A. Rojas

A therapeutic benefit from combining normobaric carbogen or oxygen with nicotinamide in fractionated X-ray treatments.

The ability of normobaric oxygen and carbogen (95% O2 + 5% CO2) combined with nicotinamide to enhance the radiosensitivity of two rodent adenocarcinomas and of mouse skin and kidneys, using a 10 fraction radiation schedule, was compared with the effect of radiation in air with and without the drug. Tumour response was assayed using local control and regrowth delay, and compared with acute skin reactions, decreased renal 51Cr-EDTA clearance and reduction in haematocrit. Nicotinamide increased the radiation sensitivity of CaNT tumours under all three different oxygen concentrations tested (21, 95 and 100% oxygen). The effect was statistically significant for oxygen and carbogen but not for air; the combination of nicotinamide with carbogen gave the greatest increase in tumour radiosensitivity. Relative to treatments in air without the drug, the enhancement ratios (ER) at the TCD50 level were 1.17, 1.65 and 1.83 for CaNT tumours irradiated in air, oxygen or carbogen and injected with nicotinamide 1 h before each fraction. The ER in CaRH tumours irradiated in carbogen plus the drug was 1.83, which was greater, but statistically not significantly different, to that seen with carbogen alone (ER = 1.68). In skin, relative to air without the drug, the increase in radiosensitivity by nicotinamide was greater in oxygen and carbogen than in air (1.29, 1.36 and 1.08, respectively). The ERs for both assays of renal damage were similar and lower than those in skin: less than or equal to 1.07, less than or equal to 1.13 and less than or equal to 1.16 for irradiations done in air, oxygen and carbogen plus nicotinamide, relative to air alone. A comparison of these results in the tumours and normal tissues showed that a significant therapeutic benefit was obtained with normobaric oxygen and carbogen combined with nicotinamide. This benefit is greater than observed with other radiosensitizers tested so far. Toxic side effects of the treatment are unlikely in a clinical situation, since prolonged administration of nicotinamide is well tolerated in man. The combination of normobaric carbogen with nicotinamide could be an effective method of enhancing tumour radiosensitivity in clinical radiotherapy where hypoxia limits the outcome of treatment.

Radiosensitization with normobaric oxygen and carbogen.

Although hypoxia is a cause of failure in clinical radiotherapy, the outcome of clinical trials with chemical radiosensitizers, such as misonidazole, has been disappointing [7,8]. Doselimiting toxicity restricts the drug dose per fraction, so that the intratumour concentrations achieved are far lower than those required for detecting a radiosensitizing effect [3]. Therefore, there is a need to identify hypoxic cell radiosensitizers which are non-toxic and effective in fractionated radiotherapy. Recent studies done with low radiation doses per fraction and clinically relevant fractionation regimes have shown, in a mouse mammary carcinoma, that normobaric 100% oxygen is an effective sensitizer [34]. Enhancement ratios (ER) * of 1.45 and 1.3 (at the TCDs0 level, i.e. the dose required to locally control 50% of tumours) were obtained for oxygen with 30 fraction given in an overall time of 6 weeks or 36 fractions in 12 days (Fig. 1). These relatively small enhancement ratios can be translated into a considerable practical gain e.g. by increasing the oxygen concentration from 21 to 100%, tumour control was increased from 0 to 80% (Fig. 1 panel c). A similar gain could be obtained in human tumours where hypoxia limits the response to treatment. Very recently, Gatenby et al. [12] have shown a remarkable and significant correlation between the degree of response to radiotherapy in metastases of the head and neck region and levels of oxygenation within these tumours. Previous studies done in a whole variety of rodent tumours, albeit with fewer fractions, also have shown significant increases in radiosensitivity for either oxygen or carbogen (a mixture of 95% 02 + 5% CO2) [10,15,18,21,32,33,37,40]. Table I and Fig. 2 summarize the ERs for oxygen and carbogen obtained by several investigators using fractionated treatments. Provided an adequate pre-irradiation breathing time (PIBT) is used, ERs in general lie between 1.3 and 1.6 for carbogen and between 1.3 and 1.45 for oxygen. The degree of sensitization achieved with normobaric gases in fractionated regimes is therefore much greater than that seen in almost all animal tumours and human tumour xenografts, with the radiosensitizer misonidazole [6,16,26,31]. Normobaric gases enhance radiosensitivity by increasing the partial pressure of oxygen (pO2) within tumours. The intracapillary saturation of haemoglobin with oxygen can be significantly enhanced in rat tumours by the administration of pure oxygen either under normoor hyperbaric conditions [28]. Improved tumour oxygenation results from increases in the amount of dissolved oxygen and by raising the systemic blood pressure [4,25]. Furthermore, carbogen enhances tumour blood flow and shifts the haemoglobin dissociation curve to the right, thus depressing the affinity of haemoglobin for oxygen [25]. Consequently, carbogen should be a more effective sensitizer than oxygen. Using a fluorescent immunologically labelled 2-nitroimidazole stain for detecting hypoxic cells, recently developed in our laboratory by Hodgkiss and collaborators [17], carbogen decreased the proportion of hypoxic cells in CaNT tumours to a greater extent than oxygen. Hypoxic fractions of 19.0 _ 1.6, 9.4 _+ 2.7 and 2.9 + 1.5% were determined for air, oxygen and carbogen, respectively [33]. These results are in agreement with the slightly higher ERs obtained with carbogen, in this same tumour model, using a local control and regrowth delay assay (Fig. 2, panel b and reference 33). However, the higher Ps0 values (i.e. the oxygen concentration at which 50% of the haemoglobin is saturated with oxygen) for rodent haemoglobin compared with that of humans [13]

The influence of dose per fraction on repair kinetics

The use of multiple fractions per day (MFD) in radiotherapy requires information about the rate of repair of radiation injury. It is important to know the minimum interval between fractions necessary for maximum sparing of normal tissue damage, whether rate of repair is dependent on the size of dose per fraction and if it is different in early and late responding tissues and in tumours. To address these questions, the rate of repair between radiation dose fractions was measured in mouse skin (acute damage), mouse kidney (late damage) and a mouse tumour (carcinoma NT). Skin and kidney measurements were made using multiple split doses of X-rays, followed by a neutron top-up. For skin, faster recovery was obtained with 4.4 Gy fractions (t1/2 = 1.29 +/- 0.35 h, 95% CL) than with 10.5 Gy fractions (t1/2 = 3.46 +/- 0.88 h). In contrast, kidney showed slower recovery at a low dose per fraction of 2 Gy (t1/2 = 1.69 +/- 0.39 h) than at a higher dose of 7 Gy per fraction (t1/2 = 0.92 +/- 0.1 h). These data show that repair rate is dependent on the size of dose per fraction, but not in a simple way. T1/2 values now available for many different tissues generally lie in the range of 1-2 h, and are not correlated with proliferation status or early versus late response to treatment. At the doses used currently in clinical MFD treatments, these data indicate that damage in almost all normal tissues would increase if interfraction intervals less than 6 h were used.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbogen and nicotinamide as radiosensitizers in a murine mammary carcinoma using conventional and accelerated radiotherapy

PURPOSE To compare the radiosensitivity of mouse tumors treated in air with conventional and accelerated radiotherapy with that of tumors treated in carbogen alone or carbogen combined with nicotinamide. METHODS AND MATERIALS CaNT mammary tumors were irradiated with either 30 x-ray fractions in 6 weeks or 40 fractions in 26 days in air, carbogen alone, or carbogen combined with 120 mg/kg of nicotinamide (NAM), the latter given intraperitonealy 30 min before each fraction. The response to treatment was assessed using local control, weight loss, and metastasis-free survival. RESULTS Both carbogen and carbogen plus nicotinamide significantly increased tumor radiosensitivity; enhancement ratios (ERs) in the 6-week regimen were similar to those seen in the accelerated schedule. The majority of the effect was achieved by carbogen alone but the addition of NAM further enhanced tumor radiosensitization (ERs of 1.5 and 1.4 for carbogen in the conventional and accelerated schedule, respectively, were significantly lower than ERs of 1.7 and 1.6 obtained with carbogen plus nicotinamide; p < or = 0.005). Treatment protraction significantly increased radioresistance, especially when tumors were treated under air. An extra 1.5 Gy per day was required in air to counterbalance proliferation; in carbogen alone and carbogen plus nicotinamide a dose loss of 0.9 and 0.6 Gy per day was observed, respectively. Compared with treatments in air alone delivered in 6 weeks, acceleration of treatment combined with carbogen and nicotinamide gave the greatest increase in tumor radiosensitization (ER = 1.9). No toxic side effects and no detrimental changes in body weight were encountered when the sensitizers were administered 30 times (one fraction per day) or 40 times (two fractions per day). In both regimens, the incidence of metastases in mice treated with carbogen or carbogen plus nicotinamide was similar to that seen in animals treated in air. There was, however, a nonsignificant trend of a higher proportion of mice with metastasis in the accelerated schedule compared with the 6-week schedule. CONCLUSIONS In both conventional and accelerated experimental radiotherapy, carbogen alone or combined with a small clinically relevant dose of NAM were well tolerated, achieved large and significant increases in radiosensitization, and did not affect the incidence of metastases. The sparing of damage, resulting from extending the overall treatment time, was less when the sensitizers were administered than when irradiations were performed in air. The study suggests that clinical radiotherapy regimens, which aim to reduce hypoxic and/or tumor clonogen proliferation, would benefit from the use of carbogen, especially if the gas is combined with nicotinamide and treatment acceleration.

