Studies on the metabolic fate of gomisin A (TJN-101). I. Absorption in rats]

Gomisin A (TJN-101) is one of the lignan components isolated from Schisandra Fruits and expected to have some efficacies in clinical treatment of hepatitis. The serum concentrations of TJN-101 and Met. B, which was identified as a demethylenated substance and one of the major metabolites of TJN-101 in rats, were investigated. After intravenous administration at doses of 1.6, 4.0 and 10 mg/kg of body weight, the serum concentration of TJN-101 decreased biphasically, and the terminal elimination half-life at each dose was about 70 min. Dose-dependency was observed for the area under the concentration-time curve (AUC). On the other hand, the serum concentration of TJN-101 increased rapidly and reached maximum within 15 to 30 min when administered orally. This result was supported by the in situ roop method. The Cmax and the AUC values were not exactly dose-dependent, but the values increased with a dose-up of TJN-101. The biotransformation of TJN-101 to Met. B, was very rapid in both intravenous and oral administrations. The AUC value of Met. B after oral administration of TJN-101 at a dose of 1.6 mg/kg was relatively larger than any other dosages. It suggested that TJN-101 was extensively underwent the first pass effect in rats. More than 80% of TJN-101 was bound with rat serum protein in vitro and in vivo. Therefore, it seems to be necessary to pay attention when it was administered concurrently with high protein binding drugs.     

Effect of Gomisin A (TJN-101) on liver regeneration.

We studied the effect of TJN-101, a lignan component of Schisandra fruits (Schisandrae fructus), on liver regeneration after partial hepatectomy. TJN-101 was given orally to male Wistar rats 30 min before partial hepatectomy. The mitotic index and the level of DNA synthesis increased after partial hepatectomy and their increase was significantly enhanced by TJN-101. Ornithine decarboxylase (ODC) activity increased in the early stages of liver regeneration and it was also significantly enhanced by TJN-101. Besides, TJN-101 enhanced the increase in hepatic putrescine. These results suggest that TJN-101 stimulates liver regeneration after partial hepatectomy by enhancing ODC activity, which is an important biochemical event in the early stages of liver regeneration.     

Determination of gomisin A (TJN-101) and its metabolite in rat serum by gas chromatography-mass spectrometry]

Gomisin A (TJN-101) is one of the lignan components isolated from Schisandra Fruits. A high sensitive and precise method for the determination of TJN-101 and its major metabolite (Met. B) in the rat serum was developed by selected ion monitoring (SIM) with gas chromatography-mass spectrometry (GC/MS) using a fused silica capillary column (SPB-1, Supelco). A 100 microliter serum sample was used for the solid phase extraction. The calibration curves of TJN-101 and Met.B both showed a good linearity between 2.0 and 2000.0 ng/ml. The analytical precision (intra-assay, C.V. less than 4.7%), recoveries (98.4 +/- 10.1%), and detection limit (2 ng/ml) of TJN-101 indicated that this system was suited for the determination of TJN-101 in biological fluid. In case of Met.B, the same results as TJN-101, were obtained. After oral administration of TJN-101 at a dose of 10 mg/kg to male rats, the average values of the maximal serum concentration of TJN-101 and Met.B were 1446.1 +/- 131.8 and 317.4 +/- 18.5 ng/ml, respectively. The serum concentrations of these substances could be monitored sufficiently for 8 h after dosing.     

Effect of gomisin A (TJN-101), a lignan compound isolated from Schisandra fruits, on liver function in rats

TJN-101 [+)-(6S, 7S, R-biar)-5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy- 6,7-dimethyl-10,11-methylenedioxy-6-dibenzo [a, c] cyclooctenol) is one of the lignan compounds isolated from Schisandra fruits. When TJN-101 was administered orally at the doses of 3-100 mg/kg/day for 4 days, bile secretion, hepatic excretion of dye or hepatic hemodynamics 24 hr after the last dose was investigated in comparison with the phenobarbital (100 mg/kg/day)-treated group. Bile flow was dose-dependently increased; in contrast, biliary concentration of bile acids was decreased in TJN-101 (30 and 100 mg/kg/day)-treated groups. Similar changes were also observed in the phenobarbital-treated group. These results suggested that the enhancement of bile secretion caused by TJN-101 or phenobarbital was due to an increase of a bile acid-independent fraction. In the bromosulfophthalein (BSP) clearance test for liver function, both TJN-101 (30 and 100 mg/kg/day) and phenobarbital accelerated the disappearance from the blood and biliary excretion of BSP. Hepatic hemodynamics was examined by the hydrogen clearance method and measurement of liver wet and dry weight. Liver blood flow tended to increase in the TJN-101 (10-100 mg/kg/day) or phenobarbital-treated group. On the other hand, TJN-101 (3-100 mg/kg/day) or phenobarbital hardly altered the water content of the liver. These results suggested that the liver enlargement caused by both compounds was not accompanied with hepatic edema and that the enhancement of bile secretion or hepatic excretion of BSP might be related to an increase of liver blood flow.     

