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1.
Rupesh Bommana Olivier Mozziconacci Y. John Wang Christian Schöneich 《Pharmaceutical research》2017,34(7):1428-1443
Purpose
The loss of potency of protein therapeutics can be linked to the oxidation of specific amino acid residues leading to a great variety of oxidative modifications. The comprehensive identification of these oxidative modifications requires high-resolution mass spectrometry analysis, which requires time and expensive resources. Here, we propose a fluorogenic derivatization method of oxidized Tyr and Phe yielding benzoxazole derivatives, as an orthogonal technique for the rapid screening of protein oxidation.Methods
Four model proteins, IgG1, human growth hormone (hGH), insulin and bovine serum albumin (BSA) were exposed to oxidation via peroxyl radicals and metal-catalyzed reactions and efficiently screened by fluorogenic derivatization of Tyr and Phe oxidation products. Complementary LC-MS analysis was done to identify the extent of methionine oxidation in oxidized proteins.Results
The Fluorogenic derivatization technique can easily be adapted to a 96-well plate, in which several protein formulations can be screened in short time. Representatively for hGH, we show that the formation of benzoxazole parallels the oxidation of Met to methionine sulfoxide which enables estimation of Met oxidation by just recording the fluorescence.Conclusions
Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.2.
Jessica Bane Olivier Mozziconacci Li Yi Y. John Wang Alavattam Sreedhara Christian Schöneich 《Pharmaceutical research》2017,34(1):229-242
Purpose
Triply oxidized histidine in an IgG1 monoclonal antibody was noticed when exposed to ICH light conditions. In order to understand the role of light source, irradiation wavelengths and primary sequence, specifically those of a nearby tryptophan, we synthesized and exposed several peptides to ICH light conditions and analyzed the products using LC-MS analysis.Methods
Protein and peptide samples were photo-irradiated under ICH conditions as well as with monochromatic light at λ?=?254 nm and analyzed using either LTQ Orbitrap or a LTQ-FT ion cyclotron resonance mass spectrometer respectively.Results
A triply oxidized His residue was detected along with a second doubly oxidized His residue in an IgG1. Both of these oxidized His residues are located near Trp residues. In order to investigate the role of Trp photosensitization in His oxidation we synthesized model peptides and Ala mutants. Peptides exposed to ICH light stress conditions revealed a small percent of triply oxidized His in the Trp-containing peptide sequences but not in their corresponding Ala mutants.Conclusions
The differences in product formation under different photo-irradiation conditions underline the importance of light source, irradiation wavelengths and primary sequence in the photosensitivity of proteins.3.
Lin Zhang Sandeep Yadav Y. John Wang Olivier Mozziconacci Christian Schӧneich 《Pharmaceutical research》2018,35(2):33
Purpose
L-Histidine (L-His) and polysorbate 20 (PS20) are two excipients frequently included in parenteral products to stabilize biotherapeutics. The objective of the current work was to investigate the impact of L-His on PS20 stability in aqueous solutions when subjected to forced oxidation and accelerated stability testing.Methods
The stability of PS20 in L-His buffer was compared with that in acetate buffer. Forced oxidation of PS20 in these two buffer systems was initiated by a free radical generator, 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH), while accelerated stability tests were carried out at 40°C. Ultra-performance liquid chromatography mass spectrometry was utilized to monitor intact PS20 and to analyze degradation products.Results
Our results demonstrate a dual effect of L-His on PS20 stability. During exposure to AAPH, L-His protects PS20 from oxidation. Stable isotope labeling of L-His with 13C was employed for mechanistic investigations. The protection of L-His was abrogated when acetate was added to L-His buffer, implying that the anti-oxidative activity of L-His may be compromised by specific counter ions. The replacement of L-His by various His derivatives led to significant changes in the protection of PS20 against AAPH-induced degradation. In contrast to forced degradation, the addition of L-His promoted oxidative PS20 degradation during accelerated storage at 40°C in solution, generating mainly short chain POE-laurates.Conclusion
L-His exhibits a dual effect on the stability profile of PS20, protecting against AAPH-induced oxidation but promoting oxidative degradation during accelerated stability testing.4.
