A complication probability planning study to predict the safety of a new protocol for intracranial tumour radiotherapy in dogs.

Technical advances make it possible to deliver radiation therapy for canine intracranial tumours in fewer fractions, under the assumption of equivalent tumour control. With the aim of estimating the late toxicity risk profile for various tumour sizes and locations, the present paper evaluates the normal tissue complication probability (NTCP) values for the intracranial organs at risk. By making isoeffect calculations, a new 10-fraction radiation protocol was developed with the same tumour control probability (TCP) as a currently used 20-fraction standard protocol, and complication risk profiles for brain, brainstem and optic chiasm were modelled using a representative population of 64 dogs with brain tumours. For >59% of cases, the new 10-fraction protocol yielded an acceptable, low risk estimate of late toxicity (<10%). Our calculations suggest that it may be safe to treat small to intermediate-sized tumours that are neither located near the optic chiasm nor at the brainstem with 10 daily fractions of 4.35 Gy.

Measurements of the neutron dose equivalent for various radiation qualities, treatment machines and delivery techniques in radiation therapy.

In radiation therapy, high energy photon and proton beams cause the production of secondary neutrons. This leads to an unwanted dose contribution, which can be considerable for tissues outside of the target volume regarding the long term health of cancer patients. Due to the high biological effectiveness of neutrons in regards to cancer induction, small neutron doses can be important. This study quantified the neutron doses for different radiation therapy modalities. Most of the reports in the literature used neutron dose measurements free in air or on the surface of phantoms to estimate the amount of neutron dose to the patient. In this study, dose measurements were performed in terms of neutron dose equivalent inside an anthropomorphic phantom. The neutron dose equivalent was determined using track etch detectors as a function of the distance to the isocenter, as well as for radiation sensitive organs. The dose distributions were compared with respect to treatment techniques (3D-conformal, volumetric modulated arc therapy and intensity-modulated radiation therapy for photons; spot scanning and passive scattering for protons), therapy machines (Varian, Elekta and Siemens linear accelerators) and radiation quality (photons and protons). The neutron dose equivalent varied between 0.002 and 3 mSv per treatment gray over all measurements. Only small differences were found when comparing treatment techniques, but substantial differences were observed between the linear accelerator models. The neutron dose equivalent for proton therapy was higher than for photons in general and in particular for double-scattered protons. The overall neutron dose equivalent measured in this study was an order of magnitude lower than the stray dose of a treatment using 6 MV photons, suggesting that the contribution of the secondary neutron dose equivalent to the integral dose of a radiotherapy patient is small.

Raman spectroscopy of tissue samples irradiated by protons.

PURPOSE: Since the number of cancer patients treated by proton irradiation has increased in the last few years, it seems appropriate to study dose-dependent effects of proton irradiation on mammalian tissues in more detail. MATERIALS AND METHODS: Tissue samples of normal skin of mouse and swine, of a human tumour model xenograph, and of normal skin and a skin tumour (basal cell carcinoma) of a human patient of about 1 mm thickness were irradiated by 24 MeV protons (uniform delivered doses of 1, 7 and 50 Gy: skin of mouse and a human tumour model xenograph, and 0.5, 5 and 50 Gy: swine and human skin). Raman spectra of non-irradiated and irradiated samples were recorded and analysed. RESULTS: Amide I, P=O and C-O bond vibrations and aromatics were sensitive to the proton irradiation dose. In the C-H stretching region, the irradiation-mediated change of Raman spectra was significant only in the case of the skin tumour. CONCLUSIONS: It has been shown that Raman spectroscopy is suited to assess the radiation damage done to biological samples by protons. Proteins of the human skin tumour seem to be more sensitive to proton irradiation than proteins of normal human skin.

The effect of proton-irradiation on the Raman spectroscopy of tissue samples.

Tumor and healthy tissue samples were irradiated by 24 MeV protons. The samples were exposed to doses from 0 to 50 Gy and subsequently examined by Raman spectroscopy. The analysis of the intensity of characteristic peaks as a function of radiation dose exhibits different trends for the two types of tissue.

[Evaluation of the electron density phantom CIRS Model 62]. / Evaluation des Elektronendichte-Phantoms CIRS Model 62.

