Modulation of the toxicity and antitumour activity of alkylating drugs by steroids.

The steroids prednisolone and progesterone significantly altered the therapeutic indices of the alkylating agents, nitrogen mustard, melphalan, cyclophosphamide, phenyl acetic mustard and chlorambucil. For nitrogen mustard, chlorambucil and phenyl acetic mustard, prednisolone reduced host toxicity in the rat and enhanced the antitumour effectiveness against alkylating-agent-resistant strains of the Yoshida sarcoma and Walker carcinosarcoma. Progesterone also increased the therapeutic index of chlorambucil in the rat by decreasing its systemic toxicity. Two other alkylating agents, melphalan and cyclophosphamide, exhibited lower therapeutic indices in combination with prednisolone against alkylating-agent-sensitive tumours. This was due to the greater host toxicity of the combination than of the alkylating agent alone. In alkylating-agent-resistant tumours, however, a significant increase in growth delay was achieved if prednisolone was combined with the alkylating agent

The Uptake and Utilization of Chlorambucil by Lymphocytes from Patients with Chronic Lymphocytic Leukaemia

It has been shown that lymphocytes isolated from the peripheral blood of patients with chronic lymphocytic leukaemia do not modify the mustard group of chlorambucil, as has been demonstrated previously in Yoshida ascites cells. However, lymphocytes from patients with an unsatisfactory clinical course or poor response to treatment were able to modify the aromatic region of the drug molecule; little change occurred in the aromatic absorption of intracellular chlorambucil in patients who responded to treatment. This simple test may provide a rapid assessment of a patient's potential response to chemotherapy

Adenosine deaminase activity in leukaemia.

Adenosine deaminase (EC 3.5.4.4., ADA) has been measured in the blast cells of 36 patients with acute lymphoblastic, acute myeloid, chronic myeloid and chronic myeloid blast crisis leukaemia. Particularly high levels were found in acute lymphoblastic and chronic myeloid blast crisis patients. The measurement of ADA may be useful diagnostically in the undifferentiated acute leukaemias and in detecting the early onset of blast crisis in chronic myeloid leukaemia. Possible reasons for the elevation of ADA in malignant cells are discussed

Criteria for the selection of second-generation platinum compounds.

Our selection of a potential second-generation platinum compound began with an initial short list of 8 compounds selected on the basis of antitumour and toxicity studies in mice. We now report further, more detailed investigations of the renal toxicity and antitumour activity of one of these compounds, cis-dichloro trans-dihydroxy bis isopropylamine platinum (IV) (CHIP), in comparison with cis-dichloro diammine platinum (II) (Neoplatin). CHIP was a more effective anti-tumour agent against both alkylating-agent sensitive and resistant strains of the Yoshida sarcoma (YSS and YSR respectively) than was Neoplatin. In addition CHIP produced negligible kidney toxicity as measured by blood urea levels. We have also compared the effects of these two drugs on nuclear-protein phosphorylation, in an attempt to gain insight into their molecular mode of action. Both Neoplatin and CHIP induced increased nuclear-protein phosphorylation in the YSS tumour cells, and loss of condensed chromatin. However, CHIP also induced increased nuclear-protein phosphorylation and loss of condensed chromatin in the YSR tumour cells. These changes correlated well with cell death. In addition Neoplatin, but not CHIP, treatment caused increased nuclear-protein phosphorylation in kidney tissues. This correlated with kidney damage as measured by blood urea levels. These selection criteria suggested that CHIP would be a more selective antitumour agent than Neoplatin, and will provide a basis for its comparison with the other 7 compounds

In vitro platinum drug chemosensitivity of human cervical squamous cell carcinoma cell lines with intrinsic and acquired resistance to cisplatin.