Studies on the metabolic fate of gomisin A (TJN-101). II. Absorption and excretion in CCl4 treated rats]

The absorption and excretion of gomisin A (TJN-101) in rats whose livers were injured by carbon tetrachloride (CCl4) were investigated. After intravenous administration of TJN-101 at a dose of 5 mg/kg, the terminal elimination half-life was 1.5 h in the CCl4-treated rats, which was two times that in normal rats. The mean area under the blood concentration-time curve (AUC) value of TJN-101 in the CCl4-treated rats was twice that in normal rats, and this difference was significant (p less than 0.05). Therefore, the total body clearance of TJN-101 in the CCl4-treated rats decreased less than half of that in normal rats. Similar results were observed when it was administered orally. In the CCl4-treated rats, the serum concentration of Met. B, which was identified as a demethylenated substance and one of major metabolites, tended to decrease more than that in normal rats. On the other hand, the cumulative biliary excretion ratio of TJN-101 in 24 h after dosing in the CCl4-treated rats was 2.5 times that in normal rats. The excretion rate of Met. B in the bile in the CCl4-treated rats tended to be delayed. However, the quantitative variance of biliary excretion of Met. B was not found in both groups. The urinary excretion of TJN-101 or Met. B in 72 h after dosing in the CCl4-treated rats was lower than that in normal rats. Similar results were also observed in excretion in the feces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fenflumizole: interactions with the arachidonic acid cascade

Fenflumizole (2-(2,4-difluorophenyl)-4,5-bis(4-methoxyphenyl)imidazole), a new non-steroidal anti-inflammatory agent, was investigated for interference with cyclo-oxygenase activity in vivo, ex vivo and in vitro in comparison with indomethacin (and aspirin). Fenflumizole was comparable to indomethacin ex vivo in inhibition of thromboxane (TX)A2 production in rabbit platelets and inhibition of prostaglandin (PG)I2 (approximately prostacyclin) generation in rabbit mesenteric arteries and in vivo as an inhibitor of PGE2 formation in inflammatory exudates in rats. Fenflumizole was 18 times less active than indomethacin in inhibition of PGE2 synthesis in vitro and 170 times weaker as an inhibitor of PGI2 generation in the rat stomach mucosa ex vivo. Fenflumizole was 20-50 times more potent than indomethacin in vivo in inhibition of arachidonic acid induced bronchoconstriction in guinea-pigs, in inhibition of platelet aggregation on tendons superfused with blood from rabbits and in vitro in inhibition of aggregation of human and rabbit platelets. Neither fenflumizole nor indomethacin inhibited TXA2-synthetase in vitro. Aspirin-when tested-was less potent than fenflumizole and indomethacin. It is concluded that fenflumizole is a potent cyclo-oxygenase inhibitor. The very potent activity of fenflumizole against platelet aggregation and bronchoconstriction suggests a selectivity in the mode of action. The weak inhibition of gastric PGI2 generation may account for the previously observed weak gastro-ulcerogenicity of fenflumizole.     

Pharmacological studies on ginger. IV. Effect of (6)-shogaol on the arachidonic cascade

(6)-Shogaol, a pungent component of ginger, which is contained in semi-dried ginger but is rarely found in fresh ginger inhibited carrageenin-induced swelling of hind paw in rats and arachidonic acid (AA)-induced platelet aggregation in rabbits. Moreover, (6)-shogaol prevented prostaglandin I2 (PGI2) release from the aorta of rats when tested as an inhibitor of platelet aggregation. These results suggest that (6)-shogaol may have an inhibitory action on the cyclo-oxygenases in both platelets and aorta. Examination of the effects of (6)-shogaol on cyclo-oxygenases in rabbit platelets and microsome fractions of rat aorta indicated that (6)-shogaol inhibited cyclo-oxygenase activities of both tissues in a concentration-dependent manner. Furthermore, when we examined the effect of (6)-shogaol on 5-lipoxygenase from RBL-1 cells, (6)-shogaol exhibited an inhibitory action on 5-lipoxygenase activity. Therefore, it seems that the inhibitory effects of (6)-shogaol on the carrageenin-induced paw edema, AA-induced platelet aggregation and PGI2 production of aorta may be caused by the inhibition of cyclo-oxygenase activity.     

