Structural investigations of stereoselective profen binding by equine and leporine serum albumins.

Serum albumin, the most abundant transport protein of mammalian blood, interacts with various nonsteroidal anti-inflammatory drugs (NSAIDs) affecting their disposition, metabolism, and excretion. A big group of chiral NSAIDs transported by albumin, profens, is created by derivatives of 2-arylpropionic acid. The chiral center in the structures of profens is adjacent to the carboxylate moiety and often determines different pharmacological properties of profen enantiomers. This study describes crystal structures of two albumins, isolated from equine and leporine serum, in complexes with three profens: ibuprofen, ketoprofen, and suprofen. Based on three-dimensional structures, the stereoselectivity of albumin is discussed and referred to the previously published albumin complexes with drugs. Drug Site 2 (DS2) of albumin, the bulky hydrophobic pocket of subdomain IIIA with a patch of polar residues, preferentially binds (S)-enantiomers of all investigated profens. Almost identical binding mode of all these drugs clearly indicates the stereoselectivity of DS2 towards (S)-profens in different albumin species. Also, the affinity studies show that DS2 is the major site that presents high affinity towards investigated drugs. Additionally, crystallographic data reveal the secondary binding sites of ketoprofen in leporine serum albumin and ibuprofen in equine serum albumin, both overlapping with previously identified naproxen binding sites: the cleft formed between subdomains IIIA and IIIB close to the fatty acid binding site 5 and the niche created between subdomains IIA and IIIA, called fatty acid site 6.

Bioavailability and corneal anti-inflammatory effect of topical suprofen.

The bioavailability in rabbit cornea and aqueous humor of an ophthalmic formulation of suprofen, a nonsteroidal anti-inflammatory drug, was evaluated following topical administration of a single dose to the eye. The drug penetrated rapidly into the uninflamed cornea with intact epithelium; highest levels occurred during the first 30 to 45 min after instillation and decreased thereafter. The bioavailability of suprofen in cornea and aqueous humor following administration of a 1.0% concentration was twice that produced by a 0.5% concentration of the drug. Topical application of multiple doses of suprofen failed to suppress polymorphonuclear leukocyte invasion of the cornea if treatment was started after the induction of inflammation. Suprofen therapy initiated prior to the induction of corneal inflammation and maintained into the post-inflammation period did produce a significant (P less than 0.01) decrease in the numbers of PMNs that invaded the inflamed cornea. There was no significant difference (P greater than 0.05) in the corneal anti-inflammatory effect achieved by the 0.5% and 1.0% concentrations of suprofen when administered according to this regimen.

Suprofen. A review of its pharmacodynamic and pharmacokinetic properties, and analgesic efficacy.

Suprofen (sutoprofen) is a non-steroidal anti-inflammatory analgesic, closely related structurally to drugs such as ibuprofen, ketoprofen and naproxen. In patients with acute pain, single oral doses of suprofen are at least as effective as: usual therapeutic doses of aspirin; codeine alone or combined with aspirin; dextropropoxyphene alone or in various combinations; oxycodone combined with aspirin; dipyrone; pentazocine; paracetamol (acetaminophen); diflunisal; ibuprofen; indomethacin; or mefenamic acid. In chronic pain due to osteoarthritis, suprofen is as effective as usual dosages of aspirin or dextropropoxyphene during long term therapy, and as effective as diclofenac, ibuprofen, indomethacin and naproxen during short term treatment. As with other non-steroidal anti-inflammatory drugs, gastrointestinal complaints are the most frequently reported side effects, although discontinuation due to gastrointestinal effects may be necessary less frequently with suprofen than with aspirin, dextropropoxyphene or combinations of the two. Suprofen appears to be a useful alternative to mild analgesics, analgesic combinations or the older more established non-steroidal anti-inflammatory drugs in the treatment of patients with acute or chronic pain. However, further definition of its efficacy and tolerability is required, especially in comparison with newer non-steroidal anti-inflammatory analgesics.

Binding of suprofen to human serum albumin. Role of the suprofen carboxyl group.

