Development and validation of the HPLC-DAD-FLD method for the analysis of alpelisib in human plasma

Rak dojke predstavlja značajan javnozdravstveni problem zbog svojeg utjecaja na živote brojnih žena širom svijeta. U posljednjih nekoliko godina, značajan napredak postignut je u liječenju raka dojke pozitivnog na hormonski receptor i negativnog na HER2 s PIK3CA mutacijom korištenjem kombinacije alpelisiba s drugim lijekovima kao što su fulvestrant ili anastrozol. Alpelisib, inhibitor PI3K-a, pokazuje velik potencijal u uspješnom liječenju ove vrste raka dojke, ali optimalni ishodi liječenja zahtijevaju učinkovito praćenje terapije. U svrhu ovog rada razvijena je i validirana HPLC-DAD-FLD metoda za analizu alpelisiba u ljudskoj plazmi. Uzorci plazme pripremljeni su jednostavnom metodom taloženja proteina pomoću organskog otapala, što je osiguralo čistu analitnu matricu pogodnu za daljnju analizu. Korištena je kolona Phenomenex Gemini C18, dimenzija 150 × 4,6 mm i veličine čestica stacionarne faze 5 µm pri konstantnoj temperaturi od 25 °C. U razvijenoj metodi korištena je gradijentna eluacija mobilnom fazom koja se sastoji od 0,1 % mravlje kiseline u vodi i 0,1 % mravlje kiseline u metanolu. Brzina protoka iznosila je 1 mL/min. Korišteni su UV-Vis detektor i fluorescencijski detektor. Validacija metode provedena je ispitivanjem parametara linearnosti, točnosti, preciznosti i utjecaja matrice. Metoda je pokazala zadovoljavajuću linearnost unutar raspona koncentracija od 300 do 3000 ng/mL (r za DAD iznosi 0,99755, a za FLD 0,99664), visoku točnost (sve dobivene koncentracije čine od -1,8 do 11,3 % stvarne vrijednosti) i preciznost (RSD vrijednosti iznose od 1,1 do 4,3 %), te minimalan utjecaj matriksa (Meff se kreće od 95,8 do 106,1 %), čime je potvrđena njezina pouzdanost za analizu alpelisiba u kliničkim uzorcima. Konačno, razvijena i validirana metoda primijenjena je za analizu alpelisiba u plazmi tri pacijentice od kojih su rezultati za dvije pacijentice unutar validiranog raspona metode, a kod treće pacijentice alpelisib nije bilo moguće detektirati. Postoji mogućnost da pacijentica nije bila adherentna ili da je koncentracija u plazmi bila preniska da bi se ovom metodom mogla detektirati. Također je moguće da postoje interindividualne varijabilnosti koje utječu na koncentraciju alpelisiba u plazmi. Razvijena HPLC-DAD-FLD metoda za analizu alpelisiba u ljudskoj plazmi predstavlja napredak u mogućnostima praćenja terapije kod pacijentica s rakom dojke. Ova metoda može se koristiti za daljnja ispitivanja farmakokinetike alpelisiba, kao i za personalizaciju liječenja na temelju interindividualnih varijabilnosti među pacijenticama. Time se može postići bolja prilagodba terapije individualnim potrebama pacijentica, što će u konačnici rezultirati poboljšanjem ishoda liječenja i kvalitete života pacijentica oboljelih od raka dojke.Breast cancer represents a significant public health problem due to its impact on the lives of numerous women worldwide. In recent years, significant progress has been made in the treatment of hormone receptor-positive and HER2-negative breast cancer with PIK3CA mutation using a combination of alpelisib with other drugs such as fulvestrant or anastrozole. Alpelisib, a PI3K inhibitor, shows great potential in successfully treating this type of breast cancer, but optimal treatment outcomes require effective therapymonitoring. For the purpose of this study, an HPLC-DAD-FLD method was developed and validated for the analysis of alpelisib in human plasma. Plasma samples were prepared using a simple protein precipitation method with an organic solvent, ensuring a clean analytical matrix suitable for further analysis. A Phenomenex Gemini C18 column, dimensions 150 × 4.6 mm and particle size 5 µm, was used at a constant temperature of 25 °C. The developed method employed gradient elution with a mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in methanol. The flow rate was 1 mL/min. Both UV-Vis and fluorescence detectors were used. Method validation was carried out by examining parameters of linearity, accuracy, precision, and matrix effect. The method demonstrated satisfactory linearity within the concentration range of 300 to 3000 ng/mL (with an r value of 0.99755 for DAD and 0.99664 for FLD), high accuracy (all obtained concentrations range from -1.8 to 11.3% of the true value) and precision (RSD values range from 1.1 to 4.3%), and minimal matrix effect (Meff ranges from 95.8 to 106.1%), confirming its reliability for the analysis of alpelisib in clinical samples.Finally, the developed and validated method was applied to analyze alpelisib in the plasma of three patients. The results for two patients were within the validated range of the method, while alpelisib could not be detected in the third patient's plasma. It is possible that the patient was not adherent to the therapy or that the plasma concentration was too low to be detected by this method. Additionally, inter-individual variability may affect the concentration of alpelisib in plasma.The developed HPLC-DAD-FLD method for analyzing alpelisib in human plasma represents an advancement in the capabilities of therapy monitoring for breast cancer patients. This method can be used for further pharmacokinetic studies of alpelisib and for the personalization of treatment based on inter-individual variability among patients. This approach can lead to better adjustment of therapy to the individual needs of patients, ultimately resulting in improved treatment outcomes and quality of life for breast cancer patients

