Taurine inhibits development of atherosclerotic lesions in apolipoprotein E-deficient mice.

1. The effects of taurine on the development of atherosclerotic lesions were investigated in apolipoprotein (apo) E-deficient mice, an animal model with severe hypercholesterolaemia and extensive atherosclerosis. These mice were fed a normal laboratory chow containing 2% taurine for 12 weeks. 2. Serum total cholesterol was significantly elevated after 12 weeks treatment with taurine. This elevation was due to increases in very low-density lipoprotein- and low-density lipoprotein-cholesterol. 3. Despite such effects on serum lipoproteins, analysis using en face oil red O staining revealed that taurine reduced the area of arterial lipid accumulation by 28%, as measured quantitatively as an index of atherosclerosis. Histological examination also demonstrated a decrease in the size of aortic lesions in taurine-treated mice. 4. Serum levels of thiobarbituric acid reactive substances (TBARS) in apoE-deficient mice were higher than in normolipidaemic C57BL/6J mice. Serum TBARS levels were significantly decreased by 12 weeks treatment of apoE-deficient mice with taurine. 5. Thus, taurine prevents the formation of atherosclerotic lesions, independently of serum cholesterol levels, and the results suggest that the anti-oxidative effects of taurine are related to its anti-atherosclerotic actions.

Heat shock protein (HSP) 47 and collagen are upregulated during neointimal formation in the balloon-injured rat carotid artery.

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is thought to be essential for the proper processing and secretion of procollagen molecules. We investigated the time course and localization of HSP47 and collagen expression after balloon catheter angioplasty in the rat carotid artery, based on the premise that accumulation of extracellular matrix components is a main feature of intimal hyperplasia in humans and in laboratory animals. Low levels of HSP47 expression were evident in uninjured carotid arteries. Northern blot analysis revealed that HSP47 mRNA expression was markedly stimulated 1--3 days after the induced injury and a high level was maintained for 7 days, followed by a gradual decline for up to 21 days after the injury. These changes in HSP47 expression paralleled changes in alpha 1(I) collagen expression. Immunohistochemical staining revealed colocalization of HSP47 and collagen in smooth muscle cells (SMCs) of the media and intima. In situ hybridization analysis showed that activated SMCs, which proliferated and migrated into the intima, expressed high levels of HSP47. In cultured human aortic SMCs, similar upregulation of HSP47 and alpha1(I) collagen by TGF-beta was noted. These results show that SMCs activated after balloon injury express high levels of HSP47 and collagen during cell proliferation and migration, hence an overproduction of collagen and development of intimal thickening. Thus, HSP47 plays a role in the formation and progression of neointima after angioplasty.

In vitro and in vivo antibacterial activity and pharmacokinetics of SC-002 and its derivative, SC-004: new oral cephalosporins.

SC-002 is a novel oral cephalosporin possessing a unique thiadiazolylethenyl moiety at the 3 position. In the present study, it was the most active against gram-positive bacteria among oral cephalosporins such as cefdinir (CFDN), cefpodoxime, cefditoren and cefaclor (CCL). It was equal to or 16 times more active than CFDN against standard and clinical strains. In particular, against clinical isolates of Morganella morganii and Haemophilus influenzae, SC-002 was 8-64 times more active than CFDN. The antibacterial activity of SC-002 against some beta-lactam-resistant strains was superior to that of CFDN. The in vivo antibacterial activity of SC-004, a pivaloyloxymethyl ester of SC-002, was 1.2-8 times more protective against systemic infections due to Streptococcus pneumoniae, Escherichia coli and Klebsiella pneumoniae than that of CFDN. The therapeutic effects of SC-004 on experimental respiratory tract infections caused by S. pneumoniae or H. influenzae were superior to those of CFDN and CCL. SC-004 showed higher and longer-lasting blood levels and higher urinary excretion in pharmacokinetics in mice.

Effects of long-term treatment with taurine in mice fed a high-fat diet: improvement in cholesterol metabolism and vascular lipid accumulation by taurine.

