPR and cisplatin and 4HPR and etoposide were more than additive in both cell lines, and the activity of 4HPR plus paclitaxel was more than additive in NCI-H446 cells. Conclusion: 4HPR's potent single-agent activity, minimal toxicity, and potential synergy with standard cytotoxic drugs will allow for the development of promising investigational regimens for the treatment of patients with SCLC. Received: 17 February 1998 / Accepted: 20 May 1998     

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     

The significance of the sequence of administration of topotecan and etoposide

 Purpose: Often the best method of integrating chemotherapeutic agents is unknown. Recently there has been interest in the use of combinations of the topoisomerase II inhibitors and the topoisomerase I inhibitors as these agents have shown individual activity in malignancies such as non-small-cell lung cancer. This study examined the interaction of the topoisomerase II inhibitor etoposide with the topoisomerase I inhibitor topotecan (Tpt) in V79 cells (hamster lung fibroblast cells) to determine the optimal method of delivering these agents. Methods and results: Cell survival was assessed by colony formation. Synergistic interactions were assessed by the median effect principle in which a combination index (CI) of less than one suggests a synergistic interaction. The V79 cells were exposed to sequential 24-h incubations with the two chemotherapeutic agents. Initially, equitoxic doses of the two agents were delivered (i.e. 0.0275 μg/ml of topotecan alone or 0.089 μg/ml of etoposide alone resulting in a surviving fraction of 70%; Tpt : etoposide ratio 1 : 3.2). It was determined that a sequence-dependent synergistic interaction (CI<1) resulted at a lower level of cytotoxicity if the etoposide exposure followed the Tpt exposure compared to the opposite sequence. This same effect was seen after treatment of cells with various concentration (μg/ml) ratios of Tpt : etoposide (1 : 4.0, 1 : 1, 2.5 : 1). Conclusions: These results suggest that maximum synergy occurs for the delivery of etoposide following Tpt exposure (compared to the opposite sequence) and these findings may have important clinical implications. Received: 29 September 1995/Accepted: 25 March 1996     

A phase I study of prolonged ambulatory infusion carboplatin with oral etoposide showing activity in prostate cancer

Purpose: This phase I study aimed to establish the dose for phase II trials of a dose-intense outpatient regimen of ambulatory carboplatin and oral etoposide. Patients and Methods: Cohorts of three patients received escalating doses of carboplatin 15, 20, and 23 mg/m²/day as a 3-week continuous ambulatory infusion with oral etoposide initially at 50 mg/day. Patients entered had prostate, colon, head and neck, breast, unknown primary cancers and mesothelioma. Results: At 23 mg/m² of carboplatin, two patients had WHO grade 3 lethargy and myelosuppression, which were the dose-limiting toxicities. Six patients were entered at the dose recommended for phase II studies, carboplatin 20 mg/m²/day and etoposide 50 mg/day for 21 days repeated every 6 weeks. This was well tolerated except for one patient with multiple bone metastases from prostate cancer experiencing grade 4 myelosuppression and a single patient with grade 3 constipation. Seven patients with hormone-resistant prostate cancer were entered into the study, one at 15 mg/m², four at 20 mg/m² and two at 23 mg/m² of carboplatin, and received a median of four cycles of treatment. The only responses were seen in prostate cancer where there were two partial responses in patients with soft tissue predominant disease. Five patients who could be evaluated with initially elevated PSA exhibited falls of ≥50% after receiving the chemotherapy. All but one patient with prostate cancer experienced significant reduction in pain levels. The median time to progression of the patients with prostate cancer was 4 months. Conclusions: Ambulatory infusion carboplatin and oral etoposide is a tolerable dose- intense outpatient regimen which warrants further testing in phase II trials including hormone-resistant prostate cancer. Received: 13 March 2000 / Accepted: 26 May 2000     

Schedule-dependent interactions between paclitaxel and etoposide in human carcinoma cell lines in vitro

 Clinical studies of paclitaxel in combination with etoposide against solid tumors have been carried out. The combination schedules used in these studies are different. We studied the cytotoxic effects of paclitaxel with etoposide against four human cancer cell lines in vitro to determine the optimal schedule of this combination at the cellular level. Cells were exposed simultaneously to paclitaxel and to etoposide for 24 h or sequentially to one drug for 24 h followed by the other for 24 h, after which they were incubated in drug-free medium for 4 and 3 days, respectively. Cell growth inhibition was determined by an MTT reduction assay. The effects of drug combinations at concentrations producing 80% inhibition (IC₈₀) were analyzed by the isobologram method of Steel and Peckham. The cytotoxic effect of paclitaxel and etoposide was cell line- and schedule-dependent. Simultaneous exposure to paclitaxel and etoposide for 24 h produced additive effects in the lung cancer cell line A549 and ovarian cancer PA1 cells, and antagonistic effects in the breast cancer cell line MCF7 and colon cancer WIDr cells. Sequential exposures to paclitaxel followed by etoposide and vice versa produced additive effects in all four cell lines. These results suggest that maximum cytotoxic effects can be obtained with sequential administration, but not simultaneous administration, of paclitaxel and etoposide. These findings may have important clinical implications for this combination. Received: 11 August 1998 / Accepted: 8 March 1999     

Tumoricidal interactions of hyperthermia with carboplatin, cisplatin and etoposide

Interactions of hyperthermia with carboplatin, cisplatin and etoposide were investigated in vitro in JM, a human, T cell, lymphoblastic cell line. Thermal enhancement ratios (TER) for carboplatin killing increased with temperature (2.3 at 40.5 degrees C, 3.2 at 41.8 degrees C) and were similar to those for cisplatin killing (2.6 at 40.5 degrees C, 3.6 at 41.8 degrees C). In a separate experiment, cytotoxicity was additive when carboplatin and cisplatin were given simultaneously at 37 degrees C and at 41.8 degrees C. Etoposide, which synergizes with platinum agents, did not have supra-additive cytotoxic interactions with hyperthermia. The data presented are relevant to the conduct of clinical hyperthermia trials.     

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     

Pharmacokinetics of paclitaxel administered in combination with cisplatin, etoposide and bleomycin in patients with advanced solid tumours

 Purpose: To evaluate the pharmacokinetics of paclitaxel and cisplatin administered in combination with bleomycin and etoposide and Granulocyte Colony-Stimulating Factor (G-CSF) in patients with advanced solid tumours. Methods: Patients were recruited to a phase I trial where escalating doses of paclitaxel (125 to 200 mg/m²) were administered in combination with etoposide 100 or 120 mg/m², and fixed dose of cisplatin 20 mg/m² and bleomycin 30 mg, with the concomitant use of G-CSF. Paclitaxel (3-h infusion) was followed by 1-h etoposide, 4-h cisplatin and 30-min bleomycin infusions, respectively. Pharmacokinetics sampling for paclitaxel analysis was performed in ten patients from dose levels II–V. Results: The mean paclitaxel area under the plasma concentration-versus-time curves (AUC) for the 125-mg/m² dose level (II) was 7.0 ± 3.6 h μmol⁻¹ l⁻¹, for the 175-mg/m² dose level (III) 10.6 ± 2.8 h μmol⁻¹ l⁻¹, for the 200-mg/m² dose level (IV) it was 16.0 ± 5.0 h μmol⁻¹ l⁻¹, and for the 175-mg/m² dose level (V) it was 12.5 ± 6.1 h μmol⁻¹ l⁻¹. The mean peak plasma concentration (C_max) values for dose levels II–V were 1.9 ± 1.1 μmol/l, 3.4 ± 1.2 μmol/l, 4.3 ± 1.0 μmol/l and 3.8 ± 1.2 h μmol/l, respectively. Conclusion: In this study, relevant pharmacokinetic parameters of paclitaxel like AUC, C_max and the paclitaxel plasma concentration above the pharmacologically relevant 0.1-μmol/l threshold concentration (t > 0.1 μM) when administered in combination with cisplatin, etoposide and bleomycin (PEB) were not statistically different from paclitaxel data of historical controls. However, given the trial design, pharmacokinetic interactions between the agents cannot be excluded. Received: 29 June 1998 / Accepted: 29 January 1999     

Efficacy of lonidamine combined with different DNA-damaging agents in the treatment of the MX-1 tumor xenograft

