Photodeamination to quinone methides in cucurbit[n]urils: potential application in drug delivery.

We demonstrate a proof of principle for a new approach in the development of a drug delivery system. A positively charged prodrug (phenol) can form a stable inclusion complex with CB[7], which enables more efficient delivery of the prodrug. After photochemical transformation (photoactivation) inside the complex, an active drug quinone methide (QM) is formed and released from the complex, since it is a neutral molecule and forms a less stable complex with CB[7].

Phase 2 study of adjuvant intravesical instillations of apaziquone for high risk nonmuscle invasive bladder cancer.

PURPOSE: We studied the safety and efficacy of multiple adjuvant apaziquone instillations in patients with high risk nonmuscle invasive bladder cancer. MATERIALS AND METHODS: Patients with high risk nonmuscle invasive urothelial carcinoma of the bladder underwent transurethral resection of all bladder tumor(s), and received 6 weekly adjuvant intravesical apaziquone instillations of 4 mg in 40 ml. Patients with carcinoma in situ received 3 further maintenance instillations at months 3, 6 and 12. Followup consisted of cystoscopy, urine cytology and observation of adverse events every 3 months for 18 months. RESULTS: A total of 53 patients were enrolled in the study. Although all patients were high risk according to the definitions used when the study was initiated, according to most recent guideline criteria, 80% and 20% of these patients would now be considered intermediate and high risk for recurrence, and 50% and 44% would be considered intermediate and high risk for progression, respectively. Intent to treat analysis of 49 patients with papillary tumors showed recurrent tumors in 34.7% and 44.9% at 12 and 18 months, respectively. One patient had progression to T2 or greater urothelial carcinoma after 9 months. There were 4 patients with carcinoma in situ who had complete responses at 3 months but discontinued treatment due to cystitis, recurrent papillary disease, urinary incontinence and dysuria. Most other side effects were mild (grade 1 to 2). CONCLUSIONS: Adjuvant intravesical instillations of apaziquone are generally well tolerated. The recurrence rates of 34.7% after 12 months and 44.9% after 18 months in these patients can be considered encouraging, and warrant further study.

Apaziquone for Nonmuscle Invasive Bladder Cancer: Where Are We Now?

Apaziquone is an interesting drug for intravesical use in patients with nonmuscle invasive bladder cancer; however, more research is needed to prove its actual benefit. Although the apaziquone trials demonstrate the potential of this new drug, the singular phase 3 trials did not reach their primary endpoint. To date, no new trials are recruiting, so the development of apaziquone seems to have stopped.

Chemoablation in Urothelial Carcinoma: A Systematic Review and Future Perspectives.

Chemoablation is an emerging treatment for urothelial carcinomas. This review provides an overview of the evidence for intracavitary chemoablation in the treatment of urothelial carcinomas. The benefits of such agents include a reduction in morbidity and diseased organ preservation. While numerous agents have shown promise, research is limited due to small patient cohorts, varying follow-up, and no standardized methodology to assess response. Therefore, to date, chemoablation has not been widely adopted. This may change as a novel mitomycin formulation has recently been approved for treating low-grade upper tract urothelial carcinoma. Future studies are ongoing which evaluate other promising chemoablation options in urothelial carcinoma.

Suppression of inflammatory responses by celastrol, a quinone methide triterpenoid isolated from Celastrus regelii.

BACKGROUND: Celastrol, a quinone methide triterpenoid isolated from the Celastraceae family, exhibits various biological properties, including chemopreventive, antioxidant and neuroprotective effects. In this study, we showed that celastrol inhibits inflammatory reactions in macrophages and protects mice from skin inflammation. MATERIALS AND METHODS: Anti-inflammatory effects of celastrol (0-1 microM) were examined in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. To investigate the effects of celastrol (0-50 microg per mice) in vivo, activation of myeloperoxidase (MPO) and histological assessment were examined in the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse ear oedema model. RESULTS: Our in vitro experiments showed that celastrol suppressed not only LPS-stimulated generation of nitric oxide and prostaglandin E(2), but also expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW264.7 cells. Similarly, celastrol inhibited LPS-induced production of inflammatory cytokines, including tumour necrosis factor-alpha and interleukin-6. In an animal model, celastrol protected mice from TPA-induced ear oedema, possibly by inhibiting MPO activity and production of inflammatory cytokines. CONCLUSIONS: Our data suggest that celastrol inhibits the production of inflammatory mediators and is a potential target for the treatment of various inflammatory diseases.

Two-year follow-up of the phase II marker lesion study of intravesical apaziquone for patients with non-muscle invasive bladder cancer.

