tirapazamine and Carcinoma--Squamous-Cell

Topics: Carcinoma, Squamous Cell; Cell Hypoxia; Cisplatin; Clinical Trials as Topic; Head and Neck Neoplasms; Humans; Positron-Emission Tomography; Prognosis; Radiation-Sensitizing Agents; Tirapazamine; Triazines

Radiotherapy and concurrent chemotherapy (CRT) is superior to radiotherapy alone for the treatment of locally advanced, nonmetastatic squamous carcinoma of the head and neck (HNC). Three issues affect the use of CRT as primary treatment for advanced HNC. The first issue is the definition of advanced stage and the initial therapeutic choice of surgery or CRT and the role of post-CRT neck dissection. Function preservation considerations should guide the choice between surgery and CRT for patients with resectable disease. Fluorodeoxyglucose-positron emission tomography scanning may identify patients who require adjuvant neck dissection. The second issue is optimization of radiotherapy and chemotherapy schedules. Ideally, concurrent chemotherapy should be incorporated into radiotherapy (RT) regimens that would constitute optimal therapy were RT to be administered as single-modality treatment. Modified fractionation schemes constitute optimal single-modality RT. Platinum schedules other than bolus dosing every 3 to 4 weeks are effective and may be less toxic. The third issue is integration of biologically targeted therapy into CRT treatment programs. Epidermal growth factor receptor blockade enhances the effectiveness of RT alone. Its role and that of angiogenic blockade in CRT are under investigation.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cetuximab; Cisplatin; Combined Modality Therapy; Consensus; Dose Fractionation, Radiation; Drug Administration Schedule; Head and Neck Neoplasms; Humans; Neck Dissection; Neoplasm Invasiveness; Neoplasm Staging; Patient Selection; Positron-Emission Tomography; Practice Guidelines as Topic; Randomized Controlled Trials as Topic; Tirapazamine; Triazines

This prospective, randomized phase III intergroup trial of the Gynecologic Oncology Group and National Cancer Institute of Canada Clinical Trials Group was designed to test the effectiveness and safety of adding the hypoxic cell sensitizer tirapazamine (TPZ) to standard cisplatin (CIS) chemoradiotherapy in locally advanced cervix cancer.. Patients with locally advanced cervix cancer were randomly assigned to CIS chemoradiotherapy versus CIS/TPZ chemoradiotherapy. Primary end point was progression-free survival (PFS). Secondary end points included overall survival (OS) and tolerability.. PFS was evaluable in 387 of 402 patients randomly assigned over 36 months, with enrollment ending in September 2009. Because of the lack of TPZ supply, the study did not reach its original target accrual goal. At median follow-up of 28.3 months, PFS and OS were similar in both arms. Three-year PFS for the TPZ/CIS/RT and CIS/RT arms were 63.0% and 64.4%, respectively (log-rank P = .7869). Three-year OS for the TPZ/CIS/RT and CIS/RT arms were 70.5% and 70.6%, respectively (log-rank P = .8333). A scheduled interim safety analysis led to a reduction in the starting dose for the TPZ/CIS arm, with resulting tolerance in both treatment arms.. TPZ/CIS chemoradiotherapy was not superior to CIS chemoradiotherapy in either PFS or OS, although definitive commentary was limited by an inadequate number of events (progression or death). TPZ/CIS chemoradiotherapy was tolerable at a modified starting dose.

Topics: Adenocarcinoma; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Canada; Carcinoma, Adenosquamous; Carcinoma, Squamous Cell; Chemoradiotherapy; Chi-Square Distribution; Cisplatin; Disease Progression; Disease-Free Survival; Female; Humans; Kaplan-Meier Estimate; Middle Aged; Neoplasm Staging; Proportional Hazards Models; Prospective Studies; Time Factors; Tirapazamine; Treatment Outcome; Triazines; United States; Uterine Cervical Neoplasms

To compare nodal response rates following chemoradiotherapy in patients with p16+ and p16- oropharyngeal squamous cell carcinoma (OPSCC).. Patients with node-positive OPSCC treated at Peter MacCallum Cancer Centre on the published phase I-III tirapazamine trials were identified. All patients had conventional assessment (clinical examination (CA), CT and/or MRI) and positron emission tomography (PET) at both baseline and 2-4 months post-treatment.. There were 30 p16+ and 18 p16- patients, the former group having significantly higher stage nodal disease (P = 0.016). The mean overall reduction in nodal size at post-treatment assessment was similar in p16+ and p16- patients (78% vs. 75%), and no statistically significant difference in nodal complete response (CR) rates was detected by either CA (50% vs. 39%, P = 0.35) or PET/PET-CT (93% vs. 83%, P = 0.19). PET was significantly more accurate in determining the true nodal CR rate in both groups, with a negative predictive value of 96%.. Nodal response rates following chemoradiotherapy appear to be similar in p16+ and p16- patients when assessed by either CA or PET/PET-CT. However, higher nodal CR was seen in PET/PET-CT compared with CA in both groups. Metabolic imaging is more accurate than CA in assessing nodal response post-treatment.

Topics: Adult; Aged; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; Chemoradiotherapy; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Male; Middle Aged; Neoplasm Proteins; Oropharyngeal Neoplasms; Radiation-Sensitizing Agents; Tirapazamine; Treatment Outcome; Triazines

High plasma osteopontin (OPN) levels have been reported to be an adverse prognostic factor in head and neck squamous cell carcinomas (HNSCC), correlate with tumor hypoxia, and be predictive of benefit from hypoxia-targeted therapy. We sought to confirm the prognostic and predictive significance of OPN in patients treated on a large international trial.. Patients with stage III/IV HNSCC were randomized to receive definitive radiotherapy concurrently with cisplatin or cisplatin plus the hypoxic cell cytotoxin, tirapazamine (TPZ). Eligibility criteria for this prospective substudy included plasma sample availability for OPN assay by ELISA and absence of major radiation therapy deviations (N = 578). OPN concentrations were analyzed for overall survival (OS) and time to locoregional failure (TTLRF), adjusting for known prognostic factors. Additional analysis was carried out in patients with available tumor p16(INK4A) staining status.. The median OPN level was 544 ng/mL (range: 7-2,640). High OPN levels were not associated with worse OS (relative HR, 1.03 for highest tertile) or TTLRF (relative HR 0.91 for highest tertile). There was no interaction between OPN and treatment arm for OS or TTLRF (P = 0.93 for OS; P = 0.87 for TTLRF). For the highest tertile the 2-year OS was 66% on control arm and 67% on TPZ arm (HR = 1.11, P = 0.67). Similarly for p16(INK4A) negative patients in the highest tertile, the 2-year OS was 61% on control arm and 63% on TPZ arm (HR = 1.05, P = 0.86).. We found no evidence that high plasma OPN levels were associated with an adverse prognosis in HNSCC, or were predictive of benefit with hypoxia targeting therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cisplatin; Cyclin-Dependent Kinase Inhibitor p16; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Head and Neck Neoplasms; Humans; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Osteopontin; Prospective Studies; Survival Rate; Tirapazamine; Treatment Outcome; Triazines

Hepatocyte growth factor (HGF) is a hypoxia-induced secreted protein that binds to cMet and regulates interleukin (IL)-8 expression. We evaluated the role of circulating HGF and IL-8 as prognostic and predictive factors for efficacy of tirapazamine (TPZ), a hypoxic cell cytotoxin.. Patients with stages III to IV head and neck cancer were randomized to receive radiotherapy with cisplatin (CIS) or CIS plus TPZ (TPZ/CIS). Eligibility for the substudy included plasma sample availability for HGF and IL-8 assay by ELISA and no major radiation deviations (N = 498). Analyses included adjustment for major prognostic factors. p16(INK4A) staining (human papillomavirus surrogate) was carried out on available tumors. Thirty-nine patients had hypoxia imaging with (18)F-fluoroazomycin arabinoside ((18)FAZA)-positron emission tomography.. Elevated IL-8 level was associated with worse overall survival (OS) irrespective of treatment. There was an interaction between HGF and treatment arm (P = 0.053); elevated HGF was associated with worse OS in the control but not in the TPZ/CIS arm. Similar trends were observed in analyses restricted to p16(INK4A)-negative patients. Four subgroups defined by high and low HGF/IL-8 levels were examined for TPZ effect; the test for interaction with arm was P = 0.099. TPZ/CIS seemed to be beneficial for patients with high HGF and IL-8 but adverse for low HGF and high IL-8. Only HGF correlated with (18)FAZA tumor standard uptake value.. IL-8 is an independent prognostic factor irrespective of treatment. There is an interaction between HGF and treatment arm. Certain subgroups based on IL-8/HGF levels seemed to do better with TPZ/CIS while others did worse, highlighting the complexity of hypoxia targeting in unselected patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Carcinoma, Squamous Cell; Chemoradiotherapy; Female; Head and Neck Neoplasms; Hepatocyte Growth Factor; Humans; Interleukin-8; Male; Middle Aged; Positron-Emission Tomography; Tirapazamine; Triazines

Promising results in a randomized phase II trial with the hypoxic cytotoxin tirapazamine (TPZ) combined with cisplatin (CIS) and radiation led to this phase III trial.. Patients with previously untreated stage III or IV (excluding T1-2N1 and M1) squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx were randomly assigned to receive definitive radiotherapy (70 Gy in 7 weeks) concurrently with either CIS (100 mg/m(2)) on day 1 of weeks 1, 4, and 7 or CIS (75 mg/m(2)) plus TPZ (290 mg/m(2)/d) on day 1 of weeks 1, 4, and 7 and TPZ alone (160 mg/m(2)/d) on days 1, 3, and 5 of weeks 2 and 3 (TPZ/CIS). The primary end point was overall survival (OS). The planned sample size was 850, estimated to result in 334 deaths, which would provide 90% power to detect a difference in 2-year survival rates of 60% v 70% for CIS versus TPZ/CIS, respectively (hazard ratio = 0.69).. Eight hundred sixty-one patients were accrued from 89 sites in 16 countries. In an intent-to-treat analysis, the 2-year OS rates were 65.7% for CIS and 66.2% for TPZ/CIS (TPZ/CIS--CIS: 95% CI, -5.9% to 6.9%). There were no significant differences in failure-free survival, time to locoregional failure, or quality of life as measured by Functional Assessment of Cancer Therapy-Head and Neck.. We found no evidence that the addition of TPZ to chemoradiotherapy, in patients with advanced head and neck cancer not selected for the presence of hypoxia, improves OS.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Combined Modality Therapy; Female; Follow-Up Studies; Head and Neck Neoplasms; Humans; International Agencies; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Quality of Life; Radiotherapy Dosage; Survival Rate; Tirapazamine; Treatment Outcome; Triazines

