Effects of the glucantime on the kinetic of biodistribution of radiopharmaceuticals in Wistar rats.

There are evidences that some drugs used for the human diseases can modify the biodistribution of radiopharmaceuticals. The N-methyl meglumine antimoniate, commercially known as glucantime (Rhodia, Brazil), is the elected drug for the treatment of all the clinical forms of leishmaniasis. As therapeutic drugs can present important toxic effects, we studied the effects of the glucantime on the kinetic of biodistribution of radiopharmaceuticals. To study the glucantime effect on the biodistribution of technetium-99m-methylenediphosphonic acid (99mTc-MDP), glucantime IM (80 mg/kg/day) was administered into male Wistar rats (3 months old age) in single dose during 7 days. 99mTc-MDP was injected 1 hr after the last dose. The animals (n = 24) were divided into two groups: treated (n = 12) and control (n = 12) and they were rapidly sacrificed, respectively, in 3 periods (5, 30 and 120 min) after administration of the 99mTc-MDP. The organs were isolated (brain, heart, thyroid, lungs, kidneys, testis, stomach, intestines, pancreas, spleen, liver, muscle, bone and bladder) and the percentages of radioactivity (%ATI) in each organ were calculated. The results were analyzed by the Wilcoxon test (p < 0.05). The analysis of the results has shown a significant increase of the %ATI after 5 min administration of the 99mTc-MDP in spleen, kidneys, testis, heart, liver and a reduction of %ATI in bladder. Thirty minutes after administration of the 99mTc-MDP, the analysis ofthe results reveals a significant reduction of the %ATI in femur, kidneys, thin bowel, lungs, heart, liver and an increase in abdominal muscle and stout bowel. One hundred-twenty min after administration of the 99mTc-MDP, the analysis of the results shows a significant reduction of the %ATI in spleen, thyroid, blood, femur, kidneys, liver and an increase in bladder, pancreas and lungs. Biochemical dosages were also performed before (control group, n = 12) and after (treated group, n = 12) treatment with glucantime. There was a significant (p < 0.05) decrease to the biochemical levels after the treatment with glucantime in following dosages: blood urea nitrogen, creatinine, alkaline phosphatase, lactic dehydrogenase, aspartate amino transferase, total creatine kinase, total protein, globulin and albumin. These results were compared with the control group, without glucantime, and statistical analyses were performed (t-student test, p < 0.05). These results could be associated with the biological effects and/or metabolization of the studied drug.     

Model to evaluate the toxic effect of drugs: vincristine effect in the mass of organs and in the distribution of radiopharmaceuticals in mice

There are evidences that the biodistribution of radiopharmaceuticals can be modified by some drugs. As chemotherapeutic drugs present important toxic effects, we studied the vincristine effect in the mass of organs and are trying to develop a model to evaluate the action of chemotherapeutic drug using the biodistribution of radiopharmaceuticals. Vincristine was administered (n=15) into female Balb/c mice, the organs isolated and their mass determined. To study the vincristine effect in the biodistribution of technetium-99m-dimercaptosuccinic acid (99mTc-DMSA) or technetium-99m-diethylenetriaminepentaacetic acid (99mTc-DTPA), vincristine (0.03 mg) was administered in the animals (n=15) in three doses. 99mTc-DMSA or 99mTc-DTPA was injected 1h after the last dose. After 0.5h, the animals were sacrificed and the percentage of radioactivity (%ATI) and the percentage of radioactivity per gram of tissue (%ATI/g) in each organ were calculated. The results have shown that the mass decreased significantly (Wilcoxon test, P<0.05) in thymus, spleen, ovary, uterus, kidneys, pancreas. The %ATI to 99mTc-DMSA increased in lung, pancreas, heart, thyroid, brain, and bone, and the %ATI/g increased in uterus, ovary, spleen, thymus, kidney, lung, liver, pancreas, heart, thyroid, brain and bone. To 99mTc-DTPA, the %ATI increased in uterus, ovary, spleen, thymus, kidney, lung, liver, stomach, heart and bone, and the %ATI/g increased in uterus, ovary, spleen, thymus, kidney, lung, liver, stomach, heart and bone. The results were statistically significant (Wilcoxon test). The results can be explained by the metabolization, therapeutic, toxicological or immunosupressive action of the vincristine. This model, probably, should be used to evaluate the toxic effect of various drugs.     

