Albumin Binding Properties Research Articles

Induced Plasma Expander-like Properties as a Function of PEG-chains on Extension Arm Facilitated PEGylation of Albumin: “Mushroom to Brush-Like” Conformational Transition of the PEG-albumin Conjugate

Plasma expander-like properties of albumin induced on hexa as well as dodecacPEGylation using Extension Arm Facilitated PEGylation platform make it an excellent resuscitation fluid. PEGylation induced changes in the structure, drug binding, and plasma expander-like properties of bovine serum albumin has been now investigated as a function of PEGylation. The molecular volume of albumin increases on PEGylation nearly linearly; in the beginning up to about six PEG chains are conjugated, then plateau off, while the viscosity and colloidal osmotic pressure change very little initially and then increase exponentially as a function of PEG chains conjugated. PEGylation has essentially no influence on the secondary structure or drug properties of albumin. Tryphtophyl fluorescence of albumin is quenched on PEGylation as a direct correlate of the changes in molecular radius of PEG-albumin. It is concluded that hexaPEGylated and dodecaPEGylated albumin belong to two different configurational states of PEG-albumin in terms of packing of PEG-chains on the molecular surface of the protein. The results suggest a transition of PEGylated albumin from the initial mushroom-like conformation to brush conformation as the PEGylation increases. The therapeutic efficacy of the two PEGylated species is needed to establish the optimum level of PEGylation to function as resuscitation fluids.

Heme Pocket Architecture in Human Serum Albumin: Regulation of O2 Binding Affinity of a Prosthetic Heme Group by Site‐Directed Mutagenesis

AbstractSummary: We present the O2 binding properties of recombinant human serum albumin (rHSA) mutants complexed with an iron(II) protoporphyrin IX as a prosthetic heme group. Iron(III) protoporphyrin IX (hemin) is bound within subdomain IB of HSA with weak axial coordination by Tyr‐161. In order to confer O2 binding capability to this naturally occurring hemoprotein: (i) a proximal histidine was introduced into position Ile‐142; and (ii) the coordinated Tyr‐161 was replaced with hydrophobic Leu using site‐directed mutagenesis. It provided a recombinant HSA double‐mutant [rHSA(I142H/Y161L) = rHSA(HL)]. The rHSA(HL)–heme formed a ferrous five‐coordinate high‐spin complex with axial ligation of His‐142 under an Ar atmosphere. This artificial hemoprotein binds O2 at room temperature. Laser flash photolysis experiments demonstrated that O2 rebinidng to rHSA(HL)–heme displays monophasic kinetics, whereas the CO recombination process obeyed a double‐exponential pattern. This might be attributable to the two different geometries of the axial imidazole coordination arising from the two orientations of the porphyrin plane in the heme pocket. The O2 binding affinity of rHSA(HL)–heme was considerably lower than those of R‐state hemoglobin (Hb) and myoglobin (Mb), principally because of the high O2 dissociation rate constant. The third mutations have been introduced into the distal side of the heme (at position Leu‐185 or Arg‐186) to increase the O2 binidng affinity. The rHSA(HL/L185N)–heme showed high O2 binding affinity ($P_{1/2}^{{\rm O}_2 } $: 1 Torr), which is 18‐fold greater than that of the original double mutant rHSA(HL)–heme and which is rather close to those of Hb (R‐state) and Mb. Furthermore, replacement of polar Arg‐186 with Leu or Phe adjusted the O2 binding affinity ($P_{1/2}^{{\rm O}_2 } $) to 10 Torr, which is almost equivalent to value for human red blood cells.

Bioreversible derivatives of phenol. 1. The role of human serum albumin as related to the stability and binding properties of carbonate esters with fatty acid-like structures in aqueous solution and biological media.

