Hypochlorous acid-mediated oxidation of lipid components and antioxidants present in low-density lipoproteins: absolute rate constants,product analysis,and computational modeling

Oxidation of low-density lipoproteins (LDL) is believed to contribute to the increased uptake of LDL by macrophages, which is an early event in atherosclerosis. Hypochlorous acid (HOCl) has been implicated as one of the major oxidants involved in these processes. In a previous study, the rates of reaction of HOCl with the reactive sites in proteins were investigated (Pattison, D. I., and Davies, M. J. (2001) Chem. Res. Toxicol. 14, 1453-1464). The work presented here expands on those studies to determine absolute second-order rate constants for the reactions of HOCl with various lipid components and antioxidants in aqueous solution (pH 7.4). The reactions of HOCl with phosphoryl-serine and phosphoryl-ethanolamine are rapid (k approximately 10(5) M(-)(1) s(-)(1)) and of comparable reactivity to many of the protein sites. The major products formed in these reactions are chloramines, which decay to give both nitrogen- and carbon-centered radicals. Subsequent reactions of these species may induce oxidation of the LDL lipid component. In contrast, phosphoryl-choline reacted much more slowly (k < 10(-)(2) M(-)(1) s(-)(1)). Reaction of HOCl with 3-pentenoic acid was used as a model of lipid double bonds and yielded k = 9 M(-)(1) s(-)(1). The reactions of the lipid-soluble antioxidants, alpha-tocopherol and ubiquinol-10, with HOCl were investigated with model compounds. For the reactions of HOCl with both Trolox and ubiquinol-0, k = 1.3 x 10(3) M(-)(1) s(-)(1); thus, these lipid soluble antioxidants are relatively ineffective as direct scavengers for HOCl as compared to water soluble antioxidants (e.g., ascorbate, k ca. 10(6) M(-)(1) s(-)(1)). The reaction of HOCl with hydroquinone (a simple model for ubiquinol-10) was also investigated both in aqueous solution (k = 45 M(-)(1) s(-)(1)) and in a less polar environment (k approximately 0.5 M(-)(1) s(-)(1) in THF). A computational model was developed using these kinetic parameters to predict which LDL targets are oxidized with varying molar excesses of HOCl, in both the absence and the presence of added ascorbate. The results from these models compare well with experimental data and can be used to predict the effects of HOCl-mediated oxidation on LDL composition.     

Kinetics of hypobromous acid-mediated oxidation of lipid components and antioxidants

Hypohalous acids are generated from the oxidation of halide ions by myeloperoxidase and eosinophil peroxidase in the presence of H2O2. These oxidants are potent antibacterial agents, but excessive production can result in host tissue damage, with this implicated in a number of human pathologies. Rate constants for HOCl with lipid components and antioxidants have been established. Here, the corresponding reactions of HOBr have been examined to determine whether this species shows similar reactivity. The second-order rate constants for the reaction of HOBr with 3-pentenoic acid and sorbate, models of unsaturated lipids, are 1.1x10(4) and 1.3x10(3) M(-1) s(-1), respectively, while those for reaction of HOBr with phosphoryl-serine and phosphoryl-ethanolamine are ca. 10(6) M(-1) s(-1). The second-order rate constants (M(-1) s(-1)) for reactions of HOBr with Trolox (6.4x10(4)), hydroquinone (2.4x10(5)), and ubiquinol-0 (2.5x10(6)) were determined, as models of the lipid-soluble antioxidants, alpha-tocopherol, and ubiquinol-10; all of these rate constants are ca. 50-2000-fold greater than for HOCl. In contrast, the second-order rate constants for the reaction of HOBr with the water-soluble antioxidants, ascorbate and urate, are ca. 10(6) M(-1) s(-1) and closer in magnitude to those for HOCl. Kinetic models have been developed to predict the sites of HOBr attack on low-density lipoproteins. The data obtained indicate that HOBr reacts to a much greater extent with fatty acid side chains and lipid-soluble antioxidants than HOCl; this has important implications for HOBr-mediated damage to cells and lipoproteins.     

Disproportionation of clozapine radical: a link between one-electron oxidation of clozapine and formation of its nitrenium cation

The primary products of one-electron oxidation of clozapine and olanzapine, very effective atypical antipsychotic drugs, have been spectroscopically characterized. The oxidation process has been studied under glassy matrix conditions and by a pulse radiolysis technique in aqueous solution. The rate constants for the oxidation of clozapine with dibromide radical anion ( k = 2 x 10 (9) M (-1) s (-1)) and azide radical ( k = 2.3 x 10 (9) M (-1) s (-1)) in aqueous solution were measured. The computational DFT results support the identification of the transient species. The mechanistic aspects of reactivity of radical cations, radicals, and nitrenium cations have been investigated. A disproportionation reaction ( k > or = 1 x 10 (8) M (-1) s (-1)) was proposed as a link between the products of one-electron oxidation and formation of the nitrenium cations of clozapine and olanzapine, products likely responsible for the pathogenesis of adverse drug reactions. The rate constants for the reactions of nitrenium cation of clozapine with glutathione ( k = 3.4 x 10 (4) M (-1) s (-1)) and cysteine ( k = 9.8 x 10 (4) M (-1) s (-1)) were determined.     

