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0King Saud University Saudi Pharmaceutical Journal
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ORIGINAL ARTICLE
Hybrid drug delivery system for oropharyngeal, cervical and colorectal cancer - in vitro and in vivo evaluation
Mohamed S. Pendekal *, Pramod K. Tegginamat
Dept of Pharmaceutics, JSS College of Pharmacy, JSS University, SS Nagar, Mysore 15, Karnataka, India
Received 3 July 2012; accepted 13 July 2012 Available online 11 August 2012
KEYWORDS
Chitosan;
Sodium alginate;
Interpolyelectrolyte complex;
Buccal pH;
Vaginal pH;
Rectal pH
Abstract The present investigation was designed with the intention to formulate a versatile 5-flu-orouracil(5-FU) matrix tablet surpassing issues associated with current conventional chemothera-peutic drug delivery systems. The novel 5-FU matrix tablet fulfills therapeutic needs by engineering matrix tablets utilizing chitosan-sodium alginate interpolyelectrolyte complex (IPEC). IPEC was characterized by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The matrix tablets were formulated utilizing IPEC alone and in combination with chitosan, sodium alginate and sodium deoxycholate as permeation enhancer. Pharmaceutical properties, swelling studies, in vitro dissolution and diffusion studies, mucoadhesive studies and in vivo studies were performed for formulated 5-FU. The selected chito-san-sodium alginate IPEC offers pH independent 5-FU release in comparison to alone or physical mixture of chitosan and sodium alginate. Furthermore, novel matrix tablets demonstrated significantly higher bioadhesive properties with controlled 5-FU release without the initial burst effect and also demonstrated a higher permeation of 5-FU. To conclude, the developed novel 5-FU matrix tablets pave way as an excellent alternative for cancer treatment which could potentially minimize the dose dependent side effects and provide better patient compliance.
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1. Introduction
5-Fluorouracil(5-FU) has been widely used as an effective che-motherapeutic agent and drug of choice in oropharyngeal ca-ner, colorectal cancer, stomach cancer and cervical cancer (Calavresi and Chabner, 1996). Chemically, 5-FU is a diprotic
* Corresponding author. Tel.: +91 0821 2548353; fax: +91 0821 2548359.
E-mail address: mohamedsaif.xlnc@gmail.com (M.S. Pendekal). Peer review under responsibility of King Saud University.
acid with pka values of 8.0 and 13.0 and is highly polar in nature (Rudy and Senkowski, 1973; Williams and Barry, 1991). After oral administration, 5-FU is poorly absorbed with erratic variation in bioavailability ranging between 0% and 80%. 5-FU after parenteral administration is rapidly eliminated with apparent terminal half life of approximately 8-20 min (Dol-lery, 1999; Singh et al., 2005). On intravenous administration 5-FU produces severe systemic toxic effects including gastrointestinal, hematological, neural, cardiac and dermatological origin (Diasio and Harris, 1989). In such cases, local administration of 5-FU would be very advantageous instead of oral or parenteral drug delivery.
Most publications on 5-flurouracil focused on only single drug delivery like buccal gels (Dhiman et al., 2008), buccal tab-
1319-0164 © 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.Org/10.1016/j.jsps.2012.07.002
lets (Libero et al., 2010),and cervical patches (Woolfson et al., 1995). Based on these, 5-FU will be suitable for transbuccal/ vaginal/rectal drug delivery. Hardly any articles reported on permeation studies and histological effects of 5-FU. Literature review revealed that there is no single drug delivery system available that can be given either through buccal, vaginal or rectal route. The prime goal has to design 5-flurourcil multipurpose tablets with greater efficacy, potency, adoptability to need, minimal toxic effects and better patient compliance than the established marketed product.
