Kinetics and Mechanism for the Reaction of Cysteine with Hydrogen Peroxide in Amorphous Polyvinylpyrrolidone Lyophiles

Purpose Peroxide impurities play a critical role in drug oxidation. In metal-free aqueous solutions, hydrogen peroxide (H₂O₂) induced thiol oxidation involves a bimolecular nucleophilic reaction to form a reactive sulfenic acid intermediate (RSOH), which reacts with a second thiol to form a disulfide (RSSR). This study examines the reaction of cysteine (CSH) and H₂O₂ in amorphous polyvinylpyrrolidone (PVP) lyophiles to explore the possible relevance of the solution mechanism to reactivity in an amorphous glass.Materials and Methods Amorphous PVP lyophiles containing CSH and H₂O₂ at varying initial ‘pH’ and reactant concentrations were prepared by methods designed to minimize reaction during lyophilization. Kinetic studies were conducted anaerobically at 25°C and reactants and products were monitored by HPLC. Products were characterized and the kinetic data were fit to models adapted from the solution mechanism.Results Key differences in the reactions in aqueous solution and amorphous PVP are: (1) while only cystine (CSSC) forms in solution, three degradants—cysteine sulfinic acid (CSO₂H), cysteine sulfonic acid (CSO₃H) and cystine (CSSC)—form in amorphous PVP; (2) simple bimolecular kinetics govern the solution reaction while initial rates in amorphous PVP suggested more complex kinetics (i.e., non-unity values for reaction order); and (3) heterogeneous (i.e., biphasic) reaction dynamics are evident in amorphous PVP. The differences in product formation and apparent reaction orders in the solid-state could be rationalized by partitioning of the same reactive intermediate to multiple products in the solid-state due to the restricted mobility of CSH. Beyond the initial rate region, the kinetics in amorphous PVP could be described by the Kohlrausch‐Williams‐Watts (KWW) stretched-exponential equation or by assuming two populations of reactant molecules having different reactivities.Conclusions When reactive intermediates are involved, differences in degradant profiles and other characteristics (e.g., rate constants, apparent reaction order) in the amorphous-state may simply reflect altered rates for individual reaction steps due to glass-induced changes in relative reactant mobilities rather than a change in overall mechanism.     

In Vitro Methionine Oxidation of Recombinant Human Leptin

Purpose. To investigate the role and importance of the four methionines in recombinant human leptin, and the effect of methionine oxidation in leptin structural stability and biological activity. Methods. Oxidized leptin derivatives were prepared in the presence of H₂O₂ and t-butylhydroperoxide, separated by RP-HPLC, and characterized by peptide mapping and LC/MS. Their biophysical and biological properties were studied. Results. Six major species of oxidized leptins were detected: two mono-oxidized, one di-oxidized, two tri-oxidized, and one tetra-oxidized. Further oxidation at cystine disulfide was also detected. Kinetic analysis indicated that oxidation at Met¹ and Met⁶⁹ proceeded first and independently. In 48 mM t-butylhydroperoxide, the pseudo first-order rate constants, k ₁ and k ₆₉, were 1.5 × 10^–3 and 2.3 × 10^–4 min^–1. No change in the secondary or tertiary structure was detected for Met¹ mono-oxidized and Met¹, Met⁶⁹ di-oxidized leptins. The Met¹ mono-oxidized leptin retained full potency as compared to native leptin. A slight decrease of thermostability and a significant loss of the in vitro bioactivity were observed for Met¹, Met⁶⁹ di-oxidized leptin. Both Met⁵⁵ and Met¹³⁷ were not oxidized in t-butylhydroperoxide but only in H₂O₂. They appeared to be much less accessible to oxidation and might interact with the hydrophobic core structure of the leptin molecule. Conclusions. The oxidation of leptin occurred in the order of Met¹ > Met⁶⁹ >> Met⁵⁵ Met¹³⁷, and the importance for maintaining leptin structural integrity was Met⁵⁵ Met¹³⁷ >> Met⁶⁹ Met¹. Met⁶⁹, but not Met¹, plays a critical role in the protein stability and activity.     