Kinetic study of the reaction of cisplatin with thiols.

The reactions of cisplatin [cis-diamminedichloroplatinum(II), CDDP] with glutathione (GSH) and drug thiols were investigated at 37 degrees C in 100 mM Tris-NO(3), pH approximately 7.4, using a clinically relevant concentration of CDDP (33 micro M), a large excess of GSH (16.5 mM), and [NaCl] of 4.62 mM. The conditions were designed to mimic passage of CDDP through the cytosol. The reactions were studied by UV-absorption spectroscopy, high-pressure liquid chromatography (HPLC), and atomic absorption spectroscopy. The initial rates, detected by UV absorbance, confirmed that the reactions are first order in [CDDP]. The HPLC peak corresponding to CDDP was analyzed for platinum content by atomic absorption spectroscopy, which decreased exponentially with time, confirming that the reactions are first order in [CDDP] and allowing determination of the pseudo first order rate constants (k(1)). For reaction of the dichloro form of CDDP with GSH, the k(1) value was approximately 2.2 x 10(-4) s(-1) (t(1/2) of approximately 53 min), giving the second order rate constant value (k(2)) of approximately 1.3 x 10(-2) M(-)1 s(-1). Reaction of a mixture of the aquated forms of CDDP with GSH gave a lower k(1) value ( approximately 0.9 x 10(-4) s(-1)). Reaction of CDDP with sodium 2-mercaptoethanesulfonate (mesna) gave a k(1) value of approximately 1.8 x 10(-4) s(-1) (t(1/2) of approximately 65 min and k(2) of approximately 1.1 x 10(-2) M(-1) s(-1)). Reaction of CDDP with S-2-(3-aminopropylamino)ethanethiol (WR-1065) gave a k(1) value of approximately 12.0 x 10(-4) s(-1) (t(1/2) of approximately 10 min and k(2) of approximately 7.3 x 10(-2) M(-)1 s(-1)). The relatively slow reaction rate of CDDP with GSH is consistent with the efficient DNA platination by CDDP in the presence of millimolar concentration of GSH in the cytosol.     

Kinetic and mechanistic studies of the peroxynitrite-mediated oxidation of oxymyoglobin and oxyhemoglobin

Kinetic studies of the peroxynitrite-mediated oxidations of oxymyoglobin (MbFeO(2)) and oxyhemoglobin (HbFeO(2)) showed that the mechanisms of these reactions are more complex than what had previously been reported; both reactions proceed in two steps. For myoglobin, we found that the small amount of deoxymyoglobin (MbFe(II)) which is in equilibrium with MbFeO(2) is first oxidized by peroxynitrous acid to ferryl myoglobin (MbFe(IV)=O). Then, in the second step, MbFe(IV)=O is reduced by peroxynitrous acid to metmyoglobin (metMb). The second-order rate constant values obtained at pH 7.3 and 20 degrees C for the two steps are (5.4 +/- 0.2) x 10(4) and (2.2 +/- 0.1) x 10(4) M(-)(1) s(-)(1), respectively. Analogous studies with hemoglobin suggest that its reaction with peroxynitrite follows the same mechanism. In this case, the second-order rate constant values measured at pH 7.0 and 20 degrees C for the two steps are (8.8 +/- 0.4) x 10(4) and (9.4 +/- 0.7) x 10(4) M(-)(1) s(-)(1), respectively. A possible mechanism in the absence as well as in the presence of CO(2) and the relevance of these reactions in vivo are discussed.     

