A screen of mammalian antibodies on snapper (Pagrus auratus, Sparidae) peripheral blood leukocytes reveals cross reactivity of an anti-human CD3 antibody with a population of mIg(-) cells.

Detailed immunological studies of the teleosts have been hampered by a lack of antibodies against cell-specific markers. Furthermore, where antibodies have been raised, in many instances they have been found to be species-specific. In comparison, many monoclonal and polyclonal antibodies exist with specificities for mammalian proteins and glycoproteins that effectively differentiate leukocyte sub-populations. In this study, we have tested a panel of 54 commercial antibodies against human and murine cell surface receptors for their ability to bind leukocytes isolated from the peripheral blood of snapper (Pagrus auratus). From this panel, one antibody, A452, which is specific for the intracytoplasmic tail of the epsilon (epsilon) chain of the T cell receptor-associated CD3 complex (CD3epsilon) bound to a subpopulation of peripheral blood leukocytes. Mutually exclusive counterstaining was observed when this antibody was used in conjunction with a monoclonal anti-snapper immunoglobulin antibody. This suggests that A452 may be binding to putative snapper T cells.

Chirality and nonsteroidal anti-inflammatory drugs.

The nonsteroidal anti-inflammatory drugs (NSAIDs) are of significant clinical importance and include congeners of many chemical classes, some of which incorporate an asymmetric or chiral carbon atom. With very few exceptions, chiral NSAIDs have been marketed for clinical use as racemates. However, it is apparent that differences, sometimes major, exist between enantiomers in terms of their pharmacological and toxicological properties. With regard to the ability of chiral NSAIDs to inhibit cyclo-oxygenase, their chief mechanism of action, major or exclusive activity is confined to enantiomers of the S-stereoconfiguration. Accordingly, it is questionable whether the R-antipodes should be included in the final drug product for use in clinic. In addition to differences between enantiomers in terms of their pharmacodynamic properties, pharmacokinetic differences are possible for chiral NSAID isomers, and these may modulate preexisting enantioselectivities at the site of action of such compounds. As a consequence, a considerably simpler pharmacological profile is likely to result from the use of single enantiomers versus racemic mixtures.

Stereoselective interactions of ketoprofen glucuronides with human plasma protein and serum albumin.

A clearance pathway common to many aryl alkanoic acids is the generation of renally eliminated ester glucuronides. These metabolites are susceptible to systemic hydrolysis which generates the parent aglycone. We have conducted in vitro studies with biosynthetic R- and S-ketoprofen glucuronides to elucidate the mechanism of this phenomenon. These conjugates were incubated in human plasma, various concentrations of human serum albumin (HSA) and protein-free buffer. It was apparent that albumin, rather than plasma esterases, catalysed the hydrolysis of the glucuronides. The albumin-catalysed hydrolysis of ketoprofen glucuronides was highly stereoselective. The mean (+/- SD) hydrolysis half-life of R-ketoprofen glucuronide in plasma (N = 4) at physiological pH and temperature was 1.37 (+/- 0.30) hr. The corresponding value for S-ketoprofen glucuronide, 3.46 (+/- 0.84) hr, was significantly different (P less than 0.005). In contrast, synthetic ethyl esters of R- and S-ketoprofen were hydrolysed by plasma esterases, but not by HSA, and with little stereoselectivity. The reversible protein binding of ketoprofen glucuronides was determined at physiological pH and temperature by a rapid ultra-filtration method. The binding of R- and S-ketoprofen glucuronide to human plasma protein was independent of concentration (P greater than 0.05) over the range of 1-20 micrograms/mL. The mean (+/- SD) percentage unbound in plasma (N = 4) of R-ketoprofen glucuronide was 12.6 (+/- 1.4)%. The corresponding value for S-ketoprofen glucuronide, 9.12 (+/- 0.54)%, was significantly different (P less than 0.005). S-Ketoprofen glucuronide was also more avidly protein bound in physiological concentrations of HSA. However, this stereoselectivity decreased in more dilute HSA solutions. Based on the hydrolysis and protein binding data for ketoprofen glucuronides, we propose the existence of separate binding and catalytic sites on the albumin molecule for these metabolites.

