international journal of innovative pharmaceutical … sunder kunwarpuriya department of...

RESEARCH ARTICLE Komal et.al / IJIPSR / 3 (5), 2015, 527-550

Department of Pharmaceutics ISSN (online) 2347-2154

Available online: www.ijipsr.com May Issue 527

FORMULATION AND EVALUATION OF SELF-

MICROEMULSIFYING DRUG DELIVERY SYSTEM OF

PRAVASTATIN SODIUM

1Nilesh Khutle,

1Komal Jayshankar Chourasia*,

1Abhijeet Sunder Kunwarpuriya

Department of Pharmaceutics, Dr.L.H.Hiranandani College of Pharmacy, Ulhasnagar-421 003,

University of Mumbai, Maharashtra, INDIA

Corresponding Author

Komal Jayshankar Chourasia

Department of Pharmaceutics,

Dr.L.H.Hiranandani College of Pharmacy,

Ulhasnagar-421 003, Mumbai, Maharashtra, INDIA

E-mail: [email protected]

Phone: +91-865548834

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

Abstract

Pravastatin Sodium (PRV) is a HMG co-A reductase inhibitor. Pravastatin sodium is BCS class III drug with high

aqueous solubility (approximately 300 mg/ml) and low permeability characteristics. It shows low absolute oral

bioavailability (approximately 17%) due to decreased permeability and high first pass extraction. Also pravastatin

sodium is found to be unstable at acidic environment of stomach (pH 1.2). Thus the aim of the present

investigation was to develop a self-microemulsifying drug delivery system (SMEDDS) to enhance the permeability

characteristics of hydrophilic drugs and to protect them from hostile environment of gut. The solubility of PRV in

various oils was determined to identify the oil phase of SMEDDS. Various surfactants and co-surfactants were

screened for their ability to emulsify the selected oil. Pseudoternary phase diagrams were constructed to identify

the efficient self-emulsifying region. The SMEDDS formulation was optimized by freeze thaw cycles, robustness

to dilution and droplet size and zeta potential tests. The optimized L-SMEDDS formulation containing PRV

(10mg), Capmul MCM C8 EP (100 mg), Cremophor RH 40 (66.66 mg) and Labrafil M 2125 (33.33mg) was

further evaluated by in-vitro and ex-vivo release studies. L-SMEDDS was further converted into T-SMEDDS by

“Liquid loading technique”. T-SMEDDS of PRV contained Neusilin®, crosspovidone, magnesium stearate and L-

SMEDDS loaded into it. The results from both L-SMEDDS and T-SMEDDS suggest the potential use of

SMEDDS to improve GI instability and intestinal permeability of BCS Class III drug PRV.

Keywords: Pravastatin sodium, low intestinal permeability, Gastro intestinal instability, Self-microemulsifying

drug delivery system, Liquid loading technique.

mailto:[email protected]




INTRODUCTION

Oral route has been the major route of drug delivery for chronic treatment of number of diseases

since many years but due to the surprising development of modern technology such as structure-

based drug design (SBDD), combinatorial chemistry, and high throughput screening (HTS),

newly synthesized compounds with customary pharmacological activity have been obscured [1].

Nowadays, an increasing number of newly launched drugs i.e. around 35-40% are categorized as

being highly water soluble and highly hydrophilic such as protein and peptides, these drugs are

considered as Class-III drugs by biopharmaceutical classification systems (BCS) [2,3]. Class-III

drugs are defined as high solubility low permeability class where the gastrointestinal permeation

is the rate controlling step in the absorption process [4]. The limited surface area and the tight

junctions present between the adjacent cells of the intestinal epithelium restrict the transport of

drug thereby responsible for its low bioavailability [5]. Due to this fact, many drug candidates fail

to reach the market, even though they exhibit prospective pharmacodynamic activity. These

capable molecules present idiosyncratic formulation and development challenges and often suffer

from poor bioavailability because the rate and degree of absorption from the gastrointestinal tract

(GIT) of such drugs are controlled and limited by intestinal permeation process [6]. Among the

various strategies, SMEDDS (Self-MicroEmulsifying Drug Delivery System) shows a great

potential for enhancing oral bioavailability of BCS class III drugs. These systems are found useful

for formulating those drugs which undergo hepatic first pass metabolism and orally administered

drugs which gain access to the systemic circulation by direct absorption into intestinal lymphatic

system[3]. Self-Micro Emulsifying Drug Delivery Systems are typically composed of oils or

lipids, surfactants and alternatively, one or more co-solvents/co-surfactants which have the ability

to undergo self-emulsification under the gentle agitation of the GI tract [7]. Due to the

spontaneous development of the emulsion in the GI tract with mild agitation provided by gastric

mobility the dissolution as well as permeation rate can be improved as this presents the drug in

solubilised form, and the small size of the formed droplets provides a large interfacial surface area

for drug absorption. Because of small globule size, the drug can be absorbed through lymphatic

pathways, thereby bypassing the hepatic first-pass effect. Also the fine oil droplets would pass

rapidly from the stomach and promote wide distribution of the drug throughout the GI tract,

thereby minimizing the irritation frequently encountered during extended contact between bulk

drug substances and gut wall. Thus for hydrophilic or BCS class III drugs with permeability rate




limited oral absorption, these systems may offer an improvement in the rate and extent of

bioavailability and more reproducible plasma concentration profiles[8]. Selection of a suitable

self‐emulsifying formulation depends upon the assessment of (1) the solubility of the drug in

various components, (2) the area of the self‐emulsifying region as obtained in the phase diagram,

and (3) the droplet size distribution of the resultant emulsion following self‐emulsification [9].

