Impact of Obesity on Hepatic Drug Clearance: What are the Influential Variables?

Drug clearance in obese subjects varies widely among different drugs and across subjects with different severity of obesity. This study investigates correlations between plasma clearance (CLp) and drug- and patient-related characteristics in obese subjects, and evaluates the systematic accuracy of common weight-based dosing methods. A physiologically-based pharmacokinetic (PBPK) modeling approach that uses recent information on obesity-related changes in physiology was used to simulate CLp for a normal-weight subject (body mass index [BMI] = 20) and subjects with various severities of obesity (BMI 25-60) for hypothetical hepatically cleared drugs with a wide range of properties. Influential variables for CLp change were investigated. For each drug and obese subject, the exponent that yields perfect allometric scaling of CLp from normal-weight subjects was assessed. Among all variables, BMI and relative changes in enzyme activity resulting from obesity proved highly correlated with obesity-related CLp changes. Drugs bound to α1-acid glycoprotein (AAG) had lower CLp changes compared to drugs bound to human serum albumin (HSA). Lower extraction ratios (ER) corresponded to higher CLp changes compared to higher ER. The allometric exponent for perfect scaling ranged from -3.84 to 3.34 illustrating that none of the scaling methods performed well in all situations. While all three dosing methods are generally systematically accurate for drugs with unchanged or up to 50% increased enzyme activity in subjects with a BMI below 30 kg/m2, in any of the other cases, information on the different drug properties and severity of obesity is required to select an appropriate dosing method for individuals with obesity.

In Search for Low-Molecular-Weight Ligands of Human Serum Albumin That Affect Its Affinity for Monomeric Amyloid ß Peptide.

An imbalance between production and excretion of amyloid ß peptide (Aß) in the brain tissues of Alzheimer's disease (AD) patients leads to Aß accumulation and the formation of noxious Aß oligomers/plaques. A promising approach to AD prevention is the reduction of free Aß levels by directed enhancement of Aß binding to its natural depot, human serum albumin (HSA). We previously demonstrated the ability of specific low-molecular-weight ligands (LMWLs) in HSA to improve its affinity for Aß. Here we develop this approach through a bioinformatic search for the clinically approved AD-related LMWLs in HSA, followed by classification of the candidates according to the predicted location of their binding sites on the HSA surface, ranking of the candidates, and selective experimental validation of their impact on HSA affinity for Aß. The top 100 candidate LMWLs were classified into five clusters. The specific representatives of the different clusters exhibit dramatically different behavior, with 3- to 13-fold changes in equilibrium dissociation constants for the HSA-Aß40 interaction: prednisone favors HSA-Aß interaction, mefenamic acid shows the opposite effect, and levothyroxine exhibits bidirectional effects. Overall, the LMWLs in HSA chosen here provide a basis for drug repurposing for AD prevention, and for the search of medications promoting AD progression.

Supramolecular prodrug of SN38 based on endogenous albumin and SN38 prodrug modified with semaglutide side chain to improve the tumor distribution.

