Effect of carrageenan on poloxamer-based in situ gel in vitro and in vivo

European Journal of Pharmaceutical Sciences 37(2009)306–312

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European Journal of Pharmaceutical

Sciences

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /e j p

s

Effect of carrageenan on poloxamer-based in situ gel for vaginal use:Improved in vitro and in vivo sustained-release properties

Yu Liu,Yi-ying Zhu,Gang Wei,Wei-yue Lu ?

Department of Pharmaceutics,School of Pharmacy,Fudan University,Shanghai,PR China

a r t i c l e i n f o Article history:

Received 22December 2008

Received in revised form 10February 2009Accepted 25February 2009Available online 14March 2009Keywords:Carrageenan Poloxamer 407Acyclovir

In situ hydrogel

Sustained-release property Vaginal use

a b s t r a c t

The aim of this work is to explore effects of carrageenan on sustained-release properties of poloxamer 407-based vaginal in situ gel.After formulation of composite gel systems composed of carrageenan and poloxamer 407,in vitro release pro?les and in vivo local drug residence after vaginal administration in mice was investigated using acyclovir as the model drug.Rheological experiment was conducted to investigate effects of carrageenan on temperature-dependent viscoelasticity of poloxamer 407-based gels.It appeared that carrageenan and poloxamer 407could form composite gel systems with good thermosensitity sim-ilar to gels containing only poloxamer 407.In the in vitro release experiment,carrageenan signi?cantly decreased the release rate of acyclovir,retarded the dissolution of poloxamer 407and slowed the gel erosion (weight loss)in a concentration-dependent manner.In vivo local drug residence experiment indicated that carrageenan signi?cantly prolonged local residence of acyclovir and further showed a syn-ergistic bioadhesive effect with acrylic acid polymers (Carbopol ?).In conclusion,carrageenan was able to improve the sustained-release properties of poloxamer 407-based in situ gel,indicating that the com-bination of carrageenan and poloxamer 407may ?nd use in the development of vaginal in situ gel drug delivery systems with prolonged local residence and thus better clinical outcome.

?2009Elsevier B.V.All rights reserved.

1.Introduction

Vagina has been used for a long time as a route for delivery of several drug classes such as antimicrobials,labor inducers,spermi-cides,and sexual hormones with the purpose of obtaining a local pharmacological effect (das Neves and Bahia,2006).From a practi-cal point of view,ideal vaginal drug delivery systems should be easy to handle,minimal leakage and long local residence after admin-istration,cost-effective,nonirritant and safe even after repeated use (Vermani and Garg,2000).In this context,most vaginal dosage forms,like liquid preparations,creams/gels,tablets,?lms and tam-pons,etc.,are not satisfactory for either dif?cult self-administration or rapid clearance from the application site (Garg et al.,2003).

In recent decades,environmentally sensitive gels,in particular thermosensitive gels,have represented an important improvement for drug delivery to special cavities including vagina (Chang et al.,2002a,b ).Geli?cation of such gels in response to environmental changes,from ambient to body temperature for thermosensitive gel,offers advantages of both liquid formulations like easy admin-istration and spread in the vagina and semi-solid formulations

?Corresponding author at:Room 815,Research Building,826Zhang Heng Road,Shanghai 201203,PR China.Tel.:+862151980090;fax:+862151980090.

E-mail address:wylu@https://www.360docs.net/doc/9d6986713.html, (W.-y.Lu).like slow clearance from local sites (das Neves and Bahia,2006).Poloxamers,especially poloxamer 407,have been the most com-monly encountered thermosensitive material for their advantages such as easy availability,simple method for gel preparation and drug loading and good compatibility with various drug and phar-maceutical excipients (Kof?et al.,2006).In recent years,some studies were carried out about clotrimazole-loaded vaginal gels using poloxamers and acrylic acid polymers to achieve a safe,con-venient,and effective treatment of vaginal candidiasis (Chang et al.,2002a,b ).Nevertheless,as demonstrated by earlier studies by us (Liu et al.,2007)and other investigators (Paavola et al.,1998;Zhang et al.,2002;Tirnaksiz and Robinson,2005),the erosion of poloxamer-based gel in aqueous medium is rather fast,leading to unsatisfactory in vivo sustained-release properties.Attempts of chemical modi?cation of poloxamer have been reported with encouraging results of slower gel erosion for injection use,but chemical modi?cation usually introduced toxic compound residue and involved complicated safety issues (Bromberg and Ron,1998;Cohn et al.,2003).On the contrary,addition of macromolecules from natural or semi-natural origin into poloxamer gel is more cost-effective and convenient,providing an alternative approach to obtain composite gel systems with improved sustained-release properties as well as good safety.However,no substantial advance has been reported to date.For example,effect of hydroxypropy-lmethylcellulose (HPMC),sodium carboxymethylcellulose (CMC)

0928-0987/$–see front matter ?2009Elsevier B.V.All rights reserved.doi:10.1016/j.ejps.2009.02.022

Y.Liu et al./European Journal of Pharmaceutical Sciences37(2009)306–312307

Table1

Model?tting of release data from poloxamer gels(20%)with different composition using Peppas(M t/M∞=k×t n)release model.

Drug content(mg/g gel)Concentration of carrageenan(%)n k Fitting equation Regression coef?cient square(r2)

100.853±0.03122.9±1.04M t/M∞=22.9t0.8530.993

10.1250.740±0.045a21.0±1.08M t/M∞=21.0t0.7400.968

10.20.452±0.015a37.2±1.03M t/M∞=37.2t0.4520.990

100.20.809±0.018b16.1±1.03M t/M∞=16.1t0.8090.995

200.20.746±0.048b11.0±1.08M t/M∞=11.0t0.7460.963

300.1250.938±0.034c 5.54±1.06M t/M∞=5.54t0.9380.988

300.20.977±0.023b 3.20±1.04M t/M∞=3.20t0.9770.995

a Signi?cantly different from the20%poloxamer407–1mg acyclovir/g gel group.P<0.05.

b Signi?cantly different from the20%poloxamer407–0.2%carrageenan–1mg acyclovir/g gel group.P<0.05.

c Signi?cantly different from the20%poloxamer407–0.125%carrageenan–1mg acyclovir/g gel group.P<0.05.

and dextran on the in vitro release of lidocaine from poloxamer407 gel was studied,but only very limited improvement was observed (Paavola et al.,1998).

Consequently,it is necessary to?nd out suitable macro-molecules to circumvent this critical defect of poloxamer gel.It is the promising microbicidal property of carrageenan that attract our interest(Coggins et al.,2000).Carrageenan,which is also usually used as a gelling agents has been proven effective against HIV infec-tion by preventing the traf?cking of HIV-infected mononuclear cells from vaginal?uids to the mucosa(Perotti et al.,2003).Considering risks of HIV infection raised by lesions in the genital tract(Low et al.,2006),the corporation of carrageenan into vaginal formulations seems of particular bene?t.Therefore carrageenan is a promising adjuvant for vaginal gels,especially which aims at prevention and treatment of infectious diseases related with virus-related sexu-ally transmitted diseases.However,combination of carrageenan and poloxamer,possessing advantages of both materials,has not been report in previous literature.

Acyclovir(9-(2-hydroxyethoxymethyl)guanine,ACV)is one of the most effective and selective antiviral drugs,widely used in clinic for the treatment of herpes viruses(Baker,1999).For vaginal use,acyclovir is commercially available as creams.Few new drug delivery systems for acyclovir vaginal administration have been reported,except for a previous report about preparation and in vitro characterization of acyclovir-loaded liposome drug delivery system which might face the problem of leakage and short local residence in vivo(Pavelic et al.,2005).

Here the aim of the present work is to explore the potential of the combination of carrageenan and poloxamer407as the matrix of thermosensitive gel for vaginal use.Acyclovir was used as model drug.Both in vitro drug release and in vivo local drug residence were evaluated.Rheological measurements were also conducted to characterize the thermo-sensitivity and viscoelastivity of the gel.

2.Methods

2.1.Materials and instruments

Acyclovir was purchased from Cheng Yi Pharmaceutical Co. (Zhe Jiang,China).Poloxamer407(Lutrol?F127,F127)was pur-chased from BASF(Ludwigshaften,Germany).?-Carrageenan was purchased from FTA Co.(Shanghai,China).Carbopol934P-NF (Carbopol?)was purchased from Noveon Inc.(Cleveland,OH,USA). Bovine serum albumin was from Sigma(St.Louis,MO,USA).All other chemicals were analytical grade and used as received.

