Pre-treatment energy status of primary rat tumours as the best predictor of response to 5-fluorouracil chemotherapy: a magnetic resonance spectroscopy study in vivo

Purpose: Fluorine-19 magnetic resonance spectroscopy (¹⁹F-MRS) studies of the pharmacokinetics of the anticancer drug 5-fluorouracil (FU) in patients at several clinical centres have shown that increased tumour retention of FU is associated with patient response. The mechanism of this increased tumour retention (FU trapping) is unknown. We used a pre-clinical model to investigate whether other MRS-measurable parameters would correlate with the response to FU treatment and, thus, help elucidate the mechanism(s) involved in FU trapping. Methods: MRS spectra were obtained using a double-tuned (³¹P/¹⁹F) surface coil from 29 N-methyl-N-nitrosourea-induced primary rat tumours. ³¹P-MRS spectra were acquired immediately prior to and at 2.5 h post-treatment with a bolus i.p. injection of FU (100 mg/kg); ¹⁹F-MRS spectra were acquired during the intervening 2.5-h period for measurement of the tumour uptake and retention of FU and of its metabolism to the cytotoxic fluoronucleotides (FNuct). From these data, four parameters were measured: tumour pH and energy status (NTP/Pi) before treatment, total FU retention, and FU anabolism to FNuct (expressed as micromoles per gram per 2.5 h). In addition, tumour response was determined at 7 days post-treatment by measurement of the percentage of change in tumour weight and was classified according to standard oncological criteria as follows: progressive (P) for a ≥25% increase, remissive (R) for a ≥50% decrease or stable (S) for values lying between these two. Results: Analysis of variance (ANOVA) for statistical assessment revealed that groups P, S and R were not distinguishable using the MRS parameters; although when S and R were combined as one group of non-progressive disease (NPD; n= 24), both the NTP/Pi ratio and the total FNuct formed were significantly greater (P= 0.04) than those observed in the P group (n= 5). Considering all 29 tumours, linear regression showed that there were positive significant correlations between the NTP/Pi ratio and (a) the percentage of response (P= 0.04), (b) the pre-treatment pH (P= 0.002) and (c) FU retention (P= 0.02), but not FNuct formation (P= 0.66). Unlike results reported in the clinic, the percentage of response and FU retention were neither significantly correlated (P= 0.22) nor associated when groups P and NPD were compared (P= 0.27, Fischer's exact test). FNuct, however, was significantly associated with response, as was the NTP/Pi ratio (P≤ 0.02). Combination of FNuct with the NTP/Pi ratio increased the significance of the association with response (P= 0.003, Fischer's exact test). Conclusions: Our results indicate that in this particular model the pre-treatment tumour NTP/Pi ratio was the best predictor of response to a bolus injection of FU, rather than FNuct formation or FU retention. An elevated NTP/Pi ratio could reflect a well-vascularised tumour with an improved capacity for energy-dependent FU uptake and metabolism to FNuct, suggesting that further investigation of this parameter could be an important line of research, which may aid the identification of tumours likely to be sensitive to FU chemotherapy in the clinic. Received: 8 June 1997 / Accepted: 20 January 1998     

A pharmacokinetic and pharmacodynamic study In vivo of human HT29 tumours using F and P magnetic resonance spectroscopy

¹⁹F-MRS (magnetic resonance spectroscopy) was used to study the pharmacokinetics of 5-fluorouracil (5-FU) in human (HT29) tumour xenografts, with and without pretreatment of the mice using either thymidine (40 min) or interferon-α (2 and 24 h). A 200 mg/kg i.p. bolus dose of 5-FU was eliminated from control tumours with a t_1/2 of 25.4 ± 2 min (mean ± SEM, n = 11), while both thymidine (500 mg/kg) and interferon (50 000 IU/mouse) significantly increased t_1/2 to 36.5 ± 6.1 (n = 5) and 48.1 ± 13.6 min (n = 4), respectively (P = 0.04, Gabriel’s ANOVA). Thymidine increased 5-FU anabolism to cytotoxic 5-fluoronucleotides, and decreased the amount of tumour catabolites; the latter probably recirculated from liver since isolated HT29 cells did not catabolise 5-FU. These in vivo observations were confirmed by ¹⁹F-MRS quantification of tumour extracts. Interferon did not significantly affect 5-FU metabolism in the tumour or liver, nor the 5-FU t_1/2 in liver. Treatment of tumours with 5-FU or interferon had no effect on tumour growth, whereas the combination strongly inhibited growth. ³¹P-MRS of HT29 tumours showed that 2 and 24 h after i.p. injections of interferon there was a significant increase in the pH_int of 0.3 ± 0.04 units (P = 0.002), while pH_ext and the tumour NTP/Pi ratio were unchanged. The large increase in the negative pH gradient (−Δ pH) across the tumour plasma membrane caused by interferon suggests the Δ pH may be a factor in tumour retention of 5-FU, as recently shown in isolated tumour cells.     

