Two fast, simple, selective and economical sample preparation methods for the determination of entecavir in biological materials available at low amounts are reported. The choice of optimal extraction techniques was performed with regard to analyte hydrophilicity, sample volumes, selectivity, method recovery and rapidity. The compatibility of the eluate with the hydrophilic interaction chromatography (HILIC) mobile phase was crucial to allow the elimination of the evaporation and reconstitution steps and to obtain acceptable peak shapes. Different types of sorbents were employed for the extraction of two biological materials (plasma and plasma ultrafiltrate). The mixed-mode polymeric sorbent MCX was chosen as a suitable one for the solid phase extraction (SPE) of plasma samples. The analytes were eluted with 1ml of the mixture of 5 % ammonium hydroxide in ACN:water (95:5). Protein precipitation (PP) with 1ml of ACN was used to remove proteins from 500μl of plasma sample prior to SPE extraction. The microextraction by packed sorbent (MEPS) was employed for the cleaning up of plasma ultrafiltrate samples due to very small volumes available for the analysis. MEPS implemented a novel sorbent based on porous graphitic carbon, semi-automatic analytical syringe and a small volume of sample (50μl). The elution step was performed using 100μl of the mixture of 5mM ammonium acetate pH 4.0:ACN (25:75). The MEPS eluate was fully compatible with HILIC mobile phase subsequently used for the analysis of entecavir, unlike SPE eluate, which had to be evaporated and reconstituted in mobile phase. Both analytical methods were validated and demonstrated good linearity in a range 1-100ng/ml (r(2)>0.9992) for plasma samples and in a range 0.5-100ng/ml (0.9991) for the plasma ultrafiltrate samples. Intra-day accuracy expressed as recovery was within the range from 80-98% for the plasma samples and 97-106% for the plasma ultrafiltrate samples. Inter-day accuracy ranged within 81-106% for the plasma and 95-101% for the plasma ultrafiltrate samples. The intra-day precision expressed as the % of RSD was lower than 4% for both matrices and inter-day precision was lower than 7% for plasma and lower than 17% for plasma ultrafiltrate. Method sensitivity reached LLOQ of 1ng/ml in plasma and 0.5ng/ml in plasma ultrafiltrate samples. The method was applied for the determination of concentration-time profiles of entecavir in plasma of the perfusate for rat kidney perfusion and for the measurement of concentration of entecavir in plasma ultrafiltrate samples. The results should be helpful in the evaluation of excretion mechanism of entecavir.

• Klíčová slova
• Antivirals, Hydrophilic compounds, MEPS, Porous graphitic carbon, Sample preparation,
• MeSH
• acetonitrily chemie   MeSH
• adsorpce MeSH
• antivirové látky krev   farmakokinetika   MeSH
• chemické techniky analytické MeSH
• chromatografie MeSH
• guanin analogy a deriváty   krev   farmakokinetika   MeSH
• hydrofobní a hydrofilní interakce MeSH
• krysa rodu Rattus MeSH
• limita detekce MeSH
• mikroextrakce na pevné fázi MeSH
• potkani Wistar MeSH
• referenční standardy MeSH
• reprodukovatelnost výsledků MeSH
• rozpouštědla MeSH
• tandemová hmotnostní spektrometrie MeSH
• ultrafiltrace MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetonitrile MeSH Prohlížeč
• acetonitrily MeSH
• antivirové látky MeSH
• entecavir MeSH Prohlížeč
• guanin MeSH
• rozpouštědla MeSH

Entecavir is a deoxyguanosine nucleotide antiviral agent with the activity against hepatitis B virus (HBV). The agent possesses a polar structure, which is predetermined for hydrophilic interaction chromatography (HILIC). Novel, fast and sensitive HILIC-UHPLC method developed in this study included separation from matrix component on BEH Amide stationary phase by isocratic elution using binary mobile phase composed of acetonitrile/5mM ammonium acetate pH 4.0 (75:25) at flow-rate 0.3 ml/min. Analysis under RP-UHPLC conditions was also possible on BEH C18 stationary phase with mostly aqueous binary mobile phase composed of (4:96) acetonitrile/0.01% formic acid. The comparison of sensitivity of the two UHPLC-MS/MS methods both using selected reaction monitoring (SRM) for quantitation revealed only slightly higher sensitivity for HILIC determination, however much better method linearity, repeatability and accuracy. HILIC separation mode provided also more convenient conditions for straightforward coupling with solid phase extraction (SPE). Entecavir was extracted on Oasis HLB cartridge (1 ml, 30 mg) and eluted by 75% acetonitrile in water, which is actually the HILIC mobile phase used in this study. Therefore the evaporation/reconstitution step was omitted, which substantially accelerated the sample preparation step. The method was validated using stable isotopically labeled internal standard entecavir-C(2)(13) N(15), which is the most appropriate internal standard. Validation results demonstrated good method accuracy (with < 5% error, and 26% at LOQ), recovery (87-114%), precision (<4% RSD), selectivity and sensitivity (LOQ=100 pg/ml). The matrix effects determined by both post-column infusion method as well as post-extraction addition method were negligible (<15%).

