A preliminary attempt to address indomethacin ’ s poor water solubility using solid self emulsifying drug delivery system as a carrier

The non-steroidal anti-inflammatory drug (NSAID), indomethacin, is disadvantaged by its poor water solubility and gastrointestinal adverse effects. The aim of this work therefore was to formulate this drug into solid self emulsifying drug delivery system (SSEDDS) as an attempt to improve its aqueous solubility and anti-inflammatory property using shea butter or its blend with Bos indicus lipid. Proximate analysis was carried out on shea butter. Indomethacin-loaded SSEDDS containing lipid (shea butter, Vitellaria paradoxa), surfactant (Tween 65) and cosurfactant (Span 85) or lipid blends (shea butter/Bos indicus fat), surfactant blends (Tween 65/Tween 80) and cosurfactant (Span 85) were formulated. The drug-loaded SSEDDS were evaluated for visual isotropicity, emulsification time, aqueous dilution, refrigeration test, loading efficiency, in vitro drug release and anti-inflammatory properties. Results showed that Shea butter contained 92.8% fat, 1.52% moisture, 2.05% protein, 9.78% carbohydrate and trace amounts of fiber and ash. Pre/post formulation isotropicity tests showed that all batches of indomethacin SSEDDS were stable. SSEDDS containing shea butter as the lipid component recorded significantly (p < 0.05) longer emulsification times than those containing lipid blends. The T85 of most of the batches was within 30 min. Shea butter-based SSEDDS demonstrated significantly (p < 0.05) higher anti-inflammatory effect than unformulated indomethacin powder. In conclusion, Shea butter conferred stability to the formulations and contributed to acceptable in vitro and superior antiinflammatory characteristics of indomethacin.


INTRODUCTION
Most commercial tablet dosage forms are produced using synthetic or semisynthetic polymers.However, these synthetic polymers could cause detrimental effects on incorporated drug during manufacturing or in vivo degradation of the polymer with attendant toxic effects (Kumar, 2000).This has warranted investigations into alternative alternative carrier systems.Among them, the proven biocompatibility, increased drug solubilization and bioavailability enhancement of lipid materials have projected their candidature as choice excipients in drug delivery (Rawat et al., 2011;Xianyi et al., 2012).Self emulsifying drug delivery system (SEDDS) is a lipid carrier with exciting *Corresponding author.E-mail: obittenick@yahoo.com.Tel: + 234 806 0532739.drug delivery characteristics.It is an isotropic mixture of oil, surfactant, co-surfactant and drug.Upon mild agitation in aqueous medium, either initiated mechanically or gastric motility-impelled, the system forms fine oil-in-water emulsions with very high surface area.Advantages of SEDDS include, more consistent drug absorption, selective targeting of drug(s) toward specific absorption window in the gastrointestinal tract, protection of drug(s) from the gut environment, control of delivery profiles, reduced variability including food effects, enhanced oral bioavailability enabling reduction in dose and high drug loading efficiency (Chudasama et al., 2011;Jingling et al., 2006).
The presence of oil makes SEDDS unique and distinguishes them from ordinary surfactant dispersions of drugs.An array of synthetic oils has been the major choice of formulators, while natural vegetable oils, though applicable, have been discredited for their compositional heterogeneity and low capacity to solubilize drugs.
Nevertheless rising interest on their potential applicability has been observed amongst researchers with regional access to ubiquitous vegetable oils.The nutritional status of these oils especially in third world countries ranks them preferred owing to nutritional acceptability and biocompatibility.These natural oils are physiologically biocompatible, nutritionally acceptable, nontoxic, economically affordable and commercially available.
Although new synthetic oils make appealing appearances at the work bench of researchers, ethical restrictions still remain a daunting hurdle to their comercial and pharmaceutical utility.In the mean time, since abundant quantities of vegetable oils are found in India, Africa and some other countries, further research efforts geared towards promoting their hidden potential usefulness in pharmaceutical formulations will be a welcome development.Our present investigation considered the applicative usefulness of shea butter and/or its blend with Bos indicus fat in the formulation of indomethacin-loaded SSEDDS.
Indomethacin is a non-steroidal anti-inflammatory drug for the management of inflammatory conditions, pain and fever.The dose-related adverse effects have been documented, hence warranting the adoption of sustained release forms to mitigate these effects (Erdal et al., 2009).It is a weekly acidic, poorly soluble drug marked by solubility and dissolution rate that are pH-dependent (Tirkkonen et al., 2009).Indomethacin has in vitro/in vivo inhibitory effect on COX-negative human colon cancer cells which is suggestive of anti tumor effect being exerted independent of its cyclooxygenase inhibitory characteristics (Wang and Zhang, 2004).Importantly, as a Biopharmaceutical Classification System class II drug, characterized by poor solubility and high permeability, attempts have been made to improve aqueous solubility via SEDDS technique (Barakat et al., 2011;Liangmei et al., 2012).Since adverse effect is dose-related, reduction of dose without compromised efficacy may be achievable with SEDDS.In traditional formulations, the 25 mg dose of indomethacin still suffers solubility constraints such that the administered dose-input may not be accountable systemically due to erratic absorption.On the other hand in aqueous medium SEDDS form droplets of drug solution which aptly guarantees consistent absorption and bioavailability, and may promote dose reduction (Sachan et al., 2011).Dose reduction of indomethacin in this work is projected to maintain optimal therapeutic effect while excluding or reducing adverse effects.Therefore, the objectives of this work were to formulate indomethacin SSEDDS using a solid biocompatible vegetable lipid, shea butter or its blend with B. indicus fat and to evaluate in vitro and anti-inflammatory properties, respectively.

