A study of ethacrynic acid as a potential modifier of melphalan and cisplatin sensitivity in human lung cancer parental and drug-resistant cell lines.

We have studied alterations in glutathione (GSH) levels and glutathione-S-transferase (GST) activity in a series of in vitro derived multidrug resistant and cisplatin resistant sublines of the human lung cancer lines NCI-H69 (small cell), COR-L23 (large cell) and MOR (adenocarcinoma). We have also investigated the effects of ethacrynic acid, a putative inhibitor of GSTs, on levels of GSH and GST activity and on cellular sensitivity to melphalan and to cisplatin. Neither GSH content nor GST activity were significantly greater in the resistant sublines compared with their respective parental lines. The only effects of treating with ethacrynic acid at doses of 1 microgram ml-1 and 3 micrograms ml-1 for 2 h were a reduction in GSH content in the cisplatin resistant subline H69/CPR at the 3 micrograms ml-1 dose, and an increase to over 140% of control at 1 microgram ml-1 and 3 micrograms ml-1 in the MOR parental line (MOR/P) and at 1 microgram ml-1 in the multidrug resistant subline MOR/R. Exposure of parental line COR-L23/P to 3 micrograms ml-1 and 6 micrograms ml-1 of ethacrynic acid for 24 h, however, increased the GSH content to over 300% and 500% of control respectively. Variable effects of ethacrynic acid on GST activity were seen in these cell lines. Doses of 1 microgram ml-1 and 3 micrograms ml-1 reduced activity to 59% and 48% of control respectively in multidrug resistant subline H69/LX4. On the other hand, activity was increased in the cisplatin resistant subline H69/CPR (to 146% and 218% of control) and in MOR/P (to 117% and 137% of control) by 1 microgram ml-1 and 3 micrograms ml-1 respectively of ethacrynic acid. Addition of ethacrynic acid (3 micrograms ml-1) to treatment of the cell lines with melphalan or with cisplatin did not alter the dose-response curves to these agents.     

Effect of culture media on in vitro susceptibility testing of fluconazole against some yeasts

Thirty clinical isolates, comprising six strains of Candida albicans, and four strains each of C. lusitaniae, C. parapsilosis, C. tropicalis, Cryptococcus neoformans, Torulopsis glabrata and Trichosporon beigelii were tested against fluconazole, using Sabouraud's dextrose (SD) broth and a high resolution (HR) medium (Pfizer Central Research, Inc.). The procedure was a standard tube (1 ml/tube) dilution, and C. albicans Y01 09 was included as a reference strain to monitor quality and reproducibility. Results indicated that the minimum inhibitory concentrations (MICs) for all isolates of C. albicans, C. lusitaniae, C. tropicalis, and Tr. beigelii were 100 micrograms ml-1 or greater in the SD medium. In the HR medium, however, the MICs for two isolates of C. albicans were 1.56 micrograms ml-1, in other four gave higher values (greater than 100 micrograms ml-1), and the results for C. lusitaniae and Tr. beigelii were in the range 1.56-3.12 micrograms ml-1. The MICs for C. tropicalis were unaffected (100 micrograms ml-1) by the medium used. All Cr. neoformans isolates yielded a uniform value (1.56 micrograms ml-1) in HR medium as compared to somewhat more variable results (MICs 0.39-1.56 micrograms ml-1) in SD broth. The MICs for T. glabrata in the SD and HR media were 3.12-12.5 and 6.25 micrograms ml-1, respectively. The data indicated that the HR medium is preferable for the in vitro susceptibility testing of C. albicans, C. lusitaniae and Tr. beigelii to fluconazole. The MICs for other yeasts were not affected by the culture medium. The reference C. albicans isolate yielded an MIC of 1.56 micrograms ml-1 throughout.     

Soluble E-cadherin fragments increased in circulation of cancer patients.

