Lipid microsphere formulation containing rifampicin targets alveolar macrophages.

The study demonstrated that lipid microspheres (LM) containing rifampicin (LM-RFP) could deliver the drug to alveolar macrophages in vitro and in vivo, and that intranasal administration to animals could achieve preferential accumulation in the lungs with less effect on the liver. The LM-RFP particles had a mean diameter of 247.2 +/- 75.7 nm, and their size remained stable when stored at 4 degrees C or 25 degrees C for at least 4 weeks. In vitro uptake of [(3)H]LM-RFP by alveolar macrophages was over 4 times higher than that of unencapsulated [(3)H]RFP, whereas the in vivo uptake was 30 times higher. Flow cytometric analysis and confocal laser scanning microscopy confirmed that LM could deliver the encapsulated drug effectively to alveolar macrophages in vitro and in vivo. Intranasal administration of [(3)H]LM-RFP to normal mice resulted in preferential pulmonary uptake of the drug and lower levels in the blood and liver compared with administration of unencapsulated [(3)H]RFP. In conclusion, LM-RFP could be a promising preparation for delivery via the respiratory tract to tuberculosis (TB) and TB/HIV patients.

Clinical and epidemiological studies of eicosapentaenoic acid (EPA) in Japan.

From 1950 to 1980, the gross alteration in dietary habit in Japan was noted. Intake of total calories has markedly increased. This could be most likely due to a remarkable increase in intake of fat, especially animal fat, egg and milk products. A marked decrease of mortality rate due to cerebral hemorrhage and in contrast a marked increase of mortality rate due to cerebral infarction and ischemic heart disease were noted. An epidemiological study of the intake of fish meat (EPA intake) and the mortality rate of adult diseases was performed in a fishing area and in a farming area in Chiba Prefecture. Intake of fish meat (EPA) by the residents of the fishing area was 2-3 times higher than by the residents of the farming area. The mortality rate due to ischemic heart disease and cerebral vascular diseases tended to be lower in the fishing area than in the farming area. EPA manufactured from sardine oil was orally given to normal subjects and to patients with cerebro- and cardiovascular diseases for 4-16 weeks. Significantly decreased platelet aggregation, decreased platelet retention, lowered whole blood viscosity, prolonged bleeding time, increased erythrocyte deformability, improvement of hyperlipidemia, and clinical improvement in some patients were noted. 12-Lipoxygenase metabolites of EPA (12-HPEPE) and arachidonic acid (12-HPETE) have an equipotent inhibitory action on platelet function.

Administration of optimum sustained-insulin release PLGA microcapsules to spontaneous diabetes-prone BB/Wor//Tky rats.

To show the possibility of sustained-release insulin formulation composed of PLGA, the optimum one was administered to BioBreeding rat, a model of spontaneous type I diabetes mellitus (IDDM). Every 2 weeks subcutaneous administration made their blood glucose level depend on the insulin release and food intake. However, all of them kept alive with little change or rather a little gain in body weight. Furthermore, some of pregnant rats with intermittent treatment bore fetuses, although additional insulin therapy seemed necessary. Therefore, the formulation could become a new tool as a provider of basal insulin for IDDM patients.

Effect of lecithinized superoxide dismutase (PC-SOD) on experimental pulmonary metastasis in mice.

The inhibitory effect of lecithinized superoxide dismutase (PC-SOD) on pulmonary metastasis in mice was investigated. In an experimental pulmonary metastasis model employing Meth A-T cells, significant and dose-dependent inhibition was observed after i.v. pre-administration of PC-SOD. Unmodified SOD (U-SOD) was also effective, but a 10-times higher dose was necessary to be significant. The pulmonary accumulation of Meth A-T cells labeled with 5-[125I]iodo-2'-deoxyuridine was not reduced by either PC-SOD or U-SOD, and neither of the compounds decreased pulmonary MPO activity. However, PC-SOD increased pulmonary SOD activity for longer, compared with U-SOD. In vitro addition of PC-SOD dose-dependently suppressed the growth of Meth A-T cells, while U-SOD had little effect. The combination of PC-SOD and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitric oxide (NO)-generating agent, had an additive effect. It was also found that PC-SOD prevented a decrease of pulmonary NOx level following tumor cell inoculation. It was concluded that PC-SOD possessed antimetastatic activity, and its potency was superior to that of U-SOD. These results suggest that PC-SOD may prevent the excessive formation of oxygen radicals and peroxynitrite (ONOO-) which cause cell damage and facilitate tumor metastasis.

In vitro effect of trichosanic acid, a major component of Trichosanthes japonica on platelet aggregation and arachidonic acid metabolism in human platelets.