With the overall objective of assessing the potential of utilizing plasma protein binding interactions in combination with the prodrug approach for improving the pharmacokinetics of drug substances, a series of model carbonate ester prodrugs of phenol, encompassing derivatives with fatty acid-like structures, were characterized in vitro. Stability of the derivatives was studied in aqueous solution, human serum albumin solution, human plasma, and rat liver homogenate at 37 degrees C. Stability of the derivatives in aqueous solution varied widely, with half-lives ranging from 31 to 1.7 x 10(4) min at pH 7.4 and 37 degrees C. The carbonate esters were subject to catalysis by plasma esterases except for the t-butyl and acetic acid derivatives, which were stabilized in both human plasma and human serum albumin solutions relative to buffer. In most cases, however, hydrolysis was accelerated in the presence of human serum albumin indicating that the derivatives interacted with the protein, a finding which was confirmed using the p-nitrophenyl acetate kinetic assay. Different human serum albumin binding properties of the phenol model prodrugs with fatty acid-like structure and neutral carbonate esters were observed. In the context of utilizing plasma protein binding in combination with the prodrug approach for optimizing drug pharmacokinetics, the esterase-like properties of human serum albumin towards the carbonate esters potentially allowing the protein to act as a catalyst of parent compound regenerations is interesting.

Physiological and pathological changes in the redox state of human serum albumin critically influence its binding properties

Binding and transport of a number of endogenous and exogenous compounds is an important function of the main plasma protein, albumin. In vivo and in vitro, albumin may be oxidatively modified in different ways with different agents at different sites. These modifications have various consequences on the physiological functions of albumin. Diabetes mellitus, liver diseases and nephropathy are just a few examples of disorders in which oxidative stress is involved and altered albumin functions have been described. This review is focussed on the consequences of oxidative modification on the binding properties of albumin. These range from no effect to decreased or increased binding affinities depending on the ligand under investigation and the type of modification. Indicators for modification include glycosylation, disulphide formation or the content of carbonyl groups. The redox state of albumin can affect the binding properties in several ways, including altered conformation and consequently altered affinities at binding sites and altered binding when the binding reaction itself is redox sensitive. The physiological or pathophysiological concentrations of different oxidatively modified albumin molecules vary over a wide range and are crucial in assessing the clinical relevance of altered ligand binding properties of a particularly modified albumin species in various disease conditions.

METHOD TO ASSESS FATTY ACID ETHYL ESTER BINDING TO ALBUMIN

To develop a method to assess the relative binding of fatty acid ethyl esters (FAEE) and free fatty acids (FFA) to albumin, and to determine if binding affinity is related to fatty acid chain length and or degree of saturation. Radiolabelled ethyl-[14C] oleate bound to albumin was challenged with various ratios of FFA to FAEE. The displacement of ethyl-[14C] oleate by FFA was visualized and quantitated through a combination of native-PAGE, autoradiography, and liquid scintillation counting (LSC). As the ratio of FFA to FAEE increased from 0:1 to 12:1, for all fatty acids tested (myristate, palmitate, stearate, oleate, linoleate, and arachidonate), ethyl-[14C] oleate displacement increased, when expressed as radioactivity (in DPM) as a percentage of control. In contrast, ethyl oleate did not displace stearate or oleate from albumin at molar ratios up to 5:1 (FAEE:FFA). The method developed gave reproducible visualization of noncovalent binding of radiolabelled FAEE to albumin. The combination of native-PAGE and autoradiography LSC works well in assessing the binding properties of albumin and radiolabelled FAEE. The data indicate preferential binding of FFA over FAEE to albumin with six different FFA displacing FAEE to an approximately equal extent.

Isolation and identification of heterogeneous nuclear ribonucleoproteins (hnRNP) from purified plasma membranes of human tumour cell lines as albumin-binding proteins

Since albumin is being developed as a drug carrier to target tumours the search for albumin-binding proteins (ABPs), which play a role in cell surface binding and endocytosis of native and conjugated albumins becomes more and more interesting. We isolated five different proteins from purified plasma membranes from three different human tumour cell lines (CCRF-CEM, MV3 and MCF7) by albumin affinity chromatography and identified them as four members of the heterogeneous nuclear ribonucleoproteins (hnRNP) family and calreticulin by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Contamination of the plasma membrane preparation by nuclear membranes was excluded with anti-nucleopore antibodies. Western blot analyses of plasma membranes showed ABPs with the same molecular weights as the albumin-affinity isolates. Tryptic digestion of intact cells was used to determine the sidedness of the albumin-binding property, which is oriented to the exterior of the cell. Localisation to the plasma membrane and albumin binding is a novel property of hnRNP.

Synthesis of 2′- O-[2-[( N, N-dialkylamino)oxy]ethyl]-modified oligonucleotides: hybridization affinity, resistance to nuclease, and protein binding characteristics

Synthesis of a series of 2′- O-[2-[( N, N-dialkylamino)oxy]ethyl]-modified 5-methyluridine nucleoside phosphoramidites and solid supports are described. Using these monomers, modified oligonucleotides containing phosphodiester linkages were synthesized in high yields. These modified oligonucleotides showed enhanced binding affinity to the complementary RNA (and not to DNA) and excellent nuclease stability with t 1/2>24 h. The human serum albumin binding properties of modified oligonucleotides have been evaluated to assess their transport and toxicity properties.

Probing the cysteine-34 position of endogenous serum albumin with thiol-binding doxorubicin derivatives. Improved efficacy of an acid-sensitive doxorubicin derivative with specific albumin-binding properties compared to that of the parent compound.

We have recently proposed a macromolecular prodrug strategy for improved cancer chemotherapy based on two features (Kratz, F.; et al. J. Med. Chem 2000, 43, 1253-1256.): (a) rapid and selective binding of thiol-reactive prodrugs to the cysteine-34 position of endogenous albumin after intravenous administration and (b) release of the albumin-bound drug in the acidic environment at the tumor site due to the incorporation of an acid-sensitive bond between the drug and the carrier. To investigate this therapeutic strategy in greater depth, four (maleinimidoalkanoyl)hydrazone derivatives of doxorubicin were synthesized differing in the length of the aliphatic spacer (1, -(CH(2))(2)-; 2, -(CH(2))(3)-; 3, -(CH(2))(5)-; 4, -(CH(2))(7)-). The albumin-binding doxorubicin prodrugs, especially the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (3), are rapidly and selectively bound to the cysteine-34 position of endogenous albumin. 3 was distinctly superior to the parent compound doxorubicin in three animal tumor models (RENCA, MDA-MB 435, and MCF-7) with respect to antitumor efficacy and toxicity.

Interactions of very long-chain saturated fatty acids with serum albumin

The remarkable binding properties of serum albumin have been investigated extensively, but little is known about an important class of fatty acids, the very long-chain saturated fatty acids (VLCFA; >18 carbons). Although VLCFA are metabolized efficiently in normal individuals, they are markers for and possibly causative agents of several peroxisomal disorders. We studied the binding of [13C]carboxyl-enriched arachidic (C20:0), behenic (C22:0), lignoceric (C24:0), and hexacosanoic (C26:0) acids to bovine serum albumin (BSA) by 13C-NMR spectroscopy. For each VLCFA, the NMR spectra showed multiple signals at chemical shifts previously identified for long-chain fatty acids (12–18 carbons), suggesting stabilization of binding by similar, if not identical, interactions of the fatty acid carboxyl anion with basic amino acid residues. The maximal binding (mol of VLCFA/mol of BSA) and the number of observed binding sites decreased with increasing chain length, from 4–5 for C20:0, 3–4 for C22:0, and 2 for C24:0; we validated our previous conclusion that BSA has only one site for C26:0 (Ho, J. K., H. Moser, Y. Kishimoto, and J. A. Hamilton. 1995. J. Clin. Invest. 96: 1455–1463). Analysis of chemical shifts suggested that the highest affinity sites for VLCFA are low affinity sites for long-chain fatty acids. In competition experiments with 13C-labeled C22:0 (3 mol/mol of BSA) and unlabeled oleic acid, C22:0 bound to BSA in the presence of up to 4 mol of oleic acid/mol of BSA, but 1 mol was shifted into a different site.Our studies suggest that albumin has adequate binding capacity for the low plasma levels of VLCFA with 20 to 26 carbons, but the protein may not be able to bind longer chain VLCFA.

Changes in the Antigenic and Immunoglobulin E–Binding Properties of Hen's Egg Albumin with the Combination of Heat and Gamma Irradiation Treatment

This study was carried out to evaluate the changes in the allergenic and antigenic properties of hen's egg albumin (ovalbumin [OVA]) with the combination of heat and gamma irradiation treatment. OVA solution samples were treated by (i) heating (sample 1), (ii) irradiation after heating (sample 2), and (iii) heating after irradiation (sample 3). Samples were isothermally heated and irradiated at the absorption dose of 10 kGy. Competitive indirect enzyme-linked immunosorbent assays (ELISAs) were performed with blood serum to test the ability of treated OVA to bind to immunoglobulin E (IgE) and mouse murine monoclonal antibody (IgG). OVA's ability to bind to mouse IgG changed upon heating at 75°C, and its ability to bind to egg-allergic IgE changed upon heating at 80°C. The ELISAs showed that egg-allergic IgE did not recognize OVA very well when heated at ≥80°C, while mouse IgG retained better activity under these conditions. Egg-allergic IgE binding was low both for OVA samples treated by heating and for samples treated by irradiation followed by heating. These results show that allergies induced by OVA could be effectively reduced by the combination of heat and gamma irradiation treatment.

