INTRODUCTION:

The reverse micelles are small, dynamic aggregates of surfactant molecules surrounding a polar core dispersed in a nonpolar continuous phase. Reverse micelle solutions are clear and thermodynamically stable; as water is added to a reverse micelle solution, a microemulsion is formed that contains nanometer-sized water droplets dispersed in a continuous oil phase¹. Many problems remain associated with insulin therapy due to its rapid enzymatic degradation on oral administration. This leads to short biological half-life and ineffectiveness of the drug. Moreover, the membrane permeability is often low due to lack of lipophilicity and higher molecular weight². Eye drops are conventional dosage forms that account for 90% of currently accessible ophthalmic formulations. Despite the excellent acceptance by patients, one of the major problem encountered is rapid precorneal loss³. Hence, there is need to develop a system that could effectively deliver the drug in a controlled fashion for prolonged period of time.

The eye has potential as an excellent route for peptide administration including insulin⁴. Blood glucose levels of test animals can successfully be lowered on instillation of insulin in eye^5,6. Upon instillation of an ophthalmic solution most of the instilled volume is eliminated from precorneal area.

This loss is mainly attributed to systemic absorption of more than 40% of instilled ocular dose, drainage of excessive fluid by naso- lachrymal duct as well as elimination by tear turn over⁷. Zaki et al. (1986) have compared the effect of viscosity on precorneal residence of solutions in rabbit and man⁸.

Carstens et al (1993) Studied transport of insulin across rabbit nasal mucosa in vitro induced by Didecanoyl-L-a-phosphatidylcholine⁹. Upadhyay et al (1997) have developed the reverse micelle-lamellar phase transition based depot preparation of rifampicin¹⁰. Rai et al. (2004) have developed reverse micellar systems for controlled ocular delivery of timolol¹¹.

Many systems have been developed and advocated to modify and optimize the drug response when delivered topically to eye such as soluble gel and emulsion, hydrophilic ocular inserts, viscous vehicle, ocular liposomes approach.

The overflow of the drug the tear may be reduced by use of reverse micellar system. The present study was aimed at occlusion of insulin in reverse micellar system based on insulin/surfactant/isopropyl myristate. Surfacants cetyltri-ammonium bromide and span-60 were used. The reverse micellar system bearing dissolved aqueous drug solution in non-polar solvent offer lipophilicity to the drug solution contained in its interior polar aqueous pool. The system was designed, developed and characterized for in-vitro characteristics.

MATERIALS:

The following materials were used; Zinc insulin (pork) (Torrent Pharmaceuticals Ltd. Ahemdabad), Isopropyl myristate (Fluka Chemie, USA), Cetyl tri-ammonium bromide (Sigma Aldrich,USA),Sorbitain monostearate (Span-60) (Robert Johnson,Mumbai), sodium Glycocholate(Fluka) and all other ingredients were of analytical grade.

Fig.1: Effect of temperature on the drug payload in reverse micellar systems.

Preparation of Reverse Micellar Systems:

An aqueous solution of insulin (5%w/v) was prepared in the double distilled water (pH 3.5). This solution (0.5mL) was injected to an organic pool consisted of Isopropyl myristate and surface active agents with continuous vigorous stirring for 1to2 hrs using magnetic stirrer at room temperature. Invariably surface-active agent/or system (when taken in combination) was added to organic bulk. The surface-active agents used are cetyl tri-ammonium bromide (CTAB) and span-60 (table1).

Table 1: Composition of reverse micellar systems.

Product Code	Surface-active agent		Insulin		Organic Solvent Isopropyl Myristate (G)
Product Code	CTAB (mM)	Span 60 (mM)	Incorpo rated (mg)	Deter mined (mg)	Organic Solvent Isopropyl Myristate (G)
CT. S-1	25	- -	25	17.53	20
CT. S-2	50	- -	25	18.70	20
CT. S-3	- -	25	25	18.30	20
CT. S-4	- -	50	25	19.82	20
CT. S-5	50	25	25	20.15	20
CT. S-6	25	50	25	20.89	20

Characterization of Reverse Miceller Systems:

Drug content:

Accurately weighed 1 mg (1.2 mL) preparations were filled in the treated dialysis tube (pieces) separately. After closing the both ends of the tubes, they were put into a beaker containing 50 mL of organic phase i.e. isopropylmyristate. The whole assembly was kept 12hrs for equilibrium dialysis with methanol. After that, each tube was taken out and preparations were evaluated by dissolving in PBS pH 7.4 with appropriate dilution. The solutions were analyzed spectrophotometrically using Shimandzu UV-2 spectrophotometer at 276 nm.

Effect of temperature on drug payload:

Accurately weighted 1 gm. reverse micellar system was filled in the treated dialysis (pieces). These tubes were kept in the 50 mL organic bulk at different temperature ranging from 25⁰C to 60⁰C. The rest of the procedure was same as described above (Fig.1).

Effect of polarity of organic phase:

The reverse micellar systems were filled in treated dialysis tubes. The tubes were kept in the external organic phase of different polarity. The polarity of organic phase was changed using methanol ranging from 1M to 5M. The rest of the procedure was same as described above (Fig.2).

In-vitro evaluation of reverse micellar systems:

In- vitro release rate of insulin from reverse micellar systems for corneal drug availability was determined using Franz diffusion cell. The pH of lachrymal fluid and the blinking rate of the eyes were the factors taken into consideration and were simulated.

Fig.2: Effect of polarity of Organic phase the drug payload in reverse micellar systems.

The receptor compartment contained 20ml of artificial tears solution (ATS,pH 7.4).One gram (1.2.ml) of the system (containing insulin 10 mg.) was kept in donor compartment over a cellophane membrane (PD 215, Dupont de Nemours) separating the system from receptor compartment. The diffusion cell was kept on magnetic stirrer and content of receptor compartment was stirred using small Teflon coated magnetic bar. The sample (2ml) was withdrawn from the receptor compartment at hourly intervals for 48 hrs.and the same volume was replaced with fresh artificial tears solution (pH7.4). Samples were analyzed spectrophotometrically at 276 nm for insulin content and data were shown in table 2.

RESULTS:

Drug content in the reverse micellar systems was determined by the equilibrium dialysis method. The drug content estimated in various reverse micellar systems was 70.12±0.5% to 83.15±1.2% based on the drug added.

Table 2: Data of cumulative percentage drug release - time profile of different reverse micellar formulations. (n=6)

Product Code

Cumulative percent drug release

1hr.

2hr.

4hr.

8hr.

12hr.

24hr.

48hr.

CT.S-1

4.5±

0.5

13.3±1.2

24.5

±1.5

43.2

±2.0

57.4

±2.2

76.0

±2.8

96.5

±3.0

CT.S-2

3.8±

0.6

10.5±1.2

19.0

±1.6

34.3

±1.8

47.9

±2.2

69.0

±2.1

80.3

±2.8

CT.S-3

1.8±

0.5

6.2±

1.1

13.0

±1.5

25.0

±2.1

35.0

±2.6

55.2

±2.2

67.5

±2.8

CT.S-4

1.0±

0.5

4.4±

1.0

9.2

±1.2

21.5

±1.8

32.0

±2.2

50.4

±2.5

60.2

±2.1

CT.S-5

2.4±

1.0

8.3±

1.5

16.3

±2.0

30.1

±2.3

39.5

±2.0

60.4

±2.8

72.5

±2.6

CT.S-6

4.1±

1.1

12.0±1.6

21.0

±1.8

36.5

±2.3

52.1

±1.4

74.0

±2.8

89.0

±3.1

An increase in the temperature noted to increase the micellar solubilization of aqueous drug solution. The relation was noted to be true upto a particular temperature for each system beyond which instead of increased percent drug payload a decrease was recorded (Fig.1). The optimum temperature for systems CT.S-1 to CT.S-6 was noted to be 35⁰C, 45⁰C, 35⁰C, 45⁰C, 50⁰C and 50⁰C respectively.

Increase in polarity of organic phase causes decrease in the drug solubilization in the pool. After addition of 5M, methanol in organic bulk, the percent drug payload for systems CT.S-1 to CT.S-6 was 18.4±0.5%, 27.0±1.5%, 33.5±0.8%, 52.7±1.8%, 46.5±2.0% and 40.2±1.5% respectively (Fig.2).

The reverse micellar systems were prepared by using surface-active agent alone or in combination were studied for their in-vitro drug release. The observations reveal that maximum retardance was offered by the system CT.S-4. While the maximum fastest release was observed in case of system CT.S-1 and CT.S-6 were 96.5±2.0% and 89.0±1.1% (Table 2&Fig.3).

DISCUSSION:

The reverse micellar systems were prepared by the method reported by Brochette et al. (1988)¹². When the surface-active agents were added in concentration above their respective critical micelle concentration they tended to form reverse micelles. Its formation was confirmed by an aqueous polarity probe method. It is essentially based on absorption maxima that are different in polar and non-polar solvent. The exhibition of maxima that corresponds to the non-polar water reveals the formation of reverse micelles. The selection of this system was made on the basis of various parameters including drug content, effect of variables on percent drug payload and in-vitro release profile.

The relation was noted to be true upto a particular temperature for each system beyond which instead of increased percent drug payload a decrease was recorded. It could be due to substantial increase in temperature provides kinematics energy too, could possibly destabilize the micellar system and mobilize the drug molecules from water pool to the organic bulk.

Fig.3: Log percentage cumulative drug release Vs time profile of different reverse micellar formulations. (n=6)

Effect of polarity was due to migration of drug into organic phase. But after a particular polarity equilibrium was established between water pool and bulk organic phase. After that there was no migration of drug molecules into organic bulk.

The reverse micellar systems were studied for their in-vitro drug release profile. The systems show approximate

first order release kinetics between log of cumulative percent drug release (Q) and time (t). The slope of the plot was used to calculate the release rate constant (k). On the basis of in-vitro release profiles the systems CT.S-1 and CT.S-6 were selected for in-vivo study.

In conclusion, the prolonged and controlled delivery of drug could be possible by using reverse micellar system because corneal surface showed charge affinity and lipoidal nature, thus facilitates the drug transport across the ocular membrane and also reduces the drug drainage with the tears.

ACKNOWLEDGEMENTS:

The authors are grateful to Torrent (India) Ltd., Ahemdabad, for providing Zinc insulin (pork) as gift sample. The facilities provided by Professor Ramesh Chandra, Ex-Vice-Chancellor, Bundelkhand University, Jhansi (U.P) India, are gratefully acknowledged.

REFERENCES:

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2. Wang,W., Sasaki, H., Chein, D.S., and Lee, V.H.L. Lipophilicity influence on rabbit: a comparison with corneal penetration.Curr.Eye Res. 1991;10, 571-579.

3. Patton, T.F. and Francoeur, M. Ocular Bioavailability and Systemic Loss of Topically Applied Ophthalmic Drugs. Amer. J. Ophthalmol. 1978; 85, 225-229.

4. Chiou, G.C.Y.,Shen. Z.F.and Zheng, Y.Q. Adjustment of Blood sugar levels with insulin and glucagons eye drops in normal and diabetic rabbits.J.Ocular Pharmacol. 1990;6,233-241.

5. Chiou, G.C.Y. Systemic Delivery of Polypeptide Drugs through Ocular Route Annu.Rev.Pharmacol.Toxicol. 1991; 31,457-467.

6. Yamamoto, T., Maitani, Y., Machida, Y. and Nagai, T. Effect of Brij-78 on systemic delivery of insulin from an ocular device. Pharm.Sci. 1994; 4, 133-138.

7. Chrai,S.S., Makoid, M.C.Eriksen, S.P. and Robinson, J.R. Drop size and initial dosing frequency problems of topically applied ophthalmic drugs. J.Pharm.Sci. 1974; 63, 333-338.

8. Zaki, I., Fitzerald, P., Hardy, J.G. and Wilson, C.G. A comparison of the effect of viscosity on the precorneal residence of solutions in rabbit and man. J. Pharm. Pharmacol. 1986;38, 463-466.

9. Carstens, S., Danielsen, G., Guldhammer,B. and Frederiksen, O. Transport of.Insulin across rabbit nasal mucosa in vitro by didecanoyl-L-a-phosphatidylcholine. Diabetes, 1993 42, 1032-1040.

10. Upadhayay, A.K., Omary, L.K., Khopade, A.J. and Jain, N.K. Study on a reverse micelle-lamellar phase transition based depot preparation of rifampicin.J.Pharmazie. 1997; 52, 961- 962.

11. Rai, A.K., Vyas, S.P. and Chitme, H.R. Reverse micelles of timolol for ocular delivery. Indian J. Pharm.Sci. 2004; 66(2), 164-170.

12. Brochette, P., Petit, C. and Pileni, M.P. Cytochrome c in sodium bis (2-ethylhexyl) sulfosuccinate reverse micelles: structure and reactivity. J. Phys. Chem. 1988; 92 (12), 3506.

Characterization of Ocular Delivery of Reverse Micelles Bearing Insulin

Fig.1: Effect of temperature on the drug payload in reverse micellar systems.

RESULTS: