Distribution of extracellular matrix proteins in indomethacin-induced lesions in the rat stomach.

INTRODUCTION: We investigated the distribution of extracellular matrix (ECM) proteins in indomethacin-induced lesions of the rat stomach. METHOD: Twenty rats received indomethacin orally at a dose of 8 mg/kg/body weight. The animals were killed at 3, 6, 12, 24, and 48 h after administration of the drug. The stomachs were removed and frozen in liquid nitrogen. Cryostat serial sections of the lesions were immunostained with antibodies to collagen III, IV, and VI, laminin, and fibronectin. RESULTS: Fibronectin was the dominant extracellular protein of the provisional ECM in deep gastric lesions and gastric ulcers. Collagen III was strongly positive in stromal cells under the necrotic material in gastric erosions. Basal membrane proteins (collagen IV and laminin) were found to originate from the muscularis mucosae at the ulcer edge. CONCLUSION: There is a typical distribution of ECM proteins in erosions and ulcers of the rat stomach. Fibronectin was most prominent in the provisional matrix of gastric erosions and ulcers.

Stereoselectivity of biliary excretion of 2-arylpropionates in rats.

To examine the stereoselectivity of biliary excretion, the optically pure enanatiomers of ketoprofen (KT), ibuprofen (IBU), and flurbiprofen (FLU) were intravenously administered to normal and bile duct-cannulated rats at 10 mg/kg. The recovery of total KT in bile was significantly higher after administration of (S)-KT than after (R)-KT [90.1 +/- 3.5% vs 68.8 +/- 8.2%, n = 3, P < 0.05]. In normal rats the terminal half-life of (R)-KT was significantly shorter than that of (S)-KT after administration of (R)-KT (2.2 +/- 0.6 h vs 14.3 +/- 4.9 h, n = 3, P < 0.05). The terminal half-life of both enantiomers was significantly shorter in rats with continuous bile drainage as compared to normal rats. No significant differences in pharmacokinetic parameters could be found between both enantiomers in bile duct-cannulated animals. The total amount of IBU in bile was slightly higher after administration of (S)-IBU than after (R)-IBU administration. The percentage of (R)-IBU after (R)-IBU administration, however, was very low [(R)-IBU: 1.5 +/- 0.9%, (S)-IBU: 23.4 +/- 5.8%]. In normal rats the clearance of (R)-IBU was significantly higher as compared to (S)-IBU. Differences in pharmacokinetic parameters between normal and bile duct-cannulated rats were not statistically significant due to high interindividual variability. The total recovery of FLU, which was excreted in bile to a lower extent than either KT or IBU, also tended to be greater after S-enantiomer administration. Only small amounts of (S)-FLU could be recovered in bile after (R)-FLU administration.(ABSTRACT TRUNCATED AT 250 WORDS)

R-flurbiprofen: isomeric ballast or active entity of the racemic compound?

The enantioselective pharmacokinetic behaviour of the flurbiprofen enantiomers was investigated following administration of optically pure R- or S-flurbiprofen to various species. Only negligible inversion (< 5%) of flurbiprofen occurred in the rat and in man. Consequently, pharmacodynamic experiments evaluating pain and inflammation as parameters have been carried out enantioselectively for both flurbiprofen enantiomers in the rat. R-flurbiprofen, which is not an inhibitor of prostaglandin synthesis in vitro, had only marginal anti-inflammatory effects as defined by the carrageenan edema of the rat paw in contrast to the S-enantiomer. In contrast, to S-flurbiprofen, R-flurbiprofen caused only marginal mucosal damage in the GI-tract. Both enantiomers, however, were of similar potency as antinociceptive drugs in the rat Randall-Selitto assay following the injection of interleukin-1 or baker's yeast. Using the pure enantiomers of flurbiprofen it appears possible to establish a more specific drug treatment: the R-enantiomer in occasional pain, the S-enantiomer in rheumatic disorders.

Variability of inversion of (R)-flurbiprofen in different species.

