pharmaceutics

Article
Stability and Safety Traits of Novel Cyclosporine A
and Tacrolimus Ophthalmic Galenic Formulations
Involved in Vernal Keratoconjunctivitis Treatment by
a High-Resolution Mass Spectrometry Approach
Daniele Giovanni Ghiglioni 1 , Piera Anna Martino 2 , Gaia Bruschi 3, *, Davide Vitali 4 ,
Silvia Osnaghi 1 , Maria Grazia Corti 1 and Giangiacomo Beretta 5
1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Via Francesco Sforza,
28, 20122 Milan (MI), Italy; daniele.ghiglioni@policlinico.mi.it (D.G.G.);
silvia.osnaghi@policlinico.mi.it (S.O.); mariagrazia.corti@policlinico.mi.it (M.G.C.)
2 Department of Veterinary Medicine, Università Degli Studi di Milano, Via Celoria 10, 20133 Milan (MI), Italy;
piera.martino@unimi.it
3 Department of Clinical and Community Sciences, University of Milan, Via della Commenda,
19, 20122 Milan (MI), Italy
4 Bruttomesso Pharmacy, Galenic Laboratory, Piazza Guglielmo Marconi, 20, 26013 Crema (CR), Italy;
lab@farmaciabruttomesso.it
5 Department of Environmental Science and Policy, Università degli Studi di Milano, Via Mangiagalli 25,
20133 Milan (MI), Italy; giangiacomo.beretta@unimi.it
* Correspondence: gaia.bruschi@unimi.it




Received: 29 February 2020; Accepted: 16 April 2020; Published: 20 April 2020


Abstract: In this study, a sensitive quantitative method based on high performance liquid chromatography
combined with high-resolution mass spectrometry, Q ExactiveTM -Orbitrap® was set up and applied for
the determination of the immunosuppressor agents cyclosporine A and tacrolimus in novel ethanol-free
ophthalmic formulations for the treatment of Vernal keratoconjunctivitis. Different storage parameters in
terms of storage temperatures and practical usage conditions were investigated to assess the stability of
all formulations during shelf life simulating the real conditions as well to confirm the feasibility of use of
ethanol-free products. The methodology was linear (r2 = 0.995) over the concentration range 0–200 ng/mL,
and its selectivity, precision, accuracy and recovery were all within the required limits. Under different
conditions (storage period 0–90 days, 5–25 ◦ C, unopened/usage simulated conditions), our results
revealed that both active pharmaceutical ingredients (API) show satisfactory stability up to 30 days of
storage/usage, with a significant and consistent concentration decline of cyclosporine A after this time
point when its hydroalcoholic formulation was kept at 25 ◦ C.

Keywords: cyclosporine A; tacrolimus; ophthalmic formulations; vernal keratoconjunctivitis

1. Introduction
Cyclosporine A and tacrolimus are immunosuppressor agents with therapeutic indications for
the treatment of several pathological conditions such as ocular Behçet’s syndrome, endogenous uveitis,
psoriasis, atopic dermatitis, rheumatoid arthritis, active Crohn’s disease and nephrotic syndrome, as
well as prophylaxis and treatment of transplant rejection. Among these pathologies, Vernal (from the
Latin word for “spring”) keratoconjunctivitis (VKC) is a bilateral, asymmetric, chronic anterior surface
disease, classified among chronic allergic eye diseases predominantly of the pediatric age, with still
unknown immunopathogenesis [1,2]. Its symptoms are similar but significantly amplified comparing to

Pharmaceutics 2020, 12, 378; doi:10.3390/pharmaceutics12040378 www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2020, 12, 378 2 of 17

those of other ocular allergic forms [3]. The subjective symptomatology is characterized, with variable
intensity, by itching, the development of a thick and filamentous mucoid secretion, photophobia,
burning, lacrimation, foreign body sensation, “redness” (hyperemia, often complained of as a “subjective
symptom”), palpebral pseudo-ptosis and also sometimes pain, which can seriously affect the quality of
life of affected children. The current VKC therapy involves as first line option the application of the
same topical agents used in the treatment of other forms of allergic conjunctivitis. However, the severity
classifications of VKC present in the literature are not always unambiguous, conditioning therapeutic
schemes that differ from center to center [4–11]. Topical antihistaminic agents, mast cell stabilizers and
dual action eye drops relieve itching and inhibit the release of mast cell mediators but are effective
only in cases of mild VKC [12] or have given conflicting results in patients from different geographical
areas [13,14]. Topical nonsteroidal anti-inflammatory drugs (NSAIDs) also reduce ocular inflammatory
signs, decreasing the use of corticosteroids, without being able to replace them; sometimes, they appear
to be associated to corneal damage [13]. Topical corticosteroids are the most effective treatment for
moderate to severe VKC forms. However, their long-term use must be strictly limited and carefully
monitored due to their potential complications (glaucoma, cataract, ocular hypertension, secondary
bacterial or viral eye infections such as herpetic keratitis) [15]. In this context, to avoid the prolonged
use of cortisone drugs, galenic ophthalmic preparations containing cyclosporine A (0.5–1% up to 2%)
and tacrolimus (0.1%)-based eye drops have been developed and studied in various double-blind
placebo trials, proving to be effective in various concentrations for the treatment of moderate and
severe VKC [16,17]. Regarding high-dose cyclosporine preparations (10–20 mg/mL), they are prepared
usually as oil ointment or artificial eye drop solution-based formulations starting from injectable
cyclosporine A preparation containing ethanol as co-solvent. The presence of ethanol leads to patients’
discomfort at product instillation, especially critical in pediatric patients. In addition, commercial
ophthalmic products containing cyclosporine A at lower concentration (e.g., 0.5 mg/mL, Restasis® ),
are of dubious efficacy in the treatment of VKC [18] and they are highly expensive (e.g., cyclosporine
1.0 mg/mL, Ikervis® ). In some cases, these preparations are not registered for the treatment of VKC or
they are recognized as VKC orphan drug (e.g., cyclosporine 1.0 mg/mL, Verkazia® ) [19,20], but still not
commercially available or highly expensive. Some researchers have already investigated some quality
traits of tacrolimus and cyclosporine-based eye drop formulations during shelf life. These studies are
limited to the elucidation of the active compound stability in unopened products or to description
of their degradation products [21]. Considering the above mentioned considerations, the aim of the
present study was to evaluate the reliability of novel ethanol-free cyclosporine A and tacrolimus
galenic eye drop formulations through the assessment of their stability and microbiological safety,
in comparison to conventional hydroalcoholic 1% cyclosporine A and 0.1% tacrolimus formulations
prepared by dilution of commercial Sandimmun® and Prograf® , respectively. Stability was monitored
under both shelf life and simulated usage conditions, using a high-performance liquid chromatography
coupled to high-resolution mass spectrometry (Q ExactiveTM Orbitrap® ). The feasibility of the use of
ethanol-free cyclosporine A and tacrolimus galenic eye drop galenic preparations represent a crucial
issue in order to minimize the side effects during the administration in children populations thus
facilitating the compliance of therapy especially when chronic treatment is required.

