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In previous studies, we have documented the potential therapeutic advantages of camptothecin analogs modified at the 7-position, i.e., 7-oxyiminomethyl derivatives. The present study was performed to explore the therapeutic potential of novel hydrophilic derivatives of this series. With one exception (ST1976), the tested camptothecins exhibited a reduced antiproliferative activity and all compounds retained ability to stabilize the topoisomerase I-mediated cleavable complex. The two analogs (ST1976 and ST1968) characterized by the presence of a free amino group in the side chain also exhibited the formation of persistent cleavable complexes. The most potent compound, ST1976 (7-(4-aminobenzyl)oxyiminomethylcamptothecin), was selected for evaluation of its preclinical profile of antitumor activity in a large panel of human tumor xenografts. As expected on the basis of the introduction of a hydrophilic substituent, the novel camptothecin was a substrate for BCRP. However, in spite of an apparent recognition by BCRP, ST1976 was effective following oral administration. The antitumor activity was evaluated using various schedules and routes of administration (i.v. and p.o.). ST1976 exhibited a remarkable activity in all tested tumors and was effective in a number of tumors which are resistant to irinotecan. The biological and pharmacological profile of ST1976 supports the therapeutic potential of camptothecins containing hydrophilic substituents at the 7-position. On the basis of its excellent activity in preclinical models, ST1976 is a promising candidate for clinical development.

Recent evidence indicates that the success of molecular targeted therapies may depend on the identification of drug targets which are essential for the survival of subsets of tumors. RET oncogenes that have been implicated in the development of thyroid carcinomas are emerging as potential therapeutic targets. In the present study, we investigated the efficacy and the cellular bases of antitumor activity of the indolinone Ret tyrosine kinase inhibitor RPI-1 against large established s.c. TT tumor xenograft, a human medullary thyroid carcinoma (MTC) harboring oncogenic MEN-2A-type RET mutation. Oral treatment with RPI-1 caused growth arrest or regression in 81% treated tumors. Following treatment suspension, tumor inhibition was maintained (51%, P

Following the realisation that DNA topoisomerase I is a useful therapeutic target to be exploited for the design of potential inhibitors, topoisomerase I inhibitors now represent an established class of effective agents. In spite of intense efforts in the field, only camptothecins have a clinical relevance. Several options in chemical manipulation of natural camptothecin have been explored to overcome the major drawbacks of the drug, which include water insolubility, lactone instability, reversibility of the drug-target interaction and drug resistance. Several analogues are currently in clinical development, including water soluble camptothecins, lipophilic camptothecins and polymer-bound camptothecins. The therapeutic advantages of novel camptothecins over the two analogues (topotecan and irinotecan) approved for clinical use remain to be defined. This article is an overview of the relevant features of the analogues that are undergoing clinical development.

ST1481, a lead compound of a novel potent 7-substituted lipophilic camptothecin series, is able to overcome several mechanisms of drug resistance and was selected for clinical development. This study was designed to examine the antitumor activity of ST1481 in the treatment of preclinical models of human p53-defective hormone-refractory prostate carcinoma (DU145, PC3, and JCA-1) and to explore the cellular bases of the efficacy of camptothecins. A cellular pharmacology study (cytotoxicity, apoptosis, cellular drug accumulation, DNA damage, and cell cycle perturbation) was performed in DU145 and PC3 cells, characterized by a different cell cycle checkpoint status. The introduction of wild-type p53 in PC3 cells appreciably decreased the drug sensitivity. The 7-substituted camptothecins exhibited a high cytotoxic potency that paralleled their relative ability to induce DNA damage and a substantially increased cellular accumulation as compared to topotecan. The cytotoxic effect of camptothecins in DU145 cells was associated with arrest in S phase and early activation of apoptosis, whereas PC3 cells responded to drugs by a persistent block in G2 phase with a cytostatic effect and a late apoptosis. The efficiency of S phase checkpoint in DU145 cells was supported by a time-dependent decrease of DNA synthesis following treatment. In spite of an apparent cytostatic response and apoptosis resistance, the PC3 tumor was more responsive to in vivo treatment with camptothecins than the DU145 model. Indeed, the therapeutic outcome did not reflect the cell susceptibility to early activation of apoptosis. We suggest that cell death in PC3 cells is a delayed event consequent to persistent arrest in G2 and insufficient repair of DNA damage. ST1481 was appreciably more effective than topotecan in all tested tumors. In conclusion, the results indicated a relevant efficacy of camptothecins against human prostate carcinoma models, in spite of p53 alterations. Although p53 status could influence DNA damage and cell cycle checkpoints, p53 mutation was not a determinant of resistance. The results support that, in addition to the extent and persistence of topoisomerase I-mediated DNA damage, cell cycle checkpoints and DNA damage signaling pathways are critical determinants of tumor responsiveness to camptothecins. A role of cell cycle checkpoints activated by DNA damage in cell response is supported by the modulation of transcriptional profile.

MEN 10755 is a disaccharide anthracycline endowed with a broader spectrum of antitumour activity than doxorubicin (DOX). To investigate the cellular and molecular basis of its action, cytotoxic activity, drug uptake, subcellular localisation, induction of DNA damage, and apoptosis were assessed in the human A2780 ovarian carcinoma cell line. Experiments with radiolabelled anthracyclines indicated that MEN 10755 exhibited reduced cellular accumulation and a different subcellular distribution (higher cytoplasmic/nuclear ratio) than DOX. In spite of the lower nuclear concentration, MEN 10755 was as potent as DOX in eliciting DNA single- and double-strand breaks, G2/M cell arrest, and apoptosis. Sequencing of drug-induced topoisomerase II cleavage sites showed a common DNA cleavage pattern for MEN 10755 and DOX. Cleavage sites were always characterised by the presence of adenine in −1 position. However, the extent of DNA cleavage stimulation induced by MEN 10755 was greater than that produced by DOX. Reversibility studies showed that MEN 10755-stimulated DNA cleavage sites were more persistent than those induced by DOX, thus suggesting a more stable interaction of the drug in the ternary complex. As a whole, the study indicated that the cellular pharmacokinetics of MEN 10755 substantially differs from that of DOX, showing a lower uptake and a different subcellular disposition. In spite of the apparently unfavourable cellular pharmacokinetics, MEN 10755 was still as potent as DOX in inducing topoisomerase-mediated DNA damage. Although the extent and persistence of protein-associated DNA breaks may contribute to the cytotoxic effects, the drug’s efficacy as apoptosis inducer and antitumour agent could not be adequately explained on the basis of DNA damage mediated by the known target (i.e. topoisomerase II), thus supporting additional cellular effects that may be relevant in cellular response.