Monoclonal antibodies were raised against human placental soluble E-cadherins and used in an immunoenzymometric assay to detect soluble E-cadherins in biological fluids. The E-cadherin assay was accurate enough to quantitate the concentration of soluble E-cadherin in the cell culture supernatants. Immunoreactive E-cadherins, identified as existing in the soluble form in normal serum, were shown to have apparent lower molecular mass (approximately 80 kDa) than intact molecules of E-cadherin. We found that the immunoreactive E-cadherin levels in the serum of the studied cancer patients were significantly elevated (mean +/- s.d. 3.80 +/- 2.36 micrograms ml-1, P < 0.0001) when compared with the normal levels (1.99 +/- 0.50 micrograms ml-1). We also found that serum E-cadherin levels in the 22 patients with gastric cancer (3.51 +/- 1.78 micrograms ml-1, P < 0.02) or the 11 patients with hepatocellular cancer (5.55 +/- 3.11 micrograms ml-1, P < 0.001) were significantly higher than those in the 26 diabetic patients (2.33 +/- 1.58 micrograms ml-1). Of the 54 cancer patients, 53.7% exhibited an elevated amount of soluble E-cadherin in serum. Thus, it is evident that soluble E-cadherin in circulation can be used as a prospective tumour marker that accurately reflects the progressive regeneration of E-cadherin at tumour sites, potentially induced by tumour-associated proteolytic degradation.     

In vitro susceptibility of yeasts for fluconazole and itraconazole. Evaluation of a microdilution test

In vitro susceptibilities were determined for a total of 159 clinical isolates and 12 reference strains of yeasts belonging to different Candida species including 94 Candida albicans strains, and further genera such as Cryptococcus, Trichosporon, Geotrichum and Saccharomyces. Minimum inhibitory concentration (MIC) values for fluconazole and itraconazole were assessed using a microdilution technique with the semisynthetic high resolution (HR) medium supplemented with glucose and asparagine but without sodium hydrogen carbonate (pH 7.0), according to a proposal of the working group 'Clinical Mycology' of the German Speaking Mycological Society. Fluconazole MIC values for C. albicans were between 0.125 and > or = 128 micrograms ml-1. Thus, the median of 1 microgram ml-1 showed that the overall fluconazole susceptibility was good. As expected, Candida krusei (seven strains) exhibited diminished in vitro susceptibility with MIC values for fluconazole of 8 to 128 micrograms ml-1 with a median of 64 micrograms ml-1. Some Candida kefyr strains seemed to be less susceptible against fluconazole which was indicated by a MIC90 of 64 micrograms ml-1. Surprisingly, no Candida glabrata isolate exhibited a MIC value greater than 16 micrograms ml-1. Other Candida species, Trichosporon cutaneum, Geotrichum candidum and Saccharomyces cerevisiae showed low MICs to fluconazole. In vitro susceptibility testing of itraconazole revealed that all Candida species except C. albicans, but also Trichosporon cutaneum, Geotrichum candidum, and Saccharomyces cerevisiae exhibited acceptable low MIC values against itraconazole (0.03-2 micrograms ml-1). Their MIC90 values for itraconazole were in the close range between 0.125 and 2 micrograms ml-1. MIC values between 0.125 and 2 micrograms ml-1 were obtained, even for C. krusei strains. On the other hand, the range of C. albicans MICs was between 0.0125 and > or = 16 micrograms ml-1 with MIC50 and MIC90 values of 0.125 and > or = 16 micrograms ml-1, respectively, indicating that a considerable number of yeast strains have high MICs. The comparative evaluation of different experimental conditions revealed that there exists a marked influence both of inoculum size and incubation time on the results of susceptibility testing. Therefore, for routine usage 10(2) CFU ml-1 and 18-24 h incubation time for this microdilution method with HR medium are recommended.     

