Synergistic effect of combination topotecan and chronomodulated radiation therapy on xenografted human nasopharyngeal carcinoma.

PURPOSE: To investigate the in vivo chronomodulated radiosensitizing effect of topotecan (TPT) on human nasopharyngeal carcinoma (NPC) and its possible mechanisms. METHODS AND MATERIALS: Xenografted BALB/c (nu/nu) NPC mice were synchronized with an alternation of 12 hours of light from 0 to 12 hours after light onset (HALO) and 12 hours of darkness to establish a unified biological rhythm. Chronomodulated radiosensitization of TPT was investigated by analysis of tumor regrowth delay (TGD), pimonidazole hydrochloride, histone H2AX phosphorylation, (γ-H2AX) topoisomerase I (Top I), cell cycle, and apoptosis after treatment with (1) TPT (10 mg/kg) alone; (2) radiation therapy alone (RT); and (3) TPT+RT at 3, 9, 15, and 21 HALO. The tumor-loaded mice without any treatment were used as controls. RESULTS: The TPT+RT combination was more effective than TPT or RT as single agents. The TPT+RT combination at 15 HALO was best (TGD = 58.0 ± 3.6 days), and TPT+RT at 3 HALO was worst (TGD = 35.0 ± 1.5 days) among the 4 TPT+RT groups (P<.05). Immunohistochemistry analysis revealed a significantly increased histone H2AX phosphorylation expression and decreased pimonidazole hydrochloride expression in the TPT+RT group at the same time point. The results suggested that the level of tumor hypoxia and DNA damage varied in a time-dependent manner. The expression of Top I in the TPT+RT group was also significantly different from the control tumors at 15 HALO (P<.05). Cell apoptosis index was increased and the proportion of cells in S phase was decreased (P<.05) with the highest value in 15 HALO and the lowest in 3 HALO. CONCLUSIONS: This study suggested that TPT combined with chronoradiotherapy could enhance the radiosensitivity of xenografted NPC. The TPT+RT group at 15 HALO had the best therapeutic effect. The chronomodulated radiosensitization mechanisms of TPT might be related to circadian rhythm of tumor hypoxia, cell cycle redistribution, DNA damage, and expression of Top I.

A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis.

Mycobacterium tuberculosis, which causes tuberculosis, is the greatest single infectious cause of mortality worldwide, killing roughly two million people annually. Estimates indicate that one-third of the world population is infected with latent M. tuberculosis. The synergy between tuberculosis and the AIDS epidemic, and the surge of multidrug-resistant clinical isolates of M. tuberculosis have reaffirmed tuberculosis as a primary public health threat. However, new antitubercular drugs with new mechanisms of action have not been developed in over thirty years. Here we report a series of compounds containing a nitroimidazopyran nucleus that possess antitubercular activity. After activation by a mechanism dependent on M. tuberculosis F420 cofactor, nitroimidazopyrans inhibited the synthesis of protein and cell wall lipid. In contrast to current antitubercular drugs, nitroimidazopyrans exhibited bactericidal activity against both replicating and static M. tuberculosis. Lead compound PA-824 showed potent bactericidal activity against multidrugresistant M. tuberculosis and promising oral activity in animal infection models. We conclude that nitroimidazopyrans offer the practical qualities of a small molecule with the potential for the treatment of tuberculosis.

Pharmacokinetics and binding of the bioreductive probe for hypoxia, NITP: effect of route of administration.