To improve the biodistribution of the drug in the tumor, a supramolecular prodrug of SN38 was fabricated in situ between endogenous albumin and SN38 prodrug modified with semaglutide side chain. Firstly, SN38 was conjugated with semaglutide side chain and octadecanedioic acid via glycine linkers to obtain SI-Gly-SN38 and OA-Gly-SN38 prodrugs, respectively. Both SI-Gly-SN38 and OA-Gly-SN38 exhibited excellent stability in PBS for over 24 h. Due to the strong binding affinity of the semaglutide side chain with albumin, the plasma half-life of SI-Gly-SN38 was 2.7 times higher than that of OA-Gly-SN38. Furthermore, with addition of HSA, the fluorescence intensity of SI-Gly-SN38 was 4 times higher than that of OA-Gly-SN38, confirming its strong binding capability with HSA. MTT assay showed that the cytotoxicity of SI-Gly-SN38 and OA-Gly-SN38 was higher than that of Irinotecan. Even incubated with HSA, the SI-Gly-SN38 and OA-Gly-SN38 still maintained high cytotoxicity, indicating minimal influence of HSA on their cytotoxicity. In vivo pharmacokinetic studies demonstrated that the circulation half-life of SI-Gly-SN38 was twice that of OA-Gly-SN38. SI-Gly-SN38 exhibited significantly reduced accumulation in the lungs, being only 0.23 times that of OA-Gly-SN38. The release of free SN38 in the lungs from SI-Gly-SN38 was only 0.4 times that from OA-Gly-SN38 and Irinotecan. The SI-Gly-SN38 showed the highest accumulation in tumors. The tumor inhibition rate of SI-Gly-SN38 was 6.42% higher than that of OA-Gly-SN38, and 8.67% higher than that of Irinotecan, respectively. These results indicate that the supramolecular prodrug delivery system can be constructed between SI-Gly-SN38 and endogenous albumin, which improves drug biodistribution in vivo, enhances tumor accumulation, and plays a crucial role in tumor growth inhibition.

Protein coupled thionine acetate probed silica nanoparticles: An integrated laser-assisted therapeutic approach for treating cancer.

Herein, we report a multifaceted nanoformulation, developed by binding thionine acetate (TA) in silica matrix to form TA loaded silica nanoparticles (STA Nps), which were characterized using various physicochemical techniques. STA NPs were spherical shaped having size 40-50 nm and exhibited good heating efficiency, improved photostability and singlet oxygen production rate than TA alone. In PDT experiment, the rate of degradation for ABDMA was enhanced from 0.1367 min-1 for TA alone to 0.1774 min-1 for STA Nps, depicting an increase in the reactive oxygen species (ROS) generation ability of STA Nps. Further, the cytotoxicity of STA Nps was investigated by carrying out the biophysical studies with Calf thymus DNA (Ct-DNA) and Human Serum Albumin (HSA). The results indicated that the binding of STA Nps to Ct-DNA causes alterations in the double helix structure of DNA and as a result, STA Nps can impart chemotherapeutic effects via targeting DNA. STA Nps showed good binding affinity with HSA without compromising the structure of HSA, which is important for STA Nps sustainable biodistribution and pharmacokinetics. Based on this study, it is suggested that because of the synergistic effect of chemo and phototherapy, STA Nps can be extensively utilized as potential candidates for treating cancer.

Circular dichroism spectrum of (R)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol in albumin: Alterations caused by complexation-Experimental and in silico investigation.

This study describes the interaction of human serum albumin (HSA) with the binol derivative (R)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (R-BrB), which has its optical activity based on the prohibitive energetic barrier for conversion into the enantiomer (S)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (S-BrB). The objective was to assess the ability of HSA to differentiate axial enantiomers based on their binding efficiency and their impact on the CD spectra. We discovered that both enantiomers were effective ligands, and the CD signal disappeared when equimolar amounts of R-BrB and S-BrB were simultaneously added, indicating no preference for either enantiomer. The complexation resulted in a significant signal increase at 250 nm and a bathochromic effect at 370 nm. Molecular docking simulations were performed, and the lower energy pose of R-BrB was selected for DFT calculations. The theoretical CD spectra of free and complexed R-BrB were obtained and showed alterations corroborating the experimental results. By comparing the difference spectrum (HSA:R-BrB minus HSA) with the spectrum of free RBrB in water or ethyl alcohol, we concluded that the CD signal intensification was due to the increased solubilization of R-BrB upon binding to HSA.

Computed Tomography-Assessed Sarcopenia Predicts Mortality in Kidney Transplant Candidates.

OBJECTIVES: Sarcopenia is common in chronic kidney disease and associated with increased mortality. We investigated the prevalence of sarcopenia, defined as low muscle mass by the psoas muscle index, in endstage renal disease patients on waiting lists for kidney transplant and determined its association with prognostic nutritional index, C-reactive protein-toalbumin ratio, cardiovascular events, and mortality. MATERIALS AND METHODS: Our study included 162 patients with end-stage renal disease and 87 agematched healthy controls. We calculated nutritional status as follows: prognostic nutritional index = (10 × albumin [g/dL]) + (0.005 × total lymphocyte count (×103/µL]) and C-reactive protein-to-albumin ratio. We gathered demographic and laboratory data from medical records. RESULTS: Patients with end-stage renal disease had a mean age of 44.7 ± 14.2 years; follow-up time was 3.37 years (range, 0.35-9.60 y). Although patients with endstage renal disease versus controls had higher prevalence of sarcopenia (16.7% vs 3.4%; P = .002) and C-reactive protein-to-albumin ratio (1.47 [range, 0.12-37.10] vs 0.74 [range, 0.21-10.20]; P < .001), prognostic nutritional index was lower (40 [range, 20.4-52.2] vs 44 [range, 36.1-53.0]; P < .001). In patients with end-stage renal disease with and without sarcopenia, prognostic nutritional index (P = .005) was lower and C-reactive protein-to-albumin ratio (P = .041) was higher in those with versus those without sarcopenia. Among 67 patients on waiting lists who received kidney transplants, those without sarcopenia had better 5-year patient survival posttransplant than those with sarcopenia (P = .001). Multivariate regression analysis showed sarcopenia and low prognostic nutritional index were independentrisk factors for mortality among patients with end-stage renal disease. CONCLUSIONS: Sarcopenia was ~5 times more frequent in patients with end-stage renal disease than in healthy controls and was positively correlated with the prognostic nutritional index. Sarcopenia was an independent risk factor for mortality in patients on transplant waiting lists.

Protein loss during membrane processes in biopharmaceutical manufacturing.

Maximizing product yield in biopharmaceutical manufacturing processes is a critical factor in determining the overall cost of goods, especially given the high value of these biological products. However, there has been relatively limited research on the quantitative analysis of protein losses due to adsorption and fouling during the different membrane filtration processes employed in typical downstream operations. This study aims to provide a comprehensive analysis of protein loss in the range of membrane systems used in downstream processing including clarification, virus removal filtration, ultrafiltration/diafiltration for formulation, and final sterile filtration, all using commercially available membranes with three model proteins (bovine serum albumin, human serum albumin, and immunoglobulin G). The correlation between protein loss and various parameters (i.e., protein type, protein concentration, throughput, membrane morphology, and protein removal mechanism) was also investigated. This study provides important insights into the nature of protein loss during membrane processes as well as a methodology for quantifying protein yield loss in bioprocesses.

Albumin levels in malaria patients: a systematic review and meta-analysis of their association with disease severity.

Albumin, a key protein in human blood plasma, has been linked to various health conditions. However, its association with malaria, particularly in assessing disease severity, remains inadequately understood. This comprehensive systematic review and meta-analysis aimed to elucidate the relationship between albumin levels and malaria severity. A comprehensive literature search was conducted across multiple databases, including Embase, Scopus, PubMed, MEDLINE, Ovid, and Google Scholar, to identify studies examining albumin levels in malaria patients. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed. Data were pooled using a random-effects model, and heterogeneity was assessed using I2 statistics. Subgroup and meta-regression analyses were performed based on publication year, study location, and Plasmodium species. A total of 37 studies were included in this review. The thematic synthesis indicated that albumin levels in malaria patients varied significantly based on geographical location. A meta-analysis of 28 studies found that albumin levels were significantly lower in malaria patients compared with non-malarial controls (P < 0.001, standardized mean differences [SMD] = -2.23, 95% CI - 3.25 to - 1.20, I2: 98%, random effects model, 28 studies). Additionally, subgroup analysis revealed variations in albumin levels based on geographical location and Plasmodium species. Regarding the association with disease severity, thematic synthesis showed that severe malaria cases generally had decreased albumin levels across various regions. However, one Brazilian study reported higher albumin levels in severe cases. A separate meta-analysis of five studies found significantly lower albumin levels in patients experiencing severe malaria relative to those with less severe forms of the disease (P < 0.001, SMD = -0.66, 95% CI - 1.07 to - 0.25), I2: 73%, random effects model, 5 studies). This study underscores the clinical significance of albumin as a potential biomarker for Plasmodium infection and the severity of malaria. The findings suggest that albumin level monitoring could be crucial in managing malaria patients, especially in assessing disease severity and tailoring treatment approaches. Additional studies are required to investigate the underlying mechanisms driving these associations and validate the clinical utility of albumin levels in malaria patient management.

