In Vitro Spectroscopic Investigation of Losartan and Glipizide Competitive Binding to Glycated Albumin: A Comparative Study

Understanding the interaction between pharmaceuticals and serum proteins is crucial for optimizing therapeutic strategies, especially in patients with coexisting chronic diseases. The primary goal of this study was to assess the potential changes in binding affinity and competition between glipizide (GLP, a second-generation sulfonylurea hypoglycemic drug) and losartan (LOS, a medication commonly prescribed for hypertension, particularly for patients with concurrent diabetes) with non-glycated (HSA) and glycated (gHSAGLC, gHSAFRC) human serum albumin using multiple spectroscopic techniques (fluorescence, UV-visible absorption, and circular dichroism spectroscopy). The results indicated that FRC is a more effective glycation agent for HSA than GLC, significantly altering the albumin structure and affecting the microenvironment around critical amino acid residues, Trp-214 and Tyr. These modifications reduce the binding affinity of LOS and GLP to gHSAGLC and gHSAFRC, compared to HSA, resulting in less stable drug–protein complexes. The study revealed that LOS and GLP interact nonspecifically with the hydrophobic regions of the albumin surface in both binary (ligand–albumin) and ternary systems (ligand–albumin–ligandconst) and specifically saturate the binding sites within the protein molecule. Furthermore, the presence of an additional drug (GLP in the LOS–albumin complex or LOS in the GLP–albumin complex) complicates the interactions, likely leading to competitive binding or displacement of the initially bound drug in both non-glycated and glycated albumins. Analysis of the CD spectra suggests mutual interactions between GLP and LOS, underscoring the importance of closely monitoring patients co-administered these drugs, to ensure optimal therapeutic efficacy and safety.


Introduction
The escalating prevalence of chronic diseases, including hypertension and type 2 diabetes mellitus, has necessitated the widespread use of polypharmacotherapy.This therapeutic approach mandates a comprehensive understanding of drug interactions and their biochemical implications, mainly when pharmaceuticals interact with modified biological molecules such as glycated human serum albumin (gHSA).gHSA, a product of non-enzymatic glycation of human serum albumin (HSA), is prevalent in diabetic patients and has significant clinical implications, due to its altered structure, biological function, and physicochemical properties [1,2].
Albumin, the primary protein in human plasma, is essential for maintaining oncotic pressure and transporting various endogenous and exogenous substances.Furthermore, HSA possesses anti-inflammatory, antioxidant, and antithrombotic properties [3].Under diabetic conditions, HSA undergoes glycation, which can markedly affect its drug-binding capabilities.Glycated albumin serves not only as a marker of short-term glycemic control but also influences the pharmacokinetics and pharmacodynamics of therapeutic agents, potentially altering their efficacy and safety profiles [4].Understanding how glycation modifies albumin's binding properties is essential for optimizing drug dosing and minimizing adverse effects in diabetic patients.
Losartan (LOS, Figure 1a) is widely used for its antihypertensive properties, primarily by inhibiting the renin-angiotensin system.By blocking angiotensin II from binding to the AT1 receptor, LOS induces vasodilation and decreases aldosterone secretion, lowering blood pressure [5,6].LOS exhibits a high affinity to HSA, predominantly binding at site II, also known as the benzodiazepine-binding site [7].This significant binding within subdomain IIIA of the HSA macromolecule ensures that a considerable portion of LOS remains inactive in the bloodstream, with only a minor fraction available as a free drug to exert its therapeutic effects.
simulation studies in silico, showing that the GLP-HSA complex is formed due to hydrogen bonds and hydrophobic interactions [8].
In combination therapy, LOS and GLP may compete for the same binding site on HSA.This competitive binding can influence their pharmacokinetics and pharmacodynamics, potentially affecting their efficacy and safety profiles.The primary aim of this study was to compare the interactions of LOS and GLP with human serum albumin-both non-glycated (HSA) and glycated by glucose (gHSAGLC) and fructose (gHSAFRC)-and to investigate their mutual binding interactions using spectroscopic techniques (fluorescence spectroscopy, UV-visible absorption spectroscopy, and circular dichroism spectroscopy-CD).These techniques are widely recognized for studying in vitro interactions of drugs with albumin, due to their sensitivity, speed, and simplicity [10][11][12].They are beneficial for calculating binding and quenching parameters, mean residue ellipticity, and characterizing proteins' secondary and tertiary structures.
By elucidating the competitive binding interactions of LOS and GLP with glycated albumin at a molecular level, this research seeks to provide insights that could inform better clinical practices in polypharmacotherapy, particularly for patients at risk of altered drug efficacy due to glycation.This understanding is imperative for optimizing therapeutic strategies in patients with comorbid hypertension and diabetes.Glipizide (GLP, Figure 1b), a second-generation sulfonylurea, is widely used to manage blood glucose levels in patients with type 2 diabetes mellitus.It exerts its pharmacological effects by stimulating insulin secretion from pancreatic β-cells.This is achieved by inhibiting ATP-sensitive potassium channels on the β-cell membrane, leading to membrane depolarization and subsequent insulin release, thereby reducing blood glucose levels [8].A study using high-performance affinity chromatography (HPAC) revealed that GLP interacts with both Sudlow's sites I and II on HSA, but with greater affinity for site II than the site I [9].Anwer et al. (2021) performed molecular docking and simulation studies in silico, showing that the GLP-HSA complex is formed due to hydrogen bonds and hydrophobic interactions [8].
In combination therapy, LOS and GLP may compete for the same binding site on HSA.This competitive binding can influence their pharmacokinetics and pharmacodynamics, potentially affecting their efficacy and safety profiles.The primary aim of this study was to compare the interactions of LOS and GLP with human serum albumin-both nonglycated (HSA) and glycated by glucose (gHSA GLC ) and fructose (gHSA FRC )-and to investigate their mutual binding interactions using spectroscopic techniques (fluorescence spectroscopy, UV-visible absorption spectroscopy, and circular dichroism spectroscopy-CD).These techniques are widely recognized for studying in vitro interactions of drugs with albumin, due to their sensitivity, speed, and simplicity [10][11][12].They are beneficial for calculating binding and quenching parameters, mean residue ellipticity, and characterizing proteins' secondary and tertiary structures.