The platinum drug chemosensitivity of five human cervical squamous cell carcinoma cell lines (HX/151, HX/155, HX/156, HX/160 and HX/171) derived from previously untreated patients has been determined. Compared to our data obtained previously using human ovarian carcinoma cell lines, all five lines were relatively resistant to cisplatin, carboplatin, iproplatin and tetraplatin. One of the lines (HX/156) was exceptionally sensitive to the novel platinum (IV) ammine/amine dicarboxylates JM216 [bis-acetatoammine dichloro (cyclohexylamine) platinum (IV)] and JM221 [ammine dibutyrato dichloro (cyclohexylamine) platinum (IV)]. The range in IC50 values across the five lines was approximately 2.5-fold for cisplatin, carboplatin and iproplatin, 13-fold for tetraplatin and JM216, and 25-fold for JM221. No significant correlation (P > 0.05) was observed between platinum drug chemosensitivity and either glutathione levels or cadmium chloride sensitivity, an indicator of metallothionein levels. In addition, there was no significant correlation (P > 0.05) between cisplatin cytotoxicity and intracellular cisplatin accumulation or JM216 cytotoxicity and intracellular JM216 accumulation over the dose range 5-100 microM (2 h exposure). The exceptional sensitivity of HX/156 to JM216 appears, at least partially, to be a result of enhanced accumulation of JM216. An 8.6-fold acquired cisplatin resistant stable variant of HX/155 has been generated in vitro. Intracellular cisplatin accumulation was reduced by 2.4 +/- 0.3-fold (mean +/- s.d.) in HX/155cisR across the dose range 1-100 microM (2 h exposure). Glutathione levels in HX/155cisR were elevated by 1.3-fold in terms of protein content and by 1.6-fold in terms of cell number. HX/155cisR was 1.9-fold resistant to cadmium chloride. Total platinum bound to DNA after cisplatin exposure (10, 25, 50 or 100 microM for 2 h) was 3.6 +/- 0.6-fold (mean +/- s.d.) lower in HX/155cisR. Hence the mechanism of acquired cisplatin resistance in HX/155cisR appears to be multifocal, with reduced intracellular drug accumulation and elevated glutathione and metallothionein levels combining to reduce DNA platination levels. While HX/155cisR was cross-resistant to tetraplatin and carboplatin, novel platinum (II) and (IV) ammine/amine complexes, including JM216 and JM221, partially circumvented resistance (resistance factors of 1.5-2). Non cross-resistance was observed to iproplatin and nine non-platinum anticancer agents. Intracellular tetraplatin accumulation was reduced by 1.8 +/- 0.1-fold (mean +/- s.d.) in HX/155cisR across the dose range 1-100 microM (2 h exposure). In contrast, after JM216 exposure (1-100 microM for 2 h), no significant difference in intracellular platinum levels between HX/155 and HX/155cisR was observed.(ABSTRACT TRUNCATED AT 400 WORDS

Acquisition of platinum drug resistance and platinum cross resistance patterns in a panel of human ovarian carcinoma xenografts.

In vivo models of acquired resistance to the platinum-based agents cisplatin (CDDP), carboplatin (CBDCA), iproplatin (CHIP) and tetraplatin have been established using a panel of six parent human ovarian carcinoma lines, two (HX/110 and PXN/87) being derived from previously untreated patients. Resistance has been generated to CDDP (three lines), CBDCA (one line), CHIP (three lines) and tetraplatin (one line) either by treatment in vivo or (for one line to CDDP) through exposure in vitro and subsequent transfer to mice. With the four tumours where resistance was generated using CDDP or CBDCA, a complete cross-resistance to the remaining platinum agents studied was observed. In contrast, in one of three lines with derived resistance to the platinum (IV) agent, CHIP, (PXN/951) a retention in sensitivity was observed with CDDP and CBDCA. Only one of the six parent tumour lines (PXN/100) was markedly sensitive to tetraplatin. Where resistance was generated to tetraplatin (PXN/100T) there was some retention of activity by CDDP. For the CDDP-resistant line established in vitro, there was a close agreement between the cross-resistance profile obtained in vitro vs that obtained in vivo. This tumour panel may be useful in the elucidation of cellular and molecular resistance mechanisms to platinum drugs operative in vivo. Moreover, as they appear to mimic the clinical observations of shared cross-resistance between CDDP, CBDCA and CHIP, they may represent valuable preclinical evaluation models for the discovery of drugs capable of conferring responses in CDDP-refractory ovarian cancer

Mechanism of action of an orally administered platinum complex [ammine bis butyrato cyclohexylamine dichloroplatinum (IV) (JM221)] in intrinsically cisplatin-resistant human ovarian carcinoma in vitro.

