International Journal of Pharmaceutics 468 (2014) 272–282

Development, characterization and application of in situ gel systems forintranasal delivery of tacrine

Shuai Qian, Yin Cheong Wong, Zhong Zuo *School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, PR China

A R T I C L E I N F O

Article history:Received 3 April 2014Received in revised form 3 April 2014Accepted 3 April 2014Available online 5 April 2014

Keywords:TacrineIn situ gelIntranasalPluronic F-127Pharmacokinetics

A B S T R A C T

The present study aimed to develop an in situ gel formulation for intranasal delivery of tacrine (THA), ananti-Alzheimer’s drug. Thermosensitive polymer Pluronic F-127 was used to prepare THA in situ gels.Sol–gel transition temperature (Tsol–gel), rheological properties, in vitro release, and in vivo nasalmucociliary transport time were optimized. The pharmacokinetics and brain dispositions of in situ gelwere compared with that from THA oral solution in rats. The in situ gel demonstrated a liquid state withNewtonian fluid behavior under 20 �C, while it exhibited as non-flowing gel with pseudoplastic fluidbehavior beyond its Tsol–gel of 28.5 �C. Based on nasal mucociliary transport time, the in situ gelsignificantly prolonged its retention in nasal cavity compared to solution form. Moreover, the in situ gelachieved 2–3 fold higher peak plasma concentration (Cmax) and area under the curve (AUC) of THA inplasma and brain tissue, but lowered Cmax and AUC of the THA metabolites compared to that of oralsolution. The enhanced nasal residence time, improved bioavailability, increased brain uptake of parentdrug and decreased exposure of metabolites suggested that the in situ gel could be an effective intranasalformulation for THA.

ã 2014 Elsevier B.V. All rights reserved.

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1. Introduction

Tacrine (THA), a reversible and non-selective acetylcholinester-ase inhibitor, was the first oral prescription medication approvedby FDA in 1993 to improve the symptoms of mild to moderateAlzheimer’s disease (AD) (Summers, 2006). Due to its flatconfiguration and medium lipophicility with a Log P of 2.71(Summers, 2000), THA has a good intestinal permeability with anapparent permeability coefficient of 2.5 �10�5 cm/s across ratjejunum (Jasti et al., 2009). Its clinical use is, however, hamperedby low oral bioavailability (17–24% in human) due to extensivefirst-pass metabolism (Hartvig et al., 1990; Madden et al., 1995)and dose-dependent hepatotoxicity (O‘Brien et al., 1991; Qizilbashet al., 1998). The hepatotoxicity of THA is probably resulted from itsreactive metabolites (Patocka et al., 2008). Therefore, it is worthexploring alternative routes of administration to avoid its first-pass

* Corresponding author at: Room 801C, School of Pharmacy, Lo Kwee-SeongIntegrated Biomedical Sciences Building, Area 39, The Chinese University of HongKong, Shatin, N.T. Hong Kong, PR China. Tel.:+852 3943 6832; fax: +852 2603 5295.

E-mail address: joanzuo@cuhk.edu.hk (Z. Zuo).

http://dx.doi.org/10.1016/j.ijpharm.2014.04.0150378-5173/ã 2014 Elsevier B.V. All rights reserved.

metabolism as well as to enhance the bioavailability and braintargeting effect of THA.

Intranasal delivery is a non-invasive and convenient methodthat could provide efficient systemic delivery for certain thera-peutic compounds (Dhuria et al., 2010). The nasal route might alsoavoid the first-pass metabolism if the nasal drug could be retainedand absorbed in the nasal cavity, thereby reducing the biotrans-formation of the parent drug to metabolites (Wong and Zuo, 2013;Wong et al., 2012; Wong and Zuo, 2010). Jogani et al. developed anasal mucoadhesive microemulsion of [99mTc]-THA and, accordingto total radioactivity measurement, it exhibited a 3-fold higherbrain disposition than that after intravenous administration of[99mTc]-THA solution (Jogani et al., 2008). This indicated that thenasal route could be an alternative way for THA administration.The limitation of radioactivity measurement is that the parent drugcould not be differentiated from its metabolites. Therefore, theexact systemic and brain dispositions of THA and its metabolitesafter intranasal THA administration remain to be established.

It was noticed that the nasal absorption of liquid dosage form isoften limited by its short residence time due to quick clearancefrom nasal cavity (Illum, 2003). Several strategies, such as addingabsorption enhancers (Illum et al., 1994) and using nasal gel or

N

NH2

N

NH2 OH

N

NH2OH

N

NH2

OH

Tacrine 1-hydroxytacrine

2-hydroxytacrine 4-hydroxytacr ine

(THA ) (1-OH -THA )

(2-OH- THA ) (4-OH -THA )

Fig. 1. Chemical structure of tacrine (THA), 1-hydroxytacrine (1-OH-THA), 2-hydroxytacrine (2-OH-THA) and 4-hydroxytacrine (4-OH-THA).

S. Qian et al. / International Journal of Pharmaceutics 468 (2014) 272–282 273

powder dosage forms (Illum et al., 2002; Wang et al., 2013), havebeen applied to prolong the contact time of drugs with the nasalmucosa or enhance drug permeability across the nasal mucosa. Inour preliminary study, addition of chitosan hydrochloride orvarious types of cyclodextrins did not show significant improve-ment of nasal permeability of THA across Calu-3 cell monolayer(data not shown), which might be due to its inherent high nasalpermeability (Papp on Calu-3 cell: 1.1 �10�5 cm/s (Qian et al., 2010))with transcellular diffusion as its major membrane transportpathway. Therefore, we speculated that composition with onlyabsorption enhancer in THA solution might not be able to providesignificant enhancement in its nasal bioavailability. Illum et al.developed a mucoadhesive microsphere powder of morphine andfound a 5–7 fold higher nasal bioavailability in sheep incomparison to its nasal solution, which could be, at least partially,attributed to the prolonged contacting time between the drug andnasal epithelium (Illum et al., 2002). Nasal gels have also beenemployed to enhance drug delivery efficiency by reducingswallowing and anterior leakage of formulation. Several productssuch as vitamin B12 nasal gel have been marketed (Suzuki et al.,2006). However, both nasal powder and ordinary gel encounterseveral problems including difficulty in accurate dosing, nasalmucosa irritation and a gritty feeling in nose (Behl et al., 1998).Recently, the nasal in situ gel appears very promising since it existsas fluid before nasal administration and thus could be easily sprayfrom nebulizer device with an accurate dose, while it forms gel filmin the nasal cavity with effective contact to nasal epithelium (Wattsand Smith, 2009).

In general, in situ gelation can be achieved by usingthermosensitive polymers which forms gel on instillation bysensing nasal temperature. Pluronic F-127, with excellent thermo-sensitive gelling properties at physiological temperature, lowtoxicity and irritation, excellent water solubility, good drug releasecharacteristics and compatibility with other chemicals (Andersonet al., 2001; Jeong et al., 2002; Li and Guan, 2011), has beenextensively investigated for developing in situ gel systems fornasal, transdermal, rectal and ocular applications (Escobar-Chavezet al., 2006).

The aim of the present study was to develop a THA nasal in situgel using Pluronic F-127 as gelling agent. The optimized THA in situgel with favorable gelation temperature, in vitro release andrheological properties would be selected to perform in vivoevaluations in rats. Systemic pharmacokinetics and brain

dispositions of THA as well as its metabolites after intranasaladministration of the THA in situ gel would be compared to thatobtained from the conventional oral route.

2. Materials and methods

2.1. Materials

THA hydrochloride was purchased from Enzo Life SciencesInc. (Farmingdale, NY, USA). 1-Hydroxytacrine (1-OH-THA),2-hydroxytacrine (2-OH-THA) and 4-hydroxytacrine (4-OH-THA)were kindly gifted from Pfizer Inc. (Groton, CT, USA) (Fig. 1).Pluronic F-68, Pluronic F-127, polyethylene glycol 8000 (PEG 8000)and indigo carmine were obtained from Sigma (St. Louis, MO, USA).Chitosan hydrochloride with average molecular weight of �200kDa and deacetylation degree of 83% was purchased from ZhejiangGolden-Shell Biomedical Co. Ltd. (Zhejiang, China). Calciumchloride and potassium chloride were purchased from BDH (Poole,England). All other reagents were of at least analytical grade andwere used without further purification. Distilled and deionizedwater was used for the preparation of solutions.

2.2. Preparation of THA in situ gels

The thermosensitive in situ gel systems were prepared by coldmethod described by Schmolka (Schmolka, 1972). Briefly, THA,Pluronic F-68, chitosan and PEG 8000 were stirred in distilledwater at room temperature until all of them completely dissolved.The mixture, together with the container, was then put into icebath followed by addition of Pluronic F-127 into the mixture. Thefinal transparent solution was stored at 4 �C for further evaluations.

2.3. Physicochemical characterizations of the in situ gels

2.3.1. Sol–gel transition temperature measurementThe sol–gel transition temperature (Tsol–gel) of the prepared in

situ gel formulations was determined as previously described byGillert et al. (Gilbert et al., 1987). Briefly, 2 ml of the preparedformulation was transferred into a test tube (10 ml) with adiameter of 1.0 cm and sealed with a parafilm. The tube was kept ina circulation water bath at 8 �C, and the temperature of water bathwas increased at an increment of 2–3 �C in the beginning (from 8 �Cto 18 �C) and then at 0.2–0.5 �C until gelation. After each setting ofthe water bath temperature, 10 min was allowed for equilibration.The test tube was then taken out and placed horizontally toobserve the state of the sample, and gelation was said to occurwhen the meniscus would no longer move upon.

2.3.2. In vitro drug release from the in situ gelsIn vitro release studies of THA in situ gel formulations were

performed in 500 ml of simulated nasal electrolyte solution(containing 1.29 mg/ml KCl, 7.45 mg/ml NaCl and 0.32 mg/mlCaCl2�2H2O, pH adjusted to 5.7 by HCl) (Callens et al., 2003) at35 � 0.5 �C using the USP II method. A dialysis bag (Spectra/Por1

membrane, MWCO: 12,000–14,000, Spectrum Laboratories Inc.,CA, USA) containing 1 g of in situ gel formulation was attached ontothe paddle by thread and the rotating speed was set at 50 rpm.Samples (2 ml) were collected through 0.22 mm syringe filters at 5,10, 30, 45, 60, 90,120,180, 240, 360, and 600 min. The same volumeof fresh medium was replaced after each sampling. The in vitrorelease experiments were run in five replicates for each formula-tion. The release of THA from its solution formulation (dissolvingTHA in normal saline) was also tested to serve as the control.

THA concentrations in the samples were determined by HPLC/UV method. The HPLC/UV system consists of Waters 600 controller(pump), Waters 717 auto sample and Waters 996 Photodiode Array

274 S. Qian et al. / International Journal of Pharmaceutics 468 (2014) 272–282

detector. Data collection was performed using a Waters Millenni-um Chromatography Manager data system (Version 3.20).Chromatographic separation was achieved by a Thermo BDSHypersil C18 analytical column (250 � 4.6 mm, 5 mm) protected bya guard column (Delta-PakTM C18 Guard-Pak, Waters). Mobilephase consisted of eluent A [50 mM sodium dihydrogen phosphateand 0.5% (v/v) triethylamine (adjusted to pH 3.0 by H3PO4) with 5%acetonitrile] and eluent B [acetonitrile]. The HPLC column wasisocratically eluted by a mixture of eluent A and eluent B at theratio of 20:80 at 1 ml/min. UV detection was performed at awavelength of 241 nm. The temperatures of column and auto-sampler were set at ambient and 4 �C, respectively. Within theconcentration range of 1.0–100.0 mg/ml, good linearity(r2 > 0.9995) was achieved. The relative standard deviation(%RSD) of intra-day and inter-day precision of the assay methodfor THA was below 5%, and the accuracy was within the range of95–105%.

