KHT Sarcoma Research Articles

Chemosensitization by lipophilic nitroimidazoles.

We have carried out experiments to determine the response of tumours and normal tissues in the C3H mouse to the combination of lipophilic nitroimidazoles and CCNU, cyclophosphamide or melphalan. The nitroimidazoles studied were Ro 07-1902 (1902) and benznidazole (Ro 07-1051, BENZO). Maximum enhancement of CCNU response in the KHT sarcoma by 2.5 mmol kg-1 1902 or 0.3 mmol kg-1 BENZO occurred at low doses of CCNU where dose modifying factors (DMF) of 2.5-3.0 and 1.5-2.0 respectively were found. The DMFs for depression of white cell count at day 3 were 1.6 and 1.2 respectively whilst the DMFs for LD50/30 were 1.5 and 1.3. There appears, therefore, to be a therapeutic gain at low doses of CCNU of about the same magnitude as produced by 2.5 mmol kg-1 misonidazole. The production of this gain at relatively low doses of BENZO is of possible clinical significance. Some sensitization of the KHT tumour to CCNU by 0.3 mmol kg-1 BENZO was maintained even with an interval of 25 h between BENZO and CCNU injection. A multiple injection regime of BENZO administration designed to maintain plasma concentrations for prolonged periods was, however, no more effective than a single dose. The response of the RIF-1 sarcoma to cyclophosphamide was not enhanced by the lipophilic sensitizers at the doses previously stated. Considerable enhancement of tumour response to melphalan (DMF 2.0) was produced by both lipophilic sensitizers. Enhancement of acute LD50 was similar in magnitude but no large enhancement by BENZO of melphalan induced white blood cell depression was observed. The evidence regarding the therapeutic potential of this combination is, therefore, equivocal.

Intercellular contact and the radiation response of cells of human melanoma xenografts

Cells from two human melanoma xenografts (E.E. and V.N.) were irradiated at various times up to 24 hours after dispersion of the tumors. The surviving fraction for cells exposed to 5.0 (E.E.) and 4.0 Gy (V.N.) did not change with time, and was not significantly different for cells kept at 4°C and 37°C in the time interval between tumor dispersion and irradiation. The complete survival curves for cells irradiated 1.5, 6, 12, and 24 hours (E.E.) and 1.5, 6, and 18 hours (V.N.) after tumor dispersion were not significantly different. It is concluded that an intercellular contact effect similar to that reported for V79 Chinese hamster spheroids and murine KHT sarcomas is not present for E.E. and V.N. human melanoma xenografts.

Modification by WR 2721 of the response to chemotherapy of tumours and normal tissues in the mouse.

The sulphydryl compound WR 2721 has been combined with a range of cytotoxic drugs in the mouse and the effects upon tumours and normal tissues determined. In the acute lethality (LD50/30) assay, mean protection factors produced by WR 2721 (200 or 400 mg kg-1) were generally less than 1.3 for cyclophosphamide (CTX), CCNU and chlorambucil (CHL) but a protection factor of 1.7 was obtained for cisplatinum (cis-P) in combination with 400 mg kg-1 of WR 2721. No protection against the depression of peripheral white cell count seen at 3 days after CTX, CCNU or cis-P was obtained with either 200 or 400 mg kg-1 of WR 2721. Significant protection of the RIF-1 sarcoma by WR 2721 against CTX and cis-P induced growth delay was seen. In the KHT sarcoma, WR 2721 produced small reductions in the growth delay caused by CCNU, melphalan and CHL but these were not statistically significant. These data show less differential normal tissue protection by WR 2721 than do a number of reports in the literature.

Hypoxia and the radiation response of tumors.

The effect of hypoxia on the in vivo radiation response of tumors is demonstrated both for the measurement of cell survival and growth delay following treatment. Results using these endpoints for the KHT Sarcoma are then presented to demonstrate the effect of various parameters which are expected to control tumor oxygenation: blood flow, blood pO2, blood oxygen carrying capacity and tissue consumption of oxygen. Intrinsic blood flow does not correlate with the level of hypoxia in the tumor, but acute reductions in blood flow do result in decreases in tumor oxygenation. Changing the blood PaO2 or the oxygen carrying capacity change the level of hypoxia as predicted, but only certain aspects have been studied. The intricate feedback mechanism which controls normal tissue oxygenation and compensates for any deficiencies cannot be assumed to work effectively in tumors.

Nitrosourea-misonidazole combination chemotherapy: effect on KHT sarcomas, marrow stem cells and gut.

C3H/HeJ mice bearing i.m. transplanted KHT sarcomas were treated with varying doses of either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin; CHLZ) as single agents or in combination with 1 mg/g of the chemical radiosensitizer, misonidazole (MISO). Using an in vivo-in vitro tumour-excision assay, the administration of MISO simultaneously with or 3 h after low doses of BCNU (less than 20 mg/kg) was found to give a dose-modification factor (DMF) of approximately 1.65 relative to BCNU alone. At higher doses of BCNU, there was less enhancement of cell kill. The DMF for tumour growth delay was likewise dependent on BCNU dose, continuously decreasing with increasing BCNU dose. In contrast, the anti-tumour activity of CHLZ, assessed by both clonogenic cell survival and tumour-growth delay, was not significantly enhanced by the addition of MISO. The enhancement of gastrointestinal toxicity and haematotoxicity by BCNU-MISO combinations was assessed by LD50/7 and CFU-S assays, respectively. MISO enhanced BCNU marrow toxicity by a factor of 1.2-1.3, whilst gut toxicity was enhanced by a factor of approximately 1.2.

Cell survival recovery kinetics in the KHT sarcoma following treatment with five alkylating agents and misonidazole

In order to determine the role of recovery of tumor cell survival in the enhanced tumor responses resulting from chemotherapeutic agent-sensitizer combinations, cell survival in KHT sarcomas as a function of time after treatment with the alkylating agents 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cyclophosphamide (CYC), phenylalanine mustard (L-PAM) and 2-[3-(2-chloroethyl)-3-nitrosoureidol-D-glucopyranose (chlorozotozin; CLZ) either alone or in combination with the radiosensitizer misonidazole (MISO) was evaluated. Tumor-bearing mice were treated with single doses of CCNU, BCNU, CYC, L-PAM and CLZ, respectively. The doses, chosen to obtain approximately the same level of cell kill with each treatment, were administered either alone or in simultaneous combination with a 1.0 mg/g dose of MISO. Tumors were excised at 1, 2, 4, 8, 16 and 24 hr after drug injection. Clonogenic cell survival was assessed at these times using an in vivo to in vitro excision assay. The results showed that following treatment with each of the chemotherapeutic agents, tumor cell survival reached a minimum at ∼2–8 hr. Subsequently cell survival remained relatively constant for CYC, but increased with time by a factor of ∼10–100 for L-PAM, CCNU, BCNU and CLZ. The addition of MISO potentiated the cell killing effects of CYC, L-PAM, CCNU and BCNU, but not of CLZ. Also, in animals treated with certain of these chemotherapeutic agents plus the sensitizer, cell survival recovery was impaired. This effect was particularly marked when the nitrosourea CCNU was used in combination with MISO (i.e. 24 hr after treatment with CCNU plus MISO, tumor cell survival was a factor of ∼50 lower than observed in tumors of mice given only CCNU). These findings indicate that the enhancement of cell killing in the KHT sarcoma observed 24 hr after treatment with chemotherapeutic agent-MISO combinations may be at least in part the result of altered recovery in tumor cell survival.

