Studies of in vitro Evaluation and Formulation of Aceclofenac Loaded PLGA Microspheres
The aim of this research was to study the influence of formulation parameters in the preparation of sustained release aceclofenac loaded PLGA microspheres by emulsion solvent diffusion technique. The methods used in this components and their concentration necessary for organogels formation were evaluated using phase diagram Solubility of aceclofenac was determined, Characterization of Poly (DL-lactide)-co-glycolide (PLGA) polymer, solubility assessment of aceclofenac, drug-excipients compatibility studies, in vitro analytical method development , preparation of aceclofenac-loaded PLGA microspheres, characterization of the formulations. Prepared microspheres were optimized and evaluated for different parameters and best formulation was subjected to in vitro drug release studies. The prepared microspheres were white, free-flowing and almost spherical in shape. In vitro drug release studies were carried out up to 24 h in three different pH media, i.e., 0.1 N HCl (pH 1.2), phosphate buffer (pH 6.8) and phosphate buffer (pH 7.4). The drug-polymer concentration of dispersed phase influences the particle size and drug release properties. In nut shell it may be concluded that sustained release aceclofenac microspheres can be successfully prepared and used parenterally with increased therapeutic value and reduced side effects.
Aceclofenac, 2-[(2,6-dichlorophenyl) amino] phenylacetoxyacetic acid, a novel
anti-inflammatory drug of choice in the treatment of osteoarthritis, rheumatoid
arthritis and ankylosing spondylitis. Researchers have attempted development
of oral drug delivery systems for aceclofenac (Yong et
al., 2005). The chronic oral administration of aceclofenac tends to
cause severe gastric irritation (Tessari et al.,
1995). Topical administration of aceclofenac offers the advantage of enhanced
drug delivery to the affected areas, by passing gastric irritation. The clinically
efficiency have evaluated of topical aceclofenac cream (1.5% w/w) in patients
(Yang et al., 2002). The formulation showed improved
therapeutic efficacy. In the formulated micro-emulsion containing aceclofenac
(3%w/w) for topical delivery (Yamazaki et al., 1997).
This action is supposed to mediate via its intracellular conversion to active
metabolites namely diclofenac, the cyclooxygenase 1 and 2 inhibitor and 4-hydroxydiclofenac,
the cyclooxygenase-2 inhibitor (Akimoto et al., 2000).
It also stimulates glycosaminoglycan synthesis in human osteoarthritic cartilage
by inhibition of interleukin-1beta (IL-1β) and suppresses cartilage degeneration
by inhibiting IL-1β mediated promatrix metalloproteinase production and
proteoglycan release (Gowda et al., 2006). Aceclofenac
is especially well-tolerated among the non-steroidal anti-inflammatory drugs
and have lower incidence of gastrointestinal (gi) adverse effects (Raber
et al., 2007). It also have lower incidence of gi bleeding, abdominal
pain and arterial hypertension than meloxicam and a lower incidence of gi bleeding,
abdominal pain, liver toxicity, thromboembolic cardiovascular events, arterial
hypertension and edema than rofecoxib (Brogden and Wiseman,
Aceclofenac is indicated and widely used for the treatment of rheumatoid arthritis,
osteoarthritis, ankylosing spondylitis and scapulohumeral periarthritis (El-Kousy,
1999; Najib et al., 2004). The usual dose
of aceclofenac is 100 mg twice daily (Kim et al.,
2001) and it is absorbed rapidly when taken orally and its analgesic effects
begin within 30 min of administration (Dashora et al.,
2006). Following oral dose, it reaches a peak plasma concentration within
1-3 h (Patil and Kasture, 2007). Since, aceclofenac
is indicated for various pains and inflammatory conditions, its accelerated
onset of action and sustained release along with decreased incidence of dose
related toxicity is desirable. Keeping in mind the above requirements we prepared
aceclofenac loaded Poly (DL-lactide)-co-glycolide (PLGA) microspheres, characterized
and evaluated for drug release pattern. PLGA is biodegradable and biocompatible
polymer and widely used in controlled release drug delivery systems. Usual therapeutic
dose is 100 mg twice daily and half life is 3-4 h; thus it is necessary to be
administered frequently in order to maintain the desired concentration. Therefore,
aceclofenac is an ideal candidate for sustained release formulation, resulting
in more reproducible drug absorption and reducing the risk of local irritations
compared to single dosage forms.
The solvent evaporation method is popularly used for microsphere preparation
because of its simplicity, reproducibility and fast processing with minimum
controllable process variables that can be easily implemented at the industrial
level (Kim et al., 2002; Dashora
et al., 2006). But it is frequently used for water-insoluble drugs,
as the entrapment efficiency of water-soluble drugs is low due to drug loss
from the organic emulsified polymeric phase before solidification of polymer
in the microspheres (Niwa et al., 1993, 1994).
The objective of this study was to encapsulate aceclofenac with biodegradable.
