Development of An HPLC Method for the Determination of Mesalazine in Human Plasma by Fluorimetric Derivatization and Application to A Prototype Pharmacokinetic Study.

In this study, a new, fast and sensitive HPLC method with fluorometric detection was developed for the determination of mesalazine in human plasma and applied to a pharmacokinetic study. Mesalazine was precolumn derivatized with NBD-Cl and the fluorescent derivative was separated on a C18 (150 × 4.6 mm × 2.6 µm) analytical column at 30 ºC using a mobile phase composed of acetonitrile-0.1% o-phosphoric acid in water (70:30, v/v) by isocratic elution with flow rate of 1.0 mL min-1. The method was based on the measurement of the derivative using fluorescence detection (λex = 280 nm, λem = 325 nm). The retention time of mesalazine is 3.08 ± 0.06 min. Nortriptiline was used as internal standard. This currently developed method was validated according to ICH criteria by evaluating the specificity, linearity, precision, accuracy and robustness. The method was determined to be linear in a concentration range of 0.25-1.5 µg mL-1 with the correlation coefficient of 0.9997. LOD and LOQ were found to be 0.075 and 0.25 µg mL-1, respectively. Intraday and interday RSD values were less than 5.92%. The plasma concentration-time profile and pharmacokinetic parameters such as AUC0-t, AUC0-∞, Cmax, tmax, t1/2, were calculated according to the assays. The presented method can certainly be used for bioequivalence and bioavailability investigations and routine analysis of the drug in plasma.

Ni-Doped ZrO2 nanoparticles decorated MW-CNT nanocomposite for the highly sensitive electrochemical detection of 5-amino salicylic acid.

In this work, Ni-doped ZrO2 nanoparticles (NPs) were used to decorate multi-walled carbon nanotubes (MWCNTs) to obtain a Ni-ZrO2/MWCNT nanocomposite, which acted as an efficient electrode material for the highly sensitive electrochemical detection of the anti-inflammatory drug 5-amino salicylic acid (5-ASA). The Ni-ZrO2 NPs were obtained through a facile co-precipitation method, and the subsequent support of these Ni-ZrO2 NPs onto MWCNTs was accomplished via an ultrasonication technique. Supporting Ni-ZrO2 NPs on MWCNTs not only results in excellent catalytic properties, but it also substantially enhances the surface area, electrical conductivity, and electron transfer process. The electrochemical activity of the synthesized Ni-ZrO2/MWCNT nanocomposite was systematically investigated via cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The constructed Ni-ZrO2/MWCNT-modified glassy carbon (GC) electrode manifests superior electrocatalytic oxidation activity toward 5-ASA, with a lower peak potential compared with Ni-ZrO2-NP- and MWCNT-modified GC electrodes. Importantly, the proposed biosensor exhibited excellent sensitivity during the detection of 5-ASA with a wide linear concentration range (0.001-500 µM) and a low detection limit of 0.0029 µM. Moreover, the biosensor demonstrated excellent repeatability, reproducibility, stability, and high specificity toward 5-ASA detection in the presence of different interfering species. Furthermore, the biosensor showed satisfactory recovery rates in complex biological samples, such as human blood serum, human urine, and 5-ASA tablet samples.

Effects of Differential Food Patterns on the Pharmacokinetics of Enteric-Coated Mesalazine Tablets in the Same Cohort of Healthy Chinese Volunteers.

This study aimed to simultaneously determine mesalazine (5-ASA) and its major metabolite N-Ac-5-ASA in the plasma and to evaluate the impact of different food patterns on the relative bioavailability and pharmacokinetics of a single oral dose of 5-ASA in healthy subjects. In this single-dose, open-label, 3-period, 3-treatment crossover study, the subjects received a single, oral dose of 500-mg enteric-coated mesalazine tablet together with either a low-fat or a high-fat breakfast or under fasting condition (reference). The pharmacokinetic parameters were determined by noncompartmental methods and analyzed with a linear mixed-effect model. The geometric least squares mean ratio for the area under the plasma concentration-time curve from zero to infinity of N-Ac-5-ASA was 1.05 (90% confidence interval [CI], 0.70-1.58) for high-fat/fasted condition and 1.06 (90%CI, 0.82-1.36) for low-fat/fasted condition. The least squares mean ratio of 5-ASA was 0.86 (90%CI, 0.65-1.14) for high-fat/fasted condition and 0.78 (90%CI, 0.60-1.02) for low-fat/fasted condition. All P values were >.05. The mean maximum plasma concentration and the time to reach the maximum plasma concentration of N-Ac-5-ASA were 2084 ng/mL, 8 hours; 2639 ng/mL, 11 hours, and 2409 ng/mL, 9 hours for fasted, high-fat, and low-fat, respectively. The values of 5-ASA were 1950 ng/mL, 7 hours; 2869 ng/mL, 9 hours; and 2837 ng/mL, 8 hours for fasted, high-fat, and low-fat condition. 5-ASA was well tolerated under all 3 conditions. Food delayed the absorption of 5-ASA, especially a high-fat meal. Therefore, enteric-coated mesalazine tablets should be taken before meals to avoid causing patients slow response and any effect of food on its efficacy.

