The Impact of Handling and Storage of Human Fecal Material on Bacterial Activity.

Fecal material prepared from human stools is frequently used for the assessment of bacterial degradation of active pharmaceutical ingredients as relevant data are useful for evaluating the potential for colonic drug delivery. The impact of handling and storage of human fecal material on bacterial activity was assessed by evaluating the degradation characteristics of metronidazole and olsalazine. Multiple freeze (-70°C)-thaw cycles should be avoided. Incubation of frozen material for about 2 h in the anaerobic workstation ensures regeneration of the highest possible bacterial activity. Material could be stored at -70°C for at least 12 months.

Degradation kinetics of metronidazole and olsalazine by bacteria in ascending colon and in feces of healthy adults.

PURPOSE: To compare the degradation kinetics of metronidazole and olsalazine by the bacteria of ascending colon and the bacteria of feces of healthy adults. METHODS: Contents of the ascending colon of seven healthy adults were collected under conditions simulating the bioavailability/bioequivalence studies in the fasted and in the fed states on a crossover basis. Material from the contents of the ascending colon was prepared by ultracentrifuging and diluting the precipitate with a volume of normal saline equivalent to that of the supernatant. Fecal material was prepared from feces of three healthy adults collected at two occasions that were separated by at least 6 months. Ex vivo drug degradation kinetics were evaluated under anaerobic conditions. RESULTS: Mean half-lives of metronidazole degradation in material from the contents of the ascending colon collected in the fasted state and in fecal material were 16.1 and 2.4 min, respectively (p<0.001). The corresponding numbers for olsalazine were 57.8 and 9.2 min, respectively (p<0.001). Both compounds were stable in material from the contents of ascending colon collected in the fed state. CONCLUSIONS: Compared with data in fecal material, degradation of metronidazole and olsalazine in material from the contents of the ascending colon is significantly slower and it becomes non-significant during the arrival of fresh food remnants in the region.

Determination of 3-methoxysalicylamine levels in mouse plasma and tissue by liquid chromatography-tandem mass spectrometry: application to in vivo pharmacokinetics studies.

We report the development of a sensitive liquid chromatography-tandem mass spectrometric assay to quantitate 3-methoxysalicylamine (3-MoSA) in biological samples. Derivatization with 1,1'-thiocarbonyldiimidazole followed by C(18) reverse-phase chromatography allowed the detection of both analyte and internal standard (hexylsalicylamine) using electrospray ionization and selected reaction monitoring (SRM) in positive ion mode. We monitored the transitions from m/z 196.7 to 65.1 and from m/z 250.1 to 77.1 for 3-MoSA and HxSA, respectively. The method is validated with respect to linearity (r(2)=0.995), precision (<17% RSD), recovery (100% for 3-MoSA and HxSA), and stability (77% after storage up to 7 month at -80°C). The LOD and LOQ were 16.12 and 48.87 µg/l, respectively and the LLOQ of 1 pg/ml. In addition, we used this assay to analyze the pharmacokinetics of 3-MoSA in mouse plasma and tissues following both intraperitoneal and oral administration, providing new information regarding the distribution of this compound in vivo.

Determination of olsalazine sodium in pharmaceuticals by differential pulse voltammetry.

The electrochemical oxidation of olsalazine sodium was investigated by cyclic, linear sweep, differential pulse and square wave voltammetry using glassy carbon disc electrode in different buffer systems. Best results were obtained for the determination of olsalazine using the differential pulse voltammetric technique in phosphate buffer at pH 7.0. The electroactive species exhibits a diffusion-controlled voltammetric wave and its differential pulse peak current shows a linear dependence on olsalazine concentration in the range between 2 x 10(-6) M and 2 x 10(-4) M. This relationship has been applied to the determination of olsalazine in commercial capsule dosage forms. The recovery study shows good accuracy and precision for the assay developed. A UV spectrophotometric assay is also reported for comparison.

Simple method for the determination of 5-aminosalicylic and N-acetyl-5-aminosalicylic acid in rectal tissue biopsies.

We describe a sensitive high-performance liquid chromatographic method for the determination of 5-aminosalicylic acid and N-acetyl-5-aminosalicylic acid in rectal tissue biopsies. Samples were derivatised using propionic anhydride and proteins were precipitated with methanol. A Supelcosil ABZ column (150x4.6 mm I.D., 5 microm silica particles) was used with a mobile phase comprising 0.1 M acetic acid, acetonitrile and triethylamine (1600:114:6, v/v/v). Fluorescence detection was employed and detection limits were 0.2 ng/mg tissue at a signal-to-noise ratio of three (measured concentration: 5-aminosalicylic acid, 0.254 (0.228-0.286) ng/mg, C.V. 10.7%; N-acetyl-5-aminosalicylic acid, 0.18 (0.154-0.198) ng/mg, C.V 9.8%). This assay was validated for use with serum, urine and faecal samples for which it proved to be both precise and accurate (C.V.<10%, measured concentration within 10%).

Distribution and concentrations of 5-aminosalicylic acid in rectosigmoid biopsy specimens after rectal administration.

PURPOSE: Using the autofluorescent properties of 5-aminosalicylic acid (5-ASA), we studied the penetration and distribution of this molecule in human colonic biopsies at different time intervals after administration of 5-ASA enemas. METHODS: Fluorescence scores of rectosigmoidal biopsy specimens were compared with 5-ASA and acetyl-5-aminosalicylic (Ac-5-ASA) concentrations, determined by high-performance liquid chromatography, in adjacent biopsies and in serum samples. RESULTS: 5-ASA penetrates into the rectal mucosa and into the epithelial cells after local application by means of an enema. We found a characteristic 5-ASA staining of two intramucosal structures that need further identification: intraepithelial triangular configurations and "5-ASA scavengers" in the lamina propria. Fluorescence scores correlate well with 5-ASA concentrations in adjacent biopsies (r = 0.67; P < 0.005) and correlate even better with serum concentrations of 5-ASA (r = 0.84; P < 0.005) and Ac-5-ASA (r = 0.80; P < 0.005), hence reflecting the amount of systemically absorbed and metabolized 5-ASA. CONCLUSION: 5-ASA penetrates into the rectal mucosa after local application. Local availability, assessed by means of fluorescence microscopy, correlates well with serum concentrations.

Simultaneous determination of 5-aminosalicylic acid, acetyl-5-aminosalicylic acid and 2,5-dihydroxybenzoic acid in endoscopic intestinal biopsy samples in humans by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method for the simultaneous determination of 5-aminosalicylic acid (5-ASA), acetyl-5-aminosalicylic acid (Ac-5-ASA) and 2,5-dihydroxybenzoic acid (5-HSA) in human endoscopic intestinal biopsy with electrochemical detection has been developed and validated. A liquid-liquid extraction procedure was used to isolate these drugs from the biological material prior to analysis. The compounds were separated on an Erbasil S reversed-phase column using methanol-critic acid-sodium hydrogenphosphate-heptane-sulfonic acid-disodium ethylenediaminetetraacetate (pH 3) as mobile phase. The method was linear from 1.0 to 300 ng ml-1 for 5-ASA, from 10 to 1000 ng ml-1 for Ac-5ASA and from 0.1 to 10 ng m-1 for 5-HSA. The limit of detection for 5-ASA and for Ac-5-ASA was 1 ng ml-1 and that for 5-HSA was 0.1 ng ml-1. This procedure is suitable for pharmacological and clinical studies of 5-ASA.

Radical-derived oxidation products of 5-aminosalicylic acid and N-acetyl-5-aminosalicylic acid.

5-Aminosalicylic acid is an agent effective in the treatment of chronic inflammatory bowel diseases. Its ability to scavenge radicals is considered to be a major factor responsible for its therapeutic efficacy. In this study oxidation products of aminosalicylates with hydroxyl radicals were produced. The compounds that could be discovered by gas chromatographic-mass spectrometric analysis originate from a 1,4-benzoquinone monoimine intermediate which subsequently undergoes multiple reactions such as hydrolysis, reductive 1,4-Michael addition, reoxidation and decarboxylation. Some of these products could represent metabolites formed under in vivo conditions and thus provide a tool for screening biological material from subjects under different clinical conditions.

Comparative bioavailability of 5-aminosalicylic acid from a controlled release preparation and an azo-bond preparation.