Detection of Ca antigen in sera from normal individuals and patients with benign and malignant breast disease

Two assay procedures, an inhibition radioimmunoassay (Inhibition-RIA) and an immunoradiometric assay (IRMA), were established for the detection of circulating tumour-associated Ca antigen. There was a good correlation between results (r = 0.987) but the Inhibition-RIA was selected for extended tests on human sera from patients with breast disease because of its greater ease and economy in use. Circulating Ca antigen was not exclusive to malignancy and the level failed to discriminate between patients with primary carcinoma and those with benign disease. Ca antigen was present in sera of 100 healthy individuals (median 7.1 micrograms ml-1, range 1.8-24.4 micrograms ml)-1, 39 patients with benign disease (median 9.9 micrograms ml-1, range 2.5- greater than 100 micrograms ml-1) and in 67 patients with primary carcinoma (median 11.0 micrograms ml-1, range 3.8- greater than 100 micrograms ml-1). Elevated Ca antigen levels were found in 50% of patients with metastatic spread (median 30.7 micrograms ml-1, range 8.2- greater than 100 micrograms ml-1) and in some patients with primary disease but further studies are needed to determine the prognostic significance. Immunochemical studies confirmed that Ca antigen is a normal serum product but its function is unclear.     

In vitro activity of rilopirox against fluconazole-susceptible and fluconazole-resistant Candida isolates from patients with HIV infection

The in vitro antifungal activity of the new hydroxypyridone antimycotic rilopirox has been evaluated against 38 fluconazole-susceptible and -resistant clinical isolates of Candida albicans together with other Candida species isolated from patients with human immunodeficiency virus (HIV) infection and oropharyngeal candidosis. Minimum inhibitory concentrations (MICs) of both rilopirox and fluconazole were measured by a microdilution method using high-resolution medium supplemented with asparagine and glucose at pH 7.0. In comparison, an agar dilution technique was carried out for susceptibility testing of the antifungal agents. Rilopirox was found to be able to inhibit growth of all clinical yeast isolates. The rilopirox MICs at which 50% and 90% of strains were inhibited (MIC50 and MIC90 respectively), as determined by the microdilution method, were 4 and 8 micrograms ml-1 respectively. The highest MIC values for rilopirox using microdilution and the agar dilution method were 32 or 25 micrograms ml-1 respectively. On the other hand, for fluconazole, the MIC50 and MIC90 achieved were 0.5 and 128 micrograms ml-1, respectively, which means that the MIC90 value of fluconazole was 16-fold higher than that of rilopirox. Using the agar dilution technique, the MIC values of rilopirox were in the range 0.006-25 micrograms ml-1 with a median of 3.12 micrograms ml-1. For fluconazole, the MIC90 value was four-fold higher than that for rilopirox, indicating a considerable proportion of yeast strains with high MICs of 100 micrograms ml-1, suggesting in vitro resistance to this azole antifungal. All strains with diminished fluconazole susceptibility were susceptible to rilopirox. Even Candida krusei and Candida glabrata exhibited good in vitro susceptibility to rilopirox. Therefore, this new antifungal agent may be used as an alternative not only in the treatment of vaginal candidosis, but also in oropharyngeal Candida infections, e.g. in AIDS patients.     

Effect of melphalan and hyperthermia on p34cdc2 kinase activity in human melanoma cells.

We previously reported that combined treatment with melphalan and mild hyperthermia (1 h at 42 degrees C) caused a synergistic cytotoxic effect in JR8 melanoma cells, paralleled by a stabilisation of a melphalan-induced G2-phase cell block. In this study, we investigated the effect of melphalan and hyperthermia on proteins that regulate G2-M transition. Neither hyperthermia nor melphalan at a concentration of 2.5 micrograms ml-1, which had no antiproliferative effect at 37 degrees C, interfered with cyclin B1 expression or p34cdc2 kinase activity. At a concentration of 8.5 micrograms ml-1, which reduced cell growth by 50% at 37 degrees C, melphalan inhibited p34cdc2 kinase activity as a consequence of an increased tyrosine phosphorylation of the protein. A similar inhibitory effect on p34cdc2 kinase was obtained when the lowest melphalan concentration (2.5 micrograms ml-1) was used under hyperthermic conditions. Our results indicate that thermal enhancement of melphalan cytotoxicity could be mediated at least in part by an inhibition of p34cdc2 kinase activity, which prevents cell progression into mitosis.     

