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Izvještaj o 27. Smotri Sveučilišta u Zagrebu koja je održana od 10. do 12. studenoga 2022. godine na Zagrebačkom velesajmu. U organizaciji predstavljanja Farmaceutsko-biokemijskog fakulteta sudjelovalo je Povjerenstvo u sastavu: dr. sc. Laura Nižić Nodilo, dr. sc. Najda Rudman i Zvonimir Mlinarić, mag. pharm., zatim studentice medicinske biokemije Monika Polić, Inja Pavlić i Mateja Pavleka te studenti farmacije Tin Rogina, Josip Cvetko, Asja Fajtović, Nikola Bišćan, Vanja Cesarec, Bruna Burić, Sara Koprivica, Ivana Babić, Klara Ćosić, Marija Kosić, Karla Kostadinov, Una Jakšić i Karla Grudenić. Predstavljanje je bilo uspješno te je privuklo veliki broj posjetitelja. Farmaceutsko-biokemijski fakultet dobio je Priznanje u kategoriji poseban doprinos u edukaciji i informiranju posjetitelja.

Nasal delivery of the anti-dementia drug donepezil hydrochloride (DH) presents an alternative route of administration directly to the brain. Nasal powders stand out as formulations with great potential for nose-to-brain delivery. However, due to close contact of powders and the mucosa, formulations can cause stinging, itching and/or burning (SIB) sensations. The slug mucosal irritation assay is a quick and accurate screening method for early prediction of nasal discomfort caused by powders. DH-loaded chitosan, chitosan/mannitol and chitosan/lactose microspheres were prepared by spray-drying (Büchi Mini Spray Dryer B-290, equipped with ultrasonic nozzle). Furthermore, DH-loaded chitosan microspheres were blended (Turbula® shaker mixer) with sieved (45-63 µm) or spray-dried mannitol and lactose, reaching the same constituent weight ratios as in spray-dried hybrid microspheres. The particle size distribution and morphology were determined by laser diffraction method (Malvern Mastersizer 3000) and scanning electron microscopy (Tescan Vega 3), respectively. The slug mucosal irritation assay was performed to screen the powders regarding their potential to cause SIB sensations. DH-loaded nasal powders were successfully prepared by spray-drying and/or powder blending. All spray-dried particles (Dv50 9.7±0.0-29.0±1.2 µm) were smaller than sieved lactose and mannitol. The tested nasal powders showed either no discomfort or mild discomfort. Statistically significant increase in irritability compared to negative control was observed only for DH-loaded chitosan microspheres and their mixture with sieved lactose. The obtained results indicate the superior sensory effects of hybrid microspheres compared to chitosan microspheres and reveal the possible combined impact of filler particle size and morphology on the powder blend irritability. Slug mucosal irritation assay proved to be a useful tool for evaluation of nasal discomfort early in the formulation development. Prepared DH powders generally showed low irritability, confirming the appropriate selection of excipients and suitability of the spray-drying process to adjust particle morphology.