2.3.3. Rheological properties of the in situ gelsThe static rheological properties of the in situ gel formulations

were investigated using a rotational viscometer (Brookfield typeDV-II+, Brookfield Engineering Laboratories Inc., MA, USA). A SC4-29 spindle with a small sample adapter was employed for themeasurements at 8 �C and 20 �C, while a RV7 spindle was used formeasurement at 32 �C due to the high viscosity after gelation. Themeasurement was conducted by increasing spindle rotationalspeed from 0.3 to 100 rpm, and the shear rate (g), shear stress (t)and viscosity (h) were recorded. All measurements wereperformed in triplicate.

2.4. In vivo evaluations of the selected THA in situ gel

Adult male Sprague-Dawley rats, weighting 230–250 g, wereprovided by Laboratory Animal Services Center of The ChineseUniversity of Hong Kong (Hong Kong, China). Animals were housedin standard cages on 12 h light-dark cycles and fed with standardanimal chow and tap water daily. All experimental procedureswere approved by the Department of Health of Hong Kong andAnimal Experimentation Ethics Committee at The ChineseUniversity of Hong Kong.

2.4.1. Nasal mucociliary transport timeTo investigate whether the in situ gel stay longer in nasal cavity

than the solution form or not, in vivo nasal mucociliary transporttime of both dosage forms was measured by a method reported byZaki et al. (Zaki et al., 2007) with modification. Briefly, five ratswere anesthetized by intramuscular injection of a mixturecontaining ketamine (80 mg/kg) and xylazine (8 mg/kg). A 10 mlof in situ gel containing the blue dye indigo carmine (5 mg/ml) wasinstilled to rat nose (5 mm depth into the right nostril) using amicropipette. Appearance of the blue dye at nasopalatine andpharyngeal was detected by swabbing the corresponding regionsin oral cavity with moistened cotton-tipped applicators, at everymin post dosing for the first 20 min and every 5 min for thefollowing 40 min. The appearance time of the blue dye wasrecorded. Indigo carmine dissolved in normal saline at the sameconcentration (5 mg/ml) was used as the control.

2.4.2. Systemic pharmacokinetics and brain dispositionRats (n = 5 per group) received jugular vein cannulation a day

before the experiment as described in our previous publication(Qian et al., 2012). On the day of experiment, rats wereanesthetized temporarily by inhalation of diethyl ether. Forintranasal administration, rats were placed in supine position,then 20 ml of the in situ gel containing 50 mg/ml THA (10 ml foreach nostril, equivalent to 4 mg/kg THA) was administrated to the

nostril (5 mm depth) with the help of a micropipette. For oraladministration, 1 ml of 1 mg/ml THA solution dissolved in saline(equivalent to 4 mg/kg THA) was orally gavaged. After dosing, ratswere returned to cage with free access to water only. Around200 ml of blood was collected via the jugular vein cannula into aheparinized centrifuge tube at predetermined time points (5, 10,20, 30, 45, 60, 90, 120, 180, 240, and 360 min) post dosing. Plasmawas obtained by centrifugation of the blood sample at 16,000 � gfor 5 min. Rats were allowed for free access to food and water 4 hafter drug administration.

To investigate the brain pharmacokinetics, another set of rats(n = 24 for each group, 4 rats/time point � 6 sampling points) withno cannulation surgery were used. After intranasal or oraladministration of THA at a single dose of 4 mg/kg according tothe procedures described above, the rat was euthanized by cervicaldislocation at predetermined time points (i.e. 15, 30, 60, 120, 240,or 360 min). The whole brain was then removed from skull, quicklyrinsed with cold normal saline (4 �C) and wiped by tissue paper toremove excess water. It was dissected into six anatomical regions(Paxinos and Watson, 2007) including (1) olfactory bulb, (2) frontalcortex, (3) hippocampus, (4) striatum, (5) occipital–temporalcortex, and (6) others (including midbrain, thalamus, hypothala-mus, and cerebellum, etc.). Meninges and blood vessels wereremoved and each tissue was accurately weighted.

All the obtained plasma and brain samples were frozen at�80 �C until analysis. The concentrations of THA and its mono-hydroxylated metabolites (1-OH-THA, 2-OH-THA and 4-OH-THA)in the plasma and brain tissue were analyzed by the HPLC/FLDmethod previous developed and validated by our research group(Qian et al., 2012). For plasma, the lower limits of quantification(LLOQs) for THA, 1-OH-THA, 2-OH-THA and 4-OH-THA were 2.5,6.7, 2.1 and 2.1 ng/ml, respectively. For brain tissue, the LLOQs forTHA,1-OH-THA, 2-OH-THA and 4-OH-THA were 12.3, 33.5,10.6 and10.5 ng/g, respectively.

2.5. Data analysis

2.5.1. Drug release kinetics model fittingIn order to illustrate the THA release mechanism from the in situ

gels, the in vitro release data in the range of 5–60% was analyzed byfitting to kinetic models (Bermudez et al., 2008) such as zero-orderequation, first-order equation, Higuchi equation, Korsmeyer–Peppas equation, and Hixson–Crowell equation (Table 2). Themodel fitting was conducted with the help of a software packageDDSolver (Zhang et al., 2010).

In all the above models, “F” refers to the amount of THA releasedat time “t”. “k0, k1, kH, kKP, and kHC” are the release constant. In theKorsmeyer–Peppas model, “n” is the release exponent indicatingdrug release mechanism (Cooke and Chen, 1995; Costa and SousaLobo, 2001). Based on the obtained n values, the drug releasemechanism from the in situ gel can be interpreted as (1) n < 0.5:quasi–Fickian diffusion; (2) n = 0.5: Higuchi kinetics with diffusionmechanism; (3) 0.5 < n < 1: anomalous (non-Fickian) diffusionincluding both diffusion and erosion; and (4) n = 1: zero orderrelease.

