Mitsuko Takenaga

Insulin-loaded biodegradable PLGA microcapsules: initial burst release controlled by hydrophilic additives

We investigated the controlled release of human insulin at an initial stage from poly(DL-lactic-co-glycolic acid) (PLGA, M(w) 6600) spherical matrices. PLGA microcapsules were prepared by the novel solvent evaporation multiple emulsion process. When the crystalline insulin was dispersed in dichloromethane as solid-in-oil (S/O) dispersion, it was found that most of insulin molecules were inlaid on the surface of PLGA microcapsules. Consequently, insulin-loaded PLGA microcapsules exhibited marked rapid release of insulin within several hours in both in vivo and in vitro experiments. On the other hand, the addition of glycerol or water in the primary dichloromethane dispersion results in drastically suppressed initial release. It was found by SEM observation that water- or glycerol-in-oil (W/O or G/O) type mini-emulsion droplets with a mean diameter of 300-500 nm were formed in this primary solution. This phenomenon can be theoretically presumed to occur because insulin and PLGA molecules, having amphiphilic properties, converge on the interface between the hydrophilic additive and dichloromethane. Hence, insulin molecules heterogeneously located in the inside of PLGA microcapsules, not on the surface, would be gradually released with PLGA hydrolytic decomposition. As an additional effect of glycerol, the initial burst was further suppressed due to the decrease of the glass transition temperature of PLGA from 42.5 to 36.7 degrees C. Since the annealing of PLGA molecules took place at around 37 degrees C, the porous structure of microspheres immediately disappeared after immersion in PBS or subcutaneous administration. The insulin diffusion through the water-filled pores would be effectively prevented. The strict controlled initial release of insulin from the PLGA microsphere suggested the possibility of utilization in insulin therapy for type I diabetic patients who need construction of a basal insulin profile.

Therapeutic Effect of Lecithinized Superoxide Dismutase against Colitis

Ulcerative colitis (UC) involves intestinal mucosal damage induced by reactive oxygen species (ROS), in particular, superoxide anion. Superoxide dismutase (SOD) catalyzes dismutation of superoxide anion to hydrogen peroxide, which is subsequently detoxified by catalase. Lecithinized SOD (PC-SOD) is a new modified form of SOD that has overcome previous clinical limitations of SOD. In this study, we examined the action of PC-SOD using an animal model of UC, dextran sulfate sodium (DSS)-induced colitis. DSS-induced colitis was ameliorated by daily intravenous administration of PC-SOD. Unmodified SOD produced a similar effect but only at more than 30 times the concentration of PC-SOD. In vivo electron spin resonance analysis confirmed that the increase in the colonic level of ROS associated with development of colitis was suppressed by PC-SOD administration. The dose-response profile of PC-SOD was bell-shaped, but simultaneous administration of catalase restored the ameliorative effect at high doses of PC-SOD. Accumulation of hydrogen peroxide was observed with the administration of high doses of PC-SOD, an effect that was suppressed by the simultaneous administration of catalase. We also found that either a weekly intravenous administration or daily oral administration of PC-SOD conferred protection. These results suggest that PC-SOD achieves its ameliorative effect against colitis through decreasing the colonic level of ROS and that its ineffectiveness at higher doses is because of the accumulation of hydrogen peroxide. Furthermore, we consider that intermittent or oral administration of PC-SOD can be applied clinically to improve the quality of life of UC patients.

A novel sustained-release formulation of insulin with dramatic reduction in initial rapid release.

To ensure a strictly controlled release of insulin, a preparation method for insulin-loaded microcapsules was designed. Microcapsules were prepared with an injectable, biodegradable polymer composed of co-poly(D,L-lactic/glycolic) acids (PLGA) (mean molecular weight 6600, LA/GA ratio 50:50). Morphological examination using scanning electron microphotography demonstrated spherical particles with a main diameter of 15-30 microm. When 3% insulin-loaded PLGA microcapsules were administered subcutaneously as a single dose (250 U/kg) to streptozotocin-induced hyperglycemic rats, plasma insulin levels increased and were sustained at levels showing hypoglycemic effects. When glycerin, ethanol, or distilled water was used throughout the preparation procedure, the resultant microcapsules dramatically reduced the initial burst. The formulation in which glycerin was added to an oil phase containing PLGA, insulin, and ZnO increased plasma insulin levels to 86.7, 108.4, and 84.9 microU/ml at 1, 2, and 6 h, respectively. The levels remained at 36.2-140.7 microU/ml from day 1 to day 9. The AUC(0-24 h)/AUC(0-336 h) ratio was calculated to be 9.7%. The formulation prepared without additives gave such a rapid insulin release that animals receiving it became transiently hypoglycemic.

