JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS

Volume 23, Number 3, 2007

Mary Ann Liebert, Inc.
DOI: 10.1089/jop.2006.130

Formulation of Sirolimus Eye Drops and Corneal

Permeation Studies
GUIDO BUECH,1 ECKART BERTELMANN,2 UWE PLEYER,2 INGO SIEBENBRODT,1
and HANS-HUBERT BORCHERT1

ABSTRACT
The aim of this study was the development of eye drops with 1 mg/mL sirolimus and the
evaluation of the drugs ability to permeate the freshly isolated pig cornea. Cyclodextrin solutions, liposomes, hydrotrope mixtures, poloxamer gels, and a microemulsion were tested
for their suitability to dissolve the extremely hydrophobic drug sirolimus (solubility in water 2.6 g/mL). The drug content in the formulations was determined by high-performance
liquid chromatography, whereas this method is not sensitive enough for the quantification
of therapeutic concentrations (712 ng/mL). Thus, the acceptor samples of the permeation tests
were examined by microparticle enzyme immunoassay. A microemulsion is a suitable vehicle to prepare eye drops with sufficient sirolimus concentrations of 1 mg/mL in a formulation with acceptable tolerance and satisfactory stability over 12 months. However, the drug
cannot permeate the intact cornea. After removal of the corneal epithelium, drug concentrations in the acceptor sample reach the lower limit of therapeutical levels. Conclusively, the
present sirolimus eye drops might be promising therapeutic tools for the immunomodulatory
treatment of ocular surface disorders, such as keratoconjunctivitis sicca, vernal conjunctivitis, or atopical blepharitis. They are not suitable to achieve therapeutic concentrations in the
aqueous humour of patients with intact cornea.

INTRODUCTION

MPORTANT OPHTHALMIC INDICATIONS for an immunosuppressive treatment are the prevention

of allograft rejections following corneal transplantation as well as chronic inflammatory disorders, such as uveitis, and especially, ocular
surface diseases, including keratoconjunctivitis
sicca, vernal conjunctivitis, or atopical blepharitis. Eye drops with glucocorticoids (i.e., prednisolone or dexamethasone) are common for this

1Institute

purpose. However, there are multiple pathologies where steroids fail, and moreover, these
drugs induce often undesired side-effects, for example, enhanced intraocular pressure (IOP).
Therefore, there is a need for further local applicable immunomodulatory agents, such as
sirolimus, with another specific mechanism of action.
The intramuscular administration of sirolimus
in extremely high doses of 10 mg/kg significantly
inhibited endotoxin-induced uveitis in rabbits.1

of Pharmacy, Free University of Berlin, Berlin, Germany.

of Ophthalmology, CharitUniversittsmedizin Berlin, Campus Virchow-Klinikum, Berlin, Ger-

2Department

many.

292

293

SIROLIMUS EYE DROPS

The systemic administration of sirolimus prolonged the allograft survival and significantly inhibited the neovascular component of rejection in
a rat model.2 The intravenous (i.v.) administration of high doses of sirolimus or ciclosporine, as
well as a low dose combination of both, could effectively prolong corneal allograft survival in
rats.3 An inhibition of retinal and choroidal neovascularization in mice was reported if sirolimus
was injected intraperitoneally with dosages of 2
or 4 mg/kg daily.4 The results of these studies
show the activity of systemically administered
sirolimus in the treatment of ophthalmic diseases,
but the sites of drug action within the eye are unclear up to now. Unfortunately, the systemical
use of sirolimus is limited to vitally important indications owing to its dose-dependent toxic effects. Therefore, an alternative option could be
the topical administration of sirolimus by means
of eye drops. The topical administration for a
prevention of allograft rejection and a treatment
of autoimmune uveitis requires the permeation
of the drug trough of the cornea. However, with
respect to its physicochemical characteristics, the
drug is not predestined for high corneal permeation rates. Sirolimus is practically insoluble in
water (2.6 g/mL), has a high molecular weight
(MW 914 Da), and contains no functional groups
that are ionizable in the pH range between 1 and
10. Sirolimus is extremely hydrophobic.
One aim of this study was the development of
an eye compatible formulation with an approached concentration of 1 mg/mL (0.1%). This
would be in accordance with nearly a four
hundredfold increase of solubility. On the other
hand, the ability of sirolimus to permeate the
freshly isolated pig cornea should be evaluated.
Possible ocular formulations for poorly soluble
drugs are cyclodextrin (CD) solutions,58 liposomes,913 hydrotrope mixtures (HM),14 polyoxyethylene-polyoxypropylene (POE-POP) block
polymers1520 (poloxamers  pluronics) or microemulsions.2128 In this study, the solubility of
sirolimus was examined in the following formulations: (i) 10% (w/v) hydroxypropyl-betacyclodextrin (HP--CD) or 10% (w/v) hydroxypropyl-gamma-cyclodextrin (HP--CD) solutions
under addition of 0.25% (w/v) of the water-soluble polymers, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose;
(ii) liposomes with a final lipid concentration of
5% (w/v) and positive and negative zeta potentials; (iii) HM with 10%40% (w/w) propylene

glycol, 2% (w/w) benzyl alcohol, and 5% (w/w)

benzoic acid/sodium benzoate; (iv) a 15% (w/w)
poloxamer 407 gel; and (v) a microemulsion. The
selection of the most suitable lipophilic component for a microemulsion, based on a dissolution test of sirolimus in isopropylmyristate,
medium chain triglycerides, and isopropylpalmitate. Some of the formulations are not only
simple solubilizer. Additionally, an increased
ocular bioavailability of drugs and their prolonged duration of action could be observed for
liposomes,1014 poloxamers,15 and microemulsions.25,26