2.5.2. Rheological model fittingThe rheograms obtained during the procedure of increasing

rotation speed were fitted to power-law rheological equation(Eq. (1)) as previously described (Bouziani and Benmounah, 2013;Hyman, 1976).

t ¼ Kgn (1)

where K is the consistency index and n is the flow index. Based onthe flow index, power-law fluids could be divided into three

14 16 18 20 22 2416

20

24

28

32

36

40T

sol-

gel (o C

)

Concen tration of Pluronic F-127 (%)

Fig. 2. Relationship between sol–gel transition temperature and Pluronic F-127concentrations.

S. Qian et al. / International Journal of Pharmaceutics 468 (2014) 272–282 275

different types of fluids: (1) n < 1 indicates pseudoplastic fluid withshear thinning behavior; (2) n = 1 indicates Newtonian behavior;and (3) n > 1 indicates dilatant fluid with shear thickening behavior(Green and Griskey, 1968; Hyman, 1976).

2.5.3. Calculation of pharmacokinetic parametersThe plasma and brain concentration-time profiles of THA

and THA metabolites of each animal were analyzed by

0 1 2 3 4 5 6 7 822

24

26

28

30

32

THA

Tso

l-ge

l (o C

)

Concen trati on (%)

a

0 1 2 3 4 522

24

26

28

30

32

c

Pluron ic F-68

Tso

l-ge

l (o C

)

Con centrati on (%)

Fig. 3. Effect of THA (a), chitosan (b), Pluronic F-68 (c) and PEG 8000 (d

non-compartmental method with aid of WinNonlin software(Pharsight Corporation, Mountain View, CA). The maximumplasma concentration (Cmax) and the time when it occurred (Tmax)were obtained by visual inspection of the plasma concentration–time curve. The terminal half-life (t1/2) was calculated as ln2/lz,where lz was the first-order rate constant associated with theterminal (log-linear) portion of the curve.

2.6. Statistical analysis

Statistical analysis was performed using the statistical softwarepackage SPSS (version 17, SPSS Inc., Chicago, IL, USA). Statisticalsignificance was estimated by one-way ANOVA. A probability levelof p < 0.05 was set as the criterion of significance.

3. Results

3.1. THA in situ gel preparation

Pluronic F-127 is an effective thermosensitive polymer, whosesolution can be transferred to gel form when temperatureincreases. As shown in Fig. 2, the Tsol–gel of Pluronic F-127 solutiondecreased sharply from 37.5 �C to 19.5 �C when its concentrationincreased from 15.5% to 24% (w/v). As previously reported, theaddition of drugs and other ingredients would change the Tsol–gel ofPluronic F-127 (Bandyopadhyay and Basu, 2010; Zaki et al., 2007).In the present study, the Tsol–gel of 20% Pluronic F-127 increasedwith the addition of THA, Pluronic F-68 and PEG 8000, but it wasnot affected by the addition of chitosan (Fig. 3). Since nasal

0.0 0.2 0.4 0.6 0.8 1.022

24

26

28

30

32

chit osan

b

Tso

l-ge

l (o C

)

Concen trati on (%)

0.0 0.5 1.0 1.5 2.0 2.522

24

26

28

30

32d

PEG 80 00

Tso

l-ge

l (o C

)

Con centrati on (%)

) on sol–gel transition temperature of 20% Pluronic F-127 solution.

Table 1Composition and sol–gel transition temperature of various in situ nasal gel formulations (F1–F8).

Ingredients (%, w/v) Formulations

F1 F2 F3 F4 F5 F6 F7 F8

THA 5 5 5 5 5 5 5 5Pluronic F-127 20 20 20 20 20 20 20 20Chitosan – 0.5 – 0.5 – 0.5 – 0.5Pluronic F-68 1 1 2 2 – – – –

PEG 8000 – – – – 0.5 0.5 1 1Tsol–gel (�C) 28.5 � 0.2 28.6 � 0.3 31.0 � 0.3 31.2 � 0.2 29.2 � 0.2 29.6 � 0.3 32.3 � 0.2 32.5 � 0.3

276 S. Qian et al. / International Journal of Pharmaceutics 468 (2014) 272–282

formulations were generally administered in small volumesranging from 100 to 200 ml in humans (Costantino et al., 2007),a high concentration of THA (5%, w/v), was adopted whendeveloping the in situ nasal gel formulations. Ideally, an in situgelling system should contain a low viscous fluid to allowreproducible administration to nose by nebulizer device underroom temperature (i.e. 20–25 �C (Burroughs and Hansen, 2011)),which transforms to a gel form with high viscosity afteradministration to nose (Lin and Sung, 2000). Since the averagetemperature in human nose was reported to be 31 �C (Jacky, 1980;Proctor et al., 1977), a suitable Tsol–gel was proposed to be rangingfrom 25 to 30 �C. In general, the viscosity of a thermosensitive insitu gel increases significantly when the environment temperatureis close to the critical temperature Tsol–gel (Chen et al., 2010). An insitu gel with a Tsol–gel higher than but close to 25 �C is still expectedto be highly viscous at room temperature. Practically, drug solutionwith low viscosity could be sprayed easily from the nebulizerdevice to provide an accurate dose. Therefore, a favorable Tsol–gelshould be close to but not exceed 30 �C. Since 20% Pluronic F-127with 5% THA had a Tsol–gel of 25.5 �C, 1–2% of Pluronic F-68 and0.5–1% of PEG 8000 were added to increase the Tsol–gel. In addition,0.5% of chitosan was also added, which was commonly used innasal formulations for enhancing nasal drug permeability andmucoadhesiveness (Illum et al., 2002; Zaki et al., 2007; Zhang et al.,2005). Therefore, formulations containing 5% THA, 20% Pluronic F-127, and various polymers (Pluronic F-68, PEG 8000 and chitosan)with different concentrations were developed and their deter-mined Tsol–gel values were shown in Table 1. Formulations such asF1, F2, F5 and F6 had suitable values of Tsol–gel within 25–30 �C. Incomparison to PEG 8000, addition of Pluronic F-68 to PluronicF-127 based in situ gel system was reported to be capable of

