European Journal of Pharmaceutical Sciences 44 (2011) 241–249

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European Journal of Pharmaceutical Sciences

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Enhanced bioavailability of nano-sized chitosan–atorvastatin conjugateafter oral administration to rats

Mohammed Anwar a, Musarrat H. Warsi a, Neha Mallick a, Sohail Akhter a, Sachin Gahoi b,Gaurav K. Jain a,⇑⇑, Sushma Talegaonkar a, Farhan J. Ahmad a,⇑, Roop K. Khar a

a Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi 110062, Indiab Senior Manager, Formulation Development Services, R&D Centre, Matrix Labs. Ltd., Andhra Pradesh, India

a r t i c l e i n f o

Article history:Received 15 April 2011Received in revised form 3 July 2011Accepted 1 August 2011Available online 16 August 2011

Keywords:ChitosanAtorvastatinNano-sizingHomogenizationBioavailabilitySustained release

0928-0987/$ - see front matter � 2011 Elsevier B.V. Adoi:10.1016/j.ejps.2011.08.001

⇑ Corresponding author. Address: Department oPharmacy, Hamdard University, Hamdard Nagar, Ne+91 09810720387; fax: +91 11 26059663.⇑⇑ Co-corresponding author. Address: DepartmentPharmacy, Hamdard University, Hamdard Nagar, New9811127909; fax: +91 11 26059663.

E-mail addresses: drgkjain@gmail.com (G.K. Jain(F.J. Ahmad).

a b s t r a c t

A novel approach to improve the bioavailability and stability of atorvastatin (AT) was developed by con-structing a nano-sized polymer–atorvastatin conjugate. Firstly, a novel chitosan–atorvastatin (CH–AT)conjugate was efficiently synthesized through amide coupling reaction. The formation of conjugatewas confirmed by 1H NMR and FT-IR spectrometry. Nano-sized conjugate with a mean size of215.3 ± 14.2 nm was prepared by the process of high pressure homogenization (HPH). Scanning electronmicroscopy (SEM) revealed that CH–AT nano-conjugate possess smooth surface whereas X-ray diffraction(XRD) spectra demonstrated amorphous nature of nano-conjugate. Further, CH–AT nano-conjugateshowed solubility enhancement of nearly 4-fold and 100-fold compared to CH–AT conjugate and pureAT, respectively. In vitro drug release studies in simulated gastric fluid and simulated intestinal fluid sug-gested sustained release of AT from the conjugate. Additionally, the nano-conjugate significantly reducedthe acidic degradation of AT. The plasma-concentration time profile of AT after oral administration ofCH–AT nano-conjugate (2574 ± 95.4 ng/mL) to rat exhibited nearly 5-fold increase in bioavailability com-pared with AT suspension (583 ± 55.5 ng/mL). Finally, variable bioavailability, as observed for AT suspen-sion was also reduced when AT was administered in form of CH–AT nano-conjugate. Taken together thesedata demonstrate that chitosan conjugate nano-prodrugs may be used as sustained polymeric prodrugsfor enhancing bioavailability.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction

Atorvastatin ([R-(R⁄,R⁄)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino) carbonyl]-1H-pyrrole-1-heptanoic acid, calcium salt (2:1) trihydrate), is a member of thedrug class known as statins, used for lowering blood cholesterol lev-els. AT is an orally administered drug used for the treatment of ele-vated total cholesterol, low density lipoprotein and triglycerides,and to elevate high density lipoprotein cholesterol (Law et al.,2003). It also stabilizes plaque and prevents strokes through anti-inflammatory and other mechanisms. Like all statins, AT works byselectively inhibiting HMG-CoA reductase, an enzyme that isinvolved in the biosynthesis of cholesterol (Law et al., 2003). AT is

ll rights reserved.

