Design, synthesis and biological activity of phenylpropiofenone aminoalcoxy derivatives

U ovoj doktorskoj disertaciji opisana je sinteza šest halkonskih intermedijera kao prekursora za sintezu šest novih aminoalkoksi derivata fenilpropiofenona i flavanona. Aminoalkoksi derivati fenilpropiofenona su strukturni analozi postojećeg antiaritmika Ic grupe, propafenona. U položaje 2 i 4 terminalnog benzenovog prstena uvođeni su supstituenti koji modifikuju hidrofobne i elektronske osobine molekula u cilju povećanja selektivnosti i potentnosti prema određenom tipu jonskog kanala. Sinteza propafenonskih derivata obuhvata pet faza: aldolnu kondenzaciju; katalitičku hidrogenizaciju; formiranje epoksida; aminolizu i građenje hidrohloridnih soli. Ako se alkalni, vodeno-etanolni rastvor trifluorometil halkona podvrgne dejstvu povišene temperature, nastaje flavanon koji hlađenjem kristališe u vidu igličastih kristala. Čistoća i struktura sintetisanih jedinjenja potvrđena je hromatografskim i spektroskopskim metodama, dok je struktura sintetisanog flavanona potvrđena i kristalografski. Vazorelaksantna aktivnost propafenona i novosintetisanih derivata (1-100 μM) ispitana je na izolovanoj aorti pacova sa i bez endotela, koja je prethodno prekontrahovana rastvorom fenilefrina (10 μM). Svih šest sintetisanih derivata, uključujući i propafenon, pokazali su doznozavisni vazorelaksantni efekat za koji je eksperimentalno potvrđeno da nije endotel zavisan. Učešće jonskih kanala u vazorelaksantnom mehanizmu dejstva derivata propafenona ispitano je u prisustvu: lidokaina, antagoniste Na+ kanala (3 mM), nifedipina, selektivnog antagoniste LCa2+ kanala (1 μM) i 4-aminopiridina (4-AP), neselektivnog antagoniste K+ kanala (3 mM). Dobijeni rezultati ukazuju da su jonski kanali uključeni u mehanizam vazorelaksantnog dejstva i da uvedene strukturne modifikacije u molekulu propafenona doprinose postizanju izvesnog stepena selektivnosti. 4-AP antagonizovao je efekat derivata koji u para položaju terminalnog benzenovog prstena ima –CH3 grupu (5PCH3). Srednja efektivna koncentracija za derivat 5PCH3 (pEC50 4,41) tri puta je veća od koncentracije propafenona (pEC50 4,97) i dva puta od koncentracije sledećeg u nizu po kompeticiji, para fluoro supstituisanog derivata (5PF). U prisustvu nifedipina, najbolji antagonizam postignut je kod orto trifluorometil supstituisanog derivata (5OCF3), što ukazuje na veću selektivnost ovog derivata (pEC50 4,55) u odnosu na propafenon (pEC50 4,90), ali i ostale derivate (pEC50 4,70 - 4,97). Lidokain je antagonizovao efekat 5PF derivata. Srednja efektivna koncentracija 5PF derivata je dva i po puta veća u odnosu na propafenon (pEC50 4,89) i druge derivate (pEC50 4,86-5,19). Docking studije koje su sprovedene uz pomoć programa AutoDock v. 4.0.1 doprinele su razjašnjenju rezultata dobijenih u in vitro ispitivanjima antagonističkog dejstva 4-AP-a i nifedipina. Docking eksperimenti sa kristalografskom strukturom bakterijskog KcsA kanala su izvedeni pod pretpostavkom da se ispitivana jedinjenja vezuju za istu aminokiselinsku sekvencu pore kanala (Thr 107, Ala 108 i Ala 111) kao i 4-AP. Na osnovu dobijenih rezultata zaključuje se, da 5OF derivat čiji vazorelaksantni efekat nije antagonizovan 4-AP-om, pokazuje najveći afinitet za vezivno mesto 4-AP-a u pori kanala. Ako se uporede rezultati dobijeni u docking eksperimentima na izdvojenom peptidu L-Ca2+ kanala, uočava se da 5OCF3 i 5OCl derivati pokazuju veći afinitet prema vezivnom mestu u poređenju sa ostalim orto i para supstituisanim derivatima. Docking eksperimenti na homologom modelu hERG kanala prethodili su in vivo eksperimentima. Najveća pažnja tokom eksperimenata bila usmerena ka interakcijama propafenona i derivata sa aminokiselinama Phe 656 i Tyr 652 za koje se smatra da su odgovorne za neželjene efekte mnogih lekova. Na osnovu dobijenih rezultata izabrana su tri halogenovana derivata (5OCl, 5OF i 5PF) za in vivo ispitivanja. Primenom akonitinskog testa, u in vivo eksperimentima na pacovima u dubokoj anesteziji, ispitivan je antiaritmijski potencijal halogenovanih derivata propafenona..

