Determination of Human Serum α1-Acid Glycoprotein and Albumin Binding of Various Marketed and Preclinical Kinase Inhibitors

1964 Current Medicinal Chemistry 2009 16 1964-1977

0929-867309 $5500+00 copy 2009 Bentham Science Publishers Ltd

Determination of Human Serum 1-Acid Glycoprotein and Albumin Bind-

ing of Various Marketed and Preclinical Kinase Inhibitors

Ferenc Zsila1 Ilona Fitos1 Gyula Bencze24 Gyoumlrgy Keacuteri245 and Laacuteszloacute rfi234

1Department of Molecular Pharmacology Institute of Biomolecular Chemistry Chemical Research Center Budapest PO Box 17 H-1525 Hungary 2Semmelweis University Rational Drug Design Laboratory CRC POB 131 1367 Budapest 5 Hungary 3Semmelweis University Department of Pharmaceutical Chemistry Hogyes E u 9 1092 Budapest Hungary 4Vichem Chemie Ltd Herman O u15 1022 Budapest Hungary 5Semmelweis University Department of Medical Chemistry Peptide Biochemistry Research Group of the Hungarian Academy of Sciences POB 260 1444 Budapest 8 Hungary

Abstract There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been mar-keted already for the treatment of cancer This time 139 validated kinase targets are in the focus of drug research of phar-maceutical companies and big efforts are made for the development of new druglike kinase inhibitors Plasma protein binding is an important factor of the ADME profiling of a drug compound Human serum albumin (HSA) and 1-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach HSA binding was also evaluated by affinity chromatography Protein binding interactions of twenty-six kinase inhibitors are characterized The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed Structural biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed

Keywords Acute phase proteins Human serum 1-acid glycoprotein Human serum albumin Induced circular dichroism Plasma protein binding Tyrosine kinase inhibitors

INTRODUCTION

Malignant diseases are characterized by several bio-chemical alterations relevant from diagnostic andor thera-peutic point of views Blood levels of positive acute phase proteins such as 1-acid glycoprotein (AAG orosomucoid) C-reactive protein ceruloplasmin haptoglobin and serum amyloid-A are increased in neoplasms and may correlate with disease severity depending on the stage and effect of therapy [1] The normal plasma concentration of human se-rum AAG is elevated in patients with malignant tumors From the normal average values ranging between 05-10 gL (20-30 M) the AAG level increases up to five fold in ma-lignant diseases [2] In contrast plasma concentration of the negative acute-phase protein human serum albumin (HSA

600 M) is markedly depressed in progressive malignan-cies [3]

One of the important determinants of the drug distribu-tion in the human body is the extent of plasma protein bind-ing Beside albumin AAG is the most important drug bind-ing protein in plasma able to transport various therapeutic agents (Table 1) [2 16 17] In several cases the minor AAG plasma component was found to be responsible for the strong plasma protein binding of some drugs [18-20] The effect of the AAG binding on drug distribution and bioavailability is more pronounced in diseases featured with elevated plasma

Address correspondence to this author at the Department of Molecular Pharmacology Institute of Biomolecular Chemistry Chemical Research Center H-1525 Budapest PO Box 17 Hungary Fax (+36) 1-325-7750 E-mail zsferichemreshu

AAG level [21-23] Anticancer compounds are especially important from this point of view since AAG serum concen-tration is increased in several neoplasia A recent finding highlights further the drug binding role of AAG showing that malignant cells themselves also can express this protein [24] Thus AAG should be considered not only a serum compo-nent but also the component of the tumor microenvironment where its drug binding ability can locally modify the tissue transfer and bioavailability of anticancer agents Neverthe-less however binding data of anticancer drugs-AAG inter-actions are scattered in the literature and no systematic stud-ies can be found (Table 2)

Kinase inhibitors are an important class of anticancer drugs being involved in a variety of cellular processes in-cluding cell-cycle regulation cellular signaling initiation of protein synthesis cell proliferationdifferentiation and apop-tosis The first protein kinases were discovered in the late 1970s and after 30 years 518 human kinases are identified [33] The protein kinases have now become the second most important group of drug targets They account for 20-30 of the drug discovery programs of big pharmaceutical compa-nies

The first inhibitors were developed in the early 1980s Staurosporine an antifungal agent that was isolated from the genus of Streptomyces was a nanomolar inhibitor of protein kinase C and this really drew the attention of pharmaceutical industry [34] Now about 60 kinase inhibitors are under clinical evaluation against different cancers inflammation diabetes and neurodegenerative diseases

At present some of the most promising drugs in devel-opment as anticancer agents are inhibitors of protein tyrosine

AAG and HSA Binding of Kinase Inhibitors Current Medicinal Chemistry 2009 Vol 16 No 16 1965

Table 1 Changes of Plasma Levels of AAG and HSA in Malignant Disorders

AAG HSA Ref Malignant disorders

plasma level changes

breast cancer 23 [4]

chronic lymphocytic leukemia 15 08 [5]

gastrointestinal carcinomas 26

30

06

[6]

