Formulation and Evaluation of Liquid Self Micro Emulsifying Drug Delivery System of Trimipramine Maleate to Enhance Aqueous Solubility

Oral route is most convenient route for non- invasive administration; mostly 40% of drugs are lipophilic in nature having poor aqueous solubility, oral bioavailability and dissolution rate. There are various methods to enhance solubility and dissolution rate they are co solvency, reduction of particle size, salt formation, hydrotrophy etc.., a novel approach has been introduced to enhance solubility and dissolution rate i.e., self emulsifying drug delivery system, which can be classified as nano emulsifying drug delivery system and micro emulsifying drug delivery system.

Solubility plays a key role in the therapeutic efficacy and bioavailability, solubility is defined as amount of solute dissolved or solubilised in a given solvent is termed as solubility.^{13, 20}

1. 1. SELF MICRO EMULSIFYING DRUG DELIVERY SYSTEM:

Self-emulsifying drug delivery system [SEDDS] is a novel approach to enhance aqueous solubility and dissolution rate. It is defined as isotropic mixtures of oils, surfactants and co surfactants some time includes co solvents upon administration which will spontaneously emulsified under mild agitation in GIT.⁵

SMEDDS can form oil in water emulsion spontaneously in GIT; SMEDDS are more stable and effective when compared to normal emulsions. SEDDS ranges about 300nm to 5µm, depending upon droplet size SEDDS are divided into SMEDDS (self micro emulsifying drug delivery system) with a droplet size of 250nm to 300nm and SNEDDS (self nano emulsifying drug delivery system) with a droplet of < 100nm or 100 to 150nm.^{14, 27}

Oral route is convenient route of administration and widely used route, it is cost effective but 40% of drugs in market are lipophilic and these dosage forms will depends upon various factors like solubility, permeability, dissolution rate and oral bioavailability.²

1.2 LIPID CLASSIFICATION SYSTEM:

Lipids are employed in SNEDDS formulation to enhance solubility, dissolution rate of drugs. Lipid classification system was developed by Pouton based on their composition as given below.

TYPE I: Lipids without any surfactants, oil based systems.

TYPE II: System with water insoluble surfactants these will forms SEDDS formulation systems.

TYPE III: System with water soluble surfactants these will forms SMEDDS or SNEDDS formulation systems.

TYPE IV: System with water soluble surfactants and co solvents, systems are oil free.¹⁴

1.3 MECHANISM OF SELF EMULSIFICATION:

Mechanism of self-emulsification was explained by REISS and WAKERLY.

According to REISS self-emulsification can be achieved by entropy change which causes dispersion which is greater than energy required to increase surface area of dispersion.

Free energy of emulsion is a direct function of energy which creates interface between oil and water phases which can be expressed by an equation as given below:

?g =∑n ∏r2s

Where: ?g = Free energy associated with process

            ∑n = Number of droplets

               r = Radius of droplets

               s = Interfacial energy       

2. MATERIALS AND METHODS

2.1 Materials:

Trimipramine maleate was obtained as gift sample from R.L.Fine chem., (Banglore, India). Capryol 90 ,Peceol , Capmul PG-8NF , Labrasol , Transcutol were donated by Gattefosse (Mumbai, India), were Almond oil donated by Hamdard Laboratories, Capmul MCM NF, Capmul PG8 , Capmul MCM C8 Ep,Captex 200 ,Labrafac PG ,Maisine CC were donated by Abitec corporation, Span 80 was donated by Research Lab Fine chem Industries , Tween 80 was donated by Oxford laboratory , PEG-400 was donated by SD Fine chem Limited, Hydrochloric acid was donated by Virat labs,mumbai and Methanol was donated by Qualigens.

2.2 Determination of Analytical methods:

Analytical methods employed in this study are UV Visible spectroscopy. UV spectroscopy used for determination of calibration curve, solubility analysis of excipients with drug (Trimipramine maleate), Determination of percentage transmittance, assay and also dissolution studies of formulation.

Trimipramine maleate is analyzed by Shimadzu UV 1800 double beam spectrophotometer shown in figure no 01.

Figure no: 01 Shimadzu UV 1800 spectrophotometer.

