In vitro and In vivo Characterisation of Piroxicam-Loaded Dika Wax Lipospheres

Purpose : To formulate piroxicam-loaded lipospheres and evaluate their in vitro and in vivo properties. Method : Piroxicam-loaded lipospheres were prepared by hot homogenization technique using dika wax and Phospholipon ® 90G (1:1, 1:2 and 2:1) as the lipid matrix. Characterisation, based on particle size and morphology, pH, drug content and encapsulation efficiency, were carried out on the lipospheres. In vitro release was evaluated in simulated intestinal fluid (pH 7.5). Anti-inflammatory and ulcerogenic properties of the piroxicam-loaded lipospheres were studied using healthy, adult Wistar rats. Result: Photomicrographs revealed spherical particles in the range of 1.66 – 3.56 µm. The results also indicated that lipospheres formulated with lipid matrix 1:1 and containing 0.25 % piroxicam had the highest encapsulation efficiency of 84 %. In vitro release data showed that lipospheres formulated with lipid matrix having higher concentration of dika wax exhibited the fastest drug release of drug with maximum release time between 60 - 70 min. The lipospheres exhibited good anti-inflammatory properties with 58.6 % oedema inhibition at 5 h. Piroxicam-loaded liposheres had an ulcer index of zero while, the reference (plain piroxicam) had an ulcer index of 15.00 ± 1.23 (p < 0.05). Conclusion: Piroxicam lipospheres formulated with a mixture of dika wax and phospholipid exhibited good in vitro and in vivo properties.


INTRODUCTION
The rapid growth in the use of lipid-based drug delivery systems is primarily due to the diversity and versatility of pharmaceutical grade lipid excipients and their compatibility with liquid, semi-solid and solid drugs [1].The increasing availability of lipidic excipients with specific characteristics offer flexibility of application with respect to improving the bioavailability of poorly water soluble drugs and manipulating their release profile [2].
Dika wax is an edible vegetable fat derived from the kernel of Irvingia gabonensis Var excelcia [3,4].It is a completely biodegradable physiological lipid, and like other lipid excipients, it has a GRAS (generally regarded as safe) status.Therefore, the danger of use of synthetic polymer matrix forming materials which may be associated with detrimental effects on incorporated drug during manufacturing of formulations or during the erosion of the polymers after application is completely avoided [5].
Lipospheres are restricted to the stabilizing material of a phospholipid layer [6].The combination of a solid inner core and a phospholipid exterior confers several advantages on the lipospheres compared with conventional microspheres, such as high dispersibility in an aqueous medium, and a release rate for the entrapped substance that is controlled by phospholipid coating and carrier [6,7].
Piroxicam is a non-steroidal anti-inflammatory drug (NSAID) with pronounced antiinflammatory, analgesic and anti-pyretic effect.The study was aimed at evaluating piroxicamloaded lipospheres formulated with dika wax and phospholipid (Phospholipon ® 90G) both in vitro and in vivo in order to improve the oral bioavailability of piroxicam and reduce its ulcerative properties.

EXPERIMENTAL Materials
The following materials were used as procured from their suppliers without further purification: piroxicam (Pfizer, Nigeria), n-hexane, ethyl acetate (Sigma-Aldrich, Germany), hydrochloric acid, sodium hydroxide, monobasic potassium phosphate and Tween 80 (Merck, Germany), purified lecithin (Phospholipon ® 90G, Phospholipid GmbH, Köln, Germany), activated charcoal (Bio-Lab, London), and sorbitol (Across Organics, Germany).Dika fat was extracted from Irvingia gabonensis seed in our laboratory.All other reagents and solvents were of analytical grade.Dried seeds of Irvingia gabonensis was milled using a hammer mill.The fat was extracted in a Soxhlet apparatus using n-hexane [8].The solvent was allowed to evaporate at room temperature and boiled distilled water, which was about twice the volume of the latter, was poured into the molten fat in order to dissolve the hydrophilic gum in the fat.The dissolved gum was removed with the aid of a separating funnel.Next, ethyl acetate was similarly used to remove the hydrophobic gum in the fat.The extracted fat was further purified by passing it through a column of activated charcoal and bentonite (2:1) at 100 o C at a ratio of 10 g fat to 1 g column material.The fat was stored in a refrigerator until used [2].

Preparation of lipid matrix
Mixtures (1:1, 1:2 and 2:1 w/w) of Phospholipon ® 90G, a purified lecithin, and dika wax were melted in a beaker and stirred at a temperature of 70 o C using a magnetic stirrer, until a homogenous, transparent yellow liquid was obtained.The homogenous mixture was stirred at room temperature until congealed to ensure adequate mixing [9].

