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Piroxicam-loaded dika wax lipospheres: In vitro and in vivo characterisation1

Brown, S.A, , 2Attama A.A. , 3Chime S.A. 3Agu C.I. and 3Onunkwo G.C.*, 11

Department of Pharmaceutics and pharmaceutical Microbiology, University of Nigeria, Port Harcourt, Nigeria

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Drug Delivery Research Unit, Department of Pharmaceutics, University of Nigeria, Nsukka, Nigeria

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Department of Pharmaceutical Technology and Industrial Pharmacy, University of Nigeria, Nsukka 410001, Nigeria *Corresponding author Tel.: + 23408037408410 E-mail address:ogo4justice@yahoo.com

Abstract The aim of the study was to formulate piroxicam-loaded lipospheres and to evaluate the in vitro and in vivo properties of the lipospheres. Piroxicam-loaded lipospheres were prepared by hot homogenization technique using dika wax and Phospholipon 90G (1:1, 1:2 and 2:1 %w/w) as the lipid matrix. Characterisation based on particle size, particle morphology, pH, drug content and encapsulation efficiency were carried out on the lipospheres. In vitro release was carried out in simulated intestinal fluid without enzymes (pH 7.5) using the USP paddle method. Antiinflammatory and ulcerogenic properties of piroxicam-loaded lipospheres were studied using healthy, adult Wistar rats. Photomicrographs revealed spherical particles within a micro meter range between 1.66 3.56 m. The results also indicated that liposheres formulated with lipid matrix 1:1 and containing 0.25 % piroxicam had the highest encapsulation of 84 % and was significantly different from the other batches of piroxicam lipospheres (p < 0.05). The in vitro release showed that lipospheres formulated with lipid matrix having higher concentration of dika wax exhibited the fastest release of drug with maximum drug release rate between 60 - 70 min. However, piroxicam-loaded lipospheres formulated with LM having equal ratios of phospholipid to dika wax (LM 1:1) showed reproducible drug release with T100 that ranged from 70 min to 80 min. The anti-inflammatory studies showed that the piroxicam-loaded lipospheres exhibited substantial anti-inflammatory effect. The results of ulcerogenic studies revealed that the issue of gastric irritation in formulations containing piroxicam could be adequately addressed using lipid drug delivery systems such as of lipospheres. Piroxicam-loaded lipospheres had good gastroprotective potentials Key words: Dika wax; Lipospheres; Piroxicam; Phospholipid; Ulcerogenicity

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1. 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 dosage forms (Attama et al., 2009). The widening 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 (Attama et al., 2005). Lipid based formulations have been shown to enhance the bioavailability drugs administered orally (Hou et al., 2005; Sarkar, 2002; Gao et al., 2004; You et al., 2005). The proven safety (biocompatibility) of lipid based carriers makes them attractive candidates for the formulation of pharmaceuticals. Lipid formulations generally provide increased drug solubilization for water in-soluble drugs. Drug suspended in lipid matrix has been shown, in most cases to have a better absorption than conventional solid dosage forms. This could be due to the ease of wetting of hydrophobic drug particles in the presence of lipid matrix. The presence of surfactant in the formulation may further promote the wetting (Joshi, et al., 2008). Furthermore, lipids are non adhesive and therefore, do not adhere to intestinal walls unlike most polymers and so present a good matrix for the formulation of NSAIDS. Dika wax is an edible vegetable fat derived from the kernel of Irvingia gabonensis Var excelcia (Ofoefule et al., 1997). Dika wax is completely biodegradable physiological lipids and like other lipid excipients, they have GRAS status. Therefore, the danger of use of synthetic polymer matrix materials which often goes along with detrimental effects on incorporated drug during manufacturing of formulations or during the erosion of the polymers after application are completely avoided (Reithmeir et al., 2001). Dika wax has been evaluated as basis for drug delivery (Chukwu et al., 1991; Okorie, 2000). An array of lipid systems such as emulsions, micellar solutions, liposomes, lipid nanoparticles, structured lipid carriers, self-emulsifying lipid formulations, solid dispersions, dry emulsions, solid-liquid compacts, and drug lipid conjugates is available to drug formulators (Attama et al., 2009). Among the various lipid systems, lipospheres have been developed to address some issues such as stability and low pay-load capacity of some lipid systems (Attama et al., 2009).

