2. MATERIALS AND METHODS
2.1. Materials
Rapamycin loaded liposomes (RL) and placebo liposomes (L) were provided
by Laboratorio Santgar (Mexico City, MX), and produced using a
proprietary methodology (23-24). Test formulations were kept under
refrigeration (2ºC-8ºC) at the experimental facility until use.
For the micronucleus test, male Hsd:ICR Specific Pathogen Free (SPF)
certified mice were purchased from UNAM-ENVIGO Center of Laboratory
Animal Production (Coyoacan, MX). Light microscopy DM2500 (Leica
Biosystems, Nussloch, DE) was used for micronucleus determinations as
well as slide observation in the subacute toxicity study.
For the HET-CAM assay, 9 day-old SPF hen’s embryos were purchased from
ALPES, S.A. de C.V. (Puebla, MX). Sodium dodecyl sulfate (SDS) from
Sigma-Aldrich (Saint Louis, USA) was used as HET-CAM positive control.
Physiologic saline solution (PhS) purchased from PISA, S.A. de C.V.
(Guadalajara, MX) was used as a negative control in this study as well
as in the Intravitreal acute retinal toxicity study (IARTS) and the
subacute toxicity study (STS).
For pyrogenicity assay and STS, healthy certified male New Zealand
rabbits were purchased from Science Animals (Mexico City, MX).
Independently, 3 animals were purchased for pyrogenicity assay and 12
animals for STS. Rectal temperature measurements were done using a Sejoy
MT 401 (Hangzhou Sejoy, CN) calibrated rectal thermometer for
pyrogenicity assessment.
SCJ injections in STS as well as intravitreal injections in IARTS were
performed using new, 30G needles, attached to 1ml sterile disposable
syringes (Becton Dickinson and Co., New Jersey, USA). Proparacaine HCl
0.5% veterinary ophthalmic solution (Paracaina®) provided by
Laboratorio Santgar (Mexico City, MX) was used as local anesthetic in
STS and IARTS. For STS, blood samples were collected using both empty
Microtainer® and Microtainer® containing EDTA K2. (Becton Dickinson and
Co., New Jersey, USA). Hematologic samples were processed in a BCVet
analyzer (KONTROLAB International Corp., Rome, IT). Hematological slides
were stained using a semiautomatic system Hematek® (Siemens Healthcare
GmbH, Munich, DE). Biochemical analysis was performed by the automated
chemistry analyzer CST-240 (DIRUI Industrial Co., Ltd., Changchun, CN).
For IARTS, 15 healthy certified New Zealand rabbits were purchased from
Soluciones MG (Mexico City, MX). Fundus photographs were recorded using
a Kowa Genesis-D Handheld Retinal Camera (Kowa American Corporation,
Torrance, USA), Cyclopentolate 1%, Phenilephrine HCl 2.5% and
Tropicamide 1% veterinary ophthalmic solution (Midriavet®) provided by
Laboratorio Santgar (Mexico City, MX) was used as local mydriatic.
Electroretinograms were recorded with a BMP 200 Retinographics
electrodiagnostic system (Dioptrix, Touluse, FR). For IVT injections,
animals were anesthetized with isoflurane Sofloran Vet® (PiSA
Agropecuaria, S.A. de C.V. Tula, MX) and sodium pentobarbital
Sedalpharma® (Pet’s Pharma, Nezahualcoyotl, MX). In all cases, animal
euthanasia was induced with an overdose of sodium pentobarbital
Pisabental® (PiSA Agropecuaria, S.A. de C.V. Tula, MX) according to
local normativity.
2.1.1 Liposome preparation:
The preparation of the proprietary liposomes has been previously
described (24-25). The liposomes were composed of lecithin and
cholesterol, preparation included dilution by injection of ethanol.
Stirring at 450-500 rpm for about 40 min was performed. Afterwards, the
formulation was heated to 45°C and then stirred at 350 rpm until the
ethanol was completely evaporated. Sterilization was done aseptically
through a polyvinylidene difluoride (PVDF) hydrophilic membrane with a
N2 pressure inlet of 2 bars and then refrigerated.
