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Synthesis and characterization of urea harmines and aminoalcohols
No full textHarmin je β-karbolinski alkaloid prirodnog podrijetla koji je u mnogim istraživanjima pokazao brojne farmakološke učinke, uključujući i antimalarijsko djelovanje. Selektivnim inhibitornim djelovanjem na protein toplinskog šoka P. falciparum (PfHsp90) u in vitro i in vivo istraživanjima uspješno je djelovao na parazita P. falciparum. Zbog specifičnog mehanizma djelovanja i mogućeg sinergizma s drugim lijekovima postoji značajan potencijal za smanjenje rezistencije na postojeću terapiju, koja je postala globalni javnozdravstveni problem. U okviru ovog rada sintetizirana su dva nova spoja, uree harmina s aminoalkoholima u položaju 9 -karbolinskog prstena (5a,b). Novosintetiziranim spojevima potvrđena je struktura te su okarakterizirani analitičkim i spektroskopskim metodama koje uključuju IR, 1H NMR, 13C NMR te MS. Novim spojevima je također određena i temperatura tališta, a u kasnijim istraživanjima bit će im ispitano i antimalarijsko djelovanje.Harmine is a naturally occurring β-carboline alkaloid which was proven to have many pharmacological effects, including antimalarial. Harmine exhibited activity against P. falciparum in in vitro and in vivo studies, possibly due to its selective and inhibitory effect on P. falciparum heat shock protein (PfHsp90). Because of the specific mechanism of action and synergy with other antimalarials, harmine possesses a potential for lowering the incidence of resistance to the available drugs, which has become a global health burden. In this thesis two novel compounds were synthesised, harmine ureas with amino alcohols, 5a and 5b. The newly synthesized compounds have been characterized using spectroscopic and analytical methods, (IR, 1H NMR, 13C NMR, MS) and their melting point was determined. Future research will focus on evaluation of their antimalarial activity
Method optimization for quantification of nicotine in tobacco by gas chromatography-mass spectrometry
No full textDuhan, Nicotiana tabacum, porodice Solanaceae, jedan je od najčešće konzumiranih sredstava ugode i ovisnosti,
najčešće pušenjem. Razlog njegove ovisnosti je nikotin, kao jedan od glavnih sastojaka samoga duhana. Ovisno
o vrstama duhana kao što su Virginia, Burley, Maryland ili Oriental, ovisit će i koncetracija nikotina u duhanu.
Nikotin djeluje na nikotinske acetilkolinske receptore (nAChR) stvarajući ovisnost, ali i brojne fiziološke te
patofiziološke učinke. Često ilegalno trgovanje duhanom, kao i njegova uzročnost brojnih požara, uz brojne
druge razloge, dovode do potrebe optimizacije metode kvantitativnog određivanja nikotina u duhanu. Tako je
metoda pripreme uzoraka duhana za kvantitativno određivanje nikotina u duhanu optimizirana Box-Behnkenovim
dizajnom temeljenim na metodi odzivne površine. Korištenjem tri varijable u spomenutom dizajnu, udjela
metanola u otapalu za ekstrakciju, vremena soniciranja te broja ekstrakcija, dobivene su optimalne vrijednosti
navedenih parametara za pripremu uzoraka duhana. Kvantitativno određivanje nikotina odrađeno je pomoću
plinske kromatografije sa spektrometrijom masa (GC-MS) koja je validirana provjerom brojnih validacijskih
parametara te zadovoljila postavljene kriterije prihvatljivosti. Razvijena metoda pokazala se prikladnom za
navedenu svrhu kvantitativnog određivanja nikotina u duhanu te će se moći koristiti u Centru za forenzična
ispitivanja, istraživanja i vještačenja „Ivan Vučetić'', MUP-a RH.Tobacco, Nicotiana tabacum, Solanaceae, is one of the most widely used stimulants and addictive substances,
consumed mainly by smoking. The reason for its addictive nature lies in nicotine, one of the main components
of tobacco. Depending on the type of tobacco, such as Virginia, Burley, Maryland or Oriental, the nicotine
concentration in tobacco also varies. Nicotine is a supstrate of nicotine acetylcholine receptors (nAChR), which
are addictive and cause many other physiological and patophysiological effects. Illegal tobacco and its causality
in numerous fire incidents, among many other reasons, have led to the need to optimize the method of
quantitative analysis of nicotine in tobacco. The method of preparing tobacco samples for quantitative analysis
of nicotine in tobacco was optimized by Box-Behnken design based on response surface modeling (RSM). In
this design, three parameters were used, namely the percentage of methanol in the extraction solution, the
duration of sonication, and the number of extractions for which optimal results were obtained, which were then
used to prepare the tobacco samples. The quantitative analysis of nicotine in tobacco samples was performed
using Gas Chromatography-Mass Spectrometry which was validated by checking numerous validation
parameters that met the established acceptance criteria. The method was thus found to be suitable for the
quantitative analysis of nicotine in tobacco and will be used at the Ivan Vučetić Forensic Science Center in
Zagreb, Croatia
Potvrda identiteta farmaceutskih supstancija u ljekarničkom laboratoriju NIR spektroskopijom
No full textCILJ ISTRAŽIVANJA Izrada magistralnih i galenskih pripravaka u ljekarničkom laboratoriju još uvijek predstavlja najpogodniju, u pojedinim slučajevima i jedinu, terapijsku opciju za pacijenta kad ne postoji industrijski lijek ili je potrebna priprema pogodnog terapijskog oblika ili doze individualno prema potrebi pacijenta. Magistralni i galenski pripravci ne prolaze strogu završnu kontrolu kvalitete kao industrijski lijekovi, a ljekarnik je u potpunosti odgovoran za kvalitetu farmaceutskih supstancija, izradu pripravaka te njihovo pravilno označavanje i izdavanje. Cilj specijalističkog rada je dati pregled regulative i smjernica o izradi magistralnih i galenskih pripravaka, obrazložiti važnost ulazne kontrole farmaceutskih supstancija (potvrde identiteta) koje se koriste za izradu pripravaka, prikazati analizu tri farmaceutske supstancije NIR spektroskopijom i objasniti njezine prednosti u potvrdi identiteta farmaceutskih supstancija te dati poticaj podizanju svijesti o potrebi uspostavljanja sustava kvalitete u ljekarničkom laboratoriju kao i podizanju kvalitete i sigurnosti izrade magistralnih i galenskih pripravaka.
MATERIJALI I METODE Istraživanja u svrhu izrade rada uključuju pregled dostupne znanstvene literature o NIR tehnici i njenoj primjeni u analitici lijekova općenito, trenutnih studija, smjernica i zakonskih regulativa vezano uz izradu i kontrolu kvalitete magistralnih i galenskih pripravaka te ulaznu potvrdu identiteta farmaceutskih supstancija.
Specijalistički rad je eksperimentalnog karaktera. U praktičnom dijelu rada korištene farmaceutske supstancije za analizu NIR spektroskopijom (eritromicin, glicerol, bijeli vazelin) su zadovoljavajućih zahtjeva kvalitete u skladu s monografijama Europske farmakopeje. Analizirane su na uređaju Apo-Ident 2 NIR spektrometru prilagođenom potvrdi identiteta supstancija u ljekarništvu. Apo-Ident 2 u svom programu QuickStep sadrži referentne NIR spektre najčešćih supstancija korištenih u izradi pripravaka u ljekarnama. Potvrda identiteta bijelog vazelina provedena je dodatno i kemijskom
reakcijom prema monografiji Hrvatske farmakopeje usklađenoj s Europskom farmakopejom. Analize su provedene u ljekarničkom laboratoriju ZU Ljekarna Marušić, u prostoriji za analize u uvjetima reguliranog tlaka, temperature i izmjene zraka.
REZULTATI Rezultati analize tri farmaceutske supstancije prikazani su u obliku NIR spektara, uz objašnjenje načela NIR tehnike i načina interpretacije rezultata mjerenja. Prikazan je rezultat i kemijske reakcije potvrde identiteta bijelog vazelina. Navode se prednosti NIR tehnike u svrhu potvrde identiteta pred nepouzdanim i nedovoljno selektivnim kemijskim reakcijama potvrde identiteta, uključujući jednostavnu i brzu analizu bez obrade uzorka koji se ne uništava mjerenjem te se dalje može koristiti u izradama pripravaka. NIR tehnika ne zahtijeva upotrebu standarda jer se identitet potvrđuje usporedbom NIR spektra ispitivane supstancije s referentnim spektrom iz baze podataka. Prikazano je kako je koncipiran sustav osiguranja kvalitete u ZU Ljekarni Marušić, zašto je važno slijediti propisane standardne operativne postupke, čuvati obaveznu dokumentaciju sirovina (certifikate analize) i analitička izvješća ulazne analize farmaceutskih supstancija. ZAKLJUČAK Magistralni i galenski pripravci još uvijek predstavljaju jedinu terapijsku opciju kada ne postoji industrijski lijek prikladnog oblika, doze, punila i slično. Ljekarnik je u potpunosti odgovoran za nabavu farmaceutskih supstancija, njihovu ulaznu analizu (potvrdu identiteta), izradu pripravaka prema smjernicama struke te pravilno izdavanje. NIR tehnika omogućava brzu i jednostavnu provjeru identiteta supstancija u tekućem, čvrstom i polučvrstom stanju, bez obrade uzorka, pripreme reagensa ili gubitaka analizirane supstancije. Potvrda identiteta u ZU Ljekarni Marušić uređajem Apo-Ident 2 NIR spektrometrom predstavlja vrlo jednostavnu, brzu i egzaktnu opciju, koja osigurava da pacijentima budu izdani sigurni i kvalitetni magistralni pripravci.OBJECTIVES Compounding of magistral and galenic preparations in compounding laboratory in pharmacy in some cases is still most suitable and only therapeutic option for patient when industrial (registered) drug is unavailable or doesn't exist on the market, or is necessary to prepare specific pharmaceutical form, or adjust therapeutic doses individually depending on patient's needs. Compounded magistral and galenic preparations are not subjected to strict final quality control testing as industrial drugs, and pharmacist is completely responsible for quality of pharmaceutical substances, compounding process, and proper labeling and dispensing of compounded preparations. The aim of this paper is to give insight in regulations and guidelines of compounding of magistral and galenic preparations, explain importance of entry quality control of pharmaceutical substances (identity confirmation) used in compounding of preparations, show results of analyses of three pharmaceutical substances analyzed with NIR spectroscopy, explain advantages of NIR spectroscopy in identity confirmation of pharmaceutical substances, raise awareness of need of quality management system in compounding laboratory in pharmacy, also putting quality and safety of compounded preparations on higher level.
