INTRODUCTION:

In recent years, much attention has been focused on lipid based formulations for delivering Biopharmaceutic Classification System (BCS) class II drugs, which suffer limited oral bioavailability, high intra- and intersubject variability and lack of dose proportionaliy.¹ Self-emulsifying system (SES) is one of the most popular and commercially viable approaches for the delivery of such drugs which exhibit solubility dissolution-rate-limited absorption.^2-6 Self emulsifying/ self-micro drug delivery systems (SEDDS/SMEDDS) are isotropic mixtures of lipid, surfactant, co-surfactant and drug substance that spontaneously form a fine oil-in-water emulsion when exposed to aqueous media under gentle stirring.^7,8,9 The digestive motility of stomach & intestine provide the agitation required for self emulsification in vivo.

The spontaneous formation of emulsion presents the drug in a dissolved form and the resultant small droplet size provide a large interfacial area for diffusion. Solidification of liquid systems has been a challenge that has attracted wide attention due to handling difficulties and machinability and stability problems that are often encountered with liquids.^10-13

In this study, an attempt an attempt was made to improve the solubility and in vitro dissolution of nifedipine by formulating it as SMEDDS. Nifedipine, Dimethyl 2, 6-dimethyl-4-(2-nitrophenyl)-1, 4 dihydropyridine - 3,5-dicarboxylate, has poor aqueous solubility resulting in low and often irregular bioavailability.^14,15,16 The present work provided nine folds improvement in dissolution rate for nifedipine when formulated as self emulsifying drug delivery systems. Self emulsifying mixture that combines good self emulsifying properties, acceptable solubilization of nifedipine and optimum surfactant, co-surfactant/co-solvent composition was selected, evaluated for droplet size, stability, dissolution, permeability and a Solid SMEDDS was prepared using Magnesium Aluminium Silicate as adsorbent. The solid SMEDDS was further evaluated by in –vitro dissolution studies, non everted sac permeability studies and characterized by Scanning electron microscopy (SEM) and Differential scanning calorimetry (DSC).

MATERIALS AND METHODS:

Materials:

Nifedipine was received as a gift sample from Shilpa Medicare Limited, Raichur, Karnataka. Corn oil was obtained from Acros Organics, New Jersey, USA. Tween 80, Tween 60, Polyethylene glycol (PEG) 400 and Magnesium aluminium silicate were obtained from CDH, New Delhi. Propylene glycol was obtained from Qualikems Fine chemicals Pvt. Limited, New Delhi. Labrasol, Transcutol P, Lauroglycol 90, Labrafil M 1944 Cs, Labrafac CC, Labrafac PG and Capryol 90 were gift samples from Gattefosse India Pvt. Limited, Mumbai. Capmul MCM EP and Captex 100 were gift samples from Abitec Corporation, Janesville, WI. All the reagents and solvents used were of analytical grade.

Methods:

Excipient Screening—Saturation Solubility Studies:

The saturation solubility of nifedipine was evaluated in various oils, surfactants, and co surfactants/ co solvents. In this study, an excess amount of nifedipine was added to 5 ml of each of vehicle in amber colored volumetric flasks and mixed using a vortex mixer (Hicon) to facilitate drug solubilization. The mixture heated to 40°C in a water bath shaker (Hicon) under continuous stirring for 6 hours. The mixture was then kept at ambient temperature for 24 h to attain equilibrium. The equilibrated sample was centrifuged (Remi RM 12C Centrifuge) at 4,000 rpm for 10 min to remove the undissolved drug which was then filtered off and the liquid was assayed through UV spectrophotometer (Shimadzu UV-1700) at 236.5 nm. (Fig. 1)

Fig.1:Solubility of Nifedipine in various oils*, surfactants** and cosurfactants/ cosolvents***

Determination of optimized composition of liquid SMEDDS (Preformulation isotropicity test):

For the determination of optimized composition of SMEDDS, different ratios of the components i.e. oil, surfactant and co surfactant/ co solvents were tested for the self-micro emulsification. The oils, surfactants and co surfactants/ co solvents chosen have high solubility for nifedipine as shown by solubility studies. The region of self-micro emulsification was assessed by visual examination. Appearance of bluish transparent color gives the indication of self- micro emulsification. The combinations (0.1 ml) were introduced into 100 ml water in a glass beaker at 37^oC and the contents were blended gently using a magnetic stirrer (Remi equipments Ltd) bar. The tendency to emulsify spontaneously and also the development of emulsion droplets were monitored. The liquid SMEDDS were optimized on two parameters, fixed ratio of surfactant and co surfactant in combination with varying amount of oil and fixed amount of oil and varying ratios of surfactant and co surfactant. All the trials were carried out in duplicate, with similar observations being made between repeats. The use of single surfactant did not give much satisfactory results so the SMEDDS were also developed using combination¹⁷ of surfactants (Table 1). Percentage transmittance of SMEDDS was obtained by using distilled water as blank at 650 nm by UV spectrophotometer (ShimadzuUV-1700).

