12-HETE Synthesis Research Articles

12-HETE/GPR31 induces endothelial dysfunction in diabetic retinopathy.

12-hydroxyeicosatetraenoic acid (12-HETE), a major metabolite of arachidonic acid, is converted by 12/15-lipoxygenase and implicated in diabetic retinopathy (DR). Our previous study demonstrated a positive correlation between 12-HETE and the prevalence of DR. However, reasons for the increased production of 12-HETE are unclear, and the underlying mechanisms through which 12-HETE promotes DR are unknown. This study aimed to elucidate the correlation between 12-HETE and DR onset, investigate potential mechanisms through which 12-HETE promotes DR, and seek explanations for the increased production of 12-HETE in diabetes. We conducted a prospective cohort study, which revealed that higher serum 12-HETE levels could induce DR. Additionally, G protein-coupled receptor 31 (GPR31), a high-affinity receptor for 12-HETE, was expressed in human retinal microvascular endothelial cells (HRMECs). 12-HETE/GPR31-mediated HRMEC inflammation occurred via the p38 MAPK pathway. 12-HETE levels were significantly higher in the retina of mice with high-fat diet (HFD)- and streptozotocin (STZ)-induced diabetes than in those with only STZ-induced diabetes and healthy controls. They were positively correlated with the levels of inflammatory cytokines in the retina, indicating that HFD could induce increased 12-HETE synthesis in patients with diabetes in addition to hyperglycemia. Conclusively, 12-HETE is a potential risk factor for DR. The 12-HETE/GPR31 axis plays a crucial role in HRMEC dysfunction and could be a novel target for DR prevention and control. Nevertheless, further research is warranted to provide comprehensive insights into the complex underlying mechanisms of 12-HETE in DR.

Disruption of phospholipid and bile acid homeostasis in mice with nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease that can develop into cirrhosis, hepatic failure, and hepatocellular carcinoma. Although several metabolic pathways are disrupted and endogenous metabolites may change in NASH, the alterations in serum metabolites during NASH development remain unclear. To gain insight into the disease mechanism, serum metabolite changes were assessed using metabolomics with ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry and a conventional mouse NASH model induced by a methionine- and choline-deficient (MCD) diet. Significant decreases in serum palmitoyl-, stearoyl-, and oleoyl-lysophosphatidylcholine (LPC) and marked increases in tauro-β-muricholate, taurocholate and 12-hydroxyeicosatetraenoic acid (12-HETE) were detected in mice with NASH. In agreement with these metabolite changes, hepatic mRNAs encoding enzymes and proteins involved in LPC degradation (lysophosphatidylcholine acyltransferase [Lpcat] 1-4), basolateral bile acid excretion (ATP-binding cassette subfamily C member [Abcc] 1/4/5 and organic solute transporter β), and 12-HETE synthesis (arachidonate 12-lipoxygenase) were significantly up-regulated. In contrast, the expression of solute carrier family 10 member 1 (Slc10a1) and solute carrier organic anion transporter family member (Slco) 1a1 and 1b2, responsible for transporting bile acids into hepatocytes, were markedly suppressed. Supplementation of the MCD diet with methionine revealed that the changes in serum metabolites and the related gene expression were derived from steatohepatitis, but not dietary choline deficiency or steatosis. Furthermore, tumor necrosis factor-α and transforming growth factor-β1 induced the expression of Lpcat2/4 and Abcc1/4 and down-regulated Slc10a1 and Slco1a1 in primary hepatocytes, suggesting an association between the changes in serum LPC and bile acids and proinflammatory cytokines. Finally, induction of hepatitis in ob/ob mice by D-galactosamine injection led to similar changes in serum metabolites and related gene expression. Phospholipid and bile acid metabolism is disrupted in NASH, likely due to enhanced hepatic inflammatory signaling.

