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The pentacyclic acridinium methosulfate salt RHPS4 induces the 3'single-stranded guanine-rich telomeric overhang to fold into a G-quadruplex structure. Stabilisation of the latter is incompatible with an attachment of telomerase to the telomere and thus G-quadruplex ligands can effectively inhibit both the catalytic and capping functions of telomerase. In this study, we examined mechanisms underlying telomere uncapping by RHPS4 in uterus carcinoma cells (UXF1138L) with short telomeres and compared the susceptibility of bulk and clonogenic cancer cells to the G-quadruplex ligand. We show that treatment of UXF1138L cells with RHPS4 leads to the displacement of the telomerase catalytic subunit (hTERT) from the nucleus, induction of telomere-initiated DNA-damage signalling and chromosome fusions. We further report that RHPS4 is more potent against cancer cells that grow as colonies in soft agar than cells growing as monolayers. Human cord blood and HEK293T embryonic kidney cell colony forming units, however, were more resistant to RHPS4. RHPS4-treated UXF1138L xenografts had a decreased clonogenicity, showed loss of nuclear hTERT expression and an induction of mitotic abnormalities compared with controls. Although single-agent RHPS4 had limited in vivo efficacy, a combination of RHPS4 with the mitotic spindle poison Taxol caused tumour remissions and further enhancement of telomere dysfunction.

AW464 (NSC 706704) is a novel benzothiazole substituted quinol compound active against colon, renal and certain breast cancer cell lines. NCI COMPARE analysis indicates possible interaction with thioredoxin/thioredoxin reductase, which is upregulated under hypoxia. Through activity on HIF1alpha, VEGF levels are regulated and angiogenesis controlled. A thioredoxin inhibitor could therefore exhibit enhanced hypoxic toxicity and indirect antiangiogenic effects. In vitro experiments were performed on colorectal and breast cancer cell lines under both normoxic and hypoxic conditions and results compared against those obtained with normal cell lines, fibroblasts and keratinocytes. Antiangiogenic effects were studied using both large and microvessel cells. Indirect antiangiogenic effects (production of angiogenic growth factors) were studied via ELISA. We show that AW464 exerts antiproliferative effects on tumour cell lines as well as endothelial cells with an IC(50) of approximately 0.5 microM. Fibroblasts are however resistant. Proliferating, rather than quiescent, endothelial cells are sensitive to the drug indicating potential antiangiogenic rather than antivascular action. Endothelial differentiation is also inhibited in vitro. Hypoxia (1% O(2) for 48 h) sensitises colorectal cells to lower drug concentrations, and in HT29s greater inhibition of VEGF is observed under such conditions. In contrast, bFGF levels are unaffected, suggesting possible involvement of HIF1alpha. Thus, AW464 is a promising chemotherapeutic drug that may have enhanced potency under hypoxic conditions and also additional antiangiogenic activity.

The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is a novel agent with potent and selective antitumour properties and, in the form of its L-lysylamide prodrug Phortress (NSC 710305), is a current candidate for early phase clinical studies. Previous findings have indicated that cytochrome P450 1A1 (CYP1A1) may play a role in the antitumour activity of molecules in the benzothiazole series including the nonfluorinated parent compound 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) (Kashiyama et al, 1999; Chua et al, 2000; Loaiza-Pérez et al, 2002). In this study, we assessed and verified that a fully functional aryl hydrocarbon receptor (AhR) signalling pathway is a necessary requisite for the induction of efficient cytotoxicity by 5F 203 in MCF-7 wild-type sensitive cells. Drug exposure caused MCF-7 sensitive cells to arrest in G(1) and S phase, and induced DNA adduct formation, in contrast to AhR-deficient AH(R100) variant MCF-7 cells. In sensitive MCF-7 cells, induction of CYP1A1 and CYP1B1 transcription (measured by luciferase reporter assay and real-time reverse transcriptase-polymerase chain reaction (RT-PCR)), and 7-ethoxyresorufin-O-deethylase (EROD) activity was demonstrated, following treatment with 5F 203. In contrast, in resistant AH(R100) cells, drug treatment did not affect CYP1A1 and CYP1B1 transcription and EROD activity. Furthermore, AH(R100) cells failed to produce either protein/DNA complexes on the xenobiotic responsive element (XRE) sequence of CYP1A1 promoter (measured by electrophoretic mobility shift assay) or DNA adducts. The data confirm that activation of the AhR signalling pathway is an important feature of the antitumour activity of 5F 203.