Table 1: Preformulation isotropicity test (*surfactant as Labrasol only)

Batch Code	S: CoS	Labrasol : Tween 60 (1:1) % w/w	Propylene glycol % w/w	PEG 400 % w/w	Captex 100 % w/w	Corn oil % w/w	Capryol 90 %w/w	Transcutol P	% Transmittance
A	1:1	22.5&22.5	-	45	10	-	-	-	99.5
B	2:1	30&30	30	-	10	-	-	-	99.7
C	1:1	22.5&22.5		45	-	10	-	-	98.4
D	2:1	30&30	-	30	-	10	-	-	98.2
E	1:1	22.5&22.5	-	45	-	-	10	-	99.4
F	1:2	15&15	-	-	-	-	10	60	99.3
G	2:1	30&30	-	-	-	-	10	30	99.4
H	1:1	45*	45	-	-	10	-	-	99.2
I	1:2	30*	60	-	-	10	-	-	99.1
J	1:2	30*	-	60	-	10	-	-	99.1
K	1:3	22.5*	-	67.5	-	10		-	99.3
L	1:2	30*	-	60	-	-	10	-	99.2

Preparation of nifedipine liquid SMEDDS:

Only those formulations having % transmittances greater than 99% were selected for preparation of Liquid SMEDDS. In the SMEDDS, the content of Nifedipine was maintained constant (10 mg dose). Components of the SMEDDS i.e. oil, surfactant and co surfactant, were weighed into a glass vial and vortexed using vortex mixer until unit dose (10 mg) of Nifedipine get dissolved completely and a transparent and monophasic solution formed. (Figure 2)

Fig. 2: Nifedipine liquid SMEDDS (left) and Nifedipine micro emulsion (right) obtained by dilution

Evaluation of Nifedipine SMEDDS^2-5

The prepared SMEDDS were evaluated by following methods:

Stability studies:

The physical stability of a lipid based formulation can be adversely affected by precipitation of drug in excipient matrix. Poor formulation physical stability can lead to phase separation of excipient and affect formulation performance. So following stability studies were performed on prepared SMEDDS.

Phase separation studies:

1 ml of SMEDDS was added to 5 ml distilled water at 25^oC. The mixture was vortexed for 2 min and then stored for 2 hrs and any phase separation was visually observed.

Heating cooling cycles:

Those formulations which passed phase separation studies were further studied. Six cycles between refrigerator temperature (4^oC) and 45^oC with storage at each temperature of not less than 48 hrs was studied. The formulations stable at these temperatures were subjected to centrifugation test.

Centrifugation studies:

The formulations were centrifuged (Remi RM 12C Centrifuge) for 3500 rpm for 30 minutes. The formulations which passed stability test were further studied.

Droplet size determination:

SMEDDS was diluted with double distilled water. The average globule size and polydispersity index of Nifedipine microemulsion were determined by the zeta sizer (Malvern instruments) from IIT, New Delhi. (Fig. 3 and Fig. 4)

Fig. 3: Particle size analysis of Formulation A

Fig. 4: Particle size analysis of Formulation B

Drug content:

Drug content analysis was done by dissolving samples equivalent to 10 mg of nifedipine in 10ml of methanol. This solution was then kept for one hour. The solution (10μg/ml) was assayed through UV spectrophotometric method at 236.5 nm.

Percentage Transmittance studies:

Percentage transmittance of SMEDDS was obtained by using distilled water as blank at 650 nm by UV spectrophotometer (ShimadzuUV-1700).

Coalescence studies:

In- vitro dissolution of optimized liquid SMEDDS was carried in distilled water (500 ml, temperature 37± 0.5^oC) using USP 2 apparatus (Hicon) at 50rpm for 24 hrs and SMEDDS were observed visually at different time intervals for clarity or turbidity.

Conversion to Solid Intermediates of Self –Micro Emulsifying Formulation^10,11,12,13

The optimized nifedipine liquid SMEDDS was converted into free flowing powder by adsorption of liquid SMEDDS onto solid carriers^10,11. The solid carrier used was Magnesium Aluminium Silicate which has high surface area, good adsorption & high disintegration characteristics. SMEDDS was adsorbed uniformly till it formed free flowing powder. Adsorbed SMEDDS was sifted with mesh no. 44 and weighed amount of blend for unit dose (10 mg) was filled in size “00” capsule.

Solid state characterization of nifedipine solid SMEDDS

Differential Scanning Calorimetry (DSC)¹⁸

Thermograms of nifedipine and Solid SMEDDS were obtained using DSC at NIPER, Mohali and IIT New Delhi.