Hypocholesterolemic effects of taurine in rats fed a high-fat and high-cholesterol diet are well established. However, there are few studies on long-term effects of taurine on cholesterol metabolism. In the present study, taurine was dissolved in drinking water and given to C57BL/6J mice during 6 months-feeding of a high fat diet. Taurine treatment significantly decreased serum LDL and VLDL cholesterol, while it significantly increased serum HDL cholesterol. In the liver, taurine decreased cholesteryl ester contents, accompanied by decrease in acyl CoA:cholesterol acyltransferase (ACAT) activity. Hepatic activity of cholesterol 7alpha-hydroxylase, a rate-limiting enzyme for bile acid synthesis, was doubled with taurine. Taurine reduced by 20% the high-fat diet-induced arterial lipid accumulation. Thus, taurine prevented elevation of serum and liver cholesterol levels, as possibly related to accelerated cholesterol elimination from the body through the stimulation of bile acid synthesis. Long-term treatment with taurine is beneficial for prevention of hypercholesterolemia and atherosclerosis.

Taurine reduces atherosclerotic lesion development in apolipoprotein E-deficient mice.

The effects of dietary taurine on development of atherosclerotic lesions were investigated using apolipoprotein E (apoE)-deficient mice. Taurine added to regular chow at 2% (w/w), was made freely available to mice for 3 months. Severe hypercholesterolemia and development of atherosclerotic lesions occurred in the apo-E-deficient mice. Taurine treatment decreased the area of Oil red-O positive lipid accumulation in the aortic valve by 31%. In contrast, taurine significantly increased serum atherogenic lipoproteins (LDL + VLDL), without changing HDL cholesterol levels. Although the levels of serum thiobarbituric acid reactive substances (TBARS) in apoE-deficient mice were significantly higher than in wild-type mice, taurine decreased TBARS by 26%. These observations mean that taurine prevents the development of atherosclerosis, independent of serum cholesterol levels. We suggest that antioxidative actions may be involved in the anti-atherosclerotic effects of taurine.

Prevention of atherosclerotic lesion development in mice by taurine.

The antiatherosclerotic effects of taurine were evaluated in two murine models. In C57BL/6J mice fed a high-fat diet, 6-month treatment with taurine decreased serum atherogenic low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) cholesterol by 44%. The same treatment increased antiatherogenic high-density lipoprotein (HDL) cholesterol by 25%. Hepatic cholesterol content was also decreased by taurine. Taurine improved the area of oil red O positive arterial lipid accumulation by 20%. Hepatic cholesterol 7 alpha-hydroxylase activity, a rate-limiting enzyme of bile acid synthesis from cholesterol, was doubled in taurine-treated mice, suggesting stimulation of cholesterol catabolism to bile acid as the cholesterol-lowering mechanism seen with taurine. Thus, taurine prevented the progression of atherosclerotic lesions, and concomitantly improved the serum lipoprotein profile. In another murine model, in apolipoprotein-E-deficient mice fed regular chow, a 3-month treatment with taurine prevented accumulation of arterial lipids by 31%, despite a significant increase in serum LDL and VLDL cholesterol levels. Serum thiobarbituric acid reactive substances (TBARS) in apolipoprotein-E-deficient mice were significantly higher than those in wild type mice and treatment with taurine lowered serum TBARS level by 26%. Thus, taurine prevents the development of atherosclerotic lesions and this antioxidative effect may play an important role in the antiatherosclerotic effect that is unrelated to serum cholesterol levels.

In vitro and in vivo antibacterial activities of clarithromycin.

The in vitro and in vivo antibacterial activities of clarithromycin (CAM), a new oral macrolide antibiotic, were compared with those of erythromycin (EM), josamycin (JM) and rokitamycin (RKM). The antibacterial spectrum and in vitro activities of CAM were similar to those of EM. Therapeutic efficacies of CAM against various experimental infections in mice--including systemic infections caused by gram-positive bacteria such as Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae, and subcutaneous abscess due to S. aureus, and bacterial pneumonia caused by S. pneumoniae--were superior to those of EM, JM and RKM. CAM exhibited higher serum levels than EM in mice after a single oral dose of 50 mg/kg.

