3. RESULTS
3.1. Evaluation of genotoxicity by in vivo micronucleus test (MPE).
According to Figure 1, there was no increase of MPE in groups treated with rapamycin liposomal formulation or empty liposomes compared with positive control, where there was an evident increase of MPE at 36 hours.
3.2. HET CAM mucous irritation potential analysis.
Irritation score of the formulations and control were <0.9, Table 3.and thus corresponds to no reaction according to standarized guidelines (27), while positive control showed a moderate reaction. These results were observed in egg-chorioallantoic membranes, Figure 2, which showed increased membrane vascular lysis, hemorrhage and coagulation when SDS was placed in contact, in contrast with RL and L formulations that had no effect.
3.3. Determination of pyrogenicity
According to the following exclusion criteria, established in the Mexican Pharmacopeia FEUM (29), pyrogen absence is warranted if:
a) No rabbit temperatures differ more than 2°C between two temperatures consecutively measured at the acclimatization period
b) No rabbit temperature is higher than 39.8°C or lower than 38.0°C
c) No rabbit temperature increases individually above 0.5°C compared to basal temperature during the test
d) No rabbit shows an increase in temperature above 0.6 °C compared to its individual basal temperature, and the sum of the maximum increment observed in the 3 rabbits does not exceed 1.4 °C.
Rapamycin loaded liposomal formulations met the mentioned requirements and can therefore be considered pyrogen free (Table 4).
3.4. Subacute toxicity in vivo in male New Zealand rabbits after subconjunctival injection.
3.4.1. Metabolic changes
No deaths or apparent adverse clinical signs were found in any group throughout the study period. There was no statistical difference in mean body weights in the comparisons made between groups. As it is shown in the results presented in Table 5, there was no discrepancy.
3.4.2. Clinical evaluation and histopathologic analysis
All the specimens were observed macroscopically at least twice a day through the entire duration of the study. Upon examination, they presented adequate general body condition. Mucous membranes had typical pale pink color and good state of preservation; however, in all groups lacrimal glands reached two times their normal size. This effect can probably be attributed to a transitory parasympathetic stimulation effect caused by the injection on the lacrimal gland processed by afferent fibers in the conjunctiva and sclera of the eye. This is supported by the fact that the effect was seen in both treatment and control groups and by that after the second subconjunctival injection, rabbits #1, 2 and 11 presented tearing that resolved within 2 days. Rabbit #1 also presented swollen eyelids and pain, which also resolved within 2 days post administration. After euthanasia of the specimens, a complete necropsy was performed. The internal general inspection detected mild to moderate pulmonary, hepatic and renal congestion in all groups. Figure 4 presents some of the observed alterations after histopathological analysis of the samples. Histopathological observations are summarized in Figure 5, p values of group comparison for each lesion demonstrate that no statistically significant differences between groups, including controls, were found.
3.4.3. Biochemical assay
Results are summarized in Table 6. No toxicity-indicative differences were found between groups, sampling time or interaction between group and sampling time in all experiments.
3.4.4 Hematologic testing
Results are summarized in Table 7. no relevant differences between groups were noted in sampling time or interaction group-sampling time.
3.5. Evaluation of acute retinal toxicity in vivo in New Zealand rabbits after intravitreal injection.
All the specimens were observed macroscopically at least once a day through the entire duration of the study by trained veterinary ophthalmologists. Upon examination, they presented adequate general condition although mild ocular and/or palpebral irritation was observed in test groups but not in control group. Fundus pictures showed no evident macroscopical alterations in retinal structure, Figure 6 presents representative pictures of basal appearance versus post injection aspect of retinas.
3.6.1. Electroretinography
No reduction in amplitude, increased implicit time or alteration in waveform was observed between the basal measurements and post injection response or in tests groups versus control group. U Mann-Whitney test (p<0.05) was performed to determine significance. Implicit times of b-wave measured in test groups compared against control group response 7 days after treatment are presented in Figure 7, statistical analysis revealed no changes associated to test products.
Representative ERG waveforms of each group 7 days after IVT injection are presented in Figure 8. Statistical difference was tested between b-wave amplitude 7 days after treatment compared with the basal measurement of each group (Fig.8). No significant detriment in amplitude was observed in any group, the group administered with 40 µg of rapamycin resulted in statistically different amplitude due to an increase in amplitude after IVT injection. Amplitude detected 7 days post injection of test groups was also compared with that of the control group, Figure 9. As well as comparative results versus basal measurements, only the group administered with 40 µg of rapamycin was statistically different from control group due to increased amplitude observed in this group after treatment.
3.6.2. Histopathology
No evident pathological alterations such as detachment or hemorrhages were found in the macroscopic observation of retinas. Nevertheless, some specimens showed signs of traumatic lens fragmentation. No other alterations were observed in the rest of the ocular structures. Figure 10A shows microscopic findings. No histopathological injury was found in the control group or the group treated with 40 µg of rapamycin. In the rest of the groups, retinal degeneration and hyperplasia was observed at least in one sample. In order to determine the significance of this findings, a one way-nonparametric Kruskal-Wallis ANOVA analysis was performed with significance at p<0.05, Fig. 10B. However, no significant differences were found between groups. Representative histologic micrographs of microscopic observations are shown in Figure 11.