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Lipospheres are restricted to the stabilizing material of a phospholipid layer (Rawat and Saraf, 2008). These have been utilized in the delivery of anti-inflammatory compounds, local anesthetics, antibiotics, anticancer agents, insect repellents, vaccines, proteins and peptides (Masters and Domb, 1998; Khopade and Jain, 1997; Amselem et al., 1996; Domb et al., 1995; Rawat and Saraf, 2008). The lipospheres are distinct from micro droplets, vesicles or liposomes since the lipospheres have solid inner core at room temperature. The combination of solid inner core with phospholipid exterior confers several advantages on the lipospheres compared with conventional microspheres and micro particles, including high dispersibility in an aqueous medium, and a release rate for the entrapped substance that is controlled by phospholipid coating and carrier. The substance to be delivered does not have to be soluble in the vehicle since it can be dispersed in the solid carrier. (Domb et al., 1996b, Rawat and Saraf, 2008). Lipospheres also have a lower risk of reaction of substance to be delivered with the vehicle than in emulsion system because the vehicle is a solid material. Moreover, the release rate of substance from the lipospheres can be manipulated by altering either or both the inner solid vesicle or the outer phospholipid layer. Lipospheres are also easier to prepare than vesicles such as liposomes, and are inherently more stable. Stability has become the major problem limiting the use of liposomes, both in terms of shelf life and after in vivo administration. The cost of the reagents for making the lipospheres (food grade) is significantly less than the cost of reagents for making liposomes, which require very pure lipids (Domb et al., 1996a). The study aimed at evaluating piroxicam-loaded lipospheres formulated with dika wax and a phospholipid (Phospholipon 90G). Piroxicam is a non-steroidal anti-inflammatory drug (NSAID) with prominent anti-inflammatory, analgesic and anti-pyretic effect. It is sparingly soluble in water and like other NSAIDS it causes severe gastric irritation ( (Burke, et al., 2006). The anti-inflammatory effect of the piroxicam loaded lipospheres and the ulcerogenicity were evaluated using laboratory animals. 2. Materials and methods The following materials were used as procured from their suppliers without further purification: piroxicam (Pfizer, Nigeria), nhexane, ethyl acetate (SigmaAldrich, Germany), hydrochloric acid, sodium hydroxide, monobasic potassium phosphate and Tween 80 (Merck,

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Germany),

Phospholipon 90G (Phospholipid GmbH, Kln, Germany), activated Charcoal

(BioLab. (UK) limited, London). Sorbitol (Across Organics, Germany), distilled water (Lion water, Nsukka, Nigeria). Dika fat was obtained from a batch processed in our laboratory. All other reagents and solvents were analytical grade and were used as supplied. 2.1 Extraction and purification of dika wax from Irvingia gabonensis Dika fat was extracted by soxhlet extraction, Irvingia gabonensis was milled in an equipment of the hammer mill type. The dika fat was extracted in a soxhlet using nhexane (Matos et al., 2009). The n-hexane was allowed to evaporate at room temperature. Boiled distilled water which was twice the volume of the fat was poured into the molten fat in order to dissolve the hydrophilic gum contained in the fat. The hydrophilic gum was removed using a separating funnel. This process was repeated three times. Ethyl acetate was equally poured into the molten fat in order to remove the hydrophobic gum from the fat. The extracted fat was further purified by passing it through a column of activated charcoal and bentonite (2:1) at 100o

C at a ratio of 10 g of fat and 1g of the column material. The fat was stored in a refrigerator until

used (Attama et al., 2005). 2.2 Preparation of simulated intestinal fluid (SIF) (pH 7.5) The solution was prepared according to pharmacopeia standard (USP, 1995).

