Solubility Of Candesartan Cilexetil In Different Vehicles Biology Essay

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The objective of present study was to explore the utility of "mixed solvency" concept to enhance the solubility of poorly-water soluble drug, candesartan cilexetil (CC) in modified solubilizer system. Also to propose an alternate system of solubilizer to provide novel surfactant/cosurfactant system, to aid traditionally involved components in formulation of SEDDS. The present study showed that "mixed solvency" concept was successfully employed in solubility enhancement of CC in TB3Mix upto 303 mg/g of blend. The study showed that alternate system of solubilizers also helped in reducing the surfactant concentration usually required to design nanoemulsions. Our study demonstrated the promising use of "mixed solvency" concept in solubility enhancement of poorly-water soluble drugs and tool to reduce the net surfactant concentration employed in designing of SEDDS.

Candesartan cilexetil was generous gift from Dr. Reddy's Laboratories Ltd., Hyderabad, India, and medium chain triglyceride oil (Capryol-90), macrogolglyceride (Labrasol), Tween 80, Labrafac CC, Lauroglycol 90, Transcutol were generous gift from Gattefosse (Mumbai), India. Capmul PG-8 was generous gift from Abitech Coroporation, USA. Acrysol K-140 was generous gift from Corel Pharma Chem, Ahmedabad, India. Cremophor RH 40, Cremophor EL and Lutrol-F68 were generous gift from BASF (Mumbai), India. L-Camphor, Vanillin, Menthol were generous gift from Shagun Pharmaceuticals (Indore), India. Soybean oil, Castor oil, Olive oil, Oleic acid were purchased from local market. Acetonitrile was of HPLC grade purchased from SRL Chemicals, India. Water, double distilled in all glass still, was used in all experiments. All other chemicals used were of analytical grade. All chemicals were used as received.

Solubility studies

The objective of solubility studies is to determine the solubilization capacity for drug in given vehicles. Vehicles which show highest solubility are then used for formulation of SEDDS. The solubility of CC in various vehicles, i.e. oils (Capryol-90, Soybean Oil, Corn Oil, Capmul PG-8, Olive Oil, Oleic acid, Castor Oil, Labrafac PG), surfactants (Acrysol, Cremophor EL, Labrasol, Tween 80, Tween 20, Span 20) and cosurfactants (PEG 400, Lauroglycol 90, Transcutol, Lutrol F-68,) was determined initially.

The solubility of CC was also determined in modified solubilizer systems (Camphor 30% in ethanol (wt/wt), Camphor 60% in ethanol (wt/wt), Menthol 30% in ethanol (wt/wt), Menthol 60% in ethanol (wt/wt), Vanillin 30% in ethanol (wt/wt), Vanillin 60% in ethanol (wt/wt), Lutrol F-68 30% in ethanol (wt/wt), Lutrol F-68 60% in ethanol (wt/wt), and combinations of thereof viz. C/V 20/20, C/V 20/40, C/V 40/20, V/L 20/20, V/L 20/40, V/L 40 /20, C/L 20/20, C/L 20/40, C/L 40/20, C/V/L 10/10/10, C/V/L 20/20/20 where C denotes Camphor, V denotes Vanillin, L denotes Lutrol F-68, and digits denotes the percentage of components (Camphor, Vanillin, Lutrol- F68) in solution in ethanol (wt/wt). For example C/V/L 20/20/20 denotes 20% Camphor, 20% Vanillin, and 20% Lutrol F-68 in ethanol (wt/wt).

A total of 5 mL of each of the selected vehicles were added to each cap vial containing an excess of CC and the mixture was gently heated at 45-60°C in a water bath under continuous stirring using vortex mixer to facilitate drug solubilization. Vials were kept at ambient temperature for 72 h to attain equilibrium. After reaching equilibrium, each vial was centrifuged at 2000 rpm for 20 min, and excess insoluble CC was discarded by filtration using syringe filter (Millipore Millex- HN Nylon 0.45 µm). Aliquots of supernatant were diluted with methanol and the concentration of solubilized CC dissolved in various vehicles was quantified by HPLC method at 254 nm.