To determine the prognostic importance of p16 and human papillomavirus (HPV) in patients with oropharyngeal cancer treated on a phase III concurrent chemoradiotherapy trial.. Patients with stage III or IV head and neck squamous cell cancer were randomly assigned to concurrent radiotherapy and cisplatin with or without tirapazamine. In this substudy, analyses were restricted to patients with oropharyngeal cancer. p16 was detected by immunohistochemistry, and HPV was detected by in situ hybridization and polymerase chain reaction.. Slides were available for p16 assay in 206 of 465 patients, of which 185 were eligible, and p16 and HPV were evaluable in 172 patients. One hundred six (57%) of 185 were p16-positive, and in patients evaluable for both p16 and HPV, 88 (86%) of 102 p16-positive patients were also HPV-positive. Patients who were p16-positive had lower T and higher N categories and better Eastern Cooperative Oncology Group (ECOG) performance status. p16-positive tumors compared with p16-negative tumors were associated with better 2-year overall survival (91% v 74%; hazard ratio [HR], 0.36; 95% CI, 0.17 to 0.74; P = .004) and failure-free survival (87% v 72%; HR, 0.39; 95% CI, 0.20 to 0.74; P = .003). p16 was a significant prognostic factor on multivariable analysis (HR, 0.45; 95% CI, 0.21 to 0.96; P = .04). p16-positive patients had lower rates of locoregional failure and deaths due to other causes. There was a trend favoring the tirapazamine arm for improved locoregional control in p16-negative patients (HR, 0.33; 95% CI, 0.09 to 1.24; P = .13).. HPV-associated oropharyngeal cancer is a distinct entity with a favorable prognosis compared with HPV-negative oropharyngeal cancer when treated with cisplatin-based chemoradiotherapy.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Australia; Biomarkers, Tumor; Carcinoma, Squamous Cell; Chemotherapy, Adjuvant; Cisplatin; Cyclin-Dependent Kinase Inhibitor p16; Disease-Free Survival; DNA, Viral; Europe; Female; Humans; Immunohistochemistry; In Situ Hybridization; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; New Zealand; North America; Oropharyngeal Neoplasms; Papillomaviridae; Polymerase Chain Reaction; Proportional Hazards Models; Radiotherapy, Adjuvant; Risk Assessment; Risk Factors; South America; Time Factors; Tirapazamine; Treatment Outcome; Triazines

Reirradiation (re-RT) with concurrent chemotherapy offers a therapeutic option in patients who have locoregional recurrence of head and neck cancer (HNC). The hypoxic cell sensitizer, tirapazamine (TPZ), has demonstrated promising results in first-line therapy for HNC. This phase I trial was designed to test the feasibility of giving TPZ in the re-RT setting.. Patients with recurrent HNC who received prior radiotherapy (RT) were enrolled and received TPZ (260 mg/m2) and cisplatin (50 mg/m2) Weeks 1, 3, and 5 concurrently with RT (72 Gy, 42 fractions over 6 weeks). TPZ (160 mg/m2) alone was added on Days 1, 3, and 5 of Week 2 (cohort 1) or Weeks 2 and 4 (cohort 2).. Twenty-five subjects were enrolled, 7 and 18 on cohorts 1 and 2, respectively. Significant toxicities included Grade 3 dermatitis (20%) and Grade 3 mucositis (40%). Dose-limiting toxicity was observed on cohort 2 (1 patient with aspiration pneumonia). Four deaths occurred during treatment. Two fatalities occurred after completing therapy as a result of carotid artery rupture. With a minimum and median follow-up of 14 and 24 months, respectively, median overall survival was 14 months with actuarial 1-year and 2-year survival of 56% and 27%, respectively.. Reirradiation with concomitant chemotherapy including TPZ in patients with unresectable recurrent HNC is feasible and results in long-term survival in a significant proportion of patients.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Combined Modality Therapy; Confidence Intervals; Feasibility Studies; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Radiation-Sensitizing Agents; Radiotherapy Dosage; Retreatment; Tirapazamine; Triazines

The objective of this article was to report the results from a randomized trial that evaluated the efficacy and toxicity of adding tirapazamine (TPZ) to chemoradiotherapy in the treatment of patients with head and neck squamous cell carcinomas (HNSCC).. Sixty-two patients with lymph node-positive, resectable, TNM Stage IV HNSCC were randomized to receive either 2 cycles of induction chemotherapy (TPZ, cisplatin, and 5-fluorouracil [5-FU]) followed by simultaneous chemoradiotherapy (TPZ, cisplatin, and 5-FU) or to receive the same regimen without TPZ. Patients who did not achieve a complete response at 50 Grays underwent surgical treatment. Stratification factors for randomization included tumor site, TNM stage, and median tumor oxygen tension. The primary endpoint was complete lymph node response.. The addition of TPZ resulted in increased hematologic toxicity. There was 1 treatment-related death from induction chemotherapy. The complete clinical and pathologic response rate in the lymph nodes was 90% and 74% for the standard treatment arm and the TPZ arm, respectively (P = .08) and 89% and 90% at the primary site in the respective treatment arms (P = .71). The 5-year overall survival rate was 59%, the cause-specific survival rate was 68%, the rate of freedom from recurrence was 69%, and the locoregional control rate was 77% for the entire group. There was no difference with regard to any of the outcome parameters between the 2 treatment arms. The significant long-term toxicity rate also was found to be similar between the 2 arms.. The addition of TPZ increased hematologic toxicity but did not improve outcomes in patients with resectable, Stage IV HNSCC using the protocol administered this small randomized study. The combination of induction and simultaneous chemoradiotherapy resulted in excellent survival in these patients.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Combined Modality Therapy; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Male; Middle Aged; Patient Compliance; Survival Rate; Tirapazamine; Treatment Outcome; Triazines

To select one of two chemoradiotherapy regimens for locally advanced squamous cell carcinoma (SCC) of the head and neck as the experimental arm for the next Trans-Tasman Radiation Oncology Group phase III trial.. One hundred twenty-two previously untreated patients with stage III/IV SCC of the head and neck were randomized to receive definitive radiotherapy (70 Gy in 7 weeks) concurrently with either cisplatin (75 mg/m(2)) plus tirapazamine (290 mg/m(2)/d) on day 2 of weeks 1, 4, and 7, and tirapazamine alone (160 mg/m(2)/d) on days 1, 3, and 5 of weeks 2 and 3 (TPZ/CIS), or cisplatin (50 mg/m(2)) on day 1 and infusional fluorouracil (360 mg/m(2)/d) on days 1 through 5 of weeks 6 and 7 (chemoboost).. Three-year failure-free survival rates were 55% with TPZ/CIS (95% CI, 39% to 70%) and 44% with chemoboost (95% CI, 30% to 60%; log-rank P = .16). Three-year locoregional failure-free rates were 84% in the TPZ/CIS arm (95% CI, 71% to 92%) and 66% in the chemoboost arm (95% CI, 51% to 79%; P = .069). More febrile neutropenia and grade 3 or 4 late mucous membrane toxicity were observed with TPZ/CIS, while acute skin radiation reaction was more severe and prolonged with chemoboost. Compliance with protocol treatment was satisfactory on both arms.. Both regimens are feasible and are associated with significant but acceptable toxicity profiles in the cooperative group setting. Based on the promising efficacy seen in this trial, TPZ/CIS is being evaluated in a large phase III trial.

Topics: Adult; Aged; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Combined Modality Therapy; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Male; Middle Aged; Patient Compliance; Radiotherapy; Survival Rate; Tirapazamine; Triazines

The purpose of the study was to evaluate [(18)F]fluoromisonidazole (FMISO) PET in advanced head and neck cancer during hypoxia-targeting therapy.. Fifteen of 16 patients in a phase I trial of chemoradiation plus tirapazamine (specific cytotoxin for hypoxic cells) in advanced (T3/4 and/or N2/3) head and neck cancer underwent serial [(18)F]fluorodeoxyglucose (FDG) and FMISO PET. We have previously reported excellent early clinical outcome of these patients and now review FMISO PET results in the context of longer follow-up of this patient cohort.. Based on blinded qualitative scoring by two readers, FMISO PET was positive in 13/15 patients at baseline: 12/15 of primary sites and 8/13 neck nodes were scored as positive. All sites of corresponding FDG and FMISO abnormality at baseline showed marked qualitative reduction of uptake within 4 weeks of commencing therapy, consistent with effective hypoxia-targeted therapy. With a median follow-up of 6.9 years, there have been only four locoregional failures, while three other patients have died of metachronous lung cancer. The 5-year overall survival was 50% (95% CI 27-73%), the 5-year failure-free survival was 44% (95% CI 22-68%) and the 5-year freedom from locoregional failure was 68% (95% CI 38-88%).. The high prevalence of hypoxia demonstrated on FMISO PET imaging is consistent with the advanced disease stage of these patients and would be expected to predict an adverse prognosis. Evidence of the early resolution of FMISO abnormality during treatment, associated with excellent locoregional control in this patient cohort, supports further investigation of hypoxia-targeting agents in advanced head and neck cancer.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Benzylamines; Carcinoma, Squamous Cell; Cell Hypoxia; Chemotherapy, Adjuvant; Cisplatin; Cohort Studies; Drug Delivery Systems; Female; Follow-Up Studies; Head and Neck Neoplasms; Humans; Male; Middle Aged; Positron-Emission Tomography; Prognosis; Radiation-Sensitizing Agents; Radiopharmaceuticals; Reproducibility of Results; Sensitivity and Specificity; Single-Blind Method; Tirapazamine; Treatment Outcome; Triazines

