Primena veze između inflamacije i tumora u razvoju jedinjenja sa antitumorskom aktivnošću

It is estimated that up to 20% of cancer-related deaths are linked with inflammation (1). Inhibition of inflammatory enzymes COX-2 and 5-LOX impacts cancer cells directly, or indirectly via tumor microenvironment. Wider anticancer potential has been investigated for a small group of COX-2 inhibitors (2), while there are no such data for dual COX-2 and 5-LOX inhibitors. The main aim of the project is to select the most promising anticancer drug candidates from a group of COX-2 and dual COX-2 and 5-LOX inhibitors (newly synthesized and previously synthesized). New compounds will be designed using structure-based and ligand-based in silico methods and synthesized. Cytotoxicity will be evaluated towards four cancer cell lines by MTT assay. Wider anticancer potential of selected compounds, which includes synergism with conventional chemotherapy and radiotherapy, inhibition of angiogenesis and activity towards multidrug resistant cancer cells, will be investigated and lead compounds will be identified. Mechanisms of action of lead compounds will be proposed after bioinformatics analysis of genes expression. In vitro evaluation of passive gastrointestinal absorption (PAMPA and BMC), binding to human serum albumin (HPLC and electrochemistry) and metabolism (human liver microsomes) will be performed. QSPR, QSRR and QSMARt models will be created and, together with analysis of metabolism, will be used for the optimization of structures of lead compounds. The project will result in the development of new anticancer drug candidates, make new and strengthen previously established scientific collaborations and give starting point for potential clinical evaluations of lead compounds.Procenjuje se da je do 20% smrtnih slučajeva koji su posledica tumora povezano sa inflamacijom (1). Inhibicija enzima inflamacije COX-2 i 5-LOX utiče na tumorske ćelije direktno ili indirektno preko tumorskog mikrookruženja. Širi antitumorski potencijal je do sada ispitan za malu grupu COX-2 inhibitora (2), dok takva istraživanja nisu do sada vršena na dualnim COX-2 i 5-LOX inhibitorima. Glavni cilj projekta je da se identifikuju najbolji kandidati za antitumorske lekove iz grupe COX-2 i grupe dualnih inhibitora COX-2 i 5-LOX (novosintetisana i prethodno sintetisana jedinjenja). Nova jedinjenja će biti dizajnirana primenom in silico metoda koje se zasnivaju na poznavanju strukture receptora i liganda, nakon čega će biti sintetisana. Citotoksičnost će biti ispitana na četiri tumorske ćelijske linije primenom MTT testa. Širi antitumorski potencijal odabranih jedinjenja, koji podrazumeva sinergističko dejstvo sa konvencionalnom hemoterapijom i radioterapijom, inhibiciju angiogeneze i aktivnost prema multidrug rezistentnim ćelijskim linijama, će biti ispitan, nakon čega će biti identifikovana vodeća (lead) jedinjenja. Mehanizam delovanja vodećih jedinjenja će biti predložen nakon bioinformatičke analize ekspresije gena. Biće izvršena in vitro procena pasivne gastrointestinalne apsorpcije (PAMPA i BMC metodama), vezivanja za humani serumski albumin (HPLC i elektrohemijkim metodama) i metabolizma primenom humanih mikrozomnih enzima jetre. QSPR, QSRR i QSMARt modeli će biti formirani i, zajedno sa analizom metabolizma, biće upotrebljeni za optimizaciju struktura vodećih jedinjenja. Rezultat projekta će biti novi kandidati za antitumorske lekove, uspostavljanje novih i jačanje postojećih naučno-istraživačkih saradnji i postavljanje polazne tačke za potencijalna klinička ispitivanja vodećih jedinjenja.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Sinteza i fizičko‐hemijska karakterizacija tri novosintetisana derivata sulfhidroksamske kiseline kao potencijalnih dualnih inhibitora enzima ciklooksigenaze‐2 i 5‐lipooksigenaze

