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

CPT-11 belongs to the class of topoisomerase I inhibitors, and it acts as a prodrug of SN-38, which is approximately 100-1000-fold more cytotoxic than the parent drug. CPT-11 has shown a broad spectrum of antitumor activity in preclinical models as well as clinically, with responses observed in various disease types including colorectal, lung, cervical, and ovarian cancer. The pharmacokinetics and metabolism of CPT-11 are extremely complex and have been the subject of intensive investigation in recent years. Both CPT-11 and SN-38 are known in an active lactone form and an inactive carboxylate form, between which an equilibrium exists that depends on the pH and the presence of binding proteins. CPT-11 is subject to extensive metabolic conversion by various enzyme systems, including esterases to form SN-38, UGT1A1 mediating glucuronidation of SN-38, as well as CYP3A4, which forms several pharmacologically inactive oxidation products. Elimination routes of CPT-11 also depend on the presence of drug-transporting proteins, notably P-glycoprotein and canalicular multispecific organic anion transporter, present on the bile canalicular membrane. The various processes mediating drug elimination, either through metabolic breakdown or excretion, likely impact substantially on interindividual variability in drug handling. Strategies to individualize CPT-11 administration schedules based on patient differences in enzyme or protein expression or by coadministration of specific agents modulating side effects are under way and may ultimately lead to more selective chemotherapeutic use of this agent.

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Clinical pharmacokinetics and metabolism of irinotecan (CPT-11)

https://flore.unifi.it/bitstream/2158/369971/1/Nobili%20Pharm%20Res%2c%202009.pdf

Natural compounds for cancer treatment and prevention

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

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.

Water-soluble analogues of the antitumor alkaloid camptothecin (1) were prepared in which aminoalkyl groups were introduced into ring A or B. Most of the analogues were prepared by oxidation of camptothecin to 10-hydroxycamptothecin (2) followed by a Mannich reaction to give N-substituted 9-(aminomethyl)-10-hydroxycamptothecins (4-12) or by subsequent modification of Mannich product 4 (13, 15, 17, 19, 21). Others were obtained by modification of the hydroxyl group of 2 (25,26) or by total synthesis (35,42,43). These analogues, as well as some of their synthetic precursors, were evaluated for inhibition of topoisomerase I, cytotoxicity, and antitumor activity. Although there was not a quantitative correlation between these assays, compounds that inhibited topoisomerase I were also cytotoxic and demonstrated antitumor activity in vivo. Further evaluation of the most active water-soluble analogue led to the selection of 9-[(dimethylamino)methyl]-10-hydroxycamptothecin (4, SK&F 104864) for development as an antitumor agent. In addition to its water solubility, ease of synthesis from natural camptothecin, and high potency, 4 demonstrated broad-spectrum activity in preclinical tumor models and is currently undergoing Phase I clinical trials in cancer patients.

Synthesis of Water-Soluble (Aminoalkyl)camptothecin Analogues: Inhibition of Topoisomerase I and Antitumor Activity.

A subline of P388 leukemia made 10-fold resistant to camptothecin (CPT) by serial passage in drug-treated mice was adapted to growth in tissue culture and made hyper-resistant to CPT by passage in the presence of increasing concentrations of the drug. Cells were obtained that were 1,000-fold resistant to CPT, compared to wild-type P388 cells. Neither topoisomerase I mRNA nor 100 kDa topoisomerase I enzyme was detectable in these cells, and topoisomerase I activity extracted from nuclei was less than 4% of that extracted from nuclei of wild-type cells. An immunoreactive 130 kDa protein that could be an altered, inactive form of topoisomerase I was evident in the hyper-resistant cells. In addition, the cells deficient in topoisomerase I contained enhanced topoisomerase II activity. Maintenance of the hyper-resistant phenotype required continued exposure to CPT; growth in its absence led to loss of hyper-resistance, increased topoisomerase I content and activity, and decreased topoisomerase II activity. The sensitivity of the cells to killing by a number of inhibitors of topoisomerases I and II was consistent with these observations. Thus, P388 cells have the potential to become highly resistant to CPT by severely curtailing topoisomerase I expression; in these circumstances, topoisomerase I and II activities are regulated coordinately.

R. P. Hertzberg

Papers

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

Synthesis of Water-Soluble (Aminoalkyl)camptothecin Analogues: Inhibition of Topoisomerase I and Antitumor Activity.

Camptothecin hyper-resistant P388 cells: drug-dependent reduction in topoisomerase I content.