To compare the Eppendorf PO2 histograph and the alkaline comet assay as methods of measuring tumor hypoxia in patients with head-and-neck squamous cell carcinomas.. As part of a larger clinical trial, 65 patients with head-and-neck squamous cell carcinoma nodal metastasis underwent tumor oxygenation measurements with Eppendorf PO2 histographs and comet assays, performed on fine-needle aspirates at 1 and 2 min after 5 Gy. Fifty-four patients had sufficient tumor cells for comet analysis at 1 min and 26 at both 1 and 2 min. Individual cells were examined for DNA single-strand breaks by alkaline gel electrophoresis, and the distribution of values was quantified using median tail moment (MTM). Nonirradiated tumor cells from pretreatment fine-needle aspirates received 5 Gy in vitro to establish the oxygenated response.. There was a significant correlation between the 1- and 2-min MTM (slope = 0.77 +/- 0.03). There was no relationship between DNA damage in tumor cells irradiated in vitro and in vivo. No correlation was found between Eppendorf PO2 measurements and comet MTM. There was a statistically significant correlation between the treatment response in the node studied and comet MTMs, whereas no correlation was observed between treatment response and Eppendorf measurements.. Comet assays are reproducible, as shown by biopsies at 1 and 2 min. Intertumor variation in the MTM is not a result of intrinsic radiosensitivity but of tumor hypoxia. There was no correlation between Eppendorf PO2 measurements and comet MTM. Comet assays were better than Eppendorf in predicting treatment response as an end point for short-term outcome. Longer follow-up is needed to determine the role of the comet assay as a predictor for locoregional tumor control and survivals.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cell Hypoxia; Cisplatin; Comet Assay; DNA Damage; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Male; Microelectrodes; Middle Aged; Oxygen; Radiation-Sensitizing Agents; Tirapazamine; Triazines

To determine the maximum-tolerated dose of tirapazamine when combined with cisplatin and radiation in patients with T3/4 and/or N2/3 squamous cell carcinoma of the head and neck.. The starting schedule was conventionally fractionated radiotherapy (70 Gy in 7 weeks) with concomitant cisplatin 75 mg/m2 and tirapazamine 290 mg/m2 (before cisplatin) in weeks 1, 4, and 7 and tirapazamine alone 160 mg/m2 three times a week in weeks 2, 3, 5, and 6. Positron emission tomography scans for tumor hypoxia (18F misonidazole) were performed before and during radiotherapy.. We treated 16 patients with predominantly oropharyngeal primary tumors, including 10 patients with T4 or N3 disease. Febrile neutropenia occurred toward the end of radiotherapy in three out of six patients treated on the initial dose level. Two of these patients also developed grade 4 acute radiation reactions. Another 10 patients were treated with the same doses, but the week 5 and week 6 tirapazamine doses were omitted. This resulted in less neutropenia and only one dose-limiting toxicity (DLT) (febrile neutropenia), and eight out of 10 patients completed treatment without any dose omissions. In these 10 patients, the acute radiation toxicities were not obviously enhanced compared with chemoradiotherapy regimens using concurrent platinum and fluorouracil. 18F misonidazole scans detected hypoxia in 14 of 15 patients at baseline, with only one patient having detectable hypoxia at the end of treatment. With a median follow-up of 2.7 years, the 3-year failure-free survival rate was 69% (SE, 12%), the 3-year local progression-free rate was 88% (SE, 8%), and the 3-year overall survival rate was 69% (SE, 12%).. DLT was due unexpectedly to febrile neutropenia, which could be overcome by omitting tirapazamine in weeks 5 and 6. The combination of tirapazamine, cisplatin, and radiotherapy resulted in remarkably good and durable clinical responses in patients with very advanced head and neck cancers. It warrants further investigation.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cell Hypoxia; Cisplatin; Combined Modality Therapy; Dose Fractionation, Radiation; Drug Administration Schedule; Fluorodeoxyglucose F18; Head and Neck Neoplasms; Humans; Middle Aged; Misonidazole; Radiation-Sensitizing Agents; Radiopharmaceuticals; Survival Analysis; Tirapazamine; Tomography, Emission-Computed; Triazines

For patients with squamous cell carcinoma of the head and neck (HNSCC), persistence of cervical adenopathy following organ-preservation therapy is a strong predictor of locoregional failure. Squamous cell granulomas of the neck may represent a regressed state of metastatic HNSCC; however, relevant clinicopathologic features of this lesion including its morphologic characteristics, association with therapy, and relationship to disease progression are not well defined.. We reviewed 866 consecutive neck dissections performed at The Johns Hopkins Hospital from 1984 to 1996. A total of eight cases showing a foreign-body giant-cell reaction to keratin in the absence of viable tumor formed the basis of this study.. All eight cases were from patients with stage III or IV HNSCC with concurrent neck masses. Patients were initially treated by chemotherapy (n = 1), radiotherapy (n = 1), or chemotherapy plus radiotherapy (n = 6); and all patients subsequently underwent neck dissection for persistence of their neck masses. Histologically, the neck lesions were characterized by a foreign-body giant-cell reaction to keratin and extensive scarring. None (0%) of the patients developed recurrent regional disease in the treated neck. Two (25%) of the patients had tumor recurrence at the primary site. Two (25%) of the patients developed widely metastatic disease.. These observations suggest that squamous cell granulomas represent histologic regression of metastatic squamous cell carcinoma in patients with HNSCC treated by chemotherapy and/or radiotherapy. Although persistent cervical adenopathy is an established risk factor for locoregional failure in this group of patients, squamous cell granulomas of the neck paradoxically may reflect enhanced regional tumor sensitivity to cytotoxic agents.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carboplatin; Carcinoma, Squamous Cell; Chemotherapy, Adjuvant; Cisplatin; Culture Techniques; Diagnosis, Differential; Female; Fibrosis; Fluorouracil; Granuloma, Foreign-Body; Head and Neck Neoplasms; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Middle Aged; Neck; Neck Dissection; Neoplasm Staging; Paclitaxel; Radiotherapy, Adjuvant; Registries; Remission Induction; Tirapazamine; Triazines

To evaluate the efficacy and toxicity of tirapazamine, a hypoxic cytotoxin, combined with conventional radiotherapy (RT) for advanced head and neck carcinomas.. From Oct. 1994 to Nov. 1996, 40 patients with stage III or IV carcinomas of the head and neck were enrolled in a Phase II trial to receive conventional RT (70 Gy in 7 weeks) with concurrent tirapazamine (159 mg/m2 intravenously, 3 times per week for 12 doses). One patient subsequently withdrew from the protocol treatment, and was excluded from analyses. Among the 39 cases, the primary sites were located in the oropharynx (n = 28), supraglottic larynx (n = 6), or hypopharynx (n = 5). Twenty-seven patients had T3 or T4, and 27 had N2 or N3 disease.. Thirty-two (82%) patients received full 12 drug doses. Thirty-two patients (82%) received full 70 Gy of RT. The most frequent drug toxicities were muscle cramps (77%) and nausea/vomiting (62%), usually grade 1 or 2. Overall, 13 patients (33%) experienced grade 3 or 4 drug-related toxicities. No excessive RT-associated acute normal tissue reactions were observed. With a median follow-up of 13 months, the 1-year and 2-year local control rate was 64% and 59% respectively.. The tirapazamine regimen was well tolerated with a compliance rate of 82%. The toxicity of RT with concurrent tirapazamine was acceptable in treating advanced head and neck carcinomas. The disease control trend was encouraging. Further clinical studies are warranted.

Topics: Adult; Aged; Antineoplastic Agents; Carcinoma, Squamous Cell; Combined Modality Therapy; Female; Follow-Up Studies; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Staging; Radiation-Sensitizing Agents; Radiotherapy Dosage; Tirapazamine; Triazines

Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxia-activated prodrugs (HAP) have been developed, including the phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia. Using genome-scale shRNA screens and a high-representation library enriched for oxidoreductases, we identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in three different genetic backgrounds. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. Knockdown or genetic knockout of POR reduced SN30000 reductive metabolism and clonogenic cell death and similarly reduced sensitivity to TH-302 under hypoxia. A retrospective evaluation of head and neck squamous cell carcinomas showed heterogeneous POR expression and suggested a possible relationship between human papillomavirus status and HAP sensitivity. Taken together, our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during the clinical development of SN30000, TH-302, and other hypoxia-directed agents.

Topics: Activation, Metabolic; Antineoplastic Agents; Biomarkers; Carcinoma, Squamous Cell; Cell Hypoxia; Cell Line, Tumor; Chemoradiotherapy; Cyclic N-Oxides; Cytochrome P-450 Enzyme System; Head and Neck Neoplasms; High-Throughput Screening Assays; Humans; Neoplasm Proteins; Nitroimidazoles; Papillomaviridae; Papillomavirus Infections; Phosphoramide Mustards; Prodrugs; Retrospective Studies; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tirapazamine; Triazines; Tumor Microenvironment; Tumor Stem Cell Assay

To assess the role of a planned neck dissection (PND) after sequential chemoradiotherapy for patients with head-and-neck cancer with N2-N3 nodal disease.. We reviewed 90 patients with N2-N3 head-and-neck squamous cell carcinoma treated between 1991 and 2001 on two sequential chemoradiotherapy protocols. All patients received induction and concurrent chemotherapy with cisplatin and 5-fluorocuracil, with or without tirapazamine. Patients with less than a clinical complete response (cCR) in the neck proceeded to a PND after chemoradiation. The primary endpoint was nodal response. Clinical outcomes and patterns of failure were analyzed.. The median follow-up durations for living and all patients were 8.3 years (range, 1.5-16.3 year) and 5.4 years (range, 0.6-16.3 years), respectively. Of the 48 patients with nodal cCR whose necks were observed, 5 patients had neck failures as a component of their recurrence [neck and primary (n = 2); neck, primary, and distant (n = 1); neck only (n = 1); neck and distant (n = 1)]. Therefore, PND may have benefited only 2 patients (4%) [neck only failure (n = 1); neck and distant failure (n = 1)]. The pathologic complete response (pCR) rate for those with a clinical partial response (cPR) undergoing PND (n = 30) was 53%. The 5-year neck control rates after cCR, cPR→pCR, and cPR→pPR were 90%, 93%, and 78%, respectively (p = 0.36). The 5-year disease-free survival rates for the cCR, cPR→pCR, and cPR→pPR groups were 53%, 75%, and 42%, respectively (p = 0.04).. In our series, patients with N2-N3 neck disease achieving a cCR in the neck, PND would have benefited only 4% and, therefore, is not recommended. Patients with a cPR should be treated with PND. Residual tumor in the PND specimens was associated with poor outcomes; therefore, aggressive therapy is recommended. Studies using novel imaging modalities are needed to better assess treatment response.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Chemoradiotherapy; Cisplatin; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neck Dissection; Neoplasm Recurrence, Local; Neoplasm, Residual; Organ Sparing Treatments; Prospective Studies; Survival Rate; Tirapazamine; Treatment Outcome; Triazines

