Theoretical and experimental study of a praziquantel and β -cyclodextrin inclusion complex using molecular mechanic calculations and H 1 -nuclear magnetic resonance

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Theoretical and experimental study of a praziquantel and β -cyclodextrin inclusion complex using molecular mechanic calculations and H 1 -nuclear magnetic resonance

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  Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 1428–1432 Short communication Theoretical and experimental study of a praziquantel and   -cyclodextrininclusion complex using molecular mechanic calculationsand  1 H-nuclear magnetic resonance Marcelo Bispo de Jesus a , Luciana de Matos Alves Pinto a , Leonardo Fernandes Fraceto a , b ,Yuji Takahata c , Antonio C.S. Lino d , Carlos Jaime d , Eneida de Paula a , ∗ a  Departamento de Bioqu´ imica, Instituto de Biologia, Universidade Estadual de Campinas, Cidade Universit´ aria Zeferino Vaz,s/n, C.P. 6109, 13083-870 Campinas, SP, Brazil b Faculdade de Farm´ acia, Universidade de Sorocaba, Sorocaba, SP, Brazil c  Departamento de F ´ isico-Qu´ imica, Instituto de Qu´ imica, Universidade Estadual de Campinas, Campinas, SP, Brazil d  Departament de Qu´ imica, Universitat Aut`onoma de Barcelona, 08193 Bellaterra, Spain Received 21 October 2005; received in revised form 23 February 2006; accepted 3 March 2006Available online 5 May 2006 Abstract Praziquantel (PZQ) is a broadly effective anthelminthic drug available for human and veterinary use, being the drug of choice for the treatmentof all forms of schistosomiasis. Nevertheless, large doses are required in order to achieve adequate concentrations at the target site due to thepoor solubility of PZQ and its significant first pass metabolism. To improve it, avoiding efficiency loss, we have designed a controlled-releasesystem, in which PZQ was encapsulated in   -cyclodextrin (  -CD). The inclusion complexes between PZQ/   -CD were studied at two differentstoichiometries 1:1 and 1:2, through experimental and theoretical analysis. Molecular modeling calculations were used to foresee the betterstoichiometry of the complex formed as well as the possible orientations of PZQ inside the   -CD cavity. The complexes prepared were analyzedthrough  1 H two-dimensional nuclear magnetic resonance ( 1 H 2D-NMR) experiments, which provide (evidences) for the 1:1 complexation of PZQ/   -CD.  1 H 2D-NMR also revealed details of PZQ/   -CD molecular interaction, in which the isoquinoline ring of praziquantel is located insidethe  -CD cavity. Finally, phase-solubility diagrams revealed a five-fold increase in praziquantel water solubility upon addition of increasing  -CDconcentrations up to 16mM, corresponding to the solubility of    -CD itself. The solubilization profile is consistent with 1:1 stoichiometry of thePZQ/   -CD complex while the solubilization effect will certainly increase the pharmacological activity of praziquantel.© 2006 Elsevier B.V. All rights reserved. Keywords:  Praziquantel;   -Cyclodextrin; Molecular mechanics; Inclusion compounds; Nuclear magnetic resonance 1. Introduction Schistosomiasis is an infectious disease with enormous pub-lic health and socio-economic importance in the developingcountries [1]. It was recently estimated that the global numberof people infected with  Schistosoma  spp. is around 200 million,with 600 million being at risk of infection and 20 million suffer-ing severe debilitating illness [2]. PZQ is the drug of choice forthetreatmentofallformsofschistosomiasis,butitsaqueoussol-ubilityisconsiderablylow,afactthatrestrictsPZQdeliveryonlyvia the oral route [3]. Searching for a controlled-release system ∗ Corresponding author.  