2.3 Preparation of lipid matrix Mixtures of (1:1, 1:2 and 2:1 w/w) Phospholipon 90G, a purified lecithin and dika wax were melted and stirred at a temperature of 70 oC using a magnetic stirrer, until a homogenous, transparent yellow melt was obtained. The homogenous mixture was stirred at room temperature until solidification to ensure adequate mixing (Friedrich et al., 2003).

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Table 1: Quantities of material used for SLMs formulation Batch Lipid matrix A1 A2 A3 B1 B2 B3 C1 C2 C3 A0 B0 C0API.

Tween 80 (ml) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

Lipid matrix (g) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

Sorbitol (%w/w) 4 4 4 4 4 4 4 4 4 4 4 4

piroxicam (%w/w) 0.25 0.50 0.75 0.25 0.50 0.75 0.25 0.50 0.75 0 0 0

Distilled water q.s (%w/w) 100 100 100 100 100 100 100 100 100 100 100 100

ratio 1:1 1:1 1:1 1:2 1:2 1:2 2:1 2:1 2:1 1:1 1:2 2:1

A0 A3: contain LM 1:1, B0- B3: contain LM 1:2, C0 C3: contain LM 2:1 and A0, B0 and C0: contain no

2.4. Preparation of lipospheres Appropriate quantities of lipid matrix, Tween 80, Sorbitol, drug and distilled water as presented in Table 1, were used for the formulation. Piroxicam-loaded lipospheres were prepared using 1:1, 1:2 and 2:1 (w/w) of the lipid matrix by hot homogenization techniques using Ultraturrax (T25 Basic Digital). 5 g of the lipid matrix was melted at 70 oC in a crucible and an appropriate amount of piroxicam was incorporated into the lipidic melt. Sorbitol was dissolved in hot distilled water at the same temperature together with Tween 80. The aqueous phase at 70 oC was poured into the lipidic melt under high shear homogenization with an ultra turrax an o/w emulsion was finally formed by phase inversion (Cortesi et al. 2003).

2.4. Evaluation of piroxicam-loaded lipospheres 2.4.1.1 Determination of particle size and morphology Small quantity of lipospheres were placed on a microscope slide, the slide was covered with a cover slip and imaged under a Hund binocular microscope (Weltzlar, Germany), attached

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with a motic image analyzer (Multicam, China) at a magnification of x 400. Different particles of the SLMs from each batch were counted (n=100), and the mean value was determined. 2.4.2. Drug Content of lipospheres Beers plot was obtained at the concentration range of (0.2, 0.4, 0.6, 0.8 and 1.0 mg %) for piroxicam in SIF. In each case 10 ml volume of each batch of the SLMs was placed in centrifuge (Chem. Lab. Instrument, UK) for 30 min at 1500 rpm; the sediment was used in the analysis of drug content. A 0.5 g of the SLMs (containing 0 %, 0.25 %, 0.5 % and 0.75 % of indomethacin) was triturated using mortar and pestle with 10 ml of SIF (pH 7.5) the solution was placed in a 100 ml volumetric flask. The flask was made up to volume; the solution was filtered through a filter paper (Whatman No.1) and analyzed spectrophotometrically at predetermined wavelength of 370 nm (Jenway 6305, Borloworld scientific Ltd.). This was repeated five times for all the batches. The drug concentrations were calculated with reference to Beers plot. 2.4.3 Drug encapsulation efficiency The quantities of the drug theoretically contained in the lipospheres were compared with the quantity actually gotten from the drug content studies. This was calculated using the equation below: Encapsulation efficiency (EE %)ADC x 100 TDC

where ADC is the actual drug content and TDC is the theoretical drug content.

2.4.4 pH analysis

The pH of the SLMs were determined in time dependent manner (24 h, 1 week, 2 months and 3 months) using pH meter ( Suntex TS 2, Taiwan).