The properties of the electron density phantom CIRS M62 were studied. Physical and electron densities were calculated for all tissue substitutes, and compared to the values given in the data sheet and to the recommendations of the ICRU Report 44. The phantom was scanned in standard-CT and spiral-CT mode with different acquisition parameters. Especially manufactured tissue substitutes for dense bone were analysed for artifacts. Hounsfield values of all tissues substitutes were determined with the treatment planning system CadPlan. Calibration curves were calculated and compared to a stoichiometric calibration for real tissue. There was good agreement between the values calculated and the values given by the manufacturer. The tissue substitutes were only approximately tissue equivalent. The tissue substitutes for dense bone showed small artifacts. The calculated calibration curves were in good agreement with the stoichiometric calibration curve.

On small angle multiple coulomb scattering of protons in the gaussian approximation.

Experimental data from the literature on small-angle multiple Coulomb scattering of protons in various materials were analysed in order to device an equation for the scattering angle in the Gaussian approximation. In comparison to Highland's well-known formula, the present approximation can be integrated to take into account energy loss in the scattering media. In addition, it is more precise than Highland's formulation for thin and thick scatterers consisting of elements with low atomic number. The simple equation obtained in this study can be used to obtain prompt answers for scattering problems which can occur, for example, in proton therapy or proton radiography.

Dosimetry of low-energy protons and light ions.

For the vertical beam facility at the 14 MV Munich tandem accelerator, various techniques for dosimetry were tested for radiation fields of low-energy protons and light ions (4He, 12C and 16O). A reference dose was determined from the fluence of particles by counting individual particles. A parallel-plate Markus chamber with a small sensitive air volume was used for beam dosimetry applying the ICRU protocol. The doses measured with the ionization chamber were compared with doses evaluated from the fluence measurements. Alternative dose measurements were performed using MTS-N LiF:Mg, Ti thermoluminescence detectors (TLDs) and a photometrically evaluated Fricke chemical dosimeter. An uncertainty of 8% was found in the determination of the dose relative to the reference method. Effects of an inhomogeneous energy loss and a finite track length of the projectiles in the sensitive detector volume of the dosimeters had to be taken into account.

CT based lung density correction verification with in vivo dosimetry using diodes.

In vivo dose measurements with diodes are easy to perform. The first aim of our study was to show whether diode measurements of the patient exit doses are precise enough for verifying inhomogeneity corrections used for treatment planning. The second aim was to assess the precision of the modified Batho Law inhomogeneity correction of the CadPlan treatment planning system. For this purpose, entrance and lait doses were measured in the thoracic region of 115 patients. Diode measurements were sufficiently precise to verify the density corrections predicted by the treatment planning system (< 0.5% of ICRU dose). The measured doses were compared with calculations of the CadPlan treatment planning system. The mean deviation of the exit dose calculations within the measurements error was zero. The present results show that measurements of exit dose even in a small number of patients are sufficient to identify systematic errors in the dose calculation.

Influence of respiration-induced organ motion on dose distributions in treatments using enhanced dynamic wedges.

The mean velocity of respiration-induced organ motion in cranio-caudal direction is of the same magnitude as the velocity of the moving jaw during a treatment with an enhanced dynamic wedge. Therefore, if organ motion is present during collimator movement, the resulting dose distribution in wedge direction may differ from that obtained for the static case, i.e., without organ motion. The position as a function of time of the moving jaw has been derived from a log-file generated during each treatment. Parameters for the respiratory cycle and information about respiration-induced motion for organs in the upper abdomen were taken from the literature. Both movements were superimposed and the resulting monitor unit distribution has been calculated in the intrinsic coordinate system of the organ. The deviations from the static case have been studied as a function of wedge angle, amplitude of organ motion, respiratory rate, asymmetry of the respiratory cycle, beam energy, and the dose rate. If an amplitude of 30 mm and a respiratory rate of 10 min(-1) are assumed, the maximum deviation in monitor units is 2.5% for a 10 degees wedge, 7% for a 30 degrees wedge, and 16% for a 60 degrees wedge. Furthermore, a dose distribution for an organ undergoing respiration-induced motion has been generated and we found dose deviations of the same magnitude as calculated with the monitor unit distribution.