Presence of glucose in dialyzing fluid and synthesis of selected lipoxygenase-derived eicosanoids during hemodialysis

Active metabolites of arachidonic acid (AA), eicosanoids, are exerting a significant influence on renal homeostasis. In our recent paper, we demonstrated that high levels of lipoxygenase (LOX)-derived AA metabolites, hydroxyeicosatetraenoic acids (HETEs), unfavorably influence post-transplant function of kidney allografts. Hence, in this study, we wanted (1) to analyze 5-, 12- and 15-HETE levels in non-diabetic chronic kidney disease (CKD) patients, already undergoing regular hemodialysis treatment, and determine factors that may influence these eicosanoids' generation, as well as, (2) to verify whether application of glucose-containing, instead of glucose-free, dialyzing fluids may be beneficial for the limitation of 5-, 12- and 15-HETE synthesis during a single hemodialysis session. Twenty-four healthy individuals and 50 CKD patients undergoing regular hemodialysis treatment were included in the study. CKD patients were divided into two subgroups depending on presence/absence of glucose in dialyzing fluid. LOX-derived HETEs were measured using liquid chromatography. Results demonstrated higher levels of examined eicosanoids in CKD patients (P<0.05 for all). Older age and higher C-reactive protein levels were associated with HETEs concentrations. Presence of glucose in dialyzing fluid significantly diminished the increase in 5- and 12-HETE synthesis (411.24% vs. 107.29%, P<0.006; 301.70% vs. 98.21%, P<0.0008, respectively), however, it did not influence 15-HETE generation (156.98% vs. 135.24%, P=0.26). Non-diabetic CKD patients have higher levels of LOX-derived HETEs, which are associated mainly with age and intensified inflammatory process. The presence of glucose in the dialysate is associated with a reduced synthesis of selected LOX-derived HETEs in these patients; hence, we speculate that the application of such a simple hemodialysis fluid modification may have a favorable influence on post-transplant outcomes.

Boswellic Acids Stimulate Arachidonic Acid Release and 12-Lipoxygenase Activity in Human Platelets Independent of Ca2+and Differentially Interact with Platelet-Type 12-Lipoxygenase

Boswellic acids inhibit the transformation of arachidonic acid to leukotrienes via 5-lipoxygenase but can also enhance the liberation of arachidonic acid in human leukocytes and platelets. Using human platelets, we explored the molecular mechanisms underlying the boswellic acid-induced release of arachidonic acid and the subsequent metabolism by platelet-type 12-li-poxygenase (p12-LO). Both beta-boswellic acid and 3-O-acetyl-11-keto-boswellic acid (AKBA) markedly enhanced the release of arachidonic acid via cytosolic phospholipase A2 (cPLA2), whereas for generation of 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], AKBA was less potent than beta-boswellic acid and was without effect at higher concentrations (> or =30 microM). In contrast to thrombin, beta-boswellic acid-induced release of ara-chidonic acid and formation of 12-H(P)ETE was more rapid and occurred in the absence of Ca2+. The Ca2+-independent release of arachidonic acid and 12-H(P)ETE production elicited by beta-boswellic acid was not affected by pharmacological inhibitors of signaling molecules relevant for agonist-induced arachidonic acid liberation and metabolism. It is noteworthy that in cell-free assays, beta-boswellic acid increased p12-LO catalysis approximately 2-fold in the absence but not in the presence of Ca2+, whereas AKBA inhibited p12-LO activity. No direct modulatory effects of boswellic acids on cPLA2 activity in cell-free assays were evident. Therefore, immobilized KBA (linked to Sepharose beads) selectively precipitated p12-LO from platelet lysates but failed to bind cPLA2. Taken together, we show that boswellic acids induce the release of arachidonic acid and the synthesis of 12-H(P)ETE in human platelets by unique Ca2+-independent routes, and we identified p12-LO as a selective molecular target of boswellic acids.

Retinoic acid induces corneal epithelial CYP4B1 gene expression and stimulates the synthesis of inflammatory 12-hydroxyeicosanoids.

Injury to the ocular surface provokes an inflammatory response that is mediated, at least in part, by corneal epithelial derived 12-hydroxyeicosanoids (HETEs) including 12-HETE and 12-HETrE; both metabolites exhibit potent inflammatory and angiogenic properties and are formed by a cytochrome P450 (CYP) 4B1. Retinoids are known to mediate wound-healing processes in many tissues and, as such, are integral components of the inflammatory response. We studied the effect of various retinoids on corneal synthesis of 12-hydroxyeicosanoids and on activation of CYP4B1 gene expression. Corneal organ cultures were used to assess the effect of retinoic acid on epithelial metabolism of arachidonic acid to 12-hydroxyeicosanoids. Luciferase reporter vectors containing different lengths of the CYP4B1 3.4 kb-5'-untranslated region were used to examine the effect of vitamin D and retinoids (9-cis-retinoic acid and all-trans retinoic acid) on transcriptional activation of CYP4B1 in transient transfection experiments with HepG2 cells. Vitamin D had no effect on CYP4B1 promoter activity, but 9-cis and all-trans retinoic acids increased promoter activity by up to 70% over control. Addition of both 9-cis and all-trans retinoic acids resulted in an additive effect increasing promoter activity by 2-fold. The increased promoter activity correlated with the presence of RAR/RXR binding motifs. Incubation of corneal organ culture for 24 hours in the presence of 9-cis and all-trans retinoic acids increased the synthesis of 12-HETE and 12-HETrE by 2-fold. The finding that retinoic acid increases the expression of the CYP4B1 gene and enhances production of the inflammatory 12-hydroxyeicosanoids in the corneal epithelium may provide a linkage between wound healing and inflammation in the ocular surface.

