Development and pharmacological evaluation of ropivacaine-2-hydroxypropyl-β-cyclodextrin inclusion complex

Development and pharmacological evaluation of ropivacaine-2-hydroxypropyl-β-cyclodextrin inclusion complex

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  european journal of pharmaceutical sciences 33 (2008) 60–71 available at journal homepage: Development and pharmacological evaluation of ropivacaine-2-hydroxypropyl-  -cyclodextrininclusion complex Daniele R. de Araujo a , Simone S. Tsuneda a , C´ ıntia M.S. Cereda a ,Fernanda Del G.F. Carvalho a , Paulo S.C. Pret´ e a , Sergio A. Fernandes b ,Fabiano Yokaichiya c , Margareth K.K.D. Franco c , Irineu Mazzaro c ,Leonardo F. Fraceto a , d , Ang´ elica de F.A. Braga e , Eneida de Paula a , ∗ a Department of Biochemistry, Institute of Biology, State University of Campinas, Campinas, SP, Brazil b Department of Chemistry, Federal University of Vic¸osa, Vic¸osa, MG, Brazil c Department of Physics, Federal University of Paran´ a, Curitiba, PR, Brazil d Department of Environmental Engineering, State University of S˜ ao Paulo, Sorocaba, SP, Brazil e Department of Anesthesiology, Faculty of Medicine, State University of Campinas, Campinas, SP, Brazil a r t i c l e i n f o  Article history: Received 18 July 2007Received in revised form11 September 2007Accepted 28 September 2007Published on line 7 October 2007 Keywords: Local anestheticRopivacaineCyclodextrinAnalgesiaDrug-delivery a b s t r a c t Ropivacaine (RVC) is an enantiomerically pure local anesthetic (LA) largely used in surgicalprocedures, which presents physico-chemical and therapeutic properties similar to thoseof bupivacaine (BPV), but associated to less systemic toxicity. This study focuses on thedevelopment and pharmacological evaluation of a RVC in 2-hydroxypropyl-  -cyclodextrin(HP-  -CD) inclusion complex. Phase-solubility diagrams allowed the determination of theassociation constant between RVC and HP-  -CD (9.46M − 1 ) and showed an increase on RVCsolubility upon complexation. Release kinetics revealed a decrease on RVC release rateand reduced hemolytic effects after complexation (onset at 3.7mM and 11.2mM for RVCand RVC HP-  -CD , respectively) were observed. Differential scanning calorimetry (DSC), scan-ning electron microscopy (SEM) and X-ray analysis (X-ray) showed the formation and themorphology of the complex. Nuclear magnetic resonance (NMR) and job-plot experimentsafforded data regarding inclusion complex stoichiometry (1:1) and topology. Sciatic nerveblockade studies showed that RVC HP-  -CD  was able to reduce the latency without increasing the duration of motor blockade, but prolonging the duration and intensity of the sensoryblockade(  p <0.001)inducedbytheLAinmice.TheseresultsidentifytheRVC HP-  -CD  complexas an effective novel approach to enhance the pharmacological effects of RVC, presenting itas a promising new anesthetic formulation.© 2007 Elsevier B.V. All rights reserved. 1. Introduction Despitetherecentadvancesinbasicandclinicalinvestigationtowards new therapeutic agents, the management of pain is ∗ Corresponding author . Tel.: +55 19 3521 6143; fax: +55 19 3521 6129.E-mail address: (E. de Paula). still a challenge. Local anesthetics (LA) are among the differ-ent classes of pharmacological compounds used to attenuateor to eliminate pain. These drugs, which are able to reversiblyblock the excitation-transmission process in axons, present 0928-0987/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejps.2007.09.010  european journal of pharmaceutical sciences 33 (2008) 60–71  61 Fig. 1 – Schematic representation of the chemical structureof ropivacaine. relatively short action and a significant toxicity to the cen-tral nervous and cardiovascular systems (Covino and Vassalo,1976; Strichartz and Ritchie, 1987; de Jong, 1994).Ropivacaine (RVC, Fig. 1), a new amide-type local anes-thetic, used as the  S -( − ) enantiomer, is the  n -propylhomologue of bupivacaine (BPV). Due