Investigation of oxidant-antioxidant status in patients treated with hirudotherapy: an experimental study.

Objectives: To examine the influence of hirudotherapy on parameters of oxidative stress. METHODS: The cross-sectional study was conducted from March 29 to September 29, 2021, at the Alanya Research and Training Hospital's Traditional and Complementary Medicine Application Centre, Turkey, and comprised adult volunteers of either gender. The participants were subjected to two sessions of hirudotherapy 4 weeks apart. Total antioxidant status, total oxidant status, oxidative stress index values, ischaemia-modified albumin level, paraoxonase 1, disulfide, native thiol, total thiol, and arylesterase levels were assessed at baseline and after the second hirudotherapy session. Data was analysed using SPSS 15. RESULTS: Of the 50 subjects, 30(60%) were females and 20(40%) were males. The overall mean age was 47.10±15.16 years. Oxidative stress, ischaemia-modified albumin and disulfide levels decreased, but not significantly (p>0.05). The reduction in disulfide levels was significant (p=0.021). CONCLUSIONS: Hirudotherapy, within its limitations, could reduce oxidative stress.

Predicting the Stability of Lyophilized Human Serum Albumin Formulations Containing Sucrose and Trehalose Using Solid-State NMR Spectroscopy: Effect of Storage Temperature on 1H T1 Relaxation Times.

In a lyophilized protein/disaccharide system, the ability of the disaccharide to form a homogeneous mixture with the protein and to slow the protein mobility dictates the stabilization potential of the formulation. Human serum albumin was lyophilized with sucrose or trehalose in histidine, phosphate, or citrate buffer. 1H T1 relaxation times were measured by solid-state NMR spectroscopy and were used to assess the homogeneity and mobility of the samples after zero, six, and twelve months at different temperatures. The mobility of the samples decreased after 6 and 12 months storage at elevated temperatures, consistent with structural relaxation of the amorphous disaccharide matrix. Formulations with sucrose had lower mobility and greater stability than formulations with trehalose.

Structure of Human Serum Albumin at a Foam Surface.

Proteins can be adsorbed on the air-water interface (AWI), and the structural changes in proteins at the AWI are closely related to the foaming properties of foods and beverages. However, how these structural changes in proteins at the AWI occur is not well understood. We developed a method for the structural assessment of proteins in the foam state using hydrogen/deuterium exchange mass spectrometry. Adsorption sites and structural changes in human serum albumin (HSA) were identified in situ at the peptide-level resolution. The N-terminus and the loop (E492-T506), which contains hydrophobic amino acids, were identified as adsorption sites. Both the structural flexibility and hydrophobicity were considered to be critical factors for the adsorption of HSA at the AWI. Structural changes in HSA were observed after more than one minute of foaming and were spread widely throughout the structure. These structural changes at the foam AWI were reversible.

Protein-Mediated Changes in Membrane Fluidity and Ordering: Insights into the Molecular Mechanism and Implications for Cellular Function.