5-Aminosalicylic Acid Azo-Coupled with a GPR109A Agonist Is a Colon-Targeted Anticolitic Codrug with a Reduced Risk of Skin Toxicity.

To develop a 5-aminosalicylic acid (5-ASA)-based anticolitic drug with enhanced therapeutic activity, a colon-targeted codrug constituting 5-ASA and a GPR109A agonist was designed. 5-ASA azo-coupled with nicotinic acid (ASA-azo-NA) was synthesized, and the colon specificity and anticolitic effects were evaluated. Approximately 89% of ASA-azo-NA was converted to 5-aminonicotinic acid (5-ANA) and 5-ASA after 24 h of incubation in the cecal contents. 5-ANA was identified as a GPR109A agonist (concentration that gives half-maximal response (EC50): 18 µM) in a cell-based assay. Upon oral gavage of ASA-azo-NA (oral ASA-azo-NA) and sulfasalazine (oral SSZ), a colon-targeted 5-ASA prodrug, cecal accumulation of 5-ASA was comparable, and 5-ANA was barely detectable in the blood, while it was detected up to 62.7 µM with oral 5-ANA. In parallel, oral ASA-azo-NA did not elicit an adverse skin response. In murine macrophage and human colon carcinoma cells, activation of GPR109A by 5-ANA elevated the level of the anti-inflammatory cytokine IL-10, suppressed NF-κB activation, and potentiated the inhibitory activity of 5-ASA on NF-κB. Oral ASA-azo-NA ameliorated rat colitis and was more effective than oral SSZ, which were substantially blunted following cotreatment with the GPR109A antagonist, mepenzolate. In conclusion, ASA-azo-NA is a colon-targeted anticolitic codrug with a reduced risk of skin toxicity induced by the GPR109A agonist, therapeutically surpassing a current 5-ASA-based anti-inflammatory bowel disease drug in a rat colitis model.

Ultrasound treated cerium oxide/tin oxide (CeO2/SnO2) nanocatalyst: A feasible approach and enhanced electrode material for sensing of anti-inflammatory drug 5-aminosalicylic acid in biological samples.

In this work, we developed cerium oxide/tin oxide (CeO2/SnO2) nanocatalyst with the assistance of urea by a simple sonochemical method and utilized as an efficient electrode material for electrochemical sensing of anti-inflammatory drug 5-aminosalicylic acid (Mesalamine, MES). The CeO2/SnO2 nanoparticles (NPs) were systematically characterized in terms of their crystal structure, morphologies, and physicochemical properties using XRD, Raman, FESEM, HR-TEM, EDX, mapping, and XPS analysis. The characterization results clearly confirmed that the prepared NPs was formed in the phase of CeO2/SnO2 without any other impurities. The electrochemical properties of CeO2/SnO2 NPs were investigated by EIS, CV, and DPV techniques. The CeO2/SnO2 NPs (9.6 µA) modified GCE demonstrated an excellent and improved electrocatalytic activity in terms of higher anodic peak current and lower peak potential when compared to bare GCE (6.7 µA) and CeO2 NPs/GCE (8.2 µA) for the sensing of MES. The CeO2/SnO2 NPs/GCE shows broader linear response range and lower detection limit of 0.02-1572 µM and 0.006 µM, respectively. Moreover, other potentially interfering compounds such as a similar functional group containing biological substances and inorganic species have no interference effect towards MES sensing. In addition, the practicability of the CeO2/SnO2 NPs/GCE was tested by real sample analysis in commercial MES tablet, human urine, and serum samples with the appreciable recovery results.