The sugar moiety is an essential component of anthracycline antibiotics for their topoisomerase poisoning activity and antitumor efficacy. Since the sugar interacts with the minor groove, modifications in this moiety could enhance the recognition potential of the drug at the target level. Based on this hypothesis, novel anthracyclines, disaccharides lacking the amino group in the first (aglycone-linked) sugar, were designed. The 3'-amino group in the first sugar was replaced by an hydroxyl group, and the second sugar residue was bound to the first sugar via an alpha (1-4) linkage. The cytotoxic and antitumor activities of disaccharide analogs of idarubicin were critically dependent on the optimal (axial) orientation of the second sugar residue. Although configurational requirements of the sugar moiety for optimal drug activity support a critical role of the external (non-intercalating) drug domains in the interaction of anthracyclines with the DNA-topoisomerase (ternary complex), the antitumor efficacy of disaccharide analogs is not fully explained by effects mediated by the nuclear enzyme target. The development of this novel disaccharide series may provide insights for a rational synthesis of anthracycline analogs with improved pharmacological profile.

A variety of cytotoxic agents effective as antitumor drugs are known to kill tumor cells through induction of apoptosis as the most relevant modality of cell death. A specific role for the protein Bcl-2 in the cell death pathway induced by antimicrotubule agents has been proposed, because Bcl-2 phosphorylation occurs in response to microtubule damage. In this study, we compared efficacy, apoptosis, and Bcl-2 phosphorylation in the Bcl-2-overexpressing MX-1 human breast carcinoma xenograft after treatment with cytotoxic agents characterized by different mechanisms of action. We demonstrated that, in addition to antimicrotubule agents, effective DNA-damaging agents were also able to induce Bcl-2 phosphorylation irrespective of the type of genotoxic lesion. A comparison of effects of drugs belonging to the same class but endowed with a different antitumor activity (i.e. cisplatin versus a novel multinuclear platinum complex and doxorubicin versus a disaccharide analogue) showed a correlation between drug efficacy, apoptotic response, and Bcl-2 phosphorylation. In conclusion, overexpression of Bcl-2 did not counteract the apoptotic effects of a number of cytotoxic agents and could not be regarded as a mechanism of cellular resistance. Since Bcl-2 phosphorylation is a common event in response to different types of cytotoxic damage and is not only related to microtubule dysfunction, we suggest that many cell death pathways converge on Bcl-2 and protein phosphorylation is a step of the signaling cascade activated by diverse stimuli and likely related to the onset of drug-induced apoptosis.

Tumor cell growth, even in advanced stages of ovarian cancer, is nearly always restricted to the peritoneal cavity; therefore, repeated intraperitoneal injections of oligodeoxynucleotides containing dinucleotides with unmethylated CpG motifs (CpG-ODN) recruiting and activating innate effector cells throughout the abdominal cavity to the tumor site might control tumor cell growth and ascites formation. After a single CpG-ODN treatment, in IGROV-1 ovarian tumor ascites-bearing athymic mice, the number of tumor cells declined rapidly and markedly, and ascites volumes declined shortly after treatment (5 h), increasing thereafter at a slower rate than in controls. When administered every 7 days for 4 weeks, CpG-ODN had only a marginal effect on survival time, whereas administration 5 days/wk for 3 or 4 weeks led to a significantly increased survival time as compared with controls (P

ST1968 is a novel hydrophilic camptothecin (CPT) derivative of the 7-oxyiminomethyl series. Because ST1968 retained ability to form remarkably stable cleavable complexes, this study was done to investigate its preclinical profile of antitumor activity in a large panel of human tumor models, including irinotecan-resistant tumors. Although less potent than SN38 in vitro, i.v. administered ST1968 caused a marked tumor inhibition, superior to that of irinotecan, in most tested models. ST1968 exhibited an impressive activity against several tumors including models of ovarian and colon carcinoma in which a high rate of cures was observed. In the most responsive tumors, complete and persistent tumor regressions were achieved even with low suboptimal doses. Even tumors derived from intrinsically resistant cells exhibited a significant responsiveness. Histologic analysis of treated tumors supports a contribution of both proapoptotic and antiangiogenic effects to ST1968 antitumor efficacy. A study done in yeast cells transformed with CPT-resistant mutant forms of topoisomerase I documented that, in contrast to other tested CPT, ST1968 was active against yeasts expressing the mutant K720E enzyme. Based on its outstanding efficacy superior to that of irinotecan and of its good therapeutic index, ST1968 has been selected for clinical development. [Mol Cancer Ther 2008;7(7):2051–9]

The monoclonal antibody trastuzumab binds to the extracellular domain of HER-2/neu and induces clinical responses in breast tumors with HER-2 gene amplification and/or protein overexpression. Its role in other tumor types remains to be investigated. We evaluated the antitumor efficacy of trastuzumab in vitro and in nude mice implanted orthotopically with cells of 3 human pancreatic tumor lines expressing only low levels of HER-2/neu, as determined by flow cytometry. Although none of the 3 cell lines showed growth inhibition when cultured directly with trastuzumab, 2 of them, GER and PaCa3, were sensitive to lysis in antibody-dependent cellular cytotoxicity assay. This pattern of response was recapitulated in tumor-bearing mice repeatedly treated with trastuzumab, in which survival was significantly prolonged as compared with controls (P=0.03 for GER and 0.0008 for PaCa3). Incidence of metastases was also reduced, especially in liver. These preclinical results indicate that trastuzumab can exert an antitumor effect against orthotopic human pancreatic cancer xenografts with low-level HER-2/neu expression and that this effect correlates with the in vitro antibody-dependent cellular cytotoxicity susceptibility, suggesting a different role for HER-2/neu in the therapy of tumor types other than breast cancer.