Nazalna primjena lijeka uobičajena je u liječenju lokalnih oboljenja nosne sluznice. Istražuje se i kao alternativni put sistemske primjene lijekova, a pruža mogućnost i izravne dostave lijeka u središnji živčani sustav. Ograničenja nazalne primjene lijeka uključuju otežanu dostavu lijeka u ciljno područje te mukocilijarni klirens koji skraćuje vrijeme kontakta lijeka i nosne sluznice. Nazalna primjena kortikosteroida česta je u liječenju bolesti nosne sluznice i paranazalnih sinusa, a u novije vrijeme predlaže se i u liječenju neuroupalnih procesa. U okviru ovog doktorskog rada, primjenjujući načela kakvoće utemeljene kroz dizajn, razvijena su tri inovativna in situ gelirajuća sustava s uklopljenim kortikosteroidom za nazalnu primjenu u obliku spreja: (i) in situ gelirajuća mikrosuspenzija flutikazonpropionata (FP-a), (ii) in situ gelirajuća nanosuspenzija FP-a pripravljena uz vlažno mljevenje te (iii) praškasti sustav mikrosfera s natrijevim deksametazonfosfatom (NDF-om) i inertnog nosača. Provedena je njihova temeljita fizičko-kemijska i biofarmaceutska karakterizacija. Polimerni sastav in situ gelirajućih mikro- i nanosuspenzija FP-a osigurao je pseudoplastično ponašanje, prikladnost za primjenu raspršivanjem (tj. prikladnu veličinu raspršenih kapljica i kut raspršenja) te svojstvo geliranja u kontaktu sa simuliranim nosnim fluidom (SNF). Mikrosfere s NDF-om, pripravljene sušenjem raspršivanjem, karakterizirane su velikom uspješnošću uklapanja lijeka, odgovarajućim sadržajem ostatne vlage te sposobnošću bubrenja u SNF-u pri čemu nastaje gel. Smjese mikrosfera s NDF-om i inertnog nosača bile su homogene te boljih svojstava tečenja i užeg kuta raspršenja od samih mikrosfera. Postignuta je uspješna depozicija sva tri farmaceutska oblika u ciljnoj regiji nosne šupljine, ovisno o željenom učinku. Razvijeni farmaceutski oblici karakterizirani su boljom mukoadhezivnošću u usporedbi s konvencionalnim oblicima. Oslobađanje lijeka iz svih razvijenih oblika bilo je prikladno za nazalnu primjenu. Nanonizacija FP-a rezultirala je većom mukoadhezivnošću formulacije te većom topljivošću i brzinom oslobađanja FP-a iz in situ gela, pružajući mogućnost smanjenja primijenjene doze. Svim sustavima potvrđena je biokompatibilnost in vitro na modelu Calu-3 stanica te fizičko-kemijska stabilnost. Praškasti sustav s NDF-om pokazao je potencijal poboljšanja permeacije lijeka kroz nazalni epitel. Prilagođavanjem procesnih, formulacijskih i/ili parametara primjene korištenjem dizajna eksperimenta razvijeni su inovativni farmaceutski oblici kortikosteroida s potencijalom produljenog zadržavanja na mjestu primjene, povoljnih biofarmaceutskih svojstava i optimirane depozicije u nosnoj šupljini. Nasal drug delivery is well estabilshed in the treatment of local nasal mucosa diseases. It is also being investigated as an alternative route of systemic drug delivery, as well as the possibility of direct drug delivery to the central nervous system. Limitations of nasal drug administration include difficult drug delivery to the target area and mucociliary clearance, which shortens the contact time between the drug and the nasal mucosa. Nasal use of corticosteroids is common in the treatment of diseases of the nasal mucosa and paranasal sinuse. Recently it has been suggested in the treatment of neuroinflammatory processes. Within this doctoral thesis, employing quality by design principles, three innovative in situ gelling pharmaceutical forms of corticosteroid for nasal spray application have been developed: (i) in situ gelling microsuspension of fluticasone propionate (FP), (ii) in situ gelling nanosuspension of FP prepared by wet media milling; and (iii) powder platform consisting of dexamethasone sodium phosphate (DSP)-loaded microspheres blended with an inert carrier. Thorough physicochemical and biopharmaceutical characterization of developed systems was performed. The polymer composition of the in situ gelling micro- and nanosuspensions of FP ensured shear thinning behavior, suitability for administration as a spray (i.e., suitable droplet size and spray angle), and immediate gelling in contact with simulated nasal fluid (SNF). Microspheres with DSP, prepared by spray drying, are characterized by high drug entrapment efficiency, adequate residual moisture content, and good swelling ability in SNF, forming a gel. Blends of microspheres with DSP and inert carrier were homogeneous and had better flow properties and a narrower spray cone angle than the microspheres alone. Successful deposition of all three pharmaceutical forms in the target region of the nasal cavity model was achieved, depending on the desired effect. Developed pharmaceutical forms are characterized by better mucoadhesiveness compared to conventional forms. Drug release from all developed forms was suitable for nasal administration. Nanonization of FP resulted in greater mucoadhesiveness of the formulation and increased solubility and release rate of FP from the in situ gel, providing the possibility of reducing the applied dose. Biocompatibility on the in vitro Calu-3 cell model and the physicochemical stability was confirmed for all developed formulations. The powder system with DSP showed the potential to improve drug permeation through the nasal epithelium. By varying process, formulation and/or administration parameters, employing design of experiments, innovative pharmaceutical forms of corticosteroids with the potential for prolonged retention at the site of administration, favourable biopharmaceutical properties and optimized deposition in the nasal cavity have been successfully developed.

Background Donepezil hydrochloride (DH) is commonly used for the treatment of symptoms of Alzheimer’s disease as an oral dosage form (1). Nasal DH administration provides the potential for direct and efficient DH delivery to the central nervous system, while reducing its systemic side effects (2). Purpose The aim of this study is to investigate the potential of spray dried DH loaded chitosan/mannitol microspheres for nasal DH delivery by relevant biopharmaceutical in vitro and ex vivo characterisation. Methods DH loaded chitosan/mannitol microspheres were characterised in terms of DH in vitro release profile, ex vivo mucoadhesiveness using porcine nasal mucosa, in vitro biocompatibility with Calu- 3 cells and DH permeability across Calu-3 cell monolayer. DH loaded chitosan microspheres, pure DH powder and/or pure mannitol were used as controls where appropriate. Results Both, chitosan and chitosan/mannitol microspheres provided prolonged DH release in comparison to pure DH powder. DH loaded chitosan/mannitol microspheres showed prominent mucoadhesive properties. Calu-3 cells exposed to microsphere suspensions prepared at chitosan concentration 10– 30 μg/mL retained viability above 90% in relation to negative control. The Papp values of DH from chitosan/mannitol and chitosan microspheres showed 1.16 and 1.30-fold higher DH permeation in relation to DH solution, respectively. Conclusion Formulating DH as chitosan/mannitol microspheres resulted in prolonged drug release, increased mucoadhesiveness and enhanced drug permeation across Calu-3 monolayer. Results on in vitro biocompatibility confirmed the formulation potential for safe and efficient DH nasal delivery.

BACKGROUND Due to disadvantages of oral administration of anti-dementia drug donepezil, nose-to-brain delivery attracts much attention as more efficient and safe route of administration (1). Nasal drug administration enables delivery to the central nervous system via olfactory nerve that innervates olfactory region of the nasal cavity. To ensure targeted delivery to the olfactory region, nasal deposition studies should be implemented in early phase of formulation development (2). PURPOSE The purpose of this study is to set the basis for the development of sprayable in situ gelling donepezil delivery system and to recognise the formulation and administration parameters with the highest impact on nasal deposition pattern. METHODS Thermosensitive chitosan (CS) based formulations for donepezil nose-to-brain delivery were prepared using CS and β-glycerophosphate as the gelling agent. In situ gelling systems were characterised in terms of rheological properties, droplet size distribution (DSD), spray cone angle (SCA) and nasal deposition pattern by using a representative 3D printed nasal cast. RESULTS Thermosensitive CS based in situ gelling donepezil formulations have been successfully prepared. Formulations showed suitable rheological characteristics and thermogelling properties. Under formulation and administration parameters employed, appropriate window of DSD and SCA was reached, resulting in targeted deposition in the olfactory region. CONCLUSION Preliminary studies revealed crucial formulation and administration parameters that resulted in suitable rheological properties and aerosol performance that paved the way for targeted nose- to-brain delivery.

