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Since activation of p53 in response to cytotoxic stress may have proapoptotic or protective effects depending on the nature of the injury, inhibitors of p53 may have therapeutic interest as modulators of chemotherapy toxicity or efficacy. In an attempt to identify novel p53 inhibitors, a quality collection of compounds structurally related to pifithrin-β were designed and synthesized as potential inhibitors of p53. The biochemical and biological evaluations supported that compounds of the tetrahydrobenzothiazole series were inhibitors of the p53 transcriptional activity and were effective in enhancing paclitaxel-induced apoptosis. In contrast, in spite of the increased cytotoxic potency, selected compounds of the benzothiazole series were not able to modulate the transcriptional activity of p53, as indicated by lack of change of p21 expression. The therapeutic interest of the compounds of the former series in combination with taxanes was confirmed in a human tumor xenograft model., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Preclinical studies support the therapeutic potential of histone deacetylases inhibitors (HDACi) in combination with taxanes. The efficacy of combination has been mainly ascribed to a cooperative effect on microtubule stabilization following tubulin acetylation. In the present study we investigated the effect of paclitaxel in combination with two novel HDACi, ST2782 or ST3595, able to induce p53 and tubulin hyperacetylation. A synergistic effect of the paclitaxel/ST2782 (or ST3595) combination was found in wild-type p53 ovarian carcinoma cells, but not in a p53 mutant subline, in spite of a marked tubulin acetylation. Such a synergistic interaction was confirmed in additional human solid tumor cell lines harboring wild-type p53 but not in those expressing mutant or null p53. In addition, a synergistic cytotoxic effect was found when ST2782 was combined with the depolymerising agent vinorelbine. In contrast to SAHA, which was substantially less effective in sensitizing cells to paclitaxel-induced apoptosis, ST2782 prevented up-regulation of p21(WAF1/Cip1) by paclitaxel, which has a protective role in response to taxanes, and caused p53 down-regulation, acetylation and mitochondrial localization of acetylated p53. The synergistic antitumor effects of the paclitaxel/ST3595 combination were confirmed in two tumor xenograft models. Our results support the relevance of p53 modulation as a major determinant of the synergistic interaction observed between paclitaxel and novel HDACi and emphasize the therapeutic interest of this combination.

The synthetic atypical retinoids containing an adamantyl group exhibit antiproliferative or proapoptotic activities. Apoptosis induction is a dose-dependent effect independent of retinoid receptors. We have reported that induction of apoptosis by the atypical retinoid, ST1926, is associated with early manifestations of genotoxic stress. Indeed, in this study performed in ovarian carcinoma cells, we show that exposure to ST1926 resulted in an increase of early markers of DNA damage, including ATM and H2AX phosphorylation. In addition, we found that a novel histone deacetylase (HDAC) inhibitor (RC307) was able to enhance sensitivity of ovarian carcinoma cells to ST1926. Under conditions where single-agent treatment caused only antiproliferative effects, the combination of the atypical retinoid and HDAC inhibitor resulted in marked apoptotic cell death with a more rapid onset in wild-type p53 ovarian carcinoma cells. The sensitization to ST1926-induced apoptosis was associated with an enhanced DNA damage response, because a prolonged expression of DNA damage markers (e.g., H2AX, p53 and RPA-2 phosphorylation) and a marked activation of DNA damage checkpoint kinases (in particular, phosphorylation of Chk1) were observed indicating an accumulation of DNA damage by the ST1926/HDAC inhibitor combination. The study provides additional support to the role of DNA damage as a primary event leading to the activation of apoptosis in ovarian carcinoma cells by adamantyl retinoids and documents the potential therapeutic efficacy of the combination of ST1926 and HDAC inhibitors of the novel series.

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<0.005) and completely controlled ascites growth without apparent toxicity, although a disorganization of lymphoid organs was observed. Bio-plex assay of cytokine levels in peritoneal fluid of ascites-bearing mice after CpG-ODN treatment revealed an increase in interleukin (IL)-6, IL-10, IL-12, and interferon-gamma at 24 hours, which returned to control mice levels at 48 to 96 hours, whereas the high levels of angiogenic factors remained unchanged. Depletion of natural killer or monocytes/macrophages only slightly influenced the CpG-ODN-induced reduction of ascites tumor cells, indicating that the antitumor activity might not be related to a specific cell/cytokine but rather to the repertoire of cells and cytokines accumulated in the peritoneal cavity. Thus, our data suggest a relevant role for repeated activation of cells and cytokines of innate immunity in the therapy of ovarian cancer patients with malignant ascites.

The 3He(alpha,gamma)7Be reaction presently represents the largest nuclear uncertainty in the predicted solar neutrino flux and has important implications on the big bang nucleosynthesis, i.e., the production of primordial 7Li. We present here the results of an experiment using the recoil separator ERNA (European Recoil separator for Nuclear Astrophysics) to detect directly the 7Be ejectiles. In addition, off-beam activation and coincidence gamma-ray measurements were performed at selected energies. At energies above 1 MeV a large discrepancy compared to previous results is observed both in the absolute value and in the energy dependence of the cross section. Based on the available data and models, a robust estimate of the cross section at the astrophysical relevant energies is proposed.

A series of hydroxamic acid-based histone deacetylase (HDAC) inhibitors were designed on the basis of a model of the HDAC2 binding site and synthesized. They are characterized by a cinnamic spacer, capped with a substituted phenyl group. Modifications of the spacer are also reported. In an in vitro assay with the isoenzyme HDAC2, a good correlation of the activity with the docking energy was found. In human ovarian carcinoma IGROV-1 cells, selected compounds produced significant acetylation of p53 and alpha-tubulin. Most compounds showed an antiproliferative activity comparable to that of SAHA. At equitoxic concentrations, the tested compounds were more effective than SAHA in inducing apoptotic cell death. Compounds selected for in vivo evaluation exhibited a significant antitumor activity on three tumor models at well tolerated doses, thus suggesting a good therapeutic index.