111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide analogues for melanoma imaging

The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized alpha-MSH peptide analogues, DOTA-CycMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]) and DOTA-GlyGlu-CycMSH (DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]), were synthesized and radiolabeled with (111)In. The internalization and efflux of (111)In-labeled CycMSH peptides were examined in B16/F1 melanoma cells. The melanoma targeting properties, pharmacokinetics, and SPECT/CT imaging of (111)In-labeled CycMSH peptides were determined in B16/F1 melanoma-bearing C57 mice. Both (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH exhibited fast internalization and extended retention in B16/F1 cells. The tumor uptake values of (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH were 9.53+/-1.41% injected dose/gram (% ID/g) and 10.40+/-1.40% ID/g at 2 h postinjection, respectively. Flank melanoma tumors were clearly visualized with (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH by SPECT/CT images at 2 h postinjection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h postinjection. There was low radioactivity (<0.8% ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h postinjection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using (111)In-labeled lactam bridge-cyclized alpha-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.     

Evaluation of the effect of mitomycin-C on the bioavailability of technetium-99m-labelled sodium pyrophosphate in mice.

We have reported that drugs alter the biodistribution of radiopharmaceuticals used in diagnostic imaging in nuclear medicine. Knowledge of such altered biodistribution is important in making diagnostic from scintigraphy. Mitomycin-C is used as component of many chemotherapeutic regimens to treat different tumors. The biological activities of mitomycin-C can be explained by its ability to inhibit deoxyribonucleic acid synthesis. Since patients on chemotherapeutic treatment can be submitted to nuclear medicine procedures, we studied the mitomycin-C effect on the bioavailability of the technetium-99m-labelled sodium pyrophosphate (9mTc-PYP) using an animal model. Mitomycin (0.45 mg) was administered by ocular plexus way Balb/c mice. One hour after the last dose, 99mTc-PYP (7.4 MBq) was administered and after 0.5 hr the animals (n = 15) were rapidly sacrificed. The organs were isolated, the radioactivity counted in a well counter and the percentage of radioactivity (%ATI) calculated. The results have shown that in the treated animals the %ATI has been decreased in spleen, thymus, heart and brain and increased in lung, liver and bone. The effect of this chemotherapeutic drug on the 99mTc-PYP biodistribution was statistically significant (Wilcoxon test, p < 0.05) and it could be explained by the metabolization or therapeutic action of mitomycin-C.     

Effect of eggplant (Solanum melongena) extract on the in vitro labeling of blood elements with technetium-99m and on the biodistribution of sodium pertechnetate in rats.

The use of eggplant has been suggested to treat different diseases. We studied the effect of eggplant extract on the labeling of red blood cells (RBC) and plasma proteins with technetium-99m (Tc-99m) and on biodistribution of sodium pertechnetate (Tc-99m) in rats. Blood was incubated with an eggplant extract (final concentrations 3.12 to 250.00 mg/ml) for 60 min. Then, stannous chloride (SnCl2) (0.06 or 1.2 microg/ml) and Tc-99m, as sodium pertechnetate, were added. Samples of RBC and plasma (P) were separated and also precipitated and soluble (SF) and insoluble (IF) fractions were isolated. The percent of radioactivity (%ATI) in the fractions was calculated. In the biodistribution study, Wistar rats were treated with eggplant extract (300 mg/ml) for 4 weeks, in drinking water. Tc-99m was administered in the rats, after 90 min they were sacrificed and organs and blood were isolated. When 0.06 microg/ml SnCl2 was used, eggplant extract: i/ inhibited the label of RBC (97.14 +/- 2.01 to 52.21 +/- 3.97%ATI), ii/ decreased the labeling in IF-P from 38.79 +/- 11.73 to 5.49 +/- 2.65%ATI, and iii/ diminished the labeling in IF-RBC from 90.04 +/- 2.65 to 46.17 +/- 9.49%ATI. This inhibitory effect was not observed with SnCl2 1.2 microg/ml. In the biodistribution study, the %ATI: i/ increased in the liver from 2.15 +/- 0.54 to 3.11 +/- 1.29 and ii/ in the other organs the Tc-99m uptake was not modified. The uptake of Tc-99m in red blood cells protein (IF-RBC) decreased from 66.62 +/- 19.67 to 31.66 +/- 8.84%. It is possible to suggest that some components of the eggplant extract present an oxidation power able to alter the fixation of the Tc-99m on the blood elements. Moreover, as eggplant is metabolized in the liver, this fact could justify the alteration of the uptake in this organ.     

64Cu-labeled alpha-melanocyte-stimulating hormone analog for microPET imaging of melanocortin 1 receptor expression

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor (melanocortin type 1 receptor, or MC1R) plays an important role in the development and growth of melanoma cells. It was found that MC1R was overexpressed on most murine and human melanoma, making it a promising molecular target for melanoma imaging and therapy. Radiolabeled alpha-MSH peptide and its analogs that can specifically bind with MC1R have been extensively explored for developing novel agents for melanoma detection and radionuclide therapy. The goal of this study was to evaluate a 64Cu-labeled alpha-MSH analog, Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys(DOTA)-NH2 (DOTA-NAPamide), as a potential molecular probe for microPET imaging of melanoma and MC1R expression in melanoma xenografted mouse models. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated NAPamide was synthesized and radiolabeled with 64Cu (t1/2=12 h) in NH4OAc (0.1 M; pH 5.5) buffered solution for 60 min at 50 degrees C. Cell culture studies reveal rapid and high uptake and internalization of 64Cu-DOTA-NAPamide in B16F10 cells. Over 90% of receptor-bound tracer is internalized at 3 h incubation. A cellular retention study demonstrates that the receptor-bound 64Cu-DOTA-NAPamide is slowly released from the B16F10 cells into the medium; 66% of the radioactivity is still associated with the cells even after 3 h incubation. The biodistribution of 64Cu-DOTA-NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16F10 melanoma tumors with high capacity of MC1R and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Tumor uptake values of 64Cu-DOTA-NAPamide are found to be 4.63 +/- 0.45% and 2.49 +/- 0.31% ID/g in B16F10 and A375M xenografted melanoma at 2 h postinjection (pi), respectively. The B16F10 tumor uptake at 2 h pi is further inhibited to 2.29 +/- 0.24% ID/g, while A375M tumor uptake at 2 h pi remains 2.20 +/- 0.41% ID/g with a coinjection of excess alpha-MSH peptide. MicroPET imaging of 64Cu-DOTA-NAPamide in B16F10 tumor mice clearly shows good tumor localization. However, low A375M tumor uptake and poor tumor to normal tissue contrast were observed. This study demonstrates that 64Cu-DOTA-NAPamide is a promising molecular probe for alpha-MSH receptor positive melanoma PET imaging as well as MC1R expression imaging in living mice.     

Laser stimulation of the acupoint ‘Zusanli’ (ST.36) on the radiopharmaceutical biodistribution in <Emphasis Type="BoldItalic">Wistar</Emphasis> rats

Laser used to stimulate acupoints is called laser acupuncture (LA). It is generally believed that similar clinical responses to manual acupuncture can be achieved. Here we analysed the effects of the laser (904 nm) at the ‘Zusanli’ acupoint (ST.36) of the stomach meridian on the biodistribution of the radiopharmaceutical Na^99mTcO₄. Wistar rats were divided into control (CG) and experimental groups (EG). The EG were exposed daily to the laser (904 nm) at ST.36 with 1 joule/min (40 mW/cm²) for 1 min. The animals of the CG were not exposed to laser at all. On the 8th day after LA, the animals were sedated and Na^99mTcO₄ was administered. After 10 min, the animals were all sacrificed and the organs removed. The radioactivity was counted in each organ to calculate the percentage of radioactivity of the injected dose per gram (%ATI/g). Comparison of the %ATI/g in EG and CG was performed by Mann-Whitney test. The %ATI/g was significantly increased in the thyroid due to the stimulation of the ST.36 by laser. It is possible to conclude that the stimulation of ST.36 does lead to biological phenomena that interfere with the metabolism of the thyroid.     

