Inherited retinal degeneration (RD), a rare neurodegenerative group of retinal blinding
disorders and is characterized by a progressive loss of retinal photoreceptor cells that
are responsible for vision. The symptoms of RD include onset of night blindness,
followed by a loss of peripheral and central vision leading to a complete loss of vision.
To date RD type diseases are untreatable, leaving a huge impact on patient population.
The lack of valid treatments can be attributed primarily to a vast genetic diversity of the
disease, obstructing identification of a common therapeutic target compound. Further,
the development of a valid treatment is hindered by a lack of effective delivery
mechanisms for potential drug candidates to be available at retinal photoreceptor cells,
primarily due to access limiting ocular barriers. The mechanism(s) involved in the
degeneration of retinal photoreceptors is still not fully understood, despite the huge
progress made over the years. One of the several mechanisms that explains the
degeneration points to a higher level of cyclic guanosine- 3′,5′-monophosphate (cGMP)
in photoreceptor cells and this mechanism has been commonly found in various forms
of RD. Thus, cGMP analogues were prepared to inhibit cGMP-activated targets within
the photoreceptor cells. DF003 (or CN03) is one such analogue that was extensively
evaluated and showed promising potential to be used in the treatment of RD.
In order to deliver the lead compound (DF003) to the retinal photoreceptors, lipid
nanocapsule formulation (LNCs) was chosen as a carrier system. LNCs have been
widely evaluated for various drug delivery applications owing to their versatile nature
and their ability to carry a wide range of drug molecules including peptides. These
nanoparticles have been shown to be promising in preclinical testing for various drug
candidates. Hence, this study aimed at understanding the suitability of LNCs as a drug
delivery carrier for DF003 to be used in ocular administration. The work includes
formulation preparation with DF003, optimization and characterization, nanoscale
evaluation of internal structure of LNCs, performance evaluation using porcine eyeballs
and retinal explants derived from retinas of diseases mouse models. This work also
includes the possibility of freeze-drying the formulation both with and without drug to
enhance the long-term storage stability.
LNCs were prepared with a phase inversion process. In Paper I, the screening of
formulation was performed by varying the composition of Labrafac™ and Kolliphor® that
forms the core and shell of the nanoparticles, respectively with an aim to study their
influence on physico-chemical attributes of the resulting LNCs. Formulations were
characterized for particle size, polydispersity index/particle size distribution (PDI), drug
loading, and entrapment efficiency. Formulation with selected composition was
characterized for additional parameters i.e., morphology using cryo-transmission
electron microscopy (cryo-TEM), stability, and in vitro drug release. It was observed that
particle size and entrapment efficiency (EE) were significantly dependent on the
proportion of Kolliphor® in the composition. With an increase in Kolliphor®, a
proportionate increase in EE and a decrease in particle size was noticed. Whereas an
increase in Labrafac™ did not change the EE significantly, however, a higher particle
size was evident owing to a lower surfactant to oil ratio. Additionally, the drug release
from the formulation was sustained for a period of up to 6 days both in phosphate
buffer and in vitreous component i.e., hyaluronic acid (HA) solution. Further, the
stability testing data indicated the absence of an unfavorable interaction between LNCs
and HA. In addition to lab-scale testing, LNCs were successfully upscaled to volumes of
1 L and 10 L using different processes and the resulted LNCs were having similar
characteristics to those prepared at lab-scale.
The selected composition was further used for developing freeze-dried LNCs and for
the work described in Paper II, and Paper III. With the objective of facilitating the
long-term storage of LNCs, the formulation was subjected to freeze-drying. The
resulting freeze-dried material was characterized after reconstitution to the initial
volume to assess the impact of freeze-drying on the physico-chemical characteristics of
the LNCs. Both unloaded and drug-loaded formulations were freeze dried with different
concentrations of cryoprotectants, namely sucrose, trehalose and raffinose. Out of
these, sucrose showed a better preservation of LNCs characteristics especially particle
size and PDI, even though a slight increase in size was evident after reconstitution.
Nevertheless, interesting findings were seen with freeze-drying of LNCs under varied
conditions.
The structure of LNCs with and without the drug presence was investigated using smallangle
scattering techniques namely, small-angle X-ray scattering (SAXS) and small-angle
neutron scattering (SANS). The findings from these techniques were combined with
DLS and cryo-TEM data to understand the internal structural information of LNCs.
Combined analysis of the data from these techniques revealed the presence of a coreshell
spherical structure that was unable to be seen with cryo-TEM. Upon drug loading,
the overall core-shell structure was retained, however, interestingly the thickness of the
shell increased in the order of 1 nm due to the predominant drug localization in the
shell region. These findings were published in Paper II.
