Drug binding sites on chicken albumin: a comparison to human albumin.

Mammalian albumins have two main structurally selective ligand binding sites. Site I binds drugs such as azapropazone, phenylbutazone and warfarin; whereas benzodiazepines, some dansyl amino acids, such as dansylsarcosine, and short chain fatty acids like octanoic acid interact with site II. However, it is not known if non-mammalian albumins have similar binding loci. In this study, drug binding sites on chicken albumin were investigated using site selective fluorescent probes (warfarin and dansylsarcosine) and p-nitrophenyl acetate (NPA); the hydrolysis of which is selectively inhibited by site II ligands. Azapropazone and phenylbutazone decreased the binding of warfarin and dansylsarcosine to a similar extent. Diazepam and octanoic acid also inhibited binding of the two fluorescent probes in a non-selective manner. However, the fluorescence intensity of the warfarin-chicken albumin complex decreased when the pH was increased from 6.0-9.0; but by contrast, the fluorescence of bound dansylsarcosine remained unchanged. Furthermore, the hydrolysis of NPA was selectively inhibited by dansylsarcosine, diazepam and octanoic acid (ligands selective for site II on mammalian albumins), but not by site I selective ligands such as azapropazone and warfarin. Overall, the results suggest that chicken albumin, like mammalian albumins, has discrete binding sites for warfarin and dansylsarcosine.

Effects of azapropazone on pain-related brain activity in human subjects.

1. The dose-related effects of azapropazone on (i) event-related and spontaneous EEG-activity and (ii) the subjects' pain ratings were investigated using an experimental human pain model based on both chemo-somatosensory event-related potentials (CSSERP) and subjects' pain ratings. 2. Healthy subjects (n = 20) participated in a placebo-controlled, randomized, double-blind, four-way cross-over study. Single doses of azapropazone (300 mg, 600 mg and 1200 mg) and placebo were administered intravenously. Each experiment consisted of five sessions (before and 1, 2, 4 and 8 h after administration of the medication). Each session lasted for approximately 40 min. In the first 20 min, pain was induced by short CO2-stimuli presented to the right nostril (phasic pain; interstimulus interval 30 s) and EEG was recorded from five positions. CSSERPs were obtained in response to painful CO2-stimuli. In the following 20 min period, tonic pain was induced by a constant stream of dry air introduced in the left nostril. Subjects rated the intensity of both phasic and tonic pain by means of a visual analogue scale. Additionally, a frequency analysis of the spontaneous EEG was performed. 3. Azapropazone reduced the pain-related CSSERP-amplitudes at frontal and parietal recording positions. This topographical pattern was observed in previous studies with opioids, while NSAIDs such as flurbiprofen and ketoprofen exerted effects at frontal and central positions. In contrast to other NSAIDs, administration of azapropazone resulted in a reduction of the frequency bands alpha 1, delta and theta of the spontaneous EEG. At the subjective level, analgesic effects of azapropazone were observed in the ratings of tonic pain. 4. Analgesic properties of azapropazone were demonstrated in man. The topographical pattern of the changes in the CSSERPs and the effects on EEG background activity suggest a central component of the analgesic action of azapropazone.

Lack of activity of azapropazone in the hot-plate test and in 5-HT1A and 5-HT2 receptor subtypes in rat brain membranes.

This study aimed to investigate the antinociceptive activity of azapropazone (AZA), a weak prostaglandin synthesis inhibitor using the hot-plate test, and its ability to modify the serotonin-binding capacity in rat brain membranes. It revealed that AZA had no antinociceptive effect in the hot-plate test at the doses of 400 and 600 mg/kg when orally administered (p.o.), and at 400, 500 and 600 mg/kg after intraperitoneal injection (i.p.). At the dose of 600 mg/kg i.p. the drug failed to modify the number and the affinity of 5-HT1A and 5-HT2 receptors in rat brain membranes. In accordance with our previous findings on a positive correlation between NSAIDs antinociception in this experimental model and changes in 5-HT receptor characteristics, these results suggest an association between the lack of AZA-mediated antinociception in the hot-plate test and the drug's inability to modify the characteristics of 5-HT1A and 5-HT2 receptor binding sites in rat brain membranes.