2.2.Preparation of thermosensitive gel

The“cold method”was adopted with minor modi?cation as described in previous reports(Zhang et al.,2002;Liu et al.,2007).The composition of each formulation was listed in Table1.The con-centration of poloxamer407,Carbopol?and carrageenan in the gel was always expressed as the weight percentage(wt.%),unless par-ticularly speci?ed.Brie?y,carrageenan was dissolved in pure water (70–80?C)to get the stock carrageenan solution at appropriate concentration.Then the required amount of F127and,if required, Carbopol?,for each formulation was carefully weighed and placed in a?at-bottomed vial,into which required weight of stock car-rageenan solution was added.The obtained mixture was placed at 4?C until poloxamer407and Carbopol?were dissolved completely and the blank gel was obtained.

For different drug content,the method of drug loading was dif-ferent.For the lowest drug content(1mg/g gel)listed in Table1, acyclovir stock solution were?rstly prepared to substitute the pure water used in preparation of the stock carrageenan solution.For the other three higher drug content(10–30mg/g gel),acyclovir powder was suspended in corresponding blank gel due to the poor solubility of acyclovir.Electromagnetic stirring was used to get homogeneous mixture.

2.3.In vitro release experiment

To better simulate the circumstance in the vagina,vaginal?uid stimulant(VFS)was used as the release medium,which was prepared according to the previous literature(Owen and Katz, 1999)with the following composition(g/L):NaCl,3.51;KOH,1.40; Ca(OH)2,0.222;bovine serum albumin,0.018;lactic acid,2.00; acetic acid,1.00;glycerol,0.160;urea,0.400;glucose,5.00.The mixed solution was adjusted to a pH of4.2using HCl.

For the release experiment,the membraneless model was selected because such release method could allow direct con-tact between the gel and release medium and take the erosion of gel into consideration.Such model is believed to be very suit-able for poloxamer407-based gels in which gel erosion usually plays a key role or even dominates in the release process of drug, thus being widely used in the research of poloxamer407-based gel systems(Liu et al.,2007;Cafaggi et al.,2008;Zhang et al., 2002).Brie?y,the?at-bottomed vial containing approximately2g acyclovir-loaded gel was placed in a water bath(37?C)until gel formed.10mL of the release medium(VFS)pre-equilibrated at 37?C was carefully layered over the surface of the gel.Then the vial was placed in a thermostatic shaker(100rpm,37?C).At prede-termined time intervals,the release medium was totally replaced by10mL fresh medium and the weight of the vial plus the gel was recorded to calculate the weight of gel dissolved.The con-centration of acyclovir in the release medium was determined by RP-HPLC(LC-10A,Shimadzu,Japan)equipped with YMC-Pack ODS-A C18column(150mm×4.5mm,5?m)at30?C with the following condition:mobile phase5%MeOH–95%water,?ow rate1mL/min, UV detector wavelength254nm.Before analysis,samples were ?ltered by0.22?m membrane.The concentration of F127in the

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release medium was determined by the colorimetric assay pre-ciously reported(Mao et al.,2004).If necessary,the sample was diluted with water before determination.

The release pro?le of acyclovir from gel was analyzed with the power law model.

M t

M∞

=kt n

where M t and M∞were the absolute cumulative amount of acy-clovir released at time t and in?nite time,respectively.k is a constant incorporating structural and geometric characteristics of the device(Tarvainen et al.,2002).By?tting data of M t,M∞and t for each formulation into this equation,the release exponent n was calculated to indicate of the mechanism of drug release(Siepmann and Peppas,2001).

2.4.Rheological tests

The rheological tests were performed in the dynamic oscilla-tion mode on a rheometer(Bohlin Gemini HR nano,Malvern,UK). The change in rheological parameters as a function of tempera-ture for three gels,20%poloxamer407,20%poloxamer407–0.2% carrageenan and20%poloxamer407–0.2%carrageenan–0.2% Carbopol?,was obtained from oscillatory measurements with the frequency?xed at1Hz from15?C to40?C at a heating rate of 1?C/min.Based on the linear viscoelastic regime obtained from pre-liminary strain/stress sweep measurements,a stress control mode with the shear stress?xed at8Pa was selected for20%poloxamer 407,while a strain control mode was selected with the strain?xed at1%was selected for the other two gels.The gelation tempera-ture was de?ned as the medial temperature between those for the solution and the gel(Jiang et al.,2008).

2.5.In vivo experiment

Female ICR mice(25±5g,provided by Department of Labo-ratory Animal Science,Fudan University Shanghai,China)were acclimated for at least1week before being used.Mice were allowed to free access to standard food and tap water.