Inflammatory mediators derived from arachidonic acid by the enzymes cyclooxygenase (COX) and lipoxygenase (LOX) are involved in the pathogenesis of various inflammatory diseases. Inhibition of the COX pathway is thought to lead to potentiation of the LOX pathway, and inhibition of both pathways represents a rational approach to the design and development of more effective and safer anti-inflammatory drugs (1). The aim of this study was the synthesis and physico-chemical characterization of 1f, 1g and 1h derivatives derived from a previously conducted 3D-QSAR study and molecular docking (2). The sulfhydroxamic acid derivative 1f was synthesized in a two-step process. Sulfonyl chloride was synthesized from commercially available sulfonic acid and thionyl chloride in the presence of a catalytic amount of DMF. Sulfhydroxamic acid was further obtained from previously synthesized sulfonyl chloride and hydroxylamine hydrochloride in the presence of 10% NaHCO3 solution. Sulfhydroxamic acid derivatives, 1g and 1h were synthesized from commercially available sulfonyl chlorides and hydroxylamine hydrochloride in the presence of 10% NaHCO3 solution. The reaction mixtures were purified by liquid-liquid extraction and preparative TLC to give derivatives 1f, 1g, 1h in yields: 26%, 53% and 63%. The structure and purity of the synthesized compounds were confirmed by determination of the melting points and spectroscopic techniques (ATR-FTIR, 1H-NMR, 13C-NMR, MS/MS). Based on a previously conducted in silico study, the voluminous sulfhydroxamic group is responsible for iron chelation within 5-LOX active center and for COX-2 selectivity, as COX-2 has wider side pocket, so potent inhibition of COX-2 and 5-LOX enzymes is expected.Inflamatorni medijatori koji nastaju iz arahidonske kiseline posredstvom enzima ciklooksigenaze (COX) i lipooksigenaze (LOX) učestvuju u patogenezi brojnih inflamatornih oboljenja. Smatra se da inhibicija COX puta dovodi do potenciranja LOX puta, te inhibicija oba puta predstavlja racionalan pristup dizajniranja i razvoja novih, efikasnijih i bezbednijih antiinflamatornih lekova (1). Cilj rada je bila sinteza i fizičko-hemijska karakterizacija derivata 1f, 1g i 1h proisteklih iz prethodno sprovedene 3D-QSAR studije i molekulskog docking-a (2). Derivat sulfhidroksamske kiseline 1f je sintetisan u dvostepenom postupku pri čemu se najpre iz komercijalno dostupne sulfonske kiseline i tionil hlorida u prisustvu katalitičke količine DMF dobija odgovarajući sulfonil hlorid. Odgovarajuća sulfhidroksamska kiselina (1f) se dobija u drugom koraku iz prethodno sintetisanog sulfonil hlorida i hidroksilamin-hidrohlorida u prisustvu 10% rastvora NaHCO3 . Derivati sulfhidroksamske kiseline, 1g i 1h su sintetisani jednostepenim postupkom iz komercijalno dostupnih sulfonil hlorida i hidroksilamin-hidrohlorida u prisustvu 10% rastvora NaHCO3 . Nakon postupka sinteze jedinjenja su prečišćena tečno-tečnom ekstrakcijom i preparativnom TLC pri čemu se dobijaju derivati 1f, 1g, 1h u prinosima 26%, 53% i 63%. Struktura i čistoća sintetisanih jedinjenja je potvrđena određivanjem temperature topljenja i spektroskopskim (ATR-FTIR, 1 H-NMR, 13 C-NMR, MS/MS) tehnikama. Na osnovu prethodno sprovedene in silico studije, voluminozni centar i sulfhidroksamska grupa sintetisanih derivata su odgovorni za COX-2 selektivnost zbog prisustva šireg vezivnog mesta unutar COX-2 enzima, dok sulfhidroksamska grupa unutar 5-LOX enzima helira gvožđe aktivnog centra i inhibira enzim, te se očekuje potentna dualna COX-2 i 5-LOX inhibitorna aktivnost sintetisanih derivata.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Primena hromatografskih tehnika u optimizaciji procesa prečišćavanja amida kortienske kiseline iz hidrokortizona i etil estra L-glicina

