The effect of selected aminoglycoside antibiotics on human serum albumin antioxidant activity: a spectroscopic and calorimetric comparative study.

BACKGROUND: Human serum albumin (HSA) is a valuable component of non-enzymatic and endogenous antioxidant mechanisms. The antioxidant activity of HSA can be modulated by ligands, including drugs. Although this is a central topic in the field of oxidation, there is still a lack of information about the protection against the effects of elevated free radical levels. METHODS: The aim of this study was to investigate the antioxidant activity of kanamycin (KAN) and neomycin (NEO) and their effect on the antioxidant potential of HSA using spectroscopic and microcalorimetric techniques. RESULTS: Despite the fact that kanamycin and neomycin interact with HSA, no changes in the secondary structure of the protein have been observed. The analysis of the aminoglycoside antibiotics showed their low antioxidant activity and a synergistic effect of the interaction, probably due to the influence of ligands (KAN, NEO) on the availability of HSA amino acid residues functional groups, such as the free thiol group (Cys-34). CONCLUSIONS: Based on the spectroscopic and microcalorimetric data, both KAN and NEO can be considered modulators of the HSA antioxidant activity.

Spectroscopic Studies of Quinobenzothiazine Derivative in Terms of the In Vitro Interaction with Selected Human Plasma Proteins: Part 2.

Synthesis of anticancer substances and studying their binding abilities towards human serum proteins as carriers are important parts of pharmaceutical and medical sciences development. The presented work is a continuation of studies of quinobenzothiazine derivatives binding with serum proteins. The main aim of this work was a spectroscopic analysis of second from benzothiazinium derivatives salt, 9-fluoro-5-alkyl-12(H)-quino [3,4-b][1,4]benzothiazinium chloride (Salt2), its interaction with carrier proteins, i.e., human serum albumin (HSA), α1-acid glycoprotein (AGP), human gamma globulin (HGG), and the study of protein secondary and tertiary structure changes using spectroscopic techniques (spectrofluorescence, UV-Vis and circular dichroism CD spectroscopy). In order to mimic in vivo conditions, control normal serum (CNS) was used. Using the Klotz method, both binding constants (Ka [M-1]) and the number of binding classes (n) were calculated. In addition, the percentage of displacement of binding site markers from HSA and AGP molecules has been defined. Based on the obtained data, it can be concluded that the main binding protein for Salt2 is AGP. HSA and HGG are also involved in the distribution of the studied substance in the bloodstream. Moreover, Salt2 very slightly interacts with CNS, which can cause strong therapeutic as well as toxic effects. The analysis of CD spectra confirms that there are no changes in the secondary structure of the main binding proteins in the presence of Salt2.

Spectroscopic Analysis of an Antimalarial Drug's (Quinine) Influence on Human Serum Albumin Reduction and Antioxidant Potential.

Quinine (Qi) is a well-known drug used in malaria therapy; it is also a potential anti-arrhythmic drug used in the treatment of calf cramps, rheumatoid arthritis, colds, and photodermatitis. Moreover, it is used in the food industry for the production of tonics. This study aimed to analyze the interaction between quinine and a transporting protein-human serum albumin (HSA)-as well as the influence of Qi on both protein reduction and antioxidant potential. It was found that Qi (via spectrofluorometric measurements and circular dichroism spectroscopy) binds to HSA with a low affinity and slightly affects the secondary structure of albumin. As demonstrated by the use of ABTS and FRAP assays, HSA has a higher antioxidant and reduction potential than Qi, while their mutual interaction results in a synergistic effect in antioxidant activity and reduction potential.

Comparison of Losartan and Furosemide Interaction with HSA and Their Influence on HSA Antioxidant Potential.

Serum albumin (HSA) is the most important protein in human body. Due to the antioxidant activity, HSA influences homeostasis maintenance and transport of drugs as well as other substances. It is noteworthy that ligands, such as popular drugs, modulate the antioxidant activity of HSA. The aim of this study was to analyze the influence of losartan (LOS) and furosemide (FUR) on HSA antioxidant properties as well as the interaction between these drugs and protein using calorimetric and spectroscopic methods. LOS and FUR showed the high affinity for human serum albumin, and the binding reactions between them were spontaneous and exothermic. LOS and FUR, separately and together in the system, have no significant impact on the secondary HSA structure; however they have significant impact on the tertiary HSA structure. LOS and FUR mixed with HSA have the ability to scavenge free radicals, and the ligand(s)-HSA interactions were synergistic.