Effects on platelet functions and pharmacokinetics of azapropazone and ketorolac tromethamine given as single parenteral doses.

The biosynthesis of thromboxane (TX) B2 and immunoreactive prostaglandin (PG) F2 alpha in clotting whole blood ex vivo as well as collagen-induced platelet aggregation were determined before and up to 72 h after intravenous injection of 600 mg azapropazone 2H2O and intramuscular injection of 30 mg ketorolac tromethamine in six healthy subjects. The drug doses were selected on the basis of comparable analgesic activity (maximal recommended analgesic dose). Both platelet aggregation and prostanoid biosynthesis were inhibited by racketorolac to a significantly greater extent and for a longer period of time than by azapropazone. Correlations between serum concentrations and the inhibitory effects on TXB2 biosynthesis were observed for both drugs. Using the sigmoidal Emax model the mean serum concentration of azapropazone inhibiting platelet TXB2 generation by 50% (EC50) was found to be 98.1 +/- 41.9 (s.d.) micrograms ml-1, a value 1000 times higher than that for rac-ketorolac. The moderate inhibition of platelet function by azapropazone as compared with rac-ketorolac might be an advantage with regard to its use as a post-operative analgesic.

Effects of chronic NSAIDs on gastric mucosal injury related to mucosal prostanoids, and plasma drug concentrations in human volunteers.

The relationship between endoscopically observed gastric mucosal damage, elicited following repeated oral intake for 7 d of four NSAIDs, to their effects on antral and fundic production of PGE2, 6-keto-PGF1 alpha and TxB2 (assayed by GC-MS), mucosal histology and plasma concentration profiles was studied in 40 normal males. Subjects received azapropazone (APZ) 600 mg b.i.d., indomethacin (IND) 50 mg t.i.d., naproxen (NAP) 500 mg b.i.d., piroxicam (PIR) 20 mg qq.d., or one placebo capsule t.i.d. (N = 8/group). Plasma NSAIDs (HPLC) levelled at 7 d. Mucosal damage occurred in the antrum region with IND and NAP. APZ and PIR exhibited no differences compared to placebo. NAP and IND reduced all three prostanoids in the antrum while APZ and PIR were ineffective. Fundic PGE2 was reduced by IND, NAP and PIR; APZ had no effects. Thus, mucosal damage relates to effects on prostanoid production in the antrum but not in the fundus.

Photoproducts of benzydamine and azapropazone: demonstration of their phototoxicity in vitro.

Red blood cell lysis, photosensitized by the products of the aerobic photolysis of benzydamine (1) and azapropazone (4), was investigated. Irradiation of a methanol solution of 1 and 4 under oxygen produces the photoproducts 3-hydroxy-benzydamine, (2), 2-(3-dimethylaminopropyl)-1-benzylindazolin-3-one (3) and 3-dimethylamino-7-methyl-1,2,4-benzotriazine (5). The mechanism of the photodegradation of 1 was examined. Photoproducts 3 and 5 produce singlet oxygen as demonstrated by trapping with 2,5-dimethylfuran. The photohemolysis rate for the photoproducts 3 and 5 was enhanced by deuterium oxide and oxygen. No change was observed in the presence of reduced glutathione. The photohemolysis rate was low under anaerobic conditions.

Quantitative contribution of endogenous compounds and hypoalbuminemia in reducing the binding of furosemide in the plasma of newborn infants.

