[Show abstract][Hide abstract] ABSTRACT: Chemotherapy using multi-drug regimens is considered more active than single-agent therapy. This may be due to synergistic interactions or, simply, a higher probability of administering an active agent. We investigated in vitro the type of drug interactions in a recognized regimen in relationship to tumour type and drug sensitivity.
The possibility of synergistic and additive interactions between individual cytotoxic drugs was investigated for the component drugs of the established FEC regimen, i.e., 5-fluorouracil, epirubicin and cyclophosphamide, in 243 patient tumour samples representing various drug sensitivity using the non-clonogenic fluorometric microculture cytotoxicity assay.
Using a cell survival of < or = 50% as a limit for drug activity and sample sensitivity, the overall response rates to the most active single drug (Dmax) and the combination were 56% and 64%, respectively, with a distribution among diagnoses similar to that in the clinic. For 86% of the samples there was concordance with respect to judgement of activity using either Dmax or the combination. For samples being sensitive to at least one single drug, 95% were also sensitive to the combination whereas for samples with insignificant Dmax effect, only 2% were sensitive to the combination. In samples with modest Dmax effects, i.e., cell survival in the range > 50%- < or = 80%, 45% responded to the combination. The effect of the combination was generally well predicted from the Dmax effect.
The superior antitumour effect of drug combinations compared with single drugs may be due to the higher chance of selecting an active agent. However, for intermediately sensitive tumours, additional interaction effects of a combination may be of clinical significance.
Annals of Oncology 10/2000; 11(10):1301-7. DOI:10.1023/A:1008332816407 · 7.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Suramin has shown promising antitumour activity against several tumour types, both in vitro and in vivo, but the clinical utility of this compound is hampered by its unfavourable toxicity profile. In the present study, the semi-automated fluorometric microculture cytotoxicity assay (FMCA) was employed for evaluation of the cytotoxicity of seven suramin analogues in vitro in a panel of human tumour cell lines and in primary cultures of tumour cells from patients. Like suramin, the analogues showed little sensitivity to resistance mechanisms involving P-glycoprotein, topoisomerase II, multidrug resistance associated protein and glutathione-mediated drug resistance. In the cell line panel, NF067 and FCE 26644 showed activity comparable with suramin. All analogues were less potent than suramin in patient cells except for FCE 26644. Correlation to suramin activity patterns in the cell line panel was highest for NF037 and low to moderate for the remaining analogues. In patient cells, high correlation coefficients were obtained for FCE 26644, NF110, NF031 and NF037. The results indicate that the cytotoxic activity of suramin on patient tumour cells is shared by the analogues with FCE 26644 being the most active. The pharmacophore for cytotoxicity in patient cells may be different from that observed in the cell lines.
European Journal of Cancer 05/2000; 36(6):803-9. DOI:10.1016/S0959-8049(00)00024-1 · 5.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In vitro tumor models could support the process of development of new cytotoxic drugs and selection of suitable drugs for the individual patient. We investigated whether the testing of tumor cells from patients with kidney or urinary bladder carcinoma by fluorometric microculture cytotoxicity assay (FMCA) could provide clinically relevant data for these tumor types.
A total of 45 tumor samples from patients with kidney or urinary bladder carcinoma were compared with 247 samples of other tumor types with respect to sensitivity to 8 standard and 6 investigational cytotoxic drugs in the FMCA, a 72 hour assay based on the concept of total cell kill. In bladder carcinomas, sensitivity to standard drugs was correlated to various tumor characteristics.
The technical success rate for kidney and bladder carcinomas was high; approximately 90% of the samples could be analyzed successfully. Kidney carcinomas were highly resistant to standard drugs and bladder carcinomas essentially as sensitive as carcinomas of the breast and ovary but with a steeper dose-response relationship. In bladder carcinoma there was no clear relationship between tumor stage, grade, ploidy, mitoses or p53 expression and drug sensitivity. Except for suramin, kidney carcinomas were poorly sensitive to the investigational drugs CdA, gemcitabine, paclitaxel, vinorelbine and topotecan. In bladder carcinomas paclitaxel, gemcitabine and suramin showed promising activity.