Cyclosporin A corrects daunorubicin resistance in Ehrlich ascites carcinoma

We have previously developed a daunorubicin resistant subline of Ehrlich ascites carcinoma (EA/DR) for studies on the reversal of daunorubicin resistance. The mean survival of untreated BALB/c mice bearing drug sensitive parental tumour (EA/DS) is 18.4 +/- 0.6 days, mice bearing EA/DS treated with five daily doses of 0.3 mg kg-1 daunorubicin greater than 60 days, and mice bearing EA/DR treated with the same daunorubicin regimen, 21.1 +/- 1.4 days. We now report complete reversal of daunorubicin resistance in EA/DR by cyclosporin A (CsA). The in vitro daunorubicin IC50, defined as that concentration of daunorubicin required to inhibit 50% of DNA synthesis, in EA/DR was 6.7 +/- 1.15 micrograms ml-1 compared to 2.8 +/- 0.72 micrograms ml-1 in EA/DS. This value was reduced to 2.8 +/- 0.52 and 2.1 +/- 0.10 micrograms ml-1 daunorubicin by 3.3 and 13.2 micrograms ml-1 CsA respectively, P less than 0.05. The MST of groups of host mice bearing EA/DR either untreated, treated with five daily doses of 0.3 mg kg-1 daunorubicin, treated with 80 mg kg-1 CsA in five divided daily doses or treated with combined daunorubicin-CsA were 19.0 +/- 1.0, 21.1 +/- 1.4, 24.0 +/- 2.6 and greater than 60 days respectively. The mean survival of groups of host mice bearing EA/DR treated with 5 mg kg-1 or 10 mg kg-1 CsA simultaneously with daunorubicin for five days was also greater than 60 days. These differences are highly significant.     

A pharmacokinetic comparison of intravenous versus intra-arterial folinic acid.

Recent clinical trials have suggested that a combination of folinic acid and 5-fluorouracil (5-FU) may improve response rates and survival in patients with advanced colorectal cancer. However, this regimen has been complicated by potentially life threatening toxicity. Regional delivery of folinic acid via a hepatic artery catheter might be expected to reduce systemic exposure and subsequent adverse effects. The present study compared the pharmacokinetic profiles of intravenous and intra-hepatic arterial infusions of folinic acid in patients with colorectal liver metastases (n = 6) who were being treated with weekly regional infusions of 5-FU. The mean area under the plasma concentration--time curve, the peak plasma concentration and the steady state volume of distribution were 163 micrograms ml-1 h-1 (SD 41), 18.5 micrograms ml-1 (SD 1.2) and 7.41 m-2 (SD 0.44) respectively following intravenous administration of folinic acid compared with 142 micrograms ml-1 h-1 (SD 45), 14.8 micrograms ml-1 (SD 2.4) and 11.21 m-2 (SD 1.22) following intra-hepatic arterial administration (P less than 0.05). Regional folinic acid was therefore associated with a statistically significant reduction in systemic exposure compared with the intravenous route.     

Case report. Osteomyelitis caused by high resistant Candida guilliermondii

We describe a case of a 57-year-old patient with osteomyelitis at a finger of his right hand caused by Candida guilliermondii. The strains isolated were highly resistant to fluconazole and itraconazole. Using the three methods, microdilution, agardilation and E-test, the highest minimum inhibitory concentrations (MICs) amounted to > 256 micrograms ml-1 for fluconazole and > 32 micrograms ml-1 for itraconazole. To our knowledge, this is the first time such high values have been described for C. guilliermondii. They correlated with the therapeutic non-response to a triazole therapy in our patient. The patient was cured by partial amputation of the affected finger.     

A phase I and pharmacokinetic study of didox administered by 36 hour infusion. The Cancer Research Campaign Phase I/II Clinical Trials Committee

