Colon-specific, mutual azo prodrug of 5-aminosalicylic acid with L-tryptophan: synthesis, kinetic studies and evaluation of its mitigating effect in trinitrobenzenesulfonic acid-induced colitis in rats

Mutual azo prodrug of 5-aminosalicylic acid with l-tryptophan was synthesized by coupling l-tryptophan with salicylic acid, for targeted drug delivery to the inflamed gut tissue in inflammatory bowel disease. The structure of synthesized prodrug was confirmed by elemental analysis, IR and NMR spectroscopy. In vitro kinetic studies in HCl buffer (pH 1.2) showed negligible release of 5-aminosalicylic acid, whereas in phosphate buffer (pH 7.4) 18% release was observed over a period of 7 h. In rat fecal matter, 87.9% of 5-aminosalicylic acid was released with a half-life of 143.6 min, following first order kinetics. The azo conjugate was evaluated for its ulcerogenic potential by Rainsford's cold stress method. The ameliorating effect of the azo conjugate and therapeutic efficacy of the carrier system was evaluated in trinitrobenzenesulfonic acid-induced experimental colitis model. The synthesized prodrug was found to be equally effective in mitigating the colitis in rats as that of sulfasalazine without the ulcerogenicity of 5-aminosalicylic acid.     

Synthesis, kinetic studies and pharmacological evaluation of mutual azo prodrug of 5-aminosalicylic acid with D-phenylalanine for colon specific drug delivery in inflammatory bowel disease

Mutual azo prodrug of 5-aminosalicylic acid with d-phenylalanine was synthesized by coupling D-phenylalanine with salicylic acid, for targeted drug delivery to the inflamed gut tissue in inflammatory bowel disease. The structure of synthesized prodrug was confirmed by elemental analysis, IR and NMR spectroscopy. In vitro kinetic studies in HCl buffer (pH 1.2) showed negligible release of 5-aminosalicylic acid, whereas in phosphate buffer (pH 7.4) only 15% release was observed over a period of 7h. In rat fecal matter the release of 5-aminosalicylic acid was almost complete (85%), with a half-life of 160.1 min, following first order kinetics. The azo conjugate was evaluated for its ulcerogenic potential by Rainsford's cold stress method. Therapeutic efficacy of the carrier system and the mitigating effect of the azo conjugate were evaluated in trinitrobenzenesulfonic acid-induced experimental colitis model. The synthesized prodrug was found to be equally effective in mitigating the colitis in rats as that of sulfasalazine without the ulcerogenicity of 5-aminosalicylic acid.     

A solution study of the interaction of the Cu(II) ions with HisGlyGlyTrp tetrapeptide and its evaluation as superoxide dismutase mimetic complex

The superoxide anion radical is a highly reactive toxic species produced during the metabolic processes. A number of copper (II) complexes with amino acids and peptides are known to show superoxide dismutase (SOD) like activity. The design and application of synthetic low molecular weight metal complexes as SOD mimics have received considerable attention during the last decade. A variety of di- and tri-peptides containing histidyl residue in different positions have been employed to bind Cu(II) and to show the activity. But reports on Cu(II) complex with tetra-peptide having histidine amino acid in this regard are limited. As the HGGGW peptide having His at its N-terminal is reported to be a potential moiety for Cu(2+) binding, in the present work the synthesis of HisGlyGlyTrp peptide and its complexation with copper (II) ions has been reported. The interaction of synthesized peptide with Cu(II) was studied by electron spray ionization-mass spectrometer (ESI-MS) and UV-Vis spectroscopic methods. The species distribution was studied by combined spectrophotometric and potentiometric methods. The studies were performed at 25 ± 0.1 °C with constant ionic strength (μ = 0.1 M NaNO(3)) in aqueous solution using Bjerrum-Calvin's pH-titration technique as adopted by Irving and Rossotti for binary systems. The solution studies suggested that the pH of the medium play important role in the different species formation of the copper complexes. Species distribution curves indicate that Cu complexation takes place at all physiological pH values from 3-11. The resultant copper (II) peptide complex at physiological pH was tested for superoxide dismutase activity using standard NBT method. The complex has SOD activity with the IC(50) value of 1.32 μM.     