Polymeric drug delivery systems are mainly designed for the efficient delivery of active drug. Among various polymeric drug delivery systems, interpolyelectrolyte complexes are the most newer and efficient form. Interpolyelectrolyte complexes (IPEC) have been used as a new class of polymeric carriers for novel drug delivery systems (Peppas and Khare, 1993; Berger et al., 2004; Nam et al., 2004; Moustanfine et al., 2005).
Chitosan, is a natural cationic polysaccharide obtained by partial deacetylation of chitin, consisting of glucosamine (GA) and N-acetyl-glucosamine (NAc-GA) linked by b-1,4 glucosidic bonds (Madihally and Matthew, 1999; Dang and Leong, 2006). Alginate is another natural anionic linear polysaccharide composed of a-L-guluronic and b-D-mannuronic acid residues (Gombotz and Wee, 1998; Augst et al., 2006). The carboxylate moieties on alginate can ionically interact with the protonated amines on chitosan, forming physical cross-linked hydro-gels known as polyelectrolyte complex (PEC) Takahashi et al., 1990; Baruch and Machluf, 2006.
Numerous 5-FU formulations have developed with various formulation strategies such as buccal gels, cervical patches, liposomes, and nanoparticles. However, these formulations are organ specific and require stringent procedures for final formulation. Cost of product development is too high as the procedures are more specific for liposomes and nanoparticles.
The versatile 5-FU matrix tablets offer a highly convenient preparation, patient compliance, minimizes toxic effects, cost effective and moreover suitable for industrial production.
2. Materials and methods
2.1. Materials
5-FU was obtained from Strides Arcolab Ltd., Bangalore, India. Chitosan (Marine chemical, Cochin, India, Deacetylation degree: 85%, viscosity: <200 mPa s, moisture content: <10%, ash content: <1%, insoluble: <1%, pH: 3-6, particle size: 80100 mesh). Sodium alginate (sigma Aldrich, nature: pH: ~7.2, viscosity: 20-400 mPa s, loss on drying: 615.0%, sulphated ash: 30.0-36.0%). Sodium deoxycholate, microcrystalline cellulose and Talc were from Zydus Cadila, India. All other chemicals and reagents used were of analytical grade.
2.2. Preparation of chitosan-sodium alginate interpolyelectrolyte complex (IPEC)
The chitosan-alginate interpolyelectrolyte complex was prepared from chitosan solution at 4.0% w/v in 1% w/w acetic acid solution and sodium alginate solution at 4.0% w/v in water. Each solution was heated separately at 70-80 0C. Both solutions were mixed with agitation until the mixture reached room temperature. Then it was left to rest for 2 h. The inter-
polyelectrolyte complex (IPEC) was thoroughly washed with distilled water and was then separated from water by centrifu-gation for 30 min at 10000 rpm. Thereafter IPEC was dried in a hot air oven and the dried complex was ground with a grinder. The powder was passed through a 200 im sieve and used for further study.
2.3. Fourier transform infrared (FT-IR) spectroscopy study
The infrared absorption spectra of Chitosan, sodium alginate and IPEC were analyzed using a FT-IR spectrophotometer (8400S, Shimadzu, Japan). The pellets were prepared by pressing the sample with potassium bromide.
2.4. Differential scanning calorimetry (DSC)
Thermal analysis was carried out using a differential scanning calorimeter (DSC 50, Shimadzu Scientific Instruments, Japan) for Chitosan, sodium alginate and IPEC. The samples were placed in an aluminum-sealed pan and preheated to 200 0C. The sample was cooled to room temperature and then reheated from 40 to 400 0C at a scanning rate of 10 0C/min.
2.5. Powder X-ray Diffraction
Powder X-ray diffraction patterns on chitosan, sodium alginate and IPEC were obtained by using an X-ray Diffractome-ter (Miniflex II Desktop X-ray Diffractometer, Rigaku Corporation, Tokyo, Japan). The samples were scanned from 60 to 400 (29) with an increment of 0.020 and measurement time of 10 s/increment.
