James H. Goldie

Drug resistance in cancer: a perspective.

Drug resistance remains the thorniest obstacle in developing improved systemic therapies for disseminated cancer. The combination of genetic instability together with the great molecular heterogeneity that are displayed by malignant cells makes constructing effective, rational treatment programs difficult in the extreme. However, new insights into the action of antitumor agents at the molecular level plus greater understanding of the relationship of drug resistant states to the fundamental abnormalities that generate malignancy point the way to producing therapies that are more specific and therapeutically effective. However, a non-trival problem is the drug development system itself which is currently poorly set up to yield patient specific drug programs in a timely fashion.

Neoadjuvant (preoperative) chemotherapy for breast cancer

Despite recent developments in the treatment of breast cancer, metastatic breast cancer remains an incurable disease. Postoperative adjuvant treatment may improve the survival of a subgroup of node positive, Stage II breast cancer patients, but the proportion of failures is still high. Preoperative adjuvant chemotherapy, an example of a new approach in scheduling of available agents, is based on sound theoretical and experimental principles. In this report, the authors summarize the background data on the rationale for preoperative adjuvant chemotherapy and present preliminary results of this study in which preoperative treatment starting with one course of cyclophosphamide, methotrexate, and 5‐fluorouracil (CMF) was given to newly diagnosed patients with breast cancer. Analysis of the first 43 patients given such treatment has shown that the side effects were comparable to those seen in patients treated with conventional postoperative chemotherapy, and that the delay time between diagnosis and starting chemotherapy has been substantially reduced compared to the historic group of patients. Additional aspects of this study include the introduction of fine‐needle aspiration as the only diagnostic method for obtaining the tissue diagnosis of breast cancer, as well as a more intensive interaction between the surgeons from private practice and the oncology institute.

Gas-chromatographic analysis of busulfan for therapeutic drug monitoring

The development and validation of a gas chromatographic assay method for determination of total and free busulfan concentrations in human plasma for pharmacokinetic studies is reported. 1,6-Bis(methanesulfonyloxy)hexane, the internal standard, and a potential metabolite, 3-hydroxysulfolane, were synthesized. Plasma and plasma ultrafiltrate samples containing busulfan and internal standard were extracted with ethyl acetate and derivatized with 2,3,5,6-tetrafluorothiophenol prior to gas chromatographic determination. The63Ni electron-capture detector provided a limit of detection of 0.0600 μg/ml with a limit of quantitation of 0.100 μg/ml busulfan in biological samples. Calibration curves were linear from 0.100 to 3.00 μg/ml in plasma (500 μl) and 0.100 to 2.00 μg/ml in plasma ultrafiltrate (100 μl). Extraction and derivatization yields ranged from 78.4% to 89.6% and 56.0% to 71.3%, respectively. Specificity of this assay for busulfan in the presence of its potential metabolites was demonstrated. Also, plasma samples containing co-administered drugs gave no response under these conditions. Clinical samples obtained following administration of a 1 mg/kg oral busulfan dose demonstrate the applicability of this method to analysis of total and free plasma concentrations.

The effect of cellular differentiation on the development of permanent drug resistance

Abstract A stem cell compartment model is utilized to simulate the growth of human tumors. This model is used to explore the effect of cell differentiation and loss on the development of spontaneous drug resistance. Cellular differentiation is found to increase the rate of development of single drug resistance, although this is balanced by the likehood that such resistant cells will subsequently become extinct. Overall the probability that singly resistant cells will develop and persist is found to be independent of the rate of cellular differentiation. Conversely, when two drugs are available, the probability that cells resistant to both drugs will persist is proportional to the rate of cellular differentiation. Approximate formulae relating the net overall mutation rate to the intrinsic mutation rates and net growth rates of the stem cell compartment are developed.

Heterogeneity in the specific activity and methotrexate sensitivity of dihydrofolate reductase from blast cells of acute myelogenous leukemia patients.

Dihydrofolate reductase activity was found to be highly heterogeneous in terms of its specific activity and methotrexate sensitivity in the blast cells of patients with acute myelogenous leukemia. None of the patients had previously been treated with methotrexate (MTX). The blast cells of four of 12 patients studied contained methotrexate-insensitive forms of dihydrofolate reductase, and the blast cells of three (distinct from the four mentioned previously) of the 12 had significantly higher dihydrofolate reductase activities than the rest. The presence of MTX-insensitive dihydrofolate reductases and high levels of enzyme activity represent intrinsic mechanisms of resistance and may explain the apparent clinical resistance of acute myelogenous leukemia to methotrexate.