Pravastatin sodium belongs to a group of drugs called HMG coA reductase inhibitors, or “statins”

used in the treatment of hypercholesterolemia and to reduce the risk of cardiovascular disease.

Pravastatin sodium is a BCS class III drug with high aqueous solubility (approximately 300

mg/ml) and low permeability characteristics. It shows low absolute oral bioavailability

(approximately 17%) due to decreased permeability and high first pass extraction. Also

pravastatin sodium is found to be unstable at acidic environment of stomach (pH 1.2)[10]. As

SMEDDS formulation claim to enhance the permeability characteristics of hydrophilic drugs and

to protect them from hostile environment of gut, these systems are selected to overcome and meet

the rate limiting properties of Pravastatin sodium i.e., low permeability, high first-pass extraction

and instability in acidic environment. Thus consequently enhances its bioavailability [11-14]. The

aim of our present study was to develop a SMEDDS formulation of pravastatin sodium and

characterize for its ability to form a microemulsion based on its particle size, zeta potential,

in‐vitro dissolution studies and ex-vivo permeability studies.

MATERIALS & METHODS

MATERIALS

PRV was obtained as a gift sample from Biocon India Pvt Ltd, Banaglore. Capmul MCM C8 EP,

Captex 200, Captex 355, Captex 500 were obtained as a gift sample from Abitec Corporation,

Janesville, USA. Capryol PGMC, Lauroglycol 90, Labrafil M 2125, Labrafil M 1944 CS were

kindly gifted by Gattefosse, France. Cremophore RH 40, Cremophore EL were gifted from

BASF, Mumbai, India. Neusilin® was obtained as gift sample from Gangwal Chemicals. Pvt.

Ltd, Thane, India. The other chemicals used were of the analytical grades.

METHODS

SATURATION SOLUBILITY STUDIES:

The saturation solubility of Pravastatin sodium was determined in various oils, surfactants and

co‐surfactants. Solubility was determined by modifying the routine shake flask method, to




overcome the practical difficulties associated with it [15]. The modified approach consists of two

stages as follows:

Stage I

Determination of approximate solubility by visual observation: In a vial containing 1gm of

prewarmed vehicles, appropriate quantity of drug was added and the mixtures were vortexed

using a cyclomixer for 4-5 min to facilitate uniform mixing. Vials containing the mixtures were

visually observed to determine the extent of solubility of first aliquot of drug. In vehicles which

showed complete solubility of the first aliquot of drug, the second aliquot was added and same

process was repeated for number of times till the vehicles get saturated by drug. The total amount

of drug added to make the vehicles saturated was noted. Only few promising vehicles which were

successful in solubilizing drug in good quantity were selected and studied further for the

quantitative estimation of solubility at stage II.

Stage II

UV-Visible Estimation: The excess amount (more than the approximate solubility) of drug was

added to each vial containing prewarmed 5gm of the selected vehicles, i.e., oil, surfactant, co-

surfactant followed by procedure same as above. The vials were shaken for 48 hrs in an incubated

orbital shaker maintained at 37± 1˚C, and these mixtures were centrifuged at 5000 rpm for 15 min

after which the excess insoluble drug was discarded by filtration. The aliquots of supernatant were

diluted and drug assay was performed [16].

EMULSIFICATION EFFICIENCY STUDY

Different surfactants and co-surfactants were screened for its emulsification ability for selected oil

phase having maximum solubility of drug. Emulsification ability of various surfactants was

screened on the basis of %T and emulsification ability [17]. Surfactant and oil in ratio of 1:1 were

mixed. This mixture was gently heated at 40-50˚C for homogenizing the components. Adequate

quantity of this isotropic mixture was accurately weighed and diluted with double distilled water

to yield fine emulsion. The ease of formation of emulsions was monitored by noting the number

of volumetric flask inversions required to give uniform emulsion. The resulting emulsions were

observed visually for the relative turbidity. The emulsions were allowed to stand for 2 hrs and

their %T was assessed at 638.2 nm by UV-visible Spectrophotometer [18]. Similar procedure is

repeated to determine the emulsification efficiency of co-surfactants.




PSEUDOTERNARY PHASE DIAGRAMS

Ternary phase diagram of oil, surfactant and co-surfactant were plotted using flask inversion

method where the concentration of each component was decided on the basis of requirements

stated by Pouton [19], for the spontaneously emulsifying systems i.e. 25% to 70 % Oily phase,

30-75% surfactant and 0-30 % co-surfactant. Based on solubility study and emulsification

efficiency, 35 different systems of this combination were prepared by varying the concentration of

oil, surfactant and co-surfactant (Table No: 8). All systems were evaluated for formation of

microemulsion where flask inversions were performed to facilitate the emulsion formation. The

obtained emulsion was allowed to stand for 2h and their transmittance was noted at 638.2 nm by

UV-Visible spectrophotometer.