[7]

[8]

laryngeal cancer [9]

lung cancer 24

24

[4]

[7]

lymphomas 27

09-04

[7]

[10]

malignant mesothelioma 30 [11]

neuroblastoma 25 [12]

ovarian cancer 16

28

[4]

[7]

various cancers 18 07

08

09

[13]

[14]

[15]

Table 2 AAG Binding Affinity of Various Anticancer Compounds Reported in the Literure Asterisks Denote nKa Values where nis the Number of Binding Sites Per a Protein Molecule and Ka is the Association Constant DACA N-[2-

(dimethylamino)ethyl]acridine-4-carboxamide

Anticancer drug Class Ka (M-1

) Ref

paclitaxel antimicrotubule agent 14 105 [25]

roscovitine cycline dependent kinase inhibitor 20 105 [26]

acridine-4-carboxamide 78 104 [27]

amsacrine 30 104 [25]

asulacrine 24 106 [25]

DACA 34 105 [25]

daunomycin 24 104 [28]

daunorubicin 28 104 [29]

doxorubicin 94 103 [29]

iododoxorubicin 34 104 [29]

mitoxantrone 20 104 [29]

pirarubicin

DNA-intercalating agents

17 104 [29]

staurosporine 11 107 [30]

UCN-01 29 108 [30]

UCN-02

protein kinase inhibitors

15 106 [30]

camptothecin 87 103 [25]

etoposide topoisomerase inhibitors

29 104 [25]

imatinib 24 106 [19]

gefitinib

tyrosine kinase inhibitors

11 105 [31]

S12363 60 105 [32]

vinblastine 94 106 [20]

vincristine Vinca alkaloids

12 105 [25]

1966 Current Medicinal Chemistry 2009 Vol 16 No 16 Zsila et al

kinases Progress in this area has been greatly influenced by seminal studies that were carried out 15-20 years ago which identified important compound classes such as quinazolines and tyrphostins which potently inhibit protein tyrosine kinases

Imatinib (Gleevec ) was the first member of a new gen-eration of kinase inhibitors approved for clinical use in the United States in 2001 Since then 12 small molecule drugs went through clinical evaluation (Table 3) Imatinib was the first from the dozen molecules which had a proven different binding mode compared to pure ATP competitive agents It is successfully used in chronic myelogenous leukemia (CML) gastrointestinal stromal tumors (GIST) and a number of other malignancies One study demonstrated that imatinib mesylate was effective in patients with systemic mastocyto-sis including those who had the D816V mutation in c-Kit

Gefitinib (Iressa ) was the first selective inhibitor of the tyrosine kinase domain of epidermal growth factor receptor (EGFR) The target protein is also sometimes referred to as Her1 or ErbB-1 Sorafenib (Nexavar ) is unique in targeting the RafMekErk pathway It was approved by the FDA and received an EU marketing authorization in 2006 The Euro-pean Commission has granted marketing authorization to Nexavar tablets for the treatment of patients with hepatocel-lular carcinoma the most common form of liver cancer in 2007 [35] Dasatinib is an oral dual BCRABL and Src fam-ily tyrosine kinases inhibitor approved for use in patients with CML after imatinib treatment and Philadelphia chromo-some-positive acute lymphoblastic leukemia It is also being assessed for use in metastatic melanoma [36] Sunitinib (Sutent ) was approved by the FDA for the treatment of re-nal cell carcinoma and imatinib-resistant GIST in 2006 It was the first cancer drug simultaneously approved for two different indications It has become the standard of care for both of these cancers and is currently being studied for the treatment of many others [37] Similarly to gefitinib er-lotinib (Tarceva ) also specifically targets the EGFR tyro-sine kinase which is highly expressed and occasionally mu-tated in non-small cell lung cancer pancreatic cancer and

several other types of cancer It binds in a reversible fashion to the ATP binding site of the receptor [38] Nilotinib has been shown to have an approximately 20-fold increased po-tency in kinase and proliferation assays compared to imatinib [39] Lapatinib is used in therapy against solid tumors such as breast cancer It inhibits receptor signal processes by bind-ing to the ATP-binding pocket of the EGFRHER2 protein kinase domain preventing self-phosphorylation and subse-quent activation of the signal mechanism [40] Vandetanib is a novel selective dual inhibitor of the vascular endothelial growth factor receptor (VEGFR) pathway and EGFR path-way

AAG has been identified as an important mediator of pharmacological resistance to imatinib in patients with acute phase CML [41] The avid binding of imatinib to AAG (Ta-ble 2) decreases free concentration of the drug resulting in reduced inhibition of Abl kinase in a dose-dependent man-ner In vitro plasma protein binding data indicated about 6-fold higher affinity of gefitinib for AAG compared to HSA which could account for the observed inter-subject differ-ences in plasma binding in cancer patients having elevated serum AAG levels [31] The unusual pharmacokinetic fea-ture of the protein kinase C-selective inhibitor UCN-01 (7-hydroxystaurosporine) was attributed to its extremely tight binding to AAG (Table 2) which strongly reduced the phar-macologically active free plasma level of the drug [18] However no specific concern has been expressed for study-ing the AAG binding of most kinase inhibitors though there is an increased understanding that the ADME profile should be investigated in the early stages of drug development to avoid failures of promising compounds in clinical trials [42] The pharmacokinetics of kinase inhibitors and other antican-cer drugs show substantial interpatient variability and their AAG binding interactions might contribute to the differences in toxicity and efficacy between individuals [31 43]

