Effects of pH on the structure and mechanical properties of dried pH-responsive latex particles.

Micrometer-sized monodisperse polystyrene (PS) particles carrying a pH-responsive poly[2-(diethylamino)ethyl methacrylate] (PDEA) colloidal stabilizer were synthesized via free radical dispersion polymerization. X-ray photoelectron spectroscopy and electrophoretic measurements verified that PDEA covered the PS particle surface. At pH 3.0 and 6.3, where the PDEA is protonated and cationically charged, the PDEA-PS particles were well dispersed in aqueous media thanks to the water soluble PDEA stabilizer and slowly sedimented due to gravity and enriched at the bottom of the glass vials. At pH 10.0, where the PDEA is non-protonated and neutral, the PDEA-PS particles weakly aggregated due to non-hydrated and collapsed PDEA. These PDEA-PS particles and aggregates sedimented to the bottom. The sediment height observed at pH 10.0 was higher than those observed at pH 3.0 and 6.3 in both wet and dry systems, which indicated that a larger porosity was formed at pH 10.0. Mechanical testing experiments confirmed that the fracture toughness of the dried materials decreased with an increase of pH. The fracture toughness was found to be correlated with the degree of particle ordering in the dried particulate materials: more ordered, dense packings lead to a higher fracture toughness compared to amorphous, less dense packings. Thus, we could tune fracture toughness and degree of particle ordering by controlling the pH.

Possible roles of the AP-1 site in the cytosolic T3 binding protein promoter and insights into its physiological significance.

Cytosolic 3,5,3'-triiodo-l-thyronine-binding protein plays pivotal roles in the regulation of intracellular 3,5,3'-triiodo-l-thyronine concentration in vivo. The expression of the protein, which is identical to µ-crystallin, is regulated by various factors. To elucidate the mechanisms of its expression, we evaluated the promoter transactivity and insulin signaling via the AP-1 site in the promoter. The isolated 600 bp human and 1976 bp mouse 5'-flanking regions were cloned in a luciferase reporter plasmid. The luciferase activity was estimated in GH3, dRLh-84, HEK293, and insulin receptor-overexpressing CHO-IR cells. The effects of 12-O-tetradecanoylphorbol 13-acetate and insulin on µ-crystallin mRNA expression were evaluated in various cells. The region between -200 and the transcriptional start site was crucial for constitutive expression in µ-crystallin-expressing dRLh-84 cells. This region contained an AP-1 site. 12-O-Tetradecanoylphorbol 13-acetate increased the level of µ-crystallin mRNA expression in HEK 293 cells. The compound also increased luciferase activity through the promoter. Mutation in the AP1 site diminished the response to the compound. The promoter was also activated by insulin treatment in CHO-IR cells. Insulin treatment increased µ-crystallin mRNA expression in Raw264.7 cells, but decreased in HEK293, P19, and dRLH-84 cells. The expression of µ-crystallin was regulated through the AP-1 site in the promoter. The signals related to AP-1 activation, such as insulin signaling may have diverse effects on µ-crystallin mRNA expression.

Selective delivery of estradiol to bone by aspartic acid oligopeptide and its effects on ovariectomized mice.

We have developed a novel osteotropic prodrug of estradiol (E(2)) conjugated with L-Asp-hexapeptide (E(2).3D(6)), which has very low affinity for estrogen receptors, and in this study, we examined its pharmacokinetic behavior and pharmacological potential. After a single iv injection of E(2) x 3D(6) to mice, the half-time for elimination from plasma was about 100 min; however, E(2) was selectively delivered to the bone and eliminated very slowly, declining to the endogenous level at about 7 days. After a single iv injection of E(2), the half-time in plasma was about 70 min, whereas E(2) was highly distributed to the uterus, and the bone concentration of E(2) was only slightly increased at 6 h. When E(2) (0.37 micromol/kg, sc, every third day) or E(2) x 3D(6) (0.11 to 1.1 micromol/kg, sc, every seventh day) was administered to OVX mice for 4 weeks, E(2) increased the bone mineral density (BMD) together with weights of liver and uterus, whereas E(2) x 3D(6) increased only the BMD, in a dose-dependent manner. E(2) x 3D(6) enhanced the expression of messenger RNAs of bone matrix proteins (osteopontin, bone sialoprotein, type I collagen alpha) of OVX mice at 4 h after administration, but E(2) did very slightly. These results indicate that the E(2) prodrug was delivered to the bone, where it gradually released E(2), thereby ameliorating bone loss. This acidic oligopeptide appears to be a good candidate for selective drug delivery to bone.

Novel drug delivery system to bone using acidic oligopeptide: pharmacokinetic characteristics and pharmacological potential.