2. Materials and Methods

2.1. Chemicals and Reagents

Methanol, ammonium formate and the internal standard Proadifen Hydrochloride (SKF-525A),
were purchased from Merck KGaA (Darmstadt, Germany). Ultra-pure water was produced using
a Milli-Q system (Millipore, Merck KGaA, Darmstadt, Germany). Tacrolimus (FarmaQuimica Sur,
Malaga, Spain), Cyclosporine A (Acef, Fiorenzuola d’Arda, Italy), Sandimmun (Novartis, Basilea,
Switzerland), Prograf (Astellas Pharma Inc., Tokyo, Japan) Lacrimart (Baif International, Genova, Italy),
Pharmaceutics 2020, 12, 378 3 of 17

polyvinylpyrrolidone (PVP; Acef, Fiorenzuola d’Arda, Italy), injectable-grade water (Fresenius Kabi,
Verona, Italy), and Cremophor RH 40 (Acef, Fiorenzuola d’Arda, Italy).

2.2. Standard Solutions

The working solutions of tacrolimus, cyclosporine A and of the internal standard for method
validation and calibration curves construction were prepared daily in methanol from corresponding
stock solutions (1 mg/mL) and stored at −20 ◦ C until use.

2.3. Cyclosporine A and Tacrolimus Ophthalmic Formulations

Different galenic formulations were investigated in this study to assess the feasibility to minimize
ethanol or lipid source used in order to prevent the children’s patient side effects at instillation phase
with better contort as final result. Several hospital pharmacies are involved in ophthalmic preparations,
based on the classical approach in which the active compounds are carried by using injectable solutions
as well as by using new promising protocols in which the eye drops are prepared as micellar by using
lipid vehicle, such as polyols castor oil as an example.
The present study design included two ophthalmic formulations based on two active substances:
high dose cyclosporine A preparations and tacrolimus comparing two different preparation protocols.

2.3.1. Cyclosporine A-based Ophthalmic Formulations (1%)

• Classical formulation (SAND): Sandimmun® (concentrated injectable solution, 50 mg/mL) was
diluted 2:8 vol/vol in commercial artificial drops Lacrimart® and homogenized under agitation to
obtain a clear lipid solution as the most common galenic protocol.
• Novel ethanol-free formulation (CSA): cyclosporine A 0.1 g, polyvinylpyrrolidone 0.2 g Cremophor
RH-40 1 g, 10 mL injectable-grade water were used as ingredients.

2.3.2. Tacrolimus-based Ophthalmic Formulations (0.1%)

• Classical formulation (PROG): Prograf® (5 mg/mL) was diluted 2:8 vol/vol in in commercial
artificial drops Lacrimart® and homogenized under agitation to obtain a clear limpid solution as
the most common galenic protocol.
• Novel ethanol-free formulation (TAC): Tacrolimus 0.01 g, polyvinylpyrrolidone 0.2 g Cremophor
RH-40 1 g, 10 mL injectable-grade water were used as ingredients.

All formulation details in terms of their composition are summarized in Table 1. The rationale
adopted for novel eye drop formulation is based on the difficulty of solubilization of organic
macromolecules such as CSA and Tacrolimus. In addition, the classical formulations involved simply
the dilution of injectable ampoules designed for other routes of administration and with an excipient
suitable for those routes (intramuscular and others). The resulting eye drops contain 100 mg of ethanol
in 10 mL of final eye drops, which can lead to a burning side effect. The first formulation step is based
on the identification of an emulsifier suitable for the ocular pathway supported by an artificial tear
with chemical characteristics that can be considered a “co-emulsifier”. In fact, polyvinyl alcohol and
Na Jaluronate are not suitable if compared to PVP, which has at its base functional ketone groups,
which is also present on the whole structure of the two active ingredients investigated.
For CSA and TAC formulation, all the constituents of the eye drops are weighed in an aseptic
chamber inside a nonsterile container Turbo emulsifier (SAMIX ES 500, Farmalabor Tech, Assago,
Milan, Italy). Once weighed, they are mixed for 5 min at 2340 rpm, the obtained emulsion is placed
under a laminar flow hood with a germicidal UV lamp for 30 min. Once the flow conditions are
activated and the aseptic environment is rendered, a 10 mL aliquot of the emulsion is taken, and filtered
by using sterile syringe equipped with a 0.22 micron Millipore filter for aqueous solutions filtration,
obtaining sterile eye drops. The final ophthalmic products obtained were clear lipid solutions. All final
ophthalmic solutions were packaged in a standard, white, opaque low-density polyethylene (LPDE)
Pharmaceutics 2020, 12, 378 4 of 17

squeezable 10-mL bottles closed with cap (ACEF, Fiorenzuola d’Arda, Italy). Containers commonly
used for ophthalmic products include glass containers and polyethylene containers. Glass containers
and polyethylene (PE) containers are recognized to be superior in maintaining stability of ophthalmic
preparations. LDPE containers were used to store all involved formulations in the present research.
The container quality represents a crucial point since the purity of a medicinal preparation may also
change during storage due to leaching of chemical or chemicals into the drug preparation from the
container materials, from the labels on the containers, or from the environment where the packaged
ophthalmic product is stored [22]. Thus, containers used for packaging medicinal preparations can
significantly affect the stability and purity of the preparations as well. All LPDE containers used
were certified for storage of ophthalmic drug regarding criteria of water loss, environmental stress
cracking resistance (ESCR). The LPDE material are certified according European Pharmacopoeia
(“Polyolefines and Polyethylene without additives for containers for preparations for parenteral use
and for ophthalmic preparations” (8th edition – 2014). They were tested by the producer in accelerated
conditions of temperature and relative humidity (40 ◦ C ± 2 ◦ C/ NMT 25% R H for plastic) during
a three-month estimation drug assay. Plastic materials were tested according the migration test to
assess the safety conditions toward compounds that can be transfer from plastic material to eyedrop.
The stability studies were finally conducted on the drug substances packaged in a container closure
system that is the same of those proposed for storage and distribution.
In order to confirm the stability of all formulations involved in the present research (Tacrolimus-
and CSA-based), they underwent High Resolution Mass Spectrometry (LC-HRMS) evaluation to detect
their concentration of active compounds at time t0 (10 mg/mL for CSA and 1 mg/mL for Tacrolimus
eyedrops). For each formulation, concentration reaching more than 99.8% of the active compound
compared with the declared one was considered as satisfactory criteria. In its final form, the ethanol-free
formulations consisted of an isotonic aqueous solution with micelles of Cremophor®solubilizing the
cyclosporine and Tacrolimus with the surfactant and polymer aiding solubility.
During all experiments, all of the multidose eyedroppers were emptied into polycarbonate test
tubes and the solutions were visually inspected under white light in front of a matte-black panel and
a non-glare white panel. Aspect and colour of the solutions were noted, and a screening for visible
particles, haziness, or gas development was performed.

