A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody.

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a(+)) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

Polarized small-angle light scattering from gels estimated in terms of a statistical approach.

To analyze polarized light scattering patterns from gels, an approach is proposed to calculate the scattered intensity. In the proposed model system, difference between polar angles of the principal axes of the and elements, which were defined with respect to the axis along the distance between two elements, was given as a correlation of the distance between the two elements. Furthermore, the azimuthal angle, which makes a projection of the principal axis onto a plane perpendicular to the principal axis of the element, was also given as a correlation of the distance between the two elements. The theoretical calculation was carried out for the scattered intensity under Hv and Vv polarization conditions. The general equations proposed for Hv and Vv scattering were based on a statistical approach for polarized light scattering system. The calculated pattern under the Hv polarization condition showed an X-type pattern and was in good agreement with the pattern observed from polymer gels prepared by quenching their solutions to the desired temperatures.

Highly stabilized amorphous 3-bis(4-methoxyphenyl)methylene-2-indolinone (TAS-301) in melt-adsorbed products with silicate compounds.

3-Bis(4-Methoxyphenyl)methylene-2-indolinone (TAS-301) is a poorly water-soluble drug showing low oral bioavailability in rats and dogs. Previously, we reported that when a physical mixture of TAS-301 and a porous calcium silicate, Florite RE (FLR), was heated at high temperature (250 degrees C), the drug melted and was adsorbed by the FLR in an amorphous state, and that the preparation (melt-adsorbed product) showed a significantly increased solubility and dissolution rate, and a significantly enhanced oral bioavailability of the drug. The aim of the present study was to elucidate important factors for preparing a melt-adsorbed product showing greater stability of drug in an amorphous state. We examined the effects of the kind of adsorbent, drug/adsorbent ratio, heating conditions, and drug particle size on converting drug crystal into an amorphous state, the stability of amorphous state, and chemical stability of the drug in the melt-adsorbed products under a high temperature and high humidity condition (60 degrees C/80% RH, open). FLR, light anhydrous silicic acid and two types of hydrated silicon dioxides were tested as adsorbents. For the batch method, TAS-301 was converted into an amorphous state by heating TAS-301/adsorbents physical mixtures above the melting point of TAS-301 for more than 2 min. The amorphous state was most stabilized when FLR was used as an adsorbent and drug/FLR ratio was 1:0.5 and more. For the continuous method using the twin screw extruder that enables significantly larger scale manufacturing than batch method, TAS-301 melt-adsorbed products were able to produce when only FLR was used as adsorbent. The heating temperature was needed to be set above the melting point of TAS-301 to convert it into an amorphous state as well as batch method. The amorphous state was stabilized when drug/FLR ratio was 1:2 and more. The micronization of the drug decreased the stability of the amorphous state. These results indicate the importance of optimizing the above factors in the preparation of melt-adsorbed product.

Inhibitory action of cilostazol, a phosphodiesterase III inhibitor, on catecholamine secretion from cultured bovine adrenal chromaffin cells.

The effect of cilostazol, a phosphodiesterase III inhibitor, on catecholamine secretion was examined using bovine adrenal chromaffin cells in culture. Catecholamine secretion evoked by acetylcholine was markedly inhibited by cilostazol. In contrast, 56 mM K+-evoked secretion was slightly inhibited by cilostazol. Cilostazol elevated the level of cyclic AMP in the cells. However, a cyclic AMP analog (dibutyryl cAMP) or an adenylate cyclase activator (forskolin) failed to inhibit secretion of catecholamine induced by acetylcholine. Cilostazol decreased the level of intracellular free Ca2+ stimulated by acetylcholine. Cilostazol thus inhibits secretion of catecholamine through its blocking action on Ca2+ movement in the adrenal chromaffin cells.

Inhibitory action of novel arginine derivative on catecholamine secretion evoked by acetylcholine from cultured bovine adrenal chromaffin cells.

A novel product, 4-amino-5-guanidinopentanoic acid 15-[(4-aminobutyl)-3-aminopropylcarbamoyl] pentadecyl ester (Arg-HSA-Spm), was synthesized based on ptilomycalin A, which is one of the extracts from marine sponge. Arg-HSA-Spm contains arginine in its chemical structure. The pharmacological action of Arg-HSA-Spm on catecholamine secretion from cultured bovine adrenal chromaffin cells was examined. Arg-HSA-Spm inhibited catecholamine secretion stimulated by the physiological secretagog acetylcholine. This inhibitory action of Arg-HSA-Spm on catecholamine secretion induced by 10(-4) M acetylcholine was dose-dependent from 10(-8) M to 10(-5) M. In the presence of 3 x 10(-7) M Arg-HSA-Spm, the stimulation of catecholamine secretion observed by increasing acetylcholine up to 10(-3) M did not reach the maximal level observed without Arg-HSA-Spm. Arg-HSA-Spm at 10(-5) M suppressed both the increase in intracellular free Ca2+ level and the influx of 45Ca2+ induced by 10(-4) M acetylcholine. The Arg-HSA-Spm-induced suppression of intracellular free Ca2+ level, the influx of 45Ca2+ and catecholamine secretion were not observed in the presence of extracellular K+ at 56 mM. The results presented in this study suggested that Arg-HSA-Spm may inhibit the influx of extracellular Ca2+ into the cells, probably through its blocking action related to acetylcholine receptors, resulting in the inhibition of catecholamine secretion in adrenal chromaffin cells.

