Near UV Light Photo-Degradation of Lactate and Related α-Hydroxycarboxylates in the Presence of Fe(III): Formation of Carbon Dioxide Radical Anion.

In recent studies we have reported on the near-UV light-induced degradation of iron complexes of various pharmaceutical excipients, such as Fe(III)-citrate and Fe(III)-amino acid complexes. Mechanistic studies revealed a common photo-degradation pattern, i.e. the formation of carbon dioxide radical anion, a potent reducing agent, via an alkoxyl/amino radical intermediate generated by light-induced ligand-to-metal charge transfer (LMCT) involving α-hydroxycarboxylates or amino acids. Herein, we confirm the proposed general photo-degradation pathways through the study of the iron complexes of other α-hydroxycarboxylates that may be present in protein formulations, such as lactate and glycolate. The results indicate that lactate generates even higher yields of •CO2- as compared to citrate, suggesting a significant potential of lactate for the promotion of photo-degradation in pharmaceutical formulations.

Near UV Photodegradation Mechanisms of Amino Acid Excipients: Formation of the Carbon Dioxide Radical Anion from Aspartate and Fe(III).

Carbon dioxide radical anion (•CO2-) is a powerful reducing agent that can reduce protein disulfide bonds and convert molecular oxygen to superoxide. Therefore, the generation of •CO2- can be detrimental to pharmaceutical formulations. Iron is among the most prevalent impurities in formulations, where Fe(III) chelates of histidine (His) can produce •CO2- upon exposure to near-UV light (Zhang and Schöneich, Eur. J. Pharm. Biopharm. 2023, 190, 231-241). Here, we monitor by spin-trapping in combination with electron paramagnetic resonance spectroscopy and/or high-performance liquid chromatography-mass spectrometry analysis the photochemical formation of •CO2- for a series of common amino acid excipients, including arginine (Arg), methionine (Met), proline (Pro), glutamic acid (Glu), glycine (Gly), aspartic acid (Asp), and lysine (Lys). Our results indicate that in the presence of Fe(III), Asp, and Glu produce significant yields of •CO2- under photoirradiation with near-UV light. Notably, Asp demonstrates the highest efficiency of •CO2- generation compared with that of the other amino acid excipients. Stable isotope labeling indicates that •CO2- exclusively originates from the α-carboxyl group of Asp. Mechanistic studies reveal two possible pathways for •CO2- formation, which involve either a ß-carboxyl radical or an amino radical cation intermediate.

Molecular Dynamics Simulation of an Iron(III) Binding Site on the Fc Domain of IgG1 Relevant for Visible Light-Induced Protein Fragmentation.

Molecular dynamics simulations were employed to investigate the interaction between Fe(III) and an iron-binding site composed of THR259, ASP252, and GLU261 on the Fc domain of an IgG1. The goal was to provide microscopic mechanistic information for the photochemical, iron-dependent site-specific oxidative fragmentation of IgG1 at THR259 reported in our previous paper. The distance between Fe(III) and residues of interest as well as the binding pocket size was examined for both protonated and deprotonated THR259. The Fe(III) binding free energy (ΔG) was estimated by using an umbrella sampling approach. The pKa shift of the THR259 hydroxyl group caused by the presence of nearby Fe(III) was estimated based on a thermodynamic cycle. The simulation results show that Fe(III) resides inside the proposed binding pocket and profoundly changes the pocket configuration. The ΔG values indicate that the pocket possesses a strong binding affinity for Fe(III). Furthermore, Fe(III) profoundly lowers the pKa value of the THR259 hydroxyl group by 5.4 pKa units.

Near UV light photo-degradation of histidine buffer: Mechanisms and role of Fe(III).

Pharmaceutical formulations are sensitive to light-induced degradation. Recent studies have attributed some of the light sensitivity to the presence of Fe(III), the most prevalent metal leachable from pharmaceutical containers. Histidine (His) can promote Fe(III) leaching from stainless steel, especially at elevated storage temperatures. Since there is the chance that combinations of His and Fe(III) are present in pharmaceutical formulations, we investigated the photo-degradation mechanisms of Fe(III)-containing His buffer during expsoure to near UV light. Our results indicate the formation of carbon dioxide radical anion (•CO2-), a powerful reductant, and other photoproducts such as aldehydes and His-derived radicals. The generation of •CO2- can be promoted by increasing concentrations of Fe(III) and inhibited by the addition of the Fe(III) chelator EDTA. Mechanistically, product formation can be rationalized by photo-induced ligand-to-metal-charge-transfer (LMCT), followed by a series of radical transformations of reaction intermediates.

Photo-induced site-specific oxidative fragmentation of IgG1 mediated by iron(III)-containing histidine buffer: Mechanistic studies and excipient effects.