By elucidating the competitive binding interactions of LOS and GLP with glycated albumin at a molecular level, this research seeks to provide insights that could inform better clinical practices in polypharmacotherapy, particularly for patients at risk of altered drug efficacy due to glycation.This understanding is imperative for optimizing therapeutic strategies in patients with comorbid hypertension and diabetes.A widely accepted assumption is that the higher the AGE fluorescence intensity, the greater the degree of glycation [17].From the experiment results, it is evident that FRC induced glycation of HSA more effectively than GLC.The higher fluorescence intensity of AGEs observed in gHSAFRC suggests that HSA undergoes a faster and more efficient Amadori rearrangement in the presence of FRC than in the HSA-GLC system.In addition to the hyperchromic effect, this study observed a hypsochromic shift in the fluorescence maxima of glycated albumin AGEs compared to HSA excited at λex = 335 nm (Δλmax = 6 ± 1.5 nm).This blue shift indicates a decrease in polarity within the environment of the A widely accepted assumption is that the higher the AGE fluorescence intensity, the greater the degree of glycation [17].From the experiment results, it is evident that FRC induced glycation of HSA more effectively than GLC.The higher fluorescence intensity of AGEs observed in gHSA FRC suggests that HSA undergoes a faster and more efficient Amadori rearrangement in the presence of FRC than in the HSA-GLC system.In addition to the hyperchromic effect, this study observed a hypsochromic shift in the fluorescence maxima of glycated albumin AGEs compared to HSA excited at λ ex = 335 nm (∆λ max = 6 ± 1.5 nm).This blue shift indicates a decrease in polarity within the environ-ment of the newly formed products (Figure 2a).Irradiation in the wavelength ranges of 320 nm to 335 nm and 325 nm to 335 nm caused the excitation of argpyrimidine (AP) and pentosidine (PEN), respectively.These compounds, which exhibit fluorescent properties, are formed due to protein modifications by methylglyoxal through interactions with Arg residues in macromolecules [18].AP emits fluorescence at an approximate wavelength of λ em ~400 nm, while PEN emits fluorescence in the range of λ em = 375 nm to 385 nm [19].The glycated albumin products gHSA GLC and gHSA FRC , when excited at λ ex = 335 nm (Figure 2a), do not display the fluorescence typical of PEN and AP.The fluorescence of AGEs at around λ em = 420 nm suggests the formation of PEN and AP in glycated proteins but also indicates the presence of additional fluorophores.This is evidenced by the shift of the fluorescence maximum towards longer wavelengths compared to the emission of PEN and AP, as described by Kessel et al. (2002) [19].An advantage of measurements at λ ex = 485 nm is the absence of interference from other fluorophores in the sample.According to Schmitt et al. (2005), fluorescence with an emission maximum at λ em = 530 nm (Figure 2b) appears to be an indicator of Arg residue modifications [20].The results presented here confirmed the efficacy of the glycation process under the experimental conditions applied in this study, providing a solid foundation for further research in this area.
Based on emission fluorescence spectra (Supplementary Materials Figures S1 and S2), fluorescence quenching curves of non-glycated (HSA) and glycated (gHSA GLC , gHSA FRC ) albumin, excited at λ ex = 275 nm and λ ex = 295 nm, were plotted to determine the interaction of losartan (LOS) and glipizide (GLP) at high-affinity binding sites of albumins in the binary systems: LOS-HSA (Figure 3a), LOS-gHSA GLC (Figure 3b), LOS-gHSA FRC (Figure 3c), GLP-HSA (Figure 3d), GLP-gHSA GLC (Figure 3e), GLP-gHSA FRC (Figure 3f).newly formed products (Figure 2a).Irradiation in the wavelength ranges of 320 nm to 335 nm and 325 nm to 335 nm caused the excitation of argpyrimidine (AP) and pentosidine (PEN), respectively.These compounds, which exhibit fluorescent properties, are formed due to protein modifications by methylglyoxal through interactions with Arg residues in macromolecules [18].AP emits fluorescence at an approximate wavelength of λem ~ 400 nm, while PEN emits fluorescence in the range of λem = 375 nm to 385 nm [19].The glycated albumin products gHSAGLC and gHSAFRC, when excited at λex = 335 nm (Figure 2a), do not display the fluorescence typical of PEN and AP.The fluorescence of AGEs at around λem = 420 nm suggests the formation of PEN and AP in glycated proteins but also indicates the presence of additional fluorophores.This is evidenced by the shift of the fluorescence maximum towards longer wavelengths compared to the emission of PEN and AP, as described by Kessel et al. (2002) [19].An advantage of measurements at λex = 485 nm is the absence of interference from other fluorophores in the sample.According to Schmitt et al. (2005), fluorescence with an emission maximum at λem = 530 nm (Figure 2b) appears to be an indicator of Arg residue modifications [20].The results presented here confirmed the efficacy of the glycation process under the experimental conditions applied in this study, providing a solid foundation for further research in this area.
Based on emission fluorescence spectra (Supplementary Materials Figures S1 and  S2), fluorescence quenching curves of non-glycated (HSA) and glycated (gHSAGLC, gHSAFRC) albumin, excited at λex = 275 nm and λex = 295 nm, were plotted to determine the interaction of losartan (LOS) and glipizide (GLP) at high-affinity binding sites of albumins in the binary systems: LOS-HSA (Figure 3a), LOS-gHSAGLC (Figure 3b), LOS-gHSAFRC (Figure 3c), GLP-HSA (Figure 3d), GLP-gHSAGLC (Figure 3e), GLP-gHSAFRC (Figure 3f).In fluorescence studies, attention is directed towards specific protein regions containing fluorophores.Using an excitation wavelength of 275 nm enables the observation of both tryptophan (Trp-214) and tyrosine (Tyr) residues, whereas a 295 nm wavelength selectively excites Trp-214 due to its unique spectral characteristics.The primary drug-binding sites are located within the IIA and IIIA subdomains of HSA.Both hydrophobic pockets contain at least one type of the mentioned amino acids, which can transfer energy to a ligand if it is close to the fluorophore [23].
In Sudlow's site I, located in the IIA subdomain, one tryptophan (Trp-214) and one tyrosine (Tyr-263) residue can participate in drug binding.Although Trp-214 is the sole tryptophan in the HSA structure, it plays a vital role in ligand interactions.The IIIA subdomain includes three tyrosine residues (Tyr-401, Tyr-411, and Tyr-497), which can transfer energy to the acceptor [24].Additionally, Tyr-401 and Tyr-411 have been identified as amino acids that stabilize the binding of numerous ligands.To verify subdomain IIIA of human serum albumin as the specific binding site for LOS and GLP, quenching curves of HSA, gHSAGLC, and gHSAFRC excited at λex = 275 nm were compared with those excited at λex = 295 nm with the addition of LOS (Figure 3a-c) and GLP (Figure 3d-f) at increasing concentrations.
As mentioned earlier, protein fluorescence quenching occurs when the distance between the chromophores in the aromatic rings of the ligand's chemical structure and the fluorophores of albumin is less than 10 nm according to Stryer [25] and less than 7 nm according to Valeur [23].This proximity enables fluorescence resonance energy transfer (FRET) from a donor (fluorophore) to an acceptor (chromophore), leading to non-radiative, direct energy transfer to the drug molecule.The quenching curves of HSA ce studies, attention is directed towards specific protein regions conres.Using an excitation wavelength of 275 nm enables the observation n (Trp-214) and tyrosine (Tyr) residues, whereas a 295 nm wavelength s Trp-214 due to its unique spectral characteristics.The primary s are located within the IIA and IIIA subdomains of HSA.Both hydrontain at least one type of the mentioned amino acids, which can transfer if it is close to the fluorophore [23].site I, located in the IIA subdomain, one tryptophan (Trp-214) and one residue can participate in drug binding.Although Trp-214 is the sole HSA structure, it plays a vital role in ligand interactions.The IIIA subhree tyrosine residues (Tyr-401, Tyr-411, and Tyr-497), which can transacceptor [24].Additionally, Tyr-401 and Tyr-411 have been identified as stabilize the binding of numerous ligands.To verify subdomain IIIA of umin as the specific binding site for LOS and GLP, quenching curves of d gHSAFRC excited at λex = 275 nm were compared with those excited at the addition of LOS (Figure 3a-c) and GLP (Figure 3d-f) at increasing d earlier, protein fluorescence quenching occurs when the distance mophores in the aromatic rings of the ligand's chemical structure and of albumin is less than 10 nm according to Stryer [25] and less than 7 nm ur [23].This proximity enables fluorescence resonance energy transfer donor (fluorophore) to an acceptor (chromophore), leading to ect energy transfer to the drug molecule.The quenching curves of HSA In fluorescence studies, attention is directed towards specific protein regions containing fluorophores.Using an excitation wavelength of 275 nm enables the observation of both tryptophan (Trp-214) and tyrosine (Tyr) residues, whereas a 295 nm wavelength selectively excites Trp-214 due to its unique spectral characteristics.The primary drug-binding sites are located within the IIA and IIIA subdomains of HSA.Both hydrophobic pockets contain at least one type of the mentioned amino acids, which can transfer energy to a ligand if it is close to the fluorophore [23].
