Analytische Ansätze zur Trennung von Deferipron von seinem Eisen (III) - Komplex und der Untersuchung seiner Wechselwirkungen mit verschiedenen essentiellen Metallionen und zielgerichteten Proteinen

Deferiprone still remains an interesting drug even after the passing of three decades from its first approval date. Recently, much attention has been on the potential benefits in the treatment of infectious diseases, neurodegenerative diseases, and cancers. This thesis details the binding affinities of deferiprone toward the essential metal ions and targeted proteins as an attempt to understand the drug behavior inside the body. The starting point was developing new chromatographic and electrophoretic methods to determine and separate deferiprone's free form from its iron (III) complex form in one single run. Thereafter, the studying of binding stability under each system has been attempted. The LC/MS method was developed to achieve a good separation through nonlinear gradient elution order, and the complex dissociation had been minimized using a high concentration of reactant partners. The CE/FA method was more robust to form a stable drug peak plateau, which separated well from the complex peak plateau. Comparing between the binding events of data that was obtained, the CE/FA method was more stable and in agreement with the literature values while LC/MS exhibited slightly weaker binding and might be attributed to chromatographic system stability, which was observed and detected in the MS system. On the other hand, MST and ESI-MS techniques have been used to characterize the binding of deferiprone toward essential metal ions. A novel MST method has been developed successfully to estimate the binding for deferiprone with five essential metal ions, including Fe3+, Cu2+, Zn2+, Co2+, and Ni2+. Fe3+ seems to bind stronger with deferiprone, followed by Cu2+ and Zn2+ and, to a lesser extent, with Co2+ and Ni2+. That being said, no binding events were estimated between deferiprone with Mn2+, Mg2+, and Ca2+. MST results were confirmed by ESI-MS and agreed with previous studies. Thereafter, MST, besides the ACE-based method, has been selected to study the binding affinities between deferiprone with targeted proteins. In this approach, MST was superior to use for characterizing deferiprone binding with proteins, whereas MST was capable of estimating the binding events for deferiprone with human serum albumin and human lactoferrin successfully. On the contrary, CE-based methods failed to estimate the binding between deferiprone with human lactoferrin. Meanwhile, both of the developed MST and CE methods estimated the binding events successfully for deferiprone with human serum albumin and are in agreement with reported values published in the literature. On the other hand, the interaction between deferiprone and human lactoferrin was reported for the first time, and the MST results indicated strong binding. Furthermore, the drug might be secreted in breast milk as a protein-bound fraction. Therefore, the later finding needs further clinical investigations to confirm the drug secretion in the absence of similar studies.Deferipron ist trotz der drei Jahrzehnte seit seiner erstmaligen Zulassung ein interessantes Medikament. Vor kurzem lag viele Aufmerksamkeit auf den potenziellen Vorteilen beider Behandlung von Infektionskrankheiten, neurodegenerativen Erkrankungen und Krebs. Diese Arbeit beschreibt die Bindungsaffinitäten von Deferipron zu essentiellen Metallionen und zielgerichteten Proteinen, um das Arzneimittelverhalten im Körper zu verstehen. Der Ausgangspunkt war die Entwicklung neuer chromatographischer und elektrophoretischer Verfahrungen zur Bestimmung und Trennung des Deferiprons in freier Form aus seiner Eisen (III) -Komplex Form in einem einzelnen Verfahren. Danach wurde versucht, die Bindungsstabilität Für jedes System zu untersuchen. Die LC/MS-Methode wurde entwickelt, um eine gute Trennung durch nicht lineare Gradientenelutionsreihenfolge zu erreichen. Die Komplexdissoziation wurde unter Verwendung einer hohen Konzentration von Reaktantenpartnern minimiert. Die CE/FA-Methode war robuster, um ein stabiles Arzneimittelpeakplateau zu bilden, welches sich gut vom komplexen Peakplateau abtrennte. Im Vergleich zwischen den Bindungsereignissen der erhaltenen Daten war die CE/FA-Methode stabiler und stimmte mit den Literaturwerten überein. Im Gegenteil hat LC/MS eine geringfügig schwächere Bindung. Diese könnte auf die Stabilität des chromatographischen Systems zurückführen, welche in dem MS-System beobachtet und nachgewiesen wurde. Andererseits wurden MST und ESI-MS Techniken verwendet, um die Bindung von Deferipron an essentielle Metallionen zu charakterisieren. Die Entwicklung einer neuen MST Verfahren erfolgt, um die Bindung von Deferipron mit fünf essentiellen Metallionen, einschließlich Fe3+, Cu2+, Zn2+, Co2+ und Ni2+, bewerten. Fe3+ bindet am stärksten mit Deferipron, gefolgt von Cu2+, Zn2+ und, in einem geringerem Maße, mit Co2+ und Ni2+. Allerdings wurden keine Bindungsereignisse zwischen Deferipron mit Mn2+, Mg2+ und Ca2+ erwartet. Die Bestätigung der MST-Ergebnisse erfolgte durch ESI-MS. Diese Ergebnisse stimmten mit früheren Studien überein. Danach werwendete MST als Wahl neben der ACE-basierten Methode, ausgewählt, um die Bindungsaffinitäten zwischen Deferipron und Zielproteinen zu Charakterisieren. In diesem Ansatz war MST für die Charakterisierung der Deferipronbindung mit Proteinen bevorzugt, da die Bestimmung der Bindungsereignisse für Deferipron mit Humanserumalbumin und humanem Lactoferrin mit MST erfolgte. Im Gegenteil, CE-basierte Methoden konnten die Bindung zwischen Deferipron und menschlichem Lactoferrin nicht erfüllen. Inzwischen erfüllen beide entwickelten MST- und CE-Methoden die Bindungsereignisse für Deferipron mit Humanserumalbumin erfolgreich und stimmen mit den in der Literatur veröffentlichten Werten überein. Andererseits wurde zum ersten Mal über die Wechselwirkung zwischen Deferipron und menschlichem Lactoferrin berichtet. Die MST-Ergebnisse zeigten eine starke Bindung an. Darüber hinaus könnte das Medikament als proteingebundener Anteil in die Muttermilch abgegeben werden

