Coincidence of Antifungal and Cytostatic Activity of Coruscanone A Derivatives and Analogues of Natural Lactones

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Inorganic and Organic Chemistry Candidate: Mgr. Lucie Tichotová Supervisor: Prof. Milan Pour Title of Doctoral Thesis: Coincidence of antifungal and cytostatic activity of coruscanone A derivatives and analogues of natural lactones This Thesis was focused on the synthesis and biological evaluation of novel analogues of a natural antifungal compound, coruscanone A. For this purpose, a catalytic version of Knoevenagel condensation of cyclopent-4-ene-1,3-dione with aldehydes was developed. Evaluation of antifungal and cytostatic activity of the new derivatives revealed that antifungal activity of many compounds is accompanied by a cytostatic effect against certain tumour cell lines (CCRF-CEM). Subsequent examination of these arylidene analogues uncovered their decomposition in water medium under the conditions of in vitro testing. Therefore, stable analogues based on maleinimide were prepared by the Mitsunobu reaction. In these compounds, antifungal and antiproliferative effects occur simultaneously as well. N-2-indanylmaleinimide displayed the highest antifungal activity against A. fumigatus, while N-benzylmaleinimide had an excellent effect against HT-29 cells (IC50 = 0.6 μmol.l-1 ). Furthermore, cytostatic activity of..

Conjugates of Butenolides with Heterocycles: Exploration of the Origin of Cytotoxicity

Katedra anorganické a organické chemieDepartment of Inorganic And Organic ChemistryFarmaceutická fakulta v Hradci KrálovéFaculty of Pharmacy in Hradec Králov

Mitochondrial Dysfunction in a High Intraocular Pressure-Induced Retinal Ischemia Minipig Model

Purpose: Retinal ischemia (RI) and progressive neuronal death are sight-threatening conditions. Mitochondrial (mt) dysfunction and fusion/fission processes have been suggested to play a role in the pathophysiology of RI. This study focuses on changes in the mt parameters of the neuroretina, retinal pigment epithelium (RPE) and choroid in a porcine high intraocular pressure (IOP)-induced RI minipig model. Methods: In one eye, an acute IOP elevation was induced in minipigs and compared to the other control eye. Activity and amount of respiratory chain complexes (RCC) were analyzed by spectrophotometry and Western blot, respectively. The coenzyme Q10 (CoQ10) content was measured using HPLC, and the ultrastructure of the mt was studied via transmission electron microscopy. The expression of selected mt-pathway genes was determined by RT-PCR. Results: At a functional level, increased RCC I activity and decreased total CoQ10 content were found in RPE cells. At a protein level, CORE2, a subunit of RCC III, and DRP1, was significantly decreased in the neuroretina. Drp1 and Opa1, protein-encoding genes responsible for mt quality control, were decreased in most of the samples from the RPE and neuroretina. Conclusions: The eyes of the minipig can be considered a potential RI model to study mt dysfunction in this disease. Strategies targeting mt protection may provide a promising way to delay the acute damage and onset of RI

Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs

Purpose: The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs. Methods: The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L-lactide-co-DL-lactide) (PDLLA). “Libechov” minipigs (12–36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE-nanofibrous carrier was conducted using general anesthesia via a custom-made injector during standard three-port 23-gauge vitrectomy, followed by silicone oil endotamponade. The observational period lasted 1, 2, 6 and 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry using the following antibodies: HNAA and STEM121 (human cell markers); Bestrophin and CRALBP (hRPE cell markers); peanut agglutining (PNA) (cone photoreceptor marker); PKCα (rod bipolar marker); Vimentin, GFAP (macroglial markers); and Iba1 (microglial marker). Results: The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans-epithelial electrical resistance on the nanofibrous PDLLA carrier. The surgical delivery of the implants in the subretinal space of the immunosuppressed minipigs was successfully achieved and monitored by fundus imaging and OCT. The implanted hRPEs were positive for HNAA and STEM121 and were located between the minipig’s neuroretina and RPE layers at week 2 post-implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post-implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time. Conclusions: The differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23-gauge vitrectomy and implantation injector. The hRPE-laden scaffolds demonstrated relatively good incorporation into the host retina over an eight-week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression