New sustainable antimicrobial chitosan hydrogels based on sulfonamides and its nanocomposites: Fabrication and characterization.

Chitosan (Ch), a linear cationic biopolymer, has broad medical applications. In this paper, new sustainable hydrogels (Ch-3, Ch-5a, Ch-5b) based on chitosan/sulfonamide derivatives 2-chloro-N-(4-sulfamoylphenethyl) acetamide (3) and/or 5-[(4-sulfamoylphenethyl) carbamoyl] isobenzofuran-1,3-dione (5) were prepared. Hydrogels (Ch-3, Ch-5a, Ch-5b) were loaded (Au, Ag, ZnO) NPs to form its nanocomposites to improve the antimicrobial efficacy of chitosan. The structures of hydrogels and its nanocomposites were characterized using different tools. All hydrogels displayed irregular surface morphology in SEM, however hydrogel (Ch-5a) revealed the highest crystallinity. The highest thermal stability was shown by hydrogel (Ch-5b) compared to chitosan. The nanocomposites represented nanoparticle sizes <100 nm. Antimicrobial activity was assayed for hydrogels using disc diffusion method exhibited great inhibition growth of bacteria compared to chitosan against S. aureus, B. subtilis and S. epidermidis as Gram-positive, E. coli, Proteus, and K. pneumonia as Gram-negative and antifungal activity against Aspergillus Niger and Candida. Hydrogel (Ch-5b) and nanocomposite hydrogel (Ch-3/Ag NPs) showed higher colony forming unit (CFU) and reduction% against S. aureus and E. coli reaching 97.96 % and 89.50 % respectively in comparison with 74.56 % and 40.30 % for chitosan respectively. Overall, fabricated hydrogels and its nanocomposites enhanced the biological activity of chitosan and it can be potential candidates as antimicrobial drugs.

Nanoscale Thermosensitive Hydrogel Scaffolds Promote the Chondrogenic Differentiation of Dental Pulp Stem and Progenitor Cells: A Minimally Invasive Approach for Cartilage Regeneration.

PURPOSE: Several scaffolds and cell sources are being investigated for cartilage regeneration. The aim of the study was to prepare nanocellulose-based thermosensitive injectable hydrogel scaffolds and assess their potential as 3D scaffolds allowing the chondrogenic differentiation of embedded human dental pulp stem and progenitor cells (hDPSCs). MATERIALS AND METHODS: The hydrogel-forming solutions were prepared by adding ß-glycerophosphate (GP) to chitosan (CS) at different ratios. Nanocellulose (NC) suspension was produced from hemp hurd then added dropwise to the CS/GP mixture. In vitro characterization of the prepared hydrogels involved optimizing gelation and degradation time, mass-swelling ratio, and rheological properties. The hydrogel with optimal characteristics, NC-CS/GP-21, was selected for further investigation including assessment of biocompatibility. The chondrogenesis ability of hDPSCs embedded in NC-CS/GP-21 hydrogel was investigated in vitro and compared to that of bone marrow-derived mesenchymal stem cells (BMSCs), then was confirmed in vivo in 12 adult Sprague Dawley rats. RESULTS: The selected hydrogel showed stability in culture media, had a gelation time of 2.8 minutes, showed a highly porous microstructure by scanning electron microscope, and was morphologically intact in vivo for 14 days after injection. Histological and immunohistochemical analyses and real-time PCR confirmed the chondrogenesis ability of hDPSCs embedded in NC-CS/GP-21 hydrogel. CONCLUSION: Our results suggest that nanocellulose-chitosan thermosensitive hydrogel is a biocompatible, injectable, mechanically stable and slowly degradable scaffold. hDPSCs embedded in NC-CS/GP-21 hydrogel is a promising, minimally invasive, stem cell-based strategy for cartilage regeneration.

Sustained-Release Injectable Hydrogel Formulations for Administration of Sodium Salicylate in Broiler Chickens.