to their similar phar-macodynamicproperties,RVCandBPVareusedinavarietyof clinical procedures (Cederholm, 1997). They present a compa-rableclinicalefficacyintermsoflatency,potencyanddurationofaction.However,amongthemainfeaturesattributedtothisnew LA, it is possible to see that RVC evokes a lower cardiactoxicity and allows an earlier motor blockade recovery in rela-tion to BPV (Knudsen et al., 1997; Dony et al., 2000; Wang etal., 2001). Nowadays, there is a strong clinical requirement forlong-acting anesthetics, as well as for LA molecules with lowsystemic uptake, leading to less toxic side effects (Mather andChang, 2001; Mcclellan and Faulds, 2001).Cyclodextrins (CD), cyclic oligosaccharides consisting of six or more   -1,4-linked  d -glucopyrannose units, are one of the most used drug-carriers. CDs are able to form inclu-sion (host–guest) complexes with a wide variety of organicmolecules, the so called guest molecules (Loftsson andDuch ˆ ene, 2007). Several reports have shown the advantagesof using    -cyclodextrin and its derivatives in pharmaceuti-cal formulations to improve the bioavailability of drugs anddecrease their toxicity (Szejtli, 1982, 1998; Rajewski and Stella,1996; Irie and Uekama, 1997; Hirayama and Uekama, 1999;Davis and Brewster, 2004).   -Cyclodextrin has been exten-sively studied despite its limited aqueous solubility. Somealkylatedderivatives,suchas2-hydroxypropyl-  -cyclodextrin(HP-  -CD),haveattractedgrowinginterestduetoitsimprovedcomplexing ability, greater water solubility and less toxic-ity than   -cyclodextrin (Carpenter et al., 1995; Gould andScott, 2005). By modifying physical, chemical and biologicalproperties (Rajewski and Stella, 1996), complexation with a CD provides a way to increase drug solubility, stability andbioavailability (Duch ˆ ene, 1987; Dollo et al., 1998, 2000; Estebeet al., 2002).Studies involving the complexation of LA molecules withCD have been reported in the literature (Freville et al., 1996;Dollo et al., 1996a,b, 1998, 2000; Estebe et al., 2002; Araujo etal., 2005, 2006; Moraes et al., 2007a,b; Karashima et al., 2007);nevertheless, none of them have employed RVC.Besides the physico-chemical stabilization, the maineffects of complexation with CD are specially observed onchanges in aqueous solubility, release kinetics, pharmacoki-netics and pharmacodynamic properties. Hence, modifying the duration and intensity of the pharmacological effects of LAmaybepotentiallyadvantageousforthepostoperativepainrelief (Freville et al., 1996; Dollo et al., 1998, 2000; Estebe et al.,2002; Araujo et al., 2005, 2006).The present study aimed to develop, to perform thephysico-chemical characterization and to evaluate the phar-macological properties of RVC inclusion complex withHP-  -CD. Phase-solubility diagrams were used to evaluate thesolubility of RVC and its association constants with HP-  -CD.Differential Scanning Calorimetry (DSC), Scanning ElectronMicroscopy (SEM) and powder X-ray (X-ray) assays were usedto observe the stoichiometry and the topology of the obtainedinclusion complex.  In vitro  release studies and hemolytic testswere carried out in order to assess the possible cellular effectsof this formulation. Finally, the motor and sensory functionswere evaluated during the sciatic blockade evoked by com-plexed RVC in relation to its plain solution. 2. Materials and methods RVC hydrochloride (attested purity of 98.5%) was donatedby Crist´alia Prod. Qu´ım. Farm. Ltda (Itapira, SP, Brazil) and HP-  -CD (Kleptose HP ® ) was obtained from Roquette Serv.Tech. Lab. (Lestrem, Cedex, France). HEPES buffer was pur-chasedfromSigmaChem.Comp.(St.Louis,MO,USA).Allotherreagents were of analytical grade. Deionized water (Elga Max-ima System, Elga, High Wycombe, UK) was used throughoutthe experiments. 