E-mail address:  depaula@unicamp.br (E. de Paula). that could improve PZQ bioavailability, we have investigatedformulationswhere  -cyclodextrin(  -CD)wasthedrug-carrier.Cyclodextrins (CDs) are cyclic oligosaccharides composed by  -(1,4)-linked glycosyl units; they are produced from starch oritsderivativesbycyclodextringlycosyltransferase(CGTase,EC2.4.1.19), a bacterial enzyme [4]. There are three different typesof natural CDs, according to the number of glucosyl residues inthe molecule:   -,   - and   -CDs (with 6, 7 or 8 glycosyl units,respectively) [5]. The toroidal shape of the CDs allows it to ac-commodate simple molecules with apolar groups into its cavity.The 7 ˚A internal cavity diameter of    -CDs [6], for instance,can accommodate guest molecules with one benzene ring:chlorhexidine [7],  p -iodophenolate [8], benzoic acid [9], benzo- caine [10,11], in a 1:1 stoichiometry as well as molecules withmore than one single ring, such as the local anesthetics bupi- 0731-7085/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jpba.2006.03.010   M.B. de Jesus et al. / Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 1428–1432  1429 vacaine [12] and the anti-inflammatory agent piroxicam [13]. Steric hindrances impede larger molecules to fit in   -CD cav-ity, favoring more complex structures such as the 1:2 (drug:CD)stoichiometries to be formed [14,15]. Here, we have employedmolecular mechanics (MM) calculations to predict the possi-ble orientations of PZQ inside the   -CD cavity at 1:1 or 1:2stoichiometries. Considering that the entropy remains approx-imately constant for the same host molecule, enthalpy of for-mation was used to determine the most stable stoichiometry of complexation[16].Thedifferencesbetweentheenthalpyoffor-mation for the inclusion compounds and the plain moleculeswere also used to determine details of the association modelof complexation. Supporting evidences for the complexation of PZQ with   -CD were also obtained from experimental NMRresults [17]. 2. Experimental and theoretical 2.1. Molecular modeling Molecular modeling was used to determine the 3D geometryof PZQ and   -CD molecules, alone and in the complex. Hy-perChem [18] was used to design the starting point molecules.MM calculations were performed in MacroModel/Maestro 7.3[19] software using MM2 force field to optimise them in wa-ter. To include the solvent effect, we used the generalized Bohrsurface area (GB/SA) protocol and Block-Diagonal Newton–Raphson gradient, with a rms less than 0.01kcalmol − 1 . ThePZQ molecule was positioned into the   -CD cavity at differentorientations. MM calculations considered the center of mass theisoquinoline ring system and denote “isoquinoline in” and “iso-quinoline out” conformers. To  -CD we used “tail” and “head”to describe the regions of the cavity (Fig. 1). According to thesedenotations, the isoquinoline system was taken as the referenceposition for PZQ molecule and “in tail” denotes the conformerthat has its ring next to the secondary hydroxyl group of the CD– the large face of the CD cavity – while “in head” refers to theconformernexttothenarrowerfaceoftheCDcavity.IntheMMcalculations, we consider the relative thermodynamic relation-ship as the proportional stability of the complex formation. Thestability of the complexes was measured assuming the propor-tionality  G ∝ H   and applying the general thermodynamicrelationship: G =− RT   ln K  (1)So that, H   ∝− RT   ln K  (2)For the general reaction of inclusion compounds, the formationof 1:1 stoichiometry can be described byS + CD ⇋ S:CD (3)where S is the guest molecule (PZQ), CD the macromolecularsystem (host) and S:CD the final inclusion compound formed.AccordingtoEq.