METHODS
Materials
Sirolimus was supplied by LC Laboratories
(Woburn, MA) and used as received. Hydroxypropyl--cyclodextrin (HP--CD) and hydroxypropyl--cyclodextrin (HP--CD) were obtained
from Wacker Chemie (Burghausen, Germany),
and hydroxyethylcellulose (HEC) hydroxypropylcellulose (HPC), and hydroxypropylmethylcellulose (HPMC) were from Caesar &
Loretz GmbH (Hilden, Germany). Phosphatidylcholine (PC, Lipoid S 100) was purchased from
Lipoid (Ludwigshafen, Germany) and stearylamine from Sigma Aldrich (Steinheim, Germany). Dihexadecylphosphate (dicetylphosphate), benzyl alcohol, benzoic acid, and sodium
benzoate were supplied by Fluka (Buchs,
Switzerland). Isopropylmyristate, isopropylpalmitate, and medium chain triglycerides came
from Caesar & Loretz GmbH. Poloxamer 407
(Lutrol F 127) was purchased from BASF (Ludwigshafen, Germany) and poloxamer 184 (Synperonic L 64) from C.H. Erbsloeh (Duesseldorf,
Germany). Triacetin was received from Alfa Aesar (Karlsruhe, Germany). Methanol and acetonitril (both gradient grade) for high-performance
liquid chromatography (HPLC) were purchased
from Merck (Darmstadt, Germany). Chloroform
was provided by Merck and Carl Roth (Karlsruhe, Germany), and propylene glycol (PG) was
provided by Wasserfuhr (Bonn, Germany). All
other chemicals were of pharmaceutical or analytical grade and were used as received from the
commercial supplier. MilliQ water (Millipore;
Bedford, MA) was used in all experiments. Hydrotrope mixtures, poloxamer gels, and the mi-

294

BUECH ET AL.

croemulsions were prepared under aseptic conditions.

Preparation of CD solutions
The CD solutions were prepared by a modification of previously described instructions.5,6
One(1) mg of sirolimus per milliliter of formulation was dissolved in methanol and evaporated
to dryness on a vacuum evaporator (Buechi;
Flawil, Switzerland) or in a vacuum drying chamber (Heraeus Instruments GmbH; Hanau, Germany) at 40C. The composition of the formulations is given in Table 1. The mixtures were
heated in an autoclave at 121C for 15 min, cooled
down to room temperature, and filtered trough a
0.8- filter (Sartorius; Goettingen, Germany) under aseptic conditions.

Preparation of liposomes
Liposomes were manufactured by a modification of the film method, which was described in.12
The lipids and the drug were dissolved in chloroform/methanol (2:1) and appropriate volumes
were combined to obtain the different lipid compositions. The following lipids were used per milliliter of the negatively charged liposomal dispersions PC() PG and PC() pH 7.4: 36.2 mg of
Lipoid S 100, 12 mg of cholesterol, 1.8 mg of
dicetylphosphate, or in the positively charged liposomal dispersions, PC(), PG, and PC(), at a
pH of 5.4: 36.5 mg of Lipoid S 100, 13 mg of cholesterol, and 0.5 mg of stearylamine. The mixtures
were evaporated to dryness in a vacuum evaporator at 40C. Residual organic solvent was removed in a vacuum drying chamber (Heraeus Instruments GmbH) at 40C and 1 mbar for 8 h. The
lipid film was hydrated in an ultrasonic bath
(Bandelin Sonorex RK 255 H; Berlin, Germany) at
60C for 15 min. The hydrophilic phase of the forTABLE 1.
Components
HP--CD (mg)
HP--CD (mg)
HPC (mg)
NaCl (mg)
Mannitol (mg)
BAC (L)
Water ad (mL)

COMPOSITION

OF

EYE DROPS

HP--CD NaCl

mulations PC(), PG, and PC() PG was water/propylene glycol 85/15 (v/v). No other
preservative was necessary for these formulations.
The formulations PC() (pH 5.4) and PC()
(pH 7.4) were hydrated with phosphate buffer,
isotonized with NaCl, and preserved by benzalkonium chloride 0.01%. The final lipid concentration was 5% (w/v) because 50 mg of lipids
were used per milliliter of formulation. The
liposomes were downsized by 21 extrusions
through a polycarbonate membrane (Nucleopore;
Pleasanton, CA) of 200 nm in pore size with a
Liposofast extruder (Avestin Inc.; Ottawa,
Canada) in a water bath (GFL 1083; Gesellschaft
fr Labortechnik, Burgwedel, Germany) at 60C.
The vesicle suspension was finally passed
through a bacteria-retentive 0.2- polycarbonate
filter (Sartorius; Goettingen, Germany).
Particle sizes, polydispersity indices, and zetapotentials of the liposomes were measured by
a Zetasizer 4 (Malvern Instruments; Worcestershire, United Kingdom).