The ingestion of a Nectandra membranacea extract changes the bioavailability of technetium-99m radiobiocomplex in rat organs

The radiobiocomplexes labeled with technetium-99m (Tc-99m) have been widely used in nuclear medicine in single photon emission computed tomography and in basic research. The aim of this study was to assess the influence of a Nectandra membranacea extract on the bioavailability of the sodium pertechnetate (Na(99m)TcO(4)) radiobiocomplex in rat organs. The animals were treated with a N. membranacea extract (30 mg/ ml), for 6 days. Na(99m)TcO(4) was injected, the organs were isolated and weighed, and the radioactivity was determined in each organ (%ATI/organ). The %ATI/organ was divided by the mass of each organ to calculate the %ATI/g. A significant increase of the %ATI/organ of Na(99m)TcO(4) was observed in muscle and thyroid as well as in the %ATI/g in the heart, kidney and thyroid. These findings could result from the interaction between components of the plant extract and the radiobiocomplex which may influence the uptake Na(99m)TcO(4) in rat organs. Therefore, precaution is suggested in the interpretation of nuclear medicine results in patients using this herb.     

Substitution of Gly with Ala enhanced the melanoma uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide

The purpose of this study was to determine the melanoma targeting property of (99m)Tc-RAD-Lys-(Arg(11))CCMSH in B16/F1 melanoma-bearing C57 mice and compare with (99m)Tc-RGD-Lys-(Arg(11))CCMSH we previously reported. (99m)Tc-RAD-Lys-(Arg(11))CCMSH exhibited rapid and high tumor uptake (19.91±4.02% ID/g at 2h post-injection) in B16/F1 melanoma-bearing C57 mice. The tumor uptake of (99m)Tc-RAD-Lys-(Arg(11))CCMSH was 1.51, 1.34 and 1.43 times the tumor uptake of (99m)Tc-RGD-Lys-(Arg(11))CCMSH at 0.5, 2 and 4h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2h post-injection using (99m)Tc-RAD-Lys-(Arg(11))CCMSH as an imaging probe. The substitution of Gly with Ala significantly enhanced the melanoma uptake of (99m)Tc-RAD-Lys-(Arg(11))CCMSH compared to (99m)Tc-RGD-Lys-(Arg(11))CCMSH in B16/F1 melanoma-bearing C57 mice, providing a new insight into the design of α-MSH peptides for melanoma targeting.     

Injection of cells and monoclonal antibodies into mice: comparison of tail vein and retroorbital routes

Organ distribution and blood concentration profiles were compared following injection of mice with radiolabeled test agents via the lateral tail vein or retroorbital venous sinus. Monoclonal antibodies directed against B16 melanoma of C57BL/6 origin were labeled with iodine-125. Thymocytes from BALB/c mice and B16 melanoma cells were labeled with technetium-99m sodium pertechnetate (Na 99mTcO4). Animals were injected with 5 microCI of iodinated antibody, 5 X 10(5) syngeneic thymocytes, 2.5 X 10(5) melanoma cells, or 10 microCi Na 99mTcO4 in 0.2 ml saline via either route. In non-tumor-bearing C57BL/6 mice radiolabeled monoclonal antibody was found primarily in the gastrointestinal tract, liver, and blood. Na 99mTcO4 localized in the gastrointestinal tract, 99mTc-labeled thymocytes in the spleen and liver, and 99mTc-labeled B16 melanoma cells in the liver and lungs. Pharmacokinetic analysis of blood samples taken 4, 8, and 12 min following injection of the labeled agents suggested that the iodinated antibody had less vascular permeability than Na 99mTcO4 and that thymocytes and B16 melanoma cells were trapped in the pulmonary vasculature as they passed through the lungs. It is noteworthy that no biologically significant differences in organ distribution patterns or blood decay profiles were found between lateral tail vein and retroorbital routes. The data clearly indicate that these routes can be used interchangeably with one another for intravenous injections.     

