Introduction

Oral drug delivery continues to be the preferred route of administration, however majority of
newly discovered and existing drugs administered by oral route frequently encounter
bioavailability problems due to several reasons like poor dissolution,unpredictable
absorption, inter and intra subject variability, and lack of dose proportionality. According to
BCS classification the class цdrug have low solubility and their absorption is dissolution rate
limited. Several strategies have been adopted for enhancing the dissolution behaviour of
insoluble drug by various techniques

In recent years, much attention has been paid to self microemulsifying drug delivery
system(SMEDDS), which have shown lots of reasonable success in improving oral
bioavailability of poorly soluble drugs. SMEDDS are usually composed of mixture of oil,
surfactant or co-surfactant and are capable of forming fine oil in water emulsion upon
gentleagitation provided by the GIT motion.

However, traditional preparation of SMEDDS are usually prepared in the liquid state which
have certain disadvantage such as high production cost, low drug incompatibility and
precipitation. Then incorporation of liquid SMEDDS in to a solid dosage form is compelling
and desirable.

Bosentan was selected as the model compound to represent class цcompounds according to
biopharmaceutical system having higher molecular weight (569.64). Bosentan belongs to
class of drugs known as endothelin receptor antagonists (ERA’S) and has slightly higher
affinity for ETA receptorthan ETB. It has been used for the treatment of pulmonary
hypertension. Absolute bioavailability of bosentan is 50%. The low bioavailability of
bosentan is mainly attributed to its poor aqueous solubility, so it is necessary to find a proper
approach that will increase drug solubility and its dissolution rate

The objective of the current investigation was to develop a novel solid SMEDDS formulation
containing bosentan to enhance its dissolution characteristics, bioavailability and stability as
compared to conventional dosage form.
Materials and methods

1) Materials:

2) Solubility studies:
The solubility of bosentan in various oils, surfactants, and co-solvent was determined.
Briefly an excess amount of bosentan was added to the various oils, surfactant and
cosurfactant and vortexed to facilitate was allowed to equilibrate for 48h in a water
bath. The equilibrated sample was centrifuged. The undissolvedbosentan settle down
at the bottom. The supernatant was taken out and diluted with methanol for
quantification of bosentan by UV spectrophotometer at λmax 273.6 nm.

3) Construction of ternary phase diagram:

To find out concentration range of components for the existing range of
microemulsion, pseudo ternary phase diagram were constructed using water titration
method. For each phase diagram surfactants and oil with respective concentration
ratio (1:9 to 9:2) were vortex mixed in 20 ml test tube for 15 min to form
homogeneous mixture. Then using water titration method titration was carried out
with distilled water with moderate stirring and mechanical shaking till turbidity as
end.

4) Preparation of liquid SMEDDS formulation :

Variable proportion of oil, surfactants and co-solvent were added into a glass vial and
bosentan was added in mixture and the mixture was facilitated to the solubilisation
using cyclomixer. After that mixture was placed for sonication using probe sonicator
for 15 min. then mixture was allowed to equilibrate for 48hr in water bath

5) Preparation of solid SMEDDS:

Aerosil 200(500mg) was suspended in 100ml ethanol by magnetic stirring. The liquid
SMEDDS (1gm) was then added with constant stirring at room temperature for 15
min to obtain a good suspension of adsorbent. The suspension was spary dried with
 spary dryer( labutima) under the following conditions: inlet temp 700C, outlet temp
450C, and feeding rate of suspension 1.5 ml / min.

6) Characterization of solid SMEDDS:

a) Droplet size of emulsion:

The average droplet size and polydispersity index of Self microemulsifying system of
emulsions of solid SMEDDS were assessed by Beckman coulter (Nano Malvern
Instruments, U.K.) at 250C . A 560 mg of S-SMEDDS was weighed add in upto 100
ml distilled water. Then beaker was placed on magnetic stirrer at 200 rpm for 5 min.
Then clear and transparent samples were taken for globule size determination. All
studies were repeated three times and the average values were used.

b) Morphological analysis of SMEDDS:

The outer macroscopic structure of the solid SMEDDS was investigated by Scanning
Electron Microscopy with a FEI Sirion-200 scanning electron microscope (FEI, the
Netherlands), operating at 10 kV. The sample was fixed on a SEM-stub using
double-sided adhesive tape and then coated with a thin layer of gold.

c) Solid state characterization of solid SMEDDS:

The physical state of Bosentan in solid SMEDDS was characterized by the
differential scanning calorimetrythermogram analysis. The samples (about 3.00 mg)
were placed in standard aluminum pans, and dry nitrogen was used as effluent gas.
All samples were scanned at a temperature ramp speed of 5 0C/min and the heat flow
from 0 to 1800C. Furthermore, X-ray powder scattering measurements were carried
out with an X’Pert PRO diffractometer (PAN alytical, the Netherlands). A voltage of
40 kV and a current of 40 mA for the generator were applied with Cu as the tube
anode material. The solids were exposed to a CuKα radiation, over a range of 2 angles
from 100–400, at an angular speed of 20 (2h)/min, a sampling interval of 0.020
 7) In vitro dissolution studies:

In vitro dissolution of tablets of sold SMEDDS was carried out by using dissolution
test apparatus USP XXII(paddle type), 900ml of phosphate buffer pH 6.8 and 0.1 N
HCl of pH 1.2 as a medium at 37±0.50c. The speed of the paddle was adjusted to 50
RPM . An aliquot of 5ml was withdrawn by means of a pipette at an interval of
every 5 min for a period of 60 min. same quantity of fresh medium was added to
maintain the sink condition. The aliquots were assayed spectrophotometrically at a
maximum of 271 nm and 274nm respectively for phosphate buffer pH 6.8 and 0.1 N
HCl of pH 1.2 by using shimadzu UV-1800 spectrophotometer. Same procedure was
carried out for marketed tablet.

