A survey of junior high school students' sleep habit and lifestyle in Okinawa.

A survey was made of the sleep and lifestyle activity patterns of 3754 students from 14 different junior high schools on Okinawa Island. The survey showed that bedtimes became progressively and significantly later as students ascended to higher grades, resulting in adolescent sleep debt. The later adolescents retired to sleep, there appeared significantly greater numbers suffering from insufficient sleep, who found difficulty in waking up, who arose later in the mornings, and who failed to eat breakfast. The study indicated that along with later bedtimes, staying up late resulted in deterioration of sleep health, poor dietary habits and feeling ill, contributing to sleepiness during class.

Survey of sleep-health and lifestyle of the elderly in Okinawa.

A total of 788 people (aged from 60 to 93 years) living in the Okinawa prefecture, renowned for the long life expectancy of its citizens, were randomly chosen as subjects. A questionnaire based on lifestyle and sleep-health was distributed. Following this, subjects were chosen in groups of nine from the good sleep-health group and then from the poor sleep-health group based on the results of the survey; their physical activities were then recorded using actigraphs for 1 week. This study suggested that lifestyle, such as exercise, walking and short naps, occupies an important position in the maintenance and improvement of sleep-health. It also verified the reliability of the sleep-health risk index and the questionnaire.

Sterility assurance of microspheres.

The International Conference on Harmonization (ICH) provides a forum for constructive dialogues between regulatory authorities and the pharmaceutical industry on the real and perceived differences in the technical requirements for product registration in the EU, US and Japan. Achievement obtained so far is beyond expectation, having strong impacts both favorable and unfavorable on Japanese regulatory authorities and the pharmaceutical industry. The ICH guidelines are very science-oriented and little consideration seems to have been paid to the cultural and legal differences among the three regions. An example of such a difference is the interpretation of the guidelines between the US and Japan. In the US, they are generally recognized as good examples, whereas in Japan, they are usually taken as minimum and rigid requirements. A more flexible approach to guidelines will be necessary in the development of new dosage forms in Japan. In this paper, how to assure sterility of a new dosage form, that is, once-monthly injectable and biodegradable microspheres of Leuprorelin, a super agonist of LHRH, whose pharmaceutical development the author was in charge of at Takeda Chemical Industries, will be introduced.

Application of a spray drying technique in the production of TRH-containing injectable sustained-release microparticles of biodegradable polymers.

Copoly (dl-lactic/glycolic acid) microparticles for sustained release of a water-soluble drug (Thyrotropin releasing hormone: TRH) were prepared by a spray drying method. A higher entrapment ratio was achieved with the spray drying method with the in-water drying method. In order to avoid agglomeration of the microparticles, a double-nozzle spray drying method was designed using mannitol as an anti-adherent. The surface of the spray-dried microparticles was coated with mannitol, and the extent of agglomeration was decreased. Acetonitrile was the most suitable solvent for microencapsulation using the double-nozzle spray drying method because the initial burst of TRH from the microparticles during the first day was the smallest. When PLGA with a weight-average molecular weight of 14,000 was used, constant release of TRH continued for one month with a small initial burst. In conclusion, the production of biodegradable microparticles by the double-nozzle spray drying method appears to be an attractive alternative to conventional microencapsulation methods.

Antitumor effect of arterial administration of a medium-chain triglyceride solution of an angiogenesis inhibitor, TNP-470, in rabbits bearing VX-2 carcinoma.

Using rabbits bearing VX-2 carcinoma on the inner side of the leg, we examined the antitumor activity of a medium-chain triglyceride (MCT) solution of an angiogenesis inhibitor, TNP-470 (AGM-1470, 6-O-(N-chloroacetylcarbamoyl)-fumagillol), following administration into the femoral artery feeding the tumor. The MCT solution of TNP-470 (1 and 5 mg) strongly suppressed tumor growth following a single intra-arterial (i.a.) injection 2 or 3 weeks after tumor inoculation. Moreover, remarkable regression of well-developed tumors, those 4 weeks after inoculation, was obtained by i.a. injection of the MCT solution containing 20 mg of TNP-470 without any influence on body weight. The antitumor effects were potentiated by coadministration of doxorubicin or mitomycin C (MMC) in the solution or microspheres containing MMC. In a shell-less chorioallantoic membrane (CAM) assay, angiogenesis was inhibited when a droplet of the MCT solution containing 25 micrograms of TNP-470 was placed on the CAM for 2 days, suggesting that the prolonged antitumor effect resulted from the inhibition of tumor neovascularization by sustained drug release from the preparation. These results indicate that i.a. injection of the MCT solution of TNP-470 is promising for treating well-developed tumors.