(2),thestabilityofthereaction,representedbythe equilibrium constant ( K  ), can be taken from the enthalpy of  Fig. 1. Schematic representation of the starting orientations of PZQ/   -CD (1:1stoichiometry) used in the molecular modeling calculations. (a) Isoquinoline intail, (b) isoquinoline in head, (c) isoquinoline out tail and (d) isoquinoline outhead. formationforallthespeciespresentedinthereaction,asfollows: H   = H  fS:CD − ( H  fS + H  fCD ) (4)The enthalpy of formation ( H  f  ) can be obtained from the MMcalculations, so that the final H   is proportional to the stabilityconstant of the inclusion compound.By the same approach, the equilibrium for the 1:2 stoichiom-etry can be described byS:CD + CD ⇋ S:CD 2  (5)where S:CD is the low energy molecular geometry of the 1:1stoichiometry, CD is the macromolecular system and S:CD 2  isthe inclusion compound formed in the 1:2 stoichiometry. In thissituation, we can calculate the enthalpy of formation from: H   = H  fS:CD 2  − ( H  fS:CD + H  fCD ) (6)In both cases the entropy contribution was considered to be thesame [20]. 2.2. Inclusion complex preparation PZQ was donated by Merck KGaA (Darmstadt, Germany)and   -CD was purchased from Sigma (St. Louis, USA). Inclu-sion complexes were obtained by mixing appropriate amountsof PZQ and   -CD, with adaptations in the protocol proposedby de Azevedo et al. [21,22]. Briefly, the protocol consists insolubilizing PZQ and   -CD for the desired molar ratios (1:1 or1:2). Then, the solutions were mixed until the equilibrium wasreached and the samples were freeze dried and stored at − 20 ◦ Cfor further use.  1430  M.B. de Jesus et al. / Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 1428–1432 2.3. Nuclear magnetic resonance NMR experiments were carried out at 298K on a Varian In-ova 500MHz spectrometer, operating at 11.7T at the NationalLaboratory of Synchrotron Radiation (Campinas, SP, Brazil).One-dimensional  1 H-NMR spectra of PZQ,   -CD and PZQ/   -CD complexes were recorded at 25 ◦ C. Sample were suspendedin 0.6mL of 99.9% D 2 O to a final concentration of 1mM;the PZQ/   -CD complex was performed in 1:1 stoichiometry.The residual water signal was used as the internal reference, at4.81ppm.Two-dimensional (2D) rotating frame Overhauser effectspectroscopy (ROESY) experiments were performed using aspin-lock field of 3kHz and 300ms of time delay. The spectrawere collected using 2048 complex data points in the F2 di-mension and 324 increments. The spectral width was 10ppm inboth dimensions and eight free induction decays were acquiredper increment. The NMR data have been processed using theNMRpipe, NMRview and VNMR 6.1 (Varian Inc.) programs[11]. 2.4. Phase solubility test  This methodology was based on the solubility of the guestmolecule [23], in which an excess of PZQ (8mM) was added toaqueoussolutions(20mL)containingincreasingconcentrationsof the CD and shook at 25 ± 0 . 5 ◦ C at 60 oscillations:min. Thistest was performed for 7 days, until equilibrium was reached.DailyaliquotswerefilteredwithaMillex-GP,0.22  mfilterunit(Millipore Carrighwohill, County Cork, Ireland) and total PZQwas analyzed by spectrophotometry (270nm). 3. Results and discussion Fig. 1 shows four starting orientations of the most stablePZQ/   -CD complexes (1:1 stoichiometry) used in the calcu-lations. In Table 1 are listed the enthalpy of formation for thesefour possibilities of 1:1 complexation, calculated according toEq. (4). The H   values indicate that the four complex types arestable in water. The most stable complexes among the four 1:1complexes, corresponding to the greatest absolute value of heatof formation ( H  ), was found to be the “isoquinoline in” com-plexes. Although the H   values for the “isoquinoline in head”were also favorable, the docking of the “isoquinoline in tail”(Fig. 2) revealed to be the most stable, with a H   numeric dataof   − 18 . 