2.6. Preparation of mucoadhesive matrix tablet
Mucoadhesive tablets were fabricated by direct compression method as shown in Table 1. The accurate quantity of 5-FU and excipients was weighed. They were passed through sieve and thoroughly mixed using mortar and pestle. The blend was lubricated and then compressed into compacts by direct compression method using 8-mm flat-faced punches in KBr press (Technosearch, Mumbai, India) at 1 ton pressure with a dwell time of 1 s.
2.7. Swelling studies
The swelling index of the prepared mucoadhesive 5-FU tablets was determined by weighing five tablets and recording their
Table 1 Formulation chart.
Ingredients F1 F2 F3 F4 F5 F6
5-Flurouracil 20 20 20 20 20 20
IPEC 40 80 80 80 80 80
Chitosan — — 10 20 20 20
Sodium alginate — — 10 20 20 20
Sodium deoxycholate — — — — 3 4.5
Microcrystalline cellulose 85 45 25 5 2 0.5
Talc 5 5 5 5 5 5
Weight in mg.
Total weight of tablet is 150 mg.
weights before placing them separately in weighed beakers. The total weight was recorded (W1). Four milliliters of phosphate buffer pH 6.8 (similarly with simulated vaginal fluid of pH 4.2 and pH 7.4) was added to each beaker and then placed in an incubator at 37 ± 0.5 0C. At time intervals of 2, 4, 6 and 8 h excess water was carefully removed, and the swollen tablets were weighed (W2). The experiment was repeated three times, and the average of W1 and W2 was reported.
The swelling index was determined from the formula.
SI = (W2 - W1)/W1 x 100 (1)
2.8. In vitro release of matrix tablets
The drug release rate from buccal compacts was studied using the orbital shaking incubator using (Remi CIS 24, India) 30 mL of phosphate buffer pH 6.8. The temperature was maintained at 37 ± 0.5 0C and 50 rpm (rotation per min). For every one hour of time interval 3 mL sample was withdrawn, filtered through a Millipore filter of 0.45 im pore size and assayed spectrophotometrically at 266 nm. Immediately after each sample withdrawal, a similar volume of phosphate buffer pH 6.8 was added to the dissolution medium.
The drug release rates from vaginal tablets were studied in 500 ml of simulated vaginal fluid pH 4.2 in type II dissolution apparatus. The temperature was maintained at 37 ± 0.5 0C and 50 rpm. For every one hour of time interval 10 mL sample was withdrawn, filtered through a Millipore filter of 0.45 im pore size and assayed spectrophotometrically at 265 nm. Immediately after each sample withdrawal, a similar volume of simulated vaginal fluid was added to the dissolution medium.
In vitro drug release for rectal tablets was performed using the dissolution apparatus I; 500 mL phosphate buffer (pH 7.4) maintained at 37 ± 0.5 0C was used as a dissolution medium. Basket was rotated at 50 rpm. 10 mL aliquots were taken at periodic time intervals and replaced by equal volume of phosphate buffer. The solution was suitably diluted and the absor-bance was taken at 267 nm using UV visible spectrophotometer.
2.9. Bioadhesive strength
Bioadhesive strength of the compacts was measured using modified physical balance as recently discussed (Deshmukh et al., 2009). In vitro bioadhesion studies were carried out using sheep buccal mucosa and modified two-armed balance. The phosphate buffer pH 6.8 was used as the moistening fluid. A glass stopper was suspended by a fixed length of thread on one side of the balance and was counter balanced with the weights on the other side. Fresh sheep buccal mucosa was collected from the slaughter house. It was scrapped off from the connective tissues and a thin layer of buccal mucosa was separated which was stored in Tris buffer until used for the bioad-hesion study. A circular piece of sheep buccal mucosa was cut and fixed to the tissue holder and was immersed in phosphate buffer pH 6.8 and the temperature was maintained at 370 ± 1 oc. Then the tablet was fixed to a glass stopper with the help of cyanoacrylate adhesive and it was placed on the buccal mucosa by using a preload of 50 gm and kept aside for 3 min to facilitate adhesion bonding. After preloading time, the preload was removed and the weights were added on the other side of the balance until tablet detaches from
the sheep buccal mucosa. The weight required to detach tablet from buccal mucosa was noted.
