- Email: [email protected]
Entecavir plasma concentrations are inversely related to HBV-DNA decrease in a cohort of treatment-naïve patients with chronic hepatitis B
International Journal of Antimicrobial Agents 48 (2016) 324–327
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j a n t i m i c a g
Short Communication
Entecavir plasma concentrations are inversely related to HBV-DNA decrease in a cohort of treatment-naïve patients with chronic hepatitis B Lucio Boglione 1,*, Amedeo De Nicolò 1, Jessica Cusato, Gabriele Bonifacio, Giuseppe Cariti, Giovanni Di Perri, Antonio D’Avolio Unit of Infectious Diseases, Department of Medical Sciences, University of Turin, Ospedale Amedeo di Savoia, Turin, Italy
A R T I C L E
I N F O
Article history: Received 3 March 2016 Accepted 28 May 2016 Keywords: Hepatitis B Entecavir Therapeutic drug monitoring Plasma concentration
A B S T R A C T
The role of therapeutic drug monitoring (TDM) of entecavir (ETV) in the treatment of patients affected by chronic hepatitis B (CHB) has not yet been defined. Here we present an interim analysis regarding the role of ETV TDM in a prospective cohort of treatment-naïve patients with CHB who received this treatment. The results from 40 patients consecutively enrolled at our centre from 2010 to 2013 are described. The primary endpoint was the evaluation of the role of ETV plasma concentrations in the kinetics of hepatitis B virus (HBV) DNA decrease. Minimum ETV concentrations (Ctrough) were measured every month after the start of therapy for the first 3 months and then every 6 months. The main result of the pharmacokinetic analysis was the significant inverse correlation of ETV concentration after 1 month of treatment and HBV-DNA decrease after 3 months of treatment (r = −0.624; P < 0.001). This correlation was also confirmed when stratifying patients on the basis of viral genotypes: A (r = −0.719; P = 0.003); C (r = −0.917; P = 0.007); and D (r = −0.760; P = 0.007). Possible explanations for this phenomenon could involve interpatient differences in liver conditions (tissue damage or inflammation) and/or genetic variability in specific drug transporters. Further investigations are needed to confirm these results quantifying ETV concentration in peripheral blood mononuclear cells as well as in a larger cohort. © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.
1. Introduction Chronic hepatitis B (CHB) affects ca. 350 million people worldwide and is a major cause of cirrhosis, liver failure and hepatocellular carcinoma (HCC) [1]. Treatment for hepatitis B e-antigen (HBeAg)-negative CHB patients includes two different approaches: long-term therapy with oral nucleos(t)ide analogues (NAs), based on serum hepatitis B virus (HBV) DNA suppression; and finite-duration therapy with standard or pegylated interferon alfa (peg-IFNα) [2]. However, IFN treatment leads to poor virological response (30–40%) with a high relapse rate and is limited by multiple side effects, whereas the major problem with NA treatment is the need for long-term (indefinite length) therapy and the risk of drug resistance [3]. Currently, the most used NAs are entecavir (ETV) and tenofovir disoproxil fumarate (TDF); however, hepatitis B surface antigen (HBsAg) loss is very rare in HBeAg-negative patients [2]. HBsAg
seroconversion was reported in 3–7% of patients treated with pegIFN, in 0.5–3% of HBeAg-positive patients treated with NAs, and in 4% and 0% of HBeAg-negative patients treated with peg-IFN and NAs, respectively [2]. HBV-DNA suppression is a virological endpoint achieved in the majority of patients with ETV or TDF administration [4]. Monitoring of quantitative HBsAg (qHBsAg) serum kinetics during treatment with NAs evidenced a small decrease in comparison with IFN therapy, even when HBV-DNA is undetectable, and the achievement of HBsAg loss could only be gained after a very long time of drug intake [5,6]. Knowledge of qHBsAg kinetics during NA use could be interesting in order to understand the time to achieve a long-term outcome of HBsAg loss. Nevertheless, the role of therapeutic drug monitoring (TDM) of NAs in HBV treatment is still undefined. The aim of the present prospective study was to evaluate the role of ETV plasma concentrations in the kinetics of HBV-DNA decrease in a cohort of treatmentnaïve patients affected by active HBeAg-negative CHB. 2. Methods
* Corresponding author. Unit of Infectious Diseases, Department of Medical Sciences, University of Turin, Ospedale Amedeo di Savoia, Turin, Italy. Fax: +39 011 439 3975. E-mail address: [email protected] (L. Boglione). 1 These two authors equally contributed to this work.