The Disposition of a Human Relaxin (hRlx-2) in Pregnant and Nonpregnant Rats

The pharmacokinetics and tissue distribution of a human relaxin were investigated after intravenous (iv) bolus administration to pregnant or nonpregnant rats. Human gene-2 relaxin (hRlx-2) serum concentrations after iv bolus administration were described as the sum of three exponentials. The pharmacokinetics were comparable in pregnant and nonpregnant rats. The serum clearance (CL) was 7.4–10.2 ml/min/kg at doses of 46–93 µg/kg and was linear in this range. The half-lives were 1.1–2.0, 15.1–16.4, and 53.7–67.9 min, respectively. The volume of the central compartment (V _c) was 48–79 ml/kg and the volume of distribution at steady state (V _ss) was 271–336 ml/kg. Increasing the dose to 463 µg/kg increased the dose-corrected area under the serum concentration–time curve and significantly decreased CL and Vss. The distribution of radioactivity in the tissues of pregnant rats was followed after iv bolus dosing with hRlx-2 internally labeled with ³⁵S-cysteine. Comparison of the extent of organ uptake of radiolabel after ³⁵S-hRlx-2 or ³⁵S-cysteine administration suggested that the kidneys were the principal site of uptake; the liver was of secondary importance. In perfusion experiments utilizing livers isolated from pregnant or nonpregnant rats, 36–52% of the dose of hRlx-2 was cleared from the perfusate in 2 hr. These studies showed that the pharmacokinetics of hRlx-2 in rats appeared to be unaffected by pregnancy and suggested that the kidneys and liver both play a role in the elimination of hRlx-2.     

Effect of Hydrogen Peroxide on the Viscosity of a Hydroxyethylcellulose-Based Gel

Pharmaceutical Research -     

新型麻醉药异丙酚的氧化动力学

采用高效液相色谱法测定了异丙酚在不同温度下及其甲醇 水（Ｖ∶Ｖ＝７０∶３０）混合溶液在不同ｐＨ值下的反应速率常数，并求出了异丙酚氧化反应的活化能，结果表明ｐＨ值与温度对异丙酚的氧化动力学均有影响     

Near-Infrared (NIR) Monitoring of H2O2 Vapor Concentration During Vapor Hydrogen Peroxide (VHP) Sterilisation

Purpose. There is an increasing use in the pharmaceutical industry of barrier systems such as transfer isolators, sterilisation tunnels and work station isolators. As Vapor Hydrogen Peroxide (VHP) sterilisation of isolators and lyophilizers becomes an important sterilisation method, there is an acute need for a VHP monitoring system to be used for in-process control and validation. In this study, near infrared (NIR) spectrofotometry was evaluated as a potential technique to monitor hydrogen peroxide. Additionally the H₂O₂ vapor permeability of different packaging materials, commonly used in steam and ethylene oxide sterilisation, was evaluated. Methods. NIR spectrofotometry, using a gas cell connected with optic fibres, was evaluated as a potential technique to monitor hydrogen peroxide vapor and water vapor during VHP sterilisation of an isolator. A NIR spectrum was taken every 30 s during VHP sterilisation of an isolator. The influence of injection rate, air flow rate, working temperature and gas distribution was investigated. The H₂O₂ vapor permeability of different packaging materials was determined by placing the gas cell in the sterilisation bags and sealing the bags hermetically. The sterilisation bag was then subjected to VHP sterilisation. Results. The NIR spectra taken at steady state sterilization conditions showed 4 absorption peaks: at 1364,1378 and 1400 nm attributed to water and at 1420 nm attributed to H₂O₂ vapor. By measuring the absorbance level at these wavelengths, the actual concentration of H₂O and H₂O₂ vapor in the isolator was calculated. The water vapor permeation of the sterilisation bags, measured with NIR, appeared to be equal for all materials tested. Whereas Tyvek^® was the most permeable material for hydrogen peroxide vapor (82.7% of the reference concentration outside the bag), only 30% was found in bags made of medical paper. Sterilisation bags consisting of laminate films and PVC sealed to medical paper showed intermediate permeability. Conclusions. Near-infrared (NIR) spectroscopy using a gascell with optic fibres is a useful technique to monitor VHP sterilisation cycles. There was a difference in H₂O₂ vapor permeability of different packaging materials, commonly used in steam and ethylene oxide sterilisation.     

