Jörgen Kristensen

Activity of cyclosporins as resistance modifiers in primary cultures of human haematological and solid tumours.

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.

Temporal effects of the novel antitumour pyridyl cyanoguanidine (CHS 828) on human lymphoma cells

CHS 828, a novel pyridyl cyanoguanidine, has shown potent antitumour activity both in vitro and in vivo and is currently undergoing phase I evaluation in humans in collaboration with the European Organization for Research and Treatment of Cancer (EORTC). Here we study the temporal effects of CHS 828 on cytotoxicity, protein and DNA synthesis, cellular morphology and ultra structure using the lymphoma cell line U-937 GTB as the primary tumour model. In vitro analysis of tumour cell survival in response to CHS 828 revealed a cytotoxic effect progressively increased as a function of exposure time with maximum efficacy observed after 72 h. Activity of CHS 828 on U-937 GTB cells grown in vivo was also found. CHS 828 induced-cell death was dependent on intact protein synthesis and most cells appeared to lose their membrane integrity in the presence of a relatively well preserved nuclear structure. The results indicate that CHS 828 induced active and delayed cell death with a non-apoptotic morphology.

Activity of CHS 828 in primary cultures of human hematological and solid tumors in vitro

CHS 828 is a pyridyl cyanoguanidine that has shown promising preclinical anticancer activity against various experimental tumor models and is presently being tested in a phase II trial in man. In the present study the fluorometric microculture cytotoxicity assay was used for in vitro evaluation of CHS 828 activity in primary cell cultures from hematological and solid tumors. In total, 156 samples from various diagnoses were tested with 72-h continuous drug exposure. CHS 828 showed high relative in vitro activity against tumor cells from chronic lymphocytic leukemia as well as from acute leukemia and high-grade lymphoma. Activity was also observed in several solid tumor cell samples, although the group as a whole appeared less responsive. CHS 828 was significantly more active against hematological malignancies compared to normal lymphocytes. Correlation analysis with standard drugs revealed low to moderate correlation coefficients. The results show that CHS 828 has potent antitumor activity against primary cultures of human tumor cells from patients and might have a unique mechanism of action.

In vitro activity of 2-chlorodeoxyadenosine (CdA) in primary cultures of human haematological and solid tumours

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.

Modeling of in vitro drug activity and prediction of clinical outcome in acute myeloid leukemia.

The objectives of this study were to develop a population pharmacodynamic model describing the in vitro drug sensitivity of tumor cells and to relate in vitro parameters to clinical outcome. Cell samples from 179 patients with acute myelocytic leukemia were exposed to cytosine arabinoside and daunorubicin, and cytotoxicity was analyzed using the fluorometric microculture cytotoxicity assay. A sigmoid Emax‐model for daunorubicin and an Emax‐model for cytosine arabinoside described the data. The model predicted drug potency (EC50) adequately from 1 concentration measurement. A logistic regression on individual in vitro parameters of 46 patients treated with the daunorubicin plus cytosine arabinoside regimen showed that the probability of complete response was significantly (P <.05) related to the product of the Emax/EC50 ratio of the two drugs. The findings demonstrate the value of population pharmacodynamic modeling of in vitro drug sensitivity data and a significant relationship between the in vitro parameters and clinical outcome.

In vitro drug resistance in B cell chronic lymphocytic leukemia : a comparison with acute myelocytic and acute lymphocytic leukemia.

The aim of the study was to evaluate cellular drug resistance in B cell chronic lymphocytic leukemia (B-CLL) in vitro, and compare it with that in acute myelocytic leukemia (AML) and acute lymphocytic leukemia (ALL). In vitro drug resistance was analyzed by the fluorometric microculture cytotoxicity assay (FMCA) in all samples from patients with leukemia sent to our laboratory between 1992 and 2001. Up to 14 standard drugs were evaluated in samples from 66 patients with B-CLL, 212 patients with AML and 80 patients with ALL. B-CLL cells were found to be more sensitive than cells from both AML and ALL to cytarabine, cladribine, fludarabine, doxorubicin, idarubicin, vincristine and cyclophosphamide (p<0.05). No difference in cellular drug resistance was found between B-CLL and ALL cells for prednisolone, whereas AML cells were more resistant (p<0.0001). In B-CLL, cells from patients who had received previous chemotherapy were more resistant to almost all tested drugs as compared to cells from treatment-naive patients. In AML and ALL, in vitro drug resistance was not related to previous chemotherapy. For all drugs, there was a good agreement between the activity in vitro and the known clinical disease-specific activity. The study also demonstrated an acquired cellular drug resistance in B-CLL, but not in the acute leukemias.

