The Role of Claudins in the Pathogenesis of Dextran Sulfate Sodium-Induced Experimental Colitis: The Effects of Nobiletin.

BACKGROUND: The pathogenesis of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease is not well understood. This study investigated the roles and regulation of the claudin-1, -2, -3, and -4 isoforms in the pathogenesis of ulcerative colitis, and the potential therapeutic effects of nobiletin. METHODS: Colitis was induced in rats by administering dextran sulfate sodium [DSS] in drinking water for seven days. Animals were treated daily with nobiletin [oral, 60 mg/Kg body weight] and studied in four groups, C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. On day seven, the animals were sacrificed, and colonic tissues were collected and analyzed. RESULTS: Both macroscopic and microscopic findings suggest the progression of colitis. In the inflamed colon, claudin-1 and -4 proteins were decreased, claudin-2 increased, while the claudin-3 protein remained unchanged. Except for claudin-1, these changes were not paralleled by mRNA expression, indicating a complex regulatory mechanism. Uniform ß-actin expression along with consistent quality and yield of total RNA indicated selectivity of these changes. Nobiletin treatment reversed these changes. CONCLUSIONS: Altered expression of the claudin isoforms -1, -2, and -4 disrupts tight junctions, exposing the lamina propria to microflora, leading to electrolyte disturbance and the development of ulcerative colitis. Nobiletin with its anti-inflammatory properties may be useful in IBD.

Synthesis, in Vitro Cholinesterase Inhibition, Molecular Docking, DFT, and ADME Studies of Novel 1,3,4-Oxadiazole-2-Thiol Derivatives.

A series of 1,3,4-oxadiazole-2-thiol derivatives bearing various alkyl or aryl moieties were designed, synthesized, and characterized using modern spectroscopic methods to yield 17 compounds (6a-6q) that were screened for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes in the search for 'lead' compounds for Alzheimer's disease treatment (AD). The compounds 6q, 6p, 6k, 6o, and 6l showed inhibitory capability against AChE and BChE, with IC50 values ranging from 11.73±0.49 to 27.36±0.29 µM for AChE and 21.83±0.39 to 39.43±0.44 µM for BChE, inhibiting both enzymes within a limited range. The SAR ascertained that the substitution of the aromatic moiety had a profound effect on the AChE and BChE inhibitory potential as compared to the aliphatic substitutions which were supported by the molecular docking studies. The drug-likeness of the most synthesized compounds was confirmed by in silico ADME investigations. These results were additionally supplemented by the molecular orbital analysis (HOMO-LUMO) and electrostatic potential maps got from DFT calculations. ESP maps expose that on all structures, there are two potential binding sites conquered by the most positive and most negative districts.

Synthesis, characterization, and computational study of copper bipyridine complex [Cu (C18H24N2) (NO3)2] to explore its functional properties.

In the present study, copper (II) complex of 4, 4'-di-tert-butyl-2,2'-bipyridine [Cu (C18H24N2) (NO3)2], 1 is investigated through its synthesis and characterization using elemental analysis technique, infra-red spectroscopy, and single-crystal analysis. The compound 1 crystallizes in orthorhombic space group P212121. The copper atom in the mononuclear complex is hexa coordinated through two nitrogen and four oxygen atoms from bipyridine ligand and nitrate ligands. The thermal analysis depicts the stability of the entitled compound up to 170 °C, and the decomposition takes place in different steps between 170 and 1000 °C. Furthermore, quantum chemical techniques are used to study optoelectronic, nonlinear optical, and therapeutic bioactivity. The values of isotropic and anisotropic linear polarizabilities of compound 1 are calculated as 41.65 × 10-24 and 23.02 × 10-24 esu, respectively. Likewise, the static hyperpolarizability is calculated as 47.92 × 10-36 esu using M06 functional compared with para-nitroaniline (p-NA) and found several times larger than p-NA. Furthermore, the antiviral potential of compound 1 is studied using molecular docking technique where intermolecular interactions are checked between the entitled compound and two crucial proteins of SARS-CoV-2 (COVID-19). Our investigation indicated that compound 1 interacts more vigorously to spike protein than main protease (MPro) due to its better binding energy of -9.60 kcal/mol compared with -9.10 kcal/mol of MPro. Our current study anticipated that the above-entitled coordination complexes could be potential candidates for optoelectronic properties and their biological activity.