Afterwards, the lipid dispertions were hydrated with prepared Phosphate
buffered saline preparation and reheated at 70°C while stirring at
750 rpm for 30 min. The mixture was then left at room temperature for 30
minutes with N2. Required sirolimus was then added. The dispersion was
filtered through a hydrophilic membrane and collected in refrigerated
glass.
This method of sirolimus liposomes preparation is proprietary to the
team and has an FDA-issued patent. It is of critical importance to
highlight the differences between this formulation and the one used by
other investigators (26). First of all, our liposomes do not employ the
thin lipid film hydration method, we also do not use chloroform in the
mixture, and do not need to place the tube under vaccum for 12 hours.
Furthermore, our formulation does not use a buffer containing 6%
trehalose. Finally, the concentration used by these authors is of 5
mg/ml, while the concentration employed by us ranges from 0.4-1 mg/ml.
2.2 Methods
All the following tests were conducted under a Quality Management System
that assures the accomplishment of Good Laboratory Practices (GLP). The
experimental facility, Preclinical Research Center of National
Autonomous University of Mexico (UNIPREC-UNAM) had registration at the
EMA/OCDE (BPL-002/15) accreditation unit, for GLPs endorsement. Also,
the center was approved by local Mexican authority (SENASICA
AUT-B-B-0919-056) for animal experimentation.
Experimental protocols were approved by the
Institutional Committee for the
Care and Use of Laboratory Animals (CICUAL) of the UNAM Faculty of
Chemistry. All animals were treated in accordance with local and
international guidelines followed by CICUAL for the ethical use of
laboratory animals in research.
Care for animals used in this research is in concordance with the
Association for Research in Vision and Ophthalmology (ARVO) statement
for the Use of Animals in Ophthalmic and Vision Research.
2.3. Evaluation of Genotoxicity by in vivo micronucleus test
The animal model and sample size were selected according to OECD
guidelines for Mammalian Erithrocyte Micronucleus Test (27). Fifteen
male mice Hsd:ICR Specific Pathogen Free, weighing between 25-30 g were
randomized into three groups: 1) positive control using
cyclophosphamide, 2) RL and 3) L. 40 ml/kg doses were administered
through intraperitoneal injection. Posterior to this, three blood
samples were taken from caudal vein as follows: before the
administration, 36 h and 72 h after administration. Samples were fixed
with ethanol at 70% for 10 minutes, stained with Giemsa 10% solution
and observed with light microscopy by a trained specialist.
Quantification of polychromatic erythrocytes for each thousand
erythrocytes and micronucleated polychromatic erythrocytes (MPE) for
each two thousand polychromatic erythrocytes were determined for each
sample time.
Results were analyzed by two ways-ANOVA with a post Hoc Tukey test. A p
value of <0.05 was considered significant using Sigmaplot
software version 13.
2.4. HET CAM mucous irritation potential analysis.
Mucous irritation potential of a drug can be evaluated by observing its
effects on the chorioallantoic membrane of a fertilized, incubated hen’s
egg. This method is considered by some to be an alternative to the
Draize eye irritation test (28). 10 day-old Specific Pathogen Free
chicken embryos were used. On the 10th day, eggshells
were carefully removed, and the exposed membranes were inoculated in
triplicate with 300 μL of SDS 1% for positive control, Phs 0.9% for
negative control, and RL & L for test product. Afterwards, for the
following 5 minutes after inoculation, membranes were monitored for
hemorrhage, vascular lysis and coagulation reaction. Time to each
reaction was recorded in seconds. The Irritation Score was calculated
according to DB-ALM:INVITTOXX protocol No. 96, as follows:
IS =
[((301-sec)(H)(0.5))+((301-sec)(L)(0.7)+((301-sec)(C)(0.9)]/(300*300*300)
Where H: hemorrhage; L: vascular lysis; C: coagulation; sec: initial
time in seconds. Assignation to severe grades on each reaction were:
0=no reaction, 1 = low, 2= moderate and 3 = severe reaction.