Dosimetry of low-energy protons on the vertical-beam facility at the Munich accelerator.

Various methods of dosimetry for protons with energies up to 25 MeV were compared for radiation fields as might be used in the skin treatment of patients or in biological experiments. The methods used were: Fluence of individually registered protons; Charge deposition in commercially available ionisation chambers; Thermoluminescence detectors, exposed in Munich and evaluated in Krakow; Photometricallly evaluated commercial Fricke dosimeter. An overall agreement within 5% was found between the absolute dose measurements. Depth-dose distributions for Bragg curves measured by a Markus chamber with a depth resolution of 10 micrometers agreed with calculations.

Atorvastatin does not alter the anticoagulant activity of warfarin.

Twelve patients chronically maintained on warfarin were administered 80 mg atorvastatin for 2 weeks. Mean prothrombin times decreased slightly, but only for the first few days of the two-week treatment period. Thus atorvastatin had no consistent effect on the anticoagulant activity of warfarin and adjustment in warfarin dosing should not be necessary.

Characterization of trimetrexate transport in human lymphoblastoid cells and development of impaired influx as a mechanism of resistance to lipophilic antifolates.

The purpose of this study was to characterize the transport properties of trimetrexate in WI-L2 human lymphoblastoid cells and determine the mode of resistance that had developed in a subline, WI-L2/TMQ, that was grown in increasing concentrations of trimetrexate. WI-L2/TMQ cells were 62-fold resistant to trimetrexate and 68- and 96-fold cross-resistant to the other lipophilic antifolates metoprine and piritrexim (BW 301U). No cross-resistance was observed with vincristine or doxorubicin, and sensitivity was not increased with 5 micrograms/ml of verapamil, indicating that it was not a typical multidrug resistance phenotype. WI-L2/TMQ exhibited a 2-fold increase in dihydrofolate reductase; however, this did not contribute significantly to the observed resistance, since these cells retained full sensitivity to methotrexate. Nor were there any kinetic alterations in dihydrofolate reductase toward trimetrexate or differences in the levels of thymidylate synthase. The major difference between the sensitive and resistant cell line was a 50% decrease in the influx rate of WI-L2/TMQ cells which produced a corresponding decrease in cellular trimetrexate at the steady state. No difference in efflux rates was detected nor were there any differences in intracellular water or metabolism of trimetrexate. Additional characterization of trimetrexate transport in WI-L2 showed that influx was nonsaturable up to 5 mM extracellular trimetrexate, relatively insensitive to sodium azide, and exhibited a Q10 of 2.7. Influx was, however, inhibited in a dose-dependent manner by concentrations of p-chloromercuribenzylsulfonate above 10 microM. Efflux studies revealed a large nonexchangeable fraction of trimetrexate that was well above the dihydrofolate reductase binding capacity and varied depending on the initial level of cell-associated drug. The intracellular exchangeable trimetrexate concentration at the steady state was always several-fold higher than the extracellular concentration. Retention of trimetrexate appeared to be coupled to some component of energy metabolism, since the presence of sodium azide stimulated this process by 2- to 3-fold. The data suggest that trimetrexate enters cells by passive diffusion but then is distributed and concentrated within the cell through more complex mechanisms which may involve energy coupling, compartmentation, or binding to macromolecules or organelles, although some type of carrier-mediated process cannot be ruled out.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of type II topoisomerase by fostriecin.

Fostriecin is a new antitumor antibiotic which is being developed further as an anticancer agent based on its marked activity in murine leukemias. Its mechanism of action, however, has thus far remained unknown. The present study demonstrates that fostriecin inhibits the catalytic activity of partially purified type II topoisomerase from Ehrlich ascites carcinoma. Under the experimental conditions employed, fostriecin completely inhibited the enzyme at 100 microM. A general kinetic analysis showed that fostriecin inhibited topoisomerase in an uncompetitive manner with a Ki,app of 110 microM and produced kinetics that were distinctly different from those of VM-26 which exhibited noncompetitive inhibition. Fostriecin did not cause DNA strand breaks in L1210 cells, suggesting that it did not stabilize a cleavable complex as do other known inhibitors of this enzyme. Fostriecin, however, did partially inhibit DNA strand breaks produced by amsacrine. An analysis by flow cytometry showed that L1210 cells exposed to 5 microM fostriecin for 12 hr caused a block in the G2 phase of the cell cycle. These studies thus suggest that the mechanism by which fostriecin produces its antitumor effects may be through inhibition of topoisomerase II and that the type of inhibition is markedly different from existing antitumor agents which inhibit this enzyme.

Biochemical pharmacology and DNA-drug interactions by Cl-958, a new antitumor intercalator derived from a series of substituted 2H-[1]benzothiopyrano[4,3,2-cd]indazoles.

Cl-958, PD 121373, and PD 114595 belong to a new class of DNA complexers, substituted 2H-[1]benzothiopyrano[4,3,2-cd] indazoles, and are being further developed as antitumor drugs based on their curative properties against murine solid tumor models. The biochemical effects of these drugs on L1210 leukemia cells and their interaction with DNA were studied and compared to clinically used intercalators. The benzothiopyranoindazoles bound to DNA with a relatively high affinity, having intrinsic association constants of between 3 and 4 x 10(5) M-1. Based on viscosity measurements, the mode of DNA binding appears to be through intercalation. Unwinding angles were calculated to be approximately 18 degrees. The benzothiopyranoindazoles were potent inhibitors of nucleic acid synthesis, reducing both DNA and RNA synthesis to the same extent at similar concentrations. Like other known intercalators, these compounds produced DNA single- and double-strand breaks in a time- and concentration-dependent manner in L1210 cells. Between one and two DNA strand breaks were formed per protein-strand crosslink. Repair of these DNA lesions after the drug was removed from the cells was either very slow or did not occur at all for at least 2 hr. Finally, since the high incidence of cardiotoxicity associated with the administration of anthracyclines has been related to the formation of reactive oxygen species, the ability of the benzothiopyranoindazoles to augment superoxide dismutase-sensitive oxygen consumption was observed in a rat liver microsomal system. These compounds produced less than 5% of the activity in this assay that doxorubicin produced.

Biochemical pharmacology and experimental chemotherapy studies with guanine-7-oxide, a novel purine antibiotic.

1. Guanine-7-oxide is a novel purine antibiotic produced by a Streptomyces species, ATCC 39364. 2. Guanine-7-oxide is cytotoxic to murine and human leukemia cells in vitro at sub-micromolar concentrations. Murine and human carcinoma cells are much less sensitive. 3. Guanine-7-oxide has significant in vivo antitumor activity, particularly against the intraperitoneal and subcutaneous L1210 leukemia systems. 4. Guanine-7-oxide, at highly cytotoxic concentrations, has little effect on biosynthesis of RNA and DNA. 5. There is preliminary evidence for an early effect of guanine-7-oxide on cellular protein synthesis. 6. Guanine, guanosine and hypoxanthine protect cells from the cytotoxicity of guanine-7-oxide. 7. Activation of guanine-7-oxide requires the presence of the enzyme hypoxanthine-guanine phosphoribosyltransferase in the target cells. 8. Cytotoxic concentrations of guanine-7-oxide do not cause depletion of cellular guanine nucleotides during a two hr incubation period. 9. Guanine-7-oxide is converted within mouse and human cells to a metabolite with chromatographic mobility corresponding to a ribonucleoside 5'-triphosphate.

Cross resistance of pleiotropically drug resistant P338 leukemia cells to the lipophilic antifolates trimetrexate and BW 301U.

Several antifolate compounds were examined for their cytotoxic activity in a pleiotropically resistant P388 cell line (P388R). The sensitivity of P388R cells to methotrexate (MTX) and the lipophilic antifols, metoprine and methotrexate gamma-mono t-butyl ester (MTX-gamma-t-butyl ester) were comparable with that activity observed in the parental cell line (P388S). P388R cells were, however, resistant to 2 other lipophilic antifols, trimetrexate (TMQ) and BW 301U. The degree of resistance to TMQ and BW 301U was 22-fold and 15-fold, respectively and could be partially overcome by the calcium channel blocker, verapamil (VER) or the detergent Tween 80. Transport studies showed that net accumulation of trimetrexate was markedly reduced in P388R cells resulting in a steady-state level which was 25% of the sensitive line. This impaired uptake was reversed by 5 micrograms/ml VER which increased the steady-state to a level comparable to P388S. P388R also exhibited a 50% reduction in the unindirectional influx rate, however, this defect could not be reversed by VER. The resistance of P388R cells to TMQ and BW 301U and their potentiation by VER extends pleiotropic resistance to yet another class of drugs which have important clinical implications.

