Jiaxin Pi,Shuya Wang,W en Li,Dereje Kebebe,c,Ying Zhang,Bing Zhang,DongliQi,Pan Guo,Nan Li,Zhidong Liu

a Tianjin State Key Laboratory ofM odern Chinese M edicine,Tianjin University ofTraditional Chinese M edicine,Tianjin 300193,China

b Engineering Research Center ofM odern Chinese M edicine Discovery and Preparation Technique,M inistry of

Education,Tianjin University ofTraditional Chinese M edicine,Tianjin 300193,China

c Institu te ofhealth sciences,Jimm a University,Jimm a,Ethiopia

Keywords:Baicalein Nano-coc rysta ls Cocrystals Bioavailability

A B S T R A C TBaica lein(BE)is one of the m ain active f lavonoids rep resen ting the variety of pharm acologica l e ffects in clud ing an tican cer,an ti-in f lam m atory and card iovascu lar p rotective ac tivities,bu t it's very low so lubility,d isso lu tion rate and poor o ra l abso rp tion lim it the therapeu tic app lication s.In th is w o rk,a nano-cocrysta l strategy w as successfu lly app lied to imp rove the d isso lu tion rate and bioavailability of BE.Baica lein-n ico tinam ide(BE-NCT)nanococ rysta ls w ere p repared by h igh p ressu re hom ogen ization and eva luated bo th in vitro and in vivo.Physical characterization resu lts in clud ing scann ing electron m icroscopy,dynam ic ligh t scattering,pow der X-ray d iffraction and d ifferen tia l scann ing calo rim etry dem onstrated that BE-NCT nano-cocrysta lsw ere changed in to am o rphous statew ith m ean partic le size of 251.53 nm.In the d isso lu tion test,the BE-NCT nano-cocrysta ls perform ed 2.17-fo ld and 2.54-fo ld enhan cem en t than BE coarse pow der in FaSSIF-V2 and FaSSGF.Upon oraladm in istration,the in tegrated AUC0-t of BE-NCT nano-coc rysta ls(6.02-fo ld)w as sign if ican tly h igher than BE coarse pow der(1-fo ld),BE-NCT cocrysta ls(2.87-fo ld)and BE nanocrysta ls(3.32-fo ld).Com pared w ith BE coarse pow der,BE-NCT coc rysta ls and BE nanocrysta ls,BENCT nano-cocrystals possessed excellen t perform ance both in vitro and in vivo evaluations.Thus,it can be seen that nano-coc rysta l is an app rop riate novel strategy for im p roving d isso lu tion rate and bioavailability of poo r so lub le natu ra l p roducts such as BE.

1. Introduction

As a sign if ican t p roblem,the poor aqueous solubility of nearly 40%of the m arketed d rugs and up to 70%of the d rug cand idates causes the po ten tia l risks of low o ra l bioavailability and exposu re issues in pharm aco logica l and toxico logica l stud ies,is im po rtan t[1].Using d ifferen tm ethods su ch as su itab le crysta l engineering techno logies o r abso rp tion-enhan ced fo rm ulations to im p rove the so lubility,d isso lu tion,and absorp tion of poorly so luble com poun ds has a great fu tu re poten tial.

Severa l pharm aceu tica l m ethods for im p rov ing so lubility o r d isso lu tion-lim ited d rug absorp tion have been developed,su ch as so lubilization,so lven t m ix tu res,in clusion com pounds,com p lexation and so on[2-4].Am ong them,cocrysta ls and nanocrysta ls are doing w e ll in overcom ing the so lubility p rob lem.

Pharm aceu tica l cocrysta ls are m u lticom ponen t system s that com posed of tw o orm ore m o lecu les at a stoich iom etric ratio and held together by H-bond ing[5,6].Pharm aceu tica l cocrysta ls in co rporate pha rm aceu tica lly accep tab le con fo rmers and the d rug substan ce in to the sam e crysta l lattice to p rovide a new com position of the active pharm aceu tical ingred ien ts(APIs).Acco rd ing to the recen t reclassif ica tion,cocrysta ls are con sidered as d rug po lym orph rather a new APIs w h ich has a sign if ican t im pact on d rug developm en t.Cocrysta llization offers an exped ien tw ay to a lter the physicochem ica l and biopharm aceu tica l p roperties of APIs in clud ing d isso lu tion rate,in trinsic so lubility,m elting poin t,hygroscop icity,comp ressibility,bu lk density and also bioavailability[7-11].