f Pharmaceutics, Faculty ofw Delhi 110062, India. Tel.:

of Pharmaceutics, Faculty ofDelhi 110062, India. Tel.: +91

), farhanja_2000@yahoo.com

a BCS class II drug, insoluble in aqueous solutions of pH 4, veryslightly soluble in distilled water and pH 7.4 phosphate buffer, andhas high intestinal permeability (Lennernas, 1997; Wu et al.,2000). AT is rapidly absorbed after oral administration, with timeto reach peak concentrations (tmax) within 1–2 h but possess poororal bioavailability (�12%) (Corsini et al., 1999). The poor oral bio-availability is attributed for its low aqueous solubility, crystallinenature, and high hepatic first-pass metabolism (Cilla et al., 1996;Lennernas, 2003). Furthermore, the bioavailability of AT is highlyvariable due to its instability in the acidic milieu of the stomach(Shah et al., 2008). Poor oral bioavailability of AT results in adminis-tration of its high doses and engenders dose related undesirable ad-verse effects such as liver abnormalities, rhabdomyolysis, arthralgia,and kidney failure. There are many existing factors limiting the suc-cessful use of orally administered AT, including problems with drugformulation due to poor aqueous solubility and more importantly,insufficient and fluctuating bioavailability obtained after oraladministration (Kim et al., 2008a,b). Therefore, it is needed to devel-op a novel approach that can resolve both the solubility and absorp-tion issues. A number of methods have been developed to improvethe oral bioavailability of AT based on improving solubility andenhancing dissolution rate. For instance, it was reported that the

242 M. Anwar et al. / European Journal of Pharmaceutical Sciences 44 (2011) 241–249

use of self-microemulsifying drug delivery system (SMEDDS) for thedelivery of AT could improve its solubility and permeability throughthe mucous membrane significantly (Shen and Zhong, 2006). Morerecently, it has been reported that the solubility and bioavailabilityof crystalline AT could be improved by physical modification suchas particle size reduction and conversion to amorphous state (Kimet al., 2008a,b; Zhang et al., 2009).

Except developing novel HMG-CoA reductase inhibitors bychemical modification, the design of drug delivery systems for stat-ins has been proven to be an efficient approach to overcome thedrawbacks of statins, such as poor solubility, limited stability, firstpass metabolism and toxicity. For instance, polymer–drug conju-gates as special type of drug delivery system have attracted consid-erable attention due to their particular therapeutic properties, suchas prolonged half-life, enhanced bioavailability, and often targetingto specific cells, tissues or organs by attaching a homing device.Drug–polymer conjugates often aim to increase the surface area,solubility and wettability of the powder particles and are thereforefocused on particle size reduction or generation of amorphousstates (Grau et al., 2000; Hancock and Zografi, 1997; Lee et al.,2009; Yang et al., 2010). Examples of polymer–drug conjugates in-clude PHEA-50-O-succinyl zidovudine with a prolonged duration ofactivity (Giammona et al., 1998), and the macromolecular prodrugof 3TC-dextran for selective antiviral delivery to the liver (Chima-lakonda et al., 2007). Very recently, it has been reported that pac-litaxel conjugate with low molecular weight chitosan exhibitedfavorable features for oral delivery including: (1) increased watersolubility of paclitaxel, (2) prolonged retention of the conjugatein the GI tract, (3) ability to bypass the P-glycoprotein mediated ef-flux, and (4) ability to bypass cytochrome P450-mediated metabo-lism, all of which led to dramatically enhanced bioavailability andantitumor efficacy in vivo (Lee et al., 2008). Further, nano-sizeddrug delivery system is a very promising way of improving drugbioavailability. Enhanced bioavailability could be observed fornanoparticles containing AT. Encouraged with the results, we sug-gested that chitosan–drug conjugate could be used as a technolog-ical platform capable of improving oral absorption of drugs. Here,we report a new nano-sized conjugate between chitosan and ATfor oral delivery of AT. Synthesis, characterization, drug releaseprofile, mucoadhesiveness of the nano-sized CH–AT conjugatewas reported. Further, the pharmacokinetics was evaluated usinga rat model.