OBJECTIVES: To study the time-to-recurrence and duration of response in non-muscle invasive bladder cancer (NMIBC) patients, with a complete ablative response after intravesical apaziquone instillations. METHODS: Transurethral resection of bladder tumour(s) (TURBT) was performed in patients with multiple pTa-T1 G1-2 urothelial cell carcinoma (UCC) of the bladder, with the exception of one marker lesion of 0.5-1.0 cm. Intravesical apaziquone was administered at weekly intervals for six consecutive weeks, without maintenance instillations. A histological confirmed response was obtained 2-4 weeks after the last instillation. Routine follow-up (FU) was carried out at 6, 9, 12, 18 and 24 months from the first apaziquone instillation. RESULTS: At 3 months FU 31 of 46 patients (67.4%) had a complete response (CR) to ablative treatment. Side-effects on the long-term were only mild. Two CR patients dropped out during FU. On intention-to-treat (ITT) analysis 49.5% of the CR patients were recurrence-free at 24 months FU, with a median duration of response of 18 months. Of 15 no response (NR) patients, only two received additional prophylactic instillations after TURBT. On ITT-analysis 26.7% of the NR patients were recurrence-free (log rank test, P = 0.155). The overall recurrence-free survival was 39% (18 of 46 patients) at 24 months FU. CONCLUSIONS: The CR of the marker lesion in 67% of patients was followed by a recurrence-free rate of 56.5% at 1-year FU, and 49.5% at 2-year FU. These long-term results are good in comparison with the results of other ablative studies.

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.

EO-9 bladder instillations: formulation selection based on stability characteristics and in vitro simulation studies.

A bladder instillation of EO-9 (EOquin) is currently used in phase II clinical trials for the treatment of superficial bladder cancer. Three alternative formulations were developed to improve its pharmaceutical properties and clinical acceptability. Freeze-dried products composed of EO-9, 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD), tri(hydroxymethyl) aminomethane (Tris), and sodium bicarbonate (NaHCO(3)) were tested. Selection of one formulation for further development was based on stability studies. These studies comprised stability of the freeze-dried products, stability after reconstitution and dilution and stability during bladder instillation in an experimental set-up. The stability study of the freeze-dried products showed that the formulation composed of EO-9/HPbetaCD/Tris (4/600/1mg/vial) was most stable. After reconstitution and dilution all products were stable for at least 8h. The product composed of EO9/HPbetaCD/NaHCO(3) (4/600/20mg/vial) was the least stable product both as freeze-dried formulation and after reconstitution and dilution. The bladder instillation simulation experiment showed that all products were stable when mixed with urine of pH 8 and unstable in urine of pH 4 and 6. The degradation products formed in urine were EO-5a and EO-9-Cl. Based on these results, the product composed of EO-9/HPbetaCD/Tris (4/600/1mg/vial) was selected for further pharmaceutical development.

Development of a bladder instillation of the indoloquinone anticancer agent EO-9 using tert-butyl alcohol as lyophilization vehicle.

The purpose of this research was to develop a stable bladder instillation of EO-9 for the treatment of superficial bladder cancer. First, stability and dissolution studies were performed. Subsequently, the freeze-drying process was optimized by determination of the freeze-drying characteristics of the selected cosolvent/water system and differential scanning calorimetry analysis of the formulation solution. Furthermore, the influence of the freeze-drying process on crystallinity and morphology of the freeze-dried product was determined with x-ray diffraction analysis and scanning electron microscopy, respectively. Subsequently, a reconstitution solution was developed. This study revealed that tert-butyl alcohol (TBA) can be used to both dramatically improve the solubility and stability of EO-9 and to shorten the freeze-drying cycle by increasing the sublimation rate. During freeze drying, 3 TBA crystals were found: TBA hydrate-ice crystals, crystals of TBA hydrate, and a third crystal, probably composed of TBA hydrate crystals containing approximately 90% to 95% TBA. Furthermore, it was shown that crystallization of TBA hydrate was inhibited in the presence of both sodium bicarbonate (NaHCO3) and mannitol. Addition of an annealing step resulted in a minor increase in the crystallinity of the freeze-dried product and formation of the delta-polymorph of mannitol. A stable bladder instillation was obtained after reconstitution of the freeze-dried product (containing 8 mg of EO-9, 20 mg of NaHCO3, and 50 mg of mannitol per vial) to 20 mL with a reconstitution solution composed of propylene glycol/water for injection (WfI)/NaHCO3/sodium edetate 60%/40%/2%/0.02% vol/vol/wt/wt, followed by dilution with WfI to a final volume of 40 mL.