MATERIALS AND METHODS Research for purpose of creating this paper includes review of available scientific literature about NIR technique and its application in analysis of drugs, current studies, guidelines and legal regulations about magistral and galenic preparations and entry identity confirmation of pharmaceutical substances.
This paper has experimental nature. In practical part of the paper, pharmaceutical substances (erythromycin, glycerol and white vaseline) used for analysis by NIR spectroscopy, meet the quality requirements according to monographs of European pharmacopoeia. They were analyzed on the
Apo-Ident 2 NIR spectrometer device adapted to confirm the identity of substances in pharmacy. Apo-Ident 2 in its QuickStep program contains the reference NIR spectrums of the most common substances used in compounding of preparations in pharmacies. Identity confirmation of white vaseline was additionally carried out by a chemical reaction according to the monograph of the Croatian Pharmacopoeia harmonized with the European Pharmacopoeia. The analyses were performed in the pharmacy laboratory of ZU Ljekarna Marušid, in the analysis room under conditions of regulated pressure, temperature and air exchange.
RESULTS The results of the analysis of three pharmaceutical substances are presented in the form of NIR spectrums, with an explanation of the principles of NIR technique and how to interpret the measurement results. The result of the chemical reaction of identity confirmation of white vaseline is also shown. The advantages of the NIR technique for the purpose of identity confirmation over unreliable and insufficiently selective chemical identity confirmation reactions are listed, including simple and fast analysis without sample processing that is not destroyed by measurement and can further be used in compounding of preparations. The NIR technique does not require the use of standards because the identity is confirmed by comparing the NIR spectrum of the tested substance with a reference spectrum from the database. The concept of quality assurance system at ZU Ljekarna Marušid is shown, also stated why it is important to follow the prescribed standard operating procedures, to keep the mandatory documentation of raw materials (certificates of analysis) and analytical reports of entry analysis of pharmaceutical substances.
CONCLUSION
Magistral and galenic preparations are still the only therapeutic option when there is no industrial drug of suitable form, dose, excipient and so on. The pharmacist is fully responsible for the purchase of pharmaceutical substances, their entry analysis (identity confirmation), compounding of
preparations according to professional guidelines, and proper dispensing. NIR technique enables a quick and simple identity confirmation of a substance in liquid, solid and semi-solid form, without sample processing, preparation of reagents or losses of the analyzed substance. Identity confirmation at ZU Ljekarna Marušid with the Apo-Ident 2 NIR spectrometer is very simple, fast and exact option, which ensures that safe and high-quality preparations are dispensed to patients
Pharmacological therapy of stable angina pectoris
No full textStabilna angina pektoris predstavlja najčešću kliničku manifestaciju ishemijske bolesti srca, a povezana je s narušenom kvalitetom života i povećanim rizikom od štetnih kardiovaskularnih događaja. Važno je prepoznati njezine simptome i započeti s liječenjem koje ima dva glavna cilja. Prvi od njih je sprječavanje ili smanjenje pojave anginoznih simptoma te poboljšanje kvalitete života, dok je drugi cilj prevencija ozbiljnih kardiovaskularnih događaja. Patofiziološku podlogu čini prisutna opstruktivna koronarna arterijska bolest, odnosno prisutnost aterosklerotskih plakova u epikardijalnim koronarnim arterijama zbog čega se javlja neravnoteža između potrebe srca za kisikom i opskrbe srca kisikom. Farmakološka terapija stabilne angine uključuje primjenu antiishemijskih lijekova, kojima se nastoji uspostaviti ravnoteža između opskrbe srca kisikom i potrebe srca za kisikom, te lijekova koji preveniraju kardiovaskularne događaje. Među antiishemijskim lijekovima razlikuju se lijekovi prve i druge linije liječenja, pri čemu se u prvu liniju ubrajaju β-blokatori, blokatori kalcijevih kanala i kratkodjelujući nitrati. Uz dugodjelujuće nitrate, lijekovima druge linije smatraju se nikorandil, ranolazin, trimetazidin i ivabradin. Lijekovi druge linije se koriste u slučaju prisutnih kontraindikacija na lijekove iz prve linije, u slučaju njihove nepodnošljivosti ili ako se prvom linijom ne može postići dobra kontrola simptoma. Pri tome je važno istaknuti kako ne postoje usporedbe između prve i druge linije koje bi pokazale superiornost jedne skupine nad drugom u pogledu učinkovitosti. Od lijekova koji preveniraju kardiovaskularne događaje značajni su antiagregacijski lijekovi (acetilsalicilna kiselina, P2Y12 antagonisti), statini i ACE inhibitori. Revaskularizacija miokarda može se razmotriti kod bolesnika koji imaju prognostički značajne lezije ili simptome unatoč optimalnoj farmakološkoj terapiji, pri čemu je moguće izvesti perkutanu koronarnu intervenciju, premoštenje koronarnih arterija ili oboje.Stable angina pectoris represents the most common