E. coli nitroreductase NfsA is a reporter gene for non-invasive PET imaging in cancer gene therapy applications.

The use of reporter genes to non-invasively image molecular processes inside cells has significant translational potential, particularly in the context of systemically administered gene therapy vectors and adoptively administered cells such as immune or stem cell based therapies. Bacterial nitroreductase enzymes possess ideal properties for reporter gene imaging applications, being of non-human origin and possessing the ability to metabolize a range of clinically relevant nitro(hetero)cyclic substrates. Methods: A library of eleven Escherichia coli nitroreductase candidates were screened for the ability to efficiently metabolize 2-nitroimidazole based positron emission tomography (PET) probes originally developed as radiotracers for hypoxic cell imaging. Several complementary methods were utilized to detect formation of cell-entrapped metabolites, including various in vitro and in vivo models to establish the capacity of the 2-nitroimidazole PET agent EF5 to quantify expression of a nitroreductase candidate. Proof-of-principle PET imaging studies were successfully conducted using 18F-HX4. Results: Recombinant enzyme kinetics, bacterial SOS reporter assays, anti-proliferative assays and flow cytometry approaches collectively identified the major oxygen-insensitive nitroreductase NfsA from E. coli (NfsA_Ec) as the most promising nitroreductase reporter gene. Cells expressing NfsA_Ec were demonstrably labelled with the imaging agent EF5 in a manner that was quantitatively superior to hypoxia, in monolayers (2D), multicellular layers (3D), and in human tumor xenograft models. EF5 retention correlated with NfsA_Ec positive cell density over a range of EF5 concentrations in 3D in vitro models and in xenografts in vivo and was predictive of in vivo anti-tumor activity of the cytotoxic prodrug PR-104. Following PET imaging with 18F-HX4, a significantly higher tumor-to-blood ratio was observed in two xenograft models for NfsA_Ec expressing tumors compared to the parental tumors thereof, providing verification of this reporter gene imaging approach. Conclusion: This study establishes that the bacterial nitroreductase NfsA_Ec can be utilized as an imaging capable reporter gene, with the ability to metabolize and trap 2-nitroimidazole PET imaging agents for non-invasive imaging of gene expression.

Radiosensitization by the combination of SR-2508 and paclitaxel in hypoxic human tumor cells in vitro.

The two radiosensitizers SR-2508 (etanidazole) and paclitaxel (taxol) have different dose-limiting toxicities in humans. Combination of the two radiosensitizers may increase radiosensitization without increasing toxicity. This study was carried out to determine the synergistic radiosensitizing effect of combination of SR-2508 and paclitaxel in two hypoxic human tumor cell lines: a breast carcinoma (MCF-7) and a carcinoma cervicis (HeLa). The 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay was used to determine the number of surviving cells. Cell cycle was evaluated by flow cytometry. Cell viability was measured by the ability of single cells to form colonies in vitro. Our data demonstrated that the radiosensitization produced by the two radiosensitizers was additive in hypoxic HeLa cells while held in the G(1) phase of the cell cycle. On the other hand, there was no synergistic radiosensitizing effect in hypoxic MCF-7 cells by combination of the two drugs. Our results suggested that the synergistic radiosensitizing effect of SR-2508 and paclitaxel may be tumor-dependent and that breast cancer may not be a good candidate. This study may provide a new combination of radiosensitizers in radiotherapy for cervical carcinoma.

In vivo performance of implantable biodegradable preparations delivering Paclitaxel and Etanidazole for the treatment of glioma.

Drug-releasing implants delivering chemotherapeutic and radio-sensitizing agents are beginning to play a major role in the post-surgical eradication of residual glioma in the brain. Benefits from early arresting of tumor growth and tumor recovery dynamics stress the impact of drug release profiles of the implants on the efficacy of the treatment. This paper examines responses of BALB/c nude mice, bearing C6 glioma tumors subcutaneously, to treatments by PLGA microspheres, microparticles and discs-delivering Paclitaxel and Etanidazole. The experimental results are used to correlate the efficacy of treatment to in vitro release profiles from the various formulations. Our study demonstrates that radio-sensitizing effects during irradiation could be achieved by double burst profiles from Etanidazole-loaded discs, when compared to controls 17 days after implantation despite the short half-life of Etanidazole (1.4h) in vivo. These results also showed inhibited tumor growth on tumor volumes of 59%, 65% and 70% over the blank placebo groups after 21 days of tumor growth for spray-dried microspheres, electrohydrodynamic atomization microparticles and spray-dried discs, respectively.