0 60 12 0 18 0 24 0 30 0 36 0 420 48 0 540 60 00

20

40

60

80

100 THA solution F1 F2

Dru

g re

leas

e (%

)

Time (min)

Zoo m in

0 15 30 45 60 75 900

20

40

60

80

100

Fig. 4. In vitro release profiles of THA from THA saline solution and nasal in situ gelformulations (F1 and F2) (n = 5).

reducing the dilution effect in the eye drug delivery system(Edsman et al., 1998). Since the nasal mucus continuouslyproduced by nasal mucosa would dilute the administered in situgel and increase its Tsol–gel, formulations containing Pluronic F-68(F1 and F2) were selected for further studies.

3.2. In vitro THA release from in situ gels F1 and F2

The results of in vitro release study are presented in Fig. 4. Incomparison to saline solution dosage, in in situ gels (F1 and F2)released THA to the medium much more slowly with only �60%of drug released in the first 90 min. The release data of F1 and F2were kinetically analyzed by different mathematic models, andthe results are shown in Table 2. According to the goodness of fit,drug releases from both F1 and F2 were well fitted with theKorsmeyer–Pappas model with the highest correlation coefficientand lowest SSE value. Since the calculated n values (0.582 and0.529 for F1 and F2, respectively) were between 0.5 and 1, theTHA release from F1 and F2 in situ gel systems underwentanomalous (non-Fickian) release mechanism, suggesting that therelease was controlled by the THA diffusion rate and therelaxation rate of polymer matrix.

3.3. Evaluation of rheological properties of in situ gels F1 and F2

The relationships of spindle rotational speed to viscosity areshown in Fig. 5. At 8 �C and 20 �C, both formulations were in liquidform. F1 exhibited no notable changes in the viscosity over a broadrange of spindle rotational speeds, while viscosity of F2 slightlydecreased when increasing the spindle rotational speed. Inaddition, the rheograms of F1 and F2 obtained at 8 �C and 20 �Cwere well fitted to power-law rheological equation (r2 > 0.999)(Fig. 6). The flow indices (n) of both F1 and F2 were close to1 (n > 0.94), indicating a Newtonian behavior at shelf condition(�8 �C) as well as during usage (�20 �C). However, at 32 �C, bothformulations formed gel with a very high viscosity and showed atypical pseudoplastic (shear-thinning) behavior (Fig. 5). An idealnasal in situ gel formulation should have an optimum viscosity thatwill allow easy spray as a liquid form, which then undergoes arapid sol–gel transition when temperature increases. In compari-son to F2, F1 demonstrated similar viscosity at 32 �C but had muchlower viscosities at 8 �C and 20 �C. In addition, it was observed thatF1 was more easily sprayed from the nebulizer device than F2under room temperature. Therefore, F1 was eventually selected forfurther in vivo evaluations.

3.4. In vivo evaluations of in situ gel F1

3.4.1. Nasal mucociliary transport timeAfter administration of the saline solution, the dye took 2.2 min

and 9.2 min to reach nasopalatine and pharyngeal (the twoconnections between nose and oral cavity), respectively. In

Table 2Model fitting and parameters of THA release from in situ gel formulations F1 and F2.

Formulation Models Equation Parameters Goodness of fit

r2 SSE

F1 Zero-order Eq. (1) k0: 0.951 0.7994 191.6First-order Eq. (2) k1: 0.012 0.9576 171.6Higuchi Eq. (3) kH: 6.365 0.9899 20.5Korsmeyer–Peppas Eq. (4) kKP: 4.573; n: 0.582 0.9960 6.87Hixson–Crowell Eq. (5) kKC: 0.004 0.8960 177.0

F2 Zero-order Eq. (1) k0: 0.694 0.6747 369.0First-order Eq. (2) k1: 0.010 0.8695 148.0Higuchi Eq. (3) kH: 5.541 0.9960 4.52Korsmeyer–Peppas Eq. (4) kKP: 4.964; n: 0.529 0.9978 2.46Hixson–Crowell Eq. (5) kKC: 0.003 0.8208 203.2

SSE: the sum of squares due to error; Eq. (1): F = k0�t; Eq. (2): F = 100�(1 – e^(�k1t)); Eq. (3): F = kH�t^0.5; Eq. (4): F = kKP�t^n; Eq. (5): F = 100�[1 � (1 � kHC�t)]^3.

S. Qian et al. / International Journal of Pharmaceutics 468 (2014) 272–282 277

contrast, the mucociliary transport time to nasopalatine andpharyngeal after administration of the in situ gel was lengthenedby at least 6-fold (33.6 min and >60 min, respectively) (p < 0.01),indicating that the in situ gel could stay in nasal cavity for a longerperiod and the clearance of drug from nasal cavity by nasalmucocilia was reduced.