DNA cleavage mediated by copper superoxide dismutase via two pathways

The known action of Cu, Zn superoxide dismutase (Cu(2)Zn(2)SOD) that converts O(2)(-) to O(2) and H(2)O(2) plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of Cu(2)Zn(2)SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much Cu(2)Zn(2)SOD may be injurious to the cells. The present study examined the DNA cleavage activity mediated by a Cu(n)SOD that contains 1-4 copper ions, in order to obtain an insight into the aberrant copper-mediated oxidative chemistry in the enzyme. A high SOD activity was observed upon metallation of the apo-form of Cu(2)Zn(2)SOD with Cu(II), indicating that nearly all of the Cu(II) in the Cu(n)SOD is as active as the Cu(II) in the copper site of fully active Cu(2)Zn(2)SOD. Using a supercoiled DNA as substrate, significant DNA cleavage was observed with the Cu(n)SOD in the presence of hydrogen peroxide or mercaptoethanol, whereas DNA cleavage with free Cu(II) ions can occur only <5% under the same conditions. Comparison with other proteins shows that the DNA cleavage activity is specific to some proteins including the Cu(n)SOD. The steady state study suggests that a cooperative action between the SOD protein and the Cu(II)may appear in the DNA cleavage activity, which is independent of the number of Cu(II) in the Cu(n)SOD. The kinetic study shows that a two-stage reaction was involved in DNA cleavage. The effects of various factors including EDTA, radical scavengers, bicarbonate anion, and carbon dioxide gas molecules on the Cu(n)SOD-mediated DNA cleavage activity were also investigated. It is proposed that DNA cleavage occurs via both hydroxyl radical oxidation and hydroxide ion hydrolysis pathways. This work implies that any form of the copper-containing SOD enzymes (including Cu(2)Zn(2)SOD and its mutants) might have the DNA cleavage activity.     

Synthesis, Copper(II) Complexation, (64)Cu-Labeling, and Bioconjugation of a New Bis(2-pyridylmethyl) Derivative of 1,4,7-Triazacyclononane

A new ligand derivative of 1,4,7-triazacyclononane (TACN), 2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic acid ( 6), has been synthesized and its complexation behavior toward Cu2+ ions investigated. The ligand 6 has been characterized by spectroscopic methods, and a molecular structure of a corresponding Cu(II) complex has been elucidated by single-crystal X-ray analysis. The suitability of 6 for conjugation to peptide substrates has been shown by amide coupling of 6 to the stabilized derivative of bombesin (BN), beta Ala-beta Ala-[Cha13, Nle14]BN(7-14), to give the conjugate 8. The free ligand 6 and the bioconjugate 8 were labeled with 64Cu2+, and the resulting complexes, 64Cu subset6 and 64Cu subset8 , were found to be stable in the presence of a large excess of a competing ligand (cyclam) or copper-seeking superoxide dismutase (SOD), as well as in rat plasma. Biodistribution studies of 64Cu subset8 in Wistar rats showed a high activity uptake into the pancreas (5.76 +/- 0.25 SUV, 5 min p.i.; 3.93 +/- 0.25 SUV, 1 h p.i.), which is the organ with high levels of gastrin-releasing peptide receptor (GRPR). This receptor is overexpressed in a large number of breast and prostate carcinomas. The novel 64Cu subset6 complex had a dominating influence on the nonspecific activity biodistribution of its BN conjugate, since the distribution data of 64Cu subset6 are similar to those of 64Cu subset8 . The 64Cu complexes exhibited a low activity accumulation in the liver tissue and an extensive renal clearance, which was distinctively different to the biodistribution of 64CuCl 2, suggesting that 64Cu subset6 does not undergo significant demetalation, but rather exhibits high in vivo stability.     

Protective effects of carnosine, homocarnosine and anserine against peroxyl radical-mediated Cu,Zn-superoxide dismutase modification

Carnosine (beta-alanyl-L-histidine), homocarnosine (gamma-amino-butyryl-L-histidine) and anserine (beta-alanyl-1-methyl-L-histidine) have been proposed to act as anti-oxidants in vivo. The protective effects of carnosine and related compounds against the oxidative damage of human Cu,Zn-superoxide dismutase (SOD) by peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) were studied. The oxidative damage to Cu,Zn-SOD by AAPH-derived radicals led to protein fragmentation, which is associated with the inactivation of enzyme. Carnosine, homocarnosine and anserine significantly inhibited the fragmentation and inactivation of Cu,Zn-SOD by AAPH. All three compounds also inhibited the release of copper ions from the enzyme and the formation of carbonyl compounds in AAPH-treated Cu,Zn-SOD. These compounds inhibited the fragmentation of other protein without copper ion. The results suggest that carnosine and related compounds act as the copper chelator and peroxyl radical scavenger to protect the protein fragmentation. Oxidation of amino acid residues in Cu,Zn-SOD induced by AAPH were significantly inhibited by carnosine and related compounds. It is proposed that carnosine and related dipeptides might be explored as potential therapeutic agents for pathologies that involve Cu,Zn-SOD modification mediated by peroxyl radicals.     

