physicochemical characterization and cyclodextrin complexation...

Research ArticlePhysicochemical Characterization and CyclodextrinComplexation of the Anticancer Drug Lapatinib

Gergy Toacuteth1 Aacutedaacutem Jaacutenoska1 Gergely Voumllgyi1 Zoltaacuten-Istvaacuten Szaboacute2

Gaacutebor Orgovaacuten1 Arash Mirzahosseini1 and Beacutela Noszaacutel1

1Department of Pharmaceutical Chemistry Semmelweis University Hogyes Endre U 9 Budapest 1092 Hungary2Department of Drugs Industry and Pharmaceutical Management University of Medicine and PharmacyGh Marinescu 38 540139 Targu Mures Romania

Correspondence should be addressed to Arash Mirzahosseini mirzahosseiniarashpharmasemmelweis-univhu

Received 24 November 2016 Accepted 9 February 2017 Published 1 March 2017

Academic Editor Jaime Villaverde

Copyright copy 2017 Gergo Toth et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Lapatinib (LAP) the tyrosine kinase inhibitor drug with moderate bioavailability was characterized in terms of physicochemicalproperties acid-base characteristics lipophilicity and solubility The highly lipophilic nature of the drug and its extremely lowwater solubility (1198780 = 082 nM) limit the development of a parenteral formulation In order to enhance solubility and bioavailabilityinclusion complex formation with cyclodextrins (CDs) is a promising method of choiceTherefore LAP-CD interactions were alsostudied by a multianalytical approach The stability constants of LAP with native cyclodextrins determined by UV spectroscopyidentified the seven-membered 120573-CD as the most suitable host Continuous variation method (Jobrsquos plot) by 1H NMR showeda 1 1 stoichiometry for the complexes The geometry of the complex was elucidated by 2D ROESY NMR measurements andmolecular modeling indicating that the partial molecular encapsulation includes the fluorophenyl ring of LAP Phase-solubilitystudies with four CDs 120573-CD (2-hydroxypropyl)-120573-cyclodextrin (HP-120573-CD) randomly methylated-120573- (RAMEB-) cyclodextrinand sulfobutylether-120573-cyclodextrin (SBE-120573-CD) show an AL type diagram and highly increased solubility via CD complexationThe results are especially promising with SBE-120573-CD exerting more than 600-fold gain in solubilityThe equilibrium and structuralinformation presented herein can offer the molecular basis for an improved drug formulation with enhanced bioavailability

1 Introduction

Lapatinib (LAP) (Figure 1) is a potent and selective inhibitorof the epidermal growth factor receptor (EGFR) and humanepidermal growth factor receptor 2 (HER2) [1]

The drug is marketed as its ditosylate salt in form of afilm-coated tablet (TyverbTykerb) It is currently used incombination therapy with capecitabine or letrozole for thetreatment of HER2-positive advanced or metastatic breastcancer [2 3] The pharmacokinetic properties of LAP arenot optimal Its oral bioavailability is moderate at best it hasconsiderable interpatient variability [4] and it is significantlyaffected by food intake Both low-fat and high-fat dietsbefore LAP administration increased its systematic exposure[4 5] Besides food intake gastric pH also influences LAPbioavailability Higher gastric pH reduces LAP absorptiontherefore concomitant use of proton-pump inhibitors (PPIs)

can decrease its bioavailability [6] The recommended doseof LAP is 1250ndash1500mg once daily which means taking5-6 tablets at once reducing patient adherence to therapyIncreasing the bioavailability of LAP could reduce the doseThus a novel formulation with high solubility and bioavail-ability is currently an unmet medical need

Enhancement of solubility and the concomitant bioavail-ability can be achieved by variousmeans such as solid disper-sion size reduction cosolvency polymorphism formation ofwater soluble prodrug or complexes [7] Cyclodextrin (CD)complexation is one of themost extensively investigated waysto improve drug solubility and bioavailability [8]

CDs are cyclic oligosaccharides comprising six seven oreight D-glucose units (120572- 120573- and 120574-CD resp) that have beenrecognized as pharmaceutical adjuvants for the past 20 yearsCDs have a hydrophilic surface responsible for their watersolubility and a hydrophobic inner cavity where lipophilic

HindawiJournal of ChemistryVolume 2017 Article ID 4537632 9 pageshttpsdoiorg10115520174537632

2 Journal of Chemistry

O

F

O

O

NH

ClN

1O

2

12

13

1415

1617

N

1920

212425

262728

29 30

31

32

3334

353640

37

38 39

3

45

67

89

10

11

2223

NH

SH3C

18

Figure 1 Chemical structure and numbering of LAP

molecules with adequate dimensions can be accommodatedforming a noncovalently bonded host-guest complex con-ferring an increased aqueous solubility for the encapsulatedmolecule Besides native CDs several modified derivativesare also available differing in nature position of substituentsand average degree of substitution in order to enhance theinclusion capacity and improve the physicochemical prop-erties of the parent native CDs [9 10] Many advantages ofinclusion complexation have been reported such as increasedsolubility enhanced bioavailability improved stability tasteand odor masking reduced volatility and modified drugrelease [11]

So far only a few attempts have been reported to improvethe bioavailability of LAP [12ndash14] To the best of our knowl-edge LAP-CD inclusion complex characterization has yetto be described despite the fact that CD complexation hasbeen proven to increase the solubility bioavailability andanticancer activity of other tyrosine kinase inhibitors erloti-nib gefitinib imatinib and so forth [15ndash19] moreoversulfobutylether-120573-CD was used for solubilizing LAP in someearlier studies [20 21]

Here we report the quantified biorelevant physicochemi-cal properties of LAP On the basis of the protonation con-stants lipophilicity and solubility parameters we focus onthe characterization and optimization of complex formationbetween various CDs and LAP The information presentedherein can be useful for the development of a new improvedLAP formulationThemethods used in this study includeUVand NMR spectroscopy electrospray ionization-mass spec-trometry (ESI-MS) phase-solubility and molecular model-ing

2 Materials and Methods

21 Materials All CDs (120572-CD 120573-CD 120574-CD (2-hydroxypro-pyl)-120573-CD (HP-120573-CD) random methylated 120573- (RAMEB-)CD and sulfobutylether-120573-CD (SBE-CD)) were the productsof Cyclolab RampD Ltd Hungary Degree of substitution (DS)

of HP-120573-CD RAMEB and SBE-CD was sim45 sim12 and65 respectively LAP was purchased from Eurasiarsquos LtdIndia Tris(hydroxymethyl)aminomethane (Tris) H3PO4KH2PO4 KOHNaOHHCl andKClwere of analytical gradefrom commercial suppliers D2O DCl dimethylsulfoxide(DMSO) hexadeuterated dimethylsulfoxide (DMSO-1198896)and n-octanol were obtained from Sigma-Aldrich whileMeOH was from Merck Ultrapure deionized water wasprepared by a Milli-Q Direct 8 Millipore system

22 Determination of Protonation Constant by UV-pH Titra-tion The log119870 value of LAP was determined by UV-pH titration using the D-PAS ultraviolet spectropho-tometer attached to a GLpKa instrument (Sirius AnalyticalInstruments Ltd Forest Row UK) Multiwavelength UVabsorbance of the sample solutionwasmonitored throughoutthe titration using the fiber-optic probe in situ [22] The UV-pH titrations were carried out under nitrogen atmosphere atconstant ionic strength (119868 = 015MKCl) and temperature(119905 = 250 plusmn 05∘C) Due to the very low solubility of LAPthe cosolvent method was used to determine its log119870 valueThe cosolvent protonation constants (log1198701015840 values) weredetermined in six differentMeOH-watermixtures within 23ndash43 (ww) A stock LAP solution of 7mM concentration wasprepared in DMSO In each experiment 75120583L of the stocksolutions was transferred into sample vials containing 15mLof 015MKCl solution to achieve the required sample concen-tration In each experiment the acidity of the sample solutionwas adjusted using 05M HCl to about pHlowast 25 and thentitrated with 05M KOH to about pHlowast 9 Spectral data wererecorded in the 200ndash700 nm region after each pH measure-ment About 20 pH readings and absorption spectra werecollected from each titration The log1198701015840 values were calcu-lated by RefinementPro software

The measured log1198701015840 values in MeOH-water solutionswere then extrapolated to aqueous condition (zero MeOHconcentration) according to the Yasuda-Shedlovsky proce-dure [23] The Yasuda-Shedlovsky extrapolation method isbased on the linear relation between log1198701015840 and the dielectricconstant (120576) of the cosolvent mixture

log1198701015840 + log [H2O] =119886120576 + 119887 (1)

where 120576 is the dielectric constant and log[H2O] is the molarwater concentration of the givenMeOH-watermixtureThreeparallel measurements were carried out

23 Determination of the Intrinsic Solubility by Chasing Equi-librium Solubility Method (Cheqsol) The thermodynamicsolubility of the nonionized form of the samples was deter-mined by the Chasing Equilibrium Solubility (CheqSol)method [24] The apparatus used to perform the solubilitydeterminations was a GLpKa titrator and a D-PAS spectrom-eter controlled by RefinementPro and CheqSol software (Sir-ius Forest Row UK) The titrations were performed undernitrogen atmosphere at 015M ionic strength and 250plusmn05∘Cusing standardized 05M HCl and 05M KOH solutions

Journal of Chemistry 3

Three parallel measurements were carried out The imple-mentation of the experiment is described in more detail inour previous articles [19 25]

24 Partition Coefficient Measurement by the Stir-FlaskMethod The distribution coefficients were measured fromthe absorbance of the molecules before and after partitioningusing standard stir-flask method as we previously reported[26]

The partition coefficient (119875) cannot be measured directlydue to the high log119875 value of LAPTherefore the distributioncoefficient (119863) was determined at acidic pH (pH = 2) wherethemolecule is in dicationic form and at two other pH valuesat pH 583 and 611Three parallel measurements were carriedout at each pH The pH was measured using a Metrohm60204100 combined pH glass electrode and a Metrohm 780pH meter The watern-octanol phase ratio varied between250 and 600 The log119875 values of each macrospecies werecalculated based on the following equation

119863pH = 120594119894119875119894 = 120594LAP2+119875LAP2+ lowast 120594LAP+119875LAP+ lowast 120594LAP119875LAP (2)

where 120594 is the mole fraction of the corresponding macrospe-cies of the charge indicated

25 Determination of Cyclodextrin Complex Stability by UVSpectroscopy UV spectroscopic measurements were carriedout on a Jasco V-550 diode-array spectrometer (Jasco Japan)with 10mm quartz cuvettes at 25∘C

LAP was titrated with a constant amount of native CDs(120572- 120573- and 120574-CD) at pH = 2 using 001MHCl as solventA stock solution of each CD was combined with differentvolumes of LAP solutions and filled to the same volume TheUV spectra were measured against a blind solution Threeparallel measurements were carried out for each CD Thecomplex formation equilibrium can be characterized by thefollowing complex stability constant

119870stab =[CD-LAP][CD] [LAP] (3)

where [CD] [LAP] and [CD-LAP] are the equilibriumconcentrations of the CD LAP and the complex respectivelySince the total concentrations of CD ([CD]119879) and LAP([LAP]119879) are known assuming 1 1 stoichiometry we can usethe following relationships

[CD]119879 = [CD] + [CD-LAP][LAP]119879 = [LAP] + [CD-LAP]

(4)

Since absorbance of any of the CDs is negligible and noassociation other than the CD-LAP formation takes placeit can be safely assumed that only LAP and the complexcontribute significantly to the absorbance therefore themeasured absorbance at any given wavelength is the weighedlinear combination of the two absorbances (1205760 and 1205761 beingthe molar extinction coefficients of LAP and the complex at agiven wavelength resp)

119860 = 1205760 [LAP] + 1205761 [CD-LAP] (5)Since the equilibrium concentration of LAP can be expressedin terms of the total LAP and the complex concentrations theabove equation takes on the following form

119860 = 1205760 [LAP]119879 + (1205761 minus 1205760) [CD-LAP] (6)

The complex concentration can be expressedwith the stabilityconstant (119870stab) and the total concentrations

119870stab =[CD-LAP]

([CD]119879 minus [CD-LAP]) ([LAP]119879 minus [CD-LAP]) (7)

Solving the above quadratic equation for [CD-LAP] one canderive an equation for absorbance (using (6)) which if fittedonto the data provides the complex stability constant

119860 = 1205760 [LAP]119879

+ (1205761 minus 1205760)(119870stab [CD]119879 + 119870stab [LAP]119879 + 1) minus radic(119870stab [CD]119879 + 119870stab [LAP]119879 + 1)2 minus 4119870stab2 [CD]119879 [LAP]119879

2119870stab

(8)

Multiple fitting was performed with Opium program

26 NMR Measurements All NMR measurements werecarried out on an AgilentVarian VNMRS spectrometer(600MHz for 1H) with a 5mm inverse-detection gradientprobehead inD2ODMSO-1198896 73 (vv)mixtures at 25∘C pDlowastwas set to 2 using DCl solution Addition of DMSO-1198896 wasnecessary due to the low solubility of LAP in aqueous solventseven at acidic pH The chemical shifts were referenced to the

solvent signal of internal DMSO Standard pulse sequencesand processing routines available in VnmrJ 22CChempack40 were used