In vitro and human pharmacoscintigraphic evaluation of an oral 5-ASA delivery system for colonic release.

5-aminosalicylic acid (5-ASA) is the most widely used drug for the treatment of ulcerative colitis. The benefits of targeted delivery of 5-ASA to the large intestine are well known, resulting in reduced systemic absorption and increased local concentrations at the disease site. In the present study, a 5-ASA colon delivery system based on the time-dependent strategy, exploiting the relatively consistent small intestinal transit time (SITT), was manufactured and evaluated in vitro as well as in vivo. The system was obtained by successive spray-coating of an immediate-release tablet core with low-viscosity HPMC and Eudragit® L. The enteric film was effective in preventing release during the acidic stage of the in vitro test, while the HPMC coating brought about reproducible lag phases prior to release in phosphate buffer medium. A γ-scintigraphy investigation pointed out that, following administration to fasted and fed volunteers, disintegration of the units never occurred prior to colon arrival. In all cases, a lag time preceded the appearance of the drug and its N-acetyl metabolite in the bloodstream, which was found to correlate with the time of disintegration in a linear mode. The plasma levels of the drug and metabolite as well as their cumulative urinary recovery were relatively low with respect to those reported when 5-ASA is delivered to the small bowel.

Determination of mesalazine, a low bioavailability olsalazine metabolite in human plasma by UHPLC-MS/MS: Application to a pharmacokinetic study.

Olsalazine sodium, salicylate derivative (prodrug) is effectively bioconverted to mesalazine (5-aminosalicylic acid; 5-ASA), which has an anti-inflammatory activity in ulcerative colitis. In this article, a novel highly sensitive and selective method was developed and validated to determine mesalazine in human plasma using a derivatization technique to enhance the signal intensity by using ultra- high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) with an electrospray ionization interface. The sample preparation consisted of a derivatization with propionyl anhydride followed by liquid liquid extraction (LLE) to remove the interference and minimize the matrix effect of human plasma. The multiple reaction monitoring (MRM) mode of the negative ion was performed and the transitions of m/z 208.1→107.0 and m/z 211.1→110.1 were used to measure the derivative of mesalazine and mesalazine-d3. The chromatographic separation was achieved using kinetex XB-C18 (100×4.6mm 2.6µ) analytical column with 0.1% formic acid in water and acetonitrile as mobile phase with a gradient elution. Nominal retention times of mesalazine and IS were 3.08 and 3.07min, respectively. Absolute recovery was found to be between 82-95% for analyte and about 78% for IS. The standard curves was linear (r(2)>0.995) in the concentration range 0.10 to 12.0ng/mL with lower limit of quantification (LLOQ) in human plasma was 0.10ng/mL. The average intra-day/inter-day precision values (%CV) were in the range from 0.6-2.9 % and 1.3-3.8 %, respectively, while the average accuracy value was 103.8-107.2%. This method has been successfully applied to the human pharmacokinetics of olsalazine sodium 250mg capsules following single oral administration.

Electrochemical sensing of mesalazine and its N-acetylated metabolite in biological samples using functionalized carbon nanotubes.

A rapid analytical method without the time-consuming separation step was developed to simultaneously determine mesalazine and its N-acetylated metabolite. A simply designed electrochemical sensor with functionalized carbon nanotubes in a Nafion matrix was constructed for this purpose. The presence of the nanocomposite modifier on the electrode surface significantly affects the voltammetric response of target analytes. The morphology of the modified surface was investigated by scanning electron microscopy. The effect of modifier amount on the sensor performance was investigated in order to obtain the most favorable response of mesalazine since it was found in lower concentration limits in real samples then its metabolite due to the rapid drug elimination and the slightly slower renal metabolite excretion. Under optimal conditions, the anodic peak currents measured by square-wave voltammetry increased linearly after short accumulation of 30s in the range of 5.0×10(-8)-2.5×10(-6)M and 1.0×10(-7)-5.0×10(-6)M for drug and metabolite, respectively. In addition to stable response, the sensor has excellent performance associated with high sensitivity (2.33×10(7) and 8.37×10(6)µAM(-1) for drug and metabolite, respectively). The synergistic effect of the carbon nanotubes and Nafion polymer film yielded detection limit of 1.2×10(-8)M for mesalazine and 2.6×10(-8)M for its metabolite that is comparable to known chromatographic methods. Due to the easy preparation and regeneration, the proposed sensor opens new opportunity for fast, simple and sensitive analysis of drug and its metabolite in human serum samples as well as direct quantification of mesalazine in delayed-release formulations.