Prediction of 5-fluorouracil cytotoxicity towards the Walker carcinosarcoma using peak integrals of fluoronucleotides measured by MRS in vivo

19F-magnetic resonance spectroscopy (MRS) can be used to non-invasively monitor metabolism of 5-fluorouracil (5FU) to cytotoxic fluoronucleotides (FNuct). We investigated whether the levels of FNuct formed from 5FU and observed in vivo by MRS in the Walker carcinosarcoma predicted cytotoxicity. Fifty mg kg-1 5FU caused tumour FNuct formation and, when repeated daily for 1 week, significant tumour growth inhibition (P less than 5%). Twenty-five mg kg-1 5FU produced less tumour FNuct (P less than 5%) and did not cause significant tumour regression. Tumour regression and tumour FNuct formation were also suppressed by 50 mg kg-1 5FU combined with a molar equivalent dose of allopurinol (P less than 2%). Tumour extracts were analysed by hplc and MRS confirming the observations in vivo and demonstrating that peak integrals in vivo were directly proportional to 5FU and FNuct concentrations. Hplc analysis of extracts showed that 50% of FNuct in tumours treated with 5FU was the cytotoxic nucleotide FUTP; this was lowered to 5% by a molar equivalent dose of allopurinol (P less than 2%). Twenty-five mg kg-1 5FU also produced significantly less FUTP (36%) than the 50 mg kg-1 dose (P less than 5%). These results suggest that MRS-detectable changes in tumour FNuct (mostly in FUTP) can be used to predict 5FU cytotoxicity.     

Response of hepatoma 9618a and normal liver to host carbogen and carbon monoxide breathing

The effects of hyperoxia (induced by host carbogen [95% oxygen/5% carbon dioxide breathing] and hypoxia (induced by host carbon monoxide [CO at 660 ppm] breathing) were compared by using noninvasive magnetic resonance (MR) methods to gain simultaneous information on blood flow/oxygenation and the bioenergetic status of rat Morris H9618a hepatomas. Both carbogen and CO breathing induced a 1.5- to 2-fold increase in signal intensity in blood oxygenation level dependent (BOLD) MR images. This was due to a decrease in deoxyhemoglobin (deoxyHb), which acts as an endogenous contrast agent, caused either by formation of oxyhemoglobin in the case of carbogen breathing, or carboxyhemoglobin with CO breathing. The results were confirmed by observation of similar changes in deoxyHb in arterial blood samples examined ex vivo after carbogen or CO breathing. There was no change in nucleoside triphosphates (NTP)/P_i in either tumor or liver after CO breathing, whereas NTP/P_i increased twofold in the hepatoma (but not in the liver) after carbogen breathing. No changes in tumor intracellular pH were seen after either treatment, whereas extracellular pH became more alkaline after CO breathing and more acid after carbogen breathing, respectively. This tumor type and the liver are unaffected by CO breathing at 660 ppm, which implies an adequate oxygen supply.     

The influence of tumor oxygenation on <Superscript>18</Superscript>F-FDG (Fluorine-18 Deoxyglucose) uptake: A mouse study using positron emission tomography (PET)

Background

This study investigated whether changing a tumor's oxygenation would alter tumor metabolism, and thus uptake of ¹⁸F-FDG (fluorine-18 deoxyglucose), a marker for glucose metabolism using positron emission tomography (PET).

Results

Tumor-bearing mice (squamous cell carcinoma) maintained at 37°C were studied while breathing either normal air or carbogen (95% O₂, 5% CO₂), known to significantly oxygenate tumors. Tumor activity was measured within an automatically determined volume of interest (VOI). Activity was corrected for the arterial input function as estimated from image and blood-derived data. Tumor FDG uptake was initially evaluated for tumor-bearing animals breathing only air (2 animals) or only carbogen (2 animals). Subsequently, 5 animals were studied using two sequential ¹⁸F-FDG injections administered to the same tumor-bearing mouse, 60 min apart; the first injection on one gas (air or carbogen) and the second on the other gas. When examining the entire tumor VOI, there was no significant difference of ¹⁸F-FDG uptake between mice breathing either air or carbogen (i.e. air/carbogen ratio near unity). However, when only the highest ¹⁸F-FDG uptake regions of the tumor were considered (small VOIs), there was a modest (21%), but significant increase in the air/carbogen ratio suggesting that in these potentially most hypoxic regions of the tumor, ¹⁸F-FDG uptake and hence glucose metabolism, may be reduced by increasing tumor oxygenation.

Conclusion

Tumor ¹⁸F-FDG uptake may be reduced by increases in tumor oxygenation and thus may provide a means to further enhance ¹⁸F-FDG functional imaging.

     

A Novel Class of Antitumor Prodrug, 1-(2'-Oxopropyl)-5-fluorouracil (OFU001), That Releases 5-Fluorouracil upon Hypoxic Irradiation

We have been developing prodrugs of anticancer agents such as 5‐fluorouracil (5‐FU) that are activated by irradiation under hypoxic conditions via one‐electron reduction. Among them, OFU001 [1‐(2′‐oxopropyl)‐5‐fluorouracil] is a prototype radiation‐activated prodrug. In this study, we investigated the radiation chemical reactivity and the biological effects of OFU001. This prodrug is presumed to release 5‐FU through incorporation of hydrated electrons into the antibonding σ* orbital of the C(1′)‐N(1) bond. Hydrated electrons are active species derived from radiolysis of water, but are readily deactivated by O₂ into superoxide anion radicals () under conditions of aerobic irradiation. Therefore, 5‐FU release occurs highly specifically upon irradiation under hypoxic conditions. OFU001 dissolved in phosphate buffer released 5‐FU with a G‐value (mol number of molecules that are decomposed or produced by 1 J of absorbed radiation energy) of 1.9×10^?7 mol/ J following hypoxic irradiation, while the G‐value for 5‐FU release was 1.0×10^?8 mol/J following aerobic irradiation. However, the G‐values for decomposition of OFU001 were almost the same, i.e., 3.4×10^?7 mol/J following hypoxic irradiation and 2.5×10^?7 mol/J following aerobic irradiation. When hypoxically irradiated (7.5–30 Gy) OFU001 was added to murine SCCVII cells for 1–24 h, a significant cell‐killing effect was observed. The degree of this cytotoxicity was consistent with that of authentic 5‐FU at the corresponding concentrations. On the other hand, cytotoxicity was minimal when the cells were treated with aerobically irradiated or unirradiated OFU001. This compound had no radiosensitizing effect against SCCVII cells under either aerobic or hypoxic conditions when the drug was removed immediately after irradiation. Since hypoxia is generally most marked in tumors and irradiation is applied at the tumor site, this concept of prodrug design appears to be potentially useful for selective tumor treatment with minimal adverse effects of anticancer agents.     

Enhanced uptake of ifosfamide into GH3 prolactinomas with hypercapnic hyperoxic gases monitored in vivo by (31)P MRS

Previously, ³¹P magnetic resonance spectroscopy (MRS) has been used to detect ifosfamide (IF) in vivo and to show that breathing carbogen (5% CO₂/95% O₂) enhances the uptake and increases the efficacy of IF in rat GH3 prolactinomas [Rodrigues LM, Maxwell RJ, McSheehy PMJ, Pinkerton CR, Robinson SP, Stubbs M, and Griffiths JR (1997). In vivo detection of ifosfamide by ³¹P MRS in rat tumours; increased uptake and cytotoxicity induced by carbogen breathing in GH3 prolactinomas. Br J Cancer 75, 62–68]. We now show that other hypercapnic and/or hyperoxic (5% CO₂ in air, 2.5% CO₂ in O₂) gas mixtures also increase the uptake of IF into tumors, measured by ³¹P MRS. All gases caused an increased uptake (C_max) of IF compared to air breathing, with carbogen inducing the largest increase (85% (P<.02) compared to 46% with 2.5% CO₂ in O₂ (P<.004) and 48% with 5% CO₂ in air (P<.004)). The T_max (time of maximum concentration in tumor posintravenous injection of IF) was significantly (P<.04) later in the cohort that breathed 5% CO₂ in air. The increased uptake of IF with carbogen breathing was selective to tumor tissue and there were no significant increases in any of the normal tissues studied, suggesting that any host tissue toxicity would be minimal. Carbogen breathing by patients causes breathlessness. There was no significant difference in IF uptake between breathing carbogen and 2.5% CO₂ in O₂ and, therefore, the ability of 2.5% CO₂ in O₂ to also increase IF uptake may be clinically useful as it causes less patient discomfort.     

The effect of hypoxia and hyperoxia on nucleoside triphosphate/inorganic phosphate, pO2 and radiation response in an experimental tumour model.

This study has evaluated the effect of breathing 100% oxygen, carbogen and carbon monoxide (at 660 p.p.m.) on the bioenergetic and oxygenation status and the radiation response of 200-mm3 C3H mammary carcinomas grown in the feet of CDF mice. Bioenergetic status was assessed by 31P magnetic resonance spectroscopy (MRS) using a 7-tesla spectrometer with both short (2 s) and long (6 s) pulse repetition times. Tumour partial pressure of oxygen (PO2) was measured with an Eppendorf polarographic electrode; the oxygenation parameters were the median pO2 and fraction of pO2 values < or = 2.5 mmHg. The radiation response was estimated using a tumour growth delay assay (time to grow three times treatment volume). Carbon monoxide breathing decreased tumour pO2 and compromised the radiation response, but the beta-nucleoside triphosphate (NTP)/Pi ratio was unchanged. Both carbogen and oxygen (100%) increased tumour pO2 and beta-NTP/Pi and enhanced the radiation response, the effects being similar under the two gassing conditions and dependent on the gas breathing time. Thus, in this tumour model, 31P-MRS can detect hyperoxic changes, but because cells can remain metabolically active even at low oxygen tensions the beta-NTP/Pi did not correlate with low tissue oxygenation. An analysis of variance showed that gas breathing time induced a significant systematic effect on beta-NTP/Pi, the MRS pulse repetition time had a significant effect on beta-NTP/Pi change under hypoxic but not under hyperoxic conditions and the type of gas that was inhaled had a significant effect on beta-NTP/Pi.     

The inhibitory effects of 5-fluorouracil on the metabolism of preribosomal and ribosomal RNA in L-1210 cells in vitro

Summary Addition of 5FU to the culture medium of mouse L-1210 cells resulted in inhibition of the maturation process of ribosomal RNA precursors in vitro. In the presence of 10^-6 M 5FU for 2 h, the 45S preribosomal RNA was processed to 32S preribosomal RNA, but 28S rRNA was not produced. The processing to 18S rRNA was intact at this drug concentration. Higher concentrations of 5FU for a longer incubation period affected the RNA processing more severely. At 10^-5 M of the drug for 24 h the processing to 18S rRNA was also inhibited, in addition to the processing to 18S rRNA and 32S preribosomal RNA. When the cells were labeled with ¹⁴C-UR for 2 h following ³H-5FU at 10^-6 M for 24 h, the radioactivities of newly synthesized RNA labeled with ¹⁴C-UR accumulated in the region of 45S and 32S preribosomal RNA, and no processing to 28S rRNA was observed. Radioactivity corresponding to ³H-5FU did not persist in the preribosomal RNA region, because further maturation proceeded in the condition of depletion of 5FU after the long incubation period. Thus, inhibition of the processing of preribosomal RNA to 28S rRNA was not brought about by the accumulation of 5FU-substituted 45S preribosomal RNA, but by some other, yet unknown, mechanism.Abbreviations used 5FU 5-fluorouracil - rRNA ribosomal RNA - FdUMP 5-fluoro-2-'deoxyuridine 5-monophosphate - FUTP 5-fluorouridine 5-triphosphate - UR uridine The work described in this paper was supported in part by a Grant-in-Aid for Cancer Research (no. 58015015) from the Ministry of Education, Science and Culture     

31P-nuclear magnetic resonance spectroscopy in vivo of six human melanoma xenograft lines: tumour bioenergetic status and blood supply.