The binding of suprofen (SP), a non-steroidal anti-inflammatory drug of the arylpropionic acid class, and its methyl ester derivative (SPM) to human serum albumin (HSA) was studied by dialysis and spectroscopic techniques. In spite of the remarkable differences in the physicochemical properties of SP and SPM, the binding of each molecule to HSA was quantitatively very similar. Thermodynamic analysis suggests that the interaction of SP with HSA may be caused by electrostatic as well as hydrophobic forces, whereas the interactions with SPM may be explained by hydrophobic and van der Waals forces. Similarities in the difference UV absorption spectra between ligand-detergent micelle and -HSA systems indicate that the SP and SPM molecules are inserted into a hydrophobic crevice on HSA. The same studies suggest that the carboxyl group of SP interacts with a cationic sub-site which is closely associated with the SP binding site. Proton relaxation rate measurements indicate that the thiophen ring and propanoate portion of the SP molecule is the major binding site for HSA. The locations of SP and SPM binding sites were identified by using fluorescence probes which bind to a known site on HSA. The displacement data implied that SP primarily binds to Site II, while the high affinity site of SPM as well as low affinity site of SP are at the warfarin binding site in the Site I area. From binding data with chemically modified HSA derivatives, it is likely that highly reactive tyrosine (Tyr) and lysine (Lys) residues, which may be Tyr-411 and Lys-195, are specifically involved in SP binding. In contrast, these two residues are clearly separated from the SPM binding site. The binding of SP and SPM is independent of conformational changes on HSA that accompany N-B transition. There is evidence that the carboxyl group may play a crucial role in the high affinity binding processes of SP to HSA.

Platelet-mediated vascular permeability in the rat: a predominant role for 5-hydroxytryptamine.

The intradermal injection in rat skin of washed, thrombin-activated platelets produces an increase in vascular permeability, the intensity of which increments with the platelet concentration. Pretreatment of the recipient animals with serotonergic antagonists, including the specific 5-HT2 receptor blocker ketanserin, potently inhibits the platelet-mediated and the 5-HT-induced vascular defect. Amine depletion of platelets or skin tissues with reserpine reduces the response to platelets. Platelet prostanoid and lipoxygenase derivatives play no major role in the vascular response to platelet. The permeability increase induced by exogenous 5-HT and by activated platelets is reduced by alpha 1-adrenergic stimulation with noradrenaline or phenylephrine and by beta 2-stimulation with terbutaline or isoprenaline, and is potentiated by adenosine; this points to a modulation of permeability by blood flow changes and to a direct beta-adrenergic effect at the endothelial cell membrane. This study demonstrates a predominant role for 5-HT in the platelet-mediated vascular permeability increase in a sensitive species like the rat.

Topically applied vitamin C and cysteine derivatives protect against UVA-induced photodegradation of suprofen in ex vivo pigskin.

Exposure of the nonsteroidal anti-inflammatory drug suprofen (SUP) to UV-radiation results in the formation of radicals, reactive oxygen species (ROS), photodecarboxylated products and photoadducts with biomacromolecules. Using an ex vivo pigskin explant model, we investigated whether topical coapplication of the water-soluble antioxidants vitamin C (Lascorbic acid, ASC), N-acetyl-L-cysteine (NAC) or L-cysteine ethylester (CYSET) with SUP reduced ultraviolet A (UVA)-induced decomposition of SUP. UVA-induced changes in antioxidant bioavailability in the stratum corneum and epidermis were also studied. Epidermal bioavailability of SUP in sham-irradiated pigskin increased 2.2- to 4.1-fold after the lowest antioxidant doses (P < 0.05). As compared with no applied antioxidant, increasing doses of all tested antioxidants resulted in increased levels of SUP and decreased levels of photoproducts (P < 0.05). A maximal protection against SUP photodegradation of 70% was found after an ASC dose of 1 micromol/cm2; these values were 60% for a NAC dose of 10 micromol/cm2 and 50% for a CYSET dose of 5 micromol/cm2. Skin antioxidant levels increased with increasing applied dose (P < 0.05); the bioavailability of CYSET was approximately three-fold lower than that of ASC and NAC. UVA exposure resulted in 30-50% consumption of the topically applied ASC or NAC in the stratum corneum, whereas CYSET was not consumed. In conclusion, the topically applied water-soluble antioxidants ASC, NAC and CYSET protect against UVA-induced decomposition of SUP by scavenging radicals and ROS. Coapplication of these antioxidants may therefore be an effective way to reduce or prevent the phototoxic effects of SUP in vivo.