The efficacy of Apaziquone in the treatment of bladder cancer.

INTRODUCTION: Bladder cancer is nowadays a common tumor. Non-muscle invasive bladder cancer (NMIBC) has still chances of recurrence and progression in spite of surgery and adjuvant treatments. New therapies are being developed to reduce these percentages with less adverse effects - Apaziquone (EO9) is an example. Areas covered: A literature search has been performed using Pubmed, UpToDate and Google verified information (mainly from Food and Drug Administration and Spectrum Pharmaceutics websites). We have included data from the most representative clinical trials and reviews published. Expert opinion: Apaziquone is considered a promising chemical agent if applied intravesically due mainly to its pharmacodynamics and safety profile. There is evidence for this with respect to adjuvant chemo ablative therapy and as a post-transurethral resection of bladder (TURB) single-dose regimen. As a result, new clinical phase III trials are needed both to evaluate its efficacy as an adjuvant therapy in the spectrum from intermediate- to high-risk non-muscle invasive bladder cancer and to select the most appropriate candidates and treatment schedule. As a conclusion, Apaziquone is a good candidate to become a better alternative as an adjuvant therapy for the treatment of NMIBC in the near future.

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.

Anticancer Activity of Apaziquone in Oral Cancer Cells and Xenograft Model: Implications for Oral Cancer Therapy.

Oral squamous cell carcinoma (OSCC) patients diagnosed in late stages have limited chemotherapeutic options underscoring the great need for development of new anticancer agents for more effective disease management. We aimed to investigate the anticancer potential of Apaziquone, [EOquin, USAN, E09, 3-hydroxy-5- aziridinyl-1-methyl-2(1H-indole-4,7-dione)-prop-ß-en-α-ol], a pro-drug belonging to a class of anti-cancer agents called bioreductive alkylating agents, for OSCC. Apaziquone treatment inhibited cell proliferation and induced apoptosis in OSCC cells in vitro. Apaziquone treated OSCC cells showed increased activation of Caspase 9 and Caspase 3, and Poly (ADP ribose) polymerase (PARP) cleavage suggesting induction of apoptosis by apaziquone in oral cancer cells. Importantly, apaziquone treatment significantly reduced oral tumor xenograft volume in immunocompromised NOD/SCID/Crl mice without causing apparent toxicity to normal tissues. In conclusion, our in vitro and in vivo studies identified and demonstrated the pre-clinical efficacy of Apaziquone, as a potential novel anti-cancer therapeutic candidate for oral cancer management.

Apaziquone as an intravesical therapeutic agent for urothelial non-muscle-invasive bladder cancer.

INTRODUCTION: Urothelial carcinoma of the bladder is a disease prone to recurrence. A new cytotoxic drug, apaziquone, is an analog of mitomycin C. Given via intravesical instillations it has the ability to specifically target cancer cells. AREAS COVERED: This article reviews the discovery and pharmacological properties of the agent and the first steps of its application in human disease. The poor performance of the drug as a systemic medication is discussed along with in vivo and in vitro studies that have led to current intravesical applications with encouraging results. In detail the limited number of clinical studies on the drug and compare them with relevant series on currently used agents. In addition, different strategies to enhance the efficacy of other agents are discussed and possible application of this experience to apaziquone is proposed. EXPERT OPINION: Apaziquone is a promising local agent for the treatment and prevention of recurrent urothelial carcinoma of the bladder. The limited number of Phase II studies has demonstrated potency and relatively low toxicity. In light of the dearth of randomized controlled trials, Phase III studies are urgently needed before any conclusive pronouncements on this agent can be made.

Enhanced resolution triple-quadrupole mass spectrometry for ultra-sensitive and quantitative analysis of the investigational anticancer agent EO9 (apaziquone) and its metabolite EO5a in human and dog plasma to support (pre)-clinical studies of EOquin given intravesically.

A highly sensitive and selective liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to quantify the experimental anticancer agent EO9 and its metabolite EO5a in biological matrices. A 200-microL aliquot of human/dog plasma was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and extracted with 1.25 mL of ethyl acetate. Dried extracts were reconstituted in 0.1 M ammonium acetate/methanol (7:3, v/v) and 20-microL volumes were injected onto the LC system. Separation was achieved on a 150 x 2.1 mm C18 column using an alkaline eluent (1 mM ammonium hydroxide/methanol (gradient system)). The detection was performed by a Finnigan TSQ Quantum Ultra equipped with an electrospray ionization source operated in positive mode and enhanced mass resolution capability. It demonstrated improved sensitivity with a factor 10-20 for EO9 and EO5a over a 3-decades dynamic range, with acceptable accuracy and precision, when compared with the previously described assay for EO9 and EO5a, developed by our group, using an API 2000. The assay quantifies a range from 0.5 to 500 ng/mL for EO9 and EO5a using 200-microL human plasma and dog samples. The described mass resolution method was successfully applied for the evaluation of the pharmacokinetic profile of EO9 and its metabolite EO5a in human and dog plasma.