The protein binding of furosemide was studied in the plasma of newborn infants and adult subjects. Plasma consisted of two pools obtained from 25 newborns and adult subjects. The concentrations of albumin were 36.8 (newborn) and 48.3 g/l (adult). The unbound fraction of furosemide was 1.38 +/- 0.15 (adult) and 2.03 +/- 0.13% (newborn; p < 0.001). After extensive dialysis of the plasma, the unbound fraction of furosemide was 1.12 +/- 0.15 (adult) and 1.39 +/- 0.09% (newborn; p < 0.0001), suggesting that dialyzable endogenous compounds interfere with the binding of furosemide. The addition of human albumin to the newborn plasma to give a final albumin concentration of 48.3 g/l yielded an unbound fraction of furosemide of 1.63 +/- 0.08 (nondialyzed plasma) and 1.17 +/- 0.08% (dialyzed plasma; p < 0.0001). The addition of albumin to the dialyzed newborn plasma, to give a final albumin concentration similar to that in adult plasma, decreased the unbound furosemide to the level of the dialyzed adult plasma. The binding defect of furosemide in newborn plasma reflects either the effects of the endogenous inhibitors or of hypoalbuminemia. The intrinsic binding properties for furosemide of newborn dialyzed plasma are similar to those of dialyzed adult plasma. This consideration corroborates our previous results on the binding of furosemide and diazepam, salicylic acid and digitoxin to newborn and adult albumin. The displacement of furosemide by salicylic acid, tolbutamide and azapropazone is 70% greater in newborn than in adult plasma. The greater displacing effect is largely due to hypoalbuminemia.

Persistent cardioprotection by azapropazone in a canine model of coronary artery thrombosis and thrombolysis.

Activated neutrophils and possibly xanthine oxidase-derived free radicals are believed to be mediators of ischemia and reperfusion-induced myocardial damage. We studied the cardioprotective effect of the neutrophil stabilizer and xanthine oxidase inhibitor azapropazone in dogs subjected to thrombotic occlusion of the left anterior descending coronary artery (LAD), induced by intracoronary introduction of a copper coil, followed 60 min later by thrombolytic treatment with intracoronary streptokinase and 4-day reperfusion; we then determined infarct size by triphenyltetrazolium stain. Azapropazone [100 mg/kg intravenously (i.v.) followed by a 24-h i.v. infusion of 10 mg/kg/h, n = 8] or vehicle (n = 10) treatments were started immediately before the streptokinase infusion. Steady-state plasma levels of azapropazone ranged from 97 to 163 micrograms/ml during the infusion. Myocardial blood flow and underperfused area at risk were determined using radiolabeled microspheres. Results were as follows (mean +/- SEM): area at risk (percentage of left ventricle) azapropazone 22.7 +/- 3.16 and vehicle 21.8 +/- 4.13; infarct size (percentage of area at risk), azapropazone 45.1 +/- 11.8 and vehicle 75.7 +/- 10.6, p less than 0.03; collateral blood flow (ml/min/g), azapropazone 0.27 +/- 0.02 and vehicle 0.23 +/- 0.02; total ischemic period (min), azapropazone 106 +/- 5.9 and vehicle 91.5 +/- 4.9. Azapropazone had no effects on heart rate (HR), blood pressure (BP), or rate/pressure product (RPP). These dta show that azapropazone limits infarct size in a canine model of coronary thrombosis and long-term reperfusion and that this cardioprotection is independent of cardiovascular parameters.

The effect of prostaglandin synthetase inhibitors on human preovulatory follicular fluid prostaglandin, thromboxane, and leukotriene concentrations.

This study evaluates the eicosanoid concentration in luteinized unruptured follicles (LUFs) on the ovaries of patients who had been treated with inhibitors of prostaglandin synthetase. Indomethacin, bromfenac, or azapropazone (or a placebo) was administered orally to 41 women during the periovulatory period. Follicular development was monitored by serial ultrasound examinations, and the onset of ovulation was regulated by an injection of hCG. Follicular fluid was aspirated during sterilization by minilaparotomy, which was performed just before the expected time of ovulation. Prostaglandin E2 and PGF2 alpha levels in the fluid were significantly reduced by indomethacin and bromfenac compared to those after placebo treatment. Bromfenac also reduced the follicular fluid leukotriene B4 level. Therefore, the development of luteinized unruptured follicles after treatment with nonsteroidal antiinflammatory drugs appears to be associated with a significant decrease in the synthesis of ovarian eicosanoids.

Evaluation of the effect of azapropazone on neutrophil migration in regional myocardial ischaemia/reperfusion injury in rabbits.