Etanidazole in pH-sensitive liposomes: design, characterization and in vitro/in vivo anti-Trypanosoma cruzi activity.

In this work, the hydrophilic, low molecular weight and trypanocidal drug etanidazole (ETZ) was loaded in pH-sensitive liposomes (L-ETZ). Liposomes were made of dioleoyl-phosphatidylethanolamine: cholesteryl hemisuccinate (DOPE:CHEMS, 6:4, mol:mol), of 380 nm size at 14% ETZ/total lipid (w/w) ratio. To follow their uptake and intracellular fate by fluorescence microscopy, pH-sensitive liposomes were loaded with the fluorophore/quencher pair HPTS/DPX. A fast and massive delivery of the liposomal aqueous content into the cytosol of murine J774 macrophages was observed. L-ETZ vesicles were phagocytosed by both uninfected and Trypanosoma cruzi-infected macrophages. A 72% of anti-amastigote activity (AA) was demonstrated on L-ETZ-treated J774 cells, whereas the same dose of free ETZ rendered 0% AA. Endovenous administration of L-ETZ at 14 microg/mouse dose provoked significant decrease in parasitemia levels of T. cruzi-infected mice. Conversely, inoculation of a 180-fold higher dose of free ETZ failed in reducing the number of bloodstream trypomastigotes. Hence, these results point to develop systems, such as L-ETZ, designed for selective delivery of drugs to the cytoplasm of phagocytic cells, thus enhancing the efficacy of molecules considered poorly active.

A phase I trial of etanidazole and hyperfractionated radiotherapy in children with diffuse brainstem glioma.

PURPOSE: To determine the toxicity and maximum tolerated dose of etanidazole administered concurrently with hyperfractionated radiation therapy (HRT) for children with brainstem glioma. METHODS AND MATERIALS: Eighteen patients with brainstem glioma were treated with etanidazole and HRT on a dose escalation protocol (Phase I trial) between 1990 and 1996. All patients had MRI confirmation of diffuse pontine glioma and signs/symptoms of cranial nerve deficit, ataxia, or long tract signs of <6 months' duration. Cervicomedullary tumors were excluded. Patients (median age: 8.5 years; 11 males, 7 females) received HRT to the tumor volume plus a 2-cm margin with parallel-opposed 6-15-MV photons. The total dose was 66 Gy in 44 fractions (1.5 Gy b.i.d., with at least 6 h between fractions) for the first 3 patients and 63 Gy in 42 fractions for the subsequent 15 patients. Etanidazole was administered as a rapid i.v. infusion 30 min before the morning fraction of HRT. Planned doses of etanidazole were 1.8 g/m(2) x 17 doses (30.6 g/m(2)) at Step 1 to a maximum of 2.4 g/m(2) x 21 doses (50.4 g/m(2)) at Step 8. Dose escalation was planned with 3 patients at each of the 8 levels. RESULTS: Three patients were treated at each dose level except Level 2, on which only 1 patient was treated. The highest dose level achieved was Level 7, which delivered a total etanidazole dose of 46.2 g/m(2). Two patients were treated at this level, and both patients experienced Grade 3 toxicity in the form of a diffuse cutaneous rash. Three patients received a lower dose of 42 g/m(2) (dose Level 6) without significant toxicity, and this represents the maximum tolerated dose (MTD). There were 23 cases of Grade 1 toxicity (10 vomiting, 5 peripheral neuropathy, 2 rash, 2 constipation, 1 weight loss, 3 others), 11 cases of Grade 2 toxicity (4 vomiting, 2 skin erythema, 2 constipation, 1 arthralgia, 1 urinary retention, 1 hematologic), and 4 Grade 3 toxicities (2 rash, 1 vomiting, 1 skin desquamation). Grade 2 or 3 peripheral neuropathy was not seen at any dose level. The median survival from the start of treatment was 8.5 months (range: 3-58 months). CONCLUSION: The MTD of etanidazole in children receiving HRT for brainstem glioma is 42 g/m(2), with cutaneous rash as the dose-limiting toxicity. This is in contrast to the adult experience, which demonstrates a 24% lower MTD of 34 g/m(2) limited by peripheral neuropathy.

