Activated oxazaphosphorines are transported predominantly by erythrocytes.

PURPOSE: Oxazaphosphorines are metabolised by a variety of pathways, one of which leads to activation and the formation of alkylating compounds. However, the transport forms conveying activated oxazaphosphorines to the tumour cell have not been fully characterised. There is increasing recognition of the importance of the erythrocyte as a carrier of compounds in the circulation, and we have recently described higher concentrations of 4-hydroxycyclophosphamide within the erythrocyte compartment compared to plasma. We have now determined the concentrations of ifosfamide and seven of its metabolites in the plasma and erythrocytes of patients receiving a six-hour intravenous infusion of ifosfamide. PATIENTS AND METHODS: Red cells from five patients, receiving a total of eight cycles of ifosfamide, were separated from plasma using the MESED instrument, and analysis of red cells and plasma performed using Gas Chromatography-Mass Spectrometry (GC/MS). RESULTS: The concentration of all compounds in the erythrocyte compartment was higher than or equal to those in plasma, and isophosphoramide mustard and carboxyifosfamide showed a particular affinity for the erythrocyte. The red cell fraction can contain as much as 77% of the total blood concentration of isophosphoramide mustard. CONCLUSIONS: Erythrocyte associated isophosphoramide mustard is an important transport form of activated ifosfamide. Red cells may have a role in the delivery of activated oxazaphosphorines to tissues.

Quantitative measurements of the efficacy of new anti-cancer agents on fresh human AML cells by using multivariate flow analysis.

The testing of new human leukemia-specific drugs for activity against primary acute myeloid leukemia (AML) blasts is severely limited by the low and variable clonogenic potential of primary human leukemias in culture. To circumvent this problem, we have modified a previously described flow cytometric approach to permit the simultaneous determination of live/dead cells, and the quantitation of the surviving cell fraction as a ratio of viable cells in treatment and control groups. The method utilizes the combination of calceinAM as a probe for intracellular esterase activity (green fluorescence,) which has the advantage over carboxyFDA of pH insensitivity and superior signal-to-noise ratio, and propidium iodide (red fluorescence) as an indicator of plasma membrane integrity. Suspension cultures of AML blood and marrow samples from patients were treated with known active agents as well as several new agents arising from a clonogenic disk assay screen. Quantitative dose-response values obtained from surviving cell fractions assayed by flow cytometry at 24 h following drug exposure demonstrated the utility of this assay for quantitating drug-induced cytotoxic effects on primary human AML cells in short-term culture.

Neurotoxicity of antineoplastic agents.

PURPOSE: to review the neurotoxicity associated with antineoplastic agents. METHODS: four hundred articles, abstracts and book chapters were selected for review. One hundred and ninety (articles, book chapters and abstracts) were identified as representative of the important aspects of neurotoxicity to be presented in this review. RESULTS: in general the dose, schedule and route of administration significantly determine the incidence and outcome of antineoplastic agents neurotoxicity. An updated and detailed review of neurotoxicity is provided with special attention to vinca alkaloids, cisplatin and biologic response modifiers. The neurotoxic side effects of some of the new approaches in cancer therapy and some of the investigational agents are discussed. Guidelines for the prevention and management of this toxicity are presented. In addition, suggestions are made in regard to the preclinical and clinical screening of new agents for neurotoxicity. CONCLUSION: quality of life issues have become a focal point in many clinical trials. Neurotoxicity associated with antineoplastic therapy clearly has an impact on the short and long term quality of the life of cancer patients. A better understanding of this toxicity requires developing reliable and predictive models to screen new agents prior to their introduction into clinical trials; a more detailed and uniform grading system; and the prospective evaluation of neurotoxicity in clinical trials of new antineoplastic agents.

A-phase II study of intravenous 6-thioguanine (NSC-752) in multiple myeloma. A Southwest Oncology Group study.

Thirty-three patients with relapsing or refractory multiple myeloma were treated with 6-Thioguanine (6TG) at a dose of 1 g/M2, with therapy given over four hours every three weeks. The major toxicity seen was myelotoxicity; thrombocytopenia was more commonly noted than neutropenia. One patient achieved a PR, two were clinically improved. 6TG in this short infusion schedule proved to be myelotoxic, but demonstrated little activity in previously treated myeloma patients.

Infused L-histidinol and cisplatin: schedule, specificity, and proliferation dependence.