The novel compound 7(-)[4'-(2-nitroimidazol-l-yl)-butyl]-theophylline (NITP) can be used as an immunologically detectable probe for hypoxic cells. Because of the limited water solubility of NITP, it has been administered dissolved in peanut oil with 10% dimethylsulphoxide (DMSO). A new aqueous formulation has been devised, based on a 50% solution of a modified beta-cyclodextrin (Molecusol HPB), which increases the water solubility of NITP 10-fold. The pharmacokinetics of NITP in plasma and tumours have been compared following oral and intraperitoneal (i.p.) administration of the NITP in Molecusol, i.p. administration of NITP dissolved in peanut oil + 10% DMSO and injection of a near-saturated aqueous solution of the drug intravenously via the tail vein or i.p. or directly into the tumours. Binding of the marker to hypoxic cells within tumours was also measured after the different routes of administration. The Molecusol vehicle was unexpectedly toxic when administered i.p., but there was no toxicity from NITP dissolved in Molecusol when administered orally. Binding of the drug within tumours was seen for both the peanut oil + 10% DMSO and Molecusol formulations and for both oral and intraperitoneal routes. Binding of NITP within tumours has also been observed following direct injection of the drug, with minimal whole-body exposure to NITP. However, the bound metabolites of NITP within tumours were localised to the injection site, suggesting that direct injection is unlikely to be a useful method of administering bioreductive hypoxia markers. The data in this paper demonstrate that bound metabolites of the hypoxia marker NITP can be detected in tumours following oral administration of an aqueous formulation of NITP, and suggest that oral administration could be a satisfactory administration route for clinical studies with NITP.

Stimulation by localized tumor hyperthermia of reductive bioactivation of 2-nitroimidazole benznidazole in mice.

We have investigated the effects of localized tumor hyperthermia (LTH; 43.5 degrees C x 30 min) on the reductive bioactivation of the 2-nitroimidazole benznidazole in C3H mouse normal tissues and KHT tumors. Mice were allocated to one of three treatment groups: (a) unrestrained controls, (b) sham tumor treatment, and (c) LTH. Concentrations of benznidazole and its amine metabolite were determined by high-performance liquid chromatography. Conscious mice were given LTH or sham treatment 2.5 h after 2.5 mmol/kg benznidazole i.p. This gave steady-state plasma benznidazole concentrations of 120-170 micrograms/ml at 2-5 h in all three groups. Plasma amine concentrations were very low at 0.1-1 micrograms/ml in all cases. Liver benznidazole concentrations were similar to plasma but amine concentrations were 30-40-fold greater at 20-40 micrograms/g in all three groups, implicating the liver as a major site of reductive metabolism. Benznidazole concentrations in tumors from unrestrained mice were comparable to those in plasma and liver, with tumor/plasma ratios of 85-113%. Tumor amine concentrations were intermediate at about 2-3 micrograms/g, indicating reductive bioactivation had occurred. Sham treatment decreased tumor benznidazole concentrations by 25-50%, particularly at later times, and amine concentrations were correspondingly increased. This may be a result of sham tumor treatment at 37 degrees C, a temperature 3-4 degrees C higher than in unrestrained controls. More importantly, LTH further decreased tumor benznidazole concentrations over sham treatment, e.g., by 59% from 114 to 47 micrograms/g (P less than 0.01) immediately after heating. Amine concentrations were correspondingly elevated, e.g., by 40% from 5.1 to 8.4 micrograms/g (P less than 0.01). These results clearly show that LTH can selectively enhance the reductive bioactivation of benznidazole in KHT tumors in mice, and support a particular role for the use of bioreductive agents with heat.

The effects of whole body hyperthermia on the pharmacokinetics and toxicity of the basic 2-nitroimidazole radiosensitizer Ro 03-8799 in mice.

We have investigated the effects of 50 min whole-body hyperthermia (WBH; 15 min equilibration followed by 41 degrees C for 35 min) on the toxicity and pharmacokinetics of the radiosensitizer Ro 03-8799 in mice. WBH markedly reduced Ro 03-8799 LD50/7d from 779 to 259 micrograms g-1 (P less than 0.001). Pharmacokinetics were studied at 175 micrograms g-1 (approximately 0.6 WBH LD50/7d) with and without heat and 437 micrograms g-1 (approximately 0.6 control LD50/7d) without heat. WBH increased Ro 03-8799 plasma concentrations and prolonged its elimination t1/2 by 26% (P less than 0.01). Total plasma area under the curve (AUC0-infinity) was increased by 22%, but was still less than 50% of the unheated high-dose value. Ro 03-8799 concentrated 300-400% in tumour and brain relative to plasma. Absolute tumour and brain levels were unaltered by WBH, giving reduced tissue/plasma ratios. WBH greatly inhibited glomerular filtration (51Cr EDTA clearance) during heating, contributing to the increased plasma Ro 03-8799 concentrations. WBH increased peak plasma concentrations of the Ro 03-8799 N-oxide metabolite Ro 31-0313 by 61% and the beta-phase AUC of i.v. administered Ro 31-0313 by 36%. Since Ro 31-0313 levels were increased to a greater extent after Ro 03-8799 and WBH than Ro 31-0313 and WBH, WBH must both increase metabolite production and decrease its plasma clearance. WBH had no effect on Ro 31-0313 tumour concentrations or its exclusion from brain. These complex effects of WBH on Ro 03-8799 pharmacokinetics may contribute to the enhanced toxicity, possibly through hyperthermia-stimulated bioreductive drug activation, but do not wholly explain it.