Simulation of intratumoral release of Etanidazole: effects of the size of surgical opening.

The efficacy of radiotherapy can be enhanced by the delivery of radiosensitizer (Etanidazole) to brain tumor from biodegradable polymer implants. This process is investigated by simulation carried out at a cut section of tumor with polymeric wafers of Etanidazole loading implanted in the resected cavity. The coupled mass and momentum equations are solved to obtain the transient solution of the drug distribution in the tumor. The polymeric delivery shows high therapeutic index, indicating the wafers' success in delivering more drugs to the tumor rather than to the tissue. The penetration distance of Etanidazole was found to decrease from 14 mm (at 5th/40th day after implantation) to 6.5 mm (at 30th/75th day), suggesting an initial high burst of drug release which cause nearby tissue toxicity and a low effective drug delivery towards the later stages. The short penetration depth is due to Etanidazole having low interstitial Peclet number and high elimination/diffusion modulus. Edema causes the interstitial pressure, velocity, and concentration to increase in all domains, and leads to enhanced convection and a lowering of therapeutic index. Simulations on the open tumor geometry show significantly lower efficacy of the drug delivery due to the uneven distribution of drug in the tumor zone.

Sustained release system for highly water-soluble radiosensitizer drug etanidazole: irradiation and degradation studies.

Etanidazole (one nitro-imidazole hypoxic radiosensitizer) is formulated as polymer matrix type controlled release devices in this study. A novel double polymer drug carrier, unlike the double wall microparticles, is fabricated for the purpose of drug delivery, with the following objectives in mind: (1) to have a high encapsulation efficiency, (2) to achieve a pusatile release profile suitable for the radiation schedule of radiotherapy, (3) to elucidate the degradation profile of these microparticles. Irradiation of the microparticles were also studied to investigate effects on release and degradation. At a dosage of 50 Gy (total dosage during a radiotherapy treatment period) showed no apparent effects on the tri-phase release profile. It consists of an initial burst in the first 72 h, followed by a slow and steady drug release phase, and finally a faster degradation controlled phase corresponding to the degradation state of the different microparticles. At 25 kGy (sterilization dosage), the release profiles of the drug carrier were drastically modified. The faster erosion of the polymer with high dosage irradiation hastened the drug release and shortened the release time span, accompanied by decreases in the polymer molecular weight and glass transition temperatures, which was not apparent from SEM imaging. Degradation studies suggested a heterogeneous degradation process, with the outer layer and inner matrix degrading at different rates. The modifiable tri-phase release profile using microparticles of different polymer blends implies that the release properties of the drug carriers can be modified for different treatment regimes.

Computer simulation of the delivery of etanidazole to brain tumor from PLGA wafers: comparison between linear and double burst release systems.

This paper presents the computer simulation results on the delivery of Etanidazole (radiosensitizer) to the brain tumor and examines several factors affecting the delivery. The simulation consists of a 3D model of tumor with poly(lactide-co-glycolide) (PLGA) wafers with 1% Etanidazole loading implanted in the resected cavity. A zero-order release device will produce a concentration profile in the tumor which increases with time until the drug in the carrier is depleted. This causes toxicity complications during the later stages of drug treatment. However, for wafers of similar loading, such release results in a higher drug penetration depth and therapeutic index as compared to the double drug burst profile. The numerical accuracy of the model was verified by the similar results obtained in the two-dimensional and three-dimensional models.

Effects of fabrication conditions on the characteristics of etanidazole spray-dried microspheres.

