E7070, a novel sulphonamide agent with potent antitumour activity in vitro and in vivo

E7070 (N-(3-Chloro-7-indolyl)-1,4-benzenedisulphonamide) was selected from our sulphonamide compound collections via antitumour screening and flow cytometric analysis. Following treatment with E7070, the cell cycle progression of P388 murine leukaemia cells was disturbed in the G1 phase. The cell-killing effect on human colon cancer HCT116 cells was found to be time-dependent. In the panel of 42 human tumour cell lines, E7070 showed an antitumour spectrum that was distinct from those of other anticancer drugs used in clinic. Animal tests using human tumour xenograft models demonstrated that E7070 could cause not only tumour growth suppression, but also tumour regression in three of five colorectal and two of two lung cancers. In the HCT116 xenograft model, E7070 was shown to be superior to 5-FU, MMC and CPT-11 (irinotecan). Furthermore, complete regression of advanced LX-1 tumours was observed in 80% of E7070-treated mice. All of these observations have promoted this drug to clinical evaluation.     

Improved targeting of platinum chemotherapeutics. the antitumour activity of the HPMA copolymer platinum agent AP5280 in murine tumour models

AP5280 is a novel N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound platinum (Pt) therapeutic designed to increase the therapeutic index relative to conventional, small-molecule platinum agents. The platinum-polymer construct accumulates in solid tumours on the basis of increased capillary permeability. The bound platinum moiety is present as an N,O-Pt chelate at the distal end of a tetrapeptide linker, glycine-phenylalanine-leucine-glycine, and the weight-average molecular weight (Mw) of the construct is 22 kDa. The antitumour activity and toxicity of AP5280 were assessed in the syngeneic murine B16F10 and Lewis lung tumour models, and in the human ovarian carcinoma 2008 and head and neck squamous carcinoma UMSCC10b xenograft models. The maximum tolerated dose (MTD) of AP5280 was 6-fold greater than that of carboplatin (CBDCA) in vivo. AP5280 was active in all four tumour models, and it displayed a higher therapeutic index than CBDCA in each of these tumour models. The antitumour effect of AP5280 given at 16% of its MTD was equivalent to that produced by a MTD of CBDCA. Thus, consistent with the design goal for this drug, and despite being less potent than CBDCA, AP5280 produced less systemic toxicity relative to its antitumour activity and thus has a greater therapeutic index. On the basis of the improved therapeutic index evidenced in these models, AP5280 has been advanced into clinical trials.     

FP3: a novel VEGF blocker with antiangiogenic effects in vitro and antitumour effects in vivo

BACKGROUND Vascular endothelial growth factor (VEGF) is a critical promoter of blood vessel growth during embryonic development and neovascularisation in tumours. VEGF serves as a logical target for antiangiogenic cancer therapy because of its fundamental role in tumour angiogenesis. This study is to investigate the inhibitory effects of FP3, a novel VEGF blocker, on angiogenesis in vitro and tumour growth in vivo. METHODS The inhibitory effects of FP3 on angiogenesis in vitro were evaluated by using human umbilical vein endothelial cells (HUVECs) and rat aortic ring. The inhibitory effects of FP3 on tumour growth and angiogenesis in vivo were evaluated in a human non-small-cell lung cancer (NSCLC) cell line A549 tumour xenograft model with the methods of tumour growth regression assay and immunohistochemical staining, respectively. RESULTS In experiments with HUVECs, FP3 inhibited cell proliferation and migration. In rat aortic ring assay, FP3 suppressed VEGF-induced vessel sprouting. In tumour growth regression assay, FP3 significantly blocked the growth of A549 tumour in the subcutaneous tumour xenograft model and dramatically decreased the vessel density of tumour. CONCLUSIONS FP3 has excellent inhibitory effects on tumour angiogenesis both in vitro and in vivo, therefore it could be used as an effective antiangiogenic agent.     