Intrinsic resistance to existing clinical platinum drugs is a major cause of treatment failure; moreover, these agents have the drawbacks of cross-resistance and intravenous administration. The mechanism of intrinsic cisplatin resistance and the mechanism of circumvention of intrinsic resistance by a member (JM221) of the ammine/amine platinum (IV) dicarboxylate class of platinum complex was studied in intrinsically resistant (SKOV-3) and sensitive (41M) human ovarian carcinoma cell lines. JM221 reduced the cisplatin resistance factor nine- to 2.7-fold, was more potent than cisplatin and showed marked time-dependent cytotoxicity. Cellular platinum accumulation was 20- to 40-fold greater (P < 0.001), and DNA platination was fourfold greater (P < 0.02), immediately following 2 h equimolar exposure to JM221, compared with cisplatin. DNA platinum levels decreased following cisplatin exposure with a half-life approximating 48 h in both lines, while no net removal of DNA-bound platinum was recorded following JM221 exposure. JM221 caused DNA interstrand cross-linking, but this was 10-20% less frequent with JM221 than with cisplatin when expressed as a proportion of total DNA platinum lesions. Cisplatin DNA interstrand cross-linking was twofold greater in the intrinsically sensitive line (41M) than in the resistant line (SKOV-3) over a range of concentrations and time-points. Neither cellular platinum accumulation, levels of DNA platination nor the rate of removal of DNA-bound platinum in the two cell lines related to their ninefold difference in cisplatin sensitivity. Intrinsic cisplatin resistance appears to be attributable to the inhibition of formation of bifunctional DNA lesions, while the circumvention of intrinsic resistance by JM221 seems to be the result of both improved transport properties and circumvention of DNA repair mechanisms

Reduced drug accumulation as a major mechanism of acquired resistance to cisplatin in a human ovarian carcinoma cell line: circumvention studies using novel platinum (II) and (IV) ammine/amine complexes.

Acquired resistance to cisplatin (cis-diamminedichloroplatinum (II)) has been generated in vitro in the 41M human ovarian carcinoma cell line, established from a previously untreated patient. Three cisplatin-resistant variants were selected at approximately 2, 4 and 6-fold resistance (in terms of 50% inhibitory concentrations), in order to study the underlying mechanisms of acquired cisplatin resistance. Compared to the parent line, platinum accumulation following exposure to equimolar concentrations of cisplatin was on average (across the entire concentration range) 2.9, 3.6 and 4.8-fold lower in the 41McisR2, 41McisR4 and 41McisR6 cell lines, respectively. Thus the difference in uptake corresponded closely with their resistance factor in the three resistant variants. Moreover, a significant reduction in platinum accumulation was observed as early as 5 min after exposure to cisplatin in the 41M vs 41McisR6 cell lines. Platinum accumulation was similar in all cell lines following exposure to equitoxic concentrations (2 h IC50) of cisplatin. Enhanced efflux of drug was not observed between the 41M and 41McisR6 cells. In addition, there was no difference in intracellular glutathione (GSH) levels. Our previous studies have shown no indication of metallothionein involvement and the decrease in cisplatin uptake in the 41McisR6 cells was reflected by a similar reduction in DNA interstrand cross-links (ISC) formation. These results suggest that the mechanism of acquired resistance to cisplatin in the 41McisR6 cell line may be predominantly due to reduced drug uptake. The 41McisR6 cells were not found to be cross-resistant to ouabain, a postulated specific inhibitor of sodium-potassium adenosine triphosphatase (Na+, K(+)-ATPase), suggesting that decreased cisplatin accumulation in these cells is probably not regulated by alterations in their Na+, K(+)-ATPase levels, and Na+ potential across the plasma membrane. Cellular accumulation of a novel class of platinum (IV) ammine/cyclohexylamine dicarboxylates, which exhibit enhanced cytotoxicity over cisplatin and completely circumvent resistance to cisplatin in the 41McisR line, was also examined. The data suggests that increased accumulation of these compounds, as a result of their enhanced lipophilicity, could account for the dramatic increase in their potency over cisplatin