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CYP2D6 genotyping - challenges in detection of copy number variants and hybrid genes
No full textSustav citokroma P450 uključen je u biotransformaciju >75% lijekova na tržištu pri čemu se oko
20% njih metabolizira s pomoću enzima CYP2D6. Gen CYP2D6 izrazito je varijabilan, do sad je
detektirano >170 varijanti, uključujući polimorfizme jednog nukleotida (SNP) i strukturne varijante
(SV). Od strukturnih varijanti javljaju se delecije, duplikacije i multiplikacije te hibridni geni
CYP2D6::CYP2D7 i CYP2D7::CYP2D6. Različite varijante gena CYP2D6 kodiraju enzime
CYP2D6 različite aktivnosti pa se pacijenti u ovisnosti o genotipu svrstavaju u fenotipske skupine
normalnih metabolizatora, intermedijarnih, sporih i vrlo brzih. Ispravna genotipizacija i
fenotipizacija ključne su za doziranje lijekova čiji učinci primarno ovise o metabolizmu putem
enzima CYP2D6, posebno ako se radi o lijekovima s uskim terapijskim rasponom. Pacijenti s
različitim fenotipom zahtijevaju različite doze takvih lijekova kako bi se s jedne strane izbjeglo
poddoziranje i izostanak željenog učinka, a s druge strane predoziranje i neželjeni učinci.
S obzirom na visoku varijabilnost gena, genotipizacija CYP2D6 je kompleksna. Poseban izazov
predstavlja detekcija hibridnih gena. PCR metode za umnažanje dugih odsječaka DNA (XL-PCR)
te TaqMan metode uspješno detektiraju SNP-ove, ali nemaju mogućnost detekcije hibridnih gena,
već se oni lažno detektiraju kao cjelovite varijante ili kao potpune delecije gena CYP2D6. CNV
analize (od eng. copy number variation), usmjerene na 2 ili više fragmenta gena, donose revoluciju
u prepoznavanju hibrida.
U ovom radu opisan je postupak farmakogenetske analize gena CYP2D6 koja uključuje analizu
polimorfizama jednog nukleotida i CNV analizu, s naglaskom na analizu strukturnih varijanti i
problematiku genotipizacije hibridnih gena. Prikazan je pregled zastupljenosti različitih alela,
genotipa i fenotipa u populaciji pacijenata upućenih na genotipizaciju na Odjel za farmakogenomiku
i individualizaciju terapije Kliničkog zavoda za laboratorijsku dijagnostiku Kliničkog bolničkog
centra Zagreb te su uspoređeni rezultati dobiveni s CNV analizom i bez nje kako bi se dobio uvid u
značaj CNV analize. Opisani su i problemi na koje nailazi CNV analiza. Njena kompleksnost
proizlazi iz činjenice da sami hibridni geni dolaze u velikom broju raznolikih varijanti te su nerijetko
uključeni u neidentične duplikacije.
U konačnici, može se reći da CNV analiza povećava kvalitetu farmakogenetske analize, međutim
metoda nije savršena. Stoga ostaju otvorena pitanja optimiranja CNV analize i mogućnosti uvođenja
drugih molekularnih metoda genotipizacije gena CYP2D6 u kliničku praksu.Cytochrome P450 system is involved in the biotransformation of >75% of the approved drugs,
where about 20% of them are metabolized by CYP2D6 enzyme. The CYP2D6 gene is extremely
variable, so far >170 variants have been detected, including single nucleotide polymorphisms (SNPs)
and structural variants (SVs). Structural variants include deletions, duplications and multiplications
and also hybrid genes CYP2D6::CYP2D7 and CYP2D7::CYP2D6. Different variants of the CYP2D6
gene encode CYP2D6 enzymes with different activities, so depending on the genotype, patients are
classified into phenotypic groups of normal, intermediate, poor and ultrarapid metabolizers.
Correctly determined genotype and phenotype are crucial for the dosing of drugs whose effects
primarily depend on metabolism via the CYP2D6 enzyme, especially for narrow therapeutic index
drugs. Patients with different phenotypes require different doses of such drugs in order to avoid on
the one hand underdosing and absence of the desired effect, and on the other hand, overdosing which
leads to side effects and toxicity.
Due to the variability of CYP2D6 gene, genotyping is complex. The detection of hybrid genes is
particulary challenging. Extra-long Polymerase Chain Reaction (XL-PCR) and TaqMan® real-time
PCR method successfully detect SNPs, but do not have the possibility of detecting hybrid genes,
instead they are falsely detected as whole genes or as deletions. CNV analysis, that detects 2 or more
CYP2D6 gene fragments, revolutionize the discovery of hybrid genes.
This paper describes the procedure of pharmacogenomic analysis of the CYP2D6 gene, which
includes the analysis of single nucleotide polymorphisms and CNV analysis, with an emphasis on
the analysis of structural variants and the difficulties in genotyping hybrid genes. An overview of the
frequencies of different allels, genotypes and fenotypes in the population of patients who underwent
pharmacogenomic analysis in the Division for Pharmacogenomics and Therapy Individualization of
the Department for Laboratory Diagnostics University Hospital Center Zagreb is presented. Also the
results of genotyping and phenotyping obtained with and without CNV analysis are compared in
order to gain insight into the significance of CNV analysis. The weaknesses of CNV analysis are also
described. Its complexity stems from the fact that the hybrid genes themselves come in a large
number of diverse variants and are often involved in non-identical duplications.
Ultimately, it can be said that CNV analysis increases the quality of pharmacogenomic analysis
of the CYP2D6 gene, however the method is not perfect. Therefore, the questions of optimizing CNV
analysis and the possibility of introducing other molecular methods for genotyping the CYP2D6 gene
into clinical practice remain open
Influence of liposome concentration and surface charge on the rheological properties of chitosan liposomal gels
No full textLiposomski gelovi (CL-CHG, PGL-CHG, CATL-CHG) pripremljeni su uklapanjem različitih vrsta AZT-liposoma u CHG pri 3 različite koncentracije (10, 20 i 30 %, m/m), Uklopljeni su liposomi bili srednjeg promjera ˂ 130 nm (CL i PGL), odnosno 330 nm (CATL). Zeta potencijal CL i PGL bio je izrazito negativan (− 43 mV, − 48 mV), dok su CATL pokazali izrazito pozitivnu vrijednost zeta potencijala (+ 59 mV). Krivulje viskoznosti dobivene za liposomske gelove pokazuju da fosfolipidni sastav liposoma, tj. površinski naboj, ima određeni utjecaj na viskoznost formulacije, koji više dolazi do izražaja pri nižim koncentracijama liposoma u sustavu. Naime, negativno nabijeni liposomi CL i PGL pri koncentraciji od 10 % ili 20 % uzrokuju povećanje viskoznosti u odnosu na izvorni CHG, dok je za pozitivno nabijene CATL pri 10 % i 20 % uočen suprotan efekt (smanjenje viskoznosti). Pri najvećoj koncentraciji liposoma u gelu (30 %), dolazi do smanjenja viskoznosti formulacije neovisno o površinskom naboju liposoma, što je vjerojatno rezultat značajnog smanjenja koncentracije kitozana u gelu uslijed razrjeđivanja dodatkom tekuće faze. Tijekom oscilacijskih testova promjene amplitude, izvorni CHG i svi liposomski gelovi pokazuju širok LVR s modulom skladištenja (G') većim od modula gubitka (G''), što predstavlja viskoelastično ponašanje karakteristično za gelove i potvrđuje da dodatak liposoma u CHG u koncentraciji ≤ 30% (m/m) ne narušava strukturu gela. Očuvana struktura gela može osigurati produljeno zadržavanje na mjestu primjene i produljeno oslobađanje lijeka, a uzorci sa širokom LVR regijom ujedno se mogu smatrati dobro dispergiranima i stabilnima.Liposomal gels (CL-CHG, PGL-CHG, CATL-CHG) were prepared by incorporating different types of AZT-liposomes into CHG at three different concentrations (10, 20, 30%, w/w). The incorporated liposomes had an average diameter ˂130 nm (CL, PGL) or 330 nm (CATL). The zeta potential of CL and PGL liposomes was strongly negative (-43 mV, -48 mV), while CATL exhibited strongly positive zeta potential (+59 mV). The viscosity curves obtained for the liposomal gels indicate that the phospholipid composition/surface charge of the liposomes, has an impact on the viscosity of the formulation, which is more pronounced at lower liposome concentrations in the formulation. Specifically, negatively charged CL and PGL liposomes at 10% or 20% cause an increase in viscosity compared to the original CHG, while for positively charged CATL at 10% and 20%, an opposite effect (viscosity decrease) was observed. At the highest liposome concentration in the gel (30 %), the viscosity of the formulation decreases regardless of the surface charge of the liposomes, which is likely the result of a significant reduction in the chitosan concentration in the gel due to dilution by the added liquid phase. During oscillatory amplitude tests, the original CHG and all liposomal gels show a wide LVR with a storage modulus (G') greater than the loss modulus (G''), which represents viscoelastic behavior characteristic of gels and confirms that the addition of liposomes to CHG at a concentration ≤ 30% (w/w) does not compromise the gel structure. The preserved gel structure can ensure prolonged retention at the site of application and extended drug release. Moreover, samples with a broad LVR region can also be considered well-dispersed and stable
Calorimetric study of the effect of synergistic anion and sialylation on the thermodynamics of the iron binding to human serum transferrin
No full textLjudski serumski transferin glavni je prenosioc željeza u organizmu. Željezo vezano na transferin doprema se iz probavnog sustava do stanica kojima je ono potrebno i otpušta s transferina promjenom pH. Transferin ima dva mjesta za vezanje željeza u kojem sudjeluje i sinergijski anion. Kod zdravih pojedinaca najčešća glikoforma transferina je glikoforma s dva biantenarna oligosaharidna lanca s ukupno četiri sijalinske kiseline, a sinergijski anion je karbonat. U in vitro uvjetima pokazano je kako drugi strukturno slični karboksilni ioni poput oksalata mogu biti sinergijski anioni. Desijalinizacija transferina primijećena je kod pacijenata s dijagnozom kroničnog alkoholizma, ali i raznih upalnih bolesti te sepse, dok su povećane serumske koncentracije oksalata često rezultat hiperoksalurije ili gastrointestinalnih bolesti. Cilj ovog doktorskog rada bio je odrediti termodinamičke parametre vezanja željeza u formi FeNTA na transferin s različitim indeksom sijalinizacije, u prisutnosti karbonata i oksalata kao sinergijskih aniona. U mjerenjima sa sijaliniziranim transferinom korišten je komercijalno dostupan nativni serumski transferin, dok je desijalinizirani transferin pripremljen djelovanjem enzima neuraminidaze u in vitro uvjetima. Nakon što je