MATERIALS AND METHODS
Indomethacin (Merck, Germany), Span 85 (FLUKA AG Chemische Buchs, Engetragene chemical Marke de chemical inc.USA), Tween 80 (Sigma Aldrich, Seelze Germany), Shea butter (from Vitellaria paradoxa tree) was procured from Owode market Offa, Kwara state, Nigeria and appropriately identified before use.B. indicus fat was obtained from Nsukka abattoir.All other reagents used were of analytical grade and were used as supplied.

Extraction and purification of homolipid from B. indicus
About 1 kg of B. indicus fat (BIF) was immersed in hot water maintained at 80 to 90°C for 45 min.It was passed through a muslin cloth to strain off extraneous matter.The fat was allowed to cool before decanting the aqueous layer.A 2% w/w suspension of a 2:1 ratio of activated charcoal and bentonite mixture in the fat was heated at 80 to 90°C for 1 h and filtered through Whatman filter paper.The fat was stored in a refrigerator until used (Attama et al., 2005).

Purification of shea butter
A 2% w/w suspension of activated charcoal in Shea butter (SB) was heated in a beaker at 80 to 90°C for an hour.The suspension was later vacuum-filtered using Buchner funnel.The purified SB was stored for further use.

Proximate analysis
The quantitative presence of protein, lipid, carbohydrate, crude fibre, moisture and ash, respectively in SB were determined using standard procedures (Association of Analytical Communities (AOAC), 1990).

Preformulation isotropicity test
Different batches of the placebo SSEDDS were prepared as shown in Table 1.Appropriate quantities of the ingredients were introduced into test tubes and stirred over a water bath at 50°C for 10 min.It was stored for 5 h at ambient temperature and subsequently evaluated for isotropicity.The formulations that passed this test were used for further studies while those that witnessed phase separation were discarded.

Drug solubility in the SSEDDS
This experiment was to determine the maximum amount of indomethacin that could be dissolved in the SSEDDS formulations without subsequent crystallization.Increasing quantities of the drug 5, 10, 15 and 20 mg, respectively were added to a 0.4 ml quantity of SSEDDS and stirred vigorously at 50°C for 30 min.The maximum drug quantity that dissolved in the SSEDDS formulations was noted and suitable dose-concentration selected for further studies.

