Effects of Simvastatin on Alveolar Regeneration and Its Relationship to Exposure in Mice with Dexamethasone-Induced Emphysema.

In the present study, the relationship between systemic exposure of simvastatin (SV) hydroxy acid (SV-acid), an active form of SV, and its alveolar regeneration rates was investigated using emphysema model mice created by postnatal treatment of dexamethasone. In a model with young animals, the mice were treated with SV for 10 d from postnatal day 42. Similar alveolar regeneration with a % mean linear intercept (Lm) recovery of 60 to 70% by histochemical observation was observed in mice after intraperitoneal administration at dose in the range of 4-100 µg/mouse. The % Lm recovery after oral administration of 20 µg/mouse was comparable with that after intraperitoneal administration at a dose of 4 µg/mouse, when their exposure of SV-acid was almost similar in both treated groups. Regardless of the route of administration, the recovery can depend on the exposure level of SV-acid, and to the maximum was about 60-70%. On the other hand, in a model with adult animals, the mice were intraperitoneally administrated SV at a dose of 4 µg/mouse for 10 d from postnatal day 152. Compared to young animals, less % Lm recovery was observed in adult mice even their systemic exposures of SV-acid were similar.

Effects of All trans-Retinoic Acid on Alveolar Regeneration in Dexamethasone-Induced Emphysema Models and Its Relationship to Exposure in ICR and FVB Mice.

During the past two decades, it has been reported that treatment with all-trans-retinoic acid (ATRA) induces alveolar regeneration in rodent emphysema models. In the present study, we investigated the regeneration by ATRA at various exposure conditions in two strains of mice with induced emphysema. The emphysema model was created by postnatal administration of dexamethasone to ICR and FVB mice, which were then treated with ATRA from postnatal day 42. The regeneration was observed in ICR mice but not in FVB mice given 10 and 40 mg/kg/d ATRA for 10 d. The concentration-time profiles of ATRA in plasma and lung were similar in both strains. These results suggest that the strain difference in the regeneration by ATRA was not caused by differences in the exposure to ATRA. On the other hand, the regeneration in ICR mice was enhanced by an increase of the intraperitoneal dose in the range of 10-40 mg/kg/d for 10 d. At an intraperitoneal dose of 40 mg/kg/d, the regeneration was observed after 10 and 20 d of treatment but not after 1 to 5 d of treatment. Meanwhile, the regeneration by intraperitoneal administration of ATRA was enhanced more than that by oral administration. Exposure to ATRA during repeated intraperitoneal administration to ICR mice was higher than that in oral administration. The results suggest that the regeneration in ICR mice requires at least 10 d of treatment with ATRA and its effects depend on the exposure to ATRA in plasma, which parallels that in lung.

Evaluation of cytochrome P450 inductions by anti-epileptic drug oxcarbazepine, 10-hydroxyoxcarbazepine, and carbamazepine using human hepatocytes and HepaRG cells.

Anti-epileptic drug oxcarbazepine is structurally related to carbamazepine, but has reportedly different metabolic pathway. Auto-induction potentials of oxcarbazepine, its pharmacologically active metabolite 10-hydroxyoxcarbazepine and carbamazepine were evaluated by cytochrome P450 (CYP) 1A2, CYP2B6 and CYP3A4 mRNA levels and primary metabolic rates using human hepatocytes and HepaRG cells. For the CYP1A2 the induction potential determined as the fold change in mRNA levels was 7.2 (range: 2.3-11.5) and 10.0 (6.2-13.7) for oxcarbazepine and carbamazepine, respectively, while 10-hydroxyoxcarbazepine did not induce. The fold change in mRNA levels for CYP2B6 was 11.5 (3.2-19.3), 7.0 (2.5-10.8) and 14.8 (3.1-29.1) for oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine, respectively. The fold change for CYP3A4 induction level by oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine was 3.5 (1.2-7.4), 2.7 (0.8-5.7) and 8.3 (3.5-14.5), respectively. The data suggest lower induction potential of oxcarbazepine and 10-hydroxyoxcarbazepine relative to carbamazepine. The results in HepaRG cells showed similar trend as the human hepatocytes. After incubation for 72 h in hepatocytes and HepaRG cells, auto-induction was evident for only carbamazepine metabolism. The 10-keto group instead of double bond at C10 position is evidently a determinant factor for limited auto-induction of P450 enzymes by oxcarbazepine.

Investigation of Enantioselective Membrane Permeability of α-Lipoic Acid in Caco-2 and MDCKII Cell.