Six human melanoma xenograft lines grown s.c. in BALB/c-nu/nu mice were subjected to 31P-nuclear magnetic resonance (31P-NMR) spectroscopy in vivo. The following resonances were detected: phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE), phosphocreatine (PCr) and nucleoside triphosphate gamma, alpha and beta (NTP gamma, alpha and beta). The main purpose of the work was to search for possible relationships between 31P-NMR resonance ratios and tumour pH on the one hand and blood supply per viable tumour cell on the other. The latter parameter was measured by using the 86Rb uptake method. Tumour bioenergetic status [the (PCr + NTP beta)/Pi resonance ratio], tumour pH and blood supply per viable tumour cell decreased with increasing tumour volume for five of the six xenograft lines. The decrease in tumour bioenergetic status was due to a decrease in the (PCr + NTP beta)/total resonance ratio as well as an increase in the Pi/total resonance ratio. The decrease in the (PCr + NTP beta)/total resonance ratio was mainly a consequence of a decrease in the PCr/total resonance ratio for two lines and mainly a consequence of a decrease in the NTP beta/total resonance ratio for three lines. The magnitude of the decrease in the (PCr + NTP beta)/total resonance ratio and the magnitude of the decrease in tumour pH were correlated to the magnitude of the decrease in blood supply per viable tumour cell. Tumour pH decreased with decreasing tumour bioenergetic status, and the magnitude of this decrease was larger for the tumour lines showing a high than for those showing a low blood supply per viable tumour cell. No correlations across the tumour lines were found between tumour pH and tumour bioenergetic status or any other resonance ratio on the one hand and blood supply per viable tumour cell on the other. The differences in the 31P-NMR spectrum between the tumour lines were probably caused by differences in the intrinsic biochemical properties of the tumour cells rather than by the differences in blood supply per viable tumour cell. Biochemical properties of particular importance included rate of respiration, glycolytic capacity and tolerance to hypoxic stress. On the other hand, tumour bioenergetic status and tumour pH were correlated to blood supply per viable tumour cell within individual tumour lines. These observations suggest that 31P-NMR spectroscopy may be developed to be a clinically useful method for monitoring tumour blood supply and parameters related to tumour blood supply during and after physiological intervention and tumour treatment. However, clinically useful parameters for prediction of tumour treatment resistance caused by insufficient blood supply can probably not be derived from a single 31P-NMR spectrum since correlations across tumour lines were not detected; additional information is needed.     

Characterization of a murine lymphoma cell line by 31P-NMR spectroscopy: In vivo monitoring of the local anti-tumor effects of systemic immune cell transfer

The intradermal ESb-MP murine T-cell lymphoma in syngeneic DBA/2 mice has been used as a model for adoptive immunotherapy (ADI). Cultured ESb-MP cells were characterized in suspension by ³¹P-NMR spectroscopy (MRS) at 11.7 T, and solid primary tumors were examined by ³¹P-MRS in vivo at 7.0 Tesla using surface-coil techniques. Growing tumors contained relatively high levels of phosphomonoesters (PME, predominantly phosphoethanolamine), nucleotides (NTP) and P₁, low levels of phosphodiesters (PDE) and no phosphocreatine. Mean tissue pH was found to be 6.7–6.9. The spectra of ESb-MP cells cultured in RPMI medium (containing choline but no ethanolamine) also showed low PDE and no phosphocreatine at an intracellular pH of 7.4; however, only a trace amount of phosphoethanolamine was detected and significant levels of nucleoside mono- and diphosphates were observed. The complete ADI treatment protocol involved low-dose irradiation (5 Gy) followed by i.v. transfer of immune spleen cells from allogeneic B10.D2 donors and resulted in 100% remission (responders); no treatment or incomplete ADI (irradiation or immune cell transfer alone) resulted in no remissions (non-responders). In vivo MRS could best discriminate between responders and non-responders on the basis of tissue pH, which increased in responders to 7.0 by day 5–6 after complete ADI. Following therapy, the sum of PME + P₁ (both absolute and as a percent of total phosphates) decreased significantly only for responders but only after a visible decrease in tumor volume was apparent. © 1996 Wiley-Liss, Inc.     

The effects of host carbogen (95% oxygen/5% carbon dioxide) breathing on metabolic characteristics of Morris hepatoma 9618a.

Characteristics of the tumour metabolic profile play a role in both the tumour-host interaction and in resistance to treatment. Because carbogen (95% oxygen/5% carbon dioxide) breathing can both increase sensitivity to radiation and improve chemotherapeutic efficacy, we have studied its effects on the metabolic characteristics of Morris hepatoma 9618a. Host carbogen breathing increased both arterial blood pCO2 and pO2, but decreased blood pH. A fourfold increase in tumour pO2 (measured polarographically) and a twofold increase in image intensity [measured by gradient recalled echo magnetic resonance (MR) imaging sensitive to changes in oxy/deoxyhaemoglobin] were observed. No changes were seen in blood flow measured by laser Doppler flowmetry. Tumour intracellular pH remained neutral, whereas extracellular pH decreased significantly (P < 0.01). Nucleoside triphosphate/inorganic phosphate (NTP/Pi), tissue and plasma glucose increased twofold and lactate decreased in both intra- and extracellular compartments, suggesting a change to a more oxidative metabolism. The improvement in energy status of the tumour was reflected in changes in tissue ions, including Na+, through ionic equilibria. The findings suggest that the metabolic profile of hepatoma 9618a is defined partly by intrinsic tumour properties caused by transformation and partly by tissue hypoxia, but that it can respond to environmental changes induced by carbogen with implications for improvements in therapeutic efficacy.     