Etanidazole, a hypoxic radiosensitizer, has potential applications in radiotherapy. Due to its high solubility in water, common methods to encapsulate etanidazole into microspheres are not feasible. In this study, a spray-drying technique was employed to encapsulate etanidazole into the biodegradable polymer, PLGA65:35. Different fabrication conditions, such as polymer concentration, inlet temperature, feed rate, compressed air flow rate, aspirator ratio, as well as drug-loading were investigated to understand their effects on the particle size and distribution, encapsulation efficiency, and release behaviour. The effect on the morphologies of microspheres were also observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). It was demonstrated that most of these fabrication conditions influence either the droplet formation process or its subsequent evaporation and particle shrinking process, thereby determining the properties of the microspheres obtained. In many cases, temperature seems to be more important among all the factors considered. The present study demonstrates good fabrication conditions for producing the etanidazole-PLGA65:35-microspheres by using DCM as a solvent. The release of etanidazole from the spray dried PLGA65:35 microspheres was very fast, with an initial burst of 47% within the first 30 min and a cumulative release of over 80% within the first 5.5 h. The encapsulation efficiency of the drug in the microspheres varied with operating conditions from 69-96%.

PEG modulated release of etanidazole from implantable PLGA/PDLA discs.

In this work, etanidazole (one type of hypoxic radiosensitizer) is encapsulated into spray dried poly(D),L-lactide-co-glycolide) (PLGA) microspheres and then compressed into discs for controlled release applications. Etanidazole is characterized by intracellular glutathione depletion and glutathione transferases inhibition, thereby enhancing sensitivity to radiation. It is also cytotoxic to tumor cells and can chemosensitize some alkylating agents by activating their tumor cell killing capabilities. We observed the release characteristics of etanidazole in the dosage forms of microspheres and discs, subjected to different preparation conditions. The release characteristics, morphology changes, particle size, and encapsulation efficiency of microspheres are also investigated. The release rate of etanidazole from implantable discs (13 mm in diameter, 1 mm in thickness, fabricated by a press) is much lower than microspheres due to the reduced specific surface. After the initial burst of 1% release for the first day, the cumulative release within the first week is less than 2% until a secondary burst of release (caused by polymer degradation) occurs after one month. Some key preparation conditions such as drug loadings, disc thickness and diameter, and compression pressure can affect the initial burst of etanidazole from the discs. However, none of them can significantly make the release more uniform. In contrast, the incorporation of polyethylene glycol (PEG) can greatly enhance the release rate of discs and also reduces the secondary burst effect, thereby achieving a sustained release for about 2 months.

Radiosensitization of a mouse tumor model by sustained intra-tumoral release of etanidazole and tirapazamine using a biodegradable polymer implant device.

BACKGROUND AND PURPOSE: Drug toxicities are often a limiting factor in long term treatment regimes used in conjunction with radiotherapy. If the drug could be localized to the tumor site and released slowly, then optimal, intra-tumoral drug concentrations could be achieved without the cumulative toxicity associated with repeated systemic drug dosage. In this paper we describe the use of a biodegradable polymer implant for sustained intra-tumoral release of high concentrations of drugs targeting hypoxic cells. MATERIALS AND METHODS: The RIF-1 tumor was implanted subcutaneously or intramuscularly in C3H mice and irradiated with 60Co gamma rays. The drug delivery device was the co-polymer CPP-SA;20:80 into which the drug was homogeneously incorporated. The hypoxic radiosensitizer Etanidazole or the bioreductive drug Tirapazamine were delivered intra-tumorally by means of implanted polymer rods containing the drugs. Tumor growth delay (TGD) was used as the end point in these experiments. RESULTS: Both Etanidazole and Tirapazamine potentiated the effects of acute and fractionated radiation in the intra-muscular tumors but neither drug was effective in sub-cutaneous tumors. Since both drugs target hypoxic cells we hypothesized that the lack of effect in the subcutaneous tumor was attributable to the smaller size of the hypoxic fraction in this tumor model. This was confirmed using the hypoxia marker EF5. CONCLUSIONS: These results indicate that the biodegradable polymer implant is an effective vehicle for the intra-tumoral delivery of Etanidazole and Tirapazamine and that, in conjunction with radiation, this approach could improve treatment outcome in tumors which contain a sub-population of hypoxic, radioresistant cells.