Bilirubin binding properties of pigeon serum albumin and its comparison with human serum albumin

Binding of bilirubin (BR) to pigeon serum albumin (PgSA) was studied by absorption, fluorescence and CD spectroscopy and results were compared with those obtained with human serum albumin (HSA). PgSA was found to be structurally similar to HSA as judged by near- and far-UV CD spectra. However, PgSA lacks tryptophan. Binding of BR to PgSA showed relatively weaker interaction compared to HSA in terms of binding affinity, induced red shift in the absorption spectrum of BR and CD spectral characteristics of BR–albumin complexes. Photoirradiation results of BR–albumin complexes also showed PgSA-bound BR more labile compared to HSA-bound BR.

Macrocyclic and polymeric lanthanide chelates: from physico-chemical studies to biomedical applications

The improvement of commonly used Gd 3+-based MRI contrast agents requires the design of new chelates with optimised in vivo efficacy, pharmacokinetic properties and specificity. The molecular parameters determining the nuclear relaxivity of the cationic chelate [Gd(DO2A)] +, as well as of some copolymers of [Gd(DTPA)] 2− with diamines, were obtained using spectroscopic techniques. In both cases, the resulting too slow inner-sphere water exchange rate limits the relaxivity increase expected, respectively, from an increased water coordination number, and some degree of rotational restriction, although in the latter case, the relaxivity shows a maximum in the 30 MHz proton frequency region. The use of the polymeric compounds as MR angiography agents is limited by their fast uptake from the blood pool of Wistar rats. The use of scintigraphic diagnostic agents critically depends on their targeting properties. 111In 3+ and 153Sm 3+ chelates of DTPA derivatives, containing albumin binding or hepato-specific synthons, show different organ specificity, albumin binding properties and excretion pathways and kinetics, as shown by biodistribution and gamma imaging studies of Wistar rats. The 111In 3+, 153Sm 3+, Gd 3+ and Tm 3+ chelates of the tetraaza-phosphonate macrocycle DOTP 8− also show high bone uptake specificity.

The glycan structure of albumin Redhill, a glycosylated variant of human serum albumin

Although human serum albumin is synthesized without carbohydrate, glycosylated variants of the protein can be found. We have determined the structure of the glycan bound to the double-mutant albumin Redhill (−1 Arg, 320 Ala→Thr). The oligosaccharide was released from the protein using anhydrous hydrazine, and its structure was investigated using neuraminidase and a reagent array analysis method, which is based on the use of specific exoglycosidases. The glycan was shown to be a disialylated biantennary complex type oligosaccharide N-linked to 318 Asn. However, a minor part (11 mol%) of the glycan was without sialic acid. The structure is principally the same as that of glycans bound to two other types of glycosylated albumin variants. Glycosylation can affect, for example, the fatty acid binding properties of albumin. Taking the present information into account, it is apparent that different effects on binding are caused not by different glycan structures but by different locations of attachment, with the possible addition of local conformational changes in the protein molecule.

Liquid chromatographic studies of the effect of phosphate on the binding properties of silica-immobilized bovine serum albumin.