Preparation of hydrotrope mixtures (HM)

The HM were prepared in accordance with previously released instructions.14 Table 2 shows the
composition of the HM. One (1) mg of sirolimus
per mL was stirred to a clear solution in a mixture of propylene glycol and benzyl alcohol. The
benzoic acid/sodium benzoate was added, and
the solution was mixed step by step with water
at 50C. Undissolved constituents and contaminating particles were removed by filtration
trough a filter with an 0.8- pore size (Sartorius).

Preparation of poloxamer gels

The poloxamer gels were prepared, modifying
the descriptions in.1517 One-point-five (1.5) g of

WITH THE

CYCLODEXTRINS HP--CD
Eye drops
HP--CD NaCl

AND

HP--CD
HP--CD Mannitol

1000
25
70

1000
25
70

100
10

100
10

HPC, hydroxypropylcellulose; BAC, benzalkonium chloride solution (1%).

1000
25
420
100
10

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SIROLIMUS EYE DROPS

TABLE 2.

COMPOSITION

OF

Components
Propylene glycol % (w/w)
Benzyl alcohol % (w/w)
Benzoic acid % (w/w)
Sodium benzoate % (w/w)
Water % (w/w)

Poloxamer 407, 0.1 g of benzyl alcohol, and 10 mg

of sirolimus were stirred at 60C by a magnetic
stirrer until all the polymer was melted. The melting was cooled down to room temperature and
phosphate buffer (pH 7.4) was added at 10.0 g.
The vessel was stored at 48C for 4872 h and
occasionally shaken until the polymer was completely dissolved. The solution was finally agitated by the magnetic stirrer. The dissolution of
eventually observable cloudings can be encouraged by heating the solution in a water bath at
60C. The solution was terminally cooled down
and filtered at 4C trough a 5- polytetrafluoroethylene (PTFE) filter (Sartorius).

Preparation of microemulsions
The preparation of the microemulsion was carried out, as described previously.22 Triacetin and
poloxamer 184 were dry-heat sterilized separately at 180C for 30 min; the propylene glycol/water mixture was steam sterilized at 121C
and 2 bar for 15 min. Ten (10) mg of sirolimus
were mixed with 2.0 g of triacetin before 1.5 g of
poloxamer was added under continous stirring
with a magnetic stirrer. A mixture of 4.0 g of
propylene glycol and 2.5 g of water was added
under continuous stirring, and the established
microemulsion was filtered trough a 5- PTFE filter (Sartorius). It should be noted that the formulation dissolves usual filter materials, such as
cellulose nitrate or acetate.

HPLC analysis
In order to quantifiy the concentrations of
sirolimus in formulations and its solubility in
pure lipids, a HPLC method was used. The structure of sirolimus offers three accumulated double bonds, which are responsible for the excellent
option for a quantification by ultraviolet (UV) detection at a wavelength of 278 nm. The HPLC conditions were modified according to previously
published instructions.29 An HPLC apparatus of

HYDROTROPE MIXTURES (HM)

HM PG 30%

HM PG 40%

30
2
2.5
2.5
63

40
2
1.5
3.5
53

Merck Hitachi was used (pump L 6200A, interface D 6000, UV/VIS detector L-4500, and autosampler AS-2000A). The Purospher column
(RP-18 125-4 mm, 5 m) was equipped with a
guard column RP-18 4-4 mm (both from Merck).
The column temperature was 70C. The mobile
phase, composed of methanol/acetonitril/water
38/34/28 (v/v), was prefiltered through a 0.45-
PTFE filter (Sartorius). The flow rate was 0.8
mL/min. The retention time of sirolimus under
these conditions was approximately 7 min. A
sample volume of 20 L was injected. A calibration curve was established by plotting the area
under the curve (AUC) versus sirolimus concentration (cSir). The calibration curve showed good
linearity from 1 to 200 g/mL (AUC  25789
cSir  5745.2, correlation coefficient r2  0.9997).