Nitrogen dioxide as an oxidizing agent of 8-oxo-7,8-dihydro-2'-deoxyguanosine but not of 2'-deoxyguanosine

The redox reactions of guanine and its widely studied oxidation product, the 8-oxo-7,8-dihydro derivative, are of significant importance for understanding the mechanisms of oxidative damage in DNA. Employing 2'-deoxyguanosine 5'-monophosphate (dGMP) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in neutral aqueous solutions as model systems, we have used nanosecond laser flash photolysis to demonstrate that neutral radicals, dGMP(-H)(*), derived by the one-electron oxidation and deprotonation of dGMP, can oxidize nitrite anions (NO2(-)) to the nitrogen dioxide radical (*)NO2. In turn, we show that (*)NO2 can give rise to a one-electron oxidation of 8-oxo-G, but not of dGMP. The one-electron oxidation of dGMP was initiated by a radical cation generated by the laser pulse-induced photoionization of a pyrene derivative with enhanced water solubility, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT). The dGMP(-H)(*) neutral radicals formed via deprotonation of the dGMP(*)(+) radical cations and identified by their characteristic transient absorption spectrum (lambda(max) approximately 310 nm) oxidize nitrite anions with a rate constant of (2.6 +/- 0.3) x 10(6) M(-1) s(-1). The 8-oxo-dG is oxidized by (*)NO2 with a rate constant of (5.3 +/- 0.5) x 10(6) M(-1) s(-1). The 8-oxo-dG(-H)(*) neutral radicals thus generated are clearly identified by their characteristic transient absorption spectra (lambda(max) approximately 320 nm). The rate constant of 8-oxo-dG oxidation (k(12)) by the (*)NO2 one-electron oxidant (the (*)NO2/NO2(-) redox potential, E degrees approximately 1.04 V vs NHE) is lower than k(12) for a series of oxidizing aromatic radical cations with known redox potentials. The k(12) values for 8-oxo-dG oxidation by different aromatic radical cations derived from the photoionization of their parent compounds depend on the redox potentials of the latter, which were in the range of 0.8-1.6 V versus NHE. The magnitude of k(12) gradually decreases from a value of 2.2 x 10(9) M(-1) s(-1) (E degrees = 1.62 V) to 5.8 x 10(8) M(-1) s(-1) (E degrees = 1.13 V) and eventually to 5 x 10(7) M(-1) s(-1) (E degrees = 0.91 V). The implications of these results, including the possibility that the redox cycling of the (*)NO2/NO2(-) species can be involved in the further oxidative damage of 8-oxo-dG in DNA in cellular environments, are discussed.     

One-electron reduction of N-chlorinated and N-brominated species is a source of radicals and bromine atom formation

Hypochlorous (HOCl) and hypobromous (HOBr) acids are strong bactericidal oxidants that are generated by the human immune system but are implicated in the development of many human inflammatory diseases (e.g., atherosclerosis, asthma). These oxidants react readily with sulfur- and nitrogen-containing nucleophiles, with the latter generating N-halogenated species (e.g., chloramines/bromamines (RR'NX; X = Cl, Br)) as initial products. Redox-active metal ions and superoxide radicals (O(2)(?-)) can reduce N-halogenated species to nitrogen- and carbon-centered radicals. N-Halogenated species and O(2)(?-) are generated simultaneously at sites of inflammation, but the significance of their interactions remains unclear. In the present study, rate constants for the reduction of N-halogenated amines, amides, and imides to model potential biological substrates have been determined. Hydrated electrons reduce these species with k(2) > 10(9) M(-1) s(-1), whereas O(2)(?-) reduced only N-halogenated imides with complex kinetics indicative of chain reactions. For N-bromoimides, heterolytic cleavage of the N-Br bond yielded bromine atoms (Br(?)), whereas for other substrates, N-centered radicals and Cl(-)/Br(-) were produced. High-level quantum chemical procedures have been used to calculate gas-phase electron affinities and aqueous solution reduction potentials. The effects of substituents on the electron affinities of aminyl, amidyl, and imidyl radicals are rationalized on the basis of differential effects on the stabilities of the radicals and anions. The calculated reduction potentials are consistent with the experimental observations, with Br(?) production predicted for N-bromosuccinimide, while halide ion formation is predicted in all other cases. These data suggest that interaction of N-halogenated species with O(2)(?-) may produce deleterious N-centered radicals and Br(?).     

Thiyl radical reaction with amino acid side chains: rate constants for hydrogen transfer and relevance for posttranslational protein modification

Thiyl radicals are prominent intermediates during biological conditions of oxidative stress and have been suggested to be involved in the mutagenic effects of thiols. While several enzymatic processes rely on the formation and selective reactions of protein thiyl radicals with substrates, such reactions may represent a source for biological damage when occurring uncontrolled during oxidative stress. For example, intramolecular hydrogen transfer reactions to protein cysteine thiyl radicals may lead to secondary amino acid oxidation products, which may represent starting points for protein aggregation and/or fragmentation. Here, we have used a kinetic NMR method to determine rate constants, k(sc), for hydrogen transfer reactions between thiyl radicals and amino acid side chain C-H bonds at 37 degrees C. Rate constants cover a range between k(sc) 相似文献   