clinical manifestation of ischemic heart disease, and is associated with impaired quality of life and increased risk of adverse cardiovascular events. It is important to recognize its symptoms and start treatment, which has two main goals. The first of them is the prevention and reduction of anginal symptoms and the improvement of quality of life, while the second goal is the prevention of serious cardiovascular events. The pathophysiological basis is the presence of obstructive coronary artery disease, that is, the presence of atherosclerotic plaques in the epicardial coronary arteries, which causes an imbalance between the heart's need for oxygen and the heart's oxygen supply. Pharmacological therapy of stable angina pectoris includes use of anti-ischemic drugs which try to restore balance between the heart's oxygen supply and the heart's need for oxygen, and drugs that prevent cardiovascular events. Among anti-ischemic drugs, first- and second-line drugs are distinguished, whereby β-blockers, calcium channel blockers and short-acting nitrates are included in the first line. In addition to long-acting nitrates, second-line drugs include nicorandil, ranolazine, trimetazidine, and ivabradine. Second-line drugs are used in case of contraindications for first-line drugs, in case of their intolerance or if good control of symptoms cannot be achieved with the first-line drug. It is important to point out that there are no comparisons between the first- and second-line drugs that would show the superiority of one group over the other in terms of effectiveness. Among the drugs that prevent cardiovascular events, antiplatelet drugs (acetylsalicylic acid, P2Y12 antagonists), statins and ACE inhibitors are important. Myocardial revascularization may be considered in patients who have prognostically significant lesions or symptoms despite optimal pharmacology therapy, where percutaneous coronary intervention, coronary artery bypass grafting or both can be performed
N-glycosylation G of immunoglobulin G in cererospinal fluid and serum in multiple sclerosis
No full textIntratekalna oligoklonska ekspanzija plazma-stanica u multiploj sklerozi (MS) podržava ozljedu središnjega
živčanog sustava (SŽS) i povezana je s aktivnom demijelinizacijom. Struktura N-glikana na konzerviranom
asparaginu 297 u svakoj od CH2 domena Fc regije imunoglobulina G (IgG) izravno utječe na njegove efektorske
funkcije. U upali se obrasci N-glikozilacije IgG-a mijenjaju i moduliraju proupalne ili protuupalne efektorske
funkcije što se potencijalno odražava na status i aktivnost MS-a. Cilj istraživanja bio je analizirati N-glikozilaciju
serumskoga i likvorskog IgG-a te ukupnih serumskih proteina u osoba s MS-om.
Presječna studija uključila je konsekutivne pacijente sa sumnjom na demijelinizacijsku bolest SŽS-a. Dijagnoza
MS-a temeljena je na McDonaldovim kriterijima iz 2017. godine, a kontrolna skupina sastojala se od osoba kod
kojih je dijagnoza MS-a isključena. Analiza fluorescentno obilježenih N-glikana izoliranih iz ukupnih proteina
seruma, serumskoga i likvorskog IgG-a, provedena je tekućinskom kromatografijom ultravisoke učinkovitosti
temeljenom na hidrofilnim interakcijama (HILIC-UPLC). N-glikani su uspoređeni s Expanded Disability Status
Score (EDSS), znakovima aktivnosti bolesti na magnetskoj rezonanciji (MR), brojem relapsa i biljezima
intratekalne upale (broj stanica, koncentracija IgG-a u likvoru i postotak intratekalne sinteze, oligoklonske trake,
pozitivan indeks specifičnih antitijela na neki od virusa morbili, rubela, varicella zoster ili herpes simpleks virusa
tipa 1 i 2 (MRZH reakcija)).
Razlike među skupinama pronađene su samo u N-glikomu IgG-a likvora. Osobe s MS-om imaju veći udio Nglikana s račvajućim N-acetilglukozaminom (P = 2,63 x 10-5) i monogalaktoziliranih N-glikana (P = 1,49 x 10-6)
koji su povezani s prisutnosti oligoklonskih traka. Svojstva N-glikozilacije, specifično strukture
monogalaktozilirane na α6 anteni: FA2[6]G1 (r = 0,56, P < 0,001) i FA2[6]BG1 (r = 0,45, P = 0,001), koreliraju
samo s postotkom intratekalne sinteze IgG-a, ali ne i ukupnom koncentracijom IgG-a u likvoru. Navedena svojstva
također su više zastupljena u MRZH pozitivnih osoba s MS-om. FA2[6]BG1 struktura je povišena u osoba s MSom s većim opterećenjem lezijama na MR-u (P = 0,0186), ali N-glikozilacija IgG-a iz likvora nije promijenjena
ovisno o prisutnosti aktivnih lezija, EDSS-u niti broju relapsa.
Promjene u N-glikozilaciji koje rezultiraju većom monogalaktozilacijom intratekalno sintetiziranih IgG-a
najistaknutija su svojstva u MS-u te su povezana s radiološkom aktivnosti bolesti.The intrathecal oligoclonal expansion of plasma cells in multiple sclerosis (MS) supports central nervous system
(CNS) injury and is associated with active demyelination. The structure of the N-glycan at the conserved
asparagine 297 in each of the CH2 domains of the Fc region of immunoglobulin G (IgG) directly affects its effector
functions. In inflammation, IgG N-glycosylation patterns change and modulate pro-inflammatory or antiinflammatory effector functions, potentially reflecting MS status and activity. The aim of the research was to
analyse N-glycosylation of serum and cerebrospinal fluid (CSF) IgG and total serum proteins in people with MS.