Purpose: Oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) are potent activators of innate and adaptive immunity. Recognition of CpG-ODN is mediated by Toll-like receptor 9 expressed by immune cells, endothelial and epithelial cells, and fibroblasts. We examined the antitumor effect of CpG-ODN and the role of administration route on human ovarian cancers growing in the peritoneal cavity of nude mice., Experimental Design: Mice implanted i.p. with human ovarian carcinoma cells were treated i.p., s.c., or i.v. and assessed for survival and tumor-free incidence. Peritoneal washings were analyzed for keratinocyte chemokine production and for functional and phenotypic profiles as indicators of the cell types involved in mediating the antitumor effects., Results: IGROV-1-bearing mice treated i.p. survived significantly longer than those treated i.v. or s.c. (P=0.0005), and nearly half of them (8 of 17) were tumor-free by the end of the experiment, a rate never achieved using a variety of chemotherapeutic drugs. High rates of tumor-free mice were observed in three other ovarian tumor xenografts treated i.p. Compared with peritoneal washings of mice treated s.c. or i.v., those from mice treated i.p. showed the highest level of serum and tissue keratinocyte chemokine, the highest number of natural killer cells and neutrophils, and the highest antiproliferative activity in vitro., Conclusions: The superior antitumor effect obtained by locoregional administration of CpG-ODN in i.p. tumor-bearing mice with a limited adaptive immune response points to the importance of innate effector cells amplification at the site of tumor growth and suggests the promise of i.p. CpG-ODN in clinical trials for ovarian cancer.

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.

ST1968, a novel hydrophilic camptothecin analogue of the 7-oxyiminomethyl series, is characterised by the formation of stable DNA-topoisomerase I cleavable complex and by a promising profile of antitumour activity. The present study was designed to extend preclinical evaluation of the novel camptothecin in human squamous cell carcinoma (SCC) models. ST1968 exhibited an impressive activity with a high cure rate in SCC models. ST1968 produced 100% of complete response without evidence of regrowth in tumours characterised by susceptibility to drug-induced apoptosis (FaDu, A431 and A2780). In contrast to irinotecan, ST1968 still showed an excellent, persisting activity in models less susceptible to apoptosis induction (KB, Caski and SiHa), in which drug treatment elicited a persistent DNA damage response, as documented by phosphorylation of p53, RPA-2 and histone H2AX, resulting in delayed apoptosis and senescence. This behaviour was associated with a marked cellular/tumour drug accumulation. In conclusion, ST1968 exhibited an outstanding antitumour activity superior to that of irinotecan against SCC. A high intracellular accumulation, resulting in fast apoptosis or DNA damage persistence, appeared to be a critical determinant of SCC sensitivity to ST1968.

In contrast to five-membered E-ring analogues, 7-oxyiminomethyl derivatives of homocamptothecins showed ability to form stable ternary complexes with DNA and topoisomerase I. The 7-oxyiminomethyl derivatives of homocamptothecins were evaluated as a racemic mixture. Following the isolation of the two enantiomers, the 20 (R)-hydroxy isomer confirms the best activity. By using a panel of human tumor cells, all tested homocamptothecins showed a potent antiproliferative activity, correlating to the persistence of the cleavable complex. No significant difference was observed between the natural scaffold and the corresponding homocamptothecin homologue. A selected compound of this series exhibited an excellent antitumor activity against human gastrointestinal tumor xenografts.

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%, p < 0.05), in contrast to intermittent treatment-gimatecan (1.5 mg/kg per os, q4d) or intravenous gemcitabine (125 and 128 T/C%, respectively, p > 0.1). 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 (P < 0.001 versus control mice), significantly superior to those of the single agent-treated mice (p < 0.05 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.

Background: The novel adamantyl retinoid ST1926 is a potent inducer of apoptosis in ovarian carcinoma cells. Since the pro-apoptotic effect is associated with activation of p53, in this study we have investigated the efficacy of combination of ST1926 with cisplatin, a DNA-damaging agent that is known to induce p53-dependent apoptosis., Materials and Methods: The efficacy of ST1926 and its combination with cisplatin was evaluated in human ovarian carcinoma models, including resistant tumors., Results: Oral treatment with ST1926 alone caused a marginal tumor growth inhibition (<50%), but the combination with cisplatin resulted in an improved efficacy, most evident in terms of tumor growth delay without a substantial increase of toxicity. The combination therapy achieved the best effects against the HOC18 ovarian carcinoma tumor, resulting in an appreciable number of animals without evidence of disease at the end of the experiment. In contrast to the marginal effect of ST1926 alone against the subcutaneous-growing tumors, loco-regional (intraperitoneal) treatment achieved a marked increase of survival of animals with ascitic IGROV-1 tumor., Conclusions: The present results document the efficacy of the combination of cisplatin with ST1926 and provide a rational basis for the design of novel, well-tolerated platinum-based treatment approaches in human ovarian carcinoma.