2.5. Determination of Pyrogenicity
The increase in temperature due to pyrogens was analyzed in vivo .
The test was performed according to compendial methodology described in
the Pharmacopoeia of the United Mexican States (FEUM) (29). Test
dilutions (1:10) were prepared by diluting test formulations with Phs
0.9% in aseptic conditions. 1.5 mL were administrated intravenously in
the marginal ear vein of three healthy male New Zealand rabbits
weighting 1.5 to 3.0 kg. Rectal temperature was measured employing a
digital thermometer. Basal temperature was recorded in the normal range
(38-39.8 ºC) prior to administration. Post administration measurements
were recorded each 30 min for 3 hours. In case no subject presented an
increase in temperature of more than 0.6 °C from its basal temperature,
and the sum of the global increments did not exceed 1.4 °C, the sample
was considered pyrogen free.
2.6. Evaluation of subacute toxicity in vivo in male New Zealand
rabbits after subconjunctival injection.
We performed a set of in vitro toxicity tests that will be later
discussed, in order to warrant in vivo experimentation. A
subacute toxicity study was performed according with US Food and Drug
Administration (FDA) Center for Veterinary Medicine Guidance for
Industry #185 (CVM GFI #185) (28) with some modifications which
scientific rationale will be later discussed.
Sample size was calculated using G-power software 3.1 version
(Heinrich-Heine-Universität Düsseldorf, DE) with a power of 80%. Twelve
young male adult New Zealand rabbits weighting 2.0 kg (+/- 0.5 kg)
received subconjunctival rapamycin doses, empty liposomes and injectable
water as control in groups of 3 rabbits each (Table1). Doses were
administrated once weekly for three weeks (0, 1, 7 and 14 days). First,
anesthesia was performed by instilling two drops of proparacaine 0.5%
in the ocular surface of the rabbits. One minute after instillation,
bulbar dorsal conjunctiva was slightly raised and the corresponding
preparation was slowly injected at the superior fornix until formation
of a bulge was observed in the inner conjunctiva.
Ophthalmologic examination was performed after each injection by trained
veterinarians using slit-lamp and fundoscopy. Changes in gait, posture
and behavior were also monitored daily by trained personnel in the
bioterium of the University (UNAM) by releasing them from enclosure and
using food to encourage the animals to walk.
2.6.1. Assesment of metabolic changes by weight monitoring
Body weight of each subject was recorded at 1, 7, 14 and 21 days.
Interaction between days and groups, comparisons between groups, initial
and final weight were analyzed with the ANOVA test
(p<0.05 ).
2.6.2 Biochemical assay
Blood samples were collected from marginal vein before treatment and on
the 10th and 22nd days of follow-up.
Previously, subjects were fasted for 4-6 hours to reduce postprandial
biochemical changes and stress. For hematologic tests, 400
µL were collected in Microtainers®
containing EDTA as anticoagulant and for biochemical assays, 700 µL were
collected in empty Microtainers®. Non-anticoagulated blood samples were
centrifuged after blood clot was formed to obtain blood serum. Samples
were analyzed using an automated chemistry analyzer to observe any renal
or hepatic alterations caused by formulation treatment. Serum glucose
(GL) levels, urea, creatinine (CR), cholesterol (CH), total bilirubin
(TB), alkaline phosphatase (AP), alanine aminotransferase (ALAT),
aspartate aminotransferase (ASAT), calcium, phosphorus (Ph), total
proteins and albumin were measured.
2.6.3. Hematology tests
Hematological parameters evaluated included hematocrit (Hat), hemoglobin
(Ham), erythrocyte count (Ert), mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), and total leucocytes (Leu). Determinations
were made using an automated hematological analyzer. Subsequently, blood
smears were performed and examined through the microscope for white
blood cell differential (WBCD), platelet estimate, and Ert morphologic
examination.
2.6.4 Necropsy
On days 21 and 22, animals were euthanized through intravenous
anesthetic overdose. All rabbits were submitted to a complete necropsy.