In vitro and in vivo evaluation of mucoadhesive microspheres prepared for the gastrointestinal tract using polyglycerol esters of fatty acids and a poly(acrylic acid) derivative.

Two types of polyglycerol ester of fatty acid (PGEF)-based microspheres were prepared: Carbopol 934P (CP)-coated microspheres (CPC-microspheres) and CP-dispersion microspheres (CPD-microspheres). Comparative studies on mucoadhesion were done with these microspheres and PGEF-based microspheres without CP (PGEF-microspheres). In an in vitro adhesion test, the CPD-microspheres adhered strongly to mucosa prepared from rat stomach and small intestine because each CP particle in the CPD-microsphere was hydrated and swelled with part of it remaining within the microsphere and part extending to the surface serving to anchor the microsphere to the mucus layer. The gastrointestinal transit patterns after administration of the CPD-microspheres and PGEF-microspheres to fasted rats were fitted to a model in which the microspheres are emptied from the stomach monoexponentially with a lag time and then transit through the small intestine at zero-order. Parameters obtained by curve fitting confirmed that the gastrointestinal transit time of the CPD-microspheres was prolonged compared with that of the PGEF-microspheres. MRT in the gastrointestinal tract was also prolonged after administration of the CPD-microspheres compared with that following the administration of the PGEF-microspheres.

In vitro cytotoxicities and in vivo distribution of transferrin-platinum(II) complex.

In vitro cytotoxic studies of protein-bound cis-diamminedichloroplatinum(II) (CDDP) against human epidermoid carcinoma A431 cells showed that transferrin (Tf)-bound CDDP (Tf-Pt, Pt/Tf 7:1 mol/mol), and human serum albumin (HSA)-bound CDDP (HSA-Pt, Pt/HSA 7:1 mol/mol) exerted antiproliferating activities with IC50 values of 7.2 and 85 microM, respectively. Tf-Pt inhibited the binding of 0.2 nM 125I-labeled human diferric transferrin (hTf(Fe)2) to A431 cells with a inhibition constant (Ki) of 42 nM, whereas HSA-Pt did not. In vivo distribution studies showed that hTf(Fe)2, the Ki of which was 5.3 nM to mouse melanoma B16 cells, was eliminated from plasma biexponentially in the B16-bearing and control mice after intravenous injection at a dose of 87 mg/kg, and AUCplasma values were 29 and 39 mg.h/mL, respectively. In the B16-bearing mice the AUCtumor was 5.6 mg.h/mL, while the AUCs of liver, kidney, and spleen were not distinguishable between the B16-bearing and control mice. Subsequently Tf-Pt (Pt/Tf 3:1 mol/mol) and free CDDP solution were administered intravenously to the B16-bearing mice. The systemic circulation of Pt was significantly prolonged by the administration of the complex. In conclusion, Tf could be a promising carrier protein for the transport of Pt to tumors.

Biodegradable microspheres in drug delivery.

General aspects of biodegradable microspheres prepared from natural and synthesized polymers used in drug delivery systems are reviewed first from various viewpoints: characteristics of biodegradable polymers (physicochemical properties, bioerosion mechanism, biocompatibility), preparation method for the microspheres, drug release from parenteral products and briefly nonparenteral products. The relationship between release pattern and pharmacological activity of therapeutic peptides and proteins and rational controlled release design are also discussed. In the latter half, successful sustained release depot formulations of peptides, leuprorelin acetate, and thyrotropin-releasing hormone (TRH), utilizing poly(lactic acid) (PLA) and poly(lactic/glycolic acid) (PLGA) microspheres are reviewed with respect to preparation, drug release, biocompatibility, pharmacological effects, and results of clinical studies. Thereafter, studies on antitumor therapy by chemoembolization using PLGA microspheres containing an angiogenesis inhibitor (TNP-470) are described as an example of targeted drug delivery with biodegradable microspheres.

Antitumor activity of a medium-chain triglyceride solution of the angiogenesis inhibitor TNP-470 (AGM-1470) when administered via the hepatic artery to rats bearing Walker 256 carcinosarcoma in the liver.