The dose and schedule requirements found for the combination of L-histidinol and 5-fluorouracil (5-FU) were concordant with those for the combination of L-histidinol and cisplatin. Furthermore, cisplatin-L-histidinol was active against colon 26 tumor, an adenocarcinoma that developed in a BALB/c female mouse and that has been grown as a solid tumor. The toxicity of cisplatin was prevented only when cisplatin was given before L-histidinol. Studies of L-histidinol and 5-FU had similar results. For (DBA/2 X BALB/c)F1 mice, 50 mg of L-histidinol per mouse was required for protection; for hematopoietic precursor cells, protection was dependent on the dose of L-histidinol. In contrast, both L1210 leukemia cells and colon 26 adenocarcinoma cells were more efficiently killed by combinations of L-histidinol and cisplatin. This effect depended on the doses of L-histidinol and cisplatin, a finding similar to the finding for hematopoietic precursor cells.

Specificity, schedule, and proliferation dependence of infused L-histidinol after 5-fluorouracil in mice.

L-Histidinol, an analogue of the amino acid L-histidine, has been reported to be able to increase the specificity of 5-fluorouracil (FUra), through both protection of normal tissues at risk and potentiation of leukemic cell killing. It is postulated that this occurs through prevention of the entry of normal cells into the cell cycle through protein deficiency, while allowing malignant cells, permissive for protein starvation, to continue to cycle, thus maintaining sensitivity for cycle specific anticancer agents. Reported in this paper is the confirmation of these L-histidinol-FUra effects. However, a modification was made by which more L-histidinol could be given and more consistent protection of whole animals demonstrated. Further, an optimal schedule of L-histidinol was defined in which FUra preceded L-histidinol infusion. Finally, the specificity and proliferation dependence of this schedule was evaluated on colony forming units-spleen in resting and proliferating state, colony forming units-granulocyte-macrophage, and L1210 leukemia. This demonstrates that the FUra/L-histidinol combination indeed protects only normal cells but that the postulated proliferation dependence is absent, indicating an alternate biological mechanism.

Two types of tumour sensitivity test compared for platinum derivatives.

For early passages of 15 human tumours grown in nude mice two types of test for prediction of sensitivity to cytostatic drugs were carried out for four platinum compounds. The subrenal capsule assay of Bogden was not found to be useful for these early passages, since the response of duplicate tests was not very different from randomly distributed results. The clonogenic assay as described by Hamburger and Salmon gave reproducible results on the drug sensitivity of those tumour cells that grew colonies in vitro. However, in a limited number of cases irregular colony growth occurred. Lower drug concentrations were apparently more effective in killing cells than higher concentrations. From the replicate test results it became clear that such results are not a reliable indicator of drug sensitivity. Furthermore, the critical drug concentrations for optimal testing of drug effectiveness is probably not well represented by a uniform relation to peak plasma level.

Tumor dependent growth kinetics of human tumor xenografts using the subrenal capsule assay.

Using the subrenal capsule assay modified by us in order to decrease the ingrowth of host cells, evaluation of the growth kinetics of three human ovarian tumors and one human lung tumor were made by multiple measurements of the tumor implantation site over the 6-day growth period. For all tumors, a lag period of 2-3 days was noticed before growth occurred in the subrenal location. In general, estimation of the composition of the fragments growing under the renal capsule did not change greatly in terms of percentage tumor of which they consisted but by day 6 most had shown a significant degree of host cell infiltration despite the effects of pre-implantation immunosuppression. We would suggest that only certain human tumors are suitable for implantation using this technique. Further, it appears that within even this group of selected tumors only some are suitable for drug studies, those having established exponential growth early enough so that a measurable endpoint can be reached within the 6-day time limit of the assay.

Improvements and limitations of the subrenal capsule assay for determining tumour sensitivity to cytostatic drugs.

Cyclophosphamide (CY) prevented the host response from occurring in treated animals, and we therefore evaluated CY and other immunosuppressive forms of pretreatment in normal mice using the subrenal capsule assay initially for transplanted and later also for primary tumours. CY pretreatment, 4 or 4.5 Gy whole-body irradiation and cortisone were superior to silica in reducing host cell infiltration, and irradiation as pretreatment has become our routine technique. The addition of cortisone acetate to irradiation was of minor benefit in only 1/3 transplanted lines. When primary tumours were tested, the irradiation was only rarely able to completely prevent cellular infiltration. Only 7/11 ovarian tumours and 3/9 lung tumours were evaluable as tumour specimens (greater than 50% tumour) in preirradiated mice. The degree of infiltration and fibrosis was similar in transplants in irradiated normal mice or in athymic nude mice, suggesting that these phenomena are largely due to properties of the tumours rather than of the host. The limitations of the technique to some cell lines and occasional primary tumours is obvious.