2.4.5 Release studies of lipospheres The USP paddle method was adopted in this study. The dissolution medium consisted of 900 ml of freshly prepared medium (SIF pH 7.5) maintained at 37 1 oC The membrane

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selected was pretreated by soaking in the dissolution medium for 24 h prior to use. A quantity of SLM equivalent to 0.020 mg piroxicam was weighed from each batch and placed in a polycarbonate dialysis membrane containing 2 ml of the dissolution medium, securely tied with a thermoresistant thread and placed in the chamber of the release apparatus. The paddle was rotated at 100 rpm, and at predetermined timed intervals, 5 ml portions of the dissolution medium was withdrawn, appropriately diluted, and analyzed for drug content in a spectrophotometer. The volume of the dissolution medium was kept constant by replacing it with 5 ml of fresh medium after each withdrawal to maintain sink condition. The amount of drug released at each time interval was determined with reference to Beers Plot. 2.4.6 Anti-inflammatory studies The antiinflammatory activity of the piroxicam-loaded lipospheres was carried out using the rat paw oedema test (Winter et al., 1962). All experimental protocols were approved by the animal ethics committee of the faculty of pharmaceutical sciences, university of Nigeria, Nsukka. The philogistic agent employed in the study was fresh undiluted egg albumin. Adult wistar rats of either sex (150 200 g) were divided into five rats per group. The animals were fasted and deprived of water for 12 hours before the experiment. The deprivation of water was to ensure uniform hydration and to minimize variability in oedematous response (Winter et al., 1963). Piroxicamloaded lipospheres equivalent to 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 eggalbumin into the sub plantar region of the right hind paw of the rats. (Ajali and okoye, 2009). The volumes of distilled water displaced by treated right hind paw of the rats were measured using plethysmometer before and at 30 min, 1, 2, 3, 4, 5 and 6 h after egg albumin injection. Average oedema at every interval was assessed in terms of difference in volume displacement (Vt - Vo) (Anosike et al., 2009). The percent inhibition of oedema was calculated using the relation, % inhibition of oedema =

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where, Vt is the volume of oedema at corresponding time and Vo is the volume of oedema in control rats at the same time (Parez, 1996; Ahmed et al., 1993; Ajali and Okoye 2009).

2.4.7 Ulcerogenicity of lipospheres The ulcerogenicity of piroxicamloaded lipospheres was determined using a method described by Chung-Chin et al (2009). All experimental protocols were approved by the animal ethics committee of the faculty of pharmaceutical sciences, university of Nigeria, Nsukka. The studies were carried out on healthy Wistar rats (180 220 g). The animals were divided into five experimental groups of five animals per group. The control group received normal saline while the reference group received 10 mg/kg pure sample of piroxicam orally. The animals were fasted for 8 hours prior to a single dose of either the control or the test compounds, given free access to food and water, and sacrificed 8 h later. The gastric mucosa of the rat was examined under a microscope using a 4 x binocular magnifier. The lesions were counted. The mean score of each treated group minus the mean score of the control group was considered as severity index of gastric damage. 2.4.8 Statistical analysis Statistical analysis was done using SPSS version 14.0 (SPSS Inc. Chicago, IL.USA). All values are expressed as mean SD. Data were analysed by one-way ANOVA. Differences between means were assessed by a two-tailed students T-test. P < 0.05 was considered statistically significant.

3. Results and discussion 3.1 Particle size analysis

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Particle size may be a function of either one or more of the following: formulation excipients, degree of homogenisation, homogenisation pressure, rate of particle size growth, crystalline habit of the particle etc. (Attama et al., 2009). The photomicrographs presented in Fig. 1 shows that piroxicam-loaded lipospheres formulated were spherical in shape. The particle size results presented in Table 2 showed that piroxicam-loaded lipospheres formulated were not affected by the ratio of the lipid matrix used. However, the concentration of the piroxicam incorporated into the lipospheres affected the particle size of piroxicam-loaded lipospheres. Increase in amount of piroxicam increased the particle size of the lipospheres, in agreement with the work done by Barakat et al (2006), who prepared carbamazepineloaded precifac lipospheres and found out that the particle size of lipospheres increased with increase in drug loading, and Joseph et al. (2002) who prepared piroxicam loaded polycarbonate microspheres. However, batch A1, prepared with LM 1:1 and containing 0.25 % piroxicam had its particle size increased with increase in encapsulation efficiency and reduced drug loading; also batch C3 formulated with LM 2:1 and loaded with 0.75 % of piroxicam had a decrease in particle size when the drug concentration was increased, this may be due to saturation of the LM with increase in drug concentration. The particle size of the control batches without piroxicam (batches A0, B0 and C0) were much lower than the particle size of SLMs with varying drug concentrations.

