International Journal of
Molecular Sciences
Review
A Clinical, Pharmacological, and Formulation Evaluation
of Melatonin in the Treatment of Ocular
Disorders—A Systematic Review
Alessia Romeo 1 , Adrienn Kazsoki 2 , Teresa Musumeci 1
Citation: Romeo, A.; Kazsoki, A.;
Musumeci, T.; Zelkó, R. A Clinical,
Pharmacological, and Formulation
Evaluation of Melatonin in the
Treatment of Ocular Disorders—A
Systematic Review. Int. J. Mol. Sci.
2024, 25, 3999. https://doi.org/
10.3390/ijms25073999
Academic Editor: Ylenia Zambito
Received: 27 February 2024
Revised: 25 March 2024
Accepted: 1 April 2024
Published: 3 April 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2024, 25, 3999. https://doi.org/10.3390/ijms25073999
and Romána Zelkó 2, *
1 Department of Drug and Health Sciences, University of Catania, Via Santa Sofia 64, 95125 Catania, Italy;
alessia.romeo@phd.unict.it (A.R.); tmusumec@unict.it (T.M.)
2 University Pharmacy Department of Pharmacy Administration, Semmelweis University,
Hőgyes Endre Street 7–9, 1092 Budapest, Hungary; kazsoki.adrienn@semmelweis.hu
* Correspondence: zelko.romana@semmelweis.hu
Abstract: Melatonin’s cytoprotective properties may have therapeutic implications in treating oc-
ular diseases like glaucoma and age-related macular degeneration. Literature data suggest that
melatonin could potentially protect ocular tissues by decreasing the production of free radicals and
pro-inflammatory mediators. This study aims to summarize the screened articles on melatonin’s
clinical, pharmacological, and formulation evaluation in treating ocular disorders. The identification
of relevant studies on the topic in focus was performed according to the Preferred Reporting Items for
Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines. The studies were searched in the
following databases and web search engines: Pubmed, Scopus, Science Direct, Web of Science, Reaxys,
Google Scholar, Google Patents, Espacenet, and Patentscope. The search time interval was 2013–2023,
with the following keywords: melatonin AND ocular OR ophthalmic AND formulation OR insert
AND disease. Our key conclusion was that using melatonin-loaded nano-delivery systems enabled
the improved permeation of the molecule into intraocular tissues and assured controlled release
profiles. Although preclinical studies have demonstrated the efficacy of developed formulations,
a considerable gap has been observed in the clinical translation of the results. To overcome this
failure, revising the preclinical experimental phase might be useful by selecting endpoints close to
clinical ones.
Keywords: melatonin; ocular drug delivery system; ophthalmic insert; clinical evaluation; pharmacology;
systematic analysis; PRISMA 2020
1. Introduction
Melatonin (MEL) is a neurohormone whose secretion occurs predominantly in the
pineal gland located in the brain. The discovery of this molecule dates to the middle of the
last century and can be attributed to dermatologist Aaron Lerner and his collaborators, who
coined the name melatonin based on its pigmentation effect on the skin (by merging the
prefix “mela-” referring to melanin and the suffix “-tonin” denoting the precursor of that
molecule, serotonin). Later, the molecule was classified as an indoleamine and identified as
N-acetyl-5-methoxytryptamine [1].
Although MEL was initially thought to be exclusively related to the dermatological
field, over time it has been shown to be involved in the regulation of a variety of physiolog-
ical processes. Lerner himself, in the late 1970s, was the first to note that the molecule was
able to affect circadian rhythms [2].
Over the years, the hormone has been associated with processes of pubertal develop-
ment and aging, the modulation of immune response, inflammation, antioxidant defenses,
neuroprotection, and the control of blood pressure and temperature [3–10].
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 2 of 23

Int. J. Mol. Sci. 2024, 25, 3999 2 of 21

MEL exerts its effects by acting on three types of receptors. Two of these are MT1 and
MT2 receptors, high-affinity transmembrane G-protein-coupled receptors that share 60%
MEL exerts
homology. MT1 isitsdistributed
effects by acting on three
in several braintypes
areasof(such
receptors.
as theTwo of these
pituitary, are MT1 and
hypothalamus,
MT2 receptors, high-affinity transmembrane G-protein-coupled
thalamus, cortex, hippocampus, cerebellum, amygdala, substantia nigra, and receptors that share 60%
nucleus
homology. MT1 is distributed in several brain areas (such as the pituitary,
accumbens) but has also been identified in ocular structures such as the cornea hypothalamus,
thalamus,
(epithelium,cortex,
stroma,hippocampus,
endothelium), cerebellum,
sclera, and amygdala, substantia
retina (retinal nigra,
vessels, andganglion
retinal nucleus
accumbens) but has also been identified in ocular structures such as the cornea (epithelium,
cells, and amacrine cells of the inner and outer plexiform layers). MT2 is largely localized
stroma, endothelium), sclera, and retina (retinal vessels, retinal ganglion cells, and amacrine
in the retina but is also present in the hippocampus, cerebellum, cortex, and
cells of the inner and outer plexiform layers). MT2 is largely localized in the retina but is
paraventricular nucleus [11,12].
also present in the hippocampus, cerebellum, cortex, and paraventricular nucleus [11,12].
The third MT3 receptor has been identified as a quinone reductase 2 enzyme and,
The third MT3 receptor has been identified as a quinone reductase 2 enzyme and,
unlike the first two, has cytoplasmic localization and low affinity. Although this enzyme
unlike the first two, has cytoplasmic localization and low affinity. Although this enzyme
is expressed in peripheral structures (heart, liver, kidney, lungs), previous studies have
is expressed in peripheral structures (heart, liver, kidney, lungs), previous studies have
shown its involvement in the modulation of intraocular pressure (IOP) [13,14].
shown its involvement in the modulation of intraocular pressure (IOP) [13,14].
In addition to the pineal gland, MEL synthesis also occurs in other extrapineal tissues
In addition to the pineal gland, MEL synthesis also occurs in other extrapineal tis-
(such as the brain, lens, ciliary body, and retina) and originates from the amino acid
sues (such as the brain, lens, ciliary body, and retina) and originates from the amino acid
precursor tryptophan [15]. The hydroxylation of tryptophan by the enzyme tryptophan
precursor tryptophan [15]. The hydroxylation of tryptophan by the enzyme tryptophan
hydroxylase (TPH)
hydroxylase converts it
(TPH) converts to 5-hydroxytryptophan.
it to 5-hydroxytryptophan. This This is then decarboxylated
is then decarboxylated by by
aromatic L-amino acid decarboxylase (AADC) to produce serotonin.
aromatic L-amino acid decarboxylase (AADC) to produce serotonin. The latter The latter undergoes
undergoes
two further
two furtherconversions,
conversions,anan acetylation
acetylation (to N-acetylserotonin)
(to N-acetylserotonin) and a and a methylation
methylation involv-
involving serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase
ing serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOT),
(HIOT), respectively,
respectively, to produce toMEL
produce MEL
(Figure 1). (Figure 1). Once synthesized,
Once synthesized, the moleculethe molecule
is released is
into
released into
capillaries andcapillaries
distributed and
todistributed
body tissues to[16].
body tissues [16].

