Inhibition of xCT by sulfasalazine alleviates the depression-like behavior of adult male mice subjected to maternal separation stress.

Maternal separation (MS) can induce emotional disorders. Our previous study reported that MS resulted in depression-like behavior. In this study, we aimed to explore the role of xCT in depression-like behavior in adult mice subjected to MS stress. Pups were divided into the control group, the control + sulfasalazine (SSZ, 75 mg/kg/day, i.p.) group, the MS group, and the MS+SSZ group. After MS, all pups were raised until PD60. Then, the depression-like behavior was detected by the novelty suppressed feeding (NSF) test, the forced swimming test (FST), and the tail suspension test (TST). The synaptic plasticity was examined by electrophysiological recordings and molecular biotechnology. The data showed that, compared with the control group, the mice in the MS group presented depression-like behavior, impairment of long-term potentiation (LTP), a reduction in the number of astrocytes, and activation of the microglia. Moreover, the expression of xCT was increased in the prefrontal cortex of MS mice, the EAAT2 and the Group Ⅱ metabotropic glutamate receptors (mGluR2/3) were decreased, and the level of pro-inflammatory factors was increased in the prefrontal cortex. After the administration with SSZ, the depression-like behavior and the impairment of LTP were alleviated, the number of astrocytes was increased, and the microglial activation was inhibited. Moreover, the levels of EAAT2 and mGluR2/3 were ameliorated, the over-activation of the microglia was mitigated, and the levels of glutamate and pro-inflammatory factors were decreased. In conclusion, the inhibition of xCT by SSZ could alleviate depression-like behavior partly via modulating the homeostasis of the glutamate system and dampening neuroinflammation.

Sulfasalazine and Chromotrope 2B reduce oxidative stress in murine bone marrow-derived mesenchymal stem cells.

BACKGROUND: With advancing age of stem cells, dysregulation of various processes at the cellular level occurs, thereby decreasing their regeneration potential. One of the changes that occurs during the aging process is the accumulation of reactive oxygen species (ROS), which accelerates the processes of cellular senescence and cell death. The aim of this study is to evaluate two antioxidant compounds; Chromotrope 2B and Sulfasalazine, for their antioxidant effects on young and old rat bone marrow mesenchymal stem cells (MSCs). METHODS AND RESULTS: Oxidative stress was induced in MSCs by 5 µM dexamethasone for 96 h and the cells were treated with Chromotrope 2B or Sulfasalazine, 50 µM each. The effects of antioxidant treatment following oxidative stress induction was evaluated by transcriptional profiling of genes involved in the oxidative stress and telomere maintenance. Expression levels of Cat, Gpx7, Sod1, Dhcr24, Idh1, and Txnrd2 were found to be increased in young MSCs (yMSCs) as a result of oxidative stress, while Duox2, Parp1, and Tert1 expression were found to be decreased as compared to the control. In old MSCs (oMSCs), the expressions of Dhcr24, Txnrd2, and Parp1 increased, while that of Duox2, Gpx7, Idh1, and Sod1 decreased following oxidative stress. In both MSC groups, Chromotrope 2B prompted decrease in the ROS generation before and after the induction of oxidative stress. In oMSCs, ROS content was significantly reduced in the Sulfasalazine treated group. CONCLUSION: Our findings suggest that both Chromotrope 2B and Sulfasalazine possess the potential to reduce the ROS content in both age groups, though the latter was found to be more potent. These compounds can be used to precondition MSCs to enhance their regenerative potential for future cell-based therapeutics.

Hydroxychloroquine Effects on THP-1 Macrophage Cholesterol Handling: Cell Culture Studies Corresponding to the TARGET Cardiovascular Trial.

Background and Objectives: Cardiovascular (CV) risk is elevated in rheumatoid arthritis (RA). RA patient plasma causes pro-atherogenic derangements in cholesterol transport leading to macrophage foam cell formation (FCF). The TARGET randomized clinical trial compares CV benefits of 2 RA drug regimens. Hydoxychloroquine (HCQ) is a key medication used in TARGET. This study examines effects of HCQ on lipid transport to elucidate mechanisms underlying TARGET outcomes and as an indicator of likely HCQ effects on atherosclerosis in RA. Materials and Methods: THP1 human macrophages were exposed to media alone, IFNγ (atherogenic cytokine), HCQ, or HCQ + IFNγ. Cholesterol efflux protein and scavenger receptor mRNA levels were quantified by qRT-PCR and corresponding protein levels were assessed by Western blot. FCF was evaluated via Oil-Red-O and fluorescent-oxidized LDL. Intracellular cholesterol and efflux were quantified with Amplex Red assay. Results: With the exception of a decrease in the efflux protein cholesterol 27-hydroxylase in the presence IFNγ at all HCQ concentrations, no significant effect on gene or protein expression was observed upon macrophage exposure to HCQ and this was reflected in the lack of change in FCF and oxidized LDL uptake. Conclusions: HCQ did not significantly affect THP1 macrophage cholesterol transport. This is consistent with TARGET, which postulates superior effects of anti-TNF agents over sulfasalazine + HCQ.