3.4.2. Systemic pharmacokinetics and brain dispositionTo evaluate the in vivo effectiveness of the developed THA nasal

in situ gel formulation (F1), the systemic pharmacokinetics andbrain disposition of orally administered THA solution were alsostudied and served as control. The plasma concentration versustime profiles of THA and its metabolites after intranasaladministration of THA in situ gel and oral administration of THAsolution are shown in Fig. 7. The calculated pharmacokineticparameters are summarized in Table 3. In comparison to oralsolution, nasal in situ gel showed a significant increase in Cmax andAUC0–6 h of parent THA for 2.0- and 2.1-fold, respectively. For THAmetabolites, 1-OH-THA and 2-OH-THA were present in quantifi-able amounts in plasma (Fig. 7b and c), while the concentration of4-OH-THA was very low and below the limit of quantification inmost plasma samples. For both 1-OH-THA and 2-OH-THA, nasalin situ gel demonstrated significantly lower Cmax and AUC0–6 h thanthat obtained from oral solution (p < 0.05). No significantdifference was detected in elimination half-life (t1/2) for bothparent THA and its metabolites between nasal in situ gel and oralsolution (p > 0.05).

In rat brain, THA was the predominant form, followed by 1-OH-THA and 4-OH-THA. 2-OH-THA was not found at the current THA

0 20 40 60 80 10 00

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250000 F1 (8 oC) F2 (8 oC)

F1 (20 oC) F2 (20 oC)

F1 (32 oC) F2 (32 oC)

Vis

cosi

ty (

cP)

Spindl e rot ational speed (rp m)

Fig. 5. Viscosity of prepared nasal in situ gel formulations (F1 and F2) at differenttemperatures (8, 20 and 32 �C).

dose (4 mg/kg). The pharmacokinetic profiles of THA and 1-OH-THA in different brain regions after THA administration viaintranasal and oral routes are shown in Fig. 8 and Fig. 9,respectively. Since THA and 1-OH-THA were evenly distributedin different brain regions after THA dosing via both routes,occipital–temporal cortex (the largest part of the brain) was takento represent the corresponding pharmacokinetic parameters ofTHA and 1-OH-THA in brain (Table 3). The concentrations of 4-OH-THA were only quantifiable in brain tissue at 1 h and 2 h afterdosing (data not shown). In all rat brain regions, rats administeredwith nasal in situ gel showed a much higher THA concentrationprofile in comparison to that received oral solution (Fig. 8). Thenasal in situ gel exhibited 3-fold higher Cmax and AUC0–6 h of THA inbrain than oral solution. In contrast to the pattern of parent THA,the brain concentrations and AUC0–6 h of 1-OH-THA wereconsistently higher throughout the whole period after oraladministration than nasal administration, although statisticalsignificance was not reached (Fig. 9 and Table 3).

In summary, the developed thermosensitive in situ gel F1exhibited higher bioavailability and brain uptake of parent THAalong with reduced exposure of its metabolites in comparison tothe THA oral solution.

4. Discussion

Pluronic F-127, the basic gelation material used in the current insitu gel formulation, is one of the poloxamer ABA block copolymersconsisting of �70% polyoxyethylene units (PEO, hydrophilic part)and �30% polyoxypropylene blocks (PPO, hydrophobic part)

Fig. 6. Rheological profiles and fitting curves of F1 and F2 at 8 and 20 �C.

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Fig. 7. Plasma concentration versus time profiles of THA (a), 1-OH-THA (b), and 2-OH-THA (c) in rats after intranasal administration of THA in situ gel (F1) and oraladministration of THA solution (4 mg/kg). Each point represents mean � s.d. (n = 5).*: p < 0.05 vs THA oral solution.

278 S. Qian et al. / International Journal of Pharmaceutics 468 (2014) 272–282

(Escobar-Chavez et al., 2006). Micelle formation and entangle-ments occur at the critical temperature as a result of PPO blockdehydration was proposed to be the gelation mechanism ofPluronic F-127 solution (Ruel-Gariépy and Leroux, 2004). At asufficiently high concentration and temperature, the micelles canpack together and form a higher ordered structure like a lattice orcubic crystalline phase, which is believed to be the driving force forgel formation from solution status. Pluronic F-127 is more solublein cold water than in hot water as a result of increased solvation

and hydrogen bonding at lower temperatures (Gilbert et al., 1986).Thus, the cold method was employed in our in situ gel preparation.In the present study, the addition of Pluronic F-68 and PEG 8000increased the Tsol–gel of Pluronic F-127 and the effect seemed to beconcentration-dependent (Fig. 3c and d). Since the dehydration ofhydrophobic PPO block controls the gelation of Pluronic F-127,reducing the ratio of PPO to PEO in solution would cause anincrease in entanglement of adjacent molecules with moreextensive intermolecular hydrogen binding, and hence increasethe Tsol–gel of Pluronic F-127. Therefore, using Pluronic F-68(containing �16% PPO and �84% PEO) with low proportion of PPOor PEG with only PEO could effectively increase the Tsol–gel ofPluronic F-127 based thermosensitive in situ gels (Baloglu et al.,2011; Varshosaz et al., 2008). Moreover, addition of THA(hydrochloride salt form) was also found to increase the Tsol–gelof Pluronic F-127 (Fig. 3a). This might be due to the hydrophilicnature of hydrochloride salt form of THA, which might modify theprocess of micellar association of Pluronic F-127 gels and therebyincreasing their gelation temperature. Such phenomenon was alsoobserved on other Pluronic F-127 based in situ gel systems afterincorporation of water soluble drugs such as metoclopramidehydrochloride (Jitendra et al., 2008), venlafaxine hydrochloride(Bhandwalkar and Avachat, 2013) and sodium salicylate (Yun et al.,1999). On the other hand, addition of chitosan did not affect theTsol–gel of Pluronic F-127 (Fig. 3b), which was consistent withprevious findings (Wagh et al., 2012). According to previousreports (Chen et al., 2010; Pereira et al., 2013), the gel strengthcould be low when the concentration of Pluronic F-127 was lowerthan 18% (w/v). However, if Pluronic F-127 was above 22%, theTsol–gel could be too low (<20 �C, Fig. 2) and further justification byPluronic F-68 or PEGs was needed. In general, the more Pluronic F-68 or PEGs added, the higher viscosity of in situ gel exhibited atroom temperature, which hampered it to be sprayed from thenebulizer device. Therefore, 18–20% of Pluronic F-127 was usuallyused in Pluronic F-127 based nasal in situ gel formulations(Bhalerao et al., 2009; Chen et al., 2010; Majithiya et al., 2006; Zakiet al., 2007). In our study, we firstly fixed the concentration ofPluronic F-127 at 20% (w/v) and the drug concentration at 5% (w/v)(Table 1) followed by optimization of the Tsol–gel by addition ofPluronic F-68 and PEG 8000. In addition, the gelation time of in situnasal gel formulations (F1–F8) was measured at 31 �C (averagehuman nasal temperature (Jacky, 1980; Proctor et al., 1977)) by gelobservation described in Section 2.3.1. It was found that thegelation time of F1, F2, F5 and F6 was within 2–3 min and that of F3and F4 was within 5–10 min, while F7 and F8 could not form gel.Such short gelation time of the selected in situ formulation F1 (forin vivo evaluation) suggested that it could quickly form gel afternasal administration in human nasal cavity.