PREPARATION OF LIQUID-SMEDDS (L-SMEDDS)

Nine different trial batches were prepared using Capmul MCM C8 EP as the oil and Cremophor

RH40 and Labrafil M 2125 as surfactant and co-surfactant respectively. The formulation batches

were differentiated from each other by varying the ratio of Oil to Smix i.e, 1:1, 1:1.5, 1:2, whereas

ratio of surfactant: co-surfactant (Smix) was used as 1:1, 2:1 and 1:2. In all the formulations, the

level of PRV was kept constant. Briefly, accurately weighed PRV was placed in a glass vial, and

oil, surfactant, and co-surfactant were added. Then the components were mixed by gentle heating

at 40ºC stirring and vortex mixing, until PRV was perfectly dissolved. The mixture was stored at

room temperature until further use (Table No: 1).

Table 1: Formulation Batches of PRV L-SMEDDS

Ingredients

(mg)*

Batch no

PLS1 PLS2 PLS3 PLS4 PLS5 PLS6 PLS7 PLS8 PLS9

PRV

10

10

10

10

10

10

10

10

10

Cap MC8

100

100

100

100

100

100

100

100

100

Cr RH 40

50

75

100

33.33

50

66.66

66.66

100

133.33

LM 2125

50

75

100

66.66

100

133.33

33.33

50

66.66

O:Smix

1:1

1:1.5

1:2

1:1

1:1.5

1:2

1:1

1:1.5

1:2

S:Co-s

1:1

1:1

1:1

1:2

1:2

1:2

2:1

2:1

2:1

Total

210

260

310

210

260

310

210

260

310 *Values represent the quantity added per unit capsule formulation




OPTIMIZATION OF L-SMEDDS OF PRV

FREEZE THAW CYCLING [20]

In order to access the thermodynamic stability of L-SMEDDS they were subjected to freeze-thaw

cycles. For this all the nine batches of L-SMEDDS were stored at 40˚C for 24 hr in incubator

followed by 24 hr at 5˚C in refrigerator. Three alternate cycles were performed and the

formulations were visually observed for any sign of phase separation and/or precipitation of drug.

The formulation batches which strongly withstand the freeze-thaw cycles were further subjected

for centrifugation test.

CENTRIFUGATION

In order to assess the physical instabilities in formulations, like phase separation, phase inversion,

aggregation, creaming and cracking, centrifugation test is carried out. After the third cycle of

freeze thaw, formulations were subjected to centrifugation at 5000 rpm for 20 min. After 20 min,

formulations were visually observed for any signs of phase separation and/or drug precipitation.

The formulation batches which shows no sign of drug precipitation and phase separation were

further evaluated and screened for their robustness to dilution ability[21-23].

ROBUSTNESS TO DILUTION [18, 24-26]

Robustness to dilution was studied by diluting L-SMEDDS of PRV by 50, 100 and 1000 times

with dissolution media i.e., 0.1N HCl (pH 1.2) and phosphate buffer (pH 6.8). The diluted

formulations were stored for 4 hr at room temperature and observed for appearance of produced

microemulsion, %T study and any signs of phase separation and/or drug precipitation.

EVALUATION OF OPTIMIZED FORMULATIONS OF PRV L-SMEDDS

GLOBULE SIZE, POLYDISPERSIBILITY INDEX AND ZETA POTENTIAL [26-28]

The mean globule size, Polydispersibility Index (P.I) and Zeta potential of the resulting

microemulsion were determined by Malvern Zeta Sizer. Prior to analysis the L-SMEDDS of

PRV, 50 mg was diluted to 50ml with distilled water, 0.1N HCl and phosphate buffer pH 6.8.

TRANSMISSION ELECTRON MICROSCOPY [21,24,29,30]

Transmission electron microscopy was employed to study the morphology of the resulting

SMEDDS.

DRUG CONTENT (ASSAY)

Accurately weighed, 210 mg of L-SMEDDS was placed in 25ml volumetric flask and volume

was made up with methanol followed by sonication in bath sonicator for 15-20 min to extract and

solubilize the PRV. 1ml of aliquot was removed and diluted further with 10ml of methanol and




analyzed with UV spectroscopy. The PRV concentration in the resulting solution was calculated

using standard calibration curve.

IN-VITRO DISSOLUTION STUDY [20,24,31]

In-vitro release studies of plain PRV powder and PRV loaded L-SMEDDS were carried out using

a standard USP type II dissolution apparatus with paddle rotating at a speed of 50 rpm and

maintained at temperature of 37˚±0.5˚C in dissolution media 0.1 N HCl with pH 1.2 (SGF) and

phosphate buffer with pH 6.8 (SIF) to examine the effect of pH on drug release from L-SMEDDS

of PRV (equivalent to 10mg of PRV base) which were filled in hard gelatin capsules. During the

study, 5 ml of aliquots were removed at predetermined time intervals i.e. 5, 10, 15, 30, 45 and 60

min from the dissolution medium and replaced with fresh buffer to maintain sink condition.

Aliquots were filtered and diluted further for UV-visible analysis to determine the amount of drug

release in dissolution media.