In Sudlow's site I, located in the IIA subdomain, one tryptophan (Trp-214) and one tyrosine (Tyr-263) residue can participate in drug binding.Although Trp-214 is the sole tryptophan in the HSA structure, it plays a vital role in ligand interactions.The IIIA subdomain includes three tyrosine residues (Tyr-401, Tyr-411, and Tyr-497), which can transfer energy to the acceptor [24].Additionally, Tyr-401 and Tyr-411 have been identified as amino acids that stabilize the binding of numerous ligands.To verify subdomain IIIA of human serum albumin as the specific binding site for LOS and GLP, quenching curves of HSA, gHSA GLC , and gHSA FRC excited at λ ex = 275 nm were compared with those excited at λ ex = 295 nm with the addition of LOS (Figure 3a-c) and GLP (Figure 3d-f) at increasing concentrations.
As mentioned earlier, protein fluorescence quenching occurs when the distance between the chromophores in the aromatic rings of the ligand's chemical structure and the fluorophores of albumin is less than 10 nm according to Stryer [25] and less than 7 nm according to Valeur [23].This proximity enables fluorescence resonance energy transfer (FRET) from a donor (fluorophore) to an acceptor (chromophore), leading to non-radiative, direct energy transfer to the drug molecule.The quenching curves of HSA (Figure 3a,d), gHSA GLC (Figure 3b,e), and gHSA FRC (Figure 3c,f) in the presence of both LOS and GLP at increasing concentrations (with molar ratios of ligand to albumin from 1:1 to 10:1 for LOS and 1:1 to 5:1 for GLP) indicate a decrease in fluorescence for non-glycated and glycated albumin at excitation wavelengths of 275 nm and 295 nm.This indicates effective energy transfer between the albumin fluorophores and ligands.After applying corrections for the inner filter effect, the observed fluorescence quenching of HSA, gHSA GLC , and gHSA FRC can be attributed to the formation of LOS-HSA, LOS-gHSA GLC , LOS-gHSA FRC , and GLP-HSA, GLP-gHSA GLC , GLP-gHSA FRC complexes.
Based on the data collected in Supplementary Materials Table S1, the percentage of non-glycated HSA fluorescence quenching, used as a control albumin, was nearly the same, reaching approximately 49.20 ± 0.32% and 45.17 ± 0.77% for LOS and 57.86 ± 0.38% and 51.46 ± 0.82% for GLP at λ ex = 275 nm and λ ex = 295 nm, respectively.For glycated albumin, weaker fluorescence quenching was observed in the presence of increasing ligand concentrations compared to the control sample, with the weakest quenching seen for albumin glycated by fructose (gHSA FRC ).LOS quenched the fluorescence of gHSA GLC by 41.67 ± 0.21% at λ ex = 275 nm and by 33.36 ± 0.19% at λ ex = 295 nm.The fluorescence of gHSA FRC decreased by 29.62 ± 0.39% at 275 nm and by 16.54 ± 0.24% at 295 nm for the same molar ratio of ligand to albumin (10:1).GLP quenched the fluorescence of gHSA GLC by 49.42 ± 0.45% at λ ex = 275 nm and by 37.41 ± 0.68% at λ ex = 295 nm, at a molar ratio GLP:albumin of 5:1.In contrast, the fluorescence of gHSA FRC at λ ex = 275 nm and λ ex = 295 nm decreased by 38.93 ± 0.54% and 21.46 ± 0.86%, respectively.Moreover, the data presented in Table S1 indicate that both LOS and GLP had a higher affinity towards non-glycated macromolecule than towards glycated proteins (gHSA GLC and gHSA FRC ).
The quenching curves of albumins excited at λ ex = 275 nm and λ ex = 295 nm in the presence of LOS (Figure 3a-c) and GLP (Figure 3d-f) at increasing drug concentrations do not overlap above the molar ratio LOS:HSA 2:1, LOS:gHSA GLC 1:1, LOS:gHSA FRC 1:1, and GLP:HSA 1.5:1, GLP:gHSA GLC 0.5:1.The exact course of the fluorescence quenching curves indicates that there was no difference in energy transfer between the tyrosyl residues and the ligand, suggesting that, initially, only the Trp-214 residue is likely involved in the interaction with LOS and GLP.This may allow the identification of subdomain IIA as a high-affinity binding site in the albumin structure.In contrast, a different trajectory of fluorescence quenching suggests that both Trp-214 residue in subdomain IIA and Tyr residues located in hydrophobic subdomains IB, IIB, IIIA, and IIIB are involved in the interaction with LOS and GLP in the binding site environment.As shown in Figure 3, the fluorescence quenching of HSA (Figure 3a,d), gHSA GLC (Figure 3b,e), and gHSA FRC (Figure 3c,f) by LOS and GLP was more pronounced when excited at λ ex = 275 nm compared to λ ex = 295 nm.This likely indicates a significant involvement not only of Trp-214 but also of Tyr residues in the interaction between the ligands and albumins.
The quenching curves of non-glycated and glycated albumin in the presence of LOS (Supplementary Materials Figure S1a,b) and GLP (Supplementary Materials Figure S1c,d) exhibit significant differences in their profiles.Specifically, these differences resulted from 7.53 ± 0.11% and 11.81 ± 0.58%, and 19.58 ± 0.09% and 28.63 ± 0.53% lower quenching of gHSA GLC and gHSA FRC by LOS relative to the LOS-HSA system at λ ex = 275 nm and λ ex = 295 nm, respectively.For the GLP-gHSA GLC and GLP-gHSA FRC systems, the differences in the quenching curve profiles amount to 8.44 ± 0.07% and 14.05 ± 0.14% for gHSA GLC , and 18.93 ± 0.16% and 30 ± 0.04% for gHSA FRC relative to the GLP-HSA system at λ ex = 275 nm and λ ex = 295 nm, respectively (Table S1).Several factors may explain these observed differences.Glycation induces conformational changes in the albumin structure, modifying the binding sites and overall protein flexibility and affecting the interaction with quenchers.Additionally, glycation alters the microenvironment around crucial amino acid residues, such as Trp-214 and Tyr, influencing their accessibility to quenchers.The binding affinity of glycated albumin for LOS and GLP may also differ due to sugar moieties, which can either hinder or facilitate quencher binding [26].Furthermore, steric hindrance from added sugar groups can reduce quenching efficiency, while new chemical interactions and potential protein aggregation further impact the quenching dynamics.These combined factors contributed to the distinct quenching behaviors observed for non-glycated and glycated albumin at different excitation wavelengths.