Boron uptake in normal melanocytes and melanoma cells and boron biodistribution study in mice bearing B16F10 melanoma for boron neutron capture therapy

Information on (10)B distribution in normal tissues is crucial to any further development of boron neutron capture therapy (BNCT). The goal of this study was to investigate the in vitro and in vivo boron biodistribution in B16F10 murine melanoma and normal tissues as a model for human melanoma treatment by a simple and rapid colorimetric method, which was validated by HR-ICP-MS. The B16F10 melanoma cell line showed higher melanin content than human melanocytes, demonstrating a greater potential for boronophenylalanine uptake. The melanocytes showed a moderate viability decrease in the first few minutes after BNCT application, stabilizing after 75 min, whereas the B16F10 melanoma showed the greatest intracellular boron concentration at 150 min after application, indicating a different boron uptake of melanoma cells compared to normal melanocytes. Moreover, at this time, the increase in boron uptake in melanoma cells was approximately 1.6 times higher than that in normal melanocytes. The (10)B concentration in the blood of mice bearing B16F10 melanoma increased until 90 min after BNCT application and then decreased after 120 min, and remained low until the 240th minute. On the other hand, the (10)B concentration in tumors was increased from 90 min and maximal at 150 min after application, thus confirming the in vitro results. Therefore, the present in vitro and in vivo study of (10)B uptake in normal and tumor cells revealed important data that could enable BNCT to be possibly used as a treatment for melanoma, a chemoresistant cancer associated with high mortality.     

Intralesional Injection of Rose Bengal Induces a Systemic Tumor-Specific Immune Response in Murine Models of Melanoma and Breast Cancer

Intralesional (IL) injection of PV-10 has shown to induce regression of both injected and non-injected lesions in patients with melanoma. To determine an underlying immune mechanism, the murine B16 melanoma model and the MT-901 breast cancer model were utilized. In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions. In a murine model of melanoma, B16 cells were injected into C57BL/6 mice to establish one subcutaneous tumor and multiple lung lesions. Treatment of the subcutaneous lesion with a single injection of IL PV-10 led to regression of the injected lesion as well as the distant B16 melanoma lung metastases. Anti-tumor immune responses were measured in splenocytes collected from mice treated with IL PBS or PV-10. Splenocytes isolated from tumor bearing mice treated with IL PV-10 demonstrated enhanced tumor-specific IFN-gamma production compared to splenocytes from PBS-treated mice in both models. In addition, a significant increase in lysis of B16 cells by T cells isolated after PV-10 treatment was observed. Transfer of T cells isolated from tumor-bearing mice treated with IL PV-10 led to tumor regression in mice bearing B16 melanoma. These studies establish that IL PV-10 therapy induces tumor-specific T cell-mediated immunity in multiple histologic subtypes and support the concept of combining IL PV10 with immunotherapy for advanced malignancies.     

Pharmacokinetics of radiopharmaceuticals.

The pharmacokinetics of various radiopharmaceuticals following i.v. administration in mice and rats has been studied and compared. Before injection the radiochemical purity (RP) of the compounds were determined by HPLC and PAGE. In all cases RP-s were higher than 90%. The biodistribution of 99mTc labelled anti CEA IgG was studied in mice bearing human colon carcinoma xenografts. Animals with different tumour weights showed different blood kinetic and tumor uptake. The pilot clinical study of the 99mTc labelled anti melanoma Fab and 111-In-DTPA labelled anti melanoma F(ab')2 showed differences in the pharmacokinetic parameters. (99mTc labelled: comp.A. 84.6%; T1/2:0.6 h, 111-In-labelled: comp.A.: 46%, T1/2 1.5 h.) The various isonitrile derivatives synthetized in our laboratory were labelled with 99mTc and the biodistribution were tested in rats. The kinetic study showed that all the three molecules have different half lives in the heart and liver (T1/2 for heart ranged 5.1-18.6 h, for liver T1/2:1.2-2.5 h). Reversed phase HPLC study of the collected bile showed the 15-100% of injected compounds are metabolized during hepatic excretion. Literature data and our recent observations confirm that the knowledge of pharmacokinetics of radiolabelled compounds both in research, development and clinical practice is of basic importance.     

Linker modification reduced the renal uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide

The purpose of this study was to examine the biodistribution of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH in B16/F1 melanoma-bearing C57 mice to determine whether the replacement of the Lys linker with an Arg linker could decrease the renal uptake of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH. ^99mTc-RAD-Arg-(Arg¹¹)CCMSH exhibited rapid and high tumor uptake (17.98 ± 4.96% ID/g at 2 h post-injection) in B16/F1 melanoma-bearing C57 mice. As compared to ^99mTc-RAD-Lys-(Arg¹¹)CCMSH, the replacement of the Lys linker with an Arg linker dramatically decreased the renal uptake of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH by 68%, 62%, 73% and 64% at 0.5, 2, 4 and 24 h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2 h post-injection using ^99mTc-RAD-Arg-(Arg¹¹)CCMSH as an imaging probe.     

Metastatic melanoma imaging using a novel Tc-99m-labeled lactam-cyclized alpha-MSH peptide

The purpose of this study was to determine the metastatic melanoma imaging property of ^99mTc(EDDA)-HYNIC-Aoc-Nle-CycMSH_hex {hydrazinonicotinamide-8-aminooctanoic acid-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂}. HYNIC-Aoc-Nle-CycMSH_hex was synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The IC₅₀ value of HYNIC-Aoc-Nle-CycMSH_hex was 0.78?±?0.13?nM for B16/F10 melanoma cells. ^99mTc(EDDA)-HYNIC-Aoc-Nle-CycMSH_hex displayed significantly higher uptake (14.26?±?2.74 and 10.45?±?2.31%?ID/g) in B16/F10 metastatic melanoma-bearing lung than that in normal lung (0.90?±?0.15 and 0.53?±?0.14%?ID/g) at 2 and 4?h post-injection, respectively. B16/F10 pulmonary metastatic melanoma lesions were clearly visualized by SPECT/CT using ^99mTc(EDDA)-HYNIC-Aoc-Nle-CycMSH_hex as an imaging probe at 2?h post-injection, underscoring its potential as an imaging probe for metastatic melanoma detection.     

IL-12 gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma

Interleukin-12 (IL-12) has been evaluated in both preclinical and clinical immunotherapy protocols as a potential therapy for melanoma. However, delivery of IL-12 in the form of recombinant protein can result in severe toxicity, and gene therapy has had limited success against B16.F10 murine melanoma. This study investigated the therapeutic effect of delivering a plasmid encoding IL-12 followed by electroporation on primary and secondary tumors. Three treatments of intratumoral (i.t.) plasmid injection and electroporation resulted in 80% of mice with B16.F10 melanoma tumors being tumor free for >100 days (cure). The "cured animals" were resistant to challenge with B16 cells. In a separate experiment, B16 cells were injected on the opposite flank of the treated tumor on the day of treatment. Eighty-seven percent of control mice developed a distant tumor while only 43.8% of mice receiving two or three i.t. electroporation treatments developed a distant tumor. For examination of tumor development in the lungs, mice were injected intravenously with B16.F10 cells then treated with i.m. injections of plasmid with or without electroporation. Only 37.5% of mice receiving i.m. injections and electroporation developed nodules in the lungs compared to 87.5% of mice in the no-treatment group. The results show that administration of a plasmid encoding IL-12 with electroporation has a therapeutic effect on primary tumors as well as distant tumors and metastases.     

Inhibition of metastasis of syngeneic murine melanoma in vivo and vasculogenesis in vitro by monoclonal antibody C11C1 targeted to domain 5 of high molecular weight kininogen

Metastasis of malignant tumors is a major cause of morbidity and mortality. Inhibition of tumor growth in distant organs is of clinical importance. We have demonstrated that C11C1, a murine monoclonal antibody to the light chain region of high molecular weight kininogen (HK), reduces growth of murine multiple myeloma in normal mice and human colon cancer in nude mice. C11C1 inhibits angiogenesis by reducing tumor microvascular density by blocking binding of HK to endothelial cells. We now evaluate the anti-metastatic effect of C11C1 on C57BL/6 mouse lung metastatic model using B16F10 melanoma cells. The tail veins of mice were injected with 0.5 × 10⁶ cells of melanoma B16F10. One group received C11C1 and the other received saline (control) intraperitoneally. When mice were killed at 28 days, 6 of 10 control mice had detectable metastatic pulmonary nodules which stained positive with an antibody against S-100 protein, a tumor antigen present in malignant melanoma cells. In the C11C1 groups, none of the mice showed metastatic foci in their lungs. We showed that C11C1 inhibits endothelial cell tube formation in a 3-D collagen fibrinogen gel model by inhibiting the rate of cleavage of HK by plasma kallikrein without changing the binding affinity for HK. These studies demonstrate that a monoclonal antibody to HK has the potential to prevent metastasis with minimal side effects.     