2.1. Patients Treatment-naïve HBeAg-negative patients with active CHB treated with ETV from 2010 to 2013 at the University Hospital Amedeo di
http://dx.doi.org/10.1016/j.ijantimicag.2016.05.016 0924-8579/© 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.
L. Boglione et al. / International Journal of Antimicrobial Agents 48 (2016) 324–327
Savoia (Turin, Italy) were prospectively included in this pharmacokinetic study. Inclusion criteria were: active CHB, defined as HBVDNA >10,000 IU/mL and alanine aminotransferase (ALT) level >50 U/L; HBeAg-negative status; and naïve for previous antiviral therapies. Exclusion criteria were: co-infection with human immunodeficiency virus (HIV), hepatitis C virus (HCV) or hepatitis D virus (HDV); HBeAg-positive status; decompensated cirrhosis; and presence of HCC. The following tests were performed before the start of therapy: evaluation of liver stiffness by FibroScan®; liver ultrasound; ALT level, qHBsAg and HBV-DNA quantification; and HBV genotype testing. The included patients underwent ETV therapy at the standard dose of 0.5 mg once daily. The primary endpoint was the evaluation of the role of ETV plasma concentrations in the kinetics of HBVDNA decrease. Secondary endpoints included evaluation of serological and biochemical response and the role of HBV genotypes according to ETV plasma concentration. After the beginning of treatment, HBV-DNA, qHBsAg, ALT levels and minimum ETV concentration (Ctrough) were measured monthly for the first 3 months and then every 6 months. This study was performed in accordance with the indications of the Ethics Committee of Hospital Amedeo di Savoia, after written informed consent was obtained. 2.2. Assays Serum HBV-DNA levels were quantified by real-time PCR using a COBAS ® AmpliPrep/COBAS ® TaqMan ® HBV Test v.2.0 (Roche Molecular Systems Inc., Branchburg, NJ). HBV genotypes were determined using an INNO-LiPA reverse hybridisation assay (Innogenetics N.V, Ghent, Belgium). HBsAg, HBeAg and hepatitis B e antibody (anti-HBe) were detected using an Elecsys instrumental platform (Roche Diagnostics, Milan, Italy). qHBsAg test was performed using ARCHITECT HBsAg qualitative assay (Abbott Diagnostics, Dublin, Ireland) with a dynamic range of 0.05–250.0 IU/mL; qHBsAg values >250.0 IU/mL were subsequently serially diluted at 1:100 and were re-tested until falling within the dynamic range. 2.3. Blood sampling and entecavir quantification Blood samples were collected 24 h (±2 h) after drug intake, immediately before the next administration (Ctrough). Samples were collected in lithium heparin tubes (7 mL) and were centrifuged at 1400 × g for 10 min at 4 °C. Plasma was stored at −20 °C until analysis (no longer than 1 month). Quantification of ETV was performed following a previously validated ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method [7]. 2.4. Statistical analysis Data were assessed for normality by Shapiro–Wilk test. Binomial groups were compared using a Mann–Whitney test. To evaluate a possible correlation between continuous data, a Pearson correlation test was adopted. Finally, a receiver operating characteristic (ROC) curve analysis was conducted to determine concentration cut-off values. Independent predictivity of factors associated with HBV-DNA decay kinetics was evaluated through a multivariate linear regression. All statistical analyses were performed using IBM SPSS Statistics for Windows v.22.0 (IBM Corp., Armonk, NY). 3. Results 3.1. Baseline description Forty CHB patients were included in the analysis. Baseline characteristics of the patients are reported in Table 1. There were 32 male
325
Table 1 Baseline characteristics of study population (n = 40). Characteristic Age (years) [median (IQR)] Male sex [n (%)] Route of transmission [n (%)] Intravenous drug use Transfusion Sexual Family history of HBV Unknown Geographical origin [n (%)] Italy Eastern Europe Africa China South America HBV genotype [n (%)] A B C D E F Liver stiffness (kPa) [median (IQR)] qHBsAg (log IU/mL) [median (IQR)] HBV-DNA (log IU/mL) [median (IQR)] ALT (U/L) [median (IQR)]
39.5 (31.5–51.0) 32 (80.0) 5 (12.5) 4 (10.0) 11 (27.5) 7 (17.5) 13 (32.5) 19 (47.5) 7 (17.5) 7 (17.5) 6 (15.0) 1 (2.5) 15 (37.5) 3 (7.5) 5 (12.5) 11 (27.5) 5 (12.5) 1 (2.5) 6.8 (6.5–10.1) 4.17 (3.95–4.43) 7.19 (5.73–8.23) 89 (76.2–122)
IQR, interquartile range; HBV, hepatitis B virus; qHBsAg, quantitative hepatitis B surface antigen; ALT, alanine aminotransferase.