Table 1. Composition of Cyclosporine A- and Tacrolimus-based galenic formulations.

Formulation Composition/Quantity Description/Function
Cyclosporine A (1%)
• Cyclosporine A 100 mg Active substance
Cyclosporine A ethanol free • Polyethoxylated castor oil - Cremophor RH 40 1000 mg Emulsifier
formulation (CSA) • Polyvinylpyrrolidone (PVP) 200 mg Polymer/Co-Emulsifier
• Ultrapure water to reach final volume
• Sandimmun® solution 50 mg/mL Active substance—
• Cyclosporine A 100 mg injectable solution
• Ethanol 556 mg Solvent
• Polyethoxylated castor oil - Cremophor 1444 mg Emulsifier
Cyclosporine A —classical galenic • Lacrimart® 8 mL: Artificial tear
(SAND) • Benzalkonium chloride Preservative
• Methyl cellulose Thickener
• NaCl e KCl Osmotic agent
• Edetic acid Chelating agent
• Ultrapure water to reach final volume
Tacrolimus (0.1%)
• Tacrolimus 10 mg Active substance
Tacrolimus ethanol free • Polyethoxylated castor oil—Cremophor RH 40 1000 mg Emulsifier
formulation (TAC) • Polyvinylpyrrolidone (PVP) 200 mg Polymer/Co-Emulsifier
• Ultrapure water to reach final volume
Pharmaceutics 2020, 12, 378 5 of 17

Table 1. Cont.
Formulation Composition/Quantity Description/Function
Cyclosporine A (1%)
• Prograf® solution 5 mg/mL Active substance—
• Tacrolimus 10 mg injectable solution
• Ethanol 1620 mg Solvent
• Olyethoxylated castor oil—Cremophor 380 mg Emulsifier
Tacrolimus— • Lacrimart® 8 mL: Artificial tear
classical galenic (PROG) • Benzalkonium chloride Preservative
• Methyl cellulose Thickener
• NaCl e KCl Osmotic agent
• Edetic acid Chelating agent
• Ultrapure water to reach final volume

2.4. Stability of Cyclosporine A and Tacrolimus Ophthalmic Formulations

Different storage conditions were tested to investigate their stability, as the literature is scarce:

- Real use of simulated conditions in opened products: all formulations underwent analysis, testing
two different temperatures (25 ◦ C as room temperature and 5 ◦ C ± 2 ◦ C as refrigerated) during
90 days of storage in which all bottles were regularly gently shaken and opened (4 s each time,
three time per day) in order to simulate the real use conditions. As partially investigated, the
role of temperature in maintaining active substances stability can play a central role for quality
of preparations.
- Shelf-life investigation on unopened products: all formulations underwent active compound
analysis during the 90-day testing. The unopened bottles were assessed to verify the decay of
cyclosporine A and tacrolimus to assess the possibility to prepare in advance the formulations
leading to several advantages for galenic pharmacies in term of stock feasibility.
- Stressed temperature conditions during delivery: all formulations underwent active compounds
analysis, simulating an unfavorable temperature condition during transport (simulating courier
time delivery), since most of the therapies are also administrated during summer period. 40 ◦ C
was selected as the stress parameter compared with the refrigerated one during 4 days by using
unopened bottles.

All bottles containing eyedroppers were stored lying down horizontally at controlled refrigerated
temperatures of (Whirlpool refrigerator) at 5 ◦ C ± 2 ◦ C (temperature measured with a Testo 175-T1
probe; Testo SARL, Forbach, France) or in a climate chamber (BINDER GmbH, Tuttlingen, Germany)
at 25 ◦ C ± 2 ◦ C and 60% residual humidity (RH), until analysis.

2.5. Sample Processing

All samples were diluted in ammonium formate 20 mM: methanol (70:30) to obtain a theoretical
final concentration of 100 ng/mL (1:10000 for tacrolimus and 1:100000 for cyclosporine preparations
respectively). Internal standard SKF-525A was added at the same concentration in the final volume of
1 mL, inserted in 2 mL glass autosampler vials and submitted to HPLC-HRMS analysis.

2.6. HPLC-HRMS Analysis

Separations were done using an HPLC system (Thermo Fisher Scientific, San Jose, CA, USA),
equipped with a Surveyor MS quaternary pump and a degasser, a Surveyor AS autosampler with
column oven, and a Rheodyne valve with a 20-µL loop. A Synergi Hydro-RP reverse-phase HPLC
column (150 × 2.0 mm, i.d. 4 µm), with a C18 guard column (4 × 3.0 mm; Phenomenex, Torrance,
CA, USA) were used. The mobile phase consisted of a binary mixture of solvents A (20 mM aqueous
ammonium formate) and B (MeOH). The elution started with 30% B, which increased to 95% in 5 min
and kept in these conditions until 10 min. The initial conditions were reached in the 11th min, with an
equilibration time of 7 min. The run was performed at 0.3 mL/min.
Pharmaceutics 2020, 12, 378 6 of 17

The detector was a Thermo Q-Exactive Orbitrap™ (Thermo Scientific, San Jose, CA, USA),
equipped with a heated electrospray ionisation (HESI) source. Capillary and vaporiser temperatures
were set at 330 ◦ C and 280 ◦ C, respectively, while the electrospray voltage was set at 3.50 kV, operating
in positive mode. The sheath and auxiliary gas were set at 35 and 15 arbitrary units (AU). Xcalibur 3.0
software (Thermo Fisher Scientific, San Jose, CA, USA) was used to control the HPLC-HRMS system.
The full scan (FS) acquisition was combined with a data-independent acquisition (DIA) strategy,
providing the MS2 spectra for a confirmatory response, based on the inclusion list. The FS resolution
was 70,000 FWHM, the selected scan range was 200–1250 m/z; the automatic gain control (AGC) was
set at 1 ×106, and the maximum injection time was 300 ms. The DIA segment operated in positive
mode at 35,000 FWHM. The AGC target was set to 2 ×104, with the maximum injection time of 100 ms.
The isolation window was of 1 m/z. Fragmentation of the precursors was obtained with two-step
normalized collision energy (10 and 15eV). Detection of the analytes was based on the retention time
(RT) of the target compounds, the calculated exact mass of the protonated molecular ions, and at least
one specific and typical fragment. Acquisition data were recorded and elaborated using Xcalibur™
software (Thermo Fisher). All determinations are conducted in triplicate.

