CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555 Vol.10 No.10, pp 60-76, 2017
Abstract : Objective : Raloxifene hydrochloride is a selective estrogen receptor modulator with a very poor oral absolute bioavailability (2%) due to high hepatic first-pass metabolism. Mucoadhesive vaginal tablets of Raloxifene hydrochloride can bypass high hepatic first pass metabolism and also improve its solubility and dissolution behaviour. Methods : Inclusion complex of drug with β-cyclodextrin was prepared by kneading method. Composition of the mucoadhesive tablet was optimized using 32 full factorial design where amount of sodium CMC (X1) and amount of Polycarbophil (X2) were taken as independent variables. Drug release at 6 hour (Q6), mucoadhesive strength and swelling index were considered as dependent variables. The formulations of design batches were characterized for weight variation, hardness, thickness, friability, drug content, swelling index, ex-vivo mucoadhesive strength, surface pH, drug release at 6 hrs, ex-vivo residence time, drug release data modelling. Optimized batch was subjected to ex-vivo permeation study and short term stability study. Results : The optimized formulation (F5) comprises 20 mg of sodium CMC and 15 mg of polycarbophil had shown mucoadhesive strength (0.343N), swelling index (36.04%) and % drug release at 6 hours (95.90%).ex-vivo permeation was found to be 47.93% at 6 hr. Results of drug release data modelling suggested zero order drug release kinetics (R2=0.9983) with case II transport release mechanism (n=0.9513) for optimised batch. Conclusion : Raloxifene hydrochloride mucoadhesive tablet is a promising approach for the effective treatment of disease as it provides control drug release and bypasses the hepatic first pass metabolism. Key words : osteoporosis, factorial design, Contour plot, β-cyclodextrin, phase solubility, Job’s plot.
Raloxifene hydrochloride is a second-generation selective estrogen-receptor modulators (SERM) endorsed by the Food and drug administration in 1997 for the treatment of osteoporosis. It is commercially available under the trade name of Evista (Eli Lilly, Indianapolis, IN) in 60-mg dose tablets. Oral bioavailability of this drug is only 2% which limits its oral administration. It is Biopharmaceutical Classification System (BCS) class II drug having low solubility and high permeability and it also undergoes extensive first pass metabolism which leads to poor bioavailability [1]. Various strategies have been reported to improve the solubility and bioavailability of raloxifene hydrochloride, such as lipid-based delivery systems, inclusion complexes, and cogrinding [2-4].
The vaginal epithelium is permeable to a wide range of drugs, like hormones, antimycotics, peptides and proteins [5]. The vagina provides a promising site for local effect as well as systemic drug delivery because of its large surface area, rich blood supply, and avoidance of the first-pass effect, relatively high permeability for many drugs and self-insertion [6, 7]. In addition, a prolonged contact of a delivery system with the vaginal mucosa may be achieved more easily than at other absorption sites like rectum or intestinal mucosa [8].
Conventional vaginal delivery systems include solutions, suspensions, gels, foams and tablets. Vaginal creams and gels provide lubrication, but tend to be messy and cluttered, and are easily washed off if they are water soluble and easily dispersive. Suspensions and solutions tend to spread widely in the vaginal cavity. Vaginal Foams are provided excessive lubrication and leakage from the vagina. Thus vaginal tablets are to be useful and most convenient dosage form as ease for application and portability [9].
The term “Mucoadhesion” describes materials that bind to biological substrates, such as mucosal members. Adhesion of bioadhesive drug delivery devices to the mucosal tissue offers the possibility of creating an intimate and prolonged contact at the site of administration. This prolonged residence time can result in enhanced absorption and in combination with a controlled release of drug also improved patient compliance by reducing the frequency of administration [10, 11].