α-Lipoic acid (LA) contains a chiral carbon and exists as two enantiomers (R-α-lipoic acid (RLA) and S-α-lipoic acid (SLA)). We previously demonstrated that oral bioavailability of RLA is better than that of SLA. This difference arose from the fraction absorbed multiplied by gastrointestinal availability (F(a) × F(g)) and hepatic availability (F(h)) in the absorption phase. However, it remains unclear whether F(a) and/or F(g) are involved in enantioselectivity. In this study, Caco-2 cells and Madin-Darby canine kidney strain II cells were used to assess the enantioselectivity of membrane permeability. LA was actively transported from the apical side to basal side, regardless of the differences in its steric structure. Permeability rates were proportionally increased in the range of 10-250 µg LA/mL, and the permeability coefficient did not differ significantly between enantiomers. Hence, we conclude that enantioselective pharmacokinetics arose from the metabolism (F(h) or F(g) × F(h)), and definitely not from the membrane permeation (F(a)) in the absorption phase.

Bioavailability of an R-α-Lipoic Acid/γ-Cyclodextrin Complex in Healthy Volunteers.

R-α-lipoic acid (R-LA) is a cofactor of mitochondrial enzymes and a very strong antioxidant. R-LA is available as a functional food ingredient but is unstable against heat or acid. Stabilized R-LA was prepared through complexation with γ-cyclodextrin (CD), yielding R-LA/CD. R-LA/CD was orally administered to six healthy volunteers and showed higher plasma levels with an area under the plasma concentration-time curve that was 2.5 times higher than that after oral administration of non-complexed R-LA, although the time to reach the maximum plasma concentration and half-life did not differ. Furthermore, the plasma glucose level after a single oral administration of R-LA/CD or R-LA was not affected and no side effects were observed. These results indicate that R-LA/CD could be easily absorbed in the intestine. In conclusion, γ-CD complexation is a promising technology for delivering functional but unstable ingredients like R-LA.

Enantioselective Pharmacokinetics of α-Lipoic Acid in Rats.

α-Lipoic acid (LA) is widely used for nutritional supplements as a racemic mixture, even though the R enantiomer is biologically active. After oral administration of the racemic mixture (R-α-lipoic acid (RLA) and S-α-lipoic acid (SLA) mixed at the ratio of 50:50) to rats, RLA showed higher plasma concentration than SLA, and its area under the plasma concentration-time curve from time zero to the last (AUC) was significantly about 1.26 times higher than that of SLA. However, after intravenous administration of the racemic mixture, the pharmacokinetic profiles, initial concentration (C0), AUC, and half-life (T1/2) of the enantiomers were not significantly different. After oral and intraduodenal administration of the racemic mixture to pyrolus-ligated rats, the AUCs of RLA were significantly about 1.24 and 1.32 times higher than that of SLA, respectively. In addition, after intraportal administration the AUC of RLA was significantly 1.16 times higher than that of SLA. In conclusion, the enantioselective pharmacokinetics of LA in rats arose from the fraction absorbed multiplied by gastrointestinal availability (FaFg) and hepatic availability (Fh), and not from the total clearance.

Effect of γ-Cyclodextrin Inclusion Complex on the Absorption of R-α-Lipoic Acid in Rats.

R-α-lipoic acid (RLA) is an endogenous organic acid, and works as a cofactor for mitochondrial enzymes and as a kind of antioxidant. Inclusion complexes of RLA with α-, ß- or γ-cyclodextrins (CD) were prepared and orally administered as a suspension to rats. Among them, RLA/γ-CD showed the highest plasma exposure, and its area under the plasma concentration-time curve (AUC) of RLA was 2.2 times higher than that after oral administration of non-inclusion RLA. On the other hand, the AUC after oral administration of non-inclusion RLA and RLA/γ-CD to pylorus-ligated rats did not differ. However, the AUC after intraduodenal administration of RLA/γ-CD was 5.1 times higher than that of non-inclusion RLA, and was almost comparable to the AUC after intraduodenal administration of RLA-Na solution. Furthermore, the AUC after intraduodenal administration of RLA/γ-CD was not affected by biliary ligation or co-administration of an amylase inhibitor. These findings demonstrated that RLA was absorbed from the small intestine effectively when orally administered as a γ-CD inclusion complex, which could be easily dissolved in the lumen of the intestine. In conclusion, γ-CD inclusion complex is an appropriate formulation for supplying RLA as a drug or nutritional supplement with respect to absorption.

Evaluation of cyanocobalamin in multivitamin tablets and their Standard Reference Material 3280 by HPLC with visible detection.

As a part of the development of an alternative to microbiological assay for vitamin B12, we performed a quantitative analysis of cyanocobalamin (CN-cbl) in a National Institute of Standards and Technology Standard Reference Material (SRM 3280) by HPLC. Using this method, the observed value (4.64 microg/g) of CN-cbl in SRM 3280 was found to be in good agreement with the certified value (4.80 microg/g). The accuracy was over 95%, with a corresponding measurement precision value of 5%. To evaluate the applicability of the method on commercial multivitamin tablets, the method was applied to a variety of these samples. The present method has a good accuracy and precision to evaluate CN-cbl with respect to all of the examined tablets.