Label-free microscale thermophoresis for the study of lactoferrin-drug interaction

In this study, a microscale thermophoretic (MST) method was developed to investigate lactoferrin-drug interaction using label-free system which depends on the intrinsic fluorescence of one interacting partner. The interaction between lactoferrin and the iron chelator deferiprone was first investigated to evaluate the excretion of deferiprone into mother milk during lactation. Furthermore, the interaction between lactoferrin and amphotericin B was investigated for possible explanation of the observed synergistic effect as antifungal effect. The experiments were performed on Monolith NT.115 LabelFree® (NanoTemper Technologies, Munich, Germany). Different concentrations of the intended drugs in the range of 0.007- 250 μM were titrated against 120 nM fixed concentration of lactoferrin which was dissolved in 0.1 M tris buffer at pH 7.4. The measurements were performed using standard capillaries at 20% excitation power and medium MST power. The results indicated a significant interaction between lactoferrin and deferiprone however, no significant interaction between lactoferrin and amphotericin B was observed. The data were analyzed using NT Analysis software which was provided by NanoTemper Technologies. The estimated binding constant for lactoferrin-deferiprone interaction was (8.9 x 10 -6 ± 1.6 SD) which is reported for the first time. This significant binding between lactoferrin and deferiprone may potentiates the drug secretion into mother milk. The technique shows fast and simple approach to study protein-drug interaction without complicated labelling procedure

Development of New Monolithic Polysaccharide-Based Columns for HPLC Enantiomeric Separation

Chondroitin sulfate A and maltodextrin have been studied as potential chiral selectors to provide new polysaccharide-based chiral stationary phases. A capillary electrophoresis method has been involved to figure out the enantiomers recognition behaviour in the presence of chiral selector. Chondroitin sulfate A 0.3% w/v and maltodextrin 10% w/v which found to be the optimal values in CE were immobilized onto monolithic silica epoxy and monolithic silica amine columns by Schiff base reaction respectively. The immobilized chondroitin sulfate A-based and maltodextrin-based CSPs have shown intial enantiomeric separation of amlodipine and verapamil as model compound of by HPLC

Immobilization of Chondroitin Sulfate A into Monolithic Epoxy Column for Chiral Separation

Chondroitin sulfate A was successfully immobilized into epoxy monolithic column at a concentration of 3% (w/v) in the presence of ethylene diamine. The epoxy group of monolithic column was first converted to aldehyde group by successive hydrolization and oxidation. A Schiff base reaction at pH 8.0 was used to attach the diamine-spacer to aldehyde group. The chondroitin sulfate A was introduced into the monolithic column by circulating the solution at a flow rate of 0.1 mL/min for 24 hours. The chondroitin sulfate A-immobilized epoxy column was evaluated for chiral separation of verapamil enantiomers under optimized HPLC conditions at a wavelength of 230 nm. As a mobile phase, 20 mM Na2HPO4 (pH 2.9) was used. A resolution (Rs) of about 1.5 was achieved for the separation of verapamil enantiomers. A good repeatability of the retention time at two concentration levels (n=8) with RSD < 1% was obtained. The linear responses of verapamil enantiomers were in the range of 1.0-3.0 ppm with R2 of about 0.994

Thermophoresis for characterizing biomolecular interaction

The study of biomolecular interactions is crucial to get more insight into the biological system. The interactions of protein-protein, protein-nucleic acids, protein-sugars, nucleic acid-nucleic acids and proteinsmall molecules are supporting therapeutics and technological developments. Recently, the development in a large number of analytical techniques for characterizing biomolecular interactions reflect the promising research investments in this field. In this review, microscale thermophoresis technology (MST) is presented as an analytical technique for characterizing biomolecular interactions. Recent years have seen much progress and several applications established. MST is a powerful technique in quantitation of binding events based on the movement of molecules in microscopic temperature gradient. Simplicity, free solutions analysis, low sample volume, short analysis time, and immobilization free are the MST advantages over other competitive techniques. A wide range of studies in biomolecular interactions have been successfully carried out using MST, which tend to the versatility of the technique to use in screening binding events in order to save time, cost and obtained high data qualit

Protein-Based Affinity Capillary Electrophoresis for Enantioseparation of Calcium Channel Blockers

The development of separation methods for chiral compounds has been an interesting field in pharmaceutical research, particularly the use of proteins as the binding agent. ln this study, the enantioselectivity of calcium channel blockers was investigated using affinity capillary electrophoresis, with human serum as a chiral selector. For this purpose, positively charged of racemic drugs, amlodipine and verapamil were selected as chiral drug models. As the sample was introduced in the capillary electrophoresis system, enantiomer bound to human serum albumin in different extents. Baseline separation was achieved in bare-fused silica capillary with a 20 mM phosphate buffer pH 7 .4, at 15 kV applied voltage and 25 oC. The difference in apparent mobility shifts of enantiomers corresponds to the resolution value of 1.0-3.9. Association constant of the enantiomers was determined using nonlinear regression. R- (+)-verapamil performed to be bound stronger to serum albumin compared with S-(-)-verapamil. The Kn value of S- (-)-amlodipine in the racemic mixture was found to be higher than its antipode. ln addition to this, a close agreement with the S-(-)- enantiomer was achieved for amlodipine

Investigation of Enantioselective Interaction and Determination of Binding Constants of Two Calcium Channel Blockers using Capillary Electrophoresis

The individual enantiomers of a racemic drug usually exhibit specific therapeutic efficacy. In order to achieve optimal therapeutic treatment, an enantioselective analytical method is needed for quality control. Capillary electrophoresis is an analytical method which shows high efficiency in enantiomeric separation. In this study, the enantioselectivity of two calcium channel blockers was investigated using chiral macromolecule as a selector in the background electrolyte of the capillary electrophoresis system. Polysaccharide-based and protein-based selectors were employed separately, each at different concentrations and conditions (e.g., pH value and applied voltage) to achieve baseline separation. Furthermore, the interaction of each enantiomer with human serum albumin as the most abundant protein in the biological system was studied. Mobility-shift affinity capillary electrophoresis was applied to perform the enantiomeric separation and the determination of binding constant. The apparent binding constant of each enantiomer was measured by nonlinear regression analysis. The R- and S- enantiomers of each drug model showed different binding constant value