The antithrombotic profile of aspirin. Aspirin resistance, or simply failure?

The antithrombotic profile of aspirin. Aspirin resistance, or simply failure?

Effect of thromboxane A2 inhibition and antagonism on prostaglandin and leukotriene synthesis in glomerular immune injury

In glomerulonephritis there is co-activation of the arachidonic acid cyclooxygenase pathway toward synthesis of prostaglandins (PG) and thromboxane (Tx) and of lipoxypenase pathways toward synthesis of hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (LTs). Cyclooxygenase inhibition with non-steroidal anti-inflammatory drugs results in enhanced glomerular LT synthesis with potentially adverse effects on the severity of the inflammation. The effect of Tx inhibition or antagonism on LT synthesis is unknown. Because TxA2 is the most abundant elcosanoid synthesized in nephritic glomeruli, and because TxA2 synthase inhibitors and receptor antagonists are now available for the treatment of glomerulonephritis, it becomes important to address this question. In this study we assessed the effect of a TxA2 synthase inhibitor, Dazmegrel, and a TxA2 receptor antagonist, SQ-29 548, on glomerular PGE2, LTB4, and 12-HETE synthesis in a model of mesangial nephritis induced in the rat by the administration of a monoclonal antibody against the Thy 1.1 antigen of rat mesangial cells. Dazmegrel, in doses sufficient to effectively block glomerular TxA2 synthesis, significantly increased 12-HETE and PGE2 synthesis without an effect on the synthesis of LTB4. SQ-29 548 had no effect on glomerular PGE2, LTB4, or 12-HETE production. Because PGE2 preserves kidney function in glomerulonephritis, and because 12-HETE inhibits 5-lipoxygenase, the enhanced PGE2 and 12-HETE production within nephritic glomerull after TxA2 synthase inhibition may be a superior anti-inflammatory strategy when compared with TxA2 receptor antagonism.

Enzyme self-inactivation is a main limitation of the preparation of eicosanoids. Enzymatic synthesis of thromboxane and 12(S)-hydroxytetraenoic acid.

Synthesis of prostanoids is accompanied by various processes reducing the product yield. These processes are also known to affect syntheses of thromboxane (TX) and 12(S)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12-HETE). Partially purified preparations of TX synthase and prostaglandin (PG) synthase were used to optimize TX synthesis with respect to concentrations of the enzymes and eicosapolyenoic acid (EPA). Conditions for the maximum product yield and the minimum consumption of enzymes were determined. Consumption of the TX synthase was large owing to its inactivation during the reaction and the nonenzymatic destruction of the intermediate product PG-endoperoxide. Separate addition of PG and TX synthases increased the product yield by preventing EPA sorption on ballast proteins. Microsomal 12-lipoxygenase (12-LO) was also shown to be inactivated during the reaction, and this process was the major limitation of 12-HETE synthesis. Lipoxygenase reaction in the presence of some reducing agents led to a considerable increase of the 12-HETE yield, supposedly by preventing further oxidation of the 12-LO reaction product 12-hydroperoxy derivative of eicosatetraenoic acid. The possibility of using human blood platelet microsomes for preparation of some derivatives of EPAs is discussed.

Platelet-derived 12-hydroxyeicosatetraenoic acid plays an important role in mediating canine coronary thrombosis by regulating platelet glycoprotein IIb/IIIa activation.

In the thrombotic process of acute coronary syndromes, the pathophysiological role of thromboxane A2 via cyclooxygenase is well established; however, the role of 12-HETE via 12-lipoxygenase is little known. Therefore, we used OPC-29030, a novel specific inhibitor of 12-HETE synthesis, to test whether platelet-derived 12-HETE is involved in mediating cyclic flow variations (CFVs) and platelet aggregation in stenosed and endothelium-injured canine coronary arteries. After developing CFVs, dogs received a vehicle or OPC-29030 intravenously. Plasma and intraplatelet 12-HETE levels increased after CFVs. OPC-29030 but not vehicle reduced CFVs, which was associated with decreases in plasma and intraplatelet 12-HETE levels. Cessation of OPC-29030 restored CFVs in association with increases in plasma and intraplatelet 12-HETE levels. ADP and U46619 induced ex vivo platelet 12-HETE production and aggregation. After OPC-29030 administration, the ADP- and U46619-induced increases in ex vivo platelet 12-HETE production and aggregation were inhibited significantly. Platelet aggregation was linearly correlated with platelet 12-HETE production. OPC-29030 suppressed activation of human platelet glycoprotein IIb/IIIa. OPC-29030 reduced intraplatelet 12-HETE levels, resulting in the inhibition of coronary thrombosis in vivo in dogs. OPC-29030 inhibited human platelet glycoprotein IIb/IIIa activation in vitro. Thus, platelet-derived 12-HETE may play an important role in mediating thrombotic process.

Aspirin increases the bleeding side effects in essential thrombocythemia independent of the cyclooxygenase pathway: role of the lipoxygenase pathway.

Acetylsalicylic acid (ASA) is currently recommended as an antithrombotic for patients with essential thrombocythemia (ET) who are at an increased risk of thrombotic events. However, ASA is also associated with an increased risk of bleeding in these patients as compared to the risk of bleeding in other patients treated with ASA. Recent data suggest that while ASA inhibits platelet thromboxane A2 (TxA2) synthesis in all individuals, ASA has little effect or inhibits the lipoxygenase pathway (i.e., 12-hydroxyeicosatetranoic acid or 12-HETE synthesis) in some individuals, and enhances 12-HETE synthesis in others. These differential effects are associated with a pronounced prolongation of the bleeding time vs. no prolongation of the bleeding time, respectively, i.e., in ASA responders and ASA nonresponders, respectively. To determine if the increased risk of ASA-induced bleeding seen in ET patients is associated with an effect on 12-HETE synthesis, we compared the relative effects of ASA on the bleeding time, platelet TxA2 and 12-HETE synthesis, and platelet aggregation and adhesion in ET patients and healthy volunteers. ASA (300 mg, taken orally) prolonged the bleeding time in 82% of the ET patients but only 27% of the healthy volunteers although platelet TxA2 synthesis and ADP- and collagen-induced aggregation were inhibited significantly in both groups. In contrast, platelet 12-HETE synthesis was unchanged and platelet adhesion was decreased in those patients and volunteers whose bleeding times were prolonged by ASA, whereas platelet 12-HETE synthesis was increased significantly and platelet adhesion was unaffected in those patients and volunteers whose bleeding times were not prolonged, and in some cases shortened by ASA. These results confirm previous data that demonstrate that ASA has different effects on platelet 12-HETE synthesis and platelet adhesion in different individuals, i.e., inhibitory or no effect in ASA responders (in whom ASA prolonged bleeding) vs. enhancing effects in ASA nonresponders (in whom ASA did not prolong bleeding). These results also indicate that there is a greater percentage of ASA responders in patients with ET than that seen in the general population, a difference that is associated with an effect of ASA on the lipoxygenase pathway. This may explain the increased bleeding side effects seen in the ET patient population.

Expression of arachidonate platelet-type 12-lipoxygenase in human rheumatoid arthritis type B synoviocytes

In the present study, we have demonstrated platelet-type 12-lipoxygenase (12-LOX) expression in human rheumatoid arthritis (RA) type B synoviocytes by reverse-transcription polymerase chain reaction (RT-PCR). The presence of 12-LOX mRNA in these cells was revealed by classical RT-PCR analysis using platelet-type 12-LOX cDNA primers and the PCR fragment (246 bp) was purified, amplified and sequenced. By sequence analysis, this fragment was determined to be 100% identical to that from platelet-type 12-LOX cDNA. Immunofluorescence data demonstrate that interleukin-1β (IL-1β) increases cellular 12-LOX protein. Other results associate specific inflammatory cytokines with the activity of 12-LOX in human RA type B synoviocytes. IL-1β increased 12 S-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) production (4-fold) and we also observed an increase in 12-HETE production (2.5-fold) after incubation of human RA type B synoviocytes with TNFα. In contrast to the action of IL-1β on 12-HETE synthesis, IL-4 and IL-6 did not enhance 12-HETE production. This is the first demonstration of platelet-type 12-LOX cDNA derived from the mRNA of cultured human RA type B synoviocytes.

Arachidonic acid metabolism in murine lymphoma cell sublines differing in radiation sensitivity

14C arachidonic acid incorporation and 14C radioactivity release as well as prostaglandin (PG) and 5-hydroxyeicosatetraenoic acid (5-HETE) synthesis were measured in the pair of murine lymphoma L5178Y (LY) cell sublines differing in radiation sensitivity. Both LY sublines, LY-R (resistant) and LY-S (sensitive), incorporated exogenous arachidonic acid and released it from membrane phospholipids. Ca 2+ ionophores (ionomycin and A23187) but not PMA stimulated the liberation of 14C arachidonic acid in LY cells. PMA did not potentiate the 14C arachidonic acid release both in the presence or in the absence of A23187; this observation suggests that protein kinase C activation is not essential for the regulation of arachidonic acid release by LY-R and LY-S cells. X-irradiation (5 Gy) did not change the uptake of 14C arachidonic acid into LY-R and LY-S cells and did not potentiate the release of its total radioactivity from the cells. PG synthesis was stimulated in irradiated LY-R cells but not in LY-S cells. The susceptibility of eicosanoid metabolism to A23187 and H 2O 2 was altered in irradiated LY-R cells. A23187 stimulated only PG and 5-HETE synthesis in irradiated LY-R cells. H 2O 2 did not stimulate the synthesis of PG from exogenous arachidonic acid in irradiated LY-R and LY-S cells and 5-HETE synthesis in LY-R cells. An implication of the increased PG synthesis in LY-R cells in the protection against radiation is discussed.

The effect of bradykinin and substance P on the arachidonate cascade of platelets

Several peptide receptors are expressed on the surface of platelets, including B1 and NK1, through which Bk and SP might influence platelet functions including their arachidonate cascade. The metabolites of the arachidonate cascade might play a regulatory role in the inter- and intracellular functions of platelets. Platelets were separated from fresh rat blood by differential centrifugation. Platelets (108 ml−1 in each sample) were preincubated with Bk or SP. The arachidonate cascade was investigated with [1–14c]arachidonic acid, as tracer substrate. The synthesised [14c] eicosanoids were isolated and quantitatively determined. Bradykinin elicited a biphasic dose-response curve in the formation of the vasoconstrictor and platelet aggregating thromboxane A2 (TxA2). Bk both inhibited (10−8 mol/1), and elevated (10−6 mol/1) the synthesis of TxA2 in the thrombocytes. The 12-HETE synthesis was inhibited by Bk (10−8, 10−7, 10−5 mol/1); 12-HETE is an endogenous regulator of prostacyclin synthesis. The formation of 12-HETE in platelets was stimulated by SP (10−11, 10−9, 10−8 mol/1). The synthesis of TxA2 in platelets was either attenuated (10–12 mol/1), or stimulated (10-9 mol/l) by SP. According to our observations Bk and SP might play a regulatory role in the activation or deactivation of platelets.

Chiral analysis of 12-hydroxyeicosatetraenoic acid formed by calf corneal epithelial microsomes.

The production of 12-hydroxyeicosatetraenoic acid by the corneal epithelium of several species has been extensively reported yet the controversy over the exclusive production of the (S) epimer (a lipoxygenase-derived metabolite) endures. Incubation of calf corneal epithelial microsomes (3 mg/ml) with arachidonic acid and NADPH resulted in the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). The synthesis of 12-HETE was inhibited by SKF-525A and clotrimazole, selective inhibitors of cytochrome P450 dependent activities, but not by indomethacin or BW-755C, inhibitors of cyclooxygenase and lipoxygenase activities, respectively. Chiral analysis revealed the presence of both enantiomers; however, the R isomer was the predominant one, i.e., 91 +/- 5% vs. 9 +/- 5% for the R and S enantiomers, respectively. Since the R enantiomer is the product of a cytochrome P450-mediated reaction, it suggests that the major metabolic activity in these microsomes is cytochrome P450-dependent and supports the claim for cytochrome P450 reactions in this ocular tissue.

The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: Implications for protection against coronary heart disease

A number of lines of evidence suggest that red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease (CHD) mortality. This protection over and above that due to ethanol itself may be explained by phenolic components with which red wines are richly endowed. We have studied the effects of the trihydroxy stilbene trans-resveratrol on human platelet aggregation and on the synthesis of three eicosanoids from arachidonate by platelets, i.e. thromboxane B 2 (TxB 2), hydroxyheptadecatrienoate (HHT) and 12-hydroxyeicosatetraenoate (12-HETE). These effects were compared with the actions of other wine phenolics (quercetin, catechin and epicatechin) and antioxidants (αtocopherol, hydroquinone and butylated hydroxytoluene). trans-Resveratrol and quercetin demonstrated a dose-dependent inhibition of both thrombin-induced and ADP-induced platelet aggregation, whereas ethanol inhibited only thrombin-induced aggregation. The other compounds tested were inactive. trans-Resveratrol also inhibited the synthesis of TxB 2, HHT, and to a lesser extent 12-HETE, from arachidonate in a dose-dependent manner. Quercetin inhibited only 12-HETE synthesis, and hydroquinone caused slight inhibition of TxB 2 synthesis, the remaining compounds being ineffective. De-alcoholized red wines inhibited platelet aggregation; their ability to inhibit the synthesis of TxB 2 but not that of 12-HETE from labelled arachidonate by washed human platelets was proportional to their trans-resveratrol concentration. These results are consistent with the notion that trans-resveratrol may contribute to the presumed protective role of red wine against atherosclerosis and CHD.

Concentrations of various arachidonic acid metabolites in menstrual fluid are associated with menstrual pain and are influenced by hormonal contraceptives.

In a pilot study we investigated the association between concentrations of various eicosanoids in menstrual blood with pain and oral contraceptive use. Menstrual fluid was collected on tampons by 12 women who did not use an oral contraceptive but suffered from slight primary dysmenorrhea and by three pain-free women who used an oral contraceptive. Eicosanoids (cyclooxygenase products: 6-ketoprostaglandin F1 alpha, thromboxane B2, prostaglandin E2, prostaglandin F2 alpha, 13,14-dihydro-15-ketoprostaglandin F2 alpha, 12-hydroxy-heptadecatrienoic acid; lipoxygenase products: 5-, 12-, 15-hydroxy-eicosatetraenoic acid (HETE), leukotriene B4, leukotriene C4, leukotriene D4, leukotriene E4) and female sex steroids (17 beta-estradiol and progesterone) were analyzed by the combined use of high-performance liquid chromatography and radioimmunoassay. 12-HETE was the main arachidonic acid metabolite. An increased metabolism of arachidonic acid was associated with pain, especially when synthesis of 12-HETE was elevated. Oral contraceptive use decreased the synthesis of prostaglandins as well as leukotrienes. The concordant changes of cyclooxygenase and lipoxygenase products in dysmenorrhea or in oral contraceptive use may be explained by an increased or decreased phospholipid metabolism, respectively.

Stereospecific pharmacodynamics and pharmacokinetics of carprofen in the dog

The non-steroidal anti-inflammatory drug (NSAID) carprofen (CPF) contains a single chiral centre. It was administered orally to Beagle dogs as a racemate (rac-CPF) at a dose of 4 mg per kg body weight and as individual (-)(R) and (+)(S) enantiomers at 2 mg per kg body weight. Each of the enantiomers achieved similar plasma bioavailability following administration as the racemate as they did following their separate administration. Only the administered enantiomers were detectable when the drug was given in the (-)(R) or (+)(S) form, indicating that chiral inversion did not occur in either direction. Higher plasma concentrations of the (-)(R) (Cmax 18 micrograms/ml, AUC0-24 118 micrograms h/ml) than the (+)(S) (Cmax 14 micrograms/ml, AUC0-24 67 micrograms h/ml) enantiomer were achieved following administration of the racemate. Both enantiomers distributed into peripheral subcutaneous tissue cage fluids, but Cmax and AUC values were lower for both transudate (non-stimulated tissue cage fluid) and exudate (induced by the intracaveal administration of the irritant carrageenan) than for plasma. Drug concentrations in transudate and exudate were similar, as indicated by Cmax and AUC values, although CPF penetrated more rapidly into exudate than into transudate. Neither rac-CPF nor either enantiomer inhibited thromboxane B2 (T x B2) generation by platelets in clotting blood (serum T x B2), or prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis in inflammatory exudate.(ABSTRACT TRUNCATED AT 250 WORDS)