For determination of complex stoichiometry Jobrsquos con-tinuous variation method was used Solutions were preparedfrom LAP and 120573-CD in complementary amounts tomake upa 15mM summary concentration The solutions were mixedin different ratios and the 1H NMR spectra were recordedafter 24 h


The average extent of penetration and the direction ofinclusion were investigated by 2D ROESYNMR Intermolec-ular NOEs between LAP and CD protons directly involvedin the host-guest interaction were detected as cross-peaksIn these experiments the samples contained 1mM LAP and2mM 120573-CD The ROESY spectrum was recorded applyinga sweep width of 6 kHz and a spinlock of 3 kHz for amixing time of 300ms 256 increments were collected with32 repetitions and the measured data matrix was processedas a matrix of 4K (1198652) by 1K (1198651) data points

27 ESI-MS Experiments High resolution accurate massof LAP-120573-CD inclusion complex was determined with anAgilent 6230 time of flight (TOF)mass spectrometer (AgilentGermany) Samples were introduced by the Agilent 1260Infinity LC system and the mass spectrometer was operatedin conjunction with a Jet Stream electrospray ion source inpositive ion mode Reference masses of mz = 121050873and 922009798 were used to calibrate the mass axis duringanalysisThe following ion source parameterswere employedflow and temperature of the drying gas 10 Lmin and 320∘Cpressure of the nebulizer gas (N2) 45 psi capillary voltage3000V nozzle voltage 500V sheath gas flow and tempera-ture 10 Lmin and 320∘C respectively TOF-MS parameterswere as follows fragmentor voltage 300V skimmer poten-tial 170V OCT 1 RF Vpp 750V 1 120583L of sample solutioncontaining 1mM LAP and 1mM 120573-CD was directly injectedinto themass spectrometer Data were acquired in a 100ndash3000mzmass range and peaks of the hypothesized complex weresearched for in the 1000ndash3000mz intervalMass spectrawereprocessed by Agilent MassHunter B0400 software

28 Phase-Solubility Studies LAP phase-solubility studieswere performed according to the method described byHiguchi and Connors [27] Excess amount of LAP (5-6mg) was added to 5mL stock solution with increasingconcentrations of different CDs (120573-CD HP-120573-CD RAMEBand SBE-CD) at pH 20 at 015M ionic strengthThe obtainedsuspensions were stirred for 24 hours After sedimentationLAP concentrations were measured from the supernatantspectrophotometrically on a Jasco V-550 instrument at269 nmThree parallel measurements were carried out

Assuming 1 1 stoichiometry the stability constant of thecomplex (119870stab) can be determined using the phase-solubilitydiagram according to the following equation

119870stab =slope

1198780 (1 minus slope) (9)

where 1198780 is the intrinsic solubility of LAP in the absence ofCDs

29 Molecular Modeling Molecular modeling study wasperformed as previously reported [19 28 29] by usingthe semiempirical PM3 method of Hyperchem Professionalsoftware (version 80) The initial structures of neutral LAPand 120573-CD were constructed with the help of Hyperchem 80and optimized with PM3 A docking procedure was used tocalculate the energy of the complex LAP was moved into

the CD cavity from the wider rim step-by-step performing ageometry optimization after each step until the conformationwith the lowest energy was achieved The docking procedurewas repeated using different starting positions and the lowestachieved energy was considered as the optimal geometry ofthe complex

The stability of the complex was estimated from theformation energy

Δ119864 = 119864complex minus (119864LAP + 119864CD) (10)

3 Results and Discussion

31 Physicochemical Characterization of LAP Determinationof the physicochemical properties of drugs is crucial tounderstand the pharmacokinetic behavior at the molecularlevel Therefore protonation constants octanolwater parti-tion coefficient and the intrinsic solubility of LAPwere deter-mined Due to the very low solubility of LAP the protonationconstants can only be determined by UV-pH titration withmixed-solvent procedure (Figure 2(a)) In ourmeasurementsthe MeOH-water cosolvent system was used where theapparent protonation constants (log1198701015840 values) were deter-mined in six different mixtures then the aqueous log119870s wereobtained by extrapolation using Yasuda-Shedlovsky method(see (1)) taking into account the dielectric constant of thedifferent cosolvent mixtures (Figure 2(b)) This way log1198701 =772 plusmn 007 and log1198702 = 508 plusmn 005 values were obtained

Using the protonation constants the distribution curvesfor protonation macrospecies of LAP can be depicted as afunction of the pH (Figure 3(a)) The diagram shows that inacidic media at the pH of the stomach the dicationic formis overwhelmingly dominant The compound exists mainlyin monocationic form at the pH of the blood (674) AbovepH 8 the neutral form dominates which plays a crucial rolein the transport mechanism in general assuming that orallyadministrated LAP is absorbed mainly from the intestine

Lipophilicity of LAP was characterized in terms of par-tition coefficient which is the most widely used descriptorof lipophilicity in QSAR studies [30] The working range ofthe used stir-flask method is minus4 to +4 in log119863 values limitedby the accuracy of the phase volume measurement Based onthis fact the octanolwater partition coefficient at pH valueswhere the nonionized form dominates cannot be measureddirectly due to the very high log119875 value of LAP Thereforethe log119863 values were measured at lower pHs At pH 2 wherethe molecule is practically 100 dicationic the measuredlog119863pH 2 = 271 plusmn 006 characterizes the lipophilicity of thedicationic species From log119863 values at pH 583 (367plusmn007)pH 611 (390 plusmn 008) and pH 2 (271 plusmn 006) the log119875 valueof the monocationic and neutral species could be calculatedusing (2)The values are summarized in Table 1 while the pH-dependent lipophilicity profile of LAP is in Figure 3(b)

These values indicate that LAP is an extremely lipophilicmolecule and even the dicationic form has the capability topenetrate membranes by passive diffusion These high log119875values also explain the ability of LAP to cross the blood-brainbarrier which could be a further therapeutic approach in


465

496

567

652

713

859

919

207

219

239

261

308

332

356

421

pH

00

02

04

06

08

10

12

14

16A

260 280 300 320 340 360 380 400 440240 420

(ＨＧ)

(a)

135 140 145 150130 160 165 170155

1000

55

60

65

70

75

80

85

90

95

100

ＦＩＡK

s+ＦＩＡ[（

2]

(b)

Figure 2 Determination of protonation constants of LAP by (a) UVpH titration of LAP in 4357 (ww) methanolwater mixture and (b)Yasuda-Shedlovsky extrapolation

00

02

04

06

08

10

X

1410 1284 620

pH

０2+ ００+

(a)