Reactive sulfur species: kinetics and mechanism of the oxidation of cystine by hypochlorous acid to give N,N'-dichlorocystine

Cystine and HOCl (a neutrophil-derived oxidant) react to form an intermediate that has a half-life of ca. 5 min at pH 7.5. The intermediate subsequently decomposes to eventually yield a mixture of cystine, higher oxides of Cys, and other uncharacterized species. Spectral titrations, transitory (1)H NMR and UV-vis spectra, and the reaction properties of the intermediate are consistent with a formulation of N,N'-dichlorocystine {NDC = [-SCH(2)CH(NHCl)(CO(2)H)](2)}. The reaction of equimolar amounts of HOCl with cystine at pH 11.3 does not yield N-chlorocystine [NCC = (-O2C)(H3N+)CHCH(2)SSCH(2)CH(NHCl)(CO(2)H)] but rather a 1:1 mixture of NDC and cystine. This result could be explained by two mechanisms: rapid disproportionation of NCC to produce NDC and cystine or a faster reaction of the second equivalent of HOCl with NCC than the first equivalent of HOCl reacts with cystine. The latter mechanism is favored because of our observation by NMR spectroscopy that NDC decomposes via a species that we have assigned as NCC. Thus, disproportionation of NCC is apparently a relatively slow process. The rates of reaction of cystine(0) = [-SCH(2)CH(NH(3)(+))(CO(2)(-))](2) degrees , cystine(1-) = [((-)O(2)C)(H(2)N)CHCH(2)SSCH(2)CH(NH(3)(+))(CO(2)(-))](-), and cystine(2-) = [-SCH(2)CH(NH2)(CO2)(-))]2(2-) have been investigated, and it is clear that cystine(0) is unreactive, whereas cystine(2-) is about four times more reactive than cystine(1-). Accordingly, the following mechanism is proposed (constants for 5 degrees C): HOCl = H+ + OCl-, pK1 = 7.47; cystine(0) = cystine(1-) + H+, pK2 = 8.15; cystine(1-) = cystine(2-) + H+, pK3 = 9.00; cystine(1-) + HOCl --> NCC(1-) + H2O, k4 = 4.3(2) x 10(6) M(-1) s(-1); cystine(2-) + HOCl --> NCC(2)(-) + H2O, k5 = 1.6(2) x 10(7) M(-1) s(-1); NCC(1-) --> NCC(2-) + H+, k6 = fast; NCC(2-) + HOCl --> NDC(2-) + H2O, k7 = fast. At physiologic pH, the k4 pathway dominates. The generation of long-lived chloramine derivatives of cystine may have physiological consequences, since such compounds are known to react with nucleophiles via mechanisms that are also characteristic of HOCl, electrophilic transfer C+.     

Kinetics and mechanism of the oxidation of the glutathione dimer by hypochlorous Acid and catalytic reduction of the chloroamine product by glutathione reductase

Oxidized glutathione (GSSG) reacts with two molar equivalents of HOCl/OCl- (a neutrophil-derived oxidant and a common biocide) to form the dichloro (bis-N-chloro-gamma-l-glutamyl) derivative (NDG). The reaction of less than two molar equivalents of HOCl with GSSG does not yield the unsymmetrical monochloro derivative (NCG) but rather a stoichiometric amount of NDG and GSSG. This result is explained by a faster reaction of the second equivalent of HOCl with NCG than that of the first equivalent of HOCl with GSSG. The rates of reaction of GSSG2-, GSSG3-, and GSSG4- (successive deprotonation of the ammonium groups) have been investigated, and it is clear that GSSG2- is unreactive, whereas GSSG4- is about twice as reactive as GSSG3-. Accordingly, the following mechanism is proposed (constants for 5 degrees C): H+ + OCl- = HOCl, pK1 = -7.47; GSSG2- = GSSG3- + H+, pK2 = 8.5; GSSG3- = GSSG4- + H+, pK3 = 9.5; GSSG3- + HOCl --> NCG3- + H2O, k4 = 2.7(2) x 106 M-1 s-1; GSSG4- + HOCl --> NCG4- + H2O, k5 = 3.5(3) x 107 M-1 s-1; NCG3- --> NDG4- + H+, k6 = fast; and NCG4- + HOCl --> NDG4- + H2O, k7 = fast. At physiologic pH, the k4 pathway dominates. NDG decomposes at pH 7.4 in a first-order process with kdec = 4.22(1) x 10-4 s-1 (t1/2 = 27 min). Glutathione reductase (EC 1.6.4.2) is capable of catalyzing the reduction of NDG by NADPH. The only NDG-derived product that is observed (by NMR) after the reduction by NADPH is GSH. Thus, in the presence of the GOR/NADPH system, GSH is capable of redox buffering a 3/2 mol equiv of HOCl rather than a 1/2 mol equiv as previously assumed.     

