S. Harada

TARGETED DELIVERY OF CHEMOTHERAPEUTIC AGENTS USING IMPROVED RADIOSENSITIVE LIQUID CORE MICROCAPSULES AND ASSESSMENT OF THEIR ANTITUMOR EFFECT

PURPOSE Radiation-sensitive microcapsules composed of alginate and hyaluronic acid are being developed. We report the development of improved microcapsules that were prepared using calcium- and yttrium-induced polymerization. We previously reported on the combined antitumor effect of carboplatin-containing microcapsules and radiotherapy. METHODS AND MATERIALS We mixed a 0.1% (wt/vol) solution of hyaluronic acid with a 0.2% alginate solution. Carboplatin (l mg) and indocyanine green (12.5 microg) were added to this mixture, and the resultant material was used for capsule preparation. The capsules were prepared by spraying the material into a mixture containing a 4.34% CaCl(2) solution supplemented with 0-0.01% yttrium. These capsules were irradiated with single doses of 0.5, 1.0, 1.5, or 2 Gy (60)Co gamma-rays. Immediately after irradiation, the frequency of microcapsule decomposition was determined using a microparticle-induced X-ray emission camera. The amount of core content released was estimated by particle-induced X-ray emission and colorimetric analysis with 0.25% indocyanine green. The antitumor effect of the combined therapy was determined by monitoring its effects on the diameter of an inoculated Meth A fibrosarcoma. RESULTS Microcapsules that had been polymerized using a 4.34% CaCl(2) solution supplemented with 5.0 x 10(-3)% (10(-3)% meant or 10%(-3)) yttrium exhibited the maximal decomposition, and the optimal release of core content occurred after 2-Gy irradiation. The microcapsules exhibited a synergistic antitumor effect combined with 2-Gy irradiation and were associated with reduced adverse effects. CONCLUSION The results of our study have shown that our liquid core microcapsules can be used in radiotherapy for targeted delivery of chemotherapeutic agents.

The antitumor effect of hyperthermia combined with fluorouracil and its analogues.

The effectiveness of 5-fluorouracil (5-FU), FT-207 and FT-207 + uracil in combination with two repetitions of 43 degrees C hyperthermia in the treatment of the Meth-A-Fibrosarcoma and Sarcoma-180 was examined in vivo in BALB/c mice. The antitumor effect was evaluated in terms of inhibition of tumor growth by measuring the tumor for 7 days. The 5-FU concentration in each tumor was also monitored. Hyperthermia combined with FT-207 or FT-207 + uracil showed a synergistic effect for the inhibition of growth of both tumors which was not observed with 5-FU. There were no significant differences in the intratumoral concentration of 5-FU in unheated or heated Sarcoma-180 for any drug treatment after the first hyperthermia treatment, except for significant decreases in the group given 5-FU with the first hyperthermia treatment. After the second hyperthermia treatment, significant decreases in the concentration of 5-FU and FT-207 + uracil were observed. In the Meth-A-Fibrosarcoma, the intratumoral concentration of 5-FU decreased significantly in the group given 5-FU and increased significantly in the group given FT-207 + uracil after the first hyperthermia treatment, while there were significant decreases in 5-FU, FT-207 and FT-207 + uracil administered with the second hyperthermia treatment. Hyperthermia combined with FT-207 or FT-207 + uracil is considered to be effective.

The correlation between spontaneous and radiation-induced apoptosis in T3B bladder cancer (histological grade G3), and the precedence between the two kinds of apoptosis for predicting clinical prognosis

PURPOSE The correlation between the frequency of spontaneous and radiation-induced apoptosis, and the precedence between those for predicting prognosis were studied at clinical level. METHODS AND MATERIALS Twenty-one patients (mean age, 65.8 years; 16 men and 5 women) with bladder cancer (transitional cell carcinoma Grade 3, T3bN0M0, Stage IIIb) underwent intraoperative radiotherapy: single 30-Gy 12-MV electron beam irradiation to bladder, followed by total cystectomy 6 h after irradiation. The specimens of pretreatment and irradiated bladder cancer were assayed for apoptosis, using TUNEL staining with counter staining of hematoxylin. The apoptotic index (AI) was calculated by dividing the number of apoptotic cells by the total number of cells and multiplying by 100. The Pearsons linear fitting was used to test the correlation between spontaneous and radiation-induced apoptosis. The Kaplan-Meier product-limit estimation was used for overall survival (OS) and freedom from recurrence (FFR). The precedence between spontaneous and radiation-induced apoptosis for predicting the clinical prognosis was estimated using the proportional hazard regression. RESULTS The mean AI of spontaneous and radiation-induced apoptosis was 1.18 +/- 0.16 and 2.63 +/- 0.45, respectively, which was significantly different. There was strong correlation between spontaneous and radiation-induced apoptosis (r(2) = 0.864, adjusted r(2) = 0.857). Radiation-induced apoptosis was estimated by equation: y (radiation-induced apoptosis) = 2.67 x (spontaneous apoptosis) -0.52. However, the proportional hazard regression test indicated that only spontaneous apoptosis was significant for predicting OS and FFR (&z.sfnc;t&z.sfnc; > 0.2), but radiation-induced apoptosis was not. CONCLUSION Estimating AI in radiation-induced apoptosis from AI in spontaneous apoptosis is possible. However, spontaneous apoptosis is more accurate in predicting clinical prognosis.

