| Abstract |
BNN27 is a novel synthetic 17-spiroepoxy derivative of the neurosteroid
Dehydroepiandrosterone (DHEA). It is one of a series of newly synthesized synthetic
derivatives of DHEA, named BNN, which are small lipophilic molecules with antiapoptotic
properties, without the undesirable estrogenic effect of DHEA. More specifically, it is one
of the three BNNs (BNN20, 23 and 27), which were found to have the most potent
neuroprotective and antiapoptotic activity. In the present thesis, BNN27 was chosen to
be studied as it is the most widely used in preclinical studies, among the three lead
molecules so far, but also the molecule that has been used in the.
The purpose of this research was to study the pharmacokinetic characteristics of
BNN27, both after systemic and local administration, in the blood, but also in the target-
tissue, the posterior segment of the eye (vitreous and retina), as well as to assess for any
retinal toxicity after local administration.
After the initial spectroscopic characterization of the three lead BNNs, BNN20, 23
and 27, the BNN27 pharmacokinetics were studied, both after systemic- intraperitoneal
administration, in the serum and in the target-tissue, the retina, as well as after local-
intravitreal (IVT) administration in the vitreous. Moreover, the presence of retinal toxicity
after intravitreal administration was investigated.
For the serum pharmacokinetics, 42 C57BL/6 mice were used. The blood was
collected post-orbitarily at time points 0, 30 min, 1h, 2, 4, 6 and 24h after one
intraperitoneal injection of 100mg/kg BNN27 and the concentrations were measured by
liquid chromatography-mass spectrometry (LC-MS). The molecule was found to have an elimination constant kel=0.465(h-1) and mean residence time (MRT) = 2.154 h in the mice
serum.
For the retinal pharmacokinetics, 48 Sprague-Dawley rats were used. The retina
was isolated, after enucleation, at 0, 15, 30min, 1h, 1h, 2, 4, 6 and 24h after one
intraperitoneal injection of 30mg/kg BNN27 and the concentrations were measured by
liquid chromatography-mass spectrometry (LC-MS). The first time point of BNN27
detection in the retinal tissue were the 30 minutes after injection. The time of the maximum
measured concentration (tcmax) was 2 hours and the maximum concentration was found
to be Cmax=1100ng/g. BNN27 was detected in the retina even after transcardiac
perfusion with Ringer's Lactate (RL), with a 2h post-injection concentration equal to 320
(70) ng/g.
For the vitreous pharmacokinetics, both eyes of 21 albino rabbits received one
intravitreal injection of 0.6mg/0.1ml BNN27. The vitreous was isolated after enucleation,
at time points 0 (euthanasia and enucleation immediately after injection), 12 h, 1 day, 2,
7, 14 and 30 days after one intravitreal injection of 6mg/ml BNN27 and concentrations
were measured by liquid chromatography-mass spectrometry (LC-MS). BNN27 was
shown to follow 1st order kinetics, with an elimination constant kel = 0.15(h-1) and a half-
life of 4.49 days. At 30 days, traces of BNN27 were detected.
To assess for any retinal toxicity, two intravitreal concentrations of BNN27 were
examined: 6mg/ml (group A) and 0.6mg/ml (group B). Each group comprised of 12
pigmented rabbits. Τhe right eye of each animal was injected with 0.1ml of the respective
BNN27 concentration (study eye) and the left eye was injected with 0.1ml of a 6% ethanol
solution (control eye-positive control), in both groups. The animals were examined at time points: 0 (before injection), 1 day, 1 week, 2 weeks and 1 month after injection. During
the experiment, the eyes were monitored macroscopically, with slit-lamp anterior segment
examination, fundoscopy and tonometry. At the end of the experiment (30 days after
injection), the eyes were studied histologically, with photon microscopy, after enucleation
and appropriate preparation. The retinal function was studied with electroretinography
(ERG) before injection and one month after the intravitreal injection.
In group A, two study eyes presented macroscopic, histological and functional
findings of posterior segment inflammation and this was potentially associated with the
BNN27 solution. In both groups, non-specific histological findings were observed, which
were not confirmed by ERG; they are not most likely associated with the BNN27.
Furthermore, in both groups, the control eyes did not show any signs of toxicity,
anatomical or functional.
In conclusion, BNN27 was proved to move rapidly from the intraperitoneal space
into the systemic circulation and thence into the retina. This is consistent with the size
and lipophilicity of the molecule. The detection of intraretinal BNN27, after transcardiac
RL perfusion, proved that BNN27 can cross the blood-retinal barrier. In addition, given
the long -term detection of the BNN27 in the vitreous after IVT administration, the present
formulation could be potentially considered for the BNN27’s slow release to the retina, in
case of future clinical use. Eventually, the low dose appears to be relatively safe for
intravitreal use and ethanol can be safely used for the BNN27 dilution at 6%
concentration.
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