Probing protein-membrane interactions is vital for understanding biological functionality for various applications such as drug development, targeted drug delivery, and creation of functional biomaterials for medical and industrial purposes. In this study, we have investigated interaction of Human Serum Albumin (HSA) with two different lipids, dipalmitoylphosphatidylglycerol (dDPPG) and dipalmitoylphosphatidylcholine (dDPPC), using Vibrational Sum Frequency Generation spectroscopy at different membrane fluidity values. In the liquid-expanded (LE) state of the lipid, HSA (at pH 3.5) deeply intercalated lipid chains through a combination of electrostatic and hydrophobic interactions, which resulted in more ordering of the lipid chains. However, in the liquid-condensed (LC) state, protein intercalation is decreased due to tighter lipid packing. Moreover, our findings revealed distinct differences in HSA's interaction with dDPPG and dDPPC lipids. The interaction with dDPPC remained relatively weak compared to dDPPG. These results shed light on the significance of protein mediated changes in lipid characteristics, which hold considerable implications for understanding membrane protein behavior, lipid-mediated cellular processes, and lipid-based biomaterial design.

Insight into the interaction of 5,6 epoxy-cholesterols with human serum albumin.

5,6-Epoxy-cholesterols has been recently revealed to control metabolic pathway in breast cancer, which makes investigating their binding interaction with human serum albumin (HSA) an attractive field of research. The main aim of this article is to examine the binding interaction of 5,6 α-epoxy-cholesterol (5,6 α EC) and 5,6 ß-epoxy-cholesterol (5,6 ß- EC) with HSA using different spectroscopic methods and molecular modeling. These compounds interact with HSA via hydrophobic interactions and hydrogen bonds with binding constants 6.3 × 105 M-1 for 5,6 α-epoxy-cholesterol and 6.9 × 105 M-1 for 5,6 ß-epoxy-cholesterol besides, the mechanism of the interaction can be attributed to static quenching. Circular dichroism data indicated that the α-helical content of HSA increased from 50.5 to 59.8 and 61.1 % after the addition of 5,6 α-ECs and 5,6 ß-EC, respectively, with a ratio of 1:2. Thermodynamic analysis revealed that binding between 5,6-epoxy-cholesterols and HSA is spontaneous and entropy-driven. The molecular docking and esterase-like activity experiments were performed to envision a link between the experimental and theoretical results. The optimal binding site of 5,6-epoxy-cholesterols with HSA was located in subdomain IIA. Moreover, theoretical calculations were performed using the B3LYP function with the 6-311++G (d,p) basis set, indicating the HOMO-LUMO energy gap of 7.874 eV for 5,6 α-epoxy-cholesterol and 7.873 eV for 5,6 ß-epoxy-cholesterol. The obtained findings are assumed to provide basic data for understanding the binding interactions of HSA with oxysterol compounds, which could help explore the pharmacokinetics and pharmacodynamics of oxysterol compounds.

Vanadium(V) complexes derived from triphenylphosphonium and hydrazides: cytotoxicity evaluation and interaction with biomolecules.

The development of coordination compounds with antineoplastic therapeutic properties is currently focused on non-covalent interactions with deoxyribonucleic acid (DNA). Additionally, the interaction profiles of these compounds with globular plasma proteins, particularly serum albumin, warrant thorough evaluation. In this study, we report on the interactions between biomolecules and complexes featuring hydrazone-type imine ligands coordinated with vanadium. The potential to enhance the therapeutic efficiency of these compounds through mitochondrial targeting is explored. This targeting is facilitated by the derivatization of ligands with triphenylphosphonium groups. Thus, this work presents the synthesis, characterization, interactions, and cytotoxicity of dioxidovanadium(V) complexes (C1-C5) with a triphenylphosphonium moiety. These VV-species are coordinated to hydrazone-type iminic ligands derived from (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride ([AH]Cl) and aromatic hydrazides ([H2L1]Cl-[H2L5]Cl). The structures of the five complexes were elucidated through single-crystal X-ray diffraction and vibrational spectroscopies, confirming the presence of dioxidovanadium(V) species in various geometries with degrees of distortion (τ = 0.03-0.50) and highlighting their zwitterionic characteristics. The molecular structural stability of C1-C5 in solution was ascertained using 1H, 19F, 31P, and 51V-nuclear magnetic resonance. Moreover, their interactions with biomolecules were evaluated using diverse spectroscopic methodologies and molecular docking, indicating moderate interactions (Kb ≈ 104 M-1) with calf thymus DNA in the minor groove and with human serum albumin, predominantly in the superficial IB subdomain. Lastly, the cytotoxic potentials of these complexes were assessed in keratinocytes of the HaCaT lineage, revealing that C1-C5 induce a reduction in metabolic activity and cell viability through apoptotic pathways.