2.7. Method Validation

Validation was performed according to European Compliance Academy (ECA) and European
Medicines Agency (EMA) guidelines [23] and in agreement with international guidelines for analytical
techniques for the quality control of pharmaceuticals [24]. Specificity and selectivity, linearity, limit of
quantification (LOQ), precision and accuracy were evaluated.

2.8. Microbiological Assay

Forty-eight ophthalmic solutions were cultured for bacteria; in particular, the solutions were tested
at the opening and after 30 days using regularly opened bottles; moreover, the packs after opening
were stored at 4 ◦ C and at room temperature and were tested at both conditions. For each solution,
20 drops were evaluated (500 µL) and placed in 4.5 mL of Brain Heart Infusion Broth (Microbiol,
Cagliari, Italy) and incubated at 35 ± 2 ◦ C for 48 h under aerobic conditions. Each test was performed
in duplicate. After the incubation time, 100 µL of the incubated broth for each sample was streaked
onto blood-agar plates (Microbiol, Cagliari, Italy) and incubated as described above.

2.9. Data Analysis

Stability for all investigated formulations was defined as the time at which 90% of the initial
concentration of Tacrolimus and Cyclosporine A active compounds remained (t90); the initial
concentration (time point 0) was considered to be 100%. Active compounds concentration was
finally expressed as the percentage of the initial active compound concentration remaining at each
sampling time. Statistical evaluation of the data was conducted by using SPSS software, Version 24;
Chicago, IL: SPSS Inc; 2002. Analysis of variance (ANOVA) was applied to the data and significant
differences were determined by one-way ANOVA and Student–Newman–Keuls (SNK) as a post-hoc
test to evaluate differences among different formulations and storage times. An effect was considered
significant at the 5% level (p < 0.05).

3. Results

3.1. Tacrolimus and Cyclosporine A Quantification in Different Galenic Preparations

The formula of the investigated active compounds cyclosporine and tacrolimus, with the exact
theoretical mass of the parents and the diagnostic transition, used to confirm them were reported in
Table 2. The extracted parent ion chromatograms, acquired from Full Scan (FS) analysis, the peak of the
main fragment and the mass spectra are also shown in Figure 1A. Bearing in mind that there is limited
information regarding the structure of main fragments obtained by high-resolution mass spectrometry,
Pharmaceutics 2020, 12, 378 7 of 17

we have proposed the fragmentation pattern for each of compounds enrolled in this study. The mild
two step collision energy (10 and 15 eV) turns to be fundamental for reproducible and characteristic
bond cleavage and molecular rearrangements (Figure 1B).
The chromatographic method used is linear for concentrations ranging from 12 to 28 µg/mL.
Concerning the validation results, the mean linear regression equation obtained was y = 67.196x + 10.069
where x is the cyclosporine A concentration and y the surface area of the corresponding peak.
The corresponding correlation
Pharmaceutics 2020, 12, 378 coefficient was 0.997. The accuracy, expressed as percentage7 of mean
of 17
calculated to nominal concentration ranged from 93% to 105% for tacrolimus and from 90% to 107% for
The corresponding
cyclosporine correlation
A. The inter-day coefficient
precision waswas
8%0.997.
and 9%Thefor
accuracy, expressed
cyclosporine andastacrolimus,
percentagerespectively.
of mean
calculated to nominal concentration ranged from 93% to 105% for tacrolimus and from 90% to 107%
for cyclosporine
Table 2. CompoundA.formula,
The inter-day precision
parent exact was fragment
mass, main 8% and mass
9% for
andcyclosporine
polarity of the and tacrolimus,
two compounds
respectively.
under study.

Parent
Table 2. Compound formula, parent exact Exact
mass, Mass
main Main
fragment Fragment
mass Massof theIonization
and polarity two
Compound Formula
compounds under study. (m/z) (m/z) Polarity
Tacrolimus C44 H69 NO12 821.51580
Parent *
exact 768.46922
Main fragment (+)
Ionization
Compound Formula
Cyclosporine A C62 H111 N11 O12 mass (m/z)*
1219.87519 mass (m/z)
1202.85098 Polarity(+)
Tacrolimus
Proadifen C 44H69NO12 821.51580 * 768.46922 (+)
Cyclosporine A CC2362HH31 NO
111N112O12
354.24276 *
1219.87519 209.13289
1202.85098 (+) (+)
(SFK-525A)
Proadifen * The parent exact masses were calculated as ammonium adducts [M+NH ]+ .
C23H31NO2 354.24276 209.13289 4 (+)
(SFK-525A)
* The parent exact masses were calculated as ammonium adducts [M+NH4]+.

(A)

Figure 1. Cont.
Pharmaceutics 2020, 12, 378 8 of 17
Pharmaceutics 2020, 12, 378 8 of 17

(B)

Figure 1. (A) Ion chromatograms extracted from full scan (FS), chromatograms of main fragment
obtained by mass spectra MS2 acquisition and mass spectra of proadifen (SKF-525A), tacrolimus and
cyclosporine A (100 ng/mL). (B) q-Exactive Orbitrap high-resolution mass spectrum and proposed
fragmentation pathway of SKF-525A, tacrolimus and cyclosporine A, acquired in Data Independent
Acquisition (DIA) mode with a two-step normalized collision energy (10 and 15 eV).
Pharmaceutics 2020, 12, 378 9 of 17

Figure 1. (A) Ion chromatograms extracted from full scan (FS), chromatograms of main fragment
obtained by mass spectra MS2 acquisition and mass spectra of proadifen (SKF-525A), tacrolimus and
cyclosporine A (100 ng/mL). (B) q-Exactive Orbitrap high-resolution mass spectrum and proposed
Pharmaceutics 2020, 12, pathway
fragmentation 378 of SKF-525A, tacrolimus and cyclosporine A, acquired in Data Independent9 of 17
Acquisition (DIA) mode with a two-step normalized collision energy (10 and 15 eV).