Formulation of indomethacin SSEDDS
Table 2 shows the various quantities of the ingredients used in the formulation of drug-loaded SSEDDS of target weight 365 mg.SB served as the oil constituent of some batches while the others were a blend of SB and BIF.In each case indomethacin was weighed and introduced into a beaker (on a water bath maintained at a temperature of 50°C) containing Span 85 and stirred for 10 min.SB or SB-BIF blend was introduced and stirring continued.Finally, Tween 65 or its blend with Tween 80 was also introduced and stirred for more 5 to 10 min until drug completely dissolved.In order to investigate the effect of an oil-soluble excipient, carbosil ® was incorporated in some batches (9 to 16) and stirred until it dissolved.

Post formulation isotropicity/stability test
The formulated SSEDDS (Table 2) were stored for 48 h at ambient temperature and observed for isotropicity (phase separation and drug precipitation).

Emulsification time test
A 100 ml quantity of 0.1 N HCl was introduced into a 250 ml beaker positioned on a hot plate and maintained at 37 ± 1°C.SEDDS (365 mg) was syringed into the beaker as the magnetic stirrer rotated at approximately 50 rpm.The time taken for complete emulsification of the SSEDDS was visually observed.

Post emulsification drug precipitation test
A 365 mg quantity of SSEDDS was emulsified as above and the emulsion stored at ambient temperature (25°C) for 4 h.Thereafter it was observed for the presence of drug precipitates.

Encapsulation of SEDDS Formulations
Using a 1 ml syringe of each dose of the SSEDDS formulations was introduced into size 0 hard gelatin capsule shells.The SSEDDSloaded capsules were later stored in a polyethylene material for further use.

Refrigeration test
Two capsules of the SSEDDS from each batch were wrapped in a polyethylene material and kept in the refrigerator for 24 h at 2°C.It was thereafter observed for drug precipitation or phase separation.

Loading efficiency
A 365 mg quantity of SSEDDS was emulsified in 100 ml of 0.1 N HCl.It was placed in a water bath with constant stirring until complete emulsification was achieved.A 0.1 ml quantity was withdrawn and made up to 10 ml with absolute ethanol.Indomethacin content was determined using a ultra violet (UV) spectrophotometer (Jenway 6305 spectrophotometer, UK) at a predetermined wavelength of 232 nm.

Drug release studies
The rotating basket dissolution apparatus (VEEGO, India) was used.Dissolution medium consisted of 900 ml of freshly prepared 0.1 N HCl.A capsule containing 365 mg quantity of SSEDDS was placed in the basket which was meant to rotate at a speed of 100 rpm.At predetermined time intervals 5 ml samples of the dissolution medium were withdrawn and assayed in a UV spectrophotometer (Jenway 6305 spectrophotometer, UK) for indomethacin content at a wavelength of 232 nm.Also, 5 ml of a fresh medium was used to refresh the dissolution medium.The experiment was run in duplicates.

Droplet size, polydispersity index and zeta potential
The droplet size, zeta potential and polydispersity index were determined using a zeta sizer (Zeta sizer 3000 HS, Malvern Instruments, Worcestershire UK).A 365 mg quantity of indomethacin SSEDDS was emulsified to form a dispersion in 300 ml phosphate buffer (PBS, pH 7.4).Droplet size was determined at 25°C at a scattering angle of 90° using cuvette containing 2.5 ml of the microemulsion in three runs with each having 10 sub-runs.On the other hand the zeta potential was determined by introducing 1 ml into capillary cuvettes by phase analysis light scattering using the same zeta sizer as in droplet size determination.The zeta potential value was a mean from two runs of 10 sub-runs each.Polydispersity index values were automatically generated by the machine.