Unraveling the Electrical and Magnetic Properties of Layered Conductive Metal-Organic Framework With Atomic Precision.

This paper describes structural elucidation of a layered conductive metal-organic framework (MOF) material Cu3 (C6 O6 )2 by microcrystal electron diffraction with sub-angstrom precision. This insight enables the first identification of an unusual π-stacking interaction in a layered MOF material characterized by an extremely short (2.73 Å) close packing of the ligand arising from pancake bonding and ordered water clusters within pores. Band structure analysis suggests semiconductive properties of the MOF, which are likely related to the localized nature of pancake bonds and the formation of a singlet dimer of the ligand. The spin of CuII within the Kagomé arrangement dominates the paramagnetism of the MOF, leading to strong geometrical magnetic frustration.

Detection of mutations in NOD2/CARD15 gene in Arab patients with Crohn's disease.

Background: Mutations in NOD2/CARD15 gene have been linked to an increased risk of Crohn's disease (CD). The objective of this study is to determine NOD2/CARD15 gene mutations, and their association with the risk of CD in Arabs in Kuwait. Methods: Four NOD2 gene mutations, including Pro268Ser (SNP5), Arg702Trp (SNP8), Gly908Arg (SNP12), and Leu1007FsinsC (SNP13) were examined in Arab CD patients (n = 103) and control subjects (n = 100). The genomic DNA was isolated and used in polymerase chain reaction (PCR) with four sets of specific primers. The PCR-amplified DNA fragments were sequenced and analyzed for the NOD2 mutations. Logistic regression was used to estimate the adjusted odds ratios (aOR) and 95% confidence intervals (CI). Results: Of the four genotyped variants, the Arg702Trp (SNP8) and Leu1007FsinsC (SNP13) variants were not informative in our study sample due to minor allele frequency of <1%. The Pro268Ser (SNP5) mutation was detected in 17 (16.5%) CD patients and 32 (32.0%) controls. The Gly908Arg (SNP12) mutation was observed in 24 (23.3%) patients and 10 (10.0%) controls. In the dominant genetic risk model (i.e. carrying at least one minor allele), CD patients compared to controls were less likely to carry either the "CT" or "TT" genotype of variant Pro268Ser (SNP5; aOR = 0.43, 95% CI: 0.22-0.84). In contrast, CD patients compared to controls were more likely to carry the homozygous for the minor allele or the heterozygous genotypes of variant Gly908Arg (SNP12; aOR = 2.67, 95% CI: 1.19-5.97). Conclusions: In this Arab population, carrying at least one copy of the minor allele of Gly908Arg (SNP12) mutation in NOD2 gene was associated with increased susceptibility to CD, while having the heterozygous or homozygous for the minor allele genotype of the Pro268Ser (SNP5) mutation provided protection against CD. Mutations in Arg702Trp (SNP8) and Leu1007FsinsC (SNP13) were not detected in this sample of the Arab population in Kuwait.

Uncoupling of Carbonic Anhydrase from Na-H exchanger-1 in Experimental Colitis: A Possible Mechanistic Link with Na-H Exchanger.

In this study, we investigated a mechanistic link between Na-H exchanger-1 (NHE-1) and carbonic anhydrase (CA) in experimental colitis induced in the rats by intrarectal administration of trinitrobenzenesulphonic acid (TNBS). Western blot analysis showed CA-I and CA-II as the major isoforms and CA-IV as a minor one in the colon, and they all are expressed as minor isoforms in the ileum. Co-immunoprecipitation and confocal immunofluorescence microscopy showed colocalization of NHE-1 with CA-I and CA-II, but not with CA-IV. TNBS significantly reduced the levels of NHE-1 and CA protein isoforms in the colon, but not in the uninflamed ileum. A similar reduction profile of the expression of CA isozymes was also obtained in ex vivo treatment of normal colon strips with TNF-α. The level of uncoupling as detected by co-immunoprecipitation was significantly more pronounced. A peptide (83 aa) from the NHE-1 C-terminus demonstrated binding of CA-II only, but not of the CA-I or CA-IV isoform. Furthermore, the profile of inflammatory test markers confirmed inflammation in the tissue used. These findings taken together suggest an inflammation-induced uncoupling of CA and NHE-1, which might be a putative mechanism for reducing the activity of NHE-1 in experimental colitis. This uncoupling might lead to an intracellular accumulation of H+, resulting in acidosis and necrosis in the inflamed colon.