Targeting of human telomerase reverse transcriptase (hTERT) by different small interfering RNAs (siRNAs) resulted in a variable degree of telomerase activity inhibition in PC-3 and DU145 prostate cancer cells. In addition, transfection with siRNA5 and siRNA41, which caused high levels ( approximately 80 and approximately 55%, respectively) of enzyme activity inhibition in both cell lines, led to a marked reduction of hTERT mRNA and protein expression and a significant inhibition of cell proliferation within a few days, without concomitant telomere shortening or telomeric 3' overhang impairment. Such an antiproliferative effect was not ascribable to the activation of non-specific responses, since siRNA5 and siRNA41 did not induce the expression of 2'-5' oligoadenylate synthetase-1 and were able to cause a significant growth impairment also in HCT 116 colon cancer cells, which have a defective interferon pathway. Cell growth inhibition was indeed associated with hTERT down-regulation, as it was almost completely rescued in siRNA-treated HCT 116 cells co-transfected with an hTERT-expressing vector. Moreover, siRNA5 and siRNA41 failed to affect the proliferation of hTERT-negative U2-OS osteosarcoma cells. Interestingly, transfection with siRNA5 significantly reduced the tumorigenic and growth potential of PC-3 cells when xenotransplanted into nude mice. Such data suggest siRNA-mediated hTERT down-regulation as an efficient strategy to impair prostate cancer cell growth.

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.

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.

A number of derivatives of camptothecin with a polyamine chain linked to position 7 of camptothecin via an amino, imino, or oxyiminomethyl group were synthesized and tested for their biological activity. All compounds showed marked growth inhibitory activity against the H460 human lung carcinoma cell line. In particular, the iminomethyl derivatives where the amino groups of the chain were protected with Boc groups exhibited a high potency, with IC50 values of approximately 10(-8) M. The pattern of DNA cleavage in vitro and the persistence of the cleavable ternary complex drug-DNA-topoisomerase I observed with polyamine conjugates containing free amino groups support a contribution of specific drug interaction with DNA as a determinant of activity. Modeling of compound 7c in the complex with topoisomerase 1 and DNA is consistent with this hypothesis. The lack of a specific correlation between stabilization of the cleavable complex and growth inhibition likely reflects multiple factors including the cellular pharmacokinetic behavior related to the variable lipophilicity of the conjugate, and the nature and linkage of the polyamine moiety.

The anti-apoptotic protein Bcl-2 has been implicated in the intrinsic resistance of melanoma to chemotherapy. The aim of this study was to investigate the effects of anti-Bcl-2 oligonucleotide oblimersen on the antitumour activity of gimatecan, a novel lipophilic camptothecin currently undergoing clinical phase II studies. Results showed a reduced sensitivity of melanoma cells to gimatecan following Bcl-2 transfection and inversely, increased cell sensitivity to gimatecan in combination with oblimersen. In in vivo studies performed in two melanoma xenografts expressing different Bcl-2 levels, the antitumour activity of oblimersen itself was modest, but the combination with gimatecan produced a significant therapeutic advantage. The combination therapy inhibited tumour growth and delayed regrowth of the two tumours tested. The enhancement of antitumour activity was observed at doses that were tolerated well. The effects of oblimersen on antitumour activity and toxicity of gimatecan were dose-dependent. The capability of oblimersen to improve the efficacy of gimatecan supports the therapeutic potential of the drug combination in the treatment of human melanoma.

To investigate the cellular/molecular basis of the activity of a novel lipophilic camptothecin, gimatecan (ST1481), against slowly proliferating cells, we performed a comparative study of topotecan and gimatecan in human bladder cancer models (HT1376 and MCR). Gimatecan was significantly more effective than topotecan in inhibiting the growth of HT1376 tumor, thus reflecting antiproliferative potency. In both HT1376 and MCR cells, gimatecan caused a persistent S-phase arrest, indicating an efficient DNA damage checkpoint. This response was consistent with a cytostatic effect, because no evidence of apoptosis was detected. In contrast to gimatecan, topotecan at equitoxic concentrations caused an early and persistent downregulation of topoisomerase I. Modulation of protein level could not be solely ascribed to the proteasome-mediated degradation of the enzyme because the proteasome inhibitor PS341 sensitized MCR but not HT1376 cells to camptothecins, suggesting alternative mechanisms of drug-induced topoisomerase I downregulation. Indeed, the two camptothecins caused a differential inhibition of topoisomerase I transcription, which is more marked in topotecan-treated cells. The HT1376 model was more sensitive to this immediate decrease of mRNA level. Our data document a marked antitumor activity of gimatecan against a bladder carcinoma model. A limited downregulation of topoisomerase I by gimatecan provides additional insights into the cellular basis of drug potency.

Purpose: Gimatecan, a novel oral lipophilic camptothecin characterized by favorable features at molecular/cellular level and by a promising profile of preclinical activity, is currently in clinical phase I/II. The aim of the study was to additionally investigate the therapeutic potential of the drug in human tumor xenografts growing in different organs as models representative of tumor growth in the clinical setting., Experimental Design: The models include two orthotopic central nervous system tumors, two melanomas growing intracranially, and an ovarian carcinoma growing i.p. In addition, gimatecan was tested against experimental lung metastases of two tumor types (lung and ovarian carcinomas). Gimatecan was delivered by oral gavage according to various schedules (daily or intermittent). The time (in days) mice required to show evident signs of disease was used as end point for drug efficacy., Results: Gimatecan was highly effective in delaying disease manifestations in all tumor systems investigated. In the intracranially growing tumors, a significant time increase (versus control mice) was achieved by the drug administered according to all of the schedules. In addition, almost all treated mice were alive and tumor-free at the end of the experiment in the metastatic models and in the ascitic ovarian tumor. The daily prolonged treatment schedule was the best one., Conclusions: In all tumor systems investigated, including orthotopic tumor growth models and lung metastases, the oral administration of gimatecan showed a therapeutic benefit in terms of survival increase. The good oral availability allowed a prolonged daily treatment regimen, which seems the most promising to exploit the therapeutic potential of the drug.