patients (80.0%) and the median patient age was 39.5 years [interquartile range (IQR) 31.5–51.0 years]. The frequencies of viral genotypes A, B, C, D, E and F were 15 (37.5%), 3 (7.5%), 5 (12.5%), 11 (27.5%), 5 (12.5%) and 1 (2.5%), respectively. Median liver stiffness was 6.8 kPa (IQR 6.5–10.1 kPa); seven patients (17.5%) had compensated cirrhosis. The Metavir score was F0 in 2 cases (5.0%), F1 in 26 cases (65.0%), F2 in 7 cases (17.5%), F3 in 3 cases (7.5%) and F4 in 2 cases (5.0%). The median baseline ALT level was 89 U/L (IQR 76.2–122 U/L). The median baseline log HBV-DNA and qHBsAg were 7.19 log IU/mL (IQR 5.73–8.23 log IU/mL) and 4.17 log IU/mL (IQR 3.95–4.43 log IU/mL), respectively. After 1, 2 and 3 months of treatment, log HBV-DNA and qHBsAg, respectively, were as follows (in log IU/mL): 5.65 (IQR 4.76–6.28) and 4.01 (IQR 3.88–4.26) at 1 month; 4.37 (IQR 3.80–5.60) and 3.94 (IQR 3.81–4.08) at 2 months; and 3.96 (IQR 0.00–4.89) and 3.94 (IQR 3.65–4.02) at 3 months. 3.2. Entecavir plasma concentration and response The median ETV plasma concentration after 1 month of standard treatment was 0.384 ng/mL (IQR 0.297–0.569 ng/mL). Strikingly, this concentration showed a significant inverse correlation with HBVDNA decrease after 3 months of treatment (r = −0.624; P < 0.001) (Fig. 1). This correlation was also confirmed when stratifying patients on the basis of viral genotypes: A (r = −0.719; P = 0.003); C (r = −0.917; P = 0.007); and D (r = −0.760; P = 0.007); genotypes B, E and F did not show the same correlation (probably for numerical reasons). Furthermore, the ETV plasma concentration was significantly correlated with patient age (r = 0.327; P = 0.039) and baseline log HBVDNA level (r = −0.380; P = 0.015). No significant correlation was observed between ETV levels after 1 month of treatment and HBsAg logarithmic decay after 3 months of treatment (r = −0.217; P = 0.178). ETV median plasma concentrations after 2 months and 3 months were 0.414 ng/mL (IQR 0.293–0.484 ng/mL) and 0.490 ng/mL (IQR 0.384–0.614 ng/mL), showing no statistically significant differences with the concentrations at 1 month (P-values of 0.748 and 0.210, respectively). ETV plasma concentrations at 2 months and 3 months of treatment were not significantly correlated with the HBV-DNA
326
L. Boglione et al. / International Journal of Antimicrobial Agents 48 (2016) 324–327
Fig. 1. Inverse correlation between entecavir (ETV) plasma concentrations after 1 month and hepatitis B virus (HBV) DNA decrease after 3 months of treatment.