High-performance liquid chromatography has been used to examine how phosphate ions affect the binding properties of bovine serum albumin (BSA) immobilized to porous silica. In doing this, the time dependence of the protein to reach conformational equilibrium is measured as a function of the concentration of phosphate in the eluent using the D- and L-isomers of tryptophan and kynurenine as solutes. The overall binding and chiral selectivity (alphaD,L) of the protein toward these solutes appear to be related to two types of effects: one being those that are site-selective and only influence the retention of the L-isomers and the other being those that are nonselective and influence the retention of both enantiomers. An interesting feature of the concentration-dependent data is a maximum in alphaD,L at intermediate phosphate concentrations (i.e., 10 to 50mM phosphate) indicative of both cooperative and antagonistic binding effects. Phosphate eluents within this concentration range provide selectivity advantages, and those at higher concentrations decrease the time required for the protein or column to reach equilibrium. A final set of studies has also been carried out using four alternate buffer systems (i.e., borate, carbonate, acetate, and arsenate eluents). Although the borate eluents affect the BSA's binding properties and alphaD,L similar to the phosphate eluents, the other buffers result in poor separations. Observations from this study are useful in helping to optimize separations carried out on immobilized BSA as well as addressing biological and mechanistic questions related to how anions influence the native binding properties of serum albumins.

Binding properties of human albumin modified by covalent binding of penicillin

Derivatisation of lysine residues in human albumin was performed in vitro by reaction with penicillin G. This modification reaction has been reported to occur in patients treated with high dosages of the antibiotic. The structure of the modified protein was characterised by mass spectrometry and circular dichroism. The number of the lysine residues involved depends on the time of incubation and on the drug/protein molar ratio. The secondary structure of the modified protein does not change significantly with respect to the native protein. Furthermore, the binding properties of the modified albumin were characterised by CD spectroscopy. Phenylbutazone, diazepam and bilirubin, known to bind to specific binding areas, were used as markers. A decrease of the affinity to the high-affinity binding sites was observed after the modification.

The Three Recombinant Domains of Human Serum Albumin: STRUCTURAL CHARACTERIZATION AND LIGAND BINDING PROPERTIES

In an attempt to systematically dissect the ligand binding properties of human serum albumin (HSA), the gene segments encoding each of its three domains were defined based on their conserved homologous structural motifs and separately cloned into a secretion vector for Pichia pastoris. We were able to establish a generally applicable purification protocol based on Cibacron Blue affinity chromatography, suggesting that each of the three domains carries a binding site specific for this ligand. Proteins were characterized by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, gel filtration, N-terminal sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as well as near- and far-UV CD. In addition to the affinity chromatography ligand Cibacron Blue, binding properties toward hemin, warfarin, and diazepam, each of which represents a standard ligand for HSA, respectively, were investigated by the measurement of induced circular dichroism. Clear experimental evidence is provided here for the location of the primary hemin binding site to be on domain I of HSA, and for the primary diazepam binding site to be on domain III. Further, secondary binding sites were found for hemin to be located on domains II and III, and for diazepam on domain I. The warfarin binding site was located primarily on domain II, while on domain I, a secondary binding site and/or parts of the primary binding site were found.

Effect of lysine modification on the conformation and indomethacin binding properties of human serum albumin

In order to study the involvement of lysine residues of human serum albumin (HSA) in the binding of indomethacin, HSA was treated with different molar excess of acetic anhydride, succinic anhydride and O-methylisourea which resulted in differently modified preparations: 30%, 62% and 87% acetylated, 20%, 34%, 64% and 78% succinylated and 21%, 43% and 86% guanidinated HSAs. All the preparations were found to be homogeneous with respect to charge as well as size as judged by polyacrylamide gel electrophoresis and gel filtration on a Seralose-6B column. Hydrodynamic and circular dichroic results showed that pronounced conformational changes (both tertiary and secondary structures) were induced in the maximally acetylated (87%) and succinylated (78%) preparations. On the other hand, guanidinated preparations showed no expansion in the hydrodynamic volume. The percent decrease in α-helical content was 34% for 87% acetylated, 31% for 78% succinylated and 10% for 86% guanidinated HSAs. A significant increase in the values of Stokes radii and frictional ratios (from 3.43 nm and 1.29 for native HSA to 4.07 nm and 1.52 for 87% acetylated and 4.35 nm and 1.60 for 78% succinylated HSAs, respectively) was also noticed in these highly modified preparations. Fluorescence quench titration results obtained at pH 7.4 and ionic strength 0.15 showed that only 54.1% and 64.7% binding of indomethacin at 4:1 drug/protein molar ratio was retained by 87% acetylated and 78% succinylated HSAs, respectively, as compared to 91% retention in binding in 86% guanidinated preparation. No reversal in the binding of drug to 87% acetylated and 78% succinylated HSA preparations was observed on increasing the ionic strength to 1.0. Therefore, it seems that one or two critical lysine residue(s) that can form salt linkage with the carboxyl group of indomethacin, was (were) probably modified in these preparations. A small decrease in the binding of drug to the guanidinated preparation also confirms the involvement of positive charge, probably contributed by lysine residue(s), in the binding of indomethacin to HSA.