The ability of melanoma cells to evade engagement of apoptosis plays a significant role in their resistance to chemotherapy. In an attempt to lower the apoptotic threshold of melanoma cells as a possible strategy to increase their drug sensitivity, we generated a hammerhead ribozyme to down-regulate the expression of the anti-apoptotic protein survivin. The JR8 human melanoma cell line was stably transfected with the active ribozyme RZsurv (targeting the 3' end of the GUC294 triplet in the exon 3 of the survivin mRNA) or the catalytically inactive ribozyme mutRZsurv (carrying a mutation in the catalytic core of RZsurv). Two polyclonal cell populations expressing the active (JR8/RZsurv) or the mutant (JR8/mutRZsurv) ribozyme were selected for the study. JR8/RZsurv cells were characterized by a markedly lower survivin protein level than JR8 parental cells, whereas a negligible reduction in survivin expression was observed in JR8/mutRZsurv cells. JR8/RZsurv cells showed a significantly increased sensitivity to the topoisomerase-I inhibitor topotecan (as detected by clonogenic cell survival) compared with JR8/mutRZsurv cells. Moreover, the extent of drug-induced apoptosis (in terms of percentage of apoptotic nuclei and level of caspase-9 and caspase-3 catalytic activity) was significantly greater in JR8/RZsurv than in JR8/mutRZsurv cells. Finally, an increased antitumor activity of oral topotecan was observed in JR8/RZsurv cells grown as xenograft tumors in athymic nude mice compared with JR8/mutRZsurv cells. These results demonstrate that attenuation of survivin expression renders human melanoma cells more susceptible to topotecan-induced apoptosis and more responsive to in vivo treatment, and support the concept that survivin is an attractive target for new therapeutic interventions in melanoma.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family, which has been implicated in inhibition of apoptosis and control of mitotic progression. The finding that survivin is overexpressed in most human tumors but absent in normal adult tissues has led to the proposal of survivin as a promising therapeutic target for anticancer therapies. We decided to evaluate the effects of a ribozyme-based strategy for survivin inhibition in androgen-independent human prostate cancer cells. We constructed a Moloney-based retroviral vector expressing a ribozyme targeting the 3' end of the CUA(110) triplet in survivin mRNA, encoded as a chimeric RNA within adenoviral VA1 RNA. Polyclonal cell populations obtained by infection with the retroviral vector of two androgen-independent human prostate cancer cell lines (DU145 and PC-3) were selected for the study. Ribozyme-expressing prostate cancer cells were characterized by a significant reduction of survivin expression compared to parental cells transduced with a control ribozyme; the cells became polyploid, underwent caspase-9-dependent apoptosis and showed an altered pattern of gene expression, as detected by oligonucleotide array analysis. Survivin inhibition also increased the susceptibility of prostate cancer cells to cisplatin-induced apoptosis and prevented tumor formation when cells were xenografted in athymic nude mice. These findings suggest that manipulation of the antiapoptotic survivin pathway may provide a novel approach for the treatment of androgen-independent prostate cancer.

ST1481 (gimatecan) is a novel lipophilic camptothecin with a promising preclinical pharmacological profile. On the basis of its high antitumor efficacy when delivered by the oral route, the compound is suitable for prolonged administration. This schedule of treatment has been reported as the most appropriate to exploit the antiangiogenic effects of cytotoxic drugs. The aim of the study was to investigate the antiangiogenic and antitumor effects of oral ST1481 in human tumor xenografts. In spite of a marginal drug effect against the s.c. growing A549 lung carcinoma following administration with an intermittent schedule (q4dx4 times, maximum tolerated dose: 2 mg/kg), tumor growth was strongly inhibited by a daily low-dose (0.5 mg/kg) prolonged administration. Immunohistochemical analysis showed a reduced number of microvessels in tumors of both treated groups versus controls and a significantly higher reduction in the daily versus the q4dx4-treated tumors (P < 0.0001, by Student's t test). In our experimental model, the relation between microvessel density and tumor size (r = 0.738, by the Spearman rank test) suggests a role of inhibition of tumor vasculature in tumor response. Significant inhibition of tumor angiogenesis (P < 0.0001 versus control tumors) was observed even with a very low drug dose (0.06 mg/kg) in the orthotopically implanted (i.d.) MeWo melanoma, under conditions causing minimal tumor growth inhibition. Additional evidences of the antiangiogenic activity of ST1481 were provided by antimotility effects on endothelial cells, in vivo inhibition of vascularization in the Matrigel assay, and down-regulation of the expression of the proangiogenic basic fibroblast growth factor in A549 tumor cells associated with inhibition of the pathway involving Akt. In conclusion, the available results support the possibility that the antiangiogenic properties of ST1481 contribute to its antitumor potential and that this effect might be enhanced by the continuous low-dose treatment.

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.

Purpose: ST1481 is the lead compound of a novel series of 7-modified camptothecins, the 7-oxyimino methyl derivatives, characterized by potent topoisomerase I inhibition and cytotoxic activity. Based on its therapeutic efficacy in a human non-small cell lung carcinoma model and its favorable pharmacological profile, the novel analogue was selected for further preclinical development., Experimental Design: We investigated the growth-inhibitory effects of ST1481 and topotecan, used as a reference compound, in a panel of human tumor cell lines of various tumor types (ovarian carcinoma, glioblastoma, osteosarcoma, and melanoma), including sublines with acquired resistance to cisplatin. We explored the antitumor efficacy in a large panel of human tumor xenografts, with particular reference to intrinsically resistant tumor types, using oral administration and an intermittent treatment schedule., Results: ST1481 showed a potent antiproliferative activity with comparable effects in all tested cell lines. Only U-87-MG glioma cells were less sensitive, presumably as a consequence of the efficiency of the S-phase checkpoint. ST1481 produced a remarkable antitumor effect (tumor volume inhibition > 85%) in 16 of 18 examined models, with an appreciable rate of complete tumor regressions in 11 of 18 models (despite the nonoptimal intermittent treatment schedule). The most impressive antitumor effects were observed against lung carcinoma, melanoma, and osteosarcoma models, as documented by the high rate of complete responses (up to 100%). The efficacy of ST1481 was significantly superior to that of topotecan in 9 of 17 tumors. The novel drug was also markedly effective against slowly growing tumors (A549 lung carcinoma and HT29 colon carcinoma) when a daily protracted treatment was used to fully exploit the therapeutic potential of camptothecins., Conclusions: The unusual potency of ST1481 in a variety of tumor cell lines suggests the ability of the drug to overcome several resistance factors. The profile of antitumor efficacy further supports the therapeutic interest in the novel analogue and provides a rational basis for clinical evaluation in selected tumor types.