Gimatecan, a potent lipophilic camptothecin, is active by oral route, which allows a comparison of different treatment schedules. The present study was designed based on previous evidence of improved antitumor efficacy of the combination of topotecan with immunostimulatory CpG-ODN. Two regimens of gimatecan, i.e., a low-dose/protracted treatment (metronomic) and a high-dose/intermittent treatment, were compared in the GER orthotopic human pancreatic tumor model in nude mice. Metronomic gimatecan (0.25 mg/kg per os, daily) significantly increased mice survival time over control mice (154 T/C%, p0.05), in contrast to intermittent treatment-gimatecan (1.5 mg/kg per os, q4d) or intravenous gemcitabine (125 and 128 T/C%, respectively, p0.1). Metronomic gimatecan combined to CpG-ODN (20 microg/mouse intraperitoneal, weekly) was well tolerated and achieved a strong antitumor effect, with a T/C% of 188 on survival time (P0.001 versus control mice), significantly superior to those of the single agent-treated mice (p0.05 versus gimatecan- or CpG-ODN-treated mice). Cellular studies indicated that TRAIL could increase the apoptotic response to gimatecan in GER tumor cells, and TRAIL was released in the peritoneal washings of CpG-ODN-treated mice. In conclusion, the combination of metronomic gimatecan with CpG-ODN was effective and tolerated, and might represent a preclinical basis for the design of clinical studies, even considering the ability of CpG-ODN to stimulate TRAIL release and the high level of TRAIL-receptors expressed in pancreatic tumor cells.

The effect of cisplatin (CDDP) in combination with etoposide (VP16) was examined on four human cell lines (one colon adenocarcinoma, LoVo; one ovarian carcinoma, IGROV-1; two small cell lung cancers, NCI-H146 and NCI-N592; and two murine leukemias, P388 and L1210). Simultaneous exposure to CDDP and subtoxic concentrations of VP16 for 1 h produced a cell killing in all cell lines comparable to that achieved by CDDP alone. Sequential exposure of NCI-H146 and NCI-N592 to CDDP for 1 h followed by VP16 for 96 h again produced an additive effect. When two of these cell lines were treated in in vivo models (i.p. P388 leukemia, s.c. NCI-N592) with suboptimal doses of the two drugs, a potentiation of the antitumor effects of the two drugs in simultaneous combination was evidenced by the increase in survival time and in the number of 'cures' in P388 leukemia bearing mice and by the inhibition of tumor size in NCI-N592. This comparative study, using the same cell lines in vitro and in vivo, indicates that the CDDP-VP16 potentiation observed in vivo does not reflect a specific interaction at the cellular-biochemical level. The results support the therapeutic interest of this combination (presumably as a result from favorable pharmacological interactions) even despite the lack of potentiation at cellular level, under comparable conditions of treatment.

To investigate the importance of natural killer (NK) and T cells in the inhibition of tumour growth by flavone acetic acid (FAA), colon 26 murine carcinoma was grafted subcutaneously in euthymic and athymic mice. FAA was active in euthymic but not in athymic mice (ratio between tumour weight in treated vs. control animals [T/C %], 27% and 92%, respectively). NK cell activity was increased in both mouse strains, indicating a lack of major involvement of this lymphocyte population in FAA efficacy. In euthymic mice tumour-specific T cells were activated, and after in vivo depletion of lymphocyte subpopulations (L3T4 and Lyt2), tumour inhibition by FAA was abrogated (T/C %, 88%). Antitumour efficacy of FAA was also reduced when the treatment was followed by injection of antitumour necrosis factor alpha (TNF alpha) antibodies. FAA toxicity depended on tumour weight at the time of treatment: 200 mg/kg caused 0 and 100% mortality in mice bearing tumour nodules under 50 and over 300 mg, respectively. When anti-TNF alpha antibodies were given after FAA treatment, the toxicity was greatly reduced (3/14 mice died compared with 10/15).

On the basis of the evidence that vacuolar H(+)-ATPase is implicated in the development of the metastatic phenotype, we have explored the possibility to target the enzyme function in an attempt to control the metastatic behavior of tumor cells. In this study, we used an indole derivative, NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], recently identified as an effective inhibitor of vacuolar H(+)-ATPase, as a potential antimetastatic agent in the treatment of NSCLC H460 xenograft, which is able to induce lung metastases in mice. Oral administration of NiK-12192 caused a significant inhibition of formation of spontaneous metastases. In contrast, the drug exhibited a negligible effect on the development of artificial metastases (i.e., after i.v. injection of tumor cells), thus supporting that the drug affects the early events of the metastatic process (e.g., migration and invasion). Cellular effects are consistent with this interpretation. In conclusion, the available results show for the first time that a vacuolar H(+)-ATPase inhibitor is effective in modulation of the metastatic behavior of a lung carcinoma, supporting its potential therapeutic interest as a novel treatment approach.

