[CANCER RESEARCH 59, 2939–2943, June 15, 1999] Homocamptothecin, an E-Ring Modified Camptothecin with Enhanced Lactone Stability, Retains Topoisomerase I-targeted Activity and Antitumor Properties1 Laurence Lesueur-Ginot, Danie`le Demarquay, Robert Kiss, Philip G. Kasprzyk, Laurent Dassonneville, Christian Bailly, Jose´Camara, Olivier Lavergne,2 and Dennis C. H. Bigg Institut Henri Beaufour, F-91966 Les Ulis, France [L. L-G., D. D., J. C., O. L., D. C. H. B]; Biomeasure Inc., Milford, Massachusetts 01757 [P. G. K.]; Laboratoire d’Histologie, Faculte´deMe´decine, Universite´ Libre de Bruxelles, B-1070 Bruxelles, Belgium [R. K.]; and Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, Institut National de la Sante´e et de la Recherche Me´dicale U-524, Institut de la Recherche sur le Cancer de Lille, F-59045 Lille, France [L. D., C. B.] ABSTRACT with CPT-sodium was due to the lack of useful activity of the ring-opened, carboxylate form adopted at that time (4), and reported Homocamptothecin (hCPT) is a semisynthetic analogue of campto- attempts to modify the a-hydroxylactone have failed to preserve Topo thecin (CPT) with a seven-membered b-hydroxylactone resulting from the I-mediated activity (5–7). Recently proposed binding modes for CPT insertion of a methylene spacer between the alcohol moiety and the carboxyl function of the naturally occurring six-membered a-hydroxylac- and Topo I-DNA cleavable complex further confirm the participation tone of CPT. This E-ring modification provides a less reactive lactone with of the lactone ring structural features to the CPT pharmacophore (8, enhanced stability and decreased protein binding in human plasma. Bio- 9). Unfortunately, the a-hydroxylactone imparts high chemical reac- logical testing against CPT revealed that, instead of being detrimental, the tivity to CPT and its analogues, which are thus intrinsically unstable modified lactone of hCPT has a positive impact on topoisomerase I (Topo molecules at physiological pH. I) poisoning properties. In vitro tests showed hCPT to fully conserve the This instability arises from the rapid hydrolysis of the a-hydroxylac- ability to stabilize Topo I-DNA cleavage complexes and to stimulate a tone in basic or neutral media to give the ring-opened, carboxylate form, higher level of DNA cleavage than CPT. A similar trend toward improve- which is essentially inactive. The reaction is reversible, and the lactone ment was also observed in antiproliferative assays with human tumor cell form predominates only at acidic pH. Pharmacokinetic studies have lines (including cells overexpressing P-glycoprotein). In two distinct in vivo shown this pH-dependent hydrolytic equilibrium to be shifted toward the models, using L1210 murine leukemia or human colon carcinoma HT29, hCPT was found to be more efficacious than CPT. The slow, but irre- carboxylate form in plasma in a species-dependent manner that is less versible, hydrolysis of hCPT, instead of the fast equilibrium of CPT, may favorable in man than in rodents (10). This latter point has been invoked account for its good in vivo activity. Overall, these results provide evidence to explain the diminished efficacy of various CPT analogues in the clinic that a highly reactive lactone is not a requisite for the Topo I-mediated compared with the spectacular results often obtained with xenograft antitumor activity of CPT analogues, and that hCPT is an interesting models (11). To circumvent the rapid opening of the lactone, the drugs pharmacological tool with improved solution behavior as well as a prom- may be administered by continuous infusion for long periods. Alterna- ising new template for the preparation of more efficacious Topo I poisons. tively, prodrugs have been proposed to achieve sustained plasma levels of CPT analogues in their lactone form. No active principle combining improved stability with good in vitro and in vivo Topo I inhibitory INTRODUCTION activity has been previously reported. The CPT3-derived Topo I inhibitors have undergone considerable We have recently described the semisynthetic preparation of a CPT b development (1) in recent years, leading to the approval of topotecan analogue bearing a seven-membered -hydroxylactone ring instead of a and irinotecan as second-line chemotherapeutic agents against resist- the naturally occurring six-membered -hydroxylactone (12). Be- ant cancers. In addition, several other molecules including CPT, cause a one-carbon ring expansion is chemically termed a homologa- 9-aminocamptothecin (9-AC), 9-nitrocamptothecin (9-NC), GG-211, tion, this new lactone ring-modified compound is a “homocampto- and DX-8951f have been clinically evaluated (2), and many other thecin” (hCPT). Because of the reduced electrophilicity of a b potent CPT analogues are in research or preclinical stage of develop- -hydroxylactone, the racemic compound showed enhanced stability ment (3). Structure-activity studies have shown the CPT skeleton to be in buffer solutions, and in vitro