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RESEARCH ARTICLE

Formulation and Evaluation of pH Sensitive Mucoadhesive Microspheres of Fluvastatin Sodium

Senthil Prabhu R *, Mohamed Asraf Ali S, Vijayalakshmi S and Abdul Hasan Sathali A

Department of pharmaceutics, College of pharmacy, Madurai Medical College, Madurai, Tamilnadu

625020, India.

*Corresponding Author E-mail: grsprabhu@yahoo.com

ABSTRACT:

The present work was aimed to develop a pH sensitive mucoadhesive microspheres sustained drug delivery system of fluvastatin sodium, a water soluble and poorly bioavailable drug (24%), unstable at gastric pH. Mucoadhesive microspheres were formulated using polymethacrylate polymer (EL 100 and ES 100) having excellent mucoadhesive property. Microspheres formed were discrete, free flowing and exhibited good mucoadhesive properties. pH sensitive mucoadhesive microspheres were formulated by w/o/o double emulsion solvent diffusion method. Parameters such as particle size analysis, drug entrapment efficiency, scanning electron microscopy (SEM) analysis, in vitro wash-off test, in vitro release studies and drug polymer compatibility studies were investigated. Results showed that the polymer concentration affected size, entrapment efficiency, mucoadhesion property and drug release from the microspheres. The good result was obtained for the formulation F10 (Drug: EL 100 (1:2.5). At this ratio, the mean particle size, entrapment efficiency, swelling index of mucoadhesive microspheres was increased and drug release was sustained when compared to the other formulations. Also this formulation showed highest mucoadhesion time when compared to other formulations. This was confirmed in vitro wash-off test. Fourier Transform-Infra Red Spectroscopy (FT-IR) and Differential Scanning Calorimetric studies (DSC) did not show any drug, polymer interactions. The results of powder X-ray diffraction (PXRD) studies showed the reduced crystal nature of the drug. Conclusively, a pH sensitive mucoadhesive drug delivery system was successfully developed that showed sustained and delayed release up to 12 hours and could be potentially useful to overcome poor bioavailability problems associated with fluvastatin sodium.

KEYWORDS: pH sensitive drug delivery system, w/o/o double emulsion solvent diffusion, Mucoadhesive microspheres, Fluvastatin sodium, Sustained drug release.

INTRODUCTION:

Oral drug administration still remains the favoured route of choice for delivery of drugs into systemic circulation. Some drugs have perfect length of stay throughout the gastro intestinal tract while the others present difficulties due to stability problems in intestinal fluids, rapid absorption in intestinal pH, poor solubility intestine and short elimination half life.

Received on 19.05.2015 Modified on 20.06.2015

Accepted on 30.06.2015 © RJPT All right reserved

Research J. Pharm. and Tech. 8(10): Oct., 2015; Page 1343-1352

DOI: 10.5958/0974-360X.2015.00241.3

So, the mucoadhesive formulations orally would be to achieve a substantial in length of stay of drug in the gastro intestinal tract and increase the bioavailability of drug. Rapid absorption and poor solubility could result in incomplete absorption from the dosage form leading to diminished efficacy of the administered dose.¹

Oral multiunit dosage forms such as microcapsules and microspheres have received much attention as modified/controlled drug delivery systems. However the success of these oral multiunit dosage forms is limited owing to their short residence time at the site of absorption. It will therefore be advantageous to have means for providing an intimate contact of the drug delivery system with the absorbing membranes. This can be achieved by coupling the bioadhesive characteristics to microcapsules and developing bioadhesive microcapsules. Bioadhesive microcapsules have advantageous such as efficient absorption and enhanced bioavailability of drugs owing to high surface to volume ratio, a much more intimate contact to mucous layer and specific targeting of drugs to the absorption site.² These systems that can be potentially minimize the first pass metabolism and consequently enhance the bioavailability.

