Stephen G. Chaney

Oxaliplatin: A review of preclinical and clinical studies

Of the new generation platinum compounds that have been evaluated, those with the 1,2-diaminocyclohexane carrier ligand-including oxaliplatin--have been focused upon in recent years. Molecular biology studies and the National Cancer Institute in vitro cytotoxic screening showed that diaminocyclohexane platinums such as oxaliplatin belong to a distinct cytotoxic family, differing from cisplatin and carboplatin, with specific intracellular target(s), mechanism(s) of action and/or mechanism(s) of resistance. In phase I trials, the dose-limiting toxicity of oxaliplatin was characterized by transient acute dysesthesias and cumulative distal neurotoxicity, which was reversible within a few months after treatment discontinuation. Moreover, oxaliplatin did not display any, auditory, renal and hematologic dose-limiting toxicity at the recommended dose of 130 mg/m2 q three weeks or 85 mg/m2 q two weeks given as a two-hour i.v. infusion. Clinical phase II experiences on the antitumoral activity of oxaliplatin have been conducted in hundreds of patients with advanced colorectal cancers (ACRC). Single agent activity reported as objective response rate in ACRC patients is 10% and 20% overall in ACRC patients with 5-fluorouracil (5-FU) pretreated/refractory and previously untreated ACRC, respectively. Synergistic cytotoxic effects in preclinical studies with thymidylate synthase inhibitors, cisplatin/carboplatin and topoisomerase I inhibitors, and the absence of hematologic dose-limiting toxicity have made oxaliplatin an attractive compound for combinations. Phase II trials combining oxaliplatin with 5-FU and folinic acid ACRC patients previously treated/refractory to 5-FU showed overall response rates ranging from 21% to 58%, and survivals ranging from 12 to 17 months. In patients with previously untreated ACRC, combinations of oxaliplatin with 5-FU and folinic acid showed response rates ranging from 34% to 67% and median survivals ranging from 15 to 19 months. Two randomized trials totaling 620 previously untreated patients with ACRC, comparing 5-FU and folinic acid to the same regimen with oxaliplatin, have shown a 34% overall response rate in the oxaliplatin group versus 12% in the 5-FU/folinic acid group for the first trial; and 51.2% vs. 22.6% in the second one. These statistically significant differences were confirmed in time to progression advantage for the oxaliplatin arm (8.7 vs. 6.1 months, and 8.7 vs. 6.1 months, respectively). A small but consistent number of histological complete responses have been reported in patients with advanced colorectal cancer treated with the combination of oxaliplatin with 5-FU/folinic acid, and secondary metastasectomy is increasingly done by oncologists familiar with the combination. Based on preclinical and clinical reports showing additive or synergistic effects between oxaliplatin and several anticancer drugs including cisplatin, irinotecan, topotecan, and paclitaxel, clinical trials of combinations with other compounds have been performed or are still ongoing in tumor types in which oxaliplatin alone showed antitumoral activity such as ovarian, non-small-cell lung, breast cancer and non-Hodgkin lymphoma. Its single agent and combination therapy data in ovarian cancer confirm its non-cross resistance with cisplatin/carboplatin. While the role of oxaliplatin in medical oncology is yet to be fully defined, it appears to be an important new anticancer agent.

Two‐polymerase mechanisms dictate error‐free and error‐prone translesion DNA synthesis in mammals

DNA replication across blocking lesions occurs by translesion DNA synthesis (TLS), involving a multitude of mutagenic DNA polymerases that operate to protect the mammalian genome. Using a quantitative TLS assay, we identified three main classes of TLS in human cells: two rapid and error‐free, and the third slow and error‐prone. A single gene, REV3L, encoding the catalytic subunit of DNA polymerase ζ (polζ), was found to have a pivotal role in TLS, being involved in TLS across all lesions examined, except for a TT cyclobutane dimer. Genetic epistasis siRNA analysis indicated that discrete two‐polymerase combinations with polζ dictate error‐prone or error‐free TLS across the same lesion. These results highlight the central role of polζ in both error‐prone and error‐free TLS in mammalian cells, and show that bypass of a single lesion may involve at least three different DNA polymerases, operating in different two‐polymerase combinations.

