The site of reduction of sulphinpyrazone in the rabbit

1. Comparison of oral and i.v. administration of sulphinpyrazone (10 mg/kg) to rabbits showed that the oral route was associated with an incomplete bioavailability and a six-fold greater formation of the active sulphide metabolite.

2. The bile was an important route of elimination of unchanged sulphinpyrazone in rabbits (18% of an i.v. dose in four hours). Only small amounts of the sulphide appeared in the bile after i.v. administration.

3. Pretreatment with oral antibiotics decreased the area under the plasma concentration-time curve (AUC) for the sulphide but increased that of the parent drug. Excretion of the p-hydroxysulphide metabolite in urine was decreased 30-fold by antibiotic treatment.

4. The contents of the caecum showed the greatest capacity for sulphinpyrazone reduction in vitro. The liver possessed a slight ability to reduce sulphinpyrazone in vitro under anaerobic, but not aerobic, conditions.

5. The gut bacteria are the main site of reduction of sulphinpyrazone to the active sulphide metabolite in the rabbit.

6. These findings contrast with those obtained for sulindac which was reduced extensively under both aerobic and anaerobic conditions by rabbit-liver soluble fraction in vitro. The sulphide metabolites of both sulphinpyrazone and sulindac were oxidized to the parent drug by rabbit-liver microsomes.     

Side effects occurring during administration of epoprostenol (prostacyclin, PGI2), in man.

1 High pressure liquid chromatographic assays for the estimation of sulphinpyrazone and its sulphide, sulphone and p-hydroxy metabolites in plasma and urine are described. 2 Five normal volunteers received 200 mg and 400 mg sulphinpyrazone orally. Sulphinpyrazone was rapidly absorbed and eliminated with a half-life of approximately 4 h irrespective of dose. Peak plasma concentrations and area under the plasma concentration-time curves (AUC) were consistent with linear pharmacokinetic behaviour. 3 Plasma concentrations of the sulphone were low and peaked before those of the sulphide; its mean half-life was 3.1 h. The sulphide, which may be the sulphinpyrazone metabolite with activity on platelets, was eliminated with a mean half-life of 13.4 h. The AUC increases with dose of both metabolites suggested non-linearity. 4 Approximately 45-50% of the administered dose was eliminated in the urine as unchanged drug or as sulphone or p-hydroxy-sulphinpyrazone. The sulphide metabolite was not detected in the urine. The renal clearance of sulphinpyrazone was approximately 18 ml min-1 and that for the sulphone was similar. Sigma minus plots of the urinary excretion yielded half-lives of 3.5 h for the sulphone and 1 h for p-hydroxy-sulphinpyrazone.     

Sulphinpyrazone metabolism during long-term therapy.

1 The plasma concentrations of sulphinpyrazone and four of its metabolites are reported together with the amounts excreted in urine. Eight insulin-requiring diabetics were investigated, all treated with sulphinpyrazone 600-800 mg day-1 for 2.5 years or more. 2 Blood samples were drawn before the first morning dose and 2 h later. The mean plasma concentrations were (t=0 h-t=2 h): sulphinpyrazone 7.1-16.0 microgram ml-1; sulphone 1.7-4.8 microgram ml-1; p-OH-sulphide 0.67-0.89 microgram ml-1; p-OH-sulphinpyrazone 0.10-0.16 microgram ml-1. Statistically significant correlations were found between the plasma concentrations at t=0 of the sulphide and the p-OH-sulphide and that of sulphinpyrazone. 3 In urine, a very wide range in excretion of unconjugated compounds was observed. Sulphinpyrazone were excreted in amounts corresponding to 1-30% of the daily dose. The metabolites were generally excreted to amounts corresponding to less than 1% of the daily dose; however, up to 3% was found as the sulphone. 4 Increases of the concentration of all compounds in urine were found after treatment with beta-glucuronidase indicating 0-conjugation with glucuronic acid. 5 Since both the sulphide and the sulphone were found more active as inhibitors of platelet function in vitro than their parent compound, they may together constitute the major part of the platelet inhibitory drug activity in plasma during long-term therapy with sulphinpyrazone.     

The reduction of sulphinpyrazone and sulindac by intestinal bacteria

1. Incubation of human or rabbit faeces with sulphinpyrazone gave greater reduction under anaerobic than under aerobic conditions. Reduction of sulindac by human faeces was more extensive than that of sulphinpyrazone.