• MeSH
• extrakce na pevné fázi MeSH
• guanin analogy a deriváty   farmakokinetika   moč   MeSH
• hydrofobní a hydrofilní interakce MeSH
• krysa rodu Rattus MeSH
• limita detekce MeSH
• lineární modely MeSH
• reprodukovatelnost výsledků MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• entecavir MeSH Prohlížeč
• guanin MeSH

The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.

• Klíčová slova
• ATP-binding cassette transporters, Concentrative nucleoside transporters, Entecavir, Equilibrative nucleoside transporters, Placental transfer,
• MeSH
• ABC transportér z rodiny G, člen 2 metabolismus   MeSH
• dítě MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• membránové transportní proteiny metabolismus   MeSH
• nádorové proteiny metabolismus   MeSH
• nádory prsu * metabolismus   MeSH
• nukleosidy metabolismus   farmakologie   MeSH
• P-glykoprotein metabolismus   MeSH
• P-glykoproteiny metabolismus   MeSH
• placenta * metabolismus   MeSH
• potkani Wistar MeSH
• protein spojený s mnohočetnou rezistencí k lékům 2 MeSH
• proteiny přenášející nukleosidy metabolismus   farmakologie   MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům metabolismus   MeSH
• těhotenství MeSH
• uridin MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 4-nitrobenzylthioinosine MeSH Prohlížeč
• ABC transportér z rodiny G, člen 2 MeSH
• ABCB1 protein, human MeSH Prohlížeč
• ABCC2 protein, human MeSH Prohlížeč
• ABCG2 protein, human MeSH Prohlížeč
• entecavir MeSH Prohlížeč
• membránové transportní proteiny MeSH
• nádorové proteiny MeSH
• nukleosidy MeSH
• P-glykoprotein MeSH
• P-glykoproteiny MeSH
• protein spojený s mnohočetnou rezistencí k lékům 2 MeSH
• proteiny přenášející nukleosidy MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům MeSH
• uridin MeSH

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.

• Klíčová slova
• antivirals, drug–drug interactions, nephrotoxicity, renal disposition,
• Publikační typ
• časopisecké články MeSH

Nucleos(t)ide analogues entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are recommended as first-line monotherapies for chronic hepatitis B (CHB). Multiple HBV genotypes/subgenotypes have been described, but their impact on treatment response remains largely elusive. We investigated the effectiveness of ETV/TDF on HBV/D-subgenotypes, D1/D2/D3/D5, studied the structural/functional differences in subgenotype-specific reverse transcriptase (RT) domains of viral polymerase, and identified novel molecules with robust inhibitory activity on various D-subgenotypes. Transfection of Huh7 cells with full-length D1/D2/D3/D5 and in vitro TDF/ETV susceptibility assays demonstrated that D1/D2 had greater susceptibility to TDF/ETV while D3/D5 exhibited poorer response. Additionally, HBV load was substantially reduced in TDF-treated CHB patients carrying D1/D2 but minimally reduced in D3/D5-infected patients. Comparison of RT sequences of D-subgenotypes led to identification of unique subgenotype-specific residues, and molecular modeling/docking/simulation studies depicted differential bindings of TDF/ETV to the active site of their respective RTs. Replacement of signature residues in D3/D5 HBV clones with corresponding amino acids seen in D1/D2 improved their susceptibility to TDF/ETV. Using high throughput virtual screening, we identified N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases, including N6-substituted (S)-FPMP derivative of 2,6-diaminopurine (DAP) (OB-123-VK), as potential binders of RT of different D-subgenotypes. We synthesized (S)-FPMPG prodrugs (FK-381-FEE/FK-381-SEE/FK-382) and tested their effectiveness along with OB-123-VK. Both OB-123-VK and FK-381-FEE exerted similar antiviral activities against all D-subgenotypes, although FK-381-FEE was more potent. Our study highlighted the natural variation in therapeutic response of D1/D2/D3/D5 and emphasized the need for HBV subgenotype determination before treatment. Novel molecules described here could benefit future design/discovery of pan-D-subgenotypic inhibitors. IMPORTANCE Current treatment of chronic hepatitis B relies heavily on nucleotide/nucleoside analogs in particular, tenofovir disoproxil fumarate (TDF) and entecavir (ETV) to keep HBV replication under control and prevent end-stage liver diseases. However, it was unclear whether the therapeutic effects of TDF/ETV differ among patients infected with different HBV genotypes and subgenotypes. HBV genotype D is the most widespread of all HBV genotypes and multiple D-subgenotypes have been described. We here report that different subgenotypes of HBV genotype-D exhibit variable response toward TDF and ETV and this could be attributed to naturally occurring amino acid changes in the reverse transcriptase domain of the subgenotype-specific polymerase. Further, we identified novel molecules and also synthesized prodrugs that are equally effective on different D-subgenotypes and could facilitate management of HBV/D-infected patients irrespective of D-subgenotype.