Fig. 1. Schematic diagram for preparation of CH–AT nano-conjugate.

2. Materials and methods

2.1. Materials

AT (Form I) was obtained as a gift sample from Lupin Ltd. (Pune,India). Chitosan (CH) (ChitoClear™, degree of deacetylation 96%;viscosity 15cp) was obtained from Primex ehf (Siglufjordur, Ice-land). 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)was purchased from Himedia Laboratories (Mumbai, India). Allother chemicals were of analytical grade and were used as receivedfrom Merck Ltd. (Mumbai, India).

2.2. Synthesis and characterization of CH–AT conjugate

As shown in Fig. 1, CH–AT conjugate was prepared by usingamide coupling reaction. A 10% (w/v) solution of AT in methanol(5 mL) was activated by EDC (125 mM, 1 mL) treatment for 4 hat room temperature to afford an ester form of AT. Separately,1% (w/v) aqueous CH solution was prepared after hydrating CHwith 1 N HCl (5 mL). The methanolic solution of AT was thenadded dropwise to the aqueous acidic CH solution undercontinuous magnetic stirring. Throughout the experiment, pH

was maintained in the range of 5–6. After stirring for 24 h at roomtemperature, the excess reagent and the corresponding acylisou-rea (by-product after coupling) was removed by washing withdistilled water. The reaction mixture was then purified usingultrafiltration, after which the CH–AT conjugate was lyophilized.The conjugate was then characterized by using 1H NMR(300 MHz, Bruker Biospin, Germany) and FT-IR spectrometry(Tensor 27, Bruker Biospin, Germany) and quantified by ultrahigh-pressure liquid chromatography (UHPLC) (Waters Acquity™,MA, USA).

2.3. Preparation of CH–AT nano-conjugate

Nano-sizing of CH–AT conjugate was achieved using HPH tech-nique. Briefly, 100 mg of the synthesized conjugate was dispersedin deionized water at a concentration of 0.1% (w/v). The suspensionthus formed was allowed to pass through a high-pressure homog-enizer (Nano DeBEE, BEE International Inc., MA, USA) to obtainnano-conjugates. CH–AT nano-conjugates were collected bylyophilization.

Fig. 2. 1H NMR spectrum of AT (A), chitosan (B), CH–AT conjugate (C), CH–AT nano-conjugate (D).

M. Anwar et al. / European Journal of Pharmaceutical Sciences 44 (2011) 241–249 243

2.4. Characterization of CH–AT nano-conjugate

2.4.1. Nano-conjugate morphologyMorphological characteristics of the nano-conjugates were ob-

served by scanning electron microscope (SEM, EVO LS 10, Zeiss,Carl Zeiss Inc., Germany) operating at an accelerating voltage of13.52 kV under high vacuum. Freshly prepared nano-conjugatesample was fixed to aluminum stubs with double-sided carbonadhesive tape, sputter-coated with conductive gold–palladiumand observed using SEM.

2.4.2. Nano-conjugate size and zeta potentialMeasurement of particle size, zeta potential and polydispersity

of nano-conjugates was done using Zetasizer (Nano ZS, MalvernInstruments, Malvern, UK), which is based on the principle of dy-namic light scattering (DLS). All DLS measurements were done intriplicate at 25 �C at a detection angle of 90�. For zeta potentialmeasurements, disposable capillary cell with a capacity of 1 mLwas used. To obtain complete dispersion, the nano-conjugateswere dispersed in Marcol 52 (Exxon Mobil Co., USA) and sonicatedfor 10 min at 120 W power (Branson 8210, Branson Ultrasonics Co.,Danbury, CT, USA).

2.4.3. Nano-conjugate crystallinityThe physical form of the lyophilized nano-conjugates was

determined by powder X-ray diffraction (XRD, X’pert pro, Pan Ana-lytical, Netherland) over a range of 2h from 5� to 60� with Ni-fil-tered Cu-Ka radiation. The scan speed was 3 min�1.