Purpose
Oxidized interferons have been shown to aggregate and cause immunogenicity. In this study, the structural mechanisms underlying oxidation-induced interferon alpha-2a (IFNA2a) aggregation and loss of function were examined.Methods
IFNA2a was oxidized using 0.037% vol/vol hydrogen peroxide. Oxidized protein was probed using biophysical methods that include denaturant melts, particle counting, proteolysis-coupled mass spectrometry, and 2D NMR.Results
Oxidized IFNA2a did not show major changes in its secondary structure, but showed minor changes in tertiary structure when compared to the unoxidized protein. In addition, a significant loss of conformational stability was observed upon oxidation. Correspondingly, increased protein aggregation was observed resulting in the formation of sub-visible particles. Oxidized protein showed decreased biological function in terms of its anti-viral potency and cytopathic inhibition efficacy. Proteolysis-coupled mass spectrometry identified five methionine residues that were oxidized with no correlation between the extent of oxidation and their accessible surface area. 2D 15N-1H HSQC NMR identified residue-level local structural changes in the protein upon oxidation, which were not detectable by global probes such as far-UV circular dichroism and fluorescence.Conclusions
Increased protein aggregation and decreased function of IFNA2a upon oxidation correlated with the site of modification identified by proteolysis-coupled mass spectrometry and local structural changes in the protein detected by 2D NMR.5.
Lin Zhang Sandeep Yadav Barthélemy Demeule Y. John Wang Olivier Mozziconacci Christian Schӧneich 《Pharmaceutical research》2017,34(1):84-100
Purpose
To investigate the mechanisms of polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways.Methods
Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate polysorbate 20 (PS20) and its degradants. 18O stable isotope labeling was implemented to produce 18O-labeled degradation products of all-laurate PS20 in H2 18O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20.Results
The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of 18O incorporation into free lauric acid indicated that hydrolysis primarily led to 18O incorporation into free lauric acid via “acyl-cleavage” of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without 18O-incorporation.Conclusions
The 18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.6.
Purpose
Colon adenocarcinoma is the most common form of gastro intestinal tract cancer, predominantly in ageing population. Chemotherapy with 5-Fluorouracil and oxaliplatin is an indispensable treatment regimen, nevertheless having limitation of systemic toxicity and lower therapeutic index. The present study is based on evaluation of anti-proliferative potential, pharmacokinetics parameters, safety profile, biodistribution and efficacy of 5-FU/oxaliplatin loaded lactoferrin nanoparticles in cell lines and wistar rats in order to overcome the above limitation.Methods
Nanoparticles were prepared by Water-in-oil process. The anti-proliferative efficacy and mode of cellular entry was evaluated in COLO-205 cells. The pharmacokinetics and biodistribution analysis were performed in healthy rats while efficacy and safety assay were performed in ACF induced rats.Results
5-FU and oxaliplatin loaded nanoparticles shows enhanced antiproliferative activity as compare to free drugs in COLO-205 cells. Lactoferrin nanoparticles also improve the pharmacokinetics profile, safety parameters and efficacy of 5-FU and Oxaliplatin.Conclusion
Lactoferrin nanoparticles demonstrated an attractive drug delivery module to manage the colon adenocarcinoma as it has improved the antiproliferative activity of 5-FU and Oxaliplatin against colon adenocarcinoma cells. Moreover, it also improves the pharmacokinetic profile and safety parameters of the same drug in wistar rat.7.
Objective
Although the organic compounds, 2-propylaniline and 4-propylaniline are frequently used in many industrial sectors, and have little information about the potential genetic toxicity, and it is covered by the Occupational Safety & Health Act (OSHAct) in Korea.Methods
The mutation test of 2-propylaniline and 4-propylaniline was evaluated in five different doses for each chemical through a well-known Ames bacterial mutation test. This test was performed regardless of metabolic activation.Results
In this assay, we obtained positive results under all tested conditions, indicating that these two chemicals have mutagenic and potentially carcinogenic properties.Conclusion
Both 2-propylaniline and 4-propylaniline were mutagenic under the conditions of these tests. This result means that all of these chemicals exhibit mutations and potential carcinogenicity.8.
Lim-Kyu Lee Seung-Min Yang Jaehong Park Junghwan Kim 《Toxicology and Environmental Health Sciences》2018,10(1):42-48
Objective
This study promotes health management activities in Y combined cycle power plants in Korea, focusing on occupational health activities, such as preventing cardiovascular disease and musculoskeletal disorders and managing work environment measurements.Methods
The results of the present study were collected from the company’s internal documents and reports in Y combined cycle power plant.Results
Diverse results for workplace activities are summarized. Furthermore, this study discusses attempts to reduce potential safety risks and to improve workers’ health conditions at the Y combined cycle power plants in Korea.Conclusion
The Y combined cycle plant discussed seeks to prevent accidents to improve workers’ health; thus, specific efforts related to onsite health and expected results for workers are evaluated.9.