Modulation of arachidonic acid turnover in macrophages by cadmium

The effects of cadmium (Cd) induced redox changes on arachidonic acid (AA) turnover in mouse resident peritoneal macrophages (pM) were studied. The pre-incubation of pM in a medium containing glutathione (GSH, 0.1 or 1 mM) for 6 h protects pM from loss of viability and AA uptake diminution induced by Cd with regard to non pre-incubated cultures. The exposure of macrophages to Cd 10 microM decreases AA uptake within 2 h and increases AA release in relation to non-exposed macrophages. It also enhances AA mobilization and reactive oxygen species (ROS) release induced by okadaic acid and opsonized zimosan and decreases those induced by lipopolysaccharide, but does not modify either AA mobilization or ROS release induced by phorbol ester. These results might suggest that redox changes induced by Cd produce an important impact on AA turnover in macrophages; information that is relevant in the understanding of the cellular toxicity of this metal.     

Inhibition of arachidonic acid cascade by extract of rye pollen

A standardized extract mainly from rye pollen (Cernilton N) was tested in vitro on the inhibition of prostaglandin and leukotrien synthesis. The determination of the prostaglandin and leukotrien synthesis from labelled arachidonic acid was done in microsomes of ram seminal vesicles resp. in rat basophilic leukemia cells (RBL-1 cells). The water soluble resp. the fat soluble extract fraction from the whole pollen extract were tested separately. The radio-TLC separation of the reaction metabolites showed a dose dependent inhibition of the cyclo-oxygenase and the 5-lipoxygenase activity by the fat soluble pollen extract fraction. The IC50-values are 0.005 mg/ml resp. 0.08 mg/ml and similar to those of the also tested diclofenac. The water soluble fractions showed no effect in this test system. According to these in vitro results and the clinical experience so far with the pollen extract its therapeutic efficacy on benign prostate diseases is best explainable by the anticongestive resp. anti-inflammatory effect of the fat soluble fraction. Due to the different actions of prostaglandins and leucotrienes also relaxant and antiproliferative effects were conceivable.     

Inhibition of the arachidonic acid cascade by aza-2-aryl-1,4-naphthoquinone derivatives

Inhibition of the arachidonic acid cascade by aza-2-aryl-1,4-naphthoquinone derivatives To find more potent 5-lipoxygenase(LO)-inhibitors than the up to now studied 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-1,4-naphthoquinone derivatives the analogous aza-1,4-naphthoquinones 14, 15, 16/17 as well as the 3-bromo precursors 7, 8, 9/10 and 11 were synthesized. The naphtho[2,1-b][1,4]thiazin derivative 21 was included in this investigation as a quinone imine. Beside 5-LO inhibition the influence on 12-LO, COX-1 and cPLA2 was determined to investigate the selectivity of the compounds within the arachidonic acid cascade. To test the biochemical properties human granulocytes (5-LO) and human platelets (12-LO/COX-1 and cPLA2) were used. All 3-bromo compounds inhibit completely the arachidonic acid cascade by blocking the cPLA2. The 3-methoxy derivatives of the quinoline quinones 12 and 13 and the 3-hydroxyisoquinoline mixture 16/17 show 5-LO selectivity. 13 inhibits 5-LO selectively, 12 is a dual 5-LO/COX-1-inhibitor and 16/17 are dual 12-LO/COX-1-inhibitors. To verify the hypothesis that the hydroxylated 2-aryl-1,4-benzoquinone structure is the pharmacophore for 5-LO-inhibition within the class of 2-aryl-1,4-naphtho- and -aza-naphthoquinones the 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-1,4-benzoquinones 24-28 were synthesized. It was shown that the 5-methoxy and 5-hydroxy compounds 24 and 27 are highly selective and potent 5-LO-inhibitors.     

