The homocamptothecin BN 80915 is a highly potent orally active topoisomerase I poison.

BN 80915, a lead compound of the homocamptothecin (hCPT) family, has entered clinical trials. BN 80915 is a difluoro-hCPT where the six-membered alpha-hydroxylactone ring of camptothecin (CPT) is replaced by a seven-membered beta-hydroxylactone ring. Preclinical data reported here show that in spite of the modification to the crucial E-ring of CPTs, BN 80915 retains topoisomerase I poisoning activity as shown in living HT29 cells as well as in cell-free assays, where BN 80915 always performs better than SN-38 or TPT. In antiproliferative assays BN 80915 is also very potent as evidenced by IC50s values consistently lower than those of SN38 in sensitive cell lines as well as in their related multidrug-resistant lines overexpressing P-glycoprotein or multidrug resistance-associated protein. Furthermore, in human plasma, in contrast to CPT analogs, the hydrolysis of BN 80915 is slow, leading to improved plasma stability, and irreversible, thus avoiding toxicity related to the accumulation of active principle during excretion in the urinary tract. These findings may account for the good in vivo efficacy observed in PC3 xenograft experiments where BN 80915 administered orally at very low doses doubled the tumor growth delay in comparison to CPT-11 administered i.p. Altogether, these results strongly support further development of BN 80915.

A novel B-ring modified homocamptothecin, 12-Cl-hCPT, showing antiproliferative and topoisomerase I inhibitory activities superior to SN-38.

We report the synthesis and pharmacological evaluation of a novel homocamptothecin (hCPT) derivative, 12-Cl-hCPT, which contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone found in camptothecin (CPT) and bears a chloro substituent at position 12. The capacity of 12-Cl-hCPT to inhibit DNA topoisomerase I was compared with that of SN-38, the active metabolite of the clinically used antitumour prodrug CPT-11. In the DNA relaxation assay, 12-Cl-hCPT proved to be slightly more potent than SN-38 at stimulating the formation of nicked plasmid DNA molecules. A series of radiolabelled DNA restriction fragments were employed to identify and compare the position of the DNA cleavage sites induced by topoisomerase I in the presence of 12-Cl-hCPT and SN-38. These sequencing studies confirm that both 12-Cl-hCPT and SN-38 strongly promote DNA cleavage by topoisomerase I and reveal that the majority of the cleavage sites are located at the same nucleotide positions for the two drugs. However, a certain number of DNA cleavage sites were found to be specific to 12-Cl-hCPT. These sites, previously characterized with unsubstituted hCPT, generally correspond to 5'-CG sites whereas the sites common to the 12-Cl-hCPT and SN-38 essentially correspond to 5'-TG sites. We also quantified the formation of drug-induced protein-DNA complexes formed in HT29 human colon carcinoma cells. Trapping of endogenous proteins onto DNA was found to be much more efficient with 12-Cl-hCPT than with SN-38. These data provide a molecular basis to account for the enhanced antiproliferative activity of 12-Cl-hCPT compared with that of SN-38. Biological evaluation on a panel of sensitive and drug-resistant cell lines revealed 12-Cl-hCPT to be more cytotoxic to tumour cells than SN-38. 12-Cl-hCPT proved 14- and 23-fold more active than SN-38 toward the K562adr and T24anp multidrug-resistant cell lines, respectively. The marked topoisomerase I inhibitory properties of 12-Cl-hCPT coupled with its interesting antiproliferative activity, in particular against cancer cells presenting multidrug resistance phenotype with overexpression of P-glycoprotein, makes 12-Cl-hCPT a valid candidate for subsequent preclinical evaluation. Collectively, the data strengthen homocamptothecin as an extremely promising template to generate novel and potent antitumour agents.

Topoisomerase I-mediated antiproliferative activity of enantiomerically pure fluorinated homocamptothecins.