2. Growth of mixed cultures of intestinal bacteria in nutrient media containing antibiotics produced a marked inhibition in their ability to reduce sulphinpyrazone. Sulphide formation was inhibited by metronidazole and lincomycin for human faeces and by tetracycline for rabbit faeces/caecal contents.

3. The formation of the sulphides of sulindac and sulphinpyrazone ex vivo was decreased in faeces from patients treated with metronidazole. Metronidazole, but not tetracycline, decreased the extent of reduction of sulphinpyrazone by rabbits in vivo. No reduction of either substrate occurred on incubation with ileostomy effluent. These data indicate that anaerobic intestinal bacteria are important in the reduction of these sulphoxide-containing drugs.

4. However, when incubated anaerobically with over 200 strains of bacteria isolated from human faeces, sulphinpyrazone was reduced by most of the aerobic but not the anaerobic organisms. Sulindac was reduced more extensively by the same aerobes and by some anaerobes.

5. The discrepancy between the apparent importance of anaerobes in vivo and in vitro may be due to their very large number present in the hind gut and to the production of an anaerobic environment suitable for the enzymic activity of other organisms, such as aerobes or facultative anaerobes.     

Effects of ischaemic heart disease, Crohn's disease and antimicrobial therapy on the pharmacokinetics of sulphinpyrazone

The renewed interest in sulphinpyrazone in recent years has arisen from its potential to inhibit platelet aggregation. In vivo much of the activity is probably due to the thioether or sulphide metabolite which has a greater potency and a longer half-life than the parent compound. The sulphide metabolite is formed exclusively by the gut microflora in man. The pharmacokinetics of sulphinpyrazone (200 mg orally) have been studied, with particular attention to the formation of the sulphide metabolite, in groups of patients who might be expected to show abnormal formation of this active metabolite due to altered delivery of the drug to the lower gut or altered gut flora. Five patients studied 1 month after a myocardial infarction did not differ markedly from young, normal volunteers with respect to either sulphinpyrazone or its metabolite. Crohn's disease in the quiescent phase did not significantly alter the pharmacokinetics or metabolism of the drug, but 1 patient who had undergone a hemicolectomy formed negligible concentrations of the active metabolite. Antimicrobial therapy produced highly variable results with almost complete suppression of sulphide formation in some subjects but no apparent effect in others.     

Renal clearance of sulphinpyrazone in man

Summary Six healthy young volunteers received a single dose of sulphinpyrazone 200 mg p.o. Plasma concentration and urinary excretion rate curves showed large intersubject variation for sulphinpyrazone and its metabolites. The sulphide metabolite could only be detected in plasma and not before 3–7 h after ingestion. The total recovery in urine of all compounds varied from 30–56% of the dose.In two subjects the mean residence time of sulphinpyrazone was twice as long as in the other subjects (10.4 h compared with 4.6 h), but the area under the plasma concentration-time curve was comparable to that in the others (mean: 3.0 mg·ml^–1·min), indicating that drug absorption was quantitatively similar but delayed.The renal clearance of sulphinpyrazone varied from 14–40 ml·min^–1 (mean: 28 ml·min^–1).In view of the very high plasma protein binding of sulphinpyrazone, active tubular secretion is the predominant mechanism in its renal clearance. The same holds for the sulphone metabolite, which has a mean renal clearance of 24 ml·min^–1, and even more for the p-hydroxysulphinpyrazone metabolite, which has a renal clearance of 118 ml·min^–1.No unambiguous evidence was found in favour of concentration-dependent renal clearance of sulphinpyrazone or its metabolites over the concentration range studied. The renal clearance, especially of sulphinpyrazone, appeared to be dependent on urine pH and not on urine flow rate.     

Hydrogen sulphide protects against NSAID-enteropathy through modulation of bile and the microbiota

Background and Purpose

Hydrogen sulphide is an important mediator of gastrointestinal mucosal defence. The use of non-steroidal anti-inflammatory drugs (NSAIDs) is significantly limited by their toxicity in the gastrointestinal tract. Particularly concerning is the lack of effective preventative or curative treatments for NSAID-induced intestinal damage and bleeding. We evaluated the ability of a hydrogen sulphide donor to protect against NSAID-induced enteropathy.

Experimental Approach

Intestinal ulceration and bleeding were induced in Wistar rats by oral administration of naproxen. The effects of suppression of endogenous hydrogen sulphide synthesis or administration of a hydrogen sulphide donor (diallyl disulphide) on naproxen-induced enteropathy was examined. Effects of diallyl disulphide on small intestinal inflammation and intestinal microbiota were also assessed. Bile collected after in vivo naproxen and diallyl disulphide administration was evaluated for cytotoxicity in vitro using cultured intestinal epithelial cells.