BACKGROUND: Knowledge of the bioavailability of 5-aminosalicylic acid (5-ASA, mesalazine) from the different 5-ASA-containing drugs is important for rational therapy of inflammatory bowel diseases. METHODS: The local and systemic bioavailability of 5-ASA from a controlled release 5-ASA preparation (Pentasa--2, 4 or 6 g/day) was investigated and compared with the azo-bond 5-ASA preparation olsalazine (Dipentum--2 g/day) in 13 healthy volunteers during steady state conditions. RESULTS: The therapeutically relevant parameter of 5-ASA at the rectal level, expressed as the mean concentration in faecal water, showed a significant trend towards higher concentrations with increasing Pentasa dose: 9.2 mmol/L, 19.0 mmol/L and 24.4 mmol/L, respectively. The concentration of olsalazine 2 g/day was 16.0 mmol/L. The concentration of the metabolite N-acetyl-5-aminosalicylic acid (Ac-5-ASA) did not rise with increasing Pentasa dose, indicating saturable presystemic acetylating capacity of 5-ASA. Total urinary excretion of 5-ASA and Ac-5-ASA, as a percentage of the daily ingested 5-ASA dose, remained constant on the three Pentasa doses, but there was a significant increase in the 5-ASA fraction. Mean steady state plasma concentrations of 5-ASA and Ac-5-ASA were significantly higher on Pentasa 4 g/day and 6 g/day than on 2 g/day. Values on Pentasa 2 g/day were comparable with those on olsalazine 2 g/day. CONCLUSIONS: The study confirmed that 5-ASA is released from Pentasa in a predictable manner, the amount released increasing with dose. Olsalazine is an excellent generator of 5-ASA in the colon.

Disposition of olsalazine and metabolites in breast milk.

This study examined the disposition of olsalazine and its metabolites into breast milk after the ingestion of a single dose of 500 mg olsalazine. Blood and serum samples were obtained for 48 hours after the ingestion of 500 mg olsalazine in a 39-year-old lactating woman. Blood samples were obtained at .0, .5, 1, 2, 4,6, 24.5, 26, and 48 hours. Maternal milk samples were obtained at .0, .5, 2, 4, 6, 14, 24, 28, 36, and 48 hours. Olsalazine and olsalazine-S underwent high-pressure liquid chromatography analysis, and 5-ASA and Ac 5-ASA underwent fluorometric detection. Acetylated-5-ASA achieved concentrations of .8, .86, and 1.24 mumol/L in breast milk at 10, 14, and 24 hours, respectively. Olsalazine, olsalazine-S, and 5-ASA were undetectable in the breast milk for 48 hours after drug administration. Clinically significant drug exposure in the breast-fed infant is unlikely after a maternal single dose of olsalazine. Idiosyncratic hypersensitivity, however, remains a possibility even if the infant is exposed to only minute quantities.

[Localization of salazopyrin in colonic mucosa patients with ulcerative colitis].

In this study SASP metabolite levels were measured in colonic mucosal specimens and plasma samples from 31 patients with ulcerative colitis (UC) under treatment with the drug. Colonic tissue specimens were obtained by endoscopically guided biopsy and plasma was isolated from peripheral blood. Measurements were performed by HPLC according to the procedure of Fischer et al. The levels of 5-ASA and SP in either of colonic tissue or plasma were significantly lower than those of Ac-5-ASA and Ac-SP, respectively. The tissue level of 5-ASA had a significant correlation to the dosage of 5-ASA. The tissue levels of 5-ASA and Ac-5-ASA were low in an active stage of UC than during a remission period and the difference observed with respect to the latter metabolite was significant. These findings suggest that acetylation of 5-ASA is inhibited in the colonic mucosa in an active stage of the disease.

Ion-pairing high-performance liquid chromatographic method for the determination of 5-aminosalicylic acid and related impurities in bulk chemical.

An ion-pairing high-performance liquid chromatographic method has been developed for the determination of 5-aminosalicylic acid (5-ASA) bulk chemical in the presence of thirteen potential synthetic process impurities. In addition, the method is suitable for the determination of the in process intermediate, 5-nitrosalicylic acid. A selective method was achieved on a Hypersil-BDS reversed-phase column using 1-heptanesulfonic acid sodium salt as the ion-pairing reagent in a 0.08 M sodium phosphate buffer (pH 2) containing 0.005 M 1-heptanesulfonic acid sodium salt and 0.07 M sodium chloride-methanol-tetrahydrofuran (85:11:4, v/v/v) isocratic mobile phase. The method was validated using a multi-day, intra-laboratory protocol. The validation addressed linearity, accuracy, precision, sensitivity, and ruggedness of the method. The validated method characterizes the purity of 5-ASA bulk chemical.