decrease at the same time points (r = −0.546, P = 0.089; and r = −0.498, P = 0.092). 3.3. Prediction of viral DNA decay By ROC curve analysis, an ETV plasma concentration of <0.4 ng/mL was found to be associated with a log HBV-DNA decrease greater than 2 log (P < 0.001; area under the ROC curve (AUROC) = 0.891; sensitivity = 80.0%; specificity = 93.3%) (Fig. 2). The positive predictive value (PPV) of this test was 95.2% and the negative predictive value (NPV) was 73.7%. Going further, genotype-specific cut-off values were identified as <0.397 ng/mL (AUROC = 0.833; P = 0.034; sensitivity = 77.8%; specificity = 83.3%) and <0.383 ng/mL (AUROC = 0.933; P = 0.018; sensitivity = 83.3%; specificity = 100.0%) for genotypes A and D, respectively: PPVs were 87.5% (NPV = 71.4%) and 83.3% (NPV = 100%) for genotypes A and D, respectively. 4. Discussion Despite ETV currently being widely used for the treatment of CHB, pharmacokinetic features remain unknown. Our group recently reported a novel UPLC-MS/MS method to quantify NAs in plasma of HBV-positive patients [7]. Following this methodological procedure, in the current study a descriptive analysis was conducted to quantify ETV plasma concentrations in a cohort of 40 treatment-naïve HBeAg-negative patients. The distribution of observed concentrations was in accordance with previous reports [8,9], with the exception of two outliers with Ctrough > 2 ng/mL; however, since we cannot automatically assume these were not real trough concentrations, these were included in the analysis anyway. Moreover, exclusion of these outliers did not change the results of the study. The main result of this interim analysis was the identification of an inverse correlation between ETV plasma concentrations and HBVDNA decrease. Strikingly, patients who showed a higher drug plasma
concentration had a lower HBV-DNA decrease than those who showed a lower concentration. Although rare, a similar paradox was already known for other drugs active on the liver and administered at a low dose, such as pravastatin [10–12]. A possible explanation of this phenomenon could reside in differences in liver condition between CHB patients: in fact, an HBV-related chronic inflammatory state, progression of liver disease and the aberrant architecture of hepatic vascular vessels could play a relevant role in drug penetration into hepatocytes. Another hypothesis could involve the effect of specific drug transporters [13–15]: altered gene expression and/or interindividual variability could influence drug entry. Considering this, higher ETV plasma concentrations could underlie lower drug penetration in the liver, thus resulting in a smaller HBV-DNA reduction during treatment. However, the reasons for these differences require more thorough investigations, by analysing drug penetration into target cells. Going further, TDM could represent a useful tool for early discrimination of CHB patients who will have an optimum response to treatment, through ROC curve analysis. In this study, a cut-off ETV plasma concentration of 0.4 ng/mL after 1 month of therapy has been identified, which appears to predict the HBV-DNA decrease after 3 months. Therefore, this cut-off concentration could be the earliest predictive factor of response for managing therapy with ETV compared with the other clinical and virological markers. Nevertheless, the clinical usefulness of this value requires analysis in a larger cohort of patients. Thus, identification of an optimal therapeutic range deserves further investigation. Since these data suggest a potential role of ETV intracellular penetration resulting in different accumulation in plasma among patients, further investigation could involve ETV quantification in ‘in vivo’ cellular models, such as peripheral blood mononuclear cells [16]. Conversely, as expected, no correlation was observed between ETV plasma concentration and serological response. These results are likely due to the pharmacodynamic properties of ETV, which inhibits HBV-DNA replication without directly interfering with covalently closed circular DNA (cccDNA) synthesis and/or transcription/ translation processes.
L. Boglione et al. / International Journal of Antimicrobial Agents 48 (2016) 324–327
327
Fig. 2. General receiver operating characteristic (ROC) curve analysis of the obtained data [P < 0.001; area under the ROC curve (AUROC) = 0.891].
In conclusion, in this work we provide the first evidence of an inverse correlation between ETV plasma concentrations at 1 month of treatment and HBV-DNA decrease after 3 months in CHB patients, highlighting a better early response in patients with a concentration <0.4 ng/mL. Funding: None. Competing interests: None declared. Ethical approval: This study was performed in accordance with the indications of the Ethics Committee of University Hospital Amedeo di Savoia (Turin, Italy), after written informed consent was obtained from patients. References [1] World Health Organization. Hepatitis B. Geneva, Switzerland: WHO; 2013 Fact sheet no. 204. [2] European Association for the Study of the Liver. EASL clinical practice guidelines: management of chronic hepatitis B virus infection. J Hepatol 2012;57:167–85. Erratum in: J Hepatol 2013;58:201. [3] Wong GL, Chan HL. Predictors of treatment response in chronic hepatitis B. Drugs 2009;69:2167–77. [4] Wong VW, Wong GL, Chim AM, Choi PC, Chan AW, Tsang SW, et al. Surrogate end points and long-term outcome in patients with chronic hepatitis B. Clin Gastroenterol Hepatol 2009;7:1113–20. [5] Boglione L, D’Avolio A, Cariti G, Gregori G, Burdino E, Baietto L, et al. Kinetics and prediction of HBsAg loss during therapy with analogues in patients affected by chronic hepatitis B HBeAg negative and genotype D. Liver Int 2013;33:580–5. [6] Jaroszewicz J, Ho H, Markova A, Deterding K, Wursthorn K, Schulz S, et al. Hepatitis B surface antigen (HBsAg) decrease and serum interferon-inducible protein-10 levels as predictive markers for HBsAg loss during treatment with nucleoside/nucleotide analogues. Antivir Ther 2011;16:915–24.