The dose of acyclovir was300?g acyclovir/10?L(30mg acy-clovir/g gel)per mouse,selected according to the dosage of commercially available acyclovir vaginal formulations used in clinical study and practice.Five formulations were tested,includ-ing:(1)acyclovir suspension group(the control group);(2) acyclovir-loaded20%poloxamer407group;(3)acyclovir-loaded 20%poloxamer407–0.2%carrageenan group;(4)acyclovir-loaded 20%poloxamer407–0.2%Carbopol?group;(5)acyclovir-loaded 20%poloxamer407–0.2%carrageenan–0.2%Carbopol?group.For each formulation,mice were divided into?ve groups to provide data for the following?ve time points:0h(immediately after administration),3h,6h,9h and12h.

Intravaginal administration of the studied thermosensitive gel was performed with a microliter syringe with a blunt needle.After insertion of the needle approximately0.5cm into the vagina,10?L of in situ gel or the control drug suspension pre-cooled to the liquid state was discharged rapidly and the animal was kept in an inverted position for about10s to assure the time for geli?cation.

To determine the local resident amount of acyclovir,the vagina was thoroughly washed by0.9%NaCl and the washing solu-tion was collected for RP-HPLC analysis which was conducted under the same condition as used for the in vitro release exper-iment.Before analysis,the collected washing solution for each mouse was diluted to1mL with water and?ltered by0.22?m membrane.2.6.Statistical analysis

The one-way analysis of variance(ANOVA)was used for compar-ison among groups.Differences of the mean values were evaluated by the Student’s unpaired t-test.A P<0.05was considered signi?-cant.

3.Result and discussion

3.1.Gel dissolution and drug release

It should be kept in mind that it is nearly impossible to totally simulate in vivo condition by in vitro release experiment,but effective and reliable comparison of different formulations can be realized by choosing suitable release medium and experiment pro-tocol.With respect to the composition and pH,the vaginal?uid is different from other body?uid in that it is acidic(pH ranges from 4to6,dependent on the menstrual cycle,age,health,etc.),rich in lactic acid and other bactericidal compounds such as hydrogen peroxide,bacteriocin-like substances and possibly biosurfactants which are produced by microorganisms speci?c for female genital tract such like Lactobacilli(Boris and Barbes,2000;das Neves and Bahia,2006).Therefore,VFS was used as the release medium in order to better simulate the actual circumstances that the gel may face after administration into the vagina.

The HPLC method for quantitation of acyclovir in the release medium was validated with respect to standard curve(y=73386x, r2=0.999),linear range(1–100?g/mL),intraday precision and interday precision(both<5%).

3.1.1.Gel dissolution

3.1.1.1.Effect of carrageenan on the gel erosion vs.time pro?les.Fig.1 presented the effect of carrageenan on the gel erosion vs.time pro?les.The control group,20%poloxamer407group,dissolved at a constant rate,in accordance with previous reports(Zhang et al.,2002;Liu et al.,2007).In comparison with the control group,the decrease in the rate of gel erosion was very signi?cant for carrageenan-containing poloxamer407gel.The effect of car-rageenan was concentration-dependent,for the gel weight for the 0.125%carrageenan group began to reduce after6h while the gel weight for the0.3%carrageenan group almost remained unchanged until10h.For the0.2%and0.3%carrageenan group,because

after

Fig.1.Erosion of thermosensitive gel composed of20%poloxamer407and different concentration of carrageenan(( )for20%poloxamer407without carrageenan,(?) for20%poloxamer407+0.125%carrageenan,( )for20%poloxamer407–0.2%car-rageenan,( )for20%poloxamer407–0.3%carrageenan)in the presence of vaginal ?uid stimulant at37?C(n=3,mean±S.D.).

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Fig.2.Dissolution of poloxamer407from thermosensitive gels composed of20% poloxamer407and different concentration of carrageenan(( )for20%polox-amer407without carrageenan,(?)for20%poloxamer407–0.125%carrageenan, ( )for20%poloxamer407–0.2%carrageenan,( )for20%poloxamer407–0.3% carrageenan)in the presence of vaginal?uid stimulant at37?C(n=3,mean±S.D.). 10h gels turned very soft and fragile,making measuring the weight of gel inaccurate,and after12–14h all the gels were detached from the bottom of vials and broken into small pieces,only data in10h are shown here.Gels containing0.3%carrageenan were too viscous under gelation temperature,which made its practical application very dif?cult,thus not included in further study.

