Ryo Matsushita

A Candidate for Cancer Gene Therapy: MIP-lα Gene Transfer to an Adenocarcinoma Cell Line Reduced T\imorigenicity and Induced Protective Immunity in Immunocompetent Mice

AbstractPurpose. To evaluate the possibility of cancer gene therapy by the gene delivery of chemokine, the effects of human macrophage inflammatory protein lα (hu-MIP-lα), murine-macrophage inflammatory protein lα (mu-MIP-lα), and human-interleukin 8 (hu-IL-8) on tumor progression and immunization were studied. Methods. Cachexia-inducing and highly tumorigenic adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid, hu-MIP-lα, mu-MIP-lα, or hu-IL-8 expression vector. The production of hu-MIP-1α reached >1.5 ng/ml in vitro when transfectant cells were cultured at a cell density of 2 × 105 cells in 7 ml for 3 days. Immunocompetent BALB/c mice were inoculated into the footpad with the tumor cells, and then primary tumor growth, morphological analyses, and tumor immunogenicity were studied. Results. The secretion of hu-MIP-lα, mu-MIP-lα, and hu-IL-8 did not affect the growth rate in vitro. Reduced tumorigenicities in vivo were observed in transfected cells with hu-MIP-lα and mu-MIP-lα. Morphologic observation of the site of inoculation of cells transfected with hu-MIP-lα showed infiltration of macrophages and neutrophils on the 5th day after the inoculation. Mice that had rejected cells transfected with hu-MIP-lα gene were immune to a subsequent challenge with the parental cells. Conclusions. The rejection of the cells depends on cytolysis and generates potent and long lasting antitumor immunity. These data suggest that tumor cells transfected with the MIP-lα gene might be useful as an effective therapy for the treatment of certain tumors.

Effect of Meropenem on Disposition Kinetics of Valproate and Its Metabolites in Rabbits

AbstractPurpose. We investigated the effect of meropenem (MEPM) on the disposition kinetics of valproate (VPA) and its metabolites in rabbits. Methods. Rabbits were given 75 mg/kg VPA intravenously with or without 300 mg/kg MEPM. Results. The plamsa total clearance of VPA was significantly increased to about 1.5 times the control (6.09 mL/min/kg vs. 4.28 mL/min/kg) by MEPM (P < 0.05). The values of the area under the plasma concentration-time curve (AUC) of 2-en-VPA, a product of β-oxidation, and VPA-glucuronide (VPA-G) were significantly decreased to about 55% and 78% of the control, respectively (P < 0.05). The cumulative urinary excretions of VPA in the control and MEPM-treated groups were 0.54% and 0.62% of the dose, respectively, whereas those of VPA-G were 45.6% and 62.5%, respectively. The urinary excretion of VPA-G was significantly increased by MEPM (P < 0.05). Further, in the case of 33.8 mg/kg VPA-G administered intravenously the AUC value of VPA-G was unchanged by MEPM, whereas that of the generated VPA was significantly decreased to about half of the control. Conclusions. The increase of the total clearance of VPA caused by MEPM appears to be a consequence of increased renal clearance of VPA-G, as well as suppression of VPA-G hydrolysis in the liver.

Pharmacist-based Donepezil Outpatient Consultation Service to improve medication persistence

Aim Donepezil is widely used to delay the progression of cognitive dysfunction in patients with Alzheimer’s disease (AD), but the efficacy of pharmacotherapy is often reduced by poor adherence to medication. In order to improve adherence by providing information about AD and the significance of pharmacotherapy, the Donepezil Outpatient Consultation Service (DOCS) was set up. The influence of this service on medication persistence was assessed in the present study. Methods Among outpatients starting donepezil therapy, we enrolled 59 patients between April 2008 and September 2010 before establishment of the DOCS (non-DOCS group) and 52 patients between October 2010 and March 2012 who attended the DOCS (DOCS group). Each patient’s and their caregiver’s understanding about the clinical features of AD and pharmacotherapy with donepezil were also assessed. Their understanding was compared before and after the DOCS, and the 1-year medication persistence rate and the reasons for discontinuation were also investigated. Results The 1-year medication persistence rate was significantly higher in the DOCS group than in the non-DOCS group (73.1% vs 49.2%, P = 0.008). We examined the association of medication persistence with age, sex, clinical dementia rating, living alone, and attending the DOCS. As a result, medication persistence was significantly higher in patients attending the DOCS. The main reasons for discontinuation of donepezil were transfer elsewhere (11) and gastrointestinal side effects (5) in the non-DOCS group, and transfer (9) and gastrointestinal side effects (3) in the DOCS group. The overall score for understanding was 2.5 ± 1.7 before attending the DOCS and it increased significantly to 5.7 ± 0.7 afterward (P < 0.001). Conclusion The DOCS consultation provided by hospital pharmacists for AD patients and their caregivers improved understanding about the clinical features of dementia and provided pharmacological knowledge about antidementia drugs, leading to better adherence to pharmacotherapy that could maximize its effect.