Table 1: Solubility of Candesartan Cilexetil in different vehicles and % Transmittance in selected Vehicles

Vehicle

Solubility (mg/gm)

% Transmittance

Vehicle

Solubility (mg/gm)

% Transmittance

OILS

Transcutol P

176.83 ± 2.28

76.2

Capryol-90

21.31 ± 3.26

-

Menthol 60%**

44.60 ± 1.31

12.7

Capmul

7.19 ± 1.19

-

Camphor 60%**

253.47 ± 2.20

70.2

Castor oil

3.8 ± 0.81

-

Vanillin 60%**

183.47 ± 0.95

29.3

Labrafac PG

1.63 ± 0.44

-

Lutrol F-68 60%**

59.22 ± 0.27

43.8

Corn oil

1.38 ± 0.81

-

C/V/L 20/20/20 (B3)**

303.79 ± 2.24

72.8

Olive oil

1.18 ± 0.27

-

TB3Mix**

282.81 ± 6.73

78.3

Oleic acid

0.66 ± 0.05

-

Camphor 30%**

145.26 ± 1.20

-

Soyabean oil

0.32 ± 0.02

-

Menthol 30%**

30.98 ± 0.18

-

SURFACTANTS

Vanillin 30%**

121.97 ± 0.86

-

Labrasol

146.07 ± 3.81

86.9

Lutrol F-68 30%**

33.34 ± 0.18

-

Tween 80

241.80 ± 9.40

24.6

C/V 20/20**

193.32 ± 2.65

-

Tween 20

217.84 ± 5.85

19.1

C/V 20/40**

219.18 ± 1.70

-

Span 20

21.93 ± 1.48

29.9

C/V 40/20**

265.34 ± 1.71

-

Cremophor EL

103.80 ± 1.99

78.5

V/L 20/20**

107.79 ± 1.86

-

Acrysol®

114.29 ± 4.32

93.7

V/L 20/40**

98.93 ± 1.40

-

COSURFACTANT/MODIFIED SOLUBILIZERS

V/L 40/20**

137.55 ± 3.48

-

Lauroglycol 90

80.51 ± 2.38

-

C/L 20/20**

134.53 ± 3.27

-

PEG 400

103.26 ± 3.37

-

C/L 20/40**

118.10 ± 1.09

-

Propylene glycol

89.51 ± 4.32

-

C/L 40/20**

214.59 ± 4.96

-

Ethanol

4.96 ± 2.78

-

C/V/L 10/10/10**

210.65 ± 1.39

-

** Solution of cosurfactant(s) in ethanol (wt/wt); C=Camphor, V=Vanillin, L=Lutrol F-68

Digits shows the % of component in solution (wt/wt); B3= 20%Camphor+20%Vanillin+20%Lutrol F-68 in ethanol (wt/wt)

TB3Mix= Transcutol P + B3 (1:1)

HPLC analysis

The HPLC analysis was carried out using Merck Lachrome high performance liquid chromatography system (Lachrome, Merck Hitachi). Chromatographic separation was accomplished using an ODS column (Lichrosphere® 100), C18, 250 mm x 4.6 mm, 5µm stainless steel column. The mobile phase consisted of a mixture of buffer (0.02 M monobasic potassium phosphate), acetonitrile, and triethylamine in the ratio of 40:60:0.2, with pH adjusted to 6.0 using phosphoric acid. The mobile phase was pumped isocratically at a flow rate of 2.0 ml/min during analysis. The amount of drug dissolved at each sampling point was estimated using UV wavelength of 254 nm.

