New Insights into Disintegrin Metalloproteases.
Explore the source record for details and available documents.
Disintegrins↗
Biomedical subjects
T J Carty
Publications and source records attributed to T J Carty.
Tenidap inhibits 5-lipoxygenase product formation in vitro, but this activity is not observed in three animal models.
OBJECTIVE AND DESIGN: The effect of tenidap on the metabolism of arachidonic acid via the 5-lipoxygenase (5-LO) pathway was investigated in vitro and in vivo. MATERIALS AND TREATMENT: In vitro (cells). Arachidonic acid (AA) stimulated rat basophilic leukemia, (RBL) cells; A23817 activated neutrophils (human rat, and rabbit), macrophages (rat), and blood (human). In vitro (enzyme activity). RBL-cell homogenate; purified human recombinant 5-LO. In vivo: Rat (Sprague-Dawley) models in which peritoneal leukotriene products were measured after challenge with zymosan (3 animals per group), A23187 (11 animals per group), and immune complexes (3-5 animals per group), respectively. METHODS: 5-Hydroxyeicosatetraenoic acid (5-HETE) and dihydroxyeicosatetraenoic acids (diHETEs, including LTB4) were measured as radiolabeled products (derived from [14C]-AA) or by absorbance at 235 or 280 nm, respectively, after separation by HPLC. Radiolabeled 5-HPETE was measured by a radio-TLC analyser after separation by thin layer chromatography (TLC). Deacylation of membrane bound [14C]-AA was determined by measuring radiolabel released into the extracellular medium. 5-LO translocation from cytosol to membrane was assessed by western analysis. Rat peritoneal fluid was assayed for PGE, 6-keto-PGF1 alpha, LTE4 or LTB4 content by EIA and for TXB2 by RIA. RESULTS: Tenidap suppressed 5-LO mediated product production in cultured rat basophilic leukemia (RBL-1) cells from exogenously supplied AA, and in human and rat neutrophils, and rat peritoneal macrophages stimulated with A23187 (IC50, 5-15 microM). In addition, tenidap was less potent in inhibiting the release of radiolabeled AA from RBL-1 cells (IC50, 180 microM), suggesting that the decrease in 5-LO derived products could not be explained by an effect on cellular mobilization of AA (i.e., phospholipase). Tenidap blocked 5-hydroxyeicosatetraenoic acid (5-HETE) production by dissociated RBL-1 cell preparations (IC50, 7 microM), as well as by a 100000 x g supernatant of 5-LO/hydroperoxidase activity, suggesting a direct effect on the 5-LO enzyme itself. In addition, tenidap impaired 5-LO translocation from cytosol to its membrane-bound docking protein (FLAP) which occurs when human neutrophils are stimulated with calcium ionophore, indicating a second mechanism for inhibiting the 5-LO pathway. Surprisingly, tenidap did not block the binding of radiolabeled MK-0591, an indole ligand of FLAP, to neutrophil membranes. Although its ability to inhibit the cyclooxygenase pathway was readily observed in whole blood and in vivo, tenidap's 5-LO blockade could not be demonstrated by ionophore stimulated human blood, nor after oral dosing in rat models in which peritoneal leukotriene products were measured after challenge with three different stimuli. The presence of extracellular proteins greatly reduced the potency of tenidap as a 5-LO inhibitor in vitro, suggesting that protein binding is responsible for loss of activity in animal models. CONCLUSIONS: Tenidap inhibits 5-lipoxygenase activity in vitro both directly and indirectly by interfering with its translocation from cytosol to the membrane compartment in neutrophils. A potential mechanism for the latter effect is discussed with reference to tenidap's ability to lower intracellular pH. Tenidap did not inhibit 5-LO pathway activity in three animal models.
6-Ketoprostaglandin F1 alpha↗
Tenidap, a structurally novel drug for the treatment of arthritis: antiinflammatory and analgesic properties.
Tenidap is a new anti-rheumatic agent which has clinical properties characteristic of a disease modifying drug combined with acute antiinflammatory and analgesic activity. This paper details tenidap's cyclooxygenase (COX) inhibitory activity and the resulting pharmacological properties in experimental animals. Tenidap inhibited calcium ionophore-stimulated prostaglandin D2 synthesis by rat basophilic leukemia cells (COX-1) with an IC50 of 20 nM. In two different in vitro human test systems, tenidap inhibited COX-1 activity more potently than COX-2, although the relative potency ratio (COX-1/COX-2) differed markedly between the two systems. Tenidap inhibited the COX pathway when added to human blood in vitro (IC50, 7.8 mu M) and when administered orally to monkeys, rats and dogs (at 5, 2.5 and 10 mg/kg p.o., respectively) and COX activity measured ex vivo in blood collected 2 to 4 hours post dose. After oral administration to rats, tenidap inhibited carrageenan-induced paw edema with an ED50 of 14 mg/kg and inhibited the glucocorticoid-resistant UV erythema in guinea pigs with an ED50 of 1.4 mg/kg. It retained antiinflammatory activity in adrenalectomized rats indicating that this property is independent of adrenal stimulation. Oral administration of tenidap inhibited the development of adjuvant-induced polyarthritis in the rat and exhibited antinociceptive activity in the murine phenylbenzoquinone and rat acetic acid abdominal constriction tests. These data indicate that tenidap is an effective antiinflammatory and analgesic agent in animal models. These cyclooxygenase-dependent pharmacologic activities do not explain tenidap's disease modifying anti-arthritic properties but add a useful symptom modifying component to its clinical profile.
Analgesics, Non-Narcotic↗
The biochemistry and pharmacology of TNF alpha and beta, 21 April 1995, New York.
Explore the source record for details and available documents.
Animals↗
Inhibitory effect of UK,74505, a potent and specific oral platelet activating factor (PAF) receptor antagonist, on airway and systemic responses to inhaled PAF in humans.
Inhaled PAF provokes bronchoconstriction, causes peripheral blood neutropenia with rebound neutrophilia, and generates urinary production of the bronchoconstrictor eicosanoids, thromboxane (TX)A2, and the cysteinyl leukotrienes. We examined the effects of an oral PAF antagonist UK,74505 on each of these responses to a single 36 micrograms dose of inhaled PAF. In a double-blind randomized placebo-controlled crossover study, 12 normal male subjects inhaled PAF on two consecutive days, 3 and 24 h after intake of two doses of UK,74505 25 mg and 100 mg, or matched placebo (P). After P, inhalation of PAF provoked bronchoconstriction, measured at regular time points for 60 min as a change in sGaw from baseline and computed as area under the curve (AUC), induced a neutropenia at 5 min and rebound neutrophilia at 2 h, and stimulated production of urinary eicosanoids. Bronchoconstriction was maximal at 5 min but had receded at 1 h; (AUC mean [95% Cl]; 20.0 [13.2, 26.8] at 3 h; 11.0 [5.3, 16.6] at 24 h) and was completely abolished by both doses of UK,74505 at 3 h and by the higher 100 mg dose at 24 h. PAF-induced neutropenia and rebound neutrophilia were abolished by both doses of drug; neutropenia at 5 min (expressed as mean [95% Cl] change from baseline; -2.5 x 10(9)/L [-2.9, -2.1] after P; -0.3 [-0.7, 0.1] after 25 mg; 0.1 [-0.3, 0.4] after 100 mg), neutrophilia at 2 h (2.0 [-1.3, 2.6] after P; -0.2 [-0.8, 0.5] after 25 mg; -0.1 [-0.8, 0.5] after 100 mg).(ABSTRACT TRUNCATED AT 250 WORDS)
Administration, Inhalation↗
Ampiroxicam, an anti-inflammatory agent which is a prodrug of piroxicam.
Ampiroxicam is a nonacidic ether carbonate prodrug of piroxicam. Our results demonstrate that, in contrast to piroxicam, ampiroxicam does not possess detectable prostaglandin synthesis inhibitory activity in vitro. Ampiroxicam, however, has similar in vivo potency to piroxicam in suppressing paw swelling in rat adjuvant arthritis. In an acute model of paw inflammation in rats, ampiroxicam is less potent than piroxicam itself: the ED50's of ampiroxicam are 9- and 3.5-fold higher than those of piroxicam following a single or multiple (5) daily oral doses, respectively. Using the phenylbenzoquinone stretching test as a method of evaluating acute analgetic activity, the ED50 for ampiroxicam is about 3-fold higher than that of piroxicam. These tests of activity share the property of being partially prostaglandin-dependent. Ampiroxicam itself is not observed in plasma after oral dosing to man, nor in the rat, dog, and monkey as reported here. Bioavailability studies show that conversion to piroxicam is about 100%, 90%, 70%, and 50% in these four species, respectively. These results indicate that ampiroxicam's anti-inflammatory activity is produced in vivo by conversion to piroxicam and support its credentials as an efficacious prodrug of piroxicam.
Animals↗
IL-1 beta maturation: evidence that mature cytokine formation can be induced specifically by nigericin.
Mouse peritoneal macrophages stimulated with LPS produce large amounts of pro-IL-1 beta. When these cells were pulse-labeled with [35S]methionine, however, little labeled cytokine appeared in the medium after a chase, and that which was externalized was not processed to its mature biologically active form. In an effort to promote proteolytic maturation of IL-1 beta, macrophages were treated with agents that were expected to compromise their viability. The calcium ionophore A23187 and the detergent saponin caused complete release of nonprocessed 35-kDa pro-IL-1 beta and liberation into the extracellular medium of the cytoplasmic marker enzyme LDH and the lysosomal enzyme beta-N-acetylglucosaminidase. Hypotonic lysis resulted in the release of a 20-kDa IL-1 beta species that was distinct from the 17-kDa mature species. Importantly, incubation of the murine macrophages with the potassium/proton ionophore nigericin led to a quantitative conversion of pro-IL-1 beta to a 17-kDa species. The N-terminus of this nigericin-derived product possessed the amino acid sequence expected for mature biologically active IL-1 beta. Monensin, an ionophore similar to nigericin, did not induce release or proteolysis of IL-1 beta. Complete release of mature IL-1 beta required concentrations of nigericin in excess of 2 microM and a minimum of 10 min of treatment. Mature 17-kDa IL-1 beta was observed within the nigericin-treated cells before their lysis. Nigericin's effect was not limited to mouse peritoneal macrophages, inasmuch as the ionophore also induced release and proteolytic maturation of IL-1 beta produced by LPS-stimulated human peripheral blood monocytes. Treatment of macrophages with LPS and nigericin, therefore, results in a unique series of intracellular events that promote formation of mature 17-kDa IL-1 beta.
Animals↗
Inhibition of 5-lipoxygenase product formation and polymorphonuclear cell degranulation by tenidap sodium in patients with rheumatoid arthritis.
We studied the effect of tenidap sodium, a new antiinflammatory/antirheumatic drug (120 mg/day for 7 days), on eicosanoid production and neutrophil degranulation in patients with rheumatoid arthritis. Endogenous prostaglandin E2 levels and ex vivo production of leukotriene B4 (LTB4) were measured in synovial fluid samples obtained at baseline and 1 week later. We measured peripheral blood polymorphonuclear cell (PMN) degranulation following surface-bound IgG stimulation, a possible 5-lipoxygenase product-mediated event, by determining lactoferrin and elastase release into the culture fluid. We found decreased levels of endogenous prostaglandin E2 as measured by radioimmunoassay, and decreased ex vivo production of LTB4 by PMN as measured by high performance liquid chromatography, in synovial fluid samples from patients who took tenidap. Release of the granule proteins lactoferrin and elastase was decreased in PMN obtained from patients receiving tenidap, as well as in the PMN incubated in vitro with tenidap. Improvement in clinical measures paralleled the biochemical changes. The unique 5-lipoxygenase inhibitory property of tenidap, as measured by LTB4 production and degranulation, suggests that it may have clinical activity which differentiates it from nonsteroidal antiinflammatory drugs.
Aged↗
1,2-Dihydro-1-oxopyrrolo[3,2,1-kl]phenothiazine-2-carboxamides and congeners, dual cyclooxygenase/5-lipoxygenase inhibitors with antiinflammatory activity.
A series of 1,2-dihydro-1-oxopyrrolo[3,2,1-kl]phenothiazine, 1,2-dihydro-1-oxopyrrolo[3,2,1-kl]phenoxazine, and 1,2-dihydro-1-oxopyrrolo[3,2,1-de]acridine-2-carboxamides were prepared by reaction of 1,2-dihydro-1-oxo-pyrrolo[3,2,1-kl]phenothiazine or other corresponding phenoxazine and acridan ethyl or methyl esters with appropriate amines. Several members of this family were found to be potent, dual inhibitors of cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism and to have in vivo antiinflammatory activity in the rat foot edema assay. Structure-activity relationships within this family of compounds are described. 1,2-Dihydro-N-(2-thiazolyl)-1-oxopyrrolo[3,2,1-kl]phenothiazine-1- carboxamide (34) was found to be one of the best compounds to display potent cyclooxygenase/5-lipoxygenase inhibition of arachidonic acid metabolism. Its IC50s against the enzymes sourced from rat basophillic leukemia-1 (RBL-1) cells were 0.07 and 1.4 microM, respectively. It was active in the rat foot edema test for antiinflammatory effect (48% inhibition at 33 mg/kg po) and in the mouse phenylbenzoquinone induced writhing test for analgesic effect (93% inhibition at 32 mg/kg po).
Animals↗
Indeno[1,2-c]pyrazolone acetic acids as semirigid analogues of the nonsteroidal anti-inflammatory drugs.
A series of 3-substituted indeno[1,2-c]pyrazol-4(1H)-one-2-acetic acids (3a-e) and 3-substituted indeno[1,2-c]pyrazol-4(1H)-one-1-acetic acids (4a-e) were synthesized as semirigid analogues of tolmetin (1). These compounds were evaluated for their anti-inflammatory action by investigating their ability to block arachidonic acid metabolism in vitro as well as the ability to block carrageenan-induced rat foot edema in vivo. No consistent pattern of biological activity was noted.
Animals↗
Meeting report. The biochemistry and pharmacology of interleukins-1 and -6.
Explore the source record for details and available documents.
Animals↗
Spectrophotometric monitoring of lipoxygenase and cyclooxygenase pathway activity using ionophore stimulated whole blood.
We have employed clotting human blood stimulated with ionophore to develop a system for measuring cyclooxygenase, 5-lipoxygenase, and 12-lipoxygenase pathway products released into the serum fraction. In a single chromatographic run, 5-HETE, 12-HETE, 12-OH-heptadecatrienoic acid (HHT), LTB4, 20-OH-LTB4, and 20-COOH-LTB4 are quantitated by UV monitoring after separation by HPLC. The kinetics of product formation/release of all fatty acid products into the serum show an apparent lag of approximately 2 min, after which time the amounts of HHT, 5-HETE, and LTB4, respectively, plateau at 10 min while 12-HETE increases over a 60 min period. The system is responsive to standard cyclooxygenase and lipoxygenase inhibitors, and is of value of evaluating prospective blockers of AA metabolism in a whole blood setting.
Calcimycin↗
The use of 12-hydroxyheptadecatrienoic acid (HHT) as an HPLC/spectrophotometric marker for cyclooxygenase pathway activity in resident rat peritoneal cells.
12-Hydroxyheptadecatrienoic acid (HHT), a UV chromophore, has been used to assess cyclooxygenase (CO) pathway metabolism of arachidonic acid (AA) by rat peritoneal cells. Simultaneous monitoring at 235 and 280 nm after HPLC permits the measurement of both CO and 5-lipoxygenase (5-LO) pathway fluxes in a single system.
Animals↗
Development of a system for evaluating 5-lipoxygenase inhibitors using human whole blood.
A reliable system for evaluating 5-lipoxygenase (5-LO) pathway inhibitors employing human whole blood stimulated by the calcium ionophore, A-23187, and yeast cell walls (YCW) is described. In developing this system, we have shown that leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) can be recovered quantitatively from whole blood, and can be measured with accuracy and a precision (standard deviation) of +/- 12%. Apparent differences in LTB4/5-HETE levels between donors can be minimized by normalizing the LTB4/5-HETE production to neutrophil number. Variability in LTB4/5-HETE production among different donors was reduced by increasing the ionophore concentration. The kinetics of ionophore stimulated product production display a 1-4 min lag which is dependent on ionophore concentration. The lag is removed by pretreatment of blood with 5 micrograms/ml cytochalasin B. Likewise, the kinetics of product formation after stimulation with yeast cell walls demonstrated a lag period, which could be shortened by prior opsonization of the YCW. The amount of LTB4 metabolism to 20-OH-LTB4 and 20-COOH-LTB4 in this system is approximately 20%. Phenidone, nordihydroguaiaretic acid, and nafazatrom, known inhibitors of the 5-LO pathway, display half-maximal inhibition points of 0.4, 1.5, and 9 micrograms/ml, respectively. In summary, we believe that this assay offers a guide for predicting systemic levels of drug needed to be achieved for effective inhibition of cellular LTB4/5-HETE synthesis/release in humans.
Arachidonate 5-Lipoxygenase↗
Radioimmunoassay for sulprostone.
The development of a radioimmunoassay for measuring subnanogram amounts of the prostaglandin uterine stimulant, sulprostone (N-methanesulfonyl 16-phenoxy-omega-tetranor PGE2 carboxamide), is described. The 9-carboxymethoxime derivative of sulprostone was coupled to keyhole limpet hemocyanin to prepare the immunogen, and to tyramine to yield a precursor suitable for radioiodination. The antiserum generated from rabbits was specific for sulprostone, showing cross reactivities of less than 0.1% against PGE2, PGF2 alpha, and known sulprostone metabolites. The range for routine assay of sulprostone was 10-300 pg which corresponded to 50-1500 pg/ml of plasma. The coefficient of variation for replicate analyses on the same sample was 8-12%. The assay was used to measure the plasma levels of sulprostone in patients who had received the drug intramuscularly.
Animals↗
The development of a radioimmunoassay for 12-L-hydroxyeicosatetraenoic acid.
Antibodies directed toward 12-L-hydroxyeicosatetraenoic acid (12-L-HETE) were generated in rabbits by immunization with conjugates of 12-L-HETE and human serum albumin. The concentration of antibodies was determined by incubating immune plasma with 12-L-HETE that had been covalently linked to a solid support, washing the 12-L-HETE support, and measuring the quantity of bound antibodies by reaction with [125I]Protein A. The addition of 0.5 ng-10 ng of fluid-phase 12-L-HETE to the standard mixture of solid-phase 12-L-HETE and anti-12-L-HETE plasma inhibited by 21-80% the binding of antibodies and consequently of [125I]Protein A to the solid support. The 12-OH function positioned between two double bonds was the immunodominant determinant of this antigen-antibody reaction, but the carboxyl function also was recognized. This radioimmunoassay was used to detect and quantitate 12-L-HETE resolved by high pressure liquid chromatography.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Piroxicam, a potent inhibitor of prostaglandin production in cell culture. Structure-activity study.
The new non-steroidal antiinflammatory (NSAI)2 agent, piroxicam [4-hydroxy-2-methyl-N-(2-pyridyl)-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide], is a highly active inhibitor of prostaglandin (PG) synthesis by methylcholanthrene transformed mouse fibroblasts (MC5-5) and rabbit synovial cells in culture. Comparison of the PG biosynthesis inhibitory activity of piroxicam with other NSAI drugs in these experiments ranks piroxicam as among the most potent agents of this type now known. Some specific modifications of piroxicam's structure result in significant loss in PG synthesis blocking activity.
Animals↗
Piroxicam, a structurally novel anti-inflammatory compound. Mode of prostaglandin synthesis inhibition.
Piroxicam is a potent inhibitor of prostaglandin biosynthesis. Experiments utilizing cell culture and microsomes derived from various sources have demonstrated that piroxicam is a selective inhibitor of the cyclooxygenase step of arachidonic acid metabolism. Little blocking activity is observed at the phospholipase, thromboxane or prostacyclin synthetase, and arachidonic acid lipoxygenase steps.
Animals↗