Mature adipocyte-derived cells, dedifferentiated fat cells (DFAT), promoted functional recovery from spinal cord injury-induced motor dysfunction in rats.

Transplantation of mature adipocyte-derived cells (dedifferentiated fat cells) led to marked functional recovery from spinal cord injury (SCI)-induced motor dysfunction in rats. When mature adipocytes were isolated from rat adipose tissue and grown in ceiling culture, transformation into fibroblast-like cells without lipid droplets occurred. These fibroblast-like cells, termed dedifferentiated fat cells (DFAT), could proliferate and could also differentiate back into adipocytes. DFAT expressed neural markers such as nestin, βIII tubulin, and GFAP. Allografting of DFAT into SCI-induced rats led to significant recovery from hindlimb dysfunction. Grafted cells were detected at the injection site, and some of these cells expressed βIII tubulin. DFAT expressed neurotrophic factors such as BDNF and GDNF prior to transplantation, and grafted cells were also positive for these factors. Therefore, these neurotrophic factors derived from grafted DFAT might have contributed to the promotion of functional recovery. These findings also suggest that mature adipocytes could become a new source for cell replacement therapy to treat central nervous system disorders.

Microparticle resins as a potential nasal drug delivery system for insulin.

The application of various resins for the nasal delivery of insulin was examined in rabbits. Intranasal administration of human insulin (28 U, 1 mg) mixed with fractionated sodium polystyrene sulfonate powder (an anionic resin with a particle size of 20-45 microns) caused a rapid increase of the plasma insulin level 413.0 +/- 71.7 microU/ml (mean +/- S.D.) after 15 min, while intranasal administration of insulin alone caused little increase. The blood glucose level decreased from 118.8 +/- 18.5 mg/dl to 65.8 +/- 13.8 mg/dl at 45 min after administration. These results were superior to those obtained with the unfractionated resin. Styrene-divinylbenzene copolymer (a nonionic resin; 20-45 microns fraction) showed similar enhancement of nasal insulin absorption. In contrast, polyacrylester (a nonionic resin; 20-45 microns fraction) and cholestyramine (a cationic resin) did not promote insulin absorption. These results suggest that some resins may be useful for nasal delivery of insulin.

Application of lipid microspheres for the treatment of cancer

Abstract Lipid microspheres can act as a carrier for antitumor agents. We incorporated a lipophilic antitumor agent, l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) into microspheres by homogenizing a soybean oil solution of BCNU with egg yolk lecithin. Lipid microsphere-encapsulated BCNU showed a significantly enhanced antitumor activity with reduced toxicity in mice with L1210 leukemia when compared to the corresponding dose of free BCNU. Lipid nanospheres, smaller particles containing BCNU with an average size of 50 nm, also showed a similar level of in vivo antitumor activity. An in vitro study showed that [ 14 C]triolein uptake by tumor cells was increased by incorporation into microspheres. The in vitro uptake of small microspheres was lower than that of standard microspheres. However, the in vivo half-life of small microspheres was longer, they avoided capture by the reticuloendothelial system and showed higher accumulation at tumor sites. Thus, lipid microspheres may be useful for delivering various lipophilic chemotherapy agents.

A stable PGE1 prodrug for targeting therapy

Abstract Lipo-PGE1 is a passively targetable prostaglandin E1 (PGE1) preparation, in which PGE1 is incorporated into lipid microspheres (LM); it has been widely used in Japan since 1988 for the treatment of various vascular diseases. Passive targeting results from the accumulation of LM in the vascular lesions following intravenous administration. However, this preparation has two major disadvantages. One is the chemical instability of PGE1 in the LM and the other is the rapid leakage of PGE1 from the LM in the blood, leading to a decrease in the targeting of this drug. We accordingly synthesized several PGE1 prodrugs and evaluated them to develop a better LM preparation. Among the PGE1 prodrugs tested, Δ8-9-O-butyryI prostaglandin F1 butyl ester (AS013) was readily hydrolyzed to PGE1 in human serum. AS013 was stable as an LM preparation and 80% of this ester was recovered unchanged even after storage as LM-ASOI3 for 4 weeks at 40°C. In contrast, PGE1 showed a recovery of only 5% from the LM under the same conditions. Moreover, AS013 was more effectively retained in the LM after incubation with human blood or serum when compared with PGE1. These results suggested that LM-AS013 would be a far superior LM preparation than lipo-PGE1 for clinical use.

Lecithinized superoxide dismutase (PC-SOD) improved spinal cord injury-induced motor dysfunction through suppression of oxidative stress and enhancement of neurotrophic factor production

PC-SOD (lecithinized superoxide dismutase) is a derivative of human Cu, Zn-SOD conjugated with 4 molecules of lecithin, yet having the enzyme activity of scavenging superoxide anion (O2-). Intravenous administration of PC-SOD promoted the recovery from spinal cord injury (SCI)-induced motor dysfunction in a dose-dependent manner in rat model, when evaluated by BBB (Basso Beattie Bresnahan) score. Even when given at 24 h after SCI, PC-SOD (1 mg/kg) significantly improved motor dysfunction. Distribution study demonstrated that PC-SOD gradually accumulated to the injured site. Enzyme-linked immunoassay revealed that PC-SOD prevented quantitative loss of neurons, astrocytes, and oligodendrocytes. PC-SOD inhibited SCI-induced oxidative stress, such as the decrease of free sulfhydryl residue, acetylcholine esterase activity, and the increase of lipid peroxidation. PC-SOD increased the production of neuroprotective factors. HIF-1alpha gene expression increased following SCI, and PC-SOD further increased it. In conclusion, PC-SOD gradually accumulated and retained at the damaged site to scavenge excessive O2-, and suppressed neuronal death through reducing oxidative stress, increasing neuroprotective factor production and HIF-1alpha gene expression.

Prolonging the in vivo residence time of prostaglandin E1 with biodegradable nanoparticles

PurposeProstaglandins have potent and diverse biologic activities, but their clinical application is severely restricted, mainly due to rapid inactivation in vivo. In order to modulate the pharmacokinetics of prostaglandin E1 (PGE1), we prepared biodegradable nanoparticles as a drug carrier.MethodsNanoparticles encapsulating PGE1 were prepared from a blend of poly(lactic acid) homopolymer and poly(ethylene glycol)-poly(lactide) block copolymer by the solvent diffusion method in the presence of iron.ResultsPGE1 was efficiently and stably embedded in the nanoparticles through interaction with iron, despite being relatively hydrophilic and having unstable chemical properties. Depending on the isomers and molecular weight of poly(lactic acid) selected, PGE1 was gradually released from the nanoparticles at various rates into diluted serum in vitro. Both stable retention of PGE1 in the nanoparticles and coating of the nanoparticles with poly(ethylene glycol) led to an extremely extended blood residence time of PGE1, as well as preferential accumulation in vascular lesions.ConclusionsThese results suggest that the present strategy is useful to advance the clinical application of PGE1 as a therapeutic agent for vascular disorders.

Marked hypotensive and blood flow-increasing effects of a new lipo-PGE1 (lipo-AS013) due to vascular wall targeting

Lipo-AS013 is being developed as an improved formulation of lipo-PGE(1), which is widely used in clinical practice in Japan and some Asian countries. We have previously reported that lipo-AS013, which is a lipid microsphere (LM) preparation of a chemically stable and lipophilic PGE(1) prodrug (AS013, Fig. 1), slowly releases small amounts of the active ingredient (AS013) in human plasma. In the present study, to estimate the vascular wall targeting ability and efficacy of lipo-AS013, we determined the hypotensive and blood flow-increasing effects of lipo-AS013, lipo-PGE(1), PGE(1)CD, and AS013. Lipo-AS013 was found to have longer-lasting hypotensive and blood flow-increasing effects than the other agents. The two LM preparations, lipo-PGE(1) and lipo-AS013, had a markedly stronger effect than PGE(1)CD and AS013 alone, demonstrating the benefit of drug delivery using LM. In spontaneously hypertensive rats (SHR), lipo-AS013 also had a significant hypotensive effect. To confirm vascular wall targeting by lipo-AS013, the localization of PGE(1) in the aorta and neovascular capillaries of rat was investigated by immunostaining. The results indicated that lipo-AS013 was more efficient at delivering the active ingredient (AS013) to the vessel wall. In conclusion, lipo-AS013 could supersede lipo-PGE(1) and PGE(1)CD in clinical use.