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.

https://bpspubs.onlinelibrary.wiley.com/doi/epdf/10.1038/sj.bjp.0707251

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

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, −(CH2)2−; 2, −(CH2)3−; 3, −(CH2)5−; 4, −(CH2)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 (R...

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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

https://www.kidney-international.org/article/S0085-2538(15)50289-5/pdf

Oxidative stress is enhanced in correlation with renal dysfunction: Examination with the redox state of albumin

Introduction. Serum proteins are potential drug carriers of antineoplastic agents due to their accumulation in tumor tissue.1 Uptake of these proteins in solid tumors is mediated by a number of factors including an increased metabolic activity of tumors, an enhanced vascular permeability of tumor blood vessels for circulating macromolecules, and a lack of a functional lymphatic drainage system in tumor tissue.2 Recently, a number of acid-sensitive albumin and transferrin conjugates with anthracyclines and the alkylating agent chlorambucil have shown promising in vitro activity.3-7 In addition, acid-sensitive doxorubicin conjugates with monoclonal antibodies and albumin doxorubicin conjugates prepared by glutaraldehyde cross-linking have shown promising antitumor efficacy in vivo.8,9 A selected acid-sensitive doxorubicin albumin conjugate that was developed in our group induced complete remissions of primary kidney tumors in murine renal carcinoma and prevented the formation of metastases in the lungs. In contrast, mice treated with doxorubicin at optimal dose manifested clearly visible kidney tumors at the end of the experiment and large numbers of lung metastases.10 This albumin doxorubicin conjugate was synthesized by coupling 1, a maleimide carboxylic hydrazone derivative of doxorubicin (see Figure 1), to thiolated albumin. 1 contains an acid-sensitive linker that allows the drug to be released at the low pH values present in lysosomes and endosomes of tumor cells. We have recently shown that 1 also binds covalently to the cysteine-34 of commercially available human serum albumin which is a mixture of mercaptalbumin and nonmercaptalbumin.11,12 Approximately 70% of circulating albumin in the blood stream is mercaptalbumin that contains an accessible cysteine-34 which is not blocked by endogenous sulfhydryl compounds such as cysteine or glutathione.12,13 Considering that free thiol groups are not found on the majority of circulating serum proteins except for albumin, cysteine-34 of endogenous albumin is a fairly unique amino acid on the surface of a circulating protein that could be exploited for developing a novel macromolecular prodrug concept. Since the maleimide group reacts specifically and selectively with thiol groups, we reasoned that it should be possible to preferentially bind maleimide drug derivatives to the HS group of the cysteine-34 position of albumin after intravenous application. In this way a macromolecular prodrug is formed after in situ coupling of a thiolbinding drug derivative to endogenous albumin in the blood circulation. Following this approach, it should be possible to avoid the ex vivo synthesis and characterization of drug albumin conjugates which are costly, are time-consuming, and rely on exogenous and possibly pathogenic albumin. The objective of the present work was to assess the feasibility and selectivity of our approach by carrying out in vitro and in vivo binding studies using the doxorubicin maleimide derivative 1. In addition, we wanted to obtain the first in vivo evidence that 1 is superior to free doxorubicin in an animal tumor model, i.e., in murine renal cell carcinoma (RENCA). In Vitro and in Vivo Binding Studies. To estimate the coupling rate and selectivity of 1 for endogenous albumin, 1 was incubated with human blood plasma at T ) 37 °C and the samples were subsequently analyzed by anion-exchange chromatography. (The ratio of drug to albumin was approximately 0.3:1; human blood plasma employed contained an albumin concentration of ∼700 μM as determined with a Vitros analyzer from Ortho-Clinical Diagnostics. 1 was synthesized previously;14 a 2000 μM solution of 1 was freshly prepared in 0.15 M NaCl, 0.004 M sodium phosphate buffer (pH 5.5), and 30 vol % of 1,2-propylene glycol for binding studies and animal experiments; doxorubicin was used as a 3400 μM stock solution in isotonic saline from Pharmacia & Upjohn.) Chromatograms after an incubation time of 5 min for 1 (Figure 2A) and for free doxorubicin (Figure 2B) under identical conditions are available as Supporting Information. Protein components were detected at 254 nm, and the anthracycline moiety was detected by simultaneous fluorescence excitation. In contrast to free doxorubicin, the major amount of 1 is associated with the albumin peak which elutes at around 23.5 min. Longer incubation periods led to a slight increase in albumin binding indicating that the coupling reaction proceeds rapidly within this short period. Incubation studies with total blood and subsequent HPLC analysis of the resulting blood plasma * To whom correspondence should be addressed: Dr. Felix Kratz, Tumor Biology Center, Department of Medical Oncology, Clinical Research, Breisacher Strasse 117, D-79106 Freiburg, FRG. Tel: 0049-761-2062176. Fax: 0049-761-2061899. E-mail: felix@ tumorbio.uni-freiburg.de. Figure 1. Structure of 1, a maleimide phenylacetylhydrazone derivative of doxorubicin. 1253 J. Med. Chem. 2000, 43, 1253-1256

A novel macromolecular prodrug concept exploiting endogenous serum albumin as a drug carrier for cancer chemotherapy.

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.

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Eiji Suzuki

Papers

High-performance liquid chromatographic studies on non-mercapt α mercapt conversion of human serum albumin. II