Microencapsulation, using mucoadhesive polymers is an extensively studied technique that is used to prolong the residence time of dosage form in the gastro intestinal tract and release the loaded drug in controlled manner.³ Most of the microencapsulation methods have been used for lipophilic drugs, since hydrophilic drugs usually showed low loading efficiency.⁴ To entrap the water soluble drugs, several formulation methods were developed such as phase separation, spray drying and solvent evaporation. But these methods are not suited for water soluble drugs because the problems of an aggregation and residual solvent in resulting microspheres and low loading efficiency. The double emulsion solvent diffusion (w/o/o) method best suited for water soluble drugs because it has high loading efficiency.⁵

Fluvastatin sodium is water soluble, fully synthetic cholesterol-lowering agent, is competitive inhibitor of HMGA CoA reductase used as hypercholesterolemia and mixed dyslipidemia, it has short biological half life and undergoes extensive first pass metabolism so that its bioavailability is low as 24%.⁶ Since fluvastatin sodium is not stable in stomach. It dissolves rapidly in intestinal fluid and reaches it maximum blood concentration within 30 minutes. Fluvastatin sodium decreases total cholesterol, LDL cholesterol, triglycerides and apolipoprotein B, while increasing HDL.

Mucoadhesive polymers selected were Eudragit S 100 and Eudragit L 100. Poly methacrylate is biocompatible polymers with low toxicity. It is a long chain, high molecular weight polymers that has property to swell by absorbing water and adheres to the mucosa through strong hydrogen bonding groups (-OH, -COOH).⁷ This is to provide longer contact time for drug transport across the mucosal membrane, before the formulation is cleared by the mucosal surface.^8,9

In the present study, an attempt was made to develop pH sensitive mucoadhesive microspheres by w/o/o double emulsion solvent diffusion method using polymethacrylate polymers in different ratios. The formulated microspheres were evaluated for drug content, entrapment efficiency, mucoadhesive property, X-ray diffraction study, surface morphology, drug polymer interaction and in vitro drug release studies.

MATERIALS AND METHODS:

Materials:

Fluvastatin sodium was obtained as a gift sample from Biocon pharmaceuticals (Bangalore, India). Eudragit S 100 and Eudragit L 100 were supplied by Evonik Pharma polymers (Mumbai, India) and hydrochloric acid procured from Nice Chemicals, Kochi, India. Acetone, ethanol, cyclohexane were procured from Central Drug House, New Delhi. Liquid paraffin, span 80 and tween 80 from Universal Scientific Appliances, India. All other solvents and reagents used were of analytical grade.

Methods:

Preparation of pH sensitive mucoadhesive microspheres

pH sensitive mucoadhesive microspheres were prepared by using water-in-oil-in-oil (w/o/o) double emulsion solvent diffusion method¹⁰ using different ratios of polymers to drug.

Table 1: Formulations of pH sensitive Mucoadhesive Microspheres of Fluvastatin Sodium (F1-F18)

Formula-tion code	Drug: Polymer	Liquid Paraffin (ml)	Drug (gm)	Eudragit S100 (gm)	Eudragit L100 (gm)	Eudragit L100and S100 (gm)	Span 80 (%)	Tween 80 (%)
F1	1:1	200	0.2	0.2	-	-	0.5	0.25
F2	1:1.5	200	0.2	0.3	-	-	0.5	0.25
F3	1:2	200	0.2	0.4	-	-	0.5	0.25
F4	1:2.5	200	0.2	0.5	-	-	0.5	0.25
F5	1:3	200	0.2	0.6	-	-	0.5	0.25
F6	1:4	200	0.2	0.8	-	-	0.5	0.25
F7	1:1	200	0.2	-	0.2	-	0.5	0.25
F8	1:1.5	200	0.2	-	0.3	-	0.5	0.25
F9	1:2	200	0.2	-	0.4	-	0.5	0.25
F10	1:2.5	200	0.2	-	0.5	-	0.5	0.25
F11	1:3	200	0.2	-	0.6	-	0.5	0.25
F12	1:4	200	0.2	-	0.8	-	0.5	0.25
F13	1:1	200	0.2	-	-	0.2	0.5	0.25
F14	1:1.5	200	0.2	-	-	0.3	0.5	0.25
F15	1:2	200	0.2	-	-	0.4	0.5	0.25
F16	1:2.5	200	0.2	-	-	0.5	0.5	0.25
F17	1:3	200	0.2	-	-	0.6	0.5	0.25
F18	1:4	200	0.2	-	-	0.8	0.5	0.25

The polymers Eudragit S100, Eudragit L100 and in combinations were used. Drug and polymer mixture were dissolved in the mixed solvent system consisting of acetone and ethanol in a 1:1 ratio using for all formulations.