Overlap in the pharmacology of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists in rat aorta.

We have previously shown that elimination of buffer Ca2+ markedly reduced maximum 5-HT-induced contractions. We have now investigated the effect of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists on 5-HT- and K+-induced contractions in rat aorta to explore the possibility of a relationship between blockade of L-type Ca2+ channels and 5-HT2 receptor antagonism. Sodium nitroprusside, felodipine, nifedipine, diltiazem, cinnarizine, verapamil, ritanserin, cyproheptadine, ketanserin and mianserin inhibited 5-HT-induced contractions of rat aorta with mean IC50 values (concentration (M) resulting in 50% inhibition) of 2.2 x 10(-11), 6.6 x 10(-11), 1.5 x 10(-9), 1.7 x 10(-9), 3.2 x 10(-7), 5.4 x 10(-7), 9.7 x 10(-10), 1.9 x 10(-8), 5.0 x 10(-7) and 6.4 x 10(-7), respectively. The same compounds antagonized K+-induced rat aortic contractions with the rank order of potency (mean IC50, M): felodipine (7.0 x 10(-11)) > nifedipine (4.8 x 10(-9)) > sodium nitroprusside (4.1 x 10(-8)) > verapamil (5.5 x 10(-8)) > cyproheptadine (6.2 x 10(-8)) > diltiazem (4.1 x 10(-7)) > cinnarizine (1.3 x 10(-6)) > ritanserin (1.8 x 10(-6)) > ketanserin (9.0 x 10(-6)) > mianserin (2.0 x 10(-5)). These data are indicative of a highly significant correlation (r=0.81, P=0.03) between potency against 5-HT-induced contraction and that against contractile response to K+ depolarization, and suggest overlap of the pharmacology of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists in rat aorta.     

Interaction of quinidine,disopyramide and metoprolol with melanin in vitro in relation to drug-induced ocular toxicity

The aim of this work was to evaluate binding capacity of quinidine, disopyramide and metoprolol to melanin in vitro. The antiarrhythmics studied cause adverse reactions to the eye. Synthetic DOPA-melanin was used in the studies and a UV spectrophotometric method was employed to determine the drugs. The studies of the kinetics of the formation of quinidine-melanin, disopyramide-melanin and metoprolol-melanin complexes indicate that for all the complexes investigated the maximum time to reach reaction equilibrium is 24 h. Binding parameters, i.e., the numbers of independent binding sites and the association constants were determined on the basis of the Scatchard plots. An analysis of the binding curves obtained supports our conclusion that both strong (n1) and weak (n2) binding sites are involved in the formation of the complexes investigated. The total numbers of binding sites in synthetic DOPA-melanin complexes with quinidine, disopyramide and metoprolol were 0.525, 0.493 and 0.387 micromol/mg, respectively. The quinidine-melanin complex is characterized by greater stability (K1 = 3.00 x 10(5) M(-1), K2 = 1.75 x 10(3) M(-1)) in comparison with biopolymer complexes with disopyramide (K1 = 1.12 x 10(4) M(-1), K2 = 6.04 x 10(2) M(-1)) and metoprolol (K1 = 1.42 x 10(4) M(-1), K2 = 7.89 x 10(2) M(-1)). The ability of these drugs to form complexes with melanin in vitro may be one of the reasons for their ocular toxicity in vivo, as a result of their accumulation in melanin in the eye.     

Absorption and disposition of a tripeptoid and a tetrapeptide in the rat

The present study is concerned with the absorption and disposition of a tripeptoid (N-substituted glycine derivative) and a tetrapeptide in the rat. The two compounds have similar backbone structures but differ with respect to the presence or absence of peptide bond. [3H]tripeptoid and [3H]tetrapeptide were administered orally (30 mg kg(-1)) and intravenously (i.v.) (30 or 3 mg kg(-1)) to Sprague Dawley rats. Blood, urine and feces were collected at designated times for radioactivity and parent drug analysis. The intestinal absorptive clearances of the tripeptoid and tetrapeptide were studied using an in situ rat intestinal perfusion model. The octanol/water partition coefficient of these two compounds was also determined. The results showed that the peptoid and peptide have similar absorptive clearance and octanol/water partitioning, but different in vivo absorption and disposition characteristics. The absorptive clearances of the tripeptoid and tetrapeptide were 6.7 and 4.8 x 10(-4) mL min(-1) cm(-1), respectively, and the corresponding octanol/water partition coefficients were 0.39 and 0.30. The extent of oral absorption of the tripeptoid was only 3-8%, consistent with its low absorptive clearance. In contrast, the apparent absorption of the tetrapeptide was > 75% of the radioactive dose. The peptide was completely metabolized within 2 h after an i.v. dose, whereas the peptoid was stable in blood and was primarily eliminated in feces as intact drug. In conclusion, the difference in in vivo absorption and disposition between the peptoid and peptide was apparently due to the presence or absence of a peptide bond. The tetrapeptide was subject to rapid metabolism in the body. Its relatively high absorption appeared to represent the absorption of metabolized radioactive fragments. The peptoid appears to have advantages over the peptide in term of metabolic stability, but its low oral absorption and rapid biliary excretion present additional challenges in the selection of an optimal drug candidate.     