Anti-inflammatory studies
The anti-inflammatory activity of the indomethacin-loaded SSEDDS was carried out using the rat paw oedema test method (Winter et al., 1962).All experimental protocols were in accordance with the animal ethics committee of the Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka.The phlogistic agent employed in the study was fresh undiluted egg albumin (Anosike et al., 2009).Adult Wistar rats of either sex (180 to 200 g) were used for the study (n = 6).The rats were fasted and deprived of water for 12 h before the experiment.The deprivation of water was to ensure uniform hydration and to minimize variability in oedematous response (Winter et al., 1963).Indomethacin SSEDDS formulation equivalent to 0.7 mg/kg body weight was administered orally to the rats using a 1 ml syringe.The negative control group received normal saline while the positive control group received pure sample of indomethacin (0.7 mg/kg).Thirty minutes post treatment oedema was induced by injection of 0.1 ml of fresh undiluted egg-albumin into the sub plantar region of the right hind paw of the rats (Ajali et al., 2009).The volumes of distilled water displaced by treated right hind paw of the rats were measured using plethysmometer before and at 1, 2, 3, 4, 5 h after injection of egg albumin.Average oedema at every interval was assessed in terms of difference in volume displacement of injected paw (Vt -Vo) (Anosike et al., 2009).The percent inhibition of oedema was calculated using the formulae (Perez, 1996): Where: a = mean paw volume of treated rats after egg albumin injection, x = mean paw volume of treated rats before egg albumin injection, b = mean paw volume of control rats after egg albumin injection and y = mean paw volume of control rats before egg albumin injection.

Statistical analysis
Statistical analysis was performed using statistical package for social sciences (SPSS) statistical package.Mean and standard error for all data were calculated.For batch comparisons, the Student's t-test was used to determine statistically significant differences at p < 0.05.

Proximate analysis
The results of proximate analysis of SB showed trace amount of fiber and ash, fat content of 92.8%, moisture content of 1.52, 9.78% carbohydrate and protein content of 2.05%.The protein content value was lower than the The Food and Agriculture Organization (FAO) recommendation value (19.8%) (FAO, 1982).Shea butter is vegetable fat extracted from the kernels of the fruit of Vitellaria paradoxa, Sapotaceae (Elias and Carney, 2004;Schreckenberg, 2004).Investigations carried out by other workers have shown a wide variation in the percent moisture content among several shea butter varieties with values ranging between 0.15 and 14.5% (Megnanou et al., 2007).Shea butter is highly regarded in the cosmetic field because of its high emolliency and moisturization capacities, but also as an occlusivity lipid replacement (Acquaye et al., 2001).Its occlusive property which may be correlated to its high lipid content (92.8%) as well as its moisture content may therefore present a favourable baseline for its usefulness in SSEDDS as a delivery vehicle for lipophylic drugs.Moreover, fat is important in diets because it promotes absorption of fat soluble vitamins (Bogert et al., 1994) and is in itself a high energy nutrient.The low protein content of 2.05% is comparably lower than those of soya beans, cowpeas, pigeon peas, melon, pumpkin and gourd seeds all ranging between 23.1 to 33.0% (Olaofe et al., 1994).The carbohydrate con-tent of 9.78% is indicative of fairly good supply source of energy requirements for the body.Overall, the outcome of the nutritional profile of shea butter indicates its potential capability of contributing minutely, in the most part to supplying some daily nutritional and energy requirements of the human or animal subject when the shea butter is developed as a SSEDDS for drug delivery purposes.

Pre/post formulation isotropicity test
Preformulation isotropicity was to determine homogenous miscibility between the surfactants and the oils.Failure of any batch at this stage would warrant outright rejection and exclusion.All the batches of SSEDDS formulated (Table 1) were isotropic since no phase separation was observed.Subsequently, they were used in the formulation of drug-loaded SSEDDS.Post formulation isotropicity test showed that all batches of the drugloaded SSEDDS were stable after 48 h upon visual observation, and there was no drug precipitation.Isotropic stability at all concentrations may be attributed to SB, BIF and 65 which are stable solids at room temperature.Thus their solid texture yielded solid SEDDS, with the dissolved drug and liquid excipients molecularly entrapped in the lipid matrix.The firmness of the formulations may have forestalled gravitational effect which liquid suspensions/dispersions are susceptible to.
It should be recalled that centrifugation is an in vitro test that may be used to evaluate in part gravitation effect on liquid SEDDS under storage.