2.1. Preparation of solid inclusion complex Inclusion complexes were prepared by shaking equimolaramounts of RVC and HP-  -CD (1:1 molar ratio) in deionizedwater at room temperature (25 ± 1 ◦ C) for 24h. After com-pletelydissolutionandreachingequilibrium,thesolutionwasfreeze-dried (Labconco-freeze dry system/Freezone ® 4.5) andstored at  − 20 ◦ C until further use. Physical mixtures wereobtained by mixing RVC and HP-  -CD powders, at the samemolar ratio (Moraes et al., 2007a). 2.2. Phase-solubility studies For the phase-solubility assays, an excess of RVC was mixedwithaqueoussolution(50mMHepeswith154mMNaClbuffer)ofincreasingHP-  -CDconcentrations(0–30mM).Thesampleswere stirred at room temperature and an aliquot was filteredthrough a 0.45  m membrane filter (Millipore). The amountof soluble RVC was determined by UV-spectrophotometry at260nm. The association constant ( K a ) was determined fromthe slope of the linear portion of the phase-solubility diagramaccording to Eq. (1) (Higuchi and Connors, 1965): K a  = slope S 0 (1 − slope) (1)where  S 0  is the aqueous solubility of RVC. All the experimentswere carried out in triplicate.Then, it was possible to calculate the complexation effi-ciency (CE) and the drug:cyclodextrin ratio (D:CD), according   62  european journal of pharmaceutical sciences 33 (2008) 60–71 to Eqs. (2) and (3) (Loftsson et al., 2005, 2007). CE = S 0 K 1:1  = [ D/ CD][CD]  = Slope1 − Slope (2) D CD  = 11 + (1 / CE) (3)where [ D  /CD] is the concentration of dissolved complex, [CD]the concentration of dissolved free cyclodextrin and the slopewas obtained from the phase-solubility profile. 2.3. Differential scanning calorimetry (DSC) The samples (10mg) were placed in aluminum pans and theexperiments run in a calorimeter (Universal V2.3D TA Instru-ments)ata10 ◦ C/minheatingrateoverawiderange(0–450 ◦ C).An empty pan served as reference and indium was used tocalibratethetemperature.Thermogramsweredeterminedforthe samples: HP-  -CD, RVC, physical mixture RVC/HP-  -CDand solid complex RVC HP-  -CD  (1:1 molar ratio). 2.4. Scanning electron microscopy (SEM) RVC, HP-  -CD, physical mixture RVC/HP-  -CD and solidcomplex RVC HP-  -CD  (1:1 molar ratio) were prepared by met-allization with gold under vacuum for 180s. Images wereanalyzed using a scanning microscope (JSM 5800LV, JEOL, Japan) in order to observe possible structural changes on theLA and CD crystal, after complexation. 2.5. X-ray powder diffractometry (X-ray) The powder diffractograms from HP-  -CD, RVC, physicalmixture RVC/HP-  -CD and solid complex RVC HP-  -CD  wereperformed in a Rigaku Wide Angle Goniometer with Co K  radiation source (Philips PW 1743), voltage and current setto 40kV and 20mA, respectively. The scan rate was 1 ◦ min − 1 ,between2   =5 ◦ and60 ◦ ina   − 2    configurationwithapyroliticgraphite as the crystal analyzer. 2.6. Nuclear Magnetic Resonance (NMR) studies The continuous variation method was adopted to determinethe stoichiometry of the complex (Djeda ¨ ıne et al., 1990).  1 HNMR spectra were obtained for a series of RVC:HP-  -CD mix-tures in which the total initial concentration of both specieswas kept constant (5mM) but the mol fraction of each compo-nent varied from 0 to 1.One-dimensional  1 H NMR spectra were recorded on a Var-ian Mercury 300MHz spectrometer, in phosphate bufferedsolution in deuterated water for RVC/HP-  -CD system. ThestocksolutionsofRVC(5mM)andHP-  -CD(5mM)weremixedin 5mm NMR tubes, giving a total sample volume of 600  L,and they were left overnight for equilibration before the NMRanalysis. The probe temperature was regulated to 298K.The  1 H NMR spectra were recorded using a simplepulse-acquire sequence with solvent pre-saturation. Typicalacquisition parameters consisted of 32K points covering asweep width of 8000Hz, a pulse width (pw 90) of 10  s, digitalzerofillingto128K;a0.5Hzexponentialfunctionwereappliedto the FID before Fourier transformation. The resonance at4.67ppm, due to residual solvent present as impurities (H 2 Oand HDO), was used as internal reference. The data werecollectedwithoutanexternalreferencetoavoidpossibleinter-actions with the HP-  -CD. The 1D-ROESY experiments wererecordedusingthefollowingparameters:themixingtimewas500ms with a radiofrequency field of 4kHz and the number of scans was 1024. 