Figure 1. The synthesis of melatonin from the essential amino acid tryptophan.

Pineal
Pineal synthesis
synthesis is is closely
closely regulated
regulated by
by the
the light/dark
light/dark cycle. During the
cycle. During the hours
hours of
of
darkness, norepinephrine released
darkness, norepinephrine released from
from postganglionic sympathetic fibers
postganglionic sympathetic fibers reaches
reaches the
the
pineal
pineal where
where itit activates
activates α-α- and β-adrenergic receptors,
and β-adrenergic receptors, increasing
increasing intracellular
intracellular cAMP
cAMP
levels, NAT enzyme transcription, and thus MEL synthesis. Conversely,
levels, NAT enzyme transcription, and thus MEL synthesis. Conversely, during during the
the day,
day,
norepinephrine release is attenuated and consequently MEL synthesis
norepinephrine release is attenuated and consequently MEL synthesis too [17]. too [17].
Although pinealocytes are the cells that synthesize most MEL in the pineal gland, a
study showed that the rat retina continues to synthesize MEL even after pinealectomy [11].
It was observed that among organelles and subcellular structures, the highest concen-
tration of MEL was found in mitochondria [18]. Karasek and colleagues observed that the
 Although pinealocytes are the cells that synthesize most MEL in the pineal gland, a
study showed that the rat retina continues to synthesize MEL even after pinealectomy
[11].
Int. J. Mol. Sci. 2024, 25, 3999 It was observed that among organelles and subcellular structures, the highest 3 of 21
concentration of MEL was found in mitochondria [18]. Karasek and colleagues observed
that the mitochondria volume was greatest during the night and corresponded to the peak
of MEL production
mitochondria [19].
volume wasThese hypotheses,
greatest during thesupported bycorresponded
night and the identification of peak
to the the enzymes
of MEL
responsible for MEL biosynthesis in the mitochondrial matrix, confirmed
production [19]. These hypotheses, supported by the identification of the enzymes thatrespon-
these
organelles would be responsible for MEL synthesis [20].
sible for MEL biosynthesis in the mitochondrial matrix, confirmed that these organelles
would Mitochondria play
be responsible forseveral important
MEL synthesis roles such as ATP production, the control of
[20].
cellular metabolism, and the regulation
Mitochondria play several important roles of programmed
such as ATP cell death. The
production, hormone
the control of
produced at the mitochondrial
cellular metabolism, level exerts
and the regulation numerous cell
of programmed beneficial effects
death. The with the
hormone aim of
produced
preserving the function
at the mitochondrial and
level structure
exerts of these
numerous organelles
beneficial (Figure
effects with2). MEL,
the aim in
of addition
preserving to
stimulating ATP synthesis and preserving membrane potential, counteracts
the function and structure of these organelles (Figure 2). MEL, in addition to stimulat- oxidative
stress
ing ATP andsynthesis
antiapoptotic events for which
and preserving membranethese potential,
subcellularcounteracts
structures are most responsible
oxidative stress and
[21–23].
antiapoptotic events for which these subcellular structures are most responsible [21–23].

effects of melatonin at the mitochondrial level.

Figure 2. Main effects

The eye
The eye isisaametabolically
metabolically active
activestructure, andand
structure, the the
visual system
visual ranksranks
system among the brain
among the
systems that in terms of ATP consume the most energy. Although
brain systems that in terms of ATP consume the most energy. Although the highest the highest metabolic
activity involves
metabolic activitythe ocularthe
involves surface
ocular(cornea
surfaceand conjunctiva),
(cornea which is the
and conjunctiva), most
which exposed
is the most
part of the eye, internal tissues (retina and optic nerve) are also
exposed part of the eye, internal tissues (retina and optic nerve) are also subject to subject to dysfunction
caused by oxidative
dysfunction caused stress [24]. Among
by oxidative stress body
[24]. tissues,
Among the retina
body and optic
tissues, nerveand
the retina are those
optic
that require the greatest energy sustainment, so they are rich in mitochondria. Therefore,
nerve are those that require the greatest energy sustainment, so they are rich in
mitochondrial dysregulation in these tissues could involve a plethora of pathophysiological
mitochondria. Therefore, mitochondrial dysregulation in these tissues could involve a
conditions and contribute to the pathogenesis of various ocular disorders [25].
plethora of pathophysiological conditions and contribute to the pathogenesis of various
In recent decades, the beneficial properties of MEL have attracted much attention
ocular disorders [25].
in the treatment and prevention of impaired ophthalmic conditions. MEL’s ability to
In recent decades, the beneficial properties of MEL have attracted much attention in
prevent oxidative damage and associated mitochondrial dysfunction has proven to be a
the treatment and prevention of impaired ophthalmic conditions. MEL’s ability to prevent
useful approach in the management of age-related macular degeneration (AMD), where
oxidative damage and associated mitochondrial dysfunction has proven to be a useful
the molecule has been able to preserve the viability of retinal pigment epithelium (RPE)
approach in the management of age-related macular degeneration (AMD), where the
cells [26,27].
molecule has been able to preserve the viability of retinal pigment epithelium (RPE) cells
MEL also exerts control over the expression of inflammatory cytokines (IL-6 and
[26,27].
TNF-α) and the pathological secretion of vascular endothelial growth factor (VEGF) at the
MEL
retinal level.also
Theexerts control
inhibition over
of the the expression
angiogenic of inflammatory
factor counteracts cytokines (IL-6 and
the neovascularization that
TNF-α) and the pathological secretion of vascular endothelial
is a manifestation of proliferative diabetic retinopathy; therefore, MEL could growth factor be
(VEGF) at the
an effective
retinal level.resource
therapeutic The inhibition
[28]. of the angiogenic factor counteracts the neovascularization
that is
Several studies have evaluated the diabetic
a manifestation of proliferative influenceretinopathy;
of melatonintherefore, MEL could
on the modulation be an
of IOP. It
effective therapeutic resource [28].
has been found that IOP and MEL follow inverse circadian rhythms; during the day, MEL
levelsSeveral
are lowstudies havehas
while IOP evaluated the influence
high values, vice versaofduring
melatonin on theThis
the night. modulation of IOP.
relationship was
It has been found that IOP and MEL follow inverse circadian rhythms;
confirmed by studies showing that MEL levels were significantly altered in ocular diseases during the day,
MEL
such aslevels are low
glaucoma [29].while IOP has in
Investigations high values,
animal viceand
models versa during
clinical the proved
studies night. This
that
relationship was confirmed by studies showing
MEL administration was effective in regulating IOP [30]. that MEL levels were significantly altered
In addition, the neuroprotection of injuries caused by ocular hypertension has shown
an added efficacy of the therapeutic benefits of this molecule in the management of glauco-
matous conditions [31]. Collectively, these mechanisms could be useful in protecting ocular
tissues from oxidative damage and subsequent pathologies that might occur.
The importance of summarizing various melatonin formulations lies in optimizing
drug delivery to the eye, overcoming bioavailability challenges, and potentially improving
Int. J. Mol. Sci. 2024, 25, 3999 4 of 21

therapeutic outcomes for ocular conditions. By exploring different formulations, researchers

aim to enhance the efficacy and bioavailability of melatonin for treating eye diseases, which
is crucial for developing effective ocular drug delivery systems. While numerous reviews
exist regarding the ocular application of MEL, a systematic review has not been put forth
thus far. Given this context, we deemed it beneficial to conduct an extensive search across
various databases, utilizing a comprehensive approach to gather, assess, and analyze
relevant empirical data through meta-analysis, aiming to offer a comprehensive overview
of the subject.