2-(4-Aminophenyl)benzothiazoles represent a potent and highly selective class of antitumour agent. In vitro, sensitive carcinoma cells deplete 2-(4-aminophenyl)benzothiazoles from nutrient media; cytochrome P450 1A1 activity, critical for execution of antitumour activity, and protein expression are powerfully induced. 2-(4-Amino-3-methylphenyl)benzothiazole-derived covalent binding to cytochrome P450 1A1 is reduced by glutathione, suggesting 1A1-dependent production of a reactive electrophilic species. In vitro, 2-(4-aminophenyl)benzothiazole-generated DNA adducts form in sensitive tumour cells only. At concentrations >100 nM, adducts were detected in DNA of MCF-7 cells treated with 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). 5F 203 (1 microM) led to the formation of one major and a number of minor adducts. However, treatment of cells with 10 microM 5F 203 resulted in the emergence of a new dominant adduct. Adducts accumulated steadily within DNA of MCF-7 cells exposed to 1 microM 5F 203 between 2 and 24 h. Concentrations of the lysylamide prodrug of 5F 203 (Phortress) > or = 100 nM generated adducts in the DNA of sensitive MCF-7 and IGROV-1 ovarian cells. At 1 microM, one major Phortress-derived DNA adduct was detected in these two sensitive phenotypes; 10 microM Phortress led to the emergence of an additional major adduct detected in the DNA of MCF-7 cells. Inherently resistant MDA-MB-435 breast carcinoma cells incurred no DNA damage upon exposure to Phortress (< or = 10 microM, 24 h). In vivo, DNA adducts accumulated within sensitive ovarian IGROV-1 and breast MCF-7 xenografts 24 h after treatment of mice with Phortress (20 mg kg(-1)). Moreover, Phortress-derived DNA adduct generation distinguished sensitive MCF-7 tumours from inherently resistant MDA-MB-435 xenografts implanted in opposite flanks of the same mouse.

Novel 2-(4-aminophenyl)benzothiazoles possess highly selective, potent antitumour properties in vitro and in vivo. They induce and are biotransformed by cytochrome P450 (CYP) 1A1 to putative active as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. The lipophilicity of these compounds presents limitations for drug formulation and bioavailability. To overcome this problem, water soluble prodrugs have been synthesised by conjugation of alanyl- and lysyl-amide hydrochloride salts to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles. The prodrugs retain selectivity with significant in vitro growth inhibitory potency against the same sensitive cell lines as their parent amine, but are inactive against cell lines inherently resistant to 2-(4-aminophenyl)benzothiazoles. Alanyl and lysyl prodrugs rapidly and quantitatively revert to their parent amine in sensitive and insensitive cell lines in vitro. Liberated parent compounds are sequestered and metabolised by sensitive cells only; similarly, CYP1A1 activity and protein expression are selectively induced in sensitive carcinoma cells. Amino acid prodrugs meet the criteria of aqueous solubility, chemical stability and quantitative reversion to parent molecule, and thus are suitable for in vivo preclinical evaluation., (Copyright 2002 Cancer Research UK)