3.2. Stability of Tacrolimus and Cyclosporine A in Eye Drop Formulations under Usage-simulated Conditions
3.2. Stability of Tacrolimus and Cyclosporine A in Eye Drop Formulations under Usage-simulated Conditions
The 90-day stability trend for all formulations was presented in order to evaluate the active
The 90-day stability trend for all formulations was presented in order to evaluate the active
substances decay, with particular attention to cyclosporine A (CSA) and tacrolimus (TAC) ethanol-free
substances decay, with particular attention to cyclosporine A (CSA) and tacrolimus (TAC)
eye drops. All observations were conducted in simulated usage conditions in which the samples
ethanol-free eye drops. All observations were conducted in simulated usage conditions in which the
were opened at time 0 and monitored during prolonged storage of 90 days as described in material
samples were opened at time 0 and monitored during prolonged storage of 90 days as described in
and method section. The relative stability of tacrolimus in both Prograf® and TAC formulations was
material and method section. The relative stability of tacrolimus in both ◦
Prograf® and TAC
evaluated under simulated usage conditions, at the storage temperatures of 25 C (room temperature)
formulations was evaluated under simulated usage conditions, at the storage temperatures of 25 °C
and 5 ◦ C (refrigeration).
(room temperature) and 5 °C (refrigeration).
In all cases, at the end of storage period, the concentration decay was within the 10–20% range
In all cases, at the end of storage period, the concentration decay was within the 10–20% range
(Figure 2A), in accordance with the results reported by Ezquer-Garin et al. [25], that described a suitable
(Figure 2A), in accordance with the results reported by Ezquer-Garin et al. [25], that described a
stability of 0.3% tacrolimus in an eye drop formulation stored at 5 ◦ C, but with a significant decline
suitable stability of 0.3% tacrolimus in an eye drop formulation stored at 5 °C, but with a significant
in its concentration (<90%) after 28 days of storage at 25 ◦ C (p < 0.05) for all formulations involved
decline in its concentration (<90%) after 28 days of storage at 25 °C (p < 0.05) for all formulations
in the present study. By contrast, a higher significant degradation rate was observed considering all
involved in the present study. By contrast, a higher significant degradation rate was observed
storage times for cyclosporine A in the Sandimmun formulation already at the first time point tested
considering all storage times for cyclosporine A in the Sandimmun formulation already at the first
(40% degradation, p < 0.05), reaching 80% after 30 days of storage at room temperature (Figure 2B,
time point tested (40% degradation, p < 0.05), reaching 80% after 30 days of storage at room
p < 0.01).
temperature (Figure 2B, p < 0.01).
Conversely, the degradation time-course of cyclosporine A in Sandimmun stored at 5 ◦ C was
Conversely, the degradation time-course of cyclosporine A in Sandimmun stored at 5 °C was
similar to that of cyclosporine A in CSA formulation, regardless of the storage temperature without
similar to that of cyclosporine A in CSA formulation, regardless of the storage temperature without
significant differences among different eye drops.
significant differences among different eye drops.
These results were
These results were in
in good
good accordance
accordance with
with those
those reported
reported by
by Chennel
Chennel et et al.
al. [22],
[22], that
that evaluated
evaluated
the stability of cyclosporine A in an eye drop formulation even if limited at
the stability of cyclosporine A in an eye drop formulation even if limited at 30 days under usage30 days under usage
conditions, observing no significant degradative effect. However, in this last study
conditions, observing no significant degradative effect. However, in this last study the evaluation of the evaluation of
active substances
active substances behavior
behavior was
was not
not extended
extended to to prolonged
prolonged storage
storage period
period and
and to to simulated usage
simulated usage
conditions as done in the present study, that is important since the frequent instillations
conditions as done in the present study, that is important since the frequent instillations required by required by
the therapy
the therapy regimen,
regimen, often
often involve
involve thethe bottle
bottle opening
opening andand closing
closing more
more than
than 33 time
time per
per day
day that
that can
can
lead to cyclosporine A and tacrolimus degradation trend more consistent especially
lead to cyclosporine A and tacrolimus degradation trend more consistent especially after 30 days. after 30 days.
Regarding visual
Regarding visual inspection,
inspection, all
all samples
samples stayed
stayed limpid
limpid and
and with
with aa slight
slight yellow
yellow tinge
tinge throughout
throughout the
the
study, for
study, for the
thetested
testedformulations
formulationsand andconservation
conservation temperatures,
temperatures,andand there waswas
there no appearance
no appearanceof any
of
visible particulate matter, haziness or gas development.
any visible particulate matter, haziness or gas development.

100
90 * * * *
80 * *
* *
Tacrolimus % remaining

70
60
PROG 5˚C
50
PROG T.amb
40
TAC 5˚C
30
TAC T.amb
20
10
0
T0 T14 T21 T28 T35 T45 T60
storage days

(A)
Figure 2. Cont.
Pharmaceutics
Pharmaceutics 2020,
2020, 12,
12, 378
378 10
10of
of17
17
Pharmaceutics 2020, 12, 378 10 of 17
100
90
100

A % remaining
80
90
A % remaining 70
80 *
60
70
* * SAND 5˚C
50
60
* SAND
SANDT.amb
Cyclosporine