2. Materials and Methods

The identification of relevant studies on the topic in focus was performed according
to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA
2020) guidelines.

2.1. Eligibility Criteria

The following criteria were established for the selection of research articles and patents
to be included in this systematic review: original research studies published in peer-
reviewed journals and published patents were included, while review articles, conference
papers, book chapters, and guidelines were excluded. The search was limited to English-
language studies published in the time range between 2013 and 2023. This review included
all research studies where MEL was administered in the eye. No eligibility criteria were
set regarding the diseases treated or regarding the in vitro and in vivo studies conducted.
Studies comparing the efficacy of MEL with that of analogs were also included, while
articles focused entirely on the delivery of analogous molecules were not considered.

2.2. Search Strategy

The search for articles and patents relevant to the administration of MEL for the
treatment of ocular disorders was systematically performed using the same keywords
specified below and their equivalent synonyms in the following databases and web search
engines: Pubmed, Scopus, Science Direct, Web of Science, Reaxys, Google Scholar, Google
Patents, Espacenet, and Patentscope. The search queries were constructed as follows:
(melatonin) AND (ocular OR ophthalmic) AND (formulation OR insert) AND (disease).
The search results were collected and tabulated as shown in the next chapter.

2.3. Data Collection and Extraction

A PRISMA 2020 flowchart was employed to tabulate and summarize the data collected
from the research. Research studies and patents obtained from the consulted databases
were exported to the Mendeley reference manager, and duplicates were removed. The
remaining articles were subjected to further screening processes by evaluating eligibility
based on the title, then the abstract, and finally the full text. Relevant residual articles were
consulted to extract the information of interest, which was tabulated under the following
items: therapeutic indication, the type of formulation, the route of administration, con-
stituent materials, the concentration of the loaded active, physicochemical characteristics
(size, polydispersion index (PDI), zeta potential (ZP), and encapsulation efficiency (EE%)),
animal models used in the in vivo studies, and properties of the considered systems. The
information extracted from the patents was as follows: the background, target pathology,
specific use of MEL, applicant/manufacturer, patent number, and publication date.

3. Results
3.1. Studies Included from the Consulted Databases
A total of 403 articles and 170 patents were collected from searches performed on
all databases, including 272 from Google Scholar, 75 from Science Direct, 37 from Reaxys,
7 from Pubmed, 6 from Scopus, and 6 from Web of Science. Of the total patents, 141 were
obtained from Patentscope, 25 from Google Patents, and 4 from Espacenet. Of the sum
 3. Results
3.1. Studies Included from the Consulted Databases
A total of 403 articles and 170 patents were collected from searches performed on all
Int. J. Mol. Sci. 2024, 25, 3999 databases, including 272 from Google Scholar, 75 from Science Direct, 37 from Reaxys, 7
5 of 21
from Pubmed, 6 from Scopus, and 6 from Web of Science. Of the total patents, 141 were
obtained from Patentscope, 25 from Google Patents, and 4 from Espacenet. Of the sum
total,
total, only
only 16
16 articles
articles and
and 77 patents
patents met
met the
the inclusion
inclusion criteria
criteria and
and thus
thus were
were included
included inin
this
this review.
review.The
Theflowchart
flowchart inin
Figure 3 illustrates
Figure the the
3 illustrates steps of identification,
steps screening,
of identification, and
screening,
inclusion.
and inclusion.

Figure
Figure 3.
3. A
A PRISMA
PRISMA 2020
2020 flow
flow diagram showing relevant
diagram showing relevant articles
articles and
and patents
patents included
includedin
inthe
thestudy.
study.
3.2. The Results of the Studies
3.2.1. Melatonin Ophthalmic Indications and Drug Delivery Strategies
Based on the above criteria, the articles considered relevant were reviewed and are
listed in Table 1. The included studies were classified according to the formulation type and
therapeutic indication; a summary of the gathered information is depicted in Figure 4. From
Figure 4A, it is evident that the largest number of studies for the ocular delivery of MEL
had designed formulations intended for the treatment of conditions such as glaucoma and
retinal degeneration. A smaller percentage also developed formulations for the treatment of
ocular disorders such as granular corneal dystrophy type 2 (GCD2), diabetic macular edema
(DME), optic neuritis, and dry eye disease (DED). In contrast, in Figure 4B, it was found that
the most investigated systems for MEL ocular delivery consisted of nanoparticles (NPs).
Int. J. Mol. Sci. 2024, 25, 3999 6 of 21

Table 1. Summary of studies on melatonin-loaded ophthalmic formulations.

Therapeutical Type of For- Route of In Vivo/Animal

Materials Loaded Drug/Concentration Size, PDI, ZP, EE% Effects/Properties References
Indication mulation Administration Model

Size < 300 nm • NPs showed suitable physicochemical features and

PLGA, PDI < 0.25 a sustained in vitro release profile.
Yes, Male albino • The tested NPs showed good ocular tolerability
Nanoparticles PLGA-PEG, Boeringher 1, 3 and 5% (w/w) ZP from −8.2 to
Topical rabbits of in vivo. [32]
(NPs) Ingelheim KG (Ingelheim to polymer −32.2 mV
New Zealand • MEL-PLGA-PEG NPs were the most effective in
am Rhein, Germany) EE% from 44.15 to
78.20% reducing IOP.

Softisan® 100, by Sasol Size = 150–300 nm • MEL elicited a significant IOP reduction in the
Cationic rabbit eye.
GmbH (Hamburg, Germany) PDI < 0.3 Yes, Male albino
solid lipid • SLN tested in vivo demonstrated good tolerability.
Topical DDAB, 0.05% (w/v) ZP from −3.99 to rabbits of [33]
nanoparti- • SLN with SA was the most effective in terms of
SA or PA, Sigma–Aldrich +61 mV New Zealand
cles (cSLNs) IOP reduction.
(Milan, Italy) EE% = 83–96%

• MSs were able to co-deliver the active compounds

in a sustained manner over 30 days with low
Glaucoma burst release.
MEL = 45.80 µg/mg MSs • In vitro studies showed MSs to be neuroprotective
Coenzyme Q10, in a glutamate-induced cytotoxicity model in the
Size: 29.04 µm
Sigma-Aldrich (St. Louis, R28 cell line.
Microspheres Intravitreal (ITV) PLGA, Evonik España EE%: CoQ10 = 96.42%; Yes, Adult male
MO., USA) • In vivo efficacy studies were performed using a [34]
(MSs) injection (Granollers, Spain) DX = 78.20%; Dark Agouti rats
(CoQ10) = 35.71 µg/mg MSs well-established rodent model of chronic ocular
MEL = 61.83%
Dexamethasone hypertension (OHT), comparing multi-loaded and
(DX) = 115.86 µg/mg MSs single-drug-loaded MSs.
• Multi-loaded MSs showed a significantly
neuroprotective effect on retinal ganglion cells.