1. The purpose of the present study was to determine the myocardial cytoprotective efficacy of azapropazone (AZA) and its potential site of action on neutrophil infiltration into reperfused/ischaemic myocardium with or without in vivo activation of neutrophils in rabbits. 2. AZA, 100 mg kg-1, was administered i.v. 10 min after occlusion of the left circumflex (LCX) artery in rabbits with and without pretreatment with phorbol myristate acetate ester (PMA). The LCX occlusion was then released at 10 min after AZA administration. Haemodynamic parameters (heart rate, LV pressure, mean arterial blood pressure and dp/dt) were monitored throughout the experiment. After 60 min reperfusion, the area at risk was delineated and the heart was then excised and divided into epi- and endocardial pieces for analysis of myeloperoxidase activity. 3. AZA inhibited neutrophil infiltration into the reperfused/ischaemic rabbit myocardium with and without PMA treatment. The inhibition of neutrophil infiltration was more apparent in the epicardium than in the endocardium. Additionally, AZA inhibited to a similar extent the in vivo PMA-stimulated neutrophil migration into the epicardium and endocardium area at risk. AZA had no significant effect on the haemodynamic parameters as compared to control. 4. AZA administered in an anaesthetized rabbit model of LCX occlusion/reperfusion resulted in the reduction of infarct size. 5. It is concluded that AZA has significant inhibitory effects on neutrophil migration which might contribute to its myocardial cytoprotective effect.

Evaluation of the effect of azapropazone on neutrophil migration in anaesthetized swine using a multichamber blister suction technique.

1. The purpose of this study was to determine the in vivo inhibitory efficacy of azapropazone on neutrophil migration. The effects of azapropazone given at a dose of 100 mg kg-1 i.v. every 2 h on the neutrophil migration into skin inflammation sites (blister fluid) as well as into an autologous serum (+/- chemoattractant) placed above the blister surface (2nd chamber) were determined. 2. Azapropazone treatment schedule maintained blood levels at 70-100 micrograms ml-1 throughout the time course (360 min) of the experiment. 3. Azapropazone significantly inhibited (48 +/- 6%) neutrophil migration into the blister fluid (as evident from the decrease in myeloperoxidase activity). 4. Azapropazone significantly inhibited the neutrophil migration into the autologous serum either with (65 +/- 5%) or (35 +/- 6%) without the chemoattractant, formyl-methionyl-leucyl-phenylalanine (FMLP). The chemoattractant, FMLP, markedly increased neutrophil migration into the autologous serum by approximately 1.5 to 2 times the non-FMLP treated group. Azapropazone was more efficacious in inhibiting the neutrophil migration in the presence of FMLP than in its absence. 5. We conclude that azapropazone is an effective inhibitor of neutrophil migration into topically inflamed sites in anaesthetized swine.

The amphiprotic character of azapropazone and its relevance to the gastric mucosa.

Since most non-steroidal anti-inflammatory drugs (NSAIDs) contain only one obvious ionisable group at physiological pH levels then they may be easily identified as having either acidic or basic character. Basic NSAIDs are simply non-acidic NSAIDs capable of accepting a proton within the physiological pH range. Within this range, however, a few NSAIDs contain two obvious ionisable groups, one acidic and the other basic. Such compounds should be described as amphiprotic, and include NSAIDs such as 4- and 5-amino substituted salicylic acids, niflumic acid, amfenac, WY 18251, and azapropazone. The aqueous ionisation equilibrium of such compounds is complex and is described by two macroscopic ionisation constants. Evidence has accumulated during the last decade to support the view that the pharmacokinetic behaviour of NSAIDs contributes not only decisively to their therapeutic effects but also to the type and incidence of their side-effects. A priori, using a physicochemical argument, certain amphiprotic NSAIDs should be better tolerated by the gastric mucosa than the classical acidic compound. Of those NSAIDs commercially available in the United Kingdom azapropazone remains the only one for which amphiprotic behaviour has been described. Following our examination of available data for azapropazone we conclude that the use of amphiprotic compounds represents a logical approach towards solving the problem of NSAID-induced gastric mucosal damage.

Comparative effects of azapropazone on cellular events at inflamed sites. Influence on joint pathology in arthritic rats, leucocyte superoxide and eicosanoid production, platelet aggregation, synthesis of cartilage proteoglycans, synovial production and actions of interleukin-1 in cartilage resorption correlated with drug uptake into cartilage in-vitro.

