The low aqueous solubility and the resultant bioavailability of poorly water soluble drugs have made their oral delivery a major challenge to drug formulators. This is because when they are administered orally the dissolution rate in the gastrointestinal tract becomes the rate limiting step in the absorption (and hence bioavailability) from the gastrointestine (Hiroshi et al., 2005). In addition since their dissolution rate is affected by diet and the flow of bile secretion these drugs are usually subject to widely variable oral bioavailability (Cakaloglu et al., 1993). Incidentally several research efforts have been geared towards the improvement of the absorption and bioavailability of these poorly water soluble drugs/insoluble drugs. These include chemical modification, lipid and surfactant dispersions, liposomes, niosomes and self emulsifying oil formulations (SEOF)/ Self Emulsifying Drug Delivery System (SEDDS) (Craig et al., 1993). Of all these the most promising technology to improve the rate and extent of these poorly water soluble drugs, which has hitherto drawn significant scientific curiosity and effort is Self Emulsifying Oil Formulations (SEOF). SEOF or SEDDS is a mixture of oil(s), surfactant(s) and if necessary a solubiliser. Self emulsification is initiated under gentle agitation following contact with aqueous phase and forms a thermodynamically stable O/W microemulsion with particle diameter of 100 nm or less (Odeberg et al., 2003; Devani et al., 2004). They are reputed to improve the oral bioavailability of poorly water soluble drugs (Shah et al., 1994) which is accomplished by rapid selfemulsification thus yielding fine O/W emulsions within which the lipophylic or hydro-lipophylic drug is present in solution or solubilised form, in small oil droplets (Constantinides, 1995). Moreover these small oil droplets occupy large interfacial area, thus facilitating drug diffusion into intestinal fluids (Erica et al., 2005).
Metronidazole, a poorly water soluble drug was used in our study as the model
drug to improve its aqueous solubility, possibly optimise its amount per dose
thus creating the opportunity for increase in its bioavailability, by incorporating
it into vegetable oil-based SEDDS. These edible oils were chosen because of
their inexpensiveness, availability and biocompatibility. Palm Kernel Oil (PKO)
and Palm Oil (PO) extracted from the hard seeds and fruits of Elaeas garbonensis
are glycerides belonging to the homolipid family of lipids. Oral lipid drug
delivery systems are usually designed to increase solubility and bioavailability
of drugs that belong to class ii and iv of the biopharmaceutical drug classification
system (ODriscoll, 2002). Medium chain triacyl glycerols, though useful
drug carriers, seem to have no significant pharmacological activity. They are
subject to intraluminal hydrolysis and are mainly absorbed as free fatty acids
(Bach and Babayan, 1982). Intraluminal digestion uptake by the mucosal cells
and transport of medium chain and long chain triacyl glycerols to the systemic
circulation are very distinct (Milan and Stanislav, 2001; Yhi-Fu, 1987). Hence
triacyl glycerols possess potential usefulness in lipid drug delivery.
The objective of this study was to formulate metronidazole, a poorly water soluble drug into SEDDS using palm kernel oil and palm oil with the hope that its aqueous solubility will be improved.
MATERIALS AND METHODS
This research was carried out in the Drug delivery unit, Department of Pharmaceutical Technology and Industrial Pharmacy, University of Nigeria, Nsukka in 2006.
Metronidazole (Evans Nigeria), Tween 65 (Fision Scientific, England), palm kernel oil, palm oil (locally sourced and further purified in our Laboratory), HCl (BDH). This research work was carried out in our Drug Delivery laboratory, Pharmaceutical Technology and Industrial Pharmacy, University of Nigeria, Nsukka.
Purification of Palm Kernel Oil (PKO) and Palm Oil (PO): A 2%w/w suspension
of activated charcoal in oil was heated in a beaker at 80-90°C for an hour.
Thereafter the suspension was vacuum-filtered using Burkners funnel. The
purified oil was stored for further use.
Drug solubility in oil: This experiment was carried out to determine the maximum amount of metronidazole that could be dissolved in the oil without precipitation. Exactly 1.75 g of metronidazole was weighed out into a 25 mL beaker. Aliquots of the oil were introduced and each time stirred at 37±1°C. The minimal amount of oil needed to completely solubilise the whole drug was noted.
Preformulation isotropicity test: Twenty four batches of SEDDS consisting
of oil:surfactant ratios of 30:70, 40:60 50:50, 60:40, 65:35, 70:30 were prepared
as shown in Table 1. The appropriate quantities of the two
ingredients were weighed out and melted with stirring at 50°C. The mixture
was then stored for 5 h at ambient temperature and later evaluated for isotropicity.
