Rakennemuunnosten ja kantajien käyttö oligonukleotidien saattamisessa vaikutuskohteeseensa : Synteesi, analyysi ja biologinen testaus

Several serious diseases remain without non-toxic curative treatments. To fill this void, one of the promising groups of medicines is that of oligonucleotides, encompassing aptamers, transcription factor decoys, and antisense therapeutics such as short interfering RNA and splice-correcting oligonucleotides. These short strands of DNA or RNA can bind to specific cellular nucleic acids or proteins and thereby inhibit or correct the function of disease-causing molecules. Extensive enzymatic degradation and poor cellular uptake are the most important obstacles for systemic oligonucleotide therapy. Numerous chemical modifications have been introduced to improve enzymatic stability, but they must be carefully optimized to avoid toxicity and to maintain target affinity. One solution is to design topological modifications, such as looped or circular oligonucleotides, which conserve the natural phosphodiester backbone but cannot be attacked by exonucleases. Cellular uptake has proven to be even more challenging. Oligonucleotides are internalized into cells by endocytosis, after which they often remain trapped in endosomes. Therefore, it would be advantageous to develop delivery vectors capable of bypassing endocytic routes of uptake or enhancing endosomal escape. Cell-penetrating peptides, for example, exploit several mechanisms of uptake, some of which lead to rapid entry without endosomal localization. In addition, encouraging results have been achieved using liposomes, gold nanoparticles, and other nanocarriers, which also shield the oligonucleotide from degrading enzymes. The aim of this work was to improve the in vitro delivery of oligonucleotides by employing chemical modifications and nanoparticle carriers. The synthesis of the compounds, their characterization by various analytical methods, and the evaluation of biological effects are described. Antisense oligonucleotides covalently linked to cell-penetrating peptides via convergent conjugation displayed improved cellular uptake but failed to inhibit reporter genes due to endosomal entrapment in cells. Circular oligonucleotides exhibited enhanced selectivity of mismatch detection and increased stability in biological fluids compared to linear oligonucleotides. Altogether 44 compounds were analyzed by electrospray ionization mass spectrometry and liquid chromatography mass spectrometry, which were found to be excellent methods for the characterization of modified oligonucleotides. Finally, we synthesized cationic gold nanoparticles modified with a Tat-related peptide, which did not adversely affect cell viability and effectively delivered short interfering RNA into cells as non-covalent complex.Monet parantumattomat sairaudet johtuvat haitallisten proteiinien syntymisestä elimistössä. Näiden proteiinien toimintaa pyritään estämään yleisimmin perinteisillä pienimolekyylisillä lääkeaineilla. Vielä tehokkaampi ja turvallisempi vaikutus voidaan saada estämällä haitallisten proteiinien syntyminen jo RNA-tasolla esimerkiksi oligonukleotidilääkkeillä. Nämä lyhyet nukleiinihappomolekyylit sitoutuvat spesifisesti kohde-RNA:han ja voivat hajottaa sen useilla eri mekanismeilla. Ne voivat myös korjata RNA:n viallista prosessointia tai sitoutua suoraan kohdeproteiiniin. Oligonukleotideja tutkitaan muun muassa syöpien ja parantumattomien geneettisten sairauksien hoitoa varten. Tähän mennessä vain yksi ei-paikallinen oligonukleotidilääke on hyväksytty kliiniseen käyttöön Yhdysvalloissa, mutta toksisuuden takia sen käyttö on tarkoin rajattu eikä sitä ole hyväksytty Euroopassa. Ongelmana hoitojen kehittämisessä on oligonukleotidien entsymaattinen hajoaminen elimistössä sekä niiden huono pääsy solujen sisään. Oligonukleotideja on muunneltu entsyyminkestäviksi muokkaamalla niiden kemiallista rakennetta, mutta tällöin toksisuus voi lisääntyä ja tehokkuus heikentyä. Oligonukleotidi voidaan myös naamioida entsyymeiltä muuttamatta luonnollista nukleotidirankaa, jos se syklisoidaan liittämällä nukleotidiketjujen päät toisiinsa. Kolmas vaihtoehto on liittää oligonukleotidi sopivaan kantajamolekyyliin, jonka läsnäolo suojaa oligonukleotidia entsyymeiltä ja lisäksi auttaa sen vaikutuspaikalleen solun sisään. Lupaavia kantajia ovat muun muassa liposomit, polymeeri- ja kultananopartikkelit sekä soluun penetroituvat peptidit, joista viimeksi mainitut on löydetty tutkittaessa virusten tehokasta pääsyä soluihin. Näiden peptidien toivotaan pystyvän ohittamaan endosytoosireitin, jonka kautta soluun kulkiessaan oligonukleotidi voi jäädä loukkuun endosomiin. Tässä väitöskirjatyössä tutkittiin kemiallisten rakennemuunnosten ja nanopartikkelikantajien mahdollisuuksia oligonukleotidien saattamisessa soluihin. Osatöissä paitsi tutkittiin muunnosten ja kantajien biologisia vaikutuksia, myös optimoitiin synteesi- ja analyysimenetelmiä. Kovalentisti liitetyt soluun penetroituvat peptidit paransivat oligonukleotidien soluunottoa, mutta antisense-vaikutusta ei silti nähty soluissa yhdisteiden juututtua endosomeihin. DNA-diagnostiikkaa varten valmistetut sykliset oligonukleotidit osoittautuivat huomattavasti lineaarisia oligonukleotideja selektiivisemmiksi emäsmutaatioiden havaitsemisessa, ja myös niiden entsyyminkestävyys oli parempi. Yhteensä 44 yhdistettä analysoitiin sähkösumutusmassaspektrometrialla ja nestekromatografia massaspektrometrialla, jotka sopivat erinomaisesti muunneltujen oligonukleotidien analysointiin. Viimeisessä osatyössä syntetisoitiin kationisia kultananopartikkeleita, joihin lisättiin peptidianalogi. Tähän kantajaan kompleksoidulla pienellä häiritsevällä RNA:lla (siRNA:lla) hiljennettiin rekombinanttisolujen ilmentämä reportterigeeni heikentämättä solujen elävyyttä

Retinal Pigment Epithelial Cell Line with Fast Differentiation and Improved Barrier Properties

Retraction published on 14 March 2022, see Pharmaceutics 2022, 14(3), 635. https://doi.org/10.3390/pharmaceutics14030635Retinal pigment epithelium (RPE) acts as an outer blood-retinal barrier that limits the access of circulating xenobiotics to the eye. In addition, the RPE limits posterior elimination of intravitreally injected drugs to circulation. Thus, permeation in the RPE has a significant effect on ocular pharmacokinetics. The RPE is also a potentially important drug target in age-related macular degeneration. Therefore, the cell models of the RPE are important tools in ocular drug development, but poor availability and problems in reproducibility limit the use of primary RPE cell cultures. Furthermore, the best and widely used human cell line ARPE19 requires specialized culture conditions and a long time for cellular differentiation. In this paper, we describe a cell population arisen from the ARPE19 culture, with fast differentiation and improved barrier properties. This cell line, LEPI, forms clear microvilli and rapidly displays RPE-like cobblestone morphology after subculture in simple culture conditions. The LEPI cells show RPE-specific functions and expression of RPE65, ezrin, and BEST1 proteins. On filter, the LEPI cells develop tighter barrier than the ex vivo bovine RPE-choroid: permeability coefficients of beta-blockers (atenolol, nadolol, timolol, pindolol, metoprolol, betaxolol) ranged from 0.4 x 10(-6 )cm/sec to 2.3 x 10(-6) cm/sec depending on the drug lipophilicity. This rapidly differentiating cell line will be an asset in ocular studies since it is easily maintained, it grows and differentiates quickly and does not require specialized culture conditions for differentiation. Thus, this cell line is suitable for both small scale assays and high throughput screening in drug discovery and development.Peer reviewe

How can we develop communication in small groups?