A series of water-soluble camptothecins obtained by linking a spermidine moiety to the 21-position of the open form through an amidic bond have been tested for their biochemical and biological activities. Growth inhibition assay on the human non–small cell lung cancer carcinoma NCI-H460 cell line revealed that the camptothecin analogues were less potent than topotecan and SN38 after 1 hour of treatment. The potency increased after 72 hours of exposure, being similar to that of reference camptothecins. The analysis of topoisomerase I–mediated DNA cleavage using the purified enzyme indicated that the novel camptothecin analogues retained ability to poison topoisomerase I and displayed the same cleavage pattern of SN38. Persistence of the DNA cleavage was comparable with that of SN38. Stabilization of the cleavable complex was not the result of hydrolysis of the N-C bond between polyamine and the drug because no free camptothecin was recovered at the end of DNA cleavage in presence of IDN5174, the analogue selected for detailed studies. IDN5174 exhibited an antitumor activity comparable with that of topotecan and irinotecan against NCI-H460 tumor xenograft. The pharmacokinetics in mice showed a favorable disposition in tumor tissue with low amount of camptothecin detectable in plasma and tumor (around 5-10%), thus supporting the efficacy of intact IDN5174. In conclusion, we found that IDN5174 maintained the biological and antitumor properties, in spite of lack of the closed E ring. The available results support the interpretation that the polyamine linked at the 21-position may allow a favorable drug interaction in the ternary complex. (Cancer Res 2006; 66(22): 10976-82)

The vacuolar-H(+)-ATPase, functionally expressed in cell membranes, is known to play a relevant role in intracellular pH regulatory mechanisms, because it is implicated in pumping protons into the extracellular environment or in sequestrating excess protons into acidic vacuolar compartments. Because tumor cells exist in a hypoxic microenvironment and produce acidic metabolites, this regulatory mechanism is recognized as a protective function. This study was designed to investigate the effect of NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], an indole derivative identified as an effective inhibitor of vacuolar-H(+)-ATPase, on the cytotoxic activity of two camptothecins, i.e., topotecan and SN-38 (7-ethyl-10-hydroxycamptothecin, the active metabolite of irinotecan). The cellular studies performed in two pairs of human colon carcinoma cell lines, i.e., LoVo and LoVo/DX (overexpressing P-glycoprotein) and HT29 and HT29/Mit (overexpressing breast cancer resistant protein), indicated an enhancement of the antiproliferative effect of camptothecins by concomitant exposure to subtoxic concentrations of NiK-12192. Studies of subcellular distribution indicated that whereas topotecan alone localized mainly in mitochondria and endoplasmic compartment, the simultaneous presence of NiK-12192 caused a cytoplasmic redistribution. In HT29/Mit cells, NiK-12192 reverted the pattern of acidification induced by topotecan. The potentiation of topotecan efficacy by NiK-12192 was documented by an increased efficacy of the combination in both the HT29 tumor xenografts, being more evident in the topotecan-resistant HT29/Mit tumor. In conclusion, the vacuolar-H(+)-ATPase inhibitor NiK-12192 was able to potentiate the cytotoxic/antitumor effects of camptothecins, either in in vitro or in in vivo systems. Such findings support a potential interest for the use of vacuolar-H(+)-ATPase inhibitors in combination therapy to improve camptothecin efficacy.

A reinvestigation of the fruiting bodies of the mushroom Leucopaxillus gentianeus, allowed the isolation of two minor cucurbitane triterpenes, namely, cucurbitacin D (5) and the new metabolite 16-deoxycucurbitacin B (6). The latter compound lacks an oxygenated substituent at C-16, an unprecedented structural feature among congeners of cucurbitacin B. The cucurbitanes present in the fruiting bodies were compared with those extracted from mycelia grown on the modified Melin-Norkans (MMN) culture medium. Cucurbitacins B (1) and D (5), as well as leucopaxillones A (3) and B (4), were isolated from both sources; in contrast, 16-deoxycucurbitacin B (6) and a mixture of fatty acid esters of cucurbitacin B (2) were absent in the mycelia. A new triterpene, 18-deoxyleucopaxillone A (7), was isolated from the mycelia, but was not detected in the fruiting bodies. The antiproliferative activity of the isolated triterpenes was determined against the NCI-H460 human tumor cell line, in comparison with the antitumor compound topotecan, a well-known topoisomerase I inhibitor.

The realization that position 7 of camptothecin allows several options in chemical manipulation of the drug has stimulated a systematic investigation of a variety of substituents in this position. These efforts resulted in the identification of a novel series of 7-oxyiminomethyl derivatives. Among compounds of this series we have selected a promising lipophilic derivative, gimatecan, for further development. The relevant features of gimatecan are: (i) marked cytotoxic potency, likely related to multiple factors, including a potent inhibition of topoisomerase I, a persistent stabilization of the cleavable complex, an increased intracellular accumulation and a peculiar subcellular localization; (ii) lack of recognition by known resistance-related transport systems; (iii) increased lactone stability and favorable pharmacokinetics; (iv) good oral bioavailability; and (v) an impressive antitumor efficacy in a large panel of human tumor xenografts, with various treatment schedules. Phase I clinical studies with oral administration support the preclinical results of the novel camptothecin. Using different schedules and dosing durations, gimatecan exhibited an acceptable toxicity profile, with myelotoxicity being the dose-limiting toxic effect. An appreciable number of tumor responses was achieved and favorable pharmacokinetics with a very long terminal half-life was observed. The clinical development of gimatecan is currently ongoing, with phase II studies in diverse tumor types (colon, lung, breast carcinoma and pediatric tumors).