Simulataneous determination of nimesulide and its impurities in pharmaceutical formulations by reversed-phase high-performance liquid chromatography

A simple, rapid and reproducible reversed-phase high-performance liquid chromatography method for the analysis of nimesulide and its impurities both in the bulk drug and pharmaceutical formulations is reported. The method is suitable for monitoring the stability of nimesulide. The presence of nimesulide impurities C (2- phenoxyaniline) and D (2- phenoxy4- nitroaniline) was observed. The best separation was achieved using an Agilent Zorbax Extend C-18 column ( 150 x 4.6 mm, particle size 5 mu m) at 40 C and flow rate of 1.0 mLmin(-1). The analytes were monitored at 230 nm. The mobile phase consisted of acetonitrile - triethylamine (TEA) - water (45:0.5:54.5 v/v/v), adjusted to pH 5.2 with formic acid. Under these conditions the retention times were of 7.11, 7.98 and 8.66 min for nimesulide, D and C, respectively. The resolution of nimesulide and impurity D was 3.20 and that of impurity D and impurity C 2.40, indicating that the compounds were well separated. Evaluation of linearity, accuracy, precision, selectivity, sensitivity and robustness of the method produced satisfactory results. The developed method was successfully applied to assay nimesulide in different solid pharmaceutical formulations

A new simple liquid chromatographic assay for gentamicin in presence of methylparaben and propylparaben

Gentamicin sulfate is a potent broad spectrum aminoglycoside antibiotic which is used against Gram- positive and Gram-negative bacteria. A simple, isocratic HPLC method for separation, identification and determination of gentamicin and parabens (methylparaben and propylparaben) was developed and validated. To our knowledge there is no report about simultaneous determination of those three ana- lytes in pharmaceutical products. The optimum chromatographic conditions were achieved on CN column with a mobile phase consisting of 0.15% triethylamine in 10 mM KH 2 PO4 aqueous solution (final pH 3.0 adjusted with H 3 PO 4 ) and methanol in the ratio 70:30 (v/v), providing selective quan- tification of analytes within 5 min. The method was successfully validated according to ICH guidelines acceptance criteria in terms of selectivity, linearity, accuracy, precision and robustness. The linearity of the method was proved in defined concentrations ranges for gentamicin (0.32–1.04 mg mL1 ), meth- ylparaben (0.0072–0.0234 mg mL1 ) and propylparaben (0.0008–0.0026 mg mL1 ). Relative standard deviations calculated for all analytes in precision testing were <2% (analysis repeatability) and <3% (intermediate precision). Recovery values were between 98.87% and 101.67%. Chromatographic pa- rameters are not significantly influenced by small variations of column temperature, pH and molarity of KH2 PO 4 . Finally, the method was successfully applied for quantitative determination of gentamicin and parabens in commercially available solution for injection. Proposed HPLC method is found to be promising in terms of simplicity, analysis times and non-use of derivatization and ion-pair agents

Development and validation of RP-HPLC method for quantification of trace levels of topical corticosteroids in ambiphilic cream

Corticosteroids are anti-inflammatory and immunosuppressant drugs. Topical corticosteroids formulations (ointments, creams, gels) are used in the treatment of different types of dermatitis and urticaria. Considering their therapeutic and whitening effects, they are frequently used for counterfeiting of cosmetic products. Corticosteroids can cause different local and systemic side effects. HPLC method is often chosen for their analysis, because it is selective, sensitive, precise, simple and fast. The aim of this study was optimization and validation of RP-HPLC method with UV detection for determination of trace levels of corticosteroids in ambiphilic creams. This method is used for qualitative and quantitative analysis of evaluated corticosteroids. Mometasone furoate, hydrocortisone acetate, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone dipropionate and triamcinolone acetonide were evaluated. Separation was performed on Inertsil® ODS-3V 250 × 4.6 mm, 5 μm chromatographic column. Mobile phase was mixture of acetonitrile and water 50:50 (v/v) with gradient elution and flow rate 1 mL min-1. Column temperature was held on 40 °C and UV detection was performed at 240 nm. Selectivity, linearity, accuracy, precision and limit of quantification (LOQ) were evaluated. Method is selective because ambiphilic cream base peaks and corticosteroids peaks were not overlapping. Linearity was confirmed since correlation coefficient was 1 for all compounds. Accuracy and precision were evaluated for hydrocortisone acetate and betamethasone dipropionate. Determined Recovery values were in range of 70-130%. Both RSD values (21.46% and 9.59%) were lower than 30%. Method is highly sensitive since LOQ concentrations were in ng mL-1 range. All evaluated parameters of validation were in accordance with regulatory requirements. Validated RP-HPLC method can be used for qualitative and quantitative analysis of selected corticosteroids in ambiphilic creams