testing showed unexpectedly good amenable to substitution, providing analogues with improved phar- inhibition of the Topo I-mediated, supercoiled DNA relaxation, as macological profiles. Most effort has targeted water-soluble and/or well as antiproliferative activity on L1210 murine leukemia cells (12). more active molecules, whereas compounds exhibiting dual activities On the basis of these encouraging results, an analogue generation involving DNA alkylation, intercalation, or minor groove binding in program has been undertaken to identify potential candidate mole- addition to Topo I inhibition have been the focus of recent studies (3). cules for clinical development (13) in parallel with further pharma- It is a generally accepted rule for CPT-derived molecules that the cological evaluation of hCPT. The biologically active enantiomer has a-hydroxylactone ring is an absolute requirement for in vitro and in been isolated in optically pure form (13) and is referred to as hCPT vivo activities (1). Indeed, the discontinuation of early clinical trials (Fig. 1), whereas dl-hCPT designates the racemic mixture. Experi- mental comparison of hCPT with CPT should contribute to under- standing of the role of lactone reactivity in biological activity. Received 1/14/99; accepted 4/14/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. MATERIALS AND METHODS 1 Supported in part by a research grant from the Association pour la Recherche sur le Cancer (to C. B.). 2 To whom requests for reprints should be addressed, at Institut Henri Beaufour, 5, Drugs. CPT was purchased from Sigma (La Verpillie`re, France). Prepara- avenue du Canada, F-91966 Les Ulis, France. Fax: 33-01-69-07-38-02; E-mail: tion and purification of racemic dl-hCPT and enantiomerically pure hCPT [email protected]. were performed according to previously published procedures (13). A three- 3 The abbreviations used are: CPT, camptothecin; hCPT, homocamptothecin; Topo I, dimensional structure obtained by X-ray diffraction on a synthetic precursor topoisomerase I; TBE, Tris-borate EDTA; T, treated; C, control; T/C index, percent increase life span or survival index; T-C index, tumor growth delay; HPLC, high- established that, as shown in Fig. 1, hCPT and CPT have identical spatial performance liquid chromatography; MTD, maximum tolerated dose. arrangements around their asymmetric carbons. 2939 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1999 American Association for Cancer Research. hCPT RETAINS TOPO I-TARGETED ACTIVITY mM). After a 10-min incubation to ensure equilibration, the reaction was initiated by the addition of 2 ml (20 units) of calf thymus Topo I (Life Sciences, Cergy Pontoise, France). Samples were incubated for 45 min at 37°C before adding SDS to 0.25% and proteinase K to 250 mg/ml to dissociate the drug-DNA-Topo I cleavable complexes. The DNA was precipitated with ethanol, resuspended in 5 ml of formamide-TBE loading buffer, denatured at 90°C for 4 min, and then chilled in ice for 4 min before loading onto the sequencing gel. DNA cleavage products were resolved by PAGE under denaturing conditions (0.3-mm thick, 8% acryl- amide containing 8 M urea). After electrophoresis (about 2.5 h at 60 W, 1600 V in TBE buffer, Life Technologies, Inc. sequencer model S2), gels were soaked in 10% acetic acid for 10 min, transferred to Whatman 3 MM paper, and dried under vacuum at 80°C. A Molecular Dynamics 425E PhosphorImager was used to collect data from the storage screens exposed to dried gels overnight at room temperature. Baseline-corrected scans were analyzed by integrating all of the densities between two selected boundaries using ImageQuant version 3.3 software. Fig. 1. Chemical structures of CPT and hCPT. Each resolved band was assigned to a particular bond within the DNA fragment by comparison of its position relative to sequencing standards generated by the treatment of the DNA with dimethylsulfate followed by piperidine-induced cleav- Drug Stability in Human Plasma. To 500-ml fractions of pooled human age at the modified guanine residues. plasma (Transfusion Sud-Est Francilien, Rungis, France), distributed in poly- Cell Growth Assay. The A427 (lung carcinoma) and PC-3 and DU145 styrene vials and preincubated at 37°C for 5 min, were added 5 ml of a solution (prostatic adenocarcinoma) cell lines were obtained from American Type Culture of the drug—hCPT or CPT—in DMSO (10 mM) and the samples were Collection (Rockville, MD). The MCF7r (breast adenocarcinoma) and K562r incubated in capped vials (to prevent CO2 loss), at 37°C. At defined times, (leukemia) cell lines were obtained from A-M. Faussat (Hoˆpital Hoˆtel-Dieu, Paris, three vials were opened, and the plasma proteins were precipitated by the France). They were derived from sensitive cell lines by prolonged exposure to addition of 2 ml of cold acetonitrile (instead of methanol, which might not be Adriamycin and have been shown by flow cytometry to overexpress a functionally inert to CPT and analogues) at 250°C.