• MEL formulated in nanomicelles showed a

longer-lasting hypotonizing effect on IOP than
MEL and agomelatine, MEL in saline solution.
Soluplus® , BASF, Size = 61 nm Yes, Rats Sprague
Nanomicelles Topical Molekula, Darlington, UK • The duration of the hypotonizing effect was further [35]
Ludwigshafen, Germany PDI = 0.068 Dawley
0.4–0.8% (w/v) increased with the addition of natural antioxidant
lipoic acid.
Int. J. Mol. Sci. 2024, 25, 3999 7 of 21

Table 1. Summary of studies on melatonin-loaded ophthalmic formulations.

Therapeutical Type of For- Route of In Vivo/Animal

Materials Loaded Drug/Concentration Size, PDI, ZP, EE% Effects/Properties References
Indication mulation Administration Model

• NPs showed a core-shell type structure, with Dex

encapsulated in the shell and MEL in the core.
Size = 468 nm • MEL release from NPs was found to be sustained
MEL 5% (w/v)
PDI = 0.283 and were nontoxic in R28 cells.
Dexamethasone, Sigma Yes, 5-week-old
NPs Topical PLGA ZP = −21.7 mV • Increased corneal penetration was obtained in [36]
Aldrich (Shanghai, China) rabbits
EE% MEL = 87.25% comparison to free Dex and ML.
(Dex) 5% (w/v)
EE% Dex = 89.41% • Multi-loaded NPs were found to be effective in
reducing IOP.
Glaucoma

• Both MEL and agomelatine penetrate the posterior

segment of the eye.
• IOP elevation was drastically reduced by
MEL and agomelatine 0.4% Yes, Rats Sprague MEL/agomelatine with higher efficacy than that of
Nanomicelles Topical Soluplus® × timolol or brimonidine. [37]
(w/v) Dawley
• Inflammation and apoptosis were partially
prevented, thus leading to RGC survival and
recovered retinal dysfunction.

• An oxidative toxicity model was used to evaluate

the neuroprotective effect of MEL on RGCs.
Injectable Yes, New Zealand • According to the results obtained from viability
ITV injection × 1 mg/mL × studies on RGCs, the antiapoptotic effect, and the [38]
solution albino rabbits
ultrastructural analysis of the retina, MEL proved
to be an efficient neuroprotectant.

MEL, Sigma-Aldrich, • MSs were able to co-deliver therapeutic active

PLGA, Resomer® 503, Schnelldorf, Germany 10% substances in a sustained manner.
Boehringer Ingelheim (w/w) to polymer Size = 21.6–24.6 µm • ITV injection led to the partial functional and
Pharma GmbH & Co, Glial cell line-derived EE% GDNF = Yes, Rhodopsin structural rescue of photoreceptors in rhodopsin
Retinal MSs ITV injection [39]
Ingelheim, Germany neurotrophic factor (GDNF, 26.4–39.5% null mice knockout mice.
degeneration
vitamin E, Sigma-Aldrich, R&D, Systems, Minneapolis, EE% MEL = 39.2% • No significant intraocular inflammatory reaction
Schnelldorf, Germany MN, USA) 0.01% (w/w)to was observed.
polymer

• The in vitro release of MEL loaded in NPs was

slower compared to the MEL solution.
Size = 147–179 nm; PDI Yes, New Zealand • The increased penetration of MEL-loaded NPs
Ethylcellulose, Colorcon® through the cornea was demonstrated by ex vivo
NPs Topical 1–2 mg/mL = 0.092–0.2; ZP = −30 white female [40]
(Buenos Aires, Argentina) and in vivo tests.
mV; EE% = 67–73% rabbits
• Efficient neuroprotective activity in a model of
retinal degeneration.
Int. J. Mol. Sci. 2024, 25, 3999 8 of 21

Table 1. Summary of studies on melatonin-loaded ophthalmic formulations.

Therapeutical Type of For- Route of In Vivo/Animal

Materials Loaded Drug/Concentration Size, PDI, ZP, EE% Effects/Properties References
Indication mulation Administration Model

• NPs showed good mucoadhesive properties and a

controlled release profile.
PLGA-PEG, Size = 190 nm • An in vitro study on a model of diabetic
Yes, Male New
Retinal DDAB, PDI = 0.2 retinopathy demonstrated neuroprotective and
NPs Topical 1% (w/w) to polymer Zealand [41]
degeneration CTAB, Merck Life Science ZP = 39.8 mV antioxidant activities.
albino rabbits
S.r.L. (Milan, Italy) EE% = 79.85% • An in vivo study confirmed no signs of
ocular irritation.

Polymeric • DDAB is the most suitable coating material for the

and hybrid PLA, Sigma (Milan, Italy) Size < 250 nm preparation of aqueous-core hybrid NCs.
No in vivo studies
aqueous- TCT, Farmalabor (Bari, Italy) PDI < 0.2 • Cationic lipid allowed for the formation of very
× 0.03% (w/w) have been [42]
core DDAB, Sigma (Milan, Italy) ZP from −36 to +30 mV stable well-defined NCs with a non-spherical
conducted
nanocap- CTAB, Sigma (Milan, Italy) EE% = 87–90% shape with sustained and prolonged drug release.
sules (NCs)
Ophthalmic
delivery • The NPs showed a nanometric size, spherical
Size = 160–180 nm shape, sustained drug release, and physical
HAS, Laboratorio de PDI = 0.06 stability.
Topical and Yes, New Zealand
NPs Hemoderivados UNC 1 mg/mL ZP = from −30 to • The topical administration of the NPs produced no [43]
subconjunctival rabbits
(Cordoba, Argentina) −40 mV irritation, while subconjunctival administration
EE% = 18–21% resulted in mild irritation 24 h after administration.

Granular • Stability studies for 60 days showed no significant

corneal HPβCD, Alfa Aesar Yes, New Zealand change in the pH, osmolarity, and MEL content.
Eye drop Topical 2.75 mg/mL × • HPβCD can reduce rabbit eye irritation and [44]
dystrophy (Haverhill, MA, USA) albino rabbits
type 2 (GCD2) increase MEL delivery into the rabbit cornea 2-fold.

• MEL prevented cell hyperpermeability and

external barrier disruption.
Diabetic No in vivo studies • MEL modulated gene expression to maintain
macular Solution × × 0.1, 0.5, and 1 mM × have been mitochondrial homeostasis. [45]
edema (DME) conducted • MEL prevented the apoptosis of retinal pigmented
epithelial cells.
• MEL may have therapeutic value in DME.
Int. J. Mol. Sci. 2024, 25, 3999 9 of 21

Table 1. Summary of studies on melatonin-loaded ophthalmic formulations.