The Role of DNA Polymerase η in Translesion Synthesis Past Platinum–DNA Adducts in Human Fibroblasts

Cisplatin, a widely used chemotherapeutic agent, has been implicated in the induction of secondary tumors in cancer patients. This drug is presumed to be mutagenic because of error-prone translesion synthesis of cisplatin adducts in DNA. Oxaliplatin is effective in cisplatin-resistant tumors, but its mutagenicity in humans has not been reported. The polymerases involved in bypass of cisplatin and oxaliplatin adducts in vivo are not known. DNA polymerase η is the most efficient polymerase for bypassing platinum adducts in vitro. We evaluated the role of polymerase η in translesion synthesis past platinum adducts by determining cytotoxicity and induced mutation frequencies at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in diploid human fibroblasts. Normal human fibroblasts (NHF1) were compared with xeroderma pigmentosum variant (XPV) cells (polymerase η-null) after treatment with cisplatin. In addition, XPV cells complemented for polymerase η expression were compared with the isogenic cells carrying the empty expression vector. Cytotoxicity and induced mutagenicity experiments were measured in parallel in UVC-irradiated fibroblasts. We found that equitoxic doses of cisplatin induced mutations in fibroblasts lacking polymerase η at frequencies 2- to 2.5-fold higher than in fibroblasts with either normal or high levels of polymerase η. These results indicate that polymerase η is involved in error-free translesion synthesis past some cisplatin adducts. We also found that per lethal event, cisplatin was less mutagenic than UVC. Treatment with a wide range of cytotoxic doses of oxaliplatin did not induce mutations above background levels in cells either expressing or lacking polymerase η, suggesting that oxaliplatin is nonmutagenic in human fibroblasts.

DNA damage inducible-gene expression following platinum treatment in human ovarian carcinoma cell lines.

Abstract Purpose: DNA damage-inducible genes, such as gadd153, gadd45, p21 and c-jun, have previously been shown to be induced by the chemotherapeutic agent cisplatin. One of these genes, gadd153, has previously been reported to be differentially expressed in cisplatin-resistant cell lines and, therefore, to be a potential prognostic indicator for tumor response to cisplatin-based chemotherapy. It is not currently known whether such damage-inducible genes are turned on by the DNA damage itself (e.g. by the formation of Pt-DNA adducts) or by the downstream biological consequences of that damage. It is also not known whether the increased expression of these DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. These experiments were initiated to characterize more fully the nature of the DNA damage-inducible response to cisplatin treatment and to determine whether any of these genes might be useful prognostic indicators of tumor response to cisplatin chemotherapy. Methods: The dose-response and time-course for the induction of the DNA damage-inducible genes gadd153, gadd45, p21 and c-jun were examined by Northern analysis in the human ovarian carcinoma cell line 2008 and its resistant subclone C13* following treatment with platinum anticancer agents. The extent of gene expression was correlated with cytotoxicity determined by growth inhibition assay, Pt-DNA adducts determined by atomic absorption spectrometry and inhibition of DNA synthesis determined by 3H-thymidine incorporation. Results: All four genes were induced maximally in both sensitive and resistant cell lines at lethal cisplatin doses (≥ ID90). Induction was maximal between 24 and 48 h following exposure to the drug for all genes except c-jun which was induced by 6 h. At 24 h following cisplatin treatment the overall levels of gadd153 were less in the resistant C13* cell line than in the parental 2008 cell line, while those of gadd45 were greater in C13* than in 2008. Maximal expression of p21 and c-jun was not significantly different in the two cell lines. The dose-response of these genes correlated with the cytotoxicity of cisplatin and the inhibition of DNA synthesis by cisplatin, rather than to the actual levels of Pt-DNA adducts. The more cytotoxic platinum analog, ormaplatin, also induced gadd153 and its induction was also based on cytotoxicity. Conclusion: These results suggest that the regulation of gadd153 and gadd45 expression occurs thorough separate pathways in the 2008 and C13* cell lines. The DNA damage-inducible gene response for all four damage-inducible genes tested appeared to be more directly correlated with downstream biologic effects of cisplatin damage than with actual Pt-DNA adduct levels. The time-course and dose-response for induction of these genes was more consistent with delayed responses such as apoptosis rather than more immediate responses such as DNA repair. Finally, these results strengthen previous suggestions that the expression of gadd153, and possibly other DNA damage-inducible genes, may be useful indicators of tumor response to cisplatin-based chemotherapy.

Specificity of platinum-DNA adduct repair.

Cell lines with resistance to cisplatin and carboplatin often retain sensitivity to platinum complexes with different carrier ligands (e.g., oxaliplatin and JM216). HeLa cell extracts were shown to excise cisplatin, oxaliplatin, and JM216 adducts with equal efficiency, suggesting that nucleotide excision repair does not contribute to the carrier-ligand specificity of platinum resistance. We have shown previously that the extent of replicative bypass in vivo is influenced by the carrier ligand of the platinum adducts. The specificity of replicative bypass may be determined by the DNA polymerase complexes that catalyze translesion synthesis past Pt-DNA adducts, by the mismatch-repair system that removes newly synthesized DNA opposite Pt-DNA adducts, and/or by DNA damage-recognition proteins that bind to the Pt-DNA adducts and block translesion synthesis. Primer extension on DNA templates containing site-specifically placed cisplatin, oxaliplatin, or JM216 Pt-GG adducts revealed that the eukaryotic DNA polymerases beta, zeta, gamma and HIV-1 RT had a similar specificity for translesion synthesis past Pt-DNA adducts (oxaliplatin > or = cisplatin > JM216). In addition, defects in the mismatch-repair proteins hMSH6 and hMLH1 led to increased replicative bypass of cisplatin adducts, but not of oxaliplatin adducts. Finally, primer extension assays performed in the presence of HMG1, which is known to recognize cisplatin-damaged DNA, revealed that inhibition of translesion synthesis by HMG1 also depended on the carrier ligand of the Pt-DNA adduct (cisplatin > oxaliplatin = JM216). These studies show that DNA polymerases, the mismatch-repair system and damage-recognition proteins can all impart specificity to replicative bypass of Pt-DNA adducts. Replicative bypass, in turn, may influence the carrier-ligand specificity of resistance.

Comparative neurotoxicity of oxaliplatin, ormaplatin, and their biotransformation products utilizing a rat dorsal root ganglia in vitro explant culture model.