A cross-sectional study included consecutive patients with suspected demyelinating disease of the CNS. The
diagnosis of MS was based on the 2017 McDonald criteria, and the control group consisted of people in whom the
diagnosis of MS was excluded. The analysis of fluorescently labelled N-glycans isolated from total serum proteins,
serum and CSF IgG was performed by ultrahigh performance liquid chromatography based on hydrophilic
interactions (HILIC-UPLC). N-glycans were compared with Expanded Disability Status Score (EDSS), signs of
disease activity on magnetic resonance imaging (MRI), number of relapses and markers of intrathecal
inflammation (cell count, IgG concentration in the CSF and percentage of intrathecal synthesis, oligoclonal bands,
positive index specific antibodies to either measles, rubella, varicella zoster or herpes simplex viruses type 1 and
2 (MRZH reaction)).
The differences between groups were found only in the N-glycome of CSF IgG. People with MS had a higher
proportion of N-glycans with bisecting N-acetylglucosamine (P = 2.63 x 10-5) and monogalactosylated N-glycans
(P = 1.49 x 10-6) which were associated with the presence of oligoclonal bands. The N-glycosylation properties,
specifically structures monogalactosylated at the α6 antenna: FA2[6]G1 (r = 0.56, P < 0.001) and FA2[6]BG1 (r =
0.45, P = 0.001), correlated only with the percentage intrathecal synthesis of IgG, but not with the total
concentration of IgG in the CSF. These structures were also more prevalent in MRZH positive people with MS.
The FA2[6]BG1 structure was elevated in MS subjects with higher MRI lesion load (P = 0.0186), but CSF IgG Nglycosylation was not altered depending on the presence of active lesions, EDSS nor the number of relapses.
Changes in N-glycosylation resulting in higher monogalactosylation of intrathecally synthesized IgGs are the most
prominent features in MS and are associated with the radiological activity of the disease
Therapeutic approaches in treating children with autism spectrum disorder
No full textPoremećaji iz spektra autizma su pervazivni razvojni poremećaji karakterizirani nepravilnom socijalnom interakcijom i komunikacijiom te prisutnošću ponavljajućih i stereotipnih ponašanja i interesa. Točan uzrok nastanka autizma još uvijek nije poznat, smatra se da je autizam kompleksni poremećaj koji je rezultat međudjelovanja okolišnih čimbenika i genetskih faktora. Autizam se često manifestira uz velik broj pridruženih simptoma kao što su anksioznost, agresija, nepažnja, hiperaktivnost, problemi sa spavanjem te gastrointestinalni problemi. Iako ne postoji specifična terapija dostupna za uklanjanje osnovnih simptoma bolesti, koriste se različiti lijekovi iz brojnih farmakoterapijskih skupina kako bi se ublažili pridruženi simptomi (atipični antipsihotici za razdražljivost i impulzivnost, inhibitori ponovne pohrane serotonina za anksioznost i opsesivno-kompulzivne simptome, psihostimulansi kod hiperaktivnosti i nepažnje). Mnogi lijekovi koji potencijalno imaju učinak na smanjenje osnovnih simptoma bolesti su trenutno u fazama istraživanja (arbaklofen i bumetanid). Nefarmakološke mjere kao što su bihevioralna terapija, terapija igrom i glazbom i nutritivne intervencije pokazale su najbolje rezultate u poboljšanju ishoda liječenja te ih treba započeti u što ranijoj dobi kako bi se maksimalizirao njihov učinak. Individualizirani pristup koji se sastoji od multidisciplinarnog tima stručnjaka i podrške obitelji obavezan je za ostvarivanje uspješno provedenih intervencija s ciljem poboljšanja kvalitete života osoba oboljelih od autizma.Autism spectrum disorder is a pervasive developmental disorder characterized by impaired social interaction, communication and repetitive and stereotyped patterns of behavior and interests. Even though the exact cause of autism is still unknown, it is considered that autism is a complex disorder resulting from the interaction of environmental and genetic factors. ASD often manifests with a wide range of associated symptoms including anxiety, aggression, inattention, hyperactivity, sleep problems and gastrointestinal disorders. Although there are no specific medications available for the treatment of core symptoms of autism, various drugs from different pharmacotherapeutic groups have shown their usefulness in associated conditions (atypical antipsychotics for irritability and impulsivity, selective serotonin reuptake inhibitors for anxiety and obsessive-compulsive disorder, psychostimulants for hyperactivity and inattention). Many drugs that potentially reduce core symptoms are currently being tested (arbaclofen and bumetanide). Nonpharmacological interventions, including behavioural therapy, play and music therapy as well as nutritive interventions proved to be effective in improving treatment outcomes and should be started at the earliest possible age to maximise their effect. An individualized approach that consists of a multidisciplinary team of experts and family support is cruical to the successful implementation of interventions with the aim of improving the quality of life in autistic individuals
The effectiveness of bioinformatics tools in predicting the pathogenicity of somatic mutations proven by next-generation sequencing technology in patients diagnosed with acute myeloid leukemia
No full textAkutna mijeloična leukemija genski je heterogena bolest kod koje se je u 97% bolesnika dokazana barem jedna
genska promjena. Posljednje smjernice za postavljanje dijagnoze i stratifikaciju rizika bolesnika oboljelih od AML
naglašavaju važnost detekcije genskih i citogenetskih promjena. Unatoč znatnom napretku dijagnostike i liječenja
AML-a, petogodišnje preživljenje ostaje vrlo nisko. Kako bi se poboljšao ishod liječenja bolesnici se na temelju
detektiranih genskih i citogenetskih promjena svrstavaju u rizične skupine na temelju kojih se procjenjuje
vjerojatnost postizanja remisije i preživljenja. Jedan od procesa određivanja mutacija u genima vezanim uz AML
je sekvenciranje sljedeće generacije. Sekvenciranjem sljedeće generacije moguće je istovremeno detektirati velik
broj genskih promjena u ispitivanim regijama genoma. Bioinformatički alati omogućuju medicinskim djelatnicima
bržu klasifikaciju i interpretaciju detektiranih varijanti. U ovom diplomskom radu uspoređena je učinkovitost
bioinformatičkih alata u predviđanju patogenosti somatskih mutacija dokazanih tehnologijom sekvenciranja
sljedeće generacije kod bolesnika s dijagnozom akutne mijeloične leukemije. Cilj ovog rada je evaluacija in house
postupka analize podataka dobivenih sekvenciranjem sljedeće generacije za mijeloidni panel, utvrđivanje stupnja
povezanosti dva bioinformatička alata za analizu podataka dobivenih tehnologijom NGS-a te utvrditi postoje li
odstupanja između njih. U prvom koraku ispitana je sposobnost detekcije mutacija, dok je u drugom koraku
uspoređena sposobnost klasifikacije varijanti VarSome Clinical algoritma u odnosu na tercijarnu analizu.
Podudaranje SureCall-a i VarSome Clinical-a u postupcima sekundarne analize iznosi 97,5%. Nadalje dokazano
je da podudaranje in house tercijarne analize i VarSome Clinical-a iznosi 84,2%.Acute myeloid leukemia (AML) is a genetically heterogeneous disease, with genetic changes being detected in
97% of all patients. The latest guidelines for the diagnosis and risk stratification of patients with AML emphasize
the importance of detecting genetic and cytogenetic changes. Despite significant advances in the diagnosis and
treatment of AML, five-year survival remains very low. In order to improve the outcome of treatment, patients
are classified into risk groups that are based on detected genetic and cytogenetic changes. One of the methods for
detecting mutations in AML-related genes is next-generation sequencing (NGS). Using NGS is possible to
simultaneously detect a large number of gene changes in the examined regions of the genome. Bioinformatic
tools enable medical professionals to classify and interpret detected variants more efficiently. This thesis aimed
to evaluate in-house procedure for analyzing data obtained by next-generation sequencing for the myeloid panel,
determine the degree of connection between two bioinformatics tools for analyzing data obtained by NGS
technology, and determine whether there are discrepancies between them. In this research, a comparison of two
bioinformatics tools (SureCall and VarSome Clinical) was carried out at two levels. Firstly, the ability to detect
mutations was tested, while in the second step, VarSome Clinical algorithm ability to classify mutations was
compared in relation to the in-house tertiary analysis. The agreement between SureCall and VarSome Clinical in
secondary analysis procedures is 97.5%. It was further proven that the agreement between the in-house tertiary
analysis and VarSome Clinical is 84.2%
Bacteriophage therapy clinical trials: hurdles and solutions
No full textAntibiotska rezistencija predstavlja globalni problem koji sve više usmjerava znanstvenu zajednicu prema istraživanju alternativnih metoda za liječenje bakterijskih infekcija. Jedan od potencijalnih pristupa u rješavanju ovog problema jest razvoj i primjena terapijskih sustava baziranih na bakteriofagima. S obzirom na to da je terapijski potencijal bakteriofaga prepoznat već gotovo stoljeće, te da postoje brojni izvještaji i studije koje potvrđuju njihovu učinkovitost u liječenju bakterijskih infekcija, cilj ovog preglednog diplomskog rada bio je istražiti razloge za ograničenu primjenu fagoterapije u kliničkoj praksi. Prema dostupnim podacima, trenutno je u tijeku ili u procesu regrutacije pacijenata ukupno 23 kliničke studije koje istražuju primjenu bakteriofaga. Ove studije provode se u Sjedinjenim Američkim Državama, Kanadi, Francuskoj, Belgiji, Danskoj, Češkoj, Gruziji, Iranu, Uzbekistanu i Kini, a ciljne bakterije uključuju ESKAPE patogene: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa i vrste Enterobacter. Među glavnim izazovima u razvoju terapijskih sustava s bakteriofagima izdvajaju se: 1) farmakokinetička svojstva bakteriofaga, koji zbog svoje kompleksne strukture izazivaju imunološke reakcije i pokazuju varijabilnu stabilnost u različitim fiziološkim uvjetima; 2) ograničena učinkovitost oralne primjene uslijed kiselog pH u želucu, dok je parenteralna primjena, osobito intraperitonealna, učinkovitija jer omogućuje bržu apsorpciju i viši titar bakteriofaga u cirkulaciji; 3) rijetke, ali potencijalne nuspojave koje mogu nastati uslijed oslobađanja endotoksina tijekom bakterijske lize; 4) mogućnost razvoja rezistencije bakterija na bakteriofage kroz mehanizme poput stvaranja biofilma, modifikacije površinskih receptora, inhibicije superinfekcije, restrikcijsko-modifikacijskih sustava, autodestrukcije stanica te CRISPR-Cas sustava adaptivne imunosti. Dodatno, primjena bakteriofaga u terapijske svrhe ograničena je nedostatkom adekvatnih regulatornih okvira u mnogim zemljama, što komplicira međunarodnu suradnju i standardizaciju. Proizvodnja bakteriofaga u skladu sa standardima Dobre proizvođačke prakse (DPP) predstavlja značajan izazov zbog sklonosti ovih virusa evolucijskim promjenama i teškoća u reproducibilnosti proizvodnih procesa. Iako bakteriofagi imaju određena ograničenja, poput mogućnosti razvoja rezistencije i poteškoća s patentiranjem, genetski modificirani bakteriofagi pružaju mogućnosti za poboljšanje njihove učinkovitosti i prevladavanje bakterijske rezistencije. Ipak, njihova primjena suočava se s dodatnim regulatornim izazovima zbog korištenja genetski modificiranih organizama, što dodatno otežava njihovu širu implementaciju u kliničku praksu.Antibiotic resistance represents a global challenge that increasingly directs the scientific community toward the exploration of alternative methods for treating bacterial infections. One potential approach to addressing this issue is the development and application of therapeutic systems based on bacteriophages. Given that the therapeutic potential of bacteriophages has been recognized for nearly a century, and considering the numerous reports and