Twelve patients were treated with didox, a new ribonucleotide reductase inhibitor, by 36 h infusion. The maximum tolerated dose was 6 g m-2, above which dose-limiting hepatic toxicity was observed. Patient tolerance was significantly better using the 36 h infusion compared to patients receiving the drug by a 30 min infusion; in particular, there were no reports of nausea or vomiting. No responses were seen in these patients. Detailed pharmacokinetics were performed at 6 g m-2 comparing the 36 h and 30 min infusions in four patients. Parent drug AUC values were lower for the 36 h infusion, 67.8 micrograms ml-1 h-1 compared to 232 micrograms ml-1 h-1 for the 30 min infusion. AUC values for the 3-hydroxy metabolite were much higher following the 36 h infusion: 55.4 compared to 18.6 micrograms ml-1 h-1. In contrast, the amide metabolite was not detected following the 36 h infusion, but AUC values of 23 micrograms ml-1 h-1 were seen after the 30 min infusion. The mean peak plasma level was 72 micrograms ml-1 following 6 g m-2 given by a 30 min infusion compared to 2.8 micrograms ml-1 following the prolonged infusion. Clearance was higher following the 36 h infusion: 97.6 versus 24.4 l h-1.     

Antifungal action of Hevea brasiliensis latex. Its effect in combination with fluconazole on Candida albicans growth.

Latex from Hevea brasiliensis and its subcellular fractions (L-serum and C-serum) were tested for antifungal activity alone or in combination with fluconazole. Candida albicans growth was inhibited with the same efficacy when yeasts were inoculated into culture medium supplemented over the total growth phase with latex as when latex was added during the exponential phase only: the minimum inhibitory concentration (MIC 80%) of H. brasiliensis latex was 123 micrograms protein ml-1. By means of a non-linear regression analysis of the experimental data, two distinct fixation sites for fluconazole (FCZ) could be determined: one of strong affinity (Kaff = 0.0162 microgram-1 protein ml) and another of low affinity (Kaff = 0.0071 microgram-1 protein ml). After addition of a mixture of FCZ and latex during the exponential phase, the affinity constant of yeasts for FCZ was calculated: when latex was in a final concentration of 21 micrograms protein ml-1 (Kaff = 1 microgram-1 protein ml) or 42 micrograms protein ml-1 (Kaff = 0.277 microgram-1 protein ml) and without latex (Kaff = 0.0502 microgram-1 protein ml). In two cases a synergistic effect between latex and FCZ was obtained. The highest efficacy was obtained with a latex concentration of 21 micrograms protein ml-1. The addition of subcellular fractions of latex, L-serum and C-serum, did not cause an antifungal effect. The indispensable role of rubber particles for raising an antifungal effect is demonstrated. Electron microscopy observations indicated a limited cell wall degradation and a high percentage of coagulated yeasts.     

An assessment of a short-term tumour chemosensitivity assay in chronic lymphocytic leukaemia

A 4-day tumour sensitivity assay of potential use in predicting tumour response to cytotoxic drugs has been investigated in patients with chronic lymphocytic leukaemia. The method comprised isolation of white cells from peripheral blood, drug exposure and incubation for 4 days. Drug-induced tumour cell kill was assessed by differential staining of dead and live cells such that the latter could be morphologically identified, with subsequent calculation of tumour cell viability. Concentrations of drug for use in the assay were chosen for chlorambucil (2 micrograms ml-1), 4-hydroperoxy-cyclophosphamide (2 micrograms ml-1)--which was used in vitro in place of cyclophosphamide--prednisolone (0.5 microgram ml-1) and vincristine (0.1 microgram ml-1), to give a scatter of values which was in good agreement with clinical expectations. In 21 cases where the in vitro result could be compared with the in vivo response, there were 4 true positive comparisons (sensitive in vitro, sensitive in vivo), 15 true negative comparisons (resistant both in vitro and in vivo) and 2 false positive comparisons (sensitive in vitro, resistant in vivo). A result was obtained in 86% (65/76) of samples received. The assay appears to show considerable promise as a tumour chemosensitivity test and warrants wider investigation, including prospective in vivo/in vitro correlations that could be based on the results presented here.     

Involvement of basic fibroblast growth factor in suramin-induced inhibition of V79/AP4 fibroblast cell proliferation.

