4. DISCUSSION
4.1. Evaluation of genotoxicity by in vivo micronucleus test.
Increased cancer risk is a serious adverse effect among patients
undergoing immunosuppressive therapy. Micronucleus frequency has been
reported to be significantly higher in pediatric patients with
immunosuppressive therapy after kidney transplant (30). Although the
dose challenged in this paper is topical and much lower, this novel RL
formulation is being proposed for a chronic disease that may imply long
term therapy, which is why assessment of genetic damage is mandatory.
The results presented in Figure 1 indicate a lack of genotoxicity
effect, which is consistent with studies reporting that unlike other
immunosuppressants, when used specifically for ocular inflammation,
rapamycin has been reported to inhibit immunosuppression-induced
neoplasia (31). This could be explained by the different mechanism of
action of rapamycin versus other immunosuppressants. Oncogenic
transformation, one of the most worrying consequences of DNA damage, is
favored by the loss of cell cycle control and the activation of growth
promoting pathways, such as the signaling path involving the mammalian
Target of Rapamycin (mTOR). Rapamycin and other inhibitors of mTOR
decelerate cell proliferation and contribute to avoid oncogenic
transformation by suppressing the signals required for cell cycle
progression, cell growth and proliferation (32). Thus, RL as well as its
liposomal vehicle, are not expected to produce genetic damage when
administered in mammals.
4.2. Evaluation of mucous irritability potential by HET CAM analysis
HET CAM test is an in vitro alternative to the in vivoDraize test, which is one of the most criticized methods of ocular
irritation testing because of the injuries inflicted on test animals.
The occurrence of vascular injury or coagulation in egg-chorioallantoic
membranes in response to a compound, has a good correlation with the
Draize rabbit test (33). This test provides information about immediate
effects after administration of a product, mainly by vascular
alterations, but also by protein interactions making it an adequate
pre-screen method of eye injury hazard potential. The results indicate
that the product is not expected to produce irritation effects on ocular
membrane when tested in vivo. The obtained results were expected,
taking into consideration previous reports of lack of ocular toxicity of
rapamycin in animals. However, testing of the vehicle irritation
potential and the combination with rapamycin was yet to be proved. These
results justify the innocuity of the product in order to be tested in
live animals, as well as confirms biocompatibility of the selected
nanocarrier.
4.3. Determination of pyrogenicity
Pyrogens are substances that can produce fever when present as
contaminants in a drug. Most pyrogens are biological substances derived
from microorganisms that trigger an immune response, they can be
life-threatening to patients because the produced systemic reaction can
go from fever to neurologic effects, shock and death. These results are
of great importance since some of the most recognized drawbacks for the
application of liposomal technology in medicine are presence of organic
solvent residues, difficult pyrogen control and sterility assurance,
poor stability and adequate size distribution. It is worth mentioning
that the formulations were also tested for sterility with good
compliance with FEUM standards (data not published), along with absence
of pyrogenic reaction. This means that proprietary methodology used for
the preparation of the samples tested in this work is suitable for
commercial production.
Aside of ruling out endotoxin contamination, absence of pyrogenicity
gives indirect information on acceptable size distribution of RL, since
it has been reported that liposomes larger than 200 nm tend to cause
non-endotoxin-dependent rise in temperature, probably due to the
metabolism of lipid mediators like prostaglandins (34). Lack of
pyrogenic reaction suggests a suitable size distribution of the RL
liposomes for intraocular injection.
4.4. Subacute toxicity in vivo in male New Zealand rabbits after
subconjunctival injection.
Subacute systemic toxicity is defined as the adverse effects occurring
after multiple or continuous exposure between 24 h and 28 days. For this
kind of studies, the FDA recommends testing the maximum dose intended
for therapeutic use, and 3 and 5 times that dose to identify effects
associated with overdosing and increased duration of administration
(35). Also, in this early stage of our development process it will help
to establish a safe dose range for future optimal dose exploration.