Yhteistoiminnalliset oppimismenetelmät, kuten pienryhmäkeskustelut tai ryhmissä suoritettavat harjoitustyöt, ovat keskeinen osa korkeakouluopetusta. Tässä työssä tarkastelemme pienryhmissä tapahtuvaa oppimista erityisesti siihen liittyvän kommunikaation kannalta. Esittelemme alan peruskäsitteitä sekä kirjallisuudessa kuvattuja käytännön tapoja rakentaa opetukseen vuorovaikutusta edellyttäviä ja sitä tukevia elementtejä. Näitä ovat esimerkiksi tutkivat oppimismenetelmät, ryhmien ohjattua vaihtelua ja/tai sarjallisuutta sisältävät keskustelumenetelmät ja opiskelijoiden toimiminen vertaistuutoreina. Pohdimme tällaisten menetelmien käytännön soveltamista oman opetuksemme eli Helsingin yliopiston mikrobiologian, farmasian ja eläinlääketieteellisen anatomian oppiaineissa annettavan pienryhmäopetuksen viitekehyksessä ja selvitämme niitä keinoja, joilla voimme opettajina auttaa opiskelijoita parantamaan ryhmien kommunikaatiota. Laadukasta ryhmässä oppimista edistää opiskelijoiden sitoutuminen tutkivan keskustelun periaatteisiin, joihin kuuluu mm. tietojen jakaminen, asioiden kyseenalaistaminen ja perustelu, vastuun jakaminen ja osallistumaan rohkaisevan ilmapiirin luominen. Opettajan taholta tärkeitä toimia ovat ryhmässä ja vertaisopettajina toimivien opiskelijoiden riittävä valmentaminen tehtäviinsä, vuorovaikutuksen strukturointi ja ryhmätehtävien suunnittelu siten, että tehtävien suorittaminen edellyttää tasapuolista ja vuorovaikutteista osallistumista. Ryhmää ohjaavan opettajan on syytä kiinnittää huomiota myös omiin viestintätapoihinsa: ollakseen tehokasta opettajan apu on tärkeä sitoa opiskelijoiden omaan ajatteluun ja tehtävänratkaisustrategiaan. Suoritimme pienimuotoisen työparien keskinäisen ja ryhmien välisen kommunikaation perustasoa kartoittavan tutkimuksen ympäristömikrobiologian laboratoriokurssilla. Tutkimus toteutettiin palautekyselyn muodossa internetpohjaisen Presemo-työkalun välityksellä. Tulosten tarkastelussa kommunikaation kehittämiskohteina nousi esille mm. kommunikaatio eri äidinkieltä puhuvien opiskelijoiden välillä ja työryhmien välinen työn koordinointi.Non peer reviewe

Corneal and conjunctival drug permeability: Systematic comparison and pharmacokinetic impact in the eye

On the surface of the eye, both the cornea and conjunctiva are restricting ocular absorption of topically applied drugs, but barrier contributions of these two membranes have not been systemically compared. Herein, we studied permeability of 32 small molecular drug compounds across an isolated porcine cornea and built a quantitative structure-property relationship (QSPR) model for the permeability. Corneal drug permeability (data obtained for 25 drug molecules) showed a 52-fold range in permeability (0.09-4.70x10(-6) cm/s) and the most important molecular descriptors in predicting the permeability were hydrogen bond donor, polar surface area and halogen ratio. Corneal permeability values were compared to their conjunctival drug permeability values. Ocular drug bioavailability and systemic absorption via conjunctiva were predicted for this drug set with pharmacokinetic calculations. Drug bioavailability in the aqueous humour was simulated to be <5% and trans-conjunctival systemic absorption was 34-79% of the dose. Loss of drug across the conjunctiva to the blood circulation restricts significantly ocular drug bioavailability and, therefore, ocular absorption does not increase proportionally with the increasing corneal drug permeability.Peer reviewe

Biopharmaceutics of Topical Ophthalmic Suspensions: Importance of Viscosity and Particle Size in Ocular Absorption of Indomethacin

Eye drops of poorly soluble drugs are frequently formulated as suspensions. Bioavailability of suspended drug depends on the retention and dissolution of drug particles in the tear fluid, but these factors are still poorly understood. We investigated seven ocular indomethacin suspensions (experimental suspensions with two particle sizes and three viscosities, one commercial suspension) in physical and biological tests. The median particle size (d50) categories of the experimental suspensions were 0.37–1.33 and 3.12–3.50 µm and their viscosity levels were 1.3, 7.0, and 15 mPa·s. Smaller particle size facilitated ocular absorption of indomethacin to the aqueous humor of albino rabbits. In aqueous humor the AUC values of indomethacin suspensions with different particle sizes, but equal viscosity, differed over a 1.5 to 2.3-fold range. Higher viscosity increased ocular absorption 3.4–4.3-fold for the suspensions with similar particle sizes. Overall, the bioavailability range for the suspensions was about 8-fold. Instillation of larger particles resulted in higher tear fluid AUC values of total indomethacin (suspended and dissolved) as compared to application of smaller particles. Despite these tear fluid AUC values of total indomethacin, instillation of the larger particles resulted in smaller AUC levels of indomethacin in the aqueous humor. This suggests that the small particles yielded higher concentrations of dissolved indomethacin in the tear fluid, thereby leading to improved ocular bioavailability. This new conclusion was supported by ocular pharmacokinetic modeling. Both particle size and viscosity have a significant impact on drug concentrations in the tear fluid and ocular drug bioavailability from topical suspensions. Viscosity and particle size are the key players in the complex interplay of drug retention and dissolution in the tear fluid, thereby defining ocular drug absorption and bioequivalence of ocular suspensions