Superoxide and hydrogen peroxide suppression by metal ions and their EDTA complexes

Redox-active metal ions such as Fe(II)\(III) and Cu(I)\(II) have been proposed to activate reactive oxygen and nitrogen species (RONS) and thus, perpetuate oxidative damage. Here, we show that concentrations of metal ions and EDTA complexes with superoxide-destroying activities equivalent to 1 U SOD are Fe(III) 5.1 microM, Mn(II) 0.77 microM, Cu(II)-EDTA 3.55 microM, Fe(III)-EDTA 2.34 microM, and Mn(II)-EDTA 1.38 microM. The most active being the aquated Cu(II) species which exhibited superoxide-destroying activity equivalent to 2U of SOD at 0.29 microM. Hydrogen peroxide-destroying activities were as follows Fe(III)-EDTA ca. 70 U/mg and aquated Fe(III) 141 U/mg. In contrast, DTPA prevented superoxide-destroying activity and significantly depleted hydrogen peroxide-destroying activity. In conclusion, non-protein bound transition metal ions may have significant anti-oxidant effects in biological systems. Caution should be employed in bioassays when chelating metal ions. Our results demonstrate that DTPA is preferential to EDTA for inactivating redox-active metal ions in bioassays.     

Copper complexes of 1,10-phenanthroline and related compounds as superoxide dismutase mimetics

In a preliminary study we tested CuSO4.5H2O, (Cu(II]2[3,5-diisopropylsalicylate]4.2H2O and a number of copper complexes of substituted 1,10-phenanthrolines for superoxide anion dismutase activity. It appeared that this activity depends on the ligands involved and might be governed by the redox potential of the Cu(I) complex/Cu(II) complex couple. The strong superoxide anion dismutase activity of Cu(II)[DMP]2 complex can be expected considering its high redox potential. Rather surprisingly is the superoxide anion dismutase activity of the Cu(I)[DMP]2 complex since it involves oxidation to Cu(II)[DMP]2 complex. From regression analysis it was established that steric and field effects of the substituents of the investigated phenanthrolines play an important role in SOD activity and therefore it is concluded that complex formation is important for the superoxide dismutase-like activity.     

Anti-inflammatory activity of superoxide dismutase conjugated with sodium hyaluronate

Superoxide dismutase (SOD) from bovine erythrocytes was conjugated with sodium hyaluronate (HA) with a mean molecular weight of 106 to have greater anti-inflammatory activity in vivo. Amino groups of SOD were coupled with carboxyl groups in the hyaluronate molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The HA-SOD conjugate was composed of 1.5 mol of SOD molecule per 1 mol of hyaluronate on the average, and retained 70% of the activity of unmodified SOD. The conjugate was essentially non-immunogenic in mice, and exhibited much higher anti-inflammatory activities than HA or SOD in models of inflammatory diseases such as ischemic oedema of the foot-pad in mice, carrageenin-induced pleurisy and adjuvant arthritis in rats. This revised version was published online in November 2006 with corrections to the Cover Date.     

Copper-catalyzed cleavage of DNA by arenes

DNA was found to be cleaved in neutral solutions containing arenes and copper (II) salts. The reaction is comparable in efficiency with the DNA cleavage by such systems as Cu(II)-phenanthroline and Cu(II)-ascorbic acid, but, in contrast to the latter, the system Cu(2+)-arene does not require the presence of an exogenous reducing agent or hydrogen peroxide. The system Cu(2+)-arene does not cleave DNA under anaerobic conditions. Catalase, sodium azide, and bathocuproine, which is a specific chelator of Cu(I), completely inhibit the reaction. The data obtained allow one to suppose that Cu(I) ions, superoxide radical, and singlet oxygen participate in the reaction. It has been shown by the EPR method using spin traps that the reaction proceeds with formation of alkoxyl radicals, which can insert breaks in the DNA molecule. For effective cleavage of DNA in the Cu(II)-o-bromobenzoic acid system, the radicals have to be generated by a specific copper-DNA-o-bromobenzoic acid complex, in which copper ions are most probably coordinated with oxygen atoms of the DNA phosphate groups. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Antioxidant response system in the short-term post-wounding effect in Mesembryanthemum crystallinum leaves

Mechanical wounding of Mesembryanthemum crystallinum leaves in planta induced a fast decrease in stomatal conductance, which was related to accumulation of hydrogen peroxide (H(2)O(2)). Higher levels of H(2)O(2) were accompanied by an increase in total activity of superoxide dismutase (SOD) and a decrease in catalase (CAT) activity. Among SOD forms, manganese SOD (MnSOD) and copper/zinc SOD (Cu/ZnSOD) seem to be especially important sources of H(2)O(2) at early stages of wounding response. Moreover, NADP-malic enzyme (NADP-ME), one of the key enzymes of primary carbon metabolism, which is also involved in stress responses, showed a strong increase in activity in wounded leaves. All these symptoms: high accumulation of H(2)O(2), high activities of Cu/ZnSOD and NADP-ME, together with the decrease of CAT activity, were also observed in the major veins of unwounded leaves. The potential role of veinal tissues as an important source of H(2)O(2) during wounding response is discussed.     

Tetracycline-Cu(II) photo-induced fragmentation of serum albumin

The protein-damaging potential of photosensitized tetracycline hydrochloride alone and in combination with the metal ion Cu(II) was assessed using serum albumin as a model protein. Exposure of tetracycline to white light in an aqueous solution triggered the generation of significant amounts of reactive oxygen species (ROS) and engendered substantial protein damage. The appearance of distinct low-molecular-mass protein bands on 10% SDS-polyacrylamide gel ascertained the tetracycline concentration-dependent fragmentation of albumin. Photoexcited tetracycline in combination with 100 microM Cu(II) enhanced the protein fragmentation process with concurrent increase in free radical production. The significant release of acid-soluble amino groups and carbonyl groups from treated albumin provided quantitative estimation of protein fragmentation at 0-1.0 mM concentrations of tetracycline. Cu(II) ions per se did not cause any perceptible protein damage. The results with free radical quenchers suggested the role of hydroxyl radicals (*OH) in tetracycline-Cu(II)-induced protein fragmentation, as no superoxide dismutase (SOD)-mediated quenching effect was noted. The generation of free radicals upon tetracycline photoexcitation and consequent protein fragmentation may be considered as important factors in augmentation of tetracycline-induced phototoxic responses.     

Catalytic superoxide scavenging by metal complexes of the calcium chelator EGTA and contrast agent EHPG

Metal ion chelators widely used in experimental protocols and clinical diagnosis are generally assumed to be inert. We previously reported that the ubiquitous chelator EDTA has high levels of superoxide suppressing activity. Here, we report that the common chelators calcium chelator EGTA and contrast agent EHPG have significant activities in suppressing superoxide levels depending on the nature of metal ion chelated. The most active species is Mn(II)-EGTA which exhibited an IC50 value of 0.19 microM for superoxide destruction. In addition, IC50 values for Mn(II)-EHPG and 2Cu(II)-EGTA were 0.69 and 0.60 microM, respectively. In conclusion, Mn(II) and Cu(II) complexes of the common chelators EGTA and EHPG exhibit considerable superoxide scavenging activities. Caution should be employed in their use in biological systems where superoxide has a key role and they may be useful for the development of catalytic anti-oxidants.     

Use of cyanide and diethyldithiocarbamate in the assay of superoxide dismutases.

Eucaryotes have two major forms of superoxide dismutase (SOD), Cu,ZnSOD and MnSOD; in most tissues Cu,ZnSOD is present in higher amounts than MnSOD. To assay MnSOD, Cu,ZnSOD can be inhibited selectively by millimolar concentrations of cyanide ion. However, calculation of MnSOD activity from the differential cyanide inhibition assay is complex and small experimental errors can cause large errors in the calculated MnSOD activity. We have assessed how interaction of cyanide and hydrogen peroxide with cytochrome c can lead to further errors in the xanthine oxidase-cytochrome c assay for SOD. Alternatively, Cu,ZnSOD can be completely inactivated by 50 mM diethyldithiocarbamate (DDC) at 30 degrees C for 1 h without affecting the activity of MnSOD. Since DDC reduces cytochrome c, the treated samples must be thoroughly dialyzed or desalted before assay. In the case of lung homogenates, dialysis is not an extra step since fresh, untreated samples must also be dialyzed or desalted before assaying by the cytochrome c method. Cu,ZnSOD activity is equal to the activity in the untreated sample minus the activity in the DDC-treated portion of the sample. Another copper chelator, triethylenetetramine, did not inactivate Cu,ZnSOD and could not be used instead of DDC. For accurate measurement of both enzymes in samples where MnSOD contributes only a small fraction of the total SOD activity, the DDC method has the advantage that it provides a direct measure of the MnSOD activity without interference by Cu,ZnSOD.     