The in vitro effect of trichosanic acid (TCA; C18:3, omega-5), a major component of Trichosanthes japonica, on platelet aggregation and arachidonic acid (AA) metabolism in human platelets was studied. TCA dose-dependently suppressed platelet aggregation of platelet rich plasma and washed platelets. TCA decreased collagen (50 micrograms/ml)-stimulated production of thromboxane B2 (TXB2) and 12-hydroxyhepta-decatrienoic acid (HHT) in a dose-dependent manner, while that of 12-hydroxyeicosatetraenoic acid (12-HETE) was rather enhanced. The conversion of exogenously added [14C]AA to [14C]TXB2 and [14C]HHT in washed platelets was dose-dependently reduced by the addition of TCA, while that to [14C]12-HETE was increased. Similar observations were obtained when linolenic acid (LNA; C18:3, omega-3) was used. These results suggest that TCA may decrease TXA2 formation in platelets, probably due to the inhibition of cyclooxygenase pathway, and thereby reduce platelet aggregation.

Endogenous calcitonin gene-related peptide suppresses vasoconstriction mediated by adrenergic nerves in rat mesenteric resistance blood vessels.

The role of perivascular calcitonin gene-related peptide (CGRP)-containing nerves in the modulation of adrenergic nerve-mediated vasoconstrictions was studied in the rat perfused mesenteric vascular bed. A frequency-dependent vasoconstriction induced by periarterial nerve stimulation (1-6 Hz) of the bed was significantly potentiated by perfusion of 1 microM CGRP-(8-37) (CGRP receptor antagonist) or to a similar extent after treatment with 500 nM capsaicin. In the preparations treated with capsaicin, CGRP-(8-37) caused a small potentiation of periarterial nerve stimulation-induced vasoconstriction. Exogenous CGRP (0.1-1 nM) concentration-dependently attenuated the augmented vasoconstriction in response to periarterial nerve stimulation after treatment with capsaicin. However, exogenous CGRP (1 nM) did not attenuate the periarterial nerve stimulation-induced vasoconstriction in the bed untreated with capsaicin. These results suggest that endogenous CGRP, which is released from CGRP-containing nerves, suppresses the adrenergic nerve function involved in mechanisms regulating the tone of resistant blood vessels.

Marked hypotensive and blood flow-increasing effects of a new lipo-PGE(1) (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.

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.

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.

Comparison of the in vitro effect of eicosapentaenoic acid (EPA)-derived lipoxygenase metabolites on human platelet function with those of arachidonic acid.

Eicosapentaenoic acid (EPA) has been reported to have a potent anti-aggregatory activity and to be efficiently metabolized by 12-lipoxygenase, not by cyclooxygenase in platelets. In vitro effect of 12-lipoxygenase metabolites of EPA on platelet function was studied and compared with those of arachidonic acid (AA). The 12-lipoxygenase metabolites of AA and EPA; 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 12-hydroperoxyeicosapentaenoic acid (12-HPEPE), and their hydroxy derivatives, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE) were prepared enzymatically using human platelet lysate. These compounds were purified by high performance liquid chromatography and identified by gas chromatography mass spectrometry. 12-HPETE and 12-HPEPE inhibited dose-dependently washed human platelet aggregation and serotonin (5-HT) release induced by AA and collagen. The potency of 12-HPEPE was almost equal to that of 12-HPETE. Their hydroxy derivatives, 12-HETE and 12-HEPE were less potent. 12-hydroperoxy derivatives of AA and EPA were the most potent in inhibiting platelet aggregation and 5-HT release among 5-, 12- and 15-hydroperoxy isomers of AA and EPA. The inhibitory effects of 12-HPETE and 12-HPEPE on platelet aggregation were additive.

Lecithinized ascorbic acid (PC-AS) effectively inhibits murine pulmonary metastasis.

In order to enhance the lipophilicity and develop the efficacy of ascorbic acid (ASA), we synthesized lecithinized ascorbic acid (PC-AS), in which a lecithin was covalently bound to ASA. Its pharmacological activity was also evaluated. The IC50 value of scavenge superoxide anions generated from hypoxanthine in combination with xanthine oxidase, indicated that the antioxidative activity of PC-AS (IC50; 22.19 microM) was about 60% of that shown by ASA (IC50; 13.35 microM). Also, PC-AS suppressed in vitro cell growth of Meth A-T, a highly metastatic cell line established by us. Although its potency (IC50; 110.0 microM) was a little lower than that of ASA, dramatic suppression was observed under serum-free culture conditions (IC50; 13.0 microM). In addition, N-acetylcysteine (NAC), an antioxidant, showed an additive inhibitory effect on cell growth in combination with PC-AS and ASA. Biodistribution studies revealed that PC-AS persisted longer in the blood (AUC0-240 min; 182.8 nmole min ml-1) than ASA (AUC0-240 min; 79.35 nmole min ml-1). It should be noted that intravenous preadministration of PC-AS significantly and dose-dependently reduced the number of colony formation in an experimental murine pulmonary metastasis model. ASA had little effect. [3H]-labeled Meth A-T cells predominantly accumulated in the lung, metastatic target organ, which was reduced by PC-AS. Our in vivo study showed that PC-AS could not totally prevent pulmonary invasion of Meth A-T cells, however, PC-AS effectively inhibited the number of metastatic colony formation. PC-AS's potency was superior to that of unmodified ASA. These findings might be in part ascribed to changes to lecithinization-induced biodistribution, antioxidative activity and cytotoxicity.

Application of lipid microspheres to prepare a thromboxane A2 receptor antagonist aerosol for inhalation.

The methyl ester of a new thromboxane A2 receptor antagonist, (+)S-145, i.e. (1R,2S,3S,4S)-(5Z)-7-(3-phenyl-sulfonyl-aminobicyclo[2,2,1]he pt-2-yl)heptenoic acid, was incorporated into lipid microspheres (lipo S-145-Me) and its pharmacological effect and tissue distribution were examined in guinea pigs following aerosol delivery. Bronchoconstrictive responses induced by intravenous injection of U46619 or the inhalation of ovalbumin were suppressed in a dose-dependent manner by aerosol inhalation of lipo S-145-Me, which was 3-10 times more potent that the unencapsulated calcium dihydrate of the original drug (S-1452). There was no significant difference in the airway tissue distribution of labelled lipo S-145-Me versus S-1452 after 2 or 5 min of inhalation, but the encapsulated drug showed marked accumulation in the lungs after 30 min of inhalation. The in vitro uptake of lipo [14C] S-145-Me by fresh human neutrophils and an eosinophil cell line was respectively 7 times and 3.5 times higher than that of [14C] S-1452. These results suggest that lipo S-145-Me has the potential to be used as an inhalational antiasthma agent, and that its effect may be partly attributable to a for inflammatory cells which are responsible for allergic airway inflammation.

Lipid microsphere preparation of a lipophilic ceramide derivative suppresses colony formation in a murine experimental pulmonary metastasis model.

Ceramide is a well-known regulator of apoptosis and cell growth. In this study, we synthesized lipophilic ceramide derivatives to incorporate into lipid microspheres (LM) and their activity was evaluated in vivo. Cera 03, a lipophilic ceramide derivative synthesized from membrane-permeable C2-ceramide, caused potent growth inhibition and DNA fragmentation of Meth A-T tumor cells in vitro. Its potency was similar to that of C2-ceramide. Both compounds increased the proportion of apoptotic cells. Cera 02, the diacetylated form of natural ceramide (Cer), also suppressed in vitro cell growth with a similar or higher potency to that of Cer, but both were far less potent than C2-ceramide and Cera 03. LM containing Cera 03 (Lipo-Cera 03) could not totally prevent metastatic incidence of Meth A-T cells, but reduced pulmonary metastatic nodules in number. Intravenous injection of Lipo-Cera 03 (1 mg/kg of Cera 03) produced about 35% inhibition, while Lipo-Cera 02 had no significant effect. In conclusion, Lipo-Cera 03 may have potential as an antimetastatic drug and may also be a useful tool for researching the role of ceramides in vivo.

Lecithinization of IL-6 enhances its thrombopoietic activity in mice.

This study was conducted to assess the merit of lecithinization of recombinant human interleukin-6 (IL-6) as a drug delivery system. IL-6 was lecithinized by covalently binding it with a phosphatidylcholine (lecithin, PC) derivative. The in-vivo thrombopoietic potency of lecithinized IL-6 (PC-IL-6) was greater than that of native IL-6 when administered subcutaneously, although the in-vitro bioactivity of PC-IL-6 was markedly reduced by lecithinization. When PC-IL-6 and native IL-6 were given in doses that produced the same level of thrombopoietic activity, the former stimulated less production of IgG1, a marker of the adverse effects of IL-6, than did the latter. Furthermore, PC-IL-6 persisted in the blood longer than native IL-6. Based on the above, PC-IL-6 appears to be useful as a drug delivery system and may also be useful in the treatment of drug-induced thrombocytopenia.

Differential hormonal profiles of adrenomedullin and proadrenomedullin N-terminal 20 peptide in patients with heart failure and effect of treatment on their plasma levels.

BACKGROUND: Adrenomedullin (AM) is a potent vasodilatory peptide discovered in human pheochromocytoma tissue. Proadrenomedullin N-terminal 20 peptide (PAMP) processed from an AM precursor is also a novel hypotensive peptide which inhibits catecholamine secretion from sympathetic nerve endings. HYPOTHESIS: The present study sought to examine the relationships between the two peptides and other clinical parameters by measuring the plasma AM and PAMP concentrations in 98 patients with heart failure. METHODS: In all, 98 patients [65 men and 33 women, aged 58.2 +/- 11.0 years, mean +/- standard deviation (SD)] with heart failure and 26 healthy volunteers (12 men and 14 women, aged 54.1 +/- 8.6 years) were examined in this study. Heart failure was secondary to previous myocardial infarction in 58 patients, valvular disease in 28, cardiomyopathy in 9, and congenital heart disease in 3. All patients were classified into two groups of class I or II (Group 1) and class III or IV (Group 2) according to the New York Heart Association (NYHA) functional classification. RESULTS: Both plasma AM and PAMP concentrations in the patients were significantly higher than those in healthy volunteers. In addition, plasma AM and PAMP concentrations in patients in class III or IV of New York Heart Association (NYHA) classification were significantly higher than those in NYHA class I or II. The elevated plasma concentrations of these peptides in patients in NYHA class III or IV significantly decreased in response to the treatment for 7 days. There was a significant correlation between plasma AM and PAMP, though the plasma concentration of PAMP was one-fifth to one-seventh of that of AM in patients and controls. The plasma AM concentration correlated significantly with the plasma concentrations of atrial and brain natriuretic peptides, epinephrine, and right atrial pressure, whereas such a relationship was not noted for the plasma PAMP concentration. CONCLUSIONS: Judging from the difference in not only the biological actions but also the hormonal profiles between AM and PAMP, they may differentially modulate the cardiovascular system in patients with heart failure, although they are processed from the same precursor.

A new phosphor Li2B4O7: Cu for TLD.

The phosphor Li2B4O7: Cu (0.03% by weight) has an effective atomic number of 7.3, which is very similar to that of tissue (7.4). This suggests that the phosphor should have excellent properties for thermoluminescent dosimetry. The phosphor prepared by a sintering method shows two glow peaks composed of a dosimetric peak at 205 degrees C and a shoulder at 120 degrees C, and a broad emission band peaking at 368 nm. The material based on the stoichiometric compound of Li2O . 2B2O3 has a good moisture resistant property. The dosimetric characteristics are as follows: (1) The sensitivity of gamma rays is about 20 times higher than that of Li2B4O2: Mn prepared by the conventional melting method. (2) The dosimetry peak of 205 degrees C fades less than 9% in intensity at 25 degrees C after 60 days in dark. (3) The TL output is linear with exposure to about 10(5) R, becoming sublinear above it. (4) The sensitivity loss caused by humidity is about 10-25% after 2-6 months of storage in air of 90% relative humidity at 25 degrees C. (5) The energy dependence of TL output for photons is flat within the limit of 10% from 40 keV to 7 MeV. (6) The light induced fading is 10% after 3 h room lighting at 1000 lux.

Construction of a composite thin-element TLD using an optical-heating method.

A composite thermoluminescent dosimeter (TLD), composed of four, thin TL elements with a high-speed reader, has been developed by employing an optical-heating method. Each TL element, which is 15 mg/cm2 thick with a 3 mm dia., is prepared by applying Li2B4O7:Cu or CaSO4:Tm to a plastic substrate 14 mg/cm2 thick. Each element can be heated to 350 degree C within 0.8 sec. by IR radiation from a tungsten lamp. The characteristics of this TLD system include the following: (1) the detection limit of the Li2B4O7:Cu is 3 mR and the limit for CaSO4:Tm is 0.1 mR; (2) the energy-dependence curves are similar to the dose-equivalent curve, showing slight under-responses by 15% near 70 KeV for Li2B4O7:Cu and over-responses by 50% at high energies for CaSO4:Tm; (3) despite quick heating, the residual dose is as low as 0.1% of the last exposure signal; (4) responses are very stable for more than 1,000 cycles of repeated exposure readings; (5) no false signal could be detected, even in the cases of sweat or soil contamination; (6) the thin Li2B4O7;Cu element can be used for skin dose monitoring; and (7) the processing time of the automatic reader for the composite dosimeter is 3 hr/500 dosimeters. This TLD system can be applied to personnel dosimetry, gate monitoring and environmental monitoring.

[Atrial septal defect with idiopathic thrombocytopenic purpura: a case of open-heart surgery].

A 40-year-old woman of atrial septal defect associated with idiopathic thrombocytopenic purpura (ITP) was successfully operated upon under cardio-pulmonary bypass. Steroids had been given preoperatively, resulting in an increase of the platelet count from 0.7 X 10(4)/mm3 to 20.6 X 10(4)/mm3. Steroids could be withdrawn before the operation. No bleeding tendency was encountered during and after surgery. This is the 6th case of open-heart surgery associated with ITP in the literature.