Ryuichiro Nishigaki

Mechanism of Cell Damage by Ultrasound in Combination with Hematoporphyrin

The mechanism of cell damage by ultrasound in combination with hematoporphyrin was studied. Mouse sarcoma 180 cell suspensions were exposed to ultrasound for up to 60 s in the presence and absence of hematoporphyrin, with and without active oxygen scavengers. The cell damage enhancement by hematoporphyrin was suppressed by adding histidine but not by mannitol. The enhancement was doubled in rate by substitution of deuterium oxide medium for normal water. Sonoluminescence was produced in a saline solution under similar acoustic conditions and observed to have spectral components that can excite hematoporphyrin molecules. These results suggest that cell damage enhancement is probably mediated via singlet oxygen generated by ultrasonically activated hematoporphyrin.

CONTRIBUTION OF OATP (ORGANIC ANION-TRANSPORTING POLYPEPTIDE) FAMILY TRANSPORTERS TO THE HEPATIC UPTAKE OF FEXOFENADINE IN HUMANS

Fexofenadine hydrochloride (FEX), a second generation H1-receptor antagonist, is mainly eliminated from the liver into bile in unchanged form. Recent studies have shown that FEX can be accepted by human MDR1 (P-glycoprotein), OATP1A2 [organic anion-transporting polypeptide (OATP)-A, and OATP2B1 (OATP-B)] expression systems. However, other transporters responsible for the hepatic uptake of FEX have not yet been identified. In the present study, we evaluated the contribution of OATP family transporters, namely OATP1B1 (OATP2/OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), to FEX uptake using transporter-expressing HEK293 (human embryonic kidney) cells. The uptake of FEX in OATP1B3-expressing cells was significantly greater than that in vector-transfected cells. On the other hand, OATP1B1- or OATP2B1-mediated uptake of FEX was not statistically significant. OATP1B3-mediated transport could be explained by a one-saturable component with a Michaelis constant (Km) of 108 ± 11 μM. The inhibitory effect of FEX on the uptake of estrone-3-sulfate (E1S), cholecystokinin octapeptide (CCK-8), and 17β-estradiol-17β-d-glucuronide (E217βG) was also examined. Both OATP1B1- and OATP1B3-mediated E217βG uptake was inhibited by FEX. The Ki values were 148 ± 61 and 205 ± 72 μM for OATP1B1 and OATP1B3, respectively. FEX also inhibited OATP1B3-mediated CCK-8 uptake and OATP1B1-mediated E1S uptake with a Ki value of 83.3 ± 15.3 and 257 ± 84 μM, respectively, suggesting that FEX could not be used as a specific inhibitor for OATP1B1 and OATP1B3, although FEX was preferentially accepted by OATP1B3. In conclusion, this is, to our knowledge, the first demonstration that OATP1B3 is thought to be a major transporter involved in hepatic uptake of FEX in humans.

Synergistic Effect of Ultrasound and Hematoporphyrin on Sarcoma 180

The antitumor effects of combined use of ultrasound (US) and a photosensitizer, hematoporphyrin (Hp), were determined in mice bearing sarcoma 180. In order to find the optimum timing of the US irradiation after the administration of Hp, the Hp concentrations in the tumor and in the plasma were determined and were analyzed pharmacokinetically. Antitumor effects were evaluated by measuring the tumor size and the tumor weight. Hp alone showed no antitumor effect but US alone showed a slight antitumor effect. The combined treatment with US and Hp showed marked synergistic effects on sarcoma 180 (inhibition ratio was 74% of the control). From these results, the enhancement of antitumor effect is thought to be caused by the sensitization of tumor cells to US mediated by Hp.

Enhancement of Ultrasonically Induced Cell Damage by a Gallium‐Porphyrin Complex, ATX‐70

Enhancement of ultrasonically induced cell damage by a gallium‐porphyrin complex [ATX‐70, 2,4‐ bis(l‐decyloxyethyl)‐Ga(III)‐1,3,5,8‐tetramethylporphyrin‐6,7‐dipropionyl diaspartic acid] was investigated. The rate of damage to isolated sarcoma 180 cells in air‐saturated suspension induced by 2 MHz ultrasound irradiation was enhanced more than four times by 80 μM ATX‐70 in contrast to only twice by the same concentration of hematoporphyrin (Hp). The enhancement was almost completely inhibited in the presence of 10 mM histidine in the suspension, but not at all by 100 mM mannitol, which suggests that the enhanced cell damage was mostly mediated by singlet oxygen. Ultrasonically induced active oxygen generation in an air‐saturated aqueous solution of ATX‐70 was studied by detecting the electron spin resonance signals of 2,2,6,6,‐tetramethyl‐4‐piperidone‐N‐oxyl produced by the reaction of 2,2,6,6‐tetramethyl‐4‐piperidone with the generated active oxygen species. The rate of ultrasonically induced nitroxide generation was enhanced five times by 80 μM ATX‐70 in contrast to only twice by Hp. The enhancement was inhibited significantly in the presence of 10 mM histidine in the suspension, but not at all by 100 mM mannitol. The singlet oxygen generation in air‐saturated aqueous solution was further confirmed by the bleaching of N, N‐dimethyl‐4‐nitrosoaniline in the presence of imidazole. The ultrasonically induced bleaching rate was enhanced six times by ATX‐70, in contrast to only twice by Hp.