desijalinizacija proteina potvrđena i udio sijalinskih kiselina je smanjen za oko 99%, termodinamički parametri određeni su korištenjem izotermne titracijske kalorimetrije. Potvrđeno je kako se dva vezna mjesta razlikuju kinetički i termodinamički te je otkriveno kako desijalinizacija utječe na termodinamičke parametre vezanja željeza u formi FeNTA na transferin tako da efektivne entalpije vezanja postaju egzotermije, a u prisutnosti karbonata pri koncentracijama koje odgovaraj onima u serumu, konstanta vezanja se povećava. Navedeno ide u prilog mogućoj povezanosti desijalinizacije i sekvestracijskog odgovora organizma na infekcije patogenom. Osim desijalinizacije, i oksalat kao sinergijski anion u in vitro uvjetima povećava konstantu vezanja željeza na transferin, a vezanje je egzergonije. Uz jače vezanje oksalata na transferin, navedeno daje osnovu za mogući nastanak ternarnog kompleksa Fe-oksalat-Tf u in vivo uvjetima, pogotovo u stanjima s povećanim koncentracijama oksalata.Human serum transferrin is the major iron transport protein in human organisms. Iron bound to transferrin is transported from the digestive system to cells that require iron. A change in the pH causes the release of iron from transferrin. Transferrin has two iron binding sites that require the presence of the synergistic anion. The most common glycoform of transferrin in healthy humans consists of two biantennary oligosaccharide chains that together contain four sialic acids with carbonate as synergistic anion. Other structurally similar carboxylic ions such as oxalate have shown to act as synergistic anions in vitro. Chronic alcoholic disease, inflammatory diseases and sepsis can cause desialylation of transferrin, and hyperoxaluria or gastrointestinal diseases can lead to higher serum oxalate levels. The aim of this thesis was to determine thermodynamic parameters for the binding of Fe in the form of FeNTA to transferrin with different sialylation index, and in the presence of carbonate and oxalate as synergistic anions. Commercially available native human serum transferrin was used as sialylated transferrin, while the desialylated transferrin was prepared in in vitro using neuraminidase enzyme. After 99% reduction in sialic acid content was confirmed, thermodynamic parameters were determined using isothermal titration calorimetry. Our data have shown that in binding sites differ in both kinetics and thermodynamics of the binding. Moreover, it was discovered that desialylation affects thermodynamic parameters of the iron binding in the form of FeNTA to transferrin, leading to a shift towards exothermic binding enthalpies. Furthermore, in the presence of carbonate at concentrations corresponding to those in human serum, the binding constant increases. These findings are in support of the possible connection between the desialylation and sequestration as an organism’s response to pathogen infections. In addition to desialylation, oxalate as a synergistic anion in in vitro conditions also increases the binding constant of iron to transferrin, and the binding becomes more exergonic. Together with the stronger prebinding constants of oxalate to human serum transferrin, the above provides the basis for the possible formation of the Fe-oxalate-Tf ternary complex in in vivo conditions, especially in conditions with increased concentration of oxalate
Systematic review of interactions of dietary supplements and drugs used in secondary therapy of myocardial infarction based on evidence
No full textInfarkt miokarda predstavlja značajno kardiovaskularno oboljenje s visokom prevalencijom širom
svijeta. U svrhu prevencije ponovnih kardiovaskularnih komplikacija, pacijentima se prepisuje
sekundarna terapija koja obuhvaća širok spektar lijekova, poput β-blokatora, antitrombotika, statina,
ACE-inhibitora, uz istovremenu kontrolu faktora rizika. Uz to, sve češće se primjećuje da pacijenti,
koji su preživjeli infarkt miokarda, mijenjaju svoje životne navike te koriste dodatke prehrani u nadi
poboljšanja općeg zdravstvenog stanja. S obzirom na kompleksnost terapije i sveprisutnu upotrebu
dodataka prehrani, postaje izrazito važno prepoznati moguće interakcije između lijekova i dodataka
prehrani. Upravo farmaceuti igraju ključnu ulogu u prevenciji, prepoznavanju i upravljanju tim
interakcijama kako bi osigurali adekvatnu skrb za pacijente. Stoga ovaj diplomski rad pruža sustavan
pregled interakcija između dodataka prehrani i lijekova korištenih u sekundarnoj terapiji infarkta
miokarda, temeljen na znanstvenim dokazima. Pritom su objašnjeni raznoliki mehanizmi interakcija,
kao što su smanjenje bioraspoloživosti, nefrotoksičnost, hepatoksičnost, te promjene
farmakokinetike lijekova. Ova tema ne samo da je relevantna zbog česte pojavnosti infarkta
miokarda, već i zbog specifičnih izazova koje donosi u ljekarničkoj praksi. Diplomski rad pridonosi
razvoju stručnosti farmaceuta u optimalnom upravljanju terapijom pacijenata nakon infarkta
miokarda, čime se direktno utječe na zdravstveno stanje i kvalitetu života pacijenata.Myocardial infarction is a significant cardiovascular disease with a high prevalence worldwide. In
order to prevent repeated cardiovascular complications, patients are prescribed secondary therapy