Key Results

Suppression of endogenous hydrogen sulphide synthesis by β-cyano-L-alanine exacerbated naproxen-induced enteropathy. Diallyl disulphide co-administration dose-dependently reduced the severity of naproxen-induced small intestinal damage, inflammation and bleeding. Diallyl disulphide administration attenuated naproxen-induced increases in the cytotoxicity of bile on cultured enterocytes, and prevented or reversed naproxen-induced changes in the intestinal microbiota.

Conclusions and Implications

Hydrogen sulphide protects against NSAID-enteropathy in rats, in part reducing the cytotoxicity of bile and preventing NSAID-induced dysbiosis.     

The effects of a synthetic diet on the pharmacokinetics of ethyl methyl sulphide and its sulphoxide and sulphone metabolites in rats.

Ethyl methyl sulphide (EMS) is a simple dialkyl sulphide, which occurs naturally and forms part structures of more complex drug molecules. EMS is oxidized to the corresponding sulphoxide (EMSO) and sulphone (EMSO2) derivatives both in vitro and in vivo. Two distinct enzymatic pathways appear to be involved in this sulphoxidation process; the flavin-containing monooxygenase (FMO) is largely responsible for the S-oxidation of EMS to its sulphoxide while both cytochrome P450 and FMO are involved in the further oxidation of the sulphoxide to the sulphone. The pharmacokinetics of EMS and its sulphoxide and sulphone metabolites were examined in male wistar rats placed on normal rat chow and those placed on a synthetic diet. Blood levels of EMS were analysed by a sensitive headspace gas chromatographic assay. A separate gas chromatographic assay was developed to monitor the blood levels of EMSO and EMSO2. The pharmacokinetics of EMS in control rats were linear from 10 to 40 mg/kg dose range. The blood concentration-time profile of EMS declined monoexponentially. EMS was rapidly eliminated from rat blood with a terminal half-life of 0.14 h and was not dytectable 1 h after administration. Following intravenous administration of EMSO (5 mg/kg), the blood concentration-time profile of EMSO declined with a terminal half-life (t 1/2) of 1.46 h, about ten times longer than that of the parent sulphide. After administration of EMSO2 (15 mg/kg), the sulphone was metabolically stable and was eliminated very slowly from the blood. The in vivo disposition of EMS and EMSO were clearly altered in rats maintained on a synthetic diet following administration of EMS or EMSO. The pharmacokinetic data were consistent with a diminished drug oxidising capacity in rats placed on the synthetic diet and could serve as a useful probe for monitoring the regulation of FMO in animals.     

Sulindac oxidation/reduction by microbial cultures; microbial models of mammalian metabolism

1. The oxidation and reduction of the sulphoxide moiety of the anti-inflammatory agent sulindac was infestigated to explore microbial systems exhibiting parallels of known mammalian metabolism.

2. Of 24 cultures initially screened, four catalysed the expected reactions in analytical studies. Arthrobacter species (ATCC 19140) and Sporobolomyces pararoseus (ATCC 11386) produced sulindac sulphide, Aspergillus alliaceus (NRRL 315) produced sulindac sulphone, and Nocardia corallina (ATCC 19070) produced both the sulphide and sulphone.

3. Preparative-scale production and full structural elucidation of metabolites was accomplished for sulindac sulphide with Arthrobacter species, and sulindac sulphone with A. alliaceus and N. corallina.

4. N. corallina also exhibited an aeration-dependent, reversible reduction of sulindac to the sulphide, and further oxidation to the sulphone. This organism thus parallels the composite of major phase-1 redox transformations of this drug observed in mammals.     

Hepatic metabolism of diallyl disulphide in rat and man

1.?The metabolism of diallyl disulphide was investigated in vitro with rat and human liver cell subfractions and ex vivo with an isolated perfused rat liver.

2.?Diallyl disulphide was oxidized to diallylthiosulphinate by rat liver microsomes with an apparent K_m = 0.86 ± 0.1?mM and an apparent V_max = 0.47 ± 0.12 nmol?min^?1?mg^?1 protein (mean ± SE). Both cytochrome P450 (CYP) and flavin-containing monooxygenases were involved, with CYP2B1/2 and CYP2E1 being the most active CYP enzymes.