Homocamptothecin (hCPT) is an E-ring modified camptothecin (CPT) analogue bearing a methylene spacer between the alcohol and carboxyl functions of the CPT lactone. Combining pronounced inhibitory activity of topoisomerase I (Topo I) with enhanced plasma stability, hCPT constitutes an attractive template for the elaboration of new anticancer agents. Fluorinated hCPT analogues, prepared in enantiomerically pure form, were assayed by their stimulation of Topo I-mediated DNA cleavage. Translation into cytotoxicity against tumor cells was evaluated on HT29 human colon adenocarcinoma and on the multidrug resistant lung and bladder tumor cell lines, A549 and T24r. Good correlation is observed between the ability of the drugs to stimulate Topo I-mediated DNA cleavage and the respective 50% inhibitory concentrations (IC(50) values) of the HT29, A549, and T24r cell growth. Fluorine substitution in the A-ring of hCPT was found to have a pronounced influence on biological activity, providing several compounds which are up to 100-fold more potent than CPT in terms of IC(50). Among these, 10,11-difluoro-hCPT has been selected for further development.

Homocamptothecin, an E-ring-modified camptothecin analogue, generates new topoisomerase I-mediated DNA breaks.

Homocamptothecin (hCPT) contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone ring found in camptothecin and its tumor active analogues, including topotecan and irinotecan. The homologation of the lactone E-ring reinforces the stability of the lactone, thus reducing considerably its conversion into a carboxylate form which is inactive. We have recently shown that hCPT is much more active than the parent compound against a variety of tumor cells in vitro and in xenograft models, suggesting that a highly reactive lactone is not essential for topoisomerase I-mediated anticancer activity [Lesueur-Ginot et al. (1999) Cancer Res. 59, 2939-2943]. In the present study, we provide further evidence that hCPT has superior topoisomerase I inhibition capacities to CPT. In particular, we show that replacement of the camptothecin lactone E-ring with a homologous seven-membered lactone ring changes the sequence-specificity of the drug-induced DNA cleavage by topoisomerase I. Both CPT and hCPT stimulate the cleavage by topoisomerase I at T( downward arrow)G sites, but in addition, hCPT stabilizes cleavage at specific sites containing the sequence AAC( downward arrow)G. At low drug concentrations, the cleavage at the T( downward arrow)G sites and at the hCPT-specific C( downward arrow)G sites is more pronounced and more stable with hCPT than with CPT. The in vitro data were confirmed in cells. Higher levels of protein-DNA complexes were detected in P388 leukemia cells treated with hCPT than those treated with CPT. Immunoblotting experiments revealed that endogenous topoisomerase I was efficiently trapped onto DNA by hCPT in cells. Finally, the use of a leukemia cell line resistant to CPT provided evidence that topoisomerase I is involved in the cytotoxicity of hCPT. Altogether, the results show that the beta-hydroxylactone ring of hCPT plays an important and positive role in the poisoning of topoisomerase I. An explanation is proposed to account for such remarkable changes in the sequence specificity of topoisomerase I cleavage consequent to the modification of the lactone. The study sheds new light on the importance of the lactone ring of camptothecins for the stabilization of topoisomerase I-DNA complexes.

BN 80927: a novel homocamptothecin with inhibitory activities on both topoisomerase I and topoisomerase II.

BN 80927, a novel homocamptothecin derivative, inhibits both topoisomerase I and topoisomerase II mediated DNA relaxation and shows pronounced cytotoxicity against HT29, SKOV-3, DU145 and MCF7 human tumor cell lines.

Flow cytometric monitoring of anthracycline accumulation after anti-neoplastic ether phospholipid treatment.

Ether phospholipids are new anti-neoplastic drugs that have been found active against a variety of tumor cell lines, including drug-resistant sublines. We have characterized the antiproliferative activity of three ether phospholipids, i.e. ET-18-OCH3 (Edelfosine), BM 41.440 (limofosine) and a new aza-derivative (BN 52205), on three leukemic cell lines, i.e. K562 (chronic myeloid leukemia, blast crisis), HL60 (promyelocytic acute leukemia) and CEM (T cell leukemia), and their respective drug-resistant sublines, i.e. K562-ADR (adryamicin resistant), HL60-DNR [daunorubicin (DNR) resistant] and CEM-VLB (vinblastin resistant). These resistant sublines have been found to express the multidrug-resistant phenotype, revealed by the presence of the P-glycoprotein (PgP) using different monoclonal antibodies. Increased cellular accumulation of the fluorescent anthracycline has been found in both sensitive and resistant cell lines after different ether phospholipid treatment times. In resistant cells, the ether phospholipid effect on DNR accumulation has also been found after blocking the PgP function by verapamil and cyclosporin A. These results confirm that the ether phospholipid action is closely linked with the membrane biochemical composition and that these new anti-tumor drugs are able to change the dynamic structural organization of the tumor cell membrane.