[Possible role of a neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) on stimulus-secretion coupling in catecholamine neuron].

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide first isolated from ovine hypothalamic tissue. This peptide stimulates adenylate cyclase activation. However, few details were known of the function of this peptide on stimulus-secretion coupling in neuronal cells. The authors have investigated the role of PACAP on catecholamine biosynthesis and secretion using cultured bovine adrenal chromaffin cells as a model for catecholamine-containing neurons. PACAP38, the 38-amino acid form of PACAP, increased cAMP formation in bovine adrenal chromaffin cells. In addition, PACAP38 increased [Ca2+]i associated with PI turnover and Ca2+ influx into the cells. The synthesis of catecholamine and the phosphorylation of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis, stimulated by the maximal effective concentration of dibutyryl cAMP or a high concentration (56 mM) of K+ were further enhanced by PACAP38. Thus PACAP38 stimulated the pathway of catecholamine biosynthesis mainly by both activation of cAMP- and Ca2(+)-dependent protein kinases. On catecholamine secretion from the cells, the effect of PACAP38 was markedly potentiated by addition of ouabain, an inhibitor of Na+/K+ ATPase. This markedly potentiated secretion was greatly reduced with Na+ omitted-sucrose medium. PACAP38 increased 22Na+ influx into the cells treated with ouabain. Thus PACAP38 with ouabain stimulated catecholamine secretion by accumulation of intracellular Na+, resulting in an increase in Ca2+ influx. These results indicate that the neuropeptide PACAP has an important role in stimulus-secretion coupling in adrenal chromaffin cells.

Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells.

This study was made to explain the mechanisms for the effects of exposure to a time varying 1.51 T magnetic field on the intracellular Ca(2+) signaling pathway. The exposure inhibited an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine chromaffin cells induced by addition of bradykinin (BK) to a Ca(2+) free medium. The exposure did not change BK induced production of inositol 1,4,5-trisphosphate (IP(3)). [Ca(2+)](i) was markedly increased in IP(3) loaded cells, and this increase was inhibited by the magnetic field exposure. A similar increase in [Ca(2+)](i) by other drugs, which stimulated Ca(2+) release from intracellular Ca(2+) stores, was again inhibited by the same exposure. However, transmembrane Ca(2+) fluxes caused in the presence of thapsigargin were not inhibited by the magnetic field exposure in a Ca(2+) containing medium. Inhibition of the BK induced increase in [Ca(2+)](i) by the exposure for 30 min was mostly recovered 1 h after exposure ended. Our results reveal that the magnetic field exposure inhibits Ca(2+) release from intracellular Ca(2+) stores, but that BK bindings to BK receptors of the cell membrane and intracellular inositol IP(3) production are not influenced.

Improvement of solubility and oral bioavailability of a poorly water-soluble drug, TAS-301, by its melt-adsorption on a porous calcium silicate.

The aim of the present study was to improve the solubility and oral bioavailability of a poorly water-soluble drug, 3-bis(4-methoxyphenyl) methylene-2-indolinone (TAS-301), by its melt-adsorption on a porous calcium silicate, Florite RE (FLR), without any solvents. The melt-adsorbed products were prepared by two methods: the small-scale batch method and the twin screw extruder method. The drug was melted and adsorbed on FLR (i.e., "melt-adsorption"), above its melting point. Crystallinity of the drug in the melt-adsorbed product was estimated by differential scanning calorimetry (DSC) and powder X-ray diffraction analysis. The dissolution test was conducted by the JP XIII paddle method. Oral absorption of the melt-adsorbed product was studied in fasted and fed dogs. The melt-adsorbed products prepared by the two methods were in powder forms. The drug existed in an amorphous state in the product and hardly recrystallized even after storing at a stressed condition (60 degrees C/80% RH for 3 days). The TAS-301 dissolution rate from the melt-adsorbed product was markedly enhanced compared with drug crystals. The area under the plasma concentration-time curve (AUC) and peak concentration (C(max)) values of the drug after dosing the melt-adsorbed product were significantly greater than those after dosing the drug crystals. The solubility and bioavailability of TAS-301 were improved by its melt-adsorption on FLR. The present findings suggest melt-adsorption is a useful technique for improving solubility and bioavailability of poorly water-soluble drugs.