Attitudes of pharmacists in Croatia towards the independent pharmacist prescribing program

Posljednjih desetljeća farmaceutska struka prolazi kroz značajne promjene diljem svijeta, s naglaskom na proširenje uloga ljekarnika izvan tradicionalnih okvira izdavanja lijekova. Jedan od smjerova takvih promjena je i koncept samostalnih farmaceuta propisivača (eng. Independent pharmacist prescriber) koji je uspješno implementiran u nekoliko zemalja svijeta. Samostalnim farmaceutima propisivačima u Velikoj Britaniji, u 2024. godini, dopušteno je procijeniti pacijente, postaviti dijagnozu i propisati lijek za bilo koje stanje ili bolest (isključujući diamorfin, kokain i dipipanon), te za bilo kojeg pacijenta unutar njihovih kliničkih kompetencija. Opseg djelovanja ovih farmaceuta širok je i obuhvaća rad na razini primarne i sekundarne zdravstvene zaštite. Cilj ovog istraživanja bio je utvrditi stavove hrvatskih ljekarnika prema programu samostalnih farmaceuta propisivača (eng. Independent pharmacist prescriber). Putem on-line anketnog upitnika ispitanici su mogli izraziti svoje stavove prema programu, njegovim prednostima i nedostatcima, ali i njegovoj potencijalnog implementaciji i izvođenju u Republici Hrvatskoj. U istraživanju je sudjelovalo 455 ispitanika, od kojih je 82,6% bilo žena, a 17,4% muškaraca, te je medijan dobi iznosio 34 godine. Većina ispitanika (85,5%) smatra da bi propisivanje lijekova od strane farmaceuta imalo pozitivan utjecaj na zdravstveni sustav. Također, svaka navedena prednost ocijenjena je većinski kao iznimno važna. „Neprihvaćanje od strane liječnika“ hrvatski su ljekarnici ocijenili kao najveću prepreku u okviru potencijalne implementacije programa, a „Vremenska ograničenja“ kao najmanju. Gotovo su svi ispitanici smatrali kako uloga farmaceuta kao propisivača lijekova predstavlja profesionalno napredovanje, a 63,5% ispitanika u potpunosti se složilo kako bi postali samostalni farmaceuti propisivači, kada bi to bila mogućnost. Dodatna obuka nakon fakulteta, specijalistički studij i kurikulum fakulteta bili su najčešći odgovori na pitanje gdje i na koji način bi se trebala odvijati obuka za stjecanje titule samostalnog farmaceuta propisivača. Zaključno, stav hrvatskih ljekarnika prema programu samostalnih farmaceuta propisivača (eng. Independent pharmacist prescriber) vrlo je pozitivan i optimističan, a približno dvije trećine ljekarnika izrazilo je želju za postizanjem titule samostalnog farmaceuta propisivača. Ovi rezultati sugeriraju želju hrvatskih ljekarnika za daljnjim napredovanjem i usavršavanjem, te upućuju na potrebu za raspravom o potencijalnim proširenjima uloga ljekarnika u sklopu hrvatskog zdravstvenog sustava.In recent decades, the pharmaceutical profession has undergone significant changes worldwide, with a focus on expanding the roles of pharmacists beyond the traditional framework of dispensing medications. One direction of these changes is the concept of independent pharmacist prescribers, which has been successfully implemented in several countries worldwide. In the UK, as of 2024, independent pharmacist prescribers are allowed to assess patients, diagnose, and prescribe medications for any condition or illness (excluding diamorphine, cocaine, and dipipanone), as well as for any patient within their clinical competence. The scope of these pharmacists' work is broad, covering both primary and secondary healthcare. The aim of this research was to determine the attitudes of Croatian pharmacists towards the independent pharmacist prescriber program. Through an online survey, respondents were able to express their views on the program, its advantages and disadvantages, as well as its potential implementation and execution in Croatia. The study involved 455 respondents, 82.6% of whom were women, and 17.4% men, with a median age of 34. Most respondents (85.5%) believe that pharmacist prescribing would have a positive impact on the healthcare system. Additionally, all listed advantages were predominantly rated as extremely important. "Lack of acceptance by doctors" was seen by Croatian pharmacists as the biggest obstacle to the potential implementation of the program, while "Time constraints" were considered the smallest obstacle. Nearly all respondents believed that the role of pharmacists as prescribers represents professional development, and 63.5% strongly agreed that they would become independent pharmacist prescribers if given the opportunity. Postgraduate training, specialist studies, and university curriculum were the most common answers regarding how and where the training for achieving the title of independent pharmacist prescriber should take place. In conclusion, the attitudes of Croatian pharmacists towards the independent pharmacist prescriber program are very positive and optimistic, with approximately two-thirds expressing a desire to obtain the title. These results suggest a strong willingness for further professional development and point to the need for discussions on potential expansions of pharmacists' roles within the Croatian healthcare system