Patent Evaluation: Hydroxylamine derivatives as lipoxygenase inhibitors

SummarySummaryNovelty: Novel substituted heteroaryl hydroxylamine derivatives are claimed to be lipoxygenase (LPO) inhibitors. They are said to be particularly effective against 5-LPO. They are potentially useful for the treatment of inflammatory and allergic disorders, asthma, dermatitis, ischaemia and endotoxin shock.Biology: 5-LPO inhibition was determined by measuring the inhibition of the synthesis of 5-HETE and LTB4 in guinea-pig polymorphonuclear leukocytes. Methods to measure antiinflammatory and anti-asthmatic activity are also described. However, no specific biological data are presented.Chemistry: A total of eighty-six compounds are disclosed. Syntheses are given in thirteen examples. Nine compounds are specifically claimed including 2-[3-(4-fluorophenyl)propyl]-6-[2-(N-aminocarbonyl-N-hydroxyamino)ethoxy]-1-oxo-1,2,3,4-tetrahydroisoquinoline.

Hemodynamic role of arachidonate 12- and 5-lipoxygenases in nephrotoxic serum nephritis

The role of arachidonate 12- and 5-lipoxygenation eicosanoids in mediating acute changes in renal hemodynamics was assessed in nephrotoxic serum nephritis (NSN) in the rat. Following a single intravenous injection of nephrotoxic serum (NTS), significant decrements in glomerular filtration rate (GFR) and renal blood flow (RBF) occurred at one hour, and were associated with increments in glomerular polymorphonuclear leukocyte (PMN) counts and in the synthesis of thromboxane (Tx) B2, leukotriene (LT) B4 and 12-hydroxyeicosatetraenoic acid (12-HETE). Pretreatment of rats with the arachidonate 12-lipoxygenase inhibitor, baicalein, partially but significantly ameliorated the decrements in GFR and RBF, and blocked the enhanced glomerular synthesis of 12-HETE following administration of NTS. Likewise, pretreatment of rats with the arachidonate 5-lipoxygenase inhibitor, U-66858, partially ameliorated the decrements in GFR and RBF induced by NTS. Combined pretreatment of rats with baicalein and U-66858 ameliorated the decrements in GFR and RBF to an extent no different to that of U-66858 alone. In rats pretreated with the LTB4 receptor antagonist, U-75302, GFR and RBF remained depressed to levels no different than in animals which received NTS alone. These observations indicate that in NSN, the acute decrements in GFR and RBF are partially mediated by 12-HETE and arachidonate 5-lipoxygenation products. Leukotrienes other than LTB4, such as LTD4 and LTC4, are the likely candidates.

Mechanisms of Glomerular Injury: Overview and Relation with Hemostasis

The mechanisms of glomerular injury can be separated into nonimmunologically mediated glomerulonephritis (GN) such as diabetes, leading to glomerular hypertension and into immunologically mediated GN. The immunologically mediated GN may induce chronic glomerulopathy such as membranous GN or proliferative GN. The final pathway of these two types of GN is proteinuria and renal failure linked to glomerulosclerosis. In inflammatory GN, most of the mediators could be synthesized either by infiltrating cells or by resident glomerular cells. They include cytokines, lymphokines, complement activation, generation of superoxyde anions, arachidonic acid metabolites, and fibrin deposition. (a) We have investigated the interaction between isolated glomeruli and platelets and have demonstrated that lipidic and proteic extracts of glomeruli enhance thromboxane B2 platelet synthesis. This fact is related to the generation by isolated glomeruli of saturated fatty acids and tissue factor. (b) We investigated the interaction between rat isolated glomeruli and peritoneal macrophages. We have demonstrated that 12-HETE synthesized by isolated glomeruli induce macrophage prostaglandin synthesis which, in turn, inhibits the 12-HETE synthesis. (c) We have demonstrated, using human glomerular epithelial cells, that alpha-thrombin, the active form of thrombin, generated before fibrin formation, is able to induce cell proliferation and abolishes the profibrinolytic activity of these cells. In summary, the mechanisms of glomerular injury are complex, certainly acting by multiple pathways. So far, the mediators leading to proteinuria and renal failure after glomerular injury remain under investigation.