At the Tsol–gel, Pluronic F-127 forms gel with aqueous pores inthe matrix. Although the 3D network of the formed hydrogel issufficiently rigid, the highly hydrated microscale environmentenables mass transfer and the release of both hydrophilicand hydrophobic drugs via the extramicellar water channels(Anderson et al., 2001). It is reported that the release of drugs(e.g. propranolol, metronidazole and cephalexin) from Pluronic F-127 gels followed zero-order kinetics with both dissolution anddiffusion controlled release mechanisms (Moore et al., 2000). Inour current in vitro release setting, a dialysis membrane was usedin which the formed gel would take up water, followed by swellingto allow the drug to diffuse out of the hydrogel matrix. Meanwhile,the water uptake would lead to erosion of the formed hydrogelmatrix, which would accelerate the drug release. The developed insitu gels (F1 and F2) followed Korsmeyer–Peppas kinetics (Table 2),further suggesting that the release was controlled by both thediffusion rate of drug from hydrogel matrix and the relaxation rateof hydrogel matrix. In comparison to F1, the rate and extent of drug

Table 3Pharmacokinetic parameters of THA and its monohydroxylated metabolites after intranasal administration of THA in situ gel F1 and oral administration of THA solution at asingle dose of 4 mg/kg.

PK parameters Plasma (n = 5) Cortex (occipital–temporal) (n = 4)

nasal in situ gel oral solution nasal in situ gel oral solution

THATmax (h) 0.7 � 0.3 1.0 � 0.3 1.0 1.0Cmax (ng/ml) 123.0 � 44.9* 63.0 � 14.3 1898.2 � 396.7* 535.6 � 169.2AUC0–6 h (ng h/ml) 236.4 � 61.5* 111.7 � 28.0 3987.4 � 335.6* 1322.9 � 255.6t1/2 (h) 1.3 � 0.5 1.2 � 0.2 1.1 1.6

1-OH-THATmax (h) 0.9 � 0.2 1.6 � 0.2 2.0 2.0Cmax (ng/ml) 191.8 � 21.0* 264.7 � 40.8 150.8 � 17.0 175.3 � 17.3AUC0–6 h (ng h/ml) 544.2 � 109.8* 757.8 � 137.9 553.6 � 40.4 775.5 � 59.2t1/2 (h) 1.8 � 0.4 1.8 � 1.0 4.1 5.0

2-OH-THATmax (h) 1.3 � 0.9 1.4 � 0.2 N.A. N.A.Cmax (ng/ml) 17.0 � 4.3* 25.7 � 10.1 N.A. N.A.AUC0–6 h (ng h/ml) 59.0 � 18.5* 89.1 � 20.8 N.A. N.A.t1/2 (h) 2.4 � 0.3 3.1 � 2.1 N.A. N.A.

N.A.: not available.* p < 0.05 vs oral solution. Data represent mean � s.d. and mean � s.e.m. for plasma and cortex pharmacokinetic parameters, respectively.

S. Qian et al. / International Journal of Pharmaceutics 468 (2014) 272–282 279

release from F2 were lower (Fig. 4), which could be attributed tothe enhanced gel strength due to incorporation of chitosan(Varshosaz et al., 2008). Under the non-physiological environ-ments, the addition of mucoadhesive polymer chitosan signifi-cantly enhanced the shear viscosity with a 8.5-fold and 4.5-foldincrease in consistency index (K) at 8 �C and 20 �C, respectively (F2versus F1 shown in Fig. 6), which agreed with the observationsfrom other studies (Cho et al., 2011; Edsman et al., 1998).

The purpose of developing in situ gel formulation is to decreasethe mucociliary clearance and hence to extend the residence timeof the drug formulation in the nasal cavity. In order to investigatethe effect of components/formulations on mucociliary clearance,several in vivo and in vitro methods have been developed, such asmonitoring the radioactive particle transport (Ojima et al., 1998),saccharin test (Corbo et al., 1989), measurement of ciliary beatfrequency on excised frog palates (Rutland and Cole, 1981) etc.However, these methods are either too costly (e.g. monitoringradioactive particle transport) or too complex with largeexperimental variations (e.g. variations in excised tissues)(Donovan and Zhou, 1995). In this study, a simple and rapidassessment of the in vivo nasal mucociliary transport time on ratswas employed. In addition to the transport time to pharyngealcommonly reported in literatures (Dondeti et al., 1996; Zaki et al.,2007), the time for the passage of the dye from the delivery site tonasopalatine was also measured in the present study. Since thenasopalatine was close to the delivery site (0.5 cm depth in thenostril), the transport time needed for the dye to reachnasopalatine was much shorter than that needed to reachpharyngeal, which served as an alternative and effective way toevaluate the nasal mucociliary transport time of administratedmaterials. On the other hand, the transport time to pharyngeal inthe present study (9.2 � 2.9 min) was consistent with the valuepreviously reported (�10 min) for in situ gel (Zaki et al., 2007). Theprolonged residence time of the in situ gel in nasal cavity could bedue to its high viscosity and bioadhesive properties of Pluronicpolymer after liquid–gel phase transition.