Sonodynamically induced effect of rose bengal on isolated sarcoma 180 cells

Purpose: The ultrasonically induced effect of rose bengal (RB) on isolated tumor cells was investigated. Methods: Sarcoma 180 cells were suspended in air-saturated phosphate-buffered saline and exposed to ultrasound in standing wave mode for up to 60 s in the presence and absence of RB. Cell viability was determined by the ability to exclude trypan blue. Results: The rate of inducing cell damage by ultrasound was enhanced two to three times with 160 μM RB, while no cell damage was observed with RB alone. This enhancement was significantly inhibited by histidine. Conclusions: Ultrasonically induced in vitro cell damage was significantly enhanced by RB. A sonochemical mechanism may be suggested since the enhancement was significantly inhibited by an active oxygen scavenger.

Uptake Is the Rate-limiting Step in the Overall Hepatic Elimination of Pravastatin at Steady-state in Rats

AbstractPurpose. Of the HMG-CoA reductase inhibitors, the hydrophilic pravastatin has been shown to exhibit relatively specific inhibition of cholesterol synthesis in the liver. As one of the reasons for this relatively specific pharmacological activity, we demonstrated that the tissue distribution of pravastatin is limited because of its high hydrophilicity, while hepatic uptake by active transport takes place at the liver surface via a multispecific anion transporter (M. Yamazaki et al., Am. J. Physiol., 264, G36-44, 1993). In this study, we examined the hepatic elimination of pravastatin at steady-state. Methods. After i.v. infusion, the plasma concentrations of pravastatin in both arterial and hepatic venous blood were measured. Results. The hepatic availability at steady-state exhibited a clear increase on increasing the infusion rate of pravastatin. The total hepatic elimination rate at steady-state exhibited Michaelis-Menten type saturation with the drug concentration in the capillary defined by typical mathematical models (i.e., well-stirred, parallel-tube and dispersion models), Km and Vmax values being comparable with those obtained from analysis of the initial uptake velocity using in vitro isolated hepatocytes. Conclusions. These results indicate that overall hepatic intrinsic clearance of pravastatin at steady-state is regulated by the uptake process, followed by rapid metabolism and/or biliary excretion with minimal efflux to the circulating blood.

Sonodynamically induced antitumor effect of gallium-porphyrin complex by focused ultrasound on experimental kidney tumor

The antitumor effect of a gallium-porphyrin complex, ATX-70, induced by focused ultrasound, on colon 26 carcinoma implanted in a mouse kidney was investigated. Colon 26 tumors were exposed to focused ultrasound at 500 kHz and 1 MHz in a progressive wave mode. Both frequency components were superimposed onto each other in the focal zone to efficiently produce cavitation in the tumor. ATX-70 was administered intravenously at the dose of 2.5 mg/kg, 24 h before the ultrasonic exposure. Antitumor effects were evaluated by histological observation 7 days after the exposure. The destruction of tumor tissue was observed with the ultrasonic treatment in combination with ATX-70, while neither the treatment with ATX-70 alone nor that with ultrasound alone caused any necrosis. These results first demonstrated that antitumor effects of a porphyrin compound can be induced by focused ultrasound in a progressive wave mode.

Recent advances in sonodynamic approach to cancer therapy

Abstract Chemical agents such as porphyrins were found to be activated by ultrasound, producing significant antitumor effects. Hematoporphyrin (Hp) enhanced ultrasonically induced damage on sarcoma cells and shown a synergistic inhibitory effect on the tumor growth in combination with ultrasound at 2 MHz. Recently, other types of porphyrins such as protoporphyrin were also found to have such sonodynamic activities. Furthermore, it was found that sonochemical reactions can be greatly accelerated by superimposing the second harmonic onto the fundamental. The highest rate of iodine release from aqueous iodide was obtained at an acoustic intensity ratio between 1 MHz and 2 MHz of 1:1 while either one of the frequency components alone could not induce significant iodine release at the same total acoustic intensity. Second-harmonic superimposition in combination with sonodynamically active antitumor agents may have the potential for selective tumor treatment.

Sonodynamically Induced Antitumor Effect of a Gallium‐Porphyrin Complex, ATX‐70

The Sonodynamically induced antitumor effect of a gallium‐porphyrin complex, ATX‐70, was evaluated in mice bearing colon 26. In order to find the optimum timing for the ultrasonic exposure after the administration of ATX‐70, the ATX‐70 concentrations in the plasma, skin, and tumor were measured and analyzed. Antitumor effect was estimated by measuring the tumor size. When used alone, ultrasound showed a slight antitumor effect, which became increasingly significant as the dose of ATX‐70 was increased, while use of ATX‐70 alone had no significant effect. At an ATX‐70 dose of 2.5 mg/kg or higher, the average tumor size decreased to smaller than a half by three days after the ultrasonic exposure. This was smaller than a third of the size of the untreated tumors on the same day. From these results, it is concluded that ATX‐70 significantly sensitizes tumors to ultrasound, demonstrating a synergistic antitumor effect.

Studies on sonodynamic cancer therapy

Chemical agents such as hematoporphyrin have been found to have a sonochemical activity to induce anti-tumor effects. The same kind of sonochemical reactions producing active oxygen species has been demonstrated to be localized with a highly focused ultrasound in a specially developed tissue-mimicking phantom. These results suggest that the focused ultrasound irradiation to a tumor combined with administration of such a sonochemically active agent can be a potential low-invasive modality for cancer therapy.