Identification of antioxidants for prevention of peroxide-mediated oxidation of recombinant human ciliary neurotrophic factor and recombinant human nerve growth factor.

Peroxides present in non-ionic surfactants used to stabilize certain recombinant protein formulations (e.g. polysorbate 80) can result in the oxidative degradation of proteins. In this study, the ability of various pharmaceutically acceptable antioxidants to prevent the oxidative degradation of two therapeutic proteins, recombinant human Ciliary Neurotrophic Factor (rhCNTF) and recombinant human Nerve Growth Factor (rhNGF), caused by alkyl hydroperoxides and hydrogen peroxide was studied. For rhCNTF, the rank order of effectiveness of the antioxidants tested was: thiols (cysteine, glutathione, thioglycerol) >> thioethers (methionine). Other parenterally acceptable antioxidants (ascorbic acid, propyl gallate and sodium bisulfite) destabilized the protein. The thiol antioxidants (cysteine and glutathione) were also the most effective antioxidants for rhNGF; however, in contrast to rhCNTF, ascorbic acid did not destabilize rhNGF. The rank order of effective antioxidants for rhNGF was: thiols (cysteine, glutathione) > > thioethers (methionine) > ascorbic acid.

Stability of interleukin 1 beta (IL-1 beta) in aqueous solution: analytical methods, kinetics, products, and solution formulation implications.

The thermal stability of IL-1 beta in aqueous solution as a function of temperature (5-60 degrees C), pH (2-9), buffer (acetate, citrate, tris, and phosphate), and cyroprotectants (sugars, HSA) was investigated in this study. The analytical methodologies included RP-HPLC, SEC, ELISA, IEF-PAGE, SDS-PAGE, and bioassay. The degradation and inactivation of IL-1 beta at or above 39 degrees C were attributed to autoxidation of the two cysteine residues in the denatured protein, followed by hydrophobic/covalent aggregation and precipitation. At or below 30 degrees C, IEF- and SDS-PAGE results suggest a possible deamidation reaction. The difference in mechanism of degradation precludes the prediction of formulation shelf life from accelerated temperature data. Nonetheless, the good stability observed at 5 degrees C suggests that a solution formulation may be feasible for IL-1 beta.

Quantitative evaluation of the stability and delivery of interleukin-1B by infusion.

The stability and delivery of IL-1 beta has been characterized in a polypropylene based syringe pump infusion system and in polyvinyl chloride based infusion bags, at concentrations ranging from 100 ng/mL to 1 microgram/mL. At higher concentrations (1 microgram/mL), minimal drug loss was observed in both systems. At low doses (100 ng/mL) in the syringe-pump system, the addition of 1% human serum albumin was necessary to prevent significant drug absorption to the polypropylene drug reservoir.

Development and characterization of a lyophilized dosage form of IL-1 beta.

The development and characterization of a lyophilized dosage form for recombinant Interleukin-1 beta is described. Included in the evaluation of the drug product are accelerated and long-term stability studies utilizing a number of biophysical techniques (reverse-phase HPLC, SDS-PAGE, isoelectric focusing and ELISA). Data collected with these methods were examined for correlations with biological activity assessments provided by an in-vitro cell culture system (mouse thymocyte proliferation). Results of these studies demonstrate that a lyophilized dosage form of Interleukin-1 beta can be prepared which retains its potency for at least 1 year when stored at ambient temperature. The analytical methodology used to assess the physicochemical integrity of the protein provided a sensitive and reproducible means of predicting changes in biological activity.

Relevance of the reaction of a manganese(III) chelate with hydroxide ion to photosynthesis: Reaction of hydroxide ion with 5,10,15,20-tetrakis(2,4,6-trimethylphenyl)porphinatomanganese(III) in ligating and nonligating solvents.

The reaction of HO(-) with 5,10,15,20-tetrakis(2,4,6-trimethylphenyl)porphinatomanganese(III) chloride [(TMP)Mn(III)(Cl)] in ligating solvents (CH(3)CN, dimethyl sulfoxide, pyridine) results in formation of (TMP)Mn(II) ( approximately 10(6) M(-1).s(-r)), which in a slower reaction is converted to a product whose structure is suggested to be that of a porphyrin manganese(III) peroxo dimer. Admittance of O(2) at any time during these reactions leads to formation of the manganese(III) peroxide (TMP)Mn(III)(O(2))(-). In nonligating solvents [CH(2)Cl(2), (CH(3))(2)CO], the reaction of HO(-) with (TMP)Mn(III)(Cl) yields (TMP)Mn(IV)(OH)(2).

Reactivity and activation of dioxygen-derived species in aprotic media (a model matrix for biomembranes).