Determination of iron and copper in encapsulated food supplements for the treatment of inflammatory bowel diseases

U današnje vrijeme sve je veća primjena biljnih dodataka prehrani koje su važan izvor biološki aktivnih sastavnica, a čije djelovanje je pozitivno na zdravlje ljudi. S obzirom da se kurkuma, indijski tamjanovac i justicija koriste kao dodaci prehrani koji ublažavanju simptoma upalnih bolesti crijeva, cilj ovog rada bio je procijeniti kvalitetu dodataka prehrani s obzirom na sadržaj željeza i bakra. Koncentracije navedenih minerala u analiziranim dodacima prehrani određene su plamenom i grafitnom atomskom apsorpcijskom spektrometrijom, a prije analize u svrhu pripreme svi su uzorci spaljeni mikrovalnom digestijom. Rezultati istraživanja su pokazali da su koncentracije željeza i bakra u dodacima prehrani koji se koriste kod upalnih bolesti crijeva bile u dozvoljenim rasponima. Primjećuje se znatna razlika u ispitanim metalima kod uzoraka u tvrdim kapsulama u odnosu na one u mekim kapsulama, te se također može primijetiti značajnija koncentracija željeza u odnosu na koncentraciju bakra.Nowadays, there is an increasing use of herbal dietary supplements, which are an important source of biologically active components, and whose effects are positive for human health. Considering that turmeric, Indian frankincense and Justicia are used as dietary supplements to alleviate the symptoms of inflammatory bowel diseases, the aim of this work was to evaluate the quality of dietary supplements regarding iron and copper content. The concentrations of the mentioned minerals in the analyzed dietary supplements were determined by flame and graphite atomic absorption spectrometry, and before the analysis for the purpose of preparation, all samples were burned by microwave digestion. The results of the research showed that the concentrations of iron and copper in dietary supplements used for inflammatory bowel diseases were within the permissible ranges. A significant difference in the tested metals can be observed for samples in hard capsules compared to those in soft capsules, and a significant concentration of iron can also be observed in relation to the concentration of copper

Optimization of fragmentation conditions for alpelisib and deuterated abemaciclib in a triple quadrupole mass spectrometer

Rak dojke je najčešća zloćudna novotvorina u žena, a njegova prevalencija širom svijeta raste. Personalizirani pristup je bitan dio liječenja, stoga je važno razviti specifične analitičke metode za istraživanje lijekova. Alpelisib i abemaciklib su se pokazali učinkovitim lijekovima u liječenju hormonski ovisnog raka dojke. Oba djeluju na signalne puteve koji su važni za stanični rast i proliferaciju te posljedično, rast tumora. Cilj ovog istraživanja je bio optimizirati uvjete fragmentacije alpelisiba i deuteriranog abemacikliba u trostrukim kvadrupolima dva različita spektrometra masa, Agilent Ultivo i Agilent 6470. Određene su optimalne vrijednosti fragmentorskih napona i kolizijskih energija, te je provedena identifikacija i kvantifikacija ključnih fragmenata specifičnih za ove spojeve. U Agilent Ultivo spektrometru masa uz pozitivnu ionizaciju elektroraspršenjem, alpelisib daje molekulski ion m/z vrijednosti 442,1 uz fragmetorski napon od 120 V. Nastaju fragmentni ioni m/z vrijednosti 328,0, 114,7, 288,0 i 273,1 uz kolizijske energije od 25, 15 45 i 60 eV. Deuterirani abemaciklib daje molekulski ion m/z vrijednosti 515,2 uz fragmentorski napon od 120 V. Nastaju fragmentni ioni m/z vrijednosti 176,8, 393,0 i 351,3 uz kolizijske energije od 105, 25 i 70 eV. U Agilent 6470 spektrometru masa uz pozitivnu ionizaciju elektroraspršenjem, alpelisib daje molekulski ion m/z vrijednosti 442,2, uz fragmentorski napon od 140 V. Nastaju fragmentni ioni m/z vrijednosti 328,1 115,0, 288,1, 273,0 i 129,0 uz kolizijske energije od 25, 15, 50, 60 i 105 eV. Deuterirani abemaciklib daje molekulski ion m/z vrijednosti 515,3 uz fragmentorski napon od 120 eV. Nastaju fragmentni ioni od 177,0, 393,1 i 244,9 uz kolizijske energije od 105, 25 i 75 eV. Provedena je analiza i uz negativnu ionizaciju elektroraspršenjem, gdje se ova metoda pokazala manje osjetljivom.Breast cancer is the most common malignant tumor in women, and its prevalence is increasing worldwide. A personalized approach is a crucial part of treatment, making it important to develop specific analytical methods for drug research. Alpelisib and abemaciclib have proven to be effective drugs in the treatment of hormone-dependent breast cancer. Both act on signaling pathways important for cell growth and proliferation, and consequently, tumor growth.The aim of this study was to optimize the fragmentation conditions for alpelisib and deuterated abemaciclib in triple quadrupoles of two different mass spectrometers, the Agilent Ultivo and Agilent 6470. Optimal values for fragmentor voltages and collision energies were determined, and identification and quantification of key fragments specific to these compounds were conducted.In the Agilent Ultivo mass spectrometer with positive electrospray ionization, alpelisib produces a molecular ion with an m/z value of 442,1 at a fragmentor voltage of 120 V. Fragment ions with m/z values of 328,0, 114,7, 288,0, and 273,1 are formed with collision energies of 25, 15, 45, and 60 eV, respectively. Deuterated abemaciclib produces a molecular ion with an m/z value of 515.2 at a fragmentor voltage of 120 V. Fragment ions with m/z values of 176,8, 393,0, and 351,3 are formed with collision energies of 105, 25, and 70 eV, respectively.In the Agilent 6470 mass spectrometer with positive electrospray ionization, alpelisib produces a molecular ion with an m/z value of 442,2 at a fragmentor voltage of 140 V. Fragment ions with m/z values of 328,1, 115,0, 288,1, 273,0 and 129,0 are formed with collision energies of 25, 15, 50, and 105 eV, respectively. Deuterated abemaciclib produces a molecular ion with an m/z value of 515,3 at a fragmentor voltage of 120 V. Fragment ions with m/z values of 177,0, 393,1, and 244,9 are formed with collision energies of 105, 25, and 75 eV, respectively. Analysis was also conducted using negative electrospray ionization, where this method proved to be less sensitive