Dose escalation of the hypoxic cell sensitizer etanidazole combined with ifosfamide, carboplatin, etoposide, and autologous hematopoietic stem cell support.

Multiple mechanisms of drug resistance contribute to treatment failure. Although high-dose therapy attempts to overwhelm these defenses pharmacologically, this approach is only successful in a fraction of treated patients. Many drug resistance mechanisms are shared between malignant and normal cells, but the expression of various drug resistance mechanisms associated with hypoxia is largely confined to tumor tissue. Thus, reversal of this mechanism is likely to provide a therapeutic advantage to the host. This study was designed to define the dose-limiting toxicities and maximum tolerated dose of etanidazole when it is given concurrently with high-dose ifosfamide, carboplatin, and etoposide (ICE), with hematopoietic stem cell support. The maximum tolerated doses of high-dose ICE were administered concurrently with dose escalations of etanidazole, a hypoxic cell sensitizer. All agents were given by 96-h continuous i.v. infusion beginning on day -7. Mesna uroprotection was provided. Autologous marrow and cytokine mobilized peripheral blood progenitor cells were reinfused on day 0. Granulocyte colony-stimulating factor was administered following reinfusion until the granulocytes recovered to > 1000/microliter. Fifty-five adults with advanced malignancies were enrolled in cohorts of five to nine patients. Four dose levels of etanidazole between 3 and 5.5 g/m2/day (12, 16, 20, and 22 g/m2 total doses) and two doses of carboplatin (1600 and 1800 mg/m2 total doses) were evaluated. Seven patients died of organ toxicity (13%); two each from veno-occlusive disease of liver and sepsis; and one each from sudden death, renal failure, and refractory thrombocytopenic hemorrhage. Five deaths occurred at the top dose level. One additional patient suffered a witnessed cardiorespiratory arrest from ventricular fibrillation and was resuscitated. Dose-dependent and largely reversible peripheral neuropathy was observed consisting of two syndromes: severe cramping myalgic/neuralgic pain, predominantly in stocking glove distribution, occurring between day -3 and day 0, and a sensory peripheral neuropathy with similar distribution peaking around day +60. The maximal achievable dose of etanidazole (16 g/m2 dose level) resulted in a mean serum level of 38 micrograms/ml (25-55 micrograms/ml). Etanidazole significantly enhanced host toxicity of high-dose ICE. Effective modulatory doses of etanidazole could not be given with acceptable toxicity using this schedule.

Intervention with the hypoxic tumor cell sensitizer etanidazole in the combined modality treatment of limited stage small-cell lung cancer. A one-institution study.

PURPOSE: We report the toxicity, patterns of failure and survival of a cohort of patients with limited disease (LD) small-cell lung cancer (SCLC) treated with combined radiation and chemotherapy. During the course of thoracic irradiation, we added intravenous (i.v.) etanidazole (SR-2508, a third-generation 2-nitroimidazole) as a hypoxic cell sensitizer in an attempt to reduce the primary local failure rate and improve survival. METHODS AND MATERIALS: Between July 1988 and August 1990, 30 consecutive patients with limited disease SCLC were enrolled and treated on a Phase II protocol receiving a standard combination chemotherapy regimen utilizing i.v. cisplatin 25 mg/m2/day x 3 days, i.v. etoposide 100 mg/m2/day x 3 days alternating with intravenous cyclophosphamide 1000 mg/m2/day, intravenous doxorubicin 15 mg/m2, and intravenous vincristine 2 mg (CAV) to a total of six cycles every 3 weeks. Radiotherapy and etanidazole were started after the first cycle of chemotherapy. Etanidazole was administered intravenously at a dose of 2 g/m2 three times per week for a total of 30 g/m2 during the course of thoracic radiation that delivered 50.00 Gy tumor dose in 25 fractions in an overall time of 6 weeks. RESULTS: The overall response rate of the primary lesion in the thorax was 96% (CR + PR), with 64% complete responses. The median time to treatment failure was 18 months. Of the patients that have relapsed, only 18% failed in the thorax (alone or concomitant with other sites). This is a marked improvement compared to the 40-50% rate reported in the literature. The 2-year crude survival was 46%. The 3- and 5-year crude survival rate with no evidence of disease was 33 and 30%, respectively. We have observed a 10% increase in the incidence of transient etanidazole related peripheral neuropathies compared to previous etanidazole studies not utilizing systemic chemotherapy. There was no increased incidence of radiation esophagitis, pulmonary toxicity, or nephro- or myelotoxicity over and above what has been routinely observed with this radio/chemotherapy regimen. There were no treatment related deaths. CONCLUSION: The moderate increase in etanidazole-related transient peripheral neuropathies could have been related to the concomitant use of etanidazole with vincristine and cisplatin. Although the almost 50% improvement in the incidence of tumor failure rate in the thorax in this small group of patients did not correlate with an equal marked improvement in their survival, the 5-year survival outcome in our series is at least equal or better than the best reports in the literature of larger clinical trials. We believe there is sufficient data from this study, particularly the improvement of local tumor control, to warrant a large randomized controlled clinical trial, using the most current systemic chemotherapy with concomitant thoracic irradiation with or without the most effective available hypoxic cell cytotoxic/sensitizer.