Effects of cigarette smoking on iodine 123 N-isopropyl-p-iodoamphetamine clearance from the lung

Iodine 123 N-isopropyl p-iodoamphetamine (123I-IMP), originally developed as a brain scanning agent, is also taken up by the lung. To evaluate the effects of cigarette smoking on the kinetics of IMP in the lung, we studied 123I-IMP clearance from the lung in 18 volunteers (8 non-smokers and 10 smokers). After the injection of 111 MBq of 123I-IMP into the medial cubital vein, the time-activity curve for 60 min and the regional activity using 1 frame per minute and a 64 × 64 matrix were obtained. The 123I-IMP clearance curve was described as follows: C (t) = A1e−k1t+ A2e−k2t (A1, A2: intercepts, and k1, k2: slopes of the exponential components). 123I-IMP clearance was delayed in smokers, and k2 was smaller in smokers. Also, a correlation between k1, k2, and the number of cigarettes smoked per day was found (r = −0.65, r = −0.74, respectively, P<0.01). In conclusion, this study suggests that the delayed clearance and retention of 123I-IMP in the lung indicate lung metabolic disorders due to cigarette smoking.

DECREASING SIZE OF RADIOSENSITIVE CAPSULES FROM MICRO TO NANO, AND ITS INCREASED ANTITUMOR EFFECT AND DECREASING ADVERSE EFFECT

We have been developing microcapsules that release anticancer drug with response to radiation. We attempted to decrease the diameter of capsules. Then, two categories were tested in VIVO in C3He mice: (1) the antitumor effect in combination with radiation and subcutaneously injected nanocapsules, (2) the kidnetics of nanocapsules when they were injected intravenously. Microcapsules were produced by spraying a mixture of 3.0 % hyaluronic acid, 2.0 % alginate, 3.0 % H2O2, and 0.3 mmol carboplatin (Pt containing anticancer drug) onto a mixture of vibrated 0.3 mol FeCl2 and 0.15 mol CaCl2. The antitumor effect was measured by measuring tumor diameter every day. The kinetics of microcapsules were expressed as the numbers of capsules in 5 views (25 × 25 μm) of micro PIXE camera and Pt concentration of quantiative PIXE. The generated microcapsules 752 ± 64 nm, which were significantly downsized relative to previous capsules. The accumulations of capsules in lungs, liver, and kidneys were decreased by downsizing, whereas those of tumors were increased. By adjusting Pt concentration in tumor, there were no significant differences in antitumor effect between not downsized and downsized microcapsules with combination with radiation. Decreased trapping of downsized microcapsules to lungs, liver, and kidneys, also increased trapping in tumors will lead to new targeted chemoradiotherapy via intravenous injection of microcapsules.

IMPROVED RADIOSENSITIVE MICROCAPSULES USING H2O2

The radiation-induced releasing of the liquid-core of the microcapsules was improved using H2O2, which produced O2 generation of H2O2 after irradiation. Further, we tested whether these microcapsules enhanced the antitumor effects and decreased the adverse effects in vivo in C3He/J mice. The capsules were produced by spraying a mixture of 3.0% hyaluronic acid, 2.0% alginate, 3.0% H2O2, and 0.3 mmol of carboplatin on a mixture of 0.3 molFeCl2 and 0.15 molCaCl2. The microcapsules were subcutaneously injected into MM46 tumors that had been inoculated in the left hind legs of C3He/J mice. The radiotherapy comprised tumor irradiation with 10 Gy or 20 Gy 60Co. The antitumor effect of the microcapsules was tested by measuring tumor size and monitoring tumor growth. Three types of adverse effects were considered: fuzzy hair, loss of body weight, and death. The size of the capsule size was 23 ± 2.4 µmɸ and that of the liquid core, 20.2 ± 2.2 µmɸ. The injected microcapsules localized drugs around the tumor. The production of O2 by radiation increased the release of carboplatin from the microcapsules. The antitumor effects of radiation, carboplatin, and released oxygen were synergistic. Localization of the carboplatin decreased its adverse effects. However, the H2O2 caused ulceration of the skin in the treated area. The use of our microcapsules enhanced the antitumor effects and decreased the adverse effects of carboplatin. However, the skin-ulceration caused by H2O2 must be considered before these microcapsules can be used clinically.