Reductive release of ferritin iron: a kinetic assay

Ferritin iron release, a process of considerable interest in biology and medicine, occurs most readily in the presence of reducing agents. Here is described a kinetic assay for measuring the rate of ferritin iron removal promoted by various reductants. The new procedure uses ferrozine as a chromophoric, high-affinity chelator for the product, Fe(II). The initial rate of iron release is quantified by continuous spectrophotometric measurement of the Fe(ferrozine)2/3+ complex which absorbs maximally at 562 nm. The initial rate of iron mobilization is dependent on reductant concentration, but not on the concentration of the chelating agent, ferrozine. Saturation kinetics are observed for all reductants, including dihydroxyfumarate, cysteine, caffeic acid, ascorbate, and glutathione. Superoxide dismutase greatly inhibits ferritin iron release by ascorbate, but has little or no effect on the reducing action of dihydroxyfumarate, cysteine, caffeic acid, or glutathione. Ferritin iron removal by dihydroxyfumarate was inhibited by various metal ions. This new assay may be used for rapid screening of test compounds for treatment of iron overload and for investigation of the mechanistic aspects of ferritin iron reduction.     

棕榈酰化超氧化物歧化酶的制备及性质研究

为了增强超氧化物歧化酶的稳定性,用棕榈酸对其进行了修饰,在修饰条件下,酶分子表面氨基修饰率为55％时,酶的活力回收为63％。修饰后的酶在耐热、耐酸、耐碱、抗有机溶剂变性和抗蛋白水解能力上均高于天然超氧化物歧化酶,为将超氧化物歧化酶作成实用药物和进一步扩大其应用范围创造了条件。     

Aberrantly increased hydrophobicity shared by mutants of Cu,Zn-superoxide dismutase in familial amyotrophic lateral sclerosis

More than 100 different mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1) cause preferential motor neuron degeneration in familial amyotrophic lateral sclerosis (ALS). Although the cellular target(s) of mutant SOD1 toxicity have not been precisely specified, evidence to date supports the hypothesis that ALS-related mutations may increase the burden of partially unfolded SOD1 species. Influences that may destabilize SOD1 in vivo include impaired metal ion binding, reduction of the intrasubunit disulfide bond, or oxidative modification. In this study, we observed that metal-deficient as-isolated SOD1 mutants (H46R, G85R, D124V, D125H, and S134N) with disordered electrostatic and zinc-binding loops exhibited aberrant binding to hydrophobic beads in the absence of other destabilizing agents. Other purified ALS-related mutants that can biologically incorporate nearly normal amounts of stabilizing zinc ions (A4V, L38V, G41S, D90A, and G93A) exhibited maximal hydrophobic behavior after exposure to both a disulfide reducing agent and a metal chelator, while normal SOD1 was more resistant to these agents. Moreover, we detected hydrophobic SOD1 species in lysates from affected tissues in G85R and G93A mutant but not wildtype SOD1 transgenic mice. These findings suggest that a susceptibility to the cellular disulfide reducing environment and zinc loss may convert otherwise stable SOD1 mutants into metal-deficient forms with locally destabilized electrostatic and zinc-binding loops. These abnormally hydrophobic SOD1 species may promote aberrant interactions of the enzyme with itself or with other cellular constituents to produce toxicity in familial ALS.     

Design and characterization of enzymosomes with surface-exposed superoxide dismutase

Superoxide dismutase (SOD) was chemically modified by covalent linkage of fatty acid chains to the accessible epsilon-amino groups of the enzyme. This acylation method gave rise to a different enzyme entity (Ac-SOD) as evidenced by different physicochemical properties such as octanol/water partition coefficient and isoelectric point (pI) as compared to SOD. Ac-SOD was incorporated in conventional and long-circulating liposomes (LCL) and characterized in terms of incorporation efficiency, protein to lipid ratio (Prot/Lip), enzymatic activity retention and zeta potential. The observation that Ac-SOD liposomes present enzymatic activity on their external surface indicates that these formulations can act independent of rate and extent of enzyme release as required in case of SOD liposomes. The decrease of superficial charge of liposomal formulations containing Ac-SOD, as compared to SOD liposomes, may be related to the negatively charged enzyme molecules localized on the liposome surface. The comparative characterization of Ac-SOD and SOD liposomal formulations evidenced that the two enzyme forms differ substantially regarding their intraliposomal location: SOD tends to be localized in the internal aqueous spaces, whereas Ac-SOD is expected to be localized in the lipid bilayers of the liposomes, partially buried into the outer surface and exposed to the external medium. These liposomal structures with surface-exposed SOD were designated as Ac-SOD enzymosomes. The properties of these enzymosomes may influence the therapeutic effect, as the release of the enzyme from extravasated vesicles is no longer a necessary requirement for achieving dismutating activity within the inflamed target site.