Preclinical pharmacokinetics of benznidazole.

Benznidazole is a lipophilic analogue of misonidazole (MISO) which shows promise as a chemosensitizer for clinical use, particularly in combination with CCNU. We have investigated the detailed pharmacokinetics of benznidazole in mice, dogs and sheep to provide a data base for the estimation of doses required for chemosensitization in man. Pharmacokinetic behaviour was linear except at high doses in mice. Absorption was fairly rapid and bioavailability was complete following both i.p. administration in mice and oral administration in dogs. Elimination t1/2 values were longer than for MISO, being 90 min in mice, 4-5 h in sheep and 9-11 h in dogs. At doses giving linear kinetics, peak whole plasma concentrations per administered mg kg-1 were 0.75 micrograms ml-1 for the i.p. route in mice and 1.8 micrograms ml-1 for the oral route in dogs. Though between 39 and 59% of plasma benznidazole was bound to protein, tissue penetration was generally good. Tissue/whole plasma ratios ranged from 59-99% for transplantable mouse tumours and from 14-70% for spontaneous dog neoplasms. Nervous tissue penetration was similar to that in tumours: brain/whole plasma ratios averaged between 61 and 76% in mice and 42% in dogs, while peripheral nerve/whole plasma ratios in dogs averaged 74%. Mean liver/whole plasma ratios were 42% and 71% in BALB/c and C3H/He mouse strains respectively. Only approximately 5% of the administered dose was excreted unchanged in the urine, indicating the likelihood of extensive metabolism. These data show that benznidazole should have suitable pharmacokinetic properties for clinical use as a chemosensitizer. Enhancement of CCNU response is likely to require circulating benznidazole concentrations of 10-30 micrograms ml-1 and we predict that these will be obtained with oral doses of 6-20 mg kg-1 in man.

Clinical trials of radiosensitizers: what should we expect?

The lack of positive results from the clinical trials undertaken so far with misonidazole (MISO) are widely considered as disappointing. This is leading to a growing sentiment that hypoxic cells may not be a significant limitation to local control of human tumors. To examine whether this is a reasonable conclusion, the relevant in vitro and in vivo data have been summarized so that predictions of the extent of radiosensitization of the hypoxic cells can be made from a knowledge of the clinically achievable levels of MISO. This analysis shows the following: First, the original curve of Adams with V-79 cells is probably over-optimistic in predicting sensitizer enhancement ratios (SERs). A new curve based on the available in vivo data predicts lower sensitization so that even at the highest MISO doses used clinically, SERs for the hypoxic cells to large single X-ray doses of only 1.45 would be expected. In a clinical trial, reoxygenation of the hypoxic cells is likely to occur, thereby considerably reducing the SER for the total tumor cell population. This, together with the problems of heterogeneous tumors and insufficient patient numbers, could well have been responsible for the negative clinical results. Second, even if tumor levels of the new radiosensitizer SR-2508 10 times those of MISO can be achieved clinically, this will still not lead to full radiosensitization of the hypoxic cells (although an SER in excess of 2.0 should be attainable). In conclusion the in vitro and in vivo data with radiosensitizers suggest that only a small effect, if any, is likely to be demonstrated in the clinical trials with MISO, even for those tumors the control of which is limited by hypoxic cells. Thus the question of whether hypoxic cells may or may not limit the local control of tumors by radiotherapy has not been addressed adequately by the presently available radiosensitizing drugs.