studies confirming their effectiveness in treating bacterial infections, the aim of this review thesis is to investigate the reasons for the limited clinical implementation of phage therapy. According to available data, there are currently 23 clinical trials investigating the application of bacteriophages, either ongoing or in the process of patient recruitment. These studies are being conducted in the United States, Canada, France, Belgium, Denmark, the Czech Republic, Georgia, Iran, Uzbekistan, and China, targeting ESKAPE pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. The main challenges in the development of bacteriophage-based therapeutic systems include: 1) the pharmacokinetic properties of bacteriophages, which, due to their complex structure, provoke immune responses and exhibit variable stability under different physiological conditions; 2) the limited efficacy of oral administration due to the acidic pH of the stomach, whereas parenteral administration, particularly intraperitoneal, is more effective as it allows for faster absorption and higher bacteriophage titers in circulation; 3) rare but potential side effects that may result from endotoxin release during bacterial lysis; 4) the possibility of bacteria developing resistance to bacteriophages through mechanisms such as biofilm formation, surface receptor modification, superinfection inhibition, restriction-modification systems, cell autodestruction, and the CRISPR-Cas adaptive immunity system. Additionally, the use of bacteriophages in therapeutic applications is constrained by the lack of adequate regulatory frameworks in many countries, complicating international collaboration and standardization. The production of bacteriophages according to Good Manufacturing Practice (GMP) standards presents a significant challenge due to their propensity for evolutionary changes and the difficulties in achieving reproducibility in manufacturing processes. Although bacteriophages have certain limitations, such as the potential for resistance development and patenting issues, genetically modified bacteriophages offer opportunities to enhance their efficacy and overcome bacterial resistance. However, their application faces additional regulatory hurdles due to the use of genetically modified organisms, further complicating their broader implementation in clinical practice
Second-tier test in newborn screening for classical homocystinuria
No full textNasljedni metabolički poremećaji većinom su uzrokovani mutacijama gena koji kodiraju enzime, transportere ili kofaktore te utječu na različite metaboličke procese. NBS predstavlja sustavno traganje za pojedinim prirođenim bolestima kod cjelokupne novorođenačke populacije određenog područja s ciljem što ranijeg otkrivanja bolesti. U program novorođenačkog probira u RH za sada je uvršteno devet bolesti, a u planu je i proširenje probira poremećaja na klasičnu homocistinuriju. Poremećaji metioninskog ciklusa uključuju manjak određenih enzima koji sudjeluju u tom ciklusu i dovode do povišenja Met i Hcy u krvi. Upravo zbog manjka ili nefunkcionalnosti enzima CBS-a, dolazi do nakupljanja Hcy i razvoja klasične homocistinurije. Postavljanje dijagnoze u što ranijoj životnoj dobi, smanjuje mogućnost nastanka ozbiljnih komplikacija poput intelektualnih poteškoća, abnormalnosti kostiju, problema s očima te kardiovaskularnih disfunkcija. Do sada se koncentracija Hcy određivala iz uzorka plazme, ali se pokazala potreba za njegovim određivanjem iz DBS-a u drugostupanjskim testovima. Drugostupanjski testovi su ključna komponenta NBS-a kojima je moguće povećati osjetljivost i specifičnost probira, a uključuju analizu iz istog uzorka specifičnijim metodama. Cilj ovog rada bio je istražiti mogućnosti određivanja koncentracije tHcy iz DBS-a LC-MS/MS tehnologijom, kao drugostupanjskog testa, u provođenju NBS-a na klasičnu homocistinuriju. U ovom radu napravljena je usporedba mjerenja koncentracije tHcy iz uzorka suhe kapi krvi na filtarskom papiru i uzorka plazme. Na temelju rezultata može se zaključiti da između te dvije metode ne postoji značajno odstupanje te se metoda određivanja tHcy u DBS-u može koristiti za rutinski rad u laboratoriju kao drugostupanjski test novorođenačkog probira na klasičnu homocistinuriju.Inherited metabolic disorders are mostly caused by mutations in genes encoding enzymes, transporters, or cofactors, affecting various metabolic processes. NBS entails systematic screening for individual congenital diseases in the entire newborn population of a specific region, aiming for early disease detection. Currently, the newborn screening program in Croatia includes nine diseases, with plans to expand the screening to include classical homocystinuria. Disorders of the methionine cycle involve deficiencies of specific enzymes participating in the cycle, leading to elevated levels of Met and Hcy in the blood. Due to the deficiency or malfunction of CBS enzyme, there is an accumulation of Hcy resulting in classical homocystinuria. Early diagnosis reduces the risk of serious complications such as intellectual disabilities, bone