Synthesis of tritium-labeled hydroxytyrosol, a phenolic compound found in olive Oil.

(3,4-Dihydroxyphenyl)ethanol, commonly known as hydroxytyrosol (1), is the major phenolic antioxidant compound in olive oil, and it contributes to the beneficial properties of olive oil. Bioavailability and metabolism studies of this compound are extremely limited, in part, related to unavailability of radiolabeled compound. Studies with radiolabeled compounds enable use of sensitive radiometric analytical methods as well as aiding elucidation of metabolic and elimination pathways. In the present study a route for the formation of hydroxytyrosol (1), by reduction of the corresponding acid 2 with tetrabutylammonium boronate, was found. Methods for the incorporation of a tritium label in 1 were investigated and successfully accomplished. Tritiated hydroxytyrosol (1t) was synthesized with a specific activity of 66 Ci/mol. The stability of unlabeled and labeled hydroxytyrosol was also investigated.

Inactivation of cytochrome P450 by the food-derived complex phenol oleuropein.

Complex phenols, such as the glycoside oleuropein and hydroxytyrosol, are found in high concentrations in the typical components of the Mediterranean diet. We have previously reported that oleuropein inhibits androstenedione 6 beta-hydroxylase activity, a CYP3A marker in human liver microsomes (Stupans, I., Stretch, G., Hayball, P., 2000. Olive oil phenols inhibit human hepatic microsomal activity. Journal of Nutrition. 130 2367-2370). Oleuropein, but not the structurally similar compounds hydroxytyrosol and secologanin, was found to be a mechanism-based inhibitor of androstenedione 6 beta-hydroxylase activity. Preincubation with 100 microM oleuropein and NADPH resulted in a significantly lower androstenedione 6 beta-hydroxylase activity when compared to preincubation carried out with oleuropein without NADPH, 0.11+/-0.01 nmol/mg microsomal protein/min compared with 0.29+/-0.03 nmol/mg microsomal protein/min (P<0.05). The inactivation exhibited pseudo-first-order kinetics. The maximal rate constant for inactivation (k(inactivation)) was calculated to be 0.09 min(-1) and the concentration of inactivator required for half maximal inactivation (Ki) was calculated to be 22.2 microM. Oleuropein was found to be a relatively weak inhibitor of CYP1A2-mediated 7-methoxyresorufin-O-deethylation (24% inhibition at 100 microM oleuropein), but not CYP2E1-mediated chlorzoxazone 6-hydroxylation. CYP1A2 did not undergo mechanism-based inactivation by oleuropein.

The processing of exogenous cholesterol in the alveolar compartment of the rat lung.

We have examined the esterification of [3H]cholesterol following the intratracheal instillation of a tracer amount into the isolated rat lung perfused with Krebs bicarbonate containing 4.5% albumin. At 5, 30 and 60 min after instillation, lungs were lavaged at 2 degrees C with 3 x 10 ml of 0.15 M NaCl, each volume instilled and withdrawn three times. Each lung was lavaged at only one time point. The saline recovered was centrifuged at 150 g (5 min) to sediment the macrophage-rich fraction, leaving the surfactant in the supernatant. The amounts and specific activity of cholesterol and cholesteryl ester were measured following isolation by high performance liquid chromatography of the free cholesterol and the hydrolyzed ester-derived cholesterol. There was a rapid fall in [3H]cholesterol in the surfactant fraction, accompanied by a reciprocal increase in [3H]cholesteryl ester in the macrophage-rich fraction, while the level of free [3H]cholesterol in that fraction remained very low. These data are consistent with exogenous cholesterol being rapidly esterified in the alveolus, and the ester then being cleared by the macrophages. We were unable to locate the actual site of esterification.