The FMCA seems suitable for cytotoxic drug sensitivity testing of urinary tract carcinomas. This technique may have a role in new drug development in these tumor types.
The Journal of Urology 01/2000; 162(6):2200-4. DOI:10.1016/S0022-5347(05)68159-1 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The fluorometric microculture cytotoxicity assay (FMCA), a short-term in vitro assay based on the concept of total tumor cell kill, was used for testing the cytotoxic drug sensitivity of tumor cells from patients with ovarian carcinoma. A total of 125 fresh specimens was obtained, 98 (78%) of which were analyzed successfully. Data from 45 patients were available for clinical correlations. The FMCA appeared to yield clinically relevant cytotoxic drug sensitivity data for ovarian carcinoma as indicated by a comparison with tumor samples obtained from patients with non-Hodgkin's lymphoma or kidney carcinoma. Considering the most active single agent in vitro actually given in vivo, and using the median drug activity among all ovarian carcinoma samples as a cut-off, the sensitivity of the assay and its specificity were 75 and 52%, respectively. Cross-resistance in vitro was frequently observed between standard drugs but not between standard drugs and Taxol. Ten percent of the specimens showed an extreme resistance for at least 4 of 6 of the drugs investigated.
International Journal of Cancer 10/1997; 72(6):1008-12. DOI:10.1002/(SICI)1097-0215(19970917)72:63.3.CO;2-9 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies indicate that Cremophor EL (CEL), the excipient for Taxol, a clinical preparation of paclitaxel, has biologic properties per se.
The cytotoxic activity of Taxol and its solvents CEL/ethanol, paclitaxel in ethanol, and 14 other cytotoxic drugs was investigated in vitro in 10 human carcinoma cell lines and 183 tumor samples from patients with tumors of various types. Cytotoxicity was determined by the fluorometric microculture cytotoxicity assay.
In the cell lines, Taxol was generally more active than paclitaxel; this may have been due to an additive effect of the diluent. This activity was pronounced in sublines expressing tubulin-associated and P-glycoprotein-mediated drug resistance, indicating involvement of these mechanisms in paclitaxel resistance and their modulation by CEL. Taxol and paclitaxel were highly cross-resistant to other tubulin-active agents, whereas the low cytotoxic effect of CEL seemed unrelated to other drugs. In the samples from patients, Taxol was less active than in the cell lines but showed a differential activity that corresponded reasonably well with that in the clinic. CEL and Taxol were similarly active, indicating that paclitaxel did not add substantially to the activity of Taxol.
Whereas the cell line data clearly confirmed the well-known properties of paclitaxel, a more valid model using tumor cells from patients demonstrated that CEL significantly contributes to the efficacy of Taxol in vitro. The clinical relevance of this finding remains to be elucidated.
Cancer 04/1997; 79(6):1225-33. · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cytotoxic activity and cross-resistance pattern of the novel topoisomerase I inhibitor topotecan (Topo) were investigated in ten cell lines, representing different mechanisms of cytotoxic drug resistance, and in 218 fresh human tumour samples using the fluorometric microculture cytotoxicity assay (FMCA). Resistance to Topo in the cell lines was associated with expression of the multidrug resistance-associated protein (MRP), whereas the cell lines with P-glycoprotein (P-gp), topoisomerase II and glutathione-associated resistance did not show decreased sensitivity to the drug. Topo was more active in haematological than in solid tumour samples, but substantial activity was observed in carcinomas of the ovary and breast, sarcoma and childhood solid tumours. Cross-resistance to standard drugs representing different mechanisms of action was generally low in patient cells. The effect of Topo was better after longer exposure, but this time-dependent effect was largely abolished when adjustment for in vitro exposure was made. Topo showed activity both in proliferative and non-proliferative cell systems. The results indicate that Topo is insensitive to major mechanisms of resistance except for MRP. Proliferation does not seem to be necessary for the effect of Topo, and no superiority for protracted dosing schedules was observed. The results also suggest that, for example, leukaemias, lymphomas, sarcomas and childhood solid tumours may be suitable targets for future phase II trials.