Synergistic antitumor interaction of human monocyte chemotactant protein-1 gene transfer and modulator for tumor-infiltrating macrophages

AbstractPurpose. In order to evaluate the possibility of synergistic antitumor gene therapy by the gene delivery of monocyte chemotactant protein-1 (MCP-1/MCAF/IE), the effect of a biological response modulater for macrophages on tumor progression of gene transfected tumor cells was studied. Methods. Cachexia-inducing adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid or MCP-1 cDNA. Results. The production of MCP-1 reached 70-80 ng/ml in vitro when transfectant cells were cultured at a cell density of 1 × 105 cells/ml for 3 days. Transfection of MCP-1 cDNA did not affect the growth ratein vitro. Also, MCP-1-transfectants formed tumors after intra-footpad inoculation similar in size to the parental cells. The number of infiltrating macrophages in the primary tumor of the transfectant rapidly increased from the 3rd to 5th day after inoculation as revealed by immunohistochemical staining using an antibody against mouse macrophages. An earlier, greater, but no longer-lasting increase in tumor-infiltrating macrophages was induced in tumors by MCP-1 transfection was compared to that induced by the parent cells. On the 10th day after the inoculation, the tumor-infiltrating macrophages in mice inoculated MCP-1 transfectants were decreased to a level similar to that of the parent cells. Groups of mice were treated intraperitoneally with LPS at different times after the inoculation. Tumor cells producing high levels of MCP-1 were significantly lysed by macrophages treated with LPS, whereas parental or control transfected cells were not. Conclusions. Combination immunotherapy can provide a rationale for the application of MCP-1 treatment to increase immunological responses to cancer.

Byakkokaninjinto prevents body water loss by increasing the expression of kidney aquaporin-2 and skin aquaporin-3 in KKAy mice.

Byakkokaninjinto (BKN) is an herbal medicine used for the relief of diuresis, thirst and dermal pruritis that are associated with diabetes. The effects of BKN on the expression of aquaporins (AQPs) in the kidney, salivary gland and skin were investigated in order to clarify the mechanism of drug action. Seven‐week‐old KKAy mice were given feed containing 4.5% BKN for 4 weeks. Compared with the control group, BKN administration did not affect the blood glucose and insulin concentration. However, water intake and urine volume were significantly reduced. AQP2 protein expression in the kidney inner medullary was significantly increased after BKN administration. AQP3 mRNA and protein expression in skin tissue was significantly increased after BKN administration. However, BKN administration did not affect AQP5 mRNA expression in the salivary gland. These results suggest that BKN treatment relieves diuresis, thirst, and dermal pruritis by increasing kidney AQP2 expression and skin AQP3 expression. Copyright

Kinetic phenotypic diagnosis of N-acetylation polymorphism in patients based on ratio of urinary metabolites of salicylazosulfapyridine.

We found that N-acetylation polymorphism can be evaluated from the disposition kinetics of sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA) and their acetylated metabolites generated by N-acetyltransferase (NAT2) after oral administration of salicylazosulfapyridine (SASP). In 126 Japanese subjects, the homozygote of NAT2*4 was the most frequent (40%), followed by heterozygotes of NAT2*4 and mutant genes (28% NAT2*4/*6A, 15% NAT2*4/*7B, and 2% NAT2*4/*5B). Combinations of mutant genes accounted for 16%. When the relationship between the molar ratio of N-acetyl-SP (Ac-SP)/SP or N-acetyl-5-ASA(Ac-5-ASA)/5-ASA in serum and five genotypes of polymorphic NAT2* was examined in patients who received multiple doses of SASP, the molar ratios of Ac-SP/SP, rather than Ac-5-ASA/5-ASA tended to decrease according to the classification of genotype. We calculated the pharmacokinetic parameters in healthy subjects with various genotypes of polymorphic NAT2* after a single p.o. administration of SASP, according to a model of the SP metabolic pathways. The molar ratios of Ac-SP/SP in serum and urine were simulated using these parameters, and the molar ratio of Ac-SP/SP in urine at 4 days after the first administration could be categorized into ranges that were specific to various NAT2* genotypes. Thus, we were able to predict the N-acetylation polymorphic genotypes of patients by measuring the molar ratio of Ac-SP/SP in urine, after administration of SASP.

Causative agent of vascular pain among photodegradation products of dacarbazine.