Immunoassay of sirolimus (IMX-system)

The HPLC was not sensitive enough for the
quantification of therapeutic concentrations of
sirolimus (712 ng/mL). The acceptor samples of
the permeation tests were, therefore, quantified
by a microparticle enzyme immunoassay (MEIA).
The IMX system (Abbott GmbH; Wiesbaden, Germany), an automated immunoassay analyzer,
was used as specified in the instruction manual.
Briefly, 150 L of the acceptor sample, calibrator,
or control solution were mixed with 300 L of
precipitation reagent in an Eppendorf centrifuge
tube on a vortex mixer (IKA MS Minishaker; IKA
works Inc., Wilmington, NC) for 15 s at the highest speed to a homogeneous mixture. The tubes
were centrifuged for 4 min at 10,000g (Eppendorf
Centrifuge 5415 C; Eppendorf-Netheler-HinzGmbH, Hamburg, Germany). The supernatant
was decanted into the sample well of an IMX reaction cell and placed into the carousel of the IMX
module for immunoassay.
The determined rates of the six calibrator solutions AF are listed in Table 3. As can be seen,
no linear relationship could be found. The assay

296

BUECH ET AL.
TABLE 3.

Calibrator
A
B
C
D
E
F

CALIBRATION

OF THE IMMUNOASSAY FOR

SIROLIMUS

c (ng/mL)

Rate 1

Rate 2

Range

Rate avr

RERR

0
3
6
12
20
30

482.0
391.2
291.9
176.4
117.4
83.0

506.9
388.3
266.5
160.7
116.0
92.9

24.9
2.9
25.4
15.7
1.4
9.9

494.5
389.8
279.2
168.6
116.7
88.0

0.0
0.7
0.8
0.3
1.9
1.4

Note. The table lists the obtained values according to the description of the manufacturer of the immunoassay.
c, concentration; rate avr, average rate; RERR, rate error; RMSE, root mean square error  0.932.

utilizes a four-parameter logistic curve fit to generate a calibration curve. The automated analyzer
approved the calibration because the following
parameters were within the acceptable ranges:
RERR (rate error)  20; RMSE (root mean
square error)  10
The measurement range is 1.530 ng/mL. Samples with higher concentrations may be diluted to
a maximum of 1:4 before quantification. The accuracy and precision of the quantification method
were proven every test series using the Mode 1
calibrator (6 ng/mL) in positions 1 and 2 of the
sample carousel for a maximum of 24 samples.

1 mL of eye drops was used as the donor. An acceptor volume of 15 mL of BR was circulated by
an Ismatec IPS 12 pump (Zuerich-Glattbrugg,
Switzerland) at a flow rate of 5 mL/min. Samples
of 5 mL in volume were removed from the acceptor after 30, 60, and 240 min and replaced by
fresh BR. The samples were stored at 20C until they were quantified on the next day. The vitality of the corneal tissue was examined by a trypan blue staining (0.4%), which colors dead cells
only. The corneas were incubated with the trypan blue solution for 5 min after the permeation
experiments. Freshly isolated corneas and
corneas treated with 1% Triton-X-100 solution
(Dow; Midland, MI) were used as the positive
and negative control, respectively.

Permeation tests
Permeation studies were performed in permeation cells (Ussing chambers, Scientific Instruments; Aachen, Germany),30 manufactured from
acrylic resin (Grndberg Kunststoffe; Roedermark, Germany) with a permeation area of 0.5
cm2. The temperature of the permeation cells
(33  1C) was maintained with a water bath
(Lauda Dr. R. Wobser GmbH & Co. KG; LaudaKoenigshofen, Germany). The eyes of freshly
slaughtered pigs were received from a veterinarian research center (LVAT Ruhlsdorf; Gro
Kreutz, Germany). The corneas were prepared as
previously described.30 Briefly, all muscular tissue was removed from the eye. Then, the eye was
rinsed with a bicarbonate ringer (BR) solution at
a pH of 7.4. Using a surgical knife and microsurgical scissors, a sclerocorneal complex was prepared. The surgical knife was used to abrade the
corneal epithelium before the preparation in experiments using corneas without epithelium. The
cornea was finally rinsed with BR and placed into
the Ussing chamber, with the epithelial side facing the donor side of the chamber. A volume of

Concentrations of sirolimus in the formulations

and solubility in pure lipids
One hundred (100) L of CD solution, HM,
poloxamer gel, or microemulsion were diluted
with methanol to a volume of 1000 L and vortexed to get a homogeneously solution. One hundred (100) L of liposomes were diluted with chloroform/methanol (1:2) to 1000 L to obtain a clear
solution. The concentrations in these mixtures were
quantified directly by HPLC. For the determination
of the solubility in isopropylpalmitate, isopropylmyristate, and medium chain triglycerides 23 mg
of the drug were added to 100 L of the pure lipid
and vortexed for 1 min. The samples were agitated
and protected from light for 24 h at room temperature on a mechanical shaker (Thys 2; Labortechnik Ilmenau, Grunsfeld, Germany) to reach an
equilibrium between the dissolved and undissolved drug. The mixtures were centrifuged at
10,000g (Eppendorf Centrifuge 5415 C; EppendorfNetheler-Hinz-GmbH). Fifty (50) L of the clear supernatant were diluted with chloroform/methanol
(1:2) to 500 L and determined by HPLC.