Effect of nonenzymatic glycation of albumin and superoxide dismutase by glucuronic acid and suprofen acyl glucuronide on their functions in vitro.

Acyl glucuronides bind irreversibly to plasma proteins, and one mechanism proposed for this covalent binding is similar to that for glycation of protein by reducing sugars. Because glycation of protein by glucose and other reducing sugars can alter protein function, this lead to the hypothesis that the glycation of proteins by acyl glucuronides may cause similar effects. When human serum albumin (HSA) was incubated with 0.5 M glucose for 5 days, the unbound fractions of diazepam and warfarin were increased by 41 and 35%, respectively, less than that caused by glucuronic acid which increased the unbound fractions by 90% for diazepam and 420% for warfarin. When HSA was incubated with suprofen glucuronide (SG) at a much lower concentration of 0.005 M for only 24 h, the effects on the unbound fractions of diazepam and warfarin to HSA were altered dramatically with increases of 340 and 230%, respectively. After incubation of superoxide dismutase (SOD) with 0.5 or 1 M reducing sugars for 14 days, the enzyme activity decreased to 82 and 61% of initial levels at day 14, respectively, whereas glucuronic acid almost completely inactivated the enzyme activity over the same period. Even at a very low concentration (0.005 M) of SG, SOD activity was reduced significantly to 11% of initial levels by day 14, which was comparable to the effect by 0.5 and 1.0 M concentrations of glucuronic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix associated laser desorption/ionization time of flight mass spectrometry indicated that several equivalents of reducing sugars or SG became attached to albumin after incubation. These results suggest that acyl glucuronides may affect the function of proteins by the formation of glycated protein in vivo and may be associated with the toxicity of xenobiotics metabolized to labile acyl glucuronides.

Mechanisms of drug photobinding to proteins: photobinding of suprofen to human serum albumin.

Photobinding of drugs to biomolecules constitutes an important early event in the onset of photoallergy. In the present work, UV irradiation of human serum albumin in the presence of either suprofen (SUP) or its major photoproduct, decarboxylated suprofen (DSUP), has been studied as a model system for drug-photosensitised protein binding. Both dark binding and binding in the presence of light were investigated since this will affect the mode, site and mechanism of drug interaction with the protein. In order to determine the binding features of SUP to albumin, competitive binding experiments were carried out using fluorescent probes specific for site I and II. Suprofen was found to selectively dark bind to site II on HSA. Photobinding of DSUP to HSA was more efficient than SUP. Parallel to this, the intrinsic tryptophan fluorescence of HSA decreased when the protein was previously irradiated in the presence of the photoactive compounds, again being DSUP more efficient compared with SUP. As fluorescence quenching involves electron transfer from the excited Trp to the ground state DSUP, it follows that the photoactive compound binding to HSA must be on (or in close proximity to) site I Trp(214) residue. It appears that photobinding of SUP is largely preceded by its photodecomposition to DSUP which, in turn, associates and photobinds to HSA.

Photoreactivity of tiaprofenic acid and suprofen using pig skin as an ex vivo model.