Fragmentation may compromise the clinical efficacy and safety profile of monoclonal antibodies (mAbs). We recently reported that Fe(III)-containing histidine (His) buffer mediates site-specific mAb fragmentation within the Fc domain when exposed to visible light (Y. Zhang and C. Schöneich, Mol. Pharm. 2023, 20, 650-662). Here, we show that this fragmentation proceeds even more efficiently under near-UV light. Several formulation strategies were applied in an attempt to reduce the photo-induced fragmentation. In solution formulations, the fragmentation can be mitigated by reducing the concentration of His buffer, adding Fe(III)-chelating agents, and replacing His with other amino acids. Fragmentation can be almost completely inhibited by formulating the protein in the lyophilized state. Mechanistically, His plays a critical role in the fragmentation process, likely due to its affinity for Fe(II), driving a photo-redox reaction towards product formation.

Visible Light Induces Site-Specific Oxidative Heavy Chain Fragmentation of a Monoclonal Antibody (IgG1) Mediated by an Iron(III)-Containing Histidine Buffer.

Fragmentation of therapeutic monoclonal antibodies represents a critical quality attribute. Here, we report a novel visible light-induced heavy chain fragmentation of IgG1 mediated by an Fe(III)-containing histidine (His) buffer. Based on non-reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis and mass spectrometry analysis, IgG1 fragments with apparent molecular weights of ∼130, ∼110, and ∼22 kDa were detected in photo-irradiated samples and were mechanistically rationalized with an oxidative cleavage at Thr259. Specifically, the reactions are proposed to involve the generation of an intermediary alkoxyl radical, which undergoes ß-cleavage to yield a glycyl radical. The latter either converts into Gly or adds oxygen and follows a peroxyl radical chemistry. The cleavage process requires the presence of His, while only negligible yields of cleavage products are formed when His is replaced by acetate, succinate, or phosphate buffer. Importantly, the fragmentation can be prevented by ethylenediaminetetraacetic acid (EDTA) only when the EDTA concentrations are in significant excess over the concentrations of Fe(III) and proteins, suggesting a strong binding between Fe(III) and IgG1.

Near-UV and Visible Light Degradation of Iron (III)-Containing Citrate Buffer: Formation of Carbon Dioxide Radical Anion via Fragmentation of a Sterically Hindered Alkoxyl Radical.

Citrate is a commonly used buffer in pharmaceutical formulations which forms complexes with adventitious metals such as Fe3+. Fe3+-citrate complexes can act as potent photosensitizers under near-UV and visible light exposure, and recent studies reported evidence for the photo-production of a powerful reductant, carbon dioxide radical anion (•CO2-), from Fe3+-citrate complexes (Subelzu, N.; Schöneich, N., Mol. Pharm. 2020, 17, 4163-4179). The mechanisms of •CO2- formation are currently unknown but must be established to devise strategies against •CO2- formation in pharmaceutical formulations which rely on the use of citrate buffer. In this study, we first established complementary evidence for the photolytic generation of •CO2- from Fe3+-citrate through spin trapping and electron paramagnetic resonance (EPR) spectroscopy, and subsequently used spin trapping in conjunction with tandem mass spectrometry (MS/MS) for mechanistic studies on the pathways of •CO2- formation. Experiments with stable isotope-labeled citrate suggest that the central carboxylate group of citrate is the major source of •CO2-. Competition studies with various inhibitors (alcohols and dimethyl sulfoxide) reveal two mechanisms of •CO2- formation, where one pathway involves ß-cleavage of a sterically hindered alkoxyl radical generated from the hydroxyl group of citrate.

Near UV and Visible Light Photo-Degradation Mechanisms in Citrate Buffer: One-Electron Reduction of Peptide and Protein Disulfides promotes Oxidation and Cis/Trans Isomerization of Unsaturated Fatty Acids of Polysorbate 80.

Citrate is a common buffer for slightly acidic pH ranges of protein formulations. In the presence of iron, citrate buffer undergoes photo-degradation induced by near UV and visible light. Recent studies (Subelzu and Schöneich, Mol. Pharm. 2020, 17, 4163-4179) have documented that such photo-degradation results in the formation of carbon dioxide radical anion (•CO2-), a strong reductant which reduces Fe3+, O2, and disulfide bonds. In the present study we show that near UV and visible light photo-degradation of citrate in the presence of iron can induce reductive peptide and protein disulfide cleavage as well as free radical damage of a surfactant, polysorbate 80 (PS80). Reductive disulfide cleavage is most likely caused by efficient electron transfer from carbon dioxide radical anions to disulfides, resulting in the generation of thiol/thiolate and thiyl radicals. The latter can react with mono- and polyunsaturated fatty acids of PS80 to cause cis/trans isomerization and/or oxidation. Representative products generated by cis/trans isomerization and oxidation of oleic acid esters have been detected by HPLC-MS analysis. Further evidence for reductive disulfide cleavage was obtained through the analysis of free thiols. The oxidation of PS80 can also be the consequence of reactive oxygen species (ROS) generation through the reduction of O2 by carbon dioxide radical anions and/or intermediary Fe2+.

