Blanka Ziomkowska

The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.

Determination of Hydroxycamptothecin Affinities to Albumin and Membranes by Steady-State Fluorescence Anisotropy Measurements

Camptothecin (CPT) and its hydroxycamptothecin analogs are fluorescent compounds exhibiting strong anticancer properties. They exist in two forms: active lactone and inactive carboxylate. The deactivation proceeds via hydrolysis in neutral and base solutions. A serious limitation to the clinical application of CPT is the strong affinity of its carboxylate form to human serum albumin (HSA) which destabilizes its active lactone form. However, binding to membranes in blood improves the stability of the lactone form of CPT and its analogs. A high-throughput screening assay based on the steady-state fluorescence anisotropy method was used to determine the protein- and membrane-binding properties of 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxycamptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67). The relative affinities of hydroxycamptothecins to HSA and model membranes in the form of DMPC liposomes were determined, and DB-67 exhibited the most desirable properties including the highest affinity to membranes in its lactone form and low affinity to HSA in its carboxylate form.

A New Approach to Determine Camptothecin and Its Analogues Affinity to Human Serum Albumin

A novel and fast method for the determination of the binding kinetic data of ligand to protein has been developed. A new tool including human serum albumin-coated magnetic beads (HSA-MB) was used to determine the affinity of camptothecin (CPT) and its analogues to HSA. From the biological activity point of view, these compounds have potential anticancer activity. However, the numerous studies indicate that some of these analogues have a strong affinity to plasma proteins stopping their effective therapy. Thus, the problem of plasma protein binding behavior of CPTs analogues was the subject of this study.

Hydroxycamptothecin Deactivation Rates and Binding to Model Membranes and HSA Determined by Fluorescence Spectra Analysis

Camptothecins (CPTs) are fluorescent compounds exhibiting anticancer activity. They can exist in two forms, a lactone and a carboxylate. In neutral and base solution, lactone forms hydrolyse and convert into carboxylates. Only the lactone forms of CPTs are biologically active. Because of strong affinity of the carboxylate form of the parent drug camptothecin to human serum albumin (HSA), this protein promotes the deactivation of this compound. On the other hand, the lactone forms of camptothecins do not hydrolyse and are stabilized when bound to membranes. The following three hydroxycamptothecins, 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxy-camptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67) were studied. Factor analysis of a set of fluorescence excitation spectra recorded during lactone hydrolysis facilitated the high-throughput determination of the deactivation rates of camptothecin and each hydroxycamptothecin in phosphate buffered saline. The fluorescence spectra of hydroxycamptothecins diluted in HSA solution or suspended in DMPC liposomes were recorded, and the association constants of these drugs to membranes and plasma proteins were calculated. Among the analysed agents, DB-67 exhibited the most desirable properties including low affinity of the carboxylate form for albumin and high affinity of its lactone form for model membranes.

PCA and FA analysis of steady-state fluorescence spectra of camptothecin

Fluorescence spectroscopy methods are widely used in biomedical research. The fluorescence spectra of camptothecin (CPT) -- anticancer agent are analyzed in this paper. CPT can exist in two forms: lactone and carboxylate. Only the lactone form is biologically active. Under physiological conditions (pH 7.4) the lactone form hydrolyses and converts into inactive carboxylate form. There are some differences in fluorescence spectra of lactone and carboxylate form of CPT -- the emission spectra of carboxylate form is slightly red-shifted and is characterized by lower intensity. Principal components analysis (PCA) fallowed by factor analysis (FA) enable to detect presence of both forms on the basis of series of time dependent set of spectra, to follow changes in their contributions and to determine these contributions.

Use of time-resolved fluorescence spectroscopy to evaluate diagnostic value of collagen degradation products.

Abstract. The concentration of collagen degradation products (CDPs) may reflect the process of left ventricular remodeling (LVR). The aim of this study was to evaluate the potential diagnostic usefulness of time-resolved fluorescence spectroscopy (TRFS) in assessment of CDPs. The preliminary experiment was designed to establish if CDPs’ characteristics might be visible by mean fluorescence lifetime (FLT) in determined conditions. The in vitro model of CDPs was prepared by conducting the hydrolysis of type III collagen. The FLT of samples was measured by the time-resolved spectrometer Life Spec II with the subnanosecond pulsed 360-nm EPLED diode. The FLTs were obtained by deconvolution analysis of the data using a multiexponential model of fluorescence decay. In order to determine the limit of traceability of CDPs, a comparison of different collagen/plasma ratio in samples was performed. The results of our study showed that the increase of added plasma to hydrolyzed collagen extended the mean FLT. Thus, the diagnosis of LVR based on measurements using TRFS is possible. However, it is important to point out the experiment was preliminary and further investigation in this field of research is crucial.