* 5˚C
40
50 *
* CSA
SAND5˚CT.amb
Cyclosporine

30
40 * * * CSA
CSAT.amb
5˚C
20 *
30
* CSA T.amb
10
20
0
10
T0 T14 T21 T28 T35 T45 T60
0
storage days
T0 T14 T21 T28 T35 T45 T60
storage days
(B)
(B)
Figure 2. (A) Active principle stability in 0.1% tacrolimus (A) eye drops formulations stored at 5 °C
and 25 °C (T.amb) under user simulated conditions (see Experimental
Figure 2. (A) Active principle stability in 0.1% tacrolimus (A) eye drops forformulations
details); * p <stored
0.05, at
T.amb
5 ◦°C
C
versus 5◦
and 25 °C °C storage temperature. (B) Active principle stability in 1.0% cyclosporine
C (T.amb) under user simulated conditions (see Experimental for details); * p < A (B) eye drops
< 0.05, T.amb
formulations
versus 5 ◦°C stored at
C storage 5 °C and 25 (B)
temperature. °C (T.amb)
(B) under user
Active principle
Active principle simulated
stability
stability conditions
in 1.0%
in (see Experimental
cyclosporine
cyclosporine A (B) eye dropsfor
details); *p < 0.05,
formulations
formulations T.amb
stored
stored at 55 versus
at ◦
°C and525
C and °C°C
25 ◦ storage
C (T.amb)
(T.amb)temperature.
under user
under user simulated
simulated conditions
conditions (see
(see Experimental
Experimental for for
details); **pp<<0.05,
details); 0.05,T.amb
T.ambversus ◦ C storage temperature.
versus55°C storage temperature.
3.3. Shelf-life of Tacrolimus and Cyclosporine A Formulations in Unopened Products
3.3. Shelf-life of Tacrolimus and Cyclosporine A Formulations in Unopened Products
3.3. Shelf-life
The 60 daysof Tacrolimus
stabilityand of Cyclosporine A Formulations
unopened formulations in Unopened
kept Products
at refrigerated storage conditions was
The 60 to
determined days stability
confirm the of unopened
suitability of formulations
eye drops to kept
be at refrigerated
stored for an storage
extend timeconditions
period. Thewas
The 60 days stability of unopened formulations kept at refrigerated storage conditions was
determined 5to°Cconfirm
temperature was the suitability
selected as it of eye
represents the drops
best to becondition
storage stored for to an extend
ensure both time period.
cyclosporine
determined to confirm the suitability of eye drops to be stored for an extend
◦ C was selected as it represents the best storage condition to ensure both cyclosporine
time period. The
AThe
andtemperature
tacrolimus 5 stability. The shelf-life of all four formulations was similar when vials were first
temperature 5 °C was selected as it represents the best storage condition to ensure both cyclosporine
A and tacrolimus
opened. stability.
The tacrolimus andThe shelf-life of
cyclosporine A all four formulations
concentration variationwas similar
was when
<20%when vials were
in respect to thefirst
T0
A and tacrolimus stability. The shelf-life of all four formulations was similar vials were first
opened. The
concentration tacrolimus
(100%) even and cyclosporine
at the A
longest storage concentration variation
time periodvariation
tested of 60was <20%
days in
(Figure respect to the
3). Thetohighest T0
opened. The tacrolimus and cyclosporine A concentration was <20% in respect the T0
concentration
and (100%)
significant(100%) even
degradation at the longest storage time period tested of 60 days (Figure 3). The highest
concentration even atrate was observed
the longest storage in CSA
time considering
period tested ofits
60 storage times,3).ifThe
days (Figure compared
highest
and significant
with other degradation
formulations (p rate
< was but
0.05), observed in CSAbelow
remaining considering
12% its storage
active compoundtimes, if compared
loss. This with
could
and significant degradation rate was observed in CSA considering its storage times, if compared
other formulations
represent a great (p <
advantage0.05), but remaining below 12% active compound loss. This could represent toa
with other formulations (p especially
< 0.05), but forremaining
hospital galenic
below pharmacies
12% active in terms of the
compound possibility
loss. This could
great
set upadvantage
product especially
stocks, savingforworking
hospital galenic pharmaciesproducts
in terms timely
of the possibility to set up product
represent a great advantage especiallytime to guarantee
for hospital galenic pharmacies inavailability.
terms of the possibility to
stocks, saving working time to guarantee products timely availability.
set up product stocks, saving working time to guarantee products timely availability.
20
18
20
16
18
14
16
% degradation

12
14 * SAND
% degradation

10
12 * CSA
SAND
8
10
68 PROG
CSA
46 TAC
PROG
24 TAC
02
T 0 days T 30 days T 60 days
0
T 0 days storage days
T 30 days T 60 days
Figure 3. Active principle stability during storage
60 days days
in 0.1% tacrolimus and 1.0% cyclosporine A unopened
eye drop formulations stored at 5 ◦ C; * p < 0.05, T.amb versus 5 ◦ C storage temperature.
Figure 3. Active principle stability during 60 days in 0.1% tacrolimus and 1.0% cyclosporine A
unopened
Figure 3. eye dropprinciple
Active formulations stored
stability at 5 °C;
during 60 *days
p < 0.05, T.amb
in 0.1% versus 5 °C
tacrolimus storage
and 1.0% temperature.
cyclosporine A
unopened eye drop formulations stored at 5 °C; * p < 0.05, T.amb versus 5 °C storage temperature.
Pharmaceutics 2020, 12,
Pharmaceutics 2020, 12, 378
378 11 of
11 of 17
17

The active compounds decay in all formulation was also investigated after 30 and 60 days of
The active compounds decay in all formulation was also investigated after 30 and 60 days of
unopened storage to assess a variation of Cyclosporine A and tacrolimus testing the real use
unopened storage to assess a variation of Cyclosporine A and tacrolimus testing the real use conditions
conditions over 30 days by opening and shacking the solutions every day. All bottles once opened
over 30 days by opening and shacking the solutions every day. All bottles once opened were subjected
were subjected to two storage conditions, at room and refrigerated temperature, monitoring the
to two storage conditions, at room and refrigerated temperature, monitoring the decay at 0, 7, 15 and
decay at 0, 7, 15 and 30 days shelf-life for formulation after 30 days unopened and at 0, 15 and 30
30 days shelf-life for formulation after 30 days unopened and at 0, 15 and 30 days for formulations
days for formulations after 60 days unopened. This is crucial to assess and confirm the feasibility to
after 60 days unopened. This is crucial to assess and confirm the feasibility to set up stock formulations
set up stock formulations saving consistent losses of active compounds. All results are reported in
saving consistent losses of active compounds. All results are reported in Figures 4 and 5.
Figures 4 and 5.

100
90
cyclosporine A % remaining

80
70 *
* *
60
SAND 5˚C
50
* SAND T,amb
40
30 CSA 5˚C
20 CSA T.amb
10
0
T0 T7 T 15 T 30
storage days

(A)
100
90
Tacrolimus % remaining

80
*
70
60
PROG 5˚C
50
PROG T.amb
40
30 TAC 5˚C
20 TAC T.amb
10
0
T0 T7 T 15 T 30
storage days

(B)
Figure 4. (A) 1.0% cyclosporine A formulations stability for 30 days shelf-life after 30 days unopened
Figure p < 1.0%
storage;4.* (A) 0.05, cyclosporine ◦ C storage temperature. (B) 0.1% tacrolimus formulations stability
T.amb versusA5 formulations stability for 30 days shelf-life after 30 days unopened
for 30 days shelf-life after 30 days unopened storage; * p < 0.05, T.amb versus 5 ◦ C storage temperature.
storage; * p < 0.05, T.amb versus 5 °C storage temperature. (B) 0.1% tacrolimus formulations stability
for 30 days shelf-life after 30 days unopened storage; * p < 0.05, T.amb versus 5 °C storage
temperature.
Pharmaceutics 2020, 12, 378 12 of 17
Pharmaceutics 2020, 12, 378 12 of 17

100

Cyclosporine A % remaining
90
*
80
70 *
60
* SAND 5˚C
50
SAND T.amb
40
30 CSA 5˚C
20 CSA T.amb
10
0
T0 T7 T 30
storage days

(A)
100
90
80 * *
Tacrolimus % remaining

70
60
PROG 5˚C
50
PROG-T.amb
40
TAC 5˚C
30
TAC-T.amb
20
10
0
T0 T7 T 30
storage days