1410 1284 620

pH

０2+

０

０+

minus6

minus4

minus2

0

2

4

6

logD

(b)

Figure 3 (a) Logarithmic distribution diagram for the LAPprotonationmacrospecies (b) Contribution of the LAPprotonationmacrospeciesto the lipophilicity profile (in black broad line)

brainmetastasis [31] As a part of our LAPprofiling its intrin-sic solubility (the equilibrium solubility of the nonionizedform) was measured by the CheqSol method which resultedin a value of only 048 ngmL LAP is a class II drug in theBiopharmaceutics Classification System being of low watersolubility and high permeability which is in good agreementwith our determined physicochemical values These data

quantify that the very poor solubility is the Achilles heelof the LAP administration It is the root cause for the lowbioavailability of the drug the necessity of taking very highdoses These problems could be alleviated by enhancing LAPsolubility through CD inclusion complexation For furtherpharmaceutical formulation development optimization ofCD-complex formation is crucial therefore interactions


times10minus3

（33

（37

（39

02 04 06 08 1000

minus40

minus30

minus20

minus10

00

Δ

(a)

0

500

1000

1500

2000

2500

3000

Rela

tive a

bund

ance

1800 2000 2200 2400 2600 2800 30001600

mz

17155157(71（96ClF439３)

(b)

Figure 4 Determination of LAP-120573-CD stoichiometry (a) Representative Job plot diagrams of selected LAP protons (b) Representative massspectrum of a LAP-120573-CD sample

Table 1 Calculated log119875 values of the different LAP macrospecies

Macrospecies log119875Dicationic 271 plusmn 007Monocationic 315 plusmn 010Neutral 549 plusmn 010

between LAP and CDs were characterized in solution witha wide variety of complementary analytical techniquesand approaches such as phase-solubility studies 1H and2D NMR techniques UV spectroscopy and electrosprayionization-time of flight-mass spectrometry (ESI-TOF-MS)

32 Determination of LAP-120573-CD Stoichiometry NMR Job Plotand ESI-TOF-MS Studies In order to draw adequate conclu-sions on the inclusion process the knowledge of selector-selectand complexation stoichiometry is a prerequisite Thegeneral method to determine complex stoichiometry is Jobrsquoscontinuous variation method In this experiment the 1HNMR chemical shifts (120575) were measured at different LAPconcentration120573-CD concentration (119888LAP119888CD) ratios whilethe sum of 119888LAP + 119888CD was kept constant The calculatedfactors (Δ120575120594LAP) were plotted as a function of LAP molarratio (120594LAP) In all cases the resulting Job plot diagramsshowed maxima at 05 indicating the typical 1 1 bindingstoichiometry Representative Job plot curves of LAP and 120573-CD are shown in Figure 4(a)

The complex stoichiometry was also studied by ESI-TOF-MS technique which provides a powerful means to studynoncovalent host-guest inclusion complexes between a CDand the guest molecule [32] A large number of publicationsprove that a simple fast ESI-MS analysis is suitable for the de-termination of inclusion complex stoichiometry [15 19 33]

however ESI-MS studies imply the question whether theobserved peaks represent actual inclusion complexes orrather electrostatic adducts formed during the electrosprayprocess [34] if however additional adequate techniquesprove that the complex formation ESI-MS is straightforwardway to determine complex stoichiometry Analyzing the MSspectra in the 100ndash1500mz range the protonated molecularion of the uncomplexed LAP (mz 5811419 [M+H]+) theirsource fragment ion (mz 4581027 [M+H]+) and the charac-teristic mz values of uncomplexed 120573-CD (mz 11353732[M+H]+ 11573531 [M+Na]+) can be observed Figure 4(b)shows the renormalized upper part (1500ndash3000 mz) of themass spectrum of a sample containing LAP and 120573-CD in 1 2molar ratio The observed signal at mz 17155157 with gen-erated C71H96ClFN4O39S molecular formula corresponds tothe LAP-120573-CD 1 1 inclusion complex whichmeans that ESI-TOF-MS confirms the NMR Job result verifying the 1 1 stoi-chiometry

33 Determination of Stability Constant by UV UV spec-troscopy can be a sensitive technique to characterize the CDinclusion complexes provided that the guestmolecule under-goes the significant absorption changes upon complexationThis technique can be used for the determination of CDstability constant of molecules with low solubility as wellUsing (8) the stability constants betweenLAP andnativeCDswere determined to select themost suitable hostThe stabilityconstants determined at pH 2 are listed in Table 2

Based on these data we can conclude that concerningthe size the seven-membered 120573-CD is the most suitable hostmolecule for complexation Thus further experiments werecarried out with 120573-CD and its derivatives

34 Structure of the LAP-120573-CD Complex 2D ROESY NMRcombined with molecular modeling is an appropriate way


41403938373635343332

H-3 H

-5 （6

（39 （37

（38（3

81 79 77 75 73 71 69 67 65 6383f2 (ＪＪＧ)

f1(Ｊ

ＪＧ)

(a) (b)

Figure 5 Determination of the LAPndash120573-CD inclusion complex geometry (a) Expansion of 2DROESYNMR spectrum (b) Energy-minimizedstructure of the complex based on the result of molecular modeling study

Table 2 Determined 119870stab values at pH 2 for different LAP-CDcomplexes with UV spectroscopy

CD 119870stab (Mminus1)120572-CD 46 plusmn 8120573-CD 121 plusmn 12120574-CD 89 plusmn 6

to characterize the geometry of inclusion complexes ROESYexperiment offers valuable information on spatial proximitybetween protons of the host and guestmolecules by observingintermolecular dipolar cross-correlations while molecularmodeling seeks the energy-minimized structure which canbe visualized at the atomic level [19 35 36]

2D NMR ROESY spectra for LAPndash120573-CD inclusion com-plex were recorded at pDlowast 2 where LAP exhibits an appro-priate solubility The expansion of this 2D spectrum inFigure 5(a) contains an intramolecular cross-peak betweenH3 (676 ppm) and H6 (352 ppm) nuclei of LAP Fromthe viewpoint of encapsulation intermolecular cross-peaksof the fluorophenyl protons (H373839) with the inner H-3(368 ppm) and H-5 (358 ppm) protons of 120573-CD are crucialDue to the fact that the H39 (749 ppm) proton of LAP givesa stronger cross-peak with the H-3 proton of the CD we canassume that the fluorophenyl moiety is accommodated in theCD cavity from the wider rim Based on ROESY datamolecularmodeling at PM3 level was carried outThe energy-minimization structure is depicted in Figure 5(b)The energyof formation of the complex was found minus107 kcalmol indi-cating that complex formation is thermodynamically favored