Thus the present work is aimed to summarize recent ex-perimental results obtained by systematic investigations of invitro AAG and HSA binding features of twenty-six kinase inhibitors already marketed or being in clinical trials (Fig 1)

Table 3 Small Molecular Kinase Inhibitors Target Proteins and the Owner Company

Name Target Company

BIBW 2992 EGFR and Erb2 Boehringer Ingelheim

Imatinib Bcr-Abl Novartis

Gefitinib EGFR AstraZeneca

Sorafenib multiple targets OnyxBayer

Dasatinib multiple targets BMS

Sunitinib multiple targets Pfizer

Erlotinib Erb1 GenentechRoche

Nilotinib Bcl-Abr Novartis

Lapatinib Erb1Erb2 GSK

Vandetanib RETVEGFR AstraZeneca

E7080 RETVEGFR Esai

AAG and HSA Binding of Kinase Inhibitors Current Medicinal Chemistry 2009 Vol 16 No 16 1967

O

O

NH2

O N

N

OH

OH

N

N

NH

O

HN Br

N

NH2

O

O

N

N

NH

N

N

SO

NH

N

N

HN

O

O

O

O N

N

HN Cl

O

O

N

O

F

NH

N

HNO O

N Cl

O

ClHN

N

N

O

O N

N

HN

N

N N

O

NH

FF

F

N

N

NH

NH

OCl

F

F

F

O

N

NH

O

1 2 3

4 5 6

78

9 10

11 12

PD98059 AG 1433 EKI-785

ER27319 10-DEBC axitinib

erlotinib gefitinib

bisindolyl-maleimide I bosutinib

nilotinib sorafenib

1968 Current Medicinal Chemistry 2009 Vol 16 No 16 Zsila et al

(Fig 1) Contdhellip

N

N

HN

F

ClHN

O

N

O

ON+

O

O

O

O

N

NO

O

HN Cl

N

N

N

S

N N

HN

NH

O Cl

Cl

N

N

HO

N

HNN

F

Cl

O

HN

ON

N

N

HN

F Br

O

O

N

N

N

HN

Cl

N

HN

N N

O

O

O

NH

O

OOH

Cl

N

HO

HO

N

NN

N

HN

Cl

OH

O

NH

OHNH

FNH

NH

O N

O

13

canertinib

14

chelerythirine

15

AG 1478

16

AG 1295

17

dasatinib

18

pelitinib

19

vandetanib

20

vatalanib21

staurosporine

22

flavopiridol

23

24

purvalanol B

sunitinib

AAG and HSA Binding of Kinase Inhibitors Current Medicinal Chemistry 2009 Vol 16 No 16 1969

(Fig 1) Contdhellip

N

N

N

N

HN

F

ClHN

HN

HN

N N

O

O

O

NH

OH

25

BIBX 1382

26

UCN-01

Fig (1) Chemical structures of kinase inhibitor drugs studied in this work

OVERVIEW OF STRUCTURAL AND BIOLOGICAL

PROPERTIES OF HUMAN SERUM 1-ACID GLYCOPROTEIN

Structure of AAG

AAG is an 36-38 kDa 1-globulin protein with five N-complex type glycans attached to asparaginyl residues at positions 15 38 54 75 and 85 [16 44] Its carbohydrate content is about 40 and 12 of the carbohydrate moiety is sialic acid residues which account for the negative charge of the protein (pI = 27-32) The single polypeptide chain of AAG stabilized by two disulfide bonds consists of 183 amino acid residues AAG exists in two main polymorphic forms called lsquoF1Srsquo (ORM1) and lsquoArsquo (ORM2) genetic vari-ants which differ from each other in 22 residues [45 46] The [rsquoF1Srsquo][rsquoArsquo] ratio in pooled normal human plasma is approximately 7030 [47] By investigating breast lung and ovary cancer groups a recent report concluded that the [rsquoF1Srsquo][rsquoArsquo] serum ratio in healthy subjects and in cancer patients were not significantly different suggesting the con-tribution of both variants to the increased plasma concentra-tion of AAG in malignant diseases [4] -sheet is dominant in the secondary structure of AAG ( 40) with a small amount of -helical structure ( 15) [48] AAG is homolo-gous with a class of proteins called lipocalins [49] in which the -barrel is a common structural motif built up from eight antiparallel -strands enclosing a central cavity Crystal structure of AAG solved very recently shows that its large -barrel cavity is divided into three distinct pockets the cen-tral deep hydrophobic site and the two adjacent smaller but negatively charged binding pockets allow the accomodation of both apolar and basic ligands providing explanation for the the broad spectrum of basic and neutral compounds known to bind to AAG [50]