that includes a wide range of drugs, such as β-blockers, antithrombotics, statins, ACE-inhibitors,
with simultaneous control of risk factors. In addition, it is increasingly noticed that patients, who
have survived a myocardial infarction, change their lifestyle and use dietary supplements with hope
of improving their general health. Considering the complexity of therapy and the ubiquitous use of
dietary supplements, it becomes extremely important to recognize possible interactions between
drugs and dietary supplements. Pharmacists play a key role in the prevention, recognition and
management of these interactions in order to ensure adequate care for patients. Therefore, this thesis
provides a systematic review of the interactions between dietary supplements and drugs used in the
secondary therapy of myocardial infarction, based on scientific evidence. Various mechanisms of
interactions, such as reduction of bioavailability, nephrotoxicity, hepatotoxicity, and changes in drug
pharmacokinetics were explained. This topic is not only relevant because of the high prevalence of
myocardial infarction, but also because of the specific challenges it brings in pharmaceutical
practice. The diploma thesis contributes to the development of the expertise of pharmacists in the
optimal management of therapy for patients after myocardial infarction, which directly affects their
health status and quality of life
Analyzing the pharmacopeial properties of the quality and antioxidant activity of chewable tablets that contain red elm bark extract
No full textKora crvenog brijesta ima široko rasprostranjenu tradicionalnu upotrebu, poglavito kod gastrointestinalnih smetnji poput želučanih tegoba i dijareje te respiratornih smetnji poput kašlja. Na tržištu su prisutni pripravci koji sadrže koru brijesta u obliku dodataka prehrani. Dodaci prehrani ne podliježu istim zakonima i strogim kontrolama kao lijekovi. Cilj ovog rada bio je ispitati kvalitetu prema zahtjevima Farmakopeje Sjedinjenih Američkih Država te procijeniti antioksidativnu aktivnost dodatka prehrani koji sadrži ekstrakt kore crvenog brijesta. Rezultati ispitivanja kvalitete pokazuju da pripravak zadovoljava kriterije kvalitete Farmakopeje Sjedinjenih Američkih Država. Antioksidativna aktivnost procijenjena je DPPH metodom i pokazano je da povećanjem koncentracije ekstrakta linearno raste i antioksidativna aktivnost u ispitivanom području koncentracija.The red elm bark has a widespread traditional use, mainly for gastrointestinal problems such as stomach disorders and diarrhea, but also for respiratory problems such as cough. Products on the market contain red elm bark in the form of dietary supplements. However, dietary supplements are not subject to the same laws and rigorous controls as medicines. This study aimed to evaluate the quality of such dietary supplement according to United States Pharmacopeia requirements and also to measure its antioxidant activity. The results of quality tests show that the supplement satisfies the United States Pharmacopeia quality criteria. The antioxidant activity was evaluated by the DPPH method, and it was shown that increasing the concentration of the extract linearly increases the antioxidant activity in the examined range of concentration
NMR study of isotopic H/D exchanges in 1,1-dimethyl-propargyl alcohol
No full textProces deuteriranja podrazumijeva izmjenu vodika (1H) u strukturi različitih spojeva za atom deuterija (2H), stabilnog, neradioaktivnog vodikovog izotopa. Ugradnjom atoma deuterija u molekule lijeka može doći do promjene svojstava apsorpcije, distribucije, metabolizma i izlučivanja tog lijeka. Deuterirani spojevi često pokazuju poboljšane farmakokinetičke profile i manju toksičnost uz zadržavanje svojih farmakodinamičkih svojstava. S obzirom da su C–D veze jače od C–H veza, H/D izmjena može značajno usporiti metabolizam spojeva, a time i povećati bioraspoloživost lijekova. Deuterirani terminalni alkini posebno su važni jer je riječ o prekursorima niza deuteriranih molekula. Znanstvenici su dugi niz godina pokušavali razviti jednostavnu, selektivnu, ekonomičnu i ekološki prihvatljivu metodu izotopske izmjene. Ovaj rad usmjeren je razvoju jednostavnog protokola koji bi primjenom blagih reakcijskih uvjeta bez korištenja katalizatora omogućio deuteriranje terminalnih alkina u relativno kratkom vremenu, uz visoku ugradnju 2H izotopa. S tim ciljem, uspješno je provedena H/D izmjena 1,1-dimetil-propargilnog alkohola u teškoj vodi kao donoru deuterija te u smjesi otapala DMSO-d6-D2O (φ(DMSO-d6) = 6,78%) pri sobnoj temperaturi, u relativno kratkom vremenu, uz visoku ugradnju izotopa (>99%). Također je izmjereno da dodatak DMSO u D2O neznatno usporava reakciju H/D izmjene, odnosno povisuje Gibbsovu aktivacijsku energiju. Svi rezultati dobiveni su uporabom NMR spektroskopije, te daju dobar temelj za nastavak istraživanja reakcije deuteriranja različitih spojeva, pa tako i lijekova, koji u strukturi imaju terminalne alkine, uz primjenu D2O te smjese otapala DMSO-D2O ili smjese teške vode s drugim organskim otapalima.The deuteration process implies the exchange of hydrogen (1H) in the structure of various compounds for an atom of deuterium (2H), a