Lipid composition and fatty acid analysis of Helicobacter pylori.

Lipids extracted from Helicobacter pylori were separated into lipid classes by thin-layer chromatography. Simple H. pylori lipids consisted of cholesterol esters, triglycerides, free fatty acids, cholesterol, diacylglycerols, and monoacylglycerols. Fatty acids were released from each lipid class by acid methanolysis, and analyzed by gas liquid chromatography and mass spectrometry. Unique methoxy fatty acids, including 11-methoxy heptadecanoic and 11-methoxy nonadecanoic acids, were the major components of the cholesterol esters and triglycerides. The predominance of methoxy fatty acids in the cholesterol esters of H. pylori may contribute to the acid-resistant characteristic of this bacillus.

Structure-activity relationship study of 6-O-methylerythromycin 9-O-substituted oxime derivatives.

In order to develop new-generation macrolide antibiotics active against erythromycin (EM)-resistant strains, a series of 6-O-methyl EM 9-O-substituted oxime derivatives was synthesized and evaluated for antibacterial activity against EM-resistant (S. aureus J-109) and susceptible (S. aureus 209P) strains. To understand how substituents affect the biological activity, the quantitative structure-activity relationships (QSAR) was analyzed using the Hansch-Fujita method. With the EM-resistant strain, the positive coefficient for log P may indicate that higher hydrophobicity of molecules is favorable for antibacterial activity. The negative coefficients of the Sterimol parameters L, B1, and B5 may indicate that long, bulky substituents are unfavorable. With the EM-susceptible strain, the negative coefficient for log P may indicate that hydrophilicity is important for antibacterial activity. A short substituent is also required to improve the activity. Based on the QSAR model, a derivative (87) having an anthracenylmethyl moiety was synthesized to reinforce and confirm the correlation. The activity of 87 against the EM-resistant strain was significant. In QSARs of 6-O-methyl EM-A 9-O-substituted oxime derivatives, the difference of the contribution of log P to the antibacterial activity between EM-resistant and susceptible strains was clearly recognized.

Suppression of IgE production in unseparated spleen cell cultures.

When B cells from BALB/c mice were cultured with lipopolysaccharide (LPS) and interleukin-4 (IL-4), a large amount of IgE was detected in the culture supernatants. The IgE production from unseparated spleen cells cultured with LPS and IL-4 was less than the amount of IgE obtained from separated B cells. When syngeneic T cells were added to separated B cells cultures, which were subsequently stimulated with LPS and IL-4, less IgE was produced, as compared to cultures without T cells. The hypothesis that T cells, or factors secreted by these cells, inhibit IgE production is supported by the fact that the degree of suppression of IgE production paralleled the number of T cells added. CD8(+)-enriched T cells were slightly more suppressive than CD4(+)-enriched T cells. Addition of exogenous IL-3 was only partially suppressive. These observations suggest that IL-4 added to unseparated spleen cells in vitro stimulates B cells for IgE production and also stimulates T cells. Lymphokines secreted by these stimulated T cells may in turn act on B cells. Some of these lymphokines, such as interferon-gamma and IL-2, may have a suppressive action on IgE production.

Lipid analysis of Helicobacter pylori.

Lipids from Helicobacter pylori were extracted, isolated by conventional DEAE-Sephadex and silica gel column chromatography, and then purified by preparative thin-layer chromatography. Simple and phospholipids were analyzed by HPTLC and quantitatively determined by densitometry scanning. The fatty acid compositions of simple lipids were estimated by gas-liquid chromatography. The simple lipid composition of H. pylori consisted of wax ester (2.5%), triglycerides (4.9%), free fatty acids (30.0%), cholesterol (6.9%), diacylglycerol (29.1%), and monoacylglycerol (2.6%). The neutral phospholipid composition consisted of phosphatidyl ethanolamine (79.1%), lysophosphatidyl ethanolamine (16%), and phosphatidyl choline (1.9%). That of acidic phospholipids consisted of phosphatidic acid (52.7%) and phosphatidyl serine (47.3%). The major fatty acids of crude lipid, wax, free fatty acids, triglycerides, diacylglycerides, and monoacyl glycerides were C19:1 (cyclo), C18:2, C16:0, C19:1 (cyclo), C22h:0, and C18:0, respectively. These results are new findings and suggest significant characteristics from the metabolic, physiologic, and chemotaxonomic point of view.