The influence of GLP on the LOS and LOS on the GLP affinity towards HSA, gHSA GLC , and gHSA FRC was studied by comparing the quenching curves of albumins in the presence of LOS in the binary LOS-HSA, LOS-gHSA GLC , LOS-gHSA FRC , and ternary complexes LOS-HSA-GLP const , LOS-gHSA GLC -GLP const , LOS-gHSA FRC -GLP const (Figure 4), and in the presence of GLP in the binary GLP-HSA, GLP-gHSA GLC , GLP-gHSA FRC , and ternary complexes GLP-HSA-LOS const , GLP-gHSA GLC -LOS const , and GLP-gHSA FRC -LOS const (Figure 5).The Supplementary Materials (Figures S3 and S4) present emission fluorescence spectra in the binary (ligand-albumin) and ternary (ligand-albumin-ligand const ) systems.Additionally, Figure S5 shows the quenching curves of HSA, gHSA GLC , and gHSA FRC in the presence of LOS and GLP at λ ex = 275 nm (Figure S5a,c) and at λ ex = 295 nm (Figure S5b,d cal interactions and potential protein aggregation further impact the quenching dynamics.These combined factors contributed to the distinct quenching behaviors observed for non-glycated and glycated albumin at different excitation wavelengths.The influence of GLP on the LOS and LOS on the GLP affinity towards HSA, gHSAGLC, and gHSAFRC was studied by comparing the quenching curves of albumins in the presence of LOS in the binary LOS-HSA, LOS-gHSAGLC, LOS-gHSAFRC, and ternary complexes LOS-HSA-GLPconst, LOS-gHSAGLC-GLPconst, LOS-gHSAFRC-GLPconst (Figure 4), and in the presence of GLP in the binary GLP-HSA, GLP-gHSAGLC, GLP-gHSAFRC, and ternary complexes GLP-HSA-LOSconst, GLP-gHSAGLC-LOSconst, and GLP-gHSAFRC-LOSconst (Figure 5).The Supplementary Materials (Figures S3 and S4) present emission fluorescence spectra in the binary (ligand-albumin) and ternary (ligand-albumin-ligandconst) systems.Additionally, Figure S5 shows the quenching curves of HSA, gHSAGLC, and gHSAFRC in the presence of LOS and GLP at λex = 275 nm (Figure S5a,c 4a), 6.64 ± 0.18% (Figure 4c), 16.69 ± 0.15% (Figure 4b), 10.04 ± 0.11% (Figure 4d), 11.76 ± 0.02% (Figure 5a), and 17.70 ± 0.21% (Figure 5b) compared to the systems with an additional ligand added to the binary system.An additional pharmaceutical in the system likely complicated the interaction between LOS-HSA and LOS-gHSA GLC (or GLP-HSA) or interfered with forming these complexes.GLP (or LOS) may cause the displacement of LOS (or GLP) from its complex with non-and glycated HSA.This effect may arise from competitive binding sites on albumin, steric hindrance, or conformational alterations to the macromolecule induced by the binding of the second ligand.Furthermore, the differing affinities and binding dynamics of GLP and LOS for albumin could result in preferential binding of one ligand over the other, thereby influencing the observed quenching effect.In contrast, the absence of differences in the quenching of intrinsic fluorescence of glycated albumin by LOS or GLP in the binary systems LOS-gHSA FRC (Figure 4e,f), GLP-gHSA GLC (Figure 5c,d), and GLP-gHSA FRC (Figure 5e,f) compared to the ternary systems LOS-gHSA FRC -GLP const (Figure 4e,f), GLP-gHSA GLC -LOS const (Figure 5c,d), and GLP-gHSA FRC -LOS const (Figure 5e,f) suggests that glycation, particularly glycation of albumin by fructose, alters the macromolecule's structure and/or binding characteristics.This modification prevents the additional drug-GLP in the LOS-gHSA FRC complex or LOS in the GLP-gHSA GLC and GLP-gHSA FRC complexes-from competing for the binding site with LOS and GLP and prevents the displacement of drugs already bound in the gHSA GLC and gHSA FRC molecules.
Additionally, the study on the LOS-HSA, LOS-gHSA GLC , GLP-HSA, and LOS-HSA-GLP const system revealed that an increase in drug concentration leads to a hypsochromic shift (∆λ max ) in the fluorescence emission band relative to the maximum emission of unbound albumin.This hypsochromic shift indicates an increase in the hydrophobic nature of the fluorophore environment due to drug interaction with albumin.It also suggests the possibility of hydrophobic interactions between the aromatic rings of LOS and GLP molecules and the aromatic rings of amino acid residues.The more pronounced hypsochromic shift upon excitation of albumin fluorescence at λ ex = 275 nm than at λ ex = 295 nm indicates a less polar environment, not only around Trp-214, but also around tyrosyl residues.The more substantial ∆λ max shift towards the blue in the double systems compared to the triple systems, i.e., in the LOS-HSA system compared to LOS-HSA-GLP const by 5 ± 1.5 nm, LOS-gHSA GLC compared to LOS-gHSA GLC -GLP const by 4 ± 1.5 nm, and GLP-HSA compared to GLP-HSA-GLP const by 6 ± 1.5 nm, and for non-modified compared to glycated albumin, may indicate a decrease in the hydrophobic nature of the environment of tryptophan or/and tyrosyl residues of albumin after glycation and in the presence of an additional drug in the drug-albumin system.
The dependence of F 0 F on the LOS or GLP concentration in the binary and ternary systems at λ ex = 275 nm (Figure 6) and λ ex = 295 nm (Supplementary Materials Figure S6) demonstrated a linear correlation for ligand-albumin complexes.The linear Stern-Volmer plots for the ligand-albumin and ligand-albumin-ligand const system may indicate a dynamic (collisional) or static quenching mechanism of fluorescence for both non-modified (HSA) and glycated albumins (gHSA GLC , gHSA FRC ).According to the literature, the ligand penetrates the macromolecule's environment in dynamic quenching, and fluorescence quenching is caused by the collision between the quencher molecule and the albumin fluorophore(s).In contrast, static quenching leads to a decrease in the intensity of the emitted fluorescence when the ligand binds to the fluorophore molecule in its ground (non-excited) state, thereby reducing the population of fluorophores capable of being excited [27][28][29].
Table 1 presents the Stern-Volmer constants K SV calculated for binary (ligand-albumin) and ternary (ligand-albumin-ligand const ) systems at λ ex = 275 nm and λ ex = 295 nm.The dependence of on the LOS or GLP concentration in the binary and ternary systems at λex = 275 nm (Figure 6) and λex = 295 nm (Supplementary Materials Figure S6) demonstrated a linear correlation for ligand-albumin complexes.The linear Stern-Volmer plots for the ligand-albumin and ligand-albumin-ligandconst system may indicate a dynamic (collisional) or static quenching mechanism of fluorescence for both non-modified (HSA) and glycated albumins (gHSAGLC, gHSAFRC).According to the literature, the ligand penetrates the macromolecule's environment in dynamic quenching, and fluorescence quenching is caused by the collision between the quencher molecule and the albumin fluorophore(s).In contrast, static quenching leads to a decrease in the intensity of the emitted fluorescence when the ligand binds to the fluorophore molecule in its ground (non-excited) state, thereby reducing the population of fluorophores capable of being excited [27][28][29].
Table 1 presents the Stern-Volmer constants  calculated for binary (ligandalbumin) and ternary (ligand-albumin-ligandconst) systems at λex = 275 nm and λex = 295 nm.The magnitude of the determined fluorescence quenching rate constants k q (ranging from 10 12 to 10 13 ; where k q = K SV τ 0 and τ 0 = 10 −9 s) for the investigated systems indicates a static fluorescence quenching mechanism in the LOS-albumin, LOS-albumin-GLP const and GLP-albumin, GLP-albumin-LOS const systems (Table 1).According to Lakowicz, for collisional fluorescence quenching, the maximum value of the constant k q in an aqueous solution is 2 × 10 10 (L•mol −1 •s −1 ) [29].The Stern-Volmer constant serves as a means to assess the accessibility of the quencher to the excited fluorophore.A higher K SV value indicates a greater availability of ligand molecules in the macromolecule, which leads to the formation of a complex in the excited state [30].Glycation of HSA by glucose and fructose results in a decrease in the Stern-Volmer constant in both binary (LOS-albumin, GLP-albumin) and ternary systems (LOSalbumin-GLP const ) at excitation wavelengths of 275 nm and 295 nm.This indicates a lower quenching efficiency of glycated albumin fluorophores compared to non-modified albumin by losartan (Table 1).In the ternary system (GLP-albumin-LOS const ), fructose-induced glycation decreased K SV , whereas glucose-induced glycation increased K SV (Table 1).The K SV values determined for the system in the presence of an additional drug (LOS-albumin-GLP const ) are lower than those for the LOS-albumin system, both for unmodified and glycated albumin at λ ex = 275 nm and λ ex = 295 nm.The K SV values determined for the system in the presence of an additional drug (GLP-albumin-LOS const complex) are also lower for HSA at λ ex = 275 nm and λ ex = 295 nm, but in contrast, for gHSA GLC and gHSA FRC , they remain unchanged at λ ex = 275 nm and λ ex = 295 nm (Table 1).At both excitation wavelengths (λ ex = 275 nm and λ ex = 295 nm), GLP complexes exhibited higher K SV values than LOS complexes, indicating a greater quenching efficiency.GLP molecules are closer to fluorophores of non-modified and glycated albumin than LOS molecules in binary and ternary complexes.It is also observed that K SV values are generally higher at λ ex = 275 nm compared to λ ex = 295 nm.