Ming Lei Cynthia Quan Y. John Wang Yung-Hsiang Kao Christian Schöneich 《Pharmaceutical research》2018,35(3):67
Purpose
Light is known to induce histidine (His) oxidation and His-His crosslinking in proteins. The crosslinking is resulted from the nucleophilic attack of a His to a photooxidized His from another protein. The goal of this work is to understand if covalent buffer adducts on His residues can be generated by light through similar mechanisms in nucleophilic buffers such as Tris and His.Methods
A model protein (DNase) was buffer exchanged into nucleophilic buffers before light exposure. Photogenerated products were characterized by tryptic peptide mapping with mass spectrometry (MS) analysis. Several buffer adductions on His residues were identified after light exposure. To understand the influencing factors of such reactions, the levels of adducts were measured for six nucleophilic buffers on all His residues in DNase.Results
The levels of adducts were found to correlate with the solvent accessibility of the His residue. The levels of adducts also correlate with the structure of the nucleophile, especially the steric restrictions of the nucleophile. The levels of adducts can be higher than that of other His photoreaction products, including photooxidation and crosslinking.Conclusions
In nucleophilic buffers, light can induce covalently-linked adducts to His residues.10.
Antony V. Samrot Ujjala Burman Padmanaban S P. Yamini Arul Maximus Rabel 《Toxicology and Environmental Health Sciences》2018,10(3):162-167
Objective
To evaluate the toxicity of the silver nanoparticle against earthworms - Eudrilus eugeniae, a model for soil organism.Methods
Silver nanoparticles were synthesised by chemical reduction and further characterised by UV Visible Spectroscopy and FeSEM. Earthworms were allowed to interact with different concentrations of the synthesized silver nanoparticles. After exposure period, histology and inductively coupled plasma optical emission spectrometry (ICP-OES) were done to determine the accumulation and toxic effects exhibited by the nanoparticle on earthworms.Results
The synthesized nanoparticle was found to be between the size of 180 and 200 nm. Histology studies revealed that silver nanoparticles to cause fibrosis, lipofuscin-like deposits and also gut disruption in earthworms.Conclusion
Silver nanoparticles were found to be toxic to Eudrilus eugeniae, which was evidenced by histology.11.
12.
Purpose
To understand hydrolysis and alcoholysis of polyvinylpyrrolidone-co-vinylacetate (PVPVA) during formulation and storage, elucidate the reaction mechanism, establish an intrinsic kinetic model, and apply this model coupled with GastroPlus? modeling to predict the amount of PVPVA degradation in vivo.Methods
The experimental approach includes the detection of the polymer reaction by solution nuclear magnetic resonance (NMR) and the measurement of reaction product concentration via gas chromatography (GC). The theoretical approach includes the establishment of the intrinsic kinetic model and the application of GastroPlus? to predict the degree of PVPVA degradation.Results
The kinetic model established is a first order reaction between PVPVA and 2-propanol (IPA) or water under an acidic condition. The application of this kinetic model shows that between 1.7 and 6.8 mg of degradant is formed in the GI tract for a 850 mg dose of PVPVA.Conclusions
The results from this application provide valuable input for process development and the risk analysis of the degradation of PVPVA.13.
Sahithi Ravuluri Rohit Bansal Nidhi Chhabra Anurag S. Rathore 《Pharmaceutical research》2018,35(7):142
Purpose
To understand non-enzymatic hydrolytic fragmentation of a monoclonal antibody therapeutic under temperature stressed conditions and investigating possible mechanism for the same.Methods
The mAb therapeutic was incubated at 50°C in phosphate buffer at pH 6.5 and fragmentation was monitored at different ionic strengths under stressed conditions. The incubated mAb was sampled at regular time intervals by analytical Size Exclusion Chromatography (SEC).Results
It was observed that 57% of the mAb product fragmented over 4 days into two fragment species – Fc-Fab and Fab with molecular weights of 97 KDa and 47 KDa, respectively, as measured by mass spectrometry (MS) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The fragmentation rate was slow initially and then accelerated with time. No change in % aggregate level was observed in this duration, implying that degradation was primarily via fragmentation at high temperature. Kinetics of hydrolytic fragmentation was hypothesized and SEC data was fitted to estimate the kinetic rate constants. While degradation of the monomer into fragment species was non-Arrhenius with a negative activation energy, further degradation of Fab-Fc fragments into Fab or Fc fragments followed Arrhenius Law with an activation energy of 2.1 and 15.38 kcal/mol, respectively.Conclusion
High temperature (50°C) causes mAb to cleave at the hinge region to form Fab-Fc and Fab/Fc, as confirmed by dynamic light scattering, SDS-PAGE, SEC, and MS. A kinetic model for hydrolytic fragmentation has been proposed. The results are expected to assist end users in formulation development as well as in monitoring stability of biotherapeutic products.14.
