Presence Of Flurbiprofen Research Articles

Mechanism of Action in NaHS‐Induced Increase in Aqueous Humor Outflow

Previous studies from our laboratory demonstrate that the hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaHS) can increase aqueous humor (AH) outflow facility in porcine trabecular meshwork tissues, ex vivo. In the present study, we investigated the mechanism/s that underlie the NaHS‐induced increase in AH outflow facilityMethodsPorcine ocular anterior segments explants were perfused with Dulbecco's Modified Eagle's Medium maintained at 37° C, 5% CO2 and constant pressure of 7.35 mmHg. Once outflow was stable (~3 hours), explants were exposed to the adenylyl cyclase inhibitor SQ22536 (100 µM), enzyme inhibitors for H2S biosynthesis [proparglyglycine (PAG, 1 mM); aminooxyacetic acid (AOA, 30 µM)], and prostanoids (flurbiprofen, 3 µM), 30 minutes prior to application of NaHS (10 µM). Outflow was then monitored for an additional 4 hours, and vehicle control (0.1% saline) was run in parallel.ResultsThe NaHS‐induced increase in AH outflow was attenuated by AOA and PAG by 79% and 56%, respectively. In the presence of flurbiprofen (3 µM), effects of NaHS on outflow was significantly (p<0.02) inhibited from [130.6 ± 10.51% of basal] to [102.9 ± 2.88% of basal]. Furthermore, the adenylyl cyclase inhibitor, SQ22536 completely blocked (p <0.05) the NaHS‐induced increase in outflow facility.ConclusionWe conclude that the ability of NaHS to increase AH outflow in porcine trabecular meshwork is dependent upon the intramural generation of H2S and prostanoids, and on the integrity of the adenylyl cyclase pathway.

Growth and Shrinkage of Pluronic Micelles by Uptake and Release of Flurbiprofen: Variation of pH

The micellization of Pluronic triblock copolymers (P103, P123, and L43) in the presence of flurbiprofen at different pH was studied by small-angle neutron scattering (SANS), pulsed-field gradient stimulated-echo nuclear magnetic resonance (PFGSE-NMR), and surface tension measurements. Addition of flurbiprofen to the Pluronic at low pH leads to an increase in the fraction of micellization, aggregation number, and the core radius of the micelles. However, changing the pH to above the pKa of flurbiprofen in an ethanol/water mixture (∼6.5) reduces the fraction of micellization and results in a weaker interaction between the drug and micelles due to the increased drug solubility in aqueous solution.

O1-2.2 Sparse instrumental variables: an integrative approach to biomarker validation

Dapsone activates CYP2C9-mediated metabolism in various expression systems and is itself metabolized by CYP2C9 to its hydroxylamine metabolite. Studies were conducted with expressed CYP2C9 to characterize the kinetic effects of dapsone (0–100 μM) on (<i>S</i>)-flurbiprofen (2–300 μM), (<i>S</i>)-naproxen (10–1800 μM), and piroxicam (5–900 μM) metabolism in 6 × 6 matrix design experiments. The influence of (<i>S</i>)-flurbiprofen on dapsone hydroxylamine formation was also studied. Dapsone increased the Michaelis-Menten-derived <i>V</i>_max of flurbiprofen 4′-hydroxylation from 12.6 to 20.6 pmol/min/pmol P450, and lowered its <i>K</i>_m from 28.9 to 10.0 μM, suggesting that dapsone activates CYP2C9-mediated flurbiprofen metabolism without displacing flurbiprofen from the active site, supporting a two-site model describing activation. Similar results were observed with piroxicam 5′-hydroxylation, as<i>V</i>_max was increased from 0.08 to 0.20 pmol/min/pmol P450 and <i>K</i>_m was decreased from 183 to 50 μM in the presence of dapsone. In addition, the kinetic profile for naproxen was converted from biphasic to hyperbolic in the presence of dapsone, while exhibiting similar decreases in<i>K</i>_m and increases in<i>V</i>_max. Kinetic parameters were also estimated using the two-site binding equation, with α values &lt;1 and β values &gt;1, indicative of activation. Additionally, dapsone hydroxylamine formation was measured from incubations containing flurbiprofen, exhibiting a kinetic profile that was minimally affected by the presence of flurbiprofen. Overall, these results suggest that dapsone activates the metabolism of multiple substrates of CYP2C9 by binding within the active site and causing positive cooperativity, thus lending further support to a two-site binding model of P450-mediated metabolism.