2.2.1 Spectrophotometric analysis of Trimipramine maleate in methanol:

2.2.1.1 Standard stock solution preparation of Trimipramine maleate in methanol:

Accurately weighed 10mg of Trimipramine maleate transferred into 10ml of volumetric flask, dissolved in methanol and volume will be made up to 10ml.

2.2.1.2 Determination of λmax of Trimipramine maleate in methanol:

After preparation of standard stock solution, from standard stock solutions 2, 4, 6, 8, 10 ppm was prepared using methanol as analytical solvent. Absorbance was noted by analyzing concentrations in UV visible spectrophotometer, absorbance was scanned from 400-200nm wavelength. Spectrum shows absorbance maxima (λmax) at 250nm, this wavelength was utilised for further analysis present in the study.

2.2.1.3 Calibration curve of Trimipramine maleate in methanol:

Standard stock solutions were prepared with concentration 2, 4, 6, 8,10 ppm using methanol as a solvent. Absorbance was measured at 250nm wavelength and standard plot was drawn by obtained data (i.e., absorbance and concentration), Slope, Regression and correlation was determined. The equation is given along with plot in figure no 02.^2,9

3. Solubility studies:

Solubility studies of Trimipramine maleate was determined in various oils, surfactants and co surfactants, Excess amount of drug added to 500mg of excipients contained in a glass vial. Mixture was then cyclomixed for 2 minutes using cyclomixer to enhance drug solubility. Mixtures were sonicated for 5-10 min to dissolve drug completely. Resultant mixtures were kept for equilibrium at room temperature in a mechanical rotary shaker at a speed of 100rpm for 72 hours.²

After 72 hours mixtures were centrifuged at 3000rpm for 15-20 minutes, these will separate dissolved drug as supernatant phase and undissolved drug as sediment phase take 0.1 ml of supernatant by utilising micropipette, supernatant were diluted with methanol which is used as a co solvent the concentrations of Trimipramine maleate in each excipient was determined spectrophotometrically at 250nm. Solubility data of Trimipramine maleate in excipients were shown in table no 04, 05, 06 and graph no 01, 02, 03.^2,3,19

3.1 Construction of pseudo ternary phase diagram:

Pseudo ternary phase diagram was constructed to determine micro emulsifying regions and also to select suitable concentrations of oils, surfactants, co surfactants Pseudo ternary phase diagram was constructed by water titration method. Surfactants and co surfactants (smix) ratios were prepared (1:4 , 4:1) by mixing accurately weighed amount of surfactant with co surfactant. Oil were mixed in different weight ratios of 9:1 to 1:9 (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9) into the glass vials. Each mixture was titrated by water until mixture turned into blue colour emulsion or milky white or coarse emulsion or gel phases. Different phases were plotted on a phase diagram using Triplot version 4.1.2 software results are shown in figure no 16, components used for construction of phase diagram are oil (Capryol 90), surfactant (Labrasol) and co surfactant (Transcutol ) and Millipore water.^20,15,2

4. Formulation of liquid self-micro emulsifying drug delivery system:

Micro emulsion regions were determined by phase diagrams, once micro emulsion region identified. SMEDDS formulations were prepared with addition of varying ratios of selected Smix (surfactant: co surfactant) and Oil. In all the formulations Trimipramine maleate was kept constant (2.5 mg). Weighed amount of drug is dissolved or solubilised in oil phase then cyclomixed for 2 minutes using cyclomixer, Surfactant and co surfactant ratios were prepared and mixed into drug oil mixture and vortexed.²

The prepared formulations were kept at room temperature for further investigation and evaluation; Composition liquid self-micro emulsifying drug delivery system was shown in Table no 18 and formulation of L-SMEDDS were shown in figure no 07.

5. Evaluation liquid self-micro emulsifying drug delivery system:

5.1 Dispersibility test:

Self-emulsification efficacy was determined by addition of 0.1ml of formulated Trimipramine maleate L- SMEDDS into 200ml of distilled water with gentle agitation using a magnetic stirrer, Formation of micro emulsion was visually observed and performance of micro emulsion was evaluated using following grading system shown in below table no 01 and results were shown in table no 11.^6,8,21

Table no: 01 Trimipramine maleate L-SMEDDS was evaluated by following grading system.

5.2 Self emulsification test:

0.1ml of Trimipramine maleate  L-SMEDDS were added into 200ml of distilled water under mild or gentle agitation using a magnetic stirrer at a speed of 100rpm temperature maintained at 37°c ± 1°c. Self-emulsification was visually assessed by changing the appearance of the formulation and self-emulsification time will be noted, if it forms clear blue emulsion which indicates formulated Ol Trimipramine maleate  L-SMEDDS was good or if it forms less clear or turbid emulsion appearance which indicates bad emulsion. Results were shown in table no 12.^1,9

5.3 Thermodynamic stability studies:

Thermodynamic stability studies were performed to determine temperature and centrifugation efficacy. The formulation were added to Millipore water at 1:20 ratio and centrifuged at 3500 rpm for 15 to 30 min and observed for changes like phase separation cracking or creaming and precipitation, Formulations are subjected to freeze thaw cycle by diluting formulations at 1:20 ratio with Millipore water at a temperature -20 to +25°c stored for 48 hrs results were shown in table no 13.^16,18

5.4 Determination of droplet size, PDI and Zeta potential:

Trimipramine maleate  L-SMEDDS were 100 times diluted with water in a test tube or volumetric flask, globule size, PDI and zeta potential were determined after 1 hour by dynamic light scattering spectroscopy using Zetasizer nano ZS 90 version 7.1 (Malvern instruments) and results were shown in table no 14.⁹

5.5 Invitro drug release by Diffusion studies :

In vitro diffusion studies were performed for all the formulations developed, using a dialysis technique. The dialyzing medium was 0.1 N HCL. One end of egg membrane was tied with thread, and then 1 ml of self-micro-emulsifying formulation was placed in it along with 0.5 ml of dialyzing medium. The other end of the tubing was also secured with thread and was allowed to rotate freely in 200 ml of dialyzing medium and stirred continuously at 100 rpm with magnetic bead on magnetic plate at 37°C.

Aliquots of 3 ml withdrawn at 5, 10, 15, 30, 45, 60, 90, 120 minutes respectively time interval with a volume of 3ml and buffer (0.1N HCL) were replaced to maintain sink conditions. These samples were analyzed quantitatively for drug dialyzed across the membrane at corresponding time by using UV-visible spectrophotometer (250nm). After 1 month diffusion studies performed and compared with diffusion data shown in table no 15.^2,14

6. RESULTS AND DISCUSSION

6.1 Spectrophotometric analysis of Trimipramine maleate in Methanol:

6.1.1 Determination of λmax and Calibration curve of Trimipramine maleate in Methanol:

λmax of Trimipramine maleate in Methanol was found to be 250nm using methanol as blank peak table with absorbance of Trimipramine maleate in Methanol were shown in table no 02, Standard graph was plotted with concentrations ranges from 2-10 µg/ml. Standard graph values were shown in table no 03.

Standard plot was constructed by taking absorbance on Y-axis and concentration on X-axis the plot is shown in figure no 02. The regression co-efficient (R2) was found to be 0.99603.

Table no: 02 Peak table showing absorbance and wavelength of Trimipramine maleate in Methanol.

No.	P/V	Wavelength	Abs.
1		398.40	0.070
2		250.40	0.324
3		210.60	1.170
4		203.60	1.150

Table no: 03 Standard graph values of Trimipramine maleate in Methanol Concentration (µg/ml).

Figure no:02 Calibration curve of Trimipramine maleate in methanol.

6.2 Solubility studies of Trimipramine maleate:

6.2.1 Solubility studies of Trimipramine maleate in various oils :

Solubility of Trimipramine maleate in various oils was determined by UV visible spectroscopy at 250nm using methanol as blank.

Table no: 04 Solubility of Trimipramine maleate in various oils.

Graph no: 01 Comparison of solubility of Trimipramine maleate in various oils

6.2.2 Solubility of Trimipramine maleate in various surfactants:

Table no:05 Solubility of Trimipramine maleate in various surfactants:

Graph no: 02 Comparison of solubility of Trimipramine maleate in various surfactants.

