Hideto To

Ternary complexes of pDNA, polyethylenimine, and γ-polyglutamic acid for gene delivery systems

We discovered a vector coated by gamma-polyglutamic acid (gamma-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic-polycytidylic acid, alpha-polyaspartic acid, alpha-polyglutamic acid, and gamma-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely high transgene efficiency although it agglutinated with erythrocytes and had extremely high cytotoxicity. Those polyanions changed the positive zeta-potential of pDNA/PEI complex to negative although they did not affect the size. They had no agglutination activities and lower cytotoxicities but most of the ternary complexes did not show any uptake and gene expression; however, the pDNA/PEI/gamma-PGA complex showed high uptake and gene expression. Most of the pDNA/PEI/gamma-PGA complexes were located in the cytoplasm without dissociation and a few complexes were observed in the nuclei. Hypothermia and the addition of gamma-PGA significantly inhibited the uptake of pDNA/PEI/gamma-PGA by the cells, although l-glutamic acid had no effect. These results strongly indicate that the pDNA/PEI/gamma-PGA complex was taken up by gamma-PGA-specific receptor-mediated energy-dependent process. Thus, the pDNA/PEI/gamma-PGA complex is useful as a gene delivery system with high transfection efficiency and low toxicity.

The development of a gene vector electrostatically assembled with a polysaccharide capsule.

The purpose of this study was to develop a gene vector electrostatically assembled with a polysaccharide capsule. We used pDNA/polyethylenimine (PEI) complexes as efficient non-viral vectors. The pDNA/PEI complex was electrostatically encapsulated with various polysaccharides such as fucoidan, lambda-carrageenan, xanthan gum, alginic acid, hyaluronic acid, and chondroitin sulfate (CS). The pDNA/PEI complex was shown as nanoparticles with positive zeta-potential, although the ternary complexes encapsulated with polysaccharides were shown as nanoparticles with negative zeta-potential. The pDNA/PEI complex showed high agglutination activity and cytotoxicity, although the ternary complexes encapsulated with polysaccharides had no agglutination activities and lower cytotoxicities. The pDNA/PEI complex showed high uptake and high transgene efficiency in B16-F10 cells. On the other hand, most of the ternary complexes show little uptake and gene expression. The ternary complex encapsulated by CS, however, showed comparable transgene efficiency to the pDNA/PEI complex. The uptake and gene expression of the ternary complex encapsulated by CS were significantly inhibited by hypothermia and the addition of CS, suggesting that the ternary complex was taken by CS-specific receptor-mediated energy-dependent process.

Chronobiological disturbances with hyperthermia and hypercortisolism induced by chronic mild stress in rats.

The chronic mild stress (CMS) model has been established as a realistic model of depressive disorder as it simulates anhedonia. In the present study, the consumption of sucrose solution was decreased in the rats exposed to CMS, which coincided with many published studies. Since depression is a multifaceted disorder, and a number of symptoms may be present, including circadian rhythm disturbances, we attempted to find the chronobiological abnormalities in CMS rats. After 4-week of the stress procedure, the rhythmic pattern of rectal temperature in the CMS group was extinguished. In particular, the temperature in the CMS group in the light phase was significantly higher than that in the control group. The plasma corticosterone levels in the CMS group were remarkably increased in the light phase compared to the control group, but not in the dark phase. It was concluded that the CMS procedure caused the disturbance of circadian rhythms with hyperthermia and hypercortisolism.

Population-Based Investigation of Valproic Acid Relative Clearance Using Nonlinear Mixed Effects Modeling: Influence of Drug-Drug Interaction and Patient Characteristics

Nonlinear mixed effects modeling (NONMEM) was used to estimate the effects of drug‐drug interaction on valproic acid relative clearance values using 792 serum levels gathered from 400 pediatric and adult patients with epilepsy (age range, 0.3–54.8 years) during their clinical routine care. Patients received valproic acid as monopharmacy or in combination with either the antiepileptic drugs, phenobarbital, or carbamazepine. The final model describing valproic acid relative clearance was CL (mL/hr/kg) = 15.6 · TBW (kg)−0.252 · DOSE (mg/kg/day)0.183 · 0.898GEN · COPB · COCBZ, where COPB equals 1.10 if the patient is treated with phenobarbital, a value of unity otherwise, and COCBZ equals 0.769 · DOSE (mg/kg/day)0.179 if the patient is treated with carbamazepine, a value of unity otherwise. Valproic acid relative clearance was highest in the very young and decreased in a weight‐related fashion in children, with minimal changes observed in adults. This pattern was consistent whether valproic acid was administered alone or coadministered with phenobarbital or carbamazepine. When valproic acid was coadministered with phenobarbital or carbamazepine, valproic acid relative clearance increased as compared with that in monopharmacy. Its magnitude in the presence of carbamazepine increased in a valproic acid daily dose‐related fashion. Concomitant administration of phenobarbital and valproic acid resulted in a 10% increase on valproic acid relative clearance. The clearance in female patients was approximately 10% less than that in male patients.

Methotrexate Chronotherapy is Effective Against Rheumatoid Arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time–dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p = .0197, 2 mos: p = .0107, 3 mos: p = .0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods. (Author correspondence: hide-to@umin.net)

Dosing time dependency of doxorubicin-induced cardiotoxicity and bone marrow toxicity in rats.

Cardiac toxicity caused by doxorubicin (adriamycin) is a serious dose‐limiting factor in the clinical situation. However, the influence of doxorubicin dosing time has not been clarified from the viewpoints of cardiotoxic development and its mechanism. In this study, we have investigated the dosing time dependency of doxorubicin‐induced cardiotoxicity and bone marrow toxicity after repeated administration of doxorubicin in rats. When doxorubicin (5 mg kg−1, i.p.) was administered every seven days (total of 30 mg kg−1) at 3, 9, 15 or 21h after the light was turned on (HALO), toxic death was significantly higher in the 9 HALO treated group than the other groups. When doxorubicin was injected every seven days for 28 days at 9 or 21 HALO, we measured the levels of creatine kinase, malondialdehyde (MDA; an index of lipid peroxide), and glutathione peroxidase (GPx) as markers of cardiotoxicity. On days 14 and 28, creatine kinase levels were significantly higher in the 9‐HALO group compared with the 21‐HALO group (P < 0.01, respectively). On day 14, MDA levels increased significantly in the 9 HALO group compared with the 21 HALO group (P < 0.01). A single dose of doxorubicin was administered at 9‐h or 21‐h after the light was turned on to investigate the dosing‐time‐dependent difference of the pharmacokinetics. The area under the plasma time‐concentration curve showed a significant increase at 9 HALO compared with 21 HALO (P < 0.05). These results suggested that the dosing‐time‐dependent difference of cardiotoxicity induced by doxorubicin was closely related to the daily variation of doxorubicin pharmacokinetics. In conclusion, the choice of optimal dosing time based on the chronopharmacokinetics of doxorubicin may decrease the cardio‐toxicity and enable the practice of effective and safe chemotherapy of doxorubicin.

Effect of Haloperidol on mPer1 Gene Expression in Mouse Suprachiasmatic Nuclei

The effect of a typical neuroleptic haloperidol (Hal) on mPer1 gene expression was investigated in mouse suprachiasmatic nuclei (SCN). Hal induced mPer1 mRNA levels both in vivo and in cultured SCN cells. For mechanisms underlying Hal-induced mPer1 expression, N-methyl-d-aspartate (NMDA) glutamate receptor subtype, the phosphorylation form of the transcription factor, and the Ser-133 phosphorylation form of cAMP-responsive element-binding protein (CREB) played an important role, because the induction of mPer1 mRNA significantly decreased after pretreatment with a non-competitive NMDA receptor antagonist, such as MK-801 or CREB antisense. These results suggest that Hal may increase CREB phosphorylation and mPer1 expression according to the activation of the NMDA receptor through the dopaminergic pathways. Although the injection of Hal during the light period increased the amplitude of mPer1 mRNA rhythmicity in a nondrug state, the injection of the drug during the dark period disturbed the rhythmic pattern of mPer1 mRNA. These results suggest that the rhythmicity of clock genes in SCN may be disturbed depending on the dosing time of Hal. On the other hand, because the induction of mPer1 mRNA by Hal seems to be at least partly caused by the NMDA receptor, showing a phase shift or resetting effect of the circadian clock, Hal may also cause such phase shift effects.

Simplified method of heterotopic rat heart transplantation using the cuff technique: application to sublethal dose protocol of methotrexate on allograft survival.

Rodent heterotopic heart transplantation (HHT) models have been developed for the study of transplantation immunology. Most of these transplantations are performed by hand‐suture techniques, requiring several months of training. We describe a modified technique of rat HHT in the neck, using a cuff method that can be mastered by beginners within a few weeks. Our main modification of the rat HHT in the neck is that the right superior vena cava of the graft is chosen as an outflow duct, while the pulmonary artery has been taken as an effluent drainage in the ordinary HHT models. The aorta of the donor is anastomosed with the carotid artery of the recipient. Donation can be completed within 5 min and vascular connections in the recipient done within 3 min, resulting in a minimum of ischemia time. Using this minimum surgical intervention model, we tested the immunosuppressive effect of a sublethal dose of methotrexate (MTX), which has been widely used in cancer therapy. Our results showed that high doses of MTX severely suppressed the recipient bone marrow, but prolonged heart allografts for more than 365 days after HHT. In conclusion, the new model simplified the rat HHT procedure and made it possible for the beginner of rodent transplantation to master this skill within a few weeks. Using this minimized intervention technique, we found that the high doses of MTX can significantly prolong the survival of fully mismatched DA heart graft in PVG/c recipient.

Therapeutic index of methotrexate depends on circadian cycling of tumour necrosis factor‐α in collagen‐induced arthritic rats and mice

Objectives Rheumatoid arthritis is an autoimmune disorder of unknown aetiology. Morning stiffness, a characteristic feature of rheumatoid arthritis, shows a 24‐h rhythm. Noticing this rhythm, we hypothesized the presence of a similar rhythm for a rheumatoid arthritis indicator, in addition to dosing‐time dependency of the anti‐rheumatic effect of methotrexate in arthritis induced by collagen in rats and mice, which reflect the symptomatology of rheumatoid arthritis patients.

Breakdown evaluation of corneal epithelial barrier caused by antiallergic eyedrops using an electrophysiologic method.

AIM The aim of this study was to examine the usefulness of an electrophysiologic method for predicting corneal epithelial breakdown by antiallergic eyedrops and comparing the results with those in other appraisal methods. METHODS Six kinds of antiallergic eyedrops, including benzalkonium chloride (BK) as an ophthalmic preservative and two kinds of BK-free antiallergic eyedrops, were used in this study. Eyedrops were applied to excise rabbit corneas and monitoring was performed according to an electrophysiologic method, using a commercially available chamber system to mimic human tear turnover. Changes in transepithelial electrical resistance (TEER) in the corneal surface were recorded. The cytotoxicity of each kind of eyedrops in a normal rabbit corneal epithelial (NRCE) cell line and a human endothelial cell line EA.hy926 was also examined. RESULTS The extent of decrease in the corneal TEER after applying antiallergic eyedrops was dependent on the concentration of the BK included as a preservative, but it was also affected by the different kinds of drugs when the BK concentration was low. Higher cytotoxicity of the eyedrops against the NRCE and EA.hy926 cell lines was observed with a reduction of TEER. CONCLUSIONS Monitoring changes in the corneal TEER, according to the electrophysiologic method with the application of antiallergic eyedrops, is useful for predicting corneal epithelial breakdown caused by their instillation.