In vivo study:

The in vivo study of two formulations of bosentan, an optimized solid SMEDDS and control
formulation was performed in rats. Male Sprague-Dawley Rat weighing 280±20g were fasted
for 10-12 hr prior to the experiments but were allowded free access to water. 12 rats were
divided into two groups. The rats in each group were administered with drug powder 2mg/kg
or 20mg os solid SMEDDS equivalent to dose 2mg of drug formulated in form of suspension.
Then 0.25 ml of blood collected from the tail method. 0.1 ml of plasma separated by
centrifuging blood sample stored at -200C until further analysis. Mix 100µL plasma with
50µL ACN:5 mM ammonium acetate : acetic acid (10:90:1), add 750 µL methanol. Mix the
supernatant with 2ml 50mM ammonium acetate buffer (pH10), wash with 2ml 20mM
phosphoric acid and then wash with 2.1 ml methanol: water (20:80). Evaporate the eluate to
dryness, reconstitute the residue with 150 µL ACN :5 mM ammonium acetate: acetic acid
(10:90:1), inject an aliquot.

8)

Result and discussion:

1) Solubility study:
Bosentan is an antihypertensive drug comes under the class ц according to BCS
classification system which has the lower solubility and higher permeability. For
 development of solid SMEDDS we screened the various oil phase, some of them are
LCT, MCT and SCT. Solubility studies clearly indicate that amongst the various oil
phase that were screened, capmul MCM could solubilize the 80mg per gram of
capmul MCM. Furthermore tween 20 was selected as a surfactant for its good
solubility. The improvement of drug loading and in the formation of spontaneous fine
emulsion.

2) Construction of pseudo ternary phase diagram:

Surfactants screening was performed on basis of efficiency of ease emulsifying
ability with selected oil. The maximum area of microemulsion formation was desired
for confirmation of best surfactant amongst the surfactants screened.Selection of
surfactant was done on the basis of Transmittance of resultant emulsion of surfactant,
rate of emulsifying ability of surfactant, highest microemulsion area forming ability
of surfactant with selected oil (Capmul MCM) and highest solubility of Drug in
surfactant and their correlation with each other. From the solubility study it was
found that Cremophor EL and Tween 20 have highest solubilising capacity for
Bosentan 71.48 mg/ml and 66.41 mg/ml respectively.Tween 20 showed highest
microemulsion region followed by CremophorEL. Hence Cremophor EL and Tween
20 were selected as surfactants for further studies.

3) Characterization of Solid SMEDDS

a) Droplet size of emulsion:
 The average droplet size and polydispersity index of reconstituted microemulsion of
S-SMEDDS was found to be 157 nm and 0.32 respectively. The droplet size of the
microemulsion from the S-SMEDDS was slightly increased, but with lack of
statistically significant difference, compared to the liquid SEDDS (P < 0.05). From
these results, adsorbing the liquid SMEDDS on Aerosil 200 by spray-drying did not
seem to have a remarkable effect on droplet size. The S-SMEDDS preserved the self-
emulsification performance of the liquid SMEDDS.
b) Morphological analysis:
From SEM study the surface of S- SMEDDS powder clearly shows the adsorption of
liquid preconcentrate on Aerosil 200 Fig 25 (c). The Fig 25 (a) indicates the
crystalline nature of drug. The crystalline state of bosentan has converted to
amorphous form or it may be molecularly dispersed. The spray drying has achieved
some spherical shape to S-SMEDDS powder.

c) Solid state characterization:

DSC curves of pure Bosentan, S-SMEDDS of Bosentan using Aerosil 200 and
Maltodextrin (1:1) and the S-SMEDDS as shown in Fig (a). Pure Bosentan showed
one sharp endothermic peak at temperatures between 101 and 124 0C at 114.20 C . No
obvious peaks for Bosentan and liquid preconcentrate (LF10) were found in the S-
SMEDDS of Bosentan Fig(c) and (e). It can be concluded that the melting behavior of
the Bosentan was changed due to its solubilisation of in liquid preconcentrate which is
adsorbed.From X-ray powder diffractograms shown in Fig, the internal physical state
of Bosentan in the solid SMEDDS was further verified. No obvious peaks
representing crystals of Bosentan were seen for the solid SMEDDS (fig). Therefore, it
could be concluded that Bosentan in the S-SMEDDS was in the amorphous or
disordered crystalline phase of a molecular dispersion state in the adsorbed liquid
preconcentrate mixture.

4) In vitro dissolution:
In the self-emulsifying systems, the free energy required to form an emulsion was
very low, thereby allowing spontaneous formationof an interface between the oil
droplets and water. It is suggestedthat the oil/surfactant/cosurfactant and water
phaseseffectively swell, decrease the oil droplet size and eventually increasethe
release rate. In vitro drug release studies were performedfor solid SMEDDS and
bosentan tablet, and areprofiled in Fig. 8. As the emulsification time is below 30 as
maximumpercentage of the drug released within 2 min from the solidSMEDDS;
however, the dissolution studies were conducted for 1 h tosee the variance or
occurrence of precipitation over a period of time. In the present investigation drug
release profile of solid SMEDDSin buffer solution showed that the formulation had
higher drugrelease profile than the bosentanpowder, ensuring that the solid SMEDDS
preserved the improvement of in vitro dissolution of liquid SMEDDS.