3.1.1.2.Effect of carrageenan on the dissolution of poloxamer407 from gel.To get more information about the mechanism of gel erosion,the dissolution of F127was monitored by a chromogenic assay based on the combination precipitation of poloxamer407 and cobalt thiocyanate(Mao et al.,2004).The standard curve was Abs=0.592concentration(poloxamer407,mg/mL)?0.0159(lin-ear range:0.1–3.0mg/mL,r2=0.992)with good interday precision and intraday precision(both<5%).The presence of carrageenan and carbopol showed no interference with the quantitation.So the chro-mogenic assay was validated for the determination of poloxamer 407in the release sample.

The release pro?les of F127from gels composed of20%F127and different concentration of carrageenan were shown in Fig.2.Similar to the case of gel dissolution,carrageenan retarded the dissolution of poloxamer407in a concentration-dependent manner.Taking the relative slow rate of the reduction in gel weight into consideration, it can be guessed that poloxamer407gradually dissolved in water and diffused into the release medium with the lapse of time,leaving carrageenan constituting the main skeleton of gel.This conjecture awaits more validation in future work.

3.1.2.Drug release

As was shown in Figs.3and4,carrageenan signi?cantly decreased the rate of acyclovir release from poloxamer407-based gel in a carrageenan concentration-dependent manner,whatever the content or loading style of acyclovir was.The drug release mechanism was also changed by the presence of carrageenan according to the values of n in Table1.Predominance of diffusion in release mechanism was observed for carrageenan-containing groups,whereas gels composed of poloxamer407alone demon-strated a release mechanism mainly controlled by the dissolution of drug(case-II transport).Such difference might be explained by the difference in the erosion rate of the gel matrix caused by the presence of

carrageenan.Fig.3.Release pro?les of acyclovir from thermosensitive gels composed of20%F127 and different carrageenan concentration(( )for no carrageenan,( )for0.125%car-rageenan,( )for0.2%carrageenan)containing1mg acyclovir/g gel in the presence of vaginal?uid stimulant at37?C(n=3,mean±S.D.).

To ful?ll the clinical requirement for acyclovir dosage of vagi-nal formulation(about3wt%.),poloxamer407-carrageenan gels loaded with higher acyclovir content were investigated.Because the content of acyclovir(10–30mg acyclovir/g gel)exceeded its solubility,acyclovir had to be suspended in the gel.The release pro-?les in Fig.4and the release exponent n listed in Table1showed the signi?cant difference among gels with different acyclovir con-tent.In contrast to the group loaded with dissolved acyclovir (1mg acyclovir/g group),gels loaded with suspended acyclovir (30mg acyclovir/g group)exhibited a case-II transport mechanism (n approximated1).For10mg acyclovir/g and20mg/g groups,the release mechanism could be viewed as anomalous transport.

3.1.3.Effect of acrylic acid polymer(Carbopol?)on drug release

Vagina is capable of self-cleaning and constantly secretes vagi-nal?uid which slowly?ushes the vagina to wash out unwanted waste and foreign matter.One of the most challenging tasks for vaginal drug delivery is to endow formulations bioadhesive prop-erty which helps to prolong the vaginal residence of formulations. For poloxamer-based in situ gel,the lack of bioadhesive

property

Fig.4.Release pro?les of acyclovir from thermosensitive gel composed of20% poloxamer407and0.2%carrageenan with different acyclovir content(( )for 1mg acyclovir/g gel,( )for10mg acyclovir/g gel,( )for20mg acyclovir/g gel,( ) 30mg acyclovir/g gel)in the presence of vaginal?uid stimulant at37?C(n=3, mean±S.D.).

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Fig.5.Effect of Carbopol?934(( )for0.2%Carbopol?and( )for0.5%Carbopol?) on the release of acyclovir from thermosensitive gel composed of20%poloxamer 407and0.2%carrageenan with the acyclovir content at30mg acyclovir/g gel in the presence of vaginal?uid stimulant at37?C(n=3,mean±S.D.).20%F127group( ) and20%poloxamer407–0.2%carrageenan group(?)were used as control.

is a unignorable drawback which necessitates the incorporation of bioadhesive materials like acrylic acid polymers when used for cavitary mucosal drug delivery(Chang et al.,2002a,b;Pavelic et al.,2005;Tirnaksiz and Robinson,2005).It has been proposed that carrageenan itself possessing certain bioadhesive property (Bertram and Bodmeier,2006)due to its abundant hydrogen bond-forming groups.Consequently,synergistic effect of bioadhesion can be expected if carrageenan can be used together with widely used bioadhesive materials like Carbopol?.Fig.5shows the release of acyclovir from20%poloxamer407–0.2%carrageenan gels contain-ing two levels of Carbopol?934(0.2%and0.5%),selected according to the usually content Carbopol?for bioadhesion.The presence of Carbopol?increased the rate of drug release,particularly for the 0.5%carbopol group in which an abrupt change was observed after 6–7h when the presence of high concentration of Carbopol?led to the sudden collapse of gel after contact with release medium for a long time.This result is in accordance with the accelerated dissolution of poloxamer407gel caused by Carbopol?observed in our preliminary study(data not shown here),perhaps because Carbopol?would interfere the microstructure poloxamer407gel which are believed to be packaging and entanglement of micelles (Klouda and Mikos,2008).Our observation about the effect of Carbopol?was opposite to previous reports in which Carbopol?retarded drug release from poloxamer407gel by increasing micro-viscosity(Zaki et al.,2007).Such discrepancy might result from the different determination method because previous literature adopted dialysis tubes which neglected the role of gel dissolution in drug release process,in contrast to the membraneless model used in our present work.

Based on the sustained-release property of poloxamer407-carrageenan composite gel which was still signi?cant even after addition of Carbopol?,it is reasonable to expect prolonged release of drug from in situ gel which would face erosion and redistribu-tion by vaginal?uid,thus maintaining high local drug concentration after administration into the vagina.

3.2.Rheological characteristics

Rheological analysis is a powerful technique to comprehen-sively investigate the gelation process and viscoelastic properties of thermosensitive gels(Chang et al.,2002a,b;Tirnaksiz and Robinson,2005;Grassi et al.,2006).From Fig.6which

presents Fig.6.Change of elastic modulus and viscous modulus as a function of temper-ature for20%poloxamer407(A),20%poloxamer407–0.2%carrageenan(B)and 20%poloxamer407–0.2%carrageenan–0.2%Carbopol?(C).On the left top are the enlarged pictures for the temperature range of gelation.

elastic modulus and viscous modulus vs.temperature pro?les for three formulations,20%poloxamer407,20%poloxamer407–0.2% carrageenan gel and20%poloxamer407–0.2%carrageenan–0.2% Carbopol?,gelation temperature could be easily determined to

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311

Fig.7.Change of viscosity(A)and complex modulus(B)as a function of tempera-ture for20%poloxamer407( ),20%poloxamer407–0.2%carrageenan( )and20% poloxamer407–0.2%carrageenan–0.2%Carbopol?( ).

be21.6?C,21.7?C and23.5?C,respectively.The gelation process of all formulations was very quick,being bene?cial for prevent-ing gel leakage immediately after administration.Carrageenan did not change the gelation temperature but Carbopol?did.The slight increase temperature of gel temperature caused by Carbopol?also suggested the possibility of microstructure change of poloxamer 407gel,consistent with the in?uence of Carbopol?on drug release as was discussed in Section3.1.2.

Fig.7provided the complex modulus and viscosity vs.tem-perature pro?les for the above-mentioned formulations.Viscosity in liquid state conferred important information for the easy use of thermosensitive gel.If the viscosity of liquid state is too low, leakage would be likely to happen immediately after administra-tion,while if the viscosity is too high,extrusion from containers would be dif?cult at the time administration.The viscosity for liquid state for the three formulations was in the order of20% poloxamer407<20%poloxamer407–0.2%carrageenan gel<20% poloxamer407–0.2%carrageenan–0.2%Carbopol?.For the latter two formulations,their viscosity was acceptable,though much higher than20%https://www.360docs.net/doc/9d6986713.html,plex modulus,de?ned here as the geometric mean of the elastic modulus and the viscous modulus,represented the anti-deformation ability of materials to resist any deformation.The value of complex modulus for the three formulations at body temperature was of the same order

of Fig.8.Residence of acyclovir in the vagine of mice after intravaginal administra-tion of different thermosensitive gel formulations loaded with30mg acyclovir/g gel (10?g/mouse).Aqueous suspension of the same acyclovir content is used as control. Each point is mean±S.E.M.from?ve to six mice.

magnitude with small difference,in the order of20%poloxamer 407>20%poloxamer407–0.2%carrageenan gel>20%poloxamer 407–0.2%carrageenan–0.2%Carbopol?,suggesting slight weak-ening of mechanic strength by carrageenan and further by Carbopol?.