Soft ('antedrug') glucocorticoids are pharmacologically active compounds which are biotransformed in a predictable and controllable way to inactive and non-toxic metabolites. Amides of cortienic acids (17(-carboxamide derivatives of glucocorticoids) are potential soft drugs with fewer side effects than traditional glucocorticoids. The purification of 17(- carboxamide derivatives of hydrocortisone was explained using the amide of hydrocortisonederived cortienic acid and ethyl ester of L-glycine as an example and performed by use of column chromatography and preparative thin-layer chromatography (TLC). The optimization of purification process was performed employing analytical TLC and reversed-phase highperformance liquid chromatography (RP-HPLC). The mobile phase that enables best chromatographic separation of the amide from impurities on TLC plate (chloroform-methanol (95:5 V/V)) was selected and modified (reduction of polarity and addition of glacial acetic acid) to be used for the column chromatography and preparative TLC purification. It was confirmed by use of RP-HPLC that purification procedures applied in this study resulted in pure (96.2 %) amide.Soft ('antedrug') glukokortikoidi su farmakološki aktivna jedinjenja koja se biotransformišu na predvidiv i kontrolisan način do neaktivnih i netoksičnih metabolita. Amidi kortienskih kiselina (17(-karboksamidni derivati glukokortikoida) su potencijalni soft lekovi sa manje neželjenih efekata u odnosu na konvencionalne glukokortikoide. Prečišćavanje 17(- karboksamidnih derivata hidrokortizona je prikazano na primeru amida kortienske kiseline iz hidrokortizona i etil estra L-glicina i vrši se primenom hromatografije na koloni i preparativne tankoslojne hromatografije (TLC). Optimizacija procesa prečišćavanja je izvršena primenom analitičke TLC i reverzno-fazne tečne hromatografije pod visokim pritiskom (RP-HPLC). Mobilna faza koja omogućuje najbolje hromatografsko razdvajanje amida od nečistoća na TLC pločici (hloroform-metanol (95:5 V/V) je odabrana i izvršena je njena modifikacija (smanjenje polarnosti, odnosno dodatak glacijalne sirćetne kliseline) u cilju prečišćavanja hromatografijom na koloni, odnosno preparativnom TLC. Primenom RP-HPLC je potvrđeno da su navedeni postupci prečišćavanja omogućili dobijanje amida stepena čistoće 96,2 %

Molecular Docking Analysis of Novel Thiourea Derivatives of Naproxen with Potential Antitumor Activity

Naproxen, as a propionic acid derivative, causes serious gastrointestinal side effects due to the presence of free carboxylic group. In that sense, masking of carboxylic group with other pharmacophores may be a promising strategy to decrease gastrointestinal toxicity. Thiourea derivatives have been intensively investigated as potential antitumor drugs, whereby their activity is based on potential inhibition of protein kinases, topoisomerases, carbonic anhydrase and sirtuins. In addition, it was shown that inhibition of certain protein kinases might reverse resistance to chemotherapeutic drugs by enhancing the cell death in the presence of low concentrations of drug. Twenty new thiourea derivatives of naproxen were designed and their binding to four selected protein kinases involved in tumor multidrug resistance (AKT2, mTOR, EGFR and VEGFR1) was estimated using two molecular docking programs (AutoDock Vina and OEDocking). According to OEDocking, the highest potential to inhibit AKT2 and mTor has derivative 1, while derivative 20 demonstrates the highest potential towards EGFR and VEGFR1. According to AutoDock Vina, the highest potential for inhibition of EGFR, AKT2 and VEGFR1 have derivatives 16 and 17. Therefore, derivatives 1, 16, 17 and 20 are potentially the most potent protein kinase inhibitors that could be further synthesized and tested for anticancer activity

Synthesis and RP-TLC lipophilicity evaluation of a novel fluocinolon acetonide soft drug derivative

Cortienic acid was obtained by periodic acid oxidation of fluocinolone acetonide, whereas corresponding amide was synthesized from the cortienic acid and ethyl ester of b-alanine by dicyclohexylcarbodiimide - hydroxybenzotriazole coupling procedure. Lipophilicity of the amide was evaluated by using reversed-phase thin-layer chromatography systems, consisting of ethanol and water in various ratios, and was higher in comparison to fluocinolone acetonide and cortienic acid

Development and Validation of a New Isocratic RP-HPLC Method for Simultaneous Determination of Sodium Metabisulfite and Sodium Benzoate in Pharmaceutical Formulation

A simple and convenient reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous separation, identification, and determination of sodium metabisulfite and sodium benzoate in pharmaceutical formulation has been developed and validated. Chromatographic separation was achieved on RP column Zorbax Extend C-18 (150 x 4.6 mm i.d., 3.5 mu m particles), and mixture of 0.1% phosphoric acid and acetonitrile in the ratio 62: 38 (v/v) was used as a mobile phase. The flow rate was set at 1.0 mL/min with detection wavelength of 275 nm. The method was successfully validated according to International Conference on Harmonization (ICH) guidelines acceptance criteria. The method is selective, as no interferences were observed at retention times corresponding to these analytes. Results of regression analyses (r) and statistical insignificance of calibration curve intercepts (p) proved linearity of the method in defined concentration ranges for sodium metabisulfite and sodium benzoate (0.05-0.15 mg/mL). Relative standard deviations calculated for both analytes in precision testing were below the limits defined for active pharmaceutical ingredients (analysis repeatability: lt 2%; intermediate precision: lt 3%). Recovery values were between 98.16% and 101.94%. According to results of robustness testing, chromatographic parameters are not significantly influenced by small variation of acetonitrile content in mobile phase, column temperature, and flow rate. Finally, the method was applied for quantitative determination of investigated preservatives in real sample analysis