Kinetics and Mechanism of Captopril Oxidation in Aqueous Solution Under Controlled Oxygen Partial Pressure

The stability of captopril in aqueous solution at 32°C was studied in the pH range 6.6 to 8.0 under controlled oxygen partial pressure (90–760 mm Hg) with and without the addition of cupric ion. The oxidation product, captopril disulfide, was found to be the sole degradation product. A change in reaction rate from first order to zero order occurs as the captopril concentration decreases. The concentration at which this transition takes place is a function of the pH, oxygen partial pressure, and cupric ion concentration. The apparent first-order rate constants show a first-order dependency on both the oxygen partial pressure and the cupric ion concentration. However, the apparent zero-order rate constants show a first-order dependency on oxygen partial pressure and a second-order dependency on cupric ion concentration. As the pH increases from 6.6 to 8.0, the first-order process becomes more predominant. A mechanism which consists of cupric ion- and molecular oxygen-catalyzed oxidation is proposed to explain those observations.     

Oxidation of Human Insulin-like Growth Factor I in Formulation Studies: Kinetics of Methionine Oxidation in Aqueous Solution and in Solid State

Purpose. The aim of this work was to study the kinetics of oxidation of methionine in human Insulin-like Growth Factor I (hIGF-I)¹ in aqueous solution and in the solid state by the aid of quantification of oxygen. Methods. The oxidized form of hIGF-I was characterized by tryptic peptide analysis, RP-HPLC and FAB-MS and quantified by RP-HPLC. The oxygen content was quantified polarographically by a Clark-type electrode. Results. Second-order kinetics with respect to amount of protein and dissolved oxygen was found to be appropriate for the oxidation of methionine in hIGF-I. The rate constants ranged from 1 to 280 M^–1 month^–l and had an activation energy of 95 (+/–4) kJ/mole. Light exposure, storage temperature and oxygen content were found to have a considerable impact on the oxidation rates. No significant difference in reaction rates was found for the oxidation of hIGF-I in aqueous solution or in the solid state. A method for decreasing the oxygen content in aqueous solution without purging is described. Conclusions. Polarographic quantification of dissolved oxygen makes it possible to establish the kinetics for oxidation of proteins. The oxidation of methionine in hIGF-I appears to follow second-order kinetics.     

The Engineering of Hydrogen Peroxide Decontamination Systems

In this article, the latest developments for designing hydrogen peroxide decontamination systems are analyzed. Specifically, focus is given to the accurate calculation of hydrogen peroxide condensation phenomena and discussion of a new correlation for its accurate prediction. A procedure for calculating the condensate composition or the dew point out of this correlation is detailed, and an h–x diagram for moist, hydrogen peroxide-laden air, which is of fundamental importance for the rational design of hydrogen peroxide decontamination systems, is proposed. Also presented are theoretical results that illustrate the effect of condensation and evaporation in these systems. Finally, some perspectives for improving hydrogen peroxide systems, and the role computational fluid dynamics (CFD) may have in this field, are provided.     

过氧化氢对FRTL细胞线粒体膜电位和超氧化物生成的影响

目的:探讨外源性过氧化氢(H2O2)对Fisher大鼠甲状腺细胞系(FRTL)线粒体膜电位(△ψ)和超氧化物生成的影响.方法:用1 mmol/L H_2O_2处理FRTL细胞10 min、30 min、24 h后,利用MitoSOX,通过活细胞影像法、流式细胞术检测线粒体超氧化物生成;利用罗丹明123(rh123),通过荧光分光光度计和荧光显微镜检测△ψ;MTT比色法检测细胞活力;光镜观察细胞形态学变化;吖啶橙(AO)染色检测细胞凋亡.结果:与对照组相比,1 mmol/L H_2O_2处理的FRTL细胞10 min、30 min、24 h,细胞内MitoSOX荧光强度明显增强,rh123荧光强度和MTT吸光度明显下降(P＜0.01),光镜下可见细胞脱壁、破碎,AO染色可见核变小、变圆,染色质浓缩、边集,核碎裂改变.结论:1 mmol/L H_2O_2急性处理(10 min,30 min)和慢性处理(24 h)均能明显增加FRTL细胞线粒体超氧化物生成,降低线粒体膜电位,造成细胞坏死和凋亡.     