EX-VIVO PERMEABILITY STUDIES STUDY BY NON-EVERTED SAC TECHNIQUE [32]

Preparation of Krebs-ringer-phosphate-buffer pH 7.2 (perfusion solution)

The perfusion solution was prepared by dissolving 7.8gm sodium chloride, 0.35gm potassium

chloride, 1.37gm sodium bicarbonate, 0.02gm magnesium chloride, 0.22gm sodium dihydrogen

phosphate and 1.48gm glucose in 1000mL of distilled water.

Preparation of PRV solution and microemulsion of PRV

The PRV solution was prepared in Krebs-Ringer‟s buffer solution to yield PRV concentration of

200μg/mL. Similarly PRV L-SMEDDS was diluted with Krebs-Ringer‟s buffer solution to

produce fine microemulsion containing 200μg/mL of PRV.

Transmittance study of resultant microemulsion in Krebs-Ringer‟s buffer solution: Robustness to

dilution of L-SMEDDS in Krebs-Ringer‟s buffer solution was assessed to determine the

efficiency of L-SMEDDS formulation to produce fine-microemulsion; this parameter was

assessed by visual observation for appearance and measuring the %T of resultant microemulsion

at 638.2nm using Krebs-Ringer‟s buffer solution as blank.

Permeability study by non-everted sac technique

Ex-vivo permeability study of L-SMEDDS of PRV was carried out by using non-everted chicken

intestinal sacs. Chicken was killed and the duodenal part of the small intestine was isolated and

washed with distilled water to remove the mucous and lumen content and then placed in cold

KRPB (Krebs-Ringer-Phosphate-Buffer, pH 7.2) solution continuously aerated with the aid of an

electrical aerator. 5-6 cm long sacs were prepared by tying up the two end of the sac either with




cotton or silk thread. 5ml microemulsion of L-SMEDDS formulation of PRV was taken inside the

sac. Intestinal sac containing only plain drug solution in KRPB was also included in this study for

comparison. The sacs were then taken into different beakers containing 100 ml of KRPB solution,

continuously bubbled with atmospheric air, maintained at 37˚ ± 0.5˚C and stirred at 50rpm.

Aliquots were withdrawn at predetermined time intervals. The permeability study was carried for

about 60 mins. Amount of PRV from L-SMEDDS microemulsion and plain drug solution

permeated across the intestine was determined by measuring the absorbance of test solution at

239nm by UV-Visible spectrophotometry using appropriated blank[33,34].

PREPARATION OF SOLID-SMEDDS(S-SMEDDS)

In order to overcome the problems associated with traditional methods of preparation of solid-

SMEDDS i.e., requirement of large amount of solid carrier to satisfy the liquid and problems of

sticking and hardness after tablet compression. Thus an alternative to above mentioned methods

and problems associated with it, a new method called „liquid-loadable tablets (LLT)‟ for

converting L-SMEDDS into Solid-SMEDDS were developed[35]. In this technique tablet blend

containing Neusilin®, Crosspovidone and magnesium stearate as the adsorbing agent,

disintegrating agent and lubricating agent respectively were mixed thoroughly and directly

compressed into tablets of uniform weight and hardness using single punch rotary table machine.

After compression, these tablets were soaked in excess of PRV L-SMEDDS until each tablet

soaked the optimized quantity of liquid formulation and the time required for the same was noted

(Figure 1). The optimized composition of PRV T-SMEDDS is mentioned in below Table No:2.

Fig.1: Liquid loading technique to develop tablet SMEDDS of PRV

Table 2: Composition of Optimized PRV Tablet-SMEDDS (PRV T-SMEDDS)

Ingredients Activity

PRV Active drug

Capmul MCM C8 EP Oil phase

Cremophor RH40 Surfactant

Labrafil M2125 Cs Co-surfactant

Neusilin US2 Adsorbent

Crosspovidone (10%) Superdisintegrant

Magnesium stearate (1%w/w) Glidant




EVALUATION OF T-SMEDDS [36-39]

TABLET THICKNESS

Average thickness of randomly selected six tablets was measured using Vernier caliper and

expressed in mm.

HARDNESS TEST

The resistance of tablets to shipping or breakage under conditions of storage, transportation and

handling before usage depends on its hardness. The mean hardness of randomly selected six

tablets was checked by using Monsanto hardness tester. It is expressed in kg/cm2.

FRIABILITY TEST

Friability of the tablets was tested using Roche friabilator. Loss of less than 1% in weight is

considered to be acceptable. The weight of randomly selected 10 tablets was noted initially (W1)

and placed in the friabilator at 20rpm. The tablets were reweighed and noted as (W2). The percent

friability is calculated using following formula and expressed as %.

% Friability = (W1 – W2)/W1 X 100

WEIGHT VARIATION TEST

Twenty tablets were selected at random and the average weight was determined. NMT two of the

individual weights deviate from the average weight by more than the percentage shown in table

and none deviates by more than twice the percentage.

DSC THERMOGRAM

Thermal properties of physical mixture of PRV and Neusilin US2 (1:1w/w), and T-SMEDDS

formulations were investigated using a Exstar SII DSC 6220. The obtained thermograph was then

compared with the thermograph of pure PRV powder to investigate the change in crystallinity of

PRV.