Role of Hydrogen Sulfide Production in Inhibitory Action of L-Cysteine on Isolated Porcine Irides

Purpose: To investigate the direct pharmacological actions of L-cysteine, a substrate for the production of H2S, on isolated porcine irides in the presence of tone induced by muscarinic receptor stimulation. Furthermore, we examined the underlying mechanism of action of L-cysteine in this smooth muscle.Methods: Isolated porcine iris muscle strips were set up in organ baths containing oxygenated Krebs buffer solution at 37°C. Longitudinal isometric tension was recorded via a grass FT03 Force-Displacement Transducer and analyzed using the PolyView computer software. The relaxant action of L-cysteine on carbachol-induced tone was studied in the absence and presence of inhibitors of enzymes of the biosynthetic pathways for H2S, and prostanoids. In addition, we also examined the effect of ATP-sensitive K+ (KATP) channel antagonist, glibenclamide on relaxations induced by L-cysteine.Results: L-cysteine (30 nM–1 mM) evoked concentration-dependent relaxations of carbachol-induced tone in isolated porcine irides, reaching a maximum inhibition of 43% at 1 mM. This response was enhanced significantly (P < 0.001) in the presence of the COX inhibitor, flurbiprofen (3 μM). Additionally,in the presence of flurbiprofen, the H2S donors, NaHS and Na2S, mimicked the relaxations produced by L-cysteine, yielding IC50 values of 5.8 μM and 180 μM, espectively. Both the inhibitor of cystathionine β-synthase, AOA (30 μM) and the KATP channel antagonist, glibenclamide (100 μM) caused significant (P < 0.001) rightward shifts in the concentration-response curves to L-cysteine and attenuated the maximum inhibitory response. Conversely, the inhibitor of cystathionine γ-lyase, PAG (1 mM) blocked only relaxations caused by high concentrations of L-cysteine (> 100 μM).Conclusions: The inhibitory action of L-cysteine in isolated porcine irides is dependent on the endogenous production of H2S by cystathionine γ-lyase and cystathionine β-synthase. Furthermore, prostanoids and KATP channels are involved in the inhibitory action of L-cysteine in this tissue.

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is up-regulated by flurbiprofen and other non-steroidal anti-inflammatory drugs in human colon cancer HT29 cells

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to inhibit prostaglandin synthetic enzyme, cyclooxygenases (COXs), as well as to exhibit anti-tumor activity although at much higher concentrations. 15-Hydroxyprostaglandin dehyrogenase (15-PGDH), a key prostaglandin catabolic enzyme, was recently shown to be a tumor suppressor. Effects of NSAIDs on 15-PGDH expression were therefore examined. Flurbiprofen and several other NSAIDs were found to induce 15-PGDH expression in human colon cancer HT29 cells. Flurbiprofen, the most active one, was also shown to induce 15-PGDH expression in other types of cancer cells. Induction of 15-PGDH expression appeared to occur at the stage of mRNA as levels of 15-PGDH mRNA were increased by flurbiprofen in HT29 cells. Levels of 15-PGDH were also found to be regulated at the stage of protein turnover. MEK inhibitors, PD98059 and U-0126, which inhibited ERK phosphorylation were shown to elevate 15-PGDH levels very significantly. These inhibitors did not appear to alter 15-PGDH mRNA levels but down-regulate matrix metalloproteinase-9 (MMP-9). This protease was shown to degrade and inactivate 15-PGDH suggesting that elevation of 15-PGDH levels could be due to inhibition of MMP-9 expression by these inhibitors. Similarly, flurbiprofen was also demonstrated to inhibit ERK activation and to down-regulate MMP-9 expression. Furthermore, flurbiprofen was shown to induce the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of MMP-9. The turnover of 15-PGDH was found to prolong in the presence of flurbiprofen as compared to that in the absence of this drug. Taken together, these results indicate that flurbiprofen up-regulates 15-PGDH by increasing the expression and decreasing the degradation of 15-PGDH in HT29 cells.

In Situ Transient Spectroscopy for the Study of Glucuronidase Activity within Serum Albumin

Laser flash photolysis (LFP) has shown to be an efficient technique for in situ determination of the glucuronidase activity of human serum albumin (HSA). After incubation of the steroisomeric flurbiprofen glucuronides (FBPGluc) during regular time intervals at the selected temperatures, in the presence of protein, regression analysis was applied to the triplet decay at lambda=360 nm. This led to a satisfactory fitting when considering a set of four lifetimes; the corresponding preexponential coefficients AIFBP, AIIFBP, AFFBPGluc, and ABFBPGluc can be correlated with the presence of flurbiprofen (FBP) within the two known binding sites (I and II), together with FBPGluc free in solution (F) and bound (B) to the protein. The new methodology based on LFP of glucuronides in the presence of HSA is fast, experimentally straightforward, and does not involve any workup. This suggests the possibility of making use of the transient triplet-triplet absorption for investigating the enzymatic-like activity of different host biomolecules and at the same time determining the distribution of the generated drug between several compartments in the protein.

Development and validation of a liquid chromatographic method for the determination of clopidogrel from pharmaceutical dosage form

A rapid, specific, precise and sensitive reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of clopidogrel from pharmaceutical dosage form in the presence of flurbiprofen as internal standard. Clopidogrel and internal standard were analyzed on Purospher start C18, 5 μm column having 250 x 4.6 mm i.d., with mobile phase containing methanol: water (80: 20; pH 3.4) was used. The flow rate was 1.0 mL min -1 and detector response was monitored at 235 nm. The retention time of clopidogrel and internal standard was 4.2 min and 6.4 min respectively. Calibration curve was linear in concentration range of 0.25-50.0 μg mL -1 with coefficient of determination (r 2 ) of 0.9994. Detection (LOD) and quantitation limits (LOQ) were 0.09 and 0.28 μg mL -1 , respectively. The intra-day accuracy ranged from 96.0-103.6% with a precision of 2.30-2.86%. Similarly, the inter-day accuracy was between 98.18 and 99.99% with a precision of 1.18-10.59%.

Involvement of organic anion transport system in transdermal absorption of flurbiprofen

We previously demonstrated that transdermal permeation of flurbiprofen is mediated by a nonlinear transport mechanism(s). Here, we aimed to characterize this transport mechanism by employing an Ussing-type chamber method with tape-stripped hairless mouse skin. Transdermal permeation of [ 3H]flurbipofen was vectorial, saturable and energy-dependent, suggesting the involvement of a carrier-mediated transport system. Transdermal permeation and uptake from the epidermal side of [ 3H]flurbiprofen were inhibited by various nonsteroidal anti-inflammatory drugs (NSAIDs). The inhibitory potency did not correlate well with lipophilicity; anionic NSAIDs tended to be more potent inhibitors than non-anionic NSAIDs. The inhibition profile of both [ 3H]flurbiprofen permeation and uptake, and the Michaelis constants, were similar for a given anionic compound. These results suggest that an organic anion transport system is involved in flurbiprofen uptake from the epidermal side during the process of transdermal absorption. Efflux of [ 3H]flurbiprofen from the skin to the epidermal side, but not to the hypodermal side, increased in the presence of flurbiprofen or several anionic compounds. Such trans-stimulation may suggest the involvement of an organic anion exchanger system. Organic anion transporter 2 (OAT2) is a candidate for the exchanger involved in uptake and/or efflux of flurbiprofen in the skin.

Effector-mediated alteration of substrate orientation in cytochrome P450 2C9.

Cytochrome P450 2C9 (CYP2C9)-mediated flurbiprofen 4'-hydroxylation is activated by the presence of dapsone resulting in reduction of the K(m) for flurbiprofen hydroxylation and an increase in V(m). Previous spectral binding studies have demonstrated that the binding of flurbiprofen with CYP2C9 is increased (decrease in K(S)) by the presence of dapsone. We hypothesized that the two compounds are simultaneously in the active site with the presence of dapsone causing flurbiprofen to be oriented more closely to the heme. T(1) relaxation rates determined by NMR were used to estimate the distances of protons on these compounds from the paramagnetic heme-iron center. Samples contained 0.014 microM CYP2C9 and 145 microM flurbiprofen in the presence and absence of 100 microM dapsone. Estimated distances of various flurbiprofen protons from the heme ranged from 4.2 to 4.5 A in the absence of dapsone and from 3.2 to 3.8 A in the presence of dapsone. The 4' proton of flurbiprofen, the site of metabolism, showed one of the greatest differences in distance from the heme in the presence of dapsone, 3.50 A, as compared to the absence of dapsone, 4.41 A. Dapsone protons were less affected, being 4.40 A from the heme in the absence of flurbiprofen and 4.00-4.01 A from the heme in the presence of flurbiprofen. Molecular modeling studies were also performed to corroborate the relative orientations of flurbiprofen and dapsone in the active site of CYP2C9. Shift of the 4' proton of flurbiprofen closer to the heme iron of CYP2C9 in the presence of dapsone may play a role in activation.

Effect of flurbiprofen on acetylcholine-induced pupillary contraction.

Preoperative treatment with topical nonsteroidal anti-inflammatory agents such as flurbiprofen (Ocufen) is used to maintain pupil dilation during cataract surgery. Flurbiprofen maintains pupil dilatation by inhibiting release of prostaglandins and other modulators of surgical miosis. Some reports suggest that these agents may reduce the miotic effect of intra-operative acetylcholine (Miochol). It is hard to explain this effect unless there is a pharmacological interaction between the two drugs. This study aimed to investigate the possibility of a direct interface by flurbiprofen with the action of acetylcholine on the pupil sphincter. Iris tissue of seven cadaver eyes were isolated and maintained in an organ bath containing Kreb's solution. Pupil contraction force induced by acetylcholine was measured in the presence and absence of flurbiprofen. Acetylcholine produced a mean pupillary contraction force of 52.4 x 10(-3) N. When it was added to the organ bath in the presence of flurbiprofen the mean contraction force was 50.5 x 10(-3) N (paired t-test P = 0.68). This study does not find any evidence to suggest that flurbiprofen reduces the pupil sphincter contraction that is induced by acetylcholine. It is likely that the apparent reduction in miosis is due to factors other than the mechanism of action of acetylcholine on the sphincter pupillae.

Effects of calcium modulators on vagally-mediated constriction in the guinea-pig isolated trachea

1. The effects of calcium modulators on tracheal constriction evoked by vagal stimulation were examined in the isolated, innervated trachea of the guinea-pig. Responses were assessed in the Krebs-filled trachea as changes in intraluminal pressure (ILP), increases and decreases reflecting constriction and dilatation, respectively. 2. Preparations had a positive resting ILP, indicating significant spontaneous tone. Verapamil and nifedipine reduced baseline ILP, whilst Bay K 8644 had mixed effects. 3. Verapamil and nifedipine attenuated vagal responses in a concentration-dependent manner. At lower concentrations attenuation was due entirely to postjunctional effects but at higher concentrations prejunctional effects may have contributed to attenuation. 4. Verapamil and nifedipine attenuated vagal responses in the absence or presence of flurbiprofen, indicating that their effects are largely independent of the generation of cyclo-oxygenase products. Nifedipine, however, was less effective in reducing responses to low frequency vagal stimulation (up to 5 Hz) when flurbiprofen was present. 5. Bay K 8644 augmented vagal responses, the degree varying widely between preparations. 6. It was concluded that influx of Ca2+ through voltage-operated Ca2+ channels contributes significantly to vagally-mediated tracheal constriction in normal trachea and in trachea where endogenous release of cyclo-oxygenase products is inhibited.