The V79/AP4 Chinese hamster fibroblasts were densely stained with the anti-basic fibroblast growth factor (bFGF) antibody demonstrating an endogenous production of the peptide. The in vitro proliferation of these cells was stimulated by exogenous bFGF and the maximum growth (259% increase in 3H-thymidine incorporation into DNA) was reached with bFGF 10 ng ml-1. Inhibition of bFGF-mediated mitogenic pathway was obtained with a 15-mer antisense oligodeoxynucleotide targeted against bFGF mRNA and with suramin, a drug which blocks the biological activity of heparin-binding growth factors. bFGF antisense oligomer reduced the synthesis of DNA by 79.5 and 89.5% at 20 and 60 microM, respectively; this effect was reversed by the addition of exogenous bFGF to the culture medium. A short-term exposure to suramin 300 micrograms ml-1 produced a modest reduction in 3H-thymidine incorporation but suppressed the mitogenic effect of bFGF on V79/AP4 cells. In cells treated with suramin 300 micrograms ml-1 the drug concentration increased linearly over 3 days, reaching 13.15 micrograms mg-1 of protein; cell proliferation was inhibited in a dose-related manner as evaluated by the colony formation assay (IC50: 344.22 micrograms ml-1) and by the number of mitoses observed in culture. Furthermore, the drug induced ultrastructural alterations, consisting of perinuclear cisternae swelling, chromatin condensation, nucleolar segregation and cytoplasmic vacuolations. These findings demonstrated that the endogenous production of bFGF plays an important role in V79/AP4 fibroblasts proliferation, and the inhibition of bFGF-mediated mitogenic signalling with bFGF antisense oligomer or suramin is an effective mean of reducing cell growth.     

Inhibitory effect of essential oils on apical growth of Aspergillus fumigatus by vapour contact

The inhibitory effect of seven essential oils on the apical growth of hyphae of Aspergillus fumigatus was studied using a bio cell tracer by vapour contact in a sealed vessel. Based on the inhibitory pattern, these essential oils were classified into three groups. The first group, composed of citron, lavender and tea tree oils, stopped the apical growth in a loading dose of 63 micrograms ml-1 air, but allowed the regrowth of the hyphae after removal of the vapour, indicating fungistatic action. The second group, consisting of perilla and lemon-grass oils, stopped the apical growth in a loading dose of 6.3 micrograms ml-1 air, and did not allow the regrowth after gaseous contact at 63 micrograms ml-1 air, indicative of fungicidal action. The third group, consisting of cinnamon bark and thyme oils, retarded the growth in a dose of 6.3 micrograms ml-1 air, stopped it in a dose of 63 micrograms ml-1 air, and incompletely suppressed regrowth of the hyphae. Gas chromatographic analysis revealed that vapours of essential oils were absorbed on fungal mycelia and agar medium most abundantly by the first group, followed by the second and third groups, reflecting the volatility of the respective groups. Suppression of the apical growth by vapour contact was ascribed to the direct deposition of essential oils on fungal mycelia, together with an indirect effect via the agar medium absorbed.     

In vitro comparative evaluations of the postantifungal effect: synergistic interaction between flucytosine and fluconazole against Candida albicans

In vitro comparative evaluations were performed to study the efficacy of combinations of flucytosine and fluconazole in producing a postantifungal effect (PAFE) on Candida albicans. Initial studies were done to determine MIC, FIC (fractional inhibitory concentration) and optimal PAFE parameters. A turbidometric method was used to measure yeast cell growth following exposure to different concentrations of the two drugs for periods of 0.5, 1 or 2 h at temperatures of 30 degrees C and 37 degrees C. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to reach the 0.5 absorbance level following removal of the drug by dilution. Ten strains of C. albicans were then assayed (30 degrees C; 2 h exposure time) and a synergistic PAFE was evidenced with the two drugs at concentrations well below their individual MICs. PAFEs ranging from 3.8 to 10.5 h, which persisted for 1.2-2.5 h longer than those achieved with either agent separately, were evidenced when flucytosine and fluconazole were combined (flucytosine: fluconazole ratios of 1:16-1:32) at concentrations ranging from 0.024 to 0.098 micrograms ml-1 and 0.78 to 1.56 micrograms ml-1 respectively. The concentrations of each agent required to produce an optimal PAFE varied according to the C. albicans strain being assayed.     