Formulation of SEDDS containing metronidazole: Formulation was limited
to only the SEDDS that passed the isotropicity test. Palm oil, palm kernel oil
and different ratios of their admixtures were respectively used. Appropriate
quantity of metronidazole was weighed and introduced into a beaker containing
weighed amount of oil and Tween 65 and stirred for 10 min on a water bath maintained
at 50°C (Hiroshi et al., 2005). Table 2 shows
the various quantities of the materials used in the formulation of drug-loaded
SEDDS of target weight 525 mg.
||Formulation of SEDDS for isotropicity test
||Formulation of drug-loaded SEDDS
|PO = Palm Kernel Oil, PKO = Palm Kernel Oil
Postformulation isotropicity test: The above prepared SEDDS were stored for 72 h at ambient temperature and observed for phase separation, drug precipitation and isotropicity. The SEDDS that passed this test were used for further tests.
Emulsification time: Five hundred and twenty five milligram of SEDDS was syringed into a 250 mL beaker containing 0.1 N HCl. The beaker was then mounted on a hot plate-magnetic stirrer assembly. The stirrer speed was maintained at approximately 50 rpm. The time for complete emulsification was visually noted.
Encapsulation of SEDDS: Five hundred and twenty five milligram of SEDDS was weighed out and introduced into a 500 mg capsule and stored for further use.
Drug release studies: A magnetic stirrer-beaker-hotplate assembly was used. Each encapsulated SEDDS was introduced into the beaker containing 500 mL of 0.1 N HCl and dissolution run at a rotation speed of 100 rpm at 37±1.0°C. At predetermined time intervals 5 mL samples were withdrawn and assayed spectrophotometrically for metronidazole content at 277 nm.
Absolute drug content: Five hundred and twenty five milligram of SEDDS was withdrawn and emulsified in 1 L of 0.1 N HCl and thereafter assayed spectrophotometrically for metronidazole content.
RESULTS AND DISCUSSION
Drug solubility in oil: Exactly 39.4 and 37.6 g of palm kernel oil and palm oil respectively were observed to effect complete dissolution of 1.75 g of metronidazole powder without precipitation. Thus the solubility of metronidazole in both oils were 4.441 and 4.654% w/w, respectively. The development of microemulsion systems for poorly water soluble drugs is critical and drug loading per formulation is a very critical design factor that depends on drug solubility in various formulation components (Natesan et al., 2004). The solvent capacity of the oil is a crucial factor, although the ability of an oil to accommodate large amount of hydrophobic drug in solution can be improved in the presence of cosurfactants and/or cosolvents. The solvent capacity of the drug for the two oils is closely similar. However, further modification of the oils may likely improve drug solubility in them. Such hydrolysed or modified vegetable oils are known to form good emulsification systems with several surfactants approved for oral administration and exhibit better drug solubility properties (Kimura et al., 1994; Hauss et al., 1998).
Preformulation isotropicity test: It was observed that formulations
containing oil:surfactant ratios of 60:40, 50:50 and some of 30:70 and 40:60
were isotropically stable (Table 3), whereas those of 70:30,
65:35 and some of 30:70 and 40:60 experienced varying degrees of phase separation.
This test is often used to determine the oil:surfactant ratio that will confer
thermodynamic stability to the formulation. Because of the unique physicochemical
properties of oils and surfactants this preformulation evaluation is critical
and expedient to avoid posformulation drug partitioning consequent upon phase
separation. Different oils may likely have varying oil: surfactant ratios that
would impart stability to the SEDDS. The oil: surfactant ratio of 60:40 is preferable
to 50:50 and the others with higher surfactant concentrations. Such higher surfactant
(Tween 65) concentration may predispose to longer emulsification times. In addition
low surfactant concentration is preferable because of the possibility of potential
toxic effects with high concentrations. Tween 65 our model surfactant is non-ionic
and therefore less affected by ionic strength and PH changes (Kawakami and Oshikawa,
2001). It has been reported that the concentration of surfactants that will
form stable SEDDS formulation range between 30-60% w/w (17). As shown in Table
3, formulations that maintained isotropicity had surfactant ratios that
fall within ref (Gursoy and Benita, 2004), in addition to batch 1C having an
oil:surfactant ratio of 30:70.
||Preformulation isotropicity test result (comprising of those
that passed the test)
||Post-formulation isotropicity/stability test results
This ratio contains 70% w/w of surfactant which is quite high. All batches
having this ratio were not isotropic with the exception of the aforementioned
1C. The reason is probably due to the high PKO:PO ratio of 75:25. PKO at temperatures
below 25°C assumes a semisolid consistency unlike PO. This therefore in
combination with Tween 65 which is also semisolid in nature will impart better
stability into the SEDDS mixture than lower PKO: PO ratios. Batches 1C and 2C
were the only two batches having oil: surfactant ratios of 40:60 that were stable;
this is also probably because of their high PKO:PO ratios of 75:25 and 60:40,
Post formulation stability test: The drug-loaded SEDDS were further
stored for 72 h. The rationale behind this test was to ascertain if the presence
of metronidazole would introduce any instability. Results obtained (Table
4) shows that batches containing palm kernel oil showed phase separation.