12kcalmol − 1 , followed by the “isoquinoline in head”( H  , − 16 . 03kcalmol − 1 ), and by the “isoquinoline out of tail”complex ( H  , − 14 . 12kcalmol − 1 ). Table 1 H   for the (1:1) PZQ/   -CD complex in water as calculated by MM2 H   (kcalmol − 1 )Isoquinoline in tail  − 18 . 12Isoquinoline in head  − 16 . 03Isoquinoline out tail  − 14 . 12Isoquinoline out head  − 9 . 34Fig. 2. 1:1 PZQ/   -CD complex by MM2 with GB/SA calculation. The values found for the 1:2 stoichiometry calculations, car-ried out with Eq. (6), were close to 0kcalmol − 1 (Table 2), indi-cating that the 1:2 complexes are not stable in water due to thesmall energy necessary to displace them.NMR studies allowed us to distinguish between inclusionand other possible external interaction processes. In fact, NMRis the most powerful technique used to determine the inclusionof a guest molecule into the hydrophobic CD cavity, in solution.It is well known that the chemical shifts of the hydrogen atomslocated in the interior of the  -CD cavity (H-3 and H-5) becomeshielded and usually show significant upfield shift in the pres-ence of a guest molecule [8,11], whereas the hydrogen atomson the outer surface (H-1, H-2, H-4 and H-6) are not affected orexperience only a marginal shift upon complexation [5].Assignment of the hydrogen NMR peaks is in good agree-ment with the literature, both for   -CD [5] and PZQ [17,24]. We have first tried to evaluate the inclusion of PZQ into the  -CD cavity analyzing changes in the chemical shifts of the hy-drogens in the complex, in comparison to free PZQ and   -CD.AlthoughmanypeaksbelongingtoPZQhydrogensshiftedaftercomplexation,the δ observedwerenotsignificant,evenforthearomatic protons ( < 0.05ppm), even though these last ones pre-sentedahigher δ thantheoneobservedforthecyclic-aliphaticprotons ( < 0.005ppm). As for the   -CD molecule, the greatestchemical shift were up-field changes, detected for H-5 and alsofor H-3 (data not shown) in a similar way as the δ reported forchlorhexidine [7].A definitive proof of the stoichiometry and insersion typeof PZQ/   -CD complex was given by two-dimensional ROESY Table 2 H   for the (1:2) PZQ/   -CD complex in water as calculated by MM2 H   (kcalmol − 1 )Tail:tail  − 1 . 23Head:tail  − 0 . 31Head:head  − 0 . 05   M.B. de Jesus et al. / Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 1428–1432  1431Fig.3. (a)ExpansionoftheROESYspectrumforthePZQ/   -CDcomplex(1:1)inD 2 Oat500MHz.Thecross-peaksrepresenttheintermolecularinteractionbetweenaromatic hydrogens of PZQ (10, 11 and 12) and  -CD hydrogens (H-3 and H-5). (b) Molecular numbering and representation of the “isoquinoline in tail” PZQ/   -CDassociation, according to MM and NMR data. experiments. As shown in Fig. 3a, Nuclear Overhauser Effects(NOE)weredetectedbetweenhydrogens10,11and12,belong-ing to the isoquinoline ring of PZQ and, H-3 and H-5 hydrogensof the   -CD cavity. ROESY spectra revealed no other inter-molecular cross-peaks between PZQ and   -CD hydrogens.The presence of NOE cross-peaks between PZQ, H-3 andH-5 hydrogens of    -CD suggests a geometry of complexationwhere the aromatic part of the PZQ isoquinoline ring is deeplyinserted in the   -CD cavity, explaining this  through-the-space intermolecular coupling (Fig. 3). This result is in accordancewith those obtained by MM calculations for the 1:1 PZQ/   -CDinclusioncomplex(Table1),whichpointedouttothe“isoquino-line in tail” conformer to be the most probable complexationarrangment.Fig. 2 depicted the “isoquinoline in tail”, preferential inser-tionofPZQintothe  -CDcavity,accordingtoMMcalculations.An analysis of this proposed model for the PZQ/   -CD complex(Fig. 3b) reveals that the ‘isoquinoline in tail’ insertion plentlysatisfies the experimental NMR results (Fig. 3).Finally, phase-solubility studies demonstrate that complexa-tion has increased ca. five-fold the amount of PZQ in water. Theincrease in solubility occurred as a linear function of   -CD con-centration ( r  = 0 . 