Fabrication of an electrochemical sensor based on computationally designed molecularly imprinted polymer for the determination of mesalamine in real samples.

A novel electrochemical sensor based on mesalamine molecularly imprinted polymer (MIP) film on a glassy carbon electrode was fabricated. Density functional theory (DFT) in gas and solution phases was developed to study the intermolecular interactions in the pre-polymerization mixture and to find the suitable functional monomers in MIP preparation. On the basis of computational results, o-phenylenediamine (OP), gallic acid (GA) and p-aminobenzoic acid (ABA) were selected as functional monomers. The MIP film was cast on glassy carbon electrode by electropolymerization of solution containing ternary monomers and then followed by Ag dendrites (AgDs) with nanobranch deposition. The surface feature of the modified electrode (AgDs/MIP/GCE) was characterized by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). Under the optimal experimental conditions, the peak current was proportional to the concentration of mesalamine ranging from 0.05 to 100 µM, with the detection limit of 0.015 µM. The proposed sensor was applied successfully for mesalamine determination in real samples.

Gene ablation of carnitine/organic cation transporter 1 reduces gastrointestinal absorption of 5-aminosalicylate in mice.

5-Aminosalicylic acid (5-ASA) is an orally administered therapeutic agent for inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease. We hypothesized that the absorption of 5-ASA is mediated by the polyspecific carnitine/organic cation transporter (OCTN1/SLC22A4), based on the similarity of chemical structure between 5-ASA and other OCTN1 substrates. Therefore, we examined the involvement of this transporter in the disposition of 5-ASA in vivo by using octn1 gene knockout (octn1(-/-)) mice. After oral administration of 5-ASA, the plasma concentrations of 5-ASA and its primary metabolite, N-acetyl-5-aminosalicylate (Ac-5-ASA), in octn1(-/-) mice were much lower than those in wild-type mice. The time required to reach maximum plasma concentration was also delayed in octn1(-/-) mice. On the other hand, the plasma concentration profiles of both 5-ASA and Ac-5-ASA after intravenous administration of 5-ASA (bolus or infusion) were similar in the two strains. Uptake of 5-ASA from the apical to the basal side of isolated small-intestinal tissues of octn1(-/-) mice, determined in an Ussing-type chamber, was lower than that in wild-type mice. Further, uptake of 5-ASA in HEK293 cells stably transfected with the OCTN1 gene, assessed as the sum of cell-associated 5-ASA and Ac-5-ASA, was higher than that in HEK293 cells transfected with the vector alone. Overall, these results indicate that OCTN1 is involved, at least in part, in the gastrointestinal absorption of 5-ASA.

Development and validation of an LC-MS/MS method for the determination of mesalazine in beagle dog plasma and its application to a pharmacokinetic study.

A simple, specific and sensitive LC-MS/MS method was developed and validated for the determination of mesalazine in beagle dog plasma. The plasma samples were prepared by protein precipitation, then the separation of the analyte was achieved on a Waters Spherisorb C6 column (150 × 4.6 mm, 5 µm) with a mobile phase consisting of 0.2% formic acid in water-methanol (20:80, v/v). The flow rate was set at 1.0 mL/min with a split ratio of 3:2. Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an electrospray source interface in positive ionization mode. Quantitation was performed using selected reaction monitoring of precursor-product ion transitions at m/z 154 → m/z 108 for mesalazine and m/z 285 → m/z 193 for diazepam (internal standard). The linear calibration curve of mesalazine was obtained over the concentration range 50-30,000 ng/mL. The matrix effect of mesalazine was within ±9.8%. The intra- and inter-day precisions were <7.9% and the accuracy (relative error) was within ±3.5%. The validated method was successfully applied to investigate the pharmacokinetics of mesalazine in healthy beagle dogs after rectal administration of mesalazine suppository.

A novel stimuli-synchronized alloy-treated matrix for space-defined gastrointestinal delivery of mesalamine in the Large White pig model.