10'-Chloro-3',4'-dihydro-2'H-spiro-[cyclo-propane-1,7'(6'H)-pyrimido[2,1-a]isoquinolin]-6'-one.

In the title compound, C(14)H(13)ClN(2)O, the fused hydro-pyrimidine ring adopts an envelope conformation with one of the methyl-ene C atoms at the flap. The three-membered ring is approximately perpendicular to the attached isoquinoline ring system, with a dihedral angle of 89.44 (11)°. In the crystal, mol-ecules are linked by a weak C-H⋯π inter-action, forming a helical chain along the c axis.

3-(2-Oxo-2,3,4,5-tetra-hydro-furan-3-yl)-1-benzofuran-2-carbonitrile.

The asymmetric unit of the title compound, C(13)H(9)NO(3), consists of two crystallographically independent mol-ecules. In each mol-ecule, the tetra-hydro-furan (THF) ring adopts an envelope conformation with one of the methyl-ene C atoms positioned at the flap. The dihedral angles between the mean plane of the THF and the benzofuran ring system are 70.85 (5) and 89.59 (6)°. In the crystal, mol-ecules are stacked in a column along the a-axis direction through C-H⋯O hydrogen bonds, with columns further linked by C-H⋯N and C-H⋯O inter-actions.

Application of a simple measuring method to evaluate the fecal microflora of dairy cows in the summer season.

OBJECTIVE: The effect of seasonality needs to be considered in designing future studies because global warming has caused a rise in ambient temperatures. The objective of the present study is to investigate the effect of high ambient temperatures on fecal score and fecal microflora in dairy cows during summer. MATERIALS AND METHODS: During the 7 days before the sampling of feces, the daily mean temperatures were 19.9°C in early summer and more than 27.5°C in late summer. Fecal samples were collected from the rectum of cows and the fecal score was evaluated on a 4-point scale. The equalized samples were used to extract the genomic deoxyribonucleic acid (DNA) of the bacteria (Escherichia coli, Salmonella, Lactobacillus, and Bifidobacterium). RESULTS: There was no significant difference in fecal scores between the sampling times in early and late summer. In the populations of the bacteria, there was no significant difference between sampling days in the DNA level of Salmonella, and E. coli in late summer increased to more than three times the level in early summer. However, both levels of Lactobacillus and Bifidobacterium in early summer significantly decreased after 2 months. CONCLUSION: These data suggest that the increase in temperature in late summer may adversely affect the populations of bacteria in the intestinal environment of dairy cows. In addition, the method used in the present study was sufficient to evaluate the changes in internal and external environmental conditions of dairy cattle.

Polycyclic N-heterocyclic compounds. Part 65: ring cleavage reactions of fused furo[2,3-c]isoquinolines and related compounds with various nucleophiles.

Reaction of fused 2,3-dihydrofuro[2,3-b]pyridines with various nucleophiles (N and O) gave dihydrofuran ring cleaved products. The scope of this reaction was investigated in detail.

Polycyclic N-heterocyclic compounds. Part 63: Improved synthesis of 5-amino-1,2-dihydrofuro[2,3-c]isoquinolines via truce-smiles rearrangement and subsequent formation to furo[2,3-c]isoquinoline.

An improved synthesis of 5-amino-1,2-dihydrofuro[2,3-c]isoquinoline has been achieved using a slight modification of reaction conditions for the Truce-Smiles rearrangement. Acid treatment of the obtained 5-amino-1,2-dihydrofuro[2,3-c]isoquinolines gave unexpected ring-opened spiro ring compounds. The previously unreported parent compound, furo[2,3-c]isoquinoline, was also synthesized.

Polycyclic N-heterocyclic compounds. Part 64: synthesis of 5-amino-1,2,6,7-tetrahydrobenzo[f]furo[2,3-c]isoquinolines and related compounds. Evaluation of their bronchodilator activity and effects on lipoprotein lipase mRNA expression.

Reaction of 1-(3-cyanopropoxy)-3,4-dihydronaphthalene-2-carbonitriles with potassium tert-butoxide gave 5-amino-1,2,6,7-tetrahydrobenzo[f]furo[2,3-c]isoquinolines via a Truce-Smiles rearrangement. The 5-amino group was transformed to the bromo derivatives which were allowed to react with aliphatic cyclic amines to produce amino derivatives. In contrast, a combination of imidazole and NaH gave a dihydrofuran ring cleaved product, the structure of which was confirmed by X-ray crystallographic analysis. Effects of the newly synthesized compounds on carbamylcholine chloride-induced contractions of trachea and lipoprotein lipase mRNA expression were also evaluated and found one promising bronchodilator.