In this study the dose range tested was limited to only 3 times the dose
intended for therapeutic use and the administration frequency was
doubled. Rationale for these modifications rely on the limitations of
the route of administration. The maximum volume generally recognized as
safe for subconjunctival injection (SCJ) is 0.5 ml, for this study the
highest dose was rounded to 0.45 ml that represents 3 times the
therapeutic dose. At first, we tried to inject 0.45 ml divided in 2
injections of 0.225 ml, considering that an injection volume of 0.2 ml
is commonly used in practice with no remarkable adverse effects.
However, significant discomfort was observed in the rabbits when
manipulated for the second injection, apparently due to the stress
caused by prolonged manipulation. Ultimately, it was decided to perform
a single injection for subsequent administrations. Regarding
administration frequency, instead of testing a higher dose, weekly
administration was established in the protocol, that frequency is twice
as frequent as the dose regimen considered for therapeutic use.
4.4.1. Metabolic changes
No difference in mean body weights in the comparisons between treatment
groups was found. Although comparisons between final and initial weight
show a slight alteration was observed, it is attributable to the innate
growth of the rabbits. Hence, no significant metabolic changes were
noted.
4.4.2. Clinical evaluation and histopathologic analysis
Macroscopic evaluation only revealed mild alterations in 3 subjects
after the second injection. Necropsy detected discrete pulmonary, renal
and hepatic congestion in all test and control animals probably related
to the euthanasia method. Other organs did not present relevant
disturbances. The microscopic examination of histologic slices after
necropsy revealed odd pathology results for each tissue, (Figure 5A).
For both right and left eyes, lymphoplasmacytic uveitis was detected
mainly in control subjects. Due to these alterations, especially in
rabbit #1, all the slices were stained with Gram. One subject (rabbit
1, group 3) was detected with presence of Encephalitozoon
cuniculi spores, (Figure 4B). This subject exhibited multifocal
lymphoplasmacytic infiltrates in stroma and ciliary body of the right
eye, this finding was also detected in right lacrimal internal gland, as
well as mild multifocal necrosis. The left eye of this specimen showed
similar injury, but the infiltration covered also the iris structure. In
the left lens slice multiple spores of Encephalitozoon cuniculiwere found, as well as in liver and kidney samples. Optical nerve
samples of this subject presented mild multifocal lymphoplasmacytic
meningitis.
Based on macroscopic and microscopic findings, it was determined that 6
subjects (2 rabbits from group 2, 3 rabbits from group 3 and 1 rabbit
from group 4) presented injuries compatible with encephalitozoonosis but
histopathological confirmation of spores was only possible in rabbit #1
from group 3. Most of the injuries observed were similar between groups,
this statement was confirmed by statistical analysis that displayed no
significant differences among treatment groups so no relationship
between treatment and organic damage could be elucidated. Figure 5
summarizes the injuries observed in the microscopic examination of the
subjects. It can be inferred that the highest injury levels were
presented in group 3 treated with liposomal vehicle. The decreased
quantity and level of injuries detected in animals administered with
rapamycin containing formulations compared with empty liposomes may be
related to the local immunosuppressant and anti-inflammatory activity of
rapamycin (36). Lymph nodes in all groups (Figure 5A) exhibited lymphoid
hyperplasia at mild multifocal level, these can be appreciated in Figure
4D where germinal centers of the lymph node present discrete
hyperplasia. Also, as mentioned, all the rabbits showed
lymphoplasmacytic dacryoadenitis. These findings are consistent with the
presence of Encephalitozoon infection (37).
Histologic examination of the liver detected hepatocellular degeneration
in all groups, Figure 5A. Group 1 showed only mild hepatocellular
degeneration and congestion. Furthermore, subjects of groups 2, 3 and 4
presented moderate periportal lymphoplasmacytic hepatitis, Figure 4E.