Therapeutical Type of For- Route of In Vivo/Animal

Materials Loaded Drug/Concentration Size, PDI, ZP, EE% Effects/Properties References
Indication mulation Administration Model

Vegetable oils, Sigma • Experimental optic neuritis caused alterations in

2.5 mm × 1 mm Yes, Male Wistar circadian physiology, and melatonin restored the
Optic neuritis Pellet Subcutaneous Chemical Co. 20 mg [46]
(diameter × length) rats circadian system misalignment.
(St Louis, MO, USA)

• NPs showed good cytocompatibility and

decreased the ROS level and apoptosis ratio.
Dry eye MEL 20% Size < 250 nm Yes, Female • Tav promoted the production of mucins on the
NPs Topical Polydopamine [47]
disease (DED) tavilermide (Tav) 2% ZP from −20 to +5 BALB/c mice ocular surface increasing the retention time of NPs.
• NPs could effectively treat DED.

Poly(D,L-lactide-co-glycolide) (PLGA); PLGA-poly(ethylenglycole) (PLGA-PEG); stearic (SA); palmitic acid (PA); cetyltrimethylammonium bromide (CTAB); didodecyldimethylam-
monium bromide (DDAB); polylactic acid (PLA); Caprylic/Capric Triglyceride (TCT); human serum albumin (HSA); 2-hydroxypropyl-β-cyclodextrins complex (HPβCD). × = no
information or not relevant information.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 12 of 23
Int. J. Mol. Sci. 2024, 25, 3999 10 of 21

Figure
Figure4.4.AA PRISMA chart
chartanalysis
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whereby the formulations showed good ocular tolerability
whereby the formulations showed good ocular tolerability in vivo. in vivo.

3.2.2.Ophthalmic
3.2.2. OphthalmicConditions
ConditionsTreated
Treatedwith
withMelatonin
Melatonin
AAsummary
summaryofofpublished
publishedstudies
studiesover
overthe
thepast
past1010years
yearsononthe
theuse
useofofMEL
MELininthe the
ocular field is discussed below. Given the important role that MEL has shown
ocular field is discussed below. Given the important role that MEL has shown in the in the control
of intraocular
control pressure,
of intraocular severalseveral
pressure, studies have investigated
studies the use
have investigated ofuse
the thisofmolecule for the
this molecule
treatment of glaucomatous conditions.
for the treatment of glaucomatous conditions.
The MEL neuroprotected effect was also tested on a model of DME, which is one of
The MEL neuroprotected effect was also tested on a model of DME, which is one of
the primary causes of blindness in diabetic retinopathy. The study in question did not
the primary causes of blindness in diabetic retinopathy. The study in question did not
involve the incorporation of MEL into any vector but aimed to investigate the protective
involve the incorporation of MEL into any vector but aimed to investigate the protective
mechanisms that MEL exerted on an in vitro model of DME using human retinal pigmented
mechanisms that MEL exerted on an in vitro model of DME using human retinal
epithelium cells. The results showed that MEL supported the integrity of the blood–retinal
pigmented epithelium cells. The results showed that MEL supported the integrity of the
barrier by suppressing angiogenesis and apoptosis, while at the mitochondrial level, MEL
blood–retinal barrier by suppressing angiogenesis and apoptosis, while at the
preserved homeostasis by regulating the expression of genes involved in fission, mitophagy,
mitochondrial level, MEL preserved homeostasis by regulating the expression of genes
and mitochondrial biogenesis [45].
involved in fission, mitophagy, and mitochondrial biogenesis [45].
A MEL-loaded plant oil-based pellet intended for subcutaneous implantation was
A MEL-loaded plant oil-based pellet intended for subcutaneous implantation was
used to evaluate the activity of the molecule on an inflammatory condition of the optic
used to evaluate the activity of the molecule on an inflammatory condition of the optic
nerve, optic neuritis. This condition involves decreases in the pupillary light reflex and
nerve, optic neuritis.
alterations Thisphysiology.
in circadian condition involves
MEL wasdecreases in the
able to both pupillarythe
counteract light reflex in
decrease andthe
alterations in circadian physiology. MEL was able to both counteract
pupillary light reflex and restore the altered circadian rhythm [46]. the decrease in the
pupillary light reflex and restore the altered circadian rhythm [46].
Int. J. Mol. Sci. 2024, 25, 3999 11 of 21

In addition to diseases affecting the posterior eye segment, MEL has also been applica-
ble in treating ocular surface disorders such as DED, a multifactorial condition that, even
when treated, often does not provide rapid relief [48]. In order to restore tear film homeosta-
sis and provide protection to ocular epithelial cells, Jin et al. proposed the co-delivery of
MEL and tavilermide, a small molecule able to preserve tear film integrity, in mesoporous
polydopamine NPs. The synergistic treatment strategy with the multifunctional eye drops
promoted mucin secretion and supported the restoration of ocular surface cells, speeding
up the alleviation of DED [47].
MEL has been shown to potentially offer therapeutic benefits for ocular surface con-
dition GCD2. It exhibits a dual antioxidant effect by safeguarding corneal fibroblasts
through direct ROS scavenging and by modulating antioxidant enzymes, as demonstrated
in studies [49].

3.2.3. Melatonin Formulations

Table 1 summarizes the materials selected to produce delivery carriers improving ad-
sorption, stability, and provided protection against the chemical and physical degradation
of the molecule. Polymeric (PLGA, PLA, PLGA-PEG, Soluplus® , and polydopamine), lipid
(Softisan® , DDAB, CTAB, TCT), protein (HSA), oligosaccharide (HPβCD), and vegetable
(vegetable oils) materials were used in the reviewed formulations. All these materials
were biodegradable and biocompatible, ideal requirements for achieving excellent in vivo
tolerability. Overall, the delivery systems examined provided enhanced drug permeation
into target tissues and the sustained release of the molecule, factors that contributed to the
improved delivery and therapeutic efficacy of MEL.
To circumvent the limitations of poor bioavailability associated with conventional
aqueous eye drops, poly(d,l-lactide-co-glycolide) (PLGA) and PLGA-poly(ethylene glycol)
(PLGA-PEG) NPs were prepared by a solvent displacement method and were studied
in vivo to assess ocular tolerability and measure IOP. The two polymers used were selected,
in addition to their proven biocompatibility and biodegradability properties, to ensure the
sustained release of MEL and particularly the PLGA-PEG copolymer to increase mucoad-
hesiveness with the ocular surface. In all formulations, both in liquid and freeze-dried
dosage forms, MEL was successfully encapsulated. Although all NPs showed suitable
technological characteristics for ophthalmic application, pegylated NPs showed superior
performance in the treatment of glaucoma. The PLGA-PEG NPs, compared with the aque-
ous solution of MEL and PLGA NPs, provided more effective (a maximum reduction of
5 mmHg) and prolonged (persistent up to 8 h) IOP lowering, confirming the hypothesis
that the mucoadhesive properties of the copolymer could have increased the contact time
with the ocular mucosa [32].
NPs of PLGA for glaucoma treatment were also successfully fabricated for the co-
delivery of MEL and dexamethasone (DX). These systems were prepared and simultane-
ously loaded with both molecules using the coaxial electrospray technique. The use of this
technique resulted in a high loading capacity for both drugs in systems with core-shell
morphology, where DX was encapsulated in the shell and MEL in the core. In vitro studies
showed sustained and nontoxic release on retinal cells for both drugs, while in vivo studies
showed that molecules loaded in NPs exhibited better corneal penetration compared to
free molecules. The use of the two molecules in combination was able to provide effective
antioxidant and hypertensive effects, which were useful in preserving the loss of retinal
ganglion cells (RGCs) and hypertension phenomena [36].
Another type of NP investigated for MEL delivery in glaucoma treatment was cationic
solid lipid NPs (SLNs). These lipid nanosystems were produced by a quasi-emulsion sol-
vent diffusion (QESD) method, which was generally used for the preparation of polymeric
NPs. The lipid matrix consisted of Softisan® 100 mixed with lipid modifiers (palmitic acid
and stearic acid) to improve the fluidity. With the aim of increasing the mucoadhesiveness
of SLNs, the surface was decorated with the cationic lipid didodecyldimethylammonium
bromide (DDAB). These nanosystems showed good ocular tolerability in addition to en-
Int. J. Mol. Sci. 2024, 25, 3999 12 of 21

