Co-am o rphou s so lid d ispersion system s o f lacid ip ine-sp irono lactone w ith im p roved d isso lu tion rate an d enhan ced physica l stability

Zhom eng Wng,M engchi Sun ,Tin Liu ,Zisen Go,Qing Ye,Xio Tn ,Ynxin Hou,Jin Sun ,Dun Wng,∗∗,Zhonggui He,∗

a W uya College of Innovation,Shenyang Pharm aceu tical University,Shenyang 110016,China

b Key Laboratory ofStructure-Based Drug Design&Discovery ofM inistry ofEducation,Shenyang Pharm aceutical University,Shenyang 110016,China

Keyw ords:Co-am o rphous so lid d ispersion Lacid ip ine Sp irono lactone Stability M o lecu lar dynam ic(MD)sim u lation s

A B S T R A C TCo-am orphou s so lid d ispersion(C-ASD)system s have attracted great atten tion to im p rove the solubility o f poorly soluble d rugs,bu t the selection o f an app rop riate stabilizer to stabilize am orphou s form s is still a huge cha llenge.Herein,C-ASD system o f tw o clin ica l combined used d rugs(lacid ip ine(LCDP)an d sp irono lactone(SPL))as stabilizers to each o ther,w as p repared by so lven t evaporation m ethod.The e ffects o f variation in m o lar ratio o f LCDP and SPL(3:1,1:1,1:3,1:6,and 1:9)on the d rug release characteristicsw ere exp lored.Po larized ligh tm icroscopy(PLM),pow der X-ray d iffrac tion(PXRD),d ifferen tial scann ing ca lo rim etry(DSC)and therm ogravim etric ana lysis(TGA)w ere em p loyed to eva luate the so lid states.Prepared C-ASDs w ere fu rther stud ied fo r their stability under the h igh hum id ity(RH 92.5%).Fu rther analysis o f C-ASDs via Fou rier-tran sform in frared spectroscopy(FTIR)and Ram an spectroscopy con f irm ed that hyd rogen bon d in teractions betw een the tw o d rugs p layed a sign if ican t ro le in m ain tain ing the stability o f the C-ASDs system s.M o reover,m o lecu lar dynam ic(MD)sim u lations p rovided a clear insigh t in to the stabilitym echan ism at them olecu lar level.Th is study dem onstrated the novel d rug-d rug C-ASDs system s is a p rom ising fo rm u lation strategy for im p roved d isso lu tion rate an d enhan ced physica l stability o f poo rly so lub le d rugs.

1. In trodu ction

Many new d rug cand idates iden tif ied by cu rren t d rug d iscovery techn iques are poo rly w ater so lub le[1].Th is d raw back obviously im pairs the o ra l bioavailability o f these cand idates[2].Thus,severa l form u lation strategies are adop ted to settle the rough issue,su ch as cyclodex trin in c lusion com p lexes[3],nanocrysta ls[4],nanoem u lsions[5]and am o rphous so lid d ispersions[6],etc.

Am o rphous so lid d ispersions(ASDs)have been a p referab lem ethod to enhan ce the so lubility an d the d isso lu tion rate[7].How ever,an am o rphous system is easily transform ed in to the crystalline form due to its h igher free energy and poor therm odynam ica l stability,w h ich w ou ld w eaken the dom inan t so lubility advan tages con ferred by am o rph ization[8].To m ain tain the stability o f th is system,a suitab le stabilizer is needed.Po lym ers are usua lly used as stabilizers for ASDs,bu t the fo llow ing p rob lem s a re found:(a)h igh hygroscop icity o f po lym ers w ou ld in crease the p lasticization e ffect o f the am orphous system s and decrease the physical stability;(b)low d rug load ing cou ld in crease the f ina l dosage un it vo lum e,lim iting the clin ic app lication;(c)large am oun ts o f po lym ers m igh t have excip ien t-associated side e ffects[9,10].

Co-am orphous so lid d ispersions(C-ASDs)system s contain ing sm a llm o lecu lar w eigh t stabilizers have been em erging as a m o re p rom ising strategy to so lve th is issue in recen t years[11].Many sm all-m o lecu le stabilizers such as saccharin,citric acid,tartaric acid,and am ino acids,have been se lected as co-form ers to stabilize the am o rphous system s[12-14].In add ition,m any pharm aco logica lly relevan t d rugd rug C-ASDs system s have also been reported.For examp le,sim vastatin and g lip izide,in dom ethacin and nap roxen,ateno lo l and hyd roch loro th iazide C-ASDs system s,show ed a huge advan tage in so lubility,d isso lu tion rate and physicalstability[15-17].

In th is study,tw o w ater-in so lub le d rugs possessing pharm aco logica l synergy,lacid ip ine(LCDP,Fig.1A)an d sp irono lactone(SPL,Fig.1B),w ere chosen to establish the C-ASDs system.LCDP is a d ihyd ropy rid ine ca lcium an tagon ist as a f irstline d rug for the therapy o f hypertension[18],and SPL is an a ldosterone an tagon ist and has d iu retic and an tihypertensive effect[19].Th is com bined use o f bo th an tihypertensive agen ts w ith d ifferen tm echan ism s have p roved few er adverse e ffects for ind ividualized treatm en t in clin ic[20].Herein,the in vitro re lease assays and a series o f so lid characterization m ethods w ere em p loyed to con f irm the fo rm ation o f the C-ASDs system s and to select the op tim a lm o lar ratio o f LCDP and SPL fo r m ain tain ing good physica l stability under h igh hum id ity.M o reover,m o lecu lar dynam ic(MD)sim u lations w ere em p loyed as a com p lem en tary too l to p rov ide insigh t in to the specif ic stabilization m echan ism at a m olecu lar level for the d rug-d rug C-ASDs system.

2. M ateria ls an d m ethod s

2.1. M aterials

Lacid ip ine w as pu rchased from Kangya o f N ingXia Pharm aceu ticals Co.Ltd.(Ningxia,Ch ina).Sp irono lactone w as pu rchased from Zhejiang Langhua Pharm aceu tica l Co.Ltd(Zhejiang,Ch ina).Tw een 80 w as obtained from Tian jin Bod i Chem ica l Ho ld ing Co.Ltd(Tian jin,Ch ina).Methano l(HPLC grade)w as pu rchased from Kangkede Tian jin Co.Ltd(Tianjin,Ch ina).Deion ized d istilled w aterw as used th roughou t the study.

2.2. Prepara tion of co-am orphous solid dispersion system s

C-ASDs sam p les w ere p repared by so lven t evaporation.LCDP and SPL in d ifferen tm o lar ratios(3:1,1:1,1:3,1:6,and 1:9)w ere d isso lved in ethano l an d ro tavaporized a t 30°C,an d the samp les w ere d ried under vacuum fo r 24 h to rem ove residua lso lven t.The resu lting am orphous m ateria l w as ground w ith a m ortar and pestle and then forcib ly passed th rough 80 m esh sieves to yie ld a un iform,free f low ing pow der.In add ition,pu re am orphous LCDP and pu re am o rphous SPL w ere p repared in the sam e p rocedu re.The am orphous physicalm ixtu re w as p repared by them ethod o f in crem en t by equa lquantity.

Fig.1-Chem ica l stru ctu res o f(A)lacid ip ine(LCDP)an d(B)sp irono lactone(SPL).

2.3. Physica l stability study

The C-ASDs system s in d ifferen tm o lar ratios(3:1,1:1,1:3,1:6,and 1:9)w ere exposed to satu rated po tassium n itrate so lutions(RH 92.5%)in a dessicator.A fter 10 d,the sam p les w ere w ithd raw n,and the stability o f the C-ASDs system s w as fu rther ana lyzed by PXRD,PLM,FTIR,an d in vitro re lease test.

2.4. Solid state characterization

2.4.1. Polarized lightm icroscopy(PLM)

Im ages of C-ASDs sam p les in d ifferen tm olar ratios(3:1,1:1,1:3,1:6,and 1:9)w ere co llected using PLM(O lym pus Co rporation.,Tokyo,Japan)at d ifferen t tim e poin ts:(i)im m ed ia te ly after obtain ing the d ried m aterial;(ii)10 d post exposed to h igh hum id ity(RH 92.5%).Sam p les w ere sp rink led on to a glass slide and then covered w ith a glass coverslip.Im ages o f C-ASDs sam p les w ere co llected using 4×m agn if ication.

2.4.2. Pow der X-ray diffraction(PXRD)

Pow der X-ray d iffraction(Rigaku Co rpo ration,Tokyo,Japan)w as used to eva luate the so lid-state characteristics o f C-ASDs system s.Cu Kαrad iation(1.54˚A;40 kV×30m A)w as used,and data w ere co llected in the range o f 3-50°2θw ith a dw e ll tim e o f 0.2 s and a step size o f 0.02°

2.4.3. Differentialscanning calorim etry(DSC)

A DSC 1(Mettler-To ledo In ternationa l In c.,Sw itzerland)w as used to ana lyze the therm a l behavio r o f crysta lline d rugs and C-ASDs sam p les.App roxim ately 4m g sam p le w as sea led in a lum inum pans w ith perfo rated lids,under the n itrogen gas at a f low rate o f 40m l/m in,the sam p les w ere heated at a rate of 10°C/m in from 25°C to 230°C to obtain DSC therm ogram s.

2.4.4. Therm ogravim etric analysis(TGA)

The TGA ana lysis w as perfo rm ed by using TGA 550(TA instrum en t-Waters LLC).Under n itrogen pu rge w ith a f low rate o f 25m l/m in,sam p les w ere heated from room tem peratu re to the desired tem peratu re at a heating rate o f 10°C/m in.

2.4.5. Fourier-transform infrared spectroscopy(FTIR)

To investigate the m o lecu lar in teractions betw een LCDP and SPL,Fou rier-transfo rm in frared spectroscopy(Bruker Co rpo ration,Sw itzerland)w as used to ob tain the spectra o f crysta lline LCDP,crysta lline SPL and C-ASDs system s in d ifferen tm o lar ratios(3:1,1:1,1:3,1:6,and 1:9)from the spectra l region 4000-400 cm-1w ith a reso lu tion o f 4 cm-1.Sam p les w ere p repared by ground ing w ith KBr gen tly and respectively.

2.4.6. Ram an spectroscopy

The existen ce form o f LCDP and SPL in d ifferen t LCDP-SPLCASDs system w as assessed by Ram an spectroscopy.C-ASDs sam p les in d ifferen tm o lar ratios(3:1,1:1,1:3,1:6,and 1:9)w ere analyzed in situ th rough a quartz sigh t w indow via a Ram an spectrom eter(in Via Laser M icro Ram an Spectroscopy,Renishaw PLC)equ ipped w ith a therm oe lectrica lly coo led CCD detector and a f iber op tic p robe.The m easu rem en ts w ere perform ed at room tem peratu re w ith a laser w avelength o f 785 nm and laser pow er o f 500m W.

2.5. In v itro release

A USP type II apparatus(ZRS-8G;Tianda Tian fa Techno logy Co.,Ltd,Tian jin,Ch ina)w as used to study the release o f crysta lline LCDP,crysta lline SPL,am o rphous physica lm ix tu re and C-ASDs system s in d ifferen tm o lar ratios(3:1,1:1,1:3,1:6,and 1:9)under physio logica l co rrelation m ed ium(0.07%Tw een 80)[21,22].The pow der sam p le 4m g(LCDP equ ivalen t)w as dispersed by f illing the pow der in a capsu le and suspend ing the capsu le in the basket in 500m lo f re leasem ed ium w ith a padd le speed o f 50 rpm.5m l sam p les w ere w ith d raw n at 5,10,20,30,45,60,and 120m in an d rep laced w ith the sam e am oun t o f fresh m ed ium to keep the vo lum e constan t.The sam p les w ere f iltered w ith 0.22μm m icropo rous m em b rane,and 1m l o f the f iltrate w as d ilu ted w ith the sam e vo lum e o fm ethano l,vo rtex b lend ing and ana lyzed by HPLC.A ll experim en ts w ere perform ed in trip licate.

2.6. M olecu lar dynam ics(MD)sim u lations

The structu re o f LCDP and SPL w as created using M arvin sketch so ftw are.The 3D structu res o f bo th d rugs w ere bu ilt by the structu ralm in im ization and the structu ral dynam ics optim ization via the Syby l 6.9.1 so ftw are package(Tripos Associates:St.Lou is,MO,2003).The param eters o f op tim izing w ere fo llow ing ou r p revious w o rk[23].A ll o ther param eters w ere m ain tained at the defau lt va lues.LCDP-SPL com p lexes w ere p red icted by m o lecu lar docking using Au toDock 4.0 so ftw are.Au toDock ing param etersw ere as fo llow s:them ax im um number ofenergy evaluationsw as 25 000 000 per run;the iterations o f So lis and W ets loca l search w ere 3000;the num ber o f generationsw as 100,and the num ber o f ind ividua ls in popu lation w as 300[24].

The LCDP-SPL com p lexes(1:1,1:3,1:6,and 1:9)w ere fu rther op tim ized w ith the Ma teria l Stud io 8.0 so ftw are.A llm o lecular dynam ics(MD)sim u lations w ere perfo rm ed by using the Am orphous Cell and Forcite m odu le o f the Material Stud io sim u lation packagew ith COMPASS.In the MD sim u lations,the am o rphous cellm odu le w as used to con stru ct a box,w h ich w as inun dated w ith 200 w aterm o lecu les and d ifferen t ratios of com p lexes.100 ns MD sim u lation s w ere perform ed un der the con stan t tem peratu re o f 200K.

2.7. HPLC conditions

The ch rom atograph ic separation w as perfo rm ed on a reverse phase Therm o Hypersil C18co lum n(250m m×4.6m m,5μm,Therm o Fisher Scien tif ic In c.)m ain tained at 30°C.Them obile phase w as com posed o fm ethano l and w ater(80:20,v/v).The f low rate w as 0.8m l/m in and the detect w avelength s o f LCDP and SPLw ere set at 284 nm and 238 nm,respectively.

2.8. Statistical ana lysis

SPSS statistics version 22 so ftw are w as used to perfo rm Unpaired t-test to determ ine any sign if ican t d ifferen ce betw een the crysta lline d rug,physica lm ix tu re an d LCDP-SPL C-ASDs system s in d ifferen tm o lar ratios(3:1,1:1,1:3,1:6,and 1:9).Differences w ere considered statistically sign if ican t w hen the P va lue w as less than 0.05(P＜0.05).

Fig.2-PXRD d iffractog ram s o f(A)(a)crysta lline LCDP,(b)crysta lline SPL,(c)crysta lline LCDP sub jected to so lven t evapo ration,(d)crysta lline SPL sub jected to so lven t evapo ration,an d their C-ASDs system in d ifferen tm o la r ratios fresh ly p repared(e)LCDP:SPL 3:1,(f)LCDP:SPL 1:1,(g)LCDP:SPL 1:3,(h)LCDP:SPL 1:6,and(i)LCDP:SPL 1:9.(B)C-ASDs system in d iffe ren tm o la r ratios ex posed to h igh hum id ity(RH 92.5%)fo r 10 days.(a)LCDP:SPL 3:1,(b)LCDP:SPL 1:1,(c)LCDP:SPL 1:3,(d)LCDP:SPL 1:6,an d(e)LCDP:SPL 1:9.

3. Resu lts an d d iscussion

3.1. Physical characterization of the LCDP-SPLC-ASDs system s

3.1.1. Pow der X-ray diffraction(PXRD)

PXRD w as comm on ly used to determ ine the so lid state of d rugs.The crysta llin ity o f crysta lline LCDP,crysta lline SPL,crysta lline LCDP sub jected to so lven t evaporation,crysta lline SPL sub jected to so lven t evapo ration and a ll the fresh ly p repared LCDP-SPLC-ASDsw ere investigated by PXRD.As seen in Fig.2A,the sharp peaks o f the d iffractogram s fo r crysta lline LCDP and crysta lline SPL w ere detected.The sing le d rug am o rphous com ponen t cou ld no t be p repared via so lven t evaporation,due to fast recrysta llization du ring the p rocess o f p reparation.The LCDP-SPL(3:1,1:1)C-ASDs a lso show ed the crysta llin ity peaks,bu t the degree o f crysta llin ity w as reduced.An am orphous halo w as observed in the d iffractogram for the investigated LCDP-SPL(1:3,1:6,and 1:9)C-ASDs system s,suggesting that the crysta lline d rugs w ere converted in to an am o rphous fo rm in these system s.

The C-ASDsw ere f irstly exposed to h igh hum id ity and then sub jected to PXRD as w e ll.In the PXRD d iffractogram s,no detectab le crysta lline peak w as observed for the LCDP-SPL(1:3,1:6,an d 1:9)C-ASDs system s,ind icating that these C-ASDs system sw ere stab le a fter exposu re to h igh hum id ity,as show n in Fig.2B.