Polycyclic N-heterocyclic compounds. Part 62: Reaction of N-(quinazolin-4-yl)amidine derivatives with hydroxylamine hydrochloride and anti-platelet aggregation activity of the products.

The reactions of N-(5,6,7,8-tetrahydroquinazolin-4-yl)amidines and their amide oximes with hydroxylamine hydrochloride gave abnormal cyclization products via a ring cleavage of pyrimidine component accompanied with a ring closure of 1,2,4-oxadiazole to give N-[2-([1,2,4]oxadiazol-5-yl)cyclohexen-1-yl]formamide oximes. Similarly, N-(quinazolin-4-yl)amidines reacted with hydroxylamine hydrochloride gave the same results. The evaluation of inhibitory activities against platelet aggregation in vitro is also described to show one derivative has potent activity.

Chloroform distribution and accumulation by combined inhalation plus oral exposure routes in rats.

The present investigation was undertaken to determine the distribution and accumulation of chloroform in the blood, liver, kidney and abdominal fat of rats after simultaneous exposure by two routes, inhalation and oral. To distinguish the contribution of each route, unmodified chloroform (CHCl3) was administered by inhalation and deuterated chloroform (CDCl3) was administered orally. Exposure by inhalation and oral administration resulted in CHCl3 and CDCl3 concentrations in the tissues which were significantly higher than when exposure was by either inhalation or oral administration alone. This is the first study to follow the contribution of each of two routes of chloroform exposure on chloroform distribution and accumulation in target tissues. Our results indicate that when assessing the toxicity and carcinogenicity of chloroform, exposure routes, especially the effects of exposure by multiple routes, must be taken into consideration.

7-Chloro-1,2-dihydro-furo[2,3-c]isoquinolin-5-amine.

In the title compound, C(11)H(9)ClN(2)O, the fused-ring system is essentially planar, with a maximum deviation of 0.0323 (16) Å. In the crystal, mol-ecules are connected by N-H⋯O hydrogen bonds forming a zigzag chain along the c axis. Mol-ecules are further stacked along the a axis through weak π-π inter-actions, the shortest distance between ring centroids being 3.6476 (8) Å.

Synthesis of a fluorine-substituted puromycin derivative for Brønsted studies of ribosomal-catalyzed peptide bond formation.

The mechanism by which the ribosome catalyzes peptide bond formation remains controversial. Here we describe the synthesis of a nucleoside that can be used in Brønsted experiments to assess the transition state of ribosome catalyzed peptide bond formation. This substrate is the nucleoside 3'-amino-3'-deoxy-3'-[(3''R)-3-fluoro-l-phenyl-alanyl]-N(6),N(6)-dimethyladenosine, which was prepared from (1R,2R)-2-amino-1-phenylpropane-1,3-diol. This substrate is active in peptide bond formation on the ribosome and is a useful probe for Brønsted analysis experiments on the ribosome.

Polycyclic N-heterocyclic compounds. Part 60: reactions of 3-(2-cyanophenyl)quinazolin-4(3H)-ones with primary amines.

The reaction of 3-(2-cyanophenyl)quinazolin-4(3H)-one with various primary alkylamines gave 3-alkylquinazolin-4(3H)-ones via an addition of the nucleophile, ring opening, and ring closure (ANRORC) mechanism. This type of reaction required hydroxy group functionality in either the solvent or reagent. When hydroxylamine was used as nitrogen nucleophile, the intermediate of this reaction was isolated and found to be an amide oxime. When ethylenediamine was used as the nucleophile, the amidine moiety of the intermediate decomposed to give a benzanilide.

Characterization of CS-023 (RO4908463), a novel parenteral carbapenem antibiotic, and meropenem as substrates of human renal transporters.

To characterize the renal handling of CS-023 (RO4908463), a novel parenteral carbapenem antibiotic, and meropenem in humans, we examined their affinities as substrates to human renal transporters. In vitro studies on the uptake of [14C]CS-023 and [14C]meropenem were conducted using HEK293 cells expressing human organic anion transporters (hOAT) 1, hOAT3, hOAT4, and the human organic cation transporters (hOCT) 1 and hOCT2. CS-023 did not serve as the substrate for any of the transporters tested. On the other hand, meropenem was transported by hOAT1 and hOAT3. The Km value of the hOAT3-mediated transport was 847 microM, and the uptake was inhibited by probenecid, p-aminohippurate and benzylpenicillin with Ki values of 3.76, 712, and 202 microM, respectively. One of the reasons why CS-023 is not a substrate of hOATs, and vice versa for meropenem, would be that a very small proportion of CS-023 exists as the anionic form at the physiological pH, whereas 50% of meropenem exists as the anionic form. These findings indicate that the lack of recognition of CS-023 by renal transporters is one of the reasons for its long plasma half-life in humans compared with meropenem which undergoes renal tubular secretion mediated by hOAT1 and hOAT3.