The skin is repeatedly exposed to solar ultraviolet radiation. Photoreaction of drugs in the body may result in phototoxic or photoallergic side effects. Non-steroidal anti-inflammatory drugs, such as tiaprofenic acid (TPA) and the closely related isomer suprofen (SUP) are frequently associated with photosensitive disorders; they may mediate photosensitised damage to lipids, proteins and nucleic acids. Using ex vivo pig skin as a model, we investigated the photodegradation of TPA and SUP, and photobinding of these drugs to protein by means of HPLC analysis and drug-directed antibodies. Both with keratinocytes, which were first isolated from the pig skin and thereafter exposed to UVA and with keratinocytes which were isolated from pig skin after the skin was UVA exposed, time-dependent photodegradation of TPA and SUP was found, beside photoadduct formation to protein. The results of this work show that: (a) TPA and SUP were photodecomposed with similar efficiency; major photoproducts detected were decarboxytiaprofenic acid (DTPA) and decarboxysuprofen (DSUP), respectively. (b) Both drugs form photoadducts, as concluded from recognition by drug-specific antibodies. Pig skin appears to be a good model for studying the skin photosensitising potential of drugs.

Pharmacokinetics and bioavailability of suprofen injection solution after intravenous application versus capsules on six healthy male volunteers.

In a three-way cross-over study six healthy male volunteers received intravenously 50 mg and 100 mg suprofen as a bolus injection and orally 200 mg as one capsule. The plasma level curves could be fitted to a two-compartimental model by means of the computer program NONLIN. The pharmacokinetic parameter estimates did not show dose dependency. The absolute bioavailability of suprofen taken as one capsule was 92.2% based on AUC-values and did not indicate a first-pass effect. The plasma input curve was determined by point-area deconvolution. Assuming first order input a half-life of 23 min could be derived based on point-area deconvolution. Different dissolution models based on USP XX are discussed in relation to the kinetic data generated.

Suprofen kinetics in healthy male volunteers after intramuscular injection of increasing dosages.

Kinetics of alpha-methyl-4-(2-thienyl-carbonyl)-phenylacetic acid (suprofen, Suprol) a non-narcotic antiphlogistic analgesic, were studied in two clinical experiments. In a bioavailability study (study A) suprofen, given as intramuscular injection was absorbed more rapidly (mean peak plasma time 20.0 min) than from the capsule formulation (mean peak plasma time 57.5 min). With respect to the amount systemically absorbed, measured as area under the plasma concentration-time curve, both dosage forms were equivalent. Individual plasma concentration profiles (study B) were described by a 2-compartmental model with a mean first order absorption rate of 0.095 min-1, a rapid mean disposition rate alpha of 0.025 min-1 and an apparent terminal plasma elimination rate beta of 0.004 min-1. There was dose-independent kinetics and a linear correlation between peak plasma concentration versus dose and area under the curve (AUC) versus dose after intramuscular injection of suprofen up to 200 mg, leading to dose proportional bioavailability data.

Stereoselective metabolism of anti-inflammatory 2-arylpropionates.

2-Arylpropionic acids (profens) are a major group of non-steroidal anti-inflammatory drugs. These compounds exist in two enantiomeric forms due to the presence of an asymmetric carbon atom alpha to the carbonyl function. In vitro tests have shown that the anti-prostaglandin synthetase activity of profens resides almost exclusively in the (+)-(S)-enantiomers, yet all profens except naproxen are marketed as racemates. The profens exhibit enantioselective pharmacokinetics, the most intriguing aspect of which is their unidirectional chiral inversion from the (-)-(R)- to the (+)-(S)-enantiomer. Since the transformation goes from the inactive to the pharmacologically active form, the (R)-enantiomer can be considered as a prodrug of its (S)-antipode. The available evidence suggests that this reaction proceeds via the formation of the acyl-CoA thioester of the 2-arylpropionates. Hutt and Caldwell have reviewed the literature describing this unusual metabolic process. The purpose of this paper is to present more recent findings from in vivo and in vitro studies and to summarize actual knowledge concerning the mechanism of the metabolic chiral inversion of profens.

Further structural analysis of urinary metabolites of suprofen in the rat.