3.1.2. Therm alanalysis

DSC w as conducted to fu rther con f irm the crysta lline natu re o f sam p les.Therm ogram s o f the crysta lline LCDP and crysta lline SPLa fter the f irst and second heating are show n in Fig.S1,w h ich revealed the Tg va lues o f 59°C and 92.16°C for LCDP and SPL,respectively.As seen in Fig.S2,crysta lline LCDP and crysta lline SPL d isp layed sharp endo therm icm elting peaks at around 185.56°C and 207.32°C,respectively.The therm ogram s o f physica lm ix tu re(1:6)and LCDP-SPL(3:1,1:1)system s ind icated that these com pounds fo rm ed a binary eu tectic system.In the tota l therm ogram s o f LCDP-SPL(1:3,1:6 and 1:9)system s,the m e lting peaks fo r LCDP and SPL w ere no t observed,an d they exh ibited a single com position-dependen t Tg va lue at 74.17°C,85.37°C,and 92.87°C,respective ly.M o re importan tly,it w as no tew o rthy that w ith h igher m o lar ratio o f SPL,the C-ASDs system s show ed Tg va lues close to Tg va lue of SPL,w h ich w as consisten t w ith the statem en t that the Tg o f the binary am o rphous system tends to be c lose to the Tg o f the com ponen t p resen t in excess them ix tu re[25].In add ition,an exotherm ic even t happened in the range of 120-150°C,fo llow ed by endo therm ic peak at around 160-185°C,w h ich m igh t be attribu ted to the recrysta llization o f am o rphous d rug and them elting o f recrysta llized d rug,respectively.

TGA experim en ts w ere carried ou t to verify the im pact o f m oistu re o r residua l so lven t on the C-ASDs system s.The TGA resu lts dem onstrated that LCDP and SPL are therm a lly stab le w ith in 189.1°C and 219.64°C,respective ly.A sm a llw eigh t loss(0.77%)at abou t 78°Cw as observed in the C-ASDs system(1:6),due to the residua l ethano l in the C-ASDs system p repa red by so lven t evapo ration(Fig.S3),w h ich m igh t lead to the low ered-Tg o f C-ASDs system.

3.1.3. Polarized lightm icroscopy(PLM)

Po larized ligh t m icroscopy(PLM)experim en t w as in tu itive ly perfo rm ed to eva luate the effect o f hum id ity on the LCDPSPL C-ASDs system s.In Fig.S4,birefringen ce phenom enon w as obviously observed in crysta lline LCDP and crysta lline SPL,w hereas no v isib le bire fringen ce phenom enon w as observed in all p repared LCDP-SPL C-ASDs system s excep t for the system w ith m o lar ratio o f 3:1 and 1:1 in Fig.3A.The resu lt w as in a good agreem en t w ith the resu lts ob tained by PXRD.For the sam p les after exposu re to h igh hum id ity in Fig.3B,LCDP-SPL(1:6,1:9)C-ASDs system s d idn’t exh ibited birefringen ce signa l,ind icating that the exce llen t stability o f both system s under th is h igh hum id ity cond itions.How ever,a few crysta ls w ere observed in the LCDP-SPL(1:3)C-ASDs system s.Th is phenom enon cou ld no t be detected by PXRD,due to the lim ited sensitivity.It w as essen tia l tha t a com bination of several so lid characterization m ethods w as adop ted to accu rate ly determ ine the crysta llin ity states.

Fig.3-PLM im ages o f LCDP-SPL C-ASDs system s(A)sam p les fresh ly p repared and(B)sam p les exposed to h igh hum id ity(RH 92.5%)fo r 10 days.(a)LCDP:SPL 3:1,(b)LCDP:SPL 1:1,(c)LCDP:SPL 1:3,(d)LCDP:SPL 1:6,and(e)LCDP:SPL 1:9.