The nature of ligand binding to albumin (specificity of binding sites within the various classes of binding sites) was determined based on the binding isotherms (saturation curves) of LOS and GLP to non-glycated and glycated HSA in binary (Figure 7a,b) and ternary systems (Figure 7c,d).
For LOS-HSA, LOS-gHSA GLC , LOS-gHSA FRC (Figure 7a), GLP-HSA, GLP-gHSA GLC , GLP-gHSA FRC (Figure 7b), LOS-HSA-GLP const , LOS-gHSA GLC -GLP const , LOS-gHSA FRC -GLP const (Figure 7c), and GLP-HSA-LOS const , GLP-gHSA GLC -LOS const , GLP-gHSA FRC -LOS const (Figure 7d) complexes at λ ex = 275 nm (Figure 7) and at λ ex = 295 nm (Supplementary Materials Figure S7), the course of the saturation curves is not linear across the entire range of ligand concentrations, as each of the binding isotherms exhibits an exponentially increasing course and does not reach a "plateau".Therefore, based on the analyzed plots, it can be inferred that LOS and GLP nonspecifically interact with the hydrophobic fragments of the surface of non-glycated and glycated albumin in both binary and ternary systems and specifically saturate the binding sites within the protein molecule, as confirmed by the literature [31].Specific binding is characterized by high affinity and low binding capacity, whereas nonspecific binding is characterized by low affinity and unlimited ligand binding capacity of the ligand [31].The physicochemical compatibility of both molecules determines the binding of ligands to serum albumin.Small structural changes in the protein molecule can influence the mutual interaction of the drug with albumin, which in turn affects the binding parameters.For LOS-HSA, LOS-gHSAGLC, LOS-gHSAFRC (Figure 7a), GLP-HSA, GLP-gHSAGLC, GLP-gHSAFRC (Figure 7b), LOS-HSA-GLPconst, LOS-gHSAGLC-GLPconst, LOS-gHSAFRC-GLPconst (Figure 7c), and GLP-HSA-LOSconst, GLP-gHSAGLC-LOSconst, GLP-gHSAFRC-LOSconst (Figure 7d) complexes at λex = 275 nm (Figure 7) and at λex = 295 nm (Supplementary Materials Figure S7), the course of the saturation curves is not linear across the entire range of ligand concentrations, as each of the binding isotherms exhibits an exponentially increasing course and does not reach a "plateau".Therefore, based on the analyzed plots, it can be inferred that LOS and GLP nonspecifically interact with the hydrophobic fragments of the surface of non-glycated and glycated albumin in both binary and ternary systems and specifically saturate the binding sites within the protein molecule, as confirmed by the literature [31].Specific binding is characterized by high affinity and low binding capacity, whereas nonspecific binding is characterized by low affinity and unlimited ligand binding capacity of the ligand [31].The physicochemical compatibility of both molecules determines the binding of ligands to serum albumin.Small structural changes in the protein molecule can influence the mutual interaction of the drug with albumin, which in turn affects the binding parameters.
Specific binding of losartan and glipizide to non-modified and glycated albumin in the complexes LOS-HSA, LOS-gHSAGLC, LOS-gHSAFRC, GLP-HSA, GLP-gHSAGLC, GLP-gHSAFRC, LOS-HSA-GLPconst, LOS-gHSAGLC-GLPconst, LOS-gHSAFRC-GLPconst and GLP-HSA-LOSconst, GLP-gHSAGLC-LOSconst, GLP-gHSAFRC-LOSconst were quantitatively analyzed by calculating the association constant  using the Scatchard equation (with ) 0.0 Specific binding of losartan and glipizide to non-modified and glycated albumin in the complexes LOS-HSA, LOS-gHSA GLC , LOS-gHSA FRC , GLP-HSA, GLP-gHSA GLC , GLP-gHSA FRC , LOS-HSA-GLP const , LOS-gHSA GLC -GLP const , LOS-gHSA FRC -GLP const and GLP-HSA-LOS const , GLP-gHSA GLC -LOS const , GLP-gHSA FRC -LOS const were quantitatively analyzed by calculating the association constant K a using the Scatchard equation (with the ligand-bound fraction concentration as the independent variable) (Figure 8), the Klotz equation (with the inverse of the free ligand fraction concentration as the independent variable) (Figure 9), and through non-linear regression based on the Levenberg-Marquardt algorithm, i.e., binding isotherms (Figure 7).Additionally, Hill's coefficients (n H ), repre- senting cooperativity, were determined using the linear Hill plot (with the logarithm of the free ligand fraction concentration as the independent variable) (Figure 10).Changes in the high-affinity binding of LOS and GLP to non-glycated and glycated albumin in binary and ternary systems, based on K a , the number of LOS and GLP molecules bound to one mole of the macromolecule at a specific binding site (n), as well as Hill's coefficient of cooperativity, are summarized in Tables 2 and 3, respectively (λ ex = 275 nm, λ ex = 295 nm).
logarithm of the free ligand fraction concentration as the independent variable) (Figure 10).Changes in the high-affinity binding of LOS and GLP to non-glycated and glycated albumin in binary and ternary systems, based on  , the number of LOS and GLP molecules bound to one mole of the macromolecule at a specific binding site (), as well as Hill's coefficient of cooperativity, are summarized in Tables 2 and 3, respectively (λex = 275 nm, λex = 295 nm).

Scatchard Method
Klotz Method Hill Method  The Scatchard model of ligand-protein interactions postulates that the protein molecule possesses a finite number of specific binding sites for ligands.In this case, the Scatchard dependence r [L f ] = f (r) is linear and intersects the x-axis of the coordinate system (the r-axis).The linear Scatchard plots for the complexes LOS-HSA, LOS-gHSA GLC , LOS-gHSA FRC (Figure 8a), GLP-gHSA GLC , GLP-gHSA FRC (Figure 8b), LOS-HSA-GLP const , LOS-gHSA GLC -GLP const , LOS-gHSA FRC -GLP const (Figure 8c) and GLP-HSA-LOS const , GLP-gHSA GLC -LOS const , GLP-gHSA FRC -LOS const (Figure 8d) at λ ex = 275 nm (Figure 8) and λ ex = 295 nm (Supplementary Materials Figure S8) indicate the existence of one class of equivalent, independent binding sites for LOS and GLP in both non-modified and glycated albumin structures (or a single binding site), characterized by the same association constant K a .A non-linear Scatchard dependence resembling a hyperbola was observed for the GLP-HSA complex excited at λ ex = 275 nm (Figure 8b) and λ ex = 295 nm (Supplementary Materials Figure S8b).This phenomenon may have resulted from the presence of more than one class of ligand-binding sites within the albumin structure (heterogeneous binding), the non-specific nature of GLP binding to HSA, and/or negative cooperativity, where the binding of the drug at one site reduces its affinity for the remaining binding sites on the macromolecule.In contrast, for the LOS-gHSA FRC -GLP const complex excited at λ ex = 295 nm (Supplementary Materials Figure S8c), a "cone-shaped" Scatchard plot was obtained, which may indicate positive cooperativity or instability of LOS.Assuming the existence of two classes of binding sites, the binding parameters for GLP-HSA and LOS-gHSA FRC -GLP const were determined by non-linear regression using the Levenberg-Marquardt algorithm (Tables 2 and 3).