Survival results from a phase I study of etanidazole (SR2508) and radiotherapy in patients with malignant glioma.

PURPOSE: To report the survival results from a previous Phase I study of etanidazole (ETA) and radiotherapy in patients with glioblastoma multiforme (GBM n = 50) or anaplastic astrocytoma (AA n = 19) and examine survival according to age, Karnofsky performance status (KPS), and implant status. PATIENTS AND METHODS: In a previous Phase I study, 70 previously untreated patients (median age 49) with malignant gliomas were accrued. One patient was excluded from analysis because pathology was unverifiable. All had KPS > or = 70. Prior to initiation of treatment, patients were stratified according to whether they were candidates for interstitial implantation. The implant patients (IMP n = 14) received accelerated fractionation radiotherapy (XRT) 2 Gy BID (6 hours apart) to 40 Gy in 2 weeks with ETA 2 gm/m2 x 6 doses, a 2 week break, and then interstitial implant for an additional 50 Gy (4-7 days) with a continuous infusion of ETA over 90-96 hours. There were 55 patients treated on two sequentially conducted non-implant arms. These patients started with accelerated fractionation XRT 2 Gy BID (6 hours apart) to 40 Gy in 2 weeks with ETA 2 gm/m2 x 4-5 doses/week. Non-IMP1 arm (n = 41) received a 2-week break before standard fractionated boost XRT of 2 Gy/day for 2 weeks to a total dose of 60 Gy with ETA. Non-IMP2 arm (n = 14) did not have the 2-week break. All patients had plasma pharmacokinetic monitoring of ETA. Subsequent follow-up study provided information regarding long-term survival status of this group of patients. The Phase I toxicity evaluation was conducted according to the RTOG toxicity scale and was found well tolerated in both groups. Overall actuarial survival was plotted for all patients, by histologic group, and by implant status. Subset analyses of GBM patients by age (< or = 49 or > 49 years), KPS (< or = 80 or > 80) and implant versus non-implant were also performed. RESULTS: Median survival of GBM patients was 1.1 years and that of anaplastic astrocytoma patients was 3.1 years (p = 0.0001). In GBM patients, KPS > 80, implanted patients, and age < or = 49 were factors found not to be associated with a statistically improved survival. CONCLUSION: The results of survival in this Phase I etanidazole study of patients with anaplastic astrocytoma are comparable to the results from other studies using bromodeoxyuridine, iododeoxyuridine, or procarbazine, lomustine (CCNU), and vincristine. The use of etanidazole with accelerated radiotherapy does not appear to improve survival in patients with glioblastoma multiforme compared to those treated with conventional therapies.

Pharmacokinetic monitoring and dose modification of etanidazole in the RTOG 85-27 phase III head and neck trial.