British Journal of Cancer 02/1997; 76(2):211-9. DOI:10.1038/bjc.1997.364 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Differential drug response in a human cell line panel representing defined types of cytotoxic drug resistance was measured using the non-clonogenic fluorometric microculture cytotoxicity assay (FMCA). In total 37 drugs were analysed; eight topoisomerase II inhibitors, eight anti-metabolites, eight alkylating agents, eight tubulin-active agents and five compounds with other or unknown mechanisms of action, including one topoisomerase I inhibitor. Correlation analysis of log IC50 values obtained from the panel showed a high degree of similarity among the drugs with a similar mechanism of action. The mean percentage of mechanistically similar drugs included among the ten highest correlations, when each drug was compared with the remaining data set, was 100%, 92%, 88% and 52% for the topoisomerase II inhibitors, alkylators, tubulinactive agents and anti-metabolites respectively. Classification of drugs into the four categories representing different mechanisms of action using a probabilistic neural network (PNN) analysis resulted in 29 (91%) correct predictions. The results indicate the feasibility of using a limited number of cell lines for prediction of mechanism of action of anti-cancer drugs. The present approach may be well suited for initial classification and evaluation of novel anti-cancer drugs and as a potential tool to guide lead compound optimisation.
British Journal of Cancer 10/1996; 74(6):888-96. DOI:10.1038/bjc.1996.453 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The feasibility of combined studies on a cell-line panel and primary cultures of patient tumor cells in the preclinical evaluation of new anticancer drugs was evaluated in a study of the activity and cross-resistance pattern in vitro of the new semi-synthetic vinca alkaloid vinorelbine (Vrb). The activity of Vrb was investigated in ten cell lines representing different resistance mechanisms and in a total of 256 fresh human tumor samples, using the fluorometric microculture cytotoxicity assay (FMCA). Resistance to Vrb in the cell lines was associated with expression of the multidrug resistance-mediating P-glycoprotein and the multidrug resistance-associated protein (MRP) and by a recently described tubulin-associated mechanism, while the cell lines with topoisomerase II- and glutathion-associated resistance did not show decreased sensitivity to the drug. Cross-resistance to vincristine (Vcr) and other tubulin-active agents was high in cell lines as well as in patient cells. As with most commonly used anti-cancer drugs, Vrb was more active in hematological than in solid tumor samples. Among the solid tumors investigated, the highest in vitro response rates were observed in ovarian cancer (27%), sarcoma (25%), non-small cell lung cancer (21%) and bladder cancer (20%), while no response was observed in renal or colorectal cancer. Compared to Vcr, Vrb appeared to be slightly more active in solid tumors and slightly less active in hematological tumors. The results show that although Vrb displays a high degree of cross-resistance to Vcr and other tubulin-active drugs, some difference in the activity spectrum could be detected and that the drug is sensitive to multiple mechanisms of resistance. The results also suggest that leukemias, ovarian cancer, sarcoma and bladder cancer are possible further targets for Vrb. The combination of studies on a cell-line panel and patient tumor cells from a broad spectrum of diagnoses to evaluate a new drug seems feasible and may give information on the mechanism of action and target diagnoses for phase II trials.