Figure 9 illustrates the linear course of the Klotz dependence 1 ) for the binary (Figure 9a,b) and ternary systems (Figure 9c,d) at λ ex = 275 nm, indicating the binding of ligands to albumins within a single class of binding sites.Notably, for the system LOS-gHSA FRC -GLP const at λ ex = 295 nm, a non-linear course of the Klotz dependence was observed (Supplementary Materials Figure S9).
In the first class of binding sites, the association constants K a determined from the linear Scatchard and Klotz dependencies for the LOS-gHSA GLC and LOS-gHSA FRC complexes when excited at λ ex = 275 nm and λ ex = 295 nm were lower than those for the LOS-HSA complex (Table 2).A comparable trend was noted in the ternary system, where the K a was lower for the complexes with glycated (LOS-gHSA GLC -GLP const , LOS-gHSA FRC -GLP const ) compared to non-modified albumin (LOS-HSA-GLP const ).This suggests that albumin glycation reduced the stability of the formed complex for both the excited Trp-214 and the Tyr residues.Losartan has the lowest affinity for fructose-glycated protein (gHSA FRC ).Moreover, the presence of an additional drug, i.e., GLP, in the LOS-albumin system weakens the binding of LOS to the macromolecules, as evidenced by a decrease in K a (Table 2).For the LOS-gHSA FRC -GLP const complex excited at λ ex = 295 nm, the non-linear course of the Klotz plot made it impossible to determine the binding parameters (Figure S9c).
The K a constants, determined by linear regression from the dependencies in the Klotz and the Scatchard equations, as well as based on binding isotherms, were significantly higher for the GLP-HSA compared to the GLP-gHSA GLC and GLP-gHSA FRC complexes (Table 3).This suggests that GLP has a greater affinity for non-modified than glycated albumin, forming a more stable complex.These findings align with previous studies by Koyama et al. (1997) [32], which used fluorescence quenching techniques to demonstrate that the binding capacity of hypoglycemic drugs to glycated albumin (G-HSA) was significantly lower than to non-modified HSA.Moreover, Wiglusz et al. (2021) [33] demonstrated that gliclazide, a popular hypoglycemic drug, binds more weakly to glycated albumin than its native form.These results confirm that glycation alters protein structure and drug-binding capacity.Furthermore, Chume et al. (2019) [34] confirmed that glycation of albumin decreases its binding capacity for hypoglycemic drugs, which has profound implications for the pharmacokinetics and pharmacodynamics of these drugs in diabetic patients.Conversely, for the ternary system, a higher K a was determined for GLP-gHSA GLC -LOS const compared to GLP-HSA-LOS const and GLP-gHSA FRC -LOS const , indicating that glucose glycation increased the stability of the formed GLP-albumin-LOS const complex.Furthermore, the presence of LOS in the GLP-albumin complex generally weakened the binding of GLP to HSA and gHSA FRC , as reflected in a decrease in K a (Table 3).However, the presence of LOS did not significantly affect the K a value in the complex with albumin glycated by GLC at λ ex = 275 nm (K a (GLP-gHSA GLC ) ≈ K a (GLP-gHSA GLC -LOS const )).
In addition, glipizide at a 5:1 GLP:albumin molar ratio had a higher affinity for nonglycated and glycated protein than losartan at a 10:1 LOS:albumin molar ratio.This effect indicates that the transfer of energy from albumin fluorophores (Trp-214 and Tyr residues) to GLP was more efficient than to LOS in both binary (ligand-albumin) and ternary (in the presence of an additional drug at a 1:1 molar ratio, ligand-albumin-ligand const ) complexes.
The number of binding sites n close to one indicates the existence of a single specific binding site for LOS and GLP in non-modified and glycated molecules (Tables 2 and 3).
To determine whether the binding of LOS and GLP to albumins affects the affinity of the ligand for other binding sites within the macromolecule, Hill's coefficient (n H ) for cooperativity was calculated based on the linear Hill plot. Figure 10 shows the linear Hill dependence log r 1−r = f log L f in the binary LOS-HSA, LOS-gHSA GLC , LOS-gHSA FRC (Figure 10a), GLP-HSA, GLP-gHSA GLC , GLP-gHSA FRC (Figure 10b), as well as the ternary systems LOS-HSA-GLP const , LOS-gHSA GLC -GLP const , LOS-gHSA FRC -GLP const (Figure 10c), and GLP-HSA-LOS const , GLP-gHSA GLC -LOS const , GLP-gHSA FRC -LOS const (Figure 10d) at λ ex = 275 nm.Supplementary Materials, Figure S10, presents Hill plots for the binary and ternary systems at λ ex = 295 nm.
For the LOS-HSA (Table 2) and GLP-HSA complexes (Table 3), the Hill coefficient values were found to be less than one (n H < 1) at both excitation wavelengths, indicating negative cooperativity.This implies that binding one LOS or GLP molecule at a binding site decreases the affinity for subsequent ligand binding at other sites on non-glycated albumin [35].Conversely, for the LOS-gHSA FRC -GLP const complex (Table 2), a Hill coefficient greater than one was observed (n H > 1), suggesting positive cooperativity.Here, the binding of one LOS molecule facilitated the binding affinity of additional molecules within the LOS-gHSA FRC -GLP const complex when excited at 295 nm.For the remaining systems, the Hill coefficient was approximately one (n H ≈ 1), indicating non-cooperative binding of LOS and GLP to the macromolecules, where the binding of one ligand molecule does not affect the binding affinity of subsequent molecules.

Investigating Ligand Interactions: Spectrophotometric Analysis of Losartan and Glipizide
There are numerous possibilities for mutual interactions between different pharmaceuticals.Some of these interactions are well-documented and thus can be easily avoided during treatment.However, other medicine interactions are often uncovered only after investigating the underlying causes of treatment failure.Understanding these interactions is crucial, as it can significantly optimize therapeutic outcomes, potentially leading to better patient care and minimizing adverse effects.In this study, in addition to spectrofluorimetry, spectrophotometric measurements were conducted to verify the hypothesis that GLP and LOS can interact, not only with non-glycated (HSA) and glycated albumin (gHSA GLC , gHSA FRC ) (as discussed in Section 2.2), but also with each other.
Figure 11 shows the absorption spectra of GLP, LOS, and the drug complex (GLP + LOS), from which the absorbance values at selected wavelengths were read.The results are presented in Table 4.There are numerous possibilities for mutual interactions between different pharmaceuticals.Some of these interactions are well-documented and thus can be easily avoided during treatment.However, other medicine interactions are often uncovered only after investigating the underlying causes of treatment failure.Understanding these interactions is crucial, as it can significantly optimize therapeutic outcomes, potentially leading to better patient care and minimizing adverse effects.In this study, in addition to spectrofluorimetry, spectrophotometric measurements were conducted to verify the hypothesis that GLP and LOS can interact, not only with non-glycated (HSA) and glycated albumin (gHSAGLC, gHSAFRC) (as discussed in Section 2.2), but also with each other.