Melatonin je neurohormon koji se primjenjuje u regulaciji cirkadijanog ritma, a u novije vrijeme istražuje se njegova uloga u liječenju pacijenata s ozbiljnijim simptomima bolesti COVID-19. Nazalna primjena pruža mogućnost jednostavne, izravne i brze dostave lijeka u središnji živčani sustav. S ciljem poboljšanja učinkovitosti nazalno primijenjenog lijeka, razvijaju se nanosuspenzije kao inovativni farmaceutski oblici poboljšanih mukoadhezivnih svojstava, povećane topljivosti i (volumenom kontrolirane) brzine otapanja lipofilnih lijekova. U ovom istraživanju pripravljene su nanosuspenzije melatonina metodom vlažnog mljevenja. Za mljevenje su korištene kuglice od cirkonijevog oksida stabiliziranog itrijem promjera 0, 1 mm, 0, 2 mm i 0, 4–0, 6 mm. Kao površinski aktivna tvar korišten je polisorbat 80 pri konstantnoj koncentraciji od 2, 86 mg/mL. Nanosuspenzije su pripravljene od melatonina i polisorbata u masenom omjeru 2:1, 1:1 i 1:2. Veličina dobivenih nanokristala i indeks polidisperznosti (PDI) određeni su metodom fotonske korelacijske spektroskopije odmah nakon priprave nanosuspenzija i tijekom 14 dana skladištenja pri 4 °C. Metoda vlažnog mljevenja uspješno je primijenjena za pripravu nanosuspenzija melatonina. Veličina čestica bila je u rasponu od 172, 2 ± 1, 0 nm do 344, 2 ± 2, 8 nm, dok je PDI bio u rasponu od 0, 126 ± 0, 016 do 0, 177 ± 0, 029. Kod formulacija s masenim omjerom melatonina i polisorbata 80 od 2:1 i 1:1 primijećen je rast nanokristala, dok su veličina čestica i PDI nanosuspenzije pripravljene pri masenom omjeru melatonina i polisorbata 80 od 1:2 ostali nepromijenjeni tijekom 14 dana skladištenja pri 4 °C. Utvrđena je prikladnost metode vlažnog mljevenja za pripravu nanosuspenzije melatonina s polisorbatom 80 kao surfaktantom u masenom omjeru 1 : 2. Dobiveni rezultati pružaju temelj za daljnji razvoj nanosustava melatonina za nazalnu primjenu.

Radionica će započeti kratkom prezentacijom o mogućnostima primjene lijeka u nos, te upoznavanjem s farmaceutskim oblicima lijeka prikladnim za tu namjenu. In vitro ispitivanja biokompatibilnosti i permeabilnosti zauzimaju važno mjesto u ranoj fazi razvoja farmaceutskog oblika lijeka. U radionici će biti prikazano korištenje humane Calu-3 stanične linije u navedenim ispitivanjima. Calu-3 stanična linija simulira fiziološka svojstva epitela sluznice nosa te omogućuje ispitivanje preživljavanja stanica koje su bile izložene novom farmaceutskom obliku kao i predviđanje uspješnosti prolaska lijeka kroz epitelnu barijeru nosne sluznice. Biokompatibilnost, odnosno citotoksičnost farmaceutskog oblika lijeka ispitat će se praćenjem metaboličke aktivnosti stanica. Permeacija lijeka iz farmaceutskog oblika kroz stanični model epitelne barijere pratit će se mjerenjem transepitelnog električnog otpora, te mjerenjem količine djelatne tvari koja je prošla kroz Calu-3 stanični monosloj.

Oral delivery of anti-dementia drug donepezil hydrochloride (DH) is related to several disadvantages such as gastrointestinal side effects and low bioavailability in the brain (1). Nasal administration of DH may provide its efficient and direct delivery to the brain via olfactory nerve that innervates olfactory region of the nasal cavity. Nasal powders have shown encouraging results in nose-to-brain drug delivery (2). This work aims to develop DH nasal powder platform by spray drying, employing Quality by Design (QbD) principles using statistical design of experiments (DoE). By coupling formulation, process and administration parameters the aim is to generate free-flowable powder with appropriate degree of swelling and spray cone angle, that will result in localised olfactory nasal deposition. DH nasal powders were prepared by spray dryer equipped with ultrasonic nozzle. Statistical design of experiments (JMP 14.0 software, USA) was employed in optimization of spray dried DH (Carbosynth Ltd., UK) loaded chitosan (Sigma Aldrich, USA) and mannitol (BDH Prolabo, UK) microspheres. DH loaded chitosan/mannitol (DH/C/M) were characterized in terms of flow properties, spray cone angle and degree of swelling. Nasal deposition studies were performed using a representative 3D printed nasal cast connected to respiratory pump simulating breathing conditions. Spray dried DH/C/M microspheres have been successfully prepared by spray drying. Powders exhibited adequate swelling: volume of simulated nasal fluid absorbed per mg of chitosan in the swelling process was between 27.7±2.8 and 67.0±0.7 mg/mL. Prepared microspheres were characterised in terms of flow properties (Hausner ratio between 1.15±0.06 and 1.32±0.07) and spray cone angle (22.5˚±0.5–28.3˚±0.9). QbD approach coupling formulation, process and administration parameters enabled optimisation of DH deposition profile reaching tremendously high percent of the applied dose deposited in the olfactory region (13.9±1.9– 65.5±0.2%). Statistical design enabled rational preparation of spray dried DH/C/M microspheres that showed favourable swelling and sprayability properties. Nasal deposition studies revealed the potential of strategy employed to deliver drug to the targeted regions of nasal cavity. References: 1. Agrawal M, et al. J. Control. Release. 2018. 281:139-177 2. Giuliani A, et al. Drug Deliv. 2018. 25:376-378