Simultaneous Enantiomeric Separation and Binding Constants Determination by Affinity Capillary Electrophoresis using Human Serum Albumin

The binding of chiral drugs to plasma proteins is potentially enantioselective and frequently exhibits different pharmacological activities. Serum albumin is one of the most important proteins in human plasma and is commonly used as protein ligand in chiral capillary electrophoresis. Furthermore, human serum albumin has been reported as a stereoselective agent for studying enantioselective interactions. The estimation of binding constant highly contributes to enantiomeric-drugs development and quality control. In this study, mobility-shift affinity capillary electrophoresis was employed for the simultaneous enantiomeric separation and determination of binding constant. The enantiomeric separation was conducted at physiological pH of phosphate buffer containing human serum albumin as background electrolyte. Accordingly, a net negative charge of protein- ligand was obtained. Therefore, positively charged racemic drugs of amlodipine and verapamil were selected as chiral model compounds. Electrophoretic parameters of resolution and effective mobility were evaluated. Subsequently, association constant was determined through nonlinear regression of effective mobilities and total ligand concentrations. In the particular separation conditions, human serum albumin in the range of 30-110 μM displayed stereoselectivity to verapamil and amlodipine. As the sample was introduced into the capillary electrophoresis system, enantiomers bound the human serum albumin to different extents. The difference in apparent mobility shifts between enantiomers corresponds to the resolution of about 1.05-3.93. Moreover, R-(+)-verapamil proved to be bound stronger to human serum albumin compared with S-(-)- verapamil. The association constant of S-(-)-amlodipine was found to be higher compared to its antipode when applying the racemic mixture. In addition to this, a close agreement with the S-(-)-enantiomer was achieved

Immobilization of Chondroitin Sulfate A onto Monolithic Epoxy Silica Column as a New Chiral Stationary Phase for High-Performance Liquid Chromatographic Enantioseparation

Chondroitin sulfate A was covalently immobilized onto a monolithic silica epoxy column involving a Schiff base formation in the presence of ethylenediamine as a spacer and evaluated in terms of its selectivity in enantioseparation. The obtained column was utilized as a chiral stationary phase in enantioseparation of amlodipine and verapamil using a mobile phase consisting of 50 mM phosphate buffer pH 3.5 and UV detection. Sample dilution by organic solvents (preferably 25% v/v acetonitrile-aqueous solution) was applied to achieve baseline enantioresolution (Rs > 3.0) of the individual drug models within 7 min, an excellent linearity (R2 = 0.999) and an interday repeatability of 1.1% to 1.8% RSD. The performance of the immobilized column for quantification of racemate in commercial tablets showed a recovery of 86–98% from tablet matrices. Computational modeling by molecular docking was employed to investigate the feasible complexes between enantiomers and the chiral selector

Biosimilars: Review of regulatory, manufacturing, analytical aspects and beyond

Biologics have more complex production processes compared to small-molecule drugs. They may even prove labile when drifting from batch-to-batch or in different production locations. The development of new similar biological product was regulated early to face the relevant challenges of this industry. As a result, since 2006 biosimilars were introduced to biotechnology arena with a massive competition in pharmaceutical industry. In this review, the aspects related to similarity testing of biosimilars to the original biological products are discussed involving manufacturing challenges to ensure the quality, safety, and efficacy of these products to the patient health. Immunogenicity studies are highlighted as an important part of the safety assessments. Additionally, several analytical methods that are usually used to evaluate biosimilars in comparison to their reference biologic are summarized and categorized in terms of the intended physicochemical and biological characterization. On the other hand, the international efforts of several regulatory agencies including the European Medicines Agency, World Health Organization and United States Food and Drug Administration for biosimilar development are discussed according to updated revised guidelines