IN VITRO ANALYSIS OF DRUG RESISTANCE IN TUMOR CELLS FROM PATIENTS WITH ACUTE MYELOCYTIC LEUKEMIA

A 72 hours fluorometric microculture cytotoxicity assay (FMCA) was used for the study of chemotherapeutic drug resistance in tumor cell suspensions from patients with acute myelocytic leukemia (AML). A marked heterogeneity with respect to sensitivity was observed for a panel of cytotoxic drugs tested in 76 samples from 60 patients with treated or untreated AML. Primary resistance to vincristine (Vcr) and prednisolone in untreated AML was observed as well as ‘acquired’ resistance to several other antileukemic drugs. Cross resistance patterns for AML active drugs revealed significant positive relationships between anthracyclines, VP 16 and amsacrine (Amsa), whereas mitoxantrone (Mitox) was more weakly correlated. Sensitivity to cytosine arabinoside was unrelated to the anthracyclines, VP16, Amsa and Mitox butshowed a significant relationship to 6-thioguanine. Several resistance modifying agents, including the novel non-imrnunosuppressive cyclosporin A analogue PSC 833, were able to potentiate the effects of doxorubicin and Vcr at concentrations achievable in the clinic. However, the pattern of activity was heterogenous and the frequency of responsive samples was higher in relapse compared tode novo cases. Individualin vitro/in vivo correlations based on quartile distributions of ail accumulated drug sensitivity data from AML patients indicated a high specifity with respect to the identification of drug resistance. The results suggest that the FMCA may provide clinically valuable information on chemotherapeutic drug resistance in AML.

Cytotoxic activity of cyclosporin A and [3-keto-Bmt1]-[Val2]-cyclosporin (SDZ PSC 833) on tumour cells from patients with haematological malignancies.

AbstractObjective and method: The fluorometric microculture cytotoxic assay was employed for characterisation of the cytotoxic effect of cyclosporin A (CsA) and its non-immunosuppressive analogue SDZ PSC 833, [3-keto-Bmt1]-[Val2]-cyclosporin (PSC) in tumour cells from patients with haematological or solid tumours. Results: Tumour cells from patients with chronic lymphocytic leukaemia (CLL) or non-Hodgkins lymphoma (NHL) were found to be more sensitive to both drugs than those of tumour cells from patients with acute lymphocytic leukaemia (ALL), acute myoblastic leukaemia (AML) and various solid tumours. There was a close correlation between the effects of the two drugs (correlation coefficient 0.71), but CsA was slightly more active than PSC in most diagnoses. No tumour cell sample showed sensitivity to PSC without also being sensitive to CsA. There was a moderate level of correlation between the activity pattern of CsA and doxorubicin (correlation coefficient 0.66), whereas the correlations with other cytostatics, such as vincristine, cytarabine and melphalan, were low (correlation coefficient −0.11 to 0.33). Conclusion: The results indicate that PSC shares the direct cytotoxic properties of CsA, but is slightly less potent. Clinical testing of the cytotoxic effect of these agents in haematological malignancies seems warranted and the apparent non-cross-resistance with standard agents makes cyclosporins a potentially useful adjunct to chemotherapy in those diagnoses.

Selective cytotoxic activity of cyclosporins against tumor cells from patients with B cell chronic lymphocytic leukemia

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.

In vitro evaluation of the efficacy of idarubicin in human tumour cells from patients with low‐grade non‐Hodgkin's lymphoma

Summary. Evaluating the potential benefit of the new anthracycline, idarubicin (Ida), in lymphoma, 58 tumour samples from patients suffering from low‐grade non‐Hodgkins lymphoma (L‐NHL), were analysed in vitro for their sensitivity to 0·5 µg/ml Ida. This was compared with the sensitivity to other anthracyclines (0·5 µg/ml), using the fluorometric microculture cytotoxicity assay. A total of 132 samples from patients with acute leukaemia and a cell‐line panel representing different resistance mechanisms was included for comparison. The median cell survival of L‐NHL cells did not differ after exposing the cells to Ida or daunorubicin (Dnr), whereas epirubicin, doxorubicin (Dox) and mitoxantrone (Mitox) were significantly less cytotoxic than Ida (P < 0·001). The median cell survival in L‐NHL cells did not differ from that of acute leukaemia cells after exposure to 0·5 µg/ml Ida, Dnr, Dox and Mitox. Cells from previously treated patients with L‐NHL had a higher median survival than cells from untreated patients after exposure to all drugs, except for Ida. In samples from previously untreated patients, Spearman rank correlations were high (Rho = 0·81–0·90) between cell survival after exposure to Ida and the other anthracyclines. The same pattern was observed in the cell‐line panel (Rho = 0·78–0·91) (P < 0·05). In contrast, low correlations (Rho = 0·24–0·42) were observed among samples from previously treated patients. Our results indicate a potential benefit of Ida in previously drug‐treated patients with L‐NHL.