We developed injectable hydrogels for the slow release of analgesic drugs in birds as an in vivo model of pharmacokinetics in wild avian species. Hydrogels loaded with sodium salicylate (NaSA) were injected subcutaneously in Ross broiler chickens. The hydrogels were made by dissolving sodium alginate and NaSA in water at 2 different concentrations (low, LALG; high, HALG) and then adding calcium chloride. In vitro drug release studies were performed by swelling the hydrogels in water and analyzing serial samples by ultraviolet-visible (UV-Vis) spectroscopy. Dried hydrogel films of the same formulations of the two alginate concentrations then were dissolved in sterile water for the in vivo pharmacokinetic study conducted in 18 chickens divided into 3 groups of 6 birds. Each of the 2 resultant NaSA hydrogel solutions were filtered with 0.2-µm syringe filters before injecting at a NaSA dose of 150 mg/kg SC in the respective LALG or HALG groups. The control group was injected SC with the same dose of NaSA dissolved in water. Pharmacokinetics parameters calculated by the compartmental and noncompartmental approaches were compared among the 3 groups by the Kruskal-Wallis test. Results of in vitro studies showed that both hydrogels released 80% of the drug during the first 3.5 hours. Results of the pharmacokinetic study indicated that NaSA concentrations remained above the minimum effective concentration (MEC) for analgesia in humans for 24 ± 8.9 (LALG) to 26 ± 4 (HALG) hours for the hydrogel formulations compared to 10 ± 5.6 hours for the aqueous formulation. These hydrogel formulations may have potential in providing long-term analgesia in avian species, but need further evaluation with pharmacodynamic or pharmacokinetic/pharmacodynamic modeling studies.

Chitosan-Based Thermosensitive Hydrogel for Controlled Drug Delivery to the Temporomandibular Joint.

Intra-articular injections of hyaluronic acid (HA) and corticosteroids have been extensively used in treating temporomandibular disorders. However, rapid clearance from the site of injection is a major concern that is commonly managed by frequent dosing, which is not without complications. This study aimed to determine the suitability of thermosensitive chitosan-based hydrogels for intra-articular controlled release of drugs in the rabbit temporomandibular joint (TMJ). A series of hydrogels were prepared using different chitosan (Ch) to ß-glycerophosphate (ß-GP) ratios. The gelation time, swelling ratio, the shape, and surface morphology of the prepared gels were investigated to select the formulation with optimum characteristics. The left TMJ in 13 adult male New Zealand white rabbits was injected with 0.2 mL of Chitosan/ß-glycerophosphate/HA while the right TMJ was injected with 0.2 mL of control solution of HA. Hyaluronic acid concentrations in experimental and control groups were measured using Hyaluronan Quantikine Enzyme-Linked Immunosorbent Assay Kit. In vitro characterization showed that both the Ch:ß-GP ratio and incorporation of HA had a significant effect on gelation time, degree of swelling, and surface morphology of the hydrogels. No morphological changes were observed in the joints in both groups. The mean concentration of HA in the experimental joints after 7 days (1339.79 ±â€Š244.98 µg/g) was significantly higher than that in the control (474.52 ±â€Š79.36 µg/g). In conclusion, the chitosan-based thermosensitive hydrogel can be considered as a promising controlled drug release system to the TMJ in a rabbit model that would potentially overcome many of the current limitations of intra-articular formulations.

Design and evaluation of a novel chitosan-based system for colon-specific drug delivery.

Tritrichomonas foetus is a flagellated protozoan parasite that colonizes the feline colon causing colitis and chronic foul smelling diarrhoea. Despite the efficacy of Ronidazole in the treatment of T. foetus, Ronidazole has been reported to cause neurotoxicity in some cats due to rapid absorption in the small intestine. A novel amphoteric derivative of chitosan was synthesised and characterized. A combination of time, pH, and an enzyme controlled system was used in a study of a new compression coated tablet for delivery of Ronidazole to the colon. Axial, radial swelling and erosion of selected tablets were carried out in various media. The effect of weight ratio, enzyme and pH on in vitro drug release profile was investigated. The results show that less than 2% of the drug was released in the physiological environment of the stomach and small intestine.

Coupled solid phase extraction and microparticle-based stability and purity-indicating immunosensor for the determination of recombinant human myelin basic protein in transgenic milk.

An optical immunosensor was developed and validated on the surface of microparticles for the determination of a biopharmaceutical protein. The recombinant human myelin basic protein (rhMBP) was produced in milk of transgenic cows as a His-tagged fusion protein. Previous work indicated exclusive association of rhMBP with milk casein micelles that hindered direct determination of the protein in milk. In this work, a solid phase extraction using a cation exchange matrix was developed in order to liberate rhMBP from casein micelles. A sandwich-type immunoassay was then used for in-process monitoring of the full-length protein in the presence of major milk proteins. The assay was successfully employed for detection of ultra-traces of rhMBP (LOD=6.04 ng mL(-1)≈0.3 n mol L(-1)) and for quantitative determination over a wide concentration range (10.00-10,000.00 ng mL(-1)). The assay was able also to detect the rhMBP in the presence of its human counterpart that lacks the His-tag. The high sensitivity along with the ability of the assay to determine the full length protein enabled monitoring of the stability of rhMBP. The testing protocol is particularly useful for intrinsically unstructured proteins that are extremely sensitive to proteolysis and lack a traceable enzymatic activity. This immunosensor provides a specific, ultrasensitive high throughput tool for in-process monitoring in biopharmaceutical industry.