Mucoadhesive vaginal drug delivery system (MVDDS) has been used for the treatment of local diseases affecting the vagina like candidiasis, sexually transmitted disease, vaginal dryness, and also can be successfully deliver drugs to systemic circulation via vaginal mucosa for treatment of various diseases like migraine and osteoporosis [12]. It can offer other numerous advantages as (i) avoiding hepatic first-pass metabolism, (ii) use of small doses, in comparison to oral administration, (iii) side effect minimization, (iv) provides intimate contact between a dosage form and the vaginal mucosa, which may result in high concentration in a local area and hence high drug flux through the vaginal mucosa and (v) easy removal [13]. The efficacy of MVDDS is affected by the biological environment and the properties of the polymer and the drug8. Acrylic acid polymers (Carbomer or polycarbophil) and cellulose derivatives (hydroxyethylcellulose, hydroxypropylcellulose or hydroxypropyl methylcellulose) have been widely used polymers in MVDDS [1417]. Degim et al successfully developed vaginal drug delivery system of insulin using chitosan gel to achieve extended release [18]. Thiolated polymers are also one of the successfully used approaches for mucosal drug delivery systems [19, 20]. Beta-cyclodextrin, citric acid, Tween 80 and Polaxamers are also added in vaginal formulation to increase drug solubility [21].
The present research work includes improvement of solubility of Raloxifene hydrochloride by complexation with cyclodextrin and development of mucoadhesive vaginal tablets of the complex using 32 full factorial design.
Raloxifene hydrochloride (R-HCl) was gifted by Aarti drugs limited, Mumbai. Β cyclodextrin, Polycarbophil, Sodium carboxy methyl cellulose, Polyox, Xanthan gum, HPMC K4M, and Microcrystalline cellulose were purchased from Lyka pharmaceuticals(Ankleshwar), Lubrizol (Belgium), Lobachemie (Mumbai), Molychem (Mumbai); respectively. Magnesium stearate and talc were purchased from Ases chemical works, Jodhpur. All reagents were used of analytical grade and were used as received.
An excess amount of Raloxifene hydrochloride (R-HCl) was placed in a 25 ml glass flask containing different concentrations of β-cyclodextrin (β-CD) (0-10 mM). Flasks were covered with cellophane membrane to avoid solvent loss and then shaken (50 agitations/min) in incubator shaker for 24 hr at 37 ± 0.5ºC. Supernatant was withdrawn and filtered through 42 µm Whatman Filter Paper. The filtrates were analyzed using a UV Visible spectrophotometer (UV 1800, Schimadzu, Japan) at 287 nm after suitable dilution against blank prepared of the same concentration of β-CD in water to cancel any interference of β-CD [22-25]. Phase solubility diagram was plotted (Figure 1) and apparent solubility constant (KC) was calculated using equation 1.
Where, S0=Solubility of the drug in the absence of β-CD.
2.2.2 Job’s plot (continuous variation method)
It was used to demonstrate the stoichiometry of R-HCl: β-CD inclusion complexes. It involves preparing an equimolar solution of drug and cyclodextrin derivative and mixing them in different proportion so that keeping the final concentration constant. Absorbance of the complex was plotted against the mole fractions of these two components (Figure 2) [26]. The maximum on the plot corresponds to the stoichiometry of the inclusion complex formation.
DSC analysis was performed to check drug excipient compatibility using Shimadzu DSC 60 (Schimadzu, Japan) using 10 mg sample. Sample was heated in aluminium pan at a rate of 10ºC/min in the temperature range of 30 to 300ºC under nitrogen flow of 40 mL/min. An empty aluminium pan was used as a reference [27].
A solid complex of R-HCl with β-CD in 1:1 equimolar ratio was prepared by kneading method. Cyclodextrin was triturated in a mortar with purified water to obtain a paste and then drug was incorporated. The resulting mixture was triturated for 1 hr in mortar followed by drying in an oven at 45°C. The dried mass was pulverized; passed through a 60-mess sieve and evaluated for drug content, saturated solubility study, DSC study and FTIR study [28, 29].
Saturated solubility of R-HCL complex in water and phosphate buffer pH 7.4 was determined by adding excess amounts of inclusion complex to water and phosphate buffer pH 7.4 at 37 ± 0.5°C; respectively [30]. Saturated solubility of pure drug in water was also determined for reference point. The solutions were equilibrated under continuous agitation for 24 h and filtered through Whatman filter paper to obtain a clear solution. The absorbance of the samples was measured by UV spectrophotometer at 287 nm and the concentrations in μg/mL were determined.
Preliminary batches P1 to P8 were prepared using 35 mg Carbopol 934, Thiolated chitosan, Sodium CMC, Polyox, Polycabophil, Sodium alginate, HPMC K4M, Xanthan gum; respectively. Each batch contained 35 mg of polymer, drug complex (196.2 mg), MCC (65.8mg), Talc (1 mg) and Magnesium stearate (2 mg). Tablets were prepared by direct compression method and polymers were selected for further study by evaluating tablets for mucoadhesive strength and swelling index.