hancing the pharmacological effect of MEL. It was reported that a single topical application
was able to effectively reduce (a maximum reduction of 7 mmHg) IOP for up to 24 h [33].
Further evidence that nanomicellar formulations of MEL were effective in the treat-
ment of glaucoma was reported in two studies conducted by Dal Monte et al [37]. In the
first study, the hypotonizing effect of MEL formulated in nanomicelles or in saline solution
was examined. MEL-loaded nanomicellular formulations were compared with nanomi-
celles loaded with the agomelatine analog, either alone or in combination with MEL. The
addition of lipoic acid, a natural antioxidant, was also evaluated. Although the molecule
alone did not show hypertensive effects on glaucoma murine models, the study aimed to
evaluate the influence on the hypotonizing effect of melatoninergic compounds [50]. The
results showed that the combination of MEL and agomelatine potentiated the magnitude
of the hypotonizing effect compared with each compound alone and that the addition of
lipoic acid ensured a longer duration of the lowering effect [35]. The second study aimed
to evaluate the neuroprotective effect of the MEL/agomelatine nanomicellular formulation
on a rat model of hypertensive glaucoma. Although the formulation was effective in
counteracting the loss of RGCs and promoting functional recovery, it was not fully clarified
whether the neuroprotective activity was direct or secondary to the hypotensive effect [37].
The formulations discussed so far were intended for topical administration and had
nanometer sizes smaller than 500 nm. Regarding the intraocular administration of drugs,
delivery systems with different sizes, selected according to the target site, pathology, and
treatment period, could be used. The intravitreal injection of microsystems (1–1000 µm) in
suspension could be useful to sustain prolonged therapeutic concentrations [51]. Among
them, microspheres (MSs) have gained increasing interest for the treatment of intraocular
disorders. However, it has been reported that the intravitreal administration of amounts
greater than 0.1 mg of PLGA MS can induce retinal dysfunction in rodents [52].
To this end, Arranz-Romera et al. designed PLGA-based MSs for co-delivery in the
treatment of glaucoma. Three molecules with anti-inflammatory and neuroprotective
activity were incorporated into the microsystems: MEL, DX, and coenzyme Q10. Neuro-
protective activity was tested in vitro on a model of glutamate-mediated neurotoxicity in a
retinal cell line and in vivo on a Morrinson rodent model of ocular hypertension. Multi-
loaded MSs ensured the simultaneous and controlled release of the three active molecules,
reducing the amount of carrier to be injected compared to formulations loaded with single
drugs [34]. This multi-therapy strategy has proven useful in treating a multifactorial disease
such as glaucoma, and its use has also been investigated in another model of retinal degen-
eration. In this case, MSs of PLGA were used for the co-delivery of glial cell line-derived
neurotrophic factor (GDNF) alone or in combination with MEL. To improve encapsulation
and to modulate the release of the biotechnological agent, vitamin E was incorporated into
the polymer matrix. The results of the study showed that a single intravitreal injection of
MS 0.5 mg in rhodopsin (rho(−/−) knockout mice was able to promote the anatomical
and functional rescue of photoreceptors. In addition, it was reported that the combination
of MEL with GDNF reduced the initial burst and the rate of delivery and prolonged the
release of the protein factor [39].
The neuroprotective efficacy of MEL administered intravitreally in a model of retinal
degeneration was also tested by Del Valle Bessone et al [38]. In vitro and in vivo tests
showed that the saline solution of MEL, in addition to exerting the neuroprotective effect
by increasing the survival of RGCs subjected to oxidative stress, was able to significantly
reduce apoptotic processes [38]. Given the promising results of this work, the following
year the same authors focused their attention on improving the delivery system. Thus,
the research was focused on the retinal delivery of MEL using nanometric systems for
noninvasive topical delivery. To promote transcorneal permeation, ethyl cellulose was
selected for its mucoadhesive properties as the main constituent of NPs. Numerous benefits
have been obtained from encapsulating MEL in NPs, from a slower release profile to
increased drug permeation and improved absorption into intraocular structures, providing
superior performance in terms of neuroprotection in the retinal degeneration model [40].
Int. J. Mol. Sci. 2024, 25, 3999 13 of 21

Given the therapeutic value the molecule has shown in the DME model, the use of MEL
was also studied in a diabetic retinopathy model. To improve the bioavailability of a topical
formulation for MEL delivery, next-generation hybrid NPs have been proposed. With the
aim of providing mucopenetrating and mucoadhesive properties to the nanosystems, the
PLGA-PEG copolymer and two cationic lipids (cetyltrimethylammonium bromide (CTAB)
and DDAB) were employed. In order to optimize the encapsulation efficiency and zeta
potential of the platforms, different lipid and MEL concentrations were examined according
to an experimental design. Lipid coating provided mucoadhesive properties to the systems
and a controlled and sustained release of MEL for up to 8 days. In vitro studies showed
that MEL loading in hybrid NPs enhanced the neuroprotective and antioxidant efficacy of
the molecule. Meanwhile, in vivo tests confirmed that topical instillation did not result in
any signs of ocular irritation [41].
The use of the cationic lipids CTAB and DDAB to functionalize systems for MEL
ophthalmic delivery was also explored by Carbone et al. Here, polylactic acid (PLA)
nanocapsules were prepared with and without lipid coating using a solvent-free method,
phase inversion temperature. The study showed that the lipid coating affected four impor-
tant properties of the nanosystems, referred to as the “4S” (the average particle size, surface
charge, shape, and stability) [42].
Recently, MEL has been delivered in protein nanosystems based on human serum
albumin (HSA). Although albumin NPs were promising ocular delivery systems, they
were susceptible to instability. Therefore, two strategies have been investigated to improve
the stability of these carriers, the addition of Eudragit® S100 as a crosslinking agent and
thermal stabilization. The latter method proved to be more efficient because, besides being
easy to implement and avoiding the use of toxic crosslinking agents, it ensured the physical
stability of the NPs at different pH levels [43].
To overcome the limitations associated with the poor stability, solubility, and ocular
absorption of MEL, Ahn et al. complexed the molecule with different cyclodextrins (CDs):
α, β, γ, and 2-hydroxypropyl-β-CD (HPβCD). Of these, it was reported that HPβCD
increased MEL delivery into the rabbit cornea 2-fold, ensuring greater therapeutic efficacy
and proving to be a promising therapy for GCD2 [44].