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A1

B1

C1Fig. 1. Photomicrographs of representative samples of piroxicam-loaded lipospheres formulated with 1:1, 1:2 and 2:1 %w/w of lipid matrix (Phospholipid: dika wax) and containing 1 % indomethacin respectively.

Table 2: Properties of piroxicam-loaded lipospheres Batch LM pH 24 hA0 A1 1:1 1:1 6.34 0.21 6.37 0.17

pH 1 week6.10 0.27 4.14 0.12

pH 1 month5.98 0.13 4.09 0.10

Particle size (m SD)a0.47 0.04 3.13 3.00

ADC (% SD)b0.00 0.21 0.27

TDC (%)0.00 0.25

EE (%)84.0

11 A2 A3 B0 B1 B2 B3 C0 C1 C2 C3a

1:1 1:1 1:2 1:2 1:2 1:2 2:1 2:1 2:1 2:1

6.46 0.14 6.48 0.27 6.11 0.19 6.37 0.11 6.43 0.10 6.57 0.11 6.12 0.17 6.38 0.12 6.41 0.07 6.51 0.09

4.62 0.17 4.62 0.15 6.07 0.11 4.15 0.20 4.38 0.17 4.94 0.21 6.08 0.20 4.20 0.16 4.37 0.17 4.90 0.12

4.15 0.11 4.38 0.15 6.00 0.25 3.94 0.23 4.09 0.15 4.21 0.11 5.97 0.17 4.88 0.12 4.10 0.13 4.88 0.27

2.09 3.13 3.08 2.88 0.50 0.03 2.89 3.03 2.89 3.03 3.56 2.95 0.51 0.07 3.10 1.02 3.24 2.91 1.66 3.18

0.12 0.32 0.13 0.47 0.00 0.19 0. 21 0.15 0.39 0.24 0.27 0.00 0.08 0.23 014 0.19 0.18 0.24

0.50 0.75 0.00 0.25 0.50 0.75 0.00 0.25 0.50 0.75

24.0 17.3 76.0 30.0 31.0 31.5 27.0 24.0

n = 100, SD = Standard deviation, bn = 5, A0 A3: contain LM 1:1, B0- B3: contain LM 1:2, C0

C3: contain LM 2:1 and A0, B0 and C0: contain no drug.

3.2 pH analysis of lipospheres The pH of different batches of lipospheres were measured in time dependent manner, 24 h, 1 week and 1 month after preparation to determine the change of pH with time. pH change could be a function of degradation of the API or excipients. A prior stable API may be affected by degradation of excipients with storage through generation of unfavorable pH (increase or decrease) or reactive species for the API (Attama et al., 2009). Table 2 shows the pH values of all the batches of piroxicam-loaded and unloaded lipospheres formulated. The pH varied from