3.1.4. FTIR spectroscopy

FTIRw as con ducted to investigate them o lecu lar in teractions in the LCDP-SPL C-ASDs system s.The FTIR spectra o f crysta lline d rugs an d C-ASDs are p resen ted in Fig.4A.The crysta lline LCDP show ed the characteristic peak at 3348.3 cm-1,belonging to the stretch ing vibration o f the am ino(-NH)groups[23].And the crysta lline SPL dem onstrated strong bands o f carbony l(C=O)groups at 1691.1 cm-1,1768.1 cm-1and 1674.1 cm-1,assigned to the stretches o f O=C-S,O=C-O and O=C-C=C,respectively.For the p repared C-ASDs system s,the peak in tensity o f am ine groups w as w eakened in LCDPSPL(3:1,1:1)C-ASDs,w h ile the d istin ctive am ine peak o f LCDP d isappeared in the LCDP-SPL(1:3,1:6 and 1:9)C-ASDs.Co rrespond ingly,the carbony l peak o f the th ioacety l group in SPL w as b roadened and in tensif ied in LCDP-SPL(1:1,1:3,1:6,and 1:9)C-ASDs system s,in d icating the fo rm ation o f in term o lecu lar hyd rogen bonds betw een the-NH group o f the LCDPand the C=O group o f th ioacety l in SPL.No in term o lecu lar hyd rogen bond in the LCDP-SPL(3:1)system s w as fo rm ed.

The C-ASDs system s a fter exposu re to h igh hum id ity cou ld be fu rther investigated using FTIR spectroscopy.As show n in Fig.4B,the in tensity o f-NH peak at 3348.1 cm-1in the LCDPSPL(1:1)C-ASDs w as enhan ced.In the LCDP-SPL(1:3)C-ASDs system,a new peak at 3348.7 cm-1w as detected,co rrespon ding to the-NH stretch ing peak o f crystalline LCDP.In add ition,the carbony l peak o f the th ioacety l group o f SPL sh ifted from 1691.1 cm-1to 1692.1 cm-1.These changes ind icated that these sam p les w ere recrysta llized,and the in term o lecu lar hyd rogen bondsm igh t be broken.In con trast,LCDP-SPL(1:6,1:9)C-ASDs system s show ed no no tab le d ifferen ces in the spectra fo llow ing storage at 92.5%RH fo r 10 d.The in term o lecu lar hyd rogen bonds in the LCDP-SPL(1:6,1:9)C-ASDs w ere w ellm ain tained.

3.1.5. Ram an spectroscopy

Ram an spectroscopy is p roved to be ano ther eff icien t too l to ana lyze so lid-state changes o f d rugs in the fo rm u lations and characterize the m o lecu la r in teractions betw een d rugs and excip ien ts[26].For the crysta lline d rug,the m o lecu le structu re is in a long-range o rder an d the Ram an-active bon ds are indeed sam e in each m o lecu le;how ever,the m o lecu le structu re o f the am orphous state is in short-range order or lack in long-range o rder and rep resen ts relative lym u ltip le regions[27].Con cern ing to the p repa red LCDP-SPL(1:1,1:3,1:6,1:9)system s,C=O peak o f the th ioacety l bond in SPL at 1691 cm-1w as broadened and sh ifted tow ards low er w avenum bers,and-NH stretch ing o f crysta lline LCDPat 3348.3 cm-1d isappeared in Fig.S5.These resu lts suggested that the changes in m o lecular con f irm ation and con f irm ed the fo rm ation o f in term o lecu lar hyd rogen bond in teractions.Specif ically,the-NH group o f the LCDP and the O=C group o f th ioacety l in SPL served as hyd rogen bond donor and accep ter,lead ing to the form ation of a heterod im er.Th is resu ltw as consisten tw ith the analysis o f FTIR.

3.2. In v itro release

Fig.4-FTIR spectra o f(A)(a)crysta lline LCDP,(b)crysta lline SPL,(c)physica lm ix tu re(1:6),an d their C-ASDs system s in d iffe ren tm o la r ratios fresh ly p repared(d)LCDP:SPL 3:1,(e)LCDP:SPL 1:1,(f)LCDP:SPL 1:3,(g)LCDP:SPL 1:6,an d(h)LCDP:SPL 1:9.(B)C-ASDs system in d ifferen tm o la r ratios ex posed to h igh hum id ity(RH 92.5%)fo r 10 d.(a)LCDP:SPL 3:1,(b)LCDP:SPL 1:1,(c)LCDP:SPL 1:3,(d)LCDP:SPL 1:6,an d(e)LCDP:SPL 1:9.