Eudragit S 100 and L 100 or both and drug were dissolved in 10 ml of acetone/ethanol mixture, followed by addition of 3 ml of aqueous phase containing 0.25% (v/v) tween 80. The drug could also be dissolved or dispersed in internal water phase and then emulsified in the polymer phase. The initial w/o emulsion was prepared by stirring the mixture for 20 seconds. The w/o emulsion was slowly added into 200ml of liquid paraffin, the second oil phase containing 0.5% (span 80) as a surfactant with stirring at 250rpm and temperature maintained at 25^oC. It was stirred for one hour and the hardened pH sensitive mucoadhesive microspheres collected by filtration. The collected pH sensitive mucoadhesive microspheres were washed with cyclohexane or petroleum ether for 4 to 5 times and dried at room temperature for 24 hours. The composition of various prepared pH sensitive mucoadhesive microspheres formulations are shown in Table 1.

Characterization of pH sensitive Mucoadhesive Microspheres

Fourier Transform-Infra Red (FT-IR) Studies

The possibilities of drug–polymer (ES100and EL100) interactions are further investigated by FT-IR. The FT-IR graph of fluvastatin sodium and combination of drug with polymer (ES 100and EL 100) are recorded. The analysis is performed by using (shimadzu FT-IR, Japan) spectrometer. The scanning range is 4000-400cm^-1 and the resolution is 4cm^-1sample is prepared in KBr pellets.¹¹

Differential Scanning Colorimetric Studies (DSC)

DSC is performed using Q200 V24.4 thermal analyzer. The instrument is calibrated with indium standard. Accurately weighed (it varies from 3mg -25mg) samples are placed in an open type ceramic sample pans. Thermo grams are obtained by heating the sample at a constant heating rate of 8˚C/minute. A dry purge of argon gas (60ml/min) is used for all runs. Samples are heated from 37˚C-9400˚C.¹²

Percentage Yield

The percentage yield of the produced pH sensitive mucoadhesive microspheres is calculated for each formulation by dividing the total weight of product (M) by the total expected weight of drug and polymer.

Weight of microspheres

Percentage yield = ------------------------------------------ x 100

Weight of drug and polymer

Particle size analysis

Particle size of pH sensitive mucoadhesive microspheres is determined by optical microscopy method using calibrated eye piece micrometer.^{13, 14} The microspheres mounted on a glass slide were placed on a mechanical stage. The microscope eye piece was fitted with a calibrated ocular micrometer and the number of microspheres in different size ranges was counted. The data was used for calculation of average particle size of microspheres.

Drug entrapment efficiency

10 mg of microspheres were crushed in a glass mortar and the powdered pH sensitive mucoadhesive microspheres were suspended in 100ml of phosphate buffer solution (pH 6.8) and it is shaking by rotary flask shaker for 24 hours.¹⁵ After 24 hours, the solution filtered, suitable dilutions are made and estimated for fluvastatin sodium content spectophotometrically at 304 nm.

Theoretical drug loading in microspheres is estimated by using the following for

Weight of drug

Theoretical drug loading (%) = ---------------------------------- x 100

Weight of microspheres

The drug entrapment efficiency was calculated using the following formula,

Experimental drug content

Entrapment efficiency (%) = ---------------------------------- x 100

Theoretical drug content

In vitro release studies

In vitro release studies are performed in USP type I basket apparatus for 12 hours.² The microspheres are placed in the dissolution medium of 900 ml of acid buffer pH 1.2 in the dissolution apparatus for first 2 hours and transferred into 900 ml of phosphate buffer pH 6.8 for next 10 hours. The basket is rotated at 50 rpm and temperature maintained at 37⁰C. 5 ml samples are withdrawn every 1 hour for first two hours and every 2 hours up to 12 hours. Samples are analyzed at 304 nm¹⁶ using UV spectrophotometer. The studies are done in triplicate.

Kinetic analysis

The kinetics of drug release is important because it is a useful tool to correlate the in vitro drug release and in vivo drug responses or to compare the results of pharmacokinetics with dissolution profiles of the formulations. Different mathematical models such as zero order, first order, higuchi and Korsemeyer–Peppas equations were applied for describing the kinetics of the drug release process from fluvastatin sodium mucoadhesive microspheres.^{17 18}

Selection of optimized formulation

The optimized microspheres formulation was selected for mucoadhesive character, X-ray diffraction and morphology studies.