The study was conducted to investigate the effects of a novel therapeutic approach, i.e. the combination of chemotherapy and immunotherapy, against a human prostate carcinoma xenograft. A topoisomerase I inhibitor, topotecan, and CpG-containing oligodeoxynucleotides (CpG-ODN) were combined. Athymic mice bearing the PC-3 human prostate carcinoma were treated with the maximum tolerated dose (MTD) of topotecan (3 weekly treatments) and with repeated treatments of CpG-ODN (40 and 20 microg/mouse); tumour growth and lethal toxicity were monitored. Topotecan effect on CpG-ODN-induced production of interleukin (IL) 12, interferon (IFN)-gamma and tumour necrosis factor-alpha was also assessed. Since topotecan pretreatment differentially influenced CpG-ODN-induced production of IL-12 and IFN-gamma, the antitumour effects of the two therapies were investigated in a sequential (full topotecan regimen followed by CpG-ODN) or in an alternating sequence (starting with CpG-ODN). Topotecan inhibited PC-3 tumour growth, inducing 95% tumour volume inhibition. All combined treatments resulted in a significant delay in tumour growth, compared to the effects in topotecan-treated mice (P0.01, by analysis of tumour growth curves). The combination regimens were well tolerated, except for the alternating sequence of 40 microg CpG-ODN and topotecan, which resulted in three out of eight toxic deaths. This alternating sequence was highly toxic even when another cytotoxic drug (doxorubicin) was used in healthy mice. In conclusion, the combination of topotecan and CpG-ODN increased antitumour effects over chemotherapy alone in the growth of a human prostate carcinoma xenograft. Administration sequence was critical to the combination toxicity: the complete regimen of the cytotoxic drug followed by repeated administrations of the immunomodulator seemed the most promising for further investigations.

Camptothecins are cytotoxic agents with a wide spectrum of antitumor activity. The unique mechanism of action, the impressive preclinical efficacy and the clinical success of irinotecan and topotecan have stimulated intensive efforts to identify novel analogues. The development of novel camptothecins was recently rationalized on the basis of the detailed knowledge of mechanism of drug-target interaction and was aimed to overcome the major limitations of these drugs (i.e. lactone ring instability and reversibility of topoisomerase I-DNA cleavage complexes). The development of novel series of analogues (7-substituted camptothecins, silatecans and homocamptothecins) resulted in identification of promising compounds, which are currently in clinical development. Considering the lack of precise correlations between preclinical activity and clinical efficacy of camptothecins, the potential advantages of novel analogs in clinical therapy remains to be documented. However, a rational basis for drug selection and development is now provided by the recognition of major limitations of these agents and by a detailed knowledge of multiple interactions between drug, cellular target and serum albumin. Inhibition of the nuclear enzyme DNA topoisomerase I has proven to be a promising strategy in the design of antitumor agents, in spite of a limited cellular basis of selectivity in cytotoxic action of camptothecins (i.e., overexpression of the target enzyme in tumor cells, and increased sensitivity of proliferating cells). The interest in topoisomerase I as a therapeutic target promoted various efforts to identify other chemotypes effective as topoisomerase inhibitors and chemical/modelling efforts to rationally design specific analogs among known inhibitors. Additional approaches, including drug delivery/formulation, optimization of dose/schedule and route of administration, are expected to improve the therapy with camptothecins and other inhibitors.

Bcl-2 blocks or delays apoptosis in many cell systems. The protein exerts its antiapoptotic effect mainly in the membrane of mitochondria. Indeed, emerging evidence supports that the mitochondrion plays an important role in the cell death pathway, integrating different pro- and antiapoptotic stimuli. Since deregulation of the expression of Bcl-2 occurs in a variety of human tumors, modulation of its function is regarded as an exploitable manipulation for pharmacological intervention in antitumor chemotherapy. Phosphorylation of Bcl-2 has been implicated as an important regulatory mechanism of its function and is a common event in response to antimitotic drugs. Recently, a similar post-transcriptional modification was observed in response to DNA-damaging agents in some tumor systems, but this is not a general finding in response to genotoxic drugs. Current investigations indicate that different signaling pathways may be involved in Bcl-2 phosphorylation, likely dependent on the kinases activated by the various stress stimuli. A better understanding of the molecular mechanisms by which Bcl-2 regulates apoptosis could provide insights for a rational design of approaches to enhance the susceptibility to drug-induced cell death.

Targeting of deregulated protein tyrosine kinases has been proposed as a new approach in the therapeutic intervention against pathological processes including proliferative disorders and cancer. Using a screening approach based on a comparative evaluation of antiproliferative effects in a panel of tumor cells with differential expression of protein tyrosine kinases, three benzoquinoid macrolidic fungal metabolites produced by Clitocybe clavipes, clavilactones A, B, and D (CA, CB, and CD) and two semisynthetic derivatives of these products, diacetyl-CA and dimethyl-CA, were identified as inhibitors of protein tyrosine kinases. Naturally occurring CA, CB, and CD showed inhibitory activity in kinase assays against the Ret/ptc1 and epidermal growth factor receptor (EGF-R) tyrosine kinases, while being less effective against the v-Abl tyrosine kinase and p34cdc2 serine/threonine kinase ( ic 50 2.8, 5.5, 81.3, and 128 μM respectively, for the most potent compound CD). CB was shown to be a non-competitive inhibitor of EGF-R with respect to ATP or poly(Glu6Ala3Tyr). CD also preferentially inhibited the growth of A431 cells, which overexpress a constitutively active EGF-R, as opposed to IGROV-1 and SKOV-3 cells, which express low levels of the receptor. Further, EGF-R was shown to be a target for clavilactones in A431 cells, since EGF-induced receptor autophosphorylation was inhibited in the presence of CB, CD, and diacetyl-CA. Both CD and diacetyl-CA displayed weak activity when administered daily (i.p.) to mice bearing ascitic A431 tumor. These findings indicate that clavilactones represent the prototypes of a new structural class of tyrosine kinase inhibitors deserving further investigation.