Fig.5-Re lease p ro f iles o f the:(A,C)LCDP and SPL from crysta lline d rug,physica lm ix tu re(1:6),and their C-ASDs system in d iffe ren tm o la r ra tios.(B,D)LCDP an d SPL from its C-ASDs system s un der h igh hum id ity(RH 92.5%)fo r 10 days.Data a re p resen ted as the m ean±SD(n=3).

As show n in Fig.5A,the release o f low-am oun t d rugs w as detected from crystalline LCDP and its physicalm ixtu re.However,the re lease o f LCDP from C-ASDs system s w ith a ll the d ifferen tm o lar ratios w as enhan ced com pared w ith its crude crysta lline pow ders.The release rate and cum u lative re lease o f LCDP from d ifferen t d rug ratios C-ASDs have the considerab le d ifferen ces w ith in 120m in.Fo r LCDP-SPLC-ASDs w ith m o lar ratios(3:1,1:1 and 1:3),the release o f LCDP w as 5.1%,21%and 56%,respectively.Th is dem onstrated that LCDP release w ou ld be enhan ced w hen the con ten t o f SPL in creased in the C-ASDs system.Itw as notew o rthy that 69%o f LCDPw as re leased from LCDP-SPL(1:6)C-ASDs system w ith in 120m in,sim ilar to that o f LCDP-SPL(1:9)C-ASDs system.Statistica l data ana lysis revea led that there w as no sign if ican t d ifferen ce betw een these tw o system s(P＞0.05).A lthough bo th system s show ed them axim um cum u lative releases,LCDP-SPL(1:6)CASDs system w as in closer p roxim ity to the c lin ica lly relevan t com bination doses o f 1:6.83(4m g/25m g).There fo re,1:6 w as considered as them ost op tim a lm o lar ratio to ach ieve the optim al release o f LCDP-SPLC-ASDs system.

Fig.6-Chem ica l stru ctu res w ith p roposed hyd rogen bond betw een(A)LCDP an d LCDP,(B)LCDP and SPL.

The re leases o f SPL from crysta lline SPL,physica lm ix tu re,and LCDP-SPLsystem w ith them o lar ratio o f 3:1w ere app rox im ately 40%(P＞0.05)in Fig.5C.The releases o f SPL from LCDPSPL(1:1,1:3,1:6,and 1:9)C-ASDs system s w ere found to be sim ilar.The cum u lative re lease o f SPL from LCDP-SPL(1:6)CASDs system w as nearly 1.5 tim es h igher than that o f the crystalline SPL.

To eva luate the physica l stability o f LCDP-SPLC-ASDs system s,the C-ASDs w ere sub jected to the in vitro release test a fter exposu re to h igh hum id ity(RH 92.5%).As seen in Fig.5B and D,the sign if ican t decrease in the release o f LCDP and SPLw ere observed in LCDP-SPL(1:1,1:3)C-ASDs system s.Th is m igh t be due to the recrysta llization o f am o rphous d rugs in CASDs.How ever,LCDPand SPL re lease p ro f iles from bo th LCDPSPL(1:6,1:9)C-ASDs system sw ere sim ilar to thato f the fresh ly p repared sam p les(P＞0.05),ind icating that LCDP-SPL(1:6,1:9)C-ASDs system s show ed excep tiona l stability under th is h igh hum id ity cond itions after 10 d.The am oun t o f SPL w as considered as a cru cia l factor in f luen cing the stability.Hen ce,it is certain ly believed that a su itab le am oun t o f stabilizer is necessary fo r a C-ASDs system to ach ieve the m axim um re lease and good physica l stability.