The anti-inflammatory activity of 2-arylpropionic acids like flurbiprofen appears to be due to the S enantiomers only. A unique characteristic of the metabolism of this class of drugs is inversion of configuration. The present study examines whether chiral inversion occurs after administration of the optically pure flurbiprofen enantiomers to various species (i.e., dogs, guinea pigs, rats, and gerbils). Concentrations of the enantiomers in plasma were analyzed by a stereoselective high-performance liquid chromatographic assay with a chiral alpha 1-acid glycoprotein column. Pharmacokinetic parameters of the flurbiprofen enantiomers were evaluated with a two-compartment computer model. Inversion of (R)-flurbiprofen to its optical antipode occurred to a variable extent in the dog [fraction inverted (Fi) = 0.39; n = 3] and the guinea pig (Fi = 1.00; n = 3) and to a much lower extent in the rat (Fi = 0.02; n = 3) and the gerbil (Fi = 0.05; n = 3). After intravenous administration of (S)-flurbiprofen to dogs, guinea pigs, rats, and gerbils, (R)-flurbiprofen was not detected in plasma (limit of quantification was 0.05 microgram/100 microL of plasma).

Aspirin-like drugs may block pain independently of prostaglandin synthesis inhibition.

Using flurbiprofen, a chiral anti-inflammatory and analgesic 2-arylpropionic acid derivative, the enantiomers of which are not converted to each other (less than 5%) in rats or man, we obtained evidence that prostaglandin synthesis inhibition is primarily mediating the anti-inflammatory activity but prostaglandin synthesis independent mechanisms contribute to the analgesic effects. Thus, the S-form inhibited prostaglandin synthesis, inflammation and nociception in rats. The R-form had much less effect on prostaglandin synthesis and did not affect inflammation. It did, however, block nociception in rats almost as potently as the S-form. S-flurbiprofen, in contrast to the R-form, was clearly ulcerogenic in the gastrointestinal mucosa. These results indicate additional molecular mechanisms of analgesia and suggest the use of R-arylpropionic acids as analgesics.

Pharmacokinetics of ibuprofen enantiomers in dogs.

Inversion of inactive (R)-ibuprofen to active (S)-ibuprofen has been suggested to occur presystemically only. In order to investigate the site of inversion in dogs we administered both enantiomers either intravenously or intraduodenally (10 mg/kg) to adult, male beagle dogs (n = 3) in a crossover design. Plasma, urine, and bile were collected for up to 6 h and analyzed stereospecifically by HPLC, according to a previously published method. Pharmacokinetic parameters were calculated using a linear computer program. Absorption after intraduodenal administration occurred rapidly, resulting in maximum plasma concentrations 0.2 h after giving the enantiomer. Approximately 70% of the (R)-enantiomer (according to AUC) was inverted to the S-enantiomer independent of route of administration. No R-ibuprofen could be detected in plasma after (S)-ibuprofen administration. Mean residence time was found to be 2 to 3 times longer for (S)- than for (R)-ibuprofen. Total systemic clearance from plasma was twice as high for (R)- than for (S)-ibuprofen. There were no differences between plasma clearances after intravenous and intraduodenal administration. Between 8 and 17% of dose was recovered in bile [especially as free and conjugated (S)-ibuprofen] and 3-12% in urine [as (S)-ibuprofen, hydroxy- and carboxyibuprofen, free and conjugated forms]. Small amounts of (R)-ibuprofen were detected in bile after intraduodenal administration of (R)-ibuprofen only (1.8% of dose). In short, the unidirectional inversion of R-ibuprofen appears to occur systemically rather than presystemically in dogs.

Towards safer nonsteroidal anti-inflammatory drugs.

More than one hundred years ago salicylic acid and its salts were introduced into the therapy of rheumatic diseases. Ninety years ago aspirin was discovered, and within the last forty years phenylbutazone, indomethacin, ibuprofen, the oxicams and many others were discovered. All of these drugs are acidic. They inhibit the prostaglandin synthetase, combine analgesic and anti-inflammatory activity and show side-effects mainly in the GI-tract, liver, bone-marrow, and kidney. Within the last twenty years, however, distinct relationships between effects and side-effects could be shown: 1. Rapid absorption beginning in the stomach goes along with intensive gastric-duodenal irritation and ulceration. 2. A high degree of enterohepatic circulation appears to be associated with ileal and jejunal ulcerations and perforations. 3. Intensive hepatic metabolisation may be related to enhanced hepatic damage. 4. Intensive intrarenal circulation of the active moiety may be related to kidney damage. These observations indicate that certain pharmacokinetic characteristics of distinct nonsteroidal anti-inflammatory drugs (NSAIDs) are responsible, at least in part, for well-known side-effects. It is obvious that modifying the pharmacokinetics of the active principle may reduce specific types of side-effects. The clinical success of these attempts are limited but altogether promising.

