Darrell Q. Brown

Polarographic needle electrode measurements of oxygen in rat prostate carcinomas : accuracy and reproducibility

PURPOSE The oxygenation status of tumors may be important for predicting tumor response to therapy. Previous studies with the anaplastic (R3327-AT) and well-differentiated (R3327-H) Dunning rat prostate tumors using indirect assays of tumor oxygenation indicated the relative hypoxic and radioresistant nature of the anaplastic tumor. We now report direct measurements of oxygen in these tumors made with the pO2 histograph to determine: (a) whether a significant difference in oxygenation status could be detected between them: (b) whether sequential measurements on the same tumor gave similar values; and (c) whether tumor oxygenation correlated with tumor volume. METHODS AND MATERIALS R3327-AT and R3327-H tumors were grown in Fischer X Copenhagen rat to volumes of 1.0-7.0 cm3. Electrode measurements (100-200) were made in tumors in anesthetized animals along two parallel tracks. Repeat measurements were made at 1-5 days along different parallel tracks. Oxygen partial pressures of muscle tissue were measured and served as a normal tissue control. Statistical analyses were applied to determine whether tumor oxygen levels were different between the two tumor histologies, whether sequential measurements in the same tumor were reproducible, and whether tumor oxygenation correlated with tumor volume. RESULTS The average median pO2 of the well-differentiated (n = 15) and the anaplastic (n = 15) tumors was 6.0 mmHg (SE +/- 1.3) and 2.2 mmHg (SE +/- 0.3), respectively. The average median pO2 of normal rat muscle (n = 15) was 23.6 mmHg (SE +/- 2.0). These values represent highly significant differences in oxygen concentration between the two tumors and rat muscle. The differences in average mean pO2 values were also highly significant. Repeat measurements in the same tumors on different days gave average median values of 4.7 and 2.2 mmHg in the R3327-H (n = 15) and R3327-AT (n = 15) tumors, respectively. For these repeat measurements, median pO2 values decreased in 15 and increased in 15 tumors, and were not significantly different from the first measurements. The average differences observed in median pO2 were 37% (SE +/- 7) and 58% (SE +/- 10) for the R3327-H and R3327-AT tumors, respectively. No significant correlation was observed between pO2 levels and the tumor volumes investigated in this study. CONCLUSIONS The median pO2 values of the anaplastic Dunning tumors were significantly lower than those of the well-differentiated tumors (p < 0.001). Oxygen levels in both tumors were significantly lower than those measured in normal rat muscle (p < 0.00005). Repeat measurements of median pO2 in the same tumors were not significantly different for either tumor model (p > 0.5). The changes observed in pO2 distributions within individual tumors from day to day may indicate true dynamics of its oxygenation status and/or the limits of electrode measurements, by sampling along only two insertion sites. The electrode measurements of pO2 in these tumor models are reproducible and confirm previously detected oxygenation differences between the anaplastic and well-differentiated tumors.

Early results of the screening program for radioprotectors

Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioc acid, is the most widely studied and most effective radioprotective drug at present, it is nevertheless clear from animal studies that it has important shortcomings as the ideal radioprotector in clinical radiotherapy. More effective and less toxic radioprotective drugs are needed. For this reason, a chemical radioprotector screening program has been initiated at the Fox Chase Cancer Center under a contract with the National Cancer Institute. Most of the 20 compounds that have now entered the screening program provide good protection of the mouse hematopoietic system as indicated by 30 day survival following the radiation LD100/30. Administration of a radioprotector dose equal to one half of the maximum tolerated dose (MTD/2) gave hematopoietic dose reduction factors (DRFs) as high as 2.3. No radioprotector appeared to be superior to WR-2721, although four others gave DRFs exceeding 1.8.

Hydrolysis of WR2721 by mouse liver cell fractions

A study of the dephosphorylation of WR2721 by broken cell preparations of mouse liver revealed the presence of at least two distinctive activities. One activity was inactivated by heat treatment and was present in the nuclear and microsomal fractions. It had an optimum pH at 9 and was inhibited by sodium vanadate, EDTA, and phenylalanine. Further subcellular fractionation demonstrated the localization of this activity in plasma membrane. A second WR2721 hydrolysis activity was detected in the cytosol fraction (postmicrosomal supernatant), which changed little with pH over the range of 5 to 10; sodium vanadate did not inhibit it. The cytosolic activity in response to heat treatment was complicated since there was an initial decrease followed by an increase in catalytic activity as a function of time at 55 degrees C. Enzyme kinetic analysis of the plasma membrane-associated activity in the microsomal fraction was performed, and Km and Vmax values of 12.5 and 69.9 nmol/min/mg protein, respectively, were obtained.