Samples of the following tissues were collected for histopathology:
Brain, liver, kidney, parotid and mandibular salivary glands, right and
left eyes, optic nerve, eyelids (lower and upper), internal and external
tear glands, as well as two lymph nodes, one maxillary and one
retropharyngeal.
Samples were placed in 10% buffered formalin, dehydrated and embedded
in paraffin. Afterwards, 3-micron thick slices were obtained. Slices
were stained with hematoxylin and eosin (H&E) and Gram stain for
further microscopic examination by a veterinary pathologist.
2.7. Evaluation of acute retinal toxicity in vivo in New Zealand
rabbits after intravitreal injection.
In order to assess the potential retinal toxicity of rapamycin
liposomes, an evaluation of retinal function and histology was performed
after administration of two doses (40 and 440 µg) of liposomal rapamycin
applied through intravitreal injection.
Sample size was calculated using the same parameters as the
subconjunctival injection group. As a result, 15 young adult New Zealand
rabbits weighting 2.0 kg (+/- 0.5 kg) were selected to receive
intravitreal injections of liposomes loaded with rapamycin, empty
liposomes and Phs as negative control (Table 2). The injection procedure
was conducted as follows: Anesthesia was induced by inhalation of
isoflurane according to standard protocol of UNIPREC-UNAM, also a drop
of topical anesthetic was applied. After one minute, 0.1 mL of the test
product was injected through a 30G needle 2 mm posterior to the limbus
directed towards the center of the vitreous cavity. Povidone-iodine
solution was instilled to prevent microbial infection. Finally, an
ophthalmologic evaluation was performed to detect immediate adverse
reactions. An ophthalmologic evaluation was conducted every 24 hours for
14 days by trained veterinarians using slit lamp and fundoscopy. Gait
and behavioral changes were monitored by trained personnel in the
bioterium releasing the animals from enclosure and using food as bait to
evaluate gait.
2.7.1. Electroretinography and fundus pictures
Basal electroretinographic measurements and fundus pictures were
registered prior to injection procedures. First, mydriasis was induced
with topical tropicamide. After a dark adaptation period of 1 hour and
topical anesthesia, an ERG of each eye was recorded. The active
electrode with golden ring was placed on the cornea, the reference
electrode was introduced subcutaneously near the lateral canthus and the
ground electrode subcutaneously in the back of the neck, scotopic flash
electroretinogram was recorded. 7 days after the experiment, a second
ERG was recorded, and new fundus pictures were taken.
2.7.1. Histopathology
On day 14, animals were euthanized by intravenous anesthetic overdose.
In the postmortem study, a macroscopic evaluation of the ocular
structures was executed and those with evidence of pathology were
considered for microscopical exam. Ocular globes were collected for
histopathology, including optic nerve and corneas.
Samples were fixed in 10% buffered formalin, dehydrated and embedded in
paraffin, then, 3-5 micron thick slices were obtained. Slices were
stained with hematoxylin and eosin (H&E) for further microscopic
examination by a veterinary pathologist.
2.8. Statistical Analysis
Shapiro Wilk and Kolmogorov-Smirnov test were applied to all results to
verify if they were normally distributed. Pearson or Spearman
Correlation test were carried out to determine if variables or
individuals were similar. Differences between groups in biochemical and
hematological parameters was evaluated by ANOVA test with significance
determination by t-Holm-Sidak post-hoc test. A t-test was
employed to compare 1:1 independent normally distributed variable. For
histopathologic comparisons in SCJ toxicity study, due to qualitative
nature of the data, a numeric score was assigned for further comparison.
Data was analyzed by a one way no parametric Kruskal-Wallis test for
multiple group comparison and Mann-Whitney test was employed to compare
1:1 independent variable. For retina IVT toxicity, correlation between
histopathologic findings and treatment was All results were evaluated atp<0.05 using SigmaStat® version 13.0 software. Plots
were edited by Origin Lab 2016® or Microsoft Excel 365®.