Cytotoxicity and p-glycoprotein modulating effects of quinolones and indoloquinazolines from the Chinese herb Evodia rutaecarpa.

The antimycobacterial quinolones 1-methyl-2-undecyl-4-quinolone, dihydroevocarpine and evocarpine as well as the indoloquinazoline alkaloids rutaecarpine and evodiamine - all from the Chinese medicinal herb Evodia rutaecarpa - were tested in two in vitro assays, for cytotoxicity and interaction with p-glycoprotein (p-gp). Cytotoxicity was measured in a cell proliferation assay against CCRF-CEM leukemia cells and their p-gp over-expressing subline CEM/ADR5000. An assay monitoring the p-gp-dependent accumulation of the dye calcein in porcine brain capillary endothelial cells (PBCECs) was used to study interactions of the test substances with this efflux pump. Rutaecarpine and evodiamine showed quite high toxicity with IC (50) values from 2.64 to 4.53 microM and were weak modulators of p-gp activity. The degrees of resistance in CEM/ADR5000 towards the saturated quinolones 1-methyl-2-undecyl-4-quinolone and dihydroevocarpine were between 3 and 4. In the calcein assay, these two quinolones were shown to be moderate modulators of p-gp activity. Evocarpine, on the other side, is not transported by p-gp, and showed only slight toxicity at the highest test concentration of 30 microM.

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.

Phase II marker lesion study with intravesical instillation of apaziquone for superficial bladder cancer: toxicity and marker response.

PURPOSE: We studied the ablative activity of intravesical apaziquone (EOquin) on a papillary marker tumor and determined the incidence of side effects. MATERIALS AND METHODS: A total of 46 patients with multiple pTa or pT1 bladder tumors underwent visible lesion resection except for 1 marker tumor. Patients were then treated with 6 instillations of apaziquone at weekly intervals. The response was determined 2 to 4 weeks after the last instillation. RESULTS: One patient withdrew informed consent and refused the last treatment due to side effects. A histologically proven complete response was seen in 30 patients. Progression to invasive stage was not observed. Local side effects in this study were comparable to those due to other chemotherapy instillations, such as mitomycin C and epirubicin, but less severe and less frequent compared to those of bacillus Calmette-Guerin instillations. CONCLUSIONS: The histological complete response rate after 6 consecutive instillations of apaziquone in patients with superficial bladder cancer was 67% (95% CI 51 to 80). Local side effects were comparable to side effects due to other chemotherapy instillations.

Effect of hyperthermia on the cytotoxicity of 4 chemotherapeutic agents currently used for the treatment of transitional cell carcinoma of the bladder: an in vitro study.

PURPOSE: Hyperthermia combined with chemotherapy is not a novel cancer treatment. However, the working mechanism of this combination therapy is not fully understood. In the current in vitro study we investigated the differences in cytotoxicity of 4 chemotherapeutic agents at 37C or 43C. MATERIALS AND METHODS: The human transitional cell carcinoma cell lines used were RT4, RT112, 253J and T24. Cells were seeded in 96-well microtiter plates. After 24 hours cells were treated for 60 minutes with increasing concentrations of mitomycin C, epirubicin, gemcitabine and EO9 at a temperature of 37C or 43C. After treatment cells were rinsed 3 times and left for 24 hours in the incubator at 37C. The influence of chemotherapy and temperature on cell survival was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. RESULTS: Decreased cell proliferation with increasing concentrations of chemotherapeutic agents was demonstrated. EO9 proved to be the most potent agent at each temperature. Hyperthermia alone did not demonstrate decreased cell proliferation. However, a synergistic effect on decreased cell proliferation was demonstrated in all cell lines and chemotherapeutic agents used, although each had a maximum at a different chemotherapy concentration and to a different extent. Synergism was most obvious in cell lines treated with low dose epirubicin. CONCLUSIONS: Synergism with hyperthermia and chemotherapy was clearly demonstrated for epirubicin, EO9, mitomycin C and to a lesser extent gemcitabine. Hyperthermia alone did not cause decreased cell proliferation. Synergism was most prominent with low drug doses and the most potent drug used in this in vitro study was EO9.