The development of drug resistance is a major obstacle to effectiveness of chemotherapeutic treatment of human tumors with cytotoxic agents. Drug resistance is described as a multifactorial phenomenon, involving the expression of defense factors and/or detoxification mechanisms, alterations in drug-target interactions, and cellular response to specific cytotoxic lesions (in particular, DNA damage). Although the proposed mechanisms may contribute to the development of a variable degree of cellular resistance, it is possible that the cell response (i.e., DNA repair or apoptosis) following DNA damage plays a critical role in determining cellular chemosensitivity. The preclinical observations that tumor response to effective drug treatments is associated with induction of apoptosis support the possibility that a decreased susceptibility to apoptosis (apoptosis resistance) is relevant to clinical resistance. A number of molecular alterations associated with transformation and/or tumor progression may also be implicated in regulation of cell death pathways and in the development of drug resistance. There is evidence that the wild-type p53 is involved in cellular response to DNA damage, including cell cycle regulation, DNA repair, and activation of the pathway leading to apoptosis. Loss of wild-type p53 function could cause resistance to DNA-damaging agents, as a consequence of abrogation of p53-dependent apoptosis. The identification of new agents able to trigger p53-independent apoptosis and the search for biochemical modulators downstream of p53 may be of clinical relevance because many tumors are deficient in p53 function due to mutation or deletion. An overview of the resistance mechanisms is presented, with particular reference to the role of p53 mutations in clinical resistance and of apoptosis-related genes in cellular chemosensitivity.

Doxorubicin is a major anticancer agent introduced to extended clinical use in the early 1970s. The fulfillment of a wide program of analogue synthesis led to the development of the better tolerated epirubicin and of a highly potent antileukemic drug, idarubicin. In recent years, on the basis of the available information on the molecular requirements for action, a new synthetic program, coupled with target-oriented pharmacological experiments, was carried out. Various interesting derivatives, namely, the 8- and 10-fluoro compounds and the disaccharides, were obtained. The latter compounds exhibited a strong dependence of biological activity on the orientation (axial vs. equatorial) of the second sugar moiety, daunosamine. A member of this group, namely, 7-O-(4'-O-alpha-L-daunosaminyl-2'-deoxy-alpha-L-fucosyl)-4-demetho xy-adriamycinone, is presently undergoing clinical trials as a third generation antitumor anthracycline.

Topotecan, a camptothecin analogue, is a specific inhibitor of topoisomerase I approved for use in the treatment of patients with refractory ovarian carcinoma. The drug's mechanism of action suggests a potential efficacy of drug combinations incorporating DNA-damaging agents. In an attempt better to define a rational basis for drug combination we examined the effect of topotecan on the cytotoxicity and antitumor activity of cisplatin in an ovarian carcinoma system growing in vitro and in vivo as a tumor xenograft. The in vitro cell system included a cisplatin-sensitive cell line, IGROV-1, and a cisplatin-resistant subline, IGROV-1/Pt0.5, which is characterized by p53 mutation and loss of normal function of the wild-type gene of the parental cell line. This cell system was chosen since the cell sensitivity to DNA-damaging agents appears to be dependent on p53 gene status. Cytotoxicity was assessed by the growth inhibition assay using different schedules: (a) a 1-h period of cisplatin exposure followed by a 24-h topotecan treatment and (b) a 1-h period of simultaneous exposure to cisplatin and topotecan. In the case of the sequential schedule, an additive interaction was observed in IGROV-1 and IGROV-1/Pt0.5 cells. When the simultaneous schedule was used, a synergistic interaction, more evident for the cisplatin-sensitive cells, was found. On the basis of these observations at a cellular level, the effect of concomitant administration of the two drugs (i.e., the most favorable schedule) was studied in the IGROV-1 tumor xenograft, which is moderately responsive to cisplatin and topotecan. Suboptimal doses of each drug (with a low dose of topotecan, 5.1 mg/kg) achieved an antitumor effect comparable with or superior to that of the optimal dose of a single treatment (tumor weight inhibition, 60%), thus indicating a pharmacological advantage of the combination over the single treatment. However, an increase in the topotecan dose (7.1 mg/kg) was associated with an evident increase in the toxicity of the combination, thereby suggesting that the drug interaction was not tumor-specific. Although the molecular basis of the drug interaction is not clear, it is likely that inhibition of topoisomerase I affects the ability of cells to repair cisplatin adducts. Such findings may have pharmacological implications since they suggest the potential clinical interest of topoisomerase I inhibitors in combination with cisplatin.

Lonidamine is an antitumor agent with a peculiar mechanism of action, since it differentially impairs the energy metabolism of normal and neoplastic cells. We investigated the effects of lonidamine on the activity of DNA-damaging antitumor agents against the MX-1 human breast carcinoma xenograft. Athymic mice bearing measurable s.c. tumors were treated by a single injection of doxorubicin (i.v.), cyclophosphamide (i.v.), or cisplatin (i.p.) followed by repeated daily injections of lonidamine (i.p. or p.o.). A potentiation of the activity of all these DNA-damaging drugs was achieved when each was given in combination with lonidamine, but for doxorubicin and cyclophosphamide the increase in antitumor activity paralleled the increase in lethal toxicity. In contrast, a therapeutic advantage of the combination was achieved for cisplatin and lonidamine as compared with cisplatin alone. Indeed, 6 mg/kg of cisplatin plus lonidamine cured all tumors, whereas the maximum tolerated dose of cisplatin alone (12 mg/kg) cured only six of eight tumors. In addition, the study indicated that the duration of lonidamine administration after injection of the cytotoxic drug influenced the tumor response and that prolonged treatment resulted in greater efficacy. These results document the ability of lonidamine to modulate the pharmacological activity of DNA-damaging drugs, thus suggesting that lonidamine may be a clinically useful cisplatin modulator.