297

SIROLIMUS EYE DROPS

Stability assessment
All formulations were stored at room temperature and protected from light. To evaluate the
long-term stability of the CD complexes and the
microemulsion, the sirolimus content was monitored over a period of 12 months.

Physicochemical characterization
The pH values were measured by a pH meter
WTW 522 (Wissenschaftlich Technische Werkstaetten; Weilheim i. OB, Germany). A Haake
Rheostress RS 100 (Thermo Haake; Karlsruhe,
Germany) was used for the rheologic characterization of poloxamer gels and of the microemulsion at 20 and 32C. Osmolarities were determined by a Osmometer 3/B (Hermann Roebling
Messtechnik; Berlin, Germany).

RESULTS
CD solutions
Hydroxypropylcellulose was identified as appropriate polymer for solubilization of sirolimus
in HP--CD and HP--CD complexes, using water as the hydrophilic phase (see Table 4). This
polymer was selected for further experiments
with isotonic solutions. It should be noted that an
enhanced solubility of sirolimus is detectable
only if the drug is dissolved in methanol and
evaporated to dryness before CDs and water are
added. Probably, sirolimus has different crystalline modifications with variable solubility. The
larger cavity diameter of HP--CDs, compared to
HP--CDs, is beneficial with respect to a higher
solubility of sirolimus. Mannitol and NaCl were
tested as isotonic reagents. A comparison of the
solubility at time zero in Figure 1 with the data
in Table 4 shows that the solubility of sirolimus
in isotonic aqueous CD solutions is lower than in
TABLE 4.
WITH

SOLUBILITY OF SIROLIMUS IN CD SOLUTIONS

0.25% OF WATER SOLUBLE POLYMERS
Solubility of sirolimus (g/mL)

Cyclodextrin derivative
HP--CD
HP--CD

HPMC

HPC

HEC

57.7
73.5

87.5
112.3

58.9
52.0

HPMC, hydroxypropylmethylcellulose; HPC, hydroxypropylcellulose; HEC, hydroxyethylcellulose.

FIG. 1. Stability of saturated cyclodextrin complexes

with sirolimus in isotonic solution (each concentration determined in triplicate, with the mean and standard deviation).

aqueous CD solutions without NaCl or mannitol.

HP--CDs achieves higher drug concentrations,
compared with HP--CDs. The drug content in
CD preparations decreased approximately 50% in
6 months (Fig. 1). Mannitol has a low benefit in
stability in comparison to NaCl. No drug was detectable after a storage of 12 months at room temperature. This result can be ascribed to the low
stability of sirolimus-CD complexes as well as to
a low stability of sirolimus in aqueous solutions.
The solubility of sirolimus in CD is approximately 6070 g/mL. The diameter of the
sirolimus molecule is presumably too large for a
complete inclusion in CD complexes. Probably,
the twentyfold increase of the solubility can be
explained by an inclusion of the side chain of the
sirolimus molecule into the CD complexes. Unfortunately, the CD formulations have low stability, low reproducibility, and high sensibility to
buffer salts and isotonic materials. No drug was
detectable in acceptor samples of cornea permeation tests owing to the low solubility of
sirolimus. Consequently, an alternative formulation should not only solubilize the drug in higher
concentrations. An improved corneal permeation
by the formulation is also preferable.
Steam sterilization at 121C and 2 bar for 15
min is a part of the manufacturing of CD solutions. Under these conditions, the degradation of
sirolimus was proved. This problem is also important for the sterilization of poloxamer gels and
microemulsions. The rate of degradation during
steam sterilization increases in the order of microemulsion (containing 25% water) and polox-

298

amer gel (85% water). Consequently, the degree

of the drug degradation is probably proportional
to the water content of the formulation.

Liposomes
Phospholipid liposomes optimized for an ophthalmic application were charged positively or negatively by the addition of stearylamin or
dicetylphosphate, respectively. Particle sizes between 100 and 165 nm were obtained in a narrow
distribution range (PI  0.25) and zeta potentials in
the positive or negative region. Zeta potentials
above 30 mV or below 30 mV are beneficial to
reach at least a medium stability. Unfortunately, no
sirolimus could be detected in these liposomes by
HPLC. It can be concluded that liposomes are not
appropriate for the solubilization of sirolimus.
Probably, the hydration of the lipid films in the
aqueous medium or the extrusion procedure is not
suitable for the preparation of formulations containing sirolimus. In contrast to this result, the immunosuppressant tacrolimus had a solubility of 4.8
mg/mL in liposomes,11 but tacrolimus is more hydrophilic than sirolimus.31 No permeation experiments were performed with sirolimus when using
liposomes as the donor solution.