Differen t from cocrysta ls,nanocrysta ls a lw ays cou ld imp rove the so lubility of poorly so lub le d rugs.Nanocrysta ls,w h ich are def ined as nano-sized d rug partic les stabilized by su rface stabilizers,have been w idely stud ied and d iscussed on their sa tu ra tion so lubility and d isso lu tion rate enhan cem en t,im p roved o ra l bioavailability and bioactivities.In genera l,nanocrysta ls a re p repared by top-dow n o r bottom-up app roaches.Top-dow n technologies consistofw etm ed iam illing and h igh p ressu re hom ogen ization w h ile bo ttom-up techno logies invo lve an ti-so lven t p recip itation.A ll those techno logies are w idely app lied to obtain nanosized crysta ls.The increase in d isso lu tion rate correspond ing w ith the decrease in the particle size w as attribu ted to the resu ltan t enhan cem en t in the su rface area and decrease in the d iffusion layer th ickness[12].Nanocrysta l is a su itab le fo rm u lation to successfu lly enhan ce the bioavailability of d rugsw here the d isso lu tion rate is the rate lim iting step in the abso rp tion[13-16];how ever,it is no t adequate to d rugs w hose satu ration so lubility is the rate lim iting step because satu ration so lubility of d rugs is not sign if ican tly im p roved by nanon ization[17,18].

Com bin ing the benef its of cocrysta l and nanocrysta l techno logies,nano-cocrysta l fo rm u lations w ere p roposed as a novel app roach to ach ieve im p roving d issolu tion rate and o ra l bioavailability.How ever,to ou r know ledge,on ly a few nano-crysta l researches had been reported,referred to the itraconazo le-ad ip ic acid[19],indom eth cin-saccharin,fu rosem ide-ca ffeine[20]and m yricetin-n icotinam ide nanococrysta l fo rm u la tions[21].De Sm et et a l.repo rted that the itraconazo le-ad ip ic acid nano-cocrysta l fo rm u lation cou ld sign if ican tly enhan ce the ora l bioavailability than that of am o rphous fo rm u lation[19].Recen tly,phenazopy rid ineph tha lim ide nano-crysta lsw ere developed and dem onstrated 2.44-fold oral bioavailability im p rovem en t than coarse suspension[22].

Baica lein(BE,show n in Fig.1)is one of the m ain active f lavonoids from natu ra l p roduct Scu te llariae Rad ix.In sp ite of the variety of pharm aco logical effects,such as an ticancer[23],an ti-in f lam m atory[24]and card iovascu lar p ro tective activities[25],its therapeu tic app lications are greatly lim ited by its very low so lubility and poo r o ra l abso rp tion.A lthough,till now,cocrysta l and nanocrystal form u lations of BE w ere ach ieved the so lubility and bioavailability im p rovem en t to som e ex ten t[10,15,26],the nano-cocrysta l strategy of BEw as still lack of research.

Therefo re,in th is w ork,w here BEw as selected as the typica l poo rly so lub le m ode l d rug,baica lein-n ico tinam ide(BENCT)nano-cocrysta lsw ere deve loped by the typ ica l top-dow n m ethod and evaluated both in vitro and in vivo to assess the po ten tia l of nanosized cocrysta ls fo r poorly so lub le d rugs as a new insigh t to so lve the p rob lem of inso lub le d rug o ra l abso rp tion in advan ce of on ly cocrysta ls o r nanocrysta ls.

2. M ateria ls and methods

2.1. Materials and anim als

Baicalein(BE,＞98%pu rity)w as pu rchased from Nan jing Zelang M ed ica l Techno logy Co.Ltd.(Nan jing,Ch ina).Po loxam er 188 w as k ind ly gifted by BASF Co.(Ludw igsha fen,Germ any).N ico tinam ide(NCT)w as pu rchased from J&K Scien tif ic Ltd.(Beijing,Ch ina).BE(98.5%pu rity,111 595-201 306),baicalin(BA,93.5%pu rity,110 715-201 619)and carbam azep ine(CBZ,98.5%pu rity,110 723-201 413)as in terna l standard(IS)w ere pu rchased from Nationa l Institu te fo r the Con tro l of Pharm aceu tical and Bio logical Products(Beijing,Ch ina).Aceton itrile and m ethano l of HPLC grade w ere offered from Fisher Scientif ic Co.(Fair Law n,New Jersey,USA).Fo rm ic acid w as supp lied from Fluka(CA,USA).A ll agen ts w ere ana ly tica l grade and pu rif ied M illi-Q w ater(M illipo re,Billerica,MA,USA)w as used th roughou t a ll experim en ts.

Sp rague-Daw ley ra ts(w eigh ting 250±20 g;Shan chuanhong Experim en tal An im al Tech Co.Ltd;Tian jin,Ch ina;Licen ce:SCXK 2014-0001,Tian jin,Ch ina)w ere housed at room tem peratu re w ith free access to d rink ing w ater bu t fasted overn igh tbe fo re the adm in istration ofd rugs.The p ro toco land any am endm en ts or p rocedu res invo lving the care or use of an im a ls in th is study w ere in acco rdan ce w ith the regu lation s for an im a l experim en tation issued by the State Comm ittee of Scien ce and Techno logy of Ch ina and app roved by TJUTCM's Institu tional An im alCare and Use Com m ittee(Docum en t num ber TCMLAEC 20170020).