3.?In rat and man, microsomal oxidation of allylmethyl sulphide to allylmethyl sulphoxide and allylmethyl sulphone also occurred, although at a low rate. Diallyl disulphide was also metabolized to allylglutathione sulphide and allylmercaptan. In addition, diallylthiosulphinate reacted non-enzymatically with glutathione to form allylglutathione sulphide.

4.?When an isolated rat liver was perfused with diallyl disulphide, the metabolites allyl mercaptan, allylmethyl sulphide, allylmethyl sulphoxide, allylmethyl sulphone and allylglutathione sulphide were detected primarily within the liver tissue, with only small amounts of metabolites found in the bile and perfusion medium. The pharmacokinetic parameters for diallyl disulphide were t_1/2 = 6.09?min; AUC_0–∞ = 4.77?min?mmol?l^?1; clearance = 34.22 ml?min^?1.

5.?A scheme for the metabolism of diallyl disulphide in rat and man is proposed.     

Comparative enantioselectivity in the sulphoxidation of albendazole in man, dogs and rats

1. H.p.l.c. analyses were performed to investigate the plasma kinetics of albendazole (ABZ), the sulphoxide (SO.ABZ) and sulphone (SO2ABZ) metabolites, as well as the chirality vs time of SO.ABZ, after oral administration to rats, dogs and man of prochiral sulphide antiparasitic drug ABZ. 2. In all three species the initial plasma concentration ratio of the enantiomers, as soon as SO.ABZ could be detected in plasma, was that of a racemate. 3. Subsequently, the ratio (+)/(-) increased linearly with time, reaching values of 13.1 and 9.3 in man and dogs, respectively, while it decreased to 0.6 in rats. 4. The (+) enantiomer represents 80%, 70% and 41% of the area under the curve of the total SO.ABZ in man, dogs and rats, respectively.     

Metabolism of trifluoperazine, fluphenazine, prochlorperazine and perphenazine in rats: in vitro and urinary metabolites

In vitro and in vivo metabolites of trifluoperazine, fluphenazine, prochlorperazine and perphenazine were isolated by solvent extraction and thin layer chromatography and quantified by u.v. spectroscopy. In liver microsomes from male rats all four drugs underwent N-dealkylation. N-oxidation, sulfoxidation and aromatic hydroxylation. The relative rates of these reactions depended on the substrate concentration, N-oxidation being favoured at higher concentrations. N-Demethylation of trifluoperazine proceeded faster than removal of the hydroxyethyl group from fluphenazine which led to the same metabolite N[γ-(2-trifluoromethyl-phenothiazinyl-10)-propyl] piperazine. The same applied to the dealkylation of prochlorperazine and perphenazine. Following oral administration of 10 mg/kg of the drugs, male rats excreted 1.8?4 per cent of the dose within the first 12 hr in urine in the form of the sulfoxide and the N-dealkylated sulfoxide. In vivo, too, the N-hydroxyethyl group was removed to a smaller extent than the N-methyl group. N-Oxides were not detected in urine at this dose level, but when 25 or 50 mg/kg prochlorperazine were administered, rats excreted small amounts of the N-oxide.     

Species differences in the disposition and metabolism of sulfinpyrazone

1. The disposition and metabolism of sulfinpyrazone have been studied in rats, guineapigs, rabbits, dogs, rhesus monkeys and miniature swine after intravenous administration of 100mg/kg of ¹⁴C-labelled drug.

2. In all species, the integrated plasma concentration (AUC, 0-24h) of total radioactivity was almost completely covered by the sum of the AUC-values of unchanged sulfinpyrazone and six metabolites, i.e. the sulphide, the sulphone, p-hydroxy-sulfinpyrazone, the p-hydroxy-sulphide, the p-hydroxy-sulphone and 4-hydroxy-sulfinpyrazone.

3. Comparison of the plasma level profiles of unchanged sulfinpyrazone and the metabolites revealed pronounced differences between the species. Unchanged sulfinpyrazone was the most prominent compound in plasma of rats, dogs, monkeys and swine, whereas the sulphide metabolite predominated in guinea-pigs. In plasma of rabbits, these two compounds were found in similar amounts.

4. Species with predominant renal excretion of the ¹⁴C dose, i.e. rabbits, dogs and monkeys, eliminated sulfinpyrazone to a high extent unchanged. The renal excretion of the sulphide metabolite was low in all species.

5. Species differences in the biotransformation of sulfinpyrazone explain previously observed differences in inhibitory effect on platelet aggregation. This effect is intensive and long-lasting in species showing high plasma concentrations of the sulphide metabolite.     