Previous studies on structure-activity relationships of anthracycline antitumor antibiotics have shown that removal of the methoxyl group at position 4 of the aglycone causes a marked increase in the potency of the compounds: 4-demethoxydaunorubicin and 4-demethoxyadriamycin had an antitumor effect similar to that of the parent compound at doses five to eight times lower, and they were active even when administered orally. This paper reports the effects of further substitutions at positions 1, 2, 3, and 4 of 4-demethoxy aglycone. The introduction of methyl groups at positions 2 and 3, or 1 and 4 resulted in decreased cytotoxicity and biological activity. The addition of a benzoyl ring at positions 2 and 3 decreased the activity further. 1,4-Dichloro-4-demethoxydaunorubicin and 2,3-dichloro-4-demethoxydaunorubicin were respectively as active and 2.5 times less active than was daunorubicin against HeLa cells in vitro while they were inactive against P388 and L1210 leukemias in vivo. 2,3-Dimethyl-4-demethoxyadriamycin showed an antitumor activity against mouse leukemias that was slightly higher than was that of adriamycin.

Reduced glutathione has been shown to be an effective protector against cisplatin-induced nephrotoxicity of potential clinical value, since it does not reduce antitumor activity of the cytotoxic drug. This paper extends previous observations on the protective potential of reduced glutathione against cisplatin-induced nephrotoxicity, in different rodent models. Following i.v. administration, glutathione protection against cisplatin-induced nephrotoxicity was found to be critically dependent on timing of thiol administration. Whereas the sulfhydryl compound provided almost complete protection in CD rats, the protective effect against toxic renal damage was only partial in mice of different strains. In spite of the modest protection against kidney toxicity, glutathione reduced lethal toxicity in the mouse. Under the same experimental conditions at protective dose levels, the tripeptide thiol did not interfere with the antitumor effectiveness of cisplatin, in any of the tumor models examined. The kidney content of non-protein sulphydryls of CD rats produced by the effective dose of glutathione was markedly higher than that found in the mouse treated with the same dose. This finding is consistent with a differential protection provided by glutathione against cisplatin-induced renal toxicity in these species.

The new anthracyclines 4-O-demethyl-11-deoxydoxorubicin, 4-O-demethyl-11-deoxydaunorubicin along with its 13-dihydro and 13-deoxo analogues are the main components of the anthracycline complex produced by cultures of Streptomyces peucetius var. aureus. They were isolated by solvent partition, separated by column chromatography and characterized by chemical and physical methods. Among these new anthracyclines, displaying antibacterial and cytotoxic activity "in vitro", 4-O-demethyl-11-deoxydoxorubicin and the corresponding daunorubicin analogue were also active against experimental tumors.

Flavone acetic acid (FAA) is a compound with proven activity against various transplantable colon cancers in mice. In this study it was evaluated against primary colon tumours, chemically induced by methylazoxymethanol in outbred CF1 mice. FAA was given i.v. at doses of 70 or 100 or 150 mg kg-1 every 7 days for 6 weeks. Only 4 out of 60 FAA treated mice died of toxicity. FAA reduced tumour number and tumour burden compared to control mice (P less than 0.05 at least), with no apparent dose-response relationship. Antitumour activity of FAA was comparable to that of 5-fluorouracil (5-FU) used as standard. Moreover, FAA was more effective that 5-FU against large tumours. FAA levels in plasma and different tissues (including colonic neoplastic lesions) after a single i.v. dose of 150 mg kg-1 were investigated. Tumour FAA levels appear insufficient to be responsible for the antitumour activity based only on a direct FAA cytotoxic effect. The results confirm clinical interest in FAA and suggest that mechanisms other than direct cytotoxicity may be involved in its activity.

Primary colon tumors of different sizes and malignancy, chemically induced by methylazoxymethanol in outbred CF-1 mice, were used to investigate the antitumor effects of 5-Fluorouracil (5FU) and cis-diammine-dichloroplatinum (DDP), given weekly i.v. as single agents or in combination. When single-drug chemotherapy was tested, DDP showed higher efficacy than 5FU. In fact, in two separate experiments a significant reduction (P less than 0.05) of tumor number (TN) and tumor burden was obtained by treatment with the optimal dose of DDP (4.5 mg/kg per injection) and not by that of 5FU (52 mg/kg). When the two drugs were combined (24-h interval), studies carried out on healthy mice treated weekly i.v. showed a lower toxicity with the same doses given in the sequence 5FU-DDP than in the opposite sequence. The two drugs, delivered in the sequence 5FU followed by DDP, statistically reduced the TN and total tumor burden compared to control mice (P less than 0.05). On the other hand, the same doses in the sequence DDP followed by 5FU did not attain significant tumor reduction. The sequence dependence of the activity and toxicity of the 5FU and DDP combination observed in this experimental model should be taken into account in the design of clinical trials.

4′-Deoxydoxorubicin has been compared with doxorubicin as regards potency, antitumor activity and toxicity in tumored and non-tumored mice treated i.v. according to different schedules. 4′-Deoxydoxorubicin was 1.5–3 times more toxic and more potent than doxorubicin. At equitoxic doses, 4′-deoxydoxorubicin was: as active as doxorubicin against Gross leukemia, mammary carcinoma and MS-2 sarcoma; slightly less active than doxorubicin against B16 melanoma; more active than doxorubicin against colon 38 adenocarcinoma. The best schedule of administration of 4′-deoxydoxorubicin in mice was the weekly treatment. The strong effectiveness against colon 38 adenocarcinoma makes 4′-deoxydoxorubicin a particularly interesting new anthracycline derivative that deserves clinical trials.

This study assessed the antitumor activity of 1-(2-chloroethyl)-3-/2-(dimethylaminosulfonyl)ethyl-1-nitrosourea (TCNU), a newly soluble nitrosourea, on NMU-1 lung tumor, a transplantable murine model poorly sensitive to BCNU. TCNU delivered orally was more effective after weekly than after biweekly treatments. When TCNU was given weekly intravenously, its therapeutic index was higher than after oral treatment. Compared with BCNU, TCNU showed superior antitumor effects on NMU-1 murine lung tumor. In this experimental model, TCNU activity was comparable to that of drugs active on human lung cancer.