Morphological Analysis of Solid SMEDDS:

Scanning Electron Microscopy (SEM)

The outer macroscopic structure of the nifedipine and nifedipine solid SMEDDS were investigated by SEM at IIT New Delhi.

Stability studies with SMEDDS:

The ageing studies¹⁹ were helpful in finding out the physico-chemical stability of SMEDDS. The stability studies of nifedipine loaded SMEDDS was assessed by keeping them at ambient temperature for 90 days. Samples were removed at 0, 30, 60 and 90 days of interval and checked for appearance and drug content.

In-Vitro release:

The release of drug from liquid SMEDDS formulation and solid intermediates filled in capsules was determined using USP apparatus 2 at 37± 0.5°C and a rotary speed of 50 rpm. The dissolution medium used was 0.1 N HCl. Samples equivalent to 10 mg of Nifedipine were taken and filled in “size 00” capsules. At specified times, 5ml samples were withdrawn, filtered, suitably diluted and assayed by the UV spectrophotometric method at 236.5 nm.

Non Everted Sac Permeability studies²⁰

The permeation studies were done by using chicken intestinal segment obtained from slaughter house. For the study, intestinal segment of 5 centimeters was chosen and separated. It was rinsed with isotonic saline (37^oC) until the outlet solution was clear. Using the aeration pump, the intestinal segments were purfused at a flow rate of 1-2 bubbles/min in Tyrode’s solution. 1.5 milliliters of tyrode’s solutions (0.1mg/ml) of nifedipine, liquid and solid SMEDDS were filled in the normal sac (mucosal side) and both ends of sac were ligated tightly. These sacs were then immersed in 40 ml of tyrode’s solution in conical flasks. The medium was prewarmed and preoxygenated. The conical flasks were kept in water bath shaker at 37^oC and bubbled with CO₂/O₂ mixture gas periodically. The nifedipine transported was measured using UV spectrophotometric method at 238 nm. The apparent permeability (P_app), in cm/sec can be calculated as

P_app = (V_A/Area× Time). [(drug)_acceptor/(drug)_donor]

Where V_A is Volume of acceptor well, area is surface area of intestinal membrane (2πrh + 2πr²) and time is total transport time i.e. 4500 seconds

Model independent approach for dissolution profile comparisons (f₁/f₂ test)²¹

A simple model independent approach using difference factor (f₁ ) and a similarity factor (f₂ ) was applied to compare dissolution profiles and permeation profiles of liquid and solid SMEDDS. The difference factor (f₁ ), percent (%) difference between the two curves at each time point is calculated as

f₁ = {[∑_t=1ⁿ | R_t-T_t |]/[∑_t=1ⁿ R_t]} ×100

where n is the number of time points, R_t is cumulative percentage dissolution /permeation values for liquid SMEDDS at time t and T_t is cumulative percentage dissolution /permeation values for solid SMEDDS at time t when comparing dissolution and permeation profiles respectively.

The similarity factor (f ₂) measures of the similarity in the percent (%) dissolution between the two curves and is calculated as

f₂ = 50×log {[1+ (1/n) ∑_t=1ⁿ (R_t-T_t)²]^-0.5 *100}

RESULTS AND DISCUSSION:

The objective of solubility studies is to identify the suitable oil, surfactants and co surfactants/ co solvents which have good solubilizing capacity for nifedipine. Based on the results of solubility studies various combinations of oil, surfactant and co-surfactant were prepared which were tested through isotropicity studies. The formulations with a surfactant combination were also prepared. Those formulations in which percentage transmittance, which is an indication of micro emulsion region, was greater than 99% (Table 1) were further studied. The effect of drug incorporation on all the combinations was observed and nifedipine liquid SMEDDS thus prepared were evaluated. Formulations A and B passed all stability tests. [Table 2]

Formulation B was selected for further studies as it had lower values of average droplet size (Fig. 4) than formulation A (Fig. 3). During coalescence studies, Formulation B nifedipine SMEDDS emulsified readily and transparency was maintained for 24 hours. The SMEDDS were classified as grade A as it formed bluish transparent emulsion within one minute. Thus, it will remain as nanoemulsion when dispersed in GIT.