Study of the biodistribution of the amantadine labelled with technetium-99m in Wistar female rats.

Amantadine (AMA) has been described as dopamine stimulant and norepineprhine release, capable to block the N-methyl-D aspartate (NMDA) glutamatergic and nicotinic receptors, enhancing the sexual behavior of the male rats and inducing hypersexuality in humans. The use of technetium-99m (99mTc) can be justified for its physical and chemical properties. The aim of this study was to label and evaluate the bioavailability of the AMA labelled with 99mTc (99mTc-AMA) in Wistar female rats. The solution of 99mTc-AMA was administered by intraperitoneal way and the animals were sacrificed in CO2 chamber 10 min after the administration of the radiotracer. Various organs were removed, weighted, their radioactivity was determined using an auto-gamma counter and the results were expressed as the percentage of the injected activity per gram of tissue (%ATI/g). In the control group only Na99mTcO4 was administered. The analysis of results shows that the highest uptakes 99mTc-AMA treated group were: ovary (7.11 +/- 1.43), spleen (3.54 +/- 1.05), thyroid (2.67 +/- 0.15), stomach (1.56 +/- 1.10), duodenum (0.87 +/- 0.52), muscular tissue (0.57 +/- 0.06), liver (0.52 +/- 0.25), and at control group: thyroid (16.45 +/- 2.57), ovary (1.28 +/- 0.12), liver (1.10 +/- 0.04), spleen (0.57 +/- 0.07) and muscular tissue (0.26 +/- 0.03). The results obtained suggest that 99mTc-AMA may be used to study the bioavailability of amantadine and evaluate its effect in sexual behavior in female rats.     

Induction of anterior chamber-associated immune deviation (ACAID) by allogeneic intraocular tumors does not require splenic metastases

Anterior chamber-associated immune deviation (ACAID), induced by intracameral injection of allogeneic tumor cells, is expressed in three distinct ways: 1) progressive growth of intraocular tumors, 2) specific suppression of systemic allograft immunity, and 3) transient growth of allogeneic tumors injected subcutaneously. Induction of ACAID requires that alloantigen presentation occur via the anterior chamber; injection by other routes failed to elicit this phenomenon. Antigenic material must remain in the anatomically intact eye for at least 10 days; removal of the injected dye before this time prevented the establishment of ACAID. The similar temporal requirement for an anatomically intact spleen confirms the validity of the concept of a camero-splenic axis for processing of intracamerally injected alloantigens. Deployment of an alternate model of ACAID, using LP/J mice injected intracamerally with B16F10 melanoma, showed the antigen-specific inductive signal for ACAID (transmitted via the camero-splenic axis) was not in the form of viable alloantigen-bearing tumor cells that metastasize to the spleen. B16F10 melanoma cells were never found in the spleens or any other extraocular sites after intracameral injection, despite the fact these mice manifested ACAID and harbored enormous ocular tumors. The data emphasize that intraocular processing of antigens is a unique and dynamic phenomenon with significant, systemic immunologic consequences.     

Further characterization of a clinically relevant model of melanoma metastasis and an effective vaccine

A major problem in evaluating the effectiveness of tumor cell vaccination and other biological therapies is the variability of experimental models. In this study we have further developed and characterized a model for metastatic melanoma that approximates the major clinical stages of metastatic dissemination: stage I-growth of the primary (local) tumor, stage II-dissemination to regional lymph nodes, and stage III-metastasis to distant organs (lungs). C57BL/6 mice were challenged subcutaneously with B16 F10 murine melanoma cells in the midtail, and within 3 weeks 100% of the mice had local tumors growing in their tails. By 5–7 weeks after challenge, most of the mice had developed metastases to the inguinal lymph nodes and subsequently had metastatic colonies in the lungs and in the bone marrow. Preimmunization of mice with a formalinized extracellular antigen vaccine, derived from B16F10 melanoma cells, provided partial inhibition of the growth of the primary melanoma tumors, as well as reducing the number of metastases to the regional (inguinal) lymph nodes and lungs along with concomitantly increasing survival time. This model for melanoma metastasis provides a reasonable and reproducible test system for the study of anti-melanoma immunity and the different cellular and humoral mechanisms involved.This work was supported in part by National Institutes of Health grants R37 CA45148 and R30 CA13943