Effect of ketoprofen and its enantiomers on the renal disposition of methotrexate in the isolated perfused rat kidney.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit the renal tubular secretion of methotrexate. However, the relative contribution of the active S- and inactive R-enantiomers is unknown. This study examined the effect of racemic ketoprofen and its enantiomers on the renal disposition of methotrexate in the isolated perfused rat kidney (IPK). Nineteen kidneys were divided between a control and three treatment groups. Controls were perfused with methotrexate alone (25 micrograms mL-1, n = 5) over three 30-min periods. Treatment groups were perfused with methotrexate (25 micrograms m-1) for the first period, followed by a second period of methotrexate (25 micrograms mL-1) plus R- (n = 5), S- (n = 5) or RS-ketoprofen (n = 4) at 25 micrograms mL-1, and a third period of methotrexate (25 micrograms mL-1) plus R-, S- or RS-ketoprofen (50 micrograms mL-1). Perfusate and urine were collected over 10-min intervals. Methotrexate was measured by HPLC and its binding in perfusate by ultrafiltration. The clearance ratio (CR) for methotrexate was obtained by dividing the renal clearance by the product of its fraction unbound and the glomerular filtration rate. During control experiments, there was no significant change in the CR over 90 min. R-, S- and RS-ketoprofen at 50 micrograms mL-1 reduced the CR of methotrexate significantly, but there was no difference between the three groups. While the enantiomers of ketoprofen reduced the renal excretion of methotrexate, the interaction was not enantioselective.

A comparative analysis of the loss of amiodarone from small and large volume PVC and non-PVC infusion systems.

The time-course of the adsorption of amiodarone hydrochloride onto flexible polyvinyl chloride (PVC) infusion bags and administration sets has been studied under ambient conditions similar to the intensive and coronary care units at the Repatriation General Hospital--Daw Park. When admixtures containing 900-1000 mg amiodarone hydrochloride in 500 ml PVC infusion bags containing glucose 5% were assayed, a minimal (2.7%) loss of amiodarone was detected at 24 hours compared with glass infusion bottles. There was no loss apparent when 100 ml PVC infusion bags were used compared with 100 ml glass bottles. When PVC administration sets were attached to 500 ml glass infusion bottles, maximal adsorption losses of up to 4.9% were observed in the effluent within one hour of the commencement of the simulated infusion. In Australia, the current recommendations by the distributor of amiodarone, Reckitt & Colman Pharmaceuticals, advise that the administration of amiodarone should be via glass infusion bottles or rigid PVC plastic containers without plasticisers together with non-PVC administration lines. These results do not support this recommendation.

Enantioselective disposition of 2-arylpropionic acid nonsteroidal anti-inflammatory drugs. III. Fenoprofen disposition.

The disposition of fenoprofen enantiomers has been studied in nine healthy rabbits. A mean (S.E.M.) of 0.73 (0.07) of R-fenoprofen was inverted to S-fenoprofen and the distribution volumes for bound plus unbound R-fenoprofen and S-fenoprofen were 50.3 (4.5) and 98.5 (5.9) ml/kg, respectively. A model was developed which predicted the area under the S-fenoprofen plasma concentration-time curve after bolus administration of racemic fenoprofen. The mean (S.E.M.) predicted area, 2.1 (0.2) mg X min/ml/kg, was within 94% of the observed area 2.2 (0.2) mg X min/ml/kg. The effect of phenobarbital on the disposition of fenoprofen enantiomers was examined in an additional eight rabbits. During the control study the glucuronidation of R-fenoprofen exceeded the corresponding clearance term for the S-enantiomer by 2.1-fold. The clearances of individual enantiomers to their respective glucuronides increased after phenobarbital pretreatment by a mean 1.6-fold for R- and 2.3-fold for S-fenoprofen. The clearance of S-fenoprofen by processes other than glucuronidation and elimination of unchanged drug in urine was increased by a mean of 2.1-fold after phenobarbital pretreatment but the fractional inversion and the inversion clearance of R- to S-fenoprofen were not affected. These data indicate that on racemic fenoprofen administration the area under the curve for the pharmacologically active S-enantiomer would be reduced by phenobarbital pretreatment.

Influence of octanoic acid on the reversible protein binding of ketorolac enantiomers to human serum albumin (HSA): comparative liquid chromatographic studies using a HSA chiral stationary phase.