Biological Properties of AAG

AAG is mainly synthesized and metabolized by the liver with a half-life of approximately 55 days [44] Its hepatic overexpression triggered by various noxa (cancer trauma infections inflammations) is regulated by pro-inflammatory cytokines including IL-1 IL-6 chemokines (IL-8) and glu-cocorticoids [51] There is a growing body of evidence however that AAG is also expressed at extra-hepatic levels such as in leukocytes [44 51-53] and even in cancer cells

[24] The principal biological function of AAG is still unre-solved although it is usually accepted as a natural immuno-modulatory and anti-inflammatory agent [51 54] Within the lipocalin family AAG is classified as immunocalin the group of small proteins which modulate immune and in-flammatory responses [55]

Drug Binding Properties of AAG

AAG binding of more than 300 pharmaceutical agents has been reported majority of which are basic (cationic) compounds such as -blockers local anesthetics and phe-nothiazine drugs [16 56] Besides basic drugs AAG binds some neutral and acidic ligands too such as steroid hor-mones [48 57 58] and coumarin type anticoagulants [47 59 60] It should be noted that the ligand binding ability of AAG is related to its polypeptide moiety and practically nei-ther the sialic acid residues nor the glycan chains are in-volved in this process [30 61 62] Ionic H-bonding and hydrophobic interactions (van der Waals - stacking) seem to play a decisive role in the ligand binding of AAG [63] eg the conserved Trp25 residue of the central cavity makes important contribution of the binding of several ligands [50 64] Genetic polymorphism of AAG however adds variabil-ity to its ligand-binding properties Although most AAG ligands show no variant selective binding some drugs ex-hibit preferential affinity to the lsquoArsquo (eg disopyramide methadone amitriptyline) or the lsquoF1Srsquo variant (eg imatinib warfarin dipyridamole) [19 47]

BRIEF SUMMARY OF THE INDUCED CIRCULAR

DICHROISM SPECTROSCOPY APPROACH USED TO STUDY THE AAG BINDING OF KINASE

INHIBITORS

Among the wide variety of biophysical techniques ap-plied for the investigation of drug-protein binding interac-tions circular dichroism (CD) spectroscopy provides bothdirect experimental evidence of the complex formation and quantitative data for estimation the binding parameters Upon inclusion of the optically inactive drug molecule into the asymmetric protein matrix it might acquire chirality and thus displays induced CD (ICD) signal It is detected in the difference CD spectrum obtained by arithmetic subtraction of the CD curve of drug-free protein solution from that of the

1970 Current Medicinal Chemistry 2009 Vol 16 No 16 Zsila et al

drug-protein mixture measured under identical experimental conditions [65] Chiroptical investigation of drug-protein complexes can be utilized to obtain the following pharmaco-logically relevant informations

bull Drug-protein binding interactions can be studied un-der a wide variety of experimental parameters (tem-perature pH ionic strength additives etc) including physiological conditions as well

bull Protein binding of achiralracemic or even chiral drugs can be quickly demonstrated by measuring of induced or modified CD bands [19 22 64-66]

bull Dependence of the amplitude of the ICD signal being proportional to the protein-bound drug concentration allows to estimate the binding parameters From se-ries of ICD spectroscopic data obtained by increasing the drug concentration in the protein solution the equilibrium association binding constant (Ka) and the number of binding sites (n) can be calculated [19 22 60]

bull This method allows to investigate protein binding of strongly hydrophobic drugs of which low aqueous solubility andor adsorption to various surfaces cause difficulties when using other techniques such as ul-trafiltrationdialysis and affinity chromatography

bull The UVVIS absorption spectra recorded simultane-ously by the CD equipment may provide additional insight into the binding process eg bathochromic (red) shift of the drug absorption maxima indicates inclusion of the drug molecule into a hydrophobic protein binding site [19 22 64 67] Less frequently

conformational alteration occurs during accomodation of the ligand at the binding site may result in hypso-chromic (blue) shift of the absorption peaks Absorp-tion band shifts might be indicative to ligand-protein interactions even in the absence of ICD signals when the ligand binds to the host by ldquoCD silentrdquo manner [22]

In our previous studies the ICD method has been suc-cessfully used to determine the AAG binding parameters of various pharmaceutical substances such as imatinib [19] and antimalarial agents [22] as well as conformational selectiv-ity diazepam protein binding [67]

The binding data of kinase inhibitors (Fig 1) summarized here were calculated from ICD spectra recorded under physiological conditions (37 ordmC pH 74 Ringer buffer) at various [drug][AAG] ratios The concentration of AAG used in the ICD titration experiments was 20-30 M which is close to the normal plasma level of the protein

AAG BINDING PROPERTIES OF KINASE INHIBITORS

Some Qualitative Features of the ICD and UVVIS Spec-tra of Kinase Inhibitor-AAG Complexes

