Paul A. Andrews

Regulatory considerations for preclinical development of anticancer drugs.

Abstract The entry of new anticancer treatments into phase I clinical trials is ordinarily based on relatively modest preclinical data. This report defines the battery of preclinical tests important for assessing safety under an Investigational New Drug application (IND) and outlines a basis for extrapolating starting doses of investigational anticancer drugs in phase I clinical trials from animal toxicity studies. Types of preclinical studies for the support of marketing of a new anticancer drug are also discussed. This report addresses differences and similarities in the preclinical development of cytotoxic drugs (including photosensitizers and targeted delivery products), drugs used chronically (chemopreventive drugs, hormonal drugs, immunomodulators), and drugs intended to enhance the efficacy (MDR-reversing agents and radiation/chemotherapy sensitizers) or diminish the toxicity of currently used anticancer therapies. Factors to consider in the design of preclinical studies of combination therapies, alternative therapies, and adjuvant therapies in the treatment of cancer, and to support changes in clinical formulations or route of administration, are also discussed.

Glutathione content but not gamma glutamyl cysteine synthetase mRNA expression predicts cisplatin resistance in head and neck cancer cell lines

Abstract Purpose: To correlate cellular glutathione content and γ-glutamyl cysteine synthetase (γGCS) mRNA expression with cisplatin sensitivity in a panel of seven head and neck squamous cancer cell lines. Methods: Cisplatin IC50 was determined for each cell line using a sodium tetreazolium (XTT) assay. Cellular glutathione content was measured by using a previously reported enzymic method. γGCS mRNA expression was measured using an RNase protection assay. Results: Total cellular glutathione was an excellent predictor of cisplatin sensitivity in this series of cell lines. The IC50 for cisplatin in the cell line with the highest glutathione concentration was approximately 90 times higher than in the cell line with the lowest glutathione concentration. Regression analysis showed a highly statistically significant positive correlation between cisplatin IC50 and cellular glutathione (coefficient of determination R2=0.81, P=0.0012). Somewhat surprisingly, in contrast to previous studies in ovarian cancer, γGCS mRNA expression in these cell lines was not significantly predictive of either total cellular glutathione or cisplatin sensitivity (R2=0.005, P=0.84). As expected, treatment of resistant cell lines with buthionine sulfoximine resulted in decreased cellular glutathione and enhanced cisplatin sensitivity. Conclusions: Our results suggest that glutathione may be an important determinant of cisplatin sensitivity in clinical head and neck cancer. Since cisplatin is the most active chemotherapy drug for the treatment of this disease, this correlation may have important clinical relevance. The lack of correlation between glutathione level and γGCS expression suggests that salvage or alternate synthetic pathways may be critical in these cells.

Characterization of binding proteins from ovarian carcinoma and kidney tubule cells that are specific for cisplatin modified DNA.

We have detected proteins in nuclear extracts from ovarian carcinoma cells and kidney tubule cells that bind specifically to platinated DNA. A 123-bp restriction fragment was platinated with cisplatin (DDP) to a formal molar platinum to nucleotide ratio of 0.05 and end-labeled with [32P]-dCTP. Incubation with nuclear extracts from 2008 human ovarian carcinoma cells caused shifts in the mobility of this probe in non-denaturing polyacrylamide gels. Proteinase K, but not ribonuclease A, destroyed the bands. Comparison of the shifted bands generated by DDP-resistant 2008 and A2780 human ovarian carcinoma cell nuclear extracts with bands from the corresponding sensitive cells showed no differences in protein levels. The affinity of the proteins for the probe was the same in sensitive and resistant 2008 nuclear extracts as determined by competition with platinated salmon sperm DNA. These proteins also bound to a probe damaged with 1,2-diaminocyclohexaneplatinum(II) dichloride but did not bind to a trans-DDP platinated probe. No differences were found in the levels of UV4 or UV5 Chinese hamster ovary cells, which were hypersensitive to DDP compared to wild-type AA8 cells. MDCK and LLC-PK1 kidney tubule cells, which were more resistant to DDP cytotoxicity than 2008 cells, exhibited decreased levels of these proteins. We conclude that, although these proteins that recognize DDP damage in DNA may be involved in excision repair, their levels did not correlate with DDP sensitivity in this panel of cell lines.