The antitumor effect of an angiogenesis inhibitor, TNP-470 (AGM-1470, 6-0-(N-chloroacetylcarbamoyl)-fumagillol), administered via the hepatic artery in a medium-chain triglyceride (MCT) solution, in which TNP-470 is very stable, was examined in rats bearing Walker 256 carcinosarcoma in the liver. The MCT solution containing 0.1 mg of TNP-470 completely suppressed tumor growth after a single arterial injection, and the solutions containing 0.5 approximately 5 mg of TNP-470 caused tumor regression function. These antitumor effects lasted for at least 2 weeks. Moreover, the administration of the MCT solution containing 5 mg of TNP-470 also caused remarkable regression of well-developed enlarged tumors 2 weeks after inoculation, indicating potential in the treatment of unresectable hepatic cancer. When the MCT solution containing radiolabeled TNP-470 was injected via the hepatic artery, the initial radioactivity in the tumor was 22 times that in the normal part of the liver and 5.7 times that in the tumor when an aqueous solution of radiolabeled TNP-470 was injected. Also, in the case of the MCT solution, the radioactivity in the tumor was maintained at a relatively high level for over 2 weeks after injection. These results indicate that the remarkable antitumor effect resulted from the selective delivery and prolonged retention of TNP-470 at the tumor site.

Insulin fragments as a carrier for peptide delivery across the blood-brain barrier.

The possibility of using insulin (INS), which is transported into the brain by receptor-mediated transcytosis, as a peptide carrier for delivery across the blood-brain barrier (BBB) was investigated. After mice received an i.v. injection of horseradish peroxidase (HRP, M.W., 40,000) conjugated with INS, the HRP activity in the brain was higher than that after HRP injection. Since INS-HRP lowered the blood glucose level, we prepared insulin fragments by chemical and enzymatic procedures in an effort to find a carrier with no hypoglycemic activity. Seven fragments were synthesized taking the binding regions into consideration, but none showed any receptor binding affinity in cultures of bovine brain microvessel endothelial cells (BMEC). However, the fragment (F007) obtained by trypsin digestion showed high affinity and scarcely any hypoglycemic activity in mice even at a dose ten times the effective dose of insulin. These results suggest that this fragment may be useful as a carrier to transport therapeutic peptides across the BBB.

pH-independent controlled-release microspheres using polyglycerol esters of fatty acids.

The release of a drug having low solubility in a certain pH range from controlled-release microspheres using tetraglycerol pentastearate and tetraglycerol monostearate in combination as the matrix base showed pH dependence. Trepibutone, an acidic drug having lower solubility in an acidic medium, was released pH-independently from the microspheres which incorporated magnesium oxide, a solid base. It might have resulted from the pH inside the matrix being kept in an optimum range for drug release due to the incorporation of a solid base. On the other hand, the addition of water soluble acidic or basic excipients was ineffective to achieve pH-independent release. For papaverine, a basic drug, pH-independent drug-release characteristics could be achieved by adding Eudragit L100-55, an enteric polymer. It is thought that the enteric polymer increased the pores for drug release by dissolving in a higher pH range, where the solubility of papaverine is low, and thereby made the release pH-independent. Further, selecting a polyglycerol ester of a fatty acid with an appropriate hydrophile-lipophile balance as the matrix could yield a drug with the desired release rate at any pH.

Anti-hypertensive effect of oral controlled-release microspheres containing an ACE inhibitor (delapril hydrochloride) in rats.

An oral controlled-release drug delivery system based on microspheres of polyglycerol esters of fatty acids (PGEFs), was applied to an anti-hypertensive, delapril hydrochloride. The in-vitro release profile was controlled by selecting a PGEF with an appropriate hydrophilic-lipophilic balance value for the matrix. The microspheres from which 80% of the drug was released in 6 h were orally administered to rats. The plasma concentration of the active metabolite was sustained after administration of the microspheres in comparison with administration of a solution. The in-vivo release profile was in good agreement with the in-vitro release profile. When the microspheres were administered, the pharmacological effect of delapril hydrochloride on the angiotensin I-induced pressor response was also sustained showing consistency with the plasma concentration-time curve.

Enhancement of the anti-tumor activity of recombinant interleukin-2 (rIL-2) by immunocomplexing with an F(ab')2 fragment of murine monoclonal antibody against rIL-2.

We have investigated the biological properties of an immune complex composed of recombinant interleukin-2 (rIL-2) and an F(ab')2 fragment of a monoclonal antibody against rIL-2 in mice for the induction of killer cells and anti-tumor activity, as well as the pharmacokinetic properties of the immune complex injected subcutaneously into mice. The immune complex demonstrated sustained serum rIL-2 levels, with a 2.4-times longer "mean-residence-time" than free rIL-2. A more significant portion of rIL-2 was detected in lymph nodes after the subcutaneous injection of the immune complex than with rIL-2 alone. Splenic lymphocytes from mice given the immune complex showed higher killer cell activity against YAC-1 and P815 cells than those from mice given rIL-2 alone. The immune complex also exerted a more significant anti-tumor effect in a dose-dependent manner in Meth-A fibrosarcoma-bearing mice than dit rIL-2 alone.

