Solid-phase microextraction - a future technique in pharmacology and coating trends.

Traditional sample preparation techniques based on liquid-liquid extraction (LLE) or solid-phase extraction (SPE) often suffer from a major error due to the matrix effects caused by significant co-extraction of matrix components. The implementation of a modern extraction technique such as solid-phase microextraction (SPME) was aimed at reducing analysis time and the use of organic solvents, as well as eliminating pre-analytical and analytical errors. Solid-phase microextraction (SPME) is an innovative technique for extracting low molecular weight compounds (less than 1500 Da) from highly complex matrices, including biological matrices. It has a wide range of applications in various types of analysis including pharmaceutical, clinical, metabolomics and proteomics. SPME has a number of advantages over other extraction techniques. Among the most important are low environmental impact, the ability to sample and preconcentrate analytes in one step, simple automation, and the ability to extract multiple analytes simultaneously. It is expected to become, in the future, another method for cell cycle research. Numerous available literature sources prove that solid-phase microextraction can be a future technique in many scientific fields, including pharmaceutical sciences. This paper provides a literature review of trends in SPME coatings and pharmacological applications.

Circular Dichroism as a Rapid Method for Analyzing the Binding of a Targeting Ligand to the Surface of Albumin Nanoparticles.

Circular dichroism (CD) is an excellent and rapid method for analysis of chiral molecules, whose mechanism is based on the absorption of left- and right-hand circularly polarized light. Albumin nanoparticles are biocompatible and easy to modify due to their structure. Tumor cell membranes are among the molecules that direct nanoparticles into the tumor microenvironment, but methods to study them except molecular biology are not well validated yet. The aim of this study was to use circular dichroism as the tool to qualitatively assess ligand binding on the surface of nanoparticles. Human serum albumin (HSA) nanoparticles with encapsulated 5-fluorouracil (5-FU) were coated on MCF-7 cell membranes and subjected to CD analysis. This study was completed using sample and separate 5-FU release analysis. The amount of encapsulated drug in nanoparticles affects the binding of cell membranes on the nanoparticle surface. In addition, it can be suspected that the alpha structure of HSA was mainly used for the interaction, which confirms the effectiveness of using CD as a rapid technique for analyzing ligand-nanoparticle interactions. The release of 5-FU from the nanoparticles proceeds in an uncontrolled manner, making this study in need of further modification and investigation.

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.

New Synthetic Quinoline (Qui) Derivatives as Novel Antioxidants and Potential HSA's Antioxidant Activity Modulators-Spectroscopic Studies.

The antioxidant activity of drugs, as well as the influence of drugs on the activity of endogenous antioxidant mechanisms in the human body is of great importance for the course of the disease and the treatment process. Due to the need to search for new therapeutic methods, the study of newly synthesized substances with potential therapeutic activity is necessary. This study aimed to designate some properties and characteristic parameters of new, synthetic quinoline three derivatives-1-methyl-3-allylthio-4-(4'-methylphenylamino)quinolinium bromide (Qui1), 1-methyl-3-allylthio-4-(3'-hydroxyphenylamino)quinolinium bromide (Qui2) as well as 1-methyl-3-allylthio-4-(4'-hydroxyphenylamino)quinolinium bromide (Qui3), including their antioxidant properties, as well as to analyse their activity as the potential modulators of Human Serum Albumin (HSA) antioxidant activity. In order to achieve the goal of the study, spectroscopic methods such as UV-Vis and circular dichroism (CD) spectroscopy have been used and based on the obtained data only slight and probably some surface interaction of quinoline derivatives (Qui1-Qui3) with HSA have been observed. The effect of Qui1-Qui3 on the HSA secondary structure was also insignificant. All analysed quinine derivatives have antioxidant activity against ABTS cation radical, in turn against DPPH radical, only Qui3 has noticeable antioxidant potential. The highest reduction potential by Qui3 as well as (Qui3 + HSA)complex has been shown. Qui3 mixed with HSA has mostly the synergistic effect against DPPH, ABTS and FRAP, while Qui1 and Qui2 in the presence of HSA mostly have a synergistic and additive effect towards ABTS, respectively. Based on the obtained results it can be concluded that Qui2 and Qui3 can be considered potential modulators of HSA antioxidant activity.

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.

Development of a thin-film solid-phase microextraction (TF-SPME) method coupled to liquid chromatography and tandem mass spectrometry for high-throughput determination of steroid hormones in white sucker fish plasma.

Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17ß-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150 ng/mL and from 0.020 to 0.500 ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract.

In Vitro Investigations of Acetohexamide Binding to Glycated Serum Albumin in the Presence of Fatty Acid.

The interaction of drugs with human serum albumin (HSA) is an important element of therapy. Albumin affects the distribution of the drug substance in the body, as well as its pharmacokinetic and pharmacodynamic properties. On the one hand, inflammation and protein glycation, directly associated with many pathological conditions and old age, can cause structural and functional modification of HSA, causing binding disorders. On the other hand, the widespread availability of various dietary supplements that affect the content of fatty acids in the body means that knowledge of the binding activity of transporting proteins, especially in people with chronic diseases, e.g., diabetes, will achieve satisfactory results of the selected therapy. Therefore, the aim of the present study was to evaluate the effect of a mixture of fatty acids (FA) with different saturated and unsaturated acids on the affinity of acetohexamide (AH), a drug with hypoglycaemic activity for glycated albumin, simulating the state of diabetes in the body. Based on fluorescence studies, we can conclude that the presence of both saturated and unsaturated FA disturbs the binding of AH to glycated albumin. Acetohexamide binds more strongly to defatted albumin than to albumin in the presence of fatty acids. The competitive binding of AH and FA to albumin may influence the concentration of free drug fraction and thus its therapeutic effect.