In aprotic media the electrochemical reduction of dioxygen yields superoxide ion (O2-), which is an effective Brønsted base, nucleophile, one-electron reductant, and one-electron oxidant of reduced transition metal ions. With electrophilic substrates (organic halides and carbonyl carbons) O2- displaces a leaving group to form a peroxy radical (ROO.) in the primary process. Superoxide ion oxidizes the activated hydrogen atoms of ascorbic acid, catechols, hydrophenazines and hydroflavins. Combination of O2- with 1,2-diphenylhydrazine yields the anion radical of azobenzene, which reacts with O2 to give azobenzene and O2- (an example of O2--induced autoxidation). With phenylhydrazine, O2- produces phenyl radicals. The in situ formation of HO2. (O2- plus a proton source) results in H-atom abstraction from allylic and other groups with weak heteroatom--H bonds (binding energy (b.e.) less than 335 kJ). This is a competitive process with the facile second-order disproportionation of HO2. to H2O2 and O2 (kbi approximately equal to 10(4) mol-1 s-1 in Me2SO). Addition of [FeII(MeCN)4] (ClO4)2 to solutions of hydrogen peroxide in dry acetonitrile catalyses a rapid disproportionation of H2O2 via the initial formation of an adduct [FeII(H2O2)2+----Fe(O)(H2O)2+], which oxidizes a second H2O2 to oxygen. In the presence of organic substrates such as 1,4-cyclohexadiene, 1,2-diphenylhydrazine, catechols and thiols the FeII-H2O2/MeCN system yields dehydrogenated products; with alcohols, aldehydes, methylstyrene, thioethers, sulphoxides, and phosphines, the FeII(H2O2)2+ adduct promotes their monoxygenation. The product from the FeO2+-H2O2 reaction, [FeII(H2O2)22+], exhibits chemistry that is closely similar to that for singlet oxygen (1O2), which has been confirmed by the stoichiometric dioxygenation of diphenylisobenzofuran, 9,10-diphenylanthracene, rubrene and electron-rich unsaturated carbon-carbon bonds (Ph2C = CPh2, PhC = CPh and cis-PhCH = CHPh). In dry ligand-free acetonitrile (MeCN), anhydrous ferric chloride (FeIIICl3) activates hydrogen peroxide for the efficient epoxidation of alkenes. The FeIIICl3 further catalyses the dimerization of the resulting epoxides to dioxanes. These observations indicate that strong Lewis acids that are coordinatively unsaturated, [FeII(MeCN)4]2+ and [FeIIICl3], activate H2O2 to form an effective oxygenation and dehydrogenation agent.(ABSTRACT TRUNCATED AT 400 WORDS)

Stabilization of higher-valent states of iron porphyrin by hydroxide and methoxide ligands: electrochemical generation of iron(IV)-oxo porphyrins.

An electrochemical study of hydroxide- and methoxide-ligated iron(III) tetraphenylporphyrins possessing ortho-phenyl substituents that block mu-oxo dimer formation has been carried out. Ligation by these strongly basic oxyanions promotes the formation of iron(IV)-oxo porphyrins upon one-electron oxidation. Further one-electron oxidation of the latter provides the iron(IV)-oxo porphyrin pi-cation radical. These results are discussed in terms of chemical model studies and the enzymatic intermediate compounds I and II of the peroxidases.

Base (O.-2, e-, or OH-)-induced autoxygenation of organic substrates: a model chemical system for cytochrome P-450-catalyzed monoxygenation and dehydrogenation by dioxygen.

When catalytic quantities of superoxide ion (O.-2; or of electrons from electrolysis or of OH-) are introduced into a dry acetonitrile solution that contains excess substrate (RH), ambient air (O2), 1,2-diphenylhydrazine (PhNHNHPh), and iron(II), the substrate is rapidly and efficiently monoxygenated (e.g., triphenylphosphine----triphenylphosphine oxide, benzyl alcohol----benzaldehyde, diphenylsulfoxide----diphenylsulfone) or dehydrogenated (1,4-cyclohexadiene----benzene). The model consists of (i) an O2-activation segment that produces H2O2 from an O.-2-initiated autoxidation of disubstituted hydrazine (a model for reduced flavin), (formula; see text) and (ii) a H2O2-activation segment via the iron(II)-induced formation of ferryl ion (FeO2+), Fe(II) + H2O2----FeO2 + H2O, an effective monoxygenating agent: FeO2+ + RH----Fe(II) + ROH. The combination of i and ii provides a catalytic system for the autoxidation of organic substrates with reaction cycles that are similar to those for cytochrome P-450 monoxygenases.