Table 3: Samples for DSC

Sl. No Sample

1 Pravastatin sodium(PRV)

2 PRV + Neusilin US2

3 T-SMEDDS


Drug content of the PRV T-SMEDDS was determined by using UV-Visible spectrophotometry

method. Ten tablets were taken randomly and powdered, the tablet powder equivalent to 10mg of

PRV was accurately weighed and transferred to 50mL volumetric flask and the volume was made

up to 50mL with methanol. Bath sonicator was used for 10min to facilitate the extraction of PRV




in methanol. The obtained solution was filtered and further diluted to determined the

concentration of PRV by UV-Spectroscopy at 239 nm.

EFFECT OF SOLIDIFICATION ON GLOBULE SIZE AND PI [24,40]

100 mg of T-SMEDDS was dissolved in 100ml of distilled water using magnetic stirrer at 500

rpm for 15-20 min. The produced dispersion is allowed to stand for 2 hr to settle down the

adsorbing agent, the supernatant microemulsion was analyzed to determine the Globule size, P.I

using Malvern zeta sizer.

SCANNING ELECTRON MICROSCOPY [40]

The outer macroscopic structure of plain PRV powder, Neusilin US2, and Solid-SMEDDS were

investigated by scanning electron microscope (SEM; JEOL 5400, Japan).

IN-VITRO DRUG RELEASE STUDY

In-vitro drug release study of T-SMEDDS of PRV was carried out in a similar manner as that of

L-SMEDDS of PRV.

RESULT & DISCUSSION

SATURATION SOLUBILITY STUDIES

Solubility studies of PRV in various oils

The components used in the SMEDDS should have high solubilization capacity for the drug,

ensuring the solubilization of the drug in the resultant dispersion. Hence, solubility studies were

aimed at identifying suitable oily phase having maximal potential to solubilize the drug under

investigation as it is very important to achieve optimum drug loading.

Table 4: Approximate estimated solubility of PRV in various oily phases

Sl.

No

OIL Amount of PRV added Visual

observation

Approx.

Solubility

(mg/gm) 10mg 10mg 10mg 10mg

1 Captex 500 - - - Slightly soluble <10mg



4 Capmul MCM C8 EP Highly soluble >40mg*

5 Miglyol 812 - - - Slightly soluble <10mg

6 Lauroglycol FCC - - - Slightly soluble <10mg

7 Maisine 35-1 - - - Slightly soluble <10mg

8 Ethyl oleate (pure) - - - Slightly soluble <10mg

9 Oleic acid (pure) Highly soluble >40mg*

10 Iso-propyl myristate - - - Slightly soluble <10mg

11 Soyabean oil - - - Slightly soluble <10mg

12 Clove oil Highly soluble >40mg*

*Added more than 40mg of PRV




Fig. 2: Solubility profile of PRV in selected Oily phases

Results from solubility studies are reported in Table No:4. As seen from the Table No:4 and

Figure 2 amongst the various oils that were screened, Capmul MCM C8 EP showed the highest

solubilization capacity for PRV, followed by Oleic acid and Clove oil. Thus, Capmul MCM C8

EP was selected as oily phase because although the other two oils showed good solubilization

capacity, they demonstrated a distinct color change at saturation concentration.

Solubility profile of PRV in Surfactant and Co-surfactant: In the present study, 20 different

surfactant and co-surfactants were screened to solubilise the PRV. This study is carried out in a

similar fashion as that used while screening oily phase. The surfactant and co-surfactant selected

namely Cremophore RH 40 (Cr-RH 40), Cremophore EL (Cr-EL), Labrafil M 2125 (LM 2125),

Labrafil M 1944 (LM 1944), Ethanol and n-butanol shows promising ability to solublise PRV

(More than or equal to 30 mg/gm) in primary screening of visual observation (Table No: 5).

Table 5: Approximate solubility of PRV in various surfactants and co-surfactants

Sl.

No.

Surfactant/ Co-

surfactant

Amount of Drug Added Visual

Observation

Approx.

Solubility

(mg/gm)

10mg 10mg 10mg 10mg

1 Cremophore EL Soluble <40

2 Cremophore RH 40 Soluble <40

3 Tween 20 - - Sparingly soluble <20

4 Tween 80 - - Sparingly soluble <20

5 Span 20 - - Sparingly soluble <20

6 Span 80 - - Sparingly soluble <20

7 Lauroglycol 90 - - Soluble ≤20

8 Lauroglycol FCC - - Soluble ≤20

9 Labrafil M 1944 CS - Soluble ≤30




10 Labrafil M 2125 CS - Soluble ≤30

11 Ethanol + - Soluble ≤30

12 n-Butanol - Sparingly soluble ≤30

13 Capryol PGMC - - Sparingly soluble <20

14 Solutol HS 15 - - - Sparingly soluble <10

15 Labrafac CC - - - Sparingly soluble <10

16 Iso-propyl Alcohol - - - Sparingly soluble <10

17 Propylene Glycol - - - Sparingly soluble <10

18 Transcutol P - - - Sparingly soluble <10

19 PEG 400 - - - Sparingly soluble <10

20 Plurol Oleique CC 497 - - - Sparingly soluble <10

Report of solubility profile by quantitative estimation is put forth in Figure 3. Result suggests that

the maximum solubility was observed in Cr-RH 40 (34 mg/gm) followed by Cr-EL (32 mg/gm),

LM 2125, LM 1944, Lauroglycol 90 and Lauroglycol FCC. In comparison to above vehicles

solubility of PRV was found lowest in Capryol PGMC, Solutol HS 15, and Labrafac CC.