Razvoj i validacija metode tečne hromatografije za određivanje acetilsalicilne i salicilne kiseline u doziranim oblicima

Acetylsalicylic acid belongs to nonsteroidal anti-inflammatory drugs with antiinflammatory, analgesic and antipyretic properties. The aim of this work was development and validation of HPLC method for qualitative and quantitative analysis of acetylsalicylic acid and its major degradation product, salicylic acid, in dosage forms. The optimal separation was achived using Zorbax Eclipse XDB-C18 Analytical column (4,6x150 mm, particle size 5 μm) thermostated at 35°C. Mobile phase consisted of eluents A and B in ratio 65:35 (V/V). As the eluent A, water of HPLC purity and 85% phosphoric acid in ratio 80:0.5 (V/V) were used, while acetonitrile was used as the eluent B. The flow rate was 1.0 mL/min and UV detection was performed at 240 nm. The method was validated in terms of selectivity, linearity, precision, accuracy and robustness for both analytes, as well as limits of detection and quantification for salicylic acid. The obtained results meet the requirements of analytical procedures validation. The proposed HPLC method was applied in qualitative and quantitative analysis of acetylsalicylic and salicylic acids in six different forms of drugs. All obtained results meet the requirements of manufacturer specifications. The established HPLC method was found to be rapid, simple, accurate and selective for simultaneous determination of acetylsalicylic and salicylic acids in dosage forms.Acetilsalicilna kiselina pripada grupi nesteroidnih antiinflamatornih lekova koji ispoljavaju antiinflamatorno, analgetičko i antipiretičko delovanje. Cilj ovog rada je bio razvoj i validacija HPLC metode za kvalitativnu i kvantitativnu analizu acetilsalicilne kiseline i njenog degradacionog proizvoda, salicilne kiseline, u doziranim oblicima. Optimalno razdvajanje ispitivanih analita postignuto je na koloni Zorbax Eclipse XDB-C18 Analytical (4,6x150 mm, veličina čestica 5 μm) na temperaturi od 35°C. Mobilnu fazu čine smeša A i B u odnosu 65:35 (V/V). Smeša A je voda HPLC čistoće i 85% fosforna kiselina u odnosu 80:0,5 (V/V), a B je acetonitril. Protok je bio 1,0 mL/min, a talasna dužina detekcije 240 nm. Metoda je validirana i ispitani su sledeći parametri validacije: selektivnost, linearnost, preciznost, tačnost i robusnost za oba analita, kao i limiti detekcije i kvantifikacije za salicilnu kiselinu. Dobijene vrednosti su u skladu sa definisanim kriterijumima za validaciju metode. Predložena HPLC metoda je primenjena za kvalitativnu i kvantitativnu analizu acetilsalicilne i salicilne kiseline u šest različitih komercijalnih preparata. Svi rezultati ispitivanja su u dozvoljenim granicama specifikacija proizvođača. Predložena HPLC metoda pod datim eksperimentalnim uslovima predstavlja brz, jednostavan, tačan i selektivan postupak za istovremeno određivanje acetilsalicilne i salicilne kiseline u doziranim oblicima

Influence of Solid Drug Delivery System Formulation on Poorly Water-Soluble Drug Dissolution and Permeability

The majority of drugs have a low dissolution rate, which is a limiting step for their absorption. In this manuscript, solid dispersions (SD), solid self-microemulsifying drug delivery systems (S-SMEDDS) and solid self-nanoemulsifying drug delivery systems (S-SNEDDS) were evaluated as potential formulation strategies to increase the dissolution rate of carbamazepine. Influence of increased dissolution rate on permeability of carbamazepine was evaluated using PAMPA test. In S-SMEDDS and S-SNEDDS formulations, the ratio of liquid SMEDDS/SNEDDS and solid carrier (Neusilin((R)) UFL2) was varied, and carbamazepine content was constant. In SD formulations, the ratio of carbamazepine and Neusilin((R)) UFL2, was varied. Formulations that showed the best dissolution rate of carbamazepine (SD_1:6, SMEDDS_1:1, SNEDDS_1:6) were mutually compared, characterization of these formulations was performed by DSC, PXRD and FT-IR analyses, and a PAMPA test was done. All formulations have shown a significant increase in dissolution rate compared to pure carbamazepine and immediate-release carbamazepine tablets. Formulation S-SMEDDS_1:1 showed the fastest release rate and permeability of carbamazepine. DSC, PXRD and FT-IR analyses confirmed that in S-SMEDDS and S-SNEDDS carbamazepine remained in polymorph form III, and that it was converted to an amorphous state in SD formulations. All formulations showed increased permeability of carbamazepine, compared to pure carbamazepine