abnormalities, eye problems, and cardiovascular dysfunctions. Hcy concentration has traditionally been determined from plasma samples, but there is a need to measure it from DBS in second-tier tests. Second-tier tests are essential components of NBS, which can increase the sensitivity and specificity of screening, involving analysis from the same sample using more specific methods. The aim of this study was to investigate the possibility of measuring tHcy concentration from DBS using LC-MS/MS technology as a second-tier test for NBS of classical homocystinuria. The study compared tHcy measurements from dried blood spot samples on filter paper and plasma samples. Based on the results, it can be concluded that there is no significant deviation between the two methods, indicating that measuring tHcy from DBS can be used for routine laboratory work as a second-tier test for newborn screening of classical homocystinuria
Primjena placeba u kliničkim ispitivanjima
No full textCilj istraživanja
Ovaj narativni pregled istražuje varijabilnost i mehanizme placebo učinka u kliničkim
ispitivanjima, s naglaskom na bol, depresiju, epilepsiju, bolesti crijeva i onkološke
bolesti. Cilj je razumjeti kliničke odluke iz placebom kontroliranih ispitivanja te etičke
principe koji osiguravaju dobrobit pacijenata i integritet znanstvenog istraživanja.
Materijali i metode
Ovaj specijalistički rad predstavlja narativni pregled literature, obuhvaćajući znanstvene i
stručne publikacije, kao i materijale relevantnih stručnih institucija poput Europske
agencije za lijekove i Američke agencije za hranu i lijekove. Za pretraživanje literature
korištene su elektronske bibliografske baze podataka PubMed, MEDLINE, Google
Scholar i ScienceDirect. Sva relevantna literatura objavljena do 1. ožujka 2024. godine
detaljno je istražena sukladno definiranim ciljevima rada.
Rezultati
Placebo učinak u kliničkim ispitivanjima uključuje kompleksne neurobiološke i
psihološke mehanizme. Placebo analgezija pokazuje interakciju psihičkih čimbenika,
očekivanja bolesnika i fizioloških procesa. Pozitivan placebo odgovor u ispitivanjima
epilepsije otežava potvrdu učinkovitosti terapije, a uzroci su nejasni. U onkologiji,
etičnost placebo kontrola ovisi o stadiju raka i dostupnosti terapija. Terapijska područja
poput upalnih bolesti crijeva, sindroma iritabilnog crijeva, depresije i epilepsije suočavaju
se s izazovima kao što su subjektivnost simptoma, varijabilnost bolesti i etičke dileme,
naglašavajući potrebu za poboljšanim dizajnom ispitivanja.
Zaključak
Razumijevanje uloge placeba ključno je za valjanost rezultata i procjenu stvarnog učinka
terapija. Etički izazovi zahtijevaju ravnotežu između novih spoznaja i individualne skrbi.
Transparentan i etički vođen proces placebom kontroliranih ispitivanja osigurava
optimalnu skrb bolesnika, uz prilagodbu pristupa ovisno o bolesti. Sveobuhvatno
razumijevanje placebo učinaka unaprjeđuje kvalitetu ispitivanja i zdravstvenih
intervencija, poboljšavajući skrb za bolesnike.Objectives
This narrative review explores the variability and mechanisms of the placebo effect in
clinical trials, with a focus on pain, depression, epilepsy, bowel disease and oncology.
The goal is to understand clinical decisions from placebo-controlled trials and ethical
principles that ensure the well-being of patients and the integrity of scientific research.
Materials and methods
This specialist paper presents a narrative literature review, including scientific and
professional publications, as well as materials from relevant professional institutions such
as the European Medicines Agency and the US Food and Drug Administration. The
electronic bibliographic databases PubMed, MEDLINE, Google Scholar and
ScienceDirect were used for the literature search. All relevant literature published until
March 1, 2024 was investigated in detail in accordance with the defined objectives of the
work.
Results
The placebo effect in clinical trials involves complex neurobiological and psychological
mechanisms. Placebo analgesia shows the interaction of psychological factors, patient
expectations and physiological processes. A positive placebo response in epilepsy trials
makes it difficult to confirm the effectiveness of therapy, and the causes are unclear. In
oncology, the ethics of placebo controls depends on the stage of the cancer and the
availability of therapies. Therapeutic areas such as inflammatory bowel disease, irritable
bowel syndrome, depression and epilepsy face challenges such as symptom subjectivity,
disease variability and ethical dilemmas, highlighting the need for improved trial design.
Conclusion
Understanding the role of placebo is essential for validating results and assessing the real
effect of therapies. Ethical challenges require a balance between new knowledge and
individual care. A transparent and ethically guided process of placebo-controlled trials
ensures optimal patient care, with adaptation of the approach depending on the disease. A
comprehensive understanding of placebo effects improves the quality of trials and
healthcare interventions, improving patient care