Elevated expression of TAM receptor tyrosine kinase in synovial fluid and synovial tissue of rheumatoid arthritis.

To investigate the expression and roles of TAM (Tyro3/Axl/Mer) receptor tyrosine kinases (TK) in synovial fluid and synovial tissue of patients with rheumatoid arthritis (RA). The expression of TAM TKs in the synovial fluid and synovial tissues of RA and osteoarthritis (OA) patients was measured by ELISA and immunohistochemistry. The relationships between soluble TAM TKs (sTAM TKs) levels and the clinical features, laboratory parameters and disease activity were analyzed in RA. The concentrations of sTAM TK in the synovial fluids of RA patients were increased in comparison to those of OA patients. Compared with OA patients, the expression of membrane Tyro3 TK (mTyro3 TK) and mMer TK in RA patient synovial tissue were significantly increased, which may partly explain the possible mechanism of elevated levels of sTAM TK in RA patient synovial fluid. sAxl TK levels were decreased in RA patients under sulfasalazine treatment and elevated in patients under Iguratimod treatment. Furthermore, sTyro3 TK levels were positively correlated with erythrocyte sedimentation rate (ESR) and negatively correlated with white blood cells (WBCs), red blood cells (RBCs), and hemoglobin (HB) in RA patients. The levels of sMer TK were positively associated with disease duration and rheumatoid factor (RF) and negatively correlated with HB, complement 3 (C3), and C4. Taken together, TAM TKs might be involved in RA synovial tissue inflammation.

Sulfasalazine exacerbates angiotensin II-induced cardiac remodelling by activating Akt signal pathway.

A thorough understanding of the pathological process underlying hypertension-induced cardiac remodelling may help in prevention and treatment of heart failure. Angiotensin II (AngII) results in cardiac fibrosis and hypertrophy partly through activation of inflammation, which increases the fibroblasts and promotes extracellular matrix production. Sulfasalazine (SASP) has evident anti-inflammatory effects and pharmacological functions on autoimmune disease. The roles of SASP in the cardiac remodelling remain unknown. In this study, we established AngII-induced cardiac remodelling mice model and then treated with SASP. Blood pressure, cardiac pump function and pathological changes of cardiac remodelling were analysed in these mice. To explore the mechanism, phosphorylated Akt was detected in vivo and vitro. In this study, we found that SASP aggravated cardiac dysfunction, hypertrophy and fibrosis after AngII infusion. In addition, SASP activated Akt in AngII-remodelled mouse hearts and cardiac cells. Our findings indicate that independent of anti-inflammatory property, SASP exacerbates AngII-induced cardiac remodelling by activation of Akt signalling pathway.

Protective antioxidative and anti-inflammatory actions of ß-caryophyllene against sulfasalazine-induced nephrotoxicity in rat.