Azapropazone (APZ) has been compared with standard NSAIDs in title systems to establish aspects of its mode of action on cellular events at inflamed sites. APZ (150 mg kg-1 day-1) given for 10-13 days exhibited a reduction in joint pathology in established adjuvant arthritis in rats comparable with that of indomethacin (2 mg kg-1 day-1) and clobuzarit (20 mg kg-1 day-1). APZ was shown to be a potent inhibitor of the production of leucocyte superoxide and synovial interleukin-1 (IL-1)-like activity and stimulated articular cartilage proteoglycan synthesis, but was ineffective as an inhibitor of platelet aggregation or IL-1 induced cartilage degradation in-vitro. These in-vitro effects may have relevance to the mode of action of this weak inhibitor of prostaglandin synthesis.

In vitro inhibition of xanthine oxidase by azapropazone and 8-hydroxy-azapropazone.

It has been demonstrated that the nonsteroidal antiinflammatory agent azapropazone (Prolixan) as well as its principal 8-hydroxy-metabolite have distinct xanthine oxidase inhibitory activity. The pharmacological spectrum of this compound has thus shown an interesting extension.

Effect of azapropazone and allopurinol on myocardial infarct size in rats.

The effect of the xanthine oxidase inhibitor allopurinol and the non-steroidal antiinflammatory agent azapropazone on infarct size in rats, subjected to 48 h of occlusion of the left anterior descending coronary artery, were studied. Allopurinol (50 mg/kg i.p., twice daily from 24 h before to 48 h after LAD occlusion) and azapropazone (100 mg/kg i.p twice daily from 24 h before to 48 h after LAD occlusion) significantly reduced infarct size when compared to saline-treated rats. These data point towards involvement of xanthine oxidase derived free radicals in evolving myocardial infarction in rats; beneficial effect of azapropazone in this model may be related to the drug's ability to inhibit xanthine oxidase as well as various key neutrophil functions.

Inhibition of rat neutrophil functional responses by azapropazone, an anti-gout drug.

Azapropazone at concentrations of 0.1 to 1 mM inhibited by 30-70% rat neutrophil migration, aggregation, and degranulation in response to the synthetic chemotactic peptide fMet-Leu-Phe. Binding studies using fNle-Leu-[3H]Phe, a radiolabeled analog of fMet-Leu-Phe, showed that azapropazone did not inhibit these responses by interfering with fMet-Leu-Phe binding. Azapropazone also decreased both the apparent rate of production and maximal levels of superoxide anion (O2-) generated by cells stimulated with 100 ng/ml phorbol-12-myristate-13-acetate (PMA). The concentrations of azapropazone that inhibit these neutrophil responses in vitro approximate those previously found in vivo after administration of therapeutic doses of drug to rats or humans. Taken together, the data suggest that the efficacy of azapropazone in gouty arthritis may be partly due to its ability to inhibit key neutrophil functional responses in vivo.

A study of the potential interactions between azapropazone and frusemide in man.

Ten healthy individuals received frusemide 40 mg orally for 7 days. Following a drug free period of 7 days they received azapropazone 600 mg twice daily for 7 days and then both treatments for a further 7 days. Sodium excretion fell from 141 +/- 16.8 mmol/day to 84.3 +/- 6.8 mmol/day (P less than 0.01) on initiation of azapropazone treatment. The natriuretic response to frusemide was unchanged by premedication with azapropazone. Urate excretion rose from 3.35 +/- 0.249 mmol/day to 4.98 +/- 0.365 mmol/day on initiation of azapropazone therapy but subsequently returned to baseline values. Plasma uric acid fell from 0.289 +/- 0.024 mmol/l to 0.167 +/- 0.0125 mmol/l (P less than 0.001) on azapropazone but rose to 0.186 +/- 0.0116 mmol/l (P less than 0.001) with the addition of frusemide. Azapropazone may cause sodium retention but after repeated administration frusemide still has a marked diuretic action. The hypouricaemic effect of azapropazone is only slightly antagonised by frusemide at the doses studied.