Possibly, hepatitis could be associated with lymphoid activity and
hepatocellular degeneration increase and necrosis could be related toEncephalitozoon cuniculi infection, Figure 4F, since these
lesions were found near the parasite spores in rabbit #1 (38). It is
difficult to attribute these injuries to the administration of the test
product since the most affected rabbits were in the control and vehicle
groups. Also, it was not possible to determine a statistically
significant difference between treatment groups. Mild to moderate
multifocal tubular degeneration was observed in the kidneys of all
groups. Additionally, lymphocytic interstitial nephritis in groups 2 and
3 was presented with one subject at level 4 and other at level 7, which
is represented in Figure 4G. Both, degeneration and nephritis could be a
consequence of the parasitic infection. No trend was observed in kidney
injuries between groups due to rapamycin administration [37].
Lymphocytic encephalitis and satellitosis were exhibited in the brain
sample of one subject of group 2 and granulomatous meningoencephalitis
was displayed in one subject of group 3. All cases were at moderate
multifocal injury level. Figure 4H displays the encephalic section of
rabbit #1 confirmed with E. cuniculi infestation, where it can be
observed an inflammatory infiltrate as well as plasmatic cells
surrounding blood vessels. The optical nerve exhibited
lymphoplasmacytic-meningitis in one rabbit of groups 2 and 3, also
satellitosis and gliosis was observed in a rabbit from group 3, Figure
4l. Those pathologies could be linked again to parasitic infection. No
damage was observed in groups 1 and 4 either in brain or optical nerve
(38-40).
Our animals were certified as healthy by the provider at the beginning
of the study, however we detected infestation in our test population.Encephalitozoon cuniculi is a common opportunistic protozoan in
laboratory animals which is very hard to identify until symptoms appear
(41). It is an obligate intracellular microsporidian parasite whose
target organs are kidney, lens and nervous tissue. Immunocompetent
animals usually are subclinical carriers, but immunocompromised hosts
often present chronic granulomatous inflammation. (42). Regarding this,
we could hypothesize that the presence of this infection could have been
associated and promoted by immunosuppressive activity of rapamycin,
however no microorganisms could be detected in the subjects from groups
1 and 2, administered with rapamycin loaded formulations. As discussed
above, parasitic infestation was confirmed only for rabbit #1,
administered with 450 µL of liposomal vehicle. Also, all kinds of
lesions encountered were statistically similar in all the treatment
groups. It has been reported that cerebral lesions can only be observed
about 8 weeks after initiation of antibody response to the infestation
which takes place within 3 weeks post-infection (43). The duration of
our experiment was of 3 weeks, with 10 additional days of quarantine
where no obvious clinical signs of disease were observed. Since the age
of the animals was of 10 weeks at the beginning of the quarantine
period, is probably that these subjects were subclinical carriers that
represented an infection focus of dissemination to the rest of the
animals from urine excretion of spores that starts 3 to 5 weeks after
antibody response (43). Thus, the rest of the animals were probably in a
primary stage of the infestation at the moment of the necropsy due to
urinary horizontal dissemination at quarantine period. This is also
consistent with the presence of kidney injury in 8 of 12 subjects,
considering that kidney damage is part of the primary stage of the
disease. (44)
Despite the infestation found in one subject, there was no relationship
between injuries and treatment according to ANOVA analysis, Figure 5B,
which means no effect can be attributable to rapamycin formulations. All
the lesions observed were attributable to encephalitozoonosis and no
other significant lesions were assessed in the high or low dose test
groups. The fact that the confirmed subject was administered with
liposomal vehicle without rapamycin suggests that other variable may
have participated in the immunosuppression of the animals. In this
manner, it has been studied that in assays with live laboratory animals,
alterations due to stress often occur during toxicity studies and may
interfere with the interpretation of the results. As discussed before,
some of the evaluated parameters suggested that the animals were
affected by stress during the study which may explain the development of
the opportunistic infection. (45)
Finally, it is worth mentioning that the etiopathological origin ofEncephalitozoon Cuniculi in rabbit lens has been reported in
literature (46). Ingestion of contaminated food or water is the most
likely origin. Trans-placental and respiratory routes are also possible
however less likely due to certification of health from the animal
provider. We also performed cultivation of the formulation, yielding
negative results to this parasite.