Comparison Between Light Induced and Chemically Induced Oxidation of rhVEGF

Purpose. The primary objective of this study was to compare the effects of light-and chemical-induced oxidation of recombinant human vascular endothelial growth factor (rhVEGF) and the impact of these reactions on protein formulation. Methods. A liquid formulation of rhVEGF was exposed to fluorescent light (2 × 10⁴ lux for up to 4 weeks), hydrogen peroxide (H₂O₂), or t-butylhydroperoxide (t-BHP) to induce oxidation of rhVEGF. All samples were then treated by tryptic digest and analyzed by reversed phase HPLC to determine the extent of oxidation. Chemically treated samples were also examined by near-UV and far-UV circular dichroism spectroscopy to determine the effect of oxidation on the structure of the protein. Results. Exposure to light for 2 weeks resulted in 8 to 40% oxidation of all 6 methionine residues of rhVEGF (Met3 > Met18 > Met55 > Met78,81 > Met94). This amount of oxidation did not affect the binding activity of rhVEGF to its kinase domain receptor (KDR). Light exposure for 4 weeks increased metsulfoxide formation at Met3 and Met18 by an additional 16%, but did not affect the other residues. This oxidation decreased the receptor binding capacity to 73%, possibly due to the role of Met18 in receptor binding. Chemical oxidation of rhVEGF resulted in a greater extent of oxidation at all 6 methionines. Complete oxidation of Met3, Met18 and Met55 was observed after treatment with H₂O₂, while these residues underwent 40 to 60% oxidation after treatment with t-BHP. The receptor binding capacity was significantly reduced to 25% and 55% after treatment with H₂O₂ and t-BHP, respectively. After chemical oxidation, no changes in the secondary or tertiary structure were observed by far-UV and near-UV CD spectroscopy, respectively. Conclusions. Methionine residues with exposed surface areas greater than 65 Ų and sulfur surface areas greater than 16 Ų were most susceptible to oxidation. Chemical oxidation resulted in higher metsulfoxide formation and decreased binding activity of the protein to KDR than light-induced oxidation. The reduction in KDR binding was not caused by measurable conformational changes in the protein. Photooxidation was dependent on the amount of energy imparted to the protein, while the ability of t-BHP or H₂O₂ to react with methionine was governed by solvent accessibility of the methionine residues and steric limitations of the oxidizing agent. Significant chemical oxidation occurred on sulfurs with minimum surface areas of 16 Ų, while increased photooxidation occurred as a function of increasing surface areas of solvent exposed sulfur atoms. Such differences in the extent of oxidation should be considered during protein formulation since it may help predict potential oxidation problems.     

衍生化HPLC法测定低分子肝素钠中过氧化氢残留

目的：建立测定低分子肝素钠中痕量过氧化氢残留的方法。方法：用4-氨基安替比林和苯酚溶液在过氧化物酶的作用下与过氧化氢的衍生化反应,以Thermo ODS-2 hypersil C18色谱柱（4.6 mm×250 mm,5μm）在流动相（甲醇-水=60∶40）的条件下,分离检测过氧化氢的浓度。结果：该方法专属性好,过氧化氢的最低检测限为0.0302μg·mL-1,定量下限为0.1007μg·mL-1,在0.1~2.0μg·mL-1的浓度范围内线性关系良好;平均回收率为97.58%,RSD为4.3%。结论：建立的方法可对低分子肝素钠中痕量过氧化氢残留进行测定。     

Application of Hydrogen Peroxide to the Control of Eutrophic Lake Systems in Laboratory Assays

We exposed water samples from a recreational lake dominated by the cyanobacterium Planktothrix agardhii to different concentrations of hydrogen peroxide (H₂O₂). An addition of 0.33 mg·L⁻¹ of H₂O₂ was the lowest effective dose for the decay of chlorophyll-a concentration to half of the original in 14 h with light and 17 h in experiments without light. With 3.33 mg·L⁻¹ of H₂O₂, the values of the chemical oxygen demand (COD) decreased to half at 36 and 126 h in experiments performed with and without light, respectively. With increasing H₂O₂, there is a decrease in the total and faecal coliform, and this effect was made more pronounced by light. Total and faecal coliform were inhibited completely 48 h after addition of 3.33 mg·L⁻¹ H₂O₂. Although the densities of cyanobacterial cells exposed to H₂O₂ did not decrease, transmission electron microscope observation of the trichomes showed several stages of degeneration, and the cells were collapsed after 48 h of 3.33 mg·L⁻¹ of H₂O₂ addition in the presence of light. Our results demonstrate that H₂O₂ could be potentially used in hypertrophic systems because it not only collapses cyanobacterial cells and coliform bacteria but may also reduce chlorophyll-a content and chemical oxygen demand.     

GS28 Protects Neuronal Cell Death Induced by Hydrogen Peroxide under Glutathione-Depleted Condition

Golgi SNAP receptor complex 1 (GS28) has been implicated in vesicular transport between intra-Golgi networks and between endoplasmic reticulum (ER) and Golgi. Additional role(s) of GS28 within cells have not been well characterized. We observed decreased expression of GS28 in rat ischemic hippocampus. In this study, we examined the role of GS28 and its molecular mechanisms in neuronal (SK-N-SH) cell death induced by hydrogen peroxide (H₂O₂). GS28 siRNA-transfected cells treated with H₂O₂ showed a significant increase in cytotoxicity under glutathione (GSH)-depleted conditions after pretreatment with buthionine sulfoximine, which corresponded to an increase of intracellular reactive oxygen species (ROS) in the cells. Pretreatment of GS28 siRNA-transfected cells with p38 chemical inhibitor significantly inhibited cytotoxicity; we also observed that p38 was activated in the cells by immunoblot analysis. We confirmed the role of p38 MAPK in cotransfected cells with GS28 siRNA and p38 siRNA in the cell viability assay, flow cytometry, and immunoblot. Involvement of apoptotic or autophagic processes in the cells was not shown in the cell viability, flow cytometry, and immunoblot analyses. However, pretreatment of the cells with necrostatin-1 completely inhibited H₂O₂-induced cytotoxicity, ROS generation, and p38 activation, indicating that the cell death is necroptotic. Collectively these data imply that H₂O₂ induces necroptotic cell death in the GS28 siRNA-transfected cells and that the necroptotic signals are mediated by sequential activations in RIP1/p38/ROS. Taken together, these results indicate that GS28 has a protective role in H₂O₂-induced necroptosis via inhibition of p38 MAPK in GSH-depleted neuronal cells.     

过氧化氢氧化降解法制备低分子玻璃酸

目的制备低分子玻璃酸 (HA)。方法过氧化氢氧化降解法。结果随着过氧化氢浓度增加 ,反应温度的升高 ,降解速率加快。中性条件下HA的氧化降解速率较快 ,而酸性或碱性时却较慢。为了方便降解过程的可控性 ,过氧化氢浓度选 0 .0 5 % ,反应温度定为 5 0℃ ,反应pH为中性。随着HA相对分子质量的降低 ,运动黏度迅速下降 ,而糖醛酸含量基本不变。不同过氧化氢浓度降解时 ,低分子HA收率基本相同。结论过氧化氢氧化降解法可用于制备低分子HA。     

过氧化氢稳定性研究

目的：研究脂肪醇聚氧乙烯醚(AEO)对过氧化氢稳定性的影响。方法：配制含有不同浓度脂肪醇聚氧乙烯醚AEO的过氧化氢溶液,混合后,采用滴定法对其含量进行检测,并利用电导仪对其进行检测。结果：通过对溶液的含量进行检测,可以看出,适量的AEO能提高过氧化氢溶液稳定性。     

Oxidation of Recombinant Human Parathyroid Hormone: Effect of Oxidized Position on the Biological Activity

Purpose. To determine the oxidation products of recombinant human parathyroid hormone (rhPTH) treated with H₂O₂, the amino acid residue oxidized, and the biological activity of the oxidation products. Methods. Oxidized residues were determined by CNBr cleavage, trypsin digestion and subsequent fast atom bombardment mass spectrometry. The biological activity of each oxidized rhPTH was examined in rat osteosarcoma cell adenylate cyclase assay. Results. Three oxidized products were isolated, namely, Met at position 8 (Met8) sulfoxide, Met at position 18 (Met 18) sulfoxide and both positions Met sulfoxide. It appears that the Met8 and Met 18 oxidized forms are intermediates in the generation of the Met doubly oxidized form. All oxidized forms possessed reduced biological activity, more so for oxidation at Met8 than at Met 18. Conclusions. The region around Met8 is important for the activity of the parathyroid hormone.     

鼻内镜手术联合双氧水冲洗治疗真菌性鼻-鼻窦炎

目的评价鼻内镜手术联合双氧水冲洗在真菌性鼻-鼻窦炎治疗中的应用价值。方法对36例真菌性鼻-鼻窦炎患者采用鼻内镜手术,术中、术后使用双氧水冲洗治疗。结果全部病例均治愈,随访6个月～4年,未见复发。结论娴熟的鼻内镜手术联合双氧水冲洗能彻底清除病变,消除真菌赖以生存的低氧微环境,是微创治疗真菌性鼻-鼻窦炎的理想方式。     

Chemical Stability of Ethyl Icosapentate Against Autoxidation. I. Effect of Temperature on Oxidation Kinetics

Pharmaceutical Research -