Journal of Experimental Therapeutics and Oncology 10/1996; 1(5):286-95.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine the in vitro cytotoxicity of calcein acetoxymethyl ester (Calcein/AM) on primary cultures derived from solid and haematological human tumours. Calcein/AM is a fluorescent dye that localises intracellularly after esterase-dependent cellular trapping and which has shown cytotoxic activity against various established human tumour cell lines at relatively low concentrations. The semi-automated fluorometric microculture cytotoxicity assay, based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate to fluorescein, in microtitre plates was used for the evaluation of Calcein/AM activity in tumour cell suspensions from patients. The cytotoxicity was measured as a survival index (SI), defined as the fluorescence as a percentage of control cultures. A total of 163 evaluable samples from various tumours were tested with continuous drug exposure. The activity of Calcein/AM was compared with representatives of six major classes of standard chemotherapeutic drugs. Calcein/AM was found to induce concentration-dependent decreases in the SI of both haematological and solid tumour cells. The ratio of solid over haematological tumour activity increased at a rate that was concentration dependent. Although it was relatively less active than cisplatin against solid tumours, Calcein/AM showed higher solid tumour activity compared to leukaemic specific agents (cytarabine and amsacrine), vincristine and doxorubicin (Dox). Among the solid tumours tested, childhood tumours, non-small cell lung cancer and sarcomas were the most sensitive to Calcein/AM. The best correlation between SI values was seen between Calcein/AM and Dox, with weaker correlations to representatives of antimetabolites, platinum compounds, topoisomerase II inhibitors, tubulin interactive agents and alkylators. Non-cytotoxic concentrations of cyclosporin A significantly potentiated calcein-induced cytotoxicity. The results show that Calcein/AM is differentially active against haematological tumours, but with substantial activity against solid tumours. The drug may represent a new class of anticancer compound with a unique means of drug delivery.
European Journal of Cancer 06/1996; 32A(5):883-7. DOI:10.1016/0959-8049(96)00015-9 · 5.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The antineoplastic activity of suramin is currently the subject of clinical trials. We therefore used the semi-automated fluorometric microculture cytotoxicity assay (FMCA) to evaluate the cytotoxicity of suramin in vitro in primary cultures of cells from patients with hematological or solid tumors. The activity patterns of some standard cytotoxic agents were included for comparison. A total of 159 samples were tested using continuous drug exposure. Suramin showed relatively high activity against solid tumors, with colorectal, adrenal and kidney carcinomas being the most sensitive, whereas hematological malignancies were more resistant. Suramin and standard drugs showed very low cross-resistance. The results indicate that suramin is differentially active against some solid tumors with comparatively little activity against hematological tumors. The study provides an experimental motive for evaluation of suramin therapy in patients with solid tumors and exploration of less toxic suramin analogues.
International Journal of Cancer 11/1995; 63(3):356-60. DOI:10.1002/ijc.2910630309 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gemcitabine (2'-deoxy-2',2'-difluorocytidine; dFdC) is a novel nucleoside analog that has shown clinical activity against solid tumors. The semiautomated fluorometric microculture cytotoxicity assay (FMCA) was used for evaluation of the cytotoxicity of gemcitabine in vitro in primary cultures of human cells from patients with hematologic or solid tumors. The activity pattern of cytarabine (ara-C), 2-chlorodeoxyadenosine (CdA), etoposide (VP-16), doxorubicin, and cisplatin were included for comparison. One hundred forty samples were tested using continuous drug exposure. Gemcitabine showed high activity against hematologic samples, whereas little or no activity was observed in the solid tumor groups. A similar pattern of activity also was observed for ara-C and CdA, whereas etoposide, cisplatin, and doxorubicin were relatively more active in solid tumors. Cross-resistance analysis between gemcitabine and the standard drugs revealed the following rank order of correlation coefficients: ara-C > doxorubicin > CdA > cisplatin > VP-16. The results indicate that gemcitabine is differentially active against hematologic tumors and that the activity pattern of gemcitabine resembles that of ara-C. However, these results also indicate that gemcitabine may be more active against some solid tumor groups in comparison to ara-C and CdA.
[Show abstract][Hide abstract] ABSTRACT: In patient tumour samples the activity in vitro of Taxol corresponded fairly well to the known clinical activity and Taxol showed low cross-resistance to standard cytotoxic drugs. However, the Taxol solvent Cremophor EL--ethanol was considerably active alone, whereas paclitaxel formulated in ethanol was less active. Taxol thus seems to contain two components active against patient tumour cells in vitro.