Hydrotrope mixtures under the addition of

cosolvents (HM)
The solubility of sirolimus in HM containing 40%
propylene glycol (HM PG 40%; see Table 2) was
1 mg/mL and in HM PG 30% between 0.31 and
0.45 mg/mL. HM contains usually 10% ethanol,
but the ocular tolerance of ethanol is low. Without
ethanol a reduction of the propylene glycol content
lower than 30% is impossible owing to the recrystallization of benzoic acid. Even in HM without
ethanol and propylene glycol contents above 30%,
a high tendency to recrystallization could be observed during a storage period of some days at
room temperature. Unfortunately, corneal opacity
could be found in the permeation experiments.
Probably, this effect can be explained by precipitated salts or proteins. It can be concluded that the
HM provides sufficient solubility features for
sirolimus, but the stability and ophthalmic compatibilty of HM were insufficient.

Poloxamer gels
The poloxamers can form semisolid gels in
situ after application. Besides the appropriate

BUECH ET AL.

opthalmic properties, this is advantageous with

respect to a prolonged retention of the formulation on the cornea. The solubility of sirolimus in
a gel containing 15% poloxamer 407 was 0.12
mg/mL. Unfortunately, this is much lower than
required. It seems that sirolimus is too hydrophobic for a solubilization in aqueous poloxamer gels. Therefore, no permeation tests were
performed. A filtration of poloxamer gels
through a bacteria-retentive filter is impossible
owing to the high viscosity of 70100 mPa s at
20C and 32003600 mPa s at 32C. A filtration
through a 5- PTFE filter is practicable at 4C to
seperate contaminating particles. An aseptic processing is difficult owing to the complex preparation procedure.

Microemulsion
Possible lipophilic components of microemulsions for an ocular application are the two waxes,
isopropylpalmitate and isopropylmyristate, as
well as the medium chain triglycerides. Unfortunately, these lipids are not suitable for the solubilization of sirolimus. The dissolution test
showed that the solubility of sirolimus in
isopropylpalmitate, isopropylmyristate, and
medium chained triglycerides was 0.5, 0.8, and
1.5 mg/mL, respectively. These results emphasized that the extremely hydrophobic sirolimus is
slightly or very slightly soluble in these apolar
lipids. The observed solubility of approximately
1 mg/mL was in good agreement with that in
corn oil.14 The drug is moderately soluble in polar lipids, such as triacetin (20 mg/mL), and water miscible solvents, such as propylene glycol
(15 mg/mL) and polyethylene glycol 400 (30
mg/mL). Sirolimus is freely soluble in polar
lipoic solvents, such as dimethylformamide
(300 mg/mL), dimethyl sulfoxide (500
mg/mL), or benzyl alcohol (400 mg/mL), but
these materials are not acceptable excipients for
eye drops. Benzyl alcohol is applicable only in
low concentrations up to 1%. Satisfying solubility of sirolimus (1 mg/ml) was achieved with
a microemulsion containing 20% triacetin and
40% propylene glycol, which was described formerly for the ocular application of other
lipophilic drugs.22 Further studies were performed using this microemulsion as a solvent for
sirolimus. The microemulsion was stable in the
temperature range of 1535C. It became cloudy
if it was stored at 48C, but the turbidity was

299

SIROLIMUS EYE DROPS

FIG. 2. Stability of sirolimus dissolved in the microemulsion (mean and standard deviation, n  3).

cleared completely at room temperature. The microemulsion should be stored at 20C and protected from light owing to the photo activity of
sirolimus. Figure 2 shows that the drug was almost stable under these conditions for 12 months.
The microemulsion had an apparent pH value of
5.855.93. The pH value is apparent because a microemulsion is no pure aqueous system. The dynamic viscosity of the microemulsion was
27.728.5 mPa s at 20C and 15.016.6 mPa s at
32C. A problem is the filtration of the microemulsion through a bacteria-retentive 0.2- PTFE
filter owing to this viscosity and/or the wettability of the filter. Therefore, an aseptic processing
is necessary. On the other hand, a microbiologic
growth can be excluded in a microemulsion containing 40% propylene glycol. A displacement of
contaminating particles is possible using 5-
PTFE filters. The in vivo toxicity data of the components used to prepare the microemulsion are
acceptable. Triacetin was affirmed as a safe human food ingredient (GRAS, generally recognized as safe) by the Food and Drug Administration. Undiluted triacetin had no, or only minor,
effects on the eye.32,33 Poloxamer 184 (Synperonic L 64, Fitz Chem Corporation; Itasca, IL) is
a well-established nonionic surfactant and was
used several times for the preparation of oral, parenteral, and ocular formulations. The toxicity of
poloxamers is generally accepted as very low
(LD50  2g/kg) but increases with decreasing
chain length and increasing hydrophobic qualities.34,* Solutions of up to 50% propylenglycol
*ICI; Wilton, Middlesborough, Cleveland, OH.

caused no irritations on the rabbit eye, whereas

the undiluted application was associated with a
weak conjunctival redness. Some osmotic irritations were observed, which were limited to a few
seconds. Ointments or solutions containing 70%
propylenglycol showed no or slight irritations on
humans, or were effective in clearing the cornea
in patients with cornal edema of various origin.35,36
Besides the good solubility of sirolimus in the
used microemulsion, is that its opthalmic compatibility is better than that of hydrotrope mixtures. It could be shown by the HET CAM tolerance test with incubated chicken eggs (the hens
egg chorio allantoic membrane test) that the microemulsion has a low irritation potential (low
blood injections in the mucous membrane but
neither coagulation nor lysis).22 Hydrolyzed triacetin or the high surfactant/cosurfactant concentration may be responsible for the low irritation. The results of the HET CAM test are
equivalent to the Draize-test on the rabbit
eye.3739