The study focussed on designing a Stimuli-Synchronized Matrix (SSM) for space-defined colonic delivery of the anti-inflammatory drug mesalamine. The configured matrix provided time-independent delivery and stimuli targeting. Formulations were optimized according to a Box-Behnken experimental design that constituted mesalamine-loaded BaSO4-crosslinked chitosan dispersed within a pectin, carboxymethylcellulose and xanthan gum complex. The complex was compressed into matrices and subsequently alloy-treated with pectin and ethylcellulose. In vitro drug release was determined in the presence and absence of colonic enzymes and the mean dissolution time was used for formulation optimization. To mechanistically elucidate the synchronous catalytic action of the enzymes pectinase and glucosidase on the matrix, computer-aided 3D modelling of active fractions of the enzyme-substrate complexes was generated to predict the orientation of residues affecting the substrate domain. Drug release profiles revealed distinct colonic enzyme responsiveness with fractions of 0.402 and 0.152 of mesalamine released in the presence and absence of enzymes, respectively after 24h. The commercial comparator product showed irreproducible release profiles over the same period (SD=0.550) compared to the SSM formulation (SD=0.037). FTIR spectra of alloy-treated matrices showed no peaks from 1589 to 1512cm(-1) after colonic enzyme exposure. With increasing enzyme exposure there were also no peaks between 1646 and 1132cm(-1). This indicated polymeric enzyme cleavage for controlled and space-defined release of mesalamine. Plasma concentration profiles in the Large White pig model produced a Cmax of 3.77±1.375µg/mL compared to 10.604±2.846µg/mL for the comparator formulation. The SSM formulation proved superior over the comparator product by providing superiorly controlled enzyme-responsive colonic drug delivery.

Absorption kinetics of 5-aminosalicylic acid in rat: influence of indomethacin-induced gastrointestinal lesions and Escherichia Coli Nissle 1917 medication.

OBJECTIVES: The therapeutic effect of probiotics has been studied in many clinical and experimental studies but no data exist concerning the influence of probiotics on pharmacokinetics of contemporary administered drugs. In this paper, we describe the influence of indomethacin-induced gastrointestinal lesions and Escherichia Coli Nissle 1917 medication on absorption of 5-aminosalicylic acid and its metabolite N-acetyl-5-aminosalicylic acid in rat. METHODS: 5-aminosalicylic acid (5-ASA) was given orally to rat using gastric probe as a suspension (25 mg/kg). The plasma time profiles of 5-ASA and its metabolite were compared between Group A (animals medicated with a suspension of Escherichia coli Nissle 1917 [EcN] in dose of 5 × 108 CFUs/day for 14 consecutive days), Group B (animals with indomethacin [IND]-induced gastrointestinal lesions; single dose of 25 mg/kg of IND), Group C (simultaneous administration of EcN and IND), and Group D (control animals without any medication). The blood samples for HPLC analysis has been taken from incannulated vena jugularis in time 30, 60, 90, 120, 180, 240, 360 min after 5-ASA administration to rat. RESULTS: The pharmacokinetics of 5-ASA was not significantly changed by EcN medication (Group A) in comparison to control animals (Group D). The significantly elevated absorption (AUC and cmax) of 5-ASA was found in animals with induced gastro-enteropathy with concurrently medicated with EcN (Group C) when compred to controls. In the case of metabolite N-acetyl-5-ASA, statistically no-significant differences were found between groups. CONCLUSIONS: Simultaneous probiotics (EcN) medication did not affect absorption 5-ASA from intestinal tract (the main site of ASAs action).

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study.

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 µL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 µm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10-10,000 ng/mL (r>0.99) for SASP and 10-1000 ng/mL (r>0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.

Development and validation of a HPLC-ESI-MS/MS method for the determination of 5-aminosalicylic acid and its major metabolite N-acetyl-5-aminosalicylic acid in human plasma.