2.2. Preparation ofcocrystals and nano-cocrystals

2.2.1. Preparation ofBE-NCT cocrystals

The BE-NCT cocrysta ls w ere fo rm ed acco rd ing to the p revious w ork[10].Brief ly,1m o l of BE and 3m o l of NCT w ere d isso lved in to 450m l of ethy l acetate.The resu lting so lu tion w as then stirred for 8 h at room tem peratu re.A fter stand ing the suspen sion over n igh t,BE-NCT cocrysta ls w ere harvested.

Fig.1-The structu res of BE(A)and BA(B).

2.2.2. Preparation of BE nanocrystals and BE-NCT nanococrystals

BE nanocrysta ls and BE-NCT nano-cocrysta ls w ere p repared using the h igh p ressu re hom ogen ization m ethod(HPH)w ith po loxam er 188 as stabilizer[13].The fo rm u lation con tain ing 2%(w/w)BE(BE-NCT cocrystals)and 0.4%(w/w)po loxam er 188,w as f irstly hom ogen ized at 15 000 rpm fo r 10m in by a lab sca le h igh shea r d ispersing em u lsif ier(FA 25,FLUKO,Germ any).High-p ressu re hom ogen ization at 900 bar w as app lied for 20 hom ogen ization cycles(AH100D,ATS Engineering Inc.Canada)to ob tain the nanosuspension of BE(BE-NCT cocrysta ls).A ll of the experim en ts w ere condu cted at room tem peratu re.

2.3. Scann ing electron m icroscope(SEM)

The su rfacem orpho logy of BE coarse pow der,BE-NCT cocrysta ls and their nanocrysta ls w ere stud ied by SEM(JSM-7500F,JEOL Ltd.,Japan)at an acceleration vo ltage of 10.0 kV.Sam p les w ere coated w ith go ld on a ho lder and d ried in vacuum and observed at d ifferen tm agn if ications.

2.4. Particle size analysis

Dynam ic ligh t scattering(DLS)m ethod w as used to eva luate the m ean particle size and po lyd ispersity index(PDI)w ithou t any d ilu tion at 25°C.Them easu rem en ts w ere carried ou t by Zetasizer(Nano ZS,Ma lvern Instrum en ts,UK)and a llsam p les w ere ana lyzed in trip licate.

2.5. Pow der X-ray d iffraction(PXRD)

PXRD pa tterns of BE coa rse pow der,BE-NCT cocrysta ls and their nanosized crysta l sam p les w ere reco rded using a Ni f iltered Cu Kαrad iation sou rce(D/MAX-2550V,Rigaku Co.,Japan).The rad iation w ave length w as 1.542?A.The samp lesw ere con tinuously scanned at 40 kV and 40m A from 5°to 80°(2-theta)using a scann ing speed of 5o/m in.The data w ere analyzed by MDIJade 6.0 softw are.

2.6. Differen tial scanning calorim etry(DSC)

The DSC therm ogram s of BEcoarse pow der,BE-NCT cocrystals and their nanosized fo rm u lations w ere determ ined by DSC(Jade DSC,Perk in Elm er In c.USA).Each sam p le(～5m g)w as heated in an a lum in ium pan at a scann ing rate of 10°C/m in in an atm osphere of n itrogen gas(30m l/m in)in the range of 30-350°C.

2.7. In vitro dissolution test

The in vitro d isso lu tion tests w ere execu ted to evaluate the d isso lu tion rate of BE,BE-NCT cocrysta ls,BE nanocrysta ls and BE-NCT nano-cocrysta ls,using basketm ethod w ith d isso lu tion testing appa ratus(DT-820,ERW EKA Co.,Germ any)w ith th ree rep licates for each group.FaSSIF-V2 and FaSSGF w ere em p loyed as d isso lu tion m ed ium.BE coarse pow der,BENCT cocrysta ls,BE nanocrysta ls and BE-NCT nano-cocrysta ls(equ iva len t to 18m g of BE)w ere separately f illed in to the hard capsu les and then imm ersed in to 900m ld isso lu tion m ed ium.The tem peratu re w as m ain tained at 37±0.5°C and the stirring rate w as 100 rpm.2m l of a liquo ts w ere w ithd raw n at 5,10,15,30,45,60,90,120,180,240,300 and 360m in,and at the sam e tim e,an equa lvo lum e of fresh d isso lu tion m ed ium w ere added.A fter f iltration of 0.45μm m icropo rousm em b rane the sam p les w ere assayed by HPLC(LC-20A,Sh im adzu,Japan).

2.8. In vivo pharm acokinetics

Tw en ty-fou r hea lthy Sp rague-Daw ley rats w eigh ing 250±20 g w ere em p loyed in th is study and a llow ed free access to w ater bu t fasted overn igh tbefore adm in istration.In pharm acokinetic study,random ized design w as app lied to d ivide a ll rats in tofou r groups of six an im a ls each.Each group w as adm in istered BEcoarse pow der suspension,BE-NCT cocrysta lsuspension,BEnanocrystals and BE-NCT nano-cocrystals at the sam e o ra l dose of 80m g/kg.Blood sam p les w ere co llected in to heparin ized tubes from the ocu li cho rioideae vein at 0.083,0.25,0.5,0.75,1,1.5,2,3,4,6,8,10,12,16,24,48 h a fter o ra l adm in istration.Then a fter cen trifugation for 10m in at4000 rpm,100μl separated p lasm a w as tran sferred in to ano ther neat tube and frozen at-20°C un til determ ination.

Plasm a sam p les w ere p repared accord ing to the following steps:200μl IS so lu tion(1200 ng/m l)w as added to 100μl p lasm a sam p le in a 1.5m l p lastic cen trifuge tube.Vo rtexm ixed for 3m in and cen trifuged a t 12 000 rpm fo r 10m in.Then 5μl aliquot w as in jected in to the LC-MS/MS system for ana lysis by a w ell va lidated m ethod as described below.

Tab le 1-M ass pa ram eters fo r the assay of BE and BA in rat p lasm a.

2.9. UPLC-MS/MS determ ination ofBE and BA in p lasm a

BEand BA in p lasm a sam p lesw ere ana lyzed by a UPLC-MS/MS system consisting of an Agilen t series 1290 UPLC system and an Agilen t 6460 trip le quad rupo lem ass spectrom eter(Agilen t Techno logies,USA).Accord ing to the a rticle w ith som e m odif ications[27],the m ethod w as estab lished and va lidated fo r specif icity,linearity,accu racy,p recision,recovery,m atrix e ffect and stability du ring the sam p les storage and p rocessing p rocedu re.

The ch rom atograph ic separation of ana ly tes and IS w as perform ed on an Acqu ity UPLC HSS T3 C18 co lum n(50mm×2.1m m,1.8μm,Waters,CA,USA)w ith the co lum n tem peratu re at 30°C.Ch rom atograph ic separation w as ach ieved w ith grad ien t elu tion using a m obile phase comp rised of 0.05%fo rm ic acid in w ater(A)and aceton itrile(B).The UPLC grad ien t p rogram w as set as fo llow s:5%→32%B at 0.0-4.0m in;32%→20%B at 4.0-4.1m in;20%→50%B at 4.1-9.0m in;50%→5%B at 9.0-9.5m in;5%→5%B a t 9.5-10.0m in.The co lum n oven w asm ain tained at 30°C.Eff icien t and symm etrical peaks w ere obtained at a f low rate of 0.3m l/m in.A vo lum e of 5μl p repared sam p les w as in jected in to the ch rom atograph ic system w ith the tem peratu re of au tospm p ler m ain tained at 4°C.

The detection of the ana ly tes w as perfo rm ed in the m u ltip le reaction m on ito ring m ode(MRM)using an e lectrosp ray positive ion ization(ESI+).The characteristic p recu rso rs[M+H]+to p roduct ions transitions for quan tif ication w ere m/z 271.06→122.9,447.09→270.8 and m/z 237.1→193.8 for BE,BA and IS,respective ly(detailed param eters w ere listed in Tab le 1).The ESI con f igu ration w as:gas tem pera tu re 350°C;gas f low rate 11 l/m in;nebu lizer 20 psi;cap illary 4000V.The fragm en to r w as op tim ized as 160V and 100V m eanw h ile the co llision energy of 14 eV and 38 eV for BEand BA,respectively.The acqu ired data w ere p rocessed by MassHun ter ana lysis for QQQ(Version 4.1,Agilen t,USA).

2.10. Sta tisticalanalysis

The pharm acokinetic param eters of BE o r BA w ere ca lcu lated acco rd ing to non-com partm en t m odel using Phoen ix W innon lin 6.4(Certara Co.,USA).To investigate and com pare the to ta l exposu re a fter o ra l adm in istration of fou r BE fo rm ulations,in tegrated pharm acok inetic pa ram eters w ere ca lculated based on the p reviously repo rted AUC based w eigh ting app roach[28].Specif ically,the w eigh ting coeff icien t(ω)w as ca lcu lated using Eq.1 o r 2.Then the in tegrated con cen trations w ere ca lcu lated by Eq.3,w here Ctrep resen ts the p lasm a concen tration at each tim e poin t.

A ll experim en ta l data w ere exp ressed as the m ean s±SD.Statistica l sign if ican ce w as estim ated by one-w ay ANOVA.

3. Resu lts and d iscussion

3.1. Preparation ofcocrystals and nano-cocrystals

M any techno logies have been reported for cocrysta l p reparation,su ch as an array of so lid state,m echanochem ica land liqu id assisted techn iques[29,30].W e p repared BEand NCT oversatu rated so lu tions to p recip itate BE-NCT cocrystals.Compared w ith m e lting m ethod,the BE-NCT cocrysta l ob tained by p recip itation had h igher pu rity and better crysta lline state.The sa tisfacto ry yield on the p rem ise of ensu ring qua lity of cocrystalsw as ach ieved by op tim izing the concen tration of BE in ethy lacetate and the ratio of BEand NCT.Thus,gram-sca led cocrysta l sam p le w as p repared in ou r w ork.

Genera lly,d rug nanocrysta l techn iques are c lassif ied as‘‘bottom up''m ethods and‘‘top dow n''m ethods.The‘‘bottom up''m ethods,such as the p recip itation techn ique,m eans that nanocrysta l can be con stru cted from them o lecu les;how ever,the‘‘top dow n''m ethods,such as the pearlm illing and hom ogen ization techn iques,m ean that the nanocrysta ls can be d isin tegrated step by step from the coarse pow der[31].In th is study,HPH w as em p loyed to p repare BE-NCT nano-cocrysta ls and BE nanocrystals.The stabilizer po loxam er 188 can contribu te to the stability of nanosuspensions.

3.2. Surfacem orphology

The su rface m orpho logy of the coarse pow der and the nanocrysta ls of BE and BE-NCT w ere p resen ted in Fig.2.BE coarse pow der(Fig.2A)appeared as b rick-shaped crysta lline,w hereas the BE-NCT cocrystals(Fig.2B)show ed a rem arkab le change in to rod shape.The BE nanocrysta ls(Fig.2C)and BENCT nano-cocrysta ls(Fig.2D)appeared as m o re sm a ll and na rrow particles w ith sho rt rod shape w ith the m ean partic le size of less than 300 nm.

Fig.2-SEM im ages of(A)BE coa rse pow der,(B)BE-NCT cocrysta ls,(C)BE nanocrysta ls and(D)BE-NCT nano-cocrysta ls at d ifferen tm agn if ica tion.

3.3. Particle size and zeta poten tial

The particle size and PDI w ere show n in Fig.3.Mean pa rtic le size of BE nanocrysta ls w as 207.33±1.62 nm and PDI w as 0.26±0.01.Fo r BE-NCT nano-cocrysta ls,the partic le size w as foun d to be 251.53±9.00 nm w ith PDI of 0.29±0.02.The zeta poten tia l va lues of BE nanocrysta ls and BE-NCT nano-cocrystalsw ere-0.39±-0.05m V and-0.68±-0.23m V,respective ly.

3.4. Crystalline state

It shou ld be bo rne in m ind that the crysta lline state is one of the factors a ffecting the d isso lu tion behavio r of d rugs[32].The eva luation of the crysta lline state is necessary to understand the po lym o rph ic changes that a d rug m igh t undergo w hen sub jected to nanosizing.

XRD and DSC are w idely used to evaluate the crystalline structu re of the d rug nanocrysta ls and w ere em p loyed in the study to assess the crysta l form s and crysta llin ity of BE coarse pow der,BE-NCT cocrysta ls,BE nanocrysta ls and BENCT nano-cocrystals.The XRD patterns of d ifferen t pow ders w ere show n in Fig.4.The BE coarse pow der(Fig.4A)exh ibited sharp,d istin ctive peaks at 2θva lues of 10.09°,11.37°,13.20°,15.31°,16.18°,16.73°,18.30°21.78°,22.95°,23.82°,24.19°,25.27°,26.22°,27.80°,34.02°and 41.28°,ind icating the crystalline natu re of BE.The XRD pa ttern of NCT(Fig.4B)w as a lso show n som e sharp peaks at 2θva lues of 11.12°,14.64°,18.90°,19.38°,19.72°,22.04°,23.18°24.54°,25.18°,25.68°,27.14°,36.78°and 38.46°.The characteristic peaks of BEw ere eviden t in BE-NCT m ix tu re(Fig.4C),revea ling no crysta l-state changes du ring the m ixing p rocess.Itw as obvious that new peaks at 2θva lues of 5.84°and 11.74°in the XRD pattern of cocrystals(Fig.4D)w h ich w ere d ifferen t from BE,NCT and their physica lm ix tu re.However,on ce nanon ized du ring h igh p ressu re hom ogen izing,the cocrysta l crysta lline sta te show ed a sign if ican t change from crystalline to am orphous state.No characteristic in ten se peak w as foun d in the XRD pattern of BE-NCT nano-cocrysta ls(Fig.4F)com pared w ith that of cocrysta l coarse pow der.In add ition,in con trast w ith BE coarse pow der,the response of BE nanocrysta ls(Fig.4E)w as clearly w eaker.

The DSC cu rves of d ifferen t pow ders w erem erged in to one p ictu re and illustrated in Fig.5.The BE coarse pow der(Fig.5A)and NCT coarse pow der(Fig.5B)exh ibited a sharp endotherm ic peak at 271.40°C and 132.30°C respective ly,attribu ted to its crysta lline natu re.A new peak in the DSC cu rves of BE-NCT cocrysta ls(Fig.5D),ind ica ting the d ifferen ce from BE,NCT and their physicalm ix tu re(Fig.5C),w as seen as the evidence of a new crysta l fo rm ation[33].The cu rves of BE nanocrysta ls(Fig.5E)and BE-NCT nano-cocrysta ls(Fig.5F)show ed a b road bu t w eak peak at fu rther reduced tem peratu re of 264.64°C and 151.76°C,ind icating an am oun t of BE and cocrystals transferred from crystalline state in to am orphous state du ring HPH p rocess.