Swelling index

An accurately weighed amount of microspheres (50 mg) was suspended in 10 ml of phosphate buffer pH 6.8 and allowed to swell after 12 hours, microspheres were again weighed and the percentage swelling (S) microspheres was calculated by using following equation,

S (%) = (Ws – Wo/Ws) ×100

Where, Wo is weight of microspheres before swelling and Ws are weight of microspheres after swelling.^{2 18}

In vitro Wash-Off test for Mucoadhesion

A freshly cut small intestine tissue obtained from local abattoir within 1 hour of killing of the goat, was cleaned by washing with isotonic saline solution. Jejunum was separated and soaked in receptor medium (phosphate buffer pH 6.8). This tissue represents a significant portion of the overall gastrointestinal tract and is therefore a good representative of the target tissue for orally administrated bioadhesive drug delivery systems. Therefore, for experimentation, a piece of jejunum mucosa (2×3 cm) was mounted onto glass slide (2 ×1) with cyanoacrylates glue. An accurate weight of microspheres (50 mg) was placed on mucosal surface. The glass slides were put in grooves of the USP tablet disintegrating test apparatus and regular up and down movement was given in a beaker containing phosphate buffer pH 6.8. The duration for complete washing of microspheres from goat intestinal mucosa was recorded and averaged from three determinations.²

X-Ray Diffraction Studies:¹⁵

Fluvastatin sodium, fluvastatin sodium loaded Eudragit S 100, Eudragit L 100 and in combination of Eudragit S 100 and Eudragit L 100 are studied X-ray diffractometer (XRD-462, Digaku, Japan). XPRD is carried out in symmetrical reflection using copper line as the source of radiation and the wavelength is set at 1.5405 A^0. Standard runs using a 40 kV and 30 mA in this process. Samples are performed with a scanning rate of 0.1000^o /min and the scanning range of the 2 θ from the initial angle 40⁰ to the final angle 90⁰.¹⁹

Morphology of microsphere by scanning electron microscopy (SEM) technique

Scanning electron microscopy is an excellent tool for physical observation of morphological features of particle both initially and degradation process. It is helpful to examine particle shape and surface characteristics such as surface area and bulk density. The formulations are poured in a circular aluminum stubs using double adhesive tape, and coated with gold in HUS – 5GB vaccum evaporator and observed in Hitachi S – 3000N SEM at an acceleration voltage of 10 Kv and a magnification of 5000X.

RESULTS AND DISCUSSION:

Fourier Transform-Infra Red (FT-IR) Studies

FT-IR spectroscopy was used to investigate the interactions between polymer and drug. The FT-IR spectral analysis of fluvastatin sodium alone showed that principal peaks were observed at wave numbers 3334 cm^-1, 3251.18 cm^-1, 1600 cm^-1, 1535.34 cm^-1, 1384.89 cm^-1 and 1215.15 cm^-1 confirming the purity of the drug.

In the FT-IR spectra of physical mixture of fluvastatin sodium, Eudragit S 100, Eudragit L 100 and their combination were studied. The major peaks of fluvastatin were observed at wave numbers 3334, 3251, 1600, 1535 and 1415 cm^-1. It was confirmed that there are no major shifting as well as any loss of functional peaks between the spectra of drug and the physical mixture. The combination of Eudragit S 100 and Eudragit L 100 peaks also confirmed, there is no interaction between the each polymer because the major functional groups Al-OH stretching 2997.38 cm^-1 is present. FT-IR spectrum of the drug and polymers are shown in the figure 1.

Differential Scanning Calorimetry (DSC)

The DSC thermo grams of pure drug and the different polymers were shown that an endothermic peak corresponding to the melting point of pure drug was important in all the drug polymer mixture, which suggested clearly that there was no interaction between the drug and the polymers and the drug was existed in its unchanged form.²²

(a)FT-IR spectrum of fluvastatin sodium (b) FT-IR spectrum of Eudragit S 100 (c) FT-IR spectrum of Eudragit L 100

(d) FT-IR spectrum of ES 100+EL 100 (e) FT-IR spectrum of Drug+ES 100 (f) FT-IR spectrum of Drug+EL 100

(g) FT-IR spectrum of Drug+ES 100+EL 100.