Influence of P-glycoprotein expression on in vitro cytotoxicity and in vivo antitumour activity of the novel pyrrolobenzodiazepine dimer SJG-136

SJG-136 is a novel pyrrolobenzodiazepine dimer analogue that acts as a minor-groove interstrand DNA cross-linking agent. The present study investigated the impact of ABCB1 (mdr-1) expression on the activity of SJG-136 using both in vitro and in vivo systems. SJG-136 was highly potent in the colon cancer cell lines HCT-116, HT-29 and SW620 (IC50 0.1-0.3 nM). However, HCT-8 and HCT-15 cells expressing significant levels of mdr-1 were less sensitive (IC50 2.3 and 3.7 nM, respectively) using a SRB assay. The cytotoxicity was increased in HCT-15 and A2780(AD) in presence of 5 microg/ml verapamil. Mdr-1 mRNA expression was determined by qRT-PCR and correlated to SJG-136 IC50s (r2=0.86, P=0.0001). Isogenic 3T3 cells expressing mdr-1 cDNA (3T3 pHamdr-1) were less sensitive to SJG-136 than the parental 3T3 cells (IC50 208 and 6.3 nM, respectively). Finally, SJG-136 (120 microg/kg/d dx5) was highly active against A2780 xenografts (SGD=275) but not A2780(AD) xenografts (SGD=67).     

A novel class of antitumour agents. II. In vitro testing.

Suspensions of Walker and HeLa cells, rat fibroblasts and human stimulated lymphocytes were incubated over various periods with the synthetic amino steroid, 2 beta,16 beta-dipiperidino-5 alpha-androstane-3 alpha,17 beta-diol dipivalate (DAP). The acute cell destroying effect was found to be exponentially time and dose dependent within a certain range. With lower nontoxic doses, decreases in the mitotic and labelling indices were observed, and clotted mitotic figures occurred. The effects were essentially the same for all experimental cell types; chromosome alterations were not seen.     

Comparative study of thermoradiosensitization by misonidazole and metronidazole in vivo: antitumour effect and pharmacokinetics

Tumour control by local hyperthermia (43–5°C, 30 min) and radiation (20 Gy) given in combination with misonidazole (MISO) or metronidazole (METRO) was studied using FSa-II murine fibrosarcoma. When MISO or METRO (5 mmol/kg) was given 30 min before heat and subsequently treated with radiation, tumour regression was observed for both agents. Radiation dose-response curves for MISO and METRO with heating at 43·5°C for 30 min were identical. Mouse foot reaction was used to evaluate local toxicity following combined heat, a nitroimidazole and radiation treatment. MISO enhanced the magnitude of foot reaction and prolonged the recovery time compared with heat plus radiation controls. There were no observable differences of foot reaction between animals treated with heat plus radiation and those animals treated with heat, radiation and METRO. Pharmacokinetics of the nitroimidazoles heated at 43·5°C for 30 min in FSa-II tumours were investigated as a possible mechanism of thermal sensitization. Local hyperthermia did not alter the pharmacokinetics of METRO. Tumour concentration and tumour/plasma ratio of MISO were slightly decreased during heating. Since the hypoxic metabolism of the nitroimidazoles did not increase significantly during the heat treatment, the thermal enhancement of MISO or METRO radiosensitization cannot be explained by the increase in hypoxic cytotoxicity of the nitroimidazoles at elevated temperature alone. The two nitroimidazoles also were not accumulated in the tumour after heating. Therefore, alternation of pharmacokinetics is not the major mechanism for the thermal enhancement of nitroimidazole radiosensitization. The METRO radiosensitization effect became identical to that of MISO at elevated temperatures is of particular importance in clinical radiosensitization. The very low local and systemic toxicity together with the high efficacy of METRO at elevated temperatures will make it an attractive candidate as a future clinical radiosensitizer.     

Comparative study of thermoradiosensitization by misonidazole and metronidazole in vivo: antitumour effect and pharmacokinetics.

Tumour control by local hyperthermia (43.5 degrees C, 30 min) and radiation (20 Gy) given in combination with misonidazole (MISO) or metronidazole (METRO) was studied using FSa-II murine fibrosarcoma. When MISO or METRO (5 mmol/kg) was given 30 min before heat and subsequently treated with radiation, tumour regression was observed for both agents. Radiation dose-response curves for MISO and METRO with heating at 43.5 degrees C for 30 min were identical. Mouse foot reaction was used to evaluate local toxicity following combined heat, a nitroimidazole and radiation treatment. MISO enhanced the magnitude of foot reaction and prolonged the recovery time compared with heat plus radiation controls. There were no observable differences of foot reaction between animals treated with heat plus radiation and those animals treated with heat, radiation and METRO. Pharmacokinetics of the nitroimidazoles heated at 43.5 degrees C for 30 min in FSa-II tumours were investigated as a possible mechanism of thermal sensitization. Local hyperthermia did not alter the pharmacokinetics of METRO. Tumour concentration and tumour/plasma ratio of MISO were slightly decreased during heating. Since the hypoxic metabolism of the nitroimidazoles did not increase significantly during the heat treatment, the thermal enhancement of MISO or METRO radiosensitization cannot be explained by the increase in hypoxic cytotoxicity of the nitroimidazoles at elevated temperature alone. The two nitroimidazoles also were not accumulated in the tumour after heating. Therefore, alternation of pharmacokinetics is not the major mechanism for the thermal enhancement of nitroimidazole radiosensitization. The METRO radiosensitization effect became identical to that of MISO at elevated temperatures is of particular importance in clinical radiosensitization. The very low local and systemic toxicity together with the high efficacy of METRO at elevated temperatures will make it an attractive candidate as a future clinical radiosensitizer.     

A novel synthetic iminoquinone,BA-TPQ,as an anti-breast cancer agent: in vitro and in vivo activity and mechanisms of action

Herein, we report our examination of the anti-breast cancer activity of a novel synthetic compound, 7-(benzylamino)-1, 3, 4, 8-tetrahydropyrrolo [4, 3, 2-de]quinolin-8(1H)-one (BA-TPQ). This agent is an analog of a naturally occurring marine compound, and was found to be the most active out of more than 40 related compounds. We investigated the in vitro activity of BA-TPQ on the survival, proliferation, and apoptosis of breast cancer cells using the MTT and BrdUrd assays, and Annexin/Annexin-PI staining and flow cytometry. The in vivo anti-cancer effects of BA-TPQ were evaluated in xenograft models of breast cancer. Finally, the mechanisms of action of the compound were also assessed by cDNA microarrays, RT–PCR and Western blotting. In a dose-dependent manner, BA-TPQ inhibited cell growth and induced apoptosis and cell cycle arrest in human MCF-7 and MDA-MB-468 breast cancer cells in vitro, and showed in vivo efficacy in mice bearing MCF-7 or MDA-MB-468 xenograft tumors. We demonstrated that BA-TPQ modifies the expression of numerous molecules involved in cell cycle progression and apoptosis. Similar changes in protein expression were observed in vitro and in vivo, as determined by examination of cells and excised xenograft tumors. Our preclinical data indicate that BA-TPQ is a potential therapeutic agent for breast cancer that has multiple hormone-, Her2-, and p53-independent mechanisms of action, providing a basis for further development of the compound as a novel anticancer agent.     

In vitro antitumour activity of the novel imidazoisoquinoline SDZ 62-434.

The novel imidazoisoquinoline SDZ 62-434, originally identified as a platelet-activating factor (PAF) antagonist, has antiproliferative activity in a range of cell lines from human solid and haematological malignancies. Using an MTT cytotoxicity assay, IC50 values of 5 microM - 111 microM were observed following a 24 h exposure. Similar results were obtained using a clonogenic assay. The HT29 colon adenocarcinoma was particularly sensitive while the MCF-7 breast carcinoma was the most resistant in our panel. Only a 2-3 fold cross-resistance was seen in the doxorubicin and cisplatin resistant variants of the A2780 ovarian carcinoma; the drug did not modulate sensitivity to doxorubicin in either parent or resistant lines. No cross-resistance to SDZ 62-434 was seen in a doxorubicin-resistant MCF-7 variant. Cytotoxicity was not due to non-specific membrane lysis. The potent PAF antagonist WEB 2086 did not modulate SDZ 62-434 cytotoxicity, indicating no role for PAF receptors. Precursor incorporation studies in A2780 cells showed that DNA synthesis was inhibited more effectively than protein synthesis while RNA synthesis was unaffected. SDZ 62-434 inhibited both bombesin and platelet-derived growth factor-induced DNA synthesis in quiescent Swiss 3T3 cells. This suggests a possible role for SDZ 62-434 as an inhibitor of signal transduction in cancer cells.     

Amrubicin, a novel 9-aminoanthracycline, enhances the antitumor activity of chemotherapeutic agents against human cancer cells in vitro and in vivo

Amrubicin, a completely synthetic 9-aminoanthracycline derivative, is an active agent in the treatment of untreated extensive disease-small-cell lung cancer and advanced non-small-cell lung cancer. Amrubicin administered intravenously at 25 mg/kg substantially prevented the growth of five of six human lung cancer xenografts established in athymic nude mice, confirming that amrubicin as a single agent was active in human lung tumors. To survey which antitumor agent available for clinical use produces a synergistic interaction with amrubicin, we examined the effects in combinations with amrubicinol, an active metabolite of amrubicin, of several chemotherapeutic agents in vitro using five human cancer cell lines using the combination index (CI) method of Chou and Talalay. Synergistic effects were obtained on the simultaneous use of amrubicinol with cisplatin, irinotecan, gefitinib and trastuzumab, with CI values after 3 days of exposure being <1. Additive effect was observed with the combination containing vinorelbine with CI values indistinguishable from 1, while the combination of amrubicinol with gemcitabine was antagonistic. All combinations tested in vivo were well tolerated. The combinations of cisplatin, irinotecan, vinorelbine, trastuzumab, tegafur/uracil, and to a lesser extent, gemcitabine with amrubicin caused significant growth inhibition of human tumor xenografts without pronouncedly enhancing body weight loss, compared with treatment using amrubicin alone at the maximum tolerated dose. Growth inhibition of tumors by gefitinib was not antagonized by amrubicin. These results suggest that amrubicin appears to be a possible candidate for combined use with cisplatin, irinotecan, vinorelbine, gemcitabine, tegafur/uracil or trastuzumab.     

Gene expression predicts differential capecitabine metabolism, impacting on both pharmacokinetics and antitumour activity

Capecitabine is converted into 5'-deoxy-5-fluorocytidine (5'DFCR), 5'-deoxy-5-fluorouridine (5'DFUR) and 5-fluorouracil (5-FU) by CES1 and 2, CDD, and TP, in both liver and tumour. 5-FU is catabolised by DPD. Gene expression analysis of these enzymes was undertaken in fresh human hepatocytes, mouse liver, colorectal cancer cell lines and xenografts. Cell lines with low CDD expression (<1.5) had 5'DFCR/5'DFUR cytotoxicity ratios>2 and cell lines with TP/DPD<0.6 had 5'DFUR IC50>50 microM (SRB assay). A pharmacokinetic/pharmacodynamic study in nude mice bearing HCT 116 xenografts and treated with capecitabine by oral gavage assessed pharmacokinetic, gene expression and antitumour activity. Low liver CDD correlated with high 5'DFCR plasma concentrations in mice. CDD expression was approximately 100-fold higher in fresh human hepatocytes than mouse liver, explaining the higher plasma 5'DFUR concentrations reported previously in humans. Tumour 5-FU concentration correlated with TP/DPD and with tumour response. These studies identify the potential utility of gene expression analysis and drug monitoring in tumour in patients.     

Antitumor activity of novel ailanthone derivatives in vitro and in vivo

The antitumor activities of two ailanthone derivatives with 15 beta-acyloxy side chains were investigated. The cytotoxic activity of 11 beta, 20-epoxy-1 beta, 11 alpha, 12 alpha-trihydroxy-15-beta-[E)-3-methyl-2-octenoyl) oxypicras-3,13(21)-diene-2,16-dione (SUN2071) and 11 beta, 20-epoxy-1 beta, 11 alpha, 12 alpha-trihydroxy-15 beta-[E)-2-undecenoyl) oxypicras-3,13(21)-diene-2,16-dione (SUN0237) was close to that of bruceantin and vincristine. SUN2071 was shown to be a potent inhibitor of protein synthesis in L1210 cultured cells. When administered i.p. to i.p. inoculated P388 leukemia mice, daily treatment with SUN2071 and SUN0237 significantly increased the lifespan (increases in lifespan in excess of 100% were achieved). These increases were comparable to those achieved with vincristine. The therapeutic ratio of SUN2071 was also close to that of vincristine. However, the compounds were ineffective when administered as a single injection. Daily i.p. treatment with SUN2071 demonstrated significant tumor growth inhibition in mice inoculated s.c. with colon-38 and moderate activity against i.p. L1210 leukemia and i.p. B16 melanoma. The compounds were ineffective when tested against the Lewis lung carcinoma and colon-26. In a preliminary toxicological study, SUN2071 at a therapeutic dose in daily consecutive i.p. injection produced leucopenia.     

In vitro antiproliferative effects, toxicity profiles in vivo in mice and antitumour activity in tumour-bearing mice of four diorganotin compounds

The in vitro antiproliferative effects, the in vivo toxicity profiles in mice and the antitumour activity in tumour bearing mice were screened for four novel di-n-butyltin carboxylates, di-n-butyltin bis(2,4-dihydroxybenzoate) [compound 1 (C1)], di-n-butyltin bis(2,5-dihydroxybenzoate) (C2), di-n-butyltin bis(pentafluorophenylacetate) (C3), and bis[di-n-butyl(pentafluorophenylacetato)tin] oxide (C4). All compounds revealed similar in vitro chemosensitivities in two cell lines, C26-10 and C26-A, two murine undifferentiated colon carcinoma cell lines. With all compounds tested, not only was cell growth inhibited in vitro, but also cell kill was achieved. At their maximum tolerated dose (MTD), C1 and C4 were inactive in vivo against colon 26 tumours in Balb/C mice when administered twice with one week interval (qd7x2 schedule). At their MTD, compound 2 (single dose administration and qd7x2 schedule) and compound 3 (qd7x2) showed slight in vivo antitumour activity with a ratio of the relative tumour size of the treated mice to that of control mice (T/C) = ca. 0.6 (T/C less than or equal to 0.6 being the cut-off level for sensitivity). However, the cut-off level for the growth delay factor (GDF) (>1) was not reached. With the exception of C2 administered with a single dose and C3 with the 2 doses protocol, treatment with these compounds did not increase the life span of the mice. Repeated administration of compound 2 did not improve the antitumour activity compared to single dose administration. This was probably due to the higher toxicity when C2 was administered a second time after one week.     

Pharmacokinetic determinants of the activity and toxicity of antitumour agents

Pharmacokinetic-pharmacodynamic relationships in cytotoxic cancer chemotherapy are reviewed. In all classes of established cytotoxic drugs there is now good evidence that pharmacokinetics are a major determinant of drug toxicity and, in some instances, activity. Attempts are now being made to exploit these relationships to improve the therapeutic utility of established cytotoxic drugs and initial results are promising. In addition, there is evidence that the early clinical evaluation of new drugs may benefit from the application of pharmacokinetic information.     

Substituted titanocenes induce caspase-dependent apoptosis in human epidermoid carcinoma cells in vitro and exhibit antitumour activity in vivo

Titanocene compounds are a novel series of agents that exhibit cytotoxic effects in a variety of human cancer cells in vitro and in vivo. In this study, the antiproliferative activity of two titanocenes (Titanocenes X and Y) was evaluated in human epidermoid cancer cells in vitro. Titanocenes X and Y induce apoptotic cell death in epidermoid cancer cells, with IC50 values that are comparable to cisplatin. Characterisation of the cell death pathway induced by titanocene compounds in A431 cells revealed that apoptosis is preceded by cell cycle arrest and the inhibition of cell proliferation. The induction of apoptosis is dependent on the activation of caspase-3 and -7 but not caspase-8. Furthermore, the antitumour activity of Titanocene Y was tested in an A431 xenograft model of epidermoid cancer. Results indicate that Titanocene Y significantly reduced the growth of A431 xenografts with an antitumour effect similar to cisplatin. These results suggest that titanocenes represent a novel series of promising antitumour agents.     

In vitro and in vivo properties of novel nucleoside transport inhibitors with improved pharmacological properties that potentiate antifolate activity.