[7] De Nicolò A, Simiele M, Pensi D, Boglione L, Allegra S, Di Perri G, et al. UPLCMS/MS method for the simultaneous quantification of anti-HBV nucleos(t)ides analogs: entecavir, lamivudine, telbivudine and tenofovir in plasma of HBV infected patients. J Pharm Biomed Anal 2015;114:127–32. [8] Yan JH, Bifano M, Olsen S, Smith RA, Zhang D, Grasela DM, et al. Entecavir pharmacokinetics, safety, and tolerability after multiple ascending doses in healthy subjects. J Clin Pharmacol 2006;46:1250–8. [9] Zhu M, Bifano M, Xu X, Wang Y, LaCreta F, Grasela D, et al. Lack of an effect of human immunodeficiency virus coinfection on the pharmacokinetics of entecavir in hepatitis B virus-infected patients. Antimicrob Agents Chemother 2008;52:2836–41. [10] Igel M, Arnold KA, Niemi M, Hofmann U, Schwab M, Lutjohann D, et al. Impact of the SLCO1B1 polymorphism on the pharmacokinetics and lipid-lowering efficacy of multiple-dose pravastatin. Clin Pharmacol Ther 2006;79:419–26. [11] Iusuf D, Sparidans RW, van Esch A, Hobbs M, Kenworthy KE, van de Steeg E, et al. Organic anion-transporting polypeptides 1a/1b control the hepatic uptake of pravastatin in mice. Mol Pharm 2012;9:2497–504. [12] van de Steeg E, Kleemann R, Jansen HT, van Duyvenvoorde W, Offerman EH, Wortelboer HM, et al. Combined analysis of pharmacokinetic and efficacy data of preclinical studies with statins markedly improves translation of drug efficacy to human trials. J Pharmacol Exp Ther 2013;347:635–44. [13] Xu Q, Wang C, Liu Q, Meng Q, Sun H, Peng J, et al. Decreased liver distribution of entecavir is related to down-regulation of Oat2/Oct1 and up-regulation of Mrp1/2/3/5 in rat liver fibrosis. Eur J Pharm Sci 2015;71:73–9. [14] Mandikova J, Volkova M, Pavek P, Navratilova L, Hyrsova L, Janeba Z, et al. Entecavir interacts with influx transporters hOAT1, hCNT2, hCNT3, but not with hOCT2: the potential for renal transporter-mediated cytotoxicity and drug–drug interactions. Front Pharmacol 2016;6:304. [15] Wang WZ, Lu LX, Li DJ, Lu JJ, He M, Liu C, et al. Effect of human concentration nucleoside transporters 1 and multi-drug resistance protein 4 gene polymorphism on response of chronic hepatitis B to nucleoside analogues treatment. Sichuan Da Xue Xue Bao Yi Xue Ban 2014;45:950–5, [in Chinese]. [16] De Nicolò A, Bonifacio G, Boglione L, Cusato J, Pensi D, Tomasello C, et al. UHPLC-MS/MS method with automated on-line solid phase extraction for the quantification of entecavir in peripheral blood mononuclear cells of HBV+ patients. J Pharm Biomed Anal 2015;118:64–9.
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j a n t i m i c a g
Short Communication
Entecavir plasma concentrations are inversely related to HBV-DNA decrease in a cohort of treatment-naïve patients with chronic hepatitis B Lucio Boglione 1,*, Amedeo De Nicolò 1, Jessica Cusato, Gabriele Bonifacio, Giuseppe Cariti, Giovanni Di Perri, Antonio D’Avolio Unit of Infectious Diseases, Department of Medical Sciences, University of Turin, Ospedale Amedeo di Savoia, Turin, Italy
A R T I C L E
I N F O
Article history: Received 3 March 2016 Accepted 28 May 2016 Keywords: Hepatitis B Entecavir Therapeutic drug monitoring Plasma concentration
A B S T R A C T
The role of therapeutic drug monitoring (TDM) of entecavir (ETV) in the treatment of patients affected by chronic hepatitis B (CHB) has not yet been defined. Here we present an interim analysis regarding the role of ETV TDM in a prospective cohort of treatment-naïve patients with CHB who received this treatment. The results from 40 patients consecutively enrolled at our centre from 2010 to 2013 are described. The primary endpoint was the evaluation of the role of ETV plasma concentrations in the kinetics of hepatitis B virus (HBV) DNA decrease. Minimum ETV concentrations (Ctrough) were measured every month after the start of therapy for the first 3 months and then every 6 months. The main result of the pharmacokinetic analysis was the significant inverse correlation of ETV concentration after 1 month of treatment and HBV-DNA decrease after 3 months of treatment (r = −0.624; P < 0.001). This correlation was also confirmed when stratifying patients on the basis of viral genotypes: A (r = −0.719; P = 0.003); C (r = −0.917; P = 0.007); and D (r = −0.760; P = 0.007). Possible explanations for this phenomenon could involve interpatient differences in liver conditions (tissue damage or inflammation) and/or genetic variability in specific drug transporters. Further investigations are needed to confirm these results quantifying ETV concentration in peripheral blood mononuclear cells as well as in a larger cohort. © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.