The pathogenesis of sulfasalazine (SFZ)-induced nephrotoxicity is unclear. Moreover, there are no reports on the protective effects of ß-caryophyllene (BCP) against SFZ-induced renal injury. Hence, in this study, we measured several oxidative stress and inflammatory regulatory molecules alongside the effects of BCP in SFZ-intoxicated rats. Male rats (n = 48) were distributed to six equal groups as follows: negative control (NC), normal rats treated with low (N-LD; 200 mg/kg/day) and high (N-HD; 400 mg/kg/day) BCP doses, and animals treated with SFZ individually (PC; 600 mg/kg/day) or combined with BCP low (P-LD) and high (P-HD) doses. All drugs were administrated for 14 consecutive days. The NC, N-LD, and N-HD groups showed comparable renal histology and biochemistry. In contrast, abnormal histology, and increased creatinine and urea alongside oliguria and proteinuria were detected in the PC group. Renal specimens from the PC group revealed increased levels of nuclear factor-kappa B (NF-κB), transforming growth factor (TGF)-ß with kidney injury molecule (KIM)-1, while the levels of nuclear factor erythroid 2-related factor 2 (Nrf2), AMP-activated protein kinase (AMPK), and protein kinase B (AKT) declined, relative to controls. The PC renal tissue also had markedly higher levels of inflammatory cytokines (tumor necrosis factor [TNF]-α/interleukin [IL]-1ß/IL-6) and pro-oxidants (malondialdehyde [MDA]/H2O2/protein carbonyls), whereas those of antioxidants (glutathione [GSH]/glutathione peroxidase [GPx]/superoxide dismutase-1 [SOD1]/catalase [CAT]) and IL-10 decreased and were associated with marked apoptosis. Both BCP regimens ameliorated renal functions and histology, and reduced NF-κB, TGF-ß, and KIM-1 levels in addition to those of oxidative stress and inflammation markers. Both protocols also augmented Nrf2, AMPK, AKT, antioxidants, and IL-10. However, P-HD showed better alleviating effects than the N-HD group. In conclusion, this study is the first to link NF-κB, TGF-ß, Nrf2, AMPK, and AKT with SFZ-induced nephrotoxicity. In addition, this is the first report to reveal antioxidative and anti-inflammatory effects for BCP against SFZ-associated nephropathy.

Determination of Sulphasalazine and Its Related Compounds by Simple, Smart, Validated, Green Spectrophotometric Methods.

BACKGROUND: Sulphasalazine (SZ) is a prodrug. Its active metabolite (mesalazine, MZ), which is also available in pharmaceutical formulations, and the major active metabolite of MZ (N-acetyl-5-aminosalicylic acid, AS) are commonly used for the treatment of inflammatory bowel diseases. OBJECTIVE: Two accurate, precise, sensitive, and specific spectrophotometric methods were developed and validated for determination of the studied components. METHODS: The first method is a modified ratio difference spectrophotometric method. In this method, SZ was determined by measuring the peak area from 410-500 nm, while MZ and AS were determined by measuring the difference of the selected amplitude values. The second method is a mean centering of ratio spectra spectrophotometric method. RESULTS: The developed methods were linear over the concentration ranges of 2-35, 2-30 and 1-25 µg/mL for SZ, MZ and AS, respectively. CONCLUSION: The developed methods were validated according to International Conference on Harmonization guidelines. They were successfully applied for determination of studied analytes. A greenness assessment was undertaken using three different tools. HIGHLIGHTS: Spectrophotometric methods were developed for determination of SZ and its related compounds for the first time. They were designated to be green and eco-friendly and their greenness profiles were evaluated using green solvents to keep the environment clean.

Quantification and Metabolite Identification of Sulfasalazine in Mouse Brain and Plasma Using Quadrupole-Time-of-Flight Mass Spectrometry.

Sulfasalazine (SAS), an anti-inflammatory drug with potent cysteine/glutamate antiporter system xc-(SXC) inhibition has recently shown beneficial effects in brain-related diseases. Despite many reports related to central nervous system (CNS) effect of SAS, pharmacokinetics (PK) and metabolite identification studies in the brain for SAS were quite limited. The aim of this study was to investigate the pharmacokinetics and metabolite identification of SAS and their distributions in mouse brain. Using in vivo brain exposure studies (neuro PK), the PK parameters of SAS was calculated for plasma as well as brain following intravenous and oral administration at 10 mg/kg and 50 mg/kg in mouse, respectively. In addition, in vivo metabolite identification (MetID) studies of SAS in plasma and brain were also conducted. The concentration of SAS in brain was much lower than that in plasma and only 1.26% of SAS was detected in mouse brain when compared to the SAS concentration in plasma (brain to plasma ratio (%): 1.26). In the MetID study, sulfapyridine (SP), hydroxy-sulfapyridine (SP-OH), and N-acetyl sulfapyridine (Ac-SP) were identified in plasma, whereas only SP and Ac-SP were identified as significant metabolites in brain. As a conclusion, our results suggest that the metabolites of SAS such as SP and Ac-SP might be responsible for the pharmacological effect in brain, not the SAS itself.

Cystine-glutamate antiporter xCT as a therapeutic target for cancer.