Role of the intestinal microflora in clonazepam metabolism in the rat

1. The metabolism of clonazepam was studied in vitro and in vivo using germ-free and ex-germ-free rats.

2. Incubation of clonazepam with rat-intestinal lumen contents gave nearly complete reduction of clonazepam to 7-aminoclonazepam. Rat-hepatic microsomes also reduced clonazepam but only under anaerobic conditions. Aerobic microsomal incubations gave 3-hydroxyclonazepam as the predominant metabolite. Both aerobic and anaerobic microsomal metabolism were induced by phenobarbital. Carbamazepine pretreatment significantly induced only 3-hydroxylation slightly; whereas β-naphthoflavone had no significant effect.

3. Extensive biliary disposition of hydroxylated clonazepam metabolites into the gut occurred. Only very low levels of clonazepam were found in bile. Using a linked-rat procedure enterohepatic recirculation of biliary metabolites was demonstrated and suppression (antibiotic treatment) or absence (germ-free) of the gut microflora nearly eliminated recycling.

4. Following oral administration of [¹⁴C]clonazepam to germ-free rats, reduced metabolites accounted for 15% of the radioactivity in the urine, with over 70% of the ¹⁴C attributed to a phenolic clonazepam metabolite. In contrast 77% of the recovered metabolites were derived from nitroreduction in the same animals following acquisition of an intestinal microflora; 7-acetamidoclonazepam was the major metabolite in these ex-germ-free animals. These studues show that clonazepam metabolism is primarily reductive in the presence of gut flora and oxidative in its absence.     

Single and multiple dose pharmacokinetics of lansoprazole in elderly subjects

Plasma concentrations of lansoprazole and of its sulphone, sulphide and 5-hydroxylated metabolites were determined after oral administration of a single 30 mg dose and after 7 days of treatment with a daily 30 mg dose in 12 elderly subjects (mean age 83 years). Results after a single dose were compared with those from a historical control group of 18 young subjects (mean age 23 years). Mean values of AUC after single dose were 2668 ng ml(-1) h in the young subjects and 5216 ng ml(-1) h in the elderly (P < 0.05). Mean t 1/2z values in young and elderly subjects were 1.4 h and 2.9 h, respectively (P < 0.001). Plasma concentrations of the metabolites were similar in both groups. However, the hydroxylated metabolite of the sulphone was detected only in elderly subjects. Steady state plasma concentrations of lansoprazole were reached after 3 days of dosing with lansoprazole. The accumulation ratio was 1.31 in the elderly subjects.     

Design and in vitro/in vivo Evaluation of Polyelectrolyte Complex Nanoparticles Filled in Enteric-Coated Capsules for Oral Delivery of Insulin

Insulin is one of the most important drugs in the treatment of diabetes. There is an increasing interest in the oral administration of insulin as it mimics the physiological pathway and potentially reduces the side effects associated with subcutaneous injection. Therefore, insulin-loaded polyelectrolyte complex (PEC) nanoparticles were prepared by the ionic cross-linking method using protamine sulfate as the polycationic and sodium alginate as the anionic polymer. Taguchi experimental design was used for the optimization of nanoparticles by varying the concentration of sodium alginate, the mass ratio of sodium alginate to protamine, and the amount of insulin. The optimized nanoparticle formulation was used for further in vitro characterization. Then, insulin-loaded PEC nanoparticles were placed in hard gelatin capsules and the capsules were enteric-coated by Eudragit L100-55 (PEC-eCAPs). Hypoglycemic effects PEC-eCAPs were determined in vivo by oral administration to diabetic rats. Furthermore, in vivo distribution of PEC nanoparticles was evaluated by fluorescein isothiocyanate (FITC) labelled nanoparticles. The experimental design led to nanoparticles with a size of 194.4 nm and a polydispersity index (PDI) of 0.31. The encapsulation efficiency (EE) was calculated as 95.96%. In vivo studies showed that PEC-eCAPs significantly reduced the blood glucose level of rats at the 8^th hour compared to oral insulin solution. It was concluded that PEC nanoparticles loaded into enteric-coated hard gelatin capsules provide a promising delivery system for the oral administration of insulin.     