2-(4-aminophenyl)benzothiazole (CJM 126) elicits potent growth inhibition in human-derived breast carcinoma cell lines, including oestrogen receptor-positive (ER+) MCF-7wt cells. Analogues substituted in the 3' position with I (DF 129), CH3 (DF 203), or Cl (DF 229) possess an extended profile of antitumour activity with remarkable selective activity in cell lines derived from solid tumours associated with poor prognosis, e.g. breast, ovarian, renal and colon. Growth inhibition occurs via unknown, possibly novel mechanism(s) of action. Two cell lines have been derived from sensitive MCF-7wt breast cancer cells (IC50 value < 0.001 microM) following long-term exposure to 10 nM or 10 microM CJM 126, MCF-7(10 nM 126) and MCF-7(10 microM 126) respectively, which demonstrate acquired resistance to this agent (IC50 > 30 microM) and cross-resistance to DF 129, DF 203 and DF 229. Sensitivity to tamoxifen, benzo[a]pyrene (BP), mitomyin C, doxorubicin and actinomycin D is retained. Resistance may, in part, be conferred by the constitutively increased expression of bcl-2 and p53 proteins detected in MCF-7(10 nM 126) and MCF-7(10 microM 126 lysates. Significantly decreased depletion of CJM 126 (30 microM) from nutrient medium of MCF-7(10 microM 126) cells was observed with predominantly cytoplasmic drug localization and negligible DNA strand breaks. N-acetyl transferase (NAT)1 and NAT2 proteins were expressed by all three MCF-7 sub-lines, but significantly higher expression of NAT2 was accompanied by enhanced acetylation efficacy in MCF-7(10 nM 126) cells. In contrast, CJM 126 (30 microM) was rapidly depleted from nutrient medium of MCF-7(10 microM 126) culture and accessed nuclei of these cells exerting damage to DNA. The major biotransformation product of CJM 126 in MCF-7(10 microM 126) cells was 2-(4-aminophenyl)-6-hydroxybenzothiazole (6-OH 126). This metabolite possessed no antitumour activity. Accordingly, in this sub-line, low constitutive expression and activity of cytochrome P450 (CYP) 1A1 was detected.

2-(4-Aminophenyl)benzothiazole molecules substituted in the 3 position of the phenyl ring with a halogen atom or methyl moiety comprise a group of compounds that potently inhibit specific human ovarian carcinoma cell lines. GI50 values fall within the nM range. Inhibition is highly selective -- whereas the GI50 value in IGROV1 cells consistently lies at < 10 nM, SK-OV-3 presents GI50 values > 10 microM. Biphasic dose-response relationships were observed in sensitive cell lines after 48-h drug exposure. COMPARE analyses revealed the very similar profiles of anti-tumour activity of 3-substituted benzothiazoles and 5-(4-dimethylaminophenylazo)quinoline, with Pearson correlation coefficients > 0.65. Anti-tumour activity extended to preliminary in vivo tests. The growth of OVCAR-3 cells in polyvinylidene fluoride (PVDF) hollow fibres implanted in the peritoneal cavity of mice was inhibited by more than 50% after intraperitoneal (i.p.) administration of 2-(4-amino-3-methylphenyl)benzothiazole (10 mg kg(-1)), 2-(4-amino-3-chlorophenyl)benzothiazole (100 mg kg(-1)) or 2-(4-amino-3-bromophenyl)benzothiazole (150 mg kg(-1)). The growth of OVCAR-3 tumours in subcutaneously (s.c.) implanted hollow fibres was retarded by more than 50% after treatment with 2-(4-amino-3-methylphenyl)benzothiazole (6.7 and 10 mg kg(-1)). In addition, the growth of s.c. OVCAR-3 xenografts was delayed after exposure to DF 203. However, the relationship between drug concentration and growth inhibition was inverse.