stable, non-radioactive hydrogen isotope. The incorporation of deuterium atoms into drug molecules can change the properties of absorption, distribution, metabolism and excretion of that drug. Deuterated compounds often show improved pharmacokinetic profiles and lower toxicity while retaining their pharmacodynamic properties. Given that C–D bonds are stronger than C–H bonds, H/D exchange can significantly slow down the metabolism of compounds, and thus increase the bioavailability of drugs. Deuterated terminal alkynes are particularly important because they are the precursors of a number of deuterated molecules. For many years, scientists have been trying to develop a simple, selective, economical and environmentally friendly method of isotopic exchange. This work is aimed at the development of a simple protocol that would enable the deuteration of terminal alkynes in a relatively short time, with a high incorporation of 2H isotopes, by applying mild reaction conditions without the use of catalysts. With this aim, the H/D exchange of 1,1-dimethyl-propargyl alcohol was successfully carried out in heavy water as a deuterium donor and in the solvent mixture DMSO-d6-D2O (φ(DMSO-d6) = 6.78%) at room temperature, in a relatively short time, with high isotope incorporation (>99%). It was also measured that the addition of DMSO to D2O slightly slows down the H/D exchange reaction, i.e. increases the Gibbs activation energy. All results were obtained using NMR spectroscopy, and they provide a good basis for continuing research on the deuteration reaction of various compounds, including drugs, which have terminal alkynes in their structure, with the use of D2O and the solvent mixture DMSO-D2O or the mixture of heavy water with other organic solvents
Agregatna onečišćenja u monoklonskim protutijelima - imunogenost i izazovi u provjeri kakvoće
No full textCilj istraživanja
Cilj rada je dati sveobuhvatnu analizu karakterizacije i kontrole agregatnih onečišćenja u monoklonskim protutijelima, radi osiguravanja odgovarajuće kakvoće i učinkovitosti te smanjivanja njihove imunogenosti.
Hipoteze istraživanja su:
- nastanak agregatnih onečišćenja u monoklonskim protutijelima se može smanjiti, ali ne i u potpunosti izbjeći;
- razumijevanje mehanizama agregacije i primjena najsuvremenijih metoda karakterizacije i kvantifikacije agregata u monoklonskim protutijelima osigurava odgovarajuću kontrolu kakvoće i smanjuje mogućnost nastanka agregata tijekom proizvodnje i čuvanja lijeka;
- agregacija proteina između referentnog i biosličnog lijeka je usporediva.
Materijal i metode
Literatura je pretraživana prema temi i predmetu istraživanja, autorima i časopisu, od općih prema specijaliziranim člancima, na temu razvoja i proizvodnje monoklonskih protutijela, nastanka agregatnih onečišćenja i njihovog imunogenog potencijala te načina kako izbjeći, odnosno minimizirati agregaciju proteina. Pri pretraživanju literature su traženi odgovori na specifična pitanja vezana uz problematiku ovog specijalističkog rada, u cilju potkrepe predloženih hipoteza.
Pretražene su bibliografska baza podataka (PubMed) i baza podataka s cjelovitim tekstom (Science Direct), kojima je pristupano elektroničkim putem preko umreženog računala koje ima
online pristup spomenutim bazama. Regulatorne smjernice i pravni dokumenti relevantni za problematiku ovog rada su istraženi pristupom mrežnih stranicama i bazama regulatornih Agencija (EMA, FDA, HALMED), Europske komisije te Europske (Ph. Eur.) i Američke farmakopeje (USP). Na temelju pretraživanih izvora su izvedeni vlastiti zaključci o predmetnoj problematici.
Rezultati
Agregacija proteina je neizbježan događaj karakterističan za gotovo sve faze u proizvodnji proteina, transport, čuvanje i primjenu lijeka te predstavlja velik izazov u proizvodnji monoklonskih protutijela. Proteinska agregacija se uglavnom može predvidjeti i donekle prevenirati još tijekom razvoja formulacije i proizvodnje monoklonskih protutijela. Agregati različitih proteina pokazuju veći imunogeni potencijal u odnosu na pojedinačne podjedinice (monomere), a pretpostavlja se da je razlog tomu razlika u veličini i vrsti agregata, njihovoj konformaciji te morfologiji. Razumijevanje mehanizama agregacije i razvoj prikladnih strategija njezine minimizacije su zadnjih desetljeća postali imperativ u razvoju proteinskih lijekova, u cilju osiguravanja njihove kliničke učinkovitosti. Zasad nije u potpunosti jasno imaju li sve vrste agregata jednak imunogeni potencijal te se ne može unaprijed znati koja je agregatna onečišćenja potrebno kontrolirati u lijeku. Prema posljednje važećim smjernicama i preporukama regulatornih agencija, svojstva svih agregata koji se mogu detektirati trebaju biti odgovarajuće karakterizirana, a sadržaj kontroliran i rutinski nadziran u svim biološkim lijekovima. Na temelju dosadašnjeg iskustva, zaključeno je kako se kvantifikacija proteinskih agregata treba provesti na temelju njihove veličine te se uobičajeno istovremeno koriste barem dvije ortogonalne metode temeljene na različitim principima rada. Usporednim ispitivanjima na razini kakvoće između biosličnih i referentnih lijekova je utvrđeno kako imaju slične profile onečišćenja, obzirom na sadržaj agregatnih onečišćenja.
