Masato Horiuchi

Method for determination of angiotensin-converting enzyme activity in blood and tissue by high-performance liquid chromatography.

A simplified method for an angiotensin-converting enzyme activity assay in biological samples was developed. Samples were incubated with hippurylhistidylleucine, an artificial substrate of angiotensin-converting enzyme. The reaction was terminated by the addition of metaphosphoric acid and liberated hippuric acid in the supernatant was quantitated directly by reversed-phase high-performance liquid chromatography. Tissues were homogenized in the presence of Nonidet-P40, a detergent, and the resulting supernatant was used for the assay of tissue angiotensin-converting enzyme activity by high-performance liquid chromatography. The present procedure made it possible to determine angiotensin-converting enzyme activity in whole blood and the total activity in tissues. A comparative study of angiotensin-converting enzyme activity in plasma, kidney and lung of five experimental animals showed a high degree of variation from species to species.

Involvement of leukotriene B4 in arthritis models.

We investigated the role of leukotriene B4 (LTB4) in murine arthritis models using a leukotriene A4 (LTA4) hydrolase inhibitor, SA6541. SA6541 inhibited the severity of collagen-induced arthritis and muramyl dipeptide (MDP)-induced hyperproliferation of synovial cells in vivo. SA6541 also inhibited LTA4-induced hyperproliferation of synovial stromal cells in vitro. These results suggest that LTB4 may play an important role in arthritis models.

Involvement of Leukotriene B4 in Murine Dermatitis Models

Leukotriene B4 (LTB4) is a product of the 5-lipoxygenase pathway of arachidonic acid (AA) metabolism. LTB4 is a potent chemotactic factor for neutrophils and has been postulated to play an important role in a variety of pathological conditions including rheumatoid arthritis, psoriasis, and inflammatory bowel disease. To investigate the role of LTB4 in dermatitis, we used S-(4-dimethylaminobenzyl)-N-[(2S)-3-mercapto-2-methylpropionyl]-L- cysteine (SA6541), a potent leukotriene A4 (LTA4) hydrolase inhibitor. SA6541 inhibited LTB4 production with an IC50 value of 270 nM in vitro. 5-Hydroperoxyeicosatetraenoic acid (5-HPETE) or AA injection induced LTB4 production and neutrophil influx in mouse ear. SA6541 inhibited 5-HPETE- and AA-induced LTB4 production and neutrophil influx in mouse ear when administered orally at a dose of 50 mg/kg. SA6541 also inhibited 5-HPETE-induced prostaglandin E2 (PGE2) production, probably by an indirect effect through the inhibition of LTB4 production. These results suggest that LTB4 may be important in the pathogenesis of dermatitides such as psoriasis.

Effects of mitogen-activated protein kinase inhibitors or phosphodiesterase inhibitors on interleukin-1-induced cytokines production in synovium-derived cells

The effects of mitogen-activated protein (MAP) kinase inhibitors or phosphodiesterase (PDE) inhibitors on interleukin (IL)-1-induced cytokines production in synovium-derived cells were investigated. Human synoviocyte (HS) or synovial sarcoma (SW982) stimulated by IL-1beta (100 ng/ml) produced various cytokines including IL-6, IL-8, GROalpha, VEGF, basic FGF and tumor necrosis factor alpha (TNFalpha) in vitro. SB202190 or SB203580, an inhibitor of p38 MAP kinase, inhibited all cytokines production in both cells. PD98059, an inhibitor of MAP kinase kinase (MEK), inhibited IL-6, IL-8 and basic FGF production in HS and all cytokines production except basic FGF in SW982. However, many of its effects were weaker than those of SB202190 or SB203580. Quazinone, an inhibitor of cyclic GMP-inhibited PDE, scarcely affected cytokines production in both cells. Rolipram or R0201724, an inhibitor of cyclic AMP-specific PDE, inhibited IL-8 and basic FGF production in HS and TNFalpha production in SW982, however, it enhanced the other cytokines production in SW982. These results suggest that the activation of MAP kinase cascade may be important for IL-1-induced cytokines production in synovium-derived cells. On the other hand, the role of cyclic AMP may be dependent on cell and cytokine types.

Effects of SA6541, a leukotriene A4 hydrolase inhibitor, and indomethacin on carrageenan-induced murine dermatitis

We investigated the effects of S-(4-dimethylaminobenzyl)-N-[(2S)-3-mercapto-2-methylpropionyl]-L- cysteine (SA6541), a potent leukotriene A4 hydrolase inhibitor. on early phase of carrageenan-induced dermatitis model. Carrageenan injection induced edema and neutrophil influx in the mouse ear. SA6541 inhibited edema formation and neutrophil influx. SA6541 also inhibited leukotriene B4 production but not prostaglandin E2 production in the mouse ear. On the other hand, indomethacin inhibited edema formation but not neutrophil influx. Indomethacin also inhibited prostaglandin E2 production but not leukotriene B4 production. Combination therapy with SA6541 and indomethacin strongly inhibited edema formation in comparison with treatment with either agent alone. These results suggest that leukotriene B4 may be important in the pathogenesis of dermatitis.