35 Phase-Solubility Studies Phase-solubility analysis accord-ing to Higuchi and Connors is an appropriate techniqueto demonstrate changes in drug solubility Moreover sta-bility constants and stoichiometry can also be determined[9] Upon addition of increasing amounts of CDs (120573-CD

Table 3119870stab values calculated from phase-solubility studies for theCDs employed

CD 119870stab (Mminus1)120573-CD 131 plusmn 9RAMEB 331 plusmn 15HP-120573-CD 537 plusmn 20SBE-CD 31622 plusmn 120

RAMEB HP-120573-CD and SBE-CD) at pH 2 linear solubilityenhancement was observed for LAP regardless of the CDemployed (Figure 6)

The resulting AL-type linear positive isotherms reveal1 1 binding stoichiometry further supporting Job plot andESI-TOF-MS results The 119870stab values calculated by (9) aresummarized in Table 3

The determined119870stab value for120573-CD byUV spectroscopyand phase-solubility is in agreement Moreover similarstability constants were determined for other cyclodextrincomplexes of tyrosine kinase inhibitors [15 16 19]The valuesin Table 3 show that the substitution of the 120573-CD enhancesinclusion complex formation stability as observed also in thecase of erlotinib [19] However in the case of imatinib themethylation of the native 120573-CD reduced the stability of thecomplex [15] which can be explained by the structural differ-ences of these anticancer drugs The largest binding constantwas observed for SBE-120573-CD which is totally ionized underthe applied conditions Thus the reason of the more stablecomplex is the additional electrostatic attractions betweenthe multiply negatively charged hosts and the dicationic LAPConcerning the solubility enhancing capabilities it can bestated that CDderivatives enhance the LAP solubility due pri-marily to the inclusion phenomenon which can be extendedby electrostatic forces The results are especially promisingwith SBE-120573-CDwheremore than 600-fold solubility increasecould be achieved


times10minus5

00

05

10

15

20

25

0000 0010 00150005 0025 00300020

-CDHP--CD

RAMEB

(ＧＩＦlowast

＞Ｇ

minus3)

c LA

P

(ＧＩＦlowast＞Ｇminus3)c＄

(a)

times10minus3

SBE-CD

00

02

04

06

08

10

0005 0015 00200000 0010

(ＧＩＦlowast

＞Ｇ

minus3)


c ０

(b)

Figure 6 Phase-solubility diagrams of LAP-CD complexes

4 Conclusion

LAP the anticancer drug was characterized in terms oflipophilicity solubility acid-base and CD-complex forma-tion properties using complementary methods such asUV NMR phase-solubility and molecular modeling Animportant practical result of this work is that CDs especiallythe substituted derivatives significantly increase the LAPsolubility which can be a starting point of a new LAPformulation of improved bioavailability

Competing Interests

The authors declare no conflict of interests

Authorsrsquo Contributions

Gergo Toth and Adam Janoska contributed equally to thiswork

Acknowledgments

The research was supported through the New NationalExcellence Program of the Ministry of Human Capacities(Adam Janoska)

References

[1] G M Higa and J Abraham ldquoLapatinib in the treatment ofbreast cancerrdquo Expert Review of Anticancer Therapy vol 7 no9 pp 1183ndash1192 2007

[2] C E Geyer J Forster D Lindquist et al ldquoLapatinib pluscapecitabine for HER2-positive advanced breast cancerrdquo New

England Journal of Medicine vol 355 no 26 pp 2733ndash27432006

[3] S Johnston J Pippen Jr X Pivot et al ldquoLapatinib combinedwith letrozole versus letrozole and placebo as first-line therapyfor postmenopausal hormone receptormdashpositive metastaticbreast cancerrdquo Journal of Clinical Oncology vol 27 no 33 pp5538ndash5546 2009

[4] H A Burris III C W Taylor S F Jones et al ldquoA phase I andpharmacokinetic study of oral lapatinib administered once ortwice daily in patients with solid malignanciesrdquo Clinical CancerResearch vol 15 no 21 pp 6702ndash6708 2009

[5] L A Devriese K M Koch M Mergui-Roelvink et al ldquoEffectsof low-fat and high-fat meals on steady-state pharmacokineticsof lapatinib in patients with advanced solid tumoursrdquo Investiga-tional New Drugs vol 32 no 3 pp 481ndash488 2014

[6] K M Koch Y-H Im S-B Kim et al ldquoEffects of esomeprazoleon the pharmacokinetics of lapatinib in breast cancer patientsrdquoClinical Pharmacology in Drug Development vol 2 no 4 pp336ndash341 2013

[7] C Leuner ldquoImproving drug solubility for oral delivery usingsolid dispersionsrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 50 no 1 pp 47ndash60 2000

[8] R L Carrier L A Miller and I Ahmed ldquoThe utility ofcyclodextrins for enhancing oral bioavailabilityrdquo Journal ofControlled Release vol 123 no 2 pp 78ndash99 2007

[9] K A Connors ldquoThe stability of cyclodextrin complexes insolutionrdquo Chemical Reviews vol 97 no 5 pp 1325ndash1358 1997

[10] J Szejtli ldquoIntroduction and general overview of cyclodextrinchemistryrdquoChemical Reviews vol 98 no 5 pp 1743ndash1753 1998

[11] T Loftsson and D Duchene ldquoCyclodextrins and their pharma-ceutical applicationsrdquo International Journal of Pharmaceuticsvol 329 no 1-2 pp 1ndash11 2007

[12] H Gao YWang C Chen et al ldquoIncorporation of lapatinib intocore-shell nanoparticles improves both the solubility and anti-glioma effects of the drugrdquo International Journal of Pharmaceu-tics vol 461 no 1-2 pp 478ndash488 2014


[13] L Zhang S Zhang S-B Ruan Q-Y Zhang Q He and H-L Gao ldquoLapatinib-incorporated lipoprotein-like nanoparticlespreparation and a proposed breast cancer-targeting mecha-nismrdquo Acta Pharmacologica Sinica vol 35 no 6 pp 846ndash8522014

[14] Y Song X Yang X Chen H Nie S Byrn and J W LubachldquoInvestigation of drug-excipient interactions in lapatinib amor-phous solid dispersions using solid-state NMR spectroscopyrdquoMolecular Pharmaceutics vol 12 no 3 pp 857ndash866 2015

[15] S Beni Z Szakacs O Csernak L Barcza andBNoszal ldquoCyclo-dextrinimatinib complexation binding mode and charge de-pendent stabilitiesrdquo European Journal of Pharmaceutical Sci-ences vol 30 no 2 pp 167ndash174 2007