A new HPLC method for the determination of 5-aminosalicylic acid (5-ASA) and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) in human plasma was developed and validated. Plasma samples were analyzed after protein precipitation with methanol and the two analytes were separated using a C18 column with a mobile phase composed of 17.5 mmol/L acetic acid (pH 3.3):acetonitrile=85:15 (v/v) at 0.2 mL/min flow rate. 4-ASA and N-Ac-4-ASA were used as internal standards. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in negative ionization mode and in multiple reaction monitoring acquisition (m/z 152-->108 for 5-ASA; m/z 194-->150 and 194-->107 for N-Ac-5-ASA). The limit of quantification (LOQ) was 50 ng/mL for both analytes (0.2 ng injected) and matrix-matched standard curves showed linearity up to 4000 ng/mL. In the entire analytical range the within- and between-batch precision (R.S.D.%) values were respectively < or = 6.3% and < or = 11% for 5-ASA and < or = 8.0% and < or = 10% for N-Ac-5-ASA. For both analytes the within- and between-batch accuracy (bias%) values ranged respectively from -8.4% to 7.9% and from -7.9% to 8.0%. The overall recoveries (n=6) at three tested concentration levels (i.e. 100, 1000 and 4000 ng/mL) were respectively >90% for 5-ASA and >95% for N-Ac-5-ASA (R.S.D.% < or = 10%). The method was applied to evaluate the pharmacokinetic of 5-ASA after a single oral dose administration of this compound (1200 mg) to 24 healthy volunteers. The mean maximum concentration levels were 680 ng/mL for 5-ASA and 1240 ng/mL for N-Ac-5-ASA and the kinetic profiles were in agreement with previous studies.

Review article: 5-aminosalicylate formulations for the treatment of ulcerative colitis--methods of comparing release rates and delivery of 5-aminosalicylate to the colonic mucosa.

BACKGROUND: Many oral 5-aminosalicylic acid (5-ASA) formulations are designed to maximize 5-ASA release in the colon where it acts topically on the colonic mucosa. Delayed-release formulations and azo-prodrugs minimize 5-ASA absorption in the upper gastrointestinal (GI) tract. AIMS: To review methods for assessing 5-ASA release and colonic distribution from oral formulations, and the potential use of this information for guiding clinical decisions. METHODS: PubMed and recent conference abstracts were searched for articles describing techniques used to assess 5-ASA release from ulcerative colitis (UC) therapies. RESULTS: In-vitro GI models, although unable to simulate more complex aspects of GI physiology, can provide useful data on 5-ASA release kinetics and bioaccessibility. Gamma-scintigraphy is useful for investigating GI disintegration of different formulations, but may not accurately reflect 5-ASA distribution. Plasma pharmacokinetic studies provide data on systemic exposure, but not on colonic distribution or mucosal uptake. Mucosal biopsies provide direct evidence of colonic distribution and may predict clinical efficacy, but must be interpreted cautiously because of considerable inter-subject variability and other confounding factors. CONCLUSION: While assessment of 5-ASA release is important, limitations of individual measurement techniques mean that randomized clinical studies in UC patients remain the best guide for dosing and treatment regimen decisions.

High-performance liquid-chromatographic determination of 5-aminosalicylic acid and its metabolites in blood plasma.

Mesalazine (5-aminosalicylic acid, 5-ASA), an anti-inflammatory agent for the treatment of inflammatory bowel diseases, is metabolized in organism to the principal biotransformation product, N-acetyl-5-ASA. Some other phase II metabolites (N-formyl-5-ASA, N-butyryl-5-ASA, N-beta-d-glucopyranosyl-5-ASA) have also been described. 5-ASA is a polar compound and besides it exhibits amphoteric properties. The extraction of this compound from biomatrices and its chromatographic analysis is complicated. In order to improve the reliability of the determination of parent 5-ASA, a derivatization of 5-ASA together with 4-ASA (added to samples as a precursor of I.S.-2) was involved into the method. More lipophilic N-propionyl-5-ASA and N-propionyl-4-ASA (I.S.-2) were obtained using propionic anhydride. These derivatives were well extractable together with N-acyl-5-ASAs (metabolites) and N-acetyl-4-ASA (I.S.-1). As the first internal standard (I.S.-1) was used for the evaluation of extracted N-acyl-metabolites, the second internal standard (I.S.-2) served for the evaluation of both derivatization and extraction steps of parent drug 5-ASA. Based on these reasonings, new HPLC bioanalytical method for the determination of 5-ASA and its metabolites in blood plasma was developed and validated. The sample preparation step consists of the deproteination of plasma by HClO(4) and the above-mentioned derivatization of ASAs followed by liquid-liquid extraction of all N-acyl-ASA-derivatives. Chromatographic analyses were performed on a 250-4 mm column containing Purospher RP-18 e, 5 microm (Merck, Darmstadt, Germany) with a precolumn (4-4 mm). The column effluent was monitored using both UV photodiode-array (lambda = 313 nm) and fluorescence detectors (lambda(exc.) = 300 nm/lambda(emiss.) = 406 nm) in tandem. The identity of individual N-acyl-ASAs in the extracts from biomatrices was verified by characteristic UV-spectra and by HPLC/MS experiments. The whole analysis lasted 23 min at the flow rate of 1 ml min(-1). LLOQ (LOD) was estimated 126 (20) pmol ml(-1) of plasma for N-acetyl-5-ASA and 318 (50) pmol ml(-1) of plasma for N-propionyl-5-ASA. The validated HPLC method was applied to pharmacokinetic studies of mesalazine in humans and animals.