Alzheimerova bolest (AB) je kronična, progresivna bolest središnjeg živčanog sustava. Lijek prvog izbora za AB je inhibitor acetilkolinesteraze donepezil koji se primjenjuje oralno u dozi od 5 ili 10 mg. Oralna primjena donepezila u pacijenata s AB-om povezuje se s nedostatcima poput poteškoća s gutanjem, slabe suradljivosti zbog gorčine i gastrointestinalnih nuspojava [1], te ograničene raspoloživosti lijeka u mozgu zbog učinka krvno- moždane barijere [2]. Nazalna primjena donepezila istražuje se kao učinkovita alternativa zbog mogućnosti izravne dostave lijeka u mozak, uz zaobilaženje probavnog sustava. Ključni čimbenik predstavlja udio doze dostavljen do olfaktorne sluznice nosne šupljine, inervirane olfaktornim i trigeminalnim živcem koji omogućuju izravnu dostavu nazalno primijenjenog lijeka u mozak. Cilj rada bio je pripraviti i karakterizirati mikrosfere s donepezilom te ispitati utjecaj parametara primjene (kut primjene u odnosu na horizontalnu ravninu i protok udahnutog zraka) na nazalnu depoziciju pripravljenih mikrosfera. Mikrosfere su pripravljene sušenjem raspršivanjem vodenih otopina donepezila i pomoćnih tvari kitozana i manitola. Mikrosfere su zadovoljile kriterije za primjenu u nosnoj šupljini s obzirom na uspješnost uklapanja, sadržaj lijeka, sadržaj vlage, veličinu čestica i reološka svojstva. U svrhu ispitivanja nazalne depozicije mikrosfera, korišten je 3D printani model nosne šupljine, izrađen prema CT snimci glave 62-godišnjeg pacijenta zdravih nosnih putova. Mikrosfere s donepezilom uspješno su dostavljene u ciljnu (olfaktornu) regiju nosne šupljine. Najučinkovitija dostava donepezila u olfaktornu regiju (68, 8 ± 7, 4 % primijenjene doze) postignuta je primjenom mikrosfera s donepezilom uređajem za nazalnu dostavu praška pri kutu primjene prema horizontalnoj ravnini od 30° i uz zadržavanje daha. Smanjenjem kuta primjene u odnosu na horizontalnu ravninu rastao je udio lijeka dostavljen u olfaktornu regiju. Protok zraka od 20 L/min smanjio je odlaganje donepezila u olfaktornoj regiji, a povećao u regijama (anteriornoj i posteriornoj) koje nisu od interesa za dostavu lijeka iz nosne šupljine u mozak. Povoljniji rezultati depozicije donepezila uz zadržavanje daha mogu biti korisni i iz pozicije pacijenta jer pojednostavljuju primjenu formulacije (izbjegnuti su problemi vezani uz koordinaciju potiska i udaha), što može povećati suradljivost pacijenta i poboljšati kliničke ishode.