(B)
Figure 5. (A) 1.0% cyclosporine A formulations stability for 30 days shelf-life after 60 days unopened
Figure p < 1.0%
storage;5.* (A) 0.05, cyclosporine ◦ C storage temperature. (B) 0.1% tacrolimus formulations stability
T.amb versusA5 formulations stability for 30 days shelf-life after 60 days unopened
for 30 days
storage; * p shelf-life after 60
< 0.05, T.amb days5unopened
versus °C storagestorage; * p < 0.05,
temperature. (B) T.amb versus 5 ◦ Cformulations
0.1% tacrolimus storage temperature.
stability
for 30 days shelf-life after 60 days unopened storage; * p < 0.05, T.amb versus 5 °C storage
Considering all storage times tested, significant losses of cyclosporine A in Sandimmun® (SAND)
temperature.
and cyclosporine A (CSA) samples were measured in vials opened both after 30 days and after 60 day
of unopened storage (p < 0.01), in formulations stored at room temperature (25 ◦ C, −60% as maximum®
Considering all storage times tested, significant losses of cyclosporine A in Sandimmun
(decay trend) confirming the role of temperature to preserve active compounds losses.
SAND) and cyclosporine A (CSA) samples were measured in vials opened both after 30 days and
Concerning Tacrolimus formulations is was been observed how all storage conditions had minor
after 60 day of unopened storage (p < 0.01), in formulations stored at room temperature (25 °C, −60%
influence even if refrigerated storage were able to guarantee the minimum decay trend both after 30
as maximum decay trend) confirming the role of temperature to preserve active compounds losses.
and 60 days of unopened conservation at 4 ◦ C there was a loss of activity of 15–20%, respectively),
Concerning Tacrolimus formulations is was been observed how all storage conditions had
confirmed by a nonsignificant variations. As final consideration, the present experiment confirms the
minor influence even if refrigerated storage were able to guarantee the minimum decay trend both
possibility to set up stock of cyclosporine A and Tacrolimus eyes drops, considering their use after
after 30 and 60 days of unopened conservation at 4 °C there was a loss of activity of 15–20%,
30 days of unopened storage assuring acceptable active compounds losses less than 20% compared to
respectively), confirmed by a nonsignificant variations. As final consideration, the present
their initial concentration.
experiment confirms the possibility to set up stock of cyclosporine A and Tacrolimus eyes drops,
considering their use after 30 days of unopened storage assuring acceptable active compounds losses
less than 20% compared to their initial concentration.

3.4. Effect of Delivery-simulated Conditions on Tacrolimus and Cyclosporine A

Pharmaceutics 2020, 12, 378 13 of 17

3.4. Effect of2020,

Pharmaceutics Delivery-simulated
12, 378 Conditions on Tacrolimus and Cyclosporine A 13 of 17

With the
With theaim
aimtotoestablish any
establish anydetrimental
detrimental effect of extreme
effect storage
of extreme conditions
storage that can
conditions thattake
canaffect
take
APIs concentration under non-refrigerated
affect APIs concentration under non-refrigerated expedition of
expeditionthe medicinal
of the products
medicinal to
productsthetopatient,
the their
patient,
stability after 24
their stability h and
after 24 72 h time-points
h and at 40 ◦ C at
72 h time-points was 40monitored. This is important
°C was monitored. since the delivery
This is important of
since the
products may be done in summer periods by courier transportation without refrigeration
delivery of products may be done in summer periods by courier transportation without refrigeration delivery
as well. The
delivery overallThe
as well. trend observed
overall trendforobserved
tacrolimus forand cyclosporine
tacrolimus and Acyclosporine
preparations,Arespectively,
preparations, is
presented in Figure
respectively, 6.
is presented in Figure 6.

100
90
80
*
Cyclosporine A % remaining

*
70
60
SAND 5 °C
50
SAND 40 °C
40
CSA 5 °C
30
CSA 40 °C
20
10
0
T 0 hours T 48 hours T 72 hours
storage days

(A)
100
90
80 *
Tacrolimus % remaining

70 *
60
PROG 5 °C
50
* PROG 40 °C
40
TAC 5 °C
30
TAC 40 °C
20
10
0
T 0 hours T 48 hours T 72 hours

storage days

(B)
Figure 6. (A) Effect of delivery simulated conditions on 1.0% cyclosporine A stability in commercial
and experimental eye drops formulations; * p < 0.05, T.amb versus 5 ◦ C storage temperature. (B) Effect
Figure 6. (A) Effect of delivery simulated conditions on 1.0% cyclosporine A stability in commercial
of delivery simulated conditions on 0.1% tacrolimus stability in commercial and experimental eye
and experimental eye drops formulations; * p◦ < 0.05, T.amb versus 5 °C storage temperature. (B)
drops formulations; * p < 0.05, T.amb versus 5 C storage temperature.
Effect of delivery simulated conditions on 0.1% tacrolimus stability in commercial and experimental
eye
Thedrops formulations;
results confirm the * pnegative
< 0.05, T.amb versus
impact 5 °C storage
of high temperature.
temperatures also during the 72 h of simulated
delivery, with significant decay (p < 0.01) observed after 48 h only for products stored at 40 ◦ C,
The results confirm the negative impact of high temperatures also during the 72 h of simulated
confirming the necessity of refrigerated storage conditions also during transportation as best practice
delivery, with significant decay (p < 0.01) observed after 48 h only for products stored at 40 °C,
to keep the active compounds of formulations stable as well.
confirming the necessity of refrigerated storage conditions also during transportation as best
practice to keep the active compounds of formulations stable as well.

3.5. Microbiological Stability

Pharmaceutics 2020, 12, 378 14 of 17

3.5. Microbiological Stability

All samples showed no bacterial charge (sterile), for all formulations tested during 30 storage
days. The results showed that no bacterial contamination was evident in all opened ophthalmic
solutions maintained both at 4 ◦ C and at environmental temperature, also assessing the feasibility
of the use of new ethanol-free formulations as well. The microbiological safety in terms of sterility
represents a critical element since it represents a mandatory requirement for possible application as
ocular preparation.
As previously reported in literature, the risk of eye drops contamination is related to reuse of these
therapeutic presidium and to its duration of use [26–28]. As such, it should be pointed out that further
investigations would be needed to evaluate whether multi-dose eye medication could be contaminated
by bacteria attributable to a potential incorrect user handling.