Modulation of arachidonic acid metabolism: focus on phospholipase A2(S)

New information has challenged our traditional concepts that the forms and functions of PLA(2) are highly homologous, suggesting now that distinct PLA(2)s may be assigned distinct functions in normal and pathological processes. The nonpancreatic type II 14-kDa PLA(2) and the recently identified type IV "cytosolic" 85-kDa PLA(2) are the two forms most studied in inflammation. Observations in the past suggested that the type II 14-kDa PLA2 is a secreted enzyme that functions extracellularly. Evidence is now emerging that the type II 14-kDa PLA(2) or its recently discovered low-molecular-weight isoforms may be localized and act intracellularly. In view of this, a more complex notion of distinctly functioning PLA(2)s in arachidonic acid release and/or eicosanoid generation can be envisioned. A comparison of the structural and biochemical features of the type II 14-kDa and the 85-kDa PLA(2)s reveals that the enzymes are more distinct than similar. These two enzymes would appear to have distinctly different genetic and biochemical regulatory mechanisms, suggesting that their functions could be quite distinct. Inhibitors of the 14-kDa PLA(2) and to a lesser extent the 85-kDa PLA(2) have been used to obtain a greater understanding of their cellular roles. The concept that the two distinct enzymes might hydrolyze arachidonic acid from different pools and/or supply distinct metabolizing systems in a single cell system has emerged. At this time an intriguing hypothesis can be formed suggesting distinct functional modalities for the two the PLA(2) enzymes in a single cell system. Evidence continues to build implicating the role of the type II 14-kDa PLA(2) in disease, providing a strong rationale for targeting this enzyme in designing novel antiinflammatory therapeutics.     

Effect of arachidonic acid on anthralin inflammation.

1 The effect of topical arachidonic acid on anthralin inflammation was studied using sequential measurements of erythema (reflectance photometry) and oedema (calipers). 2 Topical arachidonic acid in concentrations which produced a small short-lived inflammatory response greatly augmented the initial phase and depressed the later phase of the inflammatory response to anthralin. 3 The initial augmentation was inhibited by concomitant administration of alpha-tocopherol. 4 It is suggested that free radical formation by anthralin has a direct action on membrane substrates such as arachidonic acid forming inflammatory products by a non-enzymic process.     

Inhibition of the liberation of arachidonic acid by cadmium ions in rabbit alveolar macrophages

The effects of CdCl₂ on the liberation of arachidonic acid (20∶4) from membrane phospholipids of A23187-stimulated rabbit alveolar macrophages and on the activity of phospholipase A₂ (PLA₂) in a cytosolic fraction were studied. Alveolar macrophages were prelabeled with [³H]arachidonic acid (20∶4) and then treated with A23187. This treatment resulted in a remarkable increase in the liberation of [³H]20∶4 from their phospholipids. Exposure of cells to Cd²⁺ inhibited the liberation of [³H]20∶4 in a dose-dependent manner. Liberation of [³H]20∶4 from cell lipids was calcium dependent and the inhibitory effect of Cd²⁺ competed with the stimulatory effect of Ca²⁺. When Ca²⁺ was removed from the incubation medium, Cd²⁺ did not influence the liberation of [³H]20∶4. Entry of⁴⁵Ca²⁺ into cells was enhanced by treatment of A23187. However, Cd²⁺ did not influence the cellular uptake of⁴⁵Ca²⁺. Treatment with A23187 markedly enhanced entry of¹⁰⁹Cd²⁺ into cells. The effect of Cd²⁺ on the activity of phospholipase A₂ was determined with 1-palmitoyl-2-[¹⁴C]arachidonoyl-sn-glycero-3-phosphocholine as substrate. Calcium-dependent activation of PLA₂ was observed and Cd²⁺ inhibited activation in a dose-dependent manner. These results suggest that exposure of alveolar macrophages to Cd²⁺ causes a reduction in the rate of liberation of 20∶4 from cell lipids, as a possible result of the inhibition of PLA₂ activity by Cd²⁺.     

