DNA topoisomerases and their poisoning by anticancer and antibacterial drugs.

It is known that 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), a semisynthesized derivative of camptothecin (CPT), has a potent antitumor activity in vivo, but 7-ethyl-10-hydroxycamptothecin (SN-38), a metabolite of CPT-11, shows much stronger cytotoxicity in vitro than CPT-11. In this study, we demonstrated that the relaxation of SV40 DNA plasmids by type I DNA topoisomerase prepared from P388 murine leukemia cells was inhibited by 50% by SN-38 at approximately 1 microM, although CPT-11 at 1 mM slightly inhibited the relaxation. SN-38 and CPT showed strong, time-dependent inhibitory activity against DNA synthesis of P388 cells. However, CPT-11 weakly inhibited DNA synthesis independently of time with coincident inhibition of the total thymidine uptake by the cells. By alkaline and neutral elution assays, it was demonstrated that SN-38 caused much more frequent DNA single-strand breaks in P388 cells than did CPT-11. The same content of SN-38 and a similar frequency of single-strand breaks were detected in the cells treated with SN-38 at 0.1 microM or with CPT-11 at 100 microM. Therefore, single-strand breaks by CPT-11 seem to be due to SN-38 produced from CPT-11 in cells. These results indicate that CPT-11 itself possesses a marginal antiproliferative effect but that SN-38 plays an essential role in the mechanism of action of CPT-11.

https://cancerres.aacrjournals.org/content/canres/51/16/4187.full.pdf

Intracellular Roles of SN-38, a Metabolite of the Camptothecin Derivative CPT-11, in the Antitumor Effect of CPT-11

The camptothecins are a maturing class of anticancer agents. In this article, we review the pharmacology and antitumor activity of the camptothecin analogues that are approved for clinical use and those investigational agents undergoing clinical evaluation. Camptothecin is a naturally occurring cytotoxic alkaloid that has a unique intracellular target, topoisomerase I, a nuclear enzyme that reduces the torsional stress of supercoiled DNA during the replication, recombination, transcription, and repair of DNA. Topotecan and irinotecan are synthetic analogues designed to facilitate parenteral administration of the active lactone form of the compound by introducing functional groups to enhance solubility. They are now well-established components in the chemotherapeutic management of several neoplasms. Topotecan has modest activity in patients treated previously with ovarian and small cell lung cancer and is currently approved for use in the United States as second-line therapy in these diseases. Preliminary evidence of activity against hematological malignancies is also promising. Irinotecan is a prodrug that undergoes enzymatic conversion to the biologically active metabolite 7-ethyl-10-hydroxy-camptothecin. It is presently the treatment of choice when used in combination with fluoropyrimidines as first-line therapy for patients with advanced colorectal cancer or as a single agent after failure of 5-fluorouracil-based chemotherapy. Encouraging preliminary results suggest that irinotecan may have an increasing role in the treatment of other solid tumors, including small and non-small cell lung cancer, cervical cancer, ovarian cancer, gastric cancer, and malignant gliomas. Several additional camptothecin analogues are in various stages of clinical development, including 9-aminocamptothecin, 9-nitrocamptothecin, 7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin, exatecan mesylate, and karenitecin. Efforts to further optimize therapeutic effectiveness through drug delivery strategies that prolong tumor exposure to these S phase-specific agents, such as improving oral bioavailability through structure modification and innovative formulation approaches, alternative parenteral dosage forms, and administration schedules, are being actively pursued. Combining camptothecins with other anticancer drugs and treatment modalities, as well as gaining a better understanding of the factors contributing to tumor sensitivity and resistance, continues to be the object of considerable interest.

/pdf/current-perspectives-on-the-clinical-experience-pharmacology-3z73f1hwfd.pdf

Current Perspectives on the Clinical Experience, Pharmacology, and Continued Development of the Camptothecins

Elevated Expression of the Apoptotic Regulator Mcl-1 at the Time of Leukemic Relapse

PURPOSEWe conducted a phase I and pharmacokinetic trial of CPT-11 (irinotecan) to characterize the maximum-tolerated dose (MTD), toxicities, pharmacokinetic profile, and antitumor effects in patients with refractory solid malignancies.PATIENTS AND METHODSWe treated 32 patients with CPT-11 administered as a 90-minute intravenous infusion every week for 4 consecutive weeks followed by a 2-week rest period. Dose levels ranged from 50 to 180 mg/m2/wk. We determined concentrations of the lactone (active) and total (lactone plus carboxylate) forms of CPT-11 and its metabolite, SN-38, in the plasma and urine of selected patients during and after drug infusion.RESULTSGrade 4 diarrhea was the dose-limiting toxicity (DLT) at the 180-mg/m2/wk dose level. Other toxicities attributed to CPT-11 included dehydration, nausea, vomiting, and asthenia. Hematologic toxicity was mild in most patients. The terminal plasma half-life for CPT-11 (total) was 7.9 +/- 2.8 hours, for CPT-11 (lactone) 6.3 +/- 2.2 hours, for SN-38 (tot...

Phase I and pharmacokinetic trial of weekly CPT-11

A camptothecin-resistant subline of P388 leukemia (P388/CPT) was developed by repeated transplantation of P388 cells in mice treated with therapeutic doses of camptothecin. In mice bearing the resistant tumor, a maximally tolerated dose of camptothecin produced no net reduction in tumor cell burden, in contrast to a 5-log cell kill in the parental P388 (P388/S). The IC50 of camptothecin, as determined by colony formation assays of cultured cells, was 8 times greater for the cloned P388/CPT cell line than for P388/S. P388/CPT cells were not cross-resistant to other antineoplastic agents, including topoisomerase II inhibitors. The type I topoisomerases purified from P388/CPT and P388/S cells were identical with respect to molecular weight, specific activity, in vitro camptothecin sensitivity, and DNA cleavage specificity. Camptothecin induced fewer protein-associated DNA single-strand breaks in the resistant cells than in the wild-type P388 cells. Topoisomerase I mRNA, immunoreactivity, and extractable enzymatic activity were 2-4 times lower for P388/CPT cells than for P388/S cells. As resistance to camptothecin developed, topoisomerase I extractable activity decreased, concomitant with an increase in topoisomerase II extractable activity. Furthermore, the appearance of camptothecin resistance was associated with specific rearrangements of the topoisomerase I gene. These results suggest that development of resistance to inhibitors of topoisomerase I can occur by down-regulation of the target enzyme, thus reducing the production of lethal enzyme-mediated DNA damage. The enhanced topoisomerase II activity in these cells suggests that resistance to camptothecin may be overcome by co-treatment with topoisomerase II inhibitors.

Development of a stable camptothecin-resistant subline of P388 leukemia with reduced topoisomerase I content.

CHO-Cdr20 cells are 10-20 times more resistant to killing by cadmium than the parental CHO cells. Resistance has been linked to amplification of the metallothionein genes MT-I and MT-II and their coordinate induction by cadmium and other toxic metals. We studied the roles of the nuclear enzymes topoisomerase I and topoisomerase II in Cd-induced expression of MT-II. Camptothecin-induced DNA strand breakage, mediated by topoisomerase I in cells, increased by approximately 20% when the resistant cells were incubated first with 50 microM Cd and then with camptothecin. Short DNA fragments were enriched in MT-II-hybridizing sequences, indicating that topoisomerase I-associated breakage was directed in part toward the location of induced gene activity. Ten microM camptothecin inhibited Cd-induced accumulation of MT-II mRNA as well as induced and uninduced RNA synthesis in the resistant cells. These data are consistent with the notion that topoisomerase I participates in most or all forms of RNA synthesis. Topoisomerase II inhibitors which trap cleavable complexes (amsacrine, VM-26, VP-16) increased DNA strand breakage at very high concentrations (50-100 microM); the increased breakage appeared to be concentrated near the MT-II gene. This class of inhibitor did not block the accumulation of MT-II message. Novobiocin, a second type of topoisomerase II inhibitor blocked transcription at 300 microM. Merbarone, a novel, third type of topoisomerase II inhibitor, blocked MT-II transcription at 50-100 microM. The latter two inhibited total RNA synthesis in induced, but not uninduced cells. Thus, it is possible that topoisomerase II plays more than one role in transcription and that more than one form of this enzyme is involved.

K. B. Tan

Papers

Development of a stable camptothecin-resistant subline of P388 leukemia with reduced topoisomerase I content.

Evidence for the participation of topoisomerases I and II in cadmium-induced metallothionein expression in Chinese hamster ovary cells.