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between 6.11 0.19 and 6.57 0.11 within 24 h of preparation to between 3.94 0.23and 4.88 0.17 within 1 month of preparation. From Table 2, it was clear that there was a slight decrease in pH from 24 h to 1 month. However, batch B1 (formulated with LM 2:1 and containing 0.25 % piroxicam) had the highest reduction in pH after 1 month (3.94 0.23). The pH change in the piroxicam loaded lipospheres was not due to degradation of the drug since there was also a fall in pH of the unloaded lipospheres. Degradation of the free fatty acids may be implicated in the fall of pH (Attama et al., 2009). 3.3 Drug content The drug content of piroxicam-loaded lipospheres formulated with LM 1:1 and loaded with 0.25 % piroxicam were high (batch A1); also batch B1 formulated with LM 1:1 and containing 0.25 % piroxicam exhibited high drug content. However, other formulations of piroxicam-loaded lipospheres exhibited drug content values that varied widely from the amount loaded into the formulations. The drug content was significantly (p < 0.05) affected by the amount of drug loaded in the formulations and the ratio of two lipids used in formulating the lipid matrix. Increasing the amount of piroxicam in the formulations decreased the actual drug content of the lipospheres, this may be due saturation of the LM when the amount of drug was increased. Also, reducing the ratio of dika wax in the lipid matrix reduced the drug content of the lipospheres as shown in Table 2. LM 2:1, having lower ratio of dika wax exhibited very low drug content that varied widely from the amount of piroxicam incorporated into the formulations.

3.4 Encapsulation efficiency (EE%) The role of the formulated lipospheres is to deliver the API to the target tissues intact. Entrapment efficiency defines the ratio between the weight of entrapped API and the total weight of API added to the dispersion (Attama et al., 2009). Table 2 shows the EE % of the various formulations of piroxicam-loaded lipospheres. EE% depends on several parameters, such as the lipophilic properties of the API, screening of the most appropriate lipid composition/ratio and surfactant combination, as well as the production procedure used.

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From Table 2, the EE% ranged from 24 % for batch C3 lipospheres to 84 % for batch A1 lipospheres. The varied EE% may be as a result of API and matrix physicochemical and material related factors (Attama et al., 2009). Generally, the EE% was affected by the lipid composition/ratio in formulating the lipospheres. Lipospheres formulated with Lipid matrix (2:1) i.e. batches C1 C3 with lower ratio of dika wax showed very low EE% values significantly different (p < 0.05) from other batches with higher ratios of dika wax. The reason may be due presence of small amount of fat in the inner core of the lipospheres which lead to saturation of the fat core of the lipospheres by the drug incorporated in the dispersion. EE% of piroxicam-loaded lipospheres was also affected by the total amount of API in the dispersion. 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. However, batch C1 had low EE% of 31.5 % due to reasons discussed earlier. 3.5 Drug release from indomethacin-loaded lipospheres The drug release profile presented in Fig. 2 showed that piroxicam-loaded lipospheres exhibited good release of drug for all the formulations. The formulations had reproducible drug release in all the batches, without any form 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. Piroxicam-loaded lipospheres formulated with higher ratios of phospholipid (LM 2:1) i.e. batches C1 to C3 had more prolonged drug release rate with maximum drug release (T100) at 120. This may be due to enhanced physical stability caused by an increase in outer phospholipid coating of the liposphere with increased phospholipid concentration. Lipospheres formulated with lipid matrix having higher concentration of dika wax exhibited the fastest release of drug with maximum drug release between 60 min for batches B1 and B2, and 70 min for batch B3. However, piroxicam-loaded lipospheres formulated with LM having equal ratios of phospholipid to dika wax (LM 1:1) showed reproducible drug release with T100 that ranged from 70 min for batches A1 and A3 and 80 min for batch A2.

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(a)

(b)

(c)Fig. 2. Release profile of piroxicam loaded lipospheres; A1, B1and C1 were formulated with LM 1:1, 1:2 and 2:1 respectively and contain 0.25 % piroxicam; A2, B2, C2 contain 0.5 % piroxicam and A3, B3 and C3 contain 0.75 % piroxicam.

3.6 Anti-inflammatory properties The result of anti-inflammatory properties presented in Table 3 showed that piroxicam loaded lipospheres formulated exhibited good anti-inflammatory properties significantly different from the control (p < 0.05). Piroxicam-loaded lipospheres showed higher inhibition of oedema than the reference sample (piroxicam pure sample); this may be due to

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increased absorption of drug in vivo in the presence of the lipid matrix carrier. Drug suspended in lipid matrix has been shown, in most cases to have a better absorption than conventional solid dosage forms. This could be due to the ease of wetting of hydrophobic drug particles in the presence of lipid matrix. The presence of surfactant in the formulation may further promote the drug release (Attama et al., 2005). Piroxicam-loaded lipospheres (A1) had 35.3 %, 41 % and 53.3 % oedema inhibition at T60, T120, and T240, while the reference drug had 25 %, 33 % and 47 % at T60, T120, and T240 respectively.

Table 3. Anti-inflammatory properties of piroxicam-loaded lipospheres Paw volume oedema (ml SD)a and percentage inhibition of oedema (%) Groups A1 0.5 h 0.58 0.23* (27.5) B1 C1 0.56 0.37* (30.0) 0.60 0.52* (25.0) D (ref.) 0.60 0.24* (25.0) E(Cont.) 0.80 0.35 1h 0.55 0.27* (35.3) 0.58 0.45* (31.8) 0.55 0.47* (35.3) 0.57 0.43* (33.0) 0.85 0.17 2h 0.48 0.19* (40.7) 0.50 0.13* (38.3) 0.51 0.54* (37.0) 0.50 0.57* (38.3) 0.81 0.27 3h 0.42 0.29* (46.8) 0.40 0.32* (49.4) 0.43 0.39* (43.0) 0.45 0.51* (43.0) 0.79 0.13 4h 0.35 0.12* (53.3) 0.35 0.24* (53.3) 0.37 0.45* (50.7) 0.40 0.23* (46.7) 0.75 0.21 5h 0.30 0.17* (57.1) 0.29 0.21* (58.6) 0.30 1.52* (57.1) 0.31 0.27* (55.7) 0.70 0.12

*Significant at p < 0.05 compared to control. Values of oedema shown are mean SD (n = 5). Values in parenthesis are percent inhibition of oedema, A 1C2: piroxicam-loaded lipospheres, D: pure piroxicam, E: normal saline.

3.7 Ulcerogenic properties The result of ulcerogenic studies presented in Table 4 showed that piroxicam lipospheres does not have ulcer inducing potentials. The lipospheres inhibited the ulcerogenic potentials of piroxicam. This may be due to the presence of phospholipid in the formulations which may help to protect the gastric mucosa from irritations from the NSAID. However, the reference drug showed a high ulcer index of 15.00 1.23 which varied significantly (p < 0.05) from the control. The control and the lipospheres containing piroxicam showed no ulcer and had zero ulcer index.

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Table 4. Ulcerogenic properties Batch A1 B1 D (Ref.) E (Con.) Ulcer index (Mean SD)a 0.00 0.00 0.00 0.00 15.00 1.23* 0.00 0.00

*Reduction in oedema significant at p < 0.05 compared to control. an = 5; A1 and B1: piroxicam-loaded SLMs; D: piroxicam pure sample; E: normal saline.

4. Conclusion Results of the study showed that Piroxicam-loaded lipospheres formulated with dika wax and phospholipid showed good in vivo and in vitro properties. The particle size of piroxicam-loaded lipospheres formulated were not affected by the ratio of the lipid matrix used. However, the concentration of the piroxicam incorporated into the lipospheres affected the particle size of the lipospheres. Encapsulation efficiency of up to 84 % was obtained in batch A1 lipospheres. The rate of drug release was affected by the ratio/combination of the lipid used in

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preparation of the lipid matrix. Piroxicam-loaded lipospheres formulated with higher ratios of phospholipid (LM 2:1) i.e. batches C1 to C3 had more prolonged drug release rate with maximum drug release (T100) at 120. Lipospheres formulated with lipid matrix having higher concentration of dika wax exhibited the fastest release of drug with maximum drug release between 60 min for batches B1 and B2, and 70 min for batch B3. However, piroxicam-loaded lipospheres formulated with LM having equal ratios of phospholipid to dika wax (LM 1:1) showed reproducible drug release with T100 that ranged from 70 min for batches A1 and A3 and 80 min for batch A2. Piroxicam loaded lipospheres formulated exhibited good anti-inflammatory properties and also inhibited the ulcerogenic potentials of piroxicam.

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