Synthesis and in vitro evaluation of radioiodinated indolequinones targeting NAD(P)H: quinone oxidoreductase 1 for internal radiation therapy.

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase and is highly expressed in many human solid cancers. Because NQO1 can be induced immediately after exposure to ionizing radiation, we aimed to develop an NQO1-targeted radiolabeled agent to establish a novel internal radiation therapy that amplifies the therapeutic effects when combined with external radiation therapy. We designed three NQO1-targeted radioiodinated compounds including two ether linkage compounds ([(125)I]1 and [(125)I]2) and a sulfide linkage compound ([(125)I]3) based on the selective binding of indolequinone analogs to the active site of NQO1 by the stacking effect. These compounds were successfully prepared using an oxidative iododestannylation reaction with high radiochemical yields and purity. In NQO1-expressing tumor cells, [(125)I]1 and [(125)I]2 were readily metabolized to p-[(125)I]iodophenol or m-[(125)I]iodophenol and [(125)I]I(-), whereas over 85% of the initial radioactivity of [(125)I]3 was observed as an intact form at 1h after incubation. The cellular uptake of [(125)I]3 was significantly higher than those of [(125)I]1 and [(125)I]2. The uptake of [(125)I]3 was specific and was dependent on the expression of NQO1. These data suggest that the novel NQO1-targeted radioiodinated compound [(125)I]3 could be used as a novel internal radiation agent for the treatment of cancer.

Bioactivation of the cancer chemopreventive agent tamoxifen to quinone methides by cytochrome P4502B6 and identification of the modified residue on the apoprotein.

The nonsteroidal antiestrogen tamoxifen was introduced as a treatment for breast cancer 3 decades ago. It has also been approved as a chemopreventive agent and is prescribed to women at high risk for this disease. However, several studies have shown that use of tamoxifen leads to increased risk of endometrial cancer in humans. One potential pathway of tamoxifen toxicity could involve metabolism via hydroxylation to give 4-hydroxytamoxifen (4OHtam), which may be further oxidized to form a quinone methide. CYP2B6 is a highly polymorphic drug-metabolizing enzyme, and it metabolizes a number of clinically important drugs. Earlier studies from our laboratory have shown that tamoxifen is a mechanism-based inactivator of CYP2B6. The aim of the current study was to investigate the possible formation of reactive intermediates through detection of protein covalent binding and glutathione ethyl ester adduct (GSHEE) formation. The incubation of tamoxifen with 2B6 gave rise to an adduct of 4OHtam with glutathione, which was characterized as the 4OHtam quinone methide + GSHEE with an m/z value of 719, and the structure was characterized by liquid chromatography-tandem mass spectrometry. The metabolic activation of tamoxifen in the CYP2B6 reconstituted system also resulted in the formation of an adduct to the P4502B6 apoprotein, which was identified using liquid chromatography mass spectrometry. The site responsible for the inactivation of CYP2B6 was determined by proteolytic digestion and identification of the labeled peptide. This revealed a tryptic peptide ¹88FHYQDQE¹94 with the site of adduct formation localized to Gln193 as the site modified by the reactive metabolite formed during tamoxifen metabolism.

Oxidative ipso substitution of 2,4-difluoro-benzylphthalazines: identification of a rare stable quinone methide and subsequent GSH conjugate.

In vitro metabolite identification and GSH trapping studies in human liver microsomes were conducted to understand the bioactivation potential of compound 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol], an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a GSH adduct (M2). The stability of this metabolite arises from extensive resonance-stabilized conjugation of the substituted benzylphthalazine moiety. Cytochrome P450 (P450) phenotyping studies revealed that the formation of M1 and M2 were NADPH-dependent and primarily catalyzed by CYP3A4 among the studied P450 isoforms. In summary, an unusual and stable quinone methide metabolite of compound 1 was identified, and a mechanism was proposed for its formation via an oxidative ipso substitution.

Inactivation of apaziquone by haematuria: implications for the design of phase III clinical trials against non-muscle invasive bladder cancer.