Methotrexate-resistant human promyelocytic leukemia (HL-60) cells express a dihydrofolate reductase with altered properties associated with increased enzyme activity

Dihydrofolate reductase from a MTX-resistant human promyelocytic leukemia cell line (HL-60 R4-29) had previously been found to have a higher specific activity than the DHFR from the parent MTX-sensitive cell line, in the absence of enzyme overproduction (Dedhar et al, Biochem. J. 225, 609-617, 1985). The enzymes from these two cell lines have been purified to apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The dihydrofolate reductase from the sensitive cells has an apparent molecular weight of 42,000 daltons, whereas that from the resistant cells has an apparent molecular weight of 21,000 daltons. The dihydrofolate reductase activity from the resistant cells is characterized by marked heat instability and substantially higher Vmax and Km values for the dihydrofolic acid/NADPH combination. The enzyme from the resistant cells can be protected against heat inactivation by either dihydrofolic acid or NADPH, or both. A 50 fold higher concentration of methotrexate was required to totally inhibit the R4-29 dihydrofolate reductase activity as compared to the (S) activity when the enzymes were assayed using equivalent amounts of enzyme protein. These data show that the increased dihydrofolate reductase specific activity present in the (R4-29) cells is associated with an alteration in the structure of this enzyme.

Models for dose intensity

Abstract Hryniuk has recently advanced the proposal that the dose intensity of a therapeutic regimen, defined as the ratio of cumulative drug dose to therapeutic period, is an important predictor of the therapeutic outcome. We discuss two simple models of chemotherapeutic effect in cancer treatment and show how they lead to consideration of a time-dependent function, the dose-intensity function, which is a generalization of Hryniuks dose intensity. Both models show that the therapeutic outcome is dependent upon the dose-intensity function; however, the nature of the dependence varies with the model selected.

Inhibition of a methotrexate-insensitive dihydrofolate reductase from L5178Y cells by substituted triazines and quinazolines

Inhibition by a variety of substituted triazines and quinazolines of a methotrexate-insensitive form of dihydrofolate reductase from highly MTX-resistant L5178Y mouse leukemia cells was examined. Some of these compounds were significantly more potent than MTX (up to 100-fold). Two triazenes, terminally substituted with benzenesulfonylfluoride residues, were approximately 30-fold more potent than MTX. Quinazoline analogs of folic acid with alterations in different parts of the molecule varied in their potencies as inhibitors. Although none of the compounds tested was as potent as MTX against MTX-sensitive dihydrofolate reductases, these studies show that some types of folate antagonists have increased specificity against this MTX-insensitive dihydrofolate reductase. This finding increases the likelihood that it may be possible to produce compounds with marked specificity for the insensitive reductase. Such compounds might have utility in antifolate combinations designed to overcome methotrexate resistance.

Preoperative (neoadjuvant) chemotherapy

Clinical and Scientific Considerations in Preoperative (Neoadjuvant) Chemotherapy.- Experimental Adjuvant Chemotherapy: An Overview.- Theoretical Considerations Regarding the Early Use of Adjuvant Chemotherapy.- Experimental Preoperative Chemotherapy.- Implications of Certain Cell Kinetic and Biological Parameters for Preoperative Chemotherapy.- Adjuvant Therapy for Breast Cancer: A Brief Overview of the NSABP Experience and Some Thoughts on Neoadjuvant Chemotherapy.- Factors Affecting the Development of Permanent Drug Resistance and Its Impact upon Neoadjuvant Chemotherapy.- Impact of Preoperative Chemotherapy for the Surgeon.- Preoperative (Neoadjuvant) Chemotherapy for Breast Cancer: Outline of the British Columbia Trial.- Perioperative Adjuvant Chemotherapy of Breast Cancer: The Scandinavian Experience.- Perioperative and Conventionally Timed Chemotherapy in Operable Breast Cancer: The Ludwig Breast Cancer Study V.- Neoadjuvant Chemotherapy in the Conservative Management of Breast Cancer: Study of 143 Patients.- Preliminary Results of Preoperative Chemotherapy with a Combination of Platinum-Bleomycin Administered in 5-Day Cycles in Carcinoma of the Bronchus.- Impact of Primary Site of Stage III and IV Squamous Cell Carcinomas of the Head and Neck on 7-Year Survival Figures Following Initial Non-Cisplatin-Containing Combination Chemotherapy.- Chemotherapy with or Without Anticoagulation as Initial Management of Patients with Operable Colorectal Cancer: A Prospective Study with at Least 5Years Follow-up.- Preoperative Chemotherapy for Gastric Cancer: A Prospective Study with at Least 1 Year Follow-up.- Neoadjuvant Chemotherapy for Osteogenic Sarcoma: A Model for the Treatment of Other Highly Malignant Neoplasms.- Summary of Preoperative (Neoadjuvant) Chemotherapy.

Further studies on substituted quinazolines and triazines as inhibitors of a methotrexate-insensitive murine dihydrofolate reductase

Data are presented on the systematic analysis of thirty-five quinazoline and substituted triazine compounds as inhibitors of a methotrexate-insensitive form of dihydrofolate reductase purified from methotrexate-resistant L5178Y murine leukemia cells. Several of the compounds were found to be more potent inhibitors of this enzyme activity than was methotrexate. Two of the triazine compounds had IC50 values approaching 10nM, which is close to that of methotrexate for the normal drug-sensitive dihydrofolate reductase. In addition, some of these compounds, especially the triazines, exhibit a specificity of inhibition for the methotrexate-insensitive enzyme as compared to the normal methotrexate-sensitive dihydrofolate reductase derived from the same cell line. These compounds may, therefore, be potentially useful in the treatment of those methotrexate-resistant tumours which express an altered, methotrexate-insensitive dihydrofolate reductase.