The use of PAMPA for skin permeability and retention evaluation of metilprednisolone - derived cortienic acid derivatives as potential soft glucocorticoids

Amidi ili estri kortienske kiseline metilprednizolona predstavljaju potencijalne soft glukokortikoide sa manje izraženim neželjenim efektima u odnosu na konvencionalne glukokortikoide. Retencija i permeabilnost su važne osobine soft glukokortikoida za primenu na kožu koje mogu značajno uticati na njihovu aktivnost i pojavu neželjenih efekata. Jedna od in vitro metoda koja se najčešće koristi za procenu ovih osobina je PAMPA (Parallel Artificial Membrane Permeability Assay). Cilj ovog rada je procena permeabilnosti i retencije u koži pet amida (MPMA, MPMAB, MPMAIB, MPMGB i MPPA) i jednog tioestra (MPEMP) kortienske kiseline metilprednizolona primenom PAMPA testa. Procena permeabilnosti i retencije u koži izvršena je na hidrofobnoj Millipore PVDF PAMPA mikrotitarskoj ploči sa 96 odeljaka. Praćena je difuzija ispitivanih jedinjenja kroz membranu koju čine 70 % silikonsko ulje i 30 % izopropilmiristat. Koncentracije u polaznim rastvorima, donorskim i akceptorskim odeljcima određene su primenom LC‐MS/MS metode. Primenom PAMPA testa određeni su koeficijenti permeabilnosti (logPe) i retencije (R). Vrednosti logPe su u opsegu od ‐6,81 do ‐5,09, dok su vrednosti R u opsegu 1,52 ‐ 65,25. Jedinjenje sa najnižom vrednošću logPe je MPMGB, dok su za MPEMP određene najviše vrednosti parametara logPe i R, te se od ovog derivata mogu očekivati najveća permeabilnost i retencija u koži. Permeabilnost i retencija u koži pet amida (MPMA, MPMAB, MPMAIB, MPMGB i MPPA) i jednog tioestra (MPEMP) kortienske kiseline metilprednizolona procenjeni su primenom PAMPA testa. Najviše vrednosti parametara logPe i R su dobijene za MPEMP, te se ovaj derivat izdvaja kao najpovoljniji za primenu na kožu.Amides or esters of methylprednisolone‐derived cortienic acid are potential soft glucocorticoids with fewer side effects in comparison to traditional glucocorticoids. Retention and permeability are important properties of soft glucocorticoids for local skin application which could significantly influence their activity and occurrence of side effects. PAMPA (Parallel Artificial Membrane Permeability Assay) is one of the mostly used in vitro methods for the estimation of these properties. The aim of this study was estimation of skin permeability and retention of five amides (MPMA, MPMAB, MPMAIB, MPMGB and MPPA) and one thioester (MPEMP) of metilprednisolone ‐ derived cortienic acid using PAMPA. Estimation of skin permeability and retention was performed on hydrophobic Millipore PVDF PAMPA microtiter 96‐well plate. Diffusion of tested compounds through membrane, consisting of 70% silicon oil and 30% isopropyl myristate, was tested. Concentrations in starting solutions, as well as in donor and acceptor compartments were determined using LC‐MS/MS method. PAMPA permeability coefficients (logPe) and retention (R) were determined. logPe values ranged from ‐6.81 to ‐5.09, whereas R values ranged from 1.52 to 65.25. Derivative with the lowest value of logPe was MPMGB, whereas the highest values of logPe and R were determined for MPEMP. Therefore, highest skin permeability and retention could be expected from MPEMP. Skin permeability and retention of five amides (MPMA, MPMAB, MPMAIB, MPMGB and MPPA) and one thioester (MPEMP) of metilprednisolone ‐ derived cortienic acid was estimated by use of PAMPA. The highest values of logPe and R were calculated for MPEMP, which could be considered the best candidate for skin application.VII Kongres farmaceuta Srbije sa međunarodnim učešćem Zajedno stvaramo budućnost farmacije, Beograd, 10-14. oktobar 2018