Combinations of cyclophosphamide and misonidazole in the KHT sarcoma

The effect on intramuscularly growing KHT sarcomas of combinations of the chemotherapeutic agent cyclophosphamide (CYC) and the radiosensitizer misonidazole (MISO) was determined. Tumor response was evaluated using an in vivo to in vitro clonogenic cell survival assay or an in situ tumor growth delay assay. MISO, at doses ranging from 0.25 to 1.0 mg/g, was found to be effective at enhancing the tumor response to CYC when given for times of 0–4 hr before the chemotherapeutic agent. Clonogenic cell survival studies in which a 1.0 mg/g dose of MISO was administered simultaneously with CYC demonstrated that the addition of the sensitizer increased the level of tumor cell kill assessed 24 hr after treatment by a factor of ∼4–8 for CYC doses ranging from 25–150 mg/kg. Similarly, in the in situ experiments MISO enhanced the regrowth delay resulting from CYC treatments with doses up to 150 mg/kg by ∼2–4 days. Consequently, the addition of MISO to CYC was not found to be strictly dose modifying, but rather resulted in enhancement ratios ranging from 1.3 to 1.5 depending on the dose of the chemotherapeutic agent used. Normal tissue toxicity to combinations of CYC and MISO was assessed 24 hr after treatment using a spleen colony assay. When MISO (1.0 mg/g) was administered simultaneously with a range of CYC doses, a dose modifying factor of ∼1.2 for bone marrow stem cell toxicity was observed. On the basis of these observations, the addition of MISO to CYC treatment may lead to a small therapeutic gain.

Chemical properties of nitrosoureas: Implications for interaction with misonidazole

Experiments were designed to evaluate the relationship between the alkylating and/or carbamoylating properties of various nitrosoureas and the differential tumor response enhancements produced when the radiation sensitizer misonidazole (MISO) is combined with nitrosoureas for the treatment of KHT tumors. A single dose of misonidazole (1 mg/gm) was combined simultaneously with each of 3 chloroethyl-nitrosoureas (MeCCNU; 1-(2-chloroethyl)3-(2,6-dioxo-3-piperidyl)-1-nitrosourea, PCNU; and chlorozotocin, CLZ) for the treatment of intramuscularly transplanted KHT sarcomas. Using an in vivo-in vitro tumor excision assay, dose response curves and the degree of dose modification produced by combined treatment with misonidazole were determined for each nitrosourea, 24 hours after drug administration. These results were compared with those of previous studies in which MISO was combined with two other nitrosoureas, BCNU and CCNU. The dose modifying factors obtained in combination with misonidazole were 1.0, 1.5, 1.7, 1.8, and 2.4 for CLZ, PCNU, MeCCNU, BCNU, and CCNU, respectively. The magnitude of cell kill enhancement achieved by each of these nitrosourea-MISO treatments was directly ( P < 0.05) correlated with the carbamoylating activity of the nitrosourea included in the combination. Since carbamoylation has been associated with the inhibition of DNA damage repair, the correlation observed between carbamoylating activity and dose enhancement in MISO-nitrosourea combinations suggests that repair inhibition plays a significant role in the interaction between MISO and the chloroethyl-nitrosoureas in the KHT sarcoma.