• Klíčová slova
• antiviral activity, entecavir, hepatitis B virus D-subgenotypes, high throughput virtual screening, phosphonate prodrug, tenofovir,
• MeSH
• antivirové látky chemie   farmakologie   terapeutické užití   MeSH
• chronická hepatitida B farmakoterapie   virologie   MeSH
• genotyp MeSH
• guanin analogy a deriváty   chemie   farmakologie   terapeutické užití   MeSH
• inhibitory reverzní transkriptasy chemie   farmakologie   terapeutické užití   MeSH
• lidé MeSH
• mutace MeSH
• organofosfonáty chemie   farmakologie   MeSH
• prekurzory léčiv MeSH
• proteinové domény MeSH
• racionální návrh léčiv * MeSH
• reverzní transkriptasa chemie   genetika   MeSH
• tenofovir chemie   farmakologie   terapeutické užití   MeSH
• virová léková rezistence účinky léků   genetika   MeSH
• virová nálož účinky léků   MeSH
• virus hepatitidy B účinky léků   enzymologie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antivirové látky MeSH
• entecavir MeSH Prohlížeč
• guanin MeSH
• inhibitory reverzní transkriptasy MeSH
• organofosfonáty MeSH
• prekurzory léčiv MeSH
• reverzní transkriptasa MeSH
• tenofovir MeSH

Hepatitis B virus (HBV) infection remains a global public health problem with changing epidemiology due to several factors predominantly vaccination policy and migration. Chronic hepatitis B means the duration of HBV infection for more than 6 months. It is a dynamic process reflecting the interaction between HBV replication and the host immune response and not all patients with chronic HBV infection have chronic hepatitis B. All patients with chronic HBV infection are in increased risk of progression to liver cirrhosis and hepatocellular carcinoma. The long-term administration of potent nucleos(t)ide analogue with high barrier of resistance (tenofovir, entecavir) represents the treatment of choice. Pegylated interferon-α can also be considered in mid to moderate chronic hepatitis B patients.

• Klíčová slova
• chronic hepatitis B, entecavir, pegylated interferon-α, tenofovir,
• MeSH
• antivirové látky * terapeutické užití   MeSH
• chronická hepatitida B * komplikace   diagnóza   farmakoterapie   MeSH
• lidé MeSH
• virus hepatitidy B MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH

Hepatitis B virus (HBV) infection remains a global public health problem with changing epidemiology due to several factors predominantly vaccination policy and migration. Chronic hepatitis B is a dynamic process reflecting the interaction between HBV replication and the host immune response and not all patients with chronic HBV infection have chronic hepatitis B. Stopping of nucleotide or nucleoside analogues (NA) therapy is a serious resolution due the danger of reactivation of viral replication associated with increasing HBV DNA level, ALT activity and inflammatory activity in the liver histology. The safest stopping rule for NA therapy is HBsAg loss what is the sign of immune control of HBV infection.

• Klíčová slova
• chronic hepatitis B, entecavir, stopping therapy., tenofovir,
• MeSH
• antivirové látky terapeutické užití   MeSH
• chronická hepatitida B * diagnóza   farmakoterapie   MeSH
• hepatitida B - antigeny povrchové MeSH
• hepatitida B * diagnóza   farmakoterapie   epidemiologie   MeSH
• lidé MeSH
• virus hepatitidy B genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky MeSH
• hepatitida B - antigeny povrchové MeSH

Hepatitis B (HBV) is a DNA virus, which cannot be eradicated completely from the organism by treatment, only its replication can be suppressed to low levels. The pathogenesis of liver damage due to HBV is immune mediated, the infected hepatocytes represent the target structures of immune reaction. In individuals who spontaneously achieved the state of inactive carriage of the virus or even achieved HBsAg negativity, we deal only with immune control of viral replication. Chemotherapy or immunosuppressive treatment disrupt the immune control of HBV infection, the virus replication substantially increases and hepatitis B reactivates. HBV reactivation manifests as further flareup of chronic inflammation with rapid progression of liver cirrhosis or even as a fulminant hepatitis with liver failure. The risk of reactivation increases with degree of induced immunosuppression, the highest risk is associated with corticosteroid and rituximab therapy. HBV reactivation threatens patients during solid tumours treatment as well as haemato oncological malignancies, patients treated with immunosuppressive and bio-logical therapies for systemic inflammatory diseases and inflammatory bowel diseases, as well as patients on maintenance haemodialysis, after kidney transplantation and patients with HBV/ HIV co infection. HBV reactivation increases both morbidity and mortality in listed groups of patients. The patients threatened by HBV reactivation can be identified easily based on HBV serological markers assessment. Preemptive therapy with nucleos(t)ide analogues significantly reduces the risk of HBV reactivation, the effect of longterm antiviral therapy is described in detail in kidney transplant recipients in whom the 3rd generation antivirals (entecavir and tenofovir) completely obviate the negative impact of HBV on longterm survival. In oncological patients who are treated for a determined time period, we can use lamivudine, which is not suitable for longterm treatment due to high risk of resistance emergence.