Diagnosis of megaloblastic anemia.

Megaloblastic anemia can be due to cobalamin deficiency, folate deficiency, or refractory forms of bone marrow disease. This essay reviews current thinking on the diagnostic procedures available to a physician considering these disorders. The questions to be answered are as follows: Is a megaloblastic anemia present? Is there a deficiency of cobalamin or folate? If a deficiency is present, what is its cause? Various diagnostic tests are discussed with regard to the differences in their sensitivity and metabolic implications. In particular, we consider the newest diagnostic tests for cobalamin deficiency, serum homocysteine, and methylmalonate, which appear to be highly sensitive predictors of clinical morbidity. Application of these tests suggests that many more patients are cobalamin-deficient than had been supposed.

S-ibuprofen versus ibuprofen-racemate. A randomized double-blind study in patients with rheumatoid arthritis.

Ibuprofen (ibu) is a racemic 2-arylpropionic acid non-steroidal anti-inflammatory drug whose activity is due mainly to the S-enantiomer. So far only the racemic compound is in clinical use. A double-blind randomized trial was carried out for a 2-week period in 50 patients with classical rheumatoid arthritis (RA) (Steinbrocker II-III) to compare the effectiveness and tolerance of S-ibu (400 mg T.I.D.) with that of the racemic compound (600 mg T.I.D.). Ritchie-index, limitation of movement, joint pain on pressure and pain at night decreased significantly in both groups. Due to lack of effectiveness, the dose had to be increased in 3 patients from the S-ibu group as well as in 6 patients from the racemic group resulting in mean daily doses of 1220 mg S-ibuprofen and 1870 mg racemic ibu. No statistically significant difference could be found between both groups concerning efficacy and unwanted effects. Therefore, S-ibu given alone may be advantageous because the metabolic load to the human body is reduced and patients are more likely to comply with drug doses of 1.2 g/day as compared to 1.8 g/day.

Effects of sodium salicylate on elimination kinetics of indomethacin and bile production in dogs.

We investigated the effects of sodium salicylate on the elimination kinetics of indomethacin in bile duct-cannulated beagles. Indomethacin and metabolites were quantified by HPLC in plasma, bile, and urine. Indomethacin was administered as iv bolus injection and iv infusion to yield a steady-state plasma concentration of approximately 1 microgram/ml. Following sodium salicylate, given either iv (25 mg/kg) or via duodenal fistula (50 mg/kg), the indomethacin plasma level dropped instantaneously by 60-70%. Concomitantly, total systemic clearance from the plasma and biliary clearance were increased significantly. In addition, a reduced plasma protein binding of indomethacin and a significant increase in the volume of distribution were observed. The amount of indomethacin, excreted as free and conjugated drug in bile, was significantly increased temporarily by sodium salicylate. The total amount eliminated in bile (approximately 70% of the dose), however, was not changed by sodium salicylate co-administration. The bile flow was significantly enhanced for at least 4 hr. Both phase 1 metabolism and renal excretion of indomethacin remained practically unaffected by sodium salicylate treatment.

Cobalamin as coenzyme: a twisting trail of research.

Cobalamin derivatives serve as coenzymes for the body's two cobalamin-dependent enzymes--adenosylcobalamin-dependent methylmalonyl CoA mutase, and methylcobalamin-dependent methionine methyltransferase. This essay reviews, in brief form and in personal terms, the history, beginning in the mid-1950s, of how these enzymes and coenzymes were discovered and what has been learned of their reaction mechanisms. It is clear that because of the fragility of the unique carbon-cobalt bond in cobalamin coenzymes, they serve primarily as free radical formers. This accounts for their efficiency in abstracting hydrogen from substrate molecules and for a subsequent chain of events that results in the isomerization of methylmalonyl CoA, the transfer of methyl groups, and (in certain bacteria) the reduction of ribonucleotides. Some thoughts are offered on the possible evolutionary significance of these facts.