Changes in Glycated Human Serum Albumin Binding Affinity for Losartan in the Presence of Fatty Acids In Vitro Spectroscopic Analysis.

Conformational changes in human serum albumin due to numerous modifications that affect its stability and biological activity should be constantly monitored, especially in elderly patients and those suffering from chronic diseases (which include diabetes, obesity, and hypertension). The main goal of this study was to evaluate the effect of a mixture of fatty acids (FA) on the affinity of losartan (LOS, an angiotensin II receptor (AT1) blocker used in hypertension, a first-line treatment with coexisting diabetes) for glycated albumin-simulating the state of diabetes in the body. Individual fatty acid mixtures corresponded to the FA content in the physiological state and in various clinical states proceeding with increased concentrations of saturated (FAS) and unsaturated (FAUS) acids. Based on fluorescence studies, we conclude that LOS interacts with glycated human serum albumin (af)gHSA in the absence and in the presence of fatty acids ((af)gHSAphys, (af)gHSA4S, (af)gHSA8S, (af)gHSA4US, and (af)gHSA8US) and quenches the albumin fluorescence intensity via a static quenching mechanism. LOS not only binds to its specific binding sites in albumins but also non-specifically interacts with the hydrophobic fragments of its surface. Incorrect contents of fatty acids in the body affect the drug pharmacokinetics. A higher concentration of both FAS and FAUS acids in glycated albumin reduces the stability of the complex formed with losartan. The systematic study of FA and albumin interactions using an experimental model mimicking pathological conditions in the body may result in new tools for personalized pharmacotherapy.

Pre-Formulation Studies: Physicochemical Characteristics and In Vitro Release Kinetics of Insulin from Selected Hydrogels.

Insulin loaded to the polymer network of hydrogels may affect the speed and the quality of wound healing in diabetic patients. The aim of our research was to develop a formulation of insulin that could be applied to the skin. We chose hydrogels commonly used for pharmaceutical compounding, which can provide a form of therapy available to every patient. We prepared different gel formulations using Carbopol® UltrezTM 10, Carbopol® UltrezTM 30, methyl cellulose, and glycerin ointment. The hormone concentration was 1 mg/g of the hydrogel. We assessed the influence of model hydrogels on the pharmaceutical availability of insulin in vitro, and we examined the rheological and the texture parameters of the prepared formulations. Based on spectroscopic methods, we evaluated the influence of model hydrogels on secondary and tertiary structures of insulin. The analysis of rheograms showed that hydrogels are typical of shear-thinning non-Newtonian thixotropic fluids. Insulin release from the formulations occurs in a prolonged manner, providing a longer duration of action of the hormone. The stability of insulin in hydrogels was confirmed. The presence of model hydrogel carriers affects the secondary and the tertiary structures of insulin. The obtained results indicate that hydrogels are promising carriers in the treatment of diabetic foot ulcers. The most effective treatment can be achieved with a methyl cellulose-based insulin preparation.

Spectroscopic Studies of Quinobenzothiazine Derivative in Terms of the In Vitro Interaction with Selected Human Plasma Proteins. Part 1.