Effect of arachidonic acid metabolism on the release of histamine and SRS (leukotrienes) from guinea-pig lung

The effect of arachidonic acid (AA) metabolism on histamine release and SRS (leukotrienes) production has been studied in guinea-pig lung using anaphylactic reaction and Ca2+ ionophore as the triggering agents in vitro. AA and L-cysteine enhanced SRS production without any appreciable effect on histamine release. Two nonsteroid anti-inflammatory agents, indomethacin and ketoprofen, which block prostaglandin production by the cyclooxygenase pathway, stimulated SRS production but had hardly any effect on histamine release, indicating that SRS synthesis is more sensitive to prostaglandin regulation. Enhancement of SRS production was more pronounced for antigen than for Ca2+ ionophore. This might be related to different cellular origin of SRS with the two triggering agents. Using rat peritoneal cells, both mast cells and the other cells were found to produce SRS in response to Ca2+ ionophore, the amount formed by the latter type of cells being higher. Inhibition of lipoxygenase by 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid depressed SRS production, but had no effect on histamine release. SRS production triggered by Ca2+ ionophore was more sensitive, possibly because of different cellular origin of SRS in response to the two stimuli. The explanation for the discrepancy between the effect on SRS production and histamine release may also have to be sought in their different origins. SRS may mainly stem from cells, which are more sensitive to the inhibitors than the mast cell, which is the source of histamine.     

Antiplatelet effect of NQ12: a possible mechanism through the arachidonic acid cascade

NQ12, an antithrombotic agent, has been reported to display a potent antiplatelet activity. This study was undertaken to reveal the effect of NQ12 on rabbit platelet aggregation and signal transduction involved in the arachidonic acid (AA) cascade. NQ12 concentration-dependently suppressed collagen-, AA-, and U46619-induced rabbit platelet aggregation, with IC(50) values of 0.71 +/- 0.2, 0.82 +/- 0.3, and 0.45 +/- 0.1 microM, respectively. In addition, the concentration-response curve of U46619 was shifted to the right after NQ12 treatment, indicating an antagonism on thromboxane (TX) A(2) receptors. The collagen-stimulated AA liberation was inhibited by NQ12 in the same pattern as its inhibition of platelet aggregation. Further study revealed that NQ12 potently suppressed AA-mediated TXA(2) formation, but had no effect on the PGD(2) production, indicating an inhibitory effect on TXA(2) synthase, which was supported by a TXA(2) synthase activity assay indicating that NQ12 concentration-dependently inhibited TXA(2) formation converted from PGH(2). On the other hand, the AA-stimulated 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formation was also suppressed by NQ12. Taken together, these results suggest that NQ12 has a potential to inhibit TXA(2) synthase activity and TXA(2) receptors, and it can modulate AA liberation as well as 12-HETE formation in platelets. This may be a convincing mechanism for the antithrombotic action of NQ12.     

Action of bradykinin at the cyclooxygenase step in prostanoid synthesis through the arachidonic acid cascade.

Bradykinin enhances prostanoid synthesis in aorta smooth muscle cells. Free arachidonic acid also enhances prostanoid synthesis and bradykinin, unlike fatty acid releasing agents, has a synergistic effect with free arachidonic acid. Bradykinin promotes metabolite release from cells prelabeled with [14C]-arachidonic acid and this effect is blocked completely by indomethacin. High performance liquid chromatography shows increase amounts of labeled 6-keto-prostaglandin F1 alpha, prostaglandin E2 and three additional cyclooxygenase-dependent metabolites but no increase in free arachidonic acid or other metabolites either in the absence or presence of indomethacin. Fatty acid releasing agents such as A23187 and cyclosporine A have very different effects on cells. These agents enhance levels of prostanoids, a number of other cyclooxygenase-independent metabolites, and free arachidonic acid which is even more elevated with added indomethacin. Bradykinin behaves in all respects like another agent, bacterial lipopolysaccharide, and the action of both agents is consistent with a mechanism involving cyclooxygenase rather than fatty release in the arachidonic acid cascade.     

Involvement of arachidonic acid cascade in working memory impairment induced by interleukin-1 beta

The aim of the present study is to clarify the possible relevance of the arachidonic acid cascade to working memory in rats, by using a three-panel runway apparatus. Interleukin-1 beta, injected bilaterally into the dorsal hippocampus at a dose of 100 ng/side, significantly impaired working memory, and this impairment was attenuated by pretreatment with 10 mg/kg (s.c.) of diclofenac, a cyclooxygenase inhibitor. Working memory was also impaired in rats administered a bilateral intrahippocampal injection of prostaglandin E2, in a dose-dependent manner at 0.01–1 μg/side. Furthermore, an injection of 100 ng/side of interleukin-1 beta significantly increased production of prostaglandin E2 (580±32 pg to 1142±101 pg/100 mg wet tissue) in the hippocampus. Taken together, these findings suggest that the activation of the arachidonic acid cascade was causative of the working memory impairment induced by interleukin-1 beta.