PURPOSE: Despite positive responses in phase II clinical trials, the bioreductive prodrug apaziquone failed to achieve statistically significant activity in non-muscle invasive bladder cancer in phase III trials. Apaziquone was administered shortly after transurethral resection and here we test the hypothesis that haematuria inactivates apaziquone. METHODS: HPLC analysis was used to determine the ability of human whole blood to metabolise apaziquone ex vivo. An in vitro model of haematuria was developed and the response of RT112 and EJ138 cells following a 1-h exposure to apaziquone was determined in the presence of urine plus or minus whole blood or lysed whole blood. RESULTS: HPLC analysis demonstrated that apaziquone is metabolised by human whole blood with a half-life of 78.6 ± 23.0 min. As a model for haematuria, incubation of cells in media containing up to 75% buffered (pH 7.4) urine and 25% whole blood was not toxic to cells for a 1-h exposure period. Whole blood (5% v/v) significantly (p < 0.01) reduced the potency of apaziquone in this experimental model. Lysed whole blood also significantly (p < 0.05) reduced cell growth, although higher concentrations were required to achieve an effect (15% v/v). CONCLUSIONS: The results of this study demonstrate that haematuria can reduce the potency of apaziquone in this experimental model. These findings impact upon the design of further phase III clinical trials and strongly suggest that apaziquone should not be administered immediately after transurethral resection of non-muscle invasive bladder cancer when haematuria is common.

Safety and side effects of immediate instillation of apaziquone following transurethral resection in patients with nonmuscle invasive bladder cancer.

PURPOSE: We studied the safety, tolerability and pharmacokinetics of a single immediate post-transurethral resection intravesical instillation of apaziquone for patients with nonmuscle invasive bladder cancer. MATERIALS AND METHODS: Patients with cTa-T1, G1-G2 urothelial cell carcinoma of the bladder underwent transurethral resection of bladder tumor(s) followed by a single intravesical instillation of apaziquone 4 mg/40 ml for 1 hour within 6 hours of transurethral bladder tumor resection. Adverse events and safety parameters were assessed on days 8 and 15 after transurethral bladder tumor resection. Blood samples were drawn before and during the instillation for pharmacokinetic analyses. The first 10 patients with pTa-T1, G1-G2 nonmuscle invasive bladder cancer were also evaluated by cystoscopy 3 months after treatment to determine mucosal healing. RESULTS: Of 20 patients receiving apaziquone 13 (65%) reported 35 adverse events, mostly grade 1 to 2. Eight patients (40%) reported 13 adverse events related to treatment, in particular dysuria, hematuria, bladder spasm, abdominal pain, asthenia and postoperative urinary retention. Three grade 3 and 1 grade 4 event(s) occurred, but these were considered unrelated to treatment. No other significant clinical changes were observed. Apaziquone and the active metabolite EO5a were not detected with pharmacokinetic analyses at any point of time. After 3 months no evidence of impaired mucosal healing was observed. CONCLUSIONS: A single immediate post-transurethral bladder tumor resection instillation of apaziquone was well tolerated with an expected good safety profile. Apaziquone and its metabolite EO5a were not detected systemically with pharmacokinetic analyses. These results have lead to further study of a single immediate instillation of apaziquone.

Efficacy, pharmacokinetic and pharmacodynamic evaluation of apaziquone in the treatment of non-muscle invasive bladder cancer.

INTRODUCTION: Apaziquone (also known as EO9 and QapzolaTM) is a prodrug that is activated to DNA damaging species by oxidoreductases (particularly NQO1) and has the ability to kill aerobic and/or hypoxic cancer cells. Areas covered: Whilst its poor pharmacokinetic properties contributed to its failure in phase II clinical trials when administered intravenously, these properties were ideal for loco-regional therapies. Apaziquone demonstrated good anti-cancer activity against non-muscle invasive bladder cancer (NMIBC) when administered intravesically to marker lesions and was well tolerated with no systemic side effects. However, phase III clinical trials did not reach statistical significance for the primary endpoint of 2-year recurrence in apaziquone over placebo although improvements were observed. Post-hoc analysis of the combined study data did indicate a significant benefit for patients treated with apaziquone, especially when the instillation of apaziquone was given 30 min or more after surgery. A further phase III study is ongoing to test the hypotheses generated in the unsuccessful phase III studies conducted to date. Expert opinion: Because of its specific pharmacological properties, Apaziquone is excellently suited for local therapy such as NMIBC. Future studies should include proper biomarkers.

Selective photocytotoxicity of anthrols on cancer stem-like cells: The effect of quinone methides or reactive oxygen species.