Preparation of lipospheres
Piroxicam-loaded lipospheres were prepared using lipid matrix by hot homogenization technique using Ultra-Turrax ® (T25 Basic Digital, Germany).Briefly, 5 g of the lipid matrix was melted at 70 o C in a crucible and the appropriate amount of piroxicam (0.25, 0.5 or 0.75 g) was incorporated into the lipid melt.Sorbitol (4 g) was dissolved in hot distilled water at the same temperature, mixed with Tween 80 (2.5 ml) and poured at 70 o C into the molten lipid phase under high shear homogenization of 5000 rpm for 5 min.An o/w emulsion was formed by phase inversion and allowed to cool at room temperature to generate the solid lipid microparticles (SLMs) [10].The lipospheres (in emulsion form) were then stored in an amber coloured bottle until used.

Determination of liposphere particle size and morphology
A small quantity of the lipospheres were placed on a microscope slide, the slide was covered with a cover slip and imaged under a Hund ® binocular microscope (Weltzlar, Germany), fitted with a motic image analyzer (Moticam, China) at a magnification of x 400.Different portions of the SLMs from each batch were sampled and counted (n = 100) with the image analyzer, and the mean value computed.

Determination of drug content
Beer's plot was obtained at the concentration range of 0.2 -1.0 mg % for piroxicam in simulated intestinal fluid (SIF, pH 7.5).For each batch, 10 ml of SLMs was centrifuged (Chem.Lab.Instrument, UK) for 30 min at 1500 rpm.
The sediment was used in the analysis of drug content.A sample of 0.5 g of SLMs was triturated in a mortar and pestle with 10 ml of SIF (pH 7.5), transferred to a 100 ml volumetric flask and made up to volume with SIF.The solution was filtered through a filter paper (Whatman no. 1) and analyzed spectrophotometrically at a wavelength of 370 nm (Jenway 6305, UK).Drug concentrations were calculated with reference to the Beer's plot.

Drug encapsulation efficiency
Encapsulation efficiency (EE) was calculated as in Eq 1.
where ADC is the actual drug content and TDC is the theoretical drug content.

pH analysis
The pH of the SLMs were determined over a period of time (24 h, 1 week, 2 months and 3 months) using a pH meter (Suntex TS -2, Taiwan).

Evaluation of drug release studies
The USP paddle method was adopted in this study.The dissolution medium consisted of 900 ml of freshly prepared simulated intestinal fluid (SIF, pH 7.5) maintained at 37 ± 1 o C. The polycarbonate dialysis membrane (MW 5000, Spectrum Labs, Brenda, Netherlands) selected was pretreated by soaking it in the dissolution medium for 24 h prior to use.A quantity of SLM equivalent to 0.020 g piroxicam was taken from each batch, placed in a polycarbonate dialysis membrane containing 2 ml of the dissolution medium, securely tied with a thermo-resistant thread and placed in the chamber of the release apparatus.The paddle was rotated at 100 rpm, and at predetermined time intervals, 5 ml aliquots of the dissolution medium was withdrawn, appropriately diluted, and analyzed for drug content spectrophotometrically.Sink conditions were maintained by replenishing the medium with 5 ml of fresh dissolution fluid after each withdrawal.The amount of drug released at each time interval was determined with reference to Beer's Plot.

Anti-inflammatory studies
The anti-inflammatory activity of the piroxicamloaded lipospheres was evaluated using the rat paw oedema test [11].All animal experimental protocols were carried out in accordance with the guidelines of the Animal Ethics Committee of the Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka and in compliance with the Federation of European Laboratory Animal Science Association and the European Community Council Directive of November, 1986 (86/609/EEC).The phlogistic agent employed in the study was fresh undiluted egg albumin.Adult Wistar rats of either sex (150 -200 g) were divided into five groups.The animals were fasted and deprived of water for 12 h prior to the experiment.Lipospheres equivalent to piroxicam 10 mg/kg body weight was administered orally to the rats.The reference group received 10 mg/kg of pure sample of piroxicam, while the control group received normal saline 5 ml/kg.Thirty minutes post-treatment, oedema was induced by injection of 0.1 ml fresh undiluted egg albumin into the sub plantar region of the right hind paw of the rats.The volume of distilled water displaced by treated right hind paw of the rats were measured using plethysmometer before egg albumin administration and at 30 min, 1, 2, 3, 4, 5 and 6 h after egg albumin injection.Inhibition of oedema was calculated using Eq 2.
where Vt is the volume of oedema at any particular time and Vo is the volume of oedema in control rats at the same time [12,13].

Ulcerogenicity of lipospheres
The ulcerogenicity of piroxicam-loaded lipospheres was determined using a method described by Chung-Chin et al [14].The studies were carried out on healthy Wistar rats (180 -220 g).The animals were divided into four groups (n = 5).The control group received normal saline, the test group received SLMs equivalent to 10 mg/kg piroxicam (A1 and B1), while the reference group received 10 mg/kg pure sample of piroxicam orally.Prior to the test, the animals were fasted for 8 h but allowed free access to food and water, and sacrificed 8 h after administration of treatment.The gastric mucosa of each rat was examined under a microscope fitted with a 4 x binocular magnifier, and the number of lesions counted.The mean score of each treated group minus the mean score of the control group was taken as severity index of gastric damage.