Involvement of prostaglandins in the inhibitory response of adrenoceptor agonists in the rat isolated uterus

1. The possible involvement of intramurally generated prostaglandins in the responses produced by noradrenaline (NA), adrenaline (ADR) and salbutamol (SAL) has been investigated in the rat isolated uterus. 2. NA, ADR and SAL produced concentration-related inhibition of acetylcholine-induced tone in preparations from the proestrus, oestrus, metoestrus and dioestrus phases of the oestrous cycle, and from ovariectomized animals. 3. The cyclo-oxygenase inhibitor, flurbiprofen (FBF), enhanced uterine inhibitory response to all three agonists under the different hormonal conditions. 4. The dual inhibitor of cyclo-oxygenase and lipoxygenase, 3-amino-l-(m-trifluoromethyl)-phenyl-2-pyrazolone (BW 755C) also potentiated uterine response to NA, ADR and SAL, but the effect produced by BW 755C was similar to that achieved in the presence of FBF. 5. In contrast, the inhibition elicited by histamine and papaverine were unaffected by FBF treatment. 6. The above results provide pharmacological evidence that adrenoceptor agonists may influence prostaglandin production in the rat uterus both in the presence, and absence of the ovarian hormones.

Involvement of prostaglandins in the inhibitory response of adrenoceptor agonists in the rat isolated uterus

Abstract o 1. The possible involvement of intramurally generated prostaglandins in the responses produced by noradrenaline (NA), adrenaline (ADR) and salbutamol (SAL) has been investigated in the rat isolated uterus. 2. NA, ADR and SAL produced concentration-related inhibition of acetylcholine-induced tone inpreparations from the proestrus, oestrus, metoestrus and dioestrus phases of the oestrous cycle, and from ovariectomized animals. 3. The cyclo-oxygenase inhibitor, flurbiprofen (FBF), enhanced uterine inhibitory response to all threeagonists under the different hormonal conditions. 4. The dual inhibitor of cyclo-oxygenase and lipoxygenase, 3-amino-1-( m -trifluoromethyl)-phenyl-2-pyrazolone (BW 755 C) also potentiated uterine response to NA, ADR and SAL, but the effect produced by BW 755 C was similar to that achieved in the presence of FBF. 5. In contrast, the inhibition elicited by histamine and papaverine were unaffected by FBF treatment 6. The above results provide pharmacological evidence that adrenoceptor agonists may influence prostaglandin production in the rat uterus both in the presence, and absence of the ovarian hormones

Anti-leukotriene D 4 action of a new anti-asthmatic drug (KC-404) on the guinea-pig isolated trachea

1. 1. Anti-asthmatic drug, KC-404, inhibited specifically spontaneous contraction and leukotriene (LT) D 4-induced contraction of guinea-pig trachea. 2. 2. Relaxation of spontaneous contraction by KC-404 was reduced by the treatment of the guinea-pig trachea with a cyclooxygenase inhibitor, flurbiprofen. 3. 3. Some parallel shift of the concentration-action curve of LTD 4 by KC-404 was still observed in the presence of flurbiprofen. 4. 4. Specific binding of [ 3H]LTD 4 to the microsomal fraction of guinea-pig trachea was partially inhibited by KC-404. 5. 5. These results suggest that the relaxing action of trachea of KC-404 is due to the interference of interaction between LTD 4 and its receptor and to the product(s) via cyclooxygenase pathway.