3.3. Patent Studies

The use of MEL in treating ocular disorders has garnered increased attention, with
its inclusion in patents serving various purposes. Table 2 details patents incorporating
MEL. An Australian patent discusses injectable, biodegradable polymeric formulations
for the controlled release of active molecules, targeting chronic degenerative diseases like
AMD. Active agents in the patent range from monoclonal antibodies to coagulation factors,
cytokines, growth factors, peptides, and hormones like MEL. The delivery polymers are
triethylene carbonate-based with PLA and/or PGA molar fractions, supplemented with
PEG, PVA, and poloxamers to enhance injectability and modulate release. Nanoparticle
systems using copolymers improve targeted delivery, combined with injectable hydrogel
for simultaneous administration via a double syringe. Release durations vary from 30 days
to over 6 months/one year post-ITV injection, showing no anatomical changes and a
safe profile.
Int. J. Mol. Sci. 2024, 25, 3999 14 of 21

Table 2. Summary of patent studies on melatonin-loaded ophthalmic formulations.

Background
Biodegradable injectable
Chronic ocular disorder
polymer formulations that
permit an extended or
sustained release of a
bioactive agent.
Methods, compositions, and
implantable elements
comprising active cells (e.g.,
an engineered RPE cell) which
may express a therapeutic
agent useful for the treatment
of a disease.
Methods, compositions, and
implantable elements
comprising stem cells (e.g., a
mesenchymal stem function
cell (MSFC)) which produce a
therapeutic agent.
Diseases treatable with
Nanoparticle formulations for
nucleic acid complexed
delivery for increasing gene
expression in a
cicatricial pemphigoid,
targeted manner.
neuromyelitis optica).
Targeted Diseases Using MEL Applicant/Manufacturer/Assignee Patent Number Publication Date Reference
Bioactive agents include, but are
not limited to, bevacizumab,
such as age-related ranibizumab, aflibercept, and W. L. Gore & Associates, Inc.,
AU 2,021,277,631 A1 23 December 2021 [53]
macular degeneration lampalizumab. Additional Newark, DE, USA
(AMD) and others. bioactive agents are hormones
like MEL.
The therapeutic agent may be any
biological substance, such as
Neurodegenerative SIGILON THERAPEUTICS,
nucleic acid, a polypeptide (like US 20,200,263,196 A1 20 August 2020 [54]
disorders and others. Inc., Cambridge, MA, USA
MEL), a lipid, a sugar, or a
small molecule.
The therapeutic agent may be any
biological substance, such as a
Neurodegenerative SIGILON THERAPEUTICS,
nucleic acid, a polypeptide (like WO 2,019,195,056 A1 10 October 2019 [55]
disorders and others. Inc., Cambridge, MA, USA
MEL), a lipid, a sugar, or a
small molecule.
oligonucleotides
Ligands (such as MEL or others)
targeting associated
can be conjugated to the particle
mRNA (intraocular Translate Bio MA, Inc.,
to target the nucleic acid complex US 20,180,311,176 A1 1 November 2018 [48]
melanoma, ocular Lexington, MA, USA
to a specific cell type or tissue
of interest.
optic neuritis,
Int. J. Mol. Sci. 2024, 25, 3999
Table 2. Cont.
Background
The topical delivery of
therapeutic agents using
cell-penetrating peptides.
Formulations of cannabidiol
derivatives and their use as
modulators of cannabinoid
receptor type 2 in the
treatment of various diseases.
Implants of solid complexes
for the treatment of
ophthalmic diseases.
15 of 21
Targeted Diseases Using MEL Applicant/Manufacturer/Assignee Patent Number Publication Date Reference
The therapeutic agent is or
includes an anti-dyslipidemic
agent, an antioxidant (such as
AMD and other eye MEL), an anti-inflammatory agent, MacRegen, Inc.,
US 20,210,085,797 A1 25 March 2021 [56]
diseases. a complement inhibitor, a Birmingham, AL, USA
neuroprotector or an
anti-angiogenic agent, or any
combination thereof.
Pharmaceutical vehicle optionally
Demyelinating diseases EMERALD HEALTH
comprises a stabilizer, emulsifier,
(such as neuromyelitis PHARMACEUTICALS Inc., WO 2,020,163,612 A1 13 August 2020 [49]
polymer, antioxidant (such as
optica) and others. San Diego, CA, USA
MEL), or any combination thereof.
To improve the stability of the
therapeutic agent, the intraocular
implant may comprise excipients
ALLERGAN, Inc.,
Glaucoma and others. like preservatives, an antioxidant WO 2,019,023,211 A1 31 January 2019 [57]
Irvine, CA, USA
(such as MEL), buffering agents,
chelating agents, electrolytes,
and others.
Int. J. Mol. Sci. 2024, 25, 3999 16 of 21

A U.S. patent describes drug product compositions with implantable elements contain-
ing active cells (e.g., engineered RPE cells) or derivatives, capable of producing therapeutic
agents for CNS, eye, and spinal disorders. Therapeutic agents include nucleic acids,
polypeptides, sugars, small molecules, and MEL. The implantable element can be admin-
istered as an implant or injection, with evaluation based on cell viability and therapeutic
agent production levels at intervals post-administration. A similar patent was filed at the
WIPO in Geneva the year before the publication mentioned. The key difference was in the
active cell, specifically mesenchymal stem function cells (MSFCs) or its derivatives. Despite
this difference, other aspects such as indications, therapeutic agents, and implementation
forms remained identical [53].
A patent filing detailed the use of nanoparticle formulations to deliver oligonucleotides
and synthetic RNA for targeted gene expression enhancement. Materials like PLGA,
cationic lipids, anionic lipids, or PEG-conjugated lipids were used for fabrication, resulting
in nanostructures with diameters ranging from 20 to 150 nm. The lipid-to-oligonucleotide
ratios varied from 1:1 to 50:1. The protection of RNA from degradation, particularly by
exonucleases, was a key focus. Targeted delivery was achieved through ligands like anti-
bodies, drugs, proteins, and neurotransmitters, with MEL as a specific ligand for receptor
targeting. Various administration routes were explored, including ocular, intranasal, oral,
pulmonary, and intravenous routes, depending on the desired local or systemic effect.
Formulations could take the form of drops, sprays, tablets, capsules, syrups, or solutions.
For ocular delivery, topical instillation, subconjunctival application, or intraocular injection
were considered. Final formulations could include mucomimetic agents, preservatives, and
pH adjusters for stability and efficacy [48].
A patent filed in WIPO details formulations of cannabidiol derivatives targeting
cannabinoid type 2 receptors to treat various conditions, including demyelination-associated
disorders. These patented formulations are delivered using pharmaceutical vehicles en-
hancing solubility and bioavailability. The delivery methods include aqueous solvents,
co-solvents, cyclodextrin complexes, lipid carriers, or their combinations. Compositions
contain stabilizing agents, emulsifiers, polymers, antioxidants like MEL, or a mix. Lipo-
somes and polymeric reservoirs ensure slow and sustained release. These modified-release
formulations can be administered topically, intraocularly, orally, or parenterally. Intraocular
indications include neuromyelitis optica and Leber’s optic atrophy [49].
A patent deposition in WIPO detailed a novel approach for treating glaucoma us-
ing a solid complex combined with γ-cyclodextrin. The document outlined the utiliza-
tion of biodegradable polymer matrices like PLA and PLGA as carriers for this com-
plex. The degradation and release kinetics of the carrier agent were influenced by the
hydrophilic/hydrophobic nature of the end functional groups, leading to controlled release
over 1 to 6 months. The implant’s specifications (weight, diameter, length) varied based
on its placement in the eye (intracameral or intravitreal). Apart from polymers and thera-
peutic agents, the formulation could include excipients such as stabilizers, preservatives,
antioxidants (like MEL), and buffering or chelating agents, among others [57].