• MeSH
• adenin analogy a deriváty   terapeutické užití   MeSH
• aktivace viru * MeSH
• antivirové látky terapeutické užití   MeSH
• biologické markery MeSH
• chronická hepatitida B diagnóza   imunologie   prevence a kontrola   MeSH
• guanin analogy a deriváty   terapeutické užití   MeSH
• hepatitida B - antigeny povrchové imunologie   MeSH
• imunokompromitovaný pacient * MeSH
• imunosupresiva škodlivé účinky   MeSH
• lamivudin terapeutické užití   MeSH
• lidé MeSH
• myší monoklonální protilátky škodlivé účinky   MeSH
• organofosfonáty terapeutické užití   MeSH
• protinádorové látky škodlivé účinky   MeSH
• rituximab MeSH
• tenofovir MeSH
• virus hepatitidy B imunologie   MeSH
• zánět MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• adenin MeSH
• antivirové látky MeSH
• biologické markery MeSH
• entecavir MeSH Prohlížeč
• guanin MeSH
• hepatitida B - antigeny povrchové MeSH
• imunosupresiva MeSH
• lamivudin MeSH
• myší monoklonální protilátky MeSH
• organofosfonáty MeSH
• protinádorové látky MeSH
• rituximab MeSH
• tenofovir MeSH

Viral hepatitis B is a serious health problem, above all in the world's developing countries. It is estimated that two billion people will be infected with the hepatitis B virus in the course of their lives and between 350 and 400 million people are chronically infected at present. The aims of the treatment of chronic hepatitis B are sustained suppression of viral replication and remission of liver disease. For the treatment of chronic hepatitis B two drugs have been licensed worldwide: alpha-interferon and lamivudine. In chronic hepatitis B therapy there are new developments in antiviral, such as nucleoside analogues--entecavir, emtricitabine, clevudine, beta-L-nucleosides. Studies comparing pegylated interferon with lamivudine and with the combination of lamivudine and pegylated interferon are in progress. Several innovative antiviral approaches have been evaluated in vitro, and in animal models of hepadnavirus infections. These approaches are including antisense oligonucleotides, ribozymes, inhibitors blocking virus entry into hepatocytes, and decoy virus or dominant negative mutants.

• MeSH
• antivirové látky terapeutické užití   MeSH
• chronická hepatitida B farmakoterapie   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky MeSH

Mutations occurring in viral polymerase gene of hepatitis B virus (HBV) due to the use of nucleos(t)id analogs reduce the activity of the drugs by causing antiviral resistance. In this study, it was aimed to evaluate mutations responsible for drug resistance and drug resistance mutation rates in patients followed up by the diagnosis of chronic hepatitis B (CHB). A total of 318 CHB patients were included in the study. HBV mutations were detected using the INNO-LiPA commercial kit based on the reverse hybridization principle. Drug resistance mutation was detected in 46.86% (149/318) of the patients. The rates of drug resistance were found 36.79% (117/318) for lamivudine resistance, 12.58% (40/318) for entecavir (ETV), and 7.86% (25/318) for adefovir. In 10 patients, the possible tenofovir (TDF) resistance (3.14%) was found. Single-drug and double-drug resistances were detected in 34.59% and in 11.01% of the patients, respectively. Triple drug resistance was detected in only 1.26% of the patients. Unlike various studies in Turkey and in other countries, remarkable resistance to ETV and TDF were found in this study. The high rate of the probable TDF resistance was striking, with 3.14%.

• MeSH
• antivirové látky farmakologie   terapeutické užití   MeSH
• chronická hepatitida B farmakoterapie   mikrobiologie   MeSH
• dítě MeSH
• DNA virů genetika   MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mnohočetná virová léková rezistence genetika   MeSH
• mutace MeSH
• předškolní dítě MeSH
• retrospektivní studie MeSH
• reverzní transkriptasa genetika   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• virová léková rezistence genetika   MeSH
• virus hepatitidy B účinky léků   genetika   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Turecko MeSH
• Názvy látek
• antivirové látky MeSH
• DNA virů MeSH
• reverzní transkriptasa MeSH