2.10. Ex vivo permeation study
Permeation study was carried out for the optimized formulation using Franz diffusion cell. The tablet was placed in the donor compartment on the sheep mucosa. The mucosal layer is on donor compartment. The receptor compartment was filled with phosphate buffer pH 6.8. The temperature was maintained at 37 ± 0.5 oc and 50 rpm. The amount of 5-FU permeated through sheep mucosa was determined by withdrawing 3 ml of aliquots from the receptor compartment using a syringe and immediately replacing the same volume of solution.
2.11. In vivo X-ray studies
The in vivo X-ray studies were approved by the Institutional Animal Ethics Committee of JSS College of Pharmacy (Mysore, Karnataka, India). The study was performed on a healthy female rabbit, weighing between 1 and 1.5 kg. F3 formulation was modified by adding 20 mg of X-ray grade barium sulfate (20 mg 5-FU was replaced). The prepared tablet was placed in the buccal mucosa of healthy rabbit. During the study, the rabbit was not allowed to eat or drink. The rabbit was exposed to X-ray examinations and photographs were taken at 1st and 8th hr after administration of the tablet. Similar procedure was followed for vaginal and rectal delivery.
2.12. Kinetic analysis
Drug release from simple swellable systems may be described by the power law expression and is defined by the following equation.
Mt/Mi = K1tn (2)
where Mt is the amount of drug released at time t, M1 is the overall amount of drug released, K1 is the release constant; n is the release or diffusional exponent and Mt/M1 is the cumulative drug concentration released at time t (or fractional drug release).
The release exponent (n) value was used for interpretation of the release mechanism from the compacts. The dissolution data were modeled by using PCP disso v2.01 (Bharathi Vidhy-apeeth, Deemed University, Pune, Maharashtra, India).
2.13. Statistical analysis
Statistical analyses of all data were undertaken using Graph-Pad prism version 5.0 (Graphpad software Inc, San Diego, California, USA).
3. Results and discussions
In our previous paper, studies on chitosan polymers on Eudra-git RL and RS 100 on buccal patches of tizanidine were evaluated (Mohamed and Pramod, 2012a). Later, it was also shown in our laboratory that the interpolymer complex between Chitosan and Carbopol® 71G as a suitable polymer for the development of novel drug delivery of miconazole for candidiasis (Mohamed and Pramod, 2012b).
In the light of vast previous experience and literature on Chitosan, we identified that chitosan is well suitable for particular pH and alone is not suitable for novel drug delivery. In contrast, chitosan-sodium alginate interpolyelectrolyte complex showed high potential as a matrix former and used as a new class of polymeric carriers for tissue engineering in the form of scaffolds (Li et al., 2005), membranes (Chen et al., 2006), fibers (Iwasaki et al., 2004) and microcapsules (George and Abraham, 2006). Therefore based on the extensive review and previous experience on above cited polymers, chitosan-so-dium alginate interpolyelectrolyte complex is taken into present investigation.
3.1. Characterization of IPEC
The interaction between chitosan and sodium alginate has been studied by several investigators (Xiaoxia et al., 2009; Bruno et al., 2006). The studies indicated that IPEC could be formed by the electrostatic interaction between the COO-group of sodium alginate and NH3 + group of chitosan in solutions. Fig. 1 shows the superimposed IR spectra of Chitosan, sodium alginate and IPEC in 1000-2000 cm"1 and 14001800 cm"1.
The degree of deacetylation of Chitosan is 85%, the amine group of 2-aminoglucose unit and the carbonyl group of 2-aminoglucose unit of chitosan showed absorption bands at 1589 and 1656 cm"1, respectively (Tien et al., 2003). Sodium alginate FTIR spectra showed strong absorption bands at 1610 cm"1 and 1417 cm"1 due to carboxyl anion stretching vibrations(asymmetric and symmetric) and at 1124 cm"1
Figure 2 Superimposed DSC thermograms of chitosan, sodium alginate and IPEC.
Figure 3 Superimposed XRD spectra of Chitosan, sodium alginate and IPEC.
assigned to the skeletal vibrations (Lawrie et al., 2007). In IR spectra, spectra of the physical mixture of two polymers or immiscible polymers will be the sum of the spectra of the individual compounds, whereas for polymers after electrostatic interaction, there will be changes in the IR spectra such as Wavenumber shifts, band broadening and new absorption bands that are evidence of the polymers miscibility (Stuart, 2004). The interpolymer complex showed similar spectra to that of sodium alginate: a broad band at 1610 cm"1 with two other bands at 1415 cm"1 and 1454 cm"1 indicating most of the alginate in the IPEC was deprotonated. The reduction in the intensity of band at 1415 cm"1 also confirmed the protonation. The band at 1543 cm"1 could be assigned to the NH3 + group of chitosan and the broadness of the band at 1610 cm"1 to 1643 cm"1 arose from the overlapping bands from the amide of chitosan and carboxyl group of alginate.
Fig. 2 shows the DSC thermograms of chitosan, sodium alginate and chitosan-sodium alginate IPEC. The DSC ther-mograms of pure chitosan, exhibit one broad endothermic peak at 110 0C associated to the evaporation of bound water, a glass transition at 240 0C and an exothermic peak at about 320 0C attributable to the polymer degradation. This includes saccharide ring dehydration, depolymerization and decomposition of deacetylated and acetylated chitosan units (Mathew et al., 2006; Sarmento et al., 2006). These peaks are in agree-
Figure 4a Swelling studies of formulations in pH 4.2. ♦ F1, □
F2, A F3, x F4, XF5, • F6 (mean ± SD, n = 3).
Figure 4b Swelling studies of formulations in pH 6.8. ♦ F1, □ F2, A F3, x F4, XF5, • F6 (mean ± SD, n = 3).
Figure 4c Swelling studies of formulations in pH 7.4. ♦
F2, A F3, x F4, XF5, • F6 (mean ± SD, n = 3).
F1, □
Table 2 Pharmaceutical properties.
Formulation code Thickness (mm) Hardness (N) Friability (%) Weight (mg) Drug content (%)
F1 2.01 ± 0.001 F2 2.02 ± 0.001 F3 2.01 ± 0.002 F4 2.03 ± 0.003 F5 2.02 ± 0.002 73 ± 4 75 ± 4 79 ± 4 74 ± 3 75 ± 3 0.08 0.04 0.03 0.05 0.05 149.7 ± 0.523 149.6 ± 1.00 149.5 ± 0.753 150.0 ± 0.532 150.1 ± 0.512 99.96 ± 0.07 99.91 ± 0.03 99.91 ± 0.08 99.92 ± 0.09 99.89 ± 0.12
Figure 5 Mucoadhesive strength of formulations.
Figure 6c Dissolution profile of formulations F1-F4 in pH 7.4.
♦ F1, □ F2, Л F3, x F4 (mean ± SD, n = 3).
Figure 6a Dissolution profile of formulations F1-F4 in pH 4.2.
♦ F1, □ F2, Л F3, x F4 (mean ± SD, n = 3).
Figure 6b Dissolution profile of formulations F1-F4 in pH 6.8.