The urinary metabolites of 2-(4-(2-thienylcarbonyl)phenyl)propionic acid (suprofen, S) in rats were analyzed by radio-GC, GC/MS, or 1H NMR technique. Radio-GC analysis of trimethylsilylated materials after TLC separation of intact urine showed the presence of three radioactive peaks with the retention times corresponding to the authentic S, 2-(4-(2-thienylhydroxymethyl)phenyl)propionic acid, and 2-(4-carboxyphenyl)propionic acid. About 40% of the total radioactivity appearing in the 0-24-hr urine was accounted for by the three metabolites and their conjugates. The identification of these metabolites was confirmed by comparison of the MS spectra of urine, in which rats were administered an equimolar mixture of S and S[phenyl-d4], with those of synthetic standards. The labile metabolites of S, corresponding to about 32% of the total radioactivity appearing in the 0-24-hr urine, were isolated and purified by ether extraction from the fresh urine and GC/MS or HPLC. GC/MS of the methylated metabolite revealed the consistent presence of the ion peaks at m/z 304, 245, 217, and 141, indicative of a dimethylated product with monohydroxy group on the thiophene ring. Analysis of the 1H NMR spectrum demonstrated the metabolite to be 2-(4-(5-hydroxy-2-thienylcarbonyl)phenyl)propionic acid.

Absorption and excretion of suprofen in rats.

DL-2-(4-(2-Thienylcarbonyl)phenyl)propionic acid (suprofen, S) was rapidly absorbed in rats after oral administration. Blood levels after a single oral dose of 2, 10, 50, or 100 mg/kg of 3H-S reached maxima within 30 min and were dose-dependent. The major portion of the drug was shown to be absorbed from the upper part of the small intestine and a portion from the stomach. The radioactivity in rat plasma was extensively bound to the plasma protein in vivo; this was found to be unchanged S and four metabolites. Elimination of S and its metabolites from blood was rapid; 3H was mostly excreted in the urine and feces within 24 hr after oral administration of 3H-S. No significant amounts of 14CO2 were excreted in expired air after administration of 14C-S. Rat urine contained S and four metabolites found in rat plasma, accounting for about 60% of the urinary radioactivity. After rats with biliary fistulas were given an oral dose of 2 mg/kg of 3H-S, 41% of the dose was excreted in the bile during 48 hr; there was significant enterohepatic circulation. When single or 21 consecutive daily doses of 3H-S were administered to rats, the blood levels after the multiple doses were higher than those after a single dose but no significant difference was found in excretion of 3H.

Disposition of suprofen in rats following intravenous injection.

Distribution and excretion of DL-2-(4-(2-thienylcarbonyl)phenyl)propionic acid (suprofen) were evaluated in male and female rats following intravenous (i.v.) injection of labeled suprofen. The radiometric findings following i.v. administration of 2 mg/kg of 3H-suprofen to male and female rats showed similar patterns of blood level and excretion of the radioactivity. Elimination of 3H-suprofen from the blood was rapid; most of the radioactivity was excreted in the urine and a portion in the feces within 24 h after injection. After rats with biliary fistulas were given an i.v. dose of 2 mg/kg of 3H-suprofen, approximately half of the dose was excreted in the bile during 48 h. The only tissues with concentrations higher than that in the plasma were those involved in metabolism and excretion (liver and kidney); other tissue levels were all very low and there was no evidence of accumulation of drug-related material in any tissue.

Metabolic chiral inversion of anti-inflammatory 2-arylpropionates: lack of reaction in liver homogenates, and study of methine proton acidity.

1. Enrichment in the (S)-enantiomers for (R)-flurbiprofen, (R)-naproxen, (R)-suprofen and (R;S)-ibuprofen was investigated in various subcellular hepatic preparations containing coenzyme A. While such preparations were able to form hippuric acid from benzoic acid, the chiral inversion was never seen. 2. Using 2-dimethylaminoethanethiol 2-phenylpropionate (DEPP) as a model acyl thioester, the acidity of the methine proton was investigated by monitoring the proton/deuterium exchange occurring in deuterated solvents using high-resolution n.m.r. The compound was inert up to 22 h in D2O at 37 degrees C and pD 7.4. In pure methanol or a methanol-water mixture, only solvolysis was seen. In contrast, competitive hydrolysis (k = 0.005 h-1) and proton/deuterium exchange (k = 0.09 h-1) were seen in a CD3CN/D2O (50:50) mixture at 37 degrees C. 3. It is speculated that the failure to characterize chiral inversion of 2-arylpropionates in subcellular preparations may be due to the absence of a microenvironment of adequately moderate polarity.