In vitro spleen cell cytokine responses of adult mice immunized with a recombinant PorA (major outer membrane protein [MOMP]) from Campylobacter jejuni.

There is no information on cytokine profiles for use as markers of protection in Campylobacter jejuni infection. To study this, we used outer membrane protein (MOMP [PorA]) as the vaccine for protection and spleen cell cytokines as markers of protection. We cloned and expressed porA from C. jejuni111 and immunized mice by the intraperitoneal route. Subsequently, mice were orally challenged with live C. jejuni 111. The vaccine induced protection as evidenced by reduced fecal excretion of C. jejuni111. Cytokines were measured in vitro after stimulation of spleen cells with MOMP. The levels of pro-inflammatory cytokines, IL-12, TNF-α, IL-17A and IL-17F were similar in control and test mice. The levels of pro-inflammatory cytokines, IL-2 and IFN-γ were higher in control mice than in test mice, and the levels of pro-inflammatory cytokines, IL-8 and IL-1ß were higher in test mice than in control mice. Among the two anti-inflammatory cytokines, the levels were similar for IL-10 but higher for IL-4 in test mice than in control mice. Ratios of pro-inflammatory to anti-inflammatory cytokines showed a bias towards an anti-inflammatory response in favor of antibody production reflecting the role of antibodies in immunity. Cytokine production patterns by spleen cells may be used as markers of protection in the mouse model.

Synthesis, Thermal, Structural Analyses, and Photoluminescent Properties of a New Family of Malonate-Containing Lanthanide(III) Coordination Polymers.

Five new Lanthanide(III) complexes of malonic acid (HOOC-CH2-COOH); {[Gd(C3H2O4)(H2O)4]·NO3} n (1), {[Tb(C3H2O4)(H2O)4]·NO3} n (2), {[Ho(C3H2O4)(H2O)4]·NO3} n (3), [Er(C3H2O4)(C3H3O4)(H2O)2] n (4), and {[Eu2(C3H2O4)2(C3H3O4)2(H2O)6]·4H2O} n (5) were synthesized and characterized by elemental, infrared spectral, and thermal analyses. The structures of compounds 1-5 were determined by single crystal X-ray diffraction technique. The X-ray analysis reveals that compounds 1, 2, and 3 are isostructural and crystallized in the orthorhombic space group Pmn21. The lanthanide(III) ions are coordinated by four carboxylate and four water oxygen atoms adopting a distorted square antiprism geometry. The LnO8 square antiprisms are linked into infinite layers by malonate (C3H2 O 4 2 - ) dianions sandwiching sheets of nitrate counter ions. Compound 4 contains ErO8 square antiprisms linked into a two-dimensional network by hydrogen malonate (C3H3 O 4 - ) anions and malonate dianions. The europium complex, 5 is dinuclear having the two europium(III) ions (Eu1 and Eu2) bridged by carboxylate groups of hydrogen malonate ligands. The europium ions in 5 are nine-coordinate and exhibit a distorted monocapped square antiprism geometry. All the structures are consolidated by O-H⋯ O hydrogen bonds. The photoluminescence spectra of 1-5 exhibit characteristic emissions in the visible region. The IR spectra and thermal data are consistent with the structural results. The room-temperature effective magnetic moments for 1-4 are in good agreement with those expected for the free ions, while the data for 5 indicates that low-lying excited states contribute to the observed moment. The compound 1 was further subjected to quantum computational calculations to explore its optoelectronic properties including; density of states (DOS), dielectric function, refractive index, extinction coefficient, and absorption spectrum, to highlight the possible applications of such materials in the optoelectronics.

Novel pyridine-2,4,6-tricarbohydrazide thiourea compounds as small key organic molecules for the potential treatment of type-2 diabetes mellitus: In vitro studies against yeast α- and ß-glucosidase and in silico molecular modeling.

A range of novel pyridine-2,4,6-tricarbohydrazide thiourea compounds (4a-i) were synthesized in good to excellent yields (63-92%). The enzyme inhibitory potentials of these compounds were investigated against α- and ß-glucosidases because these enzymes play a crucial role in treating type-2 diabetes mellitus (T2DM). As compared to the reference compound acarbose (IC50 38.22 ± 0.12 µM), compounds 4i (IC50 25.49 ± 0.67 µM), 4f (IC50 28.91 ± 0.43 µM), 4h (IC50 30.66 ± 0.52 µM), and 4e (IC50 35.01 ± 0.45 µM) delivered better inhibition against α-glucosidase and were quite inactive/completely inactive against ß-glucosidase. The structure-activity relationship of these compounds was developed and elaborated with the help of molecular docking studies.