Compositions of batches P9 to P18 are mentioned in Table 1. Tablets were prepared by direct compression method and evaluated for drug release at 6 hour (Q6), mucoadhesive strength and swelling index.
Table 1 Composition of raloxifene hydrochloride mucoadhesive vaginal tablets (Preliminary batches P9 to P18)
Ingredients | F9 | F10 | F11 | F12 | F13 | F14 | F15 | F16 | F17 | F18 |
---|---|---|---|---|---|---|---|---|---|---|
Raloxifene+ βcyclodextrin complex | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 |
Polycarboph il | - | - | - | 25 | 15 | 10 | 15 | 20 | 25 | 15 |
Sodium Carboxy Methyl Cellulose | 25 | 25 | 25 | - | - | 25 | 25 | 25 | 25 | 15 |
Polyox 301 | 25 | 15 | 10 | - | - | - | - | - | - | - |
Hydroxy Propyl Methyl cellulose (HPMC) K4M | - | - | - | 25 | 25 | - | - | - | - | - |
Microcrystal line cellulose | 49 | 59 | 64 | 49 | 59 | 64 | 59 | 64 | 49 | 69 |
Magnesium Stearate | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
Talc | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Formulation optimization was done using 32 full factorial design [31]. In this design, two factors namely amount of sodium CMC (X1) and amount of Polycarbophil (X2) were evaluated, each at three levels (-1, 0 and +1). -1, 0 and +1 level for factor X1 represents 15, 20 and 25 mg, respectively whereas, -1, 0 and +1 level for factor X2 represents 10, 15 and 20 mg, respectively. Experimental trials were carried out for all nine possible combinations (Table 2). Drug release at 6 hour (Q6), mucoadhesive strength and swelling index were selected as dependent variables. As shown in equation (2), a statistical model incorporating interactive and polynomial terms was used to evaluate the responses.
Where, Y are the dependent variables, namely, Drug release at 6 hour (Q6) (Y1), mucoadhesive strength (Y2), and swelling index (Y3); b0 is the arithmetic mean response of the 9 runs; and b1 and b2 are the estimated coefficients for the factors X1 and X2, respectively. The main effects (X1 and X2) represent the average result of changing one factor at a time from its low to high value. b12 is the coefficient of interaction and b11 and b22 are coefficients of quadratic terms. The interaction term (X1X2) shows how the response changes when two factors are simultaneously changed. The polynomial terms (X12 and X22) are included to investigate nonlinearity.
Table 2 Compositions of R-HCl mucoadhesive vaginal tablets prepared using 32 full factorial design (Batches F1 to F9)
Ingredients | F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 |
---|---|---|---|---|---|---|---|---|---|
Raloxifene+ βcyclodextrin complex | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 | 196.2 |
Sodium Carboxy Methyl Cellulose | 15 | 20 | 25 | 15 | 20 | 25 | 15 | 20 | 25 |
Polycarbophil | 10 | 10 | 10 | 15 | 15 | 15 | 20 | 20 | 20 |
Microcrystalline cellulose | 76 | 71 | 66 | 71 | 66 | 61 | 66 | 61 | 56 |
Magnesium stearate | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
Talc | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Tablets were prepared by direct compression method hence micromeritics studies (angle of repose, bulk density, tapped density, % compressibility) of powder blend were evaluated prior to manufacturing of compression coated tablets [32].
Tablets were characterized for weight variation, thickness, hardness, friability, In vitro swelling rate, Ex-vivo mucoadhesion study, surface pH, In vitro dissolution study, Ex-vivo residence time. Hardness, thickness and friability were determined by Monsanto hardness tester, digital Vernier callipers and friabilator respectively [31, 33].
The weight of medicated tablets was determined and denoted as W1. Each tablet was placed separately in a petridish with a wire mesh to avoiding direct contact with the petridish which contained a 5 mL phosphate buffer pH 7.4. Tablets were removed at different time intervals, wiped with filter paper and reweighed (W2) it after deducting a weight of cut piece of sieve. The swelling index was calculated using equation 2 and plotted as a function of time [34].