Screening of surfactants for emulsifying ability

Emulsification ability of various surfactants was screened. Briefly, 300mg of surfactant was added to 300 mg of the selected oily phase. The mixture was gently heated at 45-60°C for homogenization. The isotropic mixture, 50 mg, was accurately weighed and diluted with double distilled water to 50 ml to yield fine emulsion. The ease of formation of emulsion was monitored by noting the number of volumetric flask inversions required to give uniform emulsion. The resulting emulsions were allow to stand for 2 h and their transmittance was assessed at 633 nm by UV-160A double beam spectrophotometer (Shimadzu, Japan) using double distilled water as blank.

Screening of cosurfactants

The turbidimetric method was used to assess relative efficacy of the cosurfactants to improve the nanoemulsification ability of the surfactant and also to select best cosurfactant from the large pool of cosurfactant available for design of formulation. Acrysol®, 200 mg was mixed with 100 mg of cosurfactant. Capryol90 (CAE), 300 mg, was added to this mixture and the mixture was homogenized with the aid of the gentle heat (45-60 â-¦C).The isotropic mixture, 50 mg, was accurately weighed and diluted to 50ml with double distilled water to yield fine emulsion. The ease of formation of emulsions was noted by noting the number of 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 h and their transmittance was measured at 638.2 nm by UV-160A double beam spectrophotometer (Shimadzu, Japan) using double distilled water as blank. As the ratio of cosurfactants to surfactants is the same, the turbidity of resulting nanoemulsions will help in assessing the relative efficacy of the cosurfactants to improve the nanoemulsification ability of surfactants.

Pseudoternary phase diagram studies

In order to identify self-emulsifying regions as well as suitable components, pseudo-ternary phase diagrams containing oil, surfactant, co-surfactant, and water were constructed by aqueous titration method. On the basis of solubility studies of CC in different vehicles, Capryol-90 were selected as the oil phase, On the basis of solubility and emulsifying ability Acrysol was selected as surfactant. The sizes of the nanoemulsion region in the diagrams were compared. Briefly, various self-emulsifying formulations were prepared by mixing oil and surfactant/co-surfactant mixture in varying volume ratio from 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, in separate glass vials. Cosurfactant system ratio containing Transcutol P and B3Mix was maintained constant at 1:1, 1:2 and 2:1. Mixtures were homogenized with the aid of gentle heat (45-60°C). Pseudo-ternary phase diagrams were developed using aqueous titration method and were mapped with the help of Sigma Plot software (version 11.0). Slow titration with aqueous phase was done to each weight ratio of oil and Smix and visual observation was carried out for transparent and easily flowable nanoemulsions. The physical state of the nanoemulsion was marked on a pseudo-three-component phase diagram with one axis representing aqueous phase, the other representing oil and the third representing a mixture of surfactant and cosurfactant at fixed weight ratios (Smix ratio). The phase diagrams are shown in Fig ….?