2.5. Solubility studies

To evaluate solubility, excess of AT, CH–AT conjugate and CH–AT nano-conjugate were added to the deionized water (10 mL) inscrew-capped tubes placed in a water-jacketed vessel linked to atemperature-controlled water bath maintained at 37 ± 0.1 �C for48 h. Continuous agitation was provided by overhead stirring. Eachsample was centrifuged (REMI, Mumbai, India) at 18,000 rpm for30 min and the respective clear supernatants containing releaseddrug were diluted with methanol and analyzed by UHPLC as de-scribed in Section 2.10.

2.6. In vitro release studies

In vitro release studies were performed using transparent gela-tin capsules containing pure AT and the formulations (CH–AT con-jugate and CH–AT nano-conjugate) equivalent to 100 mg of AT.Tests were performed employing United States Pharmacopeia(USP) paddle apparatus (Vankel apparatus, USA) using phosphatebuffer (pH 7.4), and simulated gastric fluid (SGF, pH 1.2) at37 ± 0.1 �C for up to 72 h at a rotation speed of 50 rpm. At desig-nated time points, 4 mL samples were withdrawn with replace-ment with equal volume of the fresh medium, filtered through0.11 lm nylon syringe filter, appropriately diluted with methanoland assayed for drug concentration by UHPLC method as describedunder Section 2.10. Dissolution tests were performed in triplicateand the percentage of drug dissolved at different time intervalswas estimated.

2.7. Measurements of mucoadhesiveness using small intestinalsurfaces

The mucoadhesive property of the suspension of AT, CH–ATconjugate and CH–AT nano-conjugate were evaluated by anin vitro adhesion testing method known as the wash-off method.Freshly-excised pieces of intestinal mucosa from rat were mounted

Fig. 3. FT-IR spectra of AT (A), chitosan (B), CH–AT conjugate (C), CH–AT nano-conjugate (D).

Table 1Effect of high pressure homogenization process variables on particle size.

Pressure (psi) No. of Cycles Particle size (nm)

0 0 58.9 ± 11.8 lm

20,000 1 978.6 ± 18.9*

2 710.0 ± 20.5*

3 693.7 ± 19.2*

30,000 1 770.7 ± 16.9*

2 645.2 ± 20.1*

3 648.5 ± 12.4*

244 M. Anwar et al. / European Journal of Pharmaceutical Sciences 44 (2011) 241–249

onto glass slides (3 � 1 sq. in.) with cyanoacrylate glue. Two glassslides were connected with a suitable support. About 50 lL of eachsample was spread onto each wet rinsed tissue specimen, andimmediately thereafter, the support was hung onto the arm of aUSP tablet disintegrating test machine. When the disintegratingtest machine was operated, the tissue specimen was given a slow,regular up-and-down movement in the test fluid (400 mL) at 37 �Ccontained in a 1000 mL vessel of the machine. At the end of 4 h, themachine was stopped and the remaining amount of drug adheringto the tissue was quantified by the UHPLC method.

40,000 1 392.8 ± 13.8*

2 215.3 ± 14.2*

3 214.8 ± 15.8*

Data are shown as the means ± SD, (n = 3).* p < 0.001 compare to non-homogenized sample.

2.8. Acidic degradation studies

Stability of the drug and the formulation in conditions simulat-ing the gastric environment was determined by adding 10 lg of ATand CH–AT nano-conjugate to 10 mL of 1 N HCl and mixture wasrefluxed at 80 �C. At designated time points, 3 mL of the samplewas withdrawn and assayed for drug concentration by UHPLCmethod as described in Section 2.10.