[16] Y-H Phillip Lee S Sathigari Y-J Jean Lin et al ldquoGefitinib-cyclodextrin inclusion complexes physico-chemical charac-terization and dissolution studiesrdquo Drug Development andIndustrial Pharmacy vol 35 no 9 pp 1113ndash1120 2009

[17] N Devasari C P Dora C Singh et al ldquoInclusion complexof erlotinib with sulfobutyl ether-120573-cyclodextrin preparationcharacterization in silico in vitro and in vivo evaluationrdquoCarbohydrate Polymers vol 134 pp 547ndash556 2015

[18] S M L Gontijo P P G Guimaraes C T R Viana et alldquoErlotinibhydroxypropyl-120573-cyclodextrin inclusion complexcharacterization and in vitro and in vivo evaluationrdquo Journal ofInclusion Phenomena and Macrocyclic Chemistry vol 83 no 3-4 pp 267ndash279 2015

[19] G Toth A Janoska Z-I Szabo et al ldquoPhysicochemicalcharacterisation and cyclodextrin complexation of erlotinibrdquoSupramolecular Chemistry vol 28 no 7-8 pp 656ndash664 2016

[20] D W Rusnak K Lackey K Affleck et al ldquoThe effects ofthe novel reversible epidermal growth factor receptorErbB-2tyrosine kinase inhibitor GW2016 on the growth of humannormal and tumor-derived cell lines in vitro and in vivordquoMolecular Cancer Therapeutics vol 1 no 2 pp 85ndash94 2001

[21] M J Sambade R J Kimple J T Camp et al ldquoLapatinibin combination with radiation diminishes tumor regrowthin HER2+ and basal-likeEGFR+ breast tumor xenograftsrdquoInternational Journal of Radiation Oncology Biology Physicsvol 77 no 2 pp 575ndash581 2010

[22] K Y Tam and K Takacs-Novak ldquoMulti-wavelength spec-trophotometric determination of acid dissociation constants avalidation studyrdquo Analytica Chimica Acta vol 434 no 1 pp157ndash167 2001

[23] G Volgyi R Ruiz K Box J Comer E Bosch and KTakacs-Novak ldquoPotentiometric and spectrophotometric pKadetermination of water-insoluble compounds validation studyin a new cosolvent systemrdquoAnalytica Chimica Acta vol 583 no2 pp 418ndash428 2007

[24] M Stuart and K Box ldquoChasing equilibrium measuring theintrinsic solubility of weak acids and basesrdquo Analytical Chem-istry vol 77 no 4 pp 983ndash990 2005

[25] G Volgyi E Baka K J Box J E A Comer and KTakacs-Novak ldquoStudy of pH-dependent solubility of organicbases Revisit of Henderson-Hasselbalch relationshiprdquo Analyt-ica Chimica Acta vol 673 no 1 pp 40ndash46 2010

[26] G Toth K Mazak S Hosztafi J Kokosi and B NoszalldquoSpecies-specific lipophilicity of thyroid hormones and theirprecursors in view of their membrane transport propertiesrdquoJournal of Pharmaceutical and Biomedical Analysis vol 76 pp112ndash118 2013

[27] T Higuchi and K A Connors ldquoPhase solubility studiesrdquo inAdvances in Analytical Chemistry and Instrumentation C N

Reilly Ed pp 117ndash212Wiley-Interscience NewYork NY USA1965

[28] K M Al Azzam and E Muhammad ldquoHost-guest inclusioncomplexes between mitiglinide and the naturally occurringcyclodextrins 120572 120573 and 120574 a theoretical approachrdquo AdvancedPharmaceutical Bulletin vol 5 no 2 pp 289ndash291 2015

[29] XWen F Tan Z Jing andZ Liu ldquoPreparation and study the 12inclusion complex of carvedilol with 120573-cyclodextrinrdquo Journal ofPharmaceutical and Biomedical Analysis vol 34 no 3 pp 517ndash523 2004

[30] C Hansch ldquoOn the future of QSARrdquo inMedicinal Chemistry forthe 21st Century C G Wermuth N Koga and H Konig Edspp 281ndash293 Metcalf Blackwell Oxford UK 1994

[31] AMorikawa DM PeereboomQ R Smith et al ldquoClinical evi-dence for drug penetration of capecitabine and lapatinib uptakein resected brainmetastases fromwomenwithmetastatic breastcancerrdquo Journal of Clinical Oncology vol 31 no 15 supplement1 abstract 514 2013 Proceedings of the 2013 ASCO AnnualMeeting

[32] Y Dotsikas and Y L Loukas ldquoEfficient determination andevaluation of model cyclodextrin complex binding constantsby electrospray mass spectrometryrdquo Journal of the AmericanSociety forMass Spectrometry vol 14 no 10 pp 1123ndash1129 2003

[33] N Marangoci M Mares M Silion et al ldquoInclusion complexof a new propiconazole derivative with 120573-cyclodextrin NMRESI-MS and preliminary pharmacological studiesrdquo Results inPharma Sciences vol 1 no 1 pp 27ndash37 2011

[34] J B Cunniff and P Vouros ldquoFalse positives and the detectionof cyclodextrin inclusion complexes by electrospray mass spec-trometryrdquo Journal of the American Society for Mass Spectrome-try vol 6 no 5 pp 437ndash447 1995

[35] D Salvatierra C Jaime A Virgili and F Sanchez-FerrandoldquoDetermination of the inclusion geometry for the 120573-cyclodex-trinbenzoic acid complex by NMR and molecular modelingrdquoJournal of Organic Chemistry vol 61 no 26 pp 9578ndash95811996

[36] Z-I Szabo F Mohammadhassan L Szocs et al ldquoStereoselec-tive interactions and liquid chromatographic enantioseparationof thalidomide on cyclodextrin-bonded stationary phasesrdquoJournal of Inclusion Phenomena andMacrocyclic Chemistry vol85 no 3-4 pp 227ndash236 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


O

F

O

O

NH

ClN

1O

2

12

13

1415

1617

N

1920

212425

262728

29 30

31

32

3334

353640

37

38 39

3

45

67

89

10

11

2223

NH

SH3C

18

Figure 1 Chemical structure and numbering of LAP

molecules with adequate dimensions can be accommodatedforming a noncovalently bonded host-guest complex con-ferring an increased aqueous solubility for the encapsulatedmolecule Besides native CDs several modified derivativesare also available differing in nature position of substituentsand average degree of substitution in order to enhance theinclusion capacity and improve the physicochemical prop-erties of the parent native CDs [9 10] Many advantages ofinclusion complexation have been reported such as increasedsolubility enhanced bioavailability improved stability tasteand odor masking reduced volatility and modified drugrelease [11]