2.7. Release kinetics and hemolytic assays Drug release experiments were conducted under constantstirring in a two-compartment dialysis system that uses acellulose membrane (Spectrapore, MWCO 1000Da) to sepa-rate the sample at the donor compartment (1mL capacity,containing plain RVC or complexed RVC HP-  -CD  samples) fromthe acceptor compartment (100mL, containing 20mM HEPESbuffer pH 7.4 at 37 ◦ C). Aliquots were withdrawn from theacceptorcompartmentatregularintervalsandtheLAconcen-tration was determined by UV spectrophotometry (260nm).Data were expressed as % of RVC released for each sample.Hemolytic assays were analyzed through the percent-age of hemoglobin released from human erythrocytes (0.15%hematocrit) obtained from Hemocentro/Unicamp (protocol396/03, approved by the Ethics on Research Committee,FCM/Unicamp). Red blood cells were treated with HP-  -CD,RVC or RVC HP-  -CD  complex (0.25–30mM), kept under 37 ◦ C for15min and centrifuged (1500 × g ) for 3min. The amount of hemoglobin released in the supernatant was determined at412nm.Datawereexpressedaspercentageofhemolyticeffect(Malheiros et al., 2004). 2.8. Local anesthetic activity: sciatic nerve blockade Male adult  Swiss  mice (weighting 30–35g) were obtained fromCEMIB-UNICAMP (Centro de Bioterismo, State University of Campinas). The protocols were approved by the UNICAMPInstitutional Animal Care and Use Committee (protocols 557-2, 558-1, 559-1), which follows the recommendations of theGuide for the Care and Use of Laboratory Animals.Beforethepharmacologicaltests,theabilityofeachmouseto walk normally with four limbs on both the top and invertedside of a wire mesh screen (1mm diameter wire, 5mm mesh)was evaluated and the animals that showed such behav-ior were selected for the experiments (Leszczynska and Kau,1992). After this, animals were divided in groups ( n =6): HP-  -CD and RVC or RVC HP-  -CD  at 0.125%, 0.25% and 0.5%.For the sciatic nerve blockade, drugs were administeredby infiltration (0.1mL) where, a needle was inserted into thepoplitealspace,posteriortothekneejoint,inthesciaticnervearea.Motorblockadedegreewasassessedbythelossofmotorcontrol in the injected limb according to the scores: 0 (nor-mal movement), 1 (unable to flex the limb completely) and2 (total paralysis) (Leszczynska and Kau, 1992). Any animal that could not use the injected limb to walk normally on thetop and on the inverted wire mesh screen was considered tohave a positive response to local anesthesia. The motor func-tion was evaluated from 1 to 5min and at every 10min up to1h following the injection. The parameters evaluated were:  european journal of pharmaceutical sciences 33 (2008) 60–71  63 latency (time between injection and the loss of motor func-tion), time to reach the maximum score ( T  max ), time for motorfunctionrecoveryandthetotalLAeffect(estimatedbytheareaunder the effect curve vs. time curve expressed by score/h,AUC) (Gantenbein et al., 1997; Araujo et al., 2004, 2005). Anal-ysis were made using Origin 6.0 (Microcal TM Software Inc.,Northampton, MA, USA).Evaluation of sensory blockade was performed by thepaw pressure test (Randall and Selitto, 1957) using an analgesymeter (Ugo Basile, Varese, Italy), which exerts a force(in grams) on the paw. Each animal was gently wrapped in asmalltowelsothatonlythelimbsandheadwerefree,avoiding analgesia induced by excessive stress. The withdrawal reflexwas considered representative of the pain threshold or PawWithdrawalThresholdtoPressure(PWTP).ThebaselineofthePWTP test was measured before vehicle or drug injection, inorder to determine a baseline or the pain threshold for eachanimal.Baselinevaluesof30–50gwereconsideredasthepainthreshold and animals that presented lower or higher valuesthan those were excluded. The established antinociception cut-off   value was 150g, considered to be representative of theanestheticstate(Araujoetal.,2004,2005).Afterdrugorvehicleadministration, measurements were carried out at intervalsof 15min during the first hour, 30min in the second and thirdhour and finally 60min up to 5h after treatment.After the experiments, the animals were observed for 24hin order to detect possible systemic toxic effects (such asseizuresordeaths)ornervedamage(lackofrecoveryofnormalmovement on the injected limb) caused by the procedure. 