4.4.3. Biochemical assay
Samples were analyzed to observe any renal or hepatic alteration caused
by administration of liposomal formulations with respect to water for
injection as a control. Differences in the aforementioned parameters
were analyzed at 0, 10 and 22 days after subconjunctival injection.
Biochemical measured values were between reference levels, therefore no
influence from product administration can be assumed. Differences
observed in urea and creatinine levels showing an upward trend in time
can be considered normal due to increase in body weight of the rabbits
due to normal growth. Cholesterol levels were significantly higher for
group 3, treated with placebo liposomes. This difference was found
because basal measurements in this group presented the highest values
for this analyte therefore differences that cannot be associated with
product administration. According to literature, administration of
rapamycin has been associated with cholesterol serum elevation at
rapamycin doses from 1-7 mg/day (47), however there was no increase
observed in cholesterol levels in rapamycin treated groups. This could
be explained by the very low dose that was locally administered, in
conjunction with the liposomal carrier that encapsulates the drug and
may inhibit systemic effects (48).
Concerning liver function tests, ALAT results showed difference between
groups 1 and 2 for all sampling times because group 1 presented higher
values for this parameter since basal measurements. Moreover, since
the difference was observed throughout the whole study, it is probably
not related to hepatic injury. This is also supported by a lack of
difference between treatments for ASAT and AP results, which reaffirms
that there is no evidence of damage on hepatocellular integrity.
In a similar manner, there were differences between group 1 and 3 in
serum total protein results. Since these findings were observed since
basal measurements it lacks significance considering that mean results
of each group are in the normal range. These results presented a
statistically significant gradual increment between sampling times,
associated with normal animal growth. The same upward trend was observed
for albumin and calcium, whereas for phosphorous there was observed a
downward trend.
Total bilirubin concentration was significantly decreased from basal
measurement with respect to second sampling time, but this was
considered irrelevant because mean results of each sampling time were in
reference range. Also, alkaline phosphatase results did not show
alterations, therefore concluding that biliary flux was not affected.
None of the serum biochemical parameters related to liver and kidney
function showed results indicative of toxicity due to product
administration in low or high dose, as well as the liposomal carrier.
Hematologic parameters showed significant differences in hematocrit and
erythrocyte count between groups. This difference was due to higher
values of group 4, presented in basal measurements. Bearing in mind
that this difference was found in basal measurements from the control
group, it has no physiological relevance. This may be explained as an
effect secondary to a hemoconcentration state of the animals at the
beginning of the study. Erythrocyte count was slightly out of normal
range in basal measurement of the control group, along with hematocrit
results that were in the upper limit of the normal range.
Significant differences in leucocyte count were also observed.
Specifically, there was an increase in neutrophils in groups 1, 2 and 3.
In all groups, an increase in monocytes and a decrease in lymphocytes at
the first sampling time was observed. Even though statistically
significant differences were observed, measurements were still in normal
range. It has been reported that during toxicity studies with live
laboratory animals, stress can affect diverse parameters including
hematological assays. These results also suggested the possibility of an
acute sub-clinical infectious process in all groups that will be
discussed later. Some of the most common stress-related findings are
lymphocyte depletion in thymus and spleen; resulting in altered
circulating leukocyte counts, including increased neutrophils with
decreased lymphocytes. (45) Animal stress is an inherent feature ofin vivo studies and may be triggered by many circumstances, among
them, the handling process for dosing. In this case, the subconjunctival
administration is a specialized type of injection that requires ocular
topical anesthesia and must be performed by a specialist trained for
this kind of administration. This process could have been more stressful
for the animals than expected, summed with the expected stress generated
by the sampling processes for biochemical and hematological parameters.