The retention of ketorolac enantiomers on a human serum albumin (HSA)-based HPLC chiral stationary phase (CSP) was investigated to assess the utility of immobilized protein for probing the binding of (R)- and (S)-ketorolac to native HSA. Results from the chromatographic study were compared with enantiomorph binding data obtained from HSA ultrafiltration experiments conducted both in the presence and absence of the medium chain-length fatty acid octanoic acid. Without octanoic acid in the mobile phase containing 10% propan-2-ol in 20 mM phosphate buffer at pH 6.5, racemic ketorolac was stereochemically resolved with the HSA-CSP with large enantiomeric capacity factors [106.2 and 28.7 for (R)- and (S)-ketorolac, respectively]. The inclusion of octanoic acid in the column eluent reduced the capacity factors of both isomers consistent with displacement of drug from HSA binding sites. A reduction in the capacity factor ratio [(R):(S)] was observed as the octanoate concentration increased from 0.5 to 4.0 mM. The percentage unbound of (R)- and (S)-ketorolac present separately (2.0 micrograms/ml) in 40.0 mg/ml HSA solution (22 degrees C and pH 7.4) was 0.245% and 0.643%, respectively, and both values increased as a function of increasing octanoate concentration in the HSA solution. A biphasic effect of octanoate on the percentage unbound ratio of (S):(R) was observed. In light of these findings, it would appear that silica-immobilized HSA is capable of qualitatively probing the enantioselective binding of ketorolac to HSA and moreover, more than one specific ketorolac binding site may exist on the HSA molecule.

Formulation of 1-alpha,25-dihydroxycholecalciferol topical ointment for psoriasis treatment.

A means of accurate and rapid formulation of 1-Alpha,25-dihydroxycholecalciferol topical ointment is described. The accuracy of the formulation technique is within 95% of the theoretical ointment concentration (lug/gram of petrolatum base). Moreover, the stability of the formulated ointment is examined at room temperature (21 degrees C) and refrigerated (2 degrees C) over 40 days storage. When protected from light the loss of potency is 6% at 21 degrees C and negligible at 2 degrees C.

Enantioselective pharmacodynamics of the nonsteroidal antiinflammatory drug ketoprofen: in vitro inhibition of human platelet cyclooxygenase activity.

The pharmacological activity of ketoprofen enantiomers was investigated in humans by an in vitro method. The antiplatelet effect of ketoprofen was assessed by measuring the inhibition of platelet thromboxane B2 (TXB2) generation during the controlled clotting of whole blood obtained from each of four healthy volunteers. Ketoprofen was added separately to whole blood as a range of concentrations of (1) predominantly (S)-ketoprofen, (2) racemic ketoprofen, and (3) predominantly (R)-ketoprofen. (S)-Ketoprofen was found to be solely active at inhibiting human platelet TXB2 production; (R)-ketoprofen was devoid of such activity and did not modify the potency of its optical antipode. A relationship between the percentage inhibition of TXB2 generation and the unbound concentration of (S)-ketoprofen in serum was modelled according to a sigmoidal Emax equation. The mean (+/- SD) serum unbound concentration of (S)-ketoprofen required to inhibit platelet TXB2 generation by 50% (EC50) was 0.320 (+/- 0.062) ng/ml. This value for ketoprofen is considerably lower than previously reported values for (S)-ibuprofen and (S)-naproxen.

Oleuropein, an antioxidant polyphenol from olive oil, is poorly absorbed from isolated perfused rat intestine.