Cancer stem cells (CSCs) are a subpopulation of cancer cells that share properties of embryonic stem cells like pluripotency and self-renewal and show increased resistance to chemo- and radiotherapy. Targeting CSC, rather than cancer cells in general, is a novel and promising strategy for cancer treatment. Novel therapeutic approaches, such as photodynamic therapy (PDT) have been investigated. A promising group of phototherapeutic agents are reactive intermediates - quinone methides (QMs). This study describes preparation of QM precursor, 2-hydroxy-3-hydroxymethylanthracene (2) and a detailed photochemical and photobiological investigation on similar anthracene derivatives 3 and 4. Upon photoexcitation with near visible light at λ > 400 nm 1 and 2 give QMs, that were detected by laser flash photolysis and their reactivity with nucleophiles has been demonstrated in the preparative irradiation experiments where the corresponding adducts were isolated and characterized. 3 and 4 cannot undergo photodehydration and deliver QM, but lead to the formation of phenoxyl radical and singlet oxygen, respectively. The activity of 1-4 was tested on a panel of human tumor cell lines, while special attention was devoted to demonstrate their potential selectivity towards the cells with CSC-like properties (HMLEshEcad). Upon the irradiation of cell lines treated with 1-4, an enhancement of antiproliferative activity was demonstrated, but the DNA was not the target molecule. Confocal microscopy revealed that these compounds entered the cell and, upon irradiation, reacted with cellular membranes. Our experiments demonstrated moderate selectivity of 2 and 4 towards CSC-like cells, while necrosis was shown to be a dominant cell death mechanism. Especially interesting was the selectivity of 4 that produced higher levels of ROS in CSC-like cells, which forms the basis for further research on cancer phototherapy, as well as for the elucidation of the underlying mechanism of the observed CSC selectivity based on oxidative stress activation.

Phase I/II pilot study of intravesical apaziquone (EO9) for superficial bladder cancer.

PURPOSE: The quinone based bioreductive drug apaziquone (EO9) failed to demonstrate efficacy in previous phase II studies following intravenous administration. We determined the dose of apaziquone that can be safely administered intravesically and explored its activity for superficial bladder transitional cell carcinoma. MATERIALS AND METHODS: Six patients with multifocal, Ta/T1 and G1/G2 transitional cell carcinoma of the bladder received escalating doses of apaziquone formulated as EOquintrade mark (0.5 mg/40 ml up to 16 mg/40 ml) weekly for 6 weeks. A further 6 patients received weekly apaziquone at the highest nontoxic dose established. Pharmacokinetic parameters were determined in urine and blood, and the pharmacodynamic markers NQO1 (reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase-1) and glucose transporter 1 were also characterized. Efficacy was determined against a marker lesion. RESULTS: Local toxicity (grades 2 and 3 dysuria, and hematuria) was observed at doses of 8 mg/40 ml and above but 4 mg/40 ml was well tolerated with no systemic or local side effects. Apaziquone in urine increased linearly with the dose but no apaziquone was detected in plasma. In 8 of 12 patients complete macroscopic and histological disappearance of the marker lesion occurred. A correlation between response and NQO1 and/or glucose transporter 1 expression could not be established. CONCLUSIONS: Intravesical administration of 4 mg/40 ml apaziquone was well tolerated and had ablative activity against superficial bladder cancer marker lesions.

Hypoxia: targeting the tumour.

Solid tumours contain regions of very low oxygen concentrations that are said to be hypoxic. Hypoxia is a natural phenotype of solid tumours resulting from an imperfect vascular network. There are a number of consequences associated with tumour hypoxia including: resistance to ionising radiation, resistance to chemotherapy and the magnification of mutated p53. In addition tissue hypoxia has been regarded as a key factor for tumour aggressiveness and metastasis by activation of signal transduction pathways and gene regulatory mechanisms. It is clear that hypoxia in solid tumours promotes a strong oncogenic phenotype and is a phenomenon that occurs in all solid tumours. As such this provides a significant target for drug discovery particularly for tumour-targeting agents. A range of chemical classes (N-oxides, quinones, nitro-aromatics) have been explored as bioreductive agents that target tumour hypoxia. The most advanced agent, tirapazamine, is in phase III clinical trials in combination with cis-platin. The aim of this review is to give a brief overview of the current molecules and strategies being explored for targeting tumour hypoxia.