Analysis of cor samples and identification of Fusarium genus molds

Plijesni roda Fusarium su najbrojniji rod gljivica koje kontaminiraju kukuruz. Poznat je njihov štetan učinak na zdravlje ljudi i životinja, bilo uzrokovanjem fuzarioze ili akutnim i kroničnim trovanjem mikotoksinima, a temperatura zraka i količina padalina su se pokazale kao vrlo značajni čimbenici o kojima ovisi njihova pojavnost na kukuruzu. Klimatske promjene zbog toga uvelike utječu na kontaminaciju kukuruza i prehrambenih proizvoda dovodeći do porasta koncentracija plijesni na usjevima, većih koncentracija kontaminanata u prehrambenim namirnicama i sve većem riziku za ljudsko zdravlje te smanjenoj isplativosti poljoprivrednih uzgoja. Mikološka analiza metodom razrjeđenja na hranjivim podlogama (DRBC i MGA) provedena je na 50 uzoraka kukuruza od kojih su 25 iz 2021. godine, a 25 iz 2022. godine. Istraživanje je pokazalo je da su koncentracije plijesni veće na uzorcima iz 2022. godine, za koju je prosječna vrijednost koncentracije na DRBC agaru 7,93 x 105 CFU/g dok je za 2021. srednja vrijednost koncentracije na DRBC agaru 1,32 x 105 CFU/g. Srednja vrijednost koncentracije na MGA agaru je također veća za 2022. godinu. Dobivene srednje vrijednosti su vrlo visoke, a većina uzoraka premašuje dozvoljene maksimalne koncentracije koje smiju biti prisutne u hrani. Na svim uzorcima je došlo do porasta plijesni roda Fusarium, što pokazuje na dominaciju ovog roda kao kontaminanta kukuruza. Na DRBC agaru plijesni roda Penicillium su izrasle na 96% uzoraka, a plijesni roda Aspergillus na njih 72% što ih čini sljedećima po zastupljenosti. Od ostalih plijesni pojavljuju se vrste iz roda Cladosporium, Absidia, Trichoderma i Mucor, a na 28% uzoraka je uočen porast kvasaca. Svi uzorci s kvascima poraslih na DRBC agaru su iz 2022. godine. Na MGA agaru osim fuzarija je također došlo do porasta kvasaca na nekim uzorcima, od kojih je 84% također iz 2022. godine. Izolacijom i identifikacijom plijesni roda Fusarium primjenom molekularne metode tj. sekvenciranjem genske regije ITS pokazano je da se u većini uzoraka radi o vrsti F. napiforme i/ili F. verticilloides dok je za samo 6% uzoraka identificirana vrsta F. fujikuroi i/ili F. proliferatum. Sve identificirane vrste pripadaju F. fujikuroi kompleksu vrsta. Genska regija ITS pokazala se nedovoljno specifična za razlučivanje srodnih vrsta unutar fuzarijskih kompleksa. Za razlučivanje vrsta unutar pojedinih fuzarijskih kompleksa potrebno je primijeniti više genskih markera kao što su tef1 i rpb2. Visoka učestalost i dominacija vrsta iz kompleksa F. fujikuroi u uzorcima kukuruza može predstavljati opasnost za zdravlje ljudi jer se radi o uzročnicima mikoza kao i o poznatim proizvođačima mikotoksina, poglavito iz skupine fumonizina.Molds of the genus Fusarium are the most prevalent fungi contaminating corn. Their harmful effects on human and animal health are well-documented, either through causing fusariosis or through acute and chronic poisoning by mycotoxins. Air temperature and precipitation levels have been identified as significant factors influencing their occurrence in corn. Consequently, climate change greatly impacts the contamination of corn and food products, leading to increased concentrations of molds in crops, higher contaminant levels in food products, an elevated risk to human health, and reduced profitability in agricultural production. Mycological analysis using the dilution method on nutrient media (DRBC and MGA) was conducted on 50 corn samples, 25 from 2021 and 25 from 2022. The study revealed that mold concentrations were higher in the 2022 samples, with an average concentration value on DRBC agar of 7.93 x 10⁵ CFU/g, compared to an average concentration value on DRBC agar of 1.32 x 10⁵ CFU/g for 2021. The average concentration value on MGA agar was also higher for 2022. The obtained average values are very high, with most samples exceeding the maximum allowable concentrations permitted in food. All samples showed an increase in molds of the genus Fusarium, and in the majority of samples, they were the dominant genera. On DRBC agar, molds of the genus Penicillium grew on 96% of samples, and molds of the genus Aspergillus on 72%, making them the next most prevalent. Other molds present included species from the genera Cladosporium, Absidia, Trichoderma, and Mucor, with 28% of the samples showing yeast growth. All samples with yeast growth on DRBC agar were from 2022. On MGA agar, in addition to Fusarium, yeast growth was also observed on some samples, 84% of which were from 2022. Isolation and identification of molds of the genus Fusarium using molecular methods, specifically sequencing of the ITS gene region, demonstrated that most samples contained the species F. napiforme and/or F. verticillioides, while only 6% of the isolates were identified as F. fujikuroi and/or F. proliferatum. All identified species belong to the F. fujikuroi species complex. The ITS gene region proved to be insufficiently specific for distinguishing closely related species within the Fusarium complexes. To differentiate species within specific Fusarium complexes, the application of multiple genetic markers, such as tef1α and rpb2, is required. The high prevalence and dominance of species from the F. fujikuroi complex in corn samples may pose a risk to human health, as they are known pathogens causing mycoses and are recognized producers of mycotoxins, particularly fumonisins

Determination of heavy metal content in cocoa samples using TXRF-W, TXRF-Mo and EDXRF techniques

Plod stabla kakaovca jest zrno kakaa koje je ključni sastojak u proizvodnji kakao praha i čokolade. Zbog velike zastupljenosti u ljudskoj prehrani, nužno je poznavanje sastava kakaa kako bi se osigurala kvaliteta i sigurnost samih proizvoda. Glavni cilj ovog rada jest odrediti elementni sastav kakaa te usporediti rezultate dobivene različitim analitičkim tehnikama (TXRF-W, TXRF-Mo, EDXRF). Uzorci su pripremljeni mikrovalnom digestijom te u obliku peleta te je određeno prisustvo sljedećih elemenata: željezo, bakar, cink, rubidij, stroncij, mangan, nikal i krom. Priprema uzorka u obliku peleta jeftinija je, manje zahtjevna i ekološki prihvatljivija u odnosu na mikrovalnu digestiju. Analizom je utvrđeno da se rezultati dobiveni različitim analitičkim tehnikama podudaraju (posebice između TXRF-Mo i EDXRF). Sve su se tehnike pokazale kao vrlo korisne budući da omogućavaju identifikaciju i kvantifikaciju vrlo niskih koncentracija elemenata prisutnih u uzorcima kakaa, s naglaskom na TXRF-Mo tehniku koja se pokazala kao najosjetljivija.The fruit of the cocoa tree is the cocoa bean which is a key ingredient in the production of cocoa powder and chocolate. Since cocoa is widely used in the human diet, it is necessary to know the composition of cocoa in order to ensure quality and safety of the products themselves. The main goal of this work is to determine the elemental composition of cocoa and to compare the results obtained by different analytical techniques (TXRF-W, TXRF-Mo, EDXRF). The samples were prepared by microwave digestion and in the form of pellets and the presence of the following elements was determined: iron, copper, zinc, rubidium, strontium, manganese, nickel and chromium. Sample preparation in the form of pellets is less-expensive, simpler and more environmentally friendly than microwave digestion. The analysis showed that the results obtained with the different analytical techniques were consistent (especially between TXRF-Mo and EDXRF). All techniques have proven to be very effective, as they allow the identification and quantification of very low concentrations of the elements present in cocoa samples, with the TXRF-Mo technique which proving to be the most sensitive

Synthesis, characterization and derivatization of a polysubstituted piperidino-morpholine spiro building block

