Marie Volková

Synthesis and biological activity of new salicylanilide N,N-disubstituted carbamates and thiocarbamates

The development of novel antimicrobial drugs represents a cutting edge research topic. In this study, 20 salicylanilide N,N-disubstituted carbamates and thiocarbamates were designed, synthesised and characterised by IR, (1)H NMR and (13)C NMR. The compounds were evaluated in vitro as potential antimicrobial agents against Mycobacterium tuberculosis and nontuberculous mycobacteria (Mycobacterium avium and Mycobacterium kansasii) as well as against eight bacterial and fungal strains. Additionally, we investigated the inhibitory effect of these compounds on mycobacterial isocitrate lyase and cellular toxicity. The minimum inhibitory concentrations (MICs) against mycobacteria were from 4 μM for thiocarbamates and from 16 μM for carbamates. Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, were inhibited with MICs from 0.49 μM by thiocarbamates, whilst Gram-negative bacteria and most of the fungi did not display any significant susceptibility. All (thio)carbamates mildly inhibited isocitrate lyase (up to 22%) at a concentration of 10 μM. The (thio)carbamoylation of the parent salicylanilides led to considerably decreased cytotoxicity and thus improved the selectivity indices (up to 175). These values indicate that some derivatives are attractive candidates for future research.

Interactions with selected drug renal transporters and transporter-mediated cytotoxicity in antiviral agents from the group of acyclic nucleoside phosphonates

Members of acyclic nucleoside phosphonates (ANPs) possess antiviral and antiproliferative activities. However, several clinically important ANPs may cause renal injury, most likely due to their active accumulation in the renal tubular cells. The goal of this study was to investigate in vitro relationships between the affinity of several structurally related potent ANPs to selected human transporters and their cytotoxicity. SLC (solute carrier family) transporters (hOAT1, hOCT2, hCNT2, hCNT3) and ABC (ATP-binding cassette) transporters (MDR1, BCRP), which are typically expressed in the kidney, were included in the study. The transport and toxic parameters of the tested compounds were compared to those of two clinically approved ANPs, adefovir and tenofovir. Transport studies with transiently transfected cells were used as the main method in the experiments. Most of the ANPs studied showed the potency to interact with hOAT1. GS-9191, a double prodrug of PMEG, displayed an affinity for hOAT1 comparable with that of adefovir and tenofovir. No significant interaction of the tested ANPs with hOCT2, hCNT2 and hCNT3 was observed. Only GS-9191 was found to be a strong inhibitor for both MDR1 and BCRP. PMEO-DAPy showed the potency to interact with MDR1. Most of the tested substances caused a significant decrease in cellular viability in the cells transfected with hOAT1. Only with the exclusion of GS-9191, a relatively lipophilic compound, did the in vitro cytotoxicity of the ANPs closely correspond to their potential to interact with hOAT1. The increased cytotoxicity of the studied ANPs found in OAT1 transfected cells was effectively reduced by OAT inhibitors probenecid and quercetin. The higher cytotoxicity of the compounds with affinity to hOAT1 proved in the inhibitory experiments evidences that ANPs are not only inhibitors but also substrates of hOAT1. Any clear relationship between the potency of ANPs to inhibit the studied efflux transporters and their cytotoxicity was not demonstrated. In conclusion, the study documented that among the studied transporters hOAT1 seems to be the decisive determinant for renal handling in most of the tested ANPs. This transporter may also play an important role in the mechanism of their potential cytotoxic effects. These facts are in good accordance with previous findings in the clinically used ANPs.

Entecavir Interacts with Influx Transporters hOAT1, hCNT2, hCNT3, but Not with hOCT2: The Potential for Renal Transporter-Mediated Cytotoxicity and Drug–Drug Interactions

Entecavir (ETV) is one of the most potent agents for the treatment of the hepatitis B viral infection. The drug is principally eliminated by the kidney. The goal of this study was to investigate the potential of ETV to interact in vitro with the renal SLC transporters hOAT1, hOCT2, hCNT2 and hCNT3. Potential drug–drug interactions of ETV at the renal transporters with antiviral drugs known to be excreted by the kidney (adefovir, tenofovir, cidofovir) as well as transporter-dependent cytotoxicity were also examined. Interactions with the selected transporters along with cytotoxicity were studied in several transiently transfected cellular models using specific substrates and inhibitors. ETV was found to be both a substrate and inhibitor of hOAT1 (IC50 = 175.3 μM), hCNT2 (IC50 = 241.9 μM) and hCNT3 (IC50 = 278.4 μM) transporters, although it interacted with the transporters with relatively low affinities. ETV inhibited the cellular uptake of adefovir, tenofovir, and cidofovir by hOAT1; however, effective inhibition was shown at ETV concentrations exceeding therapeutic levels. In comparison with adefovir, tenofovir, and cidofovir, ETV displayed no transporter-mediated cytotoxicity in cells transfected with hOAT1, hCNT2, and hCNT3. No significant interaction of ETV with hOCT2 was detected. The study demonstrates interactions of ETV with several human renal transporters. For the first time, an interaction of ETV with the hCNTs was proved. We show that the potency of ETV to cause nephrotoxicity and/or clinically significant drug-drug interactions related to the tested transporters is considerably lower than that of adefovir, tenofovir, and cidofovir.