Permeation studies with the microemulsion

The permeation studies were performed using
freshly isolated pig corneas. The donor volume
of 1 mL is in contact with the cornea for 240 min.
Figure 3 presents the time-dependent concentrations of sirolimus in the acceptor compartment
determined by the IMX immunoassay, which has
a limit of quantification at 1.5 ng/mL. The per-

FIG. 3. Permeation of sirolimus trough the freshly isolated pig cornea (concentrations in the acceptor compartment), using a microemulsion with 0.1% drug in the
donor compartment; (intact cornea, n  4; cornea without epithelium, n  3; mean and standard deviation, each
concentration determined in duplicate).

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BUECH ET AL.

meation through intact cornea was very slow.

Concentrations lower than 4.3 ng/ml were obtained after a time of 240 min. Acceptor concentrations between 6.7 and 9.8 ng/mL were determined after this time if the corneal epithelium
had been removed. Therefore, it can be concluded
that the hydrophilic stroma is the rate-controlling
barrier to the sirolimus permeation. The corneal
permeation of the extremely hydrophobic drug
sirolimus is nonlinear owing to the strong hydrophilicity of the stroma. No strict plateau could
be observed after 30 min, but the permeation rate
decreased after that time, probably owing to a
partial saturation of the stroma with sirolimus.
Presumably, a steady state in the permeation was
reached to permeation times longer than 240 min,
but such a long time had no practical relevance
with respect to an ocular application in vivo.
For a systemical application of sirolimus, a
whole-blood concentration of at least 712 ng/
mL was assumed to prevent renal allograft rejection.29 Although the effective sirolimus concentration in the region of the endothelium behind
the stroma and the aqueous humour to prevent
a corneal allograft rejection is currently unknown,
it could be supposed that the permeation rate
through intact cornea is too low for a topical application.
The integrity of the corneas was proved by the
vitality test using a 0.4% trypan blue solution. The
vitality tests indicated very small punctiform areas of dead cells in freshly isolated corneas that
were hard to observe macroscopically. After the
permeation time, these areas were only a little bit
larger. These observations were similar to other
results that were described with trypan blue and
a 0.5% fluorescein solution, which can color epithelial ruptures.40 Accordingly, the accuracy of
the permeation test can be stated.

DISCUSSION
In this study, an ocular delivery system for the
extremely poorly soluble immunosuppressant
sirolimus was examined. Besides a sufficient solubility for the drug, many factors are important
concerning in the development of those systems,
such as comfortableness, no significant irritation,
low toxicity, and no obstruction of the sight. The
mechanical properties are important to prevent
physical irritation, especially during a long-term

application. Aqueous systems have, therefore,

many advantages in comparison to oils, cremes,
or ointments. Their easier application, their transparency, and their lower viscosity induce a better compliance that is favorable for an adequate
response to the treatment.
The results from our study indicate that a microemulsion is a suitable vehicle to prepare eye
drops with sufficient sirolimus concentrations of
1 mg/mL and acceptable tolerance. The low viscosity of this microemulsion enables an easy application. An aseptic processing is necessary because common sterilization procedures cannot be
performed. The drug is sensitive to hydrolysis,
high temperatures, and oxidization induced by
light exposure. It is well known that microemulsions can enhance the stability of dissolved drugs
in comparison to other aqueous systems.23
Sirolimus was, in contrast to CD solutions, stable
in the microemulsion over a storage period of 12
months at 20C (Figs. 1 and 2). Accordingly, it can
be assumed that the drug location in the microemulsion is protected from oxygen and water.
It could be shown by 1H NMR studies that the
poorly soluble antibiotic chloramphenicol is entrapped into the hydrophilic shells of a microemlusion for the ocular administration.41
These hydrophilic shells are represented by
oxyethylene groups. Moreover, the high proton
mobility in methyl groups of poloxamers could
be observed in lipid formulations, such as nanoemulsions or nanostructured lipid carriers.42 It
seems that highly mobile polar groups with oxygen atoms are responsible for the good solubility
features of the lipid formulations for hydrophobic drugs. Therefore, sirolimus has an acceptable
solubility in systems containing poloxamers,
propylene glycol, benzyl alcohol, and triacetin,
which provide many polar lipid domains. This
assumption is also emphasized by our result, that
sirolimus has a low solubility in apolar lipids.
It should be noted that the drug is stabilized
by the manufacturer with lipophilic antioxidants,
such as 2,6-di-tert-butyl-4-hydroxytoluol (BHT),
in concentrations of approximately 0.2%. The
knowledge about the ocular tolerance of this substance should be improved.
The permeation study indicated that sirolimus
cannot cross the intact cornea. No more reports
about corneal penetration or permeation studies
with sirolimus could be found. Therefore, a comparison to other poorly soluble macrolid im-