Biopharmaceutics of Topical Ophthalmic Suspensions: Importance of Viscosity and Particle Size in Ocular Absorption of Indomethacin

Eye drops of poorly soluble drugs are frequently formulated as suspensions. Bioavailability of suspended drug depends on the retention and dissolution of drug particles in the tear fluid, but these factors are still poorly understood. We investigated seven ocular indomethacin suspensions (experimental suspensions with two particle sizes and three viscosities, one commercial suspension) in physical and biological tests. The median particle size (d50) categories of the experimental suspensions were 0.37–1.33 and 3.12–3.50 µm and their viscosity levels were 1.3, 7.0, and 15 mPa·s. Smaller particle size facilitated ocular absorption of indomethacin to the aqueous humor of albino rabbits. In aqueous humor the AUC values of indomethacin suspensions with different particle sizes, but equal viscosity, differed over a 1.5 to 2.3-fold range. Higher viscosity increased ocular absorption 3.4–4.3-fold for the suspensions with similar particle sizes. Overall, the bioavailability range for the suspensions was about 8-fold. Instillation of larger particles resulted in higher tear fluid AUC values of total indomethacin (suspended and dissolved) as compared to application of smaller particles. Despite these tear fluid AUC values of total indomethacin, instillation of the larger particles resulted in smaller AUC levels of indomethacin in the aqueous humor. This suggests that the small particles yielded higher concentrations of dissolved indomethacin in the tear fluid, thereby leading to improved ocular bioavailability. This new conclusion was supported by ocular pharmacokinetic modeling. Both particle size and viscosity have a significant impact on drug concentrations in the tear fluid and ocular drug bioavailability from topical suspensions. Viscosity and particle size are the key players in the complex interplay of drug retention and dissolution in the tear fluid, thereby defining ocular drug absorption and bioequivalence of ocular suspensions

Students' experiences and engagement in a flipped classroom course on pharmacokinetics

Objective: The aim of this study was to examine whether the flipped classroom method enhances the quality of students' learning by exploring the change in students' processes of understanding, their relation to study success, and students' experiences of the course. Methods: A mass pharmacokinetics course, comprising 148 second-year pharmacy students was transformed by using the flipped classroom method. Students answered a 'HowULearn' questionnaire in their first and last lecture before (n=126) and after the course (n=100) to measure their processes of understanding. Paired sample t-test, chi-square test and correlation analysis were used to analyse the change in students' scores, examine the relationship between the scales, and course grade. Students' experiences of the course were examined with open-ended questions, and these responses were analysed using qualitative content analysis. Results: The response rate to the first and second questionnaire was 68%. Surface-level processing statistically decreased significantly (t= 3.72; p Conclusions: The study showed that the flipped classroom approach resulted in decreased surface-level and increased deep-level processing. This suggests that the flipped classroom method can improve students' processes of understanding.Peer reviewe

Microflow-Based Device for In Vitro and Ex Vivo Drug Permeability Studies

This paper presents a novel microflow-based concept for studying the permeability of in vitro cell models or ex vivo tissues. Using the proposed concept, we demonstrate how to maintain physiologically relevant test conditions and produce highly reproducible permeability values for a range (31) of drug compounds. The apparent permeability coefficients (P-app) showed excellent correlation (0.89) with the values from experiments performed with a conventional Ussing chamber. Additionally, the microflow-based concept produces notably more concentrated samples than the conventional Ussing chamber-based approach, despite the fact that more than 10 times smaller quantities of test compounds and biological membranes are needed in the microflow-based concept.Peer reviewe

Retraction: Hellinen et al. Retinal Pigment Epithelial Cell Line with Fast Differentiation and Improved Barrier Properties. Pharmaceutics 2019, 11, 412

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