The photodegradation products of the anticancer drug, dacarbazine, cause adverse reactions including local venous pain when injected intravenously. In this study, we attempted to identify which of these products is responsible. We synthesized or purchased five photodegradation products of dacarbazine (dimethylamine, 5‐diazoimidazole‐4‐carboxamide (Diazo‐IC), 4‐carbamoylimidazolium‐5‐olate, 5‐carbamoyl‐2‐(4‐carbamoylimidazol‐5‐ylazo) imidazolium‐5‐olate and 2‐azahypoxanthine) and examined the pain reaction induced by their intraperitoneal administration in mice using an abdominal stretching or constriction assay. Only Diazo‐IC clearly induced pain reaction in mice in a dose‐dependent manner, the other products caused no pain reaction. The threshold concentration for pain reaction in mice was estimated to be about 0.1 mg mL−1. While diclofenac sodium significantly reduced acetic‐acid‐induced pain reaction in mice, it did not influence those induced by Diazo‐IC. This result suggests that the mechanism of Diazo‐IC‐induced pain is different from that of acetic‐acid‐induced inflammatory pain. Dacarbazine itself produced marked relaxation of rat thoracic aorta strips in a concentration‐dependent manner, but there was no difference between the activity of dacarbazine and its photo‐exposed solution, so constriction or relaxation of blood vessels is unlikely to be a factor in the pain reaction. In conclusion, Diazo‐IC generated by photodegradation of dacarbazine solution causes the side‐effect of venous pain. Dacarbazine solution that has turned pink should not be used, because Diazo‐IC is an intermediate in the formation of the reddish product, 5‐carbamoyl‐2‐(4‐carbamoylimidazol‐5‐ylazo) imidazolium‐5‐olate. Drip infusion preparations of dacarbazine should be shielded from light.

The Usefulness of Inspiratory Flow Rate during Inhalation Corticosteroid Therapy in Asthma

Background: The recently released handheld In-Check device can be used to measure the peak inspiratory flow rate (PIF) of patients and is reportedly useful in determining whether the PIF is sufficient for using inhaler devices. In this study, we evaluated the effects of instructions for the use of the device and of the device type based on measurements of the PIF in asthma. Objectives: One hundred and thirty-five asthmatic patients who used a fluticasone propionate Diskus (FP-DK) or a budesonide Turbuhaler (BUD-TH) were studied. Methods: The PIF was measured by the In-Check device. For patients without a sufficient PIF of 50 l/min, instructions for the use of the device were given, and the device was changed to hydrofluoroalkan-beclomethasone dipropionate (HFA-BDP). Results: A significant correlation between the PIF and peak expiratory flow rate (p < 0.0001) was found. In 10 patients in whom the PIF did not increase to >50 l/min after instructions, the device was changed to HFA-BDP, which resulted in significant improvements in lung function in terms of the forced expiratory volume in 1 s (p = 0.018), peak expiratory flow (p = 0.038) and the maximum expiratory flow rates at 50% (p = 0.018) and 25% (p = 0.011). Conclusions: Measurement of the PIF by the In-Check device is useful in the clinical management of asthma, to provide an appropriate device so as to improve lung function.

Synergistic effect of indomethacin and bleomycin on tumor growth produced by activating antitumor immunity

Cancer cells and macrophages produce large amounts of prostaglandin (PG) E2, which suppresses the cellular immune reaction in tumor-bearing animals (1). These findings suggest that an inhibitor of PG synthesis might be able to restore immune activity against tumors. It has been reported that indomethacin (IND) inhibits tumor growth when administered on its own and exhibits synergistic effects with several other biological response-modifiers in transplanted tumor models (2–4). In clinical studies, it has been reported that anti-inflammatory treatment may prolong the survival of undernourished patients with metastatic solid tumors (5). In addition, several studies have shown that antitumor drugs restore cellular immune responses. In one case, bleomycin (BLM) increased gamma interferon, tumor necrosis factor alpha, and nitric oxide production by macrophages (6). The above reports indicate that a marked synergistic effect on tumor growth is likely to be observed when IND and BLM are coadministered. To examine whether the tumorsuppressive effect of antitumor agents directly involves tumor growth, one well-known and useful approach is to use the severe combined immunodefficiency (SCID) mouse. The SCID mouse was discovered in 1983 and it has subsequently been shown to be defective in both T and B lymphocytes (7). In this report, in an attempt to make chemotherapy more effective, we have examined the effect of coadministration of BLM and IND on tumor growth in normal and SCID mice. MATERIALS AND METHODS

Kinetic evidence for facilitated peritoneal transport of benzoic acid in rats.

To evaluate the dose dependency in apparent peritoneal permeability (Pd) of benzoic acid as a model compound for a monocarboxylic acid transport system, a kinetic model, which involves changes in the volume and osmolality of the dialysate as well as the diffusion and convection of drugs across the peritoneum, was applied.