The formation of kinase inhibitor-AAG complexes re-sulted in ICD bands in the difference CD spectra showing various intensities and either positive or negative sign (Table 4)

In some cases AAG binding produced only a single posi-tive or negative ICD peak in the main UV absorption region

Table 4 AAG Binding Parameters of Kinase Inhibitors Estimed from ICD Data Wavelength of the Data Collection and Maxi-

mum Molar ICD Values are Indicated (37 ordmC pH 74 Ringer Buffer)

Kinase inhibitors (nm) of ICD detection max (M-1

cm-1

) Ka (M-1

) n

PD98059(1) 2920 -21 50 (plusmn08) 105 04

AG 1433(2) 3862 -20 56 (plusmn24) 104 15

10-DEBC(5) 2440 +685 94 (plusmn12) 105 06

axitinib(6) 3492 -45 26 (plusmn06) 104 12

bisindolyl-maleimide I(9) 2682 -238 26 (plusmn02) 105 06

bosutinib(10) 2726 -128 49 (plusmn07) 104 03

canertinib(13) 2934 -45 23 (plusmn04) 105 09

chelerythrine(14) 2820 -148 78 (plusmn08) 104 03

dasatinib(17) 3334 -24 37 (plusmn15) 105 09

pelitinib(18) 2694 -78 28 (plusmn02) 105 12

EKI-785(3) 2400 -191 35 (plusmn08) 104 03

ER27319(4) 2746 +72 13 (plusmn03) 105 03

erlotinib(7) 2568 -84 12 (plusmn02) 105 12

gefitinib(8) 2518 -174 12 (plusmn01) 105 06

nilotinib(11) 2900 -384 17 (plusmn04) 106 03

sorafenib(12) 2644 -105 54 (plusmn11) 105 03

tyrphostin AG 1478(15) 2540 -75 14 (plusmn05) 105 11

tyrphostin AG 1295(16) 3680 -80 50 (plusmn14) 105 18

vandetanib(19) 2518 -233 10 (plusmn01) 105 03

vatalanib(20) 2558 -58 48 (plusmn13) 105 10

AAG and HSA Binding of Kinase Inhibitors Current Medicinal Chemistry 2009 Vol 16 No 16 1971

of the drug (eg 10-DEBC(5) gefitinib(8) erlotinib(7) vandetanib(19)) while multiple ICD signals were measured with inhibitors having more complex extended chromo-phoric systems (axitinib(6) nilotinib(11) tyrphostin AG 1295(16) PD98059(1) bisindolyl-maleimide I(9) pelit-inib(18) EKI-785(3)) For kinase inhibitors having sterically restricted planar (chelerythrine(14) staurosporine(21)) or closely planar chromophores (10-DEBC(5) ER27319(4)) chiral - intermolecular exciton interactions with the neighbouring tryptophan side-chains of the protein environ-ment can be responsible for the generation of ICD activity [64] This mechanism should be important also for inhibitors having quinazoline- (eg canertinib(13) erlotinib(7) vande-tanib(19)) quinoline- (bosutinib(10)) and phthalazine-type (vatalanib(20)) chromophores (Fig 1) since principal ICD bands of these drugs were displayed in the - type UV absorption region of the planar heterocyclic rings Further-more all inhibitors possessing phenyl-1-naphtylamine-like structure (BIBX 1382(25) bosutinib(10) canertinib(13) pelitinib(18) EKI-785(3) erlotinib(7) gefitinib(8) tyr-phostin AG 1478(15) vandetanib(19) vatalanib(20)) exhibit negative ICD bands in the - absorption region of the N-heterocycles (lt280 nm Table 4) suggesting their similar steric orientation at the binding site in relation to the adjacent aromatic residue(s) Among the three tryptophan residues of AAG Trp160 is located at surface of the protein Trp25 lies in the deep of the hydrophobic ligand binding -barrel and Trp122 is positioned at open entrance of the cavity [50 57 68] Presumably Trp25 andor Trp160 are involved in the binding as well as in the generation of ICD spectra of several kinase inhibitors including BIBX 1382(25) erlotinib(7) ge-fitinib(8) staurosporine(21) tyrphostin AG 1478(15) vande-tanib(19) canertinib(13) vatalanib(20) 10-DEBC(5) chel-erythrine(14) bosutinib(10) pelitinib(18) EKI-785(3) and ER27319(4)

The hydrophobic cavity binding concept of these agents is in accordance with the changes observed in the absorption spectra Compared to the data obtained in protein-free me-dium (Ringer buffer or ethanol) bathochromic shifts of dif-ferent extent were measured in the UVVIS spectra of the drugs upon their addition into buffer solution of AAG (Table 5) Such a shift is indicative for the binding of the ligands in the protein matrix of AAG featured with hydrophobic micro-environment [19 22 50 64 67] Furthermore appearance or intensification of the vibrational fine structure of the absorp-tion bands of the AAG-bound kinase inhibitors in relation to UVVIS band characteristics measured in protein-free buffer solution also reflects the accomodation of the drugs at a hy-drophobic protein binding site [64] This phenomenon was observed for erlotinib(7) gefitinib(8) staurosporine(21) tyrphostin AG 1478(15) canertinib(13) vatalanib(20) and EKI-785(3) respectively