Polymer rosin, the effect of different formulation variables such as concentration of drug, Polymer, polyvinyl alcohol and solvent, the effect of these variables on particle size distribution, encapsulation efficiency and its in vitro release behavior. The drug was targeted to the colon and their aligned area for their local effect.
MATERIALS AND METHODS
Chemicals: Aceclofenac and Poly (DL-lactide)-co-glycolide (PLGA) were provided (2009) by Venus Medicine Research Center (VMRC), Baddi, H.P., India. Acetonitrile was HPLC grade and all other chemicals used were of analytical grade.
Characterization of Poly (DL-lactide)-co-glycolide (PLGA) polymer PLGA polymer was characterized by gel permeation chromatography for its molecular weight and molecular weight distribution with mixed bed column (SDV 10 μ, 8X300 μ, PSS polymer standard service GmbH) using chloroform (Injection volume: 100 μL) as mobile phase at 1 mL min-1 flow rate for 15 min and 30°C using Refractive index detector and GPC for Class-Vp software as analyser.
Solubility assessment of aceclofenac: The solubility of the drug was assessed in Ethyl acetate, Dichloromethane and Benzyl alcohol by serial addition of the known amount of drug into these solvents and observing visually for complete solubilisation.
Drug-excipients compatibility studies: Excipients are integral components of almost all pharmaceutical dosage forms thus it is mandatory to detect any possible physical or chemical interaction of the drug with the excipients since the excipient can affect the bioavailability and stability of the drug. The drug and the excipients must be compatible with one another to produce a product that is stable, efficacious, attractive, easy to administer and safe. If the excipients are new and have not been used in formulations containing the active substance, the compatibility studies have a considerable importance.
In vitro analytical method development: Different concentrations of aceclofenac were prepared from drug stock solution (50 μg mL-1) prepared in phosphate buffer pH 7.4 and analytical parameters were determined using Reverse Phase High-performance liquid chromatography (RP-HPLC, Waters) system consisting of a photodiode array detector. The column was a reverse phase (Inertsil ODS C18, 250x4.6 mm, 5 μm). The mobile phase was acetonitrile/10 mM phosphate buffer (pH 7.0) (40:60, v/v) and the flow rate was 1 mL/1 min. The temperature of the system was maintained at room temperature and the detection wavelength was 274 nm.
Preparation of Aceclofenac-loaded PLGA Microspheres PLGA was first dissolved in ethyl acetate (2 mL) and aceclofenac was further dissolved in polymer solution. This polymer-drug solution was then added to Poly Vinyl Acetate (PVA) surfactant solution under continuous stirring at different speeds to obtain a primary emulsion in aqueous phase. Ethyl acetate was then allowed to evaporate. The solid microspheres were washed thoroughly using distilled water, passed through sieve (BSS # 120), freeze dried and evaluated.
Characterization of the formulations
Drug content in microspheres: Microspheres (5 mg) were dissolved in 5 mL
of Dichloromethane and then evaporated under stream of nitrogen. To this 10
mL acetonitrile was added. One milliliter of this solution was then diluted
to 3 mL and filtered through 0.22 μ filter paper. The amount of drug in
the test sample was determined by RP-HPLC method.
The drug loading capacity and encapsulation efficiency of the microspheres were calculated according to the following equations:
Particle size measurements: Particle sizes of microspheres were determined using scanning optical microscopy technique.
Drug content analysis: Ten milligram accurately weighed portion of microspheres were taken in a clean 100 mL volumetric flask and dissolved in about 2 mL of acetone and the volume was made up to the mark with buffer pH 7.4. After filtration and dilution, samples were analyzed spectrophotometrically and the amounts of drug encapsulated in the microspheres were calculated. The drug content of each sample was determined in triplicate and the results were averaged. The entrapment efficiency of microspheres was calculated by dividing the actual drug content to the theoretical drug content of microspheres.
Redispersability: Prepared sodium carboxymethyl cellulose (0.5% w/v) in buffer pH 7.0. To 20 mL of sodium CMC solution SPAN 80 solution (10 mL, 1% in buffer pH 7.0) and NaCl (170 mg) was added and volume was made 100 mL in buffer pH 7.4. 300 mg of microspheres were taken and to this 1 mL of dispensing fluid was added. The microspheres were hand shaken and a small amount of sample was taken on a slide and observed under microscope.
Injectability: Microspheres were redispersed in the dispensing fluid (30% w/v) and passed through 22 G needle to determine the injectability behaviour of the microspheres.