Technique, pharmacokinetics, toxicity, and efficacy of intratumoral etanidazole and radiotherapy for treatment spontaneous feline oral squamous cell carcinoma

The histologic appearance, locoregional recurrence, and rate/site of metastases of spontaneous feline oral squamous cell carcinoma are similar to head and neck cancer in humans. A feasibility study of intratumoral Etanidazole, a hypoxic cell sensitizer, and radiation therapy were instituted in this model. Eleven cats with feline squamous cell carcinoma were treated with intratumoral Etanidazole and radiation therapy. Total Etanidazole doses were 1.5-24.0 gms/m2 (0.5-6.9 gms). The tumor partial response rate was 100% (11/11); the median volume regression was 70%. All cats have died as a result of tumor recurrence or tumor-related complications. Median survival was 116 days. Ten cats have been autopsied. Non-necrotic and necrotic tumor cells were identified at the treatment site in all cats. Pharmacokinetic studies were performed in six cats. Following intravenous infusion, the plasma elimination of the Etanidazole was biexponential. The systemic availability following intratumoral administration was 61.2 +/- 21.1%. Peak plasma Etanidazole levels were observed 14 minutes following intratumoral injection, after which elimination was biexponential. Thirty minutes following intratumoral Etanidazole administration, tumor Etanidazole levels were 62.8% of plasma levels. Feline squamous cell carcinoma appears to be a useful model of human head and neck cancer. Cats tolerate substantial doses of intratumoral and intravenous Etanidazole. Etanidazole and radiation therapy cause rapid regression, but not cure, of feline squamous cell carcinoma. There is a similarity between the intravenous kinetics of Etanidazole in humans and cats. Further studies in this model are planned.

Differential radioprotection of normal tissues by hydrophilic chemical protectors

Analogous to certain radiosensitizers which are too hydrophilic to enter tumor cells, certain radioprotectors, because of their hydrophilicity, may also be hindered from entering tumor cells and thus protect only normal tissues. In testing this hypothesis, we utilized thin layer chromatography as convenient means to measure radioprotector hydrophilicity. Dose reduction factors (DRFs) for hematopoietic radioprotection were determined in BALB/c mice given half maximum tolerated doses (MTD/2) of 11 radioprotectors 30 min prior to graded doses of gamma rays. DRFs for tumor protection were determined in MCa-11 tumor-bearing mice using a regrowth delay assay. Differential radioprotection was found to be significantly correlated (r = 0.86) with hydrophilicity. Thus, radioprotector hydrophilicity appears to be a significant factor in the differential radioprotection observed and should be useful in designing or selecting better differential radioprotectors.

Modification of WR-2721 toxicity and radioprotection by an inhibitor of alkaline phosphatase

We used levamisole, an inhibitor of alkaline phosphatase, to study the role of that enzyme in mediating the metabolic activation, toxicity, and radioprotection of WR-2721 in intact mice. We found the toxicity of WR-2721 was slightly decreased by prior subcutaneous (SQ) injection of 40 mg/kg of levamisole. In studying the effect of levamisole on WR-2721 radioprotection, we found that intraperitoneal (i.p.) injection of levamisole had little or no effect on radioprotection of the gastrointestinal and the hematopoietic systems. Even this small reduction of protection was due in part to the toxicity of levamisole as demonstrated when levamisole was injected following, rather than before, WR-2721-radiation treatment. To determine whether levamisole inhibited the activation (i.e., dephosphorylation) of WR-2721 to WR-1065, we assayed WR-1065 in the jejunum using an HPLC electrochemical assay. SQ injection of 75 mg/kg levamisole 10 min prior to WR-2721 reduced the WR-1065 observed 10 min after WR-2721 administration by 37%. In conclusion, levamisole appears to be too toxic and non-specific to be useful in studying and regulating the metabolism, toxicity and radioprotection of WR-2721.

μ-, but not δ-, opioid receptor-mediated antinociception in mice is attenuated by γ-irradiation

Abstract Mice were exposed to whole-body irradiation (500 rads) from a 137 Cs γ-source and tested 2 h later for antinociception (tail-flick test) produced by intracerebroventricular administration of morphine or the more δ-selective opioid peptide, [ d -Pen 2 , l -Pen 5 ]enkephalin (DPLPE). Irradiation significantly attenuated the antinociception produced by morphine, but not by DPLPE. These results demonstrate a differential sensitivity of μ- and δ-opioid receptors to γ-irradiation and, in addition, may be of clinical relevance for cancer patients receiving concurrent radiation therapy and opioid analgesics.

Exploitation of interstitial brachytherapy techniques for photodynamic therapy--I. Treatment planning for interstitial photoexcitation therapy: a photodosimetry treatment planning system

A photodosimetry computer program, T-PIPET, was developed to rapidly compute maps of relative light intensity within tumors illuminated by implanted optical fibers. Light attenuation was measured along radial tracks from laterally-diffusing fibers implanted into both the R3327-AT and R3327-H Dunning rat prostate carcinomas and input to the computer program. The calculations assumed (1) uniform optical property of tissue through the tumor, (2) uniform and equal illuminance from the length of optical diffuser and between fibers and (3) precise needle implantation. Values of relative light intensity were computed, color-coded and imaged for a 5 X 5 cm tumor cross section (a 150 X 150 pixel array). Illuminators consisting of seven laterally-diffusing fibers implanted as six adjacent equilateral triangles were tested. The uniformity of light fields within encompassed tumor volumes was determined as a function of fiber spacing. Measures of light intensity along specific tracks within tumors agreed well (+/- 10%) with values of relative light intensity predicted by T-PIPET. The rapid falloff of light dose beyond the illuminator will assist in minimizing normal tissue damage outside the tumor volume. To cure slid tumors, a specific light dose must be delivered throughout the tumor volume and recurrence might be expected from tumor zones which are underdosed. A 9-fiber illuminator has been constructed to deliver light more uniformly to the tumor periphery. These illuminators have been tested with R3327-AT tumors illuminated with 673 nm light after i.v. administration of a pheophorbide-base photosensitizer.