Gurpreet K Grewal Khuraijam D Singh Neha Kanojia Chitra Rawat Samiksha Kukal Ajay Jajodia Anshika Singhal Richa Misra Selvaraman Nagamani Karthikeyan Muthusamy Ritushree Kukreti 《Pharmaceutical research》2017,34(7):1444-1458
Purpose
Over expression of ATP-binding cassette transporters is considered one of the major reasons for non-responsiveness to antiepileptic drugs. Carbamazepine (CBZ), one of first line antiepileptic drug is known to influence ABCC2 expression but its exact molecular mechanism is unknown.Methods
We investigated the effect of CBZ on expression of ABCC2 and pregnane X receptor (PXR) in HepG2 cell line and compared with hyperforin (agonist of PXR) and ketoconazole (antagonist of PXR) through realtime PCR and western blot assay. Involvement of PXR was demonstrated through nuclear translocation and RNA interference and related effect of CBZ on ABCC2 through functional activity assay. Molecular docking and dynamic simulation approach was used to understand the interaction of CBZ with PXR.Results
CBZ and hyperforin increased the PXR and ABCC2 expression whereas reversed when present it in combination with ketoconazole. Experiments confirmed CBZ induced ABCC2 expression is PXR dependent. Molecular dynamic (MD) simulation and in vitro experiment indicated possibility of CBZ to be PXR agonist and PXR residue Gln285 to be important for CBZ-PXR interaction.Conclusions
CBZ alters the functional activity of ABCC2 through PXR, which in turn can interfere with therapy. Mutational analysis of residues revealed the importance of Gln285 in ligand interaction.15.
Purpose
Pulmonary infection namely tuberculosis is characterized by alveolar macrophages harboring a large microbe population. The chitosan nanoparticles exhibit fast extracellular drug release in aqueous biological milieu. This study investigated the matrix effects of chitosan nanoparticles on extracellular drug diffusion into macrophages.Methods
Oligo, low, medium and high molecular weight chitosan nanoparticles were prepared by nanospray drying technique. These nanoparticles were incubated with alveolar macrophages in vitro and had model drug sodium fluorescein added into the same cell culture. The diffusion characteristics of sodium fluorescein and nanoparticle behavior were investigated using fluorescence microscopy, scanning electron microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy techniques.Results
The oligochitosan nanoparticles enabled macrophage membrane fluidization with the extent of sodium fluorescein entry into macrophages being directly governed by the nanoparticle loading. Using nanoparticles made of higher molecular weight chitosan, sodium fluorescein permeation into macrophages was delayed due to viscous chitosan diffusion barrier at membrane boundary.Conclusion
Macrophage-chitosan nanoparticle interaction at membrane interface dictates drug migration into cellular domains.16.
Dmitry Kryndushkin Wells W. Wu Ramesh Venna Michael A. Norcross Rong-Fong Shen V. Ashutosh Rao 《Pharmaceutical research》2017,34(4):765-779
Purpose
Protein carbonylation is an irreversible modification of Lys, Arg, Thr and Pro amino acids under conditions of oxidative stress. Previous studies have reported specific carbonylated residues in purified recombinant albumins, albeit with a lack of agreement between the studies. Currently, structural factors that determine site-specific protein carbonylation are not well understood.Methods
In this study, we utilized metal-catalyzed oxidizing conditions to generate carbonylation in recombinant human serum albumin (HSA) and granulocyte-colony stimulating factor (G-CSF), two proteins with distinct metal-binding abilities. To estimate predictability of HSA carbonylation sites, the same oxidative reaction was repeated based on the previously reported conditions. For G-CSF, oxidative conditions were gradually adjusted to achieve substantial levels of protein carbonylation. Corresponding accumulation of specific oxidized residues was identified and confirmed with high-resolution mass spectrometry.Results
Our HSA dataset contained 55 carbonylated residues and showed a significant overlap with the previously published pooled data, indicating a certain level of carbonylation site specificity for albumins. Oxidation of G-CSF under multiple oxidative conditions consistently showed a highly specific carbonylation at position Pro45. We also detected a previously unreported, oxidation-induced cleavage site in G-CSF between His44 and Pro45, which might be attributed to a presence of a potential metal-binding site near residue Pro45.Conclusions
Our results show distinct patterns of protein carbonylation for HSA and G-CSF. Thus, specificity of protein carbonylation induced by metal-catalyzed oxidation is protein dependent and might be predicted by availability of transition metal binding site(s) within the protein.17.