Zaključak
Obzirom na strukturne i ostale karakteristike proteina, tijekom proizvodnje, čuvanja, transporta i primjene većine monoklonskih protutijela često dolazi do agregacije proteina. Ovisno o svojstvima, agregatna onečišćenja mogu značajno utjecati na kakvoću, djelotvornost i sigurnost monoklonskih protutijela, posebice u vidu izazivanja pojačane imunogenosti, što može dovesti do smanjenog djelovanja lijeka i značajnih nuspojava kod pacijenata. Nastanak agregatnih onečišćenja u monoklonskim protutijelima se može smanjiti, ali ne i u potpunosti izbjeći.
Razumijevanje mehanizama agregacije i razvijanje strategija smanjivanja nastanka agregatnih onečišćenja su bitni za minimiziranje nastanka imunogenog učinka monoklonskih protutijela. Isto tako, primjena sofisticiranih i specifičnih ortogonalnih analitičkih metoda za karakterizaciju i kvantifikaciju agregatnih onečišćenja te njihov daljnji razvoj osiguravaju odgovarajuću kontrolu kakvoće i smanjuje mogućnost nastanka agregata tijekom proizvodnje i čuvanja lijeka. Utvrđeno je kako su referentni biološki lijekovi i njihove bioslične inačice usporedivi obzirom na čistoću i profil onečišćenja (obzirom na fragmente, procesna onečišćenja i neglikozilirane forme), stupanj agregacije te vrstu agregata i ostalih molekula veće molekulske mase.Objectives
The objective of this work is to provide the comprehensive analysis of the characterization and quality control of the aggregate impurities in monoclonal antibodies (mABs). The aim is to ensure that mABs and other biotherapeutics are of adequate safety, quality and efficacy, with the reduced immunogenic potential as much as possible.
Hypotheses are the following:
- the formation of aggregate impurities in mABs can be decreased, but not completely avoided;
- understanding of the mechanisms of aggregation and the use of the sophisticated methods of characterization and quantification of aggregates in mABs ensure an adequate control of aggregation and reduce the possibility for the formation of aggregates during the production and storage of the drug products;
- protein aggregation between the reference drug product and the biosimilar is comparable.
Material and methods
Through this work, the importance of an adequate characterization and control of the aggregate impurities in monoclonal antibodies in a timely manner has been emphasized, in order to ensure their safety, quality and efficacy. This is especially important from the aspect of the mABs immunogenicity and its consequent possible decrease. The mechanisms of protein aggregation and the specific types of aggregates that can occur considering the protein properties and the drug product storage conditions have been described. There is a special emphasis on the
possible enhanced immune response of the patients on the drug product due to the presence of aggregate impurities.
Results
Protein aggregation is an inevitable event specific for almost all protein manufacturing stages, as well as its transport, storage and drug administration. It represents a huge challenge in the production of monoclonal antibodies. Protein aggregation is mostly predictable and in some way it can be prevented during the development of the formulation and the production of mABs. Aggregates from different proteins show a higher immunogenic potential compared to the monomers and it is assumed that the main reason for this is the difference in size and type of aggregates, their conformation and morphology. Understanding of the mechanisms of aggregation and the strategy development for its minimization have become imperative in the development of protein drug products in recent decades, all with the aim to ensure their clinical efficacy. So far, it is not entirely clear whether all types of aggregates have the same immunogenic potential and it is not possible to know which aggregate impurities should be controled in the drug product in advance. According to the recent guidelines and the recommendations from the regulatory bodies, the properties of all detectable aggregates should be properly characterized and their content routinely monitored in mABs. Based on the previous experience, it was assumed that the quantification of protein aggregates should be carried based on their size, at least with two orthogonal methods based on different principles. From the comparative studies regarding quality between biosimilars and the reference drug products it has been concluded that impurity profiles are similar, also considering the content of protein aggregates.
Conclusion
Considering the structural and other characteristics of proteins, protein aggregation occurs often during the production of the most of monoclonal antibodies, their storage, transport and administration. Depending on their properties, aggregates could significantly affect the quality, safety and efficacy of mABs, especially considering the immunogenic potential that could lead to decreased drug activity, as well as significant side effects in patients. The formation of protein aggregates in mABs can be reduced but not completely avoided. Understanding of the mechanisms of aggregation and strategy development for reduction of aggregates formation are essential for minimization of the immunogenic effects of mABs. Likewise, the application of sophisticated and specific orthogonal analytical methods for the characterization and quantification of aggregates and their further development ensure adequate quality control and reduce the possibility of aggregates formation during the production and storage of biological drugs. It has been established that the reference biological drug products and their biosimilar versions are comparable considering the purity and impurity profile (with regard to fragments, impurities and non-glycosylated forms), the level of aggregation, as well as the type of aggregates and other molecules of higher molecular weight
Sinteza novih analoga siderofora piscibactina
No full textRastući broj bakterijskih oboljenja u riba negativno utječe na sektor ribarstva i akvakulture.