Four glycosides, designated A, B, C and D, are the main components of the anthracycline complex produced by cultures of Micromonospora sp. nov. They were extracted by solvent partition, separated by column chromatography and characterized by chemical and physical methods as 11-deoxy analogues of daunorubicin. Among these new anthracyclines, displaying antibacterial and cytotoxic activity in vitro, 11-deoxydaunorubicin and 11-deoxydoxorubicin are also active against P388 leukemia in mice.

4-demethoxydaunorubicin was tested against experimental mouse leukemias, in comparison with doxorubicin and daunorubicin. 4-demethoxydaunorubicin, administered ip to mice bearing ascitic L1210 or F388 leukemia was 5 times more potent than daunorubicin and 10 times more potent than doxorubicin (potency established in relation to optimal antitumor doses). At the optimal doses, 4-demethoxydaunorubicin was as active as daunorubicin and less active than doxorubicin. 4-demethoxydaunorubicin, administered iv was 8 times more potent than daunorubicin and 4–5 times more potent than doxorubicin. At the optimal doses, 4-demethoxydaunorubicin was markedly more active than daunorubicin or doxorubicin in mice injected iv with 105 L1210 leukemia cells (early or late leukemia) or with 102 Gross leukemia cells. In mice given 2 × 106 Gross leukemia cells iv, 4-demethoxydaunorubicin was as active as doxorubicin when treatment was given iv on day 1, or on days 1 to 3 or 1 to 5. The optimal intermittent schedule was treatment on days 1, 5 and 9 for 4-demethoxydaunorubicin, and on days 1, 3 and 6 for daunorubicin and doxorubicin. 4-demethoxydaunorubicin administered orally was highly active against ascitic P388 leukemia and against L1210 and Gross leukemia inoculated iv.

New 4′-C-methyl analogues of daunorubicin, synthesized by the coupling reaction of daunomycinone with 1-chloroderivatives of protected 4-C-methyldaunosamine analogues, were chemically transformed to the corresponding doxorubicin analogues. Their cytotoxic effect against HeLa cells, ability to bind to DNA, and in vivo toxicity and antitumor activity were compared with those of daunorubicin, doxorubicin, and their 4′-O-methyl analogues. The cytotoxic effect of the new anthracyclines could be correlated with their ability to bind to DNA and with their toxicity in experimental animals; however, the antitumor effectiveness did not seem to be related to these parameters. In general all the compounds retained a remarkable antitumor activity at their optimal doses. The most active compound against P388 leukemia was 4′-O-methyldoxorubicin, which was also more active than doxorubicin against L1210 leukemia.

The effect of reduced glutathione on acute lethal toxicity and urotoxicity induced by cyclophosphamide was studied on both mice and rats. The results of this investigation indicate that reduced glutathione is an effective protective agent against bladder damage from treatment with the alkylating agent. The timing of glutathione administration (IV) with respect to cyclophosphamide treatment influenced the uroprotective efficacy of the thiol compound. A schedule-dependent protective effect of glutathione against acute lethal toxicity of the antitumor drug was also observed. This partial protection was accompanied by a reduction in body weight loss following cyclophosphamide treatment. The therapeutic activity of cyclophosphamide on two experimental tumor systems (L1210 and Gross leukemia) was not impaired by combined treatment with glutathione, even at a relatively high dose of glutathione compared with cyclophosphamide.

A new analog of doxorubicin, 4′-O-methyldoxorubicin, was previously reported to have a pronounced activity agianst L1210 leukemia, which shows a natural partial resistance to doxorubicin itself. In the present study, lines of P388 leukemia and Ehrlich ascites tumor with acquired resistance to doxorubicin were found to be cross-resistant to 4′-O-methyldoxorubicin, indicating that the natural and the acquired resistance to doxorubicin involve different mechanisms. In vitro studies on the uptake of 4′-O-methyldoxorubicin in the Ehrlich ascites tumor cells indicated that the observed cross-resistance was partly due to a decreased drug uptake in the resistant cells because of an increased extrusion of the drug, in accordance with previous findings on the mechanism of acquired resistance to doxorubicin.

This paper describes the effect of Tween 80 on the antitumor activity and on the distribution of adriamycin in mice. The dilution of adriamycin in a 10% water solution of Tween 80 produced a significant increase of the antitumor activity in mice against ascites tumors (L1210 leukemia), disseminated leukemias (transplanted leukemias originally induced by Gross leukemia virus and Moloney leukemia virus), and solid tumors (Sarcoma 180, MS-2 sarcoma). In all these experiments the drug was administered i.v., according to different schedules. Higher antitumor activity at the optimal dose and an increase of activity at lower doses were observed in different experimental systems. Toxicity was also slightly enhanced. Tissue distribution was studied in normal mice and in tumor-bearing mice (Gross leukemia and MS-2 sarcoma). In animals given i.v. adriamycin diluted in 10% Tween 80 there was a higher drug concentration in spleen, lung and kidney than there was in mice given the drug in a water solution. In all the other organs examined (heart, liver, small intestine) and in the MS-2 tumor tissue, no significant increase was observed. In L1210 leukemia-bearing mice, i.p. treatment with adriamycin diluted in 10% Tween 80 resulted in a significantly higher toxicity than that which resulted from treatment with adriamycin in a water solution; no increase of antitumor activity was observed.

A mutant strain of Streptomyces peucetius produced an anthracycline antibiotic whose structure has been established to be 4-O-demethyl-13-dihydrodaunorubicin (4), by application of spectroscopic methods and chemical degradation. A new synthesis of 4-O-demethyl-daunorubicin (carminomycin I, 2) starting from daunomycinone, together with the comparison of the antitumor activity of the anthracycline glycosides 2 and 4 are also reported.