Long duration-mild whole body hyperthermia of up to 12 hours in rats: feasibility, and efficacy on primary tumour and axillary lymph node metastases of a mammary adenocarcinoma: implications for adjuvant therapy.

The feasibility and efficacy of low temperature (40 degrees C) long duration whole body hyperthermia (LL-WBH) was investigated in rats bearing a highly metastatic mammary adenocarcinoma (MTLn3). We compared the treatment effects of various durations of LL-WBH (40 degrees C for 2-12 h) to that of conventional short duration-high temperature WBH (SH-WBH, 41.5 degrees C for 2 h). SH-WBH, 2 h LL-WBH, and 4 h LL-WBH resulted in only modest primary tumour growth delays (TGDs) of 0.9, 1.1 and 1.8 d (days) respectively. In contrast, significantly increased TGDs of 2.8, 3.2, 2.6, and 3.1 d were achieved with 6, 8, 10 and 12 h LL-WBH, respectively (p < 0.05 compared to SH-WBH, 2 h-LL-WBH, and 4 h-LL-WBH). Notably, LL-WBH reduced the incidence of axillary lymph node metastasis at 14 days post-treatment, from 100% in normothermic controls and 92% after SH-WBH, to 33, 40, 50, and 60% following 4, 6, 8 and 10 h LL-WBH respectively. When the duration of LL-WBH was extended to 12 h, no reduction in axillary lymph node metastasis was observed. Normal tissue toxicity of LL-WBH appeared to be minimal and LL-WBH durations of up to 12 h were well tolerated. These data show that LL-WBH for durations of from 4 to 10 h has greater antitumour activity than SH-WBH, against mammary adenocarcinoma, suggesting that LL-WBH may have therapeutic potential in the treatment of malignant disease.

Apoptosis and necrosis occurring during different stages of primary and metastatic tumor growth of a rat mammary adenocarcinoma.

The pattern of spontaneous apoptosis and necrosis was investigated in an untreated, transplantable rat mammary adenocarcinoma (MTLn3) throughout the natural course of primary and metastatic tumor growth. The occurrence of spontaneous apoptosis was different when comparing primary to metastatic tumor growth. In the primary MTLn3 tumor growing at the mammary fat pad inoculation site we observed an inverse association between tumor growth and apoptosis. As the primary tumor increased in size, the extent of spontaneous apoptosis decreased. In contrast, an increase in apoptosis was associated with tumor growth of MTLn3 metastases in the axillary lymph node and the lung. In regard to necrosis, a similar pattern of increased necrosis was associated with tumor progression in both primary and metastatic tumors. Differences between primary and metastatic tumors in their pattern of spontaneous apoptosis may have important implications for the design of clinical treatment strategies.

Multi-chemothermoimmunotherapy for human colon adenocarcinoma in vitro.

Effective adjunctive therapies for colorectal carcinoma are clearly needed. We evaluated the cytotoxic responses in vitro of human colon carcinoma cell lines to combined modalities: 5-fluorouracil/leucovorin (5-FU/LV), carboplatin (CP), tumor necrosis factor (TNF) and hyperthermia (HTX). Cytotoxicity was evaluated in a cell proliferation assay using crystal violet staining. 5-FU/LV was administered 2-3 days before TNF and CP, followed 1 h later by HTX. These cell lines were relatively resistant to HTX alone (42 degrees C for 2 h), but were heterogeneous in their responses to various doses of the other single agents. This heterogeneity was also evident for combined modalities: the HCT-15 cell line exhibited significant supra-additivity for selected doses of CP, TNF and 5-FU/LV, which was further enhanced by hyperthermia. In contrast, the HT-29 cell line did not demonstrate a strong pattern for supra-additivity, whereas the DLD-1 cell line had an intermediate response. Thus, our results suggest one approach to develop effective and dose-sparing multimodality therapeutic regimens for colon adenocarcinoma.

Apoptosis in tumors and normal tissues induced by whole body hyperthermia in rats.