The effect of DNA polymerase inhibitors on the cytotoxicity of cisplatin in human ovarian carcinoma cells.

We examined the effect of specific inhibitors of DNA polymerases alpha and delta, and beta, on cisplatin (DDP) cytotoxicity in DDP-sensitive and -resistant human 2008 ovarian carcinoma cells. Under conditions of continuous exposure to drug combinations, neither aphidicolin glycinate (AG) nor dideoxythymidine enhanced the cytotoxicity of DDP in either cell line as determined by clonogenic survival assays. However, when clonogenic survival was determined following short-term drug exposure, AG exhibited strong synergism with DDP in the DDP-resistant, but not the DDP-sensitive cells, as indicated by median effect analysis of the data. DNA polymerase alpha mRNA levels were the same in both cell lines under basal conditions. DDP-sensitive cells, but not DDP-resistant cells, were able to increase their expression of DNA polymerase alpha in response to DDP exposure. Levels of mRNA for DNA polymerase beta and for the human DNA repair gene ERCC-1 were not elevated in resistant cells, either under basal conditions or 18 hr after a 1 hr exposure to IC20 concentrations of DDP. In another human ovarian carcinoma cell line, A2780, AG and DDP were synergistic in both DDP-sensitive and -resistant variants in short-term exposure. We conclude that DNA polymerases alpha and/or delta play a role in the DDP sensitivity of human ovarian carcinoma cells.

Drug accumulation and DNA platination in cells exposed to aquated cisplatin species

Since CP always exists in aqueous solution as a mixture of native drug and various aqua and hydroxo species, it is conceivable that one or more of these aquated species is the main form of the drug that enters the cell. To test this hypothesis, we examined the accumulation by 2008 human ovarian carcinoma cells of CP and aquated CP in Cl-, deficient medium. After 24 h in 150 mM NaNO3, HPLC analysis indicated that 54% of the platinum in solution was accounted for by aquated species. Immediately following addition of this solution to Cl- deficient RPMI 1640 medium, the initial concentrations of the aqua and hydroxo species were calculated to be 34-2400-fold higher with pre-aquated CP than with native CP. The cellular platinum accumulation, however, was the same under both conditions and was also identical to that of native CP in normal RPMI. To confirm that aquated species were actually entering the cell, the amount of platinum reaching the DNA was determined. The total platinum levels on DNA were 1.9-fold higher when cells were exposed to pre-aquated CP in Cl- deficient medium compared to CP in regular medium. We conclude that 2008 cells do not preferentially transport an aquated form of CP under these conditions compared to native CP.

Role of Membrane Ion Transport in Cisplatin Accumulation

Cisplatin (DDP) accumulation into cells cannot be explained by passive diffusion alone. Accumulation can be modulated by energy poisons, ion concentrations, cAMP levels, osmotic strength, protein kinase C agonists, ras expression, and calmodulin antagonists1–9. Although this evidence argues for the existence of a DDP transporter, the findings that DDP accumulation cannot be saturated nor competitively inhibited with structural analogues imply that a direct carrier is not involved in DDP transport4,10–12. The observation that decreased DDP accumulation is a frequent change in cells selected for DDP-resistance in vitro suggests that the DDP accumulation mechanism might be an important determinant of the cellular response to DDP1. The biochemical basis for this decrease is not known. Our studies have been focused on the elucidation of the process by which DDP enters cells and how this has changed in resistant cells. We have found that DDP-resistant human ovarian carcinoma cells with accumulation defects have alterations in their ion transport properties. We present evidence that suggests that these changes may be linked to the DDP accumulation defect in these cells.

The effects of terbium on the accumulation of cisplatin in human ovarian cancer cells

In this investigation, we report a relationship between the terbium (Tb3+) binding protein and the accumulation of cisplatin in human ovarian cancer cells. The number of Tb3+ binding sites in cisplatin-resistant C13+ cells is significantly greater by 79% than those in cisplatin-sensitive 2008 cells. Exposure to Tb3+ also increased the cellular accumulation of cisplatin. The accumulation of cisplatin as a function of the Tb3+ concentration in the C13+ cells (0.85%/microM Tb3+) was significantly greater than the accumulation of cisplatin in 2008 cells with respect to Tb3+ (0.46%/microM Tb3+). The number of Tb3+ binding sites in revertant RH4 cells was similar to that in 2008 cells. The RH4 cells were less sensitive to the stimulatory effects of Tb3+ than the C13+ cells. Our results show that the Tb3+ binding protein correlates with cisplatin resistance, and the receptor binding of Tb3+ increases the accumulation of cisplatin in cisplatin-resistant cells.