Intestinal permeability and its relevance for absorption and elimination

Human jejunal permeability (P(eff)) is determined in the intestinal region with the highest expression of carrier proteins and largest surface area. Intestinal P(eff) are often based on multiple parallel transport processes. Site-specific jejunal P(eff) cannot reflect the permeability along the intestinal tract, but they are useful for approximating the fraction oral dose absorbed. It seems like drugs with a jejunal P(eff) > 1.5 x 10(-4) cm s(-1) will be completely absorbed no matter which transport mechanism(s) are utilized. Many drugs that are significantly effluxed in vitro have a rapid and complete intestinal absorption (i.e. >85%) mediated by passive transcellular diffusion. The determined jejunal P(eff) for drugs transported mainly by absorptive carriers (such as peptide and amino acid transporters) will accurately predict the fraction of the dose absorbed as a consequence of the regional expression. The data also show that: (1) the human intestinal epithelium has a large resistance towards large and hydrophilic compounds; and (2) the paracellular route has a low contribution for compounds larger than approximately molecular weight 200. There is a need for more exploratory in vivo studies to clarify drug absorption and first-pass extraction along the intestine. One is encouraged to develop in vivo perfusion techniques for more distal parts of the gastrointestinal tract in humans. This would stimulate the development of more relevant and complex in vitro absorption models and form the basis for an accurate physiologically based pharmacokinetic modelling of oral drug absorption.     

Honokiol is a potent scavenger of superoxide and peroxyl radicals

Honokiol, a compound extracted from Magnolia officinalis, has antitumor and antiangiogenic properties in several tumor models in vivo. Among the downstream pathways inhibited by honokiol is nuclear factor kappa beta (NFkappabeta). A prime physiologic stimulus of NFkappabeta is reactive oxygen species. The chemical structure of honokiol suggests that it may be an effective scavenger of reactive oxygen species. In this work, we have studied the reactions of honokiol with superoxide and peroxyl radicals in cell-free and cellular systems using electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques. Honokiol efficiently scavenged superoxide radicals in xanthine oxidase and cytochrome P-450 cell-free systems with the rate constant 3.2x10(5)M(-1)s(-1), which is similar to reactivity of ascorbic acid but 20-times higher than reactivity of vitamin E analog trolox. Honokiol potently scavenged intracellular superoxide within melanoma cells. In addition, honokiol scavenged peroxyl radicals generated by 2,2'-azo-bis(2-amidinopropane hydrochloride) (AAPH). The rate constant of the reaction of honokiol with peroxyl radicals (1.4x10(6)M(-1)s(-1)) was calculated from the competition with spin trap 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO), and was found close to reactivity of trolox (2.5x10(6)M(-1)s(-1)). Therefore, honokiol is an effective scavenger of both superoxide and peroxyl radicals, which may be important for physiological activity of honokiol.     

Characterization of O,O-diethylphosphoryl oximes as inhibitors of cholinesterases and substrates of phosphotriesterases.