Cystine/glutamic acid reverse transporter (System Xc - ), a member of the amino acid transporter family, consists of two subunits, light chain xCT and heavy chain 4F2hc. xCT is the cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11), which promotes cystine uptake and glutathione biosynthetic, thus protecting against oxidative stress and ferroptosis. Studies have confirmed that xCT is highly expressed in a variety of tumour and is associated with tumour proliferation, invasion, metastasis, drug resistance and ferroptosis, and can be used as a potential target for tumour treatment. This review provides insights into the biological effects of xCT and contribute to the development of new xCT-based strategies.

System Xc- Antiporter Inhibitors: Azo-Linked Amino-Naphthyl-Sulfonate Analogues of Sulfasalazine.

The cystine/glutamate antiporter system Xc- (SXc-) mediates the exchange of intracellular L-glutamate (L-Glu) with extracellular L-cystine (L-Cys2). Both the import of L-Cys2 and the export of L-Glu take on added significance in CNS cells, especially astrocytes. When the relative activity of SXc- overwhelms the regulatory capacity of the EAATs, the efflux of L-Glu through the antiporter can be significant enough to trigger excitotoxic pathology, as is thought to occur in glioblastoma. This has prompted considerable interest in the pharmacological specificity of SXc- and the development of inhibitors. The present study explores a series of analogues that are structurally related to sulfasalazine, a widely employed inhibitor of SXc-. We identify a number of novel aryl-substituted amino-naphthylsulfonate analogues that inhibit SXc- more potently than sulfasalazine. Interestingly, the inhibitors switch from a competitive to noncompetitive mechanism with increased length and lipophilic substitutions, a structure-activity relationship that was previously observed with aryl-substituted isoxazole. These results suggest that the two classes of inhibitors may interact with some of the same domains on the antiporter protein and that the substrate and inhibitor binding sites may be in close proximity to one another. Molecular modeling is used to explore this possibility.

Effects of salazosulfapyridine on the profile of cell surface proteins, revealed by biotinylation of cell surface proteins and 2-dimentional electrophoresis.

OBJECTIVE: We investigated effects of salazosulfapyridine (SASP) on the protein profile of cell surface (CS)-proteins of SW982, a human synovial sarcoma cell line, using biotinylation of CS-proteins and 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE). METHODS: SW982 cells were treated with SASP and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5ASA). Then the cells were treated with a membrane-impermeable biotinylating reagent. Biotinylated CS-proteins were isolated using NeutrAvidin-bound beads. CS-proteins affected by the drugs were detected by 2D-DIGE and subjected to mass spectrometry. RESULTS: By the 2D-DIGE analysis, in total 576 spots were detected, 29 out of which showed more than ±1.5-fold different intensity in the SASP-, SP-, and 5ASA-treated cells, compared to non-treated cells (p < 0.05). Interestingly, 7 out of the 29 spots changed their intensity only by SASP and 17 spots changed their intensity only by SP. We identified 9 protein from 15 out of the 29 spots, most of which were evidenced to exist on the cell surface by flow cytometry. CONCLUSION: We found novel effects of SASP and its metabolites on SW982 cells by the combination of biotinylation of cell surface proteins and 2D-DIGE analysis. These data would help understanding of anti-rheumatic actions of SASP. Furthermore, the combination would be a useful method for the analysis of CS-proteins in various conditions.

Effects of Various Pharmaceutical Excipients on the Intestinal Transport and Absorption of Sulfasalazine, a Typical Substrate of Breast Cancer Resistance Protein Transporter.

Breast cancer resistance protein (BCRP) transporter is an efflux transporter that utilizes energy from adenosine triphosphate hydrolysis to push its substrates, regardless of the concentration gradient. Its presence on the apical membrane of the intestinal mucosa is a major obstacle for the intestinal absorption of its substrates. In this study, we examined the effects of various pharmaceutical excipients on the intestinal transport and absorption of sulfasalazine, a BCRP substrate. Four excipients, including 0.05% and 0.075% BL-9EX, 0.01% and 0.05% Brij 97, 0.075% Labrasol, and 0.05% and 0.1% Tween 20 decreased the secretory transport of sulfasalazine in an in vitro diffusion chamber. Further investigation in an in situ closed loop experiment in rats showed that 0.05% and 0.1% BL-9EX and 0.1% Brij 97 effectively enhanced the intestinal absorption of sulfasalazine while maintaining minimal toxicity to the intestinal mucosa. However, 0.1% Brij 97 also increased the intestinal absorption of 5(6)-carboxyfluorescein, a paracellular marker compound. These findings suggest that BL-9EX might effectively inhibit the BCRP-mediated efflux of sulfasalazine in vivo, indicating that BL-9EX could improve the intestinal absorption of sulfasalazine and other BCRP substrates.