British Journal of Cancer 04/1995; 71(3):478-81. DOI:10.1038/bjc.1995.97 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The automated fluorometric microculture cytotoxicity assay (FMCA) is based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein by viable cells after a 72-hr culture period in microtiter plates. The FMCA was adopted for chemosensitivity testing of tumor cells from patients with ovarian carcinoma. Thirty-seven samples of solid tumors and malignant effusions were obtained from 35 patients at diagnosis or relapse. Tumor cells from solid samples and effusions were prepared by enzymatic digestion and centrifugation, respectively, followed by Percoll or Ficoll purification. The fluorescence was proportional to the number of cells/well and considerably higher in tumor cells than in contaminating normal cells. The effect of up to 19 cytotoxic drugs was successfully assessed in 70% of the samples and there was a good correlation between drug sensitivity data reported by the FMCA and the DiSC assay performed in parallel. The overall drug sensitivity pattern in vitro corresponded well to the clinical experience. The effect of cisplatin varied considerably between patients and resistance was found also in cases not previously exposed to cytotoxic drugs. The FMCA is a rapid and simple method that seems to report clinically relevant cytotoxic drug sensitivity data in ovarian carcinomas. In the future, this method may contribute to optimizing chemotherapy by assisting in individualized drug selection and new drug development.
[Show abstract][Hide abstract] ABSTRACT: The semiautomated fluorimetric microculture cytotoxicity assay (FMCA) was used for evaluation of the ability of cyclosporin A (CsA) and its novel non-immunosuppressive derivative SDZ PSC 833 (PSC) to modify the response to doxorubicin or vincristine in vitro in different haematological and solid human tumour types. Primary cultures of 322 tumour samples were analysed. Both cyclosporins showed resistance-modifying activity in all haematological tumours tested, and in solid tumours activity was observed in ovarian carcinoma and childhood tumours. Little or no effect was found in the remaining tumour types, including breast, renal and adrenal cortical carcinomas and adult sarcomas. In most of the responsive cases the interaction between the modifier and the cytotoxic drug was synergistic. There was a tendency to higher activity in samples from previously treated patients, and an inverse relationship between degree of cytotoxic drug resistance and resistance-modifying activity was noted. No difference in potency between CsA and PSC could be discerned. The results indicate differential in vitro resistance-modifying activity of the cyclosporins depending on tumour type. The results also suggest that treatment with resistance modifiers should be considered also for primary therapy of drug-sensitive tumours. Drug resistance assays such as the FMCA may become useful in preclinical evaluation of resistance modifiers.
British Journal of Cancer 08/1994; 70(1):11-7. DOI:10.1038/bjc.1994.242 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The semi-automated fluorometric microculture cytotoxicity assay (FMCA), based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) by viable cells, was employed for cytotoxic drug sensitivity testing of tumor cells from patients with hematological or solid tumors. In total, 390 samples from 20 diagnoses were tested-with up to 12 standard cytotoxic drugs. The technical success rate for different tumor types ranged from 67 to 95%. Fluorescence was linearly related to cell number but variably steep depending on tumor type. Samples from most solid tumors thus showed higher signal-to-noise ratios than hematological samples. A wide spectrum of in vitro drug activity was obtained, with acute leukemias and non-Hodgkin's lymphomas being sensitive to almost all tested drugs, whereas renal and adrenocortical carcinomas were essentially totally resistant. Between these extremes were samples of breast and ovarian carcinomas and sarcomas. When in vitro response was compared with known clinical response patterns, a good correspondence was observed. The results indicate that the FMCA is a rapid and efficient method for in vrtro measurement of tumor-specific drug activity both in hematological and in solid tumors. The assay may be suitable for new drug development and direction of phase-2 trials to suitable patients.
International Journal of Cancer 03/1994; 56(5):715 - 720. DOI:10.1002/ijc.2910560517 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 2-Chlorodeoxyadenosine (CdA) is a deaminase-resistant purine analogue which has shown clinical activity against various haematological tumours, and is currently undergoing phase II trials. In the present study, the semiautomated fluorometric microculture cytotoxicity assay (FMCA) was used for in vitro evaluation of CdA activity in cell suspensions from both haematological and solid tumours. A total of 133 samples from various diagnoses were successfully tested with continuous drug exposure. CdA showed high in vitro activity against samples from chronic and acute lymphocytic leukaemia and acute myelocytic leukaemia, but little or no response was observed in the solid tumour groups. Cross-resistance analysis with standard drugs revealed the following rank order of correlation coefficients: cytosine arabinoside (AraC) > daunorubicin > doxorubicin > vincristine > prednisolone > 4-hydroperoxycyclophosphamide > etoposide > cisplatin. The high correlation between CdA and AraC was maintained even if the analysis was based only on the haematological tumours. The results indicate that CdA is differentially active against haematological tumours with little or no activity against solid tumours. CdA also appears highly cross resistant with AraC. If this disease-specific information is substantiated in further clinical trials and extended to other phase I-II drugs, non-clonogenic drug resistance assays such as the FMCA may become useful in new drug evaluation, and in targeting specific diagnoses and patients for phase II trials.
European Journal of Cancer 02/1994; 30A(7):1022-6. DOI:10.1016/0959-8049(94)90136-8 · 5.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A fluorometric microculture cytotoxicity assay was employed for the study of cyclosporin A induced cytotoxicity in tumor samples from patients with B type chronic lymphocytic leukemia (B-CLL). Tumor cells from patients with B-CLL were found to be significantly more sensitive to the cytotoxic actions of cyclosporin A than normal blood mononuclear cells and tumor cells obtained from patients with different types of acute leukemia and solid tumors. The effect of cyclosporin A on B-CLL samples could be reproduced by a non-immunosuppressive cyclosporin A analogue. One B-CLL patient treated with cyclosporin A responded with a significant decrease in tumor mass and alleviation of anemia and B symptoms. The results show that cyclosporin A and its non-immunosuppressive analogues appear selectively toxic to B-CLL cells, an observation which may have clinical implications.
European Journal of Pharmacology 01/1993; 228(4-228):237-240. DOI:10.1016/0926-6917(92)90035-B · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cytotoxic activity of cyclosporin A (CsA) and the three non-immuno-suppressive CsA analogues B3-243, WO-039 and B3-665 were studied in tumor cell lines representing both classical and atypical forms of multidrug resistance (MDR): T-ALL GM3639 L100 cells selected for vincristine (vcr) resistance and displaying characteristics of classical MDR, including P-glycoprotein (pgp) expression and increased drug efflux which can be inhibited by pgp blockers (e.g. verapamil), and U-1285/ADR, a small cell lung cancer (SCLC) cell line selected for doxorubicin resistance which lacks pgp, is insensitive to pgp-blockers and shows cross resistance to cis-platinum. At 1 micrograms/ml CsA was the most active agent in reversing Vcr resistance in L100 cells followed by B3-243 and WO-039, with no effect of B3-665. Parental LO cells were only marginally sensitized to Vcr by these agents. No reversing effect of any cyclosporin was observed in the U-1285/ADR or its parental cell line. Compared to LO cells, L100 cells showed a marked hypersensitivity to CsA > B3-243 > WO-039 with B3-665 being inactive. No collateral sensitivity was observed for cyclosporins in U-1285/ADR cells. Although of different magnitude, the pattern of cytotoxic activity for the different cyclosporins alone closely parallelled that of L100 cells for U-1285, U1285/ADR and LO cells. The results indicate that not only the collateral sensitivity in classical MDR but also the cytotoxic actions of cyclosporins per se on tumor cells alone are independent of immunosuppressive activity. The results also suggest a structure-activity relationship for cyclosporin-induced cytotoxicity similar to, but independent of, MDR reversing activity.
Anticancer research 01/1992; 12(5):1581-5. · 1.83 Impact Factor