The aim of this study was to evaluate the response of ovarian cancer to intralesionally administered topotecan. Preliminary experiments were carried out in nude mice subcutaneously grafted with three different human ovarian carcinoma cells (A2780, IGROV/DDP and SKOV-3). Topotecan was administered intravenously (i.v.: 10-15 mg/kg every 4th day for 4 times) or intralesionally (i.t.: single dose of 15-20 mg/kg) and tumor size changes/drug toxicity were evaluated. The results indicate that the sensitivity of the three tumor models was different (rank: A2780 > IGROV/DDP > SKOV-3) but, for each tumor line, the pattern of response was similar after i.v. and i.t. administration. No local toxicity was detected, but appreciable systemic toxicity (animal death rate) was observed in spite of the use of a single i.t. dose. The effects of intralesional topotecan administration were then assessed in a patient with an advanced, epithelial ovarian tumor (endometroid type, poorly differentiated histologic grade), already treated with cisplatin and paclitaxel. The treatment (7.5 mg/m(2)) was repeated three times and, although drug plasma levels were in the range generally reported following i.v. administration and typical systemic toxicity occurred, no tumor regression was observed and the patient died 14 months later. We conclude that the intralesional drug delivery is effective to achieve a rapid tumor shrinkage in large tumor lesions, but in the presence of drug resistance, either intrinsic or acquired, intratumor drug administration can not be recommended.

Relevant drawbacks of the molecular structure and mechanism of the action of camptothecins are the instability of the E ring lactone and the reversibility of drug-target interaction. Such features are expected to limit the clinical efficacy of conventional camptothecins. In an attempt to overcome these limitations and to improve the pharmacological profile of camptothecins, a novel series of seven modified lipophilic analogues was synthesized based on the hypothesis that lipophilicity could promote a rapid cellular accumulation and stabilization of drug-target interaction. A novel analogue (ST1481) of the series, characterized by a potent antitopoisomerase and cytotoxic activity, was selected for preclinical development. A detailed preclinical study of ST1481 was performed in the H460 non-small cell lung tumor model using oral administration and various treatment schedules. Under all of the conditions, ST1481 exhibited an impressive efficacy in terms of tumor growth inhibition (tumor volume inhibition percentage > 99%), log(10) cell kill, rate of complete responses (including "cures"), and an improvement of the therapeutic index compared with topotecan (used as the reference drug). The cytotoxic potency was also reflected by the in vivo potency, because the drug activity was observed at doses as low as 0.25 mg/kg with the daily schedule. In contrast to topotecan, no cross-resistance to ST1481 was found in ovarian carcinoma cells overexpressing P-glycoprotein (A2780/DX). A similar trend in the improvement of activity was also observed in the same tumor model growing in vivo with a 100% rate of complete tumor regressions. A rapid intestinal absorption and good oral bioavailability were supported by in vivo distribution studies, because the peak values of drug accumulation were found from 1 to 2 h after administration. The relevant liver accumulation may account for a marked effect of ST1481 against liver metastases induced by the ovarian carcinoma IGROV-1. In conclusion, the results support the hypothesis that a potent lipophilic camptothecin with a proper substituent at the position 7 may have therapeutic advantages likely related to a rapid intracellular uptake and tissue distribution, stabilization of the drug-target complex, and good oral bioavailability. Overall, the results support the preclinical interest of ST1481 in terms of efficacy, potency, toxicity profile, and ability to overcome multidrug resistance.

In an attempt to synthesize potential anticancer agents acting by inhibition of topoisomerase I (Topo I) a new series of oxyiminomethyl derivatives in position 7 of camptothecin (CPT) was prepared. The synthesis relied on the condensation of 20S-CPT-7-aldehyde or 20S-CPT-7-ketones with alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl O-substituted hydroxylamines. The compounds were tested for their cytotoxic activity in vitro against H460 non-small lung carcinoma cell line, the activity being for 24 out of 37 compounds in the 0.01-0.3 microM range. A QSAR analysis indicated that lipophilicity is the main parameter correlated with cytotoxicity. Investigation of the DNA-Topo I-drug cleavable complex showed a rough parallelism between cytotoxicity and inhibition of Topo I. Persistence of the DNA cleavage after NaCl-mediated disruption of the ternary complex suggests that for the most potent compounds, e.g., 15, the cytotoxicity was at least in part related to stabilization of the complex, as also supported by the persistence of the DNA-enzyme complex in drug-treated cells. The in vivo antitumor efficacy of the most potent analogue (15) was evaluated in direct comparison with topotecan using human lung tumor xenograft models. In the range of optimal doses (2-3 mg/kg), the improved efficacy of 15 was documented in terms of inhibition of tumor growth and rate of complete response.