Combination of HRLC-MS with MPP and GNPS data analysis to identify major constituents of MtSK inhibitory Alpinia galanga extract.

Alpinia galanga (L.) Willd. (Zingiberaceae), or galangal, has been previously reported as active against Mycobacterium tuberculosis (TB) in vitro. The present study assessed a novel antitubercular mechanism of of galangal through M. tuberculosis shikimate kinase (MtSK) inhibitory assays. Sequential extractions of nonpolar solvents hexane and dichloromethane (DCM) were performed on galangal and screened in MtSK inhibitory assays to identify potential activity. Samples were then subjected to high resolution (HR) LC-MS chemical fingerprinting and analysis. Additionally, a novel approach was undertaken for galangal using methods such as mass professional profiler (MPP) and global natural products social (GNPS) molecular networking for structure elucidation.[Formula: see text].

Reproducible molecular networking of untargeted mass spectrometry data using GNPS.

Global Natural Product Social Molecular Networking (GNPS) is an interactive online small molecule-focused tandem mass spectrometry (MS2) data curation and analysis infrastructure. It is intended to provide as much chemical insight as possible into an untargeted MS2 dataset and to connect this chemical insight to the user's underlying biological questions. This can be performed within one liquid chromatography (LC)-MS2 experiment or at the repository scale. GNPS-MassIVE is a public data repository for untargeted MS2 data with sample information (metadata) and annotated MS2 spectra. These publicly accessible data can be annotated and updated with the GNPS infrastructure keeping a continuous record of all changes. This knowledge is disseminated across all public data; it is a living dataset. Molecular networking-one of the main analysis tools used within the GNPS platform-creates a structured data table that reflects the molecular diversity captured in tandem mass spectrometry experiments by computing the relationships of the MS2 spectra as spectral similarity. This protocol provides step-by-step instructions for creating reproducible, high-quality molecular networks. For training purposes, the reader is led through a 90- to 120-min procedure that starts by recalling an example public dataset and its sample information and proceeds to creating and interpreting a molecular network. Each data analysis job can be shared or cloned to disseminate the knowledge gained, thus propagating information that can lead to the discovery of molecules, metabolic pathways, and ecosystem/community interactions.

Screening for CYP3A4 inhibition and induction coupled to parallel artificial membrane permeability assay (PAMPA) for prediction of botanical-drug interactions: The case of açaí and maca.

BACKGROUND: The consumption of botanical dietary supplements (BDS) is a common practice among the US population. However, the potential for botanical-drug interactions exists, and their mechanisms have not been thoroughly studied. CYP3A4 is an important enzyme that contributes to the metabolism of about 60% of clinically used drugs. PURPOSE: To investigate the potential for botanical-drug interactions of Lepidium meyenii Walpers (maca) root and Euterpe oleracea Mart. (açaí) berries, two commonly used BDS, when co-administered with CYP3A4-metabolized drugs. METHODS: In an attempt to decrease the general discrepancy between in vivo and in vitro studies, the absorption profiles, particularly for passive diffusion, of plant extracts were investigated. Specifically, the parallel artificial membrane permeability assay (PAMPA) model was utilized to simulate intestinal filtration of passively diffused constituents of açaí and maca extracts. These were subsequently screened for in vitro liver CYP3A4 inhibition and induction. In the inhibition assay, midazolam was used as the probe substrate on genotyped human liver microsomes (CYP3A5 null), and the production of its 1'-substituted metabolite when co-cultured with extract treatments was monitored. In the induction assay, extract treatments were applied to human primary hepatocytes, and quantitative PCR analysis was performed to determine CYP3A4 mRNA expression. RESULTS: Passively diffused constituents of the methanol açaí extract (IC50 of 28.03 µg/µl) demonstrated the highest inhibition potential, and, at 1.5 µg/µl, induced significant changes in CYP3A4 gene expression. The composition of this extract was further investigated using the chemometric tool Mass Profiler Professional (MPP) on liquid chromatography-mass spectroscopy (LC-MS) data. Subsequently, five compounds of interest characterized by high abundance or high permeability were extracted for further study. This included efforts in effective passive permeability determination and structural elucidation by tandem mass spectrometry (MS/MS). CONCLUSION: The passively absorbable portion of a methanol açaí extract exhibited inhibition and induction effects on CYP3A4 suggesting the potential to produce botanical-drug interactions.