Tumor cell kinetics following long-term treatment with antineoplastic ether phospholipids.

Ether phospholipids are analogs of the naturally occurring 2-lysophosphatidylcholine that have been reported to have selective in vitro/in vivo antitumor activity. Their antiproliferative effect has been found against a variety of animal and human tumor cell lines. We have characterized the cytostatic activity of four ether phospholipids, the methoxy-substituted edelfosine (ET-18-OCH 3), the thio-derivative ilmofosine (BM 41.440), and two new aza-alkylphospholipids, BN 52205 and BN 52211, on a human tumor cell line derived from a colon adenocarcinoma, the HT29. A flow cytometric approach has been used and, contrary to previous studies, longer treatment times have been performed to allow multiple cell population doublings. The results confirm that the cytostatic activity of the four ether phospholipids is characterized by multiple "terminal points", as the drugs' action results in a G1 block, a slowdown of the transition from late-S to G2, followed by an accumulation of HT29 cells in the G2 phase of the cell cycle. Tumor cells in late G1 at the time of treatment progressed through S before being blocked in G2. In a similar fashion, tumor cells in late G2 at the time of treatment went through M but were then halted in G1. The long-term treatment studies indicate that the ether phospholipid cytostatic activity is partially reversible, depending on the drug concentration and the duration of the treatment.

New antineoplastic aza-phospholipids - synthesis, in-vitro cytotoxicity and differential cellular-sensitivity against 70 human tumor-cell lines.

The in vitro cytotoxicity and differential cellular sensitivity of three new synthetic anti-neoplastic aza-phospholipids has been determined in the National Cancer Institute's (NCI) primary antitumor drug screen. Based on a disease-oriented strategy, this screen incorporates seventy human cell lines representing leukemia, ovarian, brain, melanoma, colon, renal, lung, prostate and breast cancers. The analysis of the GI50 values obtained for each aza-derivative has revealed a differential cellular sensitivity among the cell lines examined. The study of the degree of differential growth inhibition has shown a statistically significant differential cell sensitivity for BN 52205 and BN 52211 for colon and melanoma tumor cells. The leukemia cell selectivity for BN 52211 was even more remarkable due to the low molar concentration at which the maximum selective effect occurred. These findings strongly encourage further investigations on the anti-neoplastic activity of aza-phospholipids.

Evaluation of combinations of antineoplastic ether phospholipids and chemotherapeutic drugs.

Combinations of drugs are used clinically for the therapeutic advantages they may provide over single agents. We have studied the cytotoxic interaction between four either phospholipids ET-18-OCH3, BM 41.440, BN 52205 and BN 52211, and several chemotherapeutic drugs (ADM, CDDP, VLB, VP-16, MMC, BLM and MTX) on two human tumor cell lines, A427 (lung) and HT29 (colon). We have used the MTT colorimetric assay to evaluate growth inhibition and performed isobologram analysis on the IC50 data. For both cell lines a synergistic effect has been found between each of the four ether phospholipids in association with CDDP and ADM. In both cell lines only BM 41.440 and BN 52211 act synergistically with VLB while, in A427 cells, only BN 52205 behaves similarly with MMC. These results show that a positive interaction exists between ether phospholipids, spindle poisons and DNA-interactive drugs.

Immunomodulatory activity of two new aza alkyl phospholipid antineoplastic drugs.

