Toshihisa Yotsuyanagi

Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: a possible use of aortic endothelial cell for in vitro BBB model.

Astrocytic contribution of endothelial cell monolayer permeability was examined in two blood-brain barrier (BBB) models, using the coculture in a double chamber system: rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in L-glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the L-glucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.

In vivo genotoxicity evaluation of dimethylarsinic acid in Muta™Mouse

Dimethylarsinic acid (DMA) induces DNA damage in the lung by formation of various peroxyl radical species. The present study was conducted to evaluate whether arsenite or its metabolite, DMA, could initiate carcinogenesis via mutagenic DNA lesions in vivo that can be attributed to oxidative damage. A transgenic mouse model, MutaMouse, was used in this study and mutations in the lacZ transgene and in the endogenous cII gene were assessed. When DMA was intraperitoneally injected into MutaMice at a dose of 10.6 mg/kg per day for 5 consecutive days, it caused only a weak increase in the mutant frequency (MF) of the lacZ gene in the lung, which was at most 1.3-fold higher than in the untreated control animals. DMA did not appreciably raise the MF in the bladder or bone marrow. Further analysis of the cII gene in the lung, the organ in which DMA induced the DNA damage, revealed only a marginal increase in the MF. Following DMA administration, no change in the cII mutation spectra was observed, except for a slight increase in the G:C to T:A transversion. Administration of arsenic trioxide (arsenite) at a dose of 7.6 mg/kg per day did not result in any increase in the MF of the lacZ gene in the lung, kidney, bone marrow, or bladder. Micronucleus formation was also evaluated in peripheral blood reticulocytes (RETs). The assay for micronuclei gave marginally positive results with arsenite, but not with DMA. These results suggest that the mutagenicity of DMA and arsenite might be too low to be detected in the MutaMouse.

Oral mucosal adhesive film containing local anesthetics: In vitro and clinical evaluation

In vitro and in vivo studies were conducted to gauge the effectiveness of a novel oral mucosa adhesive, moderately water-soluble, pliant polymer artificial dentifrice (AD) film containing dibucaine (DC) for relief of pain due to oral erosion. The film was prepared from a hydroxypropyl cellulose-M (HPC-M) ethanol solution containing varying amounts of DC, as well as polyethylene glycol. In the in vitro experiments, the disintegration of HPC-M showed a lag time of about 50 min, a much lower rate than that of drug release, which more or less leveled off after 50 min. Twenty-five percent of the DC was released from the film (0.113 and 0.225 mg/cm2) after the initial 5 min, which then reached about 80% after 50 min, the time at which the polymer began to break up. In the in vivo study, the local anesthetic effect of the film was evaluated in 23 patients (10 males, 13 females) suffering from the adverse effects of chemotherapy. When applied to the wet surface of the mucosa, the AD film absorbed moisture and showed excellent adhesion. Pain relief in patients lasted 2.2 +/- 0.21 and 4.3 +/- 0.25 h at DC doses of 0.113 and 0.225 mg/cm2, respectively. These results suggest that the AD film may cover mucositis sites of oral mucosa long enough to allow DC release and bring relief from pain arising from chemotherapy and/or radiotherapy.

Computational consideration of cisplatin hydrolysis and acid dissociation in aqueous media: effect of total drug concentrations

Cisplatin (cis-DDP) is subject to nucleophilic displacement of chloride in water, forming aquated species, subsequently liberating hydrogen ion(s) with increasing pH. This study intends to theoretically analyze the hydrolysis and polyprotic dissociation behavior of cis-DDP in various aqueous media. A mathematical model was expressed by nonlinear simultaneous equations in terms of the total drug concentration, pH and pCl based on the hydrolysis and acid dissociation constants already published. Some of the interesting simulation results include that (1) in water, cis-DDP behaves in a very complicated manner, highly depending on the total drug concentration, pH and pCl, (2) in normal saline, about 3% of the total concentration is a positively charged chloro-aqua that may be very reactive, (3) in assumed blood (pH 7.4, [Cl(-)]=0.11 mol/l, mu=0.15), the drug is stabilized at the level of 85% and the remnants are the chloro-hydroxo (11%) and the chloro-aqua (4%), (4) in assumed intracellular conditions (pH 7.1, [Cl(-)]=0.01 mol/l, mu=0.15), the drug is converted to a large extent to various species including the parent species (44%), the chloro-hydroxo (30%), hydroxo-aqua (2%), chloro-aqua (24%) diaqua (less than 1%) and dihydroxo (null). The results of this analysis may provide a useful preliminary knowledge of existing species in a system concerned and a rationale for re-evaluating the reactions between cis-DDP and various nucleophilic substances already reported while there are somewhat conflicting interpretations of some cis-DDP reactions.

Effect of trans-diamminedichloroplatinum(II) on human serum albumin : conformational changes through partial disulfide bond cleavage

Abstract trans -Diamminedichloroplatinum(II) ( trans -DDP), the trans isomer of cis -diamminedichloroplatinum(II) ( cis -DDP), was bound to human serum albumin (HSA) in pH 7.4 buffer containing 0.1 M NaCl at 37°C. The amount of bound trans -DDP per mol of HSA was found to be 21.4 mol when the protein was incubated with a 40-fold excess of trans -DDP for 6 days. In trans -DDP-treated HSA, 3.4 disulfide (S-S) bonds were cleaved, where one HSA molecule contains 17 S-S bonds. The spectral characteristics of trans -DDP-treated HSA were examined in terms of the fluorescence spectrum of its lone tryptophan (Trp-214), and molar ellipticity. The relative fluorescence intensity of platinum-bound HSA decreased to 32.4% of that of the native state, suggesting that perturbation around the Trp-214 residue took place. This was confirmed by the destruction of the warfarin-binding site containing Trp-214 observed in the metal-bound HSA. Analysis of circular dichroism (CD) spectra showed a decreasing helix content from 50.5% in the native state to 30.6% in the metal-bound HSA. These conformational changes observed in HSA may be attributed to the S-S bond rupture induced by trans -DDP. Comparison with cis -DDP, which has already been shown to cleave S-S bonds in HSA, revealed that trans -DDP binds to HSA and cleaves S-S bonds more readily than the cis isomer.