The ex-vivo mucoadhesive Strength was performed after application of the vaginal tablet on freshly cut goat buccal mucosa [35]. The fresh goat buccal mucosa was tied on the glass slide, and a mucoadhesive core side of each tablet was wetted with phosphate buffer pH 7.4 and adhered to the sheep buccal mucosa by applying a light force with a fingertip for 30 seconds. The modified physical balance was adjusted by keeping glass beaker on another side. Water was added gradually by burette and observes the weight of water needed to detach the tablet from goat buccal mucosa was recorded to measure the mucoadhesive strength in grams [36]. Force of adhesion (N) was calculated using following equation 3.
Surface pH of tablet was determined by adding 2 ml of distilled water to tablet placed in a beaker which was allowed to swell at room temperature for 2 hours. pH measurement was done by contacting the electrode with the tablet surface for 2 min [37].
Dissolution was performed in 900 ml pH 7.4 phosphate buffer using USP dissolution apparatus II equipped with a paddle operating at the speed of 50 rpm. Temperature of dissolution medium was maintained at 37 ±0.5 ºC. Five ml sample was withdrawn at regular time interval from the dissolution medium and replaced with fresh medium to maintain the sink conditions. The amount of drug released was measured at suitable time intervals using UV spectrophotometer (UV 1800, Schimadzu, Japan). The test was performed in triplicate.
Several equations are reported in the literature to identify the mechanism of drug release from the dosage from. Drug release data of optimized batch of R-HCl mucoadhesive vaginal tablets was evaluated according to the following equations:
Zero order release model (Equation 4) [38]:
First order release model (Equation 5) [39]:
Higuchi model (Equation 6) [40-42]:
Where Qt is the amount of drug dissolved in time t, Q0 is the initial amount of drug, K0 is the zero-order release constant, KH is the Higuchi rate constant, KK is a release constant, and n is the release exponent that characterizes the mechanism of drug release. For cylindrical matrix tablets, n = 0.45 indicates drug release mechanism by fickian diffusion, and if 0.45 < n <0.89, then it is non-Fickian or anomalous diffusion [45].
The tablet was applied on the goat buccal mucosa which was fixed on the glass slide with cyanoacrylate glue [5, 46]. The slide was tied to the disintegration apparatus and suspended in the beaker filled with 500 mL simulated vaginal pH 7.4. The slide was allowed to reciprocate in the medium until the tablet got detached or eroded from the mucosa. The test was performed in triplicate. Time for the detachment of the tablet was recorded as in vitro residence time.
Diffusion study was carried out to evaluate the permeability of drug across the goat buccal mucosal membrane using Franz diffusion cell. Goat buccal mucosa was obtained from a local slaughterhouse and was used within 2 h of slaughter. The tissue was stored in ice cold water, solution upon collection. The epithelium was separated from underlying connective tissues with surgical scissors and placed in between donor and receiver chambers of the diffusion cells for permeation studies. Receptor compartment contained 20 mL of pH
7.4 phosphate buffer, while donor compartment was filled with 3 mL simulated vaginal pH of 7.4. The tablet was placed on the mucosal surface in donor compartment, and 2 mL aliquots were removed at suitable intervals from the receptor compartment while the solution is being stirred continuously using magnetic stirrer, replacing it with fresh 2 mL medium each time. The absorbance was measured at 287 nm using UV visible spectrophotometer (UV 1800, Schimadzu, Japan) [47].
The stability of the optimized tablets was assessed by packing them in aluminum foil, sealed tightly and stored for 30 days at 40 ±2ºC and 75 ±5 % RH. The tablets were analyzed for drug release, mucoadhesive strength and % swelling index after 1 month of withdrawal of sample [48].
3. Results And Discussion
3.1 Phase solubility study
Solubility of R-HCL increased with increasing the amount of β-CD (Figure 1). This behaviour of linear increase in drug solubility with increased carrier concentration was indicative of the AL type of solubility phase diagram [49]. The linear host-guest correlation with slope of less than 1 suggested the formation of a 1:1complex with β-CD concentration [50]. Stability constant for β-CD was found to be 990.40 M-1 . The magnitude of apparent stability constant for several drug/CD complexes, K in M –1, ranges from 0 to 100000 [51].
Fig. 1: Plot of phase solubility study
3.2 Job’s plot (continuous variation method)
The maximum absorbance variation for R-HCl in β-CD was observed for 0.5 mole fraction, which indicated that the main stoichiometry is 1:1, in agreement with the stoichiometry suggested from the phase-solubility study [52].