4. Discussion
Although the role of MEL in ocular diseases has been widely investigated, the main
obstacle of ocular administration is the poor bioavailability in this organ. The eye has sev-
eral static and dynamic protective barriers that limit the entry and diffusion of therapeutic
agents. Ocular barriers do not present the only obstacle to effective administration; other
disadvantages that contribute to low bioavailability in ocular tissues are the enzymatic
degradation of introduced molecules, poor targeting efficiency, low retention, and perme-
ation time [58]. All these limitations contribute to providing a low dosage to the target
tissue. Many ocular diseases are chronic in nature and consequently require sustained
amounts of drug. Sustained-release pharmaceutical formulations could maintain constant
drug levels at the site of action and partially mitigate this problem [59]. In addition, sus-
tained release could improve patient compliance providing benefits for both topical and
Int. J. Mol. Sci. 2024, 25, 3999 17 of 21

intravitreal administration [60]. In the case of topical instillation, it could limit washout,
nonproductive systemic absorption, and adverse effects [61]. Concerning intravitreal injec-
tion, it could reduce the frequency of administration to ensure therapeutic levels while also
limiting the economic costs of hospitalization. In this regard, the use of reviewed delivery
systems has been helpful in providing formulations with sustained release and improved
bioavailability. The composition of these systems influenced several technological param-
eters such as the encapsulation efficiency, loading, and drug release pattern [62]. In the
case of polymeric delivery devices, polymer selection could be crucial to achieve degra-
dation rates pertinent to the desired release profiles [63]. These vehicles have additional
advantages, such as the ability to promote the crossing of precorneal barriers by surface
modification with mucoadhesive, viscosifying, and penetration enhancing agents [64–66].
Some polymer and lipid matrices themselves exhibit mucoadhesive or mucopenetrating
characteristics that enhance trans-scleral absorption and improve the delivery of the active
molecule into intraocular tissues [67,68]. In other cases, coating with cationic molecules
(CTAB and DDAB) or mucopenetrating polymers (PEG) has been useful for decorating
surfaces to enhance electrostatic interaction with the ocular mucosa and penetration of
precorneal barriers [69–72]. In addition, coating surfaces with hydrophilic polymers such as
PEG imparted “stealth” properties to delivery vehicles, reducing clearance and increasing
therapeutic efficacy [73]. The current systematic review showed that these manufacturing
strategies provided several advantages that collectively improved drug delivery to target
tissues, bioavailability, and pharmacological activity [74,75].

Future Perspectives
Eye diseases afflict millions of people worldwide and have an increasing impact on
quality of life. Given the increase in visual impairment in the world’s population, a major
challenge for researchers is directed toward the development of innovative eye delivery
systems to improve the management of these disorders [76].
Although many published and reviewed studies have demonstrated the efficacy of these
formulations, it is difficult for them to gain market availability. To date, available clinical
studies on the use of MEL for the treatment of ocular disorders are based on oral administration.
This issue may lie in the lack of robustness of the preclinical models investigated.
In preclinical testing, it is complicated to select and reproduce disease models; one of
the main difficulties consists in the complexity of the pathology. Often, ocular diseases are
multifactorial; therefore, several aspects should be captured for consideration. To improve
this aspect, the choice of preclinical endpoints should be as close as possible to that of
commonly evaluated clinical endpoints [77]. Another aspect that should be considered in
translating preclinical results to clinical is the cross-reactivity between species. Laboratory
animals used in in vivo studies are small compared with humans, and drug delivery
studies in larger animals are often prone to failure because of more complex physiological
conditions that are closer to humans. The eyes of the rodent and rabbit, which are the most
used animals for this type of preclinical experimentation, have anatomy and physiology
that are not representative and predictive of the human eye [78–80].
Considering the principle of 3Rs (reduction, replacement, and refinement) before
moving to in vivo experimentation on large primates, it might be useful to implement
advanced, elegantly designed, and more human-like ex vivo models [81]. Another recently
introduced strategy useful for supporting the 3R principle consists of in silico approaches.
Using these computational models, it has been possible to generate mathematical computer
simulations to accurately reproduce chemical–biological phenomena. The use of “qualified”
computational methods has also been recognized by regulatory agencies, which have
begun to receive and accept evidence obtained in silico for marketing the approval of new
medicinal products [82].
The combination of these shrewdnesses could reduce the deep gap between preclinical
and clinical trials to achieve successful formulations in the development of future therapies.
Int. J. Mol. Sci. 2024, 25, 3999 18 of 21

5. Conclusions
This review provided a summary of formulations for the ocular delivery of MEL.
Several ocular delivery limitations have been overcome using nano- and micrometer-sized
delivery systems, including the improved permeation of the molecule into intraocular
tissues, and sustained and controlled release profiles over time. The most strategic ap-
proaches to improve these constraints involved the fabrication of mucoadhesive and mu-
copenetrating formulations to cross ocular barriers and increase the absorption and ocular
bioavailability of MEL. Although preclinical studies have demonstrated the efficacy of
developed formulations, a huge gap has been observed with respect to the number of
formulations patented or commercially available. To reduce this gap, it might be useful to
revise the preclinical experimental phase by selecting endpoints close to clinical ones and
implementing the use of advanced ex vivo models and in silico studies to support the 3R
principle while ensuring predictive trials of efficacy in humans.

Author Contributions: Conceptualization, R.Z.; methodology, A.R.; writing—original draft prepara-

tion, A.R. and A.K.; writing—review and editing, T.M., A.K. and R.Z.; visualization, A.K.; supervision,
R.Z. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflicts of interest.

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