 Lonidamine is an antitumor agent with a peculiar mechanism of action, since it differentially impairs the energy metabolism of normal and neoplastic cells. We investigated the effects of lonidamine on the activity of DNA-damaging antitumor agents against the MX-1 human breast carcinoma xenograft. Athymic mice bearing measurable s.c. tumors were treated by a single injection of doxorubicin (i.v.), cyclophosphamide (i.v.), or cisplatin (i.p.) followed by repeated daily injections of lonidamine (i.p. or p.o.). A potentiation of the activity of all these DNA-damaging drugs was achieved when each was given in combination with lonidamine, but for doxorubicin and cyclophosphamide the increase in antitumor activity paralleled the increase in lethal toxicity. In contrast, a therapeutic advantage of the combination was achieved for cisplatin and lonidamine as compared with cisplatin alone. Indeed, 6 mg/kg of cisplatin plus lonidamine cured all tumors, whereas the maximum tolerated dose of cisplatin alone (12 mg/kg) cured only six of eight tumors. In addition, the study indicated that the duration of lonidamine administration after injection of the cytotoxic drug influenced the tumor response and that prolonged treatment resulted in greater efficacy. These results document the ability of lonidamine to modulate the pharmacological activity of DNA-damaging drugs, thus suggesting that lonidamine may be a clinically useful cisplatin modulator. Received: 26 July 1995/Accepted: 6 October 1995     

Control of cell proliferation in human glioma by glucocorticoids.

Survival and proliferation of cell cultures from human anaplastic astrocytomas were shown to be enhanced by glucocorticoids with an optimal concentration of approximately 2.5 x 10(-5)M (10 micrograms/ml). The stimulation of proliferation was only observed in a clonal growth assay and was reversed as the size of individual colonies reached approximately 50 cells. Above this size, and in regular monolayer cultures, glucocorticoids were found to inhibit cell proliferation as measured by direct cell counting and incorporation of [3H] thymidine. Cultures grown to maximum cell densities in non-limiting medium conditions reached a lower terminal cell density, and had a reduced labelling index with [3H] thymidine in the presence of glucocorticoids. Although there was little difference between the actions of beta-methasone, dexamethasone and ethyl prednisolone, methyl prednisolone was found to be more effective, both in terms of stimulation of clonal growth and inhibition of growth at high cell densities. There was no evidence of cytotoxicity with glucocorticoids up to 5 x 10(-5)M (20 micrograms/ml) and it is suggested that glucocorticoids act via a normal regulatory process, perhaps enhancing cell-cell recognition.     

Enhancement of cisplatin cytotoxicity by terbium in cisplatin-resistant MDA/CH human breast cancer cells

Purpose: The development of cisplatin resistance is a major problem in the treatment of cancer patients with cisplatin chemotherapy. The membrane binding of terbium (Tb³⁺) has been shown to increase the cellular accumulation of cisplatin in breast cancer cells. Therefore, the ability of Tb³⁺ to modulate the cytotoxicity of cisplatin was investigated in cisplatin-sensitive (MDA) and cisplatin-resistant (MDA/CH) MDA-MB-231 human breast cancer cells. Methods: The cytotoxic parameters of cisplatin were determined using live cell microfluorometry and median effect analysis. Results: MDA/CH cells (IC₅₀ = 142 ± 9 μM) were found to be approximately 3.3-fold more resistant to cisplatin than MDA cells (IC₅₀ = 43.5 ± 3.0 μM). In both cell lines, the IC₅₀ value for cisplatin was reduced two-fold in the presence of 80 μM Tb³⁺, thus indicating that the cytotoxicity of cisplatin is increased by Tb³⁺. The cytotoxic activity of cisplatin alone was observed to be 5.7 and 1.6 times more potent than that of Tb³⁺ alone in MDA and MDA/CH cells, respectively. Combination index analyses revealed that the interaction between cisplatin and Tb³⁺ was only synergistic at very low indices of cell death in MDA cells. However, in MDA/CH cells, the two drugs were synergistic up to intermediate levels of cell death. Conclusions: Our results suggest that the enhancement of cisplatin cytotoxicity by Tb³⁺ is more effective in cisplatin-resistant MDA/CH cells than in cisplatin-sensitive MDA cells. Therefore, terbium is potentially useful in cisplatin combination therapy for breast cancer patients, especially for those patients who have developed resistance to the drug. Received: 16 December 1998 / Accepted: 19 January 1999     

Carboplatin pharmacokinetics in young children with brain tumors

 Purpose: The pharmacokinetic parameters and maximal tolerated systemic exposure were determined for carboplatin in young children given in combination with cyclophosphamide and etoposide. Patients and methods: Carboplatin was administered as part of a multiagent chemotherapy regimen to 21 pediatric patients less than 5 years of age with newly diagnosed, malignant central nervous system tumors. Patients received cyclophosphamide, 1.2 g/m², on day 1 and carboplatin on day 2 followed by etoposide, 100 mg/m², each day. Carboplatin doses were calculated to achieve a targeted area under the serum concentration versus time curve (TAUC) of 5, 6.5 or 8 mg/ml . min based on each patient’s measured glomerular filtration rate (GFR). Carboplatin pharmacokinetic parameters were determined after course 1 and then after every third course of therapy. Results: The median carboplatin clearance and GFR after course 1 were 118 and 98 ml/min per m², respectively. Targeted doses based on measured GFR reliably achieved the TAUC for carboplatin. The median (range) carboplatin clearance for four children less than 1 year of age was 76 (66–84) ml/min per m², significantly lower (P=0.05) than the value of 131 (80–158) ml/min per m² for children from 1 to 4 years of age. The mean carboplatin clearance declined by 23% in 12 patients studied from course 1 to course 4 of therapy. The decrease was greater than 20% (range 20–53%) in 7 of the 12 patients studied. Conclusion: Carboplatin clearance for children aged between 1 and 4 years at diagnosis is approximately 45% higher than previously reported for pediatric patients, but declines after four courses of therapy. For children less than 1 year of age, carboplatin clearance per square meter is approximately 40% lower than patients 1 to 4 years of age. There are corresponding differences in GFR that provide a plausible explanation for the age and therapy-related changes in carboplatin clearance. Toxicity was acceptable for patients treated at a TAUC of 6.5 mg/ml . min for carboplatin given with etoposide and cyclophosphamide. The average carboplatin dose required for this AUC was 767 mg/m². Received: 13 July 1995/Accepted: 18 December 1995     

Double-platinum chemotherapy combined with etoposide in metastatic brain tumor from small cell lung carcinoma

The platinum-based chemotherapeutic agents, such as cisplatin (CDDP) and carboplatin (CBDCA), are effective for small cell lung carcinoma (SCLC). However, high dose treatment of these agents required for advanced-stage SCLC is often associated with severe toxicity. The authors used combination of lower doses of both cisplatin and carboplatin combined with etoposide (VP-16) to minimize side effects of these agents. This goal was accomplished by utilizing the facts that each agent has its own toxicity that can be controlled individually. Two patients (60- and 71-year old men) with multiple metastatic brain tumors from SCLC were treated by our chemotherapeutic regimen. After fourth chemotherapy, remarkable shrinking of brain masses was associated with significant decrease the size of original lung lesions in both cases. The two patients were discharged without any side effects of the treatment, and neurological deficits subsided in both cases. Each course provided the following schedules: carboplatin 200 mg/m² × 1 day, cisplatin 25 mg/m² × 2 days (intravenous administration), and etoposide 25 mg oral × 14 days. After second chemotherapy, the patient of Case 1 was irradiated to both brain and chest lesions, and only to brain in Case 2. The authors concluded from our two cases that the combination of these agents extremely effective to treat this malignancy with less toxicity. We named this double platinum chemotherapy as PEC, abbreviated from cisplatin, etoposide, and carboplatin.     

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     

In vitro and in vivo interaction between cisplatin and topotecan in ovarian carcinoma systems

Topotecan, a camptothecin analogue, is a␣specific inhibitor of topoisomerase I approved for use in the treatment of patients with refractory ovarian carcinoma. The drug's mechanism of action suggests a potential efficacy of drug combinations incorporating DNA-damaging agents. In an attempt better to define a␣rational basis for drug combination we examined the effect of topotecan on the cytotoxicity and antitumor activity of cisplatin in an ovarian carcinoma system growing in vitro and in vivo as a tumor xenograft. The in vitro cell system included a cisplatin-sensitive cell line, IGROV-1, and a cisplatin-resistant subline, IGROV-1/Pt0.5, which is characterized by p53 mutation and loss of normal function of the wild-type gene of the parental cell line. This cell system was chosen since the cell sensitivity to DNA-damaging agents appears to be dependent on p53 gene status. Cytotoxicity was assessed by the growth inhibition assay using different schedules: (a) a 1-h period of cisplatin exposure followed by a 24-h topotecan treatment and (b) a 1-h period of simultaneous exposure to cisplatin and topotecan. In the case of the sequential schedule, an additive interaction was observed in IGROV-1 and IGROV-1/Pt0.5 cells. When the simultaneous schedule was used, a synergistic interaction, more evident for the cisplatin-sensitive cells, was found. On the basis of these observations at a cellular level, the effect of concomitant administration of the two drugs (i.e., the most favorable schedule) was studied in the IGROV-1 tumor xenograft, which is moderately responsive to cisplatin and topotecan. Suboptimal doses of each drug (with a low dose of topotecan, 5.1 mg/kg) achieved an antitumor effect comparable with or superior to that of the optimal dose of a single treatment (tumor weight inhibition, 60%), thus indicating a␣pharmacological advantage of the combination over the single treatment. However, an increase in the topotecan dose (7.1 mg/kg) was associated with an evident increase in the toxicity of the combination, thereby suggesting that the drug interaction was not tumor-specific. Although the molecular basis of the drug interaction is not clear, it is likely that inhibition of topoisomerase I affects the ability of cells to repair cisplatin adducts. Such findings may have pharmacological implications since they suggest the potential clinical interest of topoisomerase I inhibitors in combination with cisplatin. Received: 14 June 1997 / Accepted: 18 September 1997     