Table 2: Stability studies with nifedipine liquid SMEDDS

Formulation Code	Phase separation	Heating Cooling	Centrifugation
A	ü	ü	ü
B	ü	ü	ü
E	ü	ü	×
F	ü	×	-
G	ü	ü	×
H	×	-	-
I	×	-	-
J	×	-	-
K	×	-	-
L	×	-	-

Formulations A and B were further studied. (Table 3)

Table 3: Average droplet size, drug content and % transmittance

Formulation code

Average

Droplet size (nanometers)

Drug content (%)

Transmittance (%)

27.18

99.53

99.3

20.67

99.53

99.4

Studies with solid SMEDDS:

The optimized Liquid SMEDDS B was then converted into solid SMEDDS and characterized by DSC and SEM analysis. The DSC thermograms of pure drug nifedipine (Fig. 5) showed sharp endothermic peaks at 173.36^oC indicating the drug is highly crystalline. The absence of drug peaks in the solid SMEDDS formulation (Fig. 6)indicate change in melting behavior of drug and inhibition of crystallization following granulation using lipid, surfactants and granulating materials. The SEM of pure drug nifedipine (Fig. 7(a) and 7(b))and solid SMEDDS (Fig. 7(c) and 7(d))are shown at different magnifications The SEM images of solid SMEDDS show well separated spherical particles.

Fig. 7 SEM image of nifedipine (a and b) and nifedipine solid SMEDDS (c and d) at different magnifications

Stability studies (Table 4) of liquid and solid SMEDDS indicated no decline in nifedipine content at the end of three months.

Table 4: Stability study

Days	Liquid SMEDDS		Solid SMEDDS
Days	Appearance	Drug content (%)	Appearance	Drug content (%)
0	Clear	99.53	Very Light yellow	98.76
30	Clear	99.53	No change	98.70
60	Clear	99.23	No change	98.45
90	Clear	99.23	No change	98.45

The in vitro dissolution comparisons of pure drug, liquid SMEDDS and solid SMEDDS were carried out in 0.1 N HCl (Fig. 8).

Fig. 8: Comparative Percentage cumulative drug release of drug, SMEDDS and Solid SMEDDS

The faster dissolution from SMEDDS indicates that the drug is in solubilized form and upon exposure to dissolution medium results in small droplets that can dissolve rapidly in dissolution medium. More than 55% of drug released within five minutes from liquid and solid SMEDDS as compared to only 4% dissolution from pure drug. The similarities between dissolution patterns of liquid and solid SMEDDS showed that self emulsifying properties of SMEDDS are unaffected following conversion. The permeability studies gave P_app (cm/sec) of 0.82 × 10^-4, 5.7 × 10^-4and 5.48 × 10^-4for nifedipine, liquid and solid SMEDDS respectively. Thus, nifedipine showed better permeation rate after incorporation into SMEDDS (Fig. 9).

Fig. 9: Comparative Percentage cumulative permeation of drug, SMEDDS and Solid SMEDDS

Model independent approach for dissolution profile comparisons (f₁/f₂ test):

For similarity of two curves, f₁ values need to be close to 0, and f₂ values need to be close to 100. f₁values up to 15 (0-15) and f₂ values greater than 50 (50-100) assure sameness or equivalency of two curves. The performance of the test (postchange) and reference (prechange) products is thus assured to be similar.

When comparing dissolution and permeation profiles of nifedipine liquid and solid SMEDDS f₂values were calculated as 54 and 63 for dissolution and permeation profiles respectively. Both the values were within 50-100. The results proved equivalence of liquid and solid SMEDDS for both dissolution and permeation studies. f₁ value was 7 for dissolution and permeation profiles both. This supported the results as both were within 0-15. The similarities between dissolution and permeation patterns of nifedipine liquid and solid SMEDDS showed that self emulsifying properties of SMEDDS are unaffected following conversion.

CONCLUSION:

In the present study, nifedipine SMEDDS was prepared and optimized using various parameters like particle size, in vitro release and ex vivo studies. Optimal SMEDDS consisted of captex 100 as oil phase, labrasol and tween 60 as surfactants and propylene glycol as co solvent. The formulation rapidly emulsified in aqueous media and produced mean droplet size 20.67 nm. The SMEDDS formulation and solid intermediates for capsule filling both showed faster dissolution when compared with nifedipine. The solid SMEDDS consisted of well separated spherical particles. DSC measurements suggested that nifedipine in SMEDDS was in amorphous or molecular dispersion state. Thus, the results confirm the future potential of using SMEDDS for delivery of poorly soluble drugs by oral route.

ACKNOWLEDGEMENT:

The authors are thankful to Shilpa Medicare Limited, Raichur, Karnataka, Colorcon Asia Pvt. Limited, Goa and Abitec Corporation, Janesville, WI for providing gift samples. We are also indebted to management of Raj Kumar Goel Institute of Technology, Ghaziabad for providing all possible laboratory facilities for conducting present research work. Also, we are thankful to NIPER, Mohali and I.I.T. New Delhi for providing the particle size, SEM and DSC analysis.

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Received on 15.01.2013 Modified on 30.01.2013

Research J. Pharm. and Tech. 6(3): March 2013; Page 278-284