3.3. M olecu lar dynam ic(MD)sim u la tions

Mo lecu lar dock ing w as perfo rm ed to p rovide a better understand ing o f the m o lecu lar in teractions.The hyd rogen bonds betw een LCDP and LCDP(Fig.6A),as w ell as betw een LCDP and SPL(Fig.6B)w ere obviously observed.Dock ing resu lts revea led that in term o lecu lar hyd rogen bonds w ere form ed betw een the am ine(-NH)group o f LCDPand the carbony l(C=O)o f th ioacety l group in SPL,agreeing w ellw ith the experim enta l resu lts o f IR and Ram an spectroscopy.Bu lk am o rphous cellm ode ls o f LCDP and SPLw ere constru cted(Fig.S6).In the com pu ter sim u lations,the bond length and the bind ing energy w ere used to eva luate the strength o f in teractions.The bond length o f-N-H···O=C-O betw een tw o LCDPm o lecu les w as 2.82279 pm,w hereas the bond length of-N-H···O=S-O betw een LCDPand SPLw as 2.83718 pm.Mo reover,the bind ing energy va lue o f hyd rogen bond in teraction in the LCDP-LCDP and LCDP-SPL com p lex w as-2.8 kca l/m o l an d-4.2 kca l/m o l,respectively.These resu lts p roved that LCDP had a h igher tenden cy to in teract w ith SPL th rough the fo rm ation o f hyd rogen bonds rather than aggregated betw een the ind ividualm o lecu les,w h ich u ltim ately h indered the occu rrence o f recrysta llization.Consequen tly,the-N-H···O=S-O hyd rogen bonds betw een LCDP and SPL m o lecu le w ere the dom inan t fo rce for stabilizing the LCDP-SPLC-ASDs system s.

Fig.7-Cohesive energy density o f LCDP-SPLC-ASDs system s as a fun ction o f sim u lation tim e.

In the MD sim u lation,cohesive energy density(CED)va lues w ere condu cted to estim ate the stability o f system s,w here h igher values ind icate better stability.Herein,the stability o f d ifferen t LCDP-SPL C-ASDs system s a fter exposu re to h igh hum id ity w as eva luated by CED va lues.As show n in Fig.7,for LCDP-SPL(1:1,1:3,1:6 and 1:9)C-ASDs,the CED m axim um value w as 5.89809×108J/m3,6.13701×108J/m3,6.34443×108J/m3,an d 6.38792×108J/m3,respectively.It cou ld be seen that CED va lues in creased a long w ith the in creased con ten t o f SPL in the C-ASDs system,w h ile the CED reached them axim um value in LCDP-SPL(1:6,1:9)CASDs system s.Th is trend w as consisten t w ith that o f in vitro re lease resu lts.Likew ise,th is w as a lso con f irm ed by FTIR resu lt that hyd rogen bonds in the LCDP-SPL(1:1,1:3)C-ASDs system sw ere im paired bu tw ere w ellm ain tained in LCDP-SPL(1:6,1:9)C-ASDs system s.

Taken a ll together,bo th the resu lts obtained experim enta lly and calcu lated theoretically elucidated that in term o lecu lar hyd rogen bond p lays a sign if ican t ro le in m ain tain ing the stability o f LCDP-SPLC-ASDs system.Fu rtherm o re,MD sim ulations p rovided fo rcefu leviden ce to help understand the stability m echan ism at them o lecu lar level.

4. Con clusion

LCDP-SPL C-ASDs system s w ere successfu lly estab lished by so lven t evapo ration.The LCDP-SPL(1:6,1:9)C-ASDs system s exh ibited better in vitro re lease behavio rs an d physica l stability than LCDP-SPL(1:1,1:3)C-ASDs system s.Especially,the CASDs system w ith the op tim a lm o lar ratio o f 1:6 w as close to therapeu tica lly re levan t dose.FTIR and Ram an spectroscopy revea led the form ation o f hyd rogen bond betw een the-NH o f LCDP and the O=C-S o f SPL.The MD sim u lations fu rther verif ied the fo rm a tion o f H-bonds and p rovided the ca lcu lated CED va lues fo r the C-ASDs system s in the p resen ce o f w ater m olecu les,w h ich afforded a m o lecu lar level insigh t in to the stability m echan ism o f the C-ASDs system s.

Decla ration o f in terest

The au tho rs report no con f licts o f in terest.The au tho rs a lone are responsib le fo r the con ten t and w riting o f th is article.

Acknow ledgm en ts

Th is w o rk w as f inan cia lly supported by the Nationa l Basic Research Program o f Ch ina(973 Program,No.2015CB932100),the Nationa l Natu re Scien ce Foundation o f Ch ina(No.81473164),and Key p ro jects o f Liaon ing Prov in ce Depa rtm en t o f Edu cation(No.2017LZD03).

Su pp lem en ta ry m ateria ls

Supp lem en tary m aterial associated w ith th is article can be found,in the on line version,at doi:10.1016/j.a jps.2018.11.001.