We selected a mitoxantrone-resistant HT29 colon carcinoma cell line (HT29/MIT) that exhibited a very high degree of resistance to the selecting agent and marked resistance to topotecan and SN38, but limited resistance to doxorubicin. The development of drug resistance was independent of expression of P-glycoprotein or multidrug resistance-associated protein but was associated with high up-regulation of the breast carcinoma resistance protein (BCRP) as shown by Western blot analysis. BCRP overexpression was associated with a reduced intracellular accumulation of topotecan, a known substrate for BCRP. Conversely, a lipophilic 7-modified camptothecin analogue (ST1481) displayed a complete lack of cross-resistance in HT29/MIT cells, suggesting that the drug was not a substrate for BCRP because no defects in intracellular accumulation were found. This conclusion is consistent with the antitumor efficacy of ST1481 against a BCRP-expressing tumor. These results may have therapeutic implications because the antitumor efficacy of ST1481 is in part related to a good bioavailability after oral administration, and the drug is currently under Phase I clinical evaluation.

A series of new 7-iminomethyl derivatives of camptothecin were obtained from camptothecin-7-aldehyde and aromatic, alicyclic and aliphatic amines. Their hydrogenation led to the corresponding amines. All the imines and the less polar amines showed a marked increase of the cytotoxic activity against H460 non-small lung carcinoma cell line, with respect to topotecan. The lipophilicity of the substituent in position 7 of camptothecin seems to play an important role for cytotoxic potency. The 7-phenyliminomethyl derivative showed efficacy comparable to topotecan in vivo against NSCLC H460 xenografted in athymic nude mice.

MEN10755 is a disaccharide analogue of doxorubicin (DXR) endowed with a broader spectrum of activity compared with DXR in a panel of human tumour xenografts. In an attempt to investigate the pharmacological basis of the improvement of therapeutic efficacy of the analogue, a comparative pharmacokinetic (tissue and tumour distribution) and pharmacodynamic (antitumoral activity and ability to induce apoptosis) study of MEN10755 and DXR was performed in athymic nude mice bearing a human ovarian carcinoma xenograft (A2780). Drug level was quantified by high performance liquid chromatography (HPLC) with fluorimetric detection after a single intravenous (i.v.) injection of 7 mg/kg of MEN10755 or DXR. The results indicated a reduced accumulation of MEN10755 compared with DXR in all tissues investigated (tumour, heart, kidney and liver). The reduction was more marked in normal than tumour tissues. Moreover, in spite of the reduced drug uptake by tumour tissues, the new disaccharide anthracycline given in its optimal regimen showed an enhanced antitumour efficacy, compared with DXR. The drug effects on tumour growth paralleled a marked activation of apoptosis. In conclusion, the pattern of tissue distribution and the pharmacokinetic behaviour were consistent with a better tolerability of the novel analogue which allowed a higher cumulative dose to be delivered. The superior therapeutic efficacy of the analogue over DXR, in spite of a reduced tumour accumulation, supports an increased tumour selectivity.

Cellular resistance to anthracyclines is a major limitation of their clinical use in the treatment of human tumors. Resistance to doxorubicin is described as a multifactorial phenomenon involving the overexpression of defense factors and alterations in drug-target interactions. Such changes do not account for all manifestations of drug resistance, in particular intrinsic resistance of solid tumors. Since anthracyclines can induce apoptotic cell death, an alternative promising approach to drug resistance has focused on the study of cellular response to drug-induced DNA damage, with particular reference to the relationship between cytotoxicity/antitumor efficacy and apoptotic response. The evidence that a novel disaccharide analog (MEN 10755), endowed with an improved preclinical activity over doxorubicin, was also more effective as an inducer of apoptosis provided additional insights to better understand the cellular processes that confer sensitivity to anthracyclines. Although the presence or alteration of a single apoptosis-related factor (e.g., p53, bcl-2) is not predictive of the sensitivity/resistance status, the complex interplay among DNA damage-activated pathways is likely an important determinant of tumor cell sensitivity to anthracyclines

The natural alkaloid camptothecin is the lead compound of a new class of antitumor agents with a unique mechanism of action (i.e. inhibition of DNA topoisomerase I). The pharmacological interest of these agents has generated a large number of derivatives and analogues endowed with potent cytotoxic activity, two of them being in clinical use as antitumor drugs. We have synthesized a new series of camptothecins substituted in position 7 with an alkyl or alkenyl chain bearing cyano and/or carbethoxy groups. These compounds showed potent cytotoxic activity in vitro against the human non-small-cell lung carcinoma H460 cell line, most of them exhibiting IC(50) values in the 0.05-1 microM range, more active than topotecan used as a reference compound. In particular 7-cyano-20S-camptothecin (5a) showed high in vitro cytotoxicity against a topotecan-resistant H460 cell subline (H460/TPT) and a cisplatin-resistant ovarian carcinoma subline (IGROV-1/Pt 1). In an in vivo evaluation of the antitumor activity, 5a appeared significantly more effective than topotecan in the H460 tumor model and comparable with topotecan in a small-cell lung carcinoma model and a colon carcinoma model. The efficacy and good tolerability of this compound increase interest for further preclinical development.

On the basis of their mechanism of action (cell killing during DNA replication) and the potential reversibility of the drug effects, protracted therapy with camptothecins is reported to provide optimal antitumour effects. Furthermore, oral administration may be a useful modality for optimisation of treatment. The aim of this study was to compare the therapeutic profile of topotecan given orally or intravenously in human tumours xenografted into athymic nude mice. The drug topotecan was given according to an intermittent (every fourth day, four times) or daily (qdx5/weeklyx5-10 weeks; only orally) schedule. Tumour growth inhibition and persistence of drug effects were assessed and compared with untreated mice. In a panel of seven tumour xenografts, oral topotecan was at least as effective on three and significantly more effective on four tumours. Using the intermittent schedule, the maximum tolerated dose (MTD) was comparable for the two routes (15 mg/kg), but the toxicity profile suggested a better tolerability in terms of lethal effects after oral administration. The daily oral treatment of low drug doses allowed a higher cumulative dose to be delivered with improved antitumour efficacy (2/10 cured in a large cell lung cancer) and no evidence of toxicity. In spite of the low bioavailability of oral topotecan (23.5%), the persistent plasma levels of the drug suggest that the time of exposure to the drug is more critical than the plasma concentrations for antitumour efficacy. This interpretation is consistent with the increased efficacy of prolonged daily treatment with low-dose levels. The results may have implications for the future design of clinical studies.