Intrathecal 5-fluorouracil in the rhesus monkey

Summary Because meningeal spread of both leukemia and solid tumors remains a difficult therapeutic problem, there is a compelling need to develop new agents for intrathecal administration. 5-Fluorouracil (5FU), an active anticancer agent, penetrates into the central nervous system to some degree following intravenous dosing. Significant systemic toxicity, however, is associated with this route of administration. Therefore, the pharmacokinetic behavior of 5FU following its intrathecal administration was studied in a rhesus monkey model. After a 10-mg intraventricular dose, the disappearance of the drug from ventricular cerebrospinal fluid was monoexponential, the half-life being 51 min; the area under the concentration-time curve (AUC) being greater than 18mm h^–1; and the peak ventricular 5FU concentrations ranging between 10 and 15mm. After a 1-mg intralumbar dose, the AUC was 1235 m h^–1. No toxicity was observed following intraventricular administration of 5FU. After intralumbar administration of either a 10-mg or a 1-mg dose, however, local toxicity was observed in the lumbar spinal cord. These findings suggest that intrathecal administration of 5FU is not presently a feasible means of achieving cytotoxic cerebrospinal fluid concentrations.     

Early radiation effects in highly apoptotic murine lymphoma xenografts monitored by P magnetic resonance spectroscopy

Purpose: Phosphorus-31 magnetic resonance spectra (³¹P-MRS) were obtained from highly apoptotic murine lymphoma xenografts before and up to 24 hr following graded doses of radiation ranging from 2 to 30 Gy. Radiation-induced apoptosis was also estimated up to 24 hr by scoring apoptotic cells in tumor tissue.Methods and Materials: Highly apoptotic murine lymphoma cells, EL4, were subcutaneously transplanted into C57/BL mice. At 7 days after transplantation, radiation was given to the tumor with a single dose at 3, 10, and 30 Gy. The β-ATP/Pi, PME/Pi, and β-ATP/PME values were calculated from the peak area of each spectrum. Radiation-induced apoptosis was scored with counting apoptotic cells on hematoxylin and eosin stained specimens (%apoptosis).Results: The values of % apoptosis 4, 8, and 24 hr after radiation were 21.8, 19.6, and 4.6% at 3 Gy, 35.1, 25.6, and 14.8% at 10 Gy, 38.4, 38.0, and 30.6% at 30 Gy, respectively (cf. 4.4% in control). There was no correlation between early change in β-ATP/Pi and % apoptosis at 4 hr after radiation when most of the apoptosis occurred. An early decrease in PME/Pi was observed at 4 hr after radiation dose at 30 Gy. For each dose, the values of β-ATP/Pi 24 hr after radiation were inversely related to radiation dose.Conclusion: The increase in β-ATP/Pi observed by ³¹P-MRS was linked to the degree of histological recovery from radiation-induced apoptosis.     

Early radiation effects in highly apoptotic murine lymphoma xenografts monitored by 31P magnetic resonance spectroscopy

Purpose: Phosphorus-31 magnetic resonance spectra (³¹P-MRS) were obtained from highly apoptotic murine lymphoma xenografts before and up to 24 hr following graded doses of radiation ranging from 2 to 30 Gy. Radiation-induced apoptosis was also estimated up to 24 hr by scoring apoptotic cells in tumor tissue.Methods and Materials: Highly apoptotic murine lymphoma cells, EL4, were subcutaneously transplanted into C57/BL mice. At 7 days after transplantation, radiation was given to the tumor with a single dose at 3, 10, and 30 Gy. The β-ATP/Pi, PME/Pi, and β-ATP/PME values were calculated from the peak area of each spectrum. Radiation-induced apoptosis was scored with counting apoptotic cells on hematoxylin and eosin stained specimens (%apoptosis).Results: The values of % apoptosis 4, 8, and 24 hr after radiation were 21.8, 19.6, and 4.6% at 3 Gy, 35.1, 25.6, and 14.8% at 10 Gy, 38.4, 38.0, and 30.6% at 30 Gy, respectively (cf. 4.4% in control). There was no correlation between early change in β-ATP/Pi and % apoptosis at 4 hr after radiation when most of the apoptosis occurred. An early decrease in PME/Pi was observed at 4 hr after radiation dose at 30 Gy. For each dose, the values of β-ATP/Pi 24 hr after radiation were inversely related to radiation dose.Conclusion: The increase in β-ATP/Pi observed by ³¹P-MRS was linked to the degree of histological recovery from radiation-induced apoptosis.     