2.9. Statistical analysis Release kinetics results were analyzed by two-tailed unpaired t -test. Motor blockade data (latency,  T  max , time for recovery,AUC) were analyzed by the Kruskall–Wallis test (expressed asmedians and minimum and maximum limits) and sensoryblockade results (intensity of PWTP expressed as mean andS.D.) by one-way analysis of variance (One-way ANOVA withTukey–Kramer as a post hoc test). Statistical significance wasdefined as  p <0.05. 3. Results and discussion 3.1. Phase-solubility studies The complexation phenomenon can be described by parame-ters such as time to reach solubilization, association constant( K a ) and the complexation efficiency (CE) determination(Loftsson et al., 2005, 2007).The increased aqueous solubility of RVC in the presenceof HP-  -CD was used to follow the formation of inclusioncomplexes. The determination of   K a  between drug and CDis based on the measurement of an index of changes inphysico-chemical properties of a compound upon inclusion.Most methods for determining the  K a  values are based on theanalysisoftheconcentrationdependencies,attitrationexper-imentswiththedrugmoleculeandCD(LoftssonandBrewster,1996). In this way, an important pharmaceutical applicationof CD is to enhance drug solubility in aqueous solution. The Fig. 2 – Phase-solubility diagram for RVC at increasingHP-  -CD concentrations, determined at room temperature(  n =3). increase on RVC solubility occurred as a linear function of HP-  -CD concentration (Fig. 2), corresponding to the A L -typeprofile defined by Higuchi and Connors (1965). This relation-ship suggests a first order kinetics on the complex formationbetween RVC and HP-  -CD. The  K a  value determined, was9.46M − 1 , at pH 7.4, indicating the formation of a weak (lowaffinity), but stable complex (Loukas et al., 1998).  K a  valueagrees with those determined for racemic BPV (14.7M − 1 atpH 7.4) (Araujo et al., 2005) and S( − ) BPV (13.1M − 1 at pH 7.4)(Moraes et al., 2007b).From Eqs. (2) and (3) (Section 2) a CE value of 0.0896 was found,whichisinagreementwiththeintermediatehydropho-bicity of RVC if compared to BPV (CE=0.045) or lidocaine(CE=1.508) (Loftsson et al., 2007). Besides, a drug:cyclodextrin ratio of 1:12 was calculated indicating that, on a 1:1 (RVC:HP-  -CD) complexation, just one out of 12 HP-  -CD moleculesis forming an inclusion complex with RVC (Loftsson et al.,2007). 3.2. Differential scanning calorimetry (DSC) DSC thermograms were obtained analyzing the rate of heatabsorbedbyRVC,HP-  -CD,RVC/HP-  -CDphysicalmixtureandRVC HP-  -CD  inclusion complex (1:1 molar ratio). These analysisgave supporting evidences for the complexation of RVC withHP-  -CD.Fig. 3 shows thermograms for HP-  -CD (Fig. 3A), RVC(Fig. 3B), RVC/HP-  -CD physical mixture (Fig. 3C) and solidcomplex RVC HP-  -CD  (Fig. 3D). HP-  -CD and RVC presented acharacteristic endothermic peak each corresponding to theirmelting point (336.0 ◦ C and 117.6 ◦ C, respectively) and for HP-  -CD, it was also observed a peak corresponding to water loss(50 ◦ C).The thermogram of the physical mixture RVC/HP-  -CDshows two endothermic peaks of intermediate temperatures(246.5 and 116.0 ◦ C), between those that RVC and HP-  -CD.The inclusion complex RVC HP-  -CD  presents only a single  64  european journal of pharmaceutical sciences 33 (2008) 60–71 Fig. 3 – DSC thermograms of (A) HP-  -CD, (B) RVC, (C)RVC/HP-  -CD 1:1 physical mixture, (D) RVC/HP-  -CD 1:1complex. broaden peak at 248.2 ◦ C, in a different manner than thoseobserved for the pure RVC, pure HP-  -CD or for their physicalmixture.The disappearance as well as the shift of endo or exother-mic peaks of drugs is a clear indication of the complexationphenomenon (Loukas et al., 1998) explaining the absence of  the fusion peak of pure RVC (117.6 ◦ C) in the thermogramshowed on Fig. 3D. This result is an evidence of the inclusionof the RVC molecule into the CD hydrophobic cavity. 3.3. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) is a qualitative methodused to study the structural aspects of raw materials, i.e.,CD and drugs or the products obtained by different methodsof preparation like physical mixture, solution complexation,coevaporation and others (Duch ˆ ene, 1987). SEM microghra- phies (Fig. 4) illustrate the arrangement of HP-  -CD (Fig. 4Aand B), RVC (Fig. 4C and D), RVC/HP-  -CD physical mixture(Fig. 4E) and solid complex RVC HP-  -CD  (Fig. 4F). Typical crys-tal of HP-  -CD and RVC were found in many different sizesand the inclusion complex (RVC HP-  -CD  1:1 molar ratio) sam-ple was seen as a compact and homogeneous powder-likestructure, whose dimensions were smaller than those of thecrystalofRVCorHP-  -CDalone.ThephysicalmixtureRVC/HP-  -CD revealed some similarities with the crystal of the freemolecules. The samples referring to RVC and HP-  -CD (Fig. 4Band D, respectively) were included in the assays to show thatthe crystal morphology was not affected by the lyophilizationprocess used to prepare the inclusion complex.The central cavity of the CD molecule is lined with skeletalcarbons and etheral oxygens of the dextrose residues. There-fore,itisalipophylicmoleculeandthepolarityofitscavityhasbeen estimated to be similar to that of an aqueous ethanolicsolution, providing a lipophilic microenvironment into whichsuitably sized drug molecules may enter and be included(Fr¨ omming and Szejtli, 1994). According to Davis and Brewster (2004),thecrystallineaspectofnaturalCDcrystalwaschanged(as a result of irregular molecular substitutions) and replacedbyamorphouspowders,onitssubstitutedderivatives.Overall,the SEM and DSC results indicate the formation of an inclu-sioncomplexbetweenRVCandHP-  -CDandalsodemonstratethat no complex is formed in the physical mixture of the com-pounds. 3.4. X-ray powder studies X-ray diffractograms clearly confirmed the crystalline natureof RVC while HP-  -CD was presented as an amorphous struc-ture (Fig. 5). The physical mixture RVC/HP-  -CD (Fig. 5c)showed the superposition of the crystalline pattern of RVCand the amorphous HP-  -CD diffraction (Fig. 5d and a,respectively). By contrast, the inclusion complex RVC HP-  -CD ,analysis showed that the crystalline RVC pattern has dis-appeared. Moreover, the intensity differences observed ondiffractograms from HP-  -CD and the inclusion complex inthe 5–60 (2   ) range strengthen the theory about the complex-ation phenomenon since an approximately 2-fold intensitydecrease was observed after complexation (Fig. 5b), whencompared to HP-  -CD (Fig. 5a). 3.5. Nuclear magnetic resonance The continuous variation method was employed to establishthestoichiometryofthecomplexusingNMR.Theassignmentsof   1 H-chemical shift of RVC and HP-  -CD agree with previousreportsintheliterature(Fracetoetal.,2005;Xiliangetal.,2005). The RVC-induced  1 H-chemical shifts modifications in HP-  -CD were mainly detected for hydrogens H 3  and H 5 .Ifaphysicalparameterdirectlyrelatedtotheconcentrationofthecomplexisplottedasafunctionofthemolefraction( r )of RVCorHP-  -CD,itsmaximalvaluewilloccurat r RVC  = m  /( m + n )or r HP-  -CD  = n  /( m + n ),where m and n arethemolarratiosofRVCand HP-  -CD in the complex, respectively. For a signal belong-ing to RVC and under fast motion, changes in the chemicalshift ( ı  [RVC]) will be proportional to the complex concentra-tionanditispossibletoplotthesechangesagainst r (Djeda ¨ ıne et al., 1990).
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