The activity of antimetabolite inhibitors of de novo deoxyribonucleotide biosynthesis can be compromised by the salvage of extracellular preformed nucleosides and nucleobases. Dipyridamole (DP) is a nucleoside transport inhibitor that has been used clinically in an attempt to increase antimetabolite activity; however, DP binds tightly to the serum protein alpha1-acid glycoprotein (AGP) thereby rendering this therapeutic strategy largely ineffective. Four novel DP analogues (NU3076, NU3084, NU3108, and NU3121) have been developed with substitutions at the 2,6- and 4,8-positions of the pyrimidopyrimidine ring. The novel DP analogues inhibit thymidine (dThd) uptake into L1210 cells in vitro (NU3076 IC(50), 0.25 microM; NU3084 IC(50), 0.27 microM; NU3108 IC(50), 0.31 microM; NU3121 IC(50), 0.26 microM; and DP IC(50), 0.37 microM), but, unlike DP, their activity remains largely unaffected in the presence of 5 mg/ml AGP. The four DP analogues inhibit dThd and hypoxanthine rescue from Alimta (multitargeted antifolate)-induced growth inhibition in A549 and COR L23 human lung carcinoma cell lines in the presence of 2.5 mg/ml AGP, whereas the activity of DP is completely abolished. i.p. administration of 10 mg/kg NU3108, NU3121, and DP produced peak plasma concentrations of 4.4, 2.1, and 6.7 microM, respectively, and levels were sustained above 1 microM for approximately 45 min (DP) and 120 min (NU3108 and NU3121). [3H]thymidine incorporation into COR L23 xenografts grown in CD1 nude mice was reduced by 64% (NU3108), 44% (NU3121), and 65% (DP) 2 h after administration of the nucleoside transport inhibitors. In conclusion, two novel DP analogues (NU3108 and NU3121) have been identified that do not bind to AGP and that display superior pharmacokinetic profiles in comparison to DP and inhibit [3H]thymidine incorporation into human tumor xenografts in vivo.     

Preclinical studies on toxicity,antitumour activity and pharmacokinetics of cisplatin and three recently developed derivatives

Preclinical studies were performed in mice, rats and dogs of cis-diamminedichloroplatinum(II) (CDDP) and its derivatives cis-l,l-di(aminomethyl) cyclohexane platinum(II) sulphate (TNO-6), cis-diammine-l,l-cyclobutanedicarboxylate platinum(II) (CBDCA) and cis-dichloro, trans-dihydroxybis-isopropylamine platinum(IV) (CHIP). In mice toxicity and antitumour activity were determined. All three derivatives were at least as toxic as CDDP for haemopoietic stem cells and were less active than CDDP against the mouse tumours leukaemia L1210 and osteosarcoma C22LR. Toxicology studies in rats revealed no renal toxicity after a single dose of TNO-6. Fractionated doses of TNO-6 and CBDCA did cause renal toxicity but less than CDDP. CHIP produced little or no kidney damage. In dogs, TNO-6 (1.5 mg/kg) produced more severe kidney damage—although this was reversible—than CDDP (2 mg/kg). Half-lives of distribution were 4.0–5.1 min for TNO-6 and 9.7 min for CDDP, while half-lives of elimination were 3.6–6.6 days and 5.9 days respectively. Plasma levels, normalized for the dose, were at least two times higher after TNO-6 than after CDDP. Twelve weeks after drug administration, plasma levels were undetectable, while tissue concentrations could still be measured. The platinum concentration in kidney cortex was higher after CDDP than after TNO-6.     

In vitro and in vivo activity of antifungal agents in combination with fleroxacin, a new quinolone

The in vitro and in vivo interaction of fleroxacin with amphotericin B (Amph B), flucytosine (5-FC) and azoles against Candida albicans strains was tested. In vitro the interaction between fleroxacin and various antifungals was not dependent on the incubation time. Fleroxacin neither enhances nor antagonizes the in vitro activity of Amph B at high concentration (50-100 micrograms/ml). Fleroxacin has a synergistic effect with ketoconazole (KETO), but this is not observed with itraconazole (ITRA) or fluconazole (FLU). In no instance antagonism was observed. The activity of 5-FC was antagonized by fleroxacin being generally reduced by 2-4 dilution steps. In murine candidosis the efficacies of all antifungal drugs were not influenced by addition of 100 mg/kg fleroxacin. Therefore, the effects seen in in vitro tests are most probably not relevant for the clinical use of a combination of fleroxacin with antifungal drugs.