2.9. In vivo pharmacokinetic studies

For in vivo pharmacokinetics, two groups, each containing sixfemale albino rats (0.18–0.22 kg) was used. After 12 h of fasting,the rats were allowed to administer 0.5 mL aqueous dispersion ofAT, CH–AT conjugate and CH–AT nano-conjugate (equivalent to

10 mg/mL AT) using oral feeding sonde. Blood samples (0.2 mL)were withdrawn at pre-determined time intervals through the tailvein of rats in vacutainer tubes, vortexed to mix the contents andcentrifuged at 5000 rpm for 20 min. The plasma was separatedand stored at �20 �C until drug analysis was carried out usingUHPLC method as described under section 2.10. The animal proto-col to carry out in vivo study was reviewed and approved by theInstitutional Animal Ethics Committee, Jamia Hamdard (ApprovalNo: 253) and their guidelines were followed for the studies. Data

Fig. 4. SEM images of AT (A), chitosan (B), CH–AT conjugate (C), CH–AT nano-conjugate (D).

M. Anwar et al. / European Journal of Pharmaceutical Sciences 44 (2011) 241–249 245

processing for calculating the pharmacokinetic parameters (PK)was done using Microsoft Excel software.

2.10. UHPLC analysis

AT was analyzed by UHPLC with a Waters Acquity™ UPLC sys-tem (Serial No# F09 UPB 920 M; Model Code# UPB; Waters, MA,USA). Chromatographic separation was performed on an AcquityUPLC BEH C18 (100 mm � 2.1 mm, 1.7 lm) column. The mobilephase was composed of 0.05 M NaH2PO4 buffer: methanol (30:70(v/v)), adjusted to a pH of 5.1 and a flow rate of 1.0 mL/min. Thedetection wavelength was set at 247 nm and the retention timewas 3.9 min. For the analysis of the samples from receptor solution,aliquots of 20 lL from each sample were injected via the manualinjector into a HPLC system. Plasma samples were first extractedwith ethyl acetate, vortexed and centrifuged at 10,000 rpm for15 min. The supernatant was evaporated to dryness and the resi-due was reconstituted with the mobile phase. All the samples werefiltered through a 0.11 lm pore size membrane filter before injec-tion. The assay was linear (r2 = 0.9995) in the concentration rangeof 0.01–50 lg/mL with a detection limit of 0.005 lg/mL. The per-centage recovery ranged from 98.0% to 101.2%. No interferencefrom the formulation components was observed.

3. Results and discussion

3.1. Synthesis and characterization of CH–AT conjugate

CH is a hydrophilic water soluble macromolecule with activeamine-functional groups. It is mucoadhesive in nature and is alsoknown to improve permeation of drug molecules across biologicalbarriers (Robinson et al., 1987; Smart et al., 1984). On the otherhand, AT is a hydrophobic drug consisting of free carboxylic group(Peppas and Buri, 1985). The complex between CH and AT was

attempted in order to impart hydrophilicity (increased water solu-bility of AT by conjugation to water soluble CH) and mucoadhesion(prolonged retention of the conjugate in the GI tract) to the AT.Further, it was considered that the conjugate would also be ableto prevent the degradation of AT in the acidic milieu of the stom-ach. The chemical structure of CH–AT conjugate is shown inFig. 1. AT was covalently attached to CH through an amide linkerthat is known to be cleaved under physiological conditions(Martin, 1998; Testa, 2004). The conjugation between CH and ATwas carried out using amide coupling reaction between the aminegroups of CH and activated carboxylic group of AT (Fig. 1). The car-boxylic group of AT was activated using EDC by the formation ofO-acylisourea, which could be viewed as a carboxylic ester withan activated leaving group (Fig. 1). EDC was selected because ofits solubility in a wide range of solvents and easy separation ofits by-product. The conjugate was characterized by 1H NMR, show-ing peaks corresponding to both CH and AT, and a distinctive peakat d value of 9.89 owing to amide bond formation (Fig. 2C). Thesame has been confirmed by a distinctive peak at 1700 cm�1 inFT-IR spectrum of CH–AT conjugate (Fig. 3C). Further, the absenceof unsaturated carbon–carbon double bond peaks at 1420 cm�1

(Fig. 3B) and displacement of the secondary amine deformationband from 1550 (Fig. 3A) to 1480 cm�1 (Fig. 3C), suggests thatthe coupling reaction had occurred between the amino group ofchitosan and the carboxylic group of AT. The weight percentage(% w/w) of AT in the CH–AT conjugate as quantified using theUHPLC method was �15% (w/w).