1. Introduction Chronic hepatitis B (CHB) affects ca. 350 million people worldwide and is a major cause of cirrhosis, liver failure and hepatocellular carcinoma (HCC) [1]. Treatment for hepatitis B e-antigen (HBeAg)-negative CHB patients includes two different approaches: long-term therapy with oral nucleos(t)ide analogues (NAs), based on serum hepatitis B virus (HBV) DNA suppression; and finite-duration therapy with standard or pegylated interferon alfa (peg-IFNα) [2]. However, IFN treatment leads to poor virological response (30–40%) with a high relapse rate and is limited by multiple side effects, whereas the major problem with NA treatment is the need for long-term (indefinite length) therapy and the risk of drug resistance [3]. Currently, the most used NAs are entecavir (ETV) and tenofovir disoproxil fumarate (TDF); however, hepatitis B surface antigen (HBsAg) loss is very rare in HBeAg-negative patients [2]. HBsAg
seroconversion was reported in 3–7% of patients treated with pegIFN, in 0.5–3% of HBeAg-positive patients treated with NAs, and in 4% and 0% of HBeAg-negative patients treated with peg-IFN and NAs, respectively [2]. HBV-DNA suppression is a virological endpoint achieved in the majority of patients with ETV or TDF administration [4]. Monitoring of quantitative HBsAg (qHBsAg) serum kinetics during treatment with NAs evidenced a small decrease in comparison with IFN therapy, even when HBV-DNA is undetectable, and the achievement of HBsAg loss could only be gained after a very long time of drug intake [5,6]. Knowledge of qHBsAg kinetics during NA use could be interesting in order to understand the time to achieve a long-term outcome of HBsAg loss. Nevertheless, the role of therapeutic drug monitoring (TDM) of NAs in HBV treatment is still undefined. The aim of the present prospective study was to evaluate the role of ETV plasma concentrations in the kinetics of HBV-DNA decrease in a cohort of treatmentnaïve patients affected by active HBeAg-negative CHB. 2. Methods
* Corresponding author. Unit of Infectious Diseases, Department of Medical Sciences, University of Turin, Ospedale Amedeo di Savoia, Turin, Italy. Fax: +39 011 439 3975. E-mail address: [email protected] (L. Boglione). 1 These two authors equally contributed to this work.
2.1. Patients Treatment-naïve HBeAg-negative patients with active CHB treated with ETV from 2010 to 2013 at the University Hospital Amedeo di
http://dx.doi.org/10.1016/j.ijantimicag.2016.05.016 0924-8579/© 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.
L. Boglione et al. / International Journal of Antimicrobial Agents 48 (2016) 324–327
Savoia (Turin, Italy) were prospectively included in this pharmacokinetic study. Inclusion criteria were: active CHB, defined as HBVDNA >10,000 IU/mL and alanine aminotransferase (ALT) level >50 U/L; HBeAg-negative status; and naïve for previous antiviral therapies. Exclusion criteria were: co-infection with human immunodeficiency virus (HIV), hepatitis C virus (HCV) or hepatitis D virus (HDV); HBeAg-positive status; decompensated cirrhosis; and presence of HCC. The following tests were performed before the start of therapy: evaluation of liver stiffness by FibroScan®; liver ultrasound; ALT level, qHBsAg and HBV-DNA quantification; and HBV genotype testing. The included patients underwent ETV therapy at the standard dose of 0.5 mg once daily. The primary endpoint was the evaluation of the role of ETV plasma concentrations in the kinetics of HBVDNA decrease. Secondary endpoints included evaluation of serological and biochemical response and the role of HBV genotypes according to ETV plasma concentration. After the beginning of treatment, HBV-DNA, qHBsAg, ALT levels and minimum ETV concentration (