992) up to 16mM. This concentration corre-spondstotheaqueoussolubilityof   -CD[5].Thelinearrelationbetween PZQ solubility increase and   -CD concentration cor-responds to the A L -type phase-diagram, defined by Higuchi andConnors [23] that are characteristic of 1:1 complexation [25]. In a previous work, Becket and coworkers have examinedthe complexation of    ,   and    cyclodextrins with PZQ andfoundthat  -CDwouldformthemoststablecomplexamongthenaturalCD[24].Thoseauthorsalsodescribedthephase-diagramof PZQ in   -CD as a B S  type [24]. Nevertheless, taking intoaccountthelimitingsolubilityof   -CDinwater(ca.16mM[5]),wecouldassertthatthechangeinprofileofthePZQsolubilityinexcess  -CDconcentrations(upto22mM)doesnotcharacterizethe solubilization type proposed by those authors. Instead, asdiscussed before by Fr¨omming and Szejtli [25], it is a A typephase-diagram, with enhancement in PZQ solubility up to the  -CD solubility.Herein, we show, using theoretical and experimental ap-proaches,that  -CDforms1:1complexes,increasingtheamountof PZQ solubilized in water up to its own solubility limit. 4. Conclusion ThemolecularmodelingdatasuggeststhatPZQ/   -CDinclu-sion complexes have a 1:1 stoichiometry and that the isoquino-lineringsystemofPZQisembeddedinthecavityof   -CD,witha preferential “isoquinoline in tail” mode of insertion (Fig. 3).MMwasusefultopredictthegeometryofthefinalcomplexandto calculate the energy of association between host and guestmolecules. Changes in the chemical shifts of internal hydrogensof the   -CD cavity were indicative of the insertion of PZQ into  -CD cavity. ROESY experiments detected molecular proxim-ities ( ≤ 5 ˚A) between hydrogens 10, 11 and 12 of the aromaticringofPZQisoquinolinesystemandhydrogensH-3andH-5,inthe internal cavity of    -CD (Fig 3). Moreover, phase-solubilityhas shown a five-fold increase in PZQ solubility, in agreementwith the proposed 1:1 complexation.The results reported here show a good agreement betweenMM calculation and experimental data (NMR data) revealingthat, together, these are really powerful tools for the study of the molecular details of CD inclusion complexes. This work demonstrateshowtheoretical(molecularmodelingcalculations)andexperimental(solubilityandNMRexperiments)approachescan give complementary and concordant results that revealuseful molecular details of a given drug/   -CD system. ThePZQ inclusion complex was designed as a new pharmacolog-ical formulation in the treatment of schistosomiasis, since   -CD complexation increases the solubility of PZQ. The resultsobtained prompted us to perform in vivo experiments (withmice infected with  Schistosoma mansoni ), that are now undercourse and will allow us to validate the efficiency of this novelformulation.  1432  M.B. de Jesus et al. / Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 1428–1432 Acknowledgments The finantial support given by FAPESP (Proc 02/04103-4)and FAEPEX/UNICAMP (Proc. 674/00 and 116/01) are grate-fully acknowledged. M.B.J. and E.P. received fellowships fromCNPq and A.C.S.L., a fellowship (Proc. BEX1556/04-5) fromCAPES. References [1] WHO, Geneva, 2002. http://www.who.int/topics/schistosomiasis/en/ .[2] P. Magnussen, Acta Trop. 86 (2003) 243–254.[3] D. Cioli, L. Pica-Mattoccia, Parasitol. Res. 90 (2003) S3–S9.[4] A. Biwer, G. Antranikian, E. Heinzle, Appl. Microbiol. Biotechnol. 59(2002) 609–617.[5] J. Szejtli, Cyclodextrin Technology, Kluwer Academic Publishers, Dor-drecht, 1998.[6] G.N. Kalinkova, Int. J. Pharm. 187 (1999) 1–15.[7] H. Qi, T. 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