Pharmacokinetic study revealed that the peak THA plasmalevel after intranasal administration of in situ gel was muchhigher, while the formation of THA metabolites was much lowerthan that of THA oral solution (Fig. 7), which could be due tothe avoidance of first-pass metabolism in intestine and liver

(Illum et al., 2002). Consequently, the in situ gel increased thedelivery of the parent THA to and decreased the exposure of THAmetabolites in rat brain comparing with oral THA solution.McNally et al. tested if some of the metabolites found in brain wereresulted from local brain metabolism, but no formation of 1-OH-THA or 4-OH-THA was identified by in vitro THA incubation studywith rat brain homogenates (McNally et al., 1996). Therefore, thedetected 1-OH-THA and 4-OH-THA were primarily formed insystemic circulation and entered into brain through the BBB. Onthe other hand, 2-OH-THA was not found in brain tissues, whichmight be due to its low lipophilicity leading to limited penetrationacross the BBB as well as low amount of formation (McNally et al.,1996; Qian et al., 2012). It was noticed that 2-OH-THA had a lowerlipophilicity than 1-OH-THA and 4-OH-THA, which was reflectedon the relative elution order (2-OH-THA, 1-OH-THA and 4-OH-THA) on the reversed-phase HPLC column. Such lower lipophilicityof 2-OH-THA lead to its lower penetration capacity across the BBBthan 1-OH-THA and 4-OH-THA, resulted in the brain–plasma ratioof 4-OH-THA (3.5), 1-OH-THA (<0.3) and no detection for 2-OH-THA (Qian et al., 2012). In addition, 2-OH-THA also demonstratedmuch lower plasma concentrations (�10-fold) than that of 1-OH-THA (Fig. 7) after THA administration via nasal or oral route,indicating potential lower formation of 2-OH-THA in vivo, whichmight be another reason for lack of 2-OH-THA intake in brain.Moreover, THA was evenly distributed in different brain regionswith no observable rostral-to-caudal concentration gradient (THAconcentration in the olfactory bulb was just comparable to otherbrain regions). This supported that after nasal administration THAwas absorbed into systemic circulation and then entered into brainthrough the BBB, and direct nose-to-brain transport of THA wasnot evident. Jogani et al. (Jogani et al., 2008) also studied theintranasal delivery of radiolabelled [99mTc]-THA in mice using amucoadhesive microemulsion formulation. By comparing theradioactivity in the whole brain after intranasal and IV admin-istrations, the investigators concluded that there was direct nose-to-brain transport of THA bypassing the blood–brain barrier. Theresults should be interpreted with caution since radioactivitymeasurement did not allow the differentiation of parent THA fromits metabolites nor intact THA from detached radioactivity.

Low oral bioavailability (Hartvig et al.,1990; Madden et al.,1995)and hepatotoxicity (O‘Brien et al., 1991; Qizilbash et al., 1998) are

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Fig. 8. Pharmacokinetic profiles of THA in different brain regions after intranasal administration of THA in situ gel (F1) and oral administration of THA solution (4 mg/kg).(data represents mean � s.d., n = 4 per time point).*: p < 0.05 vs THA oral solution; a: olfactory bulb; b: frontal cortex; c: hippocampus; d: striatum; e: occipital-temporal cortex; f: others including midbrain, thalamus,hypothalamus, and cerebellum.

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the two major factors hindering the clinical utility of THA. It issuggested that the hepatotoxicity is caused by the metabolites ofTHA (Madden et al., 1993; Park et al., 1994; Patocka et al., 2008;Spaldin et al., 1994). By avoiding the extensive first-pass metabo-lism of THA in intestine and liver after oral THA administration, thenasal in situ gel might indeed offer dual advantage of enhancingTHA bioavailability while reducing the formation of hepatotoxicmetabolites, which warrants further development. This alsohighlights the importance of investigating metabolite dispositionin nasal delivery studies (Wong and Zuo, 2013; Wong et al., 2012;Wong and Zuo, 2010), which deserves more attention in futureresearch. In addition, based on our pharmacokinetic studies, muchhigher exposure of THA in both plasma (�2-fold) and brain (�3-fold) were observed after nasal administration of THA in situ gel incomparison to oral THA. According to other studies that intranasaldelivery of THA in microemulsion significantly improved the brain

drug exposure and leaded faster memory regain in cognitive-impaired AD mouse model (Jogani et al., 2008), we speculated thatour developed THA nasal in situ gel would also exhibit greaterpharmacodynamic effects than that of oral THA, which warrantsfurther investigation on the diseased animal model, such as thescopolamine-induced cognitive impairment rat model etc.

5. Conclusion

In the present investigation, a Pluronic F-127 based thermo-sensitive in situ gel of THA with favorable physiochemicalproperties (suitable Tsol–gel, rheological properties and slowrelease kinetics) was developed. The enhanced residence timein nasal cavity, improved bioavailability and brain uptake of THAsuggested that in situ gel could serve as an intranasal formulationfor THA.

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Fig. 9. Pharmacokinetic profiles of 1-OH-THA in different brain regions after intranasal administration of THA in situ gel (F1) and oral administration of THA solution (4 mg/kg). (data represents mean � s.d., n = 4 per time point).*: p < 0.05 vs THA oral solution; a: olfactory bulb; b: frontal cortex; c: hippocampus; d: striatum; e: occipital-temporal cortex; f: others including midbrain, thalamus,hypothalamus, and cerebellum.

S. Qian et al. / International Journal of Pharmaceutics 468 (2014) 272–282 281

Acknowledgements

This work is supported by General Research Fund CUHK480809. The authors are grateful to Ms. Sophia Yui Kau Fong for hervaluable suggestions to the manuscript.

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