Chemotherapy Targeting Regional Lymph Nodes by Gastric Submucosal Injection of Liposomal Adriamycin in Patients with Gastric Carcinoma

We investigated the delivery of adriamycin (ADR) to the regional lymph nodes of the stomach following the gastric submucosal injection of liposomal adriamycin (Lipo‐ADR) in 34 gastric carcinoma patients, as well as following intravenous administration of free ADR (F‐ADR) in another 18 patients. Prior to radical gastrectomy, Lipo‐ADR was endoscopically injected into the gastric submucosa adjacent to the primary tumor via a needle‐tipped catheter. After Lipo‐ADR injection, the ADR concentration in the primary and secondary drainage lymph nodes was higher than in the other regional lymph nodes. Thus, the regional nodes more susceptible to metastasis showed higher levels of ADR. In contrast, the intravenous administration of F‐ADR produced a similar and far lower ADR concentration in all the nodes. Delivery of ADR to the primary drainage lymph nodes following injection of 5 ml of Lipo‐ADR was compared with delivery to the left gastric artery lymph nodes after intravenous administration of an equal dose of F‐ADR. The ADR levels (μg/g) after gastric submucosal injection were 15.1±8.30 on day 1 (n = 4); and 11.9±4.80 on day 4 (n = 6). Those after intravenous administration were 0.29±0.10 on day 1 (n = 4); and 0.36±0.0 on day 4 (n = 2). The differences between the two groups were significant (P<0.05). The ADR levels after the gastric submucosal injection were far higher than those after intravenous administration. These findings indicate that the gastric submucosal injection of Lipo‐ADR can specifically deliver ADR to the regional lymph nodes at high concentrations. Such preoperative adjuvant chemotherapy targeting the regional lymph nodes may be useful for preventing the lymph node recurrence of gastric carcinoma.

Pain relief of oral ulcer by dibucaine-film.

A water-soluble three-layered oral mucosa-adhesive film made from hydroxypropyl cellulose containing dibucaine (0.25 mg of drug/cm(2)) was designed for alleviation of severe pain due to oral ulcers, caused by chemotherapy and/or radiotherapy. We report two patients with constant severe pain ulcers treated with the dibucaine film. Patients were asked to record the time that pain was relieved while chewing following first application of the film. Pain relief lasted for 2-5 h after application of the dibucaine film.

Coagulation recovery after warfarin‐induced hypoprothrombinaemia by oral administration of liposomally‐associated vitamin K1 to rabbits

The effect of liposomally-associated vitamin K1, administered orally, was investigated using rabbits with warfarin-induced hypoprothrombinaemia, and evaluated in comparison with other dosage forms of the vitamin, including a vitamin K1 emulsion, the physical mixture of the emulsion with empty liposomes, polyoxyethylene hydrogenated castor oil (HCO-60)-stabilized emulsion and the vitamin solubilized by HCO-60. The effect on blood coagulation recovery of each preparation was estimated as the time required for the prothrombin complex activity to return to 60% (TPCA60). The coagulation recovery time of the liposomal preparation was much faster than that of the other preparations and it was compared with the response to intravenous administration in which the vitamin was considered to be 100% available. The TPCA60 for the intravenous administration was 1.9 h, that for the oral liposomal preparation was 6.2 h, HCO-60 solubilized vitamin 13.6 h, HCO-60 stabilized emulsion 19.6 h, the physical mixture 17.8 h and plain emulsion 18.2 h. The vitamin K1 dose was maintained at 12 mg kg-1 in each instance.

Disulfide bond cleavage of human serum albumin and alterations of its secondary structure by cis-diamminedichloroplatinum(II)

Abstract cw -Diamminedichloroplatinum(II) ( cis -DDP) cleaved disulfide (S-S) bonds in human serum albumin (HSA) and brought about alterations of the secondary structure. The α-helix content decreased from 50.5% (native) to approx. 33% (four S-S bonds cleaved). The tendency toward a decrease corresponded only with an increase in the β-sheet. Sulfitolysis of the S-S bonds showed a tendency similar to that of metal binding. Fluorescence and UV difference spectra changed as a function of S-S bond cleavage and led to considerable differences between the two cleaving agents.

Antitumor effect of liposome-entrapped adriamycin administered via the portal vein

We examined the distribution in tissues and antitumor effect of freeze‐dried liposome‐entrapped adriamycin (Lipo‐ADM) administered via the portal vein to rabbits bearing VX2 tumors. Liposomes composed of egg phosphatidylcholine (cholesterol 50 mol%) were used as drug carriers. The liver concentration of ADM increased after delivery and cardiac uptake decreased compared with free drug treatment. The in vivo antitumor effect of Lipo‐ADM was determined in rabbits inoculated with VX2 tumor. Repeated injections of free ADM via the portal vein prolonged the life span of tumor‐bearing rabbits. The life span was further prolonged by Lipo‐ADM treatment compared with the control group and the free ADM group. Histological examination revealed that the damage to the liver caused by Lipo‐ADM administered via the portal vein did not differ from that observed in animals treated with free ADM. These results indicate that portal vein administration of Lipo‐ADM may be more effective in dealing with liver metastases than treatment with free ADM and may be therapeutically useful without toxic side effects.