The synthesis of dihydroxybenzoperimidine derivatives, which are chromophore-modified dihydroxyanthracenediones with an additional pyrimidine ring incorporated into the chromophore, is reported. These derivatives are structurally related to the antitumor agent mitoxantrone. Their synthesis was carried out by the reaction of 6-amino-8,11-dihydroxy-7H-benzo[e]perimidin-7-one (5) or 6,8, 11-trihydroxy-7H-benzo[e]perimidin-7-one (10) with a number of respective (alkylamino)alkylamines. The dihydroxybenzoperimidine derivatives exhibited in vitro cytotoxic activity against murine leukemia L1210 and human leukemia HL60 cell lines comparable to that of mitoxantrone. These compounds also exhibited a range of in vitro activity against the human MDR-type resistant leukemia K562R cell line with the MDR phenotype. The most active compound of this series, namely 6a, exhibited potent in vitro cytotoxic activity against a panel of human cell lines. Furthermore, in contrast to both mitoxantrone and doxorubicin, it displayed little cross-resistance in cell lines characterized by a MDR phenotype. Cell cycle analysis in the sensitive HT-29 and mitoxantrone-resistant HT-29/Mx (not identified resistance mechanism) cell lines has revealed that both mitoxantrone and 6a induce a G2/M block. However, while the proportion of apoptotic cells after mitoxantrone treatment is similar for both sensitive and resistant cell lines, it is much lower for 6a. Compound 6a tested against P388 murine leukemia in vivo displayed a significant antitumor effect (%T/C 196 at an optimal dose of 10 mg/kg). The property of overcoming the cross-resistance was maintained also in in vivo efficacy studies, where no difference was observed in the antitumor activity of compound 6a against the A2780 human tumor xenograft and its MDR A2780/Dx subline. We conclude that benzoperimidines, if properly substituted, constitute a novel class of compounds that can overcome multidrug resistance.

The amino sugar is recognized to be a critical determinant of the activity of anthracycline monosaccharides related to doxorubicin and daunorubicin. In an attempt to improve the pharmacological properties of such agents, novel anthracycline disaccharides have been designed in which the amino sugar, daunosamine, is separated from the aglycone by another carbohydrate moiety. In the present study, we examined the influence of the orientation of the second sugar residue on drug biochemical and biological properties in a series of closely related analogs. This structure-activity relationship study showed that the substitution of the daunosamine for the disaccharide moiety dramatically reduced the cytotoxic potency of the drug in the 4-methoxy series (daunorubicin analogs). In contrast, in the 4-demethoxy series (idarubicin analogs), the C-4 axial, but not the equatorial, configuration conferred a cytotoxic potency and antitumor activity comparable to that of doxorubicin. The configuration also influenced the drug's ability to stimulate topoisomerase II alpha-mediated DNA cleavage. Indeed, the glycosides with the equatorial orientation were ineffective as topoisomerase II poisons, whereas the compounds with axial orientation were active, although the daunorubicin analog exhibited a lower activity than the idarubicin analog. It is conceivable that the axial orientation allows an optimal interaction of the drug with the DNA-enzyme complex only in the absence of the methoxy group. Our results are consistent with a critical role of the sugar moiety in drug interaction with the target enzyme in the ternary complex.

On the basis of a structure-activity study of a new series of anthracycline disaccharides, we recently identified a doxorubicin analogue (MEN 10755) with a promising antitumor activity. In the present study, to better support the pharmacological interest of MEN 10755, we extended the preclinical evaluation of antitumor efficacy to a large panel of 16 human tumor xenografts, which originated from different clinicopathological types. Tumors with typical multidrug-resistant phenotype were excluded because MEN 10755 was found unable to overcome resistance mediated by transport systems. In the doxorubicin-responsive series, MEN 10755 exhibited a higher activity in three of five tumors, as documented by a more marked tumor growth inhibition and an increased value of log-cell kill. In the series of doxorubicin-resistant tumors, MEN 10755 was found effective in 6 of 11 tumors (1 breast, 3 lung, and 2 prostate carcinomas). The overall response rates were 31% and 69% for doxorubicin and MEN 10755, respectively. The improvement in drug efficacy was also supported by a substantial increase in the long-term survivor rate of animals implanted with responsive tumors. Most of the tumors refractory to doxorubicin and responsive to MEN 10755 were characterized by overexpression of the antiapoptotic protein Bcl-2. In one of these tumors (MX-1 breast carcinoma), we examined the ability of MEN 10755 to induce phosphorylation of Bcl-2 after a single treatment with therapeutic doses. The results indicated that, unlike doxorubicin, MEN 10755 induced protein phosphorylation. A similar modification was produced by Taxol, which is known to be very effective against the tumor. The correlation between drug efficacy and Bcl-2 phosphorylation may underly a peculiar feature related to improvement of efficacy of the disaccharide analogue. In conclusion, the present study supports some favorable features of the novel doxorubicin analogue in terms of both efficacy and tolerability with comparison to doxorubicin, although the improvement is somewhat tumor- and schedule-dependent.