In vitro DNA strand scission and inhibition of nucleic acid synthesis in L1210 leukemia cells by a new class of DNA complexers, the anthra[1,9-cd]pyrazol-6(2H)-ones (anthrapyrazoles).

CI-937 and CI-942 belong to a new class of DNA complexers, the anthra[1,9-cd]pyrazol-6(2H)-ones (anthrapyrazoles), and are being further developed as antitumor drugs based on their curative properties against murine solid tumour models. The biochemical effects of these agents were studied in L1210 leukemia in relation to other clinically used intercalators. After a 1-hr exposure, CI-937 and CI-942 reduced the cloning efficiency of L1210 cells by 50% at 3.0 X 10(-8) and 1.5 X 10(-7) M respectively. Based on an ethidium displacement assay, these drugs bound strongly to DNA, reducing the fluorescence of an ethidium-DNA complex by 50% at concentrations of 23 and 33 nM for CI-937 and CI-942 respectively. This was comparable to mitoxantrone at 15 nM, but much more potent than Amsacrine which required over 1.3 microM. A distinct property of the anthrapyrazoles was a much more potent inhibitory effect on whole cell DNA synthesis than on RNA synthesis. After L1210 cells were exposed to drug for 2 hr the concentration needed to inhibit DNA synthesis by 50% was 0.33 and 0.57 microM for CI-937 and CI-942, respectively, whereas 2.0 and 11.3 microM were required to inhibit RNA synthesis by the same extent. This was in contrast to Adriamycin and mitoxantrone which inhibited both activities equally at similar concentrations. It was apparent that the inhibition of these processes was not due to substrate depletion since intracellular ribonucleoside and deoxyribonucleoside triphosphates either remained constant or were elevated after a 2-hr exposure to 1 or 10 microM drug. A similar discriminatory effect was observed on DNA and RNA polymerase in permeabilized cells, and the inhibition of nucleic acid synthesis in this system could be reversed by exogenously added DNA. Since the high incidence of cardiotoxicity associated with the administration of anthracyclines has been related to the formation of reactive oxygen species, the ability of the anthrapyrazoles to augment superoxide dismutase sensitive oxygen consumption was observed in a rat liver microsomal system. CI-937 and CI-942 induced 5- and 10-fold less oxygen consumption than Adriamycin, producing rates of 12.4, 24.2 and 138.9 nmoles/min/mg microsomal protein, respectively, at a drug concentration of 0.5 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemical and antitumor activity of tiazofurin and its selenium analog (2-beta-D-ribofuranosyl-4-selenazolecarboxamide).

2-beta-D-Ribofuranosyl-4-selenazolecarboxamide (selenazofurin, CI-935), the selenium analog of tiazofurin (CI-909), was 3- to 10-fold more cytotoxic to murine or human tumor cells in vitro than tiazofurin and was also more active against P388 mouse leukemia in vivo. In vitro cytotoxicity could be reversed by guanosine or guanine but not by other purine nucleosides or bases. Three human tumor cell lines selected for selenazofurin or tiazofurin resistance showed cross resistance between selenazofurin and tiazofurin. Treatment with tiazofurin, selenazofurin, or mycophenolic acid decreased guanylate pools and caused an accumulation of IMP in WIL2 human lymphoma cells. The decrease in guanylate pools was accompanied by inhibition of RNA and DNA synthesis. The NAD analogs of tiazofurin and selenazofurin were inhibitors of L1210 IMP dehydrogenase (IMP:NAD oxidoreductase, EC 1.2.1.14), and both showed uncompetitive inhibition with respect to NAD having Kii values of 5.7 X 10(-8)M and 3.3 X 10(-8)M respectively.