Malcolm F. G. Stevens

Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials

*Department of Medical Oncology, Chafing Cross Hospital, Fulham Palace Road, London W6 8RF, U.K. t Cancer Research Campaign Experimental Cancer Chemotherapy Research Group, Cancer Research Laboratories, Department of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 ZRD, U.K.

Phase I trial of temozolomide (CCRG 81045: M&B 39831: NSC 362856).

Drugs Dynamics Institute, College of Pharmacy, University of Texas at Austin, Austin, TX 78712-7074, U.S.A.

Temozolomide: A new oral cytotoxic chemotherapeutic agent with promising activity against primary brain tumours

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.

Temozolomide: Mechanisms of Action, Repair and Resistance

Temozolomide, a new oral cytotoxic agent, has been given to 28 patients with primary brain tumours. Treatment was given at a dose of 150 mg/m2/day for 5 days (i.e. total dose 750 mg/m2) escalating, if no significant myelosuppression was noted on day 22, to 200 mg/m2/day for 5 days (i.e. total dose 1000 mg/m2) for subsequent courses at 4 week intervals. A major improvement in computer tomography (CT) scan was noted in 5/10 patients with astrocytomas recurrent after radiotherapy, with a major clinical improvement but minor improvement on CT scan in one further patient. Reduction in the size of the CT lesion was also observed in 4/7 patients with newly diagnosed high grade astrocytomas given 2-3 courses of temozolomide prior to irradiation. 1 patient with recurrent medulloblastoma had a clinical response in bone metastases. Temozolomide was well tolerated with little subjective toxicity and usually predictable myelosuppression and is a promising new drug in the treatment of primary brain tumours.

Synthesis and biological properties of benzothiazole, benzoxazole, and chromen-4-one analogues of the potent antitumor agent 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610, NSC 721648).

Glioblastoma multiforme is the most common aggressive adult primary tumour of the central nervous system. Treatment includes surgery, radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. TMZ is an alkylating agent prodrug, delivering a methyl group to purine bases of DNA (O6-guanine; N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine (O6-MeG) can be removed by methylguanine methyltransferase (MGMT; direct repair) in tumours expressing this protein, or tolerated in mismatch repair-deficient (MMR-) tumours. Thus MGMT or MMR deficiency confers resistance to TMZ. Inherent- and acquired resistance to TMZ present major obstacles to successful treatment. Strategies devised to thwart resistance and enhance response to TMZ, including inhibition of DNA repair mechanisms which contribute to TMZ resistance, are under clinical evaluation. Depletion of MGMT prior to alkylating agent chemotherapy prevents O6-MeG repair; thus, MGMT pseudosubstrates O6-benzylguanine and lomeguatrib are able to sensitise tumours to TMZ. Disruption of base excision repair (BER) results in persistence of potentially lethal N7- and N3- purine lesions contributing significantly to TMZ cytoxicity particularly when O6-MeG adducts are repaired or tolerated. Several small molecule inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1), a critical BER protein are yielding promising results clinically, both in combination with TMZ and as single agent chemotherapy in patients whose tumours possess homologous recombination DNA repair defects. Another validated, but as yet preclinical protein target, mandatory to BER is abasic (AP) endonuclease-1 (APE-1); in preclinical tests, APE-1 inhibition potentiates TMZ activity. An alternative strategy is synthesis of a molecule, evoking an irrepairable cytotoxic O6-G lesion. Preliminary efforts to achieve this goal are described.

Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect

New fluorinated 2-aryl-benzothiazoles, -benzoxazoles, and -chromen-4-ones have been synthesized and their activity against MCF-7 and MDA 468 breast cancer cell lines compared with the potent antitumor benzothiazole 5. Analogues such as 9a, b and 12a, d yielded submicromolar GI50 values in both cell lines; however, none of the new compounds approached 5 in terms of antitumor potency. For 5, binding to the aryl hydrocarbon receptor appeared to be necessary but not sufficient for growth inhibition.

2-(4-Aminophenyl)benzothiazoles: novel agents with selective profiles of in vitro anti-tumour activity.

Functional telomeres are required for the replicability of cancer cells. The G-rich strand of telomeric DNA can fold into a 4-stranded structure known as the G-quadruplex (G4), whose stabilization alters telomere function limiting cancer cell growth. Therefore, the G4 ligand RHPS4 may possess antitumor activity. Here, we show that RHPS4 triggers a rapid and potent DNA damage response at telomeres in human transformed fibroblasts and melanoma cells, characterized by the formation of several telomeric foci containing phosphorylated DNA damage response factors gamma-H2AX, RAD17, and 53BP1. This was dependent on DNA repair enzyme ATR, correlated with delocalization of the protective telomeric DNA-binding protein POT1, and was antagonized by overexpression of POT1 or TRF2. In mice, RHPS4 exerted its antitumor effect on xenografts of human tumor cells of different histotype by telomere injury and tumor cell apoptosis. Tumor inhibition was accompanied by a strong DNA damage response, and tumors overexpressing POT1 or TRF2 were resistant to RHPS4 treatment. These data provide evidence that RHPS4 is a telomere damage inducer and that telomere disruption selectively triggered in malignant cells results in a high therapeutic index in mice. They also define a functional link between telomere damage and antitumor activity and reveal the key role of telomere-protective factors TRF2 and POT1 in response to this anti-telomere strategy.

Telomere uncapping by the G-quadruplex ligand RHPS4 inhibits clonogenic tumour cell growth in vitro and in vivo consistent with a cancer stem cell targeting mechanism

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 Institutes 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.

Stabilization of quadruplex DNA perturbs telomere replication leading to the activation of an ATR-dependent ATM signaling pathway.

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.

Antitumour 2-(4-aminophenyl)benzothiazoles generate DNA adducts in sensitive tumour cells in vitro and in vivo.

Functional telomeres are required to maintain the replicative ability of cancer cells and represent putative targets for G-quadruplex (G4) ligands. Here, we show that the pentacyclic acridinium salt RHPS4, one of the most effective and selective G4 ligands, triggers damages in cells traversing S phase by interfering with telomere replication. Indeed, we found that RHPS4 markedly reduced BrdU incorporation at telomeres and altered the dynamic association of the telomeric proteins TRF1, TRF2 and POT1, leading to chromosome aberrations such as telomere fusions and telomere doublets. Analysis of the molecular damage pathway revealed that RHPS4 induced an ATR-dependent ATM signaling that plays a functional role in the cellular response to RHPS4 treatment. We propose that RHPS4, by stabilizing G4 DNA at telomeres, impairs fork progression and/or telomere processing resulting in telomere dysfunction and activation of a replication stress response pathway. The detailed understanding of the molecular mode of action of this class of compounds makes them attractive tools to understand telomere biology and provides the basis for a rational use of G4 ligands for the therapy of cancer.