Apoptosis in tumor and normal tissues was examined in rats treated with whole-body hyperthermia (WBH; 41.5 degrees C for 2 h). WBH alone produced 0.5 day of tumor growth delay (TGD) in a fibrosarcoma and 5.8 days of TGD in the Ward colon carcinoma. This difference in WBH-induced TGD indicates that the fibrosarcoma is relatively resistant to WBH, whereas the Ward colon carcinoma is relatively heat sensitive. A quantitative histological assay for apoptosis demonstrated that the extent of apoptosis in the fibrosarcoma reached a maximum level of 19% 4 h after WBH and returned to the control level by 24 h. In contrast, WBH induced apoptosis with a peak value of 43% at 8 h in the Ward colon carcinoma, and the apoptotic level remained elevated above the control level until 48 h after WBH. Within normal tissues, the spleen and the lymph nodes showed WBH-induced apoptosis; however, the highest level of WBH-induced apoptosis as well as the most prolonged increase in apoptotic levels occurred in the thymus. The WBH-induced apoptosis in the thymus remained elevated above the control level until 48 h after WBH. Within the entire gastrointestinal tract, the small intestine was the most sensitive to WBH. Apoptotic cells were observed in the small bowel mucosa following WBH exposure. We also noted a minor WBH-induced increase in the apoptotic level in the bone marrow. Except for the case of the thymus, increased apoptotic levels in the normal tissues declined after peak levels at 4 h, and apoptosis above control levels was not seen beyond 12 h following WBH. Thus, within the normal tissues, WBH-induced apoptosis declined to basal levels within 12-48 h. These data indicate that both the extent and the kinetics of WBH-induced apoptosis differ between the two tumors and, meaningfully, between tumor and normal tissues. The extent and duration of apoptosis seem to correlate with tumor response to WBH.

Apoptosis in normal tissues induced by 5-fluorouracil: comparison between bolus injection and prolonged infusion.

The induction of apoptosis in normal tissues was histopathologically examined in rats treated with 5-fluorouracil (5-FU). 5-FU was administered by either bolus intravenous injection or 72-hr prolonged intravenous infusion (PIF). Bolus injection and PIF of 5-FU induced different kinetic profiles of apoptosis in the thymus, spleen and ileum. The bolus injections of 5-FU induced a greater extent of apoptosis in these tissues, compared to PIF 5-FU. These data indicate that the kinetics and extent of apoptosis induced by 5-FU depends on the schedule of the 5-FU administration, and that 5-FU-induced toxicity may be related to 5-FU-induced apoptosis in normal tissues.

Therapeutic efficacy of long duration-low temperature whole body hyperthermia when combined with tumor necrosis factor and carboplatin in rats.

This study examines the effects of a combined modality regimen of long duration-low temperature whole body hyperthermia (6 h at 40.0 degrees C; LL-WBH), recombinant human tumor necrosis factor-alpha (TNF) and carboplatin (CBDCA) on a transplantable fibrosarcoma as well as normal tissue. We compare LL-WBH with short duration-high temperature whole body hyperthermia (2 h at 41.5 degrees; SH-WBH). LL-WBH alone had no significant effect on tumor growth. Tumor growth delay (TGD) with TNF alone (0.1 days) and that with CBDCA alone (1.3 days) were significantly increased to 2.6 days (P < 0.05) and 2.8 days (P < 0.05), respectively, when combined with LL-WBH. Although TNF+CBDCA produced minimally increased TGD of 1.9 days, the combination of LL-WBH+TNF+CBDCA produced a significantly greater TGD of 5.6 days, compared to the other dual combinations (P < 0.01). There was no difference between TGDs for SH-WBH and LL-WBH in combination with TNF+CBDCA. Trimodality treatment-induced normal tissue toxicities, characterized by body weight loss, diarrhea, foot edema, and myelosuppression, were significantly greater in rats treated with SH-WBH+TNF+CBDCA, compared to LL-WBH+TNF+CBDCA. Histopathological examination also demonstrated that SH-WBH+TNF+CBDCA caused severe damage to the lymphoid tissues, intestinal tract, and peripheral microvasulature. We observed minimal histopathological changes observed in rats treated with LL-WBH+TNF+CBDCA. These data suggest that LL-WBH in combination with TNF and CBDCA has a greater therapeutic efficacy than SH-WBH.

Phase I and plasma pharmacokinetic study of infusional fluorouracil combined with recombinant interferon alfa-2b in patients with advanced cancer.