Increase in the ATP signal after treatment with cisplatin in two different cell lines studied by 31P NMR spectroscopy.

We have compared the 31P nuclear magnetic resonance (NMR) spectra of two different cisplatin resistant cell lines, one derived from human ovarian carcinoma and the other from rat lymphoma, and their respective cisplatin sensitive parental cell lines. Comparisons were made between the baseline spectra and after perfusion of the cells with 20-50 microM cisplatin for 16-20 hours. While no obvious differences were found between baseline spectra of sensitive and resistant cells, during cisplatin perfusion the sensitive cells had an increase in their ATP signals. The resistant cells also exhibited increases in their ATP signals during cisplatin perfusion but to a lesser extent than the sensitive cells. Although the significance of these ATP elevations towards the cellular pharmacology of cisplatin are not presently known, our studies demonstrate that 31P NMR spectroscopy may be useful for elucidating differences in phosphate metabolism in cells expressing the cisplatin resistant phenotype.

The effects of terbium on the cellular accumulation of cisplatin in MDA-MB-231 human breast tumor cells

Abstract Purpose: Cisplatin (DDP) is an effective antitumor agent limited in its efficacy by the development of tumor cell resistance. The defective accumulation of DDP has been shown to be a prominent feature in many DDP-resistant cell lines. In an effort to circumvent this problem, we examined the cellular accumulation of DDP in the presence of terbium (Tb3+). We also examined the effects of verapamil on the cellular accumulation of DDP in order to delineate the specific interaction of Tb3+ and DDP. All experiments were performed on DDP-sensitive or DDP-resistant MDA-MB-231 human breast tumor cells. Methods: The cellular accumulation of DDP and verapamil were determined by electrothermal atomic absorption spectrophotometry. Time-resolved luminescence spectroscopy was used to obtain equilibrium binding constants for the Tb3+/MDA cell complexes. Results: We found that 100 μM Tb3+ increased DDP accumulation in the parent MDA cell line, 5.7-fold resistant MDA/A13 and 10-fold resistant MDA/CH cells by 56.2±7.4, 71.9±9.4 and 50.8±9.4%, respectively (P<0.0001 for all MDA cell types). In contrast, 20 μM verapamil had no significant effect on DDP accumulation in the MDA cell lines. In addition, a positive correlation between the membrane binding of Tb3+ and the cellular accumulation of DDP was found to exist in the parent cell line and sublines (r=0.9). Conclusions: In agreement with earlier studies, the plasma membrane of MDA cell lines contain a specific Tb3+-binding protein. Our findings suggest that the Tb3+-binding protein may be intimately associated with the accumulation of DDP in breast tumor cells.

Toxicology and Regulatory Aspects of Platinum Drugs

The goals of preclinical toxicology studies for oncology drugs are (1) to identify a starting dose that is both safe and that minimizes the number of patients treated with ineffective doses, (2) to identify important potential clinical toxicities, and (3) to assist in the design of human dosing regimens and escalation schemes (1). Toward these goals, studies are performed in animals to estimate the human maximally tolerated dose (MTD) and characterize drug-induced toxicities. Studies in both rodents and non-rodents to support Phase I trials have been expected by the U.S. Food and Drug Adminstration (FDA) since 1982 (2,3). In contrast, since 1981 clinical trials conducted under the auspices of the Cancer Research Campaign and the European Organization for the Research and Treatment of Cancer have relied on preclinical testing in rodents only for oncology drugs (4). The European Agency for the Evaluation of Medicinal Products has recently formalized this rodent-only approach as only acceptable for entry of cytotoxic oncology drugs with known mechansims of action into Phase I trials. For drugs with novel mechanisms of action, studies in rodents and non-rodents are currently expected (5).