4. Discussion
In the present study, the stability of the immunosuppressor agents cyclosporine A and tacrolimus
in ophthalmic formulations with and without ethanol have been evaluated using a highly selective
and sensitive methodology based on high-resolution mass spectrometry.
Previous recent studies on the stability of cyclosporine A (10–20 mg/mL) in an ethanol-depleted
micellar formulation stored at 25 ◦ C and 5 ◦ C for one month with monitoring by High Performance
Liquid Chromatography (HPLC-UV), found a satisfactory stability with no significant difference in the
concentration of this API comparing to its starting concentration at the end of the storage period [23].
However, the authors reported that temperatures above 25 ◦ C may induce potential instability at
higher temperatures.
In another recent study, no significant decrease of tacrolimus in eye drop formulation (0.3%) was
found after 85 days of storage under refrigerated conditions (5–25 ◦ C). Conversely, when tacrolimus
solution was stored at 25 ◦ C, its concentration decreased below 90% of the starting concentration
already after 28 days [25]. At present, scarce information on the stability of these pharmaceutical drugs
in ethanol-free formulations, as investigated in the present work, is available. The results of the major
efforts for the definition and confirmation of cyclosporine A and tacrolimus as effective drugs for VKC
treatment (efficacy and side effects) are available from clinical studies and trials as summarized in
Table S1 (Supplementary Materials).
VKC is widespread in areas with a warm and temperate climate such as the Mediterranean basin,
north and west of Africa, the Middle East, the Anatolian peninsula, the Arabian Peninsula, parts of
India, Pakistan, Japan, and Central and South America.
Indicated among the rare diseases in the “Orphanet Journals” of January 2019 with a prevalence of
32/100000, VKC could actually have a greater prevalence also in Europe, as hypothesized for a long time;
in East Africa, the VKC affects more than 5% of children of school age [29], and in some populations
of Africa (Ethiopia) a prevalence of up to 5.2–7.3% has recently been estimated. With Orphanet’s
European estimates in the northern Italian region of Lombardy, there would be over 3000 people
affected by VKC, almost 20,000 in Italy and 240,000 in Europe. VKC mainly affects individuals of
preschool age (3–5 years) up to the end of the second decade of life, with a peak incidence between 11
and 20 years [30]. It mainly affects the male sex, with a male–female ratio ranging from 3 to 1 to 7 to 1.
A family history of atopy is present in 40% of subjects [31].
Chronic conjunctival inflammation and the release of inflammatory and keratolytic substances
determine an increased fragility and sensitivity of surface ocular tissues, predicting corneal outcomes,
such as superficial, localized or diffuse punctate keratitis, which over time can turn into an ulcer
“a shield” (mainly produced by a toxic immune-allergic mechanism) or in corneal abrasions and in
superficial proliferation of newly formed vessels [32]. “Shield” ulcers affect about 10% of patients,
appear especially in periods of greater intensity of symptoms and are considered to be feared
complications [32]. In fact, they may result in elevated astigmatism, keratoconus or more rarely in
corneal perforation. There is a variable percentage of patients from 8% to 15% that is substantially
Pharmaceutics 2020, 12, 378 15 of 17

resistant to treatment with cyclosporine A, at least for the central 3–4 months of the spring–summer
period [33]. In the most serious cases, resistant to cyclosporine A, studies on the efficacy and safety
of a local treatment with tacrolimus are in progress, prepared in a similar way to those used for
cyclosporine A, which so far have given favorable results, so much so that tacrolimus has been declared,
like cyclosporine A, an orphan drug for VKC by the EMEA [23,33].
The risks inherent to the use of galenic preparations are known and linked to the absence of a
standardization in the preparation, to which is also added the before mentioned symptomatology.
In recent years, cyclosporin dissolved in polyoxyl 40 stearate (MYS-40) has been registered for the
treatment of allergic conjunctivitis not responsive to common therapies is on the market in Japan: it
uses a 0.1% cyclosporine A concentration [34]. Due to its aqueous composition, the distribution of the
drug would seem to be about 3 times better than the emulsion in oil. This 0.1% formulation has been
tested in Japanese children with VKC and atopic keratoconjunctivitis AKC, and patients with VKC
have been shown to respond within one month of starting the administration and, after 6 months of
therapy, they have only minimal symptoms. Cyclosporine-based drugs are available on the market at
lower concentrations (0.05%) that have been found to have contrasting efficacy in VKC control [8,35].
In this context, the results recently reported by Leonardi et al. (2019) [19] and by Bremond Gignac et al.
(2019) [20] would confirm the efficacy of 0.1% cyclosporin A, due to the solution in which they are
emulsified for severe VKC treatment both in children and adolescents.
In the present research, we tested galenic preparations at a higher concentration that has proven
to be certainly effective so far (1%) [6]. Then it would be desirable to find effective preparations
characterized by a better solubility and distribution on the ocular surface level, in order to reduce
possible side effects. It also crucial to develop galenic preparations that do not cause intense local
burning (such as the galenics used so far) with lower costs than the eye drops currently available on
market to obtain patient contort and cost saving as main results.

5. Conclusions
Our study shows how the preparation of cyclosporine A powder in galenic, ethanol-free, eye drop
(ingredients) at the concentration of 1% w/vol (CSA) and tacrolimus in ethanol-free, galenic eyedrop
(0.1% w/vol, TAC) show satisfactory stability profiles; are stable, sterile and potentially as effective
as traditional formulations, and also ensure good tolerability by the patient by not containing the
alcohol commonly used to ensure the stability of the compounds. In addition, we also underline how
the galenic preparations are less expensive if compared with available commercial drugs, resulting in
pharmacies saving costs, an especially important issue when they are inserted in a hospital context.
However, it is necessary to pass to the clinical trial on patients to assess both the real efficacy of the
preparations as well as the side effect reductions reported by a clinical trial.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/12/4/378/s1,

Table S1: Research studies collection on Cyclosporine A and Tacrolimus-based ophthalmic formulations for Vernal
keratoconjunctivitis treatments.
Author Contributions: Conceptualization, D.G.G. and G.B. (Giangiacomo Beretta); methodology, G.B.
(Giangiacomo Beretta); software, G.B. (Gaia Bruschi); validation, D.G.G., G.B. (Giangiacomo Beretta) and
P.A.M.; formal analysis, investigation, eyed drops formulation D.V.; resources, M.G.C.; data curation, S.O.;
writing—original draft preparation, G.B. (Gaia Bruschi); writing—review and editing, P.A.M.; visualization, S.O.;
supervision, D.G.G.; project administration, D.V.; funding acquisition, D.G.G. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the official patient non-profit associations “OcchioallaVernal O.N.L.U.S.”,
for the project entitled: Determination of Active Ingredients in Galenic Formulations.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Pharmaceutics 2020, 12, 378 16 of 17

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