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.

The Influence of Fatty Acids on Metoprolol - Human Serum Albumin Interaction in Low Affinity Binding Sites: A Multifactorial NMR Approach.

BACKGROUND: Metoprolol (MTP) is a cardio-selective ß1-blocker used in hypertension, angina pectoris and chronic heart failure therapies. Serum albumin is the most frequently occurring protein in blood plasma. The binding of ligands to human serum albumin (HSA) has an important effect on pharmacokinetics and final clinical effects. OBJECTIVE: The objectives of this study included a detailed analysis of metoprolol - serum albumin interactions in low affinity binding sites, on the surface or within the hydrophobic subdomain of a macromolecule, as well as an analysis of the competition between MTP and fatty acids in binding with protein. METHODS: The analysis of the drug-albumin interaction was based on the observed chemical shifts in combination with correlation Times (T1 -1 = τ) [1/s], 2D NOESY 1H NMR spectra and association constants Ka [M-1]. For the determination of chemical shifts σ [ppm], relaxation times T1 [s] and for the NOESY experiment, the final concentrations of MTP and albumins (in the presence (HSA) and absence of fatty acids (dHSA)) were 5 x 10-3 M and 2 x 10-5 M - 4.55 x 10-4 M, respectively. In order to calculate the association constants, the final concentrations of MTP and both HSA and dHSA were 2.75 x 10-3 M - 6.25 x 10-2 M and 2.5 x 10-4 M, respectively. For the analysis, the MTP proton resonances of aliphatic H17, aromatic (H2/H6 and H3/H5) and the methoxy group H14 were chosen. RESULTS: Changes in the values of the 1H NMR chemical shift [ppm] are evidence of interaction between MTP, fatted (HSA) and defatted (dHSA) human serum albumin. With an increase of albumin concentration, changes in the chemical shift values were observed for the aromatic protons H2/H6 (Δσ = 0.013 ppm and 0.018 ppm) and H3/H5 (Δσ = 0.015 ppm and 0.019 ppm), the aliphatic proton H17 (Δσ = 0.018 ppm and 0.022 ppm) and the aliphatic protons of the methoxy group H14 (Δσ = 0.019 ppm and 0.022 ppm) for dHSA and HSA, respectively. Greater changes in chemical shifts in the presence of fatty acids (FA) were observed. Changes in the correlation times of MTP aromatic H2/H6 (Δτc = 0.224 1/s and 0.189 1/s) and H3/H5 (Δτc = 0.269 1/s and 0.210 1/s), aliphatic from the methoxy group H14 (Δτc = 0.472 1/s and 0.271 1/s) and aliphatic H17 protons (Δτc = 0.178 1/s and 0.137 1/s) for dHSA and HSA systems, respectively. It confirms the interaction between the drug and albumin are evidence for the dynamics of the process. In the presence of FA the relaxation time of all analyzed MTP proton resonance signals significantly increases (due to the decrease of correlation time). This phenomenon is due to the increase of electron density in the MTP protons' surroundings. Association constants for the MTP-dHSA complex in the low affinity site range between 0.29 x 102 M-1 and 0.47 x 102 M-1. The presence of FA results in a two to three-fold increase of the Ka values of protons from aromatic (H2/H6 and H3/H5), aliphatic H17 and methoxy (H14) groups. In 2D NOESY spectra proton magnetization transfer was observed between cysteine (Cys-34) and aromatic H3/H5 and H2/H6 protons. Cross-peaks were also observed between cysteine and aliphatic protons from the methoxy group. CONCLUSION: The selective changes in σ [ppm] and τc [1/s] values indicated the unequal participation of chemical groups of MTP in the interaction with HSA and dHSA. The data obtained suggest that the presence of fatty acids increases the accessibility of low affinity sites of serum albumin to MTP, which results in the higher affinity of albumin towards the drug. The results showed that the main binding site of MTP and fatty acid is probably a low affinity site in subdomain IB, where Cys-34 can be located.

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.

Synthesis, Anti-Breast Cancer Activity, and Molecular Docking Study of a New Group of Acetylenic Quinolinesulfonamide Derivatives.

In this study, a series of regioisomeric acetylenic sulfamoylquinolines are designed, synthesized, and tested in vitro for their antiproliferative activity against three human breast cacer cell lines (T47D, MCF-7, and MDA-MB-231) and a human normal fibroblast (HFF-1) by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. The antiproliferative activity of the tested acetylenic quinolinesulfonamides is comparable to that of cisplatin. The bioassay results demonstrate that most of the tested compounds show potent antitumor activities, and that some compounds exhibit better effects than the positive control cisplatin against various cancer cell lines. Among these compounds, 4-(3-propynylthio)-7-[N-methyl-N-(3-propynyl)sulfamoyl]quinoline shows significant antiprolierative activity against T47D cells with IC50 values of 0.07 µM. In addition, 2-(3-Propynylthio)-6-[N-methyl-N-(3-propynyl)sulfa-moyl]quinoline and 2-(3-propynylseleno)-6-[N-methyl-N-(3-propynyl)sulfamoyl]quinoline display highly effective atitumor activity against MDA-MB-231 cells, with IC50 values of 0.09 and 0.50 µM, respectively. Furthermore, most of the tested compounds show a weak cytotoxic effect against the normal HFF-1 cell line. Additionally, in order to suggest a mechanism of action for their activity, all compounds are docked into the binding site of two human cytochrome P450 (CYP) isoenzymes. These data indicate that some of the title compounds display significant cytotoxic activity, possibly targeting the CYPs pathways.