To report the impact of radiotherapy quality on outcome in a large international phase III trial evaluating radiotherapy with concurrent cisplatin plus tirapazamine for advanced head and neck cancer.. The protocol required interventional review of radiotherapy plans by the Quality Assurance Review Center (QARC). All plans and radiotherapy documentation underwent post-treatment review by the Trial Management Committee (TMC) for protocol compliance. Secondary review of noncompliant plans for predicted impact on tumor control was performed. Factors associated with poor protocol compliance were studied, and outcome data were analyzed in relation to protocol compliance and radiotherapy quality.. At TMC review, 25.4% of the patients had noncompliant plans but none in which QARC-recommended changes had been made. At secondary review, 47% of noncompliant plans (12% overall) had deficiencies with a predicted major adverse impact on tumor control. Major deficiencies were unrelated to tumor subsite or to T or N stage (if N+), but were highly correlated with number of patients enrolled at the treatment center (< five patients, 29.8%; > or = 20 patients, 5.4%; P < .001). In patients who received at least 60 Gy, those with major deficiencies in their treatment plans (n = 87) had a markedly inferior outcome compared with those whose treatment was initially protocol compliant (n = 502): -2 years overall survival, 50% v 70%; hazard ratio (HR), 1.99; P < .001; and 2 years freedom from locoregional failure, 54% v 78%; HR, 2.37; P < .001, respectively.. These results demonstrate the critical importance of radiotherapy quality on outcome of chemoradiotherapy in head and neck cancer. Centers treating only a few patients are the major source of quality problems.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Clinical Trials as Topic; Combined Modality Therapy; Follow-Up Studies; Guideline Adherence; Head and Neck Neoplasms; Humans; International Agencies; Neoplasm Recurrence, Local; Neoplasm Staging; Quality Assurance, Health Care; Radiotherapy Dosage; Survival Rate; Tirapazamine; Treatment Outcome; Triazines

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Hypoxia; Chemotherapy, Adjuvant; Cisplatin; Cyclin-Dependent Kinase Inhibitor p16; Disease-Free Survival; DNA, Viral; Humans; Neoplasm Recurrence, Local; Neoplasm Staging; Oropharyngeal Neoplasms; Papillomaviridae; Radiotherapy, Adjuvant; Risk Assessment; Risk Factors; Time Factors; Tirapazamine; Treatment Outcome; Triazines

The purpose of this investigation was to compare the effect on intratumour quiescent (Q) cells in vivo of hexamethylenetetramine (HMTA) or tirapazamine (TPZ) in combination with gamma-irradiation and cisplatin treatment. Squamous cell carcinoma (SCC) VII tumour-bearing mice were administered 5-bromo-2'-deoxyuridine (BrdU) continuously to label all intratumour proliferating (P) cells. The mice then received HMTA or TPZ intraperitoneally or continuously with or without gamma-irradiation or cisplatin treatment. Other tumour-bearing mice received HMTA or TPZ intraperitoneally immediately after gamma-irradiation. Immediately after gamma-irradiation or cisplatin treatment following HMTA or TPZ, or 24 h after gamma-irradiation followed by HMTA or TPZ, the response of Q cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. The response of all tumour cells (P + Q) was determined from the BrdU-non-treated tumours. HMTA was more toxic to the subset of Q cells than to the population of tumour cells as a whole, similar to the findings for TPZ. The radiosensitising effect of HMTA was similar to that of TPZ in both all cells and Q cells. The recovery-inhibiting effect of HMTA was reliable, but not as great as that of TPZ. The cisplatin sensitivity-enhancing effect of HMTA was similar to or slightly greater than that of TPZ. Continuous administration of both HMTA and TPZ resulted in higher radiosensitivity- and cisplatin sensitivity-enhancing effects than did a single i.p. administration. We concluded that, in terms of the total tumour cell killing effect, including killing of Q cells, gamma-irradiation and cisplatin treatment combined with continuous HMTA administration is a promising strategy given that HMTA is used in clinics.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Hypoxia; Cisplatin; Combined Modality Therapy; Female; Gamma Rays; Infusions, Subcutaneous; Methenamine; Mice; Mice, Inbred C3H; Radiation-Sensitizing Agents; Skin Neoplasms; Tirapazamine; Treatment Outcome; Triazines; Tumor Cells, Cultured

Well-differentiated hypoxic regions in head and neck squamous cell carcinoma like in A253 xenografts are avascular and, therefore, hinder drug delivery leading to drug resistance and tumor regrowth. Methylselenocysteine (MSC, 0.2 mg/mouse per day per oral for 35 days starting 7 days before the first irinotecan (CPT-11)) has been found to increase efficacy of a wide variety of chemotherapeutic agents including CPT-11 (100 mg/kg per week x 4 intravenously). Whereas CPT-11 leads to a 10% complete response (CR) in A253 xenografts, the combination of MSC and CPT-11 increased the CR to 70%. Surviving tumors were found to consist largely of avascular hypoxic regions. Here, we investigated the combination of tirapazamine (TPZ, 70 mg/kg per week intraperitoneal x 4 administered 3 or 72 hours before CPT-11), a bioreductive drug in clinical trial with selective toxicity for hypoxic cells, with MSC and CPT-11 in further enhancing the cure rates. Tumor response, change in tumor hypoxic regions, and DNA damage were monitored in vivo. Tirapazamine administered 3 hours before CPT-11 in combination with MSC + CPT-11 led to a lower tumor burden. Tirapazamine did not increase cure rate beyond that of MSC + CPT-11 combination and was instead found to decrease cures with no evidence of an increased DNA damage or a significant reduction in avascular hypoxic tumor regions. CD31 immunostaining in A253 demonstrated disruption of tumor vessels by TPZ that could lower cytotoxic drug delivery to carbonic anhydrase IX-positive hypoxic tumor cells and may explain at least partially these unexpected results.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Camptothecin; Carcinoma, Squamous Cell; Cell Differentiation; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cysteine; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Forkhead Transcription Factors; Head and Neck Neoplasms; Humans; Irinotecan; Maximum Tolerated Dose; Mice; Mice, Nude; Neoplasm Transplantation; Organoselenium Compounds; Selenocysteine; Tirapazamine; Triazines; Xenograft Model Antitumor Assays

The effect of vascular disrupting agent ZD6126 with time on the sensitivity to the hypoxic cytotoxin tirapazamine (TPZ) and gamma-rays was examined in large and small solid tumors.. Mice bearing SCC VII tumors 1 or 1.5 cm in diameter received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells, followed by injection with or without ZD6126. In the absence of ZD6126, or 1 or 24 h following ZD6126 injection, the response to TPZ or gamma-ray irradiation in quiescent (Q) cells was assessed in terms of induced micronucleus (MN) frequency using immunofluorescence staining for BrdU. The MN frequency in the total cell population was determined from the tumors not pretreated with BrdU. Another group of tumor-bearing mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors.. One hour after ZD6126 injection, both small and large tumors showed lower and higher sensitivity, and 24 h after, higher and lower sensitivity, to gamma-rays and TPZ, respectively, than the tumors not treated with ZD6126. Further, they showed larger and smaller HFs 1 and 24 h after ZD6126 injection, respectively. Without ZD6126 and 1 h after injection, small tumors were more sensitive to gamma-rays and less sensitive to TPZ than large tumors, probably due to the smaller HFs than large tumors. In contrast, 24 h after the injection, these differences in sensitivity and the HF between small and large tumors were reversed. The changes in sensitivity and the size of the HF were more marked in the total cell population than in Q cells.. Following ZD6126 treatment, in terms of tumor control, especially large tumors and total tumor cell population, administering TPZ 1 h later and gamma-ray irradiation 24 h later were effective. Intratumor physiologic factors such as the size of the HF, depending on the time after ZD6126 injection, have to be taken into account when combining another treatment with ZD6126.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Gamma Rays; Mice; Organophosphorus Compounds; Radiation-Sensitizing Agents; Radiotherapy Dosage; Time Factors; Tirapazamine; Triazines

We clarified the usefulness of mild temperature hyperthermia (MTH) in combination with the continuous administration of tirapazamine (TPZ) under reduced dose-rate irradiation (RDRI) using gamma-rays.. SCC VII tumour-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a 24 h continuous subcutaneous infusion of TPZ either with or without MTH under high dose-rate irradiation (HDRI) or RDRI using gamma-rays. After the irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in non-proliferating tumour cells without BrdU labeling (= quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumour cell populations was determined using tumours that were not pretreated with BrdU.. The sensitivity of both the total and Q cell populations, especially the latter, was significantly reduced with RDRI compared with HDRI. TPZ increased the sensitivity of both populations, with a slightly more remarkable increase in Q cells. Further, MTH combined with TPZ raised the sensitivity of both the total and Q cell populations, especially the latter, under RDRI more markedly than under HDRI.. From the viewpoint of solid tumour control as a whole, including intratumour Q-cell control, the use of TPZ, especially in combination with MTH, is useful for suppressing the reduction in the sensitivity of tumour cells caused by the decrease in irradiation dose rate in vivo.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Combined Modality Therapy; Fever; Gamma Rays; Humans; Mice; Radiation-Sensitizing Agents; Tirapazamine; Triazines

Topics: Antigens, Neoplasm; Biomarkers; Carbonic Anhydrase IX; Carbonic Anhydrases; Carcinoma, Squamous Cell; Cell Hypoxia; Electrodes; Etanidazole; Head and Neck Neoplasms; Humans; Hydrocarbons, Fluorinated; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Neoplasm Proteins; Nitroimidazoles; Osteopontin; Oxygen; Partial Pressure; Radiation-Sensitizing Agents; Tirapazamine; Triazines

We clarified the usefulness of the continuous administration of tirapazamine (TPZ) in combination with reduced dose-rate irradiation (RDRI) using gamma-rays or reactor thermal neutrons. Squamous cell carcinoma (SCC) VII tumour-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a single intraperitoneal injection or 24 h continuous subcutaneous infusion of TPZ in combination with conventional dose-rate irradiation (CDRI) or RDRI using gamma-rays or thermal neutrons. After irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labelling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumour cells was determined using tumours that were not pre-treated with BrdU. The sensitivity of both total and Q cells, especially of Q cells, was significantly reduced with RDRI compared with CDRI. Combination of TPZ increased the sensitivity of both populations, with a slightly more remarkable increase in Q cells. Furthermore, the continuous administration of TPZ raised the sensitivity of both total and Q cell populations, especially the former, more markedly than the single administration, whether combined with CDRI or RDRI using gamma-rays or thermal neutrons. From the viewpoint of solid tumour control as a whole, including intratumour Q-cell control, the use of TPZ, especially when administered continuously, combined with RDRI, is useful for suppressing the reduction in the sensitivity of tumour cells caused by the decrease in irradiation dose rate in vivo.