3.3 Differential scanning calorimetry (DSC) study
In DSC thermogram, melting endotherm of R-HCl was present at 265.96°C (Figure 3(a)). Figure 4 shows characteristic thermogram for individual excipients and overlay thermogram for drug with a mixture of polymeric material. Peak of drug was preserved in the same temperature region in case of drug-excipient mixtures indicated compatibility of drug with selected excipients [53]. The intensity of peak was reduced in physical mixture due to dilution effect; however it had no incompatibility issue.
Fig 3: Comparative DSC spectra of drug (a), β-cyclodextrin (b) and inclusion complex(c)
Fig. 4: DSC thermograms of B-CD(a), Polycarbophi(b), Sodium CMC, Overlay spectra of drug-excipient physical mixture
3.4 Evaluation of inclusion complex of R-HCl with β-CD using kneading method
Percentage drug content in inclusion complex was performed in triplicate and it was found to be 96.33± 0.7288%. Solubility of uncomplexed drug in water, complexed drug in phosphate buffer and complexed drug in water was found to be 20.79, 60.32 and 68.54 µg/ml; respectively which indicated enhancement of solubility of R-HCl due to ß-CD complexation. DSC thermograms of R-HCl exhibited only one endothermic peak corresponding to the melting points at 265.96°C (Figure 3 (a)). The endothermic peak at 105°C in the β-CD was observed due to the evaporation of the absorbed water (Figure 3 (b)). 50.Drug peak was completely disappeared in the inclusion complex prepared by the kneading method (Figure 3(c)) as drug was present within the cavity of the β-CD ring molecule [54].
3.5 Evaluation of R-HCl mucoadhesive vaginal tablets
3.5.1 Preliminary studies
3.5.1.1 Evaluation of R-HCl mucoadhesive vaginal tablets (Batches P1to P8)
The swelling behavior and mucoadhesive strength are the two main concerns for effective bioadhesive tablet formulations for vaginal delivery [55]. Percentage swelling and mucoadhesive strength of preliminary batches (P1 to P8) are in the range of 0.272 to 0.533 and 24.14 to 49.41, respectively. Desired mucoadhesive strength is 0.3 to 0.4 N. Xanthan gum and sodium alginate showed lower mucoadhesive strength than desired value. Lower mucoadhesion leads to less residence time of formulation in the vagina. Thiolated chitosan showed acceptable mucoadhesive strength but it failed to provide acceptable swelling. Tablets with Sodium CMC and HPMC K4 M exhibited desired mucoadhesive strength but in swelling they form rigid structure which may prolong drug release. Tablet should release more than 90% of drug within 8-10 hours to avoid vaginal irritation [56, 57]. Polycarbophil, polyox and carbopol 934 exhibited the highest mucoadhesive strength among all the selected polymers. However, Carbopol 934 showed gradual and extensive swelling and if the degree of swelling is too great, a slippy mucilage results which can be easily removed from the substrate [56, 57]. Polycarbophil and Polyox were selected as mucoadhesive polymers in further studies.
3.5.1.2 Evaluation of R-HCl mucoadhesive vaginal tablets (Batches P9 to P18)
Fig. 5: Swelling behaviour of mucoadhesive vaginal tablet
Formulation P9 having high mucoadhesive strength than desired value which may lead to local irritation in vagina [58]. Formulation P10 exhibited prolonged drug release up to 10 h because higher concentration of sodium CMC swelled rapidly when in contact with water to form a gel, prevented fast disintegration of tablet. Formulation P11 having lower mucoadhesive strength which can lead to less residence time of dosage from to vaginal mucosa. Formulation P12 containing a polycarbophil as a mucoadhesive polymer and HPMC K4M as a release retardant polymer but tablet was broken within 5h in swelling study. Formulation P13 exhibited 88.81% drug release within 4 hr followed by breaking of tablets within 5h due to presence of erodible polymer, HPMC K4M. Formulation P14 and P18 had lower mucoadhesive strength whereas formulation P16 and P17 had higher mucoadhesive strength than desired value. Formulation P15 comprising combination of polycarbophil as a mucoadhesive polymer(15 mg) and sodium CMC as a release retardant polymer (25 mg) in was considered a good candidate due to acceptable criteria of mucoadhesive strength(N), swelling index and drug release. Swelling behavior of this tablet at regular time is shown in figure 5. Fast swellability of sodium CMC prevented the premature disintegration of polycarbophil tablet and polycarbophil prevented the fast erosion of sodium CMC [59, 60].