2-(4-Aminophenyl)benzothiazole (CJM 126) elicits biphasic growth-inhibitory effects against a panel of oestrogen receptor-positive (ER+) and oestrogen receptor-negative (ER-) human mammary carcinoma cell lines in vitro, yielding IC50 values in the nM range. Substitutions adjacent to the amino group in the 2-phenyl ring with a halogen atom or methyl group enhance potency in sensitive breast lines (pM IC50 values). Transient biphasic dose responses were induced but rapidly eradicated after specific drug exposure periods. Two human prostate carcinoma cell lines were refractory to the growth-inhibitory properties of 2-(4-aminophenyl)benzothiazoles; IC50 values > 30 microM were obtained. Potency and selectivity were confirmed when compounds were examined in the National Cancer Institute's Developmental Therapeutics screen; the spectrum of activity included specific ovarian, renal, colon as well as breast carcinoma cell lines. Moreover, comparing 6-day and 48-h incubations, the exposure time-dependent nature of the biphasic response was corroborated. Differential perturbation of cell cycle distribution followed treatment of MCF-7 and MDA 468 cells with substituted 2-(4-aminophenyl)benzothiazoles. In MDA 468 populations only, accumulation of events in G2/M phase was observed. Two MCF-7 cell lines were established with acquired resistance to CJM 126 (IC50 values > 20 microM), which exhibit cross-resistance to substituted benzothiazoles, but equal sensitivity to tamoxifen and doxorubicin. Compared with standard anti-tumour agents evaluated in the National Cancer Institute in vitro cell panel, benzothiazoles revealed unique profiles of growth inhibition, suggesting a mode(s) of action shared with no known clinically active class of chemotherapeutic agents.

Temozolomide (CCRG 81045: M&B 39831: NSC 362856) is an analogue of mitozolomide displaying similar broad spectrum activity in mouse tumours, but showing considerably less myelosuppression in the toxicology screen. Temozolomide was initially studied intravenously at doses between 50-200 mg m-2 and subsequently was given orally up to 1,200 mg m-2. A total of 51 patients were entered on the single dose schedule. Temozolomide exhibits linear pharmacokinetics with increasing dose. Myelotoxicity was dose limiting. Experimentally, temozolomide activity was schedule dependent and therefore oral administration was studied as a daily x 5 schedule between total doses of 750 and 1,200 mg m-2 in 42 patients. Myelosuppression was again dose limiting. The recommended dose for Phase II trials is 150 mg m-2 po for 5 days (total dose 750 mg m-2) for the first course, and if no major myelosuppression is detected on day 22 of the 4 week cycle, the subsequent courses can be given at 200 mg m-2 for 5 days (total dose 1 g m-2) on a 4 week cycle. Mild to moderate nausea and vomiting was dose related but readily controlled with antiemetics. Clinical activity was detected using the 5 day schedule in four (2CR, 2PR; 17%) out of 23 patients with melanoma and in one patient with mycosis fungoides (CR lasting 7 months). Two patients with recurrent high grade gliomas have also had partial responses. Temozolomide is easy to use clinically and generally well tolerated. In the extended Phase I trial temozolomide only occasionally exhibited the unpredictable myelosuppression seen with mitozolomide.

Mitozolomide is a novel antitumour agent showing a broad spectrum of activity against murine tumours and is currently undergoing Phase I clinical evaluation in the UK. We have conducted an animal pharmacokinetic study using male BALB/c mice as a pre-requisite to the clinical work. Mice were dosed i.p. at 5 dose levels (0.25-20 mg kg-1) and the oral and transdermal routes of administration were investigated at 20 mg kg-1. The analytical data produced a good fit to a simple open one-compartment pharmacokinetic model with an elimination half-life of the drug from plasma of between 0.68 and 0.88 h over the 0.25-20 mg kg-1 range covered. There was no evident dose dependency over this range and studies with two formulations showed mitozolomide to have good systemic availability when administered via the oral route (F values of 0.66 and 0.81). The drug was also found to be systemically available when administered topically in dimethylsulfoxide (F = 0.47). Mitozolomide shows many biochemical and biological similarities to the clinically used nitrosoureas BCNU and CCNU but our results show that it differs markedly in its kinetics from these two agents, with mitozolomide having relatively sustained plasma levels. It is hoped that this may be of therapeutic benefit if these levels are reflected in relative tumour concentrations.