The batches that retained homogeneity i.e., those containing palm oil, palm
kernel oil or their admixtures were then stored for further tests. The loss
of stability by some of the above SEDDS in the presence of metronidazole could
be due to a complex interaction outcome of oil and surfactant in the presence
of the drug. The confirmation of SEDDS stability in the presence of drug is
needed to ensure formulation thermodynamic stability. The efficiency of addition
of drugs into a SEDDS is specific in each case depending on the physicochemical
compatibility of the drug/system (Gursoy and Benita,2004).
||Some drug release parameters
More often the drug interferes with the self-emulsification process to a certain
degree leading to a change in the optimal oil/surfactant ratio (Gursoy and Benita,
2004). Every oil:surfactant ratio often has a certain solvent capacity for drug,
beyond which drug precipitation or phase separation may result. Furthermore
stability testing of SEDDS is an evaluation strategy that assesses their vulnerability
to environmental factors during storage. The SEDDS containing palm oil and palm
oil/palmkernel oil admixtures maintained stability without phase separation.
Some reporters (Subramanian et al., 2004) have established the formulation
advantage of employing mixtures of oils in microemulsions. Incidentally the
oils used in this research that imparted stability to the SEDDS are cheap and
Emulsification Time (EMT): Table 5 shows the emulsification
time results of metronidazole SEDDS prepared with PO and PKO-PO admixtures at
the stated oil:surfactant ratios. The SEDDS containing oil:surfactant ratios
of 50:50 recorded longer EMT than the rest. Also those with the ratio of 40:60
emulsified slightly at a longer time than those of 60:40, thus still emphasizing
that higher surfactant (tween 65) concentration would lead to higher emulsification
||Dissolution studies result: Graph of % metronidazole released
vs. time in 0.1 N HCl
At a PKO:PO admixture ratio of 75:25 fastest EMT of 7 sec was recorded followed
by that of 0:100 ratio at the same oil:surfactant ratio of 60:40. It seemed
as if admixing the oils at this oil:surfactant ratio promoted faster emulsification
time than using PO alone. The EMT of batches 3B, 4D and 6D were not affected
by oil admixtures as much as surfactant concentration. The high surfactant concentration
of tween 65 whose semi solid nature at room temperature must have imbued a higher
viscosity. SEDDS with higher viscosities are likely to experience lower emulsification
rates. Carvedilol SEDDS (Lanlan et al., 2005) showed a decrease in EMT
as the tween 80 content increased from 30-40% and then increased as the tween
80 content increased from 40-60%. Emulsification rate is known to be an important
index for emulsification efficiency assessment (Pouton, 1985). Infact 2 min
(Khoo et al., 1998) has been reported as an evaluation index in the emulsification
process. With the exception of 4D the rest of the batches emulsified in less
than 2 min. Apart from viscosity, admixing of oils, the free energy of the system
has been implicated as one of the factors that affect the EMTs of SEOFS and
SMEOFS (Lanlan et al., 2005).
Drug release studies: Figure 1 shows the release profile
of metronidazole from the four batches, namely, 1C, 2C, 3B, 4C, 4D, 6C and 6D.
The onset of drug release was fast in most of the formulations but faster in
batches 1C, 4C and 6D, with t50 of 0.6, 4.4 and 3.9 min, respectively
and t85 of 21, 11.5 and 8 min, respectively (Table
5). 4C with least emulsification time of 7 sec released 50% of drug in less
than 5 min. Batch 2C with oil:surfactant ratio of 40:60 had the longest t50
of 23 min probably because of its high surfactant concentration. However this
was not consistent in batch 1C with the same oil: surfactant ratio. According
to US-FDA guidance for immediate release product 85% (t85%) of labelled
amount of drug should be released within 30 min of study. Based on this almost
all the batches conformed to this as their t85 were less than 30
min. Therefore it is perfect for them to be adjudged as having had optimum drug
release thus justifying SEDDS as a formulation option for improving the release
and anticipated bioavailability of metronidazole.
Absolute drug content: The drug contents of each of the SEDDS batches are as shown in the table below. They were within compendial limits (95-105%) for metronidazole, with the exception of 1C and 2C.
In conclusion it is safe to say that, from our studies palm oil and palm kernel oil and their admixtures are potential adjuvants in the formulation of metronidazole SEDDS for improved drug dissolution and bioavailability.