Effects of 5-fluorouracil,leucovorin, and glucarate in rat colon-tumor explants

Summary In a previous study, we showed that 5-fluorouracil (FU) is active against the dimethylhydrazine-induced colon tumor in rats; a 7-day infusion of FU at 30 mg/kg daily produced 85% tumor-free cures. The present study examined the effects of FU alone and in combination with leucovorin (LV) ord-glucarate (GT) using an ex vivo system that maintained the growth of the rat colon-tumor explants on collagen gels. The labeling index (LI) was determined by the incorporation of [³H]-thymidine and autoradiography. The mean LI of the untreated control was 64.8%±19.8%. The IC₅₀, IC₉₀, and IC₉₅ values following a 7-day exposure to FU were 0.36, 0.75, and 1.22 m, respectively. In comparison, the steady-state FU concentration required to produce 67% tumor-free cures in rats following a 7-day infusion is 1.54 m. LV alone did not produce any antiproliferative effect at concentrations as high as 10 m. The addition of LV at concentrations of 0.001–10 m did not significantly reduce the IC₅₀ of FU. The lack of effect of LV may have been due to tissue saturation with folate provided in the culture medium. GT alone reduced the tumor LI by 20%–30% at concentrations of 0.1–10 m. GT enhanced the effect of FU. As compared with FU alone, the addition of GT at concentrations of 0.1 and 1.0 m reduced the IC₅₀ of FU by 47% and 60% to 0.21 and 0.16 m, respectively. Assessment of the potentiation of the inhibitory effect of FU by GT using two-way analysis of variance and the isobologram method indicated a significant synergistic interaction between FU and GT. This interaction occurred within the FU concentration range of 0.08 and 0.4 m. In summary, these data indicate that (a) the IC values for FU are comparable in tumor explants and in rats, suggesting that the effects in cultured tumors reflect those in intact animals; (b) GT alone showed antitumor activity, albeit relatively minor as compared with FU; (c) FU and GT exhibited synergistic activity, which was most pronounced at FU concentrations that produced submaximal activity (<30% inhibition of tumor LI); and (d) GT and LV had different effects on the growth inhibition by FU, suggesting that GT acts by a mechanism different from the thymidylate synthase-directed effect of FU and LV.Abbreviations FU 5-Fluorouracil - LV leucovorin - GT d-glucarate - LI labeling index - IC inhibitory concentration - IdR thymidine Supported in part by research grants RO1 CA-43365 and RO1 CA-51757 and by Research Career Development Award K04 CA-01 497 (to J. L.-S. A.) from the National Cancer Institute, NIH, DHHS. The work of the senior author (T. D. S.) was supported in part by a fellowship from the Berlex Corporation     

In vivo 31P-NMR spectroscopy of murine tumours before and after localized hyperthermia

In vivo 31P-NMR spectroscopy was used to monitor the energy metabolism, apparent intracellular pH (pHNMR), and phospholipid turnover in subcutaneous fibrosarcomas (FSall) and mammary carcinomas (MCaIV) treated with hyperthermia (HT). Treatment consisted of elevation of tumour temperature to 43.5 degrees C for 15, 30 or 60 min (FSall) and 30 min (MCaIV). Experiments were performed on conscious mice with biologically relevant tumour sizes. Using water bath immersion, this study focused on acute heat-induced metabolic changes (up to 7 h post-HT). 31P-NMR spectra of both murine tumours were characterized by relatively high pretreatment levels of PME, Pi and NTP, and lower levels of PDE, PCr and DPDE. Following hyperthermia, NTP and PCr levels, as well as pHNMR, decreased in both tumour lines. This drop was accompanied by a prompt and dramatic increase in Pi. After heating for 15 min, the limited spectral changes observed for the high-energy phosphates and the marginal decline in pHNMR were nullified within 7 h, whereas Pi remained significantly elevated. With the exception of PME/NTP and PME/PDE, all relevant metabolic ratios (PCr/Pi, NTP/Pi, PME/Pi) decreased after heating and did not resolve thereafter. Upon longer heat exposure times the high-energy phosphates, pHNMR, NTP/Pi, PCr/Pi, and PME/Pi all decreased in a dose-dependent manner and remained at the respective lower levels. The PME/PDE ratio was increased after 43.5 degrees C/15 min whereas at longer heating times this ratio did not change. At comparable heat doses MCaIV tumours seem to exhibit changes similar to FSall tumours.     