Reactivators of organophosphate (OP)-inhibited cholinesterases (ChEs) are believed to give rise to phosphorylated oximes (POX) that reinhibit the enzyme. Diethylphosphoryl oximes (DEP-OX) that were generated in situ were demonstrated in the past to be unstable, yet were more potent inhibitors of acetylcholinesterase (AChE) than the parent OPs. In view of the inconsistencies among reported results, and the potential toxicity of POXs, it seemed important to characterize authentic DEP-OXs, and to evaluate their interference with reactivation of diethylphosphoryl-ChE (DEP-ChE) conjugates. To this end, the diethylphosphoric acid esters of 1-methyl-2-pyridinium carboxaldehyde oxime (DEP-2PAM) and 1-methyl-4 pyridinium carboxaldehyde oxime (DEP-4PAM) were synthesized and chemically defined. The half-lives of DEP-2PAM and DEP-4PAM in 10 mM Tris buffer, pH 7.8, at 29 degrees were found to be 10 and 980 sec, respectively. The two DEP-OXs inhibited ChEs with the following ranking order: for DEP-2PAM, human butyrylcholinesterase (HuBChE, k(i) = 2.03 x 10(9) M(-1) min(-1)) > mouse AChE (MoAChE) approximately equal to fetal bovine serum AChE (FBS-AChE) approximately equal to equine BChE (EqBChE); for DEP-4PAM, HuBChE (k(i) = 0.71 x 10(9) M(-1) min(-1)) > EqBChE > MoAChE > FBS-AChE. A dialkylarylphosphate hydrolase (phosphotriesterase; PTE) from Pseudomonas sp. catalyzed the hydrolysis of DEP-4PAM with k(cat)/Km = 3.56 x 10(7) M(-1) min(-1) and Km = 0.78 mM. Reactivation of DEP-ChEs was enhanced by PTE when 4-PAM-based oximes were used as reactivators, whereas reactivation with 2-PAM-based oximes was not affected by PTE. This observation is attributed primarily to the short half-life of DEP-OXs derived from the latter oximes. Relatively low doses of PTE can detoxify large quantities of DEP-OXs rapidly, and thereby augment the efficacy of antidotes that contain the oxime function in position 4 of the pyridine ring.     

Thiocyanate is an efficient endogenous scavenger of the phagocytic killing agent hypobromous acid

Second-order rate constants for the reaction of HOBr/OBr- (a putative killing agent of eosinophils and a reactive oxygen species that is implicated in mutagenesis and in human inflammatory diseases) with SCN- (an endogenous species in human physiologic fluids) are determined by stopped-flow spectroscopy. The proposed mechanism includes parallel pathways with Br+ transfer to SCN- by general acid catalysis and by direct reaction with HOBr. HOBr reacts with SCN- with a second-order rate constant (2.3 x 10(9) M(-1) s(-1)) that is 2 orders of magnitude larger than that previously measured for the reaction of HOCl with SCN- (2.3 x 10(7) M(-1) s(-1)), and very close to the diffusion limit. In contrast to OCl-, OBr- exhibits a measurable rate of reaction with SCN- (3.8 x 10(4) M(-1) s(-1)). On a molar basis, SCN- is the most effective scavenger of HOBr to be reported to date (200 times more effective than cysteine and 650 times more effective than methionine). Computational models suggest that SCN- is competitive with respect to other scavengers at physiologically relevant concentrations, which leads us to propose it may limit the lifetime of HOBr and its propensity to inflict host tissue damage during inflammatory response, especially during eosinophilia. Furthermore, the product of the nonenzymatic reaction of HOBr and SCN-, hypothiocyanite (OSCN-), is an effective antimicrobial that is relatively innocuous toward mammalian cell lines. Since one of the principal charges of eosinophil cells is to clear extracellular parasites via nonphagocytic mechanisms that involve degranulation of eosinophil peroxidase (EPO, the principal mammalian enzyme that produces HOBr), a larger role for OSCN- is suggested for parasitic infection.     

Kinetic constraints for the thiolysis of 4-methyl-5-(pyrazin-2-yl)-1,2-dithiole-3-thione (oltipraz) and related dithiole-3-thiones in aqueous solution

This report summarizes an investigation of the reactions of biological and other thiols with the cancer chemopreventive oltipraz and other dithiolethiones. Analysis of the kinetics of reaction of 4-methyl-5-(pyrazin-2-yl)-1,2-dithiole-3-thione (oltipraz) 1 with monothiols and dithiols in the range of 0.75-20 mM in aqueous 15% ethanol, at pH 7.5 (0.1 M Tris buffer) and at 37 degrees C has been undertaken. A plot of k(obsd) against [thiol] shows that reactions of mono- and dithiols are first order in thiol concentration. The dependence on pH of these reactions shows that the active species is the thiolate anion. Specific second-order rate constants, k(2) (M(-1) s(-1)) for reaction of the thiolate anions with oltipraz have been determined to be cysteine, 0.040 +/- 0.001; 2-mercaptoethanol, 2.0 +/- 0.02; glutathione, 0.099 +/- 0.001; mercaptoacetic acid anion, 4.0 +/- 0.01; dithiothreitol, 1.33 +/- 0.02; 1,3-propanedithiol, 10 +/- 0.5; 1-mercaptopropane-3-ol, 6.5 +/- 0.1; 1-mercaptopropane-2,3-diol, 1.26 +/- 0.05. A plot of pK(a) against log k(2) for monothiols shows a linear dependence of k(2) on pK(a), beta(nuc) 1.1 +/- 0.07, which accounts for most of the reportedly enhanced reactivity of dithiols over monothiols. The pseudo-first-order rate constant for the solvolysis of oltipraz has been measured as 2.2 (+/-0.2) x 10(-8) s(-1). The kinetics of reaction of three other dithiole-3-thiones with glutathione has also been studied for comparison with oltipraz. The specific second-order rate constants, k(2) (M(-1) s(-1)) are 5-phenyl-1,2-dithiole-3-thione, 4.7 x 10(-)(4); 5-(4-methoxyphenyl)-1,2-dithiole-3-thione, 4.1 x 10(-4); and 1,2-dithiole-3-thione 0.08. Important implications for the mode of biological action of these compounds and the nature of the putative biological targets of the compounds are discussed.     