Male molekule s novim mehanizmima djelovanja neophodne su u rješavanju izazovnih biomedicinskih problema današnjice poput rezistencije na postojeće lijekove za liječenje raka i zaraznih bolesti. Jedan od pristupa otkriću novih biološki aktivnih molekula uključuje sintezu usmjerenu na raznolikost (engl. diversity-oriented synthesis, DOS), koja primarno podrazumijeva razlike u strukturnoj okosnici, supstituentima i stereokemiji. Različiti lijekovi koji u svojoj strukturi sadrže morfolinski prsten imaju široku terapijsku primjenu. Privilegirani je farmakofor s mogućnošću stvaranja molekulskih interakcija s raznovrsnim biološkim metama i modificiranja farmakokinetičkih svojstava. Stoga je morfolin strukturni motiv od posebnog interesa za dizajniranje i razvoj lijekova. Istodobno je povećan interes za sintezu manje planarnih biološki aktivnih spojeva što rezultira uvođenjem spiro spojeva u nova istraživanja. Slijedom navedenih spoznaja, cilj ovog diplomskog rada bila je sinteza i karakterizacija polisupstituiranog piperidino-morfolinskog spiro gradivnog bloka te njegova derivatizacija u svrhu dobivanja novih spojeva s različitim svojstvima, korištenjem principa sinteze usmjerene na raznolikost. Osnovna strukturna okosnica sintetizirana je preko dva sintetska puta, u šest odnosno četiri reakcijska koraka, od kojih je zadnji korak intramolekulska ciklizacija u bazičnim uvjetima. U nekoliko sljedećih sintetskih koraka, iz glavnog intermedijara 6, pripravljeni su odgovarajući prekursori. Šest novih derivata 9a-c i 13a-c dobiveno je iz spojeva 8 i 12 s dvije slobodne amino-skupine, završnim reakcijama N-acetiliranja, sulfonamidacije i Eschweiler-Clarke reakcijom. Strukturna raznolikost postignuta je supstituiranjem osnovne strukturne okosnice u tri različita položaja, a strukture novosintetiziranih spojeva potvrđene su uobičajenim spektroskopskim metodama (1D i 2D NMR, MS). Dokazana je izražena trodimenzionalnost struktura finalnih spojeva 9a-c i 13a-c pomoću NOESY NMR spektara. Finalnim spojevima određena su i fizikalno-kemijska svojstva in silico.Small molecules with novel mechanisms of action are necessary in addressing today’s challenging biomedical problems such as resistance to existing drugs for the treatment of cancer and infectious diseases. One of the approaches to the discovery of new bioactive molecules includes diversity-oriented synthesis (DOS), which primarily implies differences in the molecular scaffold, substituents and stereochemistry. Various drugs containing a morpholine ring in their structure are widely used for the treatment of numerous diseases. It is a privileged pharmacophore with the possibility of forming molecular interactions with diverse biological targets and modifying pharmacokinetic properties. Therefore, morpholine is a structural motif of special interest for the drug design and development. At the same time, interest for the synthesis of less planar bioactive compounds increases, resulting in the introduction of spiro compounds into new studies. Following the stated facts, the aim of this thesis was to synthesize and characterize polysubstituted piperidino-morpholine spiro building block. Furthermore, this scaffold was derivatized in order to obtain novel compounds with different properties, using the diversity-oriented synthesis approach. The scaffold was synthesized via two synthetic routes, in six or four reaction steps, with the last step of intramolecular cyclization under basic conditions. In several subsequent chemical reactions, the corresponding precursors were prepared from the main intermediate 6. Six novel derivatives 9a-c and 13a-c were obtained from compounds 8 and 12 with two free amino groups by N-acetylation, sulfonamidation and Eschweiler-Clarke reaction respectively. Structural diversity was achieved by substituting the scaffold at three different positions. The structures of the novel compounds were confirmed by standard spectroscopic methods (1D and 2D NMR, MS). Noticeable three-dimensionality of the structures of the final compounds 9a-c and 13a-c was proved by means of NOESY NMR spectra. Additionally, the physicochemical properties of final compounds were calculated in silico

The effect of botulinum toxin type a on neural activation in the region of the trigeminal caudal nucleus in a model of inflammatory pain induced by formalin