Background
Poison hemlock (Conium maculatum) is a common plant with a significant toxicity. Data on this toxicity is sparse as there have been few case reports and never a documented poisoning after intravenous injection.Objectives
We present a case of intravenous poison hemlock injection encountered in the emergency department.Case Report
We describe a 30-year-old male who presented to the emergency department after a brief cardiac arrest after injecting poison hemlock. The patient had return of spontaneous circulation in the emergency department but had prolonged muscular weakness and encephalopathy later requiring tracheostomy.Conclusion
Intravenous injection of poison hemlock alkaloids can result in significant toxicity, including cardiopulmonary arrest, prolonged weakness, and encephalopathy.18.
Tommaso Iannitti Julio César Morales-Medina Alessandro Coacci Beniamino Palmieri 《Pharmaceutical research》2016,33(12):2879-2890
Background
In the field of aesthetic medicine there is an increasing demand for safe and effective hyaluronic acid (HA) fillers to counteract the aging process.Methods and Aims
We designed a study to evaluate the safety and histological biocompatibility of Aliaxin® Global Performance, a cross-linked HA filler and Viscoderm® Skinkò E, a product composed of non-cross-linked HA and a complex including vitamins, antioxidants, amino acids and minerals injected into the skin of guinea pigs. Then, we translated our findings into the clinical setting, administering a combination of these compounds to patients seeking a facial rejuvenation procedure targeting moderate-to-severe wrinkles affecting the nasolabial folds.Results
The animal study showed that the two compounds did not induce any significant inflammatory reactions and increased collagen and elastic fibers in the skin. In the clinical setting, injection of Aliaxin® Global Performance, followed by Viscoderm® Skinkò E, resulted in a higher improvement in nasolabial fold hydration, trans-epidermal water loss and wrinkle aesthetic appearance, if compared with a protocol based on Aliaxin® Global Performance alone.Conclusion
In summary, we show evidence on the safety and mechanism underlying two new HA-based compounds of different cross-linkage and composition, proposing that they can be safely used in combination in patients seeking facial rejuvenation procedures with long-lasting efficacy.19.
Purpose
Topical beta-blockers are efficacious for treating infantile hemangiomas, but no formulations have been specifically optimized for skin delivery. Our objective was to quantify skin concentrations and drug permeation of propranolol (a nonselective beta-blocker) after application of microemulsions to intact and microneedle pretreated skin.Methods
Four propranolol-loaded microemulsions were characterized for droplet size, surface charge, conductivity, pH, drug solubility, and drug release. Skin concentrations and drug permeation through skin were quantified using LC-MS. Skin-to-receiver ratios were used to compare the microemulsion formulations to a drug-in-PBS solution.Results
Propranolol solubility was significantly greater in microemulsions vs PBS. Cumulative drug release from the microemulsions over 24 h ranged from 13 to 26%. Skin concentrations and drug permeation through intact skin was significantly higher from PBS; however, the skin-to-receiver ratios were significantly higher for water-rich microemulsions compared to PBS or surfactant-rich microemulsions. Microneedle pretreatment significantly increased skin concentrations for all formulations. Skin-to-receiver ratios significantly increased after microneedle pretreatment for surfactant-rich microemulsions.Conclusions
Microemulsion formulation can be altered to elicit different drug delivery profiles through MN-treated skin. This could be advantageous for maximizing local skin drug concentrations and improving dosing schedules for infantile hemangioma treatment.20.
Rizzo M Lamers CT Sauer CG Ramaekers JG Bechara A Andersen GJ 《Psychopharmacology》2005,179(3):559-566