Mechanism of down regulation of Na-H exchanger-2 in experimental colitis.

BACKGROUND: The Na-H exchanger [NHE] performs an electroneutral uptake of NaCl and water from the lumen of the gastrointestinal tract. There are several distinct NHE isoforms, some of which show an altered expression in the inflammatory bowel diseases (IBD). In this study, we examined a role of NHE-2 in experimental colitis. METHODS: Colitis was induced in male Sprague-Dawley rats by intra-rectal administration of trinitrobenzenesulphonic acid (TNBS). On day 6 post-TNBS, the animals were sacrificed, colonic and ileal segments were taken out, cleaned with phosphate buffered saline and used in this study. RESULTS: There was a significant decrease in the level of NHE-2 protein as measured by ECL western blot analysis and confocal immunofluorescence microscopy. The levels of NHE-2 mRNA and heteronuclear RNA measured by an end-point RT-PCR and a real time PCR were also decreased significantly in the inflamed colon. However, there was no change in the level of NHE-2 protein in response to in vitro TNF-α treatment of uninflamed rat colonic segment. These changes were selective and localized to the colon as actin, an internal control, remained unchanged. Confocal immunofluorescence microscopy revealed co-localization of NHE-2 and NHE-3 in the brush borders of colonic epithelial cells. Inflamed colon showed a significant increase in myeloperoxidase activity and colon hypertrophy. In addition, there was a significant decrease in body weight and goblet cells' mucin staining in the TNBS treated colon. These changes were not conspicuous in the non-inflamed ileum. CONCLUSIONS: These findings demonstrate suppression of NHE-2 expression on the brush borders in the colonic epithelial cells which is regulated transcriptionally. However a role of TNF-α in the regulation of NHE-2 is discounted in the present model of colitis. This decrease in the NHE-2 expression will lead to a loss of electrolyte and water uptake thus contributing to the symptoms associated with IBD.

Organocatalyzed and mechanochemical solvent-free synthesis of novel and functionalized bis-biphenyl substituted thiazolidinones as potent tyrosinase inhibitors: SAR and molecular modeling studies.

Eluding the involvement of solvents in organic synthesis and introducing environment friendly procedures can control environmental problems. A facile and an efficient solvent free mechanochemical method (grinding) is achieved to synthesize novel bis-biphenyl substituted thiazolidinones using non-toxic and cheap N-acetyl glycine (NAG). Organocatalytic condensation of a series of Schiff's bases bearing different substituents with thioglycolic acid produces a variety of thiazolidinones derivatives in good to excellent yield. In vitro inhibition studies against mushroom tyrosinase of these thiazolidinone analogues revealed that many of them possessed good to excellent tyrosinase inhibition at low micro-molar concentrations. In particular, six compounds exhibited potent inhibitory potential with IC50 values ranging from 0.61 ± 0.31 to 21.61 ± 0.11 µM as compared with that of standard kojic acid (IC50 6.04 ± 0.11 µM). Further molecular docking studies revealed that the thiazolidinones moiety plays a key role in the inhibition mechanism by well fitting into the enzyme bounding pocket.

Antioxidant Supplements and Gastrointestinal Diseases: A Critical Appraisal.

The gastrointestinal tract digests and absorbs dietary nutrients, protects the body against physical and chemical damage from contents in its lumen, provides immunity against external antigens, and keeps an optimum environment for the gut microbiota. These functions cannot be performed normally in several diseases of which the following are discussed here: irritable bowel syndrome and inflammatory bowel disease, which includes Crohn's disease and ulcerative colitis. Because these diseases are associated with oxidative stress, a host of antioxidant supplements are used for maintenance and recovery of the gut functions. However, the benefits of these supplements have not been established. The available 80 human trials were rated for levels of confidence and for benefits of the antioxidant supplements. For Crohn's disease, the supplements for which clear benefits occurred in at least 2 studies were allopurinol, Boswellia serrata (frankincense or shallaki), Artemesia species (wormwood), Tripterygium wilfordii (léi gong téng), and omega-3 fatty acids. Similar beneficial supplements for ulcerative colitis were allopurinol, Matricaria chamomilla (chamomile), Curcuma longa (curcumin in turmeric), and omega-3 fatty acids. There was also a clear benefit for ulcerative colitis in 2 studies where a multiherbal Chinese medicine preparation and an Ayurvedic medicine preparation were used. For irritable bowel syndrome, there was only a marginal benefit of some of the antioxidant supplements. Thus, some antioxidant supplements may be beneficial at certain stages of specific diseases. This is consistent with the current concept that antioxidants act by inhibiting oxidative stress pathways in a tissue- and environment-specific manner and not by simply acting as scavengers.