Epidemiological studies have shown that the incidence of heart disease and certain cancers is lower in the Mediterranean region. This has been attributed to the high consumption of olive oil in the Mediterranean diet, which contains polyphenolic compounds with antioxidant activity. Although many in vitro studies have been performed to elucidate mechanisms by which these compounds may act, there are virtually no data relating to their fate after ingestion. Therefore, we decided to investigate the intestinal absorption of one of the major olive oil polyphenolics, oleuropein. To do this, a novel in situ intestinal perfusion technique was developed, and the absorption of oleuropein was studied under both iso-osmotic and hypotonic luminal conditions. Oleuropein was absorbed, with an apparent permeability coefficient (P:(app)) of 1.47 +/- 0.13 x 10(-6) cm/s (+/-SE) observed under iso-osmotic conditions. The mechanism of absorption is unclear but may involve transcellular transport (SGLT1) or paracellular movement. Under hypotonic conditions, the permeability of oleuropein was significantly greater (5.92 +/- 0.49 x 10(-6) cm/s, P: < 0.001). This increase is thought to be due to an increase in paracellular movement facilitated by the opening of paracellular junctions in response to hypotonicity. Overall, we determined that the olive oil polyphenolic oleuropein can be absorbed, albeit poorly, from isolated perfused rat intestine. Therefore, it is possible that it or its metabolites may confer a positive health benefit after the consumption of olive oil, most likely via an antioxidant mechanism.

Stereoselective analysis of ketorolac in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) analytical method is described for the quantification of the (R)- and (S)-enantiomers of ketorolac when present together in human plasma. The method involves derivatization with thionyl chloride/(S)-1-phenylethylamine and subsequent reversed-phase chromatography of the diastereomeric (S)-1-phenylethylamides of (R)- and (S)-ketorolac. The method is suitable for the analysis of large numbers of plasma samples and has been applied in this report to a pharmacokinetic study of ketorolac enantiomers upon intramuscular administration of racemic drug to a human subject. The limit of quantification for each enantiomer of ketorolac is 50 ng/ml (signal-to-noise ratio > 10).

Enantiospecific analysis of ketoprofen in plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) assay for the determination of the R- and S-enantiomers of ketoprofen is described. Facile ketoprofen extraction from plasma and derivatization to the diastereomeric S-1-phenylethylamides was followed by normal-phase HPLC. The ketoprofen diastereomeric amides eluted within 8 min. The limit of quantification of the assay was 0.15 mg/l of each enantiomer (signal-to-noise ratio = 5).

The pharmacokinetics of ketorolac enantiomers following intramuscular administration of the racemate.

A single dose of racemic ketorolac (30 mg of tromethamine salt, Toradol) was administered by bolus intramuscular injection to four young, healthy volunteers. The concentrations of total (bound plus unbound) (R)- and (S)-ketorolac were measured in plasma for 9 h after dosing. The mean +/- s.d. clearance of (S)-ketorolac (45.9 +/- 10.1 ml h-1 kg-1) exceeded (P = 0.0032) that of the (R)-enantiomer (19.0 +/- 5.0 ml h-1 kg-1). The mean +/- s.d. AUC ratio for (S)-ketorolac:(R)-ketorolac (0.442 +/- 0.043) was significantly different from unity (P = 0.0001). The steady-state volume of distribution of (S)-ketorolac (0.135 +/- 0.022 l kg-1) was significantly different (P = 0.0013) from that of its optical antipode (0.075 +/- 0.014 l kg-1) and the half-lives of (S)- and (R)-ketorolac (2.35 +/- 0.23 h and 3.62 +/- 0.79 h, respectively) were also significantly different (P = 0.026). These data indicate that the disposition of ketorolac in man is subject to marked enantioselectivity and, because of possible differences in biological activity of (S)- and (R)-ketorolac, emphasize the need to monitor separate stereoisomer concentrations of the drug if pharmacological data are to be interpreted correctly.

The efficacy of a commercial beta-glucan preparation, EcoActiva, on stimulating respiratory burst activity of head-kidney macrophages from pink snapper (Pagrus auratus), Sparidae.

This study investigated the in vitro effects of a commercial beta-glucan preparation, EcoActiva, on the respiratory burst activity of head-kidney macrophages isolated from pink snapper (Pagrus auratus), a marine fish cultured in Australia. Macrophages incubated with EcoActiva displayed morphological characteristics of activation, and were stimulated to produce superoxide. Pre-incubation with low levels of EcoActiva significantly increased the response to phorbol myristate acetate (PMA) and lipopolysaccharide (LPS), indicating that EcoActiva could prime these macrophages. Co-culturing macrophages with both LPS and PMA, or EcoActiva and PMA, increased burst activity compared with the response to PMA alone, however, this increase was additive and not synergistic. These results suggest that EcoActiva is able to stimulate non-specific immunity in snapper through increased respiratory burst activity of macrophages, an important component of the host defence network.

The in vivo fate of hydroxytyrosol and tyrosol, antioxidant phenolic constituents of olive oil, after intravenous and oral dosing of labeled compounds to rats.

In vitro studies have shown phenolics in olive oil to be strong radical scavengers. The absorption and elimination of two radiolabeled phenolic constituents of olive oil, hydroxytyrosol and tyrosol were studied in vivo using rats. Compounds were administered intravenously (in saline) and orally (in oil- and water-based solutions). For both compounds, the intravenously and orally administered oil-based dosings resulted in significantly greater elimination of the phenolics in urine within 24 h than the oral, aqueous dosing method. There was no significant difference in the amount of phenolic compounds eliminated in urine between the intravenous dosing method and the oral oil-based dosing method for either tyrosol or hydroxytyrosol. Oral bioavailability estimates of hydroxytyrosol when administered in an olive oil solution and when dosed as an aqueous solution were 99% and 75%, respectively. Oral bioavailability estimates of tyrosol, when orally administered in an olive oil solution and when dosed as an aqueous solution were 98% and 71%, respectively. This is the first study that has used a radiolabeled compound to study the in vivo biological fates of hydroxytyrosol and tyrosol.

Enantiospecific high-performance liquid chromatographic analysis of 2-phenylpropionic acid, ketoprofen and fenoprofen.

A high-performance liquid chromatographic method has been developed for the quantitation of the R- and S-enantiomers of 2-phenylpropionic acid, ketoprofen and fenoprofen. The assay consists of extracting the arylpropionic acid with an internal standard and measuring the total (R + S) concentration of enantiomers by reversed-phase chromatography, derivatising the chromatographic fraction corresponding to the enantiomers to form R- and S, R-2-phenylethylamide distereoisomers which are resolved by normal-phase chromatography in order to calculate the fraction of each enantiomer. The limits of sensitivity of the assay for 2-phenylpropionic acid, ketoprofen and fenoprofen are 6, 0.2 and 2.5 mg/l, respectively.

Marked enantioselective protein binding in humans of ketorolac in vitro: elucidation of enantiomer unbound fractions following facile synthesis and direct chiral HPLC resolution of tritium-labelled ketorolac.

The protein binding of the enantiomers of the nonopiate analgesic, ketorolac, was investigated in vitro using human plasma and solutions of human serum albumin (HSA) at physiological pH and temperature. In order to detect the very low levels of unbound enantiomers in protein solutions, tritium-labelled rac-ketorolac was synthesised by regiospecific isotopic exchange of the parent drug with tritiated water as the isotope donor. Radiochemical purification of this compound by reversed-phase HPLC followed by direct resolution using a chiral alpha 1-acid glycoprotein (Chiral-AGP) HPLC column afforded labelled enantiomers of high specific activity. The in vitro use of (R)- and (S)-[3H4]ketorolac enabled reproducible radiometric detection of enantiomers in protein solution ultrafiltrate. The unbound fractions of (R)- and (S)-ketorolac [fu(R) and fu(S), respectively] were determined when drug was added to various plasma or albumin solutions as either the separate enantiomers or as the racemate. Over an enantiomeric plasma concentration range of 2.0-15.0 micrograms/ml, fu(S) (mean range: 1.572-1.795%) was more than 2-fold greater (P < 0.001) than fu(R) (mean range: 0.565-0.674%). Both fu(R) and fu(S) were constant over this concentration range, and each was unaffected by the presence of the corresponding antipode (P > 0.05). At a concentration of 2.0 micrograms/ml in 40.0 g/liter fatty acid-free HSA, fu(R) and fu(S) were approximately 0.5 and 1.1%, respectively, and both values declined with increasing concentrations of the long chain fatty acid, oleic acid.(ABSTRACT TRUNCATED AT 250 WORDS)