PURPOSE: To prospectively evaluate the pharmacokinetic monitoring and drug dose adjustment of Etanidazole (Eta) in patients treated on the RTOG randomized trial for Stage III and IV head and neck cancer. METHODS AND MATERIALS: From June, 1986 to October, 1991, 521 patients were randomized to conventional RT alone or RT plus Eta. The primary goal was to determine whether the addition of Eta to conventional radiation therapy improves local-regional control and tumor-free survival. Of the 264 patients who received Eta, 233 had their drug exposure calculated and the Eta dose and schedule adjusted accordingly to prevent the occurrence of serious peripheral neuropathy. Drug exposure was assessed using the area under the curve (AUC) for a single treatment that was calculated by the integral over time of the serum concentration of Eta. The total drug exposure (total-AUC) was estimated by multiplying the AUC by the number of drug administrations. RESULTS: Eighteen percent of patients developed Grade I and 6% developed Grade II peripheral neuropathy. There was no Grade 3 or 4 peripheral neuropathy. There is a trend for an increased risk of neuropathy by single dose AUC. The minimal difference in incidence of neuropathy by single-dose AUC was due to the use of dose and schedule modification for patients with the higher values. CONCLUSIONS: The pharmacokinetics investigated in this study confirm previous work that monitoring Eta levels, with dose adjustment, allows it to be used safely in the clinic. In a subset analysis there was a statistically significant improvement in local-regional control and survival rates for patients with N0 and N1 disease, that will require confirmation (14). However, the clinical efficacy of Eta in this trial proved to be of little overall benefit.

Antitumor efficacy and pharmacokinetic analysis of 4-hydroperoxycyclophosphamide in comparison with cyclophosphamide +/- hepatic enzyme effectors.

4-Hydroperoxycyclophosphamide is an oxazaphosphorine which is readily converted without enzymatic involvement to 4-hydroxycyclophosphamide-a key intermediate in the antitumor activity of this class of drugs. The efficacy of 4-hydroperoxycyclophosphamide as a systemically administered antitumor drug was examined in mice bearing EMT-6 mammary carcinoma and in rats bearing 13762 mammary carcinoma in comparison with other oxazaphosphorines. 4-Hydroperoxycyclophosphamide was a more potent tumor cell killing agent than cyclophosphamide or ifosfamide in animals bearing the EMT-6 tumor. There were no significant differences in the toxicity to bone marrow amongst the three oxazaphosphorines. 4-Hydroperoxycyclophosphamide (90 mg/kg) on days 7, 9 and 11 produced 11.5 days of tumor growth delay compared with 10.4 days and 7.1 days for cyclophosphamide (150 mg/kg) and ifosfamide (150 mg/kg) administered on the same schedule, respectively. 4-Hydroperoxycyclophosphamide was tolerated at 90 mg/kg daily for 5 days and at 75 mg/kg twice daily for 4 days producing tumor growth delays of 14.4 days and 16.6 days, respectively. In rats bearing 13762 tumors, 4-hydroperoxycyclophosphamide (90 mg/kg) on days 8, 10 and 12 produced a tumor growth delay of 14.5 days compared with 8.9 days for cyclophosphamide (100 mg/kg) administered on the same schedule. Treatment of 13762 tumor-bearing rats with phenobarbital, pentobarbital or etanidazole increased the tumor growth delay produced by cyclophosphamide while treatment with cimetidine decreased the tumor growth delay produced by cyclophosphamide but not significantly. Administration of 4-hydroperoxycyclophosphamide (90 mg/kg) produced blood concentrations of 4-hydroxycyclophosphamide three-fold higher than those produced by administration of cyclophosphamide (100 mg/kg) at 15 min after drug injection. Treatment with phenobarbital or pentobarbital increased 4-hydroxycyclophosphamide blood concentration while pretreatment with cimetidine decreased 4-hydroxycyclophosphamide blood concentration from cyclophosphamide. 4-Hydroperoxycyclophosphamide is an effective antitumor agent worthy of further investigation.

Pilot study of local hyperthermia, radiation therapy, etanidazole, and cisplatin for advanced superficial tumours.