Topics: Animals; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Survival; Fluorescent Antibody Technique; Gamma Rays; Hyperthermia, Induced; Infusions, Parenteral; Mice; Mice, Inbred C3H; Micronucleus Tests; Neoplasm Transplantation; Neutrons; Radiation-Sensitizing Agents; Radiotherapy Dosage; Skin Neoplasms; Tirapazamine; Treatment Outcome; Triazines

Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both the hind legs of Balb/cA nude mice. Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. The mice then received a hypoxic cytotoxin, tirapazamine (TPZ) or TX-402, with or without a vascular targeting agent (VTA), ZD6126. Another group of mice given ZD6126 received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors. After each treatment, the tumor cells were isolated and incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling [quiescent (Q) cells] was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. Both hypoxic cytotoxins showed significantly greater toxicity toward SAS/mp53 and Q than SAS/neo and total tumor cells, respectively. The sensitivity to TX-402 was significantly higher than that to TPZ in both total and Q tumor cells of both tumors. The significant enhancive effect by ZD6126 combined with each hypoxic cytotoxin was similar irrespective of p53 status, and slightly greater for total than Q cells probably because of a more marked increase in the size of the HFs in total than Q cells on the use of ZD6126 in both tumors, resulting in a reduction of the difference in the sensitivity to the hypoxic cytotoxin between total and Q cells. In the treatment of conventional cancer therapy-resistant Q tumor cells or p53-mutated tumor cells, the use of hypoxic cytotoxin was very promising either alone or when VTA was co-administered. TX-402 might be more promising than TPZ, although further study of the toxicity to normal tissue is needed.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cell Survival; Combined Modality Therapy; Cyclic N-Oxides; Gamma Rays; Head and Neck Neoplasms; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Micronucleus Tests; Mutation; Organophosphorus Compounds; Quinoxalines; Radiotherapy; Tirapazamine; Triazines; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

To evaluate the usefulness of continuous administration of hypoxic cytotoxins in terms of targeting acute hypoxia in solid tumours and the significance of combination with mild temperature hyperthermia (MTH) (40 degrees C, 60 min), the cytotoxic effects of singly or continuously administered tirapazamine (TPZ) and TX-402 were examined in combination with or without MTH in vivo. Further, the effects were also analysed on total (=proliferating (P)+quiescent (Q)) and Q cell populations in solid tumours with the method for selectively detecting the Q cell response. C3H/He mice bearing SCC VII tumours received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days to label all P cells. The tumour-bearing mice then received a single intra-peritoneal injection or 24 h continuous subcutaneous infusion of hypoxic cytotoxin, TPZ or TX-402, with or without MTH. On the other hand, to detect the changes in the hypoxic fraction (HF) in the tumours by MTH, another group of mice with or without MTH received a series of test doses of gamma-rays while alive or after tumour clamping. After each treatment, the tumour cells were isolated and incubated with a cytokinesis blocker (=cytochalasin-B) and the micronucleus (MN) frequency in cells without BrdU labelling (=Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total tumour cells was determined from the tumours that were not pre-treated with BrdU. The sensitivity to TX-402 was slightly higher than that to TPZ in both total and Q tumour cells. Continuous administration elevated the sensitivity of both total and Q cells, especially total cells. MTH raised the sensitivity of Q cells more remarkably than that of total cells in both single and continuous administrations. It was thought to be probably because of the higher dose distribution of hypoxic cytotoxin in intermediately hypoxic areas derived mainly from chronic hypoxia through MTH. From the viewpoint of tumour control as a whole including both total and Q tumour cells, the continuous administration of hypoxic cytotoxin combined with MTH may be useful for sensitizing tumour cells in vivo.

Topics: Animals; Antineoplastic Agents; Bromodeoxyuridine; Carcinoma, Squamous Cell; Combined Modality Therapy; Cyclic N-Oxides; Female; Fluorescent Antibody Technique; Hyperthermia, Induced; Mice; Mice, Inbred C3H; Micronucleus Tests; Neoplasm Transplantation; Neoplasms; Quinoxalines; Tirapazamine; Triazines

Tumor hypoxia is associated with resistance to radiotherapy and anticancer chemotherapy. However, it can be exploited to therapeutic advantage by concomitantly using hypoxic cytotoxins, such as tirapazamine (TPZ). Tumor electroporation offers the means to further increase tumor hypoxia by temporarily reducing tumor blood flow and therefore increase the cytotoxicity of TPZ. The primary objective of this work was to determine whether electric pulses combined with TPZ and radiotherapy (electroradiochemotherapy) was more efficacious than radiochemotherapy (TPZ + radiation). In these studies using the SCCVII tumor model in C3H mice, electroradiochemotherapy produced up to sixfold more tumor growth delay (TGD) than TPZ + radiation. In these studies, (1) large tumors (280 +/- 15 mm3) responded better to electroradiochemotherapy than small tumors (110 +/- 10 mm3), (2) TGD correlated linearly with tumor volume at the time of electroradiochemotherapy, (3) electric pulses induced a rapid but reversible reduction in O2 saturation, and (4) the electric field was highest near the periphery of the tumor in a 3D computer model. The findings suggested that electroradiochemotherapy gained its therapeutic advantage over TPZ + radiation by enhancing the cytotoxic action of TPZ through reduced tumor oxygenation. The greater antitumor effect achieved in large tumors may be related to tumor morphology and the electric-field distribution. These results suggest that electro-pulsation of large solid tumors may be of benefit to patients treated with radiation in combination with agents that kill hypoxic cells.

Topics: Animals; Carcinoma, Squamous Cell; Combined Modality Therapy; Male; Mice; Oxygen; Radiation-Sensitizing Agents; Tirapazamine; Triazines

Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both hind legs of Balb/cA nude mice. Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. The mice then received tirapazamine (TPZ) with or without mild temperature hyperthermia (40 degrees C, 60 min) (MTH), gamma-ray irradiation with or without MTH and/or TPZ, cisplatin (CDDP) with or without MTH and/or TPZ, or paclitaxel (TXL) with or without MTH and/or TPZ. After each treatment, the tumors were excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling (i.e., quiescent (Q) cells) was determined by using immunofluorescence staining for BrdU. Meanwhile, 6 h after gamma-ray irradiation or 24 h after other cytotoxic treatments, tumor cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells. The MN frequency and apoptosis frequency in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU. On the whole, gamma-ray irradiation and CDDP injection induced a higher frequency of apoptosis and lower frequency of MN in SAS/neo cells than SAS/mp53 cells. There were no apparent differences in the induced frequency of apoptosis and MN between SAS/neo and SAS/mp53 cells after TPZ or TXL treatment. MTH sensitized cells to TPZ-inducing cytotoxicity more markedly in SAS/mp53 and Q cells than in SAS/neo cells and total cells, respectively. In gamma-ray irradiation and CDDP treatment, the enhancement in combination with MTH and/or TPZ was more remarkable in SAS/mp53 cells and Q cells than in SAS/neo and total tumor cells, respectively. Also in the case of TXL treatment, the combination with MTH and/or TPZ induced a slightly greater enhancement effect in SAS/mp53 cells and Q cells. In view of the difficulty in controlling mutated p53 status tumors and intratumor Q cells, combination treatment with MTH and/or TPZ as a cooperative modality in cancer therapy is considered to have potential for controlling solid tumors as a whole.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Hypoxia; Cisplatin; Combined Modality Therapy; DNA Damage; Gamma Rays; Genes, p53; Head and Neck Neoplasms; Humans; Hyperthermia, Induced; Mice; Mice, Inbred BALB C; Mice, Nude; Micronucleus Tests; Neoplasm Proteins; Paclitaxel; Radiation-Sensitizing Agents; Tirapazamine; Triazines; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

The penetration of anticancer agents into tumor tissue has recently attracted considerable attention. This study examines the effect of carbogen breathing on the antitumor activity of tirapazamine combined with radiation. Our hypothesis is based on the observation that the diffusion of tirapazamine through tissue is dependent on oxygen tension. We postulated that carbogen breathing might enhance the ability of tirapazamine to diffuse to hypoxic cells located distal to functional blood vessels in tumors. We first determined that carbogen breathing caused no significant change in the pharmacokinetics of tirapazamine, suggesting that any effect of carbogen breathing on the activity of tirapazamine is not attributable to modulation of pharmacokinetics. Cell survival in SCCVII and SiHa tumors after 10 Gy X rays alone was similar. However, when tirapazamine was administered 30 min after radiation treatment under air-breathing conditions, cell killing was greater in SCCVII tumors compared to SiHa tumors. Carbogen breathing during the exposure to tirapazamine did not change the cell survival in SCCVII tumors, but it enhanced cell killing in the SiHa tumors. Interestingly, carbogen breathing during radiation treatment produced greater cell killing in the SiHa tumors than in the SCCVII tumors. The vascular architecture and type of hypoxia in the two tumors probably underlie the differences in the responses of the two tumors. These findings suggest that the effectiveness of tirapazamine and other hypoxic cytotoxins may be dependent on tumor type.

Topics: Administration, Inhalation; Animals; Carbon Dioxide; Carcinoma, Squamous Cell; Drug Synergism; Female; Gamma Rays; Humans; Mice; Mice, Inbred C3H; Mice, SCID; Oxygen; Radiation-Sensitizing Agents; Tirapazamine; Triazines; Uterine Cervical Neoplasms

To evaluate the radiosensitization effect on solid tumors upon combination treatment with paclitaxel (TXL), including the effect on intratumor quiescent (Q) cells.. Mice bearing SCC VII or EL4 solid tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days to label all proliferating (P) cells. The mice then received gamma-irradiation with or without tirapazamine (TPZ) at various time points after TXL administration. Another group of mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain hypoxic fractions (HFs) in the tumors at various time points after TXL administration. Immediately after irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, the tumor cells were isolated from the solid tumors in another group of mice, and the apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU. For the measurement of the HFs, the MN or apoptosis frequency of Q cells was then used to calculate the surviving fraction of Q cells from the regression line for the relationship between the MN or apoptosis frequency and the surviving fraction of total tumor cells.. In both SCC VII and EL4 tumors, maximum values of mitotic index (MI) and apoptosis frequency were observed 9 and 24 h after TXL administration, respectively. However, on the whole, the apoptosis frequency for SCC VII was very low. gamma-Irradiation 9 h after TXL administration induced significant radiosensitization effects on the total cells of both tumors. Irradiation at 60 h had a more significant effect on total cells of EL4 tumor, but no significant effect on total cells of SCC VII tumor. Combined treatment with TXL induced no radiosensitization effect on Q cells in either tumor. The effect on Q cells was observed only after TPZ was administered. The HF of total cells in EL4 tumors decreased significantly 60 h after TXL administration.. No radiosensitization effect upon combination treatment with TXL is induced in Q tumor cells. However, the effect on P cells is produced by irradiation at the time when the maximum values of MI are induced following TXL administration. In addition, for tumors that are susceptible to apoptosis after TXL administration alone, irradiation at the time of sufficient reoxygenation in tumors after TXL administration produces a greater radioenhancement effect on P cells.

Topics: Animals; Apoptosis; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Survival; Humans; Mice; Mice, Inbred C3H; Micronucleus Tests; Neoplasms; Paclitaxel; Radiation Tolerance; Radiation-Sensitizing Agents; Radiobiology; Radiotherapy Dosage; Time Factors; Tirapazamine; Triazines

C57BL mice bearing EL4 tumors and C3H / He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Three hours after oral administration of l-p-boronophenylalanine-(10)B (BPA), or 30 min after intraperitoneal injection of sodium borocaptate-(10)B (BSH) or l-p-boronophenylalaninol (BPA-ol), a newly developed (10)B-containing alpha-amino alcohol, the tumors were irradiated with thermal neutron beams. For the combination with mild temperature hyperthermia (MTH) and / or tirapazamine (TPZ), the tumors were heated at 40 degrees C for 30 min immediately before neutron exposure, and TPZ was intraperitoneally injected 30 min before irradiation. The tumors were then excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the apoptosis frequency in Q cells. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from tumors that were not pretreated with BrdU. Without TPZ or MTH, BPA-ol increased both frequencies most markedly, especially for total cells. However, as with BPA, the sensitivity difference between total and Q cells was much larger than with BSH. On combined treatment with both MTH and TPZ, this sensitivity difference was markedly reduced, similarly to when BPA was used. MTH increased the (10)B uptake of all (10)B-compounds into both tumor cells. BPA-ol has good potential as a (10)B-carrier in neutron capture therapy, especially when combined with both MTH and TPZ.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Apoptosis; Boranes; Boron Neutron Capture Therapy; Bromodeoxyuridine; Carcinoma, Squamous Cell; Combined Modality Therapy; Cytochalasin B; Drug Screening Assays, Antitumor; Fluorescent Antibody Technique, Indirect; Hindlimb; Hyperthermia, Induced; Injections, Intraperitoneal; Interphase; Lymphoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Micronucleus Tests; Molecular Structure; Neutrons; Phenylalanine; Radiation-Sensitizing Agents; Radiometry; Tirapazamine; Triazines

The novel hypoxia-selective cytotoxin NLCQ-1, which is a weak DNA intercalator, was studied in conjunction with radiation against V79 cultured cells and EMT6 or SCCVII tumors in their syngeneic mice and compared with tirapazamine (TPZ). NLCQ-1 was a very potent and efficient radiosensitizer of hypoxic V79 cells, providing SER values of 2.27-2.56 at 20-80 microM concentration (measured at 10% survival level). Its C1.6 (concentration for an SER of 1.6 to be obtained) was 7.2+/-0.2 microM. Its in vitro therapeutic index (ThI, defined as CT50(Air),/C1.6) varied by the exposure time from 57 (1-h exposure) to 145 (4.5-h exposure). The corresponding C1.6 value for TPZ was 16.9 microM whereas its in vitro therapeutic index was 49 (3-h exposure). A schedule-dependent synergistic interaction was observed between NLCQ-1 or TPZ and 20 Gy of radiation in both tumor models examined, by using the in vivo-in vitro assay as endpoint. Optimal synergism (> 1 log) was observed in EMT6 tumors when each bioreductive drug was given between 45 and 60 min before irradiation. NLCQ-1 alone had no significant antitumor activity at 10 mg/kg (28% of its single LD50), whereas a 0.4 surviving fraction was obtained by TPZ at 30 mg/kg (38% of its single LD50). SER values of 1.52 and 1.25 were obtained with 10 mg/kg NLCQ-1 and 30 mg/kg TPZ, respectively, in EMT6 tumors. An SER value of 1.58 was obtained for both hypoxia-selective cytotoxins, at equitoxic doses, in SCCVII tumors, by using a fractionated regimen. These results suggest a possible use of NLCQ-1 or TPZ as adjuvants to radiotherapy.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Hypoxia; Cell Line; Cell Survival; Cricetinae; Cytotoxins; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Administration Schedule; Female; Imidazoles; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Neoplasms, Experimental; Quinolines; Radiation-Sensitizing Agents; Tirapazamine; Triazines; Tumor Cells, Cultured; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

C3H / He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received one of six different DNA-damaging agents with or without mild temperature hyperthermia (40 degrees C, 30 min, MTH). These agents were adriamycin (ADM), mitomycin C (MMC), cyclophosphamide (CPA), bleomycin (BLM), cisplatin (CDDP), and tirapazamine (TPZ). After the drug treatment, the tumor-bearing mice were irradiated with a series of doses of gamma-rays. Immediately after irradiation, the tumors were excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that had not been pretreated with BrdU. MTH significantly increased the MN frequency of total cells in tumors irradiated with gamma-rays combined with CPA, BLM, CDDP or TPZ, and that of Q cells in tumors irradiated with gamma-rays combined with BLM or TPZ. The sensitivity difference in the MN frequency between total and Q tumor cells was significantly decreased by the combination with TPZ. TPZ combined with radiotherapy and TPZ combined with thermo-radiotherapy at mild temperatures appear to be promising modalities for sensitizing tumor cells in vivo, including Q tumor cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Bleomycin; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cisplatin; Combined Modality Therapy; Cyclophosphamide; Dose-Response Relationship, Radiation; Doxorubicin; Hyperthermia, Induced; Mice; Mice, Inbred C3H; Micronucleus Tests; Mitomycin; Radiation-Sensitizing Agents; Tirapazamine; Triazines

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Cell Hypoxia; Cisplatin; Combined Modality Therapy; Humans; Hyperthermia, Induced; Mice; Micronucleus Tests; Radiation Tolerance; Radiation-Sensitizing Agents; Tirapazamine; Triazines

To evaluate the efficacy of the use of tirapazamine (TPZ), especially combined with mild hyperthermia (40 degrees C, 60 min), in the treatment of solid tumors following an anti-angiogenic treatment with TNP-470. In addition, we assessed the effect of TPZ and/or mild hyperthermia (MHT) combined with conventional radiotherapy or chemotherapy on TNP-470 treated tumors.. C3H/He mice bearing SCC VII tumors subcutaneously received TNP-470 at two doses of 100 mg/kg after tumor cell inoculation. At the same time, the tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received TPZ administration combined with or without MHT, gamma-ray irradiation combined with or without TPZ and/or MHT, or cisplatin injection with or without TPZ and/or MHT. Another group of mice received a series of test doses of gamma-rays while alive or after being killed to obtain hypoxic fractions (HFs) in the tumors at various time points after the above-mentioned cytotoxic treatment point. After each treatment, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (or quiescent [Q] cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. For the measurement of the HFs, the MN frequency of BrdU-unlabeled cells was then used to calculate the surviving fraction of the unlabeled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumor cells.. TPZ administration combined with TNP-470 treatment and MHT increased the MN frequency more markedly than treatment with TPZ alone, and this tendency was more remarkable in Q cells than total cells. In both total and Q cells, combined treatment with TPZ and MHT produced significant increases in MN frequencies whether gamma-rays were delivered to TNP-470 treated tumors or cisplatin was injected into the TNP-470 administered mice. Although not significantly, the HFs of total and Q cell populations within solid tumors increased after TNP-470 treatment.. Combined treatment with TPZ and MHT, whether other cytotoxic treatments such as gamma-ray irradiation or chemotherapy using cisplatin were combined or not, was useful for sensitizing tumor cells in vivo including Q cells even after TNP-470 treatment.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Squamous Cell; Cell Hypoxia; Cell Survival; Cisplatin; Combined Modality Therapy; Cyclohexanes; DNA, Neoplasm; Dose-Response Relationship, Drug; Hyperthermia, Induced; Mice; Mice, Inbred C3H; Micronucleus Tests; O-(Chloroacetylcarbamoyl)fumagillol; Radiation-Sensitizing Agents; Regression Analysis; Sesquiterpenes; Tirapazamine; Triazines

Mice bearing solid tumors received 10 intraperitoneal administrations of 5-bromo-2'-deoxyuridine (BrdU) to label the proliferating (P) tumor cells. Then, as a priming treatment, tirapazamine (TPZ) was intraperitoneally administered. Further, 0 through 48 h later, the tumor-bearing mice received TPZ again at various doses. The tumor cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequencies in cells with and without BrdU labeling, which were regarded as P and quiescent (Q) cells at the priming treatment, respectively, were determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. In addition, P cell ratios in the tumors at the second treatment were determined using immunofluorescence staining for P cell nuclear antigen. In each cell fraction, the longer the interval between the two treatments, the higher was the sensitivity to TPZ, except 1 h after the priming treatment. More than 24 h later, total and P cells, especially P cells, showed significantly higher sensitivity to TPZ than in the case of a single TPZ treatment. The longer the period between the two TPZ treatments, the lower was the P cell ratio at the second treatment. These findings were thought to indicate that the use of TPZ in the treatment of solid tumors causes a shift from the P to the Q state in vivo.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Injections, Intraperitoneal; Mice; Mice, Inbred C3H; Micronuclei, Chromosome-Defective; Neoplasm Transplantation; Tirapazamine; Triazines; Tumor Cells, Cultured