Statistical analysis
Statistical analysis of the data was carried out using SPSS, version 14.0 (SPSS Inc.Chicago, IL.USA).All values are expressed as mean ± standard deviation (SD).Data were analysed by one-way ANOVA.Differences between means were assessed by a two-tailed Student's t-test.Differences were considered statistically significant at p < 0.05.

pH of lipospheres
Table 1 shows pH values of the lipospheres over a period of 1 month.pH varied from between 6.11 ± 0.19 and 6.57 ± 0.11 within 24 h of preparation to between 5.58 ± 0.10 and 6.38 ± 0.15 over the next 1 month.

Encapsulation efficiency (EE)
As indicated in Table 1, EE of the lipospheres ranged from 24 % for batch C3 to 84 % for batch A1.Piroxicam-loaded lipospheres containing 0.25 % of piroxicam had the highest encapsulation efficiency of 76 % for batch B1 lipospheres and 84 % for batch A1 lipospheres.

Drug release from piroxicam-loaded lipospheres
The drug release profile presented in Fig. 2 show that all the piroxicam-loaded lipospheres exhibited good drug release.The lipospheres formulated with higher content of phospholipid (2:1) in the lipid matrix, i.e., batches C1 to C3 greater prolonged drug release behaviour with maximum drug release at 120 min.However, piroxicam-loaded lipospheres formulated with LM containing phospholipid: dika wax in 1:1 showed maximum drug release ranging from 70 min for batches A1 and A3 to 80 min for batch A2.

Anti-inflammatory properties
The anti-inflammatory results, presented in Table 2, show that piroxicam-loaded lipospheres exhibited good anti-inflammatory activity that was significantly higher than that of the control group (p < 0.05).The oedema inhibition ranged from 35.3 to 53.3 % for piroxicam-loaded lipospheres (A1) while the reference drug showed inhibition of 25 to 47 %.

Ulcerogenic properties
The results of ulcerogenic studies are presented in Table 3. Piroxicam lipospheres did not show ulcer-inducing potentials.However, the reference drug (plain piroxicam) showed a high ulcer index of 15.00 ± 1.23 which was significantly higher than that of the piroxicam-loaded liposheres and negative control (p < 0.05).

DISCUSSION
Increase in the amount of piroxicam encapsulated increased also the particle size of the lipospheres.This is in agreement with the findings of Barakat et al [15] who evaluated carbamazepine-loaded precifac lipospheres.
Joseph et al [16], who studied piroxicam-loaded polycarbonate microspheres made a similar observation.The formulations also exhibited stable pH from 24 h to 1 month.Therefore, there was no degradation of the API and excipients.Generally, encapsulation efficiency (EE) was affected by the lipid composition/ratio used in formulating the lipospheres.The reason may be the presence of small amounts of fat in the inner core of the lipospheres which can lead to saturation of the fat core of the lipospheres by the drug incorporated in the dispersion.EE of piroxicam-loaded lipospheres was also inversely affected by the total amount of drug in the dispersion.
The SLMs had reproducible drug release in all the batches, without any evidence of 'burst effect'.However, the rate of drug release was affected by the ratio/combination of the lipid used in preparation of the lipid matrix.Lipid matrices having higher amount of dika wax showed faster release of drug than lipospheres with higher phospholipid concentration.This may be due to diffusion of the drug from the inner core of the lipospheres through the phospholipid coating to the surface of the lipospheres.
The piroxicam-loaded lipospheres showed higher inhibition of oedema than the reference (plain piroxicam).This may be due to increased absorption of drug in vivo in the presence of the lipid matrix carrier.Drugs suspended in lipid matrix have been shown, in most cases, to be better absorbed than those incorporated in conventional solid dosage forms [1].This could be due to the ease of wetting of hydrophobic drug particles in the presence of lipid matrix [2].
The presence of surfactant in the formulation may further promote drug release [2].
Piroxicam-loaded lipospheres drastically reduced the ulcerogenic potentials of piroxicam.This may be due to the presence of the phospholipid in the formulations which might have helped to shield or protect the gastric mucosa from irritation by the drug which is a non-steroidal antiinflammatory drug (NSAID).Our finding is in agreement with that of Lichtenberger et al [17], who proposed that pre-associating NSAIDs with zwitterionic phospholipids prior to their administration should reduce the ability of the NSAIDs to associate the phospholipids in the mucus gel, and should thus reduce their ulcerogenicity.

Dika wax and Phospholipon
® 90G presented good matrix for the formulation of piroxicamloaded lipospheres.In vitro and in vivo studies undertaken with the lipospheres provided positive results, thus indicating the need for further development and optimization of the formulation.The lipospheres exhibited good antiinflammatory properties as well as minimal ulcerogenic potentials.
Piroxicam-loaded lipospheres based on dika wax matrices have advantages over the commercial formulations of piroxicam, include low excipient and processing costs.

Table 1 :
Some physicochemical properties of piroxicam-