Preparation of pH sensitive mucoadhesive microspheres

pH sensitive mucoadhesive microspheres were prepared by using water in oil-in-oil (w/o/o) double emulsion solvent diffusion method,^4,5,10,15 using different ratios of mixed polymers to fluvastatin sodium. Table 1 showed the composition of various prepared pH sensitive mucoadhesive microspheres formulations. The fluvastatin sodium and polymer (Eudragit S 100) was in the ratio of 1:1, 1:1.5, 1:2, 1:2.5, 1:3 and 1:4 for F1,F2, F3, F4, F5 and F6 respectively, fluvastatin sodium and polymer (Eudragit L 100) in the ratio of 1:1,1:1.5,1:2, 1:2.5,1:3 and 1:4 for F7, F8, F9, F10, F11 and F12 respectively, fluvastatin sodium and mixer of polymers Eudragit S 100 and Eudragit L 100 in the ratio of 1:1, 1:1.5,1:2, 1:2.5,1:3 and 1:4 for F13, F14, F15, F16, F17 and F18 respectively, followed by emulsification of this primary emulsion (w/o) in to an external oil phase (liquid paraffin containing span 80) to form a water in oil in oil (w/o/o) emulsion.

The preparation pH sensitive mucoadhesive microsphere was carried out by emulsifying an aqueous solution into solution of drug and polymers in mixed solvent system comprising of acetone and ethanol in equal volume for all formulations. The surfactant for span 80 and tween 80 are added to same concentrations of all formulations.

pH sensitive mucoadhesive microspheres were formed after a series of steps like solvent extraction, solvent evaporation and addition of a non-solvent. The solvents system was removed by a combination of extraction and evaporation. It is very important to carefully select the solvent combination and processing medium to enable the formation of double emulsion, solvent extraction and evaporation by a combination.

Acetone is a unique organic solvent which is polar, water miscible and oil immiscible and ethanol is volatile polar and water miscible. So, during the formation of microspheres ethanol was diffused by liquid paraffin containing span80 and acetone was evaporated during stirring.

Each step of microsphere preparation was intensely observed to understand the effect of particle size, total entrapment and release profile of the drug loaded microspheres. After introduction of w/o primary emulsion in to liquid paraffin, the emulsion was stirred for 1 hr using mechanical stirrer (Remi lab stirrer), during this phase it is assumed that the droplet sizes were allowed to stabilize while some amount of ethanol and acetone escaped, making the emulsion droplets become more viscous.

The cyclohexane, non solvent for the polymer added at this stage might have caused the quick precipitation of the polymer leaving the surface of microspheres smooth in nature.²¹ Water insoluble surfactant was used for stabilizing w/o primary emulsion. Span 80 (sorbitan monooleate) was used to stabilize the secondary emulsification process.

Percentage yield

The percentage yield of prepared pH sensitive mucoadhesive microspheres (F1-F18) was shown in Table 2 .Increasing the polymer concentration lead to subsequent increase in its hydrophobicity consequently, it will react better with non solvent phase (liquid paraffin) leading to more efficient precipitation of the polymer at the droplet interface with subsequent higher yield. Increasing polymer ratio in the formulation led to increase the product yield.² The low percent yield in some formulations may also due to microspheres lost during successive decantation during washing process.

Particle size analysis

Formulations F17 (1:3.5), F11 (1:3.5), F7 (1:1) and F6 (1:4) showed relatively larger particle size and formulations F1 (1:1), F2 (1:1.5), F4 (1:2.5), F10 (1:2.5) and F15 (1:3) showed relatively small particle size of pH sensitive mucoadhesive microspheres. When the polymer to drug ratio was increased, the proportion of larger particles was high, because the viscosity of the primary emulsion was increased with increase of polymer to drug ratio. Due to this increased viscosity, large emulsion droplets were formed and it was difficult to break them and, hence, they were precipitated as such leading to an increase in the mean particle size of mucoadhesive microsphere as shown in Table 2.

Entrapment efficiency

Among the different drug polymer ratios investigated, F6 (1:4), F10 (1:2.5) and F13 (1:2) was showed the maximum capacity for drug entrapment efficiency as shown in Table 2. Drug entrapment efficiency was increased with increasing polymer concentration.²³

Encapsulation efficiency of the drug depended on solubility of the drug in the solvents and continuous phase and physicochemical properties of the drug and polymer. As the high molecular weight of the polymer (methacrylate) increased its hydrophobicity increased, leading to better precipitation of the polymer at the boundary phase of the droplets.²¹ Consequently, partitioning of drug to the continuous phase (liquid paraffin) will be minimal.