C3H/He mice bearing SCC VII tumours received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received gamma-ray irradiation, or administration of tirapazamine (TPZ), cisplatin or bleomycin. At various time points after each treatment, tumour-bearing mice were irradiated with a series of test doses of gamma-rays, while alive or after being killed, to obtain hypoxic fractions (HFs) in the tumours. Immediately after gamma-ray test irradiation, the tumours were excised, minced and trypsinized. Tumour cell suspensions obtained were incubated with cytochalasin-B, a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labelling (i.e. quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. MN frequency in the total (P + Q) tumour cells was determined from the tumours that were not pre-treated with BrdU. MN frequency of BrdU-unlabelled cells was then used to calculate the surviving fraction of the unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumour cells. TPZ and cisplatin reduced the HF after treatment, especially in Q cells, and this tendency was particularly marked with TPZ. In contrast, bleomycin increased the HF after treatment. Both reoxygenation following gamma-ray irradiation or bleomycin treatment and a subsequent return to pre-treatment levels of HF following TPZ or cisplatin treatment (rehypoxiation) occurred more rapidly in total (P + Q) cells than in Q cells. Based on our previous report that total (P + Q) and Q cells within this tumour have large acutely and chronically HFs, respectively, we conclude that acute hypoxic cells play a major role in reoxygenation and rehypoxiation in SCC VII tumours.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Bleomycin; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Hypoxia; Cisplatin; Cytochalasin B; Injections; Mice; Micronucleus Tests; Tirapazamine; Triazines; Tumor Cells, Cultured

Efficient extravascular penetration is essential for the optimal activity of most anticancer drugs and is particularly relevant to bioreductive cytotoxins which target hypoxic cells that can be located distal to functional blood vessels within tumours. Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide; Triazone; SR 259075; formerly SR 4233) is a lead bioreductive cytotoxin currently undergoing clinical evaluation. It exhibits preferential cytotoxicity towards cells at reduced oxygen tension, and could complement existing anticancer therapies where hypoxic cells are believed to constitute a refractory population. We assessed the ability of tirapazamine to penetrate tumour tissue using an in vitro multilayered cell culture (MCC) model.. Diffusion of tirapazamine through oxic and hypoxic multilayered cell cultures composed of SiHa. human cervical carcinoma cells, was measured using a dual reservoir diffusion apparatus from which samples were quantified via HPLC. Drug concentration kinetics from both reservoirs were analysed using a mathematical model for diffusion and metabolism within the MCC. Results were then applied to a second mathematical model which described extravascular drug penetration within a tumour cord, the sheath of cells surrounding a blood vessel.. The diffusion coefficient of tirapazamine within SiHa MCCs was determined as 7.0+/-0.5 x 10(-7) cm2/s and the maximal metabolic rate for hypoxic MCCs, Vmax, as 1.5+/-0.4 microM/s. The thickness of individual tissue cultures was determined by diffusion of tritiated water (HTO). A linear relationship was shown to exist between tissue thickness and the inverse of permeability to HTO. Experimental results were used to simulate drug distribution within a tumour cord. These simulations indicate that, when tirapazamine is administered via intravenous infusion, a stable tirapazamine distribution throughout the cord occurs within 15 min with cells most peripheral to the blood vessel exposed to only 10% of the blood drug concentration. Under these conditions, the simulations predict cell kill to be limited to the first 75 microm of tissue surrounding a blood vessel.. This study indicates that extravascular penetration of tirapazamine to peripheral cells existing at low oxygen tension may be limited by the metabolism of tirapazamine by more proximal cells existing at moderate oxygen tension. Simulations found that tirapazamine reached only 10% of the blood concentration at cells most peripheral to blood vessels. These results indicate that tirapazamine would be significantly cytotoxic only to cells located within approximately 75 microm of blood vessels. Further MCC-based modelling of extravascular drug penetration would serve as a means of identifying new antitumour agents with location-specific activity.

Topics: Algorithms; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Survival; Chromatography, High Pressure Liquid; Diffusion; Female; Humans; Membranes, Artificial; Models, Biological; Polytetrafluoroethylene; Tirapazamine; Tissue Distribution; Triazines; Tumor Cells, Cultured; Uterine Cervical Neoplasms

The goal of this study was to compare, with a human tumor xenograft, two different strategies for increasing tumor response to fractionated irradiation, namely, oxygenating the hypoxic tumor cells with carbogen and nicotinamide, or killing these cells with the hypoxic cytotoxin, tirapazamine (TPZ). We used the human hypopharyngeal squamous cell carcinoma cell line FaDu implanted in immune-deficient SCID mice and assessed its response to radiation by cell survival and by growth delay. The tumors were irradiated either once or twice daily with 2 or 2.5 Gy/fraction with either TPZ (0.08 mmol/kg) or nicotinamide (1,000 mg/kg) with carbogen breathing. We also tested the effect of giving TPZ on alternate days, or daily during the first half of the course, the second half, or for the whole course of radiation. We found that adding TPZ or nicotinamide with carbogen to the fractionated radiation regimen enhanced the response of the human xenograft. The enhancement was somewhat greater (though not significantly so) for TPZ, especially when given with each radiation dose. In conclusion, adding TPZ, or nicotinamide plus carbogen, to fractionated irradiation enhanced the response of this human tumor xenograft to fractionated irradiation. Consistent with theoretical modeling, there was a greater enhancement of the radiation response of the tumor when TPZ was given with each radiation dose than when given with only half of the radiation doses.

Topics: Animals; Carbon Dioxide; Carcinoma, Squamous Cell; Cell Survival; Chemotherapy, Adjuvant; Combined Modality Therapy; Disease Models, Animal; Dose Fractionation, Radiation; Dose-Response Relationship, Radiation; Female; Humans; Male; Mice; Mice, SCID; Neoplasm Transplantation; Niacinamide; Oxygen; Oxygen Consumption; Pharyngeal Neoplasms; Radiation Dosage; Radiation-Sensitizing Agents; Sensitivity and Specificity; Tirapazamine; Triazines; Tumor Cells, Cultured

SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating cells. After injection of tirapazamine (TPZ), a bioreductive agent, combined with sodium borocaptate-10B (BSH) or dl-p-boronophenylalanine-10B (BPA) administration, the tumors were irradiated with thermal neutrons, and then isolated and incubated with cytochalasin-B (a cytokinesis blocker). The micronucleus (MN) frequency in cells without BrdU labeling (quiescent (Q) cells) was determined by means of immunofluorescence staining for BrdU, and that for total cells was obtained from tumors not pretreated with BrdU. Even when no 10B-compound was administered, TPZ increased the MN frequency of tumor cells including Q cells, resulting in reduction of the difference in MN frequency between total and Q cells, mainly by increasing the MN frequency of Q cells. TPZ increased the MN frequency of Q cells when combined with BPA administration, but TPZ showed no apparent effect on each cell population when combined with BSH. Namely, TPZ reduced the difference in MN frequency between total and Q cells caused by 10B-compound administration, especially when BPA was administered. From the viewpoint of the overall cell killing effect in boron neutron capture therapy (BNCT), combination with TPZ appeared to be useful in BPA-BNCT, but not in BSH-BNCT.

Topics: Animals; Boron Neutron Capture Therapy; Carcinoma, Squamous Cell; Female; Mice; Mice, Inbred C3H; Micronuclei, Chromosome-Defective; Radiation-Sensitizing Agents; Tirapazamine; Triazines

A system for growing three-dimensional cell culture has been developed which exhibits many features of solid tumours. This system comprises cells growing as a thick mat on a semipermeable membrane suspended in stirred media. SiHa cells grown as these multilayered cell cultures (MCCs) have produced cultures up to ca. 20 cell diameters in thickness. The MCCs, like solid tumours growing in vivo. develop diffusion-dependent necrosis and hypoxia and the cell packing acts as a barrier to the diffusion of drugs. These cultures can, therefore, be used to study aspects of cancer biology and drug transport that are difficult to study using other techniques.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Survival; Female; Flow Cytometry; Fluorescence; Humans; Tirapazamine; Triazines; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Mice bearing transplantable solid tumors received 10 intraperitoneal administrations of 5-bromo-2'-deoxyuridine (BrdU) to label the proliferating (P) tumor cells, and were then irradiated with 60Co gamma-rays or injected with cis-diamminedichloroplatinum (II) (cisplatin). The tumor cells were isolated and incubated with cytochalasin-B (a cytokinesis blocker). The micronucleus (MN) frequency in the cells without BrdU labeling, which were regarded as quiescent (Q) cells in the tumor, was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cell population was determined from tumors that were not pretreated with BrdU. Pretreatment with tirapazamine, a bioreductive agent, could enhance the sensitivity of tumor cells, including Q cells, to radiation more markedly than mitomycin C pretreatment as judged from an in vivo assay immediately after irradiation. Post-irradiation administration of tirapazamine produced a large post-irradiation radiosensitizing effect on both the total and Q tumor cell populations in vivo. Cisplatin treatment combined with tirapazamine demonstrated that tirapazamine also has a chemosensitizing potential for both the total and Q tumor cell populations. We confirmed that the sensitivity of Q cell populations to radiation and chemotherapy using cisplatin can be enhanced by combined treatment with tirapazamine.