AAG binding of few drugs among those listed in Fig (1)can not be determined by ICD method due to different rea-sons The purine analog cyclin-dependent kinase inhibitor purvalanol B(23) was used as a pure enantiomer Thus it exhibits own (intrinsic) CD spectrum which was not altered in the presence of AAG and no absorption band shift was observed in the UV spectrum of the drug suggesting the lack of specific binding interactions with AAG Racemic sample of the flavonoid derivative flavopiridol(22) did not show any

ICD activity in the presence of AAG and UVVIS curves of the ligand measured in AAG and in protein-free buffer solu-tions were identical No ICD bands of sunitinib(24)-AAG mixture were measured but the VIS absorption peak showed a small red shift in AAG solution (Table 5) suggesting lsquoCD-silentrsquo protein association of the drug [22] The intrinsic CD profile of the chiral staurosporine(21) was considerably modified showing sign inversion of its negative CD band at 291 nm in AAG solution compared to the CD spectrum re-corded in Ringer buffer The CD data points plotted in the function of the increasing [drug][AAG] ratios gave an ir-regular lsquoSrsquo-type curve which cannot be used to calculate the binding parameters by our method developed for simple saturation titration curves [19 69]

Distinctly from other kinase inhibitors profile of the ICD spectra and the binding stoichiometry values (n) obtained for

Table 5 Shift of the UVVIS Absorption Maxima of Kinase

Inhibitors Measured in the Presence AAG and in

Protein-Free Ringer Buffer Solution (37 ordmC) rsquoErsquo

Means Ethanolic Solution when Compounds were

Insoluble in Aqueous Medium

Kinase inhibitors max (nm)

AAG free AAG

PD98059(1) 2998 (E) 3040

AG 1433(2) 2674 367 2706

3704

10-DEBC(5) 2422 2440

axitinib(6) 3308 (E) 3338

bisindolyl-maleimide I(9) 2778 2796

bosutinib(10) 2682 2692

canertinib(13) 2506

3324

2546

3402

chelerythrine(14) 2676 2708

dasatinib(17) 3252 3264

pelitinib(18) 2592 2612

EKI-785(3) 3124

3394

3166

3472

ER27319(4) 2676 2684

erlotinib(7) 2462

3322

2478

3342

gefitinib(8) 2488

3302

2514

3346

nilotinib(11) 2686 (E) 278

staurosporine(21) 2918 2956

sunitinib(24) 4312 4334

tyrphostin AG 1478(15) 2494

3312

2516

3334

tyrphostin AG 1295(16) 2624

3516

2646

3504

vandetanib(19) 2490

3298

2508

3308

vatalanib(20) 3280 3364

1972 Current Medicinal Chemistry 2009 Vol 16 No 16 Zsila et al

tyrphostin AG 1295(16) and AG 1433(2) suggest the simul-taneous AAG association of two drug molecules It is to be noted that these compounds bear some structural resem-blance to acridine orange which associates with rsquoArsquo genetic variant of AAG in a dimeric form resulting in exciton-type ICD spectra [60] Similarly to the acridine orange-AAG complexes tyrphostin AG 1295(16) also displayed a bipha-sic ICD band pattern below 300 nm (Fig 2) which is charac-teristic to a left-handed chiral intermolecular exciton copul-ing between the - transitions of the quinoxaline rings of two drug molecules bound close to each other at the AAG binding site [65]

AAG Binding Parameters of Kinase Inhibitors Estimated from ICD Data

Binding parameter values (Ka and n) calculated from the ICD data for kinase inhibitor-AAG binding interactions are shown in Table 4 Majority of the inhibitors exhibits strong binding affinity (14 compounds with Ka gt 105 M-1) while Ka

values of only five molecules were found in the 104-105 M-1

range suggesting less avid drug-AAG binding interactions The multikinase inhibitor nilotinib(11) similarly to its struc-tural predecessor imatinib [19] showed also high-affinity AAG binding (Ka gt 106 M-1) Tricyclic compounds with ba-sic side chain (eg chlorpromazine imipramine amitrypti-line) are high-affinity drug ligands of AAG 10-DEBC(5) the AktPKB inhibitor having a related structure binds to AAG with similarly high affinity

Comparison of the binding data of bisindolylmaleimide I(9) and staurosporine(21) derivatives suggests the role of the aminosugar moiety in the very high-affinity AAG bind-ing of the latter compounds Bisindolylmaleimide I lacks the aminosugar ring and its Ka value though still high smaller by several order of magnitudes in relation to staurosporine(21) drugs

In several cases the calculated number of binding site per AAG molecule is significantly below the value of one which might indicate genetic variant binding selectivity of the ligand under study