Effect of sulphinpyrazone (Anturan®) on uric acid excretion and plasma uric acid concentration in healthy volunteers

Summary Investigations in six healthy volunteers given single oral doses of sulphinpyrazone (Anturan^®) showed that the drug had a potent uricosuric effect, even if the concentration of uric acid in the plasma was normal. In the dose range tested of 50 to 800 mg the increase in uric acid excretion was dose-related. It reached a maximum within 2 h and was still detectable 6–8 h after the drug was taken. There was a corresponding dose-related reduction in the plasma concentration of uric acid, which reached its lowest level after 8–10 h. The administration of 300 mg twice daily for four days to a further five healthy volunteers reduced plasma uric acid from 5.06 to 1.8 mg%. The findings indicate that, even if plasma uric acid levels are unknown, it would be advisable to avoid the risk of formation of urinary calculi by administering sulphinpyrazone in gradually increasing doses, ensuring adequate fluid intake and alkalinizing the urine during the first few days of treatment.     

Lack of genotoxic effect of food dyes amaranth,sunset yellow and tartrazine and their metabolites in the gut micronucleus assay in mice

The food dyes amaranth, sunset yellow and tartrazine were administered twice, at 24 h intervals, by oral gavage to mice and assessed in the in vivo gut micronucleus test for genotoxic effects (frequency of micronucleated cells) and toxicity (apoptotic and mitotic cells). The concentrations of each compound and their main metabolites (sulfanilic acid and naphthionic acid) were measured in faeces during a 24-h period after single oral administrations of the food dyes to mice. Parent dye compounds and their main aromatic amine metabolites were detected in significant amounts in the environment of colonic cells. Acute oral exposure to food dye additives amaranth, sunset yellow and tartrazine did not induce genotoxic effect in the micronucleus gut assay in mice at doses up to 2000 mg/kg b.w. Food dyes administration increased the mitotic cells at all dose levels when compared to controls. These results suggest that the transient DNA damages previously observed in the colon of mice treated by amaranth and tartrazine by the in vivo comet assay [Sasaki, Y.F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., Taniguchi, K., Tsuda, S., 2002. The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat. Res. 519, 103–119] are unable to be fixed in stable genotoxic lesions and might be partly explained by local cytotoxicity of the dyes.     

Oral Toxicity of Okadaic Acid in Mice: Study of Lethality,Organ Damage,Distribution and Effects on Detoxifying Gene Expression

In vivo, after administration by gavage to mice and rats, okadaic acid has been reported to produce lesions in liver, small intestine and forestomach. Because several reports differ in the damage detected in different organs, and on okadaic acid distribution after consumption, we determined the toxicity of this compound after oral administration to mice. After 24 hours, histopathological examination showed necrotic foci and lipid vacuoles in the livers of intoxicated animals. By immunohistochemical analysis, we detected this toxin in the liver and kidneys of intoxicated animals. Okadaic acid induces oxidative stress and can be activated in vitro into reactive compounds by the post-mitochondrial S9 fraction, so we studied the okadaic effect on the gene expression of antioxidant and phase II detoxifying enzymes in liver. We observed a downregulation in the expression of these enzymes and a reduction of protein expression of catalase and superoxide dismutase 1 in intoxicated animals.     

Lipid-core nanocapsules restrained the indomethacin ethyl ester hydrolysis in the gastrointestinal lumen and wall acting as mucoadhesive reservoirs

The aim of this work was to investigate if the indomethacin ethyl ester (IndOEt) released from lipid-core nanocapsules (NC) is converted into indomethacin (IndOH) in the intestine lumen, intestine wall or after the particles reach the blood stream. NC–IndOEt had monomodal size distribution (242 nm; PDI 0.2) and zeta potential of ?11 mV. The everted rat gut sac model showed IndOEt passage of 0.16 μmol m^?2 through the serosal fluid (30 min). From 15 to 120 min, the IndOEt concentrations in the tissue increased from 6.13 to 27.47 μmol m^?2. No IndOH was formed ex vivo. A fluorescent-NC formulation was used to determine the copolymer bioadhesion (0.012 μmol m^?2). After NC–IndOEt oral administration to rats, IndOEt and IndOH were detected in the gastrointestinal tract (contents and tissues). In the tissues, the IndOEt concentrations decreased from 459 to 5 μg g^?1 after scrapping, demonstrating the NC mucoadhesion. In plasma (peripheric and portal vein), in spleen and liver, exclusively IndOH was detected. In conclusion, after oral dosing of NC–IndOEt, IndOEt is converted into IndOH in the intestinal lumen and wall before reaching the blood stream. The complexity of a living system was not predicted by the ex vivo gut sac model.