Abrogated energy-dependent uptake of cisplatin in a cisplatin-resistant subline of the human ovarian cancer cell line IGROV-1

The parental IGROV-1 human ovarian adenocarcinoma cell line was intermittently exposed to increasing concentrations of cisplatin to obtain resistant sublines. A stable resistant subline with a resistance factor of 8.4 had been developed after 9 months and 28 passages, which was denoted IGROV_CDDP. A high correlation coefficient of 0.97 was found between the log cell survival and the DNA-adduct peak level during the process of resistance development. IGROV_CDDP was strongly cross-resistant to carboplatin and doxorubicin and moderately cross-resistant to etoposide, docetaxel, and topotecan. Only minor resistance against 5-fluorouracil was observed, whereas IGROV_CDDP was not cross-resistant to methotrexate. Intracellular accumulation of cisplatin was 65% lower in IGROV_CDDP as compared with parental IGROV-1 at 37  °C under normal conditions. Coincubation of cisplatin with the Na⁺/K⁺-ATPase inhibitor ouabain resulted in a more pronounced decrease in platinum accumulation in IGROV-1 (44% decrease) than in IGROV_CDDP (26% decrease). Under energy-depleting conditions the accumulation of cisplatin in the parental cell line was approximately 60% lower than that observed under normal (energy [i.e., ATP] rich) culture conditions. In contrast, the accumulation in IGROV_CDDP was not affected by ATP-depletion. There appeared to be no significant difference between the intracellular accumulation of platinum in the resistant and sensitive cells under conditions of energy deprivation or when the uptake was studied at 0  °C. In conclusion, abrogation of energy-dependent accumulation in IGROV_CDDP seems to be a major mechanism of resistance to cisplatin in this cell line. Received: 21 January 1997 / Accepted: 22 July 1997     

Collagen gel matrix assay as an in vitro chemosensitivity test for malignant astrocytic tumors

Background The efficacy of individual chemotherapy based on chemosensitivity has scarcely been studied. Methods We examined the chemosensitivites for four anticancer agents – 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3 (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), carboplatin, cisplatin, and etoposide – of 43 malignant astrocytic tumors (21 anaplastic astrocytomas and 22 glioblastomas) by using a collagen gel matrix assay, and we also determined the survival periods of the tumor-bearing patients. The chemosensitivity was evaluated in terms of the growth inhibition rate, using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) method. Results For the anaplastic astrocytomas, the mean growth inhibitory rate was 33.2% with cisplatin, 37.2% with carboplatin, 28.0% with ACNU, and 24.8% with etoposide. For the glioblastomas, these rates were 36.9%, 42.3%, 23.2%, and 34.8%, respectively. The median overall and progression-free survivals of anaplastic astrocytoma-bearing patients who had undergone chemotherapy with two anticancer drugs, both of which showed significant anticancer activity (growth inhibitory rate >30%) were significantly longer than those of the patients who had been treated with two drugs, one or both of which did not show significant anticancer activity. On the other hand, there was no significant difference in the overall or the progression-free survivals in the two corresponding groups of glioblastoma-bearing patients. Conclusion The collagen gel matrix assay is clinically useful to determine in vitro chemosensitivity that reflects in vivo chemosensitivity. Individual chemotherapy for malignant astrocytic tumors, based on chemosensitivity data, could contribute to longer survival, particularly in anaplastic astrocytoma-bearing patients.