Fig. 7.3: Pseudoternary Phase Diagram for Capryol-90 as

Oil Phase, Acrysol® as Surfactant and Water

Fig. 7.4: Pseudoternary Phase Diagram for Capryol-90 as

Oil Phase, Acrysol®: TB3Mix (1:1) as Smix and Water

Fig. 7.5: Pseudoternary Phase Diagram for Capryol-90 as

Oil Phase, Acrysol®: TB3Mix (2:1) as Smix and Water

Fig. 7.6: Pseudoternary Phase Diagram for Capryol-90 as

Oil Phase, Acrysol®: TB3Mix (1:2) as Smix and Water

Fig. 7.7: Comparative Pseudoternary Phase Diagram for Capryol-90 as

Oil Phase, Acrysol®: TB3Mix as Smix and Water

Construction of ternary phase diagrams

A series of self-emulsifying formulations were prepared with varying concentrations of oil, surfactant, and co-surfactant. Concentration of capryol-90 was varied from 10-55% (w/w) as an oil phase, Acrysol® from 30-75% (w/w) as surfactant and TB3Mix from 0-40% (w/w) as cosurfactant at an interval of 5%. Total of the oil, surfactant, and co-surfactant always added upto 100% in each mixture. Each formulation was homogenized with the help of gentle heat upto 45-60°C. Accurately weighed 50 mg of each of 47 mixtures was then emulsified to 50 ml with distilled water separately, under the conditions of gentle shaking and the resultant emulsion was allowed to stand undisturbed for 15 min for equilibration. The selection of emulsification range was done on the visual clearance and % transmittance. Only those compositions having % transmittance more than 70% and clear appearance were considered desirable and were used in plotting the ternary phase diagram. Ternary phase diagrams were plotted using Sigma Plot software (version 11.0). Desirable self-emulsifying region and concentration range of each component were identified as shaded are from the phase diagram shown in Fig,…. .

Fig. 7.8: Ternary Phase Diagram for Capryol-90, Acrysol® and TB3Mix

Optimization of SEDDS formulation using Mixture D-optimal design

The pre-optimization studies concluded the ranges of oil (Capryol 90), surfactant (Acrysol®) and cosurfactant, TB3Mix [Transcutol P: B3Mix (1:1)] were 10-30 %, 40-70 % and 10-40 % respectively. These concentrations were subjected to optimization using Design Expert software (Version 8.0.3) of Stat-Ease, Inc. Minneapolis, USA. A variation in concentration of any of these components causes a change in the droplet size, isotropicity, polydispersity index, drug release as well as other properties of the formulation. Thus, concentration of oil, surfactant and cosurfactant were chosen as the independent variables or factors. The lower and upper limits of each factor were selected on the basis of the pre-optimization studies as well as compatibility of possible combinations by software. The sum total of all the three components in a formulation always summed upto 100%. The variables along with their ranges are recorded in table 7.8.

Table 7.8: Independent Variables and Their Ranges for Optimization of SEDDS Formulation

S. No.

Variable

Unit

Type

Desired target

Lower

Upper

Goal

Independent Variables

1

Amount of oil

%

Numeric

10

30

2

Amount of surfactant

%

Numeric

35

60

3

Amount of cosurfactant

%

Numeric

10

45

Dependent Variables

1

Cumulative % drug release in 30 minutes

%

Numeric

55.80

94.41

Maximize

2

Average particle size

nm

Numeric

24.66

187.00

Target to 75

3

Polydispersity index

-

Numeric

0.133

0.416

Minimum

4

Turbidity

-

Numeric

0

1

minimum

Constraint Applied for Independent Variables

Amount of oil + Amount of surfactant + Amount of cosurfactant = 100%

Amount of surfactant ≥ Amount of co-surfactant

Four responses include cumulative % drug release in 30 minutes (Y1) Average droplet size (nm) (Y2), polydispersity index (Y3), and turbidity (Y4) since they are generally regarded as significant factors for assessing the qualities of SEDDS. A two-factor, two levels D-Optimal Mixture Design was undertaken to investigate the main effects and the interactions of the two factors on the four responses. The design consist of 16 runs viz. 6 model formulations, 5 runs to estimate lack of fit, and 5 replicate runs. The purpose of replication was to estimate experimental error and increase the precision. The independent and dependent variables are shown in Table3, and the experimental runs with observed responses are shown in Table1. The dissolution profiles of all formulations are given in Figure1. Based on the experimental design, the factor combinations yielded different responses.

Table 7.11: Evaluation of Optimization Batches of SEDDS Formulation

Run No.