So far only a few attempts have been reported to improvethe bioavailability of LAP [12ndash14] To the best of our knowl-edge LAP-CD inclusion complex characterization has yetto be described despite the fact that CD complexation hasbeen proven to increase the solubility bioavailability andanticancer activity of other tyrosine kinase inhibitors erloti-nib gefitinib imatinib and so forth [15ndash19] moreoversulfobutylether-120573-CD was used for solubilizing LAP in someearlier studies [20 21]

Here we report the quantified biorelevant physicochemi-cal properties of LAP On the basis of the protonation con-stants lipophilicity and solubility parameters we focus onthe characterization and optimization of complex formationbetween various CDs and LAP The information presentedherein can be useful for the development of a new improvedLAP formulationThemethods used in this study includeUVand NMR spectroscopy electrospray ionization-mass spec-trometry (ESI-MS) phase-solubility and molecular model-ing

2 Materials and Methods

21 Materials All CDs (120572-CD 120573-CD 120574-CD (2-hydroxypro-pyl)-120573-CD (HP-120573-CD) random methylated 120573- (RAMEB-)CD and sulfobutylether-120573-CD (SBE-CD)) were the productsof Cyclolab RampD Ltd Hungary Degree of substitution (DS)

of HP-120573-CD RAMEB and SBE-CD was sim45 sim12 and65 respectively LAP was purchased from Eurasiarsquos LtdIndia Tris(hydroxymethyl)aminomethane (Tris) H3PO4KH2PO4 KOHNaOHHCl andKClwere of analytical gradefrom commercial suppliers D2O DCl dimethylsulfoxide(DMSO) hexadeuterated dimethylsulfoxide (DMSO-1198896)and n-octanol were obtained from Sigma-Aldrich whileMeOH was from Merck Ultrapure deionized water wasprepared by a Milli-Q Direct 8 Millipore system

22 Determination of Protonation Constant by UV-pH Titra-tion The log119870 value of LAP was determined by UV-pH titration using the D-PAS ultraviolet spectropho-tometer attached to a GLpKa instrument (Sirius AnalyticalInstruments Ltd Forest Row UK) Multiwavelength UVabsorbance of the sample solutionwasmonitored throughoutthe titration using the fiber-optic probe in situ [22] The UV-pH titrations were carried out under nitrogen atmosphere atconstant ionic strength (119868 = 015MKCl) and temperature(119905 = 250 plusmn 05∘C) Due to the very low solubility of LAPthe cosolvent method was used to determine its log119870 valueThe cosolvent protonation constants (log1198701015840 values) weredetermined in six differentMeOH-watermixtures within 23ndash43 (ww) A stock LAP solution of 7mM concentration wasprepared in DMSO In each experiment 75120583L of the stocksolutions was transferred into sample vials containing 15mLof 015MKCl solution to achieve the required sample concen-tration In each experiment the acidity of the sample solutionwas adjusted using 05M HCl to about pHlowast 25 and thentitrated with 05M KOH to about pHlowast 9 Spectral data wererecorded in the 200ndash700 nm region after each pH measure-ment About 20 pH readings and absorption spectra werecollected from each titration The log1198701015840 values were calcu-lated by RefinementPro software

The measured log1198701015840 values in MeOH-water solutionswere then extrapolated to aqueous condition (zero MeOHconcentration) according to the Yasuda-Shedlovsky proce-dure [23] The Yasuda-Shedlovsky extrapolation method isbased on the linear relation between log1198701015840 and the dielectricconstant (120576) of the cosolvent mixture

log1198701015840 + log [H2O] =119886120576 + 119887 (1)

where 120576 is the dielectric constant and log[H2O] is the molarwater concentration of the givenMeOH-watermixtureThreeparallel measurements were carried out

23 Determination of the Intrinsic Solubility by Chasing Equi-librium Solubility Method (Cheqsol) The thermodynamicsolubility of the nonionized form of the samples was deter-mined by the Chasing Equilibrium Solubility (CheqSol)method [24] The apparatus used to perform the solubilitydeterminations was a GLpKa titrator and a D-PAS spectrom-eter controlled by RefinementPro and CheqSol software (Sir-ius Forest Row UK) The titrations were performed undernitrogen atmosphere at 015M ionic strength and 250plusmn05∘Cusing standardized 05M HCl and 05M KOH solutions












(4)



119860 = 1205760 [LAP]119879 + (1205761 minus 1205760) [CD-LAP] (6)





119860 = 1205760 [LAP]119879


2119870stab

(8)










119870stab =slope

1198780 (1 minus slope) (9)












465

496

567

652

713

859

919

207

219

239

261

308

332

356

421

pH

00

02

04

06

08

10

12

14

16A

260 280 300 320 340 360 380 400 440240 420

(ＨＧ)

(a)

135 140 145 150130 160 165 170155

1000

55

60

65

70

75

80

85

90

95

100

ＦＩＡK

s+ＦＩＡ[（

2]

(b)


00

02

04

06

08

10

X

1410 1284 620

pH

０2+ ００+

(a)

1410 1284 620

pH

０2+

０

０+

minus6

minus4

minus2

0

2

4

6

logD

(b)





times10minus3

（33

（37

（39

02 04 06 08 1000

minus40

minus30

minus20

minus10

00

Δ

(a)

0

500

1000

1500

2000

2500

3000

Rela

tive a

bund

ance

1800 2000 2200 2400 2600 2800 30001600

mz

17155157(71（96ClF439３)

(b)












41403938373635343332

H-3 H

-5 （6

（39 （37

（38（3

81 79 77 75 73 71 69 67 65 6383f2 (ＪＪＧ)

f1(Ｊ

ＪＧ)

(a) (b)













times10minus5

00

05

10

15

20

25

0000 0010 00150005 0025 00300020

-CDHP--CD

RAMEB

(ＧＩＦlowast

＞Ｇ

minus3)

c LA

P


(a)

times10minus3

SBE-CD

00

02

04

06

08

10

0005 0015 00200000 0010

(ＧＩＦlowast

＞Ｇ

minus3)


c ０

(b)


4 Conclusion


Competing Interests




Acknowledgments


References

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of












(4)



119860 = 1205760 [LAP]119879 + (1205761 minus 1205760) [CD-LAP] (6)





119860 = 1205760 [LAP]119879


2119870stab

(8)










119870stab =slope

1198780 (1 minus slope) (9)












465

496

567

652

713

859

919

207

219

239

261

308

332

356

421

pH

00

02

04

06

08

10

12

14

16A

260 280 300 320 340 360 380 400 440240 420

(ＨＧ)

(a)

135 140 145 150130 160 165 170155

1000

55

60

65

70

75

80

85

90

95

100

ＦＩＡK

s+ＦＩＡ[（

2]