3.2. Preparation and characterization of CH–AT nano-conjugate

To further enhance the solubility, CH–AT nano-conjugates wereprepared by homogenizing CH–AT conjugates using HPH. Nanoniza-tion using wet-milling by HPH was selected since the thermal energygenerated during wet-milling is lower than that generated by

Fig. 5. XRD pattern of AT (A), chitosan (B), CH–AT conjugate (C), CH–AT nano-conjugate (D).

246 M. Anwar et al. / European Journal of Pharmaceutical Sciences 44 (2011) 241–249

dry-mills as the drug is suspended in aqueous media. The influenceof operating pressure and the number of homogenization cycles onthe particle size was studied. The results of the particle size measure-ments obtained after homogenization are presented in Table 1. A sig-nificant reduction in particle size of CH–AT conjugate was observedafter homogenization. The data also showed that the particle sizehad an inverse relationship with both homogenization pressureand number of cycles and obviously mean particle size decreasedwith increase in pressure or number of homogenization cycles.

Conjugates with lowest particle size were obtained after 2 homoge-nization cycles at 40,000 psi pressure. Increasing the number ofhomogenization cycles from 2 to 3 did not result in further decreasein particle size indicating the attainment of saturation levels. Ab-sence of any chemical change during nanonization was confirmedby 1H NMR and FT-IR analysis. The 1H NMR (Fig. 2D) and FT-IR spec-tra (Fig. 3D) of CH–AT nano-conjugate were superimposable to thecorresponding spectra of CH–AT conjugate, indicating that no chem-ical change occurred during nanonization.

Table 2In vitro release studies of CH–AT nanoconjugate.

Time (h) % Drug dissolved

SGF (pH 1.2) Phosphate buffer (pH 7.4)

0.5 4.7 ± 0.5 2.7 ± 0.31 10.5 ± 1.2 5.5 ± 0.82 21 ± 1.7 12 ± 1.34 72.5 ± 4.5 28.5 ± 1.86 100 ± 6.4 47.5 ± 3.18 68 ± 4.810 92.5 ± 6.2

Results represents mean values ± standard deviation, n = 3.

Fig. 6. Acidic degradation kinetics of AT and CH–AT nano-conjugate in 1 N HCl at80 �C.

Table 3In vivo Pharmacokinetic studies of AT and CH–AT nanoconjugate.

Pharmacokinetic parameters

Parameters AT CH–AT Nano-conjugate

AUC0?t

(ng/mL h)8240.6(2180.5)

37252.9(452.8)

AUC0?1(ng/mL h)

11878.5(2250.7)

1047629.0(949.6)

Cmax

(ng/mL)583.0(55.5)

2574.0(95.4)

tmax (h) 2 4t1/2 (h) 15.8 19.3

Results represents mean values (standard deviation), n = 6.

M. Anwar et al. / European Journal of Pharmaceutical Sciences 44 (2011) 241–249 247

3.3. Morphology of CH–AT nano-conjugate

Using SEM, the homogeneity and effectiveness of the HPH mill-ing process was readily evident. SEM micrographs of AT, CH, CH–AT conjugate and CH–AT nano-conjugate are shown in Fig. 4. Inaddition to particle size analysis, the SEM micrographs further pro-vided the evidence that wet milling resulted in significant reduc-tion of particle size. The SEM micrographs of pure AT revealedlarge crystalline blocks with rough surface (Fig. 4A). The surfaceof the CH was uniform with appearance of flaws (Fig. 4B). The for-mation of CH–AT conjugate resulted in a scaffold-like structure(Fig. 4C) while on homogenization, the formation of CH–ATnano-conjugate presents a smooth surface morphology of nano-conjugates (Fig. 4D).

Fig. 7. Plasma AT concentration as a function of time after oral administratio

3.4. Crystallinity of CH–AT nano-conjugate

In order to identify the physical state and crystallinity of AT inpolymeric conjugate, the XRD spectra of pure AT, CH, CH–AT con-jugate and CH–AT nano-conjugate are presented in Fig. 5. As can beseen from the Fig. 5, pure AT is highly crystalline. CH powdershowed two major broad crystalline peaks at 2h of around 9.5�and 19.7�, respectively, while the diffraction peaks of CH–AT con-jugate were not recorded at the same position. The peak at 2h ofaround 9.5� disappeared and instead new peaks at 2h of 27.8�,32.1� and 56.9� with low intensity could be observed. The reduc-tion in crystalline peaks and formation of new peaks in CH–AT con-jugate may be attributed to a polymorph structure transformationdue to the attachment of CH to AT. In contrary to this, CH–AT nano-conjugate showed a broad amorphous peak. The possibility ofshear-induced amorphous drug formation during the millingprocess could not be ruled out as reported previously (Keck andMüller, 2006; Kipp, 2004).

3.5. Solubility studies

The aqueous solubility of pure AT, CH–AT conjugate and CH–ATnano-conjugate was found to be 23.5, 589.2 and 2410.2 lg/mL,respectively. The solubility of AT was increased by approximately25-fold after conjugation with CH. As expected, the solubility ofCH–AT nano-conjugate was approximately 4-fold greater thanthe CH–AT conjugate and nearly 100-fold higher than that of pureAT. This improved water solubility of AT for CH–AT nano-conjugatecould be attributed to the collective effect of formation of watersoluble conjugate, amorphous AT in CH–AT nano-conjugate and re-duced particle size which offer higher surface area for drug disso-lution. CH–AT nano-conjugate was selected for further studies,since it showed significantly higher solubility compared to theCH–AT conjugate.

n of aqueous dispersion of AT (A) and CH–AT nano-conjugate (B) to rats.

Fig. 8. Schematic representation of possible mechanism of drug release and bioavailability enhancement of AT through chitosan-atorvastatin nano-complex.

248 M. Anwar et al. / European Journal of Pharmaceutical Sciences 44 (2011) 241–249

3.6. In vitro release studies

To examine whether or not parent AT is released from the nano-conjugate we carried out drug release experiments at 37 �C byincubation in SGF (pH 1.2) and phosphate buffer (pH 7.4). The re-lease data clearly indicated that AT is released from the conjugateunder physiological conditions (Table 2). Complete release of ATwas attained in SGF within 6 h, whereas the similar extent of AT re-lease was observed within 10 h for phosphate buffer (92.5 ± 6.2%).Approximately, 20% of AT was released upon incubation in SGFwithin 2 h, whose amount would seem insubstantial when consid-ering the transit time in stomach (1–2 h). These observationswould be quite useful since acid catalyzed degradation of AT,which is responsible for variable bioavailability, would be signifi-cantly reduced.

3.7. Acidic degradation kinetic studies

The study was performed to determine if CH–AT nano-conju-gate would be able to prevent the acid-catalyzed degradation ofAT. Fig. 6 shows the degradation kinetics of pure AT and CH–ATnano-conjugate. From Fig. 6 it was evident that for pure AT, com-plete drug degradation occurred at 4 h time point, whereasapproximately 60% of AT was still remaining in case of CH–ATnano-conjugate. It can be anticipated that incomplete drug releasefrom the CH matrix (Fig. 6) and presence of hydrophilic coating ofCH over AT might be responsible for reduction in degradation ofAT.