Studies on cephalosporin antibiotics. VI. Synthesis, antibacterial activity and oral efficacy in mice of new 7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino)-acetamido]-3- (substituted alkylthio)cephalosporins.

A series of new 7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino)acetamido] cephalosporins (1) having various substituted alkylthio groups at the C-3 position of the cephem nucleus were prepared and evaluated for antibacterial activity and oral absorption in rats. Of these, the cephalosporin with a cyanomethylthio group (1a) showed the greatest activity against Staphylococcus aureus and Gram-negative bacteria. Its pivaloyloxymethyl ester (6a), a representative prodrug, exhibited good in vivo efficacy in mice by oral administration. The structure-activity relationships of 1 are also presented.

[Research and development of clarithromycin].

A series of O-alkylated derivatives of erythromycin (EM) has been prepared and their biological properties were evaluated. Among them, clarithromycin (CAM, 6-O-methylerythromycin) exhibits most potent in vitro and in vivo antibacterial activities, higher acid-stability than EM and favorable pharmacokinetic properties as an antibiotic. CAM was originally synthesized via methylation of 2'-O,3'-N-bis(benzyl-oxycarbonyl)-N-demethylerythromycin in low yield, because of the less selectivity of 6-O-methylation. The selective 6-O-methylation was achieved using the erythromycin 9-oxime derivative as a key intermediate. By the further investigation on the protective groups of 9-oxime and desosamine moiety, the production process of CAM on an industrial scale has been established via methylation of 2',4''-O-bis(trimethylsilyl)erythromycin 9-[O-(1-isopropoxycyclohexyl)oxime] in more than 45% overall yield. CAM has the same antibacterial spectra as EM and is active against aerobic Gram-positive bacteria, some Gram-negative bacteria, anaerobic bacteria, Mycoplasma and Chlamydia. The activity of CAM against clinical isolates was 1 to 16 times higher than that of EM. The efficacies of CAM were 6 to 15 times superior to those of EM against systemic infections due to Gram-positive bacteria in mice. CAM also showed more potent therapeutic efficacies than EM against respiratory tract infections caused by S. pneumoniae and H. influenzae. CAM was well absorbed after oral administration, and its distribution to various tissues was significantly higher than that of EM in animals. The level of CAM in the lung was extremely high, which accounted 69 times that of EM. CAM was found to be distributed predominantly in the alveolar wall, especially in the alveolar epithelial cells, by microautoradiography. After oral administration in human, the serum level and urinary excretion of CAM were 5 and 20 times higher than those of EM, respectively. The major and active metabolite of CAM in human, (14R)-14-hydroxyclarithromycin, existed in significant quantity in the serum and urine, suggesting that the metabolite contributes to the excellent clinical efficacy of CAM. This paper describes the synthesis, structure-activity relationships, antibacterial activities, metabolism and clinical efficacies of CAM, a new macrolide antibiotic.

Studies on cephalosporin antibiotics. V. Synthesis, antibacterial activity and oral absorption of new 3-[(Z)-2-methoxycarbonylvinylthio]-7 beta- [(Z)-2-(2-aminothiazol-4-yl)-2-(oxyimino)acetamido]cephalosporins.

A series of new 3-[(Z)-2-methoxycarbonylvinylthio]-7 beta-[(2- aminothiazol-4-yl)acetamido]-cephalosporins (1) having various oxyimino groups (Z-form) at the alpha position of the C-7 side chain was synthesized and evaluated for antibacterial activity and oral absorption in rats. Of these, the cephalosporin (1a) with a hydroxyimino group in the C-7 side chain showed a potent antibacterial activity against Gram-negative bacteria and Gram-positive Staphylococcus aureus as well as good oral absorption in rats. The structure-activity relationships of 1 are also presented.

Studies on cephalosporin antibiotics. IV. Synthesis, antibacterial activity and oral absorption of new 3-(2-substituted-vinylthio)-7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2- (carboxymethoxyimino)acetamido]cephalosporins.

A series of new 7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2-(carboxymethoxyimino)ace tamido] cephalosporins (1) having various substituted-vinylthio groups at the C-3 position of the cephen nucleus was synthesized and evaluated for antibacterial activity and oral absorption in rats in comparison with cefixime. Of these, the cephalosporins (1a and 1c) with a lower alkoxycarbonylvinylthio group (Z-form) at the C-3 position showed a potent antibacterial activity against Gram-negative bacteria, improved activity against Staphylococcus aureus as well as good oral absorption in rats. The structure-activity relationships of 1 are also presented.

Studies on cephalosporin antibiotics. III. Synthesis, antibacterial activity and oral absorption of new 3-(substituted-alkylthio)-7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2- (carboxymethoxyimino)acetamido]cephalosporins.

The synthesis, antibacterial activity and oral absorption in rats of new 7 beta-[(Z)-2-(2-aminothiazol-4-yl)-2-(carboxymethoxyimino)ace tamido] cephalosporins (1) having various substituted-alkylthio groups at the C-3 position of the cephem nucleus are described. Of these, the cephalosporins with a cyanomethylthio group (1d) and fluoroethylthio group (1p) at the C-3 position showed a potent in vitro antibacterial activity against Gram-positive and Gram-negative bacteria as well as good oral absorption in rats. When administered orally to mice infected with Klebsiella pneumoniae, 1d had stronger protective effect than 1p. The structure-activity relationships of 1 are also presented.

Chemical modification of erythromycins. IV. Synthesis and biological properties of 6-O-methylerythromycin B.

6-O-Methylerythromycin B has been synthesized from erythromycin B via regioselective methylation of the 6-hydroxyl group in 71% overall yield. This compound shows in vitro antibacterial activity comparable to erythromycins A and B and exhibits superior in vivo activity with improved pharmacokinetic properties.

Chemical modification of erythromycins. III. In vitro and in vivo antibacterial activities of new semisynthetic 6-O-methylerythromycins A, TE-031 (clarithromycin) and TE-032.

The in vitro and in vivo antibacterial activities of 6-O-methylerythromycin A (TE-031, A-56268, or clarithromycin) and 6,11-di-O-methylerythromycin A (TE-032) have been compared with those of erythromycin A (EM) and josamycin (JM). TE-031 and TE-032, having the same antibacterial spectra as EM, are active against aerobic Gram-positive bacteria, some Gram-negative bacteria, anaerobic bacteria, L-form bacteria and Mycoplasma pneumoniae. The activity of TE-031 against clinical isolates is equal to or two times more potent than that of EM, whereas TE-032 is slightly less active than EM. The activities of TE-031 and TE-032 are pH dependent (more active at pH 8 than at 5) and are increased by adding serum to medium. TE-031 and TE-032 show dose-related bactericidal activities against Haemophilus influenzae. The therapeutic efficacies of TE-031 and TE-032 against systemic and subcutaneous infections provoked by Gram-positive bacteria in mice are 4- to 35-fold superior to those of EM and JM. TE-031 and TE-032 have demonstrated higher and longer-lasting plasma levels than EM when administered orally to mice, rats or dogs.

Chemical modification of erythromycins. II. Synthesis and antibacterial activity of O-alkyl derivatives of erythromycin A.

A series of O-alkyl erythromycin A derivatives have been synthesized and their antibacterial activities compared with those of erythromycin A (1) and 6-O-methylerythromycin A (3). Methylation of the hydroxyl groups of erythromycin A analogue proceeded stepwise by the two main pathways beginning at the C-6 and C-11 positions, individually. O-Alkylation, other than methylation, took place at the C-11 hydroxyl group exclusively. Among O-alkyl derivatives, 6,12-di-O-methylerythromycin A (5) showed comparable in vitro antibacterial activity to those of 1 and 3. 11-O-Methylerythromycin A (8) was slightly less active than 1. O-Methylation at the C-4" position resulted in a decrease of antibacterial activity.