Fig.3-Pa rticle size of(A)BE nanocrysta ls and(B)BE-NCT nano-cocrysta ls.

Fig.4-PXRD spectra of(A)BE coa rse pow der,(B)NCT,(C)the physica lm ix tu re of BE and NCT,(D)BE-NCT cocrysta ls,(E)BE nanocrysta ls and(F)BE-NCT nano-cocrysta ls.

Fig.5-DSC cu rves of(A)BE coarse pow der,(B)NCT,(C)the physica lm ix tu re of BE and NCT,(D)BE-NCT cocrystals,(E)BE nanocrysta ls and(F)BE-NCT nano-cocrysta ls.

Fig.6-In vitro re lease p rof iles of BE coarse pow der,BE-NCT cocrysta ls,BE nanocrysta ls and BE-NCT nano-cocrysta ls in FaSSIF-V2(n=3,M ean±SD).

It w as repo rted that som e d rugs retained their crysta lline state du ring hom ogen ization,su ch as danazo l n ifed ip ine and ucb-35440-3[34].How ever,for som e other d rugs,such as BE and BE-NCT cocrysta ls,the resu lts of DSC and X-ray show ed that the am o rphous state w as generated du ring hom ogen ization.Drugs in the am o rphous state possess h igher so lubility and faster d isso lu tion rate due to the h igher inner energy.Therefore,w hen dosed th rough the o ra l rou te,the d rug nanosuspension s in am orphous rate w ou ld show m o re sign ifican t e ffects on enhan cing bioavailability than the crysta lline nanosuspen sion s p rovided the h igh energy state cou ld be kep t in the gastroin testina l tract[35].

3.5. In vitro dissolution test

An im portan t featu re of d rug nanocrystals is the in crease of the satu ration so lubility and d isso lu tion ve locity[36],w h ich m akes d rug nanocrysta ls am enab le to m any app lications.Therefore,the in vitro d isso lu tion test w as carried ou t to d isp lay the d issolu tion p rof iles of nanocrystals in FaSSIF-V2 and FaSSGF.The resu lts show n in Figs.6 and 7 exh ibited a sign if ican t enhan cem en t of BE-NCT cocrysta ls,nanosized BE crysta ls and nanosized cocrysta ls in bo th m ed ia.In FaSSIF-V2,the BE-NCT cocrysta ls,BE nanocrysta ls and BE-NCT nanococrysta ls dem onstrated 1.74-fo ld,2.01-fo ld and 2.17-fo ld increase in 360m in d isso lu tion rate(44.97%±4.35%of cocrystals,51.76%±3.59%of nanocrystals and 55.90%±3.81%of nano-cocrysta ls)com pared w ith 25.80%±1.51%d isso lu tion of BE coarse pow der.In FaSSGF,the BE coarse pow der show ed 12.60%±1.93%w h ile a 2.22-fo ld in crease 27.95%±1.08%of d isso lu tion rate w as observed by BE-NCT cocrysta ls.W hen BE coarse pow der and BE-NCT cocrysta ls w ere hom ogen ized in to～200 nm sca le,the d isso lu tion rate of their nanoform s p rof iled m uch better than coarse pow der.In the f irst 30m in,attribu ted to the resu ltan t enhancem en t in the su rface area and decrease in the d iffusion layer th ickness[37].31.04%±1.31%of BE nanocrysta ls w ere d isso lved at 360m in,and 31.94%±8.93%fo r BE-NCT nano-cocrysta ls,w h ich exh ibited 2.46-fold and 2.54-fold enhan cem en t than BEcoarse powder.

Fig.7-In v itro re lease p rof iles of BE coa rse pow der,BE-NCT cocrysta ls,BE nanocrysta ls and BE-NCT nano-cocrysta ls in FaSSGF(n=3,M ean±SD).

3.6. M ethodology verification ofp lasm a sam ple analysis

Typ ical ch rom atogram s of blank p lasm a,blank p lasm a sp iked w ith the ana ly tes and p lasm a sam p les dem onstrated that no endogenous in terferen ce w as observed at the reten tion tim e of the ana ly tes and IS in b lank p lasm a,w h ich p roved the assay specif icity.The reten tion tim e of BE,BA and IS is 8.10,4.81 and 7.27m in respective ly.Regression equation s show ed good linearity w ith co rre lation coeff icien ts greater than 0.99.In tra-day and in ter-day p recision w ere less than 4.02%and 3.81%,and the accu racy w asw ith in±6.59%in p lasm a,respectively,ind icating the overa ll rep rodu cibility of them ethod.Average ex traction recoveries of BEand BA at th ree leve ls ranged 67.81%-72.52%and 65.57%-70.50%,respectively.Satisfactory e ff icien cy of ex traction recovery w as ob tained w ith in the accep tab le lim it.Ma trix e ffect va lues ranged 68.08%-75.76%for BE,and 67.57%-71.79%fo r BA at th ree levels,respectively.A ll analytes in rat p lasm a w ere stable on storage atam bien t temperatu re fo r 6 h,on sto rage fo r 30 d under-20°C,in freezethaw stability and on sto rage in the au tosam p ler at 4°C for 24 h,respectively.The stability w ou ld satisfy the requ irem en ts of a rou tine phar m acokinetic study.More details w ere supp lied as attachm en t.