The response of mouse lung tumors to combinations of CCNU and misonidazole

The effect of combining the radiosensitizer misonidazole (MISO) and the chemotherapeutic agent 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) on mouse lung tumors of different sizes and hypoxic fractions was tested. KHT sarcoma lung colonies were treated at volumes of ∼0.7 and 2.0 mm 3. At these sizes the lung colonies contained ∼5 and 15% hypoxic tumor cells respectively. For the smaller lung colonies response to CCNU or the CCNU-MISO combination was evaluated by determining the percent of mice tumor-free 100 days after treatment. This assay could not be used when 2.0 mm 3 lung colony bearing mice were treated, since at this size only a few animals could be cured with either the single or combined modality therapy. Consequently, tumor response in these experiments was assessed using an increase in lifespan assay or a tumor growth delay assay. When 0.7 mm 3 KHT lung colonies were treated with CCNU plus 1.0 mg/g MISO, the TCD 50 100 (dose to cure 50% of the mice of their tumors in 100 days) was reduced by a factor of 1.5–1.8 compared to mice receiving only CCNU. However, because the single agent therapy was less toxic than the combination, increasing doses of CCNU ultimately resulted in the same level of cure as could be achieved with the CCNU-MISO combination. In the 2.0 mm 3 lung colonies studies, however, when compared at the maximally tolerated doses, the addition of 0.5 or 1.0 mg/g MISO to CCNU led to a percent increase in median lifespan, which was a factor of 1.4–1.5 greater than could be achieved with CCNU alone. This increase in lifespan was shown, using growth delay studies, to be a direct consequence of an enhanced tumoricidal response to the combination therapy. These findings indicate that in this lung tumor model an improved therapeutic result may be achieved through the addition of MISO to treatment with CCNU.

Enhancement by electron-affinic agents of the therapeutic effects of cytotoxic agents against the KHT tumor: Structure-activity relationships

We have studied the effectiveness of various electron-affinic and other modifying agents on the in vivo response of the KHT sarcoma to various cytotoxic agents. Misonidazole (MISO) showed good enhancement with CCNU, methyl-CCNU, BCNU and chlorambucil (CHL), a small enhancement with cyclophosphamide (CYC), but none with melphalan or HN2. With CYC a further small enhancement was obtained with a multiple-dose pretreatment, but host toxicity was also increased. Lipophilicity was more important than electron-affinity for CCNU enhancement. Several lipophilic analogues (e.g. benznidazole and Ro 07–1902) gave enhancements superior to MISO, whereas hydrophilic analogues were inferior. Similar results were obtained with CHL. Enhancement of CCNU response was seen with the nitrofuran, nitrofiuantoin, and the quinone, anthraquinone sulphonate. The non-electron affinic compound imidazole gave enhancement with CHL, but not with CCNU. The inhibitor of drug-metabolizing enzymes SKF 525A gave excellent enhancement with both CHL and CCNU.

Radiation dose fractionation studies with hypoxic cell radiosensitizers using a murine tumor

The ability of five nitroimidazoles, metronidazole (MET), misonidazole (MISO), desmethylmisonidazole (DMM), SR 2508 and SR 2555, to sensitize the KHT sarcoma to radiation treatment has been compared for drug doses in the range 0–1.5 g/Kg. Single radiation doses or two different daily fractionation schedules (4 fractions of 5 Gy each or 7 fractions of 3 Gy each) were used; the tumor cell survival was determined using either an in vivo or in vitro colony assay. Each radiation (100 kVp X rays @ 11 Gy/min) treatment was given locally, 60—70 min (MET) or 30—40 min (other drugs) after either intraperitoneal (MET, MISO, DMM) or intravenous (SR 2508, SR 2555) injection of the drugs; these times have been shown to be optimum for this tumor. For the single doses and both fractionation schedules the tumor cell survival, following the irradiation treatment, declined as the drug dose increased in the range 0 to 0.75 g/Kg for all of the drugs, but above this dose level a plateau was reached and the amount of sensitization remained essentially constant. In this plateau region the reduction in survival achieved was similar for single doses and 5 Gy fractions but was less for 3 Gy fractions, indicating that sensitization was smaller for the smaller dose fractions. For the 4 × 5 Gy fractionation schedule the plateau level of survival was lowest for MISO, DMM and SR 2508, slightly higher for SR 2555 and much higher for MET. For the 3 Gy fractions SR 2508 appeared slightly less effective than MISO and DMM. Since clinically tolerated doses of MISO are much less than that equivalent to 0.5 g/Kg, these results suggest that DMM and SR 2508 will be superior sensitizers to MISO for human tumors, provided that the lesser toxicity of these drugs in animals is confirmed by studies in man, allowing larger doses than are possible with MISO to be administered to patients.

The influence of warfarin of levamisole on the incidence of metastases following local irradiation of a solid tumor.

KHT sarcomas were implanted in the right rear legs of C3H mice. An x-ray dose of 6000 rad, delivered in ten equal fractions over 12 days, resulted in 60% local tumor control, but 83% of these mice developed metastases. Three strategies to use the tumoricidal effect of x-radiation and reduce the incidence of metastases were compared. A modification of the fractionation scheme to deliver an initial large fraction of 1800 rad followed by seven 600-rad fractions resulted in a decreased incidence of metastases compared with the same dose delivered in ten equal fractions. The use of warfarin anticoagulation during the ten-fraction course of radiation resulted in a small decrease in the incidence of metastases. Immune stimulation with levamisole, injected subcutaneously every second day during the irradiation, also resulted in a decrease in the incidence of metastases. However, when warfarin or levamisole were combined with the eight-fraction radiation scheme there were fewer metastases than following the ten-fraction scheme. The combination of the eight-fraction radiation course with levamisole also produced a significant increase in primary tumor control. In this treatment regimen, therefore, levamisole appears to act as a radiation sensitizer. An hypothesis to explain this action is proposed.

Response of murine tumours to combinations of CCNU with misonidazole and other radiation sensitizers.

The effect of combinations of the conventional chemotherapeutic agent 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and nitroimidazole radiation sensitizers was evaluated in female C3H mice. Tumour response to single-agent or combination therapy was assessed in a tumour growth-delay assay. In the KHT sarcoma the simultaneous addition of misonidazole (MISO) was found to increase significantly the tumour growth delay resulting from CCNU treatment. The observed enhancement ratios (ER) increased with MISO dose, and ranged from 1.3 to 1.9 for sensitizer doses of 0.25-1.0 mg/g. The combination of CCNU and 1.0 or 0.5 mg/g MISO in the RIF-1 tumour or the MT-1 tumour produced ERs of approximately 2.0 and approximately 1.5 respectively. In the KHT sarcoma a series of other nitroimidazole sensitizers, including Ro-05-9963, SR-2555, SR-2508 and metronidazole (METRO), were also evaluated at equimolar doses (5 mmol/kg) in combination with a 20mg/kg dose of CCNU. Unlike MISO, these compounds in general failed to enhance the CCNU cytotoxicity in this tumour model. However, SR-2508 did enhance the response of the RIF-1 tumour to large single doses of CCNU, though not as much as MISO. Normal-tissue toxicity was determined using peripheral white blood cell (WBC) counts 3 days after treatment. CCNU doses of 10-50 mg/kg given either alone or in simultaneous combination with 0.5 or 1.0 mg/g MISO were studied. WBC toxicity increased with CCNU dose, but the addition of MISO at either dose did not significantly enhance this normal-tissue toxicity.

Ara-C scheduling: theoretical and experimental considerations.

In this paper, we discuss the cytokinetic basis for optimization of cancer therapy in humans. Specifically, we define a quantitative procedure for determination of the therapeutic acceptability of therapy schedule, discuss studies of the therapeutic gain that might result from treatment of acute lymphoid leukemia (ALL) with cytosine arabinoside (Ara-C), and indicate the kinds of cytokinetic information necessary for therapy scheduling. These studies suggest 1) that substantial therapeutic improvements may result from optimal therapy scheduling, 2) that therapy schedules selected at random are not likely to be beneficial, and 3) that consideration of the cytokinetic properties of the dose limiting normal tissues is critical during therapy design. We also discuss experimental studies of the response to Ara-C of the murine KHT sarcoma. These studies illustrate the substantial cytokinetic changes occur during therapy (e.g., a substantial increase in cell cycle traverse rate and almost complete recruitment) and show the importance of obtaining mid-treatment cytokinetic information about normal and malignant tissues.

Dose local x-irradiation of a tumor increase the incidence of metastases?