The effect of chrysin binding on the conformational dynamics and unfolding pathway of human serum albumin.

Studies on the interactions between ligands and proteins provide insights into how a possible medication alters the structures and activities of the target or carrier proteins. The natural flavonoid aglycone Chrysin (CHR) has demonstrated anti-inflammatory, antioxidant, antiapoptotic, neuroprotective, and antineoplastic effects, both in vitro and in vivo. In this work, we investigated the impact of CHR binding on the as-yet-unexplored conformation, dynamics, and unfolding mechanism of human serum albumin (HSA). We determined CHR binding to HSA domain-II with the association constant (Ka) of 2.70 ± 0.21 × 105 M-1. The urea-induced sequential unfolding mechanism of HSA was used to elucidate the debatable binding location of CHR. CHR binding induced both secondary and tertiary structural alterations in the protein as studied by far-UV circular dichroism and intrinsic fluorescence spectroscopy. Red edge excitation shift (REES) indicated a decrease in conformational dynamics of the protein on the complex formation. This suggested an ordered compact and spatial arrangement of the CHR-boundmolecule. The binding of CHR was found to significantly modulate the urea-induced unfolding pathway of HSA. Urea-induced unfolding pathway of HSA became a two-state process (N-U) from a three-state process (N-I-U). The interaction of CHR is found to increase the thermal stability of the protein by ∼4 °C. This study focuses on the fundamental sciences and demonstrates how prospective medication compounds can alter the dynamics and stability of protein structure.

Lactate-to-albumin ratio: A promising predictor of 28-day all-cause mortality in critically Ill patients with acute ischemic stroke.

INTRODUCTION: Numerous diseases have been found to be associated with the lactate-to-albumin ratio (LAR), as confirmed by existing research. This study aims to investigate the relationship between LAR within 24 hours of admission and a 28-day mortality rate in patients manifesting ischemic stroke. METHODS: This retrospective cohort study utilized data from the Medical Information Mart for Intensive Care IV (MIMIC-IV, version 2.1) database. We included adult patients with acute ischemic stroke (AIS) who were admitted to the intensive care unit. The primary outcome entailed evaluating the ability of LAR to predict death at 28-day of hospital admission in patients with AIS. RESULTS: A total of 502 patients with ischemic stroke were enrolled in the study, of which 185 (36.9 %) died within 28 days after hospital admission. We identified a linear association between LAR and mortality risk. Compared with the reference group (first LAR tertile), the 28-day mortality was increased in the highest tertile; the fully adjusted HR value was 1.21 (1.08 to 1.40). the Area Under the Curve (AUC) value for LAR was 58.26 % (95 % CI: 53.05 % - 63.46 %), which was higher than that for arterial blood lactate (AUC = 56.88 %) and serum albumin (AUC = 55.29 %) alone. It was not inferior even when compared to SOFA (AUC = 56.28 %). The final subgroup analysis exhibited no significant interaction of LAR with each subgroup (P for interaction: 0.079 - 0.848). CONCLUSION: In our study, LAR emerged as a promising predictor of all-cause mortality in acute ischemic stroke patients within 28 days of admission.

Investigating the Impact of First-Line Anti-Tuberculosis Drugs Encapsulated in a Eugenol-Based Nanoemulsion on Human Serum Albumin.