Fixed-bed column studies on a modified chitosan hydrogel for detoxification of aqueous solutions from copper (II).

A new efficient, low cost chitosan based biosorbent was successfully prepared and employed for the biosorption of copper ions from an aqueous solution using a fixed bed column. Pyromellitic dianhydride crosslinked chitosan as the new adsorbent was characterized by SEM, FTIR spectroscopy, X-ray diffraction, thermogravimetric analysis and solid state (13)C NMR analysis. Scanning electron microscopy coupled with an X-ray energy dispersed analysis for the copper-equilibrated biomass confirmed the presence of Cu(II) ions on the surface of the hydrogel. Thermogravimetric analysis showed a significant improvement in the thermal stability of the new hydrogel compared to pure chitosan. Kinetic models were applied to predict the breakthrough curves. This study shows that the prepared hydrogel based on modified chitosan could be utilized as an efficient bioadsorbent for the removal of copper ions from wastewater.

Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor.

Overexpression of the Candida albicans ATP-binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ~1.89 × 10(6) member D-octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative of the D-octapeptide D-NH(2) -FFKWQRRR-CONH(2) , as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization-resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug-like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin ß9 have modes of action similar to RC21v3.

A D-octapeptide drug efflux pump inhibitor acts synergistically with azoles in a murine oral candidiasis infection model.

Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 µmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis.

New hydrogels based on symmetrical aromatic anhydrides: Synthesis, characterization and metal ion adsorption evaluation.

Unsatisfactory mechanical properties, poor heat resistance, dissolution in acidic media and high swelling ratios limit using chitosan as an adsorbent in industry. In this study, we used dianhydride derivatives famous for their exceptional mechanical and thermal characteristics and low water absorption. In this study, pyromellitic dianhydride (PMDA), benzophenone-3,3',4,4'-tetracarboxylic dianhydride (BTDA), 4,4'-oxydiphthalic dianhydride (ODPA) and 4,4'-(hexafluoroisopropylidene)diphthalic dianhydride (FDA) were utilised as crosslinking agents. Use of common crosslinking agents usually decreases the metal uptake capacity of chitosan. These novel crosslinking agents increased the metal uptake from 1.8 (ODPA/Cu2+) to 7.8 (PMDA/Zn2+) times. Langmuir and Freundlich adsorption models were applied to analyse the isotherms and isotherm constants. Kinetic, X-ray diffraction, pH, FTIR studies were also carried out.

Condensed tannins and flavonoids from the forage legume sulla (Hedysarum coronarium).

The condensed tannin concentrations and composition and the characterization of the phenolic constituents in the leaves of the forage legume sulla (Hedysarum coronarium), a biennial forage legume found in temperate agricultural regions, were studied. The colorimetric butanol-HCl assay was used for the quantitation of the seasonal condensed tannin concentrations in the leaves of sulla. Fractionation of extracts on Sephadex LH-20 using step elution with aqueous methanol, followed with aqueous acetone or gradient elution with water, aqueous methanol, and aqueous acetone, gave condensed tannin and flavonoid fractions. The chemical characteristics of the purified condensed tannin fractions were studied by acid-catalyzed degradation with benzyl mercaptan and electrospray ionization mass spectrometry (ESI-MS). Thiolysis revealed that epigallocatechin was the major extender unit (15-75%) while gallocatechin was the major terminal unit (50-66%), thus indicating the extractable sulla condensed tannin fraction as the prodelphinidin type. Condensed tannin oligomers to polymers obtained from Sephadex LH-20 gradient fractions ranged between 2.9 and 46 mDP. The homo- and heterogeneous oligomer ions in condensed tannin gradient fractions detected by ESI-MS ranged from 2 to 10 DP and are consistent with the values obtained by thiolysis (2.9-6.9 DP). Lower molecular weight phenolics, including flavonoids and phenolic acids, were characterized by liquid chromatography atmospheric pressure chemical ionization mass spectrometry (LC-APCI/MS) and ESI/MS/MS on a linear ion trap. The flavonoids extracted with aqueous acetone and methanol from sulla leaves and identified included kaempferol, rutin, quercetin-7-O-α-L-rhamnosyl-3-O-glucosylrhamnoside, quercetin-3-O-α-L-rhamnosyl-7-O-glucoside, kaempferol-3-O-ß-D-glucoside-dirhamnoside, genistein-7-O-ß-D-glucosyl-6″-O-malonate, formononetin-7-O-ß-D-glucoside-6″-O-malonate, and afrormosin and the phenolic acid chlorogenic acid.