High-performance liquid chromatographic assay for the determination of 5-aminosalicylic acid and acetyl-5-aminosalicylic acid concentrations in endoscopic intestinal biopsy in humans.

A high-performance liquid chromatographic method for the simultaneous determination of 5-amino-salicylic acid (5-ASA) and N-acetyl-5-ASA (Ac-5-ASA) concentrations in endoscopic mucosal biopsy homogenates is presented. The mean recoveries of 5-ASA and Ac-5-ASA from spiked blank biopsy homogenates ranged from 95.9 to 120% and from 92.5 to 100%, respectively. The coefficients of variation for 5-ASA and Ac-5-ASA were 0.7-8.6% and 1.4-12.9%, respectively. This method is useful for direct determination of topical availability of 5-ASA and Ac-5-ASA and probably an accurate parameter of drug bioavailability.

Clinical evidence supporting the radical scavenger mechanism of 5-aminosalicylic acid.

5-Aminosalicylic acid, the therapeutically active metabolite of sulfasalazine, was exposed to oxygen-derived free radicals produced by the Fenton reaction in vitro, and several metabolites were detected and characterized by high performance liquid chromatography and ultraviolet spectrophotometry. The majority of these metabolites were present in methanolic extracts of feces samples from sulfasalazine-treated patients with inflammatory bowel disease but not in rheumatoid arthritis patients with normal bowel function. The presence of these metabolites, which have not been demonstrated in vivo before, provides evidence of an interaction between 5-aminosalicylic acid and oxygen-derived free radicals in sulfasalazine-treated patients with inflammatory bowel disease. Since the concentration of lipid peroxides, which is dependent on the release of oxygen-derived free radicals, was significantly increased in pretreatment rectal biopsies of the patients, and further was normalized concomitantly with a significant improvement in disease activity over the 5-wk treatment period, an important role of the radical scavenger mechanism of 5-aminosalicylic acid in sulfasalazine therapy of chronic inflammatory bowel disease is strongly suggested.

Effects of mesalazine substitution on salicylazosulfapyridine-induced seminal abnormalities in men with ulcerative colitis.

Some semen characteristics of eight male patients with clinically inactive ulcerative colitis were investigated. Semen analysis was carried out twice during salicylazosulfapyridine (SASP) treatment and repeated twice after at least 3 months' treatment with mesalazine. The motility variables all showed significant improvement during mesalazine treatment: the graded motility (p less than 0.05), motility in percentage (p less than 0.01), and the penetration in egg white (p less than 0.05). The semen plasma was analyzed for mesalazine and the metabolite Ac-mesalazine during both regimens. There was no difference in the semen plasma concentration of mesalazine during the two regimens, whereas Ac-mesalazine was significantly higher during mesalazine treatment than during SASP treatment, indicating that other SASP metabolites, most likely sulfapyridine, are the agent causing the abnormal sperm characteristics. We suggest that pure mesalazine preparations are a safer alternative in young men with chronic inflammatory bowel disease.

Stability of disodium azodisalicylate (olsalazine) and metabolites in urine and faeces stored at different temperatures.

METHODS: The stability of disodium azodisalicylate, 5-aminosalicylic acid and acetyl-5-aminosalicylic acid dissolved in distilled water and in urine or mixed with faeces to which HgCl2 was added, was studied at -20 degrees C, 4 degrees C and room temperature. RESULTS: In water, no marked differences were found between the three storage regimens. In urine, disodium azodisalicylate and acetyl-5-aminosalicylic acid were stable, while the 5-ASA concentration decreased when stored at 4 degrees C and room temperature. In faeces stored during seven days, a marked decrease in 5-aminosalicylic acid concentration to about zero was found when it was kept at 4 degrees C and room temperature. No marked change in the concentration of disodium azodisalicylate and 5-aminosalicylic acid added to the faeces-HgCl2-mixtures appeared.