Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Technology 26470 Eskişehir- TURKEY

Ebru BAŞARAN, Müzeyyen DEMİREL, Yasemin YAZAN

Cyclosporine A (CsA) is a powerful immunosuppressive active agent mainly used for

autoimmune diseases and graft rejections after organ transplantations [1]. Topical

application of CsA is preferred for the ocular treatment of disorders after corneal

transplantation and dry eye syndrome [2].

Solid lipid nanoparticles (SLN) were introduced as alternative carrier systems for

controlled release of pharmaceutical and cosmetic active compounds [3]. Particles

of the system remain in the solid state at room temperature and therefore the

mobility of incorporated drug is reduced which is a prerequisite for controlled drug

release [4]. Possibility of controlled drug release and drug targeting, increased drug

stability, high drug payload, incorporation of both lipophilic and hydrophilic drugs,

avoidance of organic solvents and no problem with respect to large scale production

and sterilization were the proposed advantages of SLNs [5].

Systemic absorption of CsA is quite low and there is interindividual variation in

plasma concentrations depending on the dosage form applied and the story of the

patient [6]. Therefore, in this study, cationic SLNs were prepared, aiming the ocular

delivery of CsA with an attempt to decrease the interindividual variation and thus to

increase its topical absorption.

MATERIALSCyclosporine-A Novartis, Switzerland Active agent (Gift)

Dynasan® 116 Condea, Germany Solid lipid

Stearylamine Fluka, USA Cationic agent

Benzalconium chloride Fluka, Denmark Antimicrobial agent

Tween® 80 Merck, Germany Surfactant

METHODS

SLN formulation (Table 1) was prepared by hot homogenization technique [4].

Homogenization was achieved with Ultraturrax (T25, IKA) at a stirring rate of 13500

rpm for 5 minutes, at 85ºC±1ºC.

Table 1. Composition of the formulation

As the sterility of the ocular formulations is necessary, the formulation was sterilized

by autoclaving at 121°C for 20 minutes.

Particle size and zeta potential measurements were carried out by Malvern Nano ZS

and the structure of the solid lipid was analyzed using differential scanning

calorimetry (DSC-60, Shimadzu), X-Ray Diffractometry (XRD) (RIKAGU D/Max-3C),

Fourier Transform Infrared Spectrophotometry (FT-IR) (Perkin Elmer Spektrum 2000)

and solid state NMR Spectrophotometry (1H-NMR).

Stability of the formulation was monitored for 6 months at different conditions

(25ºC±1ºC, 40ºC±1ºC, 4ºC±1ºC).

In vivo studies were carried on the sheep. Formulation was applied topically to one

of the eyes and the other eye remained untreated as a reference. At appropriate time

intervals, sheep were sacrificed and the aqueous and vitreous humour samples were

collected and analyzed by enzyme immune assay analyzer.

Particle sizes (Figure 1) and the zeta potentials (Figure 2) of the formulation remained unchanged during

the storage at different conditions (25ºC±1ºC, 40ºC±1ºC, 4ºC±1ºC) for a period of 6 months.

Stability of the lipid structure analyzed by XRD (Figure 3), FT-IR (Figure 4) and 1H-NMR (Figure 5) showed

that these data support the DSC data (Figure 6) indicating the stability of the formulations for 6 months.

According to the in vitro tests results, cationic SLN formulation remained stable during the storage

period of 6 months. After the topical application of the formulation to the eyes, detection of CsA at the

deeper layers, and no existence of interindividual variance in the CsA concentrations showed the

efficiency of SLN formulation on the absorption of such a problematic drug.

[a] [b] [c]

Figure 6. DSC thermograms of FD4 formulation during the storage time at 25ºC±1ºC [a], 40ºC±1ºC [b] and 4ºC±1ºC [c]

Figure 4. FT-IR spectra of FD4 formulation Figure 3. XRD spectra of FD4 formulationFigure 5. 1H-NMR spectra of

FD4 formulation

In Vivo Studies

CsA was determined in the aqueous and vitreous

humour samples for 48 hours (Figure 7). Detection of

CsA in the vitreous humour showed the efficient

penetration of the drug to the deeper layers of the

eyes. Similarity in the analysis results demonstrated

that interindividual variance did not affect the

absorption level of CsA. Figure 7. CsA concentrations in aqueous and vitreous humour samples

CodeDynasan® 116

(%)

CsA

(%)

Stearylamine

(%)

Benzalconium chloride

(%)

Tween® 80

(%)

FD4 6 0.1 1.5 0.01 4

Figure 1. Particle size measurements of FD4 formulation

during the storage time

Figure 2. Zeta potential measurements of FD4 formulation

during the storage time

[1] Y-J. Lee, S-J. Chung, C-K. Shim, J. Pharm. Biomed. Anal., 22(1), 183-188 (2000). [2] S. Tamilvanan, K. Khoury, D. Gilhar, S. Benita, S.T.P Pharm. Sci., 11(6), 421-426 (2001).[3] E. Cengiz, S.A. Wissing, R.H. Müller, Y. Yazan, Int. J. Cosmet. Sci., 28, 371-378 (2006).[4] K. Manjunath, J.S. Reddy, V. Venkateswarlu, Methods Find. Exp. Clin. Pharmacol., 27(2), 127-144 (2005).[5] R.H. Müller, M. Radtke, S.A. Wissing, Adv. Drug Deliv. Rev., 54(1), 131-155 (2002).[6] M. Stettin, G. Halwachs-Baumann, B. Genser, F. Frühwırth, W. März, G.A. Khoschsorur, Talanta, 69, 1100–1105 (2006).