Plasma proteins play a fundamental role in living organisms. They participate in the transport of endogenous and exogenous substances, especially drugs. 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium salts, have been synthesized as potential anticancer substances used for cancer treatment. Most anticancer substances generate a toxic effect on the human body. In order to check the toxicity and therapeutic dosage of these chemicals, the study of ligand binding to plasma proteins is very relevant. The present work presents the first comparative analysis of the binding of one of the 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium derivatives (Salt1) with human serum albumin (HSA), α-1-acid glycoprotein (AGP) and human gamma globulin (HGG), assessed using fluorescence, UV-Vis and CD spectroscopy. In order to mimic in vivo ligand-protein binding, control normal serum (CNS) was used. Based on the obtained data, the Salt1 binding sites in the tertiary structure of all plasma proteins and control normal serum were identified. Both the association constants (Ka) and the number of binding site classes (n) were calculated using the Klotz method. The strongest complex formed was Salt1-AGPcomplex (Ka = 7.35·104 and 7.86·104 mol·L-1 at excitation wavelengths λex of 275 and 295 nm, respectively). Lower values were obtained for Salt1-HSAcomplex (Ka = 2.45·104 and 2.71·104 mol·L-1) and Salt1-HGGcomplex (Ka = 1.41·104 and 1.33·104 mol·L-1) at excitation wavelengths λex of 275 and 295 nm, respectively, which is a positive phenomenon and contributes to the prolonged action of the drug. Salt1 probably binds to the HSA molecule in Sudlow sites I and II; for the remaining plasma proteins studied, only one binding site was observed. Moreover, using circular dichroism (CD), fluorescence and UV-Vis spectroscopy, no effect on the secondary and tertiary structures of proteins in the absence or presence of Salt1 has been demonstrated. Despite the fact that the conducted studies are basic, from the scientific point of view they are novel and encourage further in vitro and in vivo investigations. As a next part of the study (Part 2), the second new synthetized quinobenzothiazine derivative (Salt2) will be analyzed and published.

The Influence of Oxidative Stress on Serum Albumin Structure as a Carrier of Selected Diazaphenothiazine with Potential Anticancer Activity.

Albumin is one of the most important proteins in human blood. Among its multiple functions, drug binding is crucial in terms of drug distribution in human body. This protein undergoes many modifications that are certain to influence protein activity and affect its structure. One such reaction is albumin oxidation. Chloramine T is a strong oxidant. Solutions of human serum albumin, both non-modified and modified by chloramine T, were examined with the use of fluorescence, absorption and circular dichroism (CD) spectroscopy. 10H-3,6-diazaphenothiazine (DAPT) has anticancer activity and it has been studied for the first time in terms of binding with human serum albumin-its potential as a transporting protein. Using fluorescence spectroscopy, in the presence of dansylated amino acids, dansyl-l-glutamine (dGlu), dansyl-l-proline (dPro), DAPT binding with two main albumin sites-in subdomain IIA and IIIA-has been evaluated. Based on the conducted data, in order to measure the stability of DAPT complexes with human (HSA) and oxidized (oHSA) serum albumin, association constant (Ka) for ligand-HSA and ligand-oHSA complexes were calculated. It has been presumed that oxidation is not an important issue in terms of 10H-3,6-diazaphenothiazine binding to albumin. It means that the distribution of this substance is similar regardless of changes in albumin structure caused by oxidation, natural occurring in the organism.

Human Serum Albumin Aggregation/Fibrillation and its Abilities to Drugs Binding.

Human serum albumin (HSA) is a protein that transports neutral and acid ligands in the organism. Depending on the environment's pH conditions, HSA can take one of the five isomeric forms that change its conformation. HSA can form aggregates resembling those in vitro formed from amyloid at physiological pH (neutral and acidic). Not surprisingly, the main goal of the research was aggregation/fibrillation of HSA, the study of the physicochemical properties of formed amyloid fibrils using thioflavin T (ThT) and the analysis of ligand binding to aggregated/fibrillated albumin in the presence of dansyl-l-glutamine (dGlu), dansyl-l-proline (dPro), phenylbutazone (Phb) and ketoprofen (Ket). Solutions of human serum albumin, both non-modified and modified, were examined with the use of fluorescence, absorption and circular dichroism (CD) spectroscopy. The experiments conducted allowed observation of changes in the structure of incubated HSA (HSAINC) in relation to nonmodified HSA (HSAFR). The formed aggregates/fibrillation differed in structure from HSA monomers and dimers. Based on CD spectroscopy, previously absent ßstructural constructs have been registered. Whereas, using fluorescence spectroscopy, the association constants differing for fresh and incubated HSA solutions in the presence of dansyl-amino acids and markers for binding sites were calculated and allowed observation of the conformational changes in HSA molecule.

Influence of Piracetam on Gliclazide-Glycated Human Serum Albumin Interaction. A Spectrofluorometric Study.

Advanced Glycation End-Products (AGEs) are created in the last step of protein glycation and can be a factor in aging and in the development or worsening of many degenerative diseases (diabetes, chronic kidney disease, atherosclerosis, Alzheimer's disease, etc.). Albumin is the most susceptible to glycation plasma protein. Modified albumin by AGEs may be more resistant to enzymatic degradation, which further increases the local accumulation of AGEs in tissues. The aim of the present study was to analyze in vitro glycation of serum albumin in the presence of piracetam (PIR) and the gliclazide (GLZ)-glycated albumin interaction. The analysis of PIR as an inhibitor and GLZ interaction with nonglycated human albumin (HSA) and glycated by fructose human albumin (gHSAFRC), in the absence and presence of piracetam (gHSAFRC-PIR), was performed by fluorescence quenching of macromolecules. On the basis of obtained data we concluded that under the influence of glycation, association constant ( K a ) of gliclazide to human serum albumin decreases and GLZ binds to HSA with less strength than under physiological conditions. PIR strongly inhibited the formation of AGEs in the system where the efficiency of HSA glycation was the largest. The analysis of piracetam influence on the GLZ-glycated albumin interaction has shown that piracetam increases the binding strength of GLZ to glycated albumin and weakens its therapeutic effect. Based on the obtained data we concluded that monitoring therapy and precautions are required in the treatment when the combinations of gliclazide and piracetam are used at the same time.

Alteration of human serum albumin binding properties induced by modifications: A review.

Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlow's site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

In Vitro Investigation of the Interaction of Tolbutamide and Losartan with Human Serum Albumin in Hyperglycemia States.

Serum albumin is exposed to numerous structural modifications which affect its stability and activity. Glycation is one of the processes leading to the loss of the original properties of the albumin and physiological function disorder. In terms of long lasting states of the hyperglycemia, Advanced Glycation End-products (AGEs) are formed. AGEs are responsible for cellular and tissue structure damage that cause the appearance of a number of health consequences and premature aging. The aim of the present study was to analyze the conformational changes of serum albumin by glycation-"fructation"-using multiple spectroscopic techniques, such as absorption (UV-Vis), fluorescence (SFM), circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy and evaluate of possible alteration of binding and competition between tolbutamide (TB, a first-generation sulfonylurea oral hypoglycemic drug) and losartan (LOS, an angiotensin II receptor (AT1) blocker used in hypertension (1st line with a coexisting diabetes)) in binding to non-glycated (HSA) and glycated (gHSAFRC) human serum albumin in high-affinity binding sites. The studies allowed us to indicate the structural alterations of human serum albumin as a result of fructose glycation. Changes in binding parameters, such as association ( K a ) or Stern-Volmer ( K S V ) constants suggest that glycation increases the affinity of TB and LOS towards albumin and affects interactions between them. The process of albumin glycation influences the pharmacokinetics of drugs, thus monitored pharmacotherapy is reasonable in the case of diabetes and hypertension polypharmacy. This information may lead to the development of more effective drug treatments based on personalized medicine for patients with diabetes. Our studies suggest the validity of monitored polypharmacy of diabetes and coexisting diseases.

Effect of Temperature on Tolbutamide Binding to Glycated Serum Albumin.