Covalent binding of suprofen acyl glucuronide to albumin in vitro.

1. Suprofen acyl glucuronide, a major metabolite of suprofen in man, is labile, undergoing acyl migration to isomeric conjugates which are not cleaved by beta-glucuronidase. 2. The pH-dependent degradation of diastereomeric suprofen glucuronides in aqueous buffer increases rapidly near physiological pH with an apparent first-order half life of 1.4 h at pH 7.4. 3. Suprofen glucuronide and its isomeric conjugates are reactive with albumin in a pH-dependent manner which corresponds to the stability of the acyl glucuronide. Several per cent of the conjugates added to albumin in solution become covalently bound. 4. The covalent binding of suprofen equivalents to albumin is greatly enhanced by the addition of either cyanide or cyanoborohydride anion, which supports the presence of an imine in the process of binding. Release of isomeric conjugates by treatment of the albumin adduct with dilute acid also supports covalent binding via an imine. 5. The covalent binding of suprofen to proteins through its reactive acyl glucuronide may be of toxicological importance and relevant to the acute renal toxicity observed for suprofen in man.

Suprofen sustained release kinetics in healthy male volunteers. 1st communication: a single dose open crossover bioavailability study of suprofen sustained release tablets versus capsules.

An open crossover study was performed in 12 healthy male volunteers to compare the bioavailability of alpha-methyl-4-(2-thienyl-carbonyl)phenylacetic acid (suprofen, Suprol) 600 mg sustained release tablets versus the suprofen capsule (2 X 200 mg). The pharmacokinetic profiles in plasma and urine were determined by a HPLC assay. In the dose range studied, the two suprofen formulations were not associated with any clinically significant effects on the blood pressure, heart rate or ECG. The results of the physical and neurological examinations showed no abnormal results. The results of haematology, clinical chemistry and urinalysis also showed no significant modifications. However, increased serum creatinine values were observed in some volunteers following suprofen administration. A drug relationship to this finding cannot be excluded with certainty. Two volunteers complained of nausea and vomiting following administration of two suprofen capsules. For this reason, volunteer no. 9 was withdrawn by the investigator from the study and replaced by an eligible substitute. In general, single doses of the two suprofen formulations investigated, were subjectively and objectively well tolerated. From the suprofen plasma-concentration time profiles, it was apparent that, whilst the elimination of suprofen was similar for both formulations, there was a marked delay in absorption of the tablet formulation. This formulation resulted in statistically significantly later maximum plasma levels and longer mean residence time (p less than 0.001). In comparison to the reference capsule formulation, the tablet had statistically significantly lower (75%) bioavailability. Measurement of suprofen concentrations in the urine indicated that less than 1% of the administered dose was excreted by this route.

Introduction to the pharmacology of suprofen.

A number of peripherally acting analgesics have become available in the past two decades for the treatment of mild to moderate pain of diverse etiology. The action of these agents is based on their putative ability to inhibit prostaglandin biosynthesis and attenuate hyperalgesia. Suprofen is a new and potent peripherally acting analgesic with anti-inflammatory and antipyretic properties which appears to inhibit prostaglandin synthetase in a tissue-selective manner. In preclinical studies, suprofen was shown to possess a wide separation of analgesic activity from gastrointestinal irritation. Suprofen appears to exert its pharmacological effect by inhibiting synthesis of prostaglandins from precursor arachidonic acid, inhibiting the pain induced by bradykinin, and by raising the threshold to pain induced by prostaglandins. The onset of action is rapid with peak analgesic effect reached in 1-2 h. Preclinical data suggest that suprofen may be an extremely valuable and versatile analgesic for the clinical management of pain.