Emulsification time
The results of emulsification times for all the batches are shown in Table 3. Batches with lipid and surfactant blends emulsified in less than 1 min and ranked the fastest.In fact formulations containing SB as the single lipid component recorded significantly (p < 0.05) higher emulsification time (EMT) than those containing lipid and surfactant blends.Furthermore, when cabosil ® was incorporated into batches 13 to 16, the EMTs significantly (p < 0.05) increased.The inclusion of liquid Tween 80 and reduction of the solid Tween 65 in the blends probably had a reduction effect on the consistency of the SSEDDS, thus facilitating dispersion in aqueous medium.The uniqueness of our formulations is the combination of solidity and propensity to undergo emulsification.Generally emulsification of SEDDS is similar to tablet disintegration, since both steps precede absorption.On the other hand whereas disintegration precedes dissolution prior to absorption, emulsification yields liquid droplets that are ready for absorption.This presupposes that SEDDS may provide a platform for faster onset of action than disintegration-bound conventional tablets.Fast emulsification is the ultimate for SEDDS intended for immediate release.However, prompt emulsification in the gastrointestinal tract may release all drug-borne droplets at a time.This may culminate in massive absorption, high bioavailability and possible overshooting of minimum effective dose for especially drugs with low safety margin.Therefore, in our present investigation we sought for solid constituents capable of minimally retarding drug release.Hence, the choice of solid lipid constituents and/or the inclusion of carbosil ® could enable increased emulsification time.
On the whole, prolonged emulsification time is thought to impose gradual droplet formation, consistent absorption and achievement of systemic drug concentration within safety margin.This may explain why the batch 6 with lipid and surfactant blends (35:45:20) had similar anti-inflammatory effect as the reference drug at the 5th hour.Its fast EMT and T50 of 23 min may have led to faster/earlier in vivo clearance before the 5th hour.On the other hand, batch 1 without blend demonstrated higher anti-inflammatory effect at the 5th hour, higher EMT (4 min) and T50 of 31 min.
Post-emulsification drug precipitation test showed absence of drug crystallization.This may connote fortified droplets with drugs dissolved in the oil surrounded by surfactant and cosurfactant layers.

Droplet size, polydispersity index and zeta potential
Droplet size results are as shown on Table 3 and Figures  3 to 6. Droplet size of batches containing SB ranged between 166 to 220 nm, while those containing SB/BIF and surfactant blends recorded droplet sizes of 193 to 238 nm.PDI generally ranged between 0.2 to 0.3, while ZP ranged between -0.6 to -0.9 mV for batches containing SB/BIF blends while that of SB (unblended) had values of -7 to -10 mV.Blending only had a significant (p < 0.05) effect on the droplet sizes of 20:60:20 SSEDDS batches.The observed range of droplet sizes is typical of SEDDS which are reported to have droplet sizes of above 100 nm compared to self microemulsifying drug delivery systems (SMEDDS) that have droplet sizes of less than 100 nm (Bo et al., 2008;Gershanik and Benita, 1996;Rajesh et al., 2010;Shafiq et al., 2007;Niedertquell and Kuentz, 2013).SEDDS may be vulnerable to drug crystallization after a period of contact with aqueous medium while SMEDDS are less vulnerable.Interestingly, results so far did not associate our formulations with drug crystallization in aqueous medium some hours after emulsification.This may suggest potential in vivo gastrointestinal stability of droplets post-emulsification.Drug leakage or crystallization from droplets jeopardizes the ultimate goal of SEDDS since the crystallized poorly water-soluble drug will suffer the same fate of traditional oral formulations.Poly dispersity index describes variations in the size of the droplets, which spans a range of 0 to 1.0.The PDI values of all the batches were reasonable enough to maintain droplet stability.High values of PDI indicate wide difference between large and small droplets.This could predispose to Oswald ripening and drug crystallization in aqueous phase.The negative zeta potential was normal for oil-in-water emulsions.

Effect of refrigeration
The effect of refrigeration was investigated in order to study the effect of low temperature on the stability of the SSEDDS formulations.The results showed that the formulations did not have any form of organoleptic changes or apparent instability.This is an indication that storage at very low temperature may be tolerated.

Loading efficiency
Table 3 shows loading efficiency values of the formulations.Most of the batches had values that fell within 90 to 100%.This means that the drug was well encapsulated within the droplets.

Group
Paw volume oedema (ml ± SD) a and percentage inhibition of oedema (%)

Release profile of indomethacin from SSEDDS
The results of in vitro drug release of indomethacin from SSEDDS are presented in Figures 1 and 2 and Table 3. Blending of the oils did not affect the release pattern in 35:45:20 batches.The prominent feature in the release profiles of the batches was that the T85 was within 30 min, with the exception of batch 4 that was 52 min.In most part, the T85 of batches containing only SB as the lipid component was slightly longer than those that had oil blends.The reverse was the case as per T50 values.For immediate release tablets, it is required that over 85% of drug be released within 30 min (United States Pharmacopeia, 2007).

Antiinflammatory studies
The anti-inflammatory properties of indomethacinloaded SSEDDS formulations are shown in Table 4. Results showed that the 20:60:20 SSEDDS formulated with SB and Tween 65 exerted significantly (p < 0.05) higher anti-inflammatory activity than the reference indomethacin powder at the 1 st ,    at the 1st, 2nd and 5th hours, respectively.In addition, the 35:45:20 SSEDDS formulated with SB and surfactant blends only demonstrated significantly (p < 0.05) higher anti-inflammatory effect over the reference (indomethacin) drug at the 2nd hour.Similarly, the 20:60:20 SSEDDS had significantly (p < 0.05) higher antiinflammatory effect over the 35:45:20 SSEDDS at the 2nd hour.In the treatment of inflammatory conditions, the goal is not only to allay pains but to achieve that promptly within a short time.This is very crucial, for instance, in cancer treatment where excruciating pains require immediate mitigation or temporary arrest.It therefore means that a formulation that can promote pain relief within 1 h should be preferred.Prompt onset of action attributed to improved solubility, absorption and bio-availability was probably the case with 20:60:20 SSEDDS compared to the reference indomethacin powder.It is possible that satisfactory anti-inflammation with reduced dose will reduce adverse effects associated with indomethacin.We posit therefore that enhanced anti-inflammatory effect of the SSEDDS formulations was due to improved aqueous solubility and bioavailability.This could open a window of possibility of adverse effect reduction through SSEDDSbased indomethacin dose reduction.

Conclusion
The SSEDDS formulations were stable and did not undergo drug precipitation in aqueous medium within 4 h.The time to achieve 85% of drug release (T85) was about 30 min for most of the batches.Shea butter-based SSEDDS demonstra-ted significantly (p < 0.05) higher anti-inflammatory effect than unformulated indomethacin powder.Therefore Shea butter and its blend with Bos indicus lipid have proved their suitability as lipid constituents of SSEDDS that improved the solubility of indomethacin and enhanced its anti-inflammatory effect.
shown are mean ± SD, a n = 6; Values in parenthesis are percent inhibition of oedema calculated relative to control; a indomethacin SSEDDS formulated with SB, surfactant and cosurfactants; b indomethacin SSEDDS formulated with blends of oil (SB and BIF) and surfactants (Tween 65/80).

Figure 1 .
Figure 1.Cummulative amount of indomethacine release against time for SEDDS formulated with varying ratios of shea butter (SBO), surfactant and co-surfactant

Table 2 .
Quantities of ingredients for the formulation of 350 mg of SSEDDS containing 15 mg of Indomethacin.

Table 3 .
Some properties of the formulated SSEDDS.