Morphological changes of Candida, albicans induced by saperconazole

The effects of a new triazole antifungal agent, saperconazole, on the morphology of Candida albicans were studied by scanning and transmission electron microscopy. An inoculum of 10(6) CFU ml-1 was exposed to saperconazole at 1 and 10 micrograms ml-1 and at different times up to 24 h samples were removed for microscopic observations. The antifungal agent caused the yeasts to become round and turgescent and to cluster; budding appeared to be affected also, as seen by scanning electron microscopy. Transmission electron microscopy showed a thickened wall, the presence of intraparietal electron-dense vesicles and of multilamellae near the plasma membrane.     

Repeated antitumour antibody therapy in man with suppression of the host response by cyclosporin A

Antibody targeted therapy of cancer results in anti-antibody production which prevents repeated treatment. Cyclosporin A (CsA) has been used to suppress this response in patients treated with a radiolabelled antibody to carcinoembryonic antigen (CEA). Patients with CEA producing tumours received a minimum of two courses consisting of an injection of radiolabelled antibody and CsA, 24 mg kg-1 day-1, for 6 days; each course was given at 2 week intervals. Two weeks after the completion of the second course the mean human antimouse antibody (HAMA) levels were 3.5 micrograms ml-1 (s.d. 2.7) in 3 patients receiving CsA and 1,998 micrograms ml-1 (s.d. 387) in 3 patients not receiving the drug. Clearance of antitumour antibody was accelerated and tumour localisation absent when HAMA levels exceeded 30 micrograms ml-1. With lower levels of HAMA in the CsA-treated patients, further antitumour antibody accumulated in the tumour after each dose. Further therapy with antitumour antibody and CsA lead to the development of HAMA, but this was less than 25% of the amount in patients not given CsA. In this preliminary study up to 4 times as many doses of antitumour antibody could be usefully given when CsA was used. This increases the potential for effective antibody targeted therapy of cancer.     

Cytostatic inhibition of endothelial cell growth by the angiogenesis inhibitor TNP-470 (AGM-1470).

Recently, we reported the anti-angiogenic action along with anti-tumour activity of TNP-470 (AGM-1470). In this study, the effect of TNP-470 on the growth of human umbilical vein endothelial (HUVE) cells was examined. TNP-470 inhibited the growth of HUVE cells in a biphasic manner. The inhibition was cytostatic in the first phase (complete inhibition at 300 pg ml-1 to 3 micrograms ml-1 with an IC50 of 15 pg ml-1) and cytotoxic in the second phase (> or = 30 micrograms ml-1). The cytostatic inhibition of HUVE cell growth by TNP-470 was durable after washing out TNP-470 in culture. Incorporation of thymidine but not uridine and leucine by HUVE cells was inhibited in the first phase, while that of all three compounds was inhibited in the second phase. Human and rat endothelial cells among various types of cells were the most sensitive to the cytostatic inhibition, while differences in the cytotoxic inhibition were minimal. These results suggest that TNP-470 exerts its specific anti-angiogenic action by inhibiting cytostatically growth of endothelial cells in a relatively specific manner.     

Flameless atomic absorption spectrophotometry analysis of cell-linked platinum after wet ashing

Wet ashing of K562 cell suspensions by means of a sulphonitroperchloric acid digestion for electrothermal atomic absorption spectrometry of platinum has been achieved. The limit of detection was about 1 ng platinum per 10(6) cells. Platinum concentrations in K562 cells were measured after exposure to platinum coordination complexes such as cis-dichlorodiamminoplatinum (DDP) and Trans-1 diamminocyclohexanooxalatoplatinum (1-OHP). Cell linked platinum was measured after a 24 hours exposure to a concentration of 6.8 nM ml-1 of both forms 1-OHP and DDP (i.e. 1.3 micrograms platinum per ml). Platinum concentrations were found to be respectively (mean +/- S.D.) 14.8 +/- 2.7 and 10.0 +/- 4.0 ng platinum per 10(6) cells. These 1-OHP and DDP concentrations were cytotoxic and about twenty times the 50 percent cell growth inhibitory concentrations (0.45 nM ml-1 and 0.33 nM ml-1 respectively).