Effect of Fatty Acids and Selected Drugs on the Albumin Binding of a Long-Acting, Acylated Insulin Analogue

□ NN304 (LysB29-tetradecanoyl, des(B30)-insulin) is a new soluble, long-acting insulin analogue that is tightly bound to human serum albumin differentiating it from human insulin. In the present study, we investigate the effect of fatty acids and selected drugs on the binding of NN304 to human serum albumin in vitro. Binding of the first fatty acid equivalent to albumin does not affect the binding of NN304. None of the tested drugs compete with the binding of NN304 at drug-to-albumin concentration ratios of <1. The binding of NN304 is shown to be independent of binding of drugs in the two major binding pockets that are located in domains IIA and IIIA of the albumin molecule. Tolbutamide and glibenclamide do not compete with NN304 for binding to albumin at therapeutic drug-to-albumin concentration ratios. High concentrations of acetylsalicylic acid and ibuprofen decrease the affinity of NN304 for albumin, but these interactions occur at drug-to-albumin concentration ratios that are higher than clinically relevant. In conclusion, NN304 is unlikely to be involved in clinically significant drug interactions at the albumin binding level. The unique ligand binding properties of serum albumin and its abundance in the extracellular fluids makes fatty acid acylation and albumin binding an attractive protraction principle for insulin and potentially also for other peptide drugs.

Synthesis, Photophysical Properties, in Vivo Photosensitizing Efficacy, and Human Serum Albumin Binding Properties of Some Novel Bacteriochlorins

The synthesis, photophysical characteristics, in vivo photosensitizing efficacy, human serum albumin (HSA) binding properties, and skin phototoxicity of some stable bacteriochlorins were investigated. The novel bacteriochlorins, obtained from chlorophyll-a, have long-wavelength absorptions in the range lambda max = 734-758 nm. Preferential migration of ethyl over methyl substituents among ketobacteriochlorins obtained in the pinacol-pinacolone rearrangements of vic-dihydroxybacteriochlorins was confirmed by NOE studies. The bacteriochlorins show relatively low fluorescence quantum yields. Among all the bacteriochlorins the triplet states were quenched by ground state molecular oxygen in a relatively similar manner, yielding comparable singlet oxygen quantum yields. In preliminary in vivo studies (DBA/2 mice, transplanted with SMT/F tumors), ketobacteriochlorins were found to be more photodynamically active than the related vic-dihydroxy analogues. Replacement of the methyl ester functionalities with di-tert-butylaspartic acids enhanced the in vivo efficacy. Site specific human serum albumin (HSA) binding studies indicated a direct correlation between the ability of the compound to bind to the diazepam binding site (albumin site II) and the in vivo photosensitizing efficacy.

Quantitative structure-retention relationships of acyclovir esters using immobilised albumin high-performance liquid chromatography and reversed-phase high-performance liquid chromatography

Acyclovir and 18 of its esters have been investigated by systematic measurement of their reversed-phase high-performance liquid chromatographic retention using differing mobile phase compositions. The methanol content of the mobile phase was varied between 5 and 95%. By linear least squares regression of the logarithmic retention factor (log k′) against methanol concentration, the slope ( S)_and intercept (log k′ 0) of the so obtained straight lines were calculated for each compound. The chromatographic hydrophobicity index ( φ 0,MeOH) calculated from the S and log k′ 0 values ( φ 0,MeOH = −log k′ 0/ S) showed significant correlation ( r > 0.96) to the calculated octanol-water partition coefficients (cLog P). The albumin-binding properties of the compounds were characterised by the log k′ HSA values obtained by using an immobilised human serum albumin (HSA) HPLC column and 1% propan-2-ol 99% aqueous 10 m M phosphate buffer pH 7.0 as mobile phase. The measured albumin-binding parameters showed significant correlations to the cLog P, φ 0MeOH, S and log k′ 0 values, establishing the importance of hydrophobic properties to the interaction of the acyclovir derivatives with HSA.