PURPOSE: Enhanced fluorouracil (FUra) cytotoxicity caused by recombinant interferon alfa-2b (rIFN-a) has been reported, but the mechanism, optimal dose, and schedule remain unknown. Therefore, a phase I and pharmacokinetic study of FUra with escalating doses of rIFN-a was initiated. PATIENTS AND METHODS: FUra (750 mg/m2/d) was given by continuous intravenous (IV) infusion for 5 days. rIFN-a (0.1 to 15 x 10(6) U/m2/d) was given subcutaneously (SC) daily for 5 days concurrent with FUra. Courses were repeated every 14 to 21 days. Forty-four patients were enrolled; 39 received at least two courses. During the first course of therapy, FUra levels before and after administration of rIFN-a were quantitated in 26 patients by high-pressure liquid chromatography. RESULTS: The maximum-tolerated dose of rIFN-a was 10 x 10(6) U/m2/d. Stomatitis was dose-limiting. Three partial and five minor responses occurred. Interpatient pharmacokinetics showed that rIFN-a did not alter steady-state plasma concentration (Css; range, 0.77 +/- 0.35 mumol/L to 1.85 +/- 0.48 mumol/L), elimination half-life (t1/2; mean, 9.7 +/- 4.3 minutes), area under the concentration-versus-time curve (AUC; range, 93 to 224 mumol/L x hours), total-body clearance (CI; range, 1,172 to 3,236 mL/min), or volume of distribution (range, 11.9 to 49.2 L) of FUra. Intrapatient data evaluation revealed a dose-independent effect of rIFN-a. The mean FUra Css after rIFN-a administration (1.31 mumol/L) was greater than that before rIFN-a administration (1.02 mumol/L, P < .0001). FUra Cl after rIFN-a administration was reduced by 20% to 35% compared with use of FUra alone (P < .0001). Patients with a greater than 20% decrease in FUra Cl had a fourfold greater incidence of diarrhea. CONCLUSION: rIFN-a reduces FUra Cl and, consequently, increases FUra-associated toxicity. Phase II studies of FUra and rIFN-a seem to be warranted.

Potential for selective enhancement of the in vivo metabolism of 1-beta-D-arabinofuranosylcytosine in rats by thymidine pretreatment.

In this study, the ability of deoxythymidine (dThd) to enhance selectively the metabolism of 1-beta-D-arabinofuranosylcytosine (ara-C) in rats bearing transplantable colon carcinoma was investigated. A steady-state plasma level of 375 microM dThd was achieved within 3 h after initiation of a 24-h infusion of dThd (7 g/kg/day) with a concomitant 80% reduction in circulating 2'-deoxycytidine levels. Complete recovery to control values occurred within 6 to 8 h after termination of the infusion. Under the conditions of dThd infusion, the intracellular levels of 2'-deoxycytidine 5'-triphosphate rose from 0.15 to 60 pmol/mg tumor tissue, from 2.5 to 15 pmol/mg intestinal tissue, and from 0.07 to 0.25 pmol/10(6) bone marrow cells. During the steady-state plasma concentration of dThd, the intracellular concentration of 2'-deoxycytidine 5'-triphosphate in tumor tissue was reduced by 50% at 6 h after the initiation of dThd treatment with a complete recovery 9 h thereafter. Differences in the capacity of tumor and host normal tissues to recover from the effects of dThd pretreatment were evaluated by measuring decreasing 1-beta-D-arabinofuranosylcytosine 5'-triphosphate formation with time following dThd infusion. The ability to accumulate 1-beta-D-arabinofuranosylcytosine 5'-triphosphate was reduced by 60 to 80% in normal tissues by 3 h after cessation of the dThd infusion but was decreased by only 15% in the tumor. These results suggested that delaying ara-C administration following dThd might result in less host toxicity while maintaining the antitumor effect. Sequential infusion of dThd (7 g/kg/day) for 24 h followed 3 h later by a 48-h infusion of ara-C (175 mg/kg/day), was as effective in reducing tumor mass as was dThd infusion immediately prior to ara-C and resulted in reduced host toxicity (less weight loss). The best schedule for the dThd-ara-C combination was two courses of alternating 24-h sequential infusions of dThd and ara-C with a 3-h delay in ara-C administration following dThd. These data show that under the conditions used, reductions in intracellular 2'-deoxythymidine 5'-triphosphate pools by dThd in vivo do not appear to correlate with the antitumor activity of the dThd-ara-C combination. Intracellular 1-beta-D-arabinofuranosylcytosine 5'-triphosphate accumulation, however, was prolonged in rat colon tumor compared to normal tissues, and selectivity of the dThd-ara-C combination in favor of the tumor could be achieved by schedule modification.

Lack of enhanced cytotoxicity of cultured L1210 cells using folinic acid in combination with sequential methotrexate and fluorouracil.

Previous studies from this laboratory have demonstrated that treatment of cultured cells with sequential methotrexate (MTX) and fluorouracil (FUra) leads to synergistic cell killing in several murine and human neoplasms in vitro. In this study leucovorin (folinic acid, LCV) was added to the MTX/FUra combination with the intention of generating elevated levels of methylenetetrahydrofolate to promote the formation of a stable fluorodeoxyuridylate-thymidylate synthetase ternary complex, thereby augmenting the cytotoxicity of the MTX-FUra sequence. The addition of 10 or 100 microM LCV concurrently with or after 10 microM FUra following MTX (1 microM) pretreatment did not augment the inhibition of L1210 cell growth or the clonigenicity compared with MTX prior to FUra without LCV. The effects of LCV scheduling on the sequential MTX and FUra-induced inhibition of thymidylate synthesis were measured by examining the rate of [6-3H] dUrd incorporation into the acid-precipitable cell fraction and by direct quantitation of the thymidylate synthetase ternary complex. Combination of 100 microM LCV with 10 microM FUra after 1 microM MTX resulted in significantly more ternary complex formation than did 1 microM MTX before 10 microM FUra alone. The inhibitory effects of FUra on thymidylate synthetase in the presence of MTX, however, could not be augmented by LCV as determined by [6-3H] incorporation into acid-precipitable material, nor did the addition of LCV result in increased cytotoxicity. Factors other than the inhibition of DNA synthesis may be critical to the cytotoxicity of sequential MTX and FUra in L1210 cells.