3.5.2 Evaluation of R-HCl mucoadhesive vaginal tablets prepared using 32 full factorial design
3.5.2.1 Pre-compression parameters
The results for bulk density, tapped density, Carr’s index, Hausner’s ratio and angle of repose of
powder blends of batches F1 to F9 indicated good flowability and compressibility of the blend [32].
3.5.2.2 Post compression parameters
The prepared tablets showed acceptable pharmaco-technical properties. For batches F1 to F9, hardness of tablets were in the range of 4-5 kg/cm2 and friability was less than 1% w/w indicated sufficient mechanical strength of tablets. All formulations were found to be within IP 2007 [61] limits as per weight variation test. Assay results of all batches were found to be in the Pharmacopoeial limits of 95–105 %.
3.5.2.3 In vitro swelling rate
Swelling study helps in analysis of important parameters like drug release mechanism from a matrix system, possibility of water penetration for drug release and lag time for insoluble drug release from matrix system. The swelling Characteristics of a polymer can also contributes to its bioadhesive behavior [62]. Swelling index of each batch (F1 to F9) is reported in table 3. Guobin Yi, et al prepared hydrogel and suggested that sodium CMC could be potential for preparation of porous and rapid swelling hydrogels [63]. Polycarbophil is an anionic polymer in with Carboxylic group of it binds hydroxyl group of oligosaccharide of mucus glycoprotein so increment in pH enhances swelling. Lower swelling behavior leads to quite deficient adhesion [64].
3.5.2.4 Ex-vivo Mucoadhesion study
Tablets of batches F3 to F7 showed acceptable mucoadhesive strength (table 3). Highest mucoadhesion was observed in batch F9. Polycarbophil is insoluble in aqueous media but in the neutral pH conditions, it has a high swelling capacity and the volume can be increased to 100 times, allowing high levels of entanglement within the mucus layer. Comprehensive adhesion and the inherent characteristics of polycarbophil, the bioadhesive effect is produced by the carboxylic acid groups binding to the mucosal surfaces via hydrogen bonding interaction. In the non-swollen state, the macromolecules are tightly coiled, so the volume and viscosity are very small. When dispersed in water, the molecules will hydrate and uncoil to some extent, though the molecular chains don't achieve the greatest degree of expansion, the viscosity of the system could be improved to a greater extent. The performance of polymers will be maximized when they are fully uncoiled and extended, which can be accomplished by neutralization or hydrogen bonding. The hydrogen bonding force makes the viscosity increased significantly [65].
3.5.2.5 Surface pH:
Surface pH of tablets of all the batches F1 to F9 was in the range of 7 to 7.4 (table 3) which indicated suitability of R-HCl tablets for vaginal route. Raloxifene approved for prevention and treatment of osteoporosis and prevention of invasive breast cancer, and ospemifene approved for treatment of dyspareunia from menopausal vaginal atrophy [66]. Vaginal pH is 6.0 to 7.5 in case of menopause [67].
3.5.2.6 In vitro dissolution study
Tablets of batch F1 to F5 exhibited desired release profile (Figure 6). In The dissolution medium, no disintegration of the tablets was observed during the test period, a fact that may be due to the persistent gel layer surrounding the tablets. It was reported that upon hydration, a mixture of MCC and sodium CMC as present in formulation generates a rheological system showing thixotropic properties over a pH range of 3.5 to 11.0. This behavior results from the formation of a three-dimensional gel structure leading to immobilization of water molecules inside it and slowing drug release in buffer. The network produced would involve hydrogen bonding between the negative oxygen of the carboxyl group of sodium CMC and hydrogen of the hydroxyl group of MCC. Moreover, the combination of MCC and sodium CMC needs little hydration time in such a medium. A higher proportion of sodium CMC, showed a slower release. The excess free sodium CMC content in formulation may form a gel layer surrounding the tablet and hindering dissolution. During storage, some water from the environment is adsorbed on the tablet surface, causing the start of ionization of exposed carboxylate groups of sodium CMC and some interaction with hydroxyl groups of MCC, and production of a gel film around the matrix with subsequent clogging of the surface pores. Consequently, the retardation of drug release in water could be due to decrease of available pores for water penetration34. Polycarbophil is reported to be used in vaginal suppository base to overcome the shortcoming of stranded short time of the site, which was observed in traditional creams, suppositories and vaginal tablets, and it can also improve the hydration of the vaginal tissue. Wang Chengwei [68] et al developed the nonoxynol vaginal sustained release gel used PCP, Carbopol 971P and glyceryl behenate, and examined the released results in vitro. The consequences showed that the preparation could prolong the contact time, release the effective dose quickly and continue for 24 h of an effective dose, which reduced the drug dose, the toxicity and adverse reactions it caused.