Preparation of three-month depot injectable microspheres of leuprorelin acetate using biodegradable polymers.

To obtain a three-month release injection of leuprorelin acetate, microspheres were prepared with copoly(DL-lactic/glycolic acid) or poly(DL-lactic acid) (PLA) using an in-water drying method, and drug release was evaluated. The content of water-soluble oligomers in the polymers was found to strongly affect the initial burst, and reducing the content to less than 0.1% was necessary to keep the first-day release below 10%. Drug loading of more than 15% also increased the initial drug release; the acceptable maximum loading was 12%. Elevation of the glass transition temperature of the microspheres was observed with an increase in drug loading. This suggests formation of a rigid structure, possibly with arrangement of the polymer around the drug cores like in a micelle. This structure provides a hydrophobic barrier against diffusion of the hydrophilic peptide, resulting in high trapping efficiency and long-term sustained release dependent on polymer erosion. The microspheres prepared with PLA having a m.w. of 12,000 to 18,000 provided linear sustained release and persistent serum levels of the drug in rats for over 3 months.

Sustained suppression of the pituitary-gonadal axis by leuprorelin three-month depot microspheres in rats and dogs.

The pharmacological effects of leuprorelin three-month depot microspheres were investigated in rats and dogs. After s.c. and i.m. injection, the microspheres provided similar linear drug release and sustained serum drug levels for 3 months. Persistent suppression of serum LH, FSH (in rats) and testosterone (in rats and dogs) for over 16 weeks was achieved when the microspheres were given at a dose of 100 (rat) and 25.6 (dog) micrograms/kg/day. These hormone release responses upon periodic challenge tests revealed that a single injection of the microspheres caused dramatic suppression of the function of the pituitary-gonadal system for 15 weeks in rats. The growth of the genital organs was also suppressed dose-dependently by injection of the microspheres over 3 months; the strongest suppression was achieved at a dose of 100 micrograms/kg/day. This three-month depot formulation is expected to be more convenient than the one-month depot with improved patient compliance and therapeutic effects.

Controlled release of thyrotropin releasing hormone from microspheres: evaluation of release profiles and pharmacokinetics after subcutaneous administration.

The drug-release kinetics of thyrotropin releasing hormone (TRH) containing copoly(dl-lactic/glycolic acid) (PLGA) microspheres were evaluated both in vitro and in vivo. The drug was encapsulated in PLGA using an in-water drying method through a water in oil in water emulsion. The drug release from the PLGA microspheres in vitro correlated well with that in vivo, and pseudo-zero-order release kinetics were observed. The pharmacokinetics of TRH following administration of this controlled-release parenteral dosage form have been also examined in rats. Following a transient increase in the plasma level due to an initial burst, steady-state plasma levels were observed. The duration of drug release estimated from the plasma level was comparable with the results in the in vitro and in vivo release studies. The steady-state plasma levels correlated well with the levels predicted from the pharmacokinetic parameters following a single subcutaneous or intravenous injection of TRH solution. The results of this study confirm the previously reported in vivo sustained release of TRH achieved with this drug-delivery system.

In vitro and in vivo evaluation of thyrotrophin releasing hormone release from copoly(dl-lactic/glycolic acid) microspheres.

In vitro and in vivo release of thyrotrophin releasing hormone (TRH) from copoly(dl-lactic/glycolic acid) (PLGA) microspheres were evaluated. Factors affecting the TRH release from the microspheres were examined to clarify the release mechanisms by changing the medium composition in the in vitro release test. The hydrolysis rate of PLGA, the matrix-forming substance in the microspheres, was faster in acidic medium than in neutral medium. The release rate of TRH from the PLGA microspheres increased with the increase in the degradation rate of PLGA. A decrease in an osmolarity of the medium also caused an increase in the TRH release rate even though no significant change in PLGA degradation was observed. The effect of osmolarity appears to be characteristic of water-soluble drug-containing microspheres composed of hydrophobic polymer. The release rate of TRH from PLGA microspheres was largely affected by the medium composition in the in vitro release test. A proper choice of medium was found to be important for the estimation of in vivo release. The in vivo release rate of TRH from the PLGA microspheres following administration to rats correlated with the in vitro release in pH 7, 1/30 M buffer.

Pharmacokinetic alteration in rats of recombinant interleukin-2 (rIL-2) by immunocomplexing with a monoclonal antibody against rIL-2.

We have investigated the pharmacokinetic alteration in rats of recombinant interleukin-2 (rIL-2) by immunocomplexing with a monoclonal antibody against rIL-2. Serum rIL-2 levels after the intravenous administration of the immune complex at a dose of 100 micrograms/rat as rIL-2 were significantly higher than those after intravenous administration of rIL-2 alone at the same dose. Pharmacokinetic analysis indicated that the distribution volume of rIL-2 decreased from 74.0 to 10.3 ml/rat, while the elimination rate of rIL-2 was little changed by immunocomplexing with the antibody. On the other hand, serum rIL-2 levels after the subcutaneous administration of the immune complex at a dose of 100 micrograms/rat as rIL-2 were sustained longer than those after the subcutaneous administration of rIL-2 alone at the same dose, and Tmax shifted from 0.83 to 3.0 h by immunocomplexing with the antibody. Pharmacokinetic analysis also revealed that the mean-residence-time of rIL-2 increased from 1.98 to 6.52 h, and the area-under-the curve of rIL-2 decreased slightly, from 834 to 548 ng.h/ml, by immunocomplexing with the antibody.

Membrane modification by negatively charged stearyl-polyoxyethylene derivatives for thermosensitive liposomes: reduced liposomal aggregation and avoidance of reticuloendothelial system uptake.

In order to avoid reticuloendothelial system (RES) uptake and prolong systemic circulation of cisplatin (CDDP)-encapsulating thermosensitive liposomes, stearylpolyoxyethylene (POE) derivatives [SnC, stearyl-O-(CH2CH2O)n-CH2COONa] were incorporated as membrane modifiers into lipid bilayers composed of dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC). The incorporation of S2C, S5C, S10C or S15C [lipid/SnC = 10/2 (w/w)] greatly reduced liposomal aggregation without impairing liposomal stability. After being intravenously administered to rats, the liposomes remained longer in the systemic circulation and showed lower RES levels than the control liposomes. When incorporated into liposomes [DPPC/DSPC = 7/3 (w/w)], S10C provided the greatest increase in systemic circulation time and the RES-avoiding activity among the modifiers tested. The systemic elimination rate (the ratio of the percent of the dose systemically eliminated to the AUC of the liposome level) for this type of liposome was 0.24/hr, about one fourth the rate for the control liposomes, and the RES uptake rate (the ratio of the percent of the dose taken up by the RES to the AUC) was 0.04/hr, one seventh the rate for the control liposomes. The RES uptake rate for S10C 7/3-liposomes was similar to the rate reported for GM1 liposomes, although the systemic elimination rate was double that for the GM1 liposomes. The obtained RES avoidance activity can be attributed to decreased liposomal aggregation and increased surface hydrophilicity. This type of thermosensitive liposome should be more useful in hyperthermia-mediated targeted tumor drug delivery systems than the thermosensitive liposomes without the modifiers to avoid RES uptake.

Rates of systemic degradation and reticuloendothelial system (RES) uptake of thermosensitive liposome encapsulating cisplatin in rats.

The systemic degradation and reticuloendothelial system (RES) uptake of cisplatin (CDDP)-encapsulated thermosensitive liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC) (DPPC/DSPC = 9/1, 7/3, and 5/5, w/w) after intravenous administration to rats were examined by measuring the platinum (Pt) levels in the blood and RES (liver and spleen). The blood liposome level profile showed first-order rate elimination for each liposome administration. The elimination rate (Kel) was faster when the content of DSPC was lower (Kel: 1.3/hr for 9/1-liposomes, 0.7/hr for 7/3-liposomes, 0.5/hr for 5/5-liposomes). On the other hand, the RES liposome level profile showed distribution of liposomes followed by elimination therefrom. The RES level of the liposomes was lower when the content of DSPC was smaller (maximal level: 25% for 9/1-liposomes at 1 hr, 32% for 7/3-liposomes at 1 hr, 37% for 5/5-liposomes at 2 hr). The kinetic analysis demonstrated that the RES uptake rate (Kres) was almost the same among the liposomes (0.4/hr), while the systemic degradation rate (Kdeg; Kel-Kres) became larger as the content of DSPC decreased (0.9/hr for 9/1-liposomes, 0.3/hr for 7/3-liposomes, and 0.1/hr for 5/5-liposomes) and that the RES liposome distribution amount was dependent not only on the Kres but also on the Kdeg and the rate of RES liposome degradation. The Kdeg for each type of liposome corresponded with the systemic CDDP release rate.