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SIROLIMUS EYE DROPS

munosuppressants with a similar structure could

be meaningful. A similar structure to sirolimus
have everolimus (MW 958 Da), tacrolimus (MW
822 Da), and pimecrolimus (MW 810 Da). As
can be seen, the lower molecular weights of
tacrolimus and pimecrolimus are more favorable
for a sufficient corneal permeation than those of
sirolimus and everolimus. The distribution coefficient between hydrophilic and lipophilic phases
is also important to estimate the permeation rates
of drugs. Unfortunately, previously reported distribution coefficients of the immunosuppressants
cannot be directly compared to each other owing
to different measurement systems. Using a chromatographic method, an experimental distribution coefficient between octanol and buffer solution at a pH of 7.4 (Elog DOct) was determined
with 6.09 and 6.99 for tacrolimus and pimecrolimus, respectively.43 It was concluded that
tacrolimus is approximately eight times more
hydrophilic than pimecrolimus. Using a twophase system composed of hexane/tert-butylmethylether/methanol/water (1:3:6:5), distribution coefficients of K  0.917 (log K  0.038),
K  0.996 (log K  0.002), and K  1.401 (log
K  0.146) were determined for tacrolimus,
everolimus, and sirolimus, respectively.31 Log K
was calculated from the difference of the logarithmic concentrations in the lipophilic and hydrophilic phase. Accordingly, tacrolimus is somewhat more hydrophilic than everolimus, whereas
sirolimus is much more hydrophobic. In conclusion, it seems that tacrolimus and everolimus
are less hydrophobic than pimecrolimus and
sirolimus. Although little is known about the permeation of everolimus, under consideration of
distribution coefficients and molecular weights,
the permeation rates of the other macrolid immunosuppressants across the cornea can be assumed in the following order: tacrolimus 
pimecrolimus  sirolimus. This order is in good
agreement with previously published reports
about the ocular application of pimecrolimus and
tacrolimus. An amount of 1% pimecrolimus dissolved in a mixture of corn oil/2,2-dimethyl-1,3dioxalan-4-methanol (24/1) was used for a topical treatment of keratoconjunctivitis sicca and
chronic superficial keratitis in dogs.44 Accordingly, a relatively high concentration of pimecrolimus was tested for a treatment of ocular surface diseases only. Therefore, it can be assumed
that pimecrolimus is too hydrophobic to obtain

sufficient permeation rates through the cornea.

Our permeation test indicated similar results for
sirolimus. In contrast to pimecrolimus and
sirolimus, the permeation of tacrolimus through
the cornea could be observed. Tacrolimus was detected in therapeutic concentrations in all ocular
tissues, and particularly, in the aqueous and vitreous humor of rabbit eyes.11 Tacrolimus could
permeate only if it was encapsulated in a concentration of 4.8 mg/mL in liposomes but not if
it was dissolved in olive oil. Liposomal
tacrolimus could also significantly inhibit the endotoxin-induced uveitis in rats.45 Furthermore, it
could be shown that a twice-daily administration
of an aqueous 0.02% tacrolimus suspension effectively increased the tear production in dogs
with keratoconjunctivitis sicca.46 In summary, liposomal encapsulated tacrolimus in a higher concentration of 4.8 mg/mL can permeate the cornea
and could, therefore, be a promising tool for a
topical treatment of corneal allograft rejections,
as well as autoimmune uveitis. The drugs, pimecrolimus and sirolimus, as well as a low-dose suspension of tacrolimus can be used for a topical
treatment of ocular surface disorders only. For
these indications, the safety margins of topical applied pimecrolimus and sirolimus are higher than
that of tacrolimus because a drug transfer into the
circulation is undesired to prevent unwanted
side-effects.

CONCLUSIONS
In conclusion, a microemulsion was successfully used to dissolve the virtually insoluble drug,
sirolimus, in a concentration of 1 mg/mL. The eye
drops were not suitable to achieve sufficient concentrations of sirolimus in the corneal endothel
and the aqueous humour. Accordingly, they cannot be used for the prevention of allograft rejections following corneal transplantation as well as
autoimmune uveitis, but they should be applicable for the treatment of inflammatory disorders
of the ocular surface, such as keratoconjunctivitis sicca, vernal conjunctivitis, or atopical blepharitis. In the future, the ocular permeation of
the synthetic sirolimus derivative, everolimus,
should be investigated. Everolimus was developed to overcome the low bioavailability of
sirolimus and has an eight times higher solubility in water.

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BUECH ET AL.

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Received: November 2, 2006

Accepted: January 25, 2007

Reprint Requests:

Hans-Hubert Borchert
Institute of Pharmacy
Free University of Berlin
Kelchstr. 31
12169 Berlin
Germany

E-mail: hhb@zedat.fu-berlin.de.