6.2.3 Solubility of Trimipramine maleate in various co surfactants:

Table no: 06 Solubility of Trimipramine maleate in various co surfactants:

Graph no: 03 Comparison of solubility of Trimipramine maleate in various co surfactants:

6.3 Pseudo ternary phase diagram:

From pseudo ternary phase diagram, it was found that systems containing Capryol 90 as oil phase, Labrasol as surfactant and Transcutol as co surfactant showed good micro emulsifying property.

Smix (CLT1:4, CLT4:1) ratios were prepared with oil ratios from 9:1 to 1:9 i.e.,  9:1,8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9.

• Smix (1:4) formulations of 1:9 to 2:8 showed bluish white emulsions (BWE) ,as 3:7 to 5:5 showed milky white emulsions (MWE) and 6:4 to 9:1 showed clear phase separation (PS) showed in table no 07 and figure no 03.
• Smix (4:1) formulation of 2:8 to 5:5 showed milky white emulsion (MWE) and 1:9 showed bluish white emulsion (BWE), and 6:4 to 9:1 showed clear phase separation (PS) showed in table no 08 and figure no 05.

Table no: 07 Percentage composition of Capryol 90, Labrasol and Transcutol upon water Titration method.

Figure no:03 Physical appearance of CL1T4(Smix1:4) with water titration method.

Figure no:04 Pseudo ternary phase diagram of CL1T4(Smix1:4)

Table no: 08 Percentage composition of Capryol 90, Labrasol and Transcutol upon water Titration method.

Figure no:05 Physical appearance of CL4T1(Smix4:1) with water titration method.

Figure no:6 Pseudo ternary phase diagram of CL4T1(Smix4:1)

6.4 Selection of Formulation:

Table no:09 Selection composition of formulation.

7. Formulation of Trimipramine maleate L-SMEDDS:

Smix (Labrasol: Transcutol) ratios were prepared, Trimipramine maleate was added into oil (Capryol 90) and the mixtures were cyclomixed for 2min, Trimipramine maleate L-SMEDDS were shown below.

Figure no 7: Formulation of Trimipramine maleate.

7.1 Composition of prepared Trimipramine maleate L-SMEDDS formulations:

Table no:10 Composition of Trimipramine maleate L-SMEDDS formulations.

8. Evaluation of Trimipramine maleate L-SMEDDS:

8.1 Dispersibility test: Visual observation of Trimipramine maleate liquid self-micro emulsifying drug delivery system showed that formulations are grade A, Rapidly emulsion forming within less than minute with clear appearance. Formulations were assessed by grading system results were shown in table no 11.

Table no: 11 Dispersibility test values of Trimipramine maleate L-SMEDDS

8.2 Self emulsification time: Trimipramine maleate L-SMEDDS was dispersed completely and quickly when subjected to aqueous dilution under mild agitation results were shown in table no 12.

It was clear that the formulations were self-emulsified within 30 ± 1.75 to 35 ± 1.85 seconds and indicates good micro emulsion formation.

Table no:12 Self emulsification time of Trimipramine maleate L-SMEDDS

All values are expressed as Mean±SD(n=3)

8.3 Thermodynamic stability studies:

No phase separation or precipitation is observed for formulations CL1T4(2:8) and CL4T1(1:9) which indicate that the formulations are stable formulations under temperature results were showni in table no13.

Table no:13 Thermodynamic stability studies of Trimipramine maleate L-SMEDDS.

All values are expressed as Mean±SD(n=3)

8.4 Determination of Globule size and Zeta potential:

Table no: 14 Globule size, Zeta potential and PDI values of Trimipramine maleate L-SMEDDS.

All values are expressed as Mean±SD(n=3)

Figure no 08 : Globule size of CL1T4(2:8)

Figure no 09 : Zeta potential of CL1T4(2:8)

Figure no 10 : Globule size of CL4T1(1:9)

Figure no 11 : Zeta potential of CL4T1(1:9)

8.5 Invitro drug release:

Figure no 12 : In vitro diffusion studies.

Table no:15 In vitro diffusion studies of Trimipramine maleate L-SMEDDS.

Graph no 04: % CDR of Trimipramine maleate im 0.1NHcl.