HSA Binding of Kinase Inhibitors Studied by Affinity Chromatography

Binding affinity of the drugs to HSA can be evaluated from their retentions on a HSA-Sepharose column since elu-tion volumes correlate with the overall binding a nity val-ues ( nKa) Oxazepam acetate and lorazepam acetate enanti-omers as well as diazepam were used as reference com-pounds with known binding affinities [70] The binding af-finities evaluated for the binding of kinase inhibitors to HSA are shown in Table 6

According to the chromatographic data most kinase in-hibitors showed considerable binding affinity for HSA Weak binding was detected only for ER27319(4) It is to be noted that in case of gefitinib(8) the evaluated HSA affinity is substantially higher compared to the published parameter of 185 104 M-1 [31] Due to poor aqueous solubility soraf-enib(12) could not be investigated by this method but the ICD approach was successfully applied to obtain the binding parameters which indicated low-affinity HSA association (Table 6)

Exceedingly high binding affinities (Ka gt 106 M-1) were found for nilotinib(11) and PD98059(1) The PD98059(1) analogue tyrphostin AG 1295(16) as well as EKI-785(3) also exhibited very strong binding The surprisingly high-affinity of nilotinib(11) was also checked by ICD method in HSA solution and the result (nKa = 13 106 M-1) confirmed the chromatographic parameter (Table 6)

CD measurements were also performed with tyrphostin AG 1295(16) in HSA solution since its ICD band profile in

Fig (2) ICD and UV absorption spectra of tyrphostin AG 1295(16) in the presence of AAG and HSA [drug][protein] ratio is 08 in both cases (pH 74 Ringer buffer 37 ordmC)

AAG and HSA Binding of Kinase Inhibitors Current Medicinal Chemistry 2009 Vol 16 No 16 1973

the presence of AAG revealed its dimeric binding mode ICD curves in Fig (2) suggest that tyrphostin AG 1295(16) binds to HSA also in dimeric form (n 19) though just in opposite (right-handed) chiral arrangement of the quinox-aline rings compared to the AAG-bound state The very strong albumin binding of its dihydroxy derivative AG 1433(2) however occurs in monomeric form (n 09)

The moderate HSA binding affinity obtained for fla-vopiridol(22) agrees well with that of related flavonoid com-pounds [71]

Comparison of AAG and HSA Binding of Kinase Inhibi-tors Pharmacokinetic Implications

In general plasma protein binding is unimportant for drugs showing less than 90 of bound fraction Naturally

besides the extent of protein binding other pharmacokinetic parameters play also role in determination of the unbound level of drugs in plasma which is thought to correlate the clinical effects in vivo [89] In case of large volume of distri-bution (VD) and short plasma elimination half-life (t12) the extensive plasma protein binding of drugs may be in-significant from therapeutic point of view Most kinase in-hibitors studied here are highly protein-bound drugs and HSA makes significant contributions in their serum protein binding (Table 7) It was found that many inhibitors bind to AAG stronger by a one order of magnitude than to HSA but it seems that the ratio of the nKa values (15-77) are not large enough to compensate the high excess of plasma con-centration of HSA ( 600 M) over AAG (20-30 M) This is true even if the increased AAG and reduced HSA plasma levels in malignant diseases (Table 1) are taken into consid-

Table 6 Elution Volumes (Ve) and Corresponding Binding Parameters (nKa) of Kinase Inhibitors Obtained on a HSA-Sepharose

Column (Eluent pH 74 Ringer Buffer Room Temperature)

Sample Ve (mL) nKa (M-1

)

solvent 65 no binding

(R)-oxazepam acetate 11 10 104

(S)-oxazepam acetate 26 80 104

(R)-lorazepam acetate 13 20 104

(S)- lorazepam acetate 185 40 104

diazepam 46 18 105

PD98059(1) 21 60 104

AG 1433(2) 250 11 106

10-DEBC(5) 295 90 104

axitinib(6) 65 25 105

bisindolyl-maleimide I(9) 19 50 104

bosutinib(10) 35 11 105

canertinib(13) 23 66 104

chelerythrine(14) 23 66 104

dasatinib(17) 19 50 104

pelitinib(18) 37 12 105

EKI-785(3) 110 47 105

ER27319(4) 10 10 104

erlotinib(7) 19 50 104

flavopiridol(22) 21 60 104

gefitinib(8) 24 70 104

nilotinib(11) 320 14 106

purvalanol B(23) 25 70 104

sorafenib(12) ndash a21 104

staurosporine(21) 62 21 105

sunitinib(24) 21 60 104

tyrphostin AG 1478(15) 52 21 105

tyrphostin AG 1295(16) 145 62 105

vandetanib(19) 19 50 104

vatalanib(20) 42 16 105

aMeasured by ICD method

1974 Current Medicinal Chemistry 2009 Vol 16 No 16 Zsila et al

eration Clinically relevant effects of AAG binding on the pharmacokinetics of drugs can only be expected at much higher differences in the binding affinities as seen in the case of UCN-01(26) staurosporine(21) and imatinib (Table 7) Furthermore the large VD and low t12 values of inhibitors including axitinib(6) canertinib(13) dasatinib(17) er-lotinib(7) sunitinib(24) vandetanib(19) vatalanib(20) tyr-phostin AG 1478(15) and flavopiridol(22) suggest that plasma protein binding does not limit significantly the tissue availability of these compounds (Table 7) It is to be noted that the long plasma half-life of vandetanib(19) is related to the lack of significant metabolism instead of its extensive plasma protein binding [86]