Fig. 6: In vitro drug release profile of study design batches F1 to F9
3.5.2.7 Ex-vivo residence time
The tablet softened inside the vaginal tube after absorbing simulated vaginal fluid and became a swollen structure, helping it to adhere to the vaginal mucosa. Swollen bioadhesive polymers held the solid content of the tablet inside the vagina; at the same time preventing premature leakage. As the concentration of bioadhesive polymer increased, the residence time also increased. This examination reveals the mucoadhesive capacity of polymers used in formulations. Polycarbophil had much more effect on the retention time than sodium CMC and formulation containing higher concentration of polycarbophil showed higher retention time.
Table 3: Results of swelling index, mucoadhesion strength, surface PH and Ex vivo residence time of tablets of batches F1 to F9
Batch No. | Swelling index | Mucoadhesive strength (N) | Surface pH | Ex vivo residence time |
---|---|---|---|---|
F1 | 33.73±0.38% | 0.267±0.28 | 7.0±0.2 | 5.22 |
F2 | 35.23±0.27% | 0.294±0.31 | 7.2±0.3 | 5.38 |
F3 | 37.73±0.25 % | 0.304±0.25 | 7.4±0.4 | 5.45 |
F4 | 34.23±0.61 % | 0.338±0.61 | 7.0±0.1 | 5.55 |
F5 | 36.04±0.13% | 0.343±0.19 | 7.4±0.1 | 6.10 |
F6 | 38.4±0.34 % | 0.362±0.34 | 7.2±0.2 | 6.25 |
F7 | 35.02±0.22% | 0.389±0.64 | 7.0±0.2 | 6.40 |
F8 | 37.17±0.15% | 0.412±0.39 | 7.2±0.1 | 6.55 |
F9 | 40.64±0.41% | 0.43±0.56 | 7.4±0.2 | 7.11 |
3.6 Statistical Data Analysis
3.6.1 Data analysis for Mucoadhesive strength (Y1)
The observed values of response Y1 for different batches showed wide variation i.e., values ranged from a minimum of 0.267 to a maximum 0.43 N. There was not much difference between actual and predicted values. The response (Y1) obtained at three levels of the two independent variables (X1 and X2) were subjected to multiple regression to yield a polynomial equation 4 (full model). The coefficients b1 and b2 were found to be significant having p < 0.05 whereas, the coefficient for b11, b22 and b12 were insignificant having p> 0.05. Reduced model equation was generated by omitting insignificant terms (Equation 5) [69].
Positive sign of coefficient indicate a synergistic effect while negative sign indicate an antagonistic effect on the response. In the present study, coefficients b1 and b2 possessed positive sign which indicated agonistic effect of both variables X1 and X2 on response Y1. Among the two independent variables, X2 (amount of Polycarbophil) has prominent effect (b2 = 0.061 and p = 1.34E-07) on mucoadhesive strength after that to some extent X1 (amount of sodium CMC) affects the results (b1 = 0.017 and p = 0.000229) Hence increasing the concentration of X2 polymer in tablet formulation will mainly influence to achieve desired mucoadhesive strength
3.6.2 Data analysis for % swelling (Y2)
The observed values of response Y2 for different batches showed wide variation i.e., values ranged from a minimum of 33.73 to a maximum 40.64%. There was not much difference between actual and predicted values. Full model equation for the response (Y2) obtained at three levels of the two independent variables (X1 and X2) is given as equation 6. The coefficients b1, b2 and b11 were found to be significant having p < 0.05 whereas, the coefficient for b22 and b12 were insignificant having p> 0.05. Reduced model equation was generated by omitting insignificant terms (Equation 7).