The present work reports the modulation of immunocompetent cell functions by two aza alkyl phospholipids (AAP), BN 52205 and BN 52211. Each compound was compared with 1-O-octadecyl-2-O-rac-glycero-3-phosphocholine (ET-18-OCH3) and/or three drugs used for cancer treatment, i.e. cisplatyl (CIS), 5-fluorouracil (5-FU) and cytosine arabinoside (ARA-C). Interleukin (IL)-1 release from P388D1 cells was increased 2-fold in the presence of 5 micrograms/ml BN 52205 or BN 52211. However, these stimulations were lower than those obtained with ARA-C, 5-FU and CIS. Compared with ET-18-OCH3, CIS and 5-FU, BN 52205 and BN 52211 were more efficient in increasing tumor necrosis factor production induced by lipopolysaccharide (LPS) from human monocytes. In vitro, all compounds exhibited similar activity in enhancing IL-6 production from human monocytes stimulated with LPS, with the exception of 5-FU and CIS that were inactive. At 20 mg/kg (i.v.), a peak of IL-6 production was reached 2 h after injection of ET-18-OCH3 [> 1280 U/ml (n = 4, p < 0.001) versus 3.5 +/- 0.2 U/ml (n = 7)], whereas BN 52211 induced a maximum of IL-6 production after 4 h (77 +/- 27 U/ml, n = 5, p < 0.001). BN 52205 induced peaks of IL-6 production after 3 and 6 h (90 +/- 62 and 68 +/- 35 U/ml, respectively, p < 0.001, n = 4). The proliferation of rat splenocytes was abolished in the presence of BN 52205 and BN 52211 at 10 micrograms/ml, corresponding to only a partial reduction of IL-2 production at the same concentration. The production of interferon-gamma was stimulated 6- to 10-fold in the presence of 1-5 micrograms/ml BN 52205, BN 52211 and ARA-C. BN 52211 and BN 52205 were also potent enhancers of IL-3 production, whereas 5-FU and ARA-C were inhibitory. These results indicate that in addition to a direct antitumoral effect, AAP may also exhibit immunomodulatory activity both in vitro and in vivo.

Effect of serum concentration on the cytotoxicity and sister chromatid exchange induction by a chemical carcinogen and by a diesel particulate extract in V79 hamster cells.

The role of serum concentration on the cytotoxicity and on the sister chromatid exchange (SCE)-induction by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and by a diesel particulate extract (DPE), a complex mixture, has been carried out on V79 cells. An increase of the serum concentration in the medium decreases the toxicity of these chemicals, and especially when they are dispersed first in serum. Although no influence of serum concentration on the number of spontaneous SCEs occurring in control cells has been observed, the increase of serum concentration leads to a decrease in SCE's induction in treated cells. Our results show that serum can protect cells from the cytotoxic and mutagenic action of MNNG and diesel extract.

Tumour-initiating activities of dihydrodiols of dibenz[a,c]anthracene.

The tumor-initiating activities of dibenz[a,c]anthracene (DBA) and of the related trans-1,2-, 3,4- and 10,11-dihydrodiols have been examined on mouse skin subsequently promoted with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The 1,2- and 10,11-dihydrodiols were active and were more active than equivalent doses of either the parent hydrocarbon or the 3,4-dihydrodiol. The data are discussed in relation to possible mechanisms that may be involved in the metabolic activation of DBA.

The uptake and release of benzo[a]pyrene and benzo[e]pyrene in vitro by Syrian hamster embryo cells as a function of serum concentration.

A quantitative study on the in vitro uptake of benzo[a]pyrene (B[a]P) and benzo[e]pyrene (B[e]P) by Syrian hamster embryo cells and the induction of sister chromatid exchange (SCE) has been carried out. The amounts of B[a]P and B[e]P taken up by the cells decreases as does the induction of SCEs by B[a]P when the concentration of serum in the culture medium increases. It appears that serum prevents (B[a]P or B[e]P uptake. We have observed no significant differences between the two hydrocarbons regarding uptake by cells; chromatographic results show however that B[a]P is metabolized by these cells, while B[e]P is not. Our results suggest that serum inhibits B[a]P and B[e]P uptake and hence decreases the number of SCEs.