Antimikrobna rezistencija postaje jedan od najzabrinjavajućih problema u zdravstvenom sektoru
diljem svijeta, rezultirajući hitnom potrebom za implementacijom novih strategija kako bi se zarazne
bolesti prevenirale i zaustavile. Javlja se i potreba za izumom djelotvornih antibiotika koji bi
pomogli u borbi protiv rezistencije među bakterijama. Piscibactin (Pcb) je siderofor fenolnog tipa
koji se sastoji od dva tiazolinska i jednog tiazolidinskog prstena. Olakšava unos Fe3+ iona u bakterije
kao što su Photobacterium damselae subsp. piscicida i Vibrio anguillarum. Međutim, ograničena
stabilnost narušava potencijalnu primjenu u medicinske i biotehnološke svrhe. Stoga je razvoj
stabilnih Pcb analoga ključan za daljnji napredak. Ovaj rad izlaže sintetski put pojednostavljenog
Pcb analoga s promijenjenim strukturnim značajkama, uzevši u obzir računske rezultate. Oksazoloksazolin-
tiazol intermedijer analoga piscibactina uspješno je sintetiziran i mogao bi služiti kao
napredni intermedijer u budućoj sintezi analoga, kao i u farmaceutskoj industriji. Svi sintetizirani
spojevi karakterizirani su pomoću tehnika NMR-a i masene spektrometrije.The fishery and aquaculture sector are being negatively impacted by a rising number of bacterial
diseases in fish. Additionally, antimicrobial resistance is becoming one of the most concerning
problems in the health sector globally. As a result, there is an urgent need of implementing new
strategies to prevent and stop infectious diseases, as well as inventing effective antibiotics, which
would help fight the resistance amongst bacteria. Piscibactin (Pcb) is a phenolate-type siderophore,
comprising two thiazoline and one thiazolidine rings. It facilitates Fe3+ ion uptake in bacteria, such
as Photobacterium damselae subsp. piscicida and Vibrio anguillarum. However, its limited stability
impairs the potential medical and biotechnological applications. Therefore, the development of
stable Pcb analogues is crucial for further advancement. This study proposes the design of a
simplified analogue of Pcb with alternative structural features, guided by computational results. The
oxazole-oxazoline-thiazole Pcb analogue intermediate was successfully synthesised, and it could be
used as an advanced intermediate in further analogue synthesis, as well as in pharmaceutical
industry. All the synthesised compounds were characterised by both NMR and mass spectrometry
techniques
The effect of digestion on properties of selenium nanoparticles
No full textRazvoj novih oblika nutraceutika za per os primjenu korištenjem zelenih i održivih formulacijskih pristupa koji se uklapaju u načela kružne ekonomije vrlo je dinamično i aktualno područje istraživanja. U ovom radu istražene su mogućnosti zelene sinteze nanočestica Se korištenjem pomoćnih tvari koje je moguće jednostavnim i zelenim postupcima izolirati iz prehrambenog otpada te je provedena funkcionalna karakterizacija dobivenih nanosustava s posebnim naglaskom na gastrointestinalnu stabilnost. U postupku sinteze kao stabilizacijska sredstva korišteni su pektini različitog stupnja čistoće (sirovi i pročišćeni) te iz različitih izvora (kora mandarine i komina rajčice), a u svrhu dodatne funkcionalizacije površine nastalih nanosustava korišten je ekstrakt komine masline bogat polifenolima. Svojstva nanočestica prije i nakon in vitro simulacije probave karakterizirana su određivanjem raspodjele veličine čestica, zeta potencijala, indeksa polidisperznosti te ukupnog redukcijskog potencijala.Predloženi zeleni postupci sinteze nanočestica rezultirali su nastankom stabilnih nanosustava odgovarajućih fizikalno-kemijskih svojstava. Polifenoli masline uspješno su primijenjeni za funkcionalizaciju površine nanočestica selena te su značajno povećali redukcijski potencijal sustava. Tijekom probave dolazi do promjena fizikalno-kemijskih karakteristika nanosustava, međutim sve istražene nanočestice ostaju stabilne i primjenjive za per os primjenu. Dobiveni rezultati pridonijet će trenutnim saznanjima o mogućnostima održivih postupaka formulacije visokovrijednih oblika selena za peroralnu primjenu poboljšanih biofarmaceutskih svojstava.Development of innovative nutraceuticals for oral administration by using green and sustainable formulation approaches that fit into the principles of the circular economy is particularly dynamic and contemporary area of research.This research investigated the possibilities of green synthesis of Se nanoparticles using excipients that can be isolated from food waste by applying simple and green procedures. Functional characterization of the obtained nanosystems was carried out with special emphasis put on the investigation of on gastrointestinal stability. In the synthesis process pectins of different degrees of purity (raw and purified) and from different sources (tangerine peel and tomato pomace) were used as stabilizing agents, and for the purpose of additional functionalization of the surface of the resulting nanosystems, olive pomace extract rich in polyphenols was used. The properties of nanoparticles before and after in vitro digestion simulation were characterized by determining the particle size distribution, zeta potential, polydispersity index and total reduction potential.The proposed green synthesis procedure resulted in the creation of stable selenium nanosystems with appropriate physicochemical properties. Olive waste derived polyphenols were successfully applied to functionalize the surface of selenium nanoparticles and significantly increase the reduction potential of obtained nanoparticles. Even though digestion process altered particular physicochemical characteristics of the investigated nanosystems, all investigated nanoparticles remained stable and applicable for per os application.Obtained results will contribute to current knowledge about the possibilities of sustainable formulations of innovative per os selenium formulations with improved biopharmaceutical properties