Formulation batch code

Factor-1

Factor-2

Factor-3

Response-1

Response-2

Response-3

Response-4

Amount of Oil (%)(wt/wt)

Amount of Surfactant (%) (wt/wt)

Amount of Cosurfactant (%) (wt/wt)

Release in 30 Min (%)

Avg. Droplet Size (nm)

Polydispersity Index (PDI)

Turbidity*

1

CCRUN 1

10

60

30

84.41

25.31

0.221

0

2

CCRUN 2

20

40

40

71.19

59.00

0.362

0

3

CCRUN 3

20

50

30

62.87

38.41

0.147

0

4

CCRUN 4

10

45

45

78.84

96.70

0.221

0

5

CCRUN 5

20

60

20

64.50

134.00

0.183

1

6

CCRUN 6

30

60

10

55.80

182.00

0.176

1

7

CCRUN 7

15

51.25

33.75

70.68

65.88

0.182

0

8

CCRUN 8

25

55

20

62.00

165.00

0.291

1

9

CCRUN 9

20

45

35

69.00

137.00

0.269

1

10

CCRUN 10

10

45

45

81.90

99.80

0.227

0

11

CCRUN 11

30

47.5

22.5

70.32

71.23

0.327

0

12

CCRUN 12

30

35

35

75.85

48.31

0.219

0

13

CCRUN 13

30

47.5

22.5

72.54

73.62

0.416

0

14

CCRUN 14

30

60

10

57.92

187.00

0.133

1

15

CCRUN 15

30

35

35

77.2

48.25

0.212

0

16

CCRUN 16

10

60

30

82.96

24.66

0.227

0

* Turbidity, 0 = clear emulsion and 1 = turbid emulsion

The mathematical relationships in the form of polynomial equations for the measured responses are listed in Table2. The statistical summary of response variables is summarized in Table.

Table : Mathematical Relationship For Measured Responses As Polynomial Equation

Cumulative % Drug Release in 30 Minutes

=

+7.24431 *Oil

+1.18051 *Surfactant

+0.34100 *Cosurfactant

-0.12168 *Oil*Surfactant

-0.07951 *Oil*Cosurfactant

+0.01482 *Surfactant*Cosurfactant

Avg. droplet size (nm)

=

+7.24431 *Oil

+1.18051 *Surfactant

+0.34100 *Cosurfactant

-0.12168*Oil*Surfactant

-0.07951*Oil*Cosurfactant

+0.01482 *Surfactant *Cosurfactant

Polydispersity Index (PDI)

=

-0.04192 * Oil

+0.00512 * Surfactant

+0.28316 * Cosurfactant

+0.00160* Oil * Surfactant

-0.006050* Oil * Cosurfactant

-0.00587* Surfactant * Cosurfactant

+0.00009 * Oil * Surfactant * Cosurfactant

+0.00004 * Oil * Surfactant * (Oil-Surfactant)

-0.00002 * Oil * Cosurfactant * (Oil-Cosurfactant)

+0.00005 * Surfactant * Cosurfactant *

(Surfactant-Cosurfactant)

Turbidity

=

-0.30455 * Oil

+0.03350* Surfactant

+0.07390* Cosurfactant

+0.00440* Oil * Surfactant

+0.00390* Oil * Cosurfactant

-0.00276* Surfactant * Cosurfactant

Table 7.13: Statistical Summary for the Response Variable

Response Variable

Model

F Value

Df

P-Value Prob>F

Adjusted R-Square

Predicted R-Square

Adequate Precision

Y1

Quadratic

42.91

5

0.0001

0.9332

0.8921

6.248

Y2

Quadratic

7.66

5

0.0034

0.6895

0.6023

11.184

Y3

Cubic

4.64

9

0.0376

0.6862

0.9705

8.820

Y4

Quadratic

5.12

5

0.0137

0.5789

0.4749

7.463

The results obtained were statistically analyzed for response variables by using Design expert software (8.0.3 version) of Stat-Ease, Inc. Minneapolis, USA. After performing response surface analysis, numerical optimization was done by setting the criteria for optimization which are reported in table 7.14. The optimized batches were selected on the basis of desirability function. Those formulations having desirability factor near 1.0 were selected. The desirability plot, overlay plot, and selected optimized batches are presented in fig. 7.14, fig. 7.15 and table 7.16 respectively.