SERS and MEF: promising techniques for biomedicine

SERS and MEF phenomena are the result of resonance excitation of localized surface plasmons in nanoparticles of some metals. By adequate choice of shapes and sizes of metallic nanoparticles, kind of adsorbed molecules and wavelength of exciting light, it is possible to achieve enhancement of intensity of Raman scattering equal to 1012-1014. It means that only few molecules inside exciting light beam are sufficient to record the Raman spectrum. Then SERS can be very sensitive tools of trace analysis and very useful in recording Raman spectra of extremely low concentrated biomolecules. The MEF technique can become very useful in study of low fluorescent compounds. In contraire to SERS system for MEF has to be specially prepared to avoid quenching of fluorescence i.e. between the metal nanoparticles and fluorophores should exist the distance layer formed by dielectric or by adsorbed nonfluorescent macromolecules. Increase of fluorescence intensity of low fluorescent compound and possibility of record fluorescence of extremely low concentrated compound cause that MEF can become very promising techniques in biomedical research.

The comparison of biophysical properties of DB-67 and its ester DB-67-4ABTFA determined by fluorescence spectroscopy methods.

Fluorescence spectroscopy methods are applied to the study of camptothecin analogue DB-67 and its ester DB-67-4ABTFA (trifluoroacetic acid salt of 20(S)-aminobutyrate substituted DB-67). Camptothecin and many of its analogues exhibit anticancer properties. They are fluorescent compounds, so using the method of fluorescence anisotropy measurements and fluorescence spectra recording many biophysical properties can be determined including affinity to proteins and membranes. One can also observe the process of conversion of the ester into DB-67. Active lactone form of camptothecin in fluids at pH 7.4 hydrolyses and converts into inactive carboxylate. Process of camptothecin deactivation is accelerated in plasma and after about 2h the total conversion to carboxylate form occurs. It is caused by fast and irreversible binding of carboxylate form to the human serum albumin (HSA). Camptothecin carboxylate bound to HSA does not lactonise. On the other hand, camptothecin lactone binding to membranes is reversible, but as long as lactone form bound to membranes does not hydrolyse. Knowledge of binding properties to proteins and membranes permits to select among many camptothecin analogues the ones exhibiting desirable behavior in physiological conditions: high affinity of lactone form to membranes and low affinity of carboxylate form to albumin. The studied DB-67 and DB-67-4ABTFA fulfill these requirements.

Membranes affinity of promising anticancer agent DB-67 determined by fluorescence spectra analysis

Camptothecins are fluorescent compounds which exhibit anticancer properties. A disadvantage which seriously limits application of camptothecins in antitumor chemotherapy is the hydrolysis of these compounds. They convert into inactive carboxylate forms. The process of hydrolysis is inhibited when the molecules of camptothecin are bound to cell membranes. So it is desirable that camptothecins molecules bind easily to membranes. A quantitative measure of drugs affinity to membranes is the association constant. To determine the association constant to membranes the lipid bilayers i.e. liposomes are used as model membranes. In this work affinity of hydroxycamptothecin DB-67 to model membranes is determined. Fluorescence spectra of this analogue change in presence of liposomes: the fluorescence intensity is bigger and besides green band the blue band appears. The spectra of hydroxycamptothecins change over lipids concentration. On the basis of this changes the association constant to membranes is calculated.

Properties of Ultraviolet Exposed Camptothecin Studied by Using Optical Spectroscopy Methods.

Camptothecin (CPT) and its analogs as inhibitors of topoisomerase I are anticancer compounds. Their antitumor potency is seriously limited due to hydrolysis of lactone form of camptothecins in solutions at pH>5.5, which leads to the formation of inactive carboxylate form with open lactone ring. Furthermore, the clinical application of CPT is also restricted by strong affinity of its carboxylate form to human serum albumin which destabilizes the active lactone form. By UV irradiation of the CPT carboxylate authors of this paper received camptothecin compound which has biophysical properties similar to the lactone form. The specific objective of the project is to determine the properties using the methods of absorption and steady-state fluorescence spectra analysis, fluorescence lifetime measurements as well as steady-state fluorescence anisotropy. The results suggest that the UV exposed camptothecin carboxylate changes the chemical structure. The high-throughput assays based on the steady state fluorescence anisotropy measurements proved that the form obtained as a result of UV irradiation of CPT carboxylate exhibits weaker affinity to albumin than CPT carboxylate however stronger than CPT lactone. This property is very desirable from the point of view of clinical applications.