In vitro comparative assessment of the scavenging activity against three reactive oxygen species of non-steroidal anti-inflammatory drugs from the oxicam and sulfoanilide families

The study of the interaction of non-steroidal anti-inflammatory drugs (NSAIDs) with several reactive oxygen species is of great interest in inflammatory conditions where an uncontrolled release of these potentially damaging intermediates has been documented. This study focused on the scavenging of three species (hydroxyl radical, hydrogen peroxide and hypochlorous acid) with several members of the oxicam family and with the sulfoanilide nimesulide. Reaction with hydroxyl radical was assessed by the modified deoxyribose assay, and rate constants were calculated showing values between 0.8 and 1.1 x 10(10) M(-1) s(-1) for oxicams and of about 0.9 x 10(10) M(-1) s(-1) for nimesulide and ibuprofen. These were consistent with those of the literature but in the same range as those for other NSAIDs and for several thiol-containing molecules. The study of hydrogen peroxide scavenging by the horseradish peroxidase (HRP) assay lacked specificity but no interaction could be evidenced by the glutathione peroxidase assay. The scavenging of hypochlorous acid was finally investigated by the recently developed para-aminobenzoic acid assay which demonstrated better performances for meloxicam (1.7 x 10(4) M(-1) s(-1)) as compared to the other oxicams (tenoxicam: 4.0 x 10(4) M(-1) s(-1), piroxicam: 3.6 x 10(4) M(-1) s(-1), lornoxicam: 4.3 x 10(4) M(-1) s(-1)) and nimesulide (2.3 x 10(3) M(-1) s(-1)). These rate constants were, however, lower than those for thiol-containing molecules and ascorbate. These results suggest that the antioxidant properties of NSAIDs could be influenced by a proper pharmacomodulation as far as the scavenging of hypochlorous acid is concerned while the interest is quite limited for the scavenging of hydroxyl radical.     

Dualistic actions of cromakalim and new potent 2H-1,4-benzoxazine derivatives on the native skeletal muscle K ATP channel

1 New 2H-1,4-benzoxazine derivatives were synthesized and tested for their agonist properties on the ATP-sensitive K(+) channels (K(ATP)) of native rat skeletal muscle fibres by using the patch-clamp technique. The novel modifications involved the introduction at position 2 of the benzoxazine ring of alkyl substituents such as methyl (-CH(3)), ethyl (-C(2)H(5)) or propyl (-C(3)H(7)) groups, while maintaining pharmacophore groups critical for conferring agonist properties. 2 The effects of these molecules were compared with those of cromakalim in the presence or absence of internal ATP (10(-4) M). In the presence of internal ATP, all the compounds increased the macropatch K(ATP) currents. The order of potency of the molecules as agonists was -C(3)H(7) (DE(50)=1.63 x 10(-8) M) >-C(2)H(5) (DE(50)=1.11 x 10(-7) M)>-CH(3) (DE(50)=2.81 x 10(-7) M)>cromak-slim (DE(50)= 1.42 x 10(-5) M). Bell-shaped dose-response curves were observed for these compounds and cromakalim indicating a downturn in response when a certain dose was exceeded. 3 In contrast, in the absence of internal ATP, all molecules including cromakalim inhibited the K(ATP) currents. The order of increasing potency as antagonists was cromakalim (IC(50)=1.15 x 10(-8) M)> or =-CH(3) (IC(50)=2.6 x 10(-8) M)>-C(2)H(5) (IC(50)=4.4 x 10(-8) M)>-C(3)H(7) (IC(50)=1.68 x 10(-7) M) derivatives. 4 These results suggest that the newly synthesized molecules and cromakalim act on muscle K(ATP) channel by binding on two receptor sites that have opposite actions. Alternatively, a more simple explanation is to consider the existence of a single site for potassium channel openers regulated by ATP which favours the transduction of the channel opening. The alkyl chains at position 2 of the 2H-1,4-benzoxazine nucleus is pivotal in determining the potency of benzoxazine derivatives as agonists or antagonists.