An Efficient Synthesis of bi-Aryl Pyrimidine Heterocycles: Potential New Drug Candidates to Treat Alzheimer's Disease.

A series of 13 novel pyrimidine-based sulfonamides 6a-m were synthesized in short periods of time under microwave conditions in good to excellent yield (54-86%). The chemical structures of these heterocycles consist of a central pyrimidine ring having a phenyl group and pyrimidine groups with sulfonamide motifs. The enzyme inhibitory potential of these compounds was investigated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) because these enzymes play a crucial role in the treatment of Alzheimer's disease. As compared to the reference compound eserine (IC50 = 0.04 ± 0.0001 µM for AChE and IC50 = 0.85 ± 0.0001 µM for BChE), the IC50 values of the synthesized compounds ranged from 3.73 ± 0.61 µM to 57.36 ± 0.22 µM for AChE and 4.81 ± 0.16 µM to 111.61 ± 0.53 µM for BChE. Among these tested compounds, 6j having a -CH3 group was found to be the most potent one against both enzymes (AChE, IC50 = 3.73 ± 0.61 µM; BChE, IC50 = 4.81 ± 0.16 µM). Quantitative structure-activity relationship (QSAR) and molecular docking studies of the synthesized compounds were also performed.

Novel biphenyl bis-sulfonamides as acetyl and butyrylcholinesterase inhibitors: Synthesis, biological evaluation and molecular modeling studies.

A series of new biphenyl bis-sulfonamide derivatives 2a-3p were synthesized in good to excellent yield (76-98%). The inhibitory potential of the synthesized compounds on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was investigated. Most of the screened compounds showed modest in vitro inhibition for both AChE and BChE. Compared to the reference compound eserine (IC50 0.04 ± 0.0001 µM for AChE) and (IC50 0.85 ± 0.0001 µM for BChE), the IC50 values of these compounds were ranged from 2.27 ± 0.01 to 123.11 ± 0.04 µM for AChE and 7.74 ± 0.07 to <400 µM for BuChE. Among the tested compounds, 3p was found to be the most potent against AChE (IC50 2.27 ± 0.01 µM), whereas 3g exhibited the highest inhibition for BChE (IC50 7.74 ± 0.07 µM). Structure-activity relationship (SAR) of these compounds was developed and elaborated with the help of molecular docking studies.

Immunization with a Double-Mutant (R192G/L211A) of the Heat-Labile Enterotoxin of Escherichia coli Offers Partial Protection against Campylobacter jejuni in an Adult Mouse Intestinal Colonization Model.

We have previously shown that antibodies to cholera toxin (CT) reacted with the major outer membrane proteins (MOMPs) from Campylobacter jejuni strains on Western blot. Further, oral immunization with CT significantly protected against challenge with C. jejuni in an adult mouse colonization model of infection. CT and the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli are structurally and functionally related. LT and its mutants including the double-mutant LT (R192G/L211A) (dmLT), are powerful mucosal adjuvants. Unlike LT which is reactogenic, dmLT has been shown to be safe for human use. In the current study, we determined whether rabbit anti-dmLT antibodies reacted with MOMPs from C. jejuni strains and whether immunization with dmLT would afford protection against C. jejuni. On Western blot, the MOMPs from C. jejuni 48 (Penner serotype O:19), C. jejuni 75 (O:3) and C. jejuni 111 (O:1,44) were probed with rabbit antibodies to dmLT or LT-E112K (a non-toxic LT mutant), which showed a lack of reaction. Adult BALB/c mice were orally immunized with dmLT and orally challenged with C. jejuni 48 or 111. Protection from colonization with the challenge bacteria was studied by enumerating Campylobacter colonies in feces daily for 9 days. Vaccination produced robust serum and stool antibody responses to dmLT and no antibody responses to C. jejuni MOMP. Vaccinated mice showed reduced colonization and excretion of both challenge strains compared to control mice. However, the differences were not statistically significant. The protective efficacy of the dmLT vaccine varied from 9.1% to 54.5%. The lack of cross-reaction between the MOMP and dmLT suggests that protection is not mediated by cross-reacting antibodies, but may be due to activation of innate immunity. As dmLT is safe for humans, it could be incorporated into a C. jejuni vaccine to enhance its efficacy.