This is consistent with the fact that lymphocytes and monocytes count
alterations were also observed in the control group that was handled in
the same manner, table 7.
4.5. Evaluation of acute retinal toxicity in vivo in New Zealand
rabbits by intravitreal injection.
Intravitreal injection of rapamycin has shown good tolerability in some
animal models so far [9,10,14,15]. However, the toxicity evaluation
of every new formulation is mandatory being that the excipients of the
formulation are not always compatible with the intraocular route (49).
In this study, no statistically significant retinal acute toxicity was
observed in electrical function evaluation or histological tissue
examination. This is consistent with previous efforts to assess
intravitreal toxicity with this drug. Fundus pictures showed no evident
macroscopic alteration in retinal structure, however traumatic cataract
formation was detected in some subjects 3 days after intravitreal
administration. By slit-lamp examination, cataract formation was
attributed to accidental lens traumatism during intravitreal injection
technique since no trend or correlation could be determined between
rapamycin amount and lens damage.
3.6.1. Electroretinography
Retinal damage can lead to vision loss due to lack of transmission of
visual signal and has been in the top 5 most important causes of new
drug candidate dismissal during drug development process (50).
Dark-adapted ERG was used as a non-invasive in vivo evaluation of
the electrical response of retinal cells. A significant decrease in
amplitude of a or b-waves and/or prolongation of implicit times of these
waves are indicative of retinal toxicity. In our study no statistically
significant (p<0.05) reduction in amplitude, increased
implicit time or alteration in waveform was observed between the basal
measurements and post injection response. The same lack of significant
difference was observed when comparing ERG results of tests groups
versus control group 7 days after IVT administration.
This results are of great importance because we are challenging a new
formulation of a drug that has been previously proved as safe for
intravitreal injection by many other independent research groups, but
also, this same drug has also been found to be toxic to the ocular
structures when formulated in certain excipients with specific
deterioration of retinal function evidenced by unfavorable
electroretinography results (50). Interestingly, the b-wave response in
the ERG of the group administered with 40 µg of rapamycin was
statistically different from control group and of its original basal
measurements due to increased amplitude observed in this group after
treatment. These may suggest a potential mechanism to improve impaired
visual signaling in the retina. This amplitude augmenting effect has
been previously reported at least once to our knowledge by De Paivaet al (2019), with the use of sustained-release rapamycin systems
and was considered transient and clinically irrelevant (52). Also, a
protective effect of rapamycin on visual impairment during inflammation
has been reported as the attenuation in a- and b-wave reduction due to
induced inflammation (53). Further studies to explore its effects on
human retinal signal transmission would be interesting.
3.6.2. Clinical evaluation and histopathologic analysis
Light microscopy showed normal tissue organization and cellularity of
retinas in most subjects. Although, non-statistically significant
alterations were observed, some subjects showed mild to severe injuries,
specifically tissue degeneration and hyperplasia. Also, as can be
appreciated in Fig. 11vi, lens fragmentation and presence of Morgagnian
globules due to vacuole formation. This was consistent with slit-lamp
examination and is probably related to histologic alterations observed
since those subjects with cataract formation presented higher levels of
histologic injury. In fact, it is noteworthy that the most severe
injuries were observed in subjects treated with the intermediate dose of
rapamycin, while only mild lesions were observed at the highest
rapamycin dose. Despite these not significant (p<0.05)
injuries, functionality of retinas was conserved from 40 to 440 µg of
liposomal rapamycin as proved by ERG evaluations.
Limitations of this study include the small number of eyes included in
each experiment and unexpected development of a subclinical disease
common in laboratory animals. Further research in order to elucidate
intraocular pharmacokinetics of the formulation and ocular
biodistribution of the drug will be determining to achieve aim of
clinical testing for the treatment of eye immune mediate diseases. A 40
µg liposomal rapamycin dose appears to have the best toxicity profile to
be used by intraocular route, further research of its clinical
effectivity is warranted.