KHT sarcomas were implanted into the right rear legs of C3H mice. Animals receiving no treatment died at 32 days with a 67% incidence of metastatic tumors. A dose of 6000 rads in ten fractions over 12 days, starting ten days after implant, delivered to the primary tumor (with the remainder of the body shielded) produced 55% local tumor control. However, 83% of the mice with local tumor control, subsequently developed metastatic lesions. Amputation of the tumor-bearing leg either prior to the start of radiation treatment, at the end of treatment or at intervals during treatment or sham treatment, indicated that 1) metastases occurred predominantly during the initial three radiation fractions and 2) the increased incidence of metastases following irradiation was a result of the manipulations associated with the irradiation and not the irradiation per se. Irradiation did not change the distribution of organ sites involved with metastatic tumors compared to untreated or sham-treated tumors.

Cell subpopulations dispersed from solid tumours and separated by centrifugal elutriation.

The degree of non-neoplastic host-cell infiltration was assessed in 3 in vivo-in vitro tumour models commonly used in radiobiological studies: EMT6/Ro mammary carcinoma, 9L/Ro tumour and KHT sarcoma. While the 2 former tumour models have been shown to be moderately to highly immunogenic when grown s.c., the KHT sarcoma is apparently non-immunogenic. Using differential staining on single-cell suspensions from enzymatically dissociated solid tumours, all 3 tumour types were found to contain large proportions (30-60%) of non-neoplastic host cells. The actual host-cell component found in the cell suspensions differed both in type and percentage for the 3 tumours studied. These host and neoplastic cells in the cell suspensions prepared from the solid tumours could be readily separated by centrifugal elutriation. After separation the clonogenic potential of the neoplastic cells was assessed, and was found to be higher than the clonogenic capacity of the unseparated cell suspension by a factor directly related to the host/neoplastic cell ratio. Even after the removal of the host cells, the clonogenic capacities of the neoplastic EMT6 and 9L tumour cells were lower than that of the corresponding in vitro sublines (approximately 30 vs 75%). However, in the KHT sarcoma the removal of the host cell component raised the plating efficiency to approximately 60%, which was similar to the value for the in vitro cell subline of this tumour.

Modification of tumour and host response to cyclophosphamide by misonidazole and by WR 2721.

The effect has been studied of adding either misonidazole (MISO) or the radioprotective drug, WR 2721, to cyclophosphamide (CY) treatment of mice bearing either the RIF-1 or KHT sarcomas. In RIF-1, the growth delay due to CY was increased by the addition of 1 mg/g of MISO. At doses below 75 mg/kg of CY, the effect was dose modifying but, at higher doses, the curves were parallel. When the MISO dose was reduced to 0.33 mg/g, the effect was reduced but not entirely lost. Only a small enhancement of CY response in the KHT tumour was seen with single doses, but the enhancement was greater with fractionated doses. The growth delay produced by CY in both tumour systems was reduced if WR 2721 (400 mg/kg) was given 30 min earlier. At a CY dose of 75-100 mg/kg the dose-modifying factor (DMF) was approximately 0.7-0.8 but, at least in the RIF-1 tumour, was not so low at higher doses of CY. Determination of the LD50 for CY showed a DMF of approximately 1.2-1.3 for MISO (0.33 mg/g) and approximately 0.8 for WR 2721 (400 mg/kg). Neither modifying agent appeared to cause any consistent change in the pattern of body-weight loss after CY, but WR 2721 reduced the myelosuppression seen at 3-4 days after CY. The data suggest that modification of tumour response to CY by the addition of MISO varies from tumour to tumour, and is very dependent upon the MISO dose. The protective effect of WR 2721 when combined with CY is not confined to normal tissues, and at a dose of 400 mg/kg may be as great in terms of tumour response as in terms of acute LD50 in this system. At a lower dose of WR 2721, however, some differential protection may occur.

Combinations of Radiation and Misonidazole in a Murine Lung Tumor Model

The response of KHT sarcoma cells growing as microscopic lung colonies to radiation treatments given with or without the hypoxic cell sensitizer misonidazole was evaluated using an in vivo to in vitro clonogenic cell survival assay. For comparison the survival curve for well-oxygenated KHT sarcoma cells was obtained by excising the colony-containing lungs from their hosts, dispersing the lung colonies to a single-cell suspension, and irradiating the cells in vitro. By increasing the time between excision and irradiation from 2 to 24 hr it was possible to demonstrate for these lung colony-derived cells an intercellular contact effect denoted by a reduction in the shoulder of the cell survival curve over this period. In vivo radiation treatments were given at two different lung colony volumes, 0.7 and 1.8 mm/sup 3/. The addition of misonidazole prior to irradiation sensitized the hypoxic cell compartment at both colony sizes and reduced the final slope in each case to the same degree (factor of approx. 1.8). At a cell survival level of 10/sup -3/ this resulted in an enhancement ratio of approx. 1.2 to approx. 1.5. By comparison this dose of misonidazole did not enhance the radiation-induced pulmonary toxicity determined using a LD/sub 50/180/ assay.

In vivo combination of misonidazole and the chemotherapeutic agent CCNU.

The response of intramuscularly growing KHT sarcomas to the chemotherapeutic agent (1-(2-cloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) alone or simultaneously with the chemical radio-sensitizer misonidazole (MISO) was assessed using either a tumour growth-delay assay or an in vivo-in vitro tumour-excision assay. Median tumour growth delay following the combination of 20 mg/kg CCNU and either 0.5 or 1.0 mg/g MISO was 19.5 and 21.5 days, compared to 10 days for this CCNU dose alone. A similar degree of enhanced tumour response by MISO (factor of approximately 2 in tumour growth delay) was seen in RIF-1 tumours treated with 20 mg/kg CCNU plus 1.0 mg/g MISO. Clonogenic cell-survival studies with KHT sarcomas demonstrated that MISO at doses of 0.25, 0.5 or 1.0 mg/g given simultaneously with a range of CCNU doses produced dose-modifying factors (DMFs) of 1.9, 2.1 and 2.4 respectively. Normal tissue toxicity assessed by an LD50/7 assay led to DMFs of 1.2 and 1.4 for CCNU doses combined with 0.5 and 1.0 mg/g MISO. Thus in this animal tumour model the combination of CCNU and MISO appears to lead to a potential gain by a factor of approximately 1.7.

In vivo response of KHT sarcomas to combination chemotherapy with radiosensitizers and BCNU.

Female C3H/HeJ mice bearing intramuscularly transplanted KHT sarcomas were treated with a single dose of 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU, 30 mg/kg, i.p.) alone or in combination with a single dose of misonidazole (MISO, 1.0 mg/g, i.p.) or its desmethylated metabolite Ro-05-9963 (2.0 mg/g, i.p.). The effectiveness of drug therapy was assessed by a tumour growth-delay assay (i.e. measuring the median time required for tumours to grow to treatment size x 4). The relative efficacy of administering the nitroimidazoles in various schedules ranging from 12 h before to 12 h after BCNU administration also was evaluated. Untreated control KHT tumours grew to the initial size x 4 in a median time of 4 days. No significant growth delay was seen in mice treated with either nitroimidazole alone, whilst treatment with BCNU alone produced a median growth delay of 7 days. Combination chemotherapy with 9963 administration 3 h after BCNU significantly increased the median tumour growth delay to 9 days. However, no significant growth delay was produced in any of the other combinations of these agents. The median growth delay was significantly reduced to 5 days when MISO was administered 3 h before BCNU, whereas MISO administered simultaneously 3,6, or 12 h after BCNU significantly enhanced delays ( 9 days). These results indicate that both MISO and 0063 may be combined with conventional therapeutic agents, in this particular case a nitrosourea, to produce an enhanced tumour response. The production of such a response appears to be nitroimidazole as well as schedule dependent.