1. INTRODUCTION
Rapamycin, also called sirolimus, is a macrolide with antifungal,
antineoplastic, and immunosuppressive properties discovered in 1975 (1).
It was approved by the United States Food and Drug Administration in
1999 for the prophylaxis of organ rejection in human patients older than
13 years at a dose of 2 mg/day (2-3). It is practically insoluble in
water (2.6 µg/mL), yields high liposolubility (log PO/W 5.77), very
unstable in ionic medium and has demonstrated a high rate of degradation
under UV light (4-6). Despite its liposolubility, the drug has low
corneal penetration, and has therefore not been able to show a high
effectivity rate for the treatment of autoimmune ocular surface
disorders (7).
Some of the advantages of rapamycin as an immunosuppressant derive from
its unique mechanism of action, its improved side-effect profile, and
its ability to synergize with other immunosuppressive agents
(calcineurin inhibitors) due to the shared metabolism by CYP 3A (8).
Because of this, its potential clinical uses range from organ
transplantation to ocular disorders, including non-infectious uveitis,
diabetic macular edema, autoimmune non-necrotizing anterior sclerosis
and Sjögren syndrome (9-12)
Rapamycin pharmacokinetics display large inter- and intra-patient
variability, due to different disease stages and use of concurrent
immunosuppressants or other interacting drugs, as well as polymorphisms
in the genetic configuration of CYP 3A. Due to the long half-life of
rapamycin, dose adjustments should ideally be based on levels obtained
5–7 days after initiation of therapy or dosage change (13).
Unfortunately, although rapamycin-induced toxicity has been reported
extensively in patients with organ rejection, there are few reports
concerning specific ocular toxicity. Adult horse and rabbit models with
different ophthalmologic disorders have been used for this purpose.
Administration through intravitreal (IVT) and subconjunctival (SCJ)
injection at doses ranging from 20 µg to 10 mg have reported no evidence
of tissue damage or toxicity (14-15). The safety profile of rapamycin in
adult human patients has also been evaluated, no toxicity was reported
at doses ranging from 44 to 1760 µg. (10-11)
Carriers can be used to enhance the intraocular concentrations of drugs,
as well as minimize the dose, and therefore side effects of medications.
One type of such carriers are liposomes. Liposomes are microscopic
vesicles composed of lipid bilayers surrounding an aqueous core (16-17).
They can improve the solubilization and absorption of a lipophilic drug
encapsulated inside the vesicle, as well as the bioavailability in the
ocular environment while protecting it from degradation (18-20). Since
liposomes have a similar composition as cell membranes, they are
expected to be biocompatible and biodegradable (21). Furthermore, due to
the fact that liposomes release constant low doses of drugs instead of
the usual single complete dose in one take, they are expected to be able
to reduce nonspecific side-effects and toxicity. (22). Nevertheless, the
thorough study of the toxicologic profile of new pharmaceutical
formulations is always important in the drug development process. Taking
into consideration the scarcity of in vivo studies demonstrating
the assumed non-toxicity of liposomes prepared through non-conventional
solvent free methodologies only tested in vitro in non-ocular cell lines
by other authors (23), the aim of
this study is to describe the results of an in vitro and in
vivo toxicity evaluation of liposomes loaded with rapamycin using a
proprietary methodology, administered through subconjunctival and
intravitreal injections to support their potential as a treatment of
ocular inflammatory autoimmune disorders.