Coefficients b1 and b2 possessed positive sign which indicated agonistic effect of both variables X1 and X2 on response Y2. Among the two independent variables, X1 (amount of sodium CMC) has prominent effect (b1 = 2.299 and p = 4.44E-05) on % swelling after that to some extent X2 (amount of polycarbophil) affects the results (b2 = 1.023 and p = 0.002017).
3.6.3 Data analysis for Q6 (Y3)
Full model equation and reduced model equation for the response (Y3) is given as equation 8 and 9, respectively. The high values of the coefficient of determination indicate a good fit i.e. good agreement between the dependent and independent variables.
Coefficients b1 and b2 possessed negative sign which indicated antagonistic effect of both variables X1 and X2 on response Y3. Among the two independent variables, X2 (amount of polycarbophil) has prominent effect (b2 =22.29 and p = 8.5E-06) on Q6 after that to some extent X1 (amount of sodium CMC) affects the results (b1 = 6.65 and p = 0.002688).
3.7 Contour plots and response surface analysis
Two-dimensional contour plots and three-dimensional response surface plots are very useful to study the interaction effects of the factors on the responses. These types of plots are useful in study of the effects of two factors on the response at one time. Six contour plots and response surface plots were generated using software. Representative figure for only response for mucoadhesive strength are given in Figure 7. Non-linear relationship was observed between two factors (X1 and X2) with all three responses (Mucoadhesive strength, swelling index and Q6).
Fig. 7: (a) Contour plot and (b) 3D surface polo for response mucoadhesive strength
3.8 Optimization of formulation
The optimum formulation was selected based on the criteria of attaining the minimum, target and maximum range of the dependent variables in minitab 17 software. Optimized batch was prepared using amount of X1(sodium CMC) 20 mg and amount of X2(polycarbophil) 15 mg. Desirability of optimized batch was found to be 0.9880.
3.9 Prediction of Release Mechanism
Batches F1 to F9 showed n value between 0.5 to 1, so drug released by non-Fickian transport mechanism (anomalous transport) which is controlled by a combination of diffusion and chain relaxation mechanism and having a correlation coefficient value fitted in zero order drug release profile.
3.10 Ex-vivo permeation study
Result of ex-vivo permeation of the optimized formulation is displayed in figure 8. This result can give idea about systemic absorption of the drug. The vagina stands as an important alternative to the oral route for those systemic drugs that are poorly absorbed orally or are rapidly metabolized by the liver. Drug permeation through the vaginal tissue can be estimated by using in vitro, ex vivo and in vivo models. The latter ones, although more realistic, assume ethical and biological limitations due to animal handling. Therefore, in vitro and ex vivo models have been developed to predict drug absorption through the vagina route [70].
Fig. 8: Result of ex-vivo permeation of the optimized formulation
3.11 Validation of design model
To confirm the validity of design, the optimized batch was prepared; three responses were measured and % relative error was calculated which was found to be less than the 5% which was indicated goodness of fit in model. It was found that both factor had statistically significant influence on all dependent variables as P <
0.05. Thus both reasons were confirm the validity of design.
3.12 Short-term stability study
The formulation retained the pale yellow appearance. No remarkable change was observed in Surface pH, Mucoadhesive strength, Swelling index and Drug release after 6 hr (%). There was small increase in swelling index, which led to slightly lower the drug release after 6hr, but changes were insignificant. Negligible difference was observed in results obtained during optimization and those after stability study. Thus the formulation retained the good stability at accelerated condition of temperature and humidity.
4. Conclusion
Raloxifene hydrochloride is a selective estrogen receptor modulator with very poor oral absolute bioavailability (2%) due to high hepatic first-pass metabolism. In present research, Raloxifene hydrochloride mucoadhesive vaginal tablets were developed for the effective treatment of disease as it provides control drug release and bypasses the hepatic first pass metabolism. Raloxifene hydrochloride was complexed with βcyclodextrin to improve the solubility of drug. Amount of sodium carboxymethyl cellulose and amount of polycarbophil showed agonistic effect on mucoadhesive strength and percentage swelling whereas, antagonistic effect on drug release at 6 hr. Based on experimental results, applied statistics and response surface methodology; 20 mg sodium carboxymethyl cellulose and 15 mg polycarbophil was selected as optimized batch to formulate tablets.
5. References
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