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®.