Fig.3: Estimated solubility of PRV in selected Surfactants/Co-surfactants

EMULSIFICATION EFFICIENCY STUDY

Based on the reports obtained from Table No:6 and Table No:7, Cremophor RH40 and Labrafil M

2125 was selected as surfactant and co-surfactant respectively as they show higher %T values

then others.

Table 6: Emulsification efficiency of surfactants

Sl. No Surfactant No. of flask inversion %T Appearance

1 Cremophore EL 8 98.14 Transparent

2 Cremophore RH 40 7 99.20 Transparent

3 Lauroglycol 90 31 60.73 Colloidal

4 Lauroglycol FCC 54 51.32 Turbid

5 Labrafil M 1944 27 72.45 Colloidal




CS

6 Labrafil M 2125

CS

30 76.82 Colloidal

7 Ethanol 25 81.49 Colloidal

8 n-Butanol 29 75.61 Colloidal

9 Labrafac CC 49 69.38 Colloidal

Table 7: Spontaneity of emulsification by co-surfactants for surfactant-Cr RH40

Sl.

No

Co-surfactant Surfactant: Cr- RH40 Appearance

No. of flask inversion %T

1 Lauroglycol 90 6 93.48 Transparent

2 Lauroglycol FCC 8 91.84 Transparent

3 Ethanol 5 96.52 Transparent

4 N-Butanol 5 97.35 Transparent

5 Transcutol P 5 95.48 Transparent

6 Plurol Oleique CC 497 15 72.89 Colloidal

7 Labrafil M 2125 Cs 2 100.20 Transparent

8 Labrafil M 1944 Cs 2 97.55 Transparent

PSEUDOTERNARY PHASE DIAGRAM

Based on emulsification ability of surfactants and their spontaneity enhancement by co-

surfactants, Cap MC8: Cr-RH 40: LM 2125 system was selected to plot ternary phase diagram to

identify the possible mixture point producing microemulsion. The phase diagrams of Cap MC8:

Cr-RH 40: LM 2125 systems are shown in Figure No:4. The outer parallelogram indicates the

area, which was explored for locating microemulsification region. The captions in the diagrams

indicates the system possessing ability to produce fine microemulsion, coarse emulsion and turbid

emulsion (No emulsion). Observations were made based on the visual observations of the

appearance of emulsion and its % transmittance values. From the phase diagram it is evident that

the system CapMC8:Cr-RH40:LM2125 possesses the ability to produce fine microemulsion

(>95%T) for the compositions that had as high as 55% (w/w) of oily phase; Cap MC8.

Table 8: Data of Ternary phase diagram for System (Cap MC8: Cr-RH40: LM 2125) in

distilled water Sl

No.

Cap MC8

(%)

Cr-RH40

(%)

LM 2125

(%)

No. of

FI

Transmittance

(%)

Grade Remark

A1 25 75 0 <6 100.34 I ME

A2 25 70 5 <6 100.27 I ME

A3 25 65 10 <6 100.21 I ME

A4 25 60 15 <6 100.04 I ME

A5 25 55 20 <6 99.86 I ME

A6 25 50 25 <6 99.73 I ME

A7 25 45 30 <6 99.52 I ME




B1 35 65 0 <8 98.21 I ME

B2 35 60 5 <6 98.69 I ME

B3 35 55 10 <6 98.62 I ME

B4 35 50 15 <6 98.29 I ME

B5 35 45 20 <6 98.15 I ME

B6 35 40 25 <6 98.01 I ME

B7 35 35 30 <6 97.92 I ME

C1 45 55 0 <6 96.39 I ME

C2 45 50 5 <6 96.58 I ME

C3 45 45 10 <6 96.11 I ME

C4 45 40 15 <6 95.83 I ME

C5 45 35 20 <6 95.64 I ME

C6 45 30 25 <6 95.33 I ME

D1 50 50 0 <8 94.11 II ME

D2 50 45 5 <7 94.61 II ME

D3 50 40 10 <7 95.44 I ME

D4 50 35 15 <7 95.17 I ME

D5 50 30 20 <6 95.06 I ME

E1 55 45 0 <7 93.88 II ME

E2 55 40 5 <7 94.62 II ME

E3 55 35 10 <7 94.51 II ME

E4 55 30 15 <6 92.65 II ME

F1 60 40 0 >9 91.36 II ME

F2 60 35 5 >15 91.19 III CE

F3 60 30 10 >20 90.30 III CE

J1 65 35 0 >20 60.71 IV CE

J2 65 30 5 >35 54.23 IV NE

K1 70 30 0 >40 41.72 IV NE

Fig. 4 Ternary phase diagram of Cap MC8 EP: Cr-RH40: LM 2125 in distilled water

OPTIMIZATION OF PRV L-SMEDDS FORMULATION

FREEZE THAW CYCLES AND CENTRIFUGATION STUDY:

The comparison of nine test formulations for freeze thaw cycles and centrifugation study is

tabulated in Table No:9 and Table No:10. The report of current investigations shows that all the

other formulations were able to withstand the freeze thaw cycles and centrifugation test except

formulations PLS2, PLS4, PLS5 and PLS9.




Table 9: Optimisation of PRV L-SMEDDS formulation by Freeze thaw cycle

Batch No. Freeze thaw cycles

Remarks 1

st Cycle 2

nd Cycle 3

rd Cycle

PLS1 Stable Stable Stable Passes

PLS2 Stable Unstable Unstable Fails







PLS9 Stable Stable Unstable Fails

Table 10: Optimisation of PRV L-SMEDDS formulation by centrifugation study

Batch No. Centrifugation test (5000 rpm)

Remarks Phase separation Precipitation

PLS1 Stable Clear Passes





ROBUSTNESS TO DILUTION STUDY

Effect of dilution and pH of dilution media on SMEDDS containing PRV is explained in Table

No:11. PRV L-SMEDDS were diluted with aqueous phases differing in pH. The clarity of

microemulsions was measured in terms of %T as SMEDDS forms o/w microemulsion. From the

results, formulation PLS7 was observed to be robust to dilution as % T value was greater than

98% and appeared clear or slightly bluish and there were no drug precipitation observed even

after 4hr of standing irrespective of pH of dilution media. Thus, based on above study formulation

code PLS7 was selected as an optimized formula to produce L-SMEDDS of PRV and evaluated

in further studies.

Table 11: Data of robustness to dilution study of PRV L-SMEDDS formulations

Dilution

Media Dilution

% Transmittance

Batch No

PLS1 PLS3 PLS6 PLS7 PLS8

Distilled

water (D.W)

50 97.11 96.19 94.23 99.21 96.28

100 96.49 95.61 93.24 99.57 95.64

1000 96.72 95.43 92.25 99.23 96.45

0.1N HCl

(1.2 pH)

50 95.29 95.21 93.45 98.13 96.25

100 95.83 95.34 92.28 98.64 96.76

1000 95.39 95.26 92.11 99.49 96.04

Phosphate

buffer

(6.8 pH)

50 96.24 96.09 93.61 97.73 95.29

100 96.29 96.73 93.09 98.56 95.81

1000 96.38 96.53 92.83 98.33 96.21




EVALUATION OF OPTIMIZED PRV L-SMEDDS

GLOBULE SIZE ANALYSIS

The droplet size of the emulsion is a crucial factor in the self‐emulsification performance because

it determines the rate and extent of drug release as well as drug absorption and distribution. Also,

it has been reported that the smaller particle size of the emulsion droplets may lead to more rapid

absorption and improve the bioavailability.

Table 12: Data representing the Globule size, Polydispersity Index (P.I.) and Zeta potential

of PLS7 in various dilution media Media #Globule size *Polydispersibility Index (P.I) *Zeta potential

Distilled water (D.W) 21.07 ± 1.28 0.37 -8.19

0.1N HCl (pH 1.2) 38.12 ± 1.45 0.41 -9.27

Phosphate buffer (pH 6.8) 35.85 ± 1.21 0.49 -5.49

# Globule size is expressed as Mean ± Standard deviation of two replicates.

* P.I. and Zeta

potential are expressed as mean of two replicates.

Fig.5: Globule size distribution and P.I. obtained from PRV L-SMEDDS

Table No:12 shows the mean globule size obtained from optimized L-SMEDDS of PRV in the

range of 20-40 nm. The globule size in SGF (38.12 ± 1.45 nm) was found to be large as compare

to DW and SIF. The polydispersity index of PRV L-SMEDDS in various media was in the range

of 0.37 to 0.49. These results indicate that the optimal PRV L-SMEDDS produced a resultant

emulsion with a small mean size and a narrow particle size distribution (Figure 5 and Figure 6).

Fig. 6: Zeta potential distribution obtained from PRV L-SMEDDS




TRANSMISSION ELECTRON MICROSCOPY (TEM)

Fig. 7: TEM image obtained from 1000 fold dilution of PRV L-SMEDDS in Distilled water

Figure 7 confirms the ability of PRV L-SMEDDS to produce spherical oil globule of nano size,

the oil droplets are equally distributed all over the film. This observation of TEM image is in

agreement with the result obtained from droplet size analysis.

DIFFERENTIAL SCANNING CALORIMETRIC EVALUATION

The DSC thermograms of pure PRV, physical mixture of PRV and Neusilin US2, and T-

SMEDDS formulation are shown in Figure 8. Pure PRV showed sharp endothermic peaks at

172.4°C indicating that the drug is highly crystalline. The physical mixture comprising equal

amounts of Neusilin US2 and PRV showed a less intense melting point peak at 210.7°C due to

presence of crystalline PRV. The absence of obvious PRV peaks in the solid-SMEDDS

formulation indicates change in the melting behavior of PRV and inhibition of crystallization

following solubilisation using lipid surfactants and physical mixing with solid carrier.

Fig. 8: DSC thermograms of (a) Plain PRV

(b) PRV + Neusilin US2 (NU2) physical mixture and

(c) T-SMEDDS





Table 13: Observation of drug content of L-SMEDDS

Batch No. Drug content (%)*

PLS7 99.60 ± 2.24%

*Values are expressed as Mean ± Standard deviation of 3 replicas

IN-VITRO DISSOLUTION STUDY

The results of in-vitro dissolution profiles of plain PRV powder and optimized PRV L-SMEDDS

in various dissolution media are provided in respective Figure 9 and Figure 10.