Determination of the precision of differential blood count preparation using digital morphology on the Sysmex DI-60 (Cellavision) analyzer and light microscopy
No full textIzrada DKS-a uobičajeno se provodi primjenom triju metoda: automatiziranom metodom na hematološkim
brojačima, svjetlosnom mikroskopijom i digitalnom morfološkom analizom stanica. Izrada DKS-a na
hematološkim brojačima predstavlja prvi korak u analizi DKS-a periferne krvi. U slučaju prisutnosti patoloških
stanica i stanica s morfološkim abnormalnostima, analizator daje upozorenje o patološkom nalazu što upućuje na
potrebu izrade DKS-a svjetlosnom mikroskopijom i/ili digitalnom morfološkom analizom. Bez obzira na
tehnološki napredak, izrada DKS-a svjetlosnom mikroskopijom i dalje predstavlja zlatni standard. Međutim,
prednost primjene uređaja za digitalnu morfologiju je ušteda vremena uz visoku preciznost i točnost. Cilj ovog
diplomskog rada bio je ispitati i usporediti preciznost izrade DKS-a metodom svjetlosne mikroskopije i digitalne
morfološke analize (postupak preklasifikacije i klasifikacije) u razmazima periferne krvi sa i bez patologije,
primjenom uređaja Sysmex DI-60. Ukupno je analizirano sedam razmaza periferne krvi: tri razmaza rutinskih
uzoraka u kojima je ispitana preciznost svjetlosne mikroskopije i digitalne morfološke analize, te četiri razmaza iz
programa vanjske procjene kvalitete u kojima je ispitana preciznost digitalne morfološke analize. Temeljem
dobivenih rezultata potvrđena je hipoteza o većoj preciznosti digitalne morfološke analize u odnosu na metodu
svjetlosne mikroskopije. Iznimka su bili patološki razmazi s akutnim leukemijama u kojima se metoda svjetlosne
mikroskopije pokazala preciznijom u brojenju patoloških, nezrelih stanica periferne krvi. Nadalje, preciznost
dobivena postupkom klasifikacije na uređaju Sysmex DI-60 nije uvijek bila veća u odnosu na postupak
preklasifikacije zbog nejasno definiranih morfoloških kriterija i subjektivnosti u njihovoj procjeni. Također, u
postupku preklasifikacije na uređaju Sysmex DI-60, uočen je veći udio neidentificiranih stanica u patološkim
razmazima periferne krvi u odnosu na razmaze bez patologije, što potvrđuje nužnost primjene svjetlosne
mikroskopije u takvim slučajevima. Najmanja preciznost uočena je za nesegmentirane granulocite za sve tri
metode i za ponovljivost i za međupreciznost u razmazima sa i bez patologije.Preparation of a differential blood count (DBC) is commonly carried out using three methods: automated analysis
on hematology analyzers, light microscopy, and digital morphological cell analysis. Performing a DBC on
hematology analyzers is the first step in the analysis of peripheral blood DBC. In the presence of pathological cells
and cells with morphological abnormalities, the analyser provides a warning indicating a pathological finding,
suggesting the need for DBC analysis using light microscopy and/or digital morphological analysis. Despite
technological advancements, DBC performed using light microscopy remains the gold standard. However, the
advantage of using digital morphology analyzers lies in time savings, along with high precision and accuracy. The
aim of this thesis was to examine and compare the precision of DBC performed by light microscopy and digital
morphological analysis (reclassification and classification processes) on peripheral blood smears with and without
pathology, using the Sysmex DI-60 analyzer. A total of seven peripheral blood smears were analyzed: three smears
of routine samples in which the precision of light microscopy and digital morphological analysis was examined,
and four smears from the external quality assessment program in which the precision of digital morphological
analysis was assessed. Based on the obtained results, the hypothesis of greater precision of digital morphological
analysis compared to the light microscopy method was confirmed. Exceptions were pathological smears with acute
leukemias, in which light microscopy proved to be more precise in counting pathological, immature peripheral
blood cells. Furthermore, the precision obtained by the classification process on the Sysmex DI-60 analyzer was
not always higher compared to the reclassification process, due to poorly defined morphological criteria and
subjectivity in their assessment. Additionally, in the reclassification process on the Sysmex DI-60 analyzer, a
higher proportion of unidentified cells was observed in pathological peripheral blood smears compared to nonpathological
smears, confirming the necessity of using light microscopy in such cases. The lowest precision was
observed for non-segmented granulocytes across all three methods, for both repeatability and intermediate
precision, in smears with and without pathology