Background: Although doxorubicin remains one of the most effective agents for the treatment of solid tumors, there is an intensive effort to synthesize doxorubicin analogues (compounds with similar chemical structures) that may have improved antitumor properties. We have synthesized a novel doxorubicin disaccharide analogue (MEN 10755) and have characterized some of its relevant biochemical, biologic, and pharmacologic properties., Methods: The antitumor activity of this compound (MEN 10755) was studied in a panel of human tumor xenografts, including xenografts of A2780 ovarian tumor cells, MX-1 breast carcinoma cells, and POVD small-cell lung cancer cells. MEN 10755 was compared with doxorubicin according to the optimal dose and schedule for each drug. The drug's cytotoxic effects, induction of DNA damage, and intracellular accumulation were studied in A2780 cells. DNA cleavage mediated by the enzyme topoisomerase II was investigated in vitro by incubating fragments of simian virus 40 DNA with the purified enzyme at various drug concentrations and analyzing the DNA cleavage-intensity patterns. Drug-induced apoptosis (programmed cell death) in tumors was determined with the use of MX-1 and POVD tumor-bearing athymic Swiss nude mice., Results: MEN 10755 was more effective than doxorubicin as a topoisomerase II poison and stimulated DNA fragmentation at lower intracellular concentrations. In addition, MEN 10755 exhibited striking antitumor activity in the treatment of human tumor xenografts, including those of the doxorubicin-resistant breast carcinoma cell line MX-1., Conclusions: The high antitumor activity of MEN 10755 in human tumor xenografts, including doxorubicin-resistant xenografts, and its unique pharmacologic and biologic properties make this disaccharide analogue a promising candidate for clinical evaluation.

Two novel porphyrin-bisacridine conjugates (1 and 2) were designed as bifunctional antitumour agents to combine the DNA-binding character of the acridines and the photosensitizing capacity of porphyrin, and have been subjected to biophysical and biological evaluation. The interactions of the conjugates with calf thymus DNA were evaluated using viscometric, spectrophotometric and stopped-flow sodium dodecyl sulphate (SDS) sequestration methods. Both conjugates acted as bis-intercalators via the two acridine chromophores and displayed a longer residence time on DNA relative to the parent acridine ligand. Their biological activity in vitro was studied against the C6 rat glioma, MCF-7, GBM and A431 cell lines. Both conjugates were cytotoxic to all four cell lines. The ID50 (C6 glioma) was essentially the same as that of the parent acridine for one conjugate, but was increased 20-fold for the other, while both conjugates were approximately 10-fold more cytotoxic than the parent porphyrin component. The tissue distribution of the two conjugates was assessed in nude mice xenografted with a human small cell lung carcinoma (POVD). There were large differences in the tissue distribution of the two conjugates, with conjugate 2 localizing 8-fold more in the tumour than conjugate 1.

In an attempt to identify novel compounds useful for the optimization of Photodynamic Therapy (PDT), the tissue localization of new synthetic porphyrins was compared with Photofrin II in nude mice xenografted with a human small cell lung cancer (POVD). Three haematoporphyrin analogues were selected for this study based on prior in vitro photosensitivity screening of a series of 15 such derivatives, as well as on the basis of improved localization in C6 gliomas in mice. Two of the porphyrins yielded better tumour:normal lung ratios than Photofrin II and, of these two, one (P13) is known to exhibit good photosensitization properties both in vitro and in vivo, and is therefore a good candidate as a lead compound for the development of porphyrins suitable for the photodynamic treatment of lung tumours.

5'-Deoxy-5-fluorouridine (doxifluridine) is a prodrug of 5-fluorouracil (5FU) selectively activated by tumor cells. Since in clinical studies the side effects of doxifluridine differed after intravenous (i.v.) or oral administration, and oral route was the most promising in preclinical studies with murine models, in this study the drug was tested orally against a panel of human colorectal tumor xenografts with varying degrees of sensitivity to 5FU. Doxifluridine efficacy was comparable to that of 5FU when it was delivered according to a weekly schedule, but it was statistically higher when it was delivered more frequently. Impressive tumor inhibition (between 90 and 97%) was achieved in 4 out of 5 tumor lines after treatments delivered twice a week or daily 5 times a week. No difference in 5FU activity was observed between weekly and biweekly treatments, or between oral and i.v. injections. Moreover, in one tumor line in which different dosages of doxifluridine were investigated, a marked antitumor effect was obtained with a wide range of tolerated doses (4000-8000 mg/kg). Overall, these data indicated that doxifluridine is well tolerated when given orally and frequently. Using an adequate schedule, the prodrug has a better therapeutic efficacy against a variety of human colon cancer models than 5FU.

A cell line, GBM, was established from a human malignant glioblastoma and was characterized with particular reference to its response to conventional drugs. The GBM cell line exhibited a 73 +/- 7 h doubling time in monolayer cultures. Expression of glial fibrillary acidic and S-100 proteins was observed. Karyotype analysis of GBM cells at early passages revealed the presence of two near-triploid clones (A and B) with multiple chromosome rearrangements; a 100% frequency for clone B was observed in the established cell line. GBM cells had tumorigenic properties, since the s.c. injection of cultured cells into nude mice gave rise to slowly growing tumors. The morphology of GBM cells was retained during in vitro and in vivo passages, as judged by light microscopy. GBM cells were relatively resistant to most conventional drugs; among the tested drugs, only taxol exhibited a marked cytotoxic effect comparable to that found in cells of a different tumor type. GBM cells were found positive for the epidermal growth factor receptor, HER2-neu and P-glycoprotein by flow cytometry of cells labelled with monoclonal antibodies. In spite of the expression of relatively high gamma-glutamyltransferase activity, the intracellular glutathione level was comparable to that of other chemosensitive tumor cells. This glioblastoma cell line is a suitable model for the identification and preclinical studies of new agents and provides an additional system to explore the molecular basis of the intrinsic drug resistance of glioblastoma.

The presence of glycosaminoglycans and hyaluronate was histochemically detected in connective tissue surrounding the intraductal carcinoma of human breast. The implications of the observation in the interaction between neoplasia and host tissues are discussed.