A phase I clinical tolerance study of rDNA alpha 2 human interferon in patients with non-reticuloendothelial system malignancies.

Twenty-seven patients with non-reticuloendothelial malignancies were treated with a single intramuscular injection of recombinant leukocyte alpha 2 interferon (rIFN) to assess clinical tolerance and define a maximum tolerated dose. A single patient in each of six increasing dosage groups (0.3 X 10(6) IU, 1 X 10(6) IU, 3 X 10(6) IU, 10 X 10(6) IU, 30 X 10(6) IU, 100 X 10(6) IU) received a low dose (0.01 X 10(6) IU) and served as a control for subjective and objective toxicity measurements. Severe fatigue proved dose-limiting at 100 X 10(6) IU, and all dosages above 3.0 X 10(6) IU produced one or more signs or symptoms, which typically resembled a 'flu-like' syndrome. Objective toxicity was mild to moderate (leukopenia, thrombopenia) and no toxicities were found not already known from work with interferon obtained directly from leukocytes. Evidence of an antitumor effect was apparent in 3/19 evaluable patients.

Comparison between macroscopic and microscopic evaluation of tumour responsiveness using the subrenal capsule assay.

Using the subrenal capsule assay in normal mice, a histologic evaluation was made of 8 human primary ovarian tumours and 3 human colon, 2 lung and 5 ovarian carcinomas growing in serial passage in nude mice. The results of the evaluation indicated that there is a tumour- and drug-dependent correlation between the macroscopically and microscopically evaluated effects, with cyclophosphamide demonstrating excellent concordance but adriamycin and cisplatin both demonstrating consistently more tumour cell killing on histologic analysis than could be appreciated macroscopically. Leukocyte infiltration and fibrosis were greatly increased by the latter 2 drugs, leading to unrepresentative macroscopic measurements. Variable amounts of host cell infiltration can also be demonstrated in the untreated control when normal mice are used. The use of nude mice decreases the discrepancy between macroscopic and microscopic evaluation.

Model studies in adjuvant chemotherapy.

We have focused on three aspects of adjuvant chemotherapy applied to mice with one of the metastasizing tumors: Lewis lung carcinoma (LL) or mammary carcinoma 2661 (M2661). The first aspect concerned the timing of adjuvant chemotherapy. To investigate this, tumor-bearing mice were treated postoperatively with cyclophosphamide using a standard regimen. In M2661, adjuvant therapy was marginally effective in contrast to the clearly significant results obtained in LL. Any delay in the initiation of adjuvant therapy decreased the efficacy of the treatment. The effect of administering chemotherapy before surgery was also studied; normally, marginally effective adjuvant therapy was found to become effective when started preoperatively in M2661. In LL, effective adjuvant therapy was found to become less effective when started preoperatively. The second aspect considered was the comparability between the increase in relapse-free survival time and the increase in cure rate as alternate goals of adjuvant therapy. To study this, mice with small, medium, or large residual tumor loads were subjected to surgery and subsequently treated with cyclophosphamide. While the effect of adjuvant therapy on the cure rate increased proportionally with decreasing tumor load, the increase in lifespan in nonsurvivors was not related to tumor load. The final aspect of study was the selection procedure for drugs to be applied in adjuvant treatment in our models. Taking the volume response of large tumors as being predictive for the successful use of the same agent in adjuvant therapy, we obtained both false-negative and false-positive results in our tumor lines.

Cell kinetic factors, single drugs and combination.

This communication focuses on the problems that exist in the application of cell kinetic information in cancer chemotherapy. From selected historical work, the following conclusions can be drawn. 1) Theoretically optimal drug sequences may fail to show the expected effect. 2) Similar drug sequences cause different effects in different tumors, even when the tumors have similar proliferation characteristics. The unpredictability and the heterogeneity in response make the extrapolation to clinical cancer chemotherapy very difficult. Better prediction may be made from the integration of results from studies combining cell kinetic factors, cellular pharmacokinetics, and biochemistry of drug exposure.