BACKGROUND: Eugenol exhibits broad-spectrum antibacterial and anti-inflammatory properties. However, cytotoxicity at high concentrations limits the full utilization of eugenol-based drug complexes. Formulations of multidrug-loaded eugenol-based nanoemulsions have reduced cytotoxicity; however, it remains crucial to understand how these eugenol complexes interact with primary human carrier proteins to design and develop therapeutic alternatives. Consequently, this study primarily aims to investigate the impact on Human Serum Albumin (HSA) when it interacts with eugenol-based complexes loaded with first-line anti-tuberculosis drugs. METHODS: This study used various spectroscopic such as UV-visible spectroscopy, Fluorescence spectroscopy, Fourier-transform infrared spectroscopy and computational methods such as molecular docking and 100 ns molecular simulation to understand the impact of eugenol-based first-line anti-tuberculosis drug-loaded nanoemulsions on HSA structure. RESULTS: The binding of the HSA protein and eugenol-based complexes was studied using UV-visible spectroscopic analysis. Minor changes in the fluorophores of the protein further confirmed binding upon interaction with the complexes. The Fourier-transform infrared spectra showed no significant changes in protein structure upon interaction with eugenol-based multidrug-loaded nanoemulsions, suggesting that this complex is safe for internal administration. Unlike eugenol or first-line anti-tuberculosis alone, molecular docking revealed the strength of the binding interactions between the complexes and the protein through hydrogen bonds. The docked complexes were subjected to a 100 ns molecular dynamics simulation, which strongly supported the conclusion that the structure and stability of the protein were not compromised by the interaction. CONCLUSIONS: From the results we could comprehend that the eugenol (EUG)-drug complex showed greater stability in HSA protein structure when compared to HSA interacting with isoniazid (INH), rifampicin (RIF), pyrazinamide (PYR), or ethambutol (ETH) alone or with EUG alone. Thus, inferring the potential of EUG-based drug-loaded formulations for a safer and efficient therapeutic use.

Contrast Agents Based on Human Serum Albumin and Nitroxides for 1H-MRI and Overhauser-Enhanced MRI.

In cancer diagnostics, magnetic resonance imaging (MRI) uses contrast agents to enhance the distinction between the target tissue and background. Several promising approaches have been developed to increase MRI sensitivity, one of which is Overhauser dynamic nuclear polarization (ODNP)-enhanced MRI (OMRI). In this study, a macromolecular construct based on human serum albumin and nitroxyl radicals (HSA-NIT) was developed using a new synthesis method that significantly increased the modification to 21 nitroxide residues per protein. This was confirmed by electron paramagnetic resonance (EPR) spectroscopy and matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF) mass spectrometry. Gel electrophoresis and circular dichroism showed no significant changes in the structure of HSA-NITs, and no oligomers were formed during modification. The cytotoxicity of HSA-NITs was comparable to that of native albumin. HSA-NITs were evaluated as potential "metal-free" organic radical relaxation-based contrast agents for 1H-MRI and as hyperpolarizing contrast agents for OMRI. Relaxivities (longitudinal and transversal relaxation rates r1 and r2) for HSA-NITs were measured at different magnetic field strengths (1.88, 3, 7, and 14 T). Phantoms were used to demonstrate the potential use of HSA-NIT as a T1- and T2-weighted relaxation-based contrast agent at 3 T and 14 T. The efficacy of 1H Overhauser dynamic nuclear polarization (ODNP) in liquids at an ultralow magnetic field (ULF, B0 = 92 ± 0.8 µT) was investigated for HSA-NIT conjugates. The HSA-NITs themselves did not show ODNP enhancement; however, under the proteolysis conditions simulating cancer tissue, HSA-NIT conjugates were cleaved into lower-molecular-weight (MW) protein fragments that activate ODNP capabilities, resulting in a maximum achievable enhancement |Emax| of 40-50 and a radiofrequency power required to achieve half of Emax, P1/2, of 21-27 W. The HSA-NIT with a higher degree of modification released increased the number of spin probes upon biodegradation, which significantly enhanced the Overhauser effect. Thus, HSA-NITs may represent a new class of MRI relaxation-based contrast agents as well as novel cleavable conjugates for use as hyperpolarizing contrast agents (HCAs) in OMRI.