Glycation process occurs in protein and becomes more pronounced in diabetes when an increased amount of reducing sugar is present in bloodstream. Glycation of protein may cause conformational changes resulting in the alterations of its binding properties even though they occur at a distance from the binding sites. The changes in protein properties could be related to several pathological consequences such as diabetic and nondiabetic cardiovascular diseases, cataract, renal dysfunction and Alzheimer's disease. The experiment was designed to test the impact of glycation process on sulfonylurea drug tolbutamide-albumin binding under physiological (T = 309 K) and inflammatory (T = 311 K and T = 313 K) states using fluorescence and UV-VIS spectroscopies. It was found in fluorescence analysis experiments that the modification of serum albumin in tryptophanyl and tyrosyl residues environment may affect the tolbutamide (TB) binding to albumin in subdomain IIA and/or IIIA (Sudlow's site I and/or II), and also in subdomains IB and IIB. We estimated the binding of tolbutamide to albumin described by a mixed nature of interaction (specific and nonspecific). The association constants Ka (L∙mol-1) for tolbutamide at its high affinity sites on non-glycated albumin were in the range of 1.98-7.88 × 104 L∙mol-1 (λex = 275 nm), 1.20-1.64 × 104 L∙mol-1 (λex = 295 nm) and decreased to 1.24-0.42 × 104 L∙mol-1 at λex = 275 nm (T = 309 K and T = 311 K) and increased to 2.79 × 104 L∙mol-1 at λex = 275 nm (T = 313 K) and to 4.43-6.61 × 104 L∙mol-1 at λex = 295 nm due to the glycation process. Temperature dependence suggests the important role of van der Waals forces and hydrogen bonding in hydrophobic interactions between tolbutamide and both glycated and non-glycated albumin. We concluded that the changes in the environment of TB binding of albumin in subdomain IIA and/or IIIA as well as in subdomains IB and IIB influence on therapeutic effect and therefore the studies of the binding of tolbutamide (in diabetes) to transporting protein under glycation that refers to the modification of a protein are of great importance in pharmacology and biochemistry. This information may lead to the development of more effective drug therapy in people with diabetes.

Fluorometric Investigation on the Binding of Letrozole and Resveratrol with Serum Albumin.

Breast cancer is the most common cancer in women worldwide. Combination of drugs during long-therm cancer therapy can increase free, biological active form of the drug and cause dangerous side effects. The 21^st century is a period of searching for a progress in cancer chemotherapy. The simultaneous dosage of drugs and natural agents isolated from fruits and vegetables used in breast cancer treatment could be more effective and less toxic. The aim of the study was to determine the binding sites of both letrozole (LET) and polyphenol product, resveratrol (RES) in tertiary structure of human serum albumin (HSA) based on the fluorescence spectroscopy. The binding of LET and RES to HSA was studied by monitoring the changes in emission fluorescence spectra of albumin in the presence of ligands at 280 nm and 295 nm excitation wavelengths. To identify the binding sites of LET and RES on HSA, warfarin (WAR) and 5-(Dimethylamino)-1-naphthalenesulfonamide (DNSA) were used as site probes for binding site I, while dansyl-L-proline (DP) was studied as a marker for binding site II. The binding sites for LET and RES in HSA were found to be located in subdomain IIIA. Based on the Stern - Volmer and binding isotherm using non-linear regression methods, the formation of complexes of LET and RES with HSA and association constants were obtained. The binding analysis showed that the association constants indicated a stronger interaction of HSA with RES than LET and the presence of RES in the tertiary system alters the stability of LET-albumin complex. This conclusion points to the necessity of precaution and monitoring therapy when resveratrol as a natural compound and letrozole are used together.

Analysis of Aged Human Serum Albumin Affinity for Doxazosin.

Structural changes of human serum albumin (HSA) caused by old age and coexisting diseases result in differences in the binding of doxazosin (DOX). DOX is a postsynaptic α1- adrenoreceptor antagonist used for treatment of hypertension and benign prostatic hyperplasia. In elderly people suffering from various renal or hepatic diseases the significant portion of N-form of human serum albumin (normal) is converted to A-form (aged). The differences in binding of doxazosin to N- and Aform of albumin are an important factor, which may determines therapeutic dosage and toxicity of the test drug. To indicate these differences, the technique of fluorescence spectroscopy was used. The association constant (Ka) obtained from fluorescence quenching demonstrated that doxazosin has higher affinity for AHSA than for HSA. In order to describe the cooperativity in binding process, the values of the Hill's coefficient has been analysed. For DOX-HSA system (λex 295 nm) Hill's coefficient is close to 1 and it indicates that there is a single class of binding sites. For DOX-HSA (λex 275 nm) and DOX-AHSA (λex 275 nm and λex 295 nm) systems we observed positive cooperativity (nH>1). A greater red shift of fluorescence emission maximum of AHSA than HSA in the presence of DOX was observed. This suggests that the binding of DOX to AHSA was accompanied by a stronger increase in polarity around the fluorophores in comparison to HSA. The binding interaction between DOX and HSA has been also studied by molecular docking simulation.