Pyridine sulfonamide as a small key organic molecule for the potential treatment of type-II diabetes mellitus and Alzheimer's disease: In vitro studies against yeast α-glucosidase, acetylcholinesterase and butyrylcholinesterase.

This paper presents the efficient high yield synthesis of novel pyridine 2,4,6-tricarbohydrazide derivatives (4a-4i) along with their α-glucosidase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition activities. The enzymes inhibition results showed the potential of synthesized compounds in controlling both type-II diabetes mellitus and Alzheimer's disease. In vitro biological investigations revealed that most of compounds were more active against yeast α-glucosidase than the reference compound acarbose (IC50 38.25±0.12µM). Among the tested series the compound 4c bearing 4-flouro benzyl group was noted to be the most active (IC50 25.6±0.2µM) against α-glucosidase, and it displayed weak inhibition activities against AChE and BChE. Compound 4a exhibited the most desired results against all three enzymes, as it was significantly active against all the three enzymes; α-glucosidase (IC50 32.2±0.3µM), AChE (IC50 50.2±0.8µM) and BChE (IC50 43.8±0.8µM). Due to the most favorable activity of 4a against the tested enzymes, for molecular modeling studies this compound was selected to investigate its pattern of interaction with α-glucosidase and AChE targets.

SENSITIVITY PATTERNS OF PSEUDOMONAS AERUGINOSA ISOLATES OBTAINED FROM CLINICAL SPECIMENS IN PESHAWAR.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is a highly virulent opportunistic pathogen and a leading cause of nosocomial infections. Affected patients are often hospitalized in an intensive care unit, and are immuno-compromised as a result of disease and treatment. Suspected P. aeruginosa require timely, adequate and empirical antibiotic therapy to ensure improved outcomes. The purpose of the study was to find the sensitivity and resistance pattern of P. aeruginosa to various groups of drugs, in clinical isolates collected from two major tertiary care hospitals of Peshawar. METHODS: Different clinical isolate were taken from patients admitted in various wards of Khyber Teaching Hospital and Lady Reading Hospital Peshawar. RESULTS: A total of 258 clinical isolates were positive for P. aeruginosa out of 2058 clinical isolates. Pseudomonas showed high degree of resistance to third generation Cephalosporins (Ceftazidime, and Ceftriaxone) and moderate degree of resistance to Quinolones and Aminoglycosides (Ofloxacin, Ciprofloxacin, Levofloxacin and Amikacin). Low resistance was observed to different combinations (Cefoperazone+Sulbactum, Piperacillin+Tazobactum). Meropenem and Imipenem had negligible resistance. CONCLUSION: There is growing resistance to different classes of antibiotics. Combination drugs are useful approach for empirical treatment in suspected Pseudomonas infection. Imipenem and Meropenem are extremely effective but should be in reserve.

Stability indicating RP-HPLC method for simultaneous determination of piroxicam and ofloxacin in binary combination.

A simple and precise RP-HPLC method was developed for simultaneous determination of piroxicam and ofloxacin in pharmaceutical formulations and human serum. Optimum separations of piroxicam, ofloxacin and stress-induced degradation products were achieved by use of Hypersil BDS C8 column (250 x 4.6mm, 5 µm). The mobile phase was a mixture of acetonitrile: 0.012M K2HPO4: 0.008M sodium citrate (both buffers mixed and pH adjusted to 2.8) (50:25:25 v/v/v) delivered at flow rate of 1.5 mL min⁻¹ using DAD at 254 nm. Response was linear function of concentration over the ranges of 70-130 mg mL⁻¹ for piroxicam and ofloxacin (r² ≥ 0.999). The method efficiently separated the analytical peaks from degradation products with acceptable tailing and resolution. The developed method was successfully used for concurrent analysis of piroxicam and ofloxacin in pharmaceutical formulations, human serum and in vitro drug interaction studies.