Preparation and characterization of optimized batches of Candesartan Cilexetil SEDDS

The optimized formulations obtained by the Design-Expert software, were prepared by spontaneous emulsification method. All the three components of the system were accurately weighed in the required amounts in glass vials. They were then homogenized by gentle heating upto 45-60°C. The mixtures were then stirred using vortex stirrer for 5 min for proper mixing of the components. Sixteen mg of drug was added to each formulation and mixed using vortex stirrer for 10 min for proper solubilization of drug and development of a homogeneous formulation.

Formulation containing 16 mg of the drug was finally filled in size '2' capsule with the help of a micropipette. The capsule shell was then sealed by applying 1% gelatin solution and subsequently 70% w/v solution of alcohol on the joint and cooling. Compositions of the optimized batches are recorded in table 7.17.

Table 7.17: Composition of Optimized SEDDS Formulations

of Candesartan Cilexetil

Sr No.

Ingredients of SEDDS

Formulation Batch Code

FCC- 1

FCC- 2

FCC- 3

FCC- 4

1

Amount of Capryol-90 (mg)

38.7

37.4

45.0

25.8

2

Amount of Acrysol® (mg)

55.6

63.7

68.7

68.0

3

Amount of Transcutol P(mg)

27.8

24.4

18.2

28.1

4

Amount of Camphor (mg)

5.6

4.9

3.6

5.6

5

Amount of Vanillin (mg)

5.6

4.9

3.6

5.6

6

Amount of Lutrol F-68 (mg)

5.6

4.9

3.6

5.6

7

Amount of Ethanol (mg)

11.1

9.8

7.3

11.3

SEDDS Characterization

Determination of droplet size and zeta-potential

Droplet size of SEDDS is a critical step in the pathway of enhancing drug bioavailability. To investigate the globule size of resultant emulsion, fifty mg of the formulations was diluted to 50 ml with distilled water and was allowed to equilibrate for 15 min. Droplet size, distribution and zeta potential of the resulting emulsion was then measured by laser particle size analyzer (Malvern Zetasizer Nano S, Malvern Co., UK). The detection range was from 2 to 5000 nm. Each sample was analyzed in triplicate. Result

Cloud point determination

The coloud point is the temperature above which the formulation clarity turns into cloudiness.

At higher temperatures, phase separation can occur due to dehydration of polyethyleneoxide moiety of the non-ionicsurfactant. Since both drug solubilization and formulation stability will decline with this phase separation, the cloud point of the formulation should be over 37â-¦C. The cloud point value is affected by factors such as drug hydrophobicity, kind, combination, mixing ratio and amount of each of the oils, surfactants and co-surfactants used (Itohetal.,2002;Zhangetal.,2008). To measure the cloud point, 1 ml of the formulation was diluted with 250 ml distilled water, and temperature of the resulting emulsion was gradually increased at increments of 2°C. The temperature at which turbidity appeared was noted down. In this study, cloud point of F4 formulation was very high as reported in Table5

In vitro release studies

An in-vitro drug release study for the optimized formulations was performed using USP paddle apparatus. The dissolution media used for study is recommended by USFDA, comprising 900 ml of 0.35% polysorbate 20 in 0.05 M phosphate buffer of pH 6.5 at 50 rpm (paddle rotation). A 166 mg aliquot of the formulation (equivalent to 16 mg of candesartan cilexetil with 10.7 % drug loading in 150 mg formulation blend) in prefilled capsule shell was placed in dissolution media and temperature was maintained at 37°C±0.5°C. Placebo formulations were also tested to check interference, if any. Samples were collected periodically and replaced with fresh dissolution medium. Samples after filtration through syringe filter (Millipore Millex-HN, Nylon 0.45 µm) were analyzed by HPLC method at 254 nm for candesartan cilexetil content. 100µl samples were drawn out at the predetermined intervals, and the same volume of fresh dissolution medium was replenished. The release of candesartan cilexetil from SMEDDS formulation was compared with the marketed tablet of candesartan cilexetil containing the same quantity of drug. A sample (20µl) was injected into HPLC.

Results and discussion

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