Bol kroničnog trajanja je veliki problem u svijetu i u mnogim se slučajevima ona ne može uspješno kontrolirati konvencionalnim analgeticima. Tijekom posljednja tri desetljeća u nekliničkim i kliničkim istraživanjima pokazan je potencijal botulinum toksina tipa A (BT-A) u nanogramskim dozama u smanjenju i dugotrajnoj kontroli kronične boli različite etiologije. Međutim, njegov mehanizam djelovanja na patološku bolnu preosjetljivost nije do kraja poznat. Dosadašnja istraživanja pokazala su da BT-A s perifernog mjesta primjene retroaksonalnim transportom putuje u središnji živčani sustav gdje modulira djelovanje različitih neurotransmiterskih sustava, da bi najnovija istraživanja sugerirala i njegov među-sinaptički prijenos. U ovom diplomskom radu je istraženo središnje antinociceptivno djelovanje periferno jednostrano primjenjenog BT-A u modelu upalne boli izazvane bilatralnom primjenom formalina u području njuške štakora. Praćenjem ekspresije c-Fos-a pokazano da BT-A smanjuje neuralnu aktivaciju u neuronima TNC-a na strani na kojoj je primjenjen, ali i na suprotnoj strani, pri čemu neutralizirajući antitoksin za BT-A poništava to djelovanje na obje strane. Rezultati ovog rada se slažu s do sada provedenim istraživanjima i upućuju na među-sinaptički prijenos kao važan čimbenik u kompleksnom mehanizmu antinociceptivnog djelovanja BT-A.Chronic pain is big health-care issue worldwide, and in many cases, it cannot be completely resolved with conventional analgetics. Over the past three decades, non-clinical and clinical research demonstrated that in nanogram concentrations, botulinum toxin type A (BT-A) has potential to alleviate and long-term control chronic pain of various etiologies. However, its mechanism of action on pathological pain hypersensitivity is not fully understood. So far, researchers have shown that BT-A travels from peripheral application site to central nervous system via retrograde axonal transport, where it modulates different neurotransmitter systems, and the latest research suggests transynaptic transmission. In this master’s thesis, the central antinocieptive action of unilaterally, peripherally applied BT-A, was investigated in the inflammatory pain model caused by bilateral application of formalin to the rat whisker pad. By monitoring expression of c-Fos as a marker of neural activation, it was shown that BT-A reduces neural activation in the neurons of the TNC region on the side of the application as well as on the opposite side, whilst neutralizing BT-A antitoxin reverses BT-A effect on both sides. Obtained results in this master’s thesis are consistent with previous studies and indicate that transynaptic transmission is one of the important factors in the complex antinociceptive mechanism of BT-A

Influence of rice sample preparation on elemental composition - application of total reflection X-ray fluorescence spectroscopy

Riža je namirnica koja se u velikim količinama konzumira svakodnevno diljem svijeta. Zbog zagađenosti okoliša, može biti kontaminirana teškim metalima te je stoga određivanje elementarnog sastava riže od iznimne važnosti za očuvanje ljudskog zdravlja. Glavni cilj ovoga rada jest odrediti utjecaj različite predobrade uzoraka riže na elementarni sadržaj te usporediti učinkovitost korištenih tehnika u određivanju sadržaja metala (TXRF-Mo, ICP-MS). Prikupljeni uzorak riže prije postupka mikrovalne digestije korišten je i) bez prethodne obrade, ii) nakon što se tri puta isprao s ultra čistom vodom, iii) nakon što je usitnjen u mlinu. Svi uzorci su nakon predobrade podvrgnuti postupku mikrovalne digestije te analizirani primjenom TXRF-Mo i ICP-MS. U svim ispitanim uzorcima određeni su slijedeći elementi: željezo, bakar, mangan, cink, stroncij i nikal. Dobiveni rezultati za sadržaj elemenata u uzorcima samljevene riže i riže tri puta isprane s ultračistom vodom uspoređeni su sa sadržajem elemenata u uzorcima riže bez prethodne obrade. Sadržaj analiziranih elemenata određenih s TXRF tehnikom usporediv je s rezultatima ICP-MS tehnike. U usporedbi s netretiranim uzorkom, rezultati analize su utvrdili da ispiranje zrna riže, za razliku od mljevenja, uzrokuje značajnije promjene sadržaja za sve određene elemente. Prethodna obrada uzorka je pokazala najveći utjecaj na sadržaj željeza, a najmanji na mangan. Unatoč tome, nije utvrđena statistički značajna razlika kod dodatne obrade uzorka te je stoga donesen zaključak kako predpriprema uzorka nije potrebna. Analiza TXRF tehnikom iziskuje vrlo male količine uzoraka i reagensa, proizvodi malo kemijskog otpada, ne iziskuje rashladne sustave i plinove te uključuje jednostavnu kvantifikaciju dodatkom unutarnjeg standarada što ju čini bržom, jednostavnijom te ekološki i ekonomski prihvatljivijom tehnikom od ICP-MS-a.Rice is consumed daily in large quantities all over the world. Due to environmental pollution, rice can be contaminated with heavy metals, which is why controlling the elemental composition of rice grains is extremely important for the preservation of human health. The main objective of this work is to determine the influence of different pretreatments of rice samples on elemental analysis and to compare the effectiveness of the techniques used to determine the metal content (TXRF-Mo, ICP-MS). The rice sample collected before microwave digestion was used i) without pretreatment, ii) after washing three times with ultrapure water and iii) after milling in a mill. After processing, all samples were subjected to microwave digestion and analyzed by TXRF-Mo and ICP-MS. The following elements were determined in all analyzed samples: iron, copper, manganese, zinc, strontium and nickel. The results for the content of elements in samples of milled rice and rice washed three times with ultrapure water were compared with the content of elements in samples of rice without prior treatment. The content of the elements analyzed with the TXRF technique corresponds to the ICP-MS element content. Compared to the untreated samples, the results showed that washing the rice grains, as opposed to milling, caused more significant changes in the content of all analyzed elements. The pre-treatment of the sample had the greatest influence on the iron content and the least on the manganese content. Regardless of this, no statistically significant difference was found with additional sample preparation, so it was concluded that pretreatment of the samples is not necessary. Analysis using the TXRF technique requires very small amounts of samples and reagents, generates little chemical waste, does not require cooling systems or gasses and involves simple quantification with internal standards, making it a faster, simpler and more environmentally and economically acceptable technique than ICP-MS