Synthesis and biological evaluation of N-mercaptoacylcysteine derivatives as leukotriene A4 hydrolase inhibitors.

We studied synthetic modifications of N-mercaptoacylamino acid derivatives to develop a new class of leukotriene A(4) (LTA(4)) hydrolase inhibitors. S-(4-Dimethylamino)benzyl-l-cysteine derivative 2a (SA6541) showed inhibitory activity against LTA(4) hydrolase (IC(50), 270nM) and selectivity over other metallopeptidases except angiotensin-converting enzyme (ACE, IC(50), 520nM). Modification at the para-substituent of the phenyl ring of compound 2a improved LTA(4) hydrolase inhibitory activity as well as selectivity over ACE. Finally, we obtained S-(4-cyclohexyl)benzy-l-cysteine derivatives 11l and 16i as potent and selective LTA(4) hydrolase inhibitors.

Synthesis and pharmacological evaluation of 1,1,3-substituted urea derivatives as potent TNF-α production inhibitors

A three substituted urea derivative, SA13353 (compound 1a), exhibited potent inhibitory activity against lipopolysaccharide (LPS)-induced TNF-alpha production. We focused on the 1,1-substituted moiety (R(1) and R(2)) of SA13353 and investigated substituent effects of this moiety on LPS-induced TNF-alpha production by oral administration in rats. The synthesis of the urea derivatives was performed rapidly in a one-pot manner using a manual synthesizer. Several compounds containing hydrophobic substituents at this moiety showed more potent inhibitory activities than SA13353.

Application of a drug delivery system to a steroidal ophthalmic preparation with lipid microspheres

The applicability of a drug delivery system to an ophthalmic preparation was examined using lipid microspheres containing hydrocortisone 17-butyrate 21-propionate (HBP). A 3H-labelled HBP ophthalmic suspension and 3H-labelled HBP lipid microspheres were applied to rabbit eyes, which were then enucleated at fixed intervals to determine the level of 3H-labelled HBP in ocular tissues. The lipid microspheres were shown to deliver the drug to the anterior ocular tissues more effectively than the ophthalmic suspension. It is suggested that a lipid microsphere ophthalmic preparation of various lipophilic drugs including steroids may be useful as a drug delivery system for ophthalmic therapy.

Synthesis and biological evaluation of N-mercaptoacylproline and N-mercaptoacylthiazolidine-4-carboxylic acid derivatives as leukotriene A4 hydrolase inhibitors.

We studied the synthetic modification of structurally similar N-mercaptoacyl-L-proline and (4R)-N-mercaptoacylthiazolidine-4-carboxylic acid to obtain potent leukotriene A(4) (LTA(4)) hydrolase inhibitors. An N-mercaptoacyl group, (2S)-3-mercapto-2-methylpropionyl group, was effective for both scaffolds. Additional introduction of a large substituent such as 4-isopropylbenzylthio (3f), 4-tert-butylbenzylthio (3l) or 4-cyclohexylbenzylthio group (3m) with (S)-configuration at the C(4) position of proline yielded much more potent LTA(4) hydrolase inhibitors (IC(50); 52, 31, and 34 nM, respectively) than captopril (IC(50); 630,000 nM).

Determination of a new calcium antagonist, sesamodil fumarate (SD-3211), and its metabolite in plasma by liquid chromatography with electrochemical detection

A sensitive and selective high-performance liquid chromatographic method is described for the determination of a novel calcium antagonist, (+)-(R)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-[(3,4- methylenedioxy)phenoxy]ethyl]amino]propoxy]phenyl]-4-methyl-3-oxo-2H- 1,4-benzothiazine hydrogen fumarate (sesamodil fumarate; JAN, SD-3211, I), and its N-desmethylated metabolite (II) in plasma. Compounds I and II and an internal standard were isolated from plasma by solid-phase and liquid-liquid extraction. The extract was chromatographed on a reversed-phase C18 column, and the compounds of interest were detected by dual coulometric electrodes operated in an oxidative screen mode. The limit of determination for both I and II was at least 0.4 ng/ml in plasma. The utility of the assay was demonstrated by determining plasma levels of I and II in five dogs administered an oral dose of 60 mg of the drug.