Figure 9: In-vitro dissolution profile of Plain drug PRV in various buffer solutions

Figure 10: In-vitro dissolution profile of PRV L-SMEDDS in various dissolution media

It is evident from the observation that PRV L-SMEDDS showed a dramatic improvement in the

in-vitro dissolution profile compared to the pure PRV in both the dissolution media especially in

SGF. Thus optimized L-SMEDDS presents the PRV in fine globule form which disperses

spontaneously irrespective of the pH of dissolution media. The rate and extend of PRV release

from L-SMEDDS was excellent suggested that L-SMEDDS improves the oral bioavailability of

PRV.




EX-VIVO DRUG PERMEABILITY STUDY USING NON-EVERTED SAC TECHNIQUE

Figure 11: Ex-vivo permeability profile of Plain PRV solution and PRV L-SMEDDS microemulsion

The produced microemulsion appeared as clear and transparent which indicated that the L-

SMEDDS produced fine-microemulsion in Krebs-Ringer buffer solutions which was further

confirmed by %T value of 99.12.

Significant increase in permeability of PRV was observed from L-SMEDDS as compared to plain

PRV. From Figure 11, it was observed that after 60min of study, only 30% of PRV was

transported through intestinal lumen from PRV solution, on the other hand, 75.98% of PRV was

transported through intestinal lumen from microemulsion produced from L-SMEDDS

formulation. Such a dramatic improvement of permeability of PRV was attributed mainly to the

formation of uniformly dispersed globules with nano size in which PRV is present in the

dissolved state. These fine globule size increases the surface area and thus facilitates the

permeability of drug.

EVALUATION OF T-SMEDDS:

The prepared T-SMEDDS was evaluated for hardness, friability, weight variation and

disintegration time.

Table 14: Evaluation of T-SMEDDS

Evaluation parameter Results

Diameter (mm) 10

Hardness*(kg/cm2) 2-2.5

Friability* 0.60%

Weight variation(%) 2.46

Disintegration time 1 min 25sec

*Values are expressed as mean of 3 observations





Table 15: Observation of drug content

Batch No. Drug content (%)*

PLS7 101.31 ± 1.34%

*Values are expressed as Mean ± Standard deviation of three replicates

SCANNING ELECTRON MICROSCOPY

Photo image obtained by SEM analysis is presented in Figure 12 which shows that micrographs

of PRV appeared to be made of irregular crystalline structures, Neusilin US2 appeared to be

spherical porous particles and PRV S-SMEDDS shows Liquid SMEDDS adsorbed onto the

spherical surface of Neusilin US2 particles. Crystalline structural characteristic of PRV are not

seen in S-SMEDDS micrographs suggesting that the drug is present in completely dissolved state

in the solid-SMEDDS.

Fig. 12: SEM images of A)Plain PRV B)Neusilin US2 (NU2) 3)PRV T-SMEDDS

EFFECT OF SOLIDIFICATION ON GLOBULE SIZE PROPERTIES OF PRV SMEDDS

FORMULATION

Effect of solidification on L-SMEDDS formulation by adsorbing on Neusilin US2 was evaluated

by measuring Globule size and P.I. of solution of T-SMEDDS. Table No:16 and Figure 13 shows

the results observed. The mean globule size (in DW) obtained from L-SMEDDS was 21.07 ± 1.28

nm (Figure 5), while the mean droplet size obtained from Solid SMEDDS was 129.8 ± 1.24 nm.

This increase in globule size after conversion to T-SMEDDS was attributed to the presence of

Neusilin US2 particles in the sample.

Figure 13: Globule size distribution of PRV T-SMEDDS




Table 16: Data representing the Globule size, Polydispersity Index (P.I.) of optimized formulation of

PRV T-SMEDDS Formulation

*Globule size (nm) *Polydispersibility Index (P.I)

129.8 ± 1.24 nm 0.431

*Globule size is expressed as Mean ± Standard deviation of two replicate and P.I. is expressed as mean of

two replicates

IN-VITRO DISSOLUTION PROFILE OF PRV T-SMEDDS:

Fig. 14: In-vitro dissolution profile of PRV T-SMEDDS in various media

In-vitro dissolution profile of T-SMEDDS of PRV show similar dissolution release profile as that

of L-SMEDDS of PRV. Thus converting liquid formulation into tablet form does not affect its

release characteristics.

CONCLUSION

A SMEDDS formulation of poorly permeable drug, Pravastatin sodium was formulated and

subsequently converted into tablet-SMEDDS by “Liquid loading technique”. The formulation

PLS7 was found to be the optimized L-SMEDDS formulation on the basis of results of

pseudoternary phase diagram, in vitro drug release, droplet size and zeta potential. The optimized

formulation showed rapid self-emulsification in aqueous media, 0.1N HCl (pH 1.2) and phosphate

buffer (pH 6.8) and also protect Pravastatin sodium from degrading in acidic media. Thus results

from intro-vitro and ex-vivo studies show the utility of SMEDDS to enhance permeability

characteristics thereby increasing the bioavailability of BCS Class III drugs like Pravastatin

sodium.

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