INTRODUCTION Epilepsy requires a rapid and effective treatment, targeting the brain. In this area, the intranasal administration of lipid nanosystems, such as nanostructured lipid carriers (NLC) was suggested as a promising strategy. This route allows drug passage directly from the nose to the brain, bypassing the blood-brain barrier [1]. However, nasal administration faces some physiological barriers, such as the mucociliary clearance, which decreases the residence time of the formulation in the nasal cavity, reducing drug absorption. To circumvent this limitation, the use of ion sensitive polymers, such as pectin, which interacts with the calcium ions of the nasal mucus, increases the residence time of the NLC formulation within the nasal cavity [2]. The aim of this work was to develop an ion sensitive in situ hydrogel containing an optimized diazepam- loaded NLC formulation and to study its nasal deposition using a 3D nasal cavity model. The experiments started with the determination of the in situ hydrogel gelation time and the spray cone angle, using two types of pectin, at different concentrations. Subsequently, the nasal deposition in a 3D nasal cavity model was evaluated at with an airflow of 0 L/min and different administration angles. EXPERIMENTAL Preparation and characterization of in situ hydrogels containing diazepam-loaded NLC Costa et al. [3] previously optimized the diazepam-loaded NLC composition and preparation method. For the in situ hydrogels preparation, pectin CF005 and pectin CF025 were added at concentrations of 0.6% and 1.2% to the aqueous phase of the diazepam-loaded NLC, under continuous stirring, at 20.0±0.5ºC. Evaluation of the gelation time The gelation time of the in situ hydrogels was determined by the oscillatory gelation time test (Modular Compact Rheometer 102, Anton Paar GmbH, Austria) after mixing the diazepam-loaded NLC suspension containing pectin with simulated nasal fluid, in a volume ratio of 1:1. For this test, changes in the storage modulus (G´) and loss modulus (G´´) were recorded. The experiment was performed at 34ºC, using a parallel plate 50 (PP50) measuring system. The defined measuring position was 0.5 mm and the angular frequency was fixed at 6.28 rad s-1. Three replicate measurements were conducted for each formulation and the results are presented as mean values ± standard deviation (SD). Spray cone angle determination The spray cone angle was measured using a virtual protractor, after spraying the formulation against a dark background. The formulation was first placed in a Spray Pump 3K (Aeropump, Germany) and sprayed three times. A camera recorded the emitted plume and the spray cone angle was analyzed for each sample. Evaluation of the formulation deposition profile in a 3D nasal cavity model The 3D nasal cavity model was carried out using a computed tomography (CT) scan of a 62-year old patient with healthy nasal passages airways, obtained from the database of University Hospital Center “Sestre milosrdnice” (Ethics Committees approval codes: EP-9941/19-3 class: 003-06/21- 02/001, registry number:251-29- 11/1-21-01-1 ; PEER class: 643-02/21-03/01, registry number 251-62-03- 21-8) [4]. The nasal model is divided in (Figure 1): anterior region, turbinate region with detachable olfactory part, septum with detachable olfactory part, and nasopharynx. For the nasal deposition profile, the 3D nasal model was covered with Sar-gel®, which turns purple in contact with water and has calcium ions that induce pectin gelation. The administration of the formulations was performed on the right nostril of the 3D model. The fractional spray deposition pattern was determined weighting the amount of deposited formulation in all the nasal regions. Three replicates were conducted for each formulation and the results are presented as mean values ± SD. RESULTS Evaluation of the gelation time For both concentrations of pectin CF025, the G´ was higher than G´´ during the entire measuring period, which indicates that the gelation occurred immediately after the contact with the nasal fluid. The same was not observed with the formulations with pectin CF005. For this reason, further experiments were only performed with pectin CF025. Spray cone angle determination The spray cone angle of the in situ hydrogels was 34.3±0.2º and 16.3±0.4º for the formulation with 0.6% and 1.2% of pectin, respectively, which shows that spray cone angle decreased with the increase of pectin concentration. It was reported that narrow cone angles could increase the drug deposition on the olfactory region. However, if the angle is too narrow, the impact of the sprayed jet might cause patient discomfort. For this reason, only the formulation with 0.6% pectin was chosen for the nasal deposition studies [5]. Evaluation of the formulation deposition profile in a 3D nasal cavity model The deposition in the olfactory region (olfactory part of septum + olfactory part of turbinates) at different administration angles, from the vertical plane (0 and 20º) and horizontal plane (60 and 75º) are shown in Table 1. Table 1. Deposition profile of the in situ hydrogels containing diazepam-loaded NLC in the 3D nasal cavity model, administered at angles of 0 and 20° from the vertical plane and 60 and 75° from the horizontal plane. AHP: angle from horizontal plane ; AVP: angle from vertical plane OS: olfactory part of septum ; OT: olfactory part of turbinates ; R: recovery. Based on the results of Table 1, it was concluded that the angles of administration from the horizontal and vertical planes affect the nasal deposition in the olfactory region. The highest olfactory deposition was obtained for the administration angles of 75º from the horizontal plane and 0º from the vertical plane, with 28.10% of the formulation found in the olfactory region (Figure 2). In addition, it was observed that olfactory deposition increases with the increase of the administration angle from the horizontal plane and with the decrease of administration angle from the vertical plane. CONCLUSION The in situ hydrogel containing diazepam-loaded NLC with 0.6% of pectin CF025 presented an adequate gelation time and provided a suitable spray cone angle. About 28% of the formulation was deposited in the olfactory region, which is considered high. From the results of this study, we conclude that angles of administration from horizontal and vertical planes have impact on olfactory deposition. Currently, more factors that may also interfere, including the presence of airflow and the use of different nasal devices, are being studied by our research group. ACKNOWLEDGEMENTS This work was supported by Fundação para a Ciência e a Tecnologia (FCT) (SFRH/136177/2018, Portugal), by the Applied Molecular Bio-sciences Unit-UCIBIO, which is financed by national funds from FCT (UIDP/04378/2020 and UIDB/04378/2020) and CIBB (FCT reference: UIDB/04539/2020). Also, this work has been supported in part by Croatian Science Foundation under the project UIP-2017-05-4592". REFERENCES 1. Costa, C. et al. Nose-to-brain delivery of lipid-based nanosystems for epileptic seizures and anxiety crisis, Journal of Controlled Release. 187-200 (2019). 2. Nižić, L. et al. Innovative sprayable in situ gelling fluticasone suspension: Development and optimization of nasal deposition. International Journal of Pharmaceutics. 563, 445-456 (2019). 3. Costa, C.P. et al. Quality by design (QbD) optimization of diazepam-loaded nanostructured lipid carriers (NLC) for nose-to-brain delivery: Toxicological effect of surface charge on human neuronal cells. International Journal of Pharmaceutics. 607, 120933 (2021). 4. Nižić Nodilo, L. et al. A Dry Powder Platform for Nose-to-Brain Delivery of Dexamethasone: Formulation Development and Nasal Deposition Studies. Pharmaceutics. 13, 795 (2021). 5. Maaz, A. et al. In Vitro Evaluation of Nasal Aerosol Depositions: An Insight for Direct Nose to Brain Drug Delivery. Pharmaceutics. 13, 1079 (2021).