(b)


00

02

04

06

08

10

X

1410 1284 620

pH

０2+ ００+

(a)

1410 1284 620

pH

０2+

０

０+

minus6

minus4

minus2

0

2

4

6

logD

(b)





times10minus3

（33

（37

（39

02 04 06 08 1000

minus40

minus30

minus20

minus10

00

Δ

(a)

0

500

1000

1500

2000

2500

3000

Rela

tive a

bund

ance

1800 2000 2200 2400 2600 2800 30001600

mz

17155157(71（96ClF439３)

(b)












41403938373635343332

H-3 H

-5 （6

（39 （37

（38（3

81 79 77 75 73 71 69 67 65 6383f2 (ＪＪＧ)

f1(Ｊ

ＪＧ)

(a) (b)













times10minus5

00

05

10

15

20

25

0000 0010 00150005 0025 00300020

-CDHP--CD

RAMEB

(ＧＩＦlowast

＞Ｇ

minus3)

c LA

P


(a)

times10minus3

SBE-CD

00

02

04

06

08

10

0005 0015 00200000 0010

(ＧＩＦlowast

＞Ｇ

minus3)


c ０

(b)


4 Conclusion


Competing Interests




Acknowledgments


References

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






119870stab =slope

1198780 (1 minus slope) (9)












465

496

567

652

713

859

919

207

219

239

261

308

332

356

421

pH

00

02

04

06

08

10

12

14

16A

260 280 300 320 340 360 380 400 440240 420

(ＨＧ)

(a)

135 140 145 150130 160 165 170155

1000

55

60

65

70

75

80

85

90

95

100

ＦＩＡK

s+ＦＩＡ[（

2]

(b)


00

02

04

06

08

10

X

1410 1284 620

pH

０2+ ００+

(a)

1410 1284 620

pH

０2+

０

０+

minus6

minus4

minus2

0

2

4

6

logD

(b)





times10minus3

（33

（37

（39

02 04 06 08 1000

minus40

minus30

minus20

minus10

00

Δ

(a)

0

500

1000

1500

2000

2500

3000

Rela

tive a

bund

ance

1800 2000 2200 2400 2600 2800 30001600

mz

17155157(71（96ClF439３)

(b)












41403938373635343332

H-3 H

-5 （6

（39 （37

（38（3

81 79 77 75 73 71 69 67 65 6383f2 (ＪＪＧ)

f1(Ｊ

ＪＧ)

(a) (b)













times10minus5

00

05

10

15

20

25

0000 0010 00150005 0025 00300020

-CDHP--CD

RAMEB

(ＧＩＦlowast

＞Ｇ

minus3)

c LA

P


(a)

times10minus3

SBE-CD

00

02

04

06

08

10

0005 0015 00200000 0010

(ＧＩＦlowast

＞Ｇ

minus3)


c ０

(b)


4 Conclusion


Competing Interests




Acknowledgments


References

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


465

496

567

652

713

859

919

207

219

239

261

308

332

356

421

pH

00

02

04

06

08

10

12

14

16A

260 280 300 320 340 360 380 400 440240 420

(ＨＧ)

(a)

135 140 145 150130 160 165 170155

1000

55

60

65

70

75

80

85

90

95

100

ＦＩＡK

s+ＦＩＡ[（

2]

(b)


00

02

04

06

08

10

X

1410 1284 620

pH

０2+ ００+

(a)

1410 1284 620

pH

０2+

０

０+

minus6

minus4

minus2

0

2

4

6

logD

(b)





times10minus3

（33

（37

（39

02 04 06 08 1000

minus40

minus30

minus20

minus10

00

Δ

(a)

0

500

1000

1500

2000

2500

3000

Rela

tive a

bund

ance

1800 2000 2200 2400 2600 2800 30001600

mz

17155157(71（96ClF439３)

(b)












41403938373635343332

H-3 H

-5 （6

（39 （37

（38（3

81 79 77 75 73 71 69 67 65 6383f2 (ＪＪＧ)

f1(Ｊ

ＪＧ)

(a) (b)













times10minus5

00

05

10

15

20

25

0000 0010 00150005 0025 00300020

-CDHP--CD

RAMEB

(ＧＩＦlowast

＞Ｇ

minus3)

c LA

P


(a)

times10minus3

SBE-CD

00

02

04

06

08

10

0005 0015 00200000 0010

(ＧＩＦlowast

＞Ｇ

minus3)


c ０

(b)


4 Conclusion


Competing Interests




Acknowledgments


References

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


times10minus3

（33

（37

（39

02 04 06 08 1000

minus40

minus30

minus20

minus10

00

Δ

(a)

0

500

1000

1500

2000

2500

3000

Rela

tive a

bund

ance

1800 2000 2200 2400 2600 2800 30001600

mz

17155157(71（96ClF439３)

(b)












41403938373635343332

H-3 H

-5 （6

（39 （37

（38（3

81 79 77 75 73 71 69 67 65 6383f2 (ＪＪＧ)

f1(Ｊ

ＪＧ)

(a) (b)













times10minus5

00

05

10

15

20

25

0000 0010 00150005 0025 00300020

-CDHP--CD

RAMEB

(ＧＩＦlowast

＞Ｇ

minus3)

c LA

P


(a)

times10minus3

SBE-CD

00

02

04

06

08

10

0005 0015 00200000 0010

(ＧＩＦlowast

＞Ｇ

minus3)


c ０

(b)


4 Conclusion


Competing Interests




Acknowledgments


References

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


41403938373635343332

H-3 H

-5 （6

（39 （37

（38（3

81 79 77 75 73 71 69 67 65 6383f2 (ＪＪＧ)

f1(Ｊ

ＪＧ)

(a) (b)













times10minus5

00

05

10

15

20

25

0000 0010 00150005 0025 00300020

-CDHP--CD

RAMEB

(ＧＩＦlowast

＞Ｇ

minus3)

c LA

P


(a)

times10minus3

SBE-CD

00

02

04

06

08

10

0005 0015 00200000 0010

(ＧＩＦlowast

＞Ｇ

minus3)


c ０

(b)


4 Conclusion


Competing Interests




Acknowledgments


References

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


times10minus5

00

05

10

15

20

25

0000 0010 00150005 0025 00300020

-CDHP--CD

RAMEB

(ＧＩＦlowast

＞Ｇ

minus3)

c LA

P


(a)

times10minus3

SBE-CD

00

02

04

06

08

10

0005 0015 00200000 0010

(ＧＩＦlowast

＞Ｇ

minus3)


c ０

(b)


4 Conclusion


Competing Interests




Acknowledgments


References

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

physicochemical characterization and cyclodextrin complexation...

Documents