Chemotherapeutic efficacy of 5-fluorouracil with concurrent thymidine infusion against transplantable colon tumors in rodents.

The therapeutic efficacy of 5-fluorouracil (FU) given concomitantly with thymidine (TdR) versus that of FU alone at an equitoxic dose was evaluated when these agents were given by 72-h continuous i.v. infusion or as an i.v. push dose through the tail vein to Fischer rats bearing a transplantable colon carcinoma or to BALB/c or C57BL/6J mice bearing murine colon tumors no. 26 or 38, respectively. In the presence of TdR, the dose of FU maximally tolerated by normal rats and mice was reduced by approximately half. In tumor-bearing rats, infusion of FU alone (150 mg/kg X 72 h) was as effective as concurrent infusion of FU (100 mg/kg X 72 h) with TdR (5 g/kg X 72 h), as evaluated in terms of tumor-free survivors (7/22 and 6/20, respectively). In addition, both regimens were more effective than an i.v. push dose of FU (200 mg/kg) or FU (80 mg/kg) and TdR (2 g/kg), which resulted in 2/18 and 0/18 tumor-free survivors, respectively. No significant differences in tumor growth inhibition were seen using either murine colon tumor, whether FU or the FU with TdR combination was administered as an i.v. push dose or as an infusion. Quantitation of the levels of TdR and thymine (T) in rat plasma obtained during infusion of various doses of TdR showed dose-dependent levels of TdR and T, indicating conversion of TdR to T in vivo. These data showed that, under the conditions employed, TdR did not modify significantly the antitumor activity of FU against rodent colon tumors. The toxicity of FU, however, could be enhanced by the coadministration of TdR, probably due, in part, to a reduced degradation of FU resulting from competition by T.

Changes in ribo- and deoxyribonucleoside triphosphate pools within the cell cycle of a synchronized mouse fibroblast cell line.

Intracellular pool levels of ribo- and deoxyribonucleoside triphosphates were monitored throughout the cell cycle of C3H10T1/2 mouse embryo fibroblast cells synchronized by isoleucine deprivation. Absolute pool sizes of ribonucleoside triphosphates were approximately 30 fold greater than those of the corresponding deoxyribonucleoside triphosphates. Of the ribonucleoside triphosphates, pool sizes of ATP exhibited the greatest change, increasing from a low of 32.7 nmol/10(7) cells during G1 to a high of 81.6 nmol/10(7) cells 2 h prior to mid S-phase. Levels of ATP subsequently declined to 40.2 nmol/10(7) cells during late S-phase, followed by a second peak of 65.8 nmol/10(7) with the onset of cell division. No significant changes in the pool sizes of UTP and GTP were found throughout the cell cycle. Of the deoxyribonucleoside triphosphates, pool sizes of pyrimidine deoxyribonucleoside triphosphates were approx. 5-10 fold greater than those of purine deoxyribonucleoside triphosphates. Low levels of deoxyribonucldoside triphosphates during G1 (0.3-1.3 pmol/10(7) cells) increased coordinately with the initiation of DNA synthesis to an initial peak during mid S-phase (0.5-6.4 pmol/10(7) cells). Declining levels of deoxyribonucleoside triphosphates during late S-phase were followed by a subsequent larger second peak (1.7-10.7 pmol/10(7) cells) during G2-M.

A method for continuous drug infusion in unrestrained rats: its application in evaluating the toxicity of 5-fluorouracil/thymidine combinations.

A method for continuous infusion of drugs into the tail vein of rats was developed. This simple procedure has the advantage of permitting freedom of movement of the animal during extended drug exposure. Utilization of an aluminum sheath as a tail cover prevents destruction of the cannulating apparatus without placing additional stress upon the animal. This method produced no adverse effects upon rats during prolonged infusion (up to 7 days) and may be useful in the routine evaluation of agents having a short plasma half-life. The lethality of FU alone or in combination with TdR was evaluated with this technique. Infusion of up to 10 gm/kg/72 hr of TdR produced no mortality. Co-administration of TdR at 1 gm/kg/72 hr with FU, however, potentiated the toxicity of FU. These data indicate that the toxicity of FU may be modified by provision of TdR with this method.