When poloxamer407is alone in aqueous solution,its gelation is believed to be the result of organization of poloxamer micelles.It is reasonable to assume that the gelation mechanism might change due to the addition of carrageenan and/or Carbopol?.Carrageenan chains,for instance,might form a network in which poloxamer407 micelles were dispersed.Charges on chains of Carbopol?might also interact with poloxamer407micelles.Nevertheless,it is only after fully characterization like differential scanning calorimetry or small angle X-ray diffraction that the elucidation of gelation mechanism of such composite gel systems would become possible.

3.3.In vivo local residence

The analytical method of HPLC for acyclovir in the vaginal washing?uid was validated with respect to the standard curve (y=0.110x,r2=0.994),linear range(0.5–200?g/mL),recovery(all >80%for three levels,S.D.<5%),intraday precision(S.D.<4%)and interday precision(S.D.<5%).

Fig.8presents the local residence of acyclovir after vaginal administration in female mice.Vaginal washing is usually used for diagnostic tests of genital diseases and also for quanti?cation of interested compounds in the vagina(Si-Mohamed et al.,2001).As is shown in Fig.8,more than85%of drug administered could be recovered in the washing?uid obtained immediately after adminis-tration(0h),con?rming that this method could constantly recover most of the drug in the vagina with good repeatability.The aqueous suspension group(control group)did not result in signi?cant res-idence of drug even after only3h.The drug residence for the20% poloxamer407group fell to nearly zero after6h.This case was also true for20%poloxamer407–0.2%Carbopol?group,indicating that Carbopol?alone could not signi?cantly improve the residence of drug,perhaps related with the accelerated drug release caused by Carbopol?.By contrast,the20%poloxamer407–0.2%carrageenan group achieved obvious drug residence after6h,but failed to main-tain to9h.The signi?cant prolongation of drug residence caused by carrageenan might be attributed to both the slowdown in gel erosion and the bioadhesive property of carrageenan which was suggested by a previous report on the interaction between car-

312Y.Liu et al./European Journal of Pharmaceutical Sciences37(2009)306–312

rageenan and mucin in vitro(Bertram and Bodmeier,2006).20% poloxamer407–0.2%carrageenan–0.2%carbopol group achieved the longest local residence of drug(>12h,not longer than24h), indicating signi?cant synergistic bioadhesive effect between car-rageenan and Carbopol?.More detailed investigation about the nature of such synergistic bioadhesive effect will be performed in the future.

Further work including investigation on the anti-viral ef?cacy in the mouse HSV infection model,the toxicity of carrageenan on the cell level,and local irritation to the vagina of rabbits is under-going.The safety of carrageenan for vaginal use has been proved by data from a phase I preliminary safety study on a carbopol gel containing carrageenan as the microbicidal agent(Coggins et al., 2000).Therefore,we are optimistic about the outcomes of ef?cacy and safety of the in situ gel formulation reported here.

4.Conclusion

To summarize,addition of carrageenan signi?cantly decreased the erosion rate of poloxamer407-based gel,improved the sustained-release properties of acyclovir and exhibited synergistic bioadhesive effect with Carbopol?in vivo.It is especially interest-ing that carrageenan not only helped to circumvent drawbacks of poloxamer407gel alone like fast erosion,but also did not adversely affect its thermosensitive behavior,which promised easy vaginal administration as well as avoidance of leakage after vaginal admin-istration.The ease in drug loading and the nearly zero-order release kinetics for high drug content proved the suitability of such com-posite in situ gel systems as the vehicle for most drugs,in particular in cases when active drug has to be suspended in semi-solid for-mulations due to high dosage or low drug solubility.Therefore,the combination of carrageenan and poloxamer407may be of great use in future development of vaginal in situ gel sustained-release drug delivery systems.

Acknowledgements

The?nancial support from China Postdoctoral Science Foun-dation(China Postdoctoral Fund20070420608)is gratefully acknowledged.The authors also wish to thank Mr.Kim Wang (Malvern Instruments,China)for his skillful technical assistance in rheological measurements.

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