Antinociceptive effect of botulinum toxin type A on CGRP release in the trigeminal ganglion in an animal model of formalin-induced pain

Botulinum toksin tipa A (BT-A) potentan je neurotoksin kojeg proizvodi bakterija Clostridium botulinum. Mehanizam djelovanja mu se temelji na sprječavanju otpuštanja neurotransmitera u sinaptičku pukotinu proteolitičkim kidanjem SNARE proteina zaduženih za njihovu egzocitozu. Dugo se vremena smatralo da je djelovanje BT-A ograničeno samo na periferna područja u kojima je i primijenjen, zbog čega mu je terapijska primjena bila ograničena na liječenje autonomnih poremećaja, lokalizirane spastičnosti i hiperkinetičkih poremećaja pokreta. S vremenom je bilo ustanovljeno kako BT-A ima izraženo antinociceptivno djelovanje i na središnjoj razini uz minimalne nuspojave, dugotrajan učinak i izostanak tolerancije za razliku od ostalih analgetika sa sistemskim djelovanjem što ga čini izuzetno privlačnim kandidatom za daljnja istraživanja, posebice u vidu liječenja boli kroničnog tijeka. Cilj ovog diplomskog rada bio je istražiti utjecaj BT-A na ekspresiju CGRP-a, neuropeptidnog medijatora boli i upale, u području trigeminalnog ganglija, u modelu upalne orofacijalne boli uzrokovane primjenom formalina u štakora. BT-A (7 i.j./kg) bio je unilateralno primijenjen u područje desnog brka (ipsilateralno) Wistar štakora. Šest dana kasnije u područje lijevog brka (kontralateralno) uzrokovana je bol primjenom 50 μL 2,5 % formalina nakon čega su životinje žrtvovane te su analizirani prerezi njihovih trigeminalnih ganglija. U negativnoj kontrolnoj skupini se umjesto BT-A koristila fiziološka otopina primijenjena bilateralno, dok se u pozitivnoj kontrolnoj skupini fiziološka otopina primijenila ipsilateralno, a 50 μL 2,5 % formalina kontralateralno. Imunohistokemijskom analizom i statističkom obradom praćen je i kvantificiran intenzitet signala CGRP-a u poprečnim prerezima trigeminalnih ganglija. U eksperimentalnoj skupini primijećeno je statistički značajno smanjenje ekspresije CGRP-a u odnosnu na kontrolnu skupinu. Dobiveni rezultati sugeriraju da BT-A djeluje i na suprotnoj strani od mjesta primjene, što upućuje na njegovu mogućnost aksonalnog transporta i potencijalnog među-sinaptičkog prijenosa (transcitoze). Zaključno, ovaj diplomski rad pokazuje da BT-A može djelovati ne samo na lokalnoj razini, već i na razini središnjeg živčanog sustava putem moguće transcitoze što otvara prostor za daljnja istraživanja njegovog središnjeg antinociceptivnog djelovanja.Botulinum toxin type A (BT-A) is a potent neurotoxin produced by the bacterium Clostridium botulinum. Its mechanism of action is based on preventing the release of neurotransmitters into the synaptic cleft by proteolytically cleaving SNARE proteins responsible for their exocytosis. For a long time, it was believed that the effects of BT-A were limited to the peripheral areas where it was applied, restricting its therapeutic use to treating autonomic disorders, localized spasticity, and hyperkinetic movement disorders. Over time, it was established that BT-A has significant antinociceptive effects at the central level, with minimal side effects, long-lasting impact, and no development of tolerance compared to other systemically acting analgesics, making it an exceptionally attractive candidate for further research, particularly in the treatment of chronic pain. The aim of this thesis was to investigate the influence of BT-A on the expression of CGRP, a neuropeptide mediator of pain and inflammation, in the trigeminal ganglion in a model of inflammatory orofacial pain induced by formalin application in rats. BT-A (7 i.j./kg) was unilaterally administered to the area of the right facial vibrissae (ipsilateral) in Wistar rats. Six days later, pain was induced in the area of the left facial vibrissae (contralateral) by applying 50 μL of 2.5% formalin, after which the animals were sacrificed and sections of their trigeminal ganglia were analyzed. In the negative control group, saline was used instead of BT-A, administered bilaterally, while in the positive control group, saline was administered ipsilaterally, and 50 μL of 2.5% formalin was applied contralaterally. Immunohistochemical analysis and statistical processing were used to monitor and quantify the intensity of CGRP signals in transverse sections of the trigeminal ganglia. In the experimental group, a statistically significant reduction in CGRP expression was observed compared to the control group. The obtained results suggest that BT-A also acts on the opposite side of the application site, indicating its potential for axonal transport and possible trans-synaptic transfer (transcytosis). In conclusion, this thesis demonstrates that BT-A can act not only at the local level but also at the central nervous system level through potential transcytosis, opening up the possibility for further research into its central antinociceptive effects