3.7. In vivo pharm acokinetics

Ow ing to the m etabo lic transfo rm ation from BE in to BA a fter ora l adm in istration[38],both BE and BA w as detected in rat p lasm a to eva luate the pha rm acok inetics a fter o ra l adm in istration of d ifferen t BE form u lations.The m ean p lasm a con cen tration versus tim e p lots of BE and BA a fter o ra l BE coarse pow der suspension,BE-NCT cocrysta l suspen sion,BE nanocrysta ls and BE-NCT nano-cocrysta ls a t the sam e dose of 80m g/kg are p resen ted in Fig.8 and 9.The co rrespon d ingm ajor pharm acokinetic param eters of non-com partm en tm odel fo r BE and BA in rats are listed in Tab le 2.

Fig.8-M ean baica lein p lasm a con cen tration-tim e p rof iles a fter oral adm in istration of BE coarse pow der,BE-NCT cocrysta ls,BE nanocrysta ls and BE-NCT nano-cocrysta ls suspension to rats(n=6,M ean±SD).

Fig.9-M ean baica lin p lasm a con cen tration-tim e p rof iles a fter oral adm in istration of BE coarse pow der,BE-NCTcocrysta ls,BE nanocrysta ls and BE-NCT nano-cocrysta ls suspension to rats(n=6,m ean±SD).

The con cen tration of BA show ed m u ch larger than BE,w h ich eviden ced the f irst-pass m etabo lism in the in testine[39].Fo llow ing the oral adm in istration of BE,BE-NCT cocrysta ls,BE nanocrysta ls or BE-NCT nano-cocrysta ls,the doub le peak show n in the m ean p lasm a con cen tration versus tim e p lo ts of bo th BE and BA w as typ ica l acco rd ing to the in testinalm etabo lism m en tioned in the references[40,41].The AUC of BE group w as 17.95±5.24 h·μg/m l.The AUC s of cocrystals and nanocrystals w ere 52.32±9.27 h·μg/m l and 59.21±6.95 h·μg/m l w h ich rep resen ted 2.91-fo ld and 3.30-fo ld than that of BE.The h ighest AUC w as observed by BE-NCT nanococrysta ls 109.34±18.73 h·μg/m l am ong the fou r groups w h ich w as 6.09-fo ld than BE.

Table 2 - Main pharmacokinetic parameters of BE and BA in plasma after oral BE (Mean±SD, n=6).

Fig.10-In teg rated p lasm a con cen tration-tim e p rof iles a fter o ra l adm in istration of BE coarse pow der,BE-NCT cocrysta ls,BE nanocrysta ls and BE-NCT nano-cocrysta ls suspension to rats(n=6,M ean±SD).

Tofu rther investigate the to ta lexposu re a fter o ra ladm in istration of BE fo rm u la tions,the in tegrated pharm acokinetic app roach w as carried ou tw ith the AUC-based w eigh tingm ethod and the to ta l d rug con cen tration m ethod[28,42]and in tegra l con cen tration in p lasm a and in tegra l pharm acok inetic param eters w ere thus ca lcu la ted.

The in tegrated p lasm a concen tration-tim e p rof iles w ere p resen ted in Fig.10.The AUC w eigh ted facto rs(ω)of the BEand BA in rat p lasm a a fter o ra l adm in istration of BE coarse powder,BE-NCT cocrysta ls,BE nanocrysta ls and BE-NCT nanococrystals w ere show n in Table 3.The in tegral pharm acokinetic param eters for o ra l BE w ere sum m arized in Tab le 4.Fo llow ing the o ra l adm in istration of BE coarse suspension,the peak con cen tration reached a t 2880.83±775.45 ng/m l.A fter adm in istration of the sam e dose of cocrystals,nanocrysta ls,or nano-cocrysta ls,the p lasm a d rug con cen tration peaks in creased in to 4048.49±374.71 ng/m l,7993.59±1257.51 ng/m l and 6568.72±1229.92 ng/m l,respective ly.BE coarse pow der group gained the f irst peak at 1.14 h and the second at 6.50 h w h ile fo r cocrysta ls at 1.04 h and 9.00 h.BE nanocrysta ls gained the f irst peak at 0.28 h,m u ch earlier than the o ther th ree groups.BE-NCT nano-cocrysta ls gained the f irst peak at 1.58 h and the second at 12.67 h,w h ich m ay be related to the specia l p roperty of BE-NCT nano-cocrysta ls.From the com parison of AUCs show n in Tab le 4,w e cou ld no tice that AUC0-tsign if ican tly increased from 17.73±5.18 h·μg/m l for BE coarse suspension to 50.87±9.01 h·μg/m l fo r BENCT cocrysta ls,58.84±6.91 h·μg/m l fo r BE nanocrysta ls and 106.77±18.28 h·μg/m l fo r BE-NCT nano-cocrysta ls(P＜0.01),ind icating that the relative bioavailability enhancem en t of cocrysta ls,nanocrysta ls and nano-cocrysta ls w ere 2.87,3.32 and 6.02-fo ld(the ratio betw een AUC0-t)than BE coarse suspen sion,respectively.

Tab le 3-AUC0-t and AUCw eigh ted facto rs(ω)of the BEand BA in rat p lasm a a fte r o ra ladm in istration of BE coarse pow der,BE-NCT cocrysta ls,BE nanocrysta ls and BE-NCT nano-cocrysta ls(M ean±SD,n=6).

Tab le 4-In tegrated pha rm acok inetic pa ram eters of BE and BA in p lasm a a fter o ra l BE(M ean±SD,n=6).

The resu lts of in tegra l pharm acok inetic param eters exh ibited nanocrysta llizaton form as w e ll as cocrysta llization fo rm cou ld im p rove the abso rp tion of BE,w h ich re fers to their enhan ced disso lu tion rate.In the f irst tw o hou rs after adm in istration,the in tegra l con cen tration in p lasm a and AUC of BE nanocrysta ls,w h ich w ere m uch h igher than BE coarse powder,revea led the advan tages of d rug nanocrysta ls on the ora l d rug delivery of poorly so luble d rugs.Mechan ism s con tribu ted fo r the im p roved abso rp tion cou ld be m a jo rly sum m arized as tw o poin ts:i)im p roved so lubility and d isso lu tion rate;and ii)bioadhesion to the in testina l w a ll[43].Differen t from BE and BE nanocrysta ls,the pharm acok inetic p rof ile of BE-NCT cocrysta ls exh ibited the second peak at 10 h larger than the f irst peak at 1 h,w h ich revea led that BE-NCT cocrysta l fo rm im p roved the bioavailability and m od if ied the absorp tion or m etabo lic p rocess to som e ex ten t,attribu ted to the un ique p roperties of cocrysta ls beyond to ou r know ledge.Com pared w ith nanocrysta l and cocrysta l fo rm u la tion,AUC of BE-NCT nano-cocrystal from in tegral con cen tration exh ibited sign if ican t in crease(P＜0.01).Nano-sized BE-NCT cocrysta ls sim u ltaneously possessed the advan tages of bo th BE nanocrysta ls w ith a rap id and successfu l abso rp tion in the f irst tw o hou rs and BE-NCT cocrystalsw ith a rising absorp tion at 10 h.Therefo re,BE-NCT nano-cocrysta ls w ere p roved for its h igh potentia l to im p rove the ora l abso rp tion and bioavailability of BE in con trast w ith BE nanocrysta ls and BE-NCT cocrysta ls.W hen the particle size is redu ced to nano-scale,the th ickness of the d iffusion layer decreases and the d isso lu tion rate in creases sign if ican tly.In add ition,nanoparticles are m o re easily attached to the in testina lm ucosa,thereby in creasing d rug residence tim e in the gastroin testinal tract and fu rther p rom oting the abso rp tion of poo rly so lub le d rugs.Th is p rovidesm o re su ff icien t tim e fo r the d issociation,d isso lu tion and abso rption of the d rug cocrysta lm o lecu les.Th ism ay a lso be an importan t reason for the synergistic increase of the total exposu re of BEbased on the in creasing o ra labso rp tion m echan ism of nanocrysta l and cocrysta l.How ever,the specif ic m echan ism of nano-cocrysta l p rom o ting ora l abso rp tion has not yet been clearly con f irm ed,and fu rther in tensive study is needed.

4. Con clu sion

In th is study,the BE-NCT nano-cocrysta ls w ere su ccessfu lly p repared and characterized.Com pared w ith BE coarse pow der,BE-NCT cocrysta ls and BE nanocrysta ls,BE-NCT nano-cocrysta ls exh ibited sign if ican tly-in creasing perfo rm an ce both in vitro and in vivo eva luation s,suggesting that the nano-cocrystals cou ld be p roposed as an advanced strategy fo r d isso lu tion rate and bioavailability enhan cem en t of poor so lub le natu ra l p roducts such as BE.

Con f licts of in terest

The au thors declare that there is no con f licts of in terest.

Sup p lem en tary m ateria ls

Supp lem en tary m ateria l associated w ith th is article can be found,in the on line version,at doi:10.1016/j.a jps.2018.04.009.