Pharmacokinetics of nicotinamide and its effect on blood pressure, pulse and body temperature in normal human volunteers

The pharmacokinetics of nicotinamide were studied in four human volunteers after oral doses of 1-6 g. Plasma concentrations and clearance rates of the vitamin were found to be dose-dependent, with a half-life of approximately 7-9 h for the two highest doses administered (4 and 6 g), approximately 4 h with 2 g and approximately 1.5 h with a 1-g dose. Peak concentrations ranged from 0.7 to 1.1 mumol.ml-1 after a 6-g dose. The time to reach peak plasma concentration was dose independent with a broad range from 0.73 to 3 h. In this study, nicotinamide had no detectable effect on blood pressure, pulse or body temperature.

Glutathione depletion in tissues after administration of buthionine sulphoximine

Buthionine sulphoximine (BSO) an inhibitor of glutathione (GSH) biosynthesis, was administered to mice in single and repeated doses of 0.5, 1 and 5 mmol kg-1 (i.p.). The resultant pattern of GSH depletion was studied in liver, kidney, skeletal muscle and three types of murine tumor. Liver and kidney exhibited a rapid depletion to GSH levels of ca. 20% of controls after single doses of 1-5 mmol kg-1 BSO. Muscle was depleted to a similar level, but at a slower rate after a single dose. All three tumors required repeated administration of BSO over several days to obtain a similar degree of depletion to that shown in the other tissues.

Radioprotection of mouse skin by WR-2721: The critical influence of oxygen tension

The epidermal clone assay has been used to study the radioprotective effect of WR-2721 on mouse skin under different conditions of oxygenation and under anoxia. The skin has shown a progressive decrease in sensitivity as the inspired gas was changed from 100% oxygen towards 0% oxygen. Compared with mice breathing 100% oxygen, those breathing air are partially protected. The inspired oxygen concentration to given half the full oxygen effect is 10--12%. The radioprotection observed with 400 mg/kg WR-2721 is markedly dependent on the ambient oxygen concentration. The protection factor is 1.1 or less in mice breathing 5%, 1% of 0% oxygen. Protection is maximal (1.95) in air and in 50% oxygen and diminishes to 1.6 at higher oxygen tensions.

ARCON: accelerated radiotherapy with carbogen and nicotinamide in head and neck squamous cell carcinomas. The experience of the Co-operative Group of Radiotherapy of the European Organization for Research and Treatment of Cancer (EORTC)☆

BACKGROUND Since there is increasing evidence that both acute (perfusion-limited) and chronic (diffusion-limited) hypoxia, and tumor repopulation may prejudice the outcome of radiotherapy, the combination of carbogen (95% oxygen-5% carbon dioxide) and nicotinamide with accelerated radiotherapy (ARCON) should reduce the impact of these factors of radioresistance. AIM This clinical study was aimed at determining the feasibility, as well as the qualitative and quantitative toxic effects of a therapeutic approach based on ARCON, and assessing the tumor response rates that can be achieved with this regime in patients with locally advanced tumors of the head and neck. METHODS A phase I/II study conducted between 1993 and 1996 by the Co-operative Group of Radiotherapy of the EORTC included three consecutive steps: accelerated fractionation (AF) combined with carbogen (11 analyzable patients), AF combined with the daily administration of nicotinamide (n=10), and AF with both carbogen and nicotinamide (n=17). Radiotherapy was based on an accelerated regime (72 Gy in 5.5 weeks). Nicotinamide was delivered 90 min before the first irradiation session, at a daily dose of 6 g. Carbogen breathing started 5 min before irradiation and lasted throughout the entire radiotherapy sessions. RESULTS No significant difference in loco-regional toxicity was found among the three study steps, when carbogen and nicotinamide, either alone or in combination, were combined with AF. The feasibility of the ARCON protocol, as proposed in the present EORTC study, appears to be significantly impaired when nicotinamide is added, at a daily dose of 6 g, to AF and carbogen, in an unselected group of patients. More than 20% of patients experienced grade 2 or 3 emesis. It also demonstrates, in unselected groups of patients, no significant difference in tumor response and local control when carbogen and nicotinamide, either alone or in combination, are added to accelerated radiotherapy. The percentages of objective response at 2 months were 81, 70 and 87%, respectively. CONCLUSION Future ARCON trials should target selected head and neck tumor localizations and stages, and a lower nicotinamide dose is needed to reduce severe upper gastro-intestinal toxicity.

ARCON: accelerated radiotherapy with carbogen and nicotinamide in non small cell lung cancer: a phase I/II study by the EORTC

BACKGROUND Non small cell lung cancers (NSCLC) are rapidly proliferating tumours, which are characterized by the presence of extensive hypoxic components, especially in patients with advanced loco-regional disease. Previous studies suggest a deleterious impact of acute (perfusion-limited) hypoxia on the outcome of radiotherapy for these tumours. AIM This pilot study was aimed at determining the feasibility and tumour response rates that can be achieved with an ARCON regime in patients with locally advanced, staged IIIA or B, NSCLC tumours. METHODS The phase I/II study included three steps: accelerated fractionation (AF) combined with carbogen (ten analysable patients), AF together with the daily administration of nicotinamide (n = 11 ) and AF with both carbon and nicotinamide (n = 14). Radiotherapy was based on a large daily dose per fraction (2.75 Gy up to 55 Gy in 4 weeks). Nicotinamide was administered at a dose of 6 g per patient per treatment day and carbogen was inhaled for 5 min before and during radiotherapy. RESULTS The incidence of grade 3 + acute toxicity during the irradiation did not exceed 10%, neither in the lung parenchyma nor in the mediastinum. No significant difference was found in loco-regional, radio-induced toxicity among the three study steps. Although a similar fraction of patients showed grade 2 or 3 emesis in all the steps, of the 25 patients entered in the two Nicotinamide containing steps 10 (40%) developed grade 2 or greater reactions which significantly detracted from their quality of life. There was no significant difference in tumour clearance rate among the three steps. The percentage of objective responses at 2 months was 60, 54 and 57% in steps 1, 2 and 3, respectively. CONCLUSION The feasibility of this ARCON protocol, using 2.75 Gy doses per fraction over 4 weeks, is good as regards radiotherapy-related side effects but it appears necessary in future to reduce the dose of Nicotinamide to reduce the incidence of nausea and vomiting. There was no significant difference in time to progression among the three study steps.

Radioprotection of two mouse tumors by WR-2721 in single and fractionated treatments

The radioprotective effect of WR-2721 has been studied in two murine tumors using single doses or five daily fractions. Single dose irradiations of the SA FA resulted in a highly variable radio-protective response. In one experiment large protection factors (1.2-2.5) were obtained, with the greatest protection at low X ray doses. In later experiments with the same tumor, there was little or no radioprotection. The Ca MT was significantly protected against single dose irradiation with both 250 mg/kg and 400 mg/kg of the drug. In a five fraction schedule the extent of radioprotection for CA MT was greater than with single X ray doses for the same drug dose per fraction. Tumor protection factors from the present work and from the literature are compared with published protection factors for normal tissues. Significant tumor radioprotection is seen in most studies. The data indicate more variability in the extent of tumor protection for a given drug dose than is seen in normal tissues. Tumor protection is often greatest at low X ray doses which may be a result of preferential protection of the better-oxygenated tumor cells.