We synthesized fifteen oligopeptides consisting of Asp or Glu conjugated with a fluorescent probe, 9- fluorenylmethylchloroformate (Fmoc). In the in vitro binding assay to putative hydroxyapatite (HA), the affinities of these conjugates depended only on the number of amino acid residues, not on their optical characters (L or D) or their species (Asp or Glu). In an in vivo experiment involving a single i.v. injection of Fmoc-D-Asp oligopeptides into mice, peptides consisting of over six Asp residues were selectively distributed to the bone. Then, we synthesized estradiol-17 beta-succinate-(L-Asp)6 [E2-(L-Asp)6] and studied its pharmacokinetic characteristics and its antiosteoporotic effects on ovariectomized (OVX) mice. Although the distribution volume of E2-(L-Asp)6 was significantly smaller than that of E2, E2-(L-Asp)6 was selectively distributed in the bone after i.v. injection and gradually decreased during 7 days. E2-(L-Asp)6 effectively prevented OVX-induced bone loss, without altering the uterine weight, in the dosage range of 0.11 to 1.1 mumol/kg once a week, while E2 increased both the bone mineral density and uterine weight at 0.37 mumol/kg every third day. The results suggest that acidic oligopeptide may be useful for drug delivery to bone and E2-(L-Asp)6 is a good candidate as an anti-osteoporosis drug without the adverse side effects of E2.

Hepatic drug metabolizing enzymes induced by clofibrate in rasH2 mice.

Hepatic drug metabolizing enzyme activities were determined, after treatment with clofibrate, in transgenic mice carrying human c-Ha-ras (rasH2 mice). Changes in the drug metabolizing enzyme activities in these mice by gene integration were also evaluated. Male and female rasH2 mice (Tg) and the litter mates not carrying the gene (non-Tg) received orally 500 mg/kg of clofibrate or the vehicle for 12 consecutive days. Liver homogenate and microsomes were prepared and the contents and activities of cytochrome P450 (CYP), cytochrome b5 content and enzyme activities related to peroxisome proliferation were determined. Relative liver weights, CYP4A and activities of catalase and carnitine palmitoyl transferase increased to the same extent in Tg and non-Tg mice treated with clofibrate. In Tg and non-Tg groups that received vehicle, contents and activities of CYP and cytchrome b5 contents were comparable. It was concluded that gene integration did not alter drug metabolizing enzymes and responses to clofibrate.

Time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic drug-metabolizing enzyme activity in rats.

The time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic cytochrome P450-dependent drug-metabolizing capacity (cytochrome P450 and b5 content, activity of aminopyrine N-demethylase, p-nitroanisole O-demethylase, aniline hydroxylase and benzphetamine N-demethylase) and on the pharmacokinetics of antipyrine have been determined in rats. Measurement of enzyme activity and antipyrine (after intravenous injection of 20 mg kg(-1)) were performed 2, 24 and 96 h after a single intraperitoneal injection of endotoxin (1 mg kg(-1)) and after repeated doses (once daily for 4 days). The contribution of tumour necrosis factor alpha (TNFalpha) to the endotoxin-induced changes was also examined in rats pretreated with granulocyte colony-stimulating factor (G-CSF). The systemic clearance of antipyrine and the activity of hepatic cytochrome P450-dependent drug-metabolizing enzymes were dramatically reduced 24 h after a single injection of endotoxin, but had returned to control levels by 96h. The magnitudes of these decreases in these measurements after repeated doses of endotoxin were similar to those seen 24h after the single dose. The systemic clearance of antipyrine correlated significantly with cytochrome P450 content and aminopyrine N-demethylase activity. In histopathological experiments, moderate hypertrophy of Kupffer cells was observed, with no evidence of severe liver-tissue damage. G-CSF pretreatment suppressed the increased plasma concentrations of TNFalpha produced 2 h after single endotoxin injection, but did not eliminate the endotoxin-induced decrease in the systemic clearance of antipyrine, suggesting that TNFalpha is not the sole component responsible for the reduction of cytochrome P450-mediated drug-metabolizing enzyme activity. These results provide evidence that a single intraperitoneal injection of 1.0 mgkg(-1)K. pneumoniae endotoxin in rats reduces hepatic P450 and b5 levels, and reduces the activity of various cytochrome P450-mediated drug-metabolizing enzymes without causing severe liver-tissue damage. This suggests that the effect of endotoxin on hepatic cytochrome P450-mediated drug-metabolizing isozymes is non-selective.

[General toxicity study of gadobenate dimeglumine formulation (E7155) (2)--Single dose intravenous toxicity study in dogs].