The higher entrapment of the fluvastatin sodium to the polymer blend (Eudragit S 100, Eudragit L 100 and combination of Eudragit S 100, Eudragit L 100) may be attributed to faster precipitation of polymer at sphere interface at these drug: polymer ratio(1:1,1:2.5 and 1:.2), consequently, higher amount of drug was entrapped.

Table 2: percentage yield, drug content, entrapment efficiency and particle size

Formulation Code	Percentage Yield (%)	Theoretical Drug Loading	Experimental Drug Content	Entrapment Efficiency	Mean Particle Size Range In mm
F1	75.05	15.62	52.00 ± 1.591	89.96±0.533	28.02
F2	91.22	19.62	35.00 ± 1.380	79.75±1.771	44.88
F3	83.86	39.76	39.33 ± 0.212	58.04±1.766	63.96
F4	69.27	20.67	22.52 ± 1.102	81.15±1.501	31.63
F5	86.04	49.01	23.22 ± 1.766	55.72±0.922	68.48
F6	93.74	15.03	15.71 ± 1.088	90.01±1.281	31.63
F7	76.00	39.69	51.55 ± 0.324	59.55±0.612	79.13
F8	95.18	20.72	37.39 ± 1.051	77.05±2.039	78.16
F9	70.00	33.12	23.72 ± 1.944	68.38±1.350	78.49
F10	90.00	24.32	27.19 ± 0.575	91.71±1.356	33.67
F11	52.02	14.41	23.74 ± 0.367	90.80±0.855	82.42
F12	89.54	14.10	11.91 ± 0.122	93.68±1.628	40.13
F13	67.83	39.10	33.88 ± 1.360	57.55±0.612	74.00
F14	80.21	38.13	27.92 ± 1.294	55.53±2.574	77.98
F15	89.06	36.10	29.62 ± 0.118	76.04±2.746	29.67
F16	62.61	34.12	24.95 ± 0.311	52.95±0.608	93.55
F17	81.62	35.28	31.2 ± 0.367	65.18±1.894	82.52
F18	90.02	35.02	11.51 s ± 0.110	65.84±2.692	49.67

The higher drug loading typically results in lower entrapment efficiency due to higher concentration gradients resulting the drug to diffuse out of the polymer/solvent droplets to the external medium. And also the viscosity of the polymer solution at higher drug loading was very high and is responsible for the formation of larger polymer/solvent droplets. It caused a decrease rate of entrapment efficiency of the drug due to slower hardening of larger particles, which tend to decrease entrapment efficiency.^20.

In vitro drug release studies

Fluvastatin sodium is unstable at gastric pH and therefore the microspheres of the drug was capsulated in hard gelatin capsules and studied for release of drug in pH 1.2 for 2 hours followed by release in pH 6.8. Eudragit S 100 and Eudragit L 100 being a pH sensitive polymer, is soluble above pH 7.0, thus pH 6.8 was selected for the study because if the microspheres were able to sustain drug at the pH 6.8, there will be able to sustain the release at lower intestinal pH values as well. Another biorelevent consideration is that transit time of a drug through the absorptive area of the gastrointestinal tract is between 9 to 12 hours this includes 2-3 hours of gastric residence time. The release of fluvastatin sodium from microspheres made with Eudragit S 100 showed cumulative percentage drug release in the range of 74.01%-90.55% and Eudragit L 100 showed drug release in the range of 69.94%-94.16% and Eudragit S 100 and Eudragit L 100 combination polymers showed in the range of 70.13%-90.33%.

The cumulative percentage of pH sensitive mucoadhesive microspheres as shown in figures 2.

Figure 2 (a) Comparison of in vitro drug release profile of fluvastatin sodium loaded Eudragit S 100

Figure 2 (b) Comparison of in vitro drug release profile of fluvastatin sodium loaded Eudragit L 100

Figure 2(c). Comparison of in vitro drug release profile of fluvastatin sodium loaded Eudragit S100andL100

Table 3.Kinetics analysis of pH sensitive mucoadhesive microspheres of fluvastatin sodium

Formula-tion code	Zero order Kinetics		First order Kinetics		Higuchi Model		Korsemeyer-Peppas model		Hixson Crowell
	R^2 value	K0 (mg/h-1)	R^2 value	K1(h-1)	R^2 value	KH(mg/ h-1)	R^2value	n value	R^2 value	KHC(h-1/3)
FI	0.909	8.518	0.991	0.099	0.962	40.01	0.947	0.821	0.981	0.244
F2	0.839	7.108	0.969	0.067	0.918	33.95	0.992	0.510	0.938	0.181
F3	0.892	7.309	0.986	0.067	0.953	34.50	0.941	0.704	0.945	0.182
F4	0.870	7.500	0.965	0.068	0.937	35.54	0.862	0.798	0.940	0.187
F5	0.873	7.500	0.974	0.063	0.941	33.69	0.921	0.682	0.949	0.173
F6	0.937	10.65	0.864	0.059	0.905	32.38	0.816	0.779	0.856	0.162
F7	0.866	8.318	0.989	0.102	0.905	32.38	0.951	0.643	0.972	0.245
F8	0.924	8.158	0.972	0.089	0.970	38.15	0.971	0.778	0.984	0.225
F9	0.916	8.038	0.984	0.086	0.965	37.68	0.946	0.764	0.984	0.220
F10	0.912	8.336	0.991	0.092	0.963	39.13	0.920	0.830	0.984	0.232
F11	0.917	7.909	0.988	0.083	0.966	37.07	0.982	0.719	0.984	0.213
F12	0.822	6.514	0.914	0.054	0.909	31.30	0.893	0.621	0.881	0.151
F13	0.931	8.111	0.986	0.089	0.974	37.90	0.959	0.796	0.991	0.225
F14	0.945	8.085	0.992	0.083	0.979	37.58	0.966	0.885	0.993	0.215
F15	0.904	8.005	0.993	0.083	0.960	37.68	0.942	0.770	0.979	0.215
F16	0.936	7.892	0.988	0.079	0.975	36.78	0.960	0.844	0.988	0.208
F17	0.930	7.825	0.991	0.078	0.972	36.52	0.947	0.821	0.987	0.205
F18	0.937	10.65	0.914	0.054	0.925	31.85	0.879	0.741	0.896	0.154

Kinetic analysis

The release kinetics of all the formulations are followed by first order release mechanism with r² ranging from 0.993 to 0.991.But the ES100 using formulation F6 (1:4) and combinations of ES100 and EL100 polymers using formulation F18 (1:4) are followed by zero order kinetics release mechanism.

The release kinetics of all the formulations is best fitted the Higuchi model and r² values ranges from 0.905 to 0.976. It showed purely diffusion controlled.

The Korsemeyer-peppas release exponent (n) was analyzed to confirm the release mechanism. The release mechanism of all the formulations is best fitted the Korsemeyer release mechanism with n values ranges from 0.510 to 0.885. From this Korsemeyer release mechanism showed non-fickian diffusion. In the present study, the values of n and the coefficients of determination (r²) obtained from the drug release profiles are listed in Table 3.

Selection of best formulations

The best formulations were selected is based on the entrapment efficiency, in vitro drug release and in vitro kinetics analysis studies.

Formulations F1, F10 and F15 were selected on the basis high percentage efficiency (89.96%,91.71% and 76.04%) and these formulations demonstrated delayed and sustained drug release till 12 hours with diffusion controlled release mechanism. These formulations were subjected to mucoadhesive character and X-ray diffraction studies.

Evaluation of best formulations

Mucoadhesive property

The mucoadhesive property of the microspheres was evaluated by swelling studies and in vitro wash off test. All microspheres swelled in phosphate buffer pH 6.8 with a characteristic swelling pattern. Poor initial swelling in the first 4 hours was followed by relatively higher swelling till the end of the period for Eudragit S 100 microspheres. Gradual swelling was observed for Eudragit L 100 loaded microspheres during end of the period of time. Quantitative assessment of swelling reported as percent swelling was found to be in the range of 48.54% for Eudragit S 100 (F1), 69.12% for Eudragit L 100(F10) and 50% for combination polymers of Eudragit S 100 and Eudragit L 100 (F15).