Purpose: Neurotoxicity is one of the major toxicities of platinum-based anticancer drugs, especially oxaliplatin and ormaplatin. It has been postulated that biotransformation products are likely to be responsible for the toxicity of platinum drugs. In our preceding pharmacokinetic study, both oxaliplatin and ormaplatin were observed to produce the same types of major plasma biotransformation products. However, while the plasma concentration of ormaplatin was much lower than that of oxaliplatin at an equimolar dose, one of their common biotransformation products, Pt(dach)Cl2, was present at 29-fold higher concentrations in the plasma following the i.v. injection of ormaplatin than of oxaliplatin. Because ormaplatin has severe neurotoxicity and Pt(dach)Cl2 is very cytotoxic, we have postulated that Pt(dach)Cl2 is likely to be responsible for the differences in neurotoxicity between ormaplatin and oxaliplatin. In order to test this hypothesis, we compared the neurotoxicity of oxaliplatin, ormaplatin, and their biotransformation products. Since the dorsal root ganglia (DRGs) have been suggested to be the likely targtet for platinum drugs and in vitro DRG explant cultures have been suggested to be a valid model for studying cisplatin-associated neurotoxicity, our comparative neurotoxicity study was conducted with DRG explant cultures in vitro. Methods: Based on the previous studies of cisplatin neurotoxicity, we established our in vitro DRG explant culture utilizing DRGs dissected from E-19 embryonic rats. Rat DRGs were incubated for 30 min with different platinum compounds to mimic in vivo exposure conditions; this was by followed by a 48-h incubation in culture medium at 37 °C. At the end of the incubation, the neurites were fixed and stained with toluidine blue, and neurite outgrowth was quantitated by phase-contrast microscopy. The inhibition of neurite outgrowth by platinum compounds was used as an indicator of in vitro neurotoxicity. Since an in vivo study has indicated that the order of neurotoxicity is ormaplatin > cisplatin ≥ oxaliplatin > carboplatin as measured by morphometric changes to rat DRGs, we initially validated our DRG explant culture model by comparing the in vitro neurotoxicity of ormaplatin, cisplatin, oxaliplatin, and carboplatin. After observing the same neurotoxicity rank between this study and a previous in vivo study, we further compared the neurotoxicity of oxaliplatin, ormaplatin, and their biotransformation products including Pt(dach)Cl2, Pt(dach)(H2O)Cl, Pt(dach)(H2O)2, Pt(dach)(Met), and Pt(dach)(GSH) utilizing the DRG explant culture model. Results: Our study indicated that Pt(dach)Cl2 and its hydrolysis products were more potent at inhibiting neurite outgrowth than the parent drugs oxaliplatin and ormaplatin. In contrast, no detectable inhibition of neurite outgrowth was observed for DRGs dosed with Pt(dach)(Met) and Pt(dach)(GSH). Conclusion: This study suggests that biotransformation products such as Pt(dach)Cl2 and its hydrolysis products are more neurotoxic than the parent drugs oxaliplatin and ormaplatin. The different neurotoxicity profiles of oxaliplatin and ormaplatin are more likely due to the different plasma concentrations of their common biotransformation product Pt(dach)Cl2 than to differences in their intrinsic neurotoxicity.

Oxaliplatin: A Review of Evolving Concepts

Department of Oncology, Weiler Hospital/Montefiore Medical Center and the Albert Einstein Cancer Center of the Albert Einstein College of Medicine, Room 2S-63, 1825 Eastchester Road, Bronx, NY 10461 Department of Clinical Pharmacokinetics and Drug Metabolism, Sanofi-Synthelabo, 9 Great Valley Parkway, Malvern, Pennsylvania Department of Pathology, Albert Einstein College of Medicine, Bronx, New York Cancer Therapy Evaluation Program of the National Cancer Institute, Bethesda, Maryland Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Cell cycle changes associated with formation of Pt-DNA adducts in human ovarian carcinoma cells with different cisplatin sensitivity

The possible correlation between alterations in cytokinetic response to cisplatin (CP) treatment and drug resistance in human ovarian carcinoma cell lines was examined. Using dual parameter flow cytometry, we performed detailed time-course and dose-response analysis of cell cycle modifications in the parental A2780 and resistant A2780/CP cells exposed to CP. The data suggested that drug treatment resulted in similar types of cell cycle alterations in cells with different CP sensitivity. Rapid normalization of the cytokinetic pattern in both cell lines at low doses of CP was observed. At higher drug concentrations reversible S phase delay predominated, accompanied by blocks in both G1/S and G2/M and followed by complete normalization of cytokinetic patterns in the surviving cells. CP treatment by lethal doses resulted in almost complete S phase block. The surviving cells at 72 h accumulated in G2 phase. CP-induced cell cycle perturbations, among which the most pronounced were alterations in the S phase populations, correlated with the level of DNA damage, but not with cell survival in these cell lines. However, at identical levels of DNA damage, the resistant A2780/CP cell line demonstrated decreased p53 induction and decreased apoptosis compared to the parental cell line. Thus, at equivalent levels of DNA damage, resistance in this model system correlated with a diminished p53-dependent apoptotic pathway rather than with differences in cell cycle response.

High-performance liquid chromatographic separation of the biotransformation products of oxaliplatin

A novel single reversed-phase HPLC system was developed for separating oxaliplatin and its biotransformation products formed in rat plasma. The major stable biotransformation products of oxaliplatin formed in rat plasma were identified as Pt(dach)(Cys)2, Pt(dach)(Met) and free dach. The minor biotransformation products Pt(dach)Cl2, Pt(dach)(GSH) and Pt(dach)(GSH)2 could also be resolved from other Pt-dach complexes. Among these biotransformation products, the identification of Pt(dach)(Met) was further confirmed by LC-ESI-MS, and the identification of Pt(dach)(Cys)2, Pt(dach)(GSH), Pt(dach)(GSH)2 and free dach was confirmed by atomic absorption and double isotope labeling. This HPLC technique should prove useful for separating and identifying the biotransformation products of Pt-dach drugs such as oxaliplatin, ormaplatin and Pt(dach)(mal) in biological fluids. This will allow a more complete characterization of the pharmacokinetics and biotransformations of these Pt-dach drugs, which should in turn lead to a better understanding of the mechanisms leading to their toxicity and efficacy.

Displacement of the bidentate malonate ligand from (d,l-trans-1,2-diaminocyclohexane)malonatoplatinum(II)by physiologically important compounds in vitro

Previous studies of platinum(II) compounds with bidentate leaving ligands have emphasized the contrast between the stability of the bidentate leaving ligand in vitro (T1/2 greater than 11 days in water) and the apparent reactivity of these bidentate platinum compounds in vivo. However, none of these studies actually measured the stability of these compounds in tissue culture medium (or in any other reaction mixture resembling in vivo conditions). The experiments described in this paper were designed to measure the stability and fate of (d,l-trans-1,2-diaminocyclohexane)malonatoplatinum(II) [Pt(mal)(trans-dach)] in RPMI-1640 tissue culture medium. The T1/2 for displacement of the malonate ligand in this medium was 9.5 hr at 37 degrees. Of the inorganic anions present in the medium, chloride accounted for the greatest displacement of the malonate ligand. However, at the concentrations with which it is found in tissue culture medium and in blood, bicarbonate was nearly as effective as chloride at displacing the malonate ligand. This observation is of particular significance because the bicarbonatoplatinum complex is unstable and the bicarbonate displacement reaction appears to represent a major non-enzymatic pathway for the formation of the biologically active aquated platinum complexes. At the concentrations with which they occur inside the cell, phosphates may play a similar role. Of the amino acids present in the medium, glutathione and the sulfur-containing amino acids were 50- to 400-fold more effective at displacing the malonate ligand than the other amino acids in RPMI-1640 medium. In the case of methionine, the reaction with Pt(mal)(trans-dach) was shown to be a direct displacement (SN2) reaction at physiological methionine concentrations. When Pt(mal)(trans-dach) was incubated at 37 degrees for 24 hr in RPMI-1640 medium, the major transformation products formed were (d,l-trans-1,2-diaminocyclohexane)methionineplatinum(II) (38%), other amino acid-platinum complexes (19%), and (d,l,-trans-1,2-diaminocyclohexane)dichloroplatinum(II) (14%). Eleven percent of the Pt(mal)(trans-dach) remained intact. Mass spectrometry and 1H-NMR indicated that the (d,l-trans-1,2-diaminocyclohexane)methionineplatinum(II) complexes that formed in RPMI-1640 medium consisted of approximately 60% of the bidentate mono-methionine complex coordinated to platinum at the sulfur and alpha-amino positions and 40% of the bis-methionine complex, presumably coordinated at the sulfurs.(ABSTRACT TRUNCATED AT 400 WORDS)