Figure 11 shows the absorption spectra of GLP, LOS, and the drug complex (GLP + LOS), from which the absorbance values at selected wavelengths were read.The results are presented in Table 4.If the absorbance of a mixture of two substances deviates from the mathematical sum of their individual absorbances, this may indicate potential mutual interactions, as suggested by Ren et al. (2019) [36].By observing differences in the absorbance values of GLP and LOS in their combined mixture (GLP + LOS), compared to the expected mathematical sum of their absorbances (Figure 11, Table 4), it can be inferred that GLP and LOS interact with each other.These interactions emphasize the importance of closely monitoring patients co-administered LOS and GLP, mainly focusing on blood glucose levels, blood pressure, and renal function, to ensure safe and effective therapy.

Spectropolarimetric Analysis of Glycation and Losartan Influence on Macromolecule Secondary Structure
Circular dichroism (CD) spectroscopy was used to evaluate the impact of the glycation process and the presence losartan (LOS) on the secondary structure of human serum albumin (Figure 12).CD spectroscopy is a valuable analytical technique for assessing the secondary structure of chiral molecules, particularly proteins.It can monitor conformational protein changes, such as folding/unfolding, ligand binding, and protein-protein interactions [37].CD measurements in the far-UV region can provide quantitative assessments of secondary structure, which can be compared to findings from X-ray crystallography or NMR studies [38].

Spectropolarimetric Analysis of Glycation and Losartan Influence on Macromolecule Secondary Structure
Circular dichroism (CD) spectroscopy was used to evaluate the impact of the glycation process and the presence of losartan (LOS) on the secondary structure of human serum albumin (Figure 12).CD spectroscopy is a valuable analytical technique for assessing the secondary structure of chiral molecules, particularly proteins.It can monitor conformational protein changes, such as folding/unfolding, ligand binding, and proteinprotein interactions [37].CD measurements in the far-UV region can provide quantitative assessments of secondary structure, which can be compared to findings from X-ray crystallography or NMR studies [38].
Tables 5 and 6 present the value of non-glycated and glycated albumin mean residue ellipticity [θ] and the percentage content (%) of albumin secondary structure elements in the sample without (HSA, gHSAGLC, gHSAFRC) and in the presence of losartan at molar ratio of LOS:albumin 10:1 (LOS-HSA, LOS-gHSAGLC, LOS-gHSAFRC).Tables 5 and 6 present the value of non-glycated and glycated albumin mean residue ellipticity [θ] mrw and the percentage content (%) of albumin secondary structure elements in the sample without (HSA, gHSA GLC , gHSA FRC ) and in the presence of losartan at molar ratio of LOS:albumin 10:1 (LOS-HSA, LOS-gHSA GLC , LOS-gHSA FRC ).According to the data presented in Tables 5 and 6, it can be inferred that both nonglycated and glycated albumin primarily display an α-helical structure.The CD spectrum of HSA is characterized by a strong double minimum at λ min = 209.2nm and λ min = 221.2nm, which shifts slightly towards shorter wavelengths due to albumin glycation (Figure 12a, Table 5).Absorption in the region below λ = 240 nm is primarily attributed to the peptide bond, featuring a weak but broad n → π* transition centered around λ = 220 nm and a more intense π → π* transition near λ = 190 nm [38].The reduction in mean residue ellipticity [θ] mrw and CD band intensity observed, especially in glycated gHSA FRC compared to non- glycated HSA (Figure 12a, Table 5), may indicate a decrease in α-helix content (gHSA FRC 16.65 ± 0.16%) and an increase in the β-sheet content of albumin after glycation by fructose (gHSA FRC 6.55 ± 0.21%) (Table 6).Mou et al. (2022) observed significant changes in the secondary structure of bovine serum albumin (BSA) after glycation by ribose (RBSA).They indicated that an increase in β-sheet content in RBSA is pivotal for protein aggregation [39].On the other hand, some authors have proposed that glycation may induce aggregation, not by unfolding, but through overall stabilization of the macromolecule, enhancing protein stability and prolonging its lifespan [40].
The formation of LOS-albumin complexes did not substantially impact the wavelength at which the spectrum reached its minimum or the mean residue ellipticity values (Table 5).As demonstrated in Table 6, the binding of LOS to HSA and gHSA GLC did not lead to significant alterations in the macromolecule's secondary structure beyond 2%.Similarly, as in this work, Żurawska-Płaksej et al. (2018) observed only slight changes in the secondary structure of non-glycated and glycated BSA upon binding with gliclazide (GLICL).The authors emphasized that the decrease in α-helix content indicated a certain degree of structural unfolding, suggesting that GLICL is a weak modifier of BSA's secondary structure [41].However, in the presented study, the α-helix content of gHSA FRC increased from 20.00 ± 0.42% to 29.90 ± 0.14% upon interaction with LOS (Table 6).This result may indicate that LOS stabilizes the secondary structure of the protein glycated by fructose.This stabilization could result from forming new hydrogen bonds or other interactions between the ligand and albumin, leading to more significant structural organization and increased α-helix content.Based on the DSSP (Dictionary of Secondary Structure of Proteins) method, Moeinpour et al. (2016) observed that LOS subtly impacts hydrogen bonds and, consequently, the secondary structure of HSA [42].It should be emphasized that studies using CD spectroscopy to investigate the secondary structure of proteins after glycation are inconclusive.Some researchers noted glucose-concentration-dependent changes in CD spectra, while others observed partial denaturation and structural disintegration [43].The results presented in this study indicate that various sugars, as glycation inducers, can affect the secondary structure of human serum albumin differently.

Sample Preparation
HSA Glycation: Before the in vitro glycation of human serum albumin (HSA), all glassware and spatulas were sterilized to prevent bacterial contamination.Glycated albumins (gHSA GLC and gHSA FRC ) were prepared by exposing HSA solutions (5 × 10 −6 mol•L −1 ) to D(+)-glucose (GLC) (0.05 mol•L −1 ) and D(-)-fructose (FRC) (0.05 mol•L −1 ) in a phosphate buffer (0.05 mol•L −1 ) preserved with sodium azide (0.015 mol•L −1 ) for 21 days incubation at a temperature of t = 37 • C. A control sample (HSA) was prepared in the same manner, but without the addition of reducing sugars.After the incubation period, the non-glycated HSA and glycated albumins were extensively dialyzed against phosphate buffer (pH = 7.4 ± 0.1) for 24 h to remove the excess unbound GLC and FRC, then passed through a sterile Millex-GP syringe filter with 0.2 µm pores.The pH of the buffer solution, HSA, gHSA GLC , and gHSA FRC was confirmed using a pH meter (FEP20 Mettler Toledo, Columbus, Ohio, USA).The absorbance ratio of HSA, gHSA GLC , and gHSA FRC at λ = 255 nm and λ = 280 nm was less than 0.5, indicating the purity of the prepared albumin solutions.
Investigating Ligand Interactions-Spectrophotometric Analysis of LOS and GLP: The solutions of LOS and GLP at a concentration of 2.5 × 10 −5 mol•L −1 , as well as the drug mixture (GLP + LOS; GLP:LOS molar ratio 1:1), were prepared in phosphate buffer (pH = 7.4 ± 0.1, 0.05 mol•L −1 ), by diluting the stock solutions of the drugs (2.5 × 10 −3 mol•L −1 ).Spectropolarimetric Analysis of Glycation and LOS Influence on Macromolecule Secondary Structure: In this study, the samples (HSA, gHSA GLC , and gHSA FRC at a concentration of 5 × 10 −6 mol•L −1 each, as well as the LOS-HSA, LOS-gHSA GLC , LOS-gHSA FRC complexes, where the molar ratio of LOS to albumin was 10:1) were prepared according to the procedure described above.
Due to the inner filter effect resulting from the presence of the drugs, the recorded fluorescence of HSA, gHSA GLC , and gHSA FRC in the binary and ternary systems was corrected using Equation (1) [44].This equation is applicable provided that the increase in absorbance within the system does not exceed approximately 0.3.
where F cor and F obs are the corrected and observed fluorescence intensities, respectively; and Abs ex and Abs em are the absorbance at the excitation and emission wavelengths, respectively.
The samples' absorbance measurements were recorded using a model V-760 JASCO spectrophotometer (Easton, MD, USA) equipped with a thermostat bath, using quartz cuvettes of dimensions 1.0 cm × 1.0 cm × 4.0 cm.The apparatus has a wavelength correction error of ±0.3 nm and a photometric correction error of ±0.002 Abs at 0.5 Abs.
The absorption spectra of GLP and LOS at a concentration of 2.5 × 10 −5 mol•L −1 , as well as the GLP + LOS system at GLP:LOS 1:1 (v/v) molar ratio, were determined in the wavelength range of λ = 235-300 nm at t = 37 • C.
Far-UV CD spectra of HSA, gHSA GLC , gHSA FRC , and LOS-HSA, LOS-gHSA GLC , and LOS-gHSA FRC complexes were recorded using a JASCO model J-1500 CD spectropolarimeter (Hachioji, Tokyo, Japan) equipped with a thermostatic Peltier cell holder (accuracy t = ±0.05• C).All spectra were measured in a 0.1 cm path length quartz cuvette and scanned from λ = 200 to 250 nm at wavelength intervals of 0.2 nm, with a bandwidth set at 2.0 nm and a D.I.T. of 4 s.CD intensity was expressed as mean residue ellipticity at wavelength λ ([θ] mrw ) (deg•cm 2 •dmol −1 ) according to Equation (2) [38]: where MRW is the mean residue weight (MRW HSA = 113.7 Da); θ λ is the observed ellipticity at wavelength λ (deg); d is the optical path length (cm); c is the protein concentration (g•cm −3 ).The content of the samples' secondary structure elements was calculated using the Secondary Structure Estimation program (version 2.13.00) with Yang's reference model.
The fluorescence, absorption, and Far-UV CD spectra presented in this study were corrected by smoothing using the Savitzky and Golay method with a convolution width of 15, using the Spectra Analysis program (version 1.53.07,JASCO, Easton, MD, USA).
Based on the calculated fluorescence emission intensities, the quenching curves ( F F 0 vs. drug:albumin molar ratio, where F, F 0 are the fluorescence intensities at the maximum wavelength in the presence and absence of the quencher, respectively) of non-glycated and glycated human serum albumin in the presence of losartan (LOS) or glipizide (GLP) (binary system: LOS-HSA, LOS-gHSA GLC , LOS-gHSA FRC and GLP-HSA, GLP-gHSA GLC , GLP-gHSA FRC ) or in the presence of both drugs (ternary system: LOS-HSA-GLP const , LOS-gHSA GLC -GLP const , LOS-gHSA FRC -GLP const and GLP-HSA-LOS const , GLP-gHSA GLC -LOS const , GLP-gHSA FRC -LOS const ) were plotted.The fluorescence quenching mechanism (static and/or dynamic) of HSA, gHSA GLC , and gHSA FRC in the absence or presence of LOS and GLP in the binary and ternary systems was analyzed based on the Stern-Volmer equation (Equation ( 3)) [29]: where k q is the bimolecular quenching rate constant (L•mol −1 •s −1 ), k q = K SV τ 0 ; τ 0 is the average fluorescence lifetime of albumin without a quencher, τ 0 = 6.0 × 10 −9 s [28]; K SV is the Stern-Volmer constant (L•mol −1 ); [L] is the ligand concentration (mol•L −1 ); where [L b ] and [L f ] are the bound and unbound (free) drug concentra- tions, respectively.
The association constant (K a ) and the number of binding sites classes (n) in the ligand- albumin or ligand-albumin-ligand const complexes were determined using the Scatchard (Equation ( 4)) [45] and Klotz (Equation ( 5)) equations [46]: ), and ∆F is the difference between F 0 and F.
For two classes of binding sites in albumin structure (in this study, GLP-HSA and LOS-gHSA FRC -GLP const complexes), the binding isotherms were drawn using non-linear regression analysis according to Equation (6), and the association constants (K a1 , K a2 ) and the number of binding sites (n 1 , n 2 ) were calculated [47]: Hill's coefficient n H was determined on the basis of the Hill method (Equation ( 6)) [35]:

Statistics
The experiments were conducted in triplicate, and the results are presented as the mean ± relative standard deviation (mean ± RSD).Linear regression analysis (R 2 ) was performed by fitting the experimental data to the appropriate equation using OriginPro version 8.5 SR1 (Northampton, MA, USA).

Conclusions
The main goal of this project was to analyze the interactions of losartan (LOS) and glipizide (GLP) with non-glycated (HSA) and glycated human serum albumin (gHSA GLC and gHSA FRC ) and to investigate their mutual competition in the binding sites of the macromolecule using multiple spectroscopic techniques.
The analysis of the emission fluorescence spectra of glycation products (AGEs) indicated that fructose (FRC) is a more effective glycation agent for HSA than glucose (GLC).This conclusion is supported by the observation that glycation in the presence of FRC leads to a faster and more efficient Amadori rearrangement.Glycation induces conformational changes in the albumin structure, particularly altering the microenvironment around crucial amino acid residues (Trp-214 and Tyr), thus influencing their accessibility to quenchers.The results showed that LOS and GLP had a higher affinity for non-glycated HSA than glycated gHSA GLC and gHSA FRC , forming a more stable complex.Additionally, LOS and GLP were found to interact nonspecifically with the hydrophobic fragments of the surface of albumins in binary (ligand-albumin) and ternary systems (ligand-albumin-ligand const ), and to specifically saturate the binding sites within the protein molecule.An additional drug (GLP in the LOS-albumin complex or LOS in the GLP-albumin complex) complicated the interaction, likely leading to competitive binding or displacement of the initially bound drug in both non-and glycated albumins.Analysis of the UV spectra revealed that GLP and LOS interact with each other.This finding emphasizes the importance of closely monitoring patients co-administered these drugs, focusing on blood glucose levels, blood pressure, and renal function, to ensure safe and effective therapy.
In conclusion, the significant impact of glycation on the drug-binding capacity of albumin, as revealed by this study, has profound implications for patient care.Particularly for diabetic patients, the altered binding properties of glycated albumin could potentially compromise the efficacy and safety of drug therapy.Therefore, the need for careful monitoring, and potentially adjusted dosing regimens, is urgent, to ensure optimal therapeutic outcomes.Further clinical studies are imperative, to develop comprehensive guidelines for the concurrent use of LOS and GLP in polypharmacotherapy.

10 *
Relative standard deviation;(a) determined by non-linear regression using binding isotherms;(b)  impossible to determine.
) where r = [L b ] [P] is the number of ligand moles bound to one mole of protein; [P] is the total protein concentration (mol•L −1 ); [L b ] = ∆F ∆F max •[P], ∆F max is maximal fluorescence change with complete saturation (evaluated from the linear part of the 1 ∆F vs. 1 [L] ), respectively.

Table 4 .
The values of maximum absorbance of GLP, LOS, and drug mixture at the selected wavelengths λ = 235 nm, λ = 253 nm, and λ = 282 nm.

Table 6 .
The percentage content (%) of non-glycated and glycated albumin secondary structure elements in the sample without (HSA, gHSA GLC , gHSA FRC ) and in the presence of losartan (LOS-HSA, LOS-gHSA GLC , LOS-gHSA FRC ) based on Yang's reference model.