Synthesis and in vitro antimycobacterial activity of 2-methoxybenzanilides and their thioxo analogues

A new series of N-(3/4-substituted phenyl) 4/5-chloro-2-methoxybenzamides and their thioxo analogues have been synthesised and evaluated for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv, as well as the two atypical strains Mycobacterium kansasii and Mycobacterium avium. Five of the most active compounds were evaluated for cytotoxicity and their ability to inhibit mycobacterial isocitrate lyase, which is responsible for latent survival of Mycobacterium. The results showed that benzthioanilides were more active than the corresponding benzanilides. The most active compound, 4-chloro-2-methoxy-N-(3,4-dichlorophenyl)benzothioamide (4e), had a minimal inhibition concentration (MIC) against M. tuberculosis of 2 μmol L(-1), which was better than the activity of the previously published corresponding salicylanilide.

Modulatory Effects of Quercetin and Rutin on the Activity, Expression and Inducibility of CYP1A1 in Intestinal HCT-8 Cells

Flavonoids, plant secondary metabolites present in fruits and vegetables, show antioxidant and anti‐tumorigenic effects in vitro, but their poor absorption from gastrointestinal tract limits their systemic efficacy in humans. On the other hand, flavonoids could protect intestinal cells against carcinogens by their potential to inhibit the enzymes metabolizing pre‐carcinogenic compounds (e.g. CYP1A) to reactive ones. This work was designed to test the effect of quercetin (the most abundant flavonoid) and rutin (the most abundant glycosidic form) on the activity, expression and inducibility of CYP1A in intestinal HCT–8 cells. CYP1A enzymatic activity was measured by ethoxyresorufin‐O‐deethylase (EROD) activity, CYP1A protein expression was detected by western blotting. The effect of flavonoids on viability of cells was examined by neutral red uptake test. No cytotoxic effect of flavonoids up to 50 μM concentration was observed. Quercetin significantly inhibited EROD activity in the cells, where CYP1A had been preinduced by β‐naphthoflavone and methylcholanthrene, and it also significantly reduced the CYP1A induction mediated by these model inducers. The effect of rutin was substantially weaker and mostly insignificant in all conducted experiments. The results suggest that quercetin may have a potential to limit the CYP1A‐mediated activation of pre‐carcinogens in intestinal cells. Copyright

The involvement of selected membrane transport mechanisms in the cellular uptake of 177Lu-labeled bombesin, somatostatin and gastrin analogues

INTRODUCTION Radiolabeled receptor-targeting peptides are a useful tool for the diagnostic imaging and radiotherapy of some malignancies. However, the retention of radioactivity in the kidney may result in renal radiotoxic injury. This study seeks to evaluate the role of endocytic receptor megalin, renal SLC influx transporters and fluid phase endocytosis (FPE) in the cellular accumulation of radiolabeled peptides. METHODS In vitro transport cellular studies using megalin ligands (RAP, albumin), fluid phase endocytosis (FPE) inhibitor rottlerin and low temperature were employed to evaluate the transport mechanisms of the peptides. Cells transfected with hOAT1 or hOCT2 were used to analyze the role of these SLC transporters. Somatostatin ((177)Lu-DOTA-[Tyr(3)]octreotate, (177)Lu-DOTA-[1-Nal(3)]octreotide), gastrin ((177)Lu-DOTA-sargastrin) and bombesin ((177)Lu-DOTA-[Pro(1),Tyr(4)]bombesin, (177)Lu-DOTA-[Lys(3)]bombesin, (177)Lu-PCTA-[Lys(3)]bombesin) analogues were involved in the study. RESULTS RAP, albumin and low temperature decreased the accumulation of all the studied peptides significantly. With one exception, rottlerin caused the concentration dependent inhibition of the cellular accumulation of the radiopeptides. No significant differences in the uptake of the peptides between the control cells and those transfected with hOAT1 or hOCT2 were observed. CONCLUSION The study showed that active transport mechanisms are decisive for the cellular accumulation in all tested (177)Lu-labeled somatostatin, gastrin and bombesin analogues. Besides receptor-mediated endocytosis by megalin, FPE participates significantly in the uptake. The tested types of renal SLC transporters are not involved in this process.

Renal Handling of Amphotericin B and Amphotericin B-Deoxycholate and Potential Renal Drug-Drug Interactions with Selected Antivirals

ABSTRACT Amphotericin B (AmB) is excreted via the renal excretion route. This excretion process may result in nephrotoxicity. However, relevant information on the precise renal excretion mechanisms is not available. The aim of the study was to analyze the possible interaction of AmB or its prodrug AmB deoxycholate (AmB-DOC) with the typical renal organic anion transporters (OATs) and organic cation transporters (OCTs), using cellular and organ models. The relevant transport systems were then investigated in terms of the drug-drug interactions of AmB-DOC with antivirals that might potentially be used concomitantly. To analyze the renal excretion mechanisms of [3H]AmB, perfused rat kidney was employed. HeLa and MDCK II cells transiently transfected with human OAT1 (hOAT1) or hOCT2 were used as the cellular models. A significant tubular secretion of AmB was demonstrated in the perfused rat kidney. The cellular studies performed confirmed the active transport of AmB into cells. AmB did not interact with hOAT1 but strongly inhibited hOCT2. In contrast, AmB-DOC inhibited both hOAT1 and hOCT2. However, [3H]AmB cellular uptake by hOAT1 and hOCT2 was not found. AmB-DOC interacted significantly with adefovir, tenofovir, and cidofovir in hOAT1-transfected cells at supratherapeutic concentrations. In conclusion, the significant potency of AmB and AmB-DOC for inhibiting the transporters was demonstrated in this study. The secretion of AmB in the renal tubules is likely not related to the transporters here, since the drug was not proven to be a substrate for them. Drug-drug interactions of AmB and the antivirals used in this study on the investigated transporters are not probable.

The in vivo disposition and in vitro transmembrane transport of two model radiometabolites of DOTA-conjugated receptor-specific peptides labelled with (177) Lu.

In vivo metabolism of the radiolabelled receptor-specific peptides has been described; however, information regarding the pharmacokinetic behaviour of the degradation products within the body is very scarce. The present study was designed to obtain new knowledge on the disposition and elimination of low-molecular radiometabolites of receptor-specific peptides in the organism and to reveal the potential involvement of selected membrane transport mechanisms in the cellular uptake of radiometabolites, especially in the kidney. The study compared pharmacokinetics of two radiometabolites: a final metabolite of somatostatin analogues, (177)Lu-DOTA-DPhe, and a tripeptide metabolite of (177)Lu-DOTA-minigastrin 11, (177)Lu-DOTA-DGlu-Ala-Tyr. Their pharmacokinetics was compared with that of respective parent (177)Lu-radiopeptide. Both radiometabolites exhibited relative rapid clearing from most body tissues in rats in vivo along with predominant renal excretion. The long-term renal retention of the smaller radiometabolite (177)Lu-DOTA-DPhe was lower than that of (177)Lu-DOTA-DGlu-Ala-Tyr. An uptake of (177)Lu-DOTA-DPhe by human renal influx transporter organic cation transporter 2 was found in vitro in a cellular model. The study brings the first experimental data on the in vivo pharmacokinetics of radiometabolites of receptor-specific somatostatin and gastrin analogues. The found results may indicate a negative correlation between the degree of decomposition of the parent peptide chain and the renal retention of the metabolite.

Determination of receptor protein binding site specificity and relative binding strength using a time-resolved competition assay

INTRODUCTION Competitive binding assays can be used to decipher not only the binding kinetics of studied ligands but also the binding site preference. Such assays are an essential step in the characterization of radioligands. However, the currently used competition assays require high concentrations of usually expensive ligands and still provide only binding site preference. By employing the time-resolved competition assay presented in this paper, binding characteristics including binding site preference can be obtained using less ligand. METHODS To demonstrate the appropriateness of the time-resolved competition assay, we developed an assay in which the ligand binding was interrupted with a competitor. Experiments were performed on human carcinoma cell lines expressing epidermal growth factor receptor (EGFR). The targeting of the receptor was performed with radio-iodinated epidermal growth factor (EGF). The employed competitors involved either natural ligand transforming growth factor alpha (TGF-α) or anti-EGFR antibodies cetuximab and panitumumab targeting the same EGFR domain. RESULTS Radio-iodinated EGF bound to EGFR was displaced with either low concentrations of cetuximab or high concentrations of panitumumab. In the case of TGF-α, we observed no competitive displacement of bound EGF at either high or low concentrations. When comparing the time-resolved competition assay with a manual competition assay, the resulting data of measured inhibition constants were in agreement. DISCUSSION The results summarised in this study confirm the appropriateness of the time-resolved competition assay for assessing ligand binding properties. The assay has the potential to complement or replace conventional competition assays for determining binding site preference in the future.