5-aminosalicylic acid release from a new controlled-release mesalazine formulation during gastrointestinal transit in healthy volunteers.

BACKGROUND: Mesalazine (5-aminosalicylic acid, 5-ASA) containing formulations represent a cornerstone in the treatment of inflammatory bowel diseases. A novel formulation with an Eudragit L/S mixture coating has been developed to provide selective release of 5-ASA to the ileo-caecal region and the colon. AIM: To determine the release of 5-ASA during the gastrointestinal transit. METHODS: A single oral dose of mesalazine EC 500 mg gastroresistant tablets (Asamax) was administered to eight healthy male volunteers. Gastrointestinal transit and tablet disintegration were monitored by scintigraphy. 5-ASA release was verified by assessing plasma pharmacokinetics. RESULTS: Initial tablet disintegration was observed 5.65 +/- 0.86 h after dosing, corresponding to the detection of 5-ASA in plasma. This occurred in the ileo-caecal region in three subjects and the ascending colon in the remaining five. The relative percentage of 5-ASA absorption was more pronounced in the ascending colon (41 +/- 27.4%) than the ileo-caecal region (6.6 +/- 9.2%). CONCLUSION: This mesalazine EC gastroresistant tablets release locally active 5-ASA specifically in the ileo-caecal region and the ascending colon.

Delayed-release oral mesalamine at 4.8 g/day (800 mg tablet) for the treatment of moderately active ulcerative colitis: the ASCEND II trial.

BACKGROUND AND AIMS: Preliminary data have shown that delayed release oral mesalamine (Asacol) dosed at 4.8 g/day provided additional efficacy benefit compared to 1.6 g/day in patients with mildly to moderately active ulcerative colitis. Additionally, Asacol dosed at 2.4 g/day has been proved to be more effective than 1.6 g/day. Whether 4.8 g/day of mesalamine (dosed with an investigational 800 mg tablet) is more effective than Asacol 2.4 g/day (dosed with a 400 mg tablet) in patients with moderately active ulcerative colitis is unknown. METHODS: A randomized, double-blind, controlled trial (ASCEND II) was conducted to evaluate the efficacy of 4.8 g/day of mesalamine in adults with active ulcerative colitis. Three hundred eighty-six patients with mild to moderate ulcerative colitis were randomized for treatment with mesalamine 2.4 g/day (400 mg tablet) or 4.8 g/day (800 mg tablet) for 6 wk. The primary efficacy population was 268 patients with moderately active ulcerative colitis treated with 2.4 g/day (n = 139) or 4.8 g/day (n = 129). The primary endpoint was the proportion of patients in each treatment group that achieved overall improvement ("treatment success," defined as either complete remission or a clinical response to therapy) from baseline at week 6. RESULTS: Seventy-two percent of patients receiving 4.8 g/day of mesalamine for moderate ulcerative colitis (89/124 patients) achieved treatment success at week 6, compared with 59% of those who received 2.4 g/day (77/130 patients) (p= 0.036). Both regimens were well tolerated. Adverse events and clinically significant changes in laboratory results were similar in both treatment groups. CONCLUSIONS: Patients with moderately active ulcerative colitis treated with 4.8 g/day of mesalamine (800 mg tablet) are significantly more likely to achieve overall improvement at 6 wk compared to patients treated with 2.4 g/day.

A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma.

A high-performance liquid chromatographic method was developed, validated and applied to the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its acetylated metabolite (acetyl-5-ASA) in human plasma. The method involves liquid-liquid extraction with methanol followed by isocratic reversed-phase chromatography on a Kromasil KR100 C(18) column with electrochemical detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of 5-ASA, acetyl 5-ASA and internal standard were investigated. Limits' of detection were 5 ng/mL for 5-ASA and 10 ng/mL for acetyl-5-ASA, respectively. The method can be used for supporting therapeutical drug monitoring and pharmacokinetic studies.