Improved Fmoc synthesis of bradykinin.

Two arginine side-chain protecting groups, N(G)-4-methoxy-2,3,6-trimethylbenzensulfonyl group (Mtr) and N(G)-2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), have been investigated at both the Arg(1) and/or Arg(9) position of the bioactive peptide, Bradykinin using Fluorenylmethyloxycarbonyl (Fmoc) Solid Phase Peptide Synthesis. A more efficient synthesis of the peptide has been found when a combination of Arg(Mtr) is present at position 1 and Arg(Pmc) is present at position 9 giving a cleaved pure yield of 52%.

Probing the interaction between recombinant human myelin basic protein and caseins using surface plasmon resonance and diffusing wave spectroscopy.

An intrinsically unstructured human myelin basic protein (hMBP) was expressed in the milk of transgenic cows (TGmilk) and found exclusively associated with the casein micellar phase. The interaction between the recombinant protein and milk caseins was investigated using surface plasmon resonance (SPR). An anti-human myelin basic protein antibody was covalently immobilized to the surface of the sensor chip. Subsequently the interaction between the recombinant protein (captured by this antibody) and caseins was studied in comparison to that noted with its human counterpart. Results showed a calcium-mediated interaction between the recombinant protein and caseins. The order of magnitude of this interaction was in agreement with the number of phosphorylated residues carried by each type of casein (alpha(s)- > beta- > kappa-casein). This selective interaction was not noted between the human protein and milk caseins indicating that the recombinant protein was phosphorylated to a higher extent than the human protein. The obtained results indicated that the co-expression of the recombinant protein and caseins by the mammary gland along with the recombinant protein's ability to form calcium bridges played a key role in the association of the recombinant human myelin basic protein (rhMBP) with the casein micelles of milk. Despite this association between the recombinant protein and milk caseins, light scattering investigations using diffusing wave spectroscopy (DWS) showed no significant differences between the milks of the transgenic and the non-transgenic control cows, with respect to both the average micelle size and surface charges. This was attributed to the low expression levels of the recombinant protein in milk.

Preparation, characterization and in vitro biological study of biomimetic three-dimensional gelatin-montmorillonite/cellulose scaffold for tissue engineering.

This work focused on studying the effect of blending gelatin (Gel) with Cellulose (Cel), in the presence of montmorillonite (MMT), on the swelling behavior, in vitro degradation and surface morphology. Additionally, the effect of the prepared biocomposites on the characteristics of the human osteosarcoma cells (Saos-2), including proliferation, scaffold/cells interactions, apoptosis and their potential of the cells to induce osteogenesis and differentiation was evaluated. The crosslinked biocomposites with glutaraldehyde (GA) or N,N-methylene-bisacrylamide (MBA) was prepared via an intercalation process and freeze-drying technique. Properties including SEM morphology, X-ray diffraction characterization and in vitro biodegradation were investigated. The successful generation of 3-D biomimetic porous scaffolds incorporating Saos-2 cells indicated their potential for de novo bone formation that exploits cell-matrix interactions. In vitro studies revealed that the scaffolds containing 12 and 6% MMT crosslinked by 5 and 0.5% GA seem to be the two most efficient and effective biodegradable scaffolds, which promoted Saos-2 cells proliferation, migration, expansion, adhesion, penetration, spreading, and differentiation, respectively. MMT improved cytocompatibility between the osteoblasts and the biocomposite. In vitro analysis indicated good biocompatibility of the scaffold and presents the scaffold as a new potential candidate as suitable biohybrid material for tissue engineering.

On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows.

Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni2+ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.

Peptide motifs for cell-surface intervention: application to anti-infective and biopharmaceutical development.