3D tehnika digitalne obrade svetlosti (DLP) primenjena u izradi dvoslojnih tableta: koncept kombinovane polipilule

Ever since 3D printing was introduced to the field of pharmacy, it has caused a paradigm shift from the manufacturing of large-scale to small batches of medicines tailored accordingly to the specific needs of patients. This study aimed to formulate and fabricate two-layered 3D tablets using the digital light processing (DLP) technique. Hydrochlorothiazide (HHT,5%,w/w) and warfarin sodium (WS,5%,w/w) were selected as model drugs. The printing process was initiated with 0.1% of photoinitiator, at a constant ratio of poly(ethylene glycol)diacrylate and poly(ethylene glycol) 400, 1:1, with the addition of water (10%,w/w). Single-layered tablets of 8.00 mm diameter and 1.50 mm thickness, containing HHT and WS respectively, were successfully printed, as well as combined two-layered 3D tablets, with each of the active substances in separate layers. Dissolution tests of single-layered tablets showed immediate, but incomplete release of WS (81.47±1.47%, after 45min), and prolonged and complete release of HHT (98.17±3.11%, after 8h), while significantly slower and incomplete release of both drugs from the combined two-layered 3D tablets was observed. The absence of drug-polymer interaction and presence of a layered cross-sectional tablet structure were confirmed. DLP technique enables simple and rapid fabrication of combined two-layered 3D tablets, while further optimization of formulation factors is necessary to achieve complete drug release.Uvođenje tehnologije 3D štampe u oblasti farmacije uslovilo je razvoj fundamentalnih promena, pri čemu serijska proizvodnja velikih šarži pretenduje da bude zamenjena malim serijama lekova prilagođenih prema specifičnim potrebama pacijenata. Cilj istraživanja bio je da se formulišu i izrade dvoslojne tablete primenom tehnike digitalne obrade svetlosti (DLP). Hidrohlortiazid (HHT, 5%, m/m) i varfarin-natrijum (WS, 5%, m/m) odabrani su kao model lekovite supstance. Proces štampanja sproveden je u prisustvu 0,1% fotoinicijatora, pri konstantnom masenom odnosu poli(etilen glikol)diakrilata i poli(etilen glikola) 400, 1:1, uz dodatak 10% vode. Jednoslojne 3D tablete prečnika 8,00 mm i debljine 1,50 mm, koje sadrže HHT, odnosno WS, kao i kombinovane dvoslojne 3D tablete, sa svakom od aktivnih supstanci u posebnom sloju, uspešno su odštampane. Prilikom ispitivanja brzine rastvaranja lekovite supstance iz jednoslojnih tableta, došlo je do trenutnog (81,47±1,47%, nakon 45 min), ali nepotpunog oslobađanja WS, i produženog i potpunog oslobađanja HHT (98,17±3,11%, nakon 8 h), dok je iz kombinovanih tableta zapaženo znatno sporije i nepotpuno oslobađanje obe lekovite supstance. Potvrđeno je odsustvo interakcija i prisustvo slojevite strukture. DLP tehnika pruža mogućnost jednostavne i brze izrade kombinovanih tableta, pri čemu je dalja optimizacija formulacionih faktora neophodna u cilju postizanja potpunog oslobađanja lekovite supstance