Gadobenate dimeglumine formulation (E7155) was given by single intravenous injection to 4-5 month-old beagle dogs at doses of 2 or 6 mmol/kg. Treatment was followed by a 14-day observation period in order to evaluate the test article's toxicity. The male and female dogs at 6 mmol/kg vomited and showed reddened gums and ears as clinical signs. One male dog at 6 mmol/kg was euthanized approximately 23 hr after administration due to its very poor clinical condition, which included an unwillingness to move, pale gums and weak pulse. Body weight was decreased at 6 mmol/kg, and also slightly at 2 mmol/kg. Decreased food consumption was noted both at 2 and 6 mmol/kg. Hematology for the euthanized male at 6 mmol/kg showed increases in the total white blood cell count, packed cell volume, hemoglobin and red cell count and a decrease in the platelet count. Biochemistry showed a dose-related increase in alkaline phosphatase, GPT and GOT at 2 and 6 mmol/kg. Males and females at 6 mmol/kg showed increases in bilirubin, calcium and urea, and a reduction in glucose. Females at 6 mmol/kg also showed a reduction in total protein. Urinalysis showed an increase in pH at 2 mmol/kg and above. For females at 6 mmol/kg, an increase in urine volume and a decrease in specific gravity and osmolality were noted. An increase in relative liver and kidney weights was recorded for males and females dosed at 6 mmol/kg. For the euthanized male at 6 mmol/kg, postmortem examination revealed a pale liver with rounded edges and an accentuated lobular pattern, and dark material on the gastro-intestinal mucosal surface. In macroscopic pathology, the male at 6 mmol/kg revealed single liver cell necrosis, minimal early hyperplasia in small biliary ductules, inflammatory cells in the sinusoidal and portal tracts, centrilobular inflammatory cells, diffuse vacuolation of the hepatocytes and sinusoidal dilatation in the liver, and cortical tubular vacuolation in the kidneys. In the female dog treated at 6 mmol/kg, hyperplasia in the small biliary ductules, inflammatory cells in the portal tracts, diffuse vacuolation of hepatocytes and sinusoidal dilatation were seen in the liver, and increases in the severity of cortical tubular basophilia, cortical tubular dilatation and cortical tubular casts were detected in the kidney. Based on these results, the lethal dose of E7155 was set at 6 mmol/kg. It is also concluded that a dose of 2 mmol/kg was tolerated in the beagle dog after a single injection followed by a 14-day observation period.

Transdermal delivery and intramuscular injection of trimethoprim/sulphadiazine in sucking piglets.

The pharmacokinetics of a combination of trimethoprim (TMP) and sulphadiazine (SDZ) after topical application to sucking piglets was compared with the pharmacokinetics after intramuscular injection. A long-lasting and fairly constant SDZ/TMP concentration ratio in plasma was obtained after topical application. The mean plasma concentration of TMP ranged from 0.091 to 0.17 micrograms/ml and that of SDZ from 0.72 to 1.1 micrograms/ml for at least 24 h. TMP and SDZ had different half-lives after intramuscular injection. Transdermal delivery of a combined preparation of TMP/SDZ may be usable for colibacillosis of sucking piglets, although the bioavailability of the drugs is poor.

Case studies for statistical analysis of toxicokinetic data.

The present paper discusses aspects of the statistical analysis of toxicokinetic data by reference to 102 case studies. In toxicokinetic studies, dose dependence of exposure is the most important subject. For this purpose, and because variability in concentration is closely related to the mean value, before beginning the standard procedures of analysis of variance and regression analysis concentration data should be transformed into logarithms. This data transformation also assists assessment of nonlinear kinetics. The paper discusses a special issue in the analysis of experiments for which measurement on each subject has been repeated. A simple procedure is proposed for estimating the daily exposure level in rodent studies when test chemicals have been mixed in diet and exposure level is measured on different animals at each time point. Experimental designs are classified into three types, and these are illustrated by data from representative case studies. Conclusions from 102 case studies are summarized, with attention to how often sex differences, change in exposure level during repeated dosing, and how often evidence is strong for nonlinear kinetics. Various key points of study design are discussed.

Effect of E5510 on anastomotic intimal hyperplasia and platelet aggregation in dogs.

We examined the effect of an antiplatelet agent, E5510, which inhibits both platelet aggregation and release of platelet-derived growth factor (PDGF), on anastomotic intimal hyperplasia and platelet aggregation. Twenty Beagle dogs underwent infrarenal aortic reconstruction with an expanded polytetrafluoroethylene (ePTFE) graft 5 mm in diameter and 3 cm long. The dogs were divided into three groups: placebo (control group, 7 dogs), E5510 1 mg/day (1-mg group, 6 dogs), and E5510 4 mg/day (4-mg group, 7 dogs). E5510 was administered orally 2 h before operation and once daily for 3 months after operation. Grafts were harvested 3 months after operation. All 13 grafts in the treated groups remained patent without evidence of intimal hyperplasia, whereas only 4 of 7 grafts (57%) remained patent in the control group, including 1 graft with > 50% stenosis. Three occluded grafts showed severe intimal hyperplasia at the anastomoses. The platelet aggregation ratio (PAR) with collagen (100 micrograms/ml) before drug administration at 3 months in the 4-mg group was significantly lower than that in the control and 1-mg groups. PAR after drug administration at 3 months in the 1- and 4-mg groups was significantly lower than that in the control group. Intimal thickness at the distal anastomosis was 817 +/- 190 microns in the control group, 240 +/- 80 microns in the 1-mg group, and 197 +/- 28 microns in the 4-mg group. Intimal thickness in the control group was significantly greater than that in the 1- and 4-mg groups. Smooth muscle cell (SMC) values in the intima at the distal anastomosis were 65.6 +/- 4.4% extinction (%E) in the control group, 47.6 +/- 3.4%E in the 1-mg group, and 51.3 +/- 3.5%E in the 4-mg group. SMC value in the control group was significantly greater than that in the 1- and 4-mg groups. E5510 inhibited PAR and reduced the degree of anastomotic intimal hyperplasia.