Topics: Animals; Antineoplastic Agents; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Division; Cisplatin; Cobalt Radioisotopes; Cytochalasin B; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Gamma Rays; Mice; Mice, Inbred C3H; Micronuclei, Chromosome-Defective; Micronucleus Tests; Mitomycin; Mitotic Index; Tirapazamine; Triazines

Tirapazamine, a new bioreductive agent currently advancing through clinical trials, may have a valuable role to play in cancer therapy. In vitro, the drug shows markedly more toxicity to hypoxic cells than to aerobic cells, and preferential activity against hypoxic cells of solid tumors in vivo also can be inferred in many investigations. However, we have previously reported that tirapazamine has minimal activity against cells in the center of hypoxic spheroids, raising concerns with regard to whether the drug may be bioreductively inactivated before reaching chronically hypoxic tumor cells. We consequently examined the oxygen-dependent differential activity of tirapazamine in solid tumors in vivo by using fluorescence-activated cell sorting with clonogenicity assays for cell viability or with the comet assay for DNA damage. The preferential activity of tirapazamine against hypoxic vs. aerobic tumor cells in vivo was approximately threefold, much less than the factors of 50-500 typically seen in vitro. Interestingly, we also found that tirapazamine administration often modified tumor blood flow in the murine models, an effect that could be of clinical utility in sufficiently sensitive tumor cells. Taken together, our observations suggest that sequencing of tirapazamine with other agents requires careful consideration in the clinic.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Hypoxia; Cell Separation; Cell Survival; Drug Screening Assays, Antitumor; Flow Cytometry; Mice; Mice, SCID; Neoplasm Transplantation; Tirapazamine; Triazines

Development of strategies to eradicate radioresistant hypoxic cells would be of great benefit for clinical radiotherapy. In the present study, the in vivo effects of a promising hypoxic cytotoxin, tirapazamine (3-amino-1,2,4-benzotriazine 1,4-di-N-oxide), were examined in comparison with those of KU-2285, one of the best hypoxic cell radiosensitizers, in combination with both single and fractionated irradiation. The tumor response was assessed by the standard in vivo-in vitro clonogenic assay using SCCVII tumors in C3H mice and EMT-6/KU tumors in Balb/c mice with different characteristics of tumor hypoxia. With single-dose irradiation (18 Gy), both tirapazamine and KU-2285 showed significant enhancement of cell killing in a dose-dependent manner, but tirapazamine was more effective for SCCVII tumors with acutely hypoxic cells, while KU-2285 was more effective for EMT-6/KU tumors predominantly with chronically hypoxic cells. In fractionated irradiation regimens (4 fractions of 5 Gy at 12 h intervals), tirapazamine showed more marked combined effects at 10 and 20 mg/kg than KU2285 at 100-200 mg/kg in both SCCVII and EMT-6/KU tumors. We concluded that the effectiveness of KU-2285 and tirapazamine was correlated with the nature of tumor hypoxia with single-dose irradiation, whereas tirapazamine appeared more potent than KU-2285 with fractionated irradiation. These findings suggest the potential usefulness of tirapazamine in clinical fractionated radiotherapy.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Combined Modality Therapy; Female; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Neoplasms, Experimental; Nitroimidazoles; Radiotherapy Dosage; Tirapazamine; Triazines

Tirapazamine, a bioreductive drug preferentially toxic to hypoxic cells, produces significant numbers of DNA single-strand breaks that can be detected using the alkaline comet assay. Our goal was to determine whether single-strand breaks measured using this assay could act as a surrogate end point for cell killing in multicell spheroids and solid tumors in mice. In spheroids composed of Chinese hamster V79 cells, WiDr human colon carcinoma cells, or SiHa human cervical carcinoma cells, histograms of tail moments (indicators of DNA damage in the comet assay) could be used to identify the percentage of cells that sustained sufficient DNA damage to cause cell death after treatment with tirapazamine. The proportion of comets with tail moments

Topics: Animals; Carcinoma, Squamous Cell; Cell Hypoxia; Cell Survival; Cricetinae; Cricetulus; DNA Damage; DNA, Neoplasm; DNA, Single-Stranded; Drug Resistance, Neoplasm; Electrophoresis, Agar Gel; Fibroblasts; Humans; Image Processing, Computer-Assisted; Male; Mice; Mice, Inbred C3H; Mice, SCID; Microchemistry; Spheroids, Cellular; Tirapazamine; Triazines; Tumor Cells, Cultured

The alkaline comet assay was applied to individual cells from mice exposed to two bioreductive drugs, tirapazamine and RSU 1069, with the goal of comparing DNA damage to tumours and normal tissues. More DNA single-strand breaks (SSBs) and a greater heterogeneity in DNA damage were observed in tumour cells than in spleen and marrow cells of mice exposed to 10-100 mg/kg tirapazamine, consistent with the presence of hypoxic cells and the greater bioreductive capacity of tumours. In mice injected with 25-200 mg/kg RSU 1069, aerobic cells exhibited large numbers of SSBs while toxic DNA interstrand crosslinks were produced only in hypoxic cells. Cells from bone marrow and spleen showed extensive numbers of SSBs, but minimal crosslinking compared to tumours where 10-20% of cells were heavily crosslinked. DNA damage produced by these two bioreductive drugs may be useful in estimating the range of individual cell oxygen contents within tumours and normal tissues.

Topics: Aerobiosis; Animals; Antineoplastic Agents; Bone Marrow; Carcinoma, Squamous Cell; Cell Hypoxia; DNA Damage; DNA, Single-Stranded; Dose-Response Relationship, Radiation; Electrophoresis, Agar Gel; Hydrogen-Ion Concentration; Male; Mice; Mice, Inbred C3H; Misonidazole; Organ Specificity; Oxidation-Reduction; Radiation-Sensitizing Agents; Spleen; Tirapazamine; Triazines

The possibility of using tirapazamine (SR 4233) to identify hypoxic cells in multicell spheroids and murine tumours was examined by measuring tirapazamine-induced DNA damage to individual cells from multicell spheroids and SCCVII murine tumours. Fluorescence microscopy and image analysis were used to measure the extent of migration of DNA from individual cells embedded in agarose and exposed to an electric field. Using both the alkaline and neutral versions of the comet assay, at least 20 times more single-strand breaks were observed in cells from fully anoxic than fully oxic Chinese hamster V79 spheroids exposed to 30 microM tirapazamine, and about 10 times more single- than double-strand breaks were observed. Cells from spheroids containing about 50% radiobiologically hypoxic cells showed a pattern of tirapazamine breaks which translated to approximately 30% well-oxygenated in SCCVII tumors growing in C3H mice was also demonstrated. Cells close to tumour blood vessels showed less DNA damage by 20 mg kg-1 tirapazamine than cells distant from blood vessels. Rejoining of single-strand breaks was exponential, with a half-time of about 1 h under aerobic conditions, but rejoining half-time increased to 2 h for cells allowed to repair under anoxic conditions. While tirapazamine damage to DNA measured using the comet assay cannot provide a direct measure of hypoxic fraction, the degree of heterogeneity in DNA damage can be used to estimate the range and distribution of individual cell oxygen contents within spheroids and tumours.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Carcinoma, Squamous Cell; Cell Hypoxia; Cells, Cultured; Cricetinae; Cricetulus; DNA; DNA Damage; DNA Repair; DNA, Neoplasm; Fibroblasts; Flow Cytometry; Fluorescent Dyes; Kinetics; Male; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Tirapazamine; Triazines

To test the hypothesis that, following preferential killing of tumor hypoxic cells, the fraction of hypoxic cells in the tumor will reestablish itself to pretreatment levels (rehypoxiation) with the same kinetics as for reoxygenation.. Mouse squamous cell carcinoma VII (SCCVII) tumors were treated with a single dose of 10 Gy or a single dose of the bioreductive hypoxic cell cytotoxin, tirapazamine (SR 4233, 0.2 mmol/kg), which preferentially kills hypoxic cells within the tumor. Hypoxic fractions were determined by the paired survival curve technique using the in vivo-in vitro clonogenic assay 0-24 h after treatment.. Immediately after irradiation with 10 Gy, the hypoxic fraction of the tumors increased to 80% and rapidly returned to pretreatment levels 3-6 h later. Within 1 h of injecting tirapazamine, the hypoxic fraction fell to 0.57% (about 7% of pretreatment levels) and returned to pretreatment levels 3-5 h later.. The return to pretreatment levels of hypoxia among tumor cells surviving a single dose of radiation (reoxygenation) and of the hypoxic cell toxin tirapazamine (rehypoxiation) was rapid and occurred with similar kinetics for the two processes. These data support the hypothesis that reoxygenation and rehypoxiation are different manifestations of the same phenomenon and result from fluctuating tumor blood flow which creates acute hypoxia.

Topics: Animals; Carcinoma, Squamous Cell; Cell Hypoxia; Female; Mice; Mice, Inbred C3H; Oxygen; Tirapazamine; Triazines

The bioreductive cytotoxic agent SR 4233 (1,2,4-benzotriazine 3-amine 1,4-dioxide) has been shown to markedly potentiate the cell killing of mouse tumours when combined with fractionated radiation therapy. Differential metabolism under oxic compared to hypoxic conditions results in SR 4233 exhibiting selective cytotoxicity to hypoxic cells. This is thought to result from the production of a cytotoxic free radical which is generated predominantly in the absence of oxygen. We have examined a way of enhancing the effectiveness of this antitumour agent in vivo by artificially increasing the hypoxic fraction of tumours by hypoxic breathing. Mice are placed in a chamber containing 10% Oxygen 90% Nitrogen for 1 h after each administration of SR 4233. Our results in the SCCVII tumour model indicate that this manoeuvre results in a 10-fold increase in antitumour effectiveness of SR 4233 when administered in a fractionated regime with radiotherapy (8 x 2.5 Gy and 0.08 mmol kg-1), but not when a single treatment regime (1 x 20 Gy and 0.3 mmol kg-1) is used. Mathematical modelling of this effect is used to illustrate this phenomenon and can be used to predict the dependence of this type of therapy on the modification of tumour oxygenation.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Hypoxia; Female; Male; Mice; Mice, Inbred C3H; Radiotherapy Dosage; Tirapazamine; Triazines; Tumor Cells, Cultured

The present study describes a new way in which tumors may be made more sensitive to fractionated irradiation without affecting the sensitivity of surrounding normal tissues. It involves exploiting the cycling or intermittent hypoxia that occurs in at least some solid tumors, but not in normal tissues, using a new drug SR 4233, a benzotriazine di-N-oxide, which is rapidly metabolized in hypoxic cells to a product that kills these cells. Using this approach with a rodent tumor in a fractionated x-ray treatment regimen similar to that used in human radiotherapy, the addition of SR 4233 produced a large enhancement of the radiation response of the tumor with no change in the sensitivity of normal mouse skin. Under identical circumstances, there was no effect of the hypoxic cell radiosensitizer SR 2508, showing that SR 4233 with intermittent hypoxia was superior to a protocol which sensitized the hypoxic cells to doses of 2.5 Gy per fraction.

Topics: Animals; Carcinoma, Squamous Cell; Cell Hypoxia; Cell Survival; Female; Mice; Neoplasms, Experimental; Oxygen; Radiation Tolerance; Radiation-Sensitizing Agents; Skin; Tirapazamine; Triazines