The kinetics of Fe and Ca for the development of radiation-induced apoptosis by micro-PIXE imaging

Abstract To study the interactions between the induction of radiation-induced apoptosis and trace elements kinetics, human leukemia cells were irradiated in vitro by 60 Co γ rays, after which the cells were evaluated for the detection of apoptosis and trace element (Fe, Ca, Zn) imaging was carried out. The frequency of apoptosis, i.e. the number of apoptotic bodies per 100 nuclei, was obtained by microscopic assay using TUNEL staining at 400× magnification. The trace element distribution in the cell was determined by micro-PIXE using 2 MeV proton beams. In the early phase of apoptosis, the maximum level of Fe accumulation was observed in the cell stroma. In the mid to end phase, Fe accumulation was diminished, and instead, Ca accumulation increased and Zn decreased in the nucleus. There appear to be two steps for the development of apoptosis: (1) the signaling from cell stroma to nucleus by Fe or an Fe-containing enzyme; and (2) the degeneration of the nucleus by Ca-dependent enzyme, and release of Zn from digested nucleus. Those strong accumulations may be new markers for apoptosis.

Targeted concurrent chemoradiotherapy, by using improved microcapsules that release carboplatin in response to radiation, improves detectability by computed tomography as well as antitumor activity while reducing adverse effect in vivo

PURPOSE The effect of alginate-hyaluronate microcapsules that release carboplatin in response to radiation was improved by adding ascorbic acid (AA). MATERIALS AND METHODS Four measures of the effectiveness of the microcapsules were evaluated: 1) release of carboplatin in response to radiation in vitro and in vivo; 2) detectability of their accumulation by computed tomography (CT) in vivo; 3) enhancement of antitumor effects in vivo; and 4) reduction of adverse effects in vivo. RESULTS There were significant increases in the rupture of microcapsules by adding AA in vitro. Subcutaneously injected microcapsules around the tumor could be detected by using CT and the alteration of CT-values correlated with the accumulation of the microcapsules. Those microcapsules released carboplatin and resulted in synergistic antitumor effect with concomitant radiation. With the encapsulation of carboplatin, chemotherapeutic effects were still observed two weeks after treatment. However, addition of AA did not result in increased antitumor effect in vivo. A reduction in adverse effects was observed with the encapsulation of carboplatin, through localization of carboplatin around the tumor. CONCLUSION Addition of AA to the materials of microcapsules did not result in increasing antitumor effect. However encapsulation of carboplatin will be useful as a clinical cancer-therapy option.

THE TWO INVERSE PATHWAYS : THE APOPTOSIS AND THE MULTINUCLEATED CELL SWITCHED BY THE MAGNESIUM/ZINC BALANCE IN IRRADIATED SARCOMA IN VIVO

Abstract The Mg dependence on apoptosis, and the Zn dependence on the multinucleated cell were evaluated in irradiated Sarcoma-180 in vivo in BALB/c mice. The kinetics of total concentration of Mg strongly correlated with the frequency of apoptosis from 6 to 24 h after irradiation and that of Zn strongly correlated with the frequency of the multinucleated cell from 12 to 48 h after irradiation under the 10 or 20 Gy irradiation. There was an inverse proportion of frequency of apoptosis to that of the multinucleated cell. The administration of Mg significantly increased the frequency of apoptosis and significantly decreased the frequency of the multinucleated cell, from 6 to 12 h after the 20 Gy irradiation. In contrast, the administration of Zn significantly increased the frequency of the multinucleated cell and significantly decreased the frequency of apoptosis from 6 to 12 h under the 20 Gy irradiation. The combined administration of Mg and Zn did not change the frequency of apoptosis and the multinucleated cell. The administration of these elements did not change those correlation coefficients. The two inverse pathways were considered: the one for apoptosis; and another for the multinuclear cell, switched by the Mg/Zn balance.

INCREASING ANTITUMOR EFFECTS OF CHEMORADIOTHERAPY BY DRUG EFFLUX INHIBITION WITH ENCAPSULATED ANTI-RLIP-76

Microencapsulated anti-RLIP76 was tested in vivo using C3He/J mice to determine the increasing of antitumor effects by chemotherapeutic agent efflux inhibition during chemoradiotherapy. Microcapsules were produced by spraying a mixture of 3.0% hyaluronic acid, 2.0% alginate, 3.0% H2O2, and 0.3 mmol carboplatin onto a mixture of 0.3 mol FeCl2 and 0.15 mol CaCl2. Microcapsules were subcutaneously injected into MM46 tumors previously inoculated into the left hind legs of C3He/J mice. Subsequent radiotherapy consisted of tumor irradiation with 10 Gy or 20 Gy60Co. The antitumor effects of microcapsules were tested by measuring tumor size and monitoring tumor growth. Three types of adverse effects were considered: fuzzy hair, loss of body weight, and mortality. Carboplatin levels were monitored using particle-induced X-ray emission (PIXE) and a micro-PIXE camera. Anti-RLIP76 inhibited the efflux of carboplatin from tumor tissue, which led to an increase in the concentration of carboplatin. Higher carboplatin concentration significantly increased the combined antitumor effect of radiation and chemotherapy. A significant decrease in adverse effects was also observed with microencapsulated anti-RLIP76.