5-Fluorouracil and folinic acid with or without CPT-11 in advanced colorectal cancer patients: A multicenter randomised phase II study of the Southern Italy Oncology Group

Purpose:The combination regimen CPT-11 plus bolus and infusion5-fluorouracil (5-FU) with high-dose leucovorin (hybrid regimen LV5FU2) hasbeen tested for activity and toxicity against advanced colorectal carcinomain a randomised, multicenter phase II trial. Patients and methods:A total of 102 chemotherapy-naïvepatients were randomised in a 1 : 2 fashion to receive: leucovorin 100mg/m² administered as a two-hour infusion before 5-FU 400mg/m² as an intravenous bolus, and FU 600 mg/m² as a22-hour infusion immediately after 5-FU bolus injection repeated on days 1 and2 (LV5FU2 regimen, arm A, 34 patients) or CPT-11 at 180 mg/m² (150mg/m² for patients of age 70 and <75 years) only on day 1immediately before LV5FU2 therapy (LV5FU2 + CPT-11 regimen, arm B 68patients). Both treatments were repeated every two weeks. The presence of acalibration arm assured consistency and more realistic evaluation of resultsachieved with the LV5FU2 + CTP-11 regimen. Results:Thirty-three and sixty-four patients were evaluable inarm A and B, respectively. The overall response rate was 18% in arm A(95% CI: 7%–34%) and 40% in arm B(95% CI: 28%–52%). Median time to progression,median duration of response and survival were similar in both groups.Responders (CR + PR) survived statistically longer than non-responders onlyin arm B (20 vs. 10 months, P = 0.0016). All patients were evaluablefor toxicity which was mild in both groups; gastrointestinal disturbances werethe most common. There were no treatment-related deaths. Grade 3–4toxicity was uncommon in both arms. Conclusions:The addition of CPT-11 to the hybrid LV5FU2 regimenprovided a significant overall response rate (40%) with relatively mildtoxicity. The overall response rate was 18% in patients treated withLV5FU2 alone in the calibration arm. Thus, considering other encouraging datafrom the literature, the CPT-11 + FU–LV combination therapy can beregarded as a new, very effective treatment option for first-line treatmentof advanced colorectal cancer patients.     

Effects of nicotinamide and carbogen on tumour oxygenation, blood flow, energetics and blood glucose levels

Both host carbogen (95% oxygen/5% carbon dioxide) breathing and nicotinamide administration enhance tumour radiotherapeutic response and are being re-evaluated in the clinic. Non-invasive magnetic resonance imaging (MRI) and 31P magnetic resonance spectroscopy (MRS) methods have been used to give information on the effects of nicotinamide alone and in combination with host carbogen breathing on transplanted rat GH3 prolactinomas. Gradient recalled echo (GRE) MRI, sensitive to blood oxygenation changes, and spin echo (SE) MRI, sensitive to perfusion/flow, showed large signal intensity increases with carbogen breathing. Nicotinamide, thought to act by suppressing the transient closure of small blood vessels that cause intermittent tumour hypoxia, induced a small increase in blood oxygenation but no detectable change in perfusion/flow. Carbogen combined with nicotinamide was no more effective than carbogen alone. Both carbogen and nicotinamide caused significant increases in the nucleoside triphosphate/inorganic phosphate (betaNTP/Pi) ratio, implying that the tumour cells normally receive sub-optimal substrate supply, and is consistent with either increased glycolysis and/or a switch to more oxidative metabolism. The most striking observation was the marked increase in blood glucose (twofold) induced by both nicotinamide and carbogen. Whether this may play a role in tumour radiosensitivity has yet to be determined.     

Antibiotic C3368-A,a fungus-derived nucleoside transport inhibitor,potentiates the activity of antitumor drugs

Antibiotic C3368-A (CA) is produced by a fungus strain from a soil sample collected in Antarctica. CA markedly inhibited radiolabeled thymidine and uridine transport in mouse Ehrlich carcinoma cells, its 50% inhibitory concentration (IC₅₀) being 4.6 and 7.7 M, respectively. In clonogenic assay, CA displayed a synergistic effect with methotrexate (MTX), mitomycin C (MMC), 5-fluorouracil (5FU), and Adriamycin (ADR) against human oral epidermoid carcinoma KB cells. CA also markedly enhanced the inhibitory effect of 5FU and ADR on the proliferation of human hepatoma BEL-7402 cells as determined by thep-nitrophenyl-N-acetyl--d-glucosaminide (NAG) enzyme-reaction assay. 5FU or ADR cytotoxicity was not augmented by CA in human fetal lung 2BS cells. In vivo, CA significantly potentiated the inhibitory effect of MMC against colon carcinoma 26 in mice. No significant augmentation of toxicity by the combination was found in treated mice. The results suggest that CA, the newly found nucleoside-tranport inhibitor, may be useful in potentiation of the effect of antitumor drugs.