3.8. Evaluation of mucoadhesive properties of CH–AT nano-conjugate

The binding responses of pure AT and CH–AT nano-conjugate onintestinal membrane after 4 h were found to be 10.2% and 68.9%,respectively. The mucoadhesive nature of the CH–AT nano-conju-gate was due to the presence of CH, which is known to be mucoad-hesive. This result suggests clearly that CH–AT nano-conjugateretains mucoadhesiveness of parent CH. We also speculate thatmucoadhesiveness observed for pure AT might be due to its hydro-phobicity, resulting in enhanced interaction with the intestinalepithelium.

3.9. Pharmacokinetics after oral administration of CH–AT nano-conjugates to rats

Fig. 7A and B depict the plot of AT concentration in plasma as afunction of time individually for each rat in the group, after admin-istration of AT suspension and CH–AT nano-conjugate solution,respectively. Plasma AT concentration vs. time plots obtained afteroral administration of AT suspension to rats (Fig. 7A) clearly indi-cates that bioavailability is highly variable probably due to acid-catalyzed degradation of AT or P-glycoprotein-mediated efflux. Incontrast to this, the plasma AT concentration vs time plots obtainedafter oral administration of CH–AT nano-conjugate to rats exhibitednearly similar profile (Fig. 7B) indicating a reduction in variability inbioavailability. This could be either due to the prevention of acid-catalyzed degradation by CH–AT nano-conjugate as demonstratedby acid degradation kinetic study or due to the ability of CH–ATnano-conjugate to bypass the P-glycoprotein-mediated efflux as re-ported previously for oral delivery of paclitaxel–chitosan conjugate(Lee et al., 2008). The relevant pharmacokinetic parameters arelisted in Table 3. While AT suspension showed plasma half-life(t1/2) of 15.8 h, CH–AT nano-conjugate group exhibited delayedt1/2 value of 19.3 h suggesting that AT is released from CH–ATnano-conjugate in a sustained manner over prolonged period oftime. In addition, the Cmax of the nano-conjugate (2574 ± 95.4 ng/mL) was greater than that of AT suspension (583 ± 55.5 ng/mL).As expected, a marked increment by 5-fold was observed inAUC0–1 of CH–AT nano-conjugate as compared to AT suspensiongroup. To the best of our knowledge, the oral bioavailability of ATfrom CH–AT nano-conjugate is the highest among others reportedin the literatures to date (Kim et al., 2008a,b; Shen and Zhong,2006; Zhang et al., 2009). This unprecedented high absorptionmay be attributed to enhanced solubility of amorphous AT inCH–AT nano-complex, the known ability of CH to be mucoadhesiveand open tight junctions in intestinal epithelial cells. Furthermore,CH–AT nano-conjugate may also be able to bypass both P-glycopro-tein-mediated efflux (displayed on intestinal epithelial cells) andcytochrome P450-mediated drug metabolism (hepatic clearance)as demonstrated previously for oral delivery of paclitaxel in theform of conjugate with chitosan (Lee et al., 2008). The possiblemechanism of drug release and bioavailability enhancement of ATthrough CH–AT nano-complex is depicted in Fig. 8.

M. Anwar et al. / European Journal of Pharmaceutical Sciences 44 (2011) 241–249 249

4. Conclusion

In the present work, we synthesized a nano-sized CH–AT conju-gate, and reported the physicochemical characteristics and phar-macokinetics of the new prodrug. CH–AT nano-conjugate showedmarkedly enhanced water solubility (�100 times) and better sta-bility of AT in simulated gastric milieu. In vitro drug release studiesindicated that the polymeric conjugate prodrug released AT for aprolonged period. Compared to AT suspension, CH–AT nano-conjugate exhibited less variable and 5-fold higher oral bioavail-ability. Taken together, CH-based conjugate system may be usedas a promising oral delivery platform for sparingly soluble APIs.We are currently synthesizing other CH-based conjugates with dif-ferent types of drugs in the same direction.

Acknowledgment

The authors are thankful to Ms. Priya Ahuja, Department ofChemistry, Hamdard University, New Delhi, for her inputs regard-ing synthesis of the conjugate.

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