Five patients (six hyperthermia sites) with advanced superficial tumours were treated with combined etanidazole, cisplatin, local hyperthermia, and radiation therapy as part of a Phase I pilot study. Treatment was given once weekly and consisted of etanidazole 3 gm/m2 IV bolus, cisplatin 50 mg/m2 IV bolus, hyperthermia for 60 min with a target temperature of 43 degrees C, and radiation therapy 500 cGy/fraction (median total dose 3000 cGy) for a total of six weeks. Blood levels of etanidazole were taken during treatment at week 1 and week 4. Etanidazole drug exposure was calculated using the trapezoidal rule and expressed as the area under the curve (AUC) of plasma concentration x time. Five of six treatment sites had received prior irradiation. Prior chemotherapy had been given in three patients and tamoxifen therapy given in the other two patients. The median follow-up time is 34 months; 3/5 patients have died of disease. The most significant toxicity was grade I or II nausea and vomiting associated with 19/32 treatments (59%) and a second degree burn in 2/6 fields. None of the five patients experienced peripheral neuropathy, skin ulceration, or needed surgical repair. In addition, there was mild renal toxicity; pharmacokinetic analysis showed a 28-75% increase in the week 1 to week 4 AUC in three patients, all of whom had a decrease in creatinine clearance over the same time of 15-47%. This pilot study suggests this combined modality therapy can be delivered without major complications and that renal function, determined by creatinine clearance, affects clearance of etanidazole and alters the AUC. Therefore, monitoring renal function is important in patients receiving etanidazole in addition to other nephrotoxic agents such as cisplatin. The impact of etanidazole on the therapeutic index of hyperthermia, radiation therapy and cisplatin may be worth of study, especially since a positive interaction between these modalities is found in laboratory models.

Cytotoxicity of antitumor platinum complexes with L-buthionine-(R,S)-sulfoximine and/or etanidazole in human carcinoma cell lines sensitive and resistant to cisplatin.

Human 2008 ovarian carcinoma cells and the C13 CDDP-resistant subline and human MCF-7 breast carcinoma cells and the MCF-7/CDDP CDDP-resistant subline were exposed to L-buthionine-(S,R)-sulfoximine (50 microM) for 48 h prior to and during exposure for 1 h to the antitumor platinum complexes, cis-diamminedichloroplatinum(II), carboplatin or D,L-tetraplatin and/or to etanidazole (1 mM) for 2 h prior to and during exposure for 1 to the antitumor platinum complexes. These modulators alone did not significantly alter the cytotoxicity of CDDP toward either parental line. A twofold enhancement in cytotoxicity was observed with carboplatin in the 2008 cells and with D,L-tetraplatin in both parental lines with the single modulators. The modulator combination (buthionine sulfoximine/etanidazole) was very effective along with D,L-tetraplatin in both the MCF-7 parent and MCF-7/CDDP cell lines where at the higher platinum complex concentrations there was 1.5 to 3 logs increased killing of cells by the drug plus the modulators compared with the drug alone. Similarly, when C13 cells were exposed to CDDP (100 microM) or D,L-tetraplatin (100 microM) along with buthionine sulfoximine and etanidazole there was a 2-log increase in cell killing compared with exposure to the platinum complex alone. Treatment of each of the four cell lines with buthionine sulfoximine decreased both the non-protein and total sulfhydryl content of the cells. Treatment with the combination of modulators did not produce a further decrease in cellular sulfhydryl content compared with buthionine sulfoximine alone. The total sulfhydryl content in MCF-7 cells and 2008 cells exposed to buthionine sulfoximine and etanidazole was 58% and 31% of normal and the total sulfhydryl content of MCF-7/CDDP cells and C13 cells treated the same way was 54% and 23% of normal, respectively. DNA alkaline elution was used to assess the impact of exposure to the modulators, buthionine sulfoximine and etanidazole, alone and in combination on the cross linking of DNA by the antitumor platinum complexes in the MCF-7 and MCF-7/CDDP cell lines. Overall, the increases in DNA cross linking factors were greater in the MCF-7 cells than in the MCF-7/CDDP cells. These results indicate a possible clinical potential for this modulator combination.