♦ F1, □ F2, Л F3, x F4 (mean ± SD, n = 3).
ment with other reported studies (Neto et al., 2005; Sankalia et al., 2007). Sodium alginate thermogram exhibits one endo-thermic and exothermic peak at 100 0C and 250 0C. The first endothermic peak is a broad peak assigned to the evaporation of water from hydrophilic groups in the polymers and the second exothermic peak resulted from degradation of polyelec-trolytes due to dehydration and depolymerization reactions most probably to the partial decarboxylation of the proton-ated carboxylic groups and oxidation reactions of the polyelec-trolytes (Mimmo et al., 2005; Soares et al., 2004). Chitosan-sodium alginate physical mixture shows an endothermic peak at 110 0C that can be probably explained due to the coales-
cence of both isolated endothermic polymer peaks, whereas exothermic peaks at 245 0C and 300 0C resulted from individual contribution of alginate and chitosan, respectively. In IR spectra of IPEC, it could be seen that the peaks of the IPEC were shifted from those of physical mixture. Exothermic peak of IPEC appeared at 290 0C, an intermediate and broader peak value compared with isolated chitosan and sodium alginate, which represents an interaction between both polymers. Endo-thermic peak is also shifted to a higher temperature at 130 0C with a relative broader and shorter peak. This represents that the formed IPEC is a different chemical entity than the mixture of two polymers.
The X-ray diffraction of chitosan, sodium alginate and IPEC is shown in Fig. 3. The powder X-ray diffraction pattern of chitosan powder showed two prominent diffraction peaks at 10.60 (29) and 19.640 (29). a shoulder peak appears at 21.740 and also a minor peak appears at 26.620. The two prominent crystalline peaks at 10.60 and 19.640 are typical fingerprints of chitosan which were related to the hydrated and anhydrous crystals respectively (Mimmo et al., 2005) (Wan et al., 2003). Sodium alginate shows peaks at 13.360 and 21.380, whereas IPEC showed a peak at 21.660. The typical peaks of chitosan disappeared and the IPEC showed an amorphous morphology after complexing. The integration of sodium alginate into chitosan disrupted the crystalline structure of chitosan, which is due to the elimination of hydrogen bonding between amino groups and hydroxyl groups in chitosan (Kim et al., 1993).
3.2. Pharmaceutical properties
The results of pharmaceutical properties are summarized in Table 2. The matrix tablets showed a diameter of 8 mm with negligible variation and therefore not included in the table. All the formulations showed satisfactory values within the limits of conventional oral tablets stated in the Indian Pharmacopoeia (Indian pharmacopeia, 2007).
3.3. Swelling studies
In vaginal pH 4.2, F2 formulation showed maximum swelling at 8 h. F1 formulation exhibited less swelling than F2. F2 formulation was found to have high 662% of swelling at 8 h. Further, the addition of chitosan and sodium alginate in F3 and F4 formulations reduces the swelling than F1 and F2 formulations. Nearly the same degree of swelling was observed for F5
Table 3 Drug release kinetic parameters for the formulations.
Formulation Zero order Matrix Korsmeyer-Peppas
R K R K n K
Vaginal pH 0.9700 14.328 0.9788 34.1764 0.7705 21.8835
Buccal pH 0.9906 1.5205 0.9563 3.5852 0.9291 1.7538
Rectal pH 0.9770 14.3480 0.7753 34.1419 0.7726 21.6978
Vaginal pH 0.9980 11.9137 0.9235 27.7433 0.9464 12.8462
Buccal pH 0.9964 1.2622 0.9103 2.9246 1.0865 1.0718
Rectal pH 0.9967 12.7421 0.9445 29.9076 0.8613 16.1300
Vaginal pH 0.9799 12.2565 0.9740 29.1354 0.7648 18.7170
Buccal pH 0.9063 1.6313 0.9920 3.9438 0.4840 4.0109
Rectal pH 0.9882 11.4832 0.8846 26.3793 1.1759 8.1070
Vaginal pH 0.9871 11.8275 0.8809 27.1174 1.3717 6.0614
Buccal pH 0.9982 1.3597 0.9235 3.1648 1.0372 1.2634
Rectal pH 0.9874 11.3880 0.8830 26.1553 1.1348 8.5792
0 2 4 6 S 10
Time in hrs
Figure 7 Ex vivo permeation studies of F5 & F6 formulation. ♦ F5, ■ F6. (mean ± SD, n = 3).
and F6 as that of the formulation F4. The addition of sodium deoxycholate does not significantly alter the swelling in F5 and F6. The comparison of degree of swelling of all formulations in vaginal pH 4.2 is shown in Fig. 4a. F3 formulation exhibited highest swelling in pH 6.8, IPEC along with chitosan and sodium alginate increases the swelling to 698% whereas F1 and F2 formulations showed 623 and 654% swelling degrees respectively. F4 formulation containing IPEC, chitosan and sodium alginate in higher concentration than F3 formulation showed decreased swelling than F3 formulation. Formulation F4 was found to have 417% of swelling that is little lesser than swelling in vaginal pH 4.2. This may be explained by the ability of forming a gel layer in acidic pH which is lacking in buccal pH 6.8. Similar swelling profile was seen for F5 and F6 formulations as of F4 formulation. Swelling profiles of formulations in pH 6.8 are shown in Fig. 4b. Even in pH 7.4, the formulation F2 is found to be having highest swelling. The swelling of F3 formulation was found to be 663%. F4 formulation exhibited swelling of 492% that is as nearly similar in pH 4.2 and pH 6.8. Nearly same degree of swelling was observed for F5 and F6 as that of the formulation F4. Swelling profiles of formulations in pH 7.4 are shown in Fig. 4c.
These findings indicate that the presence of IPEC alone in the matrix tablet exhibits a slow uniform pH independent swelling degree and also the presence of chitosan and sodium alginate in IPEC matrix tablets alters the swelling degree. Therefore the mechanism of drug release from IPEC matrix tablets was affected by the presence of chitosan and sodium alginate.
3.4. Bioadhesion studies
The mucoadhesive strength for all the formulations is shown in Fig. 5. F1 formulation containing only IPEC shows the least detachment force this may be explained due to the lacking of free functional groups which were probably involved in the adhesion of the mucosa. F2 formulation having a higher concentration of IPEC still exhibited the least detachment force, further addition of chitosan and sodium alginate to IPEC concentration in F3 formulation enhances the detachment force. F4 formulation shows the highest detachment force, this may be due to the availability of free functional groups. F5 & F6 formulations containing sodium deoxycholate does not have any impact on the detachment force.
These findings indicate that the presence of IPEC alone does not exhibit sufficient bioadhesion hence the presence of other polymers is necessary for the development of bioadhesive matrix tablets. Therefore a suitable combination of polymers along with IPEC is selected for producing sufficient bioadhe-sion without altering properties of IPEC.
3.5. In vitro drug release studies
In vitro drug release study as dissolution profiles of formulation F1-F4 in pH 4.2, pH 6.8 and pH 7.4 is shown in Fig. 6a, Fig. 6b and Fig. 6c respectively. 5-FU matrix tablets were initially prepared from IPEC alone and the in vitro drug release was investigated in buccal, vaginal and rectal pH. Formulations containing only IPEC (F1& F2) in all pH exhibited controlled release properties. But F3 formulation retarded
Figure 8 X-ray radiographic images of buccal, vaginal and rectal cavity at 1 and 8 h after ingestion of BaSO4-loaded optimized F4 matrix tablet in rabbits.
drug release (86% of drug released at 8th hour) this may be explained due to the addition of chitosan which forms gel at acidic pH. Further, the increase in concentration of chitosan in F4 formulation retards the drug release but later releases the drug up to 95% that is desirable, this may be due to the presence of sodium alginate that inhibits the gel forming ability of chitosan in higher concentration. In case of buccal pH 6.8, F3 formulation exhibits the initial burst effect about 45% of the drug is released at the 1st hour that is due to the presence of chitosan and sodium alginate inhibiting the controlled release effect of IPEC. Whereas in case of F4 formulation, the initial burst effect is abolished with increase in the concentration of sodium alginate that forms a stiff gel along with IPEC
and hence retards the drug release. In pH 7.4, all the formulations exhibit the controlled drug release without the initial burst effect. Among all formulations, F4 formulation exhibits an excellent and desirable drug release profile. The presence of chitosan and sodium alginate does not alter properties hence desirable drug release profile was seen for F4 formulation in pH 7.4. Similar drug profile was seen as in buccal and vaginal pH. F5 & F6 formulations containing SDC exhibited similar drug release profile as F4 formulation (Data not mentioned).
To confirm the similarity of F4 formulation dissolution profiles in buccal, vaginal and rectal pH, the similarity factor (F2) was used and was found above 80. Since the F2 values were higher than 50, these results confirmed that the drug re-
lease profiles were almost similar for F4 formulation for buccal, vaginal pH and rectal pH.
Kinetic parameters determined are shown in Table 3.
To ascertain drug release mechanism and release rate, the release data were fitted into release models using PCP Disso V2.01 dissolution software. The models selected were of zero order, Higuchi matrix and Korsmeyer-Peppas.
3.6. Ex vivo permeation studies
5-FU permeation from formulations F5 and F6 across sheep mucosa over a period of 8 h is shown in Fig. 7. The maximum permeation of 5-FU from F5 was 60% at 8 h compared with 98% from F6. Regression of the linear portions of the two plots gave slopes and intercepts from which the permeation flux (slope divided by mucosal surface area) of F5 and F6 was calculated to be 5.01 and 8.6mg/cm2/h, respectively.
In formulation F5 addition of SDC 2% increased the cumulative percentage of drug permeated to 60%. This may be due to SDC extracted only the mucosal lipid from the intercellular spaces. Thus, this enhances the diffusivity of the 5-FU via the paracellular or polar route. Further increase in concentration of SDC (F6), i.e., 3%, increased the drug permeation up to 98% thus SDC in 3% extracts lipids from the cell membranes, along with the extraction of mucosal lipid from the intercellular spaces by the formation of micelles. This resulted in enhancing passive diffusivity of the 5-FU via transcellular (crossing the cell membranes and entering the cell) and para-cellular routes (Hoogstraate et al., 1997). It was mentioned that SDC can also cause the uncoiling and extension of the protein helices, which leads to opening of the polar pathways for diffusion (Gandhi and Robinson, 1992). All these effects might contribute to enhancing the permeation of the drug.
3.7. In vivo X-ray studies
After administration of the optimized formulation (F4), developed by using barium sulfate, the duration of the tablet in the buccal, vaginal and rectal cavity was monitored by radiograms (Fig. 8). The tablet adheres to the buccal, vaginal and rectal mucosa. The buccal tablet swells and is retained till 8 h with little reduction in tablet size, as the tablet swells certain part of the tablet was swallowed by the rabbit thereby the tablet size reduces. Whereas the tablet in the vaginal cavity swells in 1 h and little reduction of size at 8 h. In the rectal cavity tablet remained in the cavity till 8 h.
4. Conclusion
The IPEC between Chitosan and sodium alginate demonstrated not only as a potential excipient for control release in the matrix system but also possesses pH independent drug release for the delivery of 5-FU. The matrix system with a suitable combination of chitosan and sodium alginate was able to produce desired drug release, bioadhesion and swelling. The desired bioadhesion can be achieved only with the addition of chitosan and sodium alginate; hence asuitable combination played a key role in the bioadhesion and subsequently maintains the pH independent drug release without initial burst release pattern. The addition of Sodium deoxycholate to the
matrix tablets is also necessary to attain optimum drug permeation.
Acknowledgment
The authors wish to thank the JSS University, Mysore, India
for providing the facilities to complete this work.
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