The discovery of new antimicrobial and anticancer drugs, and overcoming the problem of resistance to current anti-infective and anticancer drug therapies require innovation in the pharmaceutical and scientific research community. A further challenge of drug design is to make the therapeutic agent specific, long lasting, of minimal toxicity, and affordable. Microbial and cancer cell surfaces present molecular features that can differentially prefocus drugs within the human host. This property can localize drugs near cell-surface targets, thereby reducing opportunities for adverse effects, or the emergence of drug resistance caused by intracellular drug and target modification and by the induction of drug efflux pumps. The solubility demands on cell-surface targeting drugs should also be less stringent than for those drugs requiring transmembrane transport or internalization in order to reach intracellular targets. Cationic peptides have provided an increasingly important research focus in this regard. Although the cationic antimicrobial peptides are distributed widely in nature and provide localized primary defenses against microbial attack, the susceptibility of L-peptides to proteolysis and the known properties of successful antimicrobials have led to a focus on circularized peptides, D,L-peptides, and peptides containing unusual amino acids. New on the scene as lead antifungal agents are D-octapeptides and their derivatives that were developed from a combinatorial library produced through solid-phase peptide synthesis protocols. These peptides contain an amidated C-terminal tri-arginine motif, which confers membrane impermeability and focuses the peptides near the fungal cell surface. To date, the octapeptides and their derivatives also require some aromaticity, preferably the indole ring of tryptophan. In some cases, a single 4-methoxy-2,3,6-trimethylbenzenesulfonyl moiety remaining on the peptide after incomplete cleavage of the peptide from the solid phase produces a peptide with activity, whereas the parent shows little or no activity in the screen. Recent research advances that support the polycationic cell surface approach include the RGD (Arg-Gly-Asp) tripeptide and its mimetics, as well as aminoglycoside arginine drugs (e.g. neomycin coupled to small arginine polymers) and prodrugs. In the case of polycationic peptides, D-peptides could be used for intravenous injection and direct-surface drug applications, but mimetics will probably be needed for oral delivery.

Surface-active fungicidal D-peptide inhibitors of the plasma membrane proton pump that block azole resistance.

A 1.8-million-member D-octapeptide combinatorial library was constructed in which each member comprised a diversity-containing N-terminal pentapeptide and a C-terminal amidated triarginine motif. The C-terminal motif concentrated the library members at the fungal cell surface. A primary screen for inhibitors of Saccharomyces cerevisiae and Candida albicans growth, together with an in vitro secondary screen with the S. cerevisiae plasma membrane ATPase (Pma1p) as a target, identified the antifungal D-octapeptide BM0 (D-NH(2)-RFWWFRRR-CONH(2)). Optimization of BM0 led to the construction of BM2 (D-NH(2)-RRRFWWFRRR-CONH(2)), which had broad-spectrum fungicidal activity against S. cerevisiae, Candida species, and Cryptococcus neoformans; bound strongly to the surfaces of fungal cells; inhibited the physiological activity of Pma1p; and appeared to target Pma1p, with 50% inhibitory concentrations in the range of 0.5 to 2.5 microM. At sub-MICs (<5 microM), BM2 chemosensitized to fluconazole (FLC) S. cerevisiae strains functionally hyperexpressing fungal lanosterol 14alpha-demethylase and resistance-conferring transporters of azole drugs. BM2 chemosensitized to FLC some FLC-resistant clinical isolates of C. albicans and C. dubliniensis and chemosensitized to itraconazole clinical isolates of C. krusei that are intrinsically resistant to FLC. The growth-inhibitory concentrations of BM2 did not cause fungal cell permeabilization, significant hemolysis of red blood cells, or the death of cultured HEp-2 epithelial cells. BM2 represents a novel class of broad-spectrum, surface-active, Pma1p-targeting fungicides which increases the potencies of azole drugs and circumvents azole resistance.

Isolation and characterisation of renal metabolites of gamma-glutamylfelinylglycine in the urine of the domestic cat (Felis catus).

The renal metabolism of the tripeptide, gamma-glutamylfelinylglycine, which our group recently identified in the blood of domestic cats (Felis catus), was investigated. To test our hypothesis that this unique tripeptide is metabolised by the kidney in a similar manner to glutathione-S-conjugates in other animal species, [(35)S]cysteine was administered intraperitoneally to an entire male cat, and urine collected at 1, 4 and 8 h post-injection. Radiolabelled fractions were isolated from the urine following reversed-phase (RP) HPLC. Four [(35)S]radiolabelled fractions were identified and characterised by amino acid analysis, mass spectrometry and comparison of retention times with synthetic compounds (felinine, N-acetyl felinine, felinylglycine, gamma-glutamylfelinylglycine). In addition to the previously described presence of free felinine, we showed the presence of several felinine-containing metabolites, including N-acetyl felinine, felinylglycine and unaltered gamma-glutamylfelinylglycine in cat urine. The results show that renal metabolism of gamma-glutamylfelinylglycine in cats, generally occurs in a similar manner to glutathione S-conjugates in other animal species, although the detection of felinylglycine indicates that subtle differences may exist. Additionally, our research indicates that previously reported estimates of felinine excretion in male cats need to be increased by as much as 54% to account for other felinine containing metabolites in the urine.