Nasal powders are promising platforms in nose-to- brain drug delivery (1). Spray drying is a single- step, fast and scalable dry powder preparation method (2). Design of experiments (DoE) is often used in product-related optimization of the spray drying process (2). The potential of ultrasonic nozzle for the preparation of nasal powders has not yet been deeply investigated. The aim of this study is to prepare donepezil loaded chitosan/mannitol microspheres by spray drying using an ultrasonic nozzle. Employing Quality by Design approach will enable elucidating and understanding the impact of formulation and process parameters on spray drying yield and microspheres physicochemical properties. Statistical DoE (JMP 14.0 software, USA) was employed in optimisation of spray dried donepezil hydrochloride (DH ; Carbosynth Ltd, UK) loaded chitosan (Sigma Aldrich, USA) and mannitol (BDH Prolabo, UK) microspheres. The following properties were observed as responses: process yield, entrapment efficiency (high performance liquid chromatography ; Perkin Elmer, USA), particle size (Mastersizer 3000 ; Malvern Instruments Ltd, UK) and moisture content (TA Instruments Q500 ; TA Instruments, USA). Microspheres morphology and surface properties were observed by scanning electron microscopy (SEM) using a Tescan Vega 3 microscope (Czech Republic). Spray dried chitosan/mannitol microspheres with high DH entrapment efficiency and adequate moisture content were successfully prepared using an ultrasonic nozzle, fitting the size requirements for targeted nasal delivery (majority of particles larger than 10 µm). Process yield was in line with literature reports. Regression modelling revealed the impact of both, formulation and process parameters on yield, moisture content and particle size. The obtained microspheres were uniform and spherical with smooth surface, as showed by SEM micrographs. This study confirmed the suitability of ultrasonic nozzle for preparation of microspheres for nasal drug delivery. Predictive power of regression model obtained for observed responses enables the development of donepezil loaded chitosan/mannitol nasal powder platform with built-in quality, maximizing cost and time savings. References: 1. Giuliani A, Balducci AG, Zironi E, Colombo G, Bortolotti F, Lorenzini L, Galligioni V, Pagliuca G, Scagliarini A, Calzà L, Sonvico F. Drug Deliv. 2018. 25:376-387 2. Ziaee A, Albadarin AB, Padrela L, Femmer T, O’Reilly E, Walker G. Spray drying of pharmaceuticals and biopharmaceuticals: Critical parameters and experimental process optimization approaches. Eur J Pharm Sci. 2019 ; 12: 300–318.

INTRODUCTION Donepezil hydrochloride (DH) is widely used for the symptomatic treatment of Alzheimer’s disease (AD), currently available only in form for oral administration. Oral administration of DH presents low bioavailability in the brain, first-pass metabolism and unwanted gastrointestinal side effects [1]. Nasal delivery of DH proposes an alternative route of administration directly to central nervous system. Nasal powders have shown encouraging results in nose-to-brain drug delivery [2]. The purpose of this study is to set the basis in the development of powder delivery platform suitable for DH nasal delivery. MATERIALS AND METHODS DH nasal powders were prepared by spray drying using Büchi Mini Spray Dryer B-290, equipped with ultrasonic nozzle. Spray dried solutions contained DH, low molecular weight chitosan and/or mannitol or lactose monohydrate. Microspheres were characterized in terms of entrapment efficiency, particle size and zeta potential, flow properties and spray cone angle. Nasal deposition studies were performed using a representative 3D printed nasal cast connected to respiratory pump simulating breathing conditions. RESULTS DH nasal powders have been successfully prepared. Under formulation, process and administration parameters employed, appropriate window of particle size (greater than 10 µm), flow properties (Hausner ratio between 1.16 and 1.25) and spray cone angle (17.5-19.8º) was reached resulting in targeted deposition pattern within the nasal cavity. CONCLUSION Preliminary studies revealed the formulation parameters to be included in experimental design aiming to enlighten a relation of DH nasal powder properties to deposition pattern. Such an approach potentiates the development of DH nasal powder delivery platform with built-in qualities, maximizing cost and time saving. Acknowledgement This work has been supported in part by Croatian Science Foundation under the project UIP-2017-05-4592 and European Social Fund under the Croatian Science Foundation project DOK- 2020-01-2473. REFERENCES 1. Agrawal M, et al. J. Control. Release (2018) 281:139-177. 2. Giuliani A, et al. Drug Deliv. (2018) 25:376- 378.

Radionica je započela kratkom prezentacijom o mogućnostima primjene lijeka u nos, s naglaskom na praškastim sustavima koji se mogu primijeniti u obliku spreja. Sudionici su imali priliku promatrati sastavljanje modela nosne šupljine iz više dijelova i upoznati se s ciljnim područjima nazalne dostave lijeka. Nadalje, promatrala se sudbina praškastog sustava u nosnoj šupljini uz korištenje respiratorne pumpe koja oponaša disanje.

INTRODUCTION Strategies to improve nasal drug delivery include development of formulations which reduce mucociliary clearance [1] and careful selection of nasal delivery device [2]. In situ gelling nanosuspensions present combined approach to increase mucoadhesion and prolong drug retention at nasal mucosa [1]. The aim of this study is to develop an in situ gelling fluticasone propionate nanosuspension and assess its nasal deposition after spraying with jet-flow nebulizer. MATERIALS AND METHODS Fluticasone propionate nanosuspensions prepared by wet media milling, were mixed with pectin and sodium hyaluronate solutions at different ratios. Formulations were characterised in terms of particle size, zeta- potential, surface tension, rheological properties, in vitro biocompatibility using Calu-3 cells, mucoadhesiveness with porcine mucosa and nasal deposition pattern using a jet- flow nebulizer and a 3D printed nasal cast. RESULTS In situ gelling fluticasone propionate nanosuspensions were characterised by particle diameter between 140.3 to 166.8 nm, zeta-potential between –89.5 and –83.2 mV and surface tension of about 37 mN m-1 at 25 °C. Rheological studies confirmed the gelation of formulations when mixed with simulated nasal fluid. The formed gel showed appropriate strength and stability profile. All formulations were biocompatible (cell viability > 90%) and mucoadhesive. Spraying the formulations with jet-flow nebulizer resulted in targeted deposition within the 3D printed nasal cast. CONCLUSION Biocompatible in situ gelling fluticasone propionate nanosuspensions have been successfully prepared. Their rheological properties imply instant gelling in contact with nasal fluid, which, combined with mucoadhesive properties, indicate the potential for extended residence time at nasal mucosa. Nasal deposition profiles proved the suitability of jet-flow nebulizer for nasal delivery of in situ gelling nanosuspensions. Acknowledgement This work has been supported in part by Croatian Science Foundation under the project UIP-2017-05-4592 and European Social Fund under the Croatian Science Foundation project DOK- 2020-01-2473. REFERENCES 1. Alshweiat, A, et al. Int J Pharm (2020) 579:119-166 2. Xi, J, et al. Pharm Res (2016) 33:1527- 1541

Introduction: Nasal route of administration offers a variety of therapeutic opportunities. Recently, it has been proposed as a route of choice for direct nose-to-brain glucocorticoid delivery to control neuroinflammation in patients with severe Covid-19 (1). Nasal powders have shown encouraging results in nose-to-brain drug delivery (2). This work presents the development of nasal powder delivery platform for dexamethasone sodium phosphate (DSP), employing Quality by Design (QbD) principles and including consideration of nasal deposition in the early phase of formulation development. Methods: QbD approach was employed in optimization of spray-dried DSP-loaded pectin and hypromellose microspheres. The optimized microspheres were blended with lactose monohydrate or mannitol at particle size of 45-63 µm as an inert carrier. Powder blends were characterized in terms of homogeneity, flow properties, spray cone angle, in vitro biocompatibility using Calu-3 cells and mucoadhesion using porcine nasal mucosa. Nasal deposition studies were performed using a 3D printed nasal cast connected to respiratory pump. Results: Swellable DSP-loaded polymeric microspheres (Dv50=2.88±0.01 mm) ensuring diffusion-controlled drug release completed in 90 minutes were developed based on QbD approach. Optimised microspheres/inert carrier powder blends were shown to be homogenous and suitable for administration by spraying. SEM micrographs of powder blends revealed microspheres adhered to carrier particles, fitting into the surface within the capsule upon device actuation (1.7-6.3% vs. 14.8%) and narrower spray cone angle (19.6-22.0° vs. 26.5°), which, coupled with appropriate paricle size range, resulted in improved nasal deposition profile. Microspheres were proven to be mucoadhesive showing 12-fold higher work of adhesion in relation to pure drug powder. Blending with inert carriers did not impair their mucoadhesive properties. Powder blends were shown to be biocompatible, inducing no decrease in cell viability. Conclusion/Impact: QbD approach enabled rational desing of spray-dried DSP-loaded polymeric microspheres. Optimised microspheres/inert carrier powder blends showed favourable biopharmaceutcal and sprayability properties. Nasal deposition studies revealed the potential of strategy employed to deliver drug to the targeted regions of nasal cavity.

Nazalna primjena lijekova široko je zastupljena u liječenju oboljenja sluznice nosa. U odnosu na tekuće farmaceutske oblike, nazalni prašci omogućuju veću koncentraciju i produljeno zadržavanje djelatne tvari na mjestu primjene te bolju stabilnost. Većina nazalnih prašaka temeljena je na mukoadhezivnim polimerima sa sposobnošću bubrenja, a od posebnog interesa su polimerne mikrosfere s uklopljenim lijekom pripravljene sušenjem raspršivanjem [1]. Mikrosfere natrijevog deksametazon fosfata (NDF), pektina i hipromeloze pripravljene su sušenjem raspršivanjem uz variranje protoka uzorka te pri konstantnim postavkama kapaciteta aspiratora, ulazne temperature i protoka komprimiranog zraka. Mikrosferama su određeni sadržaj i uspješnost uklapanja lijeka, veličina te stupanj bubrenja u pročišćenoj vodi i umjetnom nosnom fluidu (SNF). Odabrane mikrosfere pomiješane su s inertnim nosačem (α-laktoza monohidrat ili manitol veličine čestica 45–63 μm) u omjerima 1:1, 1:2, 1:4, 1:7 te 1:9 (m/m) pomoću mješača prašaka. Tako dobivenim prašcima određeni su homogenost, svojstva tečenja i stupanj bubrenja. Mikrosfere pektina i hipromeloze (srednjeg volumnog promjera Dv50 od 2, 15 ± 0, 01 do 13, 65 ± 0, 45 μm), sadržaja lijeka od 1, 30 ± 0, 02 do 33, 50 ± 0, 44 % (m/m), uspješno su pripravljene metodom sušenja raspršivanjem. Veličina mikrosfera povećavala se s koncentracijom sastavnica u sušenoj otopini. Mikrosfere su apsorbirale značajno manji volumen SNF-a po jediničnoj masi u usporedbi s pročišćenom vodom, uslijed umrežavanja pektina ionima kalcija iz SNF-a. Sposobnost bubrenja mikrosfera u SNF-u povećavala se s porastom udjela hipromeloze u polimernom matriksu i sa smanjenjem sadržaja uklopljenog lijeka. Praškasti sustavi odabranih mikrosfera i nosača bili su prikladne homogenosti te boljih reoloških svojstava u odnosu na same mikrosfere. Dodatak nosača povećao je volumen apsorbiranog medija u donorskom odjeljku po miligramu mikrosfera, proporcionalno udjelu nosača. Mikrosfere pektina i hipromeloze primjenjive su u razvoju praškastog farmaceutskog oblika za nazalnu primjenu NDF-a. Utvrđena je prikladnost laktoze monohidrata i manitola kao nosača mikrosfera. Praškasti sustavi masenog omjera mikrosfera i inertnog nosača 1:9 najprikladniji su za dodatna in vitro te in vivo ispitivanja. Ovaj rad sufinancirala je Hrvatska zaklada za znanost projektom UIP-2017-05-4592. [1] L. Tiozzo Fasiolo, M. D. Manniello, E. Tratta, F. Buttini, A. Rossi, F. Sonvico, F. Bortolotti, P. Russo, G. Colombo. Opportunity and challenges of nasal powders: Drug formulation and delivery. Eur J Pharm Sci 113 (2018) 2–17.

U videozapisu je prikazan proces priprave praškastog sustava za nazalnu primjenu te biofarmaceutska karakterizacija koja uključuje ispitivanje in vitro oslobađanja i mukoadhezivnosti.

Introduction: Nanosuspensions present innovative formulations with enhanced solubility and volume- controlled dissolution rate of poorly water- soluble drugs as well as improved particle mucoadhesive properties(1). Developing of in situ gelling systems presents the strategy to reduce the mucociliary clearance and extend the contact time between the drug and the nasal mucosa (2). In order to ensure targeted delivery of such systems, deposition studies should be implemented in early phase of formulation development (3). The aim of this study is to prepare in situ gelling fluticasone propionate (FP) nanosuspension with rheological properties suitable for nasal delivery and develop a regression model for nasal deposition pattern using Quality by Design(QbD) principles. Methods: QbD approach was employed in development of in situ gelling FP nanosuspensions prepared by wet media milling (1), with Tween 80 as a suspending agent, and pectin and sodium hyaluronate (SH) as mucoadhesive and/or in situ gelling polymers. The in situ gelling FP nanosuspensions were characterised in terms of particle size and zeta- potential, sprayability and rheological properties. Nasal deposition pattern was studied using 3D printed nasal cast connected to respiratory pump. Results: In situ gelling FP nanosuspensions (d=133.0–213.7 nm, PDI=0.227-0.386 ; zeta-potential=(-93.1) - (-77.2) mV) have been successfully prepared and showed suitable stability profile during 1-month storage at 4°C. They showed shear-thinning behaviour and instant gelation upon mixing with simulated nasal fluid. Regression modelling revealed Tween 80 concentration, SH concentration and interaction between FP and SH concentration to be the key factors determining sprayability of developed nanosuspensions. Formulation and administration parameters employed resulted in suitable range of droplet size distribution (Dv10=16.9-30.4 μm ; Dv50=38.7-100.7 μm ; Dv90=98.2- 219.3 μm) and spray cone angle (17.0-36.2°) ensuring targeted deposition within the nasal cavity. Conclusions/Impact: The formulation strategy employed showed the potential of coupling inherent advantages of nanosuspensions with benefits of shear-thinning in situ gelling platforms including appropriate sprayability, targeted deposition in the regions beyond the nasal valve and potential to ensure prolonged retention at the site of delivery. QbD approach enabled recognition of crucial parameters in formulation and nasal administration of in situ gelling FP nanosuspensions.

Purpurna rudbekija (Ehinacea purpurea (L.) Moench, Asteraceae) se tradicionalno koristi za pomoć kod prehlade i gripe te u terapiji kožnih oboljenja i manjih rana (1). Nadzemni dijelovi sadrže različite bioaktivne tvari među kojima se posebno ističu fenolne kiseline, uglavnom derivati kavene kiseline (2). U radu je razvijena ekološki prihvatljiva metoda ultrazvučne ekstrakcije purpurne rudbekije pomoću glicerola, određeni su optimalni uvjeti ekstrakcije te je ispitan učinak priređenih ekstrakata na cijeljenje rane in vitro. Biljni materijal je donirala tvrtka Suban. Identitet je potvrđen od strane autora. Ultrazvučna ekstrakcija je optimirana korištenjem Box-Behnken dizajna. Neovisne varijable bile su udio glicerola (50-90 %, m/m), temperatura (40-70 C), snaga ultrazvuka (72-360 W) i vrijeme ekstrakcije (20-60 min). Ovisne varijable bile su sadržaj derivata kavene kiseline i antiradikalna aktivnost ekstrakata. Određivanje fenolnih kiselina provedeno je korištenjem HPLC-DAD metode. Antiradikalna aktivnost određena je spektrofotometrijski, pomoću DPPH slobodnog radikala. Učinak ekstrakata priređenih pri optimalnim uvjetima na vijabilnost stanica i cijeljenje rane ispitan je in vitro provođenjem MTT odnosno ''scratch'' testa na HaCaT stanicama. Utvrđeno je da su optimalni uvjeti za ekstrakciju fenolnih kiselina iznosili: 70 % glicerola (m/m), 60 C, 72 W te 60 min. Najbolju antiradikalnu aktivnost pokazao je ekstrakt priređen korištenjem 50 % glicerola (m/m), 33 C, 72 W tijekom 40 min. U koncentracijama nižim od 25 % priređeni ekstrakti nisu negativno utjecali na vijabilnost stanica. Oba glicerolna ekstrakta ubrzala su zatvaranje pukotine u konfluentnom sloju stanica, tj. cijeljenje rane in vitro, pri čemu je utjecaj otapala, u usporedbi s ekstraktima, bio zanemariv. Provedena istraživanja ukazuju na značajan potencijal glicerolnih ekstrakata E. purpurea u liječenju rana i manjih kožnih oštećenja.

The presentation provided an overview of project aims and scopes in the field of development and nasal deposition studies of innovative drug delivery systems. Diverse considerations and characterisation procedures were presented, highlighting the need for interdisciplinary approach.

Na videoprezentaciji je ukratko predstavljena primjena silikonskog modela nosne šupljine u ispitivanju depozicije lijeka u nosnoj šupljini raspršenog pomoću sprej pumpice i nebulizatora.