The biliary elimination and enterohepatic circulation of ibuprofen in rats.

The biliary and urinary excretion of ibuprofen and its metabolites were determined in rats after intravenous and peroral administration of 25 and 100 mg/kg of the drug. Within 24 hours 48% of the low i.v. dose and 59% of the high i.v. dose were eliminated via bile as ibuprofen and its metabolites. Following oral administration 40 to 41% of the dose were recovered in bile, whereas 16 to 32% of the dose were eliminated in urine, resulting in an overall drug recovery of 66 to 79% within 24 hours. Upon infusion of bile containing ibuprofen and its metabolites into the duodenum substantial enterohepatic cycling of the drug occurred in the rat.

Gastrointestinal ulcerations induced by anti-inflammatory drugs in rats. Physicochemical and biochemical factors involved.

Aspirin, diclofenac, diflunisal, ibuprofen and indomethacin were given orally or intravenously to fasted or fed rats. The resulting gastric and intestinal damage was assessed using standard methods. The same drugs were administered to rats with biliary fistulas, and the fraction of drug excreted in bile was quantified using HPLC methods. We found that gastric damage occurred only in the fasted animals and was found to be dose-dependent and related to the amount (r = 0.871) and solubility (r = 0.909) of the individual drug. As far as acute gastric toxicity is concerned, neither the potency of a drug as an inhibitor of cyclo-oxygenase nor the fraction of unchanged or conjugated agent excreted in bile appeared to be relevant. Secondly, ulcerations of the small intestine occurred in fed animals only. The degree of damage was related to the amount of unchanged or conjugated drug excreted in bile and cyclo-oxygenase inhibitory potency (r = 0.873). The administered dose (within the range investigated) and drug solubility appeared not to contribute to intestinal toxicity. It is concluded that, in the rat, acute gastric and intestinal toxicity of non-steroidal anti-inflammatory drugs are due to different mechanisms. Whereas gastric toxicity is strongly influenced by the amount of drug dissolved under the pH conditions in the stomach, intestinal toxicity appears to depend on biliary excretion and enterohepatic circulation of a drug as well as on its potency as an inhibitor of prostaglandin synthesis.

Cobalamin analogues in plasma. An in vitro phenomenon?

In the course of investigations on the source of cobalamin analogues in human plasma six kits for measuring serum cobalamins were studies to determine if analogues can arise in vitro. High pressure liquid chromatography (HPLC) were used to estimate tracer stability during extraction procedures. Results showed that up to 60 percent of the radioactive cyanocobalamin was degraded. Alkaline pH increases the degradation; this effect is further intensified by addition of dithiothreitol. Implications for the cobalamin assay is discussed. Determination of cobalamin analogues by the difference of concentrations determined from assays using haptocorrin ("total corrinoids") and instrinsic factor ("true cobalamins") as binders may be misleading because the affinity of possible analogues for the assay binder is unknown. Thus quantification of the analogues could be erroneous. The need for definitive assay methods for cobalamin and its analogues is stressed.

Coenzyme function of cobalamin analogues from calf kidney.

Calf kidney has been used as a tissue source for the isolation of cobalamin analogues, which are defined here as cobalt-containing compounds of distinctive chromatographic behavior that are extractable from tissues by methods conventionally used to extract cobalamin and which, in radioisotope dilution assays, are more active with R-protein as binder than intrinsic factor and are relatively less active in microbiologic assays. Preparatory methods employed reverse affinity chromatography or a series of chemical extractions for the isolation of corrin followed by Dowex-50 chromatography. An analogue-containing fraction (peak 2) was eluted by acetate buffers between pH 4 and 5. This material was shown to contain cobalt, to migrate differently than the four cobalamins in Dowex-50 and paper chromatography, and to display a pattern of properties that is compatible with the above definition of cobalamin analogues. These analogues stimulated crude preparations of N5-methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13) from Escherichia coli and rat liver at far lower concentrations (1-40 nmol/L) than the major cobalamins. No evidence of enzymatic conversion of cobalamin to analogue could be demonstrated.