In vitro drug release studies: In vitro release of STP
from microspheres was evaluated in phosphate buffer (pH 7.4). Amount of microspheres
equivalent to 20 mg of STP were transferred to the prewarmed dissolution media
(20 mL) and maintained at 37±0.5°C under stirring at 50 rpm. Samples
were withdrawn every hour up to 6 hours and the volume was replaced immediately
by fresh phosphate buffer. The sample withdrawn was centrifuged (3000 rpm, 15
min). The supernatant solution was filtered and analyzed for STP content by
measuring absorbance in a UV-spectrophotometer (Shimadzu UV-1700, Pharmaspec)
at 229.5 nm by the first-derivative spectrophotometric method (Saudagar
et al., 2007) using phosphate buffer (pH 7.4) as blank. Results were
expressed as mean±SD of 3 experiments. Statistical models are extensively
used in diversified areas to strengthen the art of drug formulation (Dubey
and Parikh, 2004; Govender et al., 2005).
Characterization of poly (DL-lactide)-co-glycolide (PLGA) polymer: Unimodal
and symmetrical GPC trace was obtained (Fig. 1) thus confirming
the purity of the polymer. The polymer PLGA was having weight average molecular
weight of 130 kDa, number average molecular of 76 kDa and polydispersity of
1.7 which was sufficient enough for current application. Optimization of drug:
Polymer concentration, PVA concentration and speed are shown in Table
1. Solubility studies of aceclofenac revealed soloubility of aceclofenac
in following order Benzyl alcohol>Ethyl acetate> Dichloromethane and are
shown in Table 2. In vitro analytical method development
by validation parameters for calibration over a range of 1 to 50 mg mL-1
is summarized. The rapid diffusion of acetone into the aqueous phase causes
a remarkable decrease in interfacial tension between the organic and aqueous
phase and hence finer microspheres are obtained (Niwa et
At least one stabilizer is necessary for microsphere formation and suspension
stabilization. Tween 20, Tween 40 and Tween 80 were used as surfactants to prepare
blank microspheres; however, using Tween 20 and Tween 40, microsphere formation
was not achieved successfully independent of their concentration. Microspheres
with superior topographical characteristics were obtained when Tween 80 was
used as the emulsifier (Fig. 2). This might be due to better
emulsification capability of Tween 80 as compared with Tween 20 or Tween 40
(Kibbe, 2000) The pH of Tween 80 aqueous solution is in
the range of 6.0 to 6.5 and hence did not warrant pH adjustment (Salamone
and Wodzinski, 1997).
||Representative gel permeation chromatogram of PLGA (75: 25)
|| Optimization of drug: polymer concentration, PVA concentration
|| Solubility of aceclofenac in different solvents
||Scanning electron micrograph (SEM) of cellulose acetate microspheres
||Particle size distribution of drug loaded microspheres
|| In vitro release of acelofenac from microsphere
In vitro studies showed in this batch of Aceclofenac Loaded PLGA Microsphere
showed cumulative percentage release of 10 to 65% respectively for 40 h (Fig.
Evaluation of formulation variables: As mentioned previously, the effect of different formulation variables on microspheres properties including entrapment efficiency and particle size distribution were evaluated. Variables studied in this investigation were as follows. Drug polymer ratio, volume of dichloromethane and polyvinyl alcohol concentration in the external phase of the emulsion.
Particle size measurements: All the particles of the selected batch V15 were spherical in shape (Fig. 2) and the particle size was in the range of 20- 110 μm (Fig. 3), of which 53% particles were in the range of 30-60 μm and 75% of the particles were in the range of 20-70 μm which is very much suitable for intramuscular parenteral drug delivery system.
Redispersability: The microspheres were found to be uniformly dispersed in the dispensing fluid. Further they were present as single particles when observed under microscope. It was also observed that particles were suspended in the fluid for more than 3 min which is very much sufficient for injection.
Injectability: The suspension of the microsphere was easily passed through 22 G needle therefore no problem will arise due to injectability properties of the prepared formulations.
This research study indicates that aceclofenac loaded PLGA microspheres can
be successfully formulated for parenteral drug delivery of the drug. All the
preparation having ratio 1:1 showed good release properties. As the amount of
polymer increased, drug release was decreased. This is because smaller the particle
size, larger the surface area available for drug release. This may be attributed
to the film forming ability of the lipospheres pellet which reduces transepidermal
water loss similar to solid lipid nanoparticles (Mei et
al., 2003). The major finding of this study can be concluded as aceclofenac
loaded PLGA microspheres with mean size of about 54 μm can be prepared
using solvent-evaporation technique with PVA as stabilizer and ethyl acetate
as solvent. Microspheres belong to the above category and are utilized for controlled
drug delivery. Microspheres were prepared by the emulsion solvent evaporation
method and evaluated. Aceclofenac is a novel anti-inflammatory drug with multidisciplinary
therapeutic activity in pain and inflammatory conditions. The loading capacity
increases with increase in initial drug polymer ratio. The formulated microspheres
can be used for IM delivery of the drug with sustained effect. Dose of the drug
is reduced with consequently decrease in dose related toxicity of the drug.
Authors are thankful to my department of Pharmaceutical Sciences, Jaipur National University, Jaipur and CGM, Domestic operations of Venus Remedies Limited for providing the samples of drug, polymer and facility for completion of this research.
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