To understand the behavior of these nanoparticles in relation to their ocular
administration, several in vitro and ex vivo tests were performed. These include
permeability testing across excised tissues from isolated porcine eye balls using Franzdiffusion
cell system, ocular distribution of fluorescently labelled LNCs in intact porcine
eye balls following intravitreal (IVT) injection, toxicity and efficacy evaluation of
unloaded and drug-loaded LNCs, respectively using retinal explant cultures obtained
from mouse models. Data from the permeability testing showed that this formulation is
not well suited for topical administration as eye drops. This led us to further evaluate
the alternative administration routes. IVT administration of DiO-loaded LNCs
demonstrated the distribution of LNCs across the vitreous, with the DiO signal evident
from most of the ocular tissues. These nanoparticles showed well tolerability with only a
slight increase in the number of dying photoreceptors after ex vivo exposure to
concentrations of 10 mg/mL in the retinal cultures. Furthermore, the drug-loaded LNCs
showed similar pharmacological effect to that of free drug solution upon testing with
retinal explants derived from retinal degeneration rd1 mouse model, despite the fact
that most of the drug is associated with the nanoparticles. These findings were
published in Paper III. Taking together the information from these papers, LNCs
showed considerable potential for ocular application and are therefore suitable for
further evaluation.
In summary, this thesis gives a comprehensive information about the understanding of
LNCs as a drug carrier system and its suitability for ocular application, in particular for
the retinal delivery of DF003.
Arftengd sjónhimnuhrörnun (retinal degeneration, RD) er samheiti á sjaldgæfum og
ólæknandi taugahrörnunarsjúkdómum í sjónhimnu sem einkennast af stig vaxandi tapi á
sjónviðtakafrumum. Í byrjun verður sjúklingurinn var við næturblindu en síðan tapi á
útlægri og miðlægri sjón sem að lokum leiðir til blindu. Erfiðleika við meðhöndlunar
sjúkdómsins má rekja til mikils erfðafræðilegs fjölbreytileika hans og takmarkaðrar
þekkingu á orsökum sjúkdómsins sem gerir lyfjaþróun erfiða, sem og erfiðleika við að
koma lyfjum til sjónhimnunnar. Það er talið að í sumum tilfellum megi rekja sjúkdóminn
til hækkunar á styrk hringlaga gúanósíns-3',5'-mónófosfats (cyclic guanosine-3′,5′-
monophosphate, cGMP) í ljósviðtakafrumum. Því voru cGMP hemlar (cGMP inhibitors)
samtengdir sem eiga að hindra cGMP viðtaka. Einn slíkur er DF003 og hefur virkni
hans verið sannreynd í RD rannsóknum. Fitusækin nanó-hylki (lipid nanocapsules,
LNCs) voru valin sem flutningskerfi fyrir DF003. Slík hylki hafa áður verið notuð við
lyfjagjafir, meðal annars fyrir peptíð-lyf. Rannsóknirnar fólust í eðlisefnarfæðilegum
rannsóknum á nanó-hylkjunum og hvernig slík flutningskerfi virka fyrir DF003. Til að ná
sem bestum árangri var samsetning nanó-hylkjanna aðlöguð að lyfinu DF003. Við
rannsóknirnar var m.a. notast við svínsaugu og einangraðar sjónhimnur úr músum.
Fyrsta greinin (grein I) lýsir hvernig samsetning nanó-hylkjanna var aðlöguð að lyfinu
DF003. Nanó-hylkin voru rannsökuð með tilliti til kornastærðar, stærðardreifingar
(polydispersity index, PDI), lyfjahleðslu og upptöku lyfs (entrapment efficiency). Lyfið
losnaði með jöfnum hraða úr hylkjunum í a.m.k. 6 daga, bæði í vatni og
gerviglerhlaupi (þ.e. hýalúrónsýru lausn). Einnig var framleiðsla á nanó-hylkjunum aukin
frá örfáum millilítrum upp í einn lítra. Nanó-hylkin voru einnig frostþurrkuð til að auka
geymsluþol þeirra. Bæði óhlaðnar og lyfjahlaðnar samsetningar voru frostþurrkaðar
með mismunandi styrk af frostvarnarefnum, þ.e. súkrósa, trehalósa og raffínósa. Í grein
II er ítarleg rannsókn á uppbyggingu nanó-hylkja lýst þar sem beitt var tækni sem
byggist á endurvörpun geisla (small-angle scattering). Rannsóknirnar sýndu að bæði
óhlaðin og lyfjahlaðin nanó-hylki hafa kjarna-skel uppbyggingu. Til að skilja hegðun
nanó-hylkjanna við augnlyfjagjöf voru framkvæmdar nokkrar in vitro og ex vivo
rannsóknir þar sem m.a. lyfjaflutningur í gegnum himnur úr svínsaugum sem komið
hafði verið fyrir Franz-sellum var mældur. Einnig var dreifing lyfs í svínsaugum eftir
innsprautun í glerhlaup (intravitreal injection) mæld auk þess sem mat var lagt á
eituráhrif og virkni á óhlöðnum og lyfjahlöðnum nanó-hylkjum í vefjasýnum úr músum.
Þessar niðurstöður voru birtar í grein III.
Í stuttu máli sýndu rannsóknirnar að hægt er að nota fitusækin nanó-hylki sem
augnlyfjaferjur og að þau geta ferjað cGMP hemlann DF003 til sjónhimnunnar.
Læst til 31.12.2024