Nilotinib(11) which is successfully used for treatment of CML in imatinib resistant patients showed the highest affin-ity binding for AAG among the drugs studied here its Ka

value (17 106 M1) is close to that of imatinib [19] Dis-

tinctly from imatinib however this drug binds also to HSA with comparable high affinity suggesting that HSA is the principal binding protein of nilotinib(11) in plasma This result is in contrast with the data provided by the manufac-turer saying that bdquoThe 1-acid glycoprotein (AAG) was found to be the primary binding protein as compared to se-rum albumin in human plasmardquo [81]

Based on the data of Table 7 the extent of human plasma protein binding of kinase inhibitors in drug developmental phase can be evaluated Upon comparison with the reported binding data of marketed inhibitors our results suggest more than 90 plasma protein binding for all these agents except for ER27319(4) Among these compounds the strong AAG binding of canertinib(13) vatalanib(20) AG 1433(2) pelit-inib(18) ER27319(4) 10-DEBC(5) and bisindolyl-maleimide I(9) may have a clinical significance depending on the actual VD and t12 values and drug metabolism

Table 7 Pharmacokinetic Parameters of Kinase Inhibitors Studied here and of Some Reference Compounds

Compound name nKa (AAG)

nKa (HSA)

Plasma

protein

binding

Principal binding

matrix in plasma VD t12 (h) Ref

PD98059(1) 33 gt90a HSA AAG

AG 1433(2) 01 gt99a HSA

10-DEBC(5) 63 gt95a AAG HSA

axitinib(6) 02 gt99a HSA 129-263 L 17-48 [72]

BIBX 1382(25) 46 gt95a AAG HSA

bisindolyl-maleimide I(9) 31 gt95a HSA AAG

bosutinib(10) 01 gt95a HSA 17-21 [73]

canertinib(13) 31 gt90a HSA AAG 1330 L 4 [74]

chelerythrine(14) 04 gt90a HSA

dasatinib(17) 67 94 AAG HSA gt3 Lkg lt4 [75 76]

pelitinib(18) 28 gt95a HSA AAG

EKI-785(3) lt01 gt99a HSA

ER27319(4) 39 lt90 HSA AAG

erlotinib(7) 29 93 HSA AAG 232 L 36 [77]

flavopiridol(22) ndash gt95a HSA 65 Lm2 52 [78]

gefitinib(8) 10 91 HSA 1400 L 48 [79]

imatinib 52b 89-96 AAG 244 L80kg 18 [80]

nilotinib(11) 04 98 HSA AAG 17 [81]

sorafenib(12) 77 995 AAG HSA 223 L 24-48 [82]

staurosporine(21) 48c gt99a AAG HSA

sunitinib(24) ndash 95 HSA 2230 L 40-60 [83]

tyrphostin AG 1478(15) 07 gt99a HSA 23-36 Lkg 05-08 [84]d

tyrphostin AG 1295(16) 15 gt99a HSA

UCN-01(26) gt10000e gt99 AAG 0113 Lkg 581 [18]

vandetanib(19) 06 95 HSA 2680 L 120 [85 86]

vatalanib(20) 30 gt95a HSA AAG 235-316 L 35-5 [87]

Abbreviations t12 plasma elimination half life VD apparent volume of distribution Plasma protein binding VD and t12 values were obtained from references listed in the right column apredicted value (see text) bfrom [19] cnKa for AAG obtained from [30] din rats eestimated from data of [88]

AAG and HSA Binding of Kinase Inhibitors Current Medicinal Chemistry 2009 Vol 16 No 16 1975

Besides plasma AAG is present in the extravascular space expressed by both normal and malignant cells thus bioavailability of drugs showing high-affinity AAG-binding (Kagt105 M-1) might be affected at tissue level as well From this point of view strong AAG binding of several kinase inhibitors demonstrated here points out the possible role of this protein in decreasing the free fraction of inhibitors in malignant tissues

ACKNOWLEDGEMENT

This work was supported by the research grants of OTKA K69213 OM-000552005 OMFB-006242007 OM-000412007 OM-000812008 and National Office for Research and Technology No 2006ALAP3-014342006 Skilful technical assistance by Ilona Kawka is appreciated

ABBREVIATIONS

AAG = Human serum 1-acid glycoprotein

CD = Circular dichroism

CML = Chronic myelogenous leukemia

EGFR = Epidermal growth factor receptor

GIST = Gastrointestinal stromal tumor

HSA = Human serum albumin

ICD = Induced circular dichroism

UVVIS = Ultraviolet-visible

VEGFR = Vascular endothelial growth factor receptor

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Received January 03 2009 Revised March 05 2009 Accepted March 06 2009