Mucoadhesive chitosan hydrogels as rectal drug delivery vessels to treat ulcerative colitis.

Mucoadhesive drug delivery systems stick to mucosal tissues and prolong the local retention time of drugs. Since the colon is covered by a mucosal layer, mucoadhesive rectal formulations may improve treatment of such diseases as hypertension or colon cancer. Ulcerative colitis (UC) is an inflammatory bowel disease characterized by chronic inflammation of the colonic mucosa. It is commonly treated with sulfasalazine (SSZ), which is metabolized by the intestinal flora into the therapeutic 5-aminosalicylic acid (5-ASA) and a toxic by-product sulfapyridine (SP). SSZ can be administered orally or rectally. The latter route avoids unintended absorption of the drug or its degradation products in the upper gastrointestinal tract, but often fails due to limited retention time. Here, we propose a mucoadhesive hydrogel to improve the efficacy of rectal SSZ administration. The gel is made of catechol modified-chitosan (Cat-CS) crosslinked by genipin. After loading the gel with SSZ, we evaluated its efficacy in a mouse model of UC. Compared to oral SSZ treatment, rectal SSZ/Cat-CS delivery was more therapeutic, showed equivalent histological scores, and induced a lower plasma concentration of the potentially toxic SP by-product. These results show SSZ/Cat-CS rectal hydrogels are more effective and safer formulations for UC treatment than oral SSZ. STATEMENT OF SIGNIFICANCE: Ulcerative colitis affects the colon by causing chronic inflammation on the mucosa. One of the most common drugs to treat mild to moderate UC is sulfasalazine, which can be administrated both orally and rectally. Rectal formulations are preferable, since their therapeutic effect happens topically, and they prevent side effects related to absorption of the drug in the small intestine. However, the efficacy of rectal sulfasalazine formulations is decreased by their limited colon residence time. Here we propose a chitosan-catechol mucoadhesive gel that allows delivering sulfasalazine more effectively and safely than oral administration. Our results bring new insights into the field of mussel-inspired catechol hydrogels, showing their potential as drug delivery systems to treat a widespread disease such as ulcerative colitis.

Jump into a New Fold-A Homology Based Model for the ABCG2/BCRP Multidrug Transporter.

ABCG2/BCRP is a membrane protein, involved in xenobiotic and endobiotic transport in key pharmacological barriers and drug metabolizing organs, in the protection of stem cells, and in multidrug resistance of cancer. Pharmacogenetic studies implicated the role of ABCG2 in response to widely used medicines and anticancer agents, as well as in gout. Its Q141K variant exhibits decreased functional expression thus increased drug accumulation and decreased urate secretion. Still, there has been no reliable molecular model available for this protein, as the published structures of other ABC transporters could not be properly fitted to the ABCG2 topology and experimental data. The recently published high resolution structure of a close homologue, the ABCG5-ABCG8 heterodimer, revealed a new ABC transporter fold, unique for ABCG proteins. Here we present a structural model of the ABCG2 homodimer based on this fold and detail the experimental results supporting this model. In order to describe the effect of mutations on structure and dynamics, and characterize substrate recognition and cholesterol regulation we performed molecular dynamics simulations using full length ABCG2 protein embedded in a membrane bilayer and in silico docking simulations. Our results show that in the Q141K variant the introduced positive charge diminishes the interaction between the nucleotide binding and transmembrane domains and the R482G variation alters the orientation of transmembrane helices. Moreover, the R482 position, which plays a role the substrate specificity of the transporter, is located in one of the substrate binding pockets identified by the in silico docking calculations. In summary, the ABCG2 model and in silico simulations presented here may have significant impact on understanding drug distribution and toxicity, as well as drug development against cancer chemotherapy resistance or gout.

Sulfasalazine and its metabolites inhibit platelet function in patients with inflammatory arthritis.

The purpose of this study is to assess the effect of sulfasalazine and its metabolites on platelet function in patients with inflammatory arthritis (IA). One hundred thirty-five consecutive patients with an established diagnosis of IA were screened. Those with a history of cardiovascular disease (CVD), taking anti-platelet agents or non-steroidal anti-inflammatory drugs (NSAIDs) were excluded. A total of 32 patients were investigated, 15 taking sulfasalazine and 17 taking other disease-modifying anti-rheumatic drugs (DMARDs) and no sulfasalazine. These two cohorts were compared to 15 patients with stable CVD on long-term aspirin. The effect of sulfasalazine and its metabolites on arachidonic acid (AA)-induced platelet aggregation was also tested in vitro in samples from healthy donors (n = 18). Demographics, CVD risk factors and disease activity indices were similar in the sulfasalazine and other DMARD groups. AA-induced platelet aggregation was significantly inhibited in the sulfasalazine group (9 ± 7 %) and comparable to that in the aspirin group (10 ± 6 %). In contrast, there was no effect on AA-induced platelet aggregation in the other DMARDs group (77 ± 12 %) (p < 0.001). Furthermore, sulfasalazine therapy had no effect on platelet aggregation in response to multiple other agonists. Sulfasalazine and its metabolites (5-aminosalicylic acid and sulfapyridine) exerted an additive and dose-dependent inhibitory effect on AA-induced platelet aggregation in vitro (p < 0.001). The inhibition of AA-induced platelet aggregation by sulfasalazine is comparable to that achieved by aspirin and is dependent on both sulfasalazine and its metabolites. This represents a potential mechanism that may contribute to the known cardioprotective effect of sulfasalazine in patients with IA.

Spectroscopic analysis of the impact of oxidative stress on the structure of human serum albumin (HSA) in terms of its binding properties.

Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis (RA). Reactive oxygen species (ROS) are produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative bursts, exceed the physiological buffering capacity and result in oxidative stress. ROS result in oxidation of serum albumin, which causes a number of structural changes in the spatial structure, may influence the binding and cause significant drug interactions, particularly in polytherapy. During the oxidation modification of amino acid residues, particularly cysteine and methionine may occur. The aim of the study was to investigate the influence of oxidative stress on human serum albumin (HSA) structure and evaluate of possible alterations in the binding of the drug to oxidized human serum albumin (oHSA). HSA was oxidized by a chloramine-T (CT). CT reacts rapidly with sulfhydryl groups and at pH 7.4 the reaction was monitored by spectroscopic techniques. Modification of free thiol group in the Cys residue in HSA was quantitatively determined by the use of Ellman's reagent. Changes of albumin conformation were examined by comparison of modified (oHSA) and nonmodified human serum albumin (HSA) absorption spectra, emission spectra, red-edge shift (REES) and synchronous spectroscopy. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region of 200-250 nm involve structural alterations in peptide backbone conformation. Synchronous fluorescence spectroscopy technique confirmed changes of position of tryptophanyl and tyrosyl residues fluorescent band caused by CT. Moreover analysis of REES effect allowed to observe structural changes caused by CT in the region of the hydrophobic pocket containing the tryptophanyl residue. Effect of oxidative stress on binding of anti-rheumatic drugs, sulfasalazine (SSZ) and sulindac (SLD) in the high and low affinity binding sites was investigated by spectrofluorescence, ITC and (1)H NMR spectroscopy, respectively. SSZ and SLD change the affinity of each other to the binding site in non- and modified human serum albumin. The presence of SLD causes the increase of association constant (Ka) of SSZ-oHSA system and the strength of binding and the stability of the complexes has been observed while in the presence of SSZ a displacement of SLD from the SLD-HSA has been recorded. The analysis of (1)H NMR spectral parameters i.e. changes of chemical shifts of the drug indicate that the presence of SSZ and SLD have a mutual influence on changes in the affinity of human serum albumin binding site and this competition takes place not only due to the additional drug but also to the oxidation of HSA.

Influence of feeding regimens on rat gut fluids and colonic metabolism of diclofenac-ß-cyclodextrin.

Feeding states may affect the performance of colonic prodrugs. The aim is to investigate the influence of feeding regimen in Wistar rats on: (i) distribution and pH contents along the gut and (ii) metabolism of two colonic prodrugs, diclofenac-ß-cyclodextrin and a commercially available control, sulfasalazine, within the caecal and colonic contents. Male Wistar rats were subject to four different feeding regimens, the gut contents characterized (mass and pH) and the metabolism of prodrugs investigated. The feeding regimen affects gut contents (mass and pH), more specifically in the stomach and lower intestine, and affects the rate of metabolism of diclofenac-ß-cyclodextrin, but not that of sulfasalazine. The latter's degradation is much faster than that of diclofenac-ß-cyclodextrin while the metabolism of both prodrugs is faster in colonic (versus caecal) contents. Fasting results in most rapid degradation of diclofenac-ß-cyclodextrin, possibly due to lack of competition (absence of food) for microbial enzymatic activity.