Higher swelling of Eudragit L 100 having greater ability of polymeric chains of uncoil into a extended structure facilitating interpenetration and entanglement and consequently allowing binding groups come together²⁴. The highest drug: polymer ratio F10 (1:2.5) showed highest swelling capacity for the simple reason that water uptake/binding ability of microspheres increases with increase in polymer concentration. The swelling index profiles are shown Table 4.

The time period for in which microspheres adhere to intestinal mucosa was estimated by in vitro wash- off test that is another useful assessment of mucoadhesive character. The microspheres made with Eudragit L 100 (F10) demonstrated higher mucoadhesion time. Whereas F1, F15 showed 1.07 hours and 2.33 hours respectively.

Mucoadhesion of pH sensitive mucoadhesive polymers Eudragit S 100 and Eudragit L 100 is favoured when majority of carboxylic groups are in unionized form that occurs at pH below its pKa. The highest drug : polymer ratio showed highest mucoadhesion time F10 (1:2.5), the amount of polymers are directly affects mucoadhesion time that ensures prolonged residence time at the absorption site to facilitate intimate contact with the absorption surface and thereby improve and enhance the bioavailability.² The mucoadhesion time of fluvastatin sodium pH sensitive mucoadhesive microspheres were shown Table: 4.

Table 4. Mucoadhesion Time and Percent Swelling of Mucoadhesive Microspheres of Fluvastatin Sodium. The values reported are mean ± s.d. (n=3)

Formulation Code	Percentage swelling after 12 hours	Mucoadhesion Time (h)
F1	48.54±0.332	1.07±0.048
F10	69.12±0.387	3.33±0.053
F15	50±0.353	2.33±0.116

X-ray Diffraction study

Drug polymorphism can have a significant impact on pharmaceutical properties such as apparent solubility, dissolution rate, and density. These properties can directly impact on the quality and performance of drug properties, by impacting stability, dissolution, and in some case bioavailability.¹⁹

In order to determine the physical state of the drug whether amorphous or crystalline before and after pH sensitive mucoadhesive microspheres formulation, X-ray examinations were conducted for the pure drug, and the formulations of pH sensitive mucoadhesive microspheres. The X-ray study of pure drug and formulated microspheres are shown in figure 3.

From X-ray patterns it was observed that the pure drug exhibited crystalline characteristics peaks, polymers ES 100 and EL 100 also known its amorphous form and after formulations showed reduced crystalline peaks of pure drug. The fluvastatin sodium made with EL100 is completely reduced to the crystalline form.

The amorphous state of microspheres release the drug less rapidly than the crystalline.²⁵ Therefore, the lack of polymer crystllinity suggests better drug dispersion and increased drug polymer interactions. The drug release rate can be tailored by manipulating the degree of crystallnity; reduced crystallnity is favorable when slow release is desired.

Figure 3 (a). X-ray Diffraction pattern of fluvastatin Sodium pure drug

Figure 3 (b) X-ray Diffraction study of F1 using Eudragit S 100 1:1 ratio

Figure 3 (c) X-ray Diffraction study of F10 using Eudragit L 100 1:2.5 ratio

Figure 3 (d) X-ray Diffraction study of F15 combination Eudragit S 100 and Eudragit L 100 in 1.2.5 ratios.

Morphology studies

The best formulation F 10 are subjected for morphology study, because it has high mucoadhesive property and completely to reduce the crystal nature of drug.

Scanning Electron Microscopy

The SEM photomicrograph of microsphere showed that the microspheres were almost spherical, globular, dense and smooth surface.

Smooth surface was observed on the surface of microspheres shell due to the rapid diffusion of the solvent, the morphology may be attributed to the preference of the polymer to orient itself towards the external oil phase. SEM photographs were shown in Figure 4.

Figure 4 (a)

Figure 4 (b)

Figure 4 (c)

Figure 4 (d)

CONCLUSION:

From these studies it was concluded that pH sensitive mucoadhesive microspheres of fluvastatin sodium loaded polymethacrylate polymers prove to be a successful intestinal mucoadhesive oral delivery of the poor bioavailable drug. Since the drug was incorporated into polymethacrylate and the particle size was reduced to micro, increase contact time of microsphere to the intestinal mucosa and increased surface area. The presence of mucoadhesive polymer results in adhesion of microspheres to the intestinal mucosa resulting in targeted drug delivery. The formulated system showed delayed and sustained release up to 12 hours and the system is potentially useful to overcome poor bioavailability problems associated with fluvastatin sodium.

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