Razvoj i validacija RP-HPLC metode za analizu višekomponentnog sirupa za kašalj

In this study a reversed phase HPLC method for rapid and simultaneous identification and quantification of doxylamine succinate, ephedrine sulfate, dextrometorphane hydrobromide, paracetamole and sodium benzoate in cough-cold syrup formulation was described. Separation was carried out on XTerraTM RP 18, Waters (150 mm x 4.6 mm column, 5 μm particle size). For the analysis of investigated substances gradient elution was used employing water, pH adjusted at 2.5 with 85 % ortophosphoric acid as the mobile phase A and acetonitrile as the mobile phase B. Detection was carried out by UV absorbance at 210 nm for doxylamine succinate, ephedrine sulfate, dextromethorphane hydrobromide and sodium benzoate and at 270 nm for paracetamole. The method was validated statistically for selectivity, linearity, precision, accuracy.U ovom radu opisana je brza, efikasna, ekonomična reverzno fazna HPLC metoda za identifikaciju i određivanje doksilamin-sukcinata, efedrin-hidrohlorida, dekstrometorfanhidrobromida, paracetamola kao aktivnih komponenti i natrijum-benzoata kao konzervansa u sirupu za kašalj. Razdvajanje komponenata, njihova identifikacija i određivanje postignuto je na C18 stacionarnoj fazi (XTerraTM RP 18, Waters (150 mm x 4,6 mm, 5 μm veličine čestica) uz gradijentno eluiranje sa mobilnom fazom koju čine voda (čiji je pH podešen na 2,5 sa ortofosfornom kiselinom) i acetonitril kao organski rastvarač. Za detekciju ispitivanih jedinjenja korišćen je UV/VIS detektor podešen na 210 nm (doksilamin sukcinata, efedrin-hidrohlorida, dekstrometorfan-hidrobromida i natrijum-benzoata) tj. 270 nm (paracetamol). Kako je definisana metoda namenjena za identifikaciju i određivanje aktivnih supstanci i konzervansa u sirupu za kašalj, od parametara validacije ispitani su: selektivnost/specifičnost, linearnost, tačnost i preciznost. Svaki od parametara je statistički potvrđen. Dobijene vrednosti statističkih parametara (r³ 0,999, CV£ 2 % i Recovery od 98 % - 102 %) ukazuju da je definisana RP- HPLC metoda pogodna za identifikaciju i određivanje doksilamin-sukcinata, efedrinhidrohlorida, dekstrometorfan-hidrobromida, paracetamola i natrijum-benzoata u sirupu za kašalj

Elektrohemijsko ponašanje ivermektina na elektrodi od staklastog ugljenika

Ivermectin (IVM) is a drug from the group of anthelmintics used in veterinary and human medicine. Recently, it has been used in the treatment of some malignant diseases, as well as viral infections caused by Zika virus, HIV-1, SARS-CoV-2, which has increased the interest in this medicine (1). Electrochemical characterization of IVM was done with the aim of better understanding the redox behavior of the molecule, as well as to predict potential transformations during interactions with other electroactive biomolecules. Electrochemical behavior was examined by cyclic (CV), differential pulse (DPV) and square wave voltammetry (SWV) using a glassy carbon electrode (GCE). The effect of pH was examined in the pH range 2–10. The results confirmed that IVM is electroactive in the entire study area. IVM shows the main oxidation peak at a potential of about 1.0 V, which is most intense in a neutral environment, and an additional (weakly expressed) peak, due to oxidation of the reduced drug form. In voltammograms, there is also a peak of low intensity, which originates from the reduction of the previously oxidized form of the drug. Based on the shape of the voltammogram, it is concluded that all these processes are irreversible. The analysis of the influence of the rate of change of potential (pH 4.6 and 7.0) is also responsible for the adsorption of the active component on the electrode. The presented results indicate the advantage of applying electrochemical methods in drug analysis due to low cost, high speed and ease of execution.Ivermektin (IVM) je lek iz grupe antihelmintik koji se primenjuje u veterinarskoj i humanoj medicini. U novije vreme našao je primenu i kao lek u terapiji nekih malignih oboljenja, kao i virusnih infekcija prouzrokovanih virusom Zika, HIV-1, SARS-CoV-2 , čime je ponovo poraslo interesovanje za ovaj lek (1). Elektrohemijska karakterizacija IVM je rađena sa ciljem boljeg razumevanja redoks ponašanja samog molekula, kao i za predviđanje potencijalnih transformacija tokom interakcija sa drugim elektroaktivnim lekovima ili biomolekulima. Elektrohemijsko ponašanje IVM-a ispitivano je cikličnom (CV), diferencijalnom pulsnom (DPV) i voltametrijom pravougaonih talasa (SWV) korišćenjem elektrode od staklastog ugljenika (GCE). Uticaj pH na oksidaciju i redukciju IVM ispitan je u opsegu pH 2–10. Rezultati su potvrdili da je IVM elektroaktivan u celoj ispitivanoj oblasti. IVM pokazuje glavni oksidacioni pik na potencijalu oko 1,0 V koji je najintenzivniji u neutralnoj sredini i dodatni (slabo izražen) pik koji je posledica oksidacije redukovanog oblika leka . U voltamogramima IVM-a javlja se i pik slabog intenziteta koji potiče od redukcije prethodno oksidovane forme leka. Na osnovu oblika voltamograma zaključuje se da su svi navedeni procesi ireverzibilni. Analizom uticaja brzine promene potencijala (pH 4,6 i 7,0) određena je priroda redoks procesa: oksidacija IVM-a je difuziono kontrolisan proces, dok je pored difuzije za redukciju IVM-a odgovorna i adsorpcija aktivne komponente na elektrodi. Pored svog fundamentalnog značaja, prikazani rezultati ukazuju na prednost primene elektrohemijskih metoda u analizi lekova zbog niske cene, velike brzine i jednostavnosti izvođenja.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Antiaritmički efekti novosintetisanih derivata propafenona

It is well known that the presence of different chemical groups in drug molecules influences their pharmacological properties. The aim of our study is to investigate whether newly synthesized derivatives of propafenone, with changes in benzyl moiety, have a different effect upon arrhythmia, compared to propafenone. 5OCl-PF and 5OF-PF are derivatives of propafenone with-Cl or –F substituent on the ortho position of the benzyl moiety. For verification of their antiarrhythmic effect, we used an in vivo rat model of aconitine-induced arrhythmia. 5OCl-PF speeded the appearance of supraventricular premature beats (SVPB) and death more than aconitine. All animals treated with 5OCl-PF developed ventricular premature beats in salvos (VPBS), bigeminies (VPBB) and paroxysmal ventricular tachycardia (PVT). 5OF-PF had a negative chronotropic effect and potentiated atrial excitability (more SVPB). It had a positive effect on the occurrence and onset time of supraventricular tachycardia, VPBS, and PVT. Based on the obtained results, it can be concluded that newly synthesized propafenone derivatives have no better antiarrhythmic effect than the parent compound. In the future, our research will be focused on the synthesis of different derivatives and examining their antiarrhythmic effects.Dobro je poznato da prisustvo različitih hemijskih grupa u molekulima leka utiče na njegova farmakološka svojstva. Cilj našeg istraživanja je ispitati da li novosintetisani derivati propafenona, s promenama u benzilnoj grupi, imaju drugačiji efekat na aritmiju u odnosu na propafenon. 5OCl-PF i 5OF-PF su derivati propafenona sa -Cl ili –F supstituentom na orto položaju benzilnog dela. Za proveru njihovog antiaritmičnog efekta koristili smo in vivo model na pacovima sa aritmijom izazvanom akonitinom. 5OCl-PF je ubrzao pojavu supraventrikularnih prevremenih otkucaja (SVPB) i smrt više nego akonitin. Sve životinje lečene sa 5OCl-PF razvile su ventrikularne prevremene otkucaje (VPBS i VPBB) i paroksizmalnu ventrikularnu tahikardiju (PVT). 5OF-PF je imao negativan hronotropni efekat i potencirao atrijalnu ekscitabilnost (više SVPB). Pozitivno je uticao na pojavu i vreme početka supraventrikularne tahikardije, VPBS i PVT. Na osnovu dobijenih rezultata se može zaključiti da novosintetisani derivati propafenona nemaju bolji antiaritmijski efekat od polaznog jedinjenja. U budućnosti, istraživanje će biti usmereno ka sintezi hemijski drugačijih derivata i ispitivanju njihovog antiaritmijskog efekta

Electrochemical Characterisation and Confirmation of Antioxidative Properties of Ivermectin in Biological Medium

Ivermectin (IVM) is a drug from the group of anthelmintics used in veterinary and human medicine. Recently, interest in IVM has increased as it has been used for the treatment of some malignant diseases, as well as viral infections caused by the Zika virus, HIV-1 and SARS-CoV-2. The electrochemical behaviour of IVM was investigated using cyclic (CV), differential pulse (DPV) and square wave voltammetry (SWV) at glassy carbon electrode (GCE). IVM showed independent oxidation and reduction processes. The effect of pH and scan rate indicated the irreversibility of all processes and confirmed the diffusion character of oxidation and reduction as an adsorption-controlled process. Mechanisms for IVM oxidation at the tetrahydrofuran ring and reduction of the 1,4-diene structure in the IVM molecule are proposed. The redox behaviour of IVM in a biological matrix (human serum pool) showed a pronounced antioxidant potential similar to that of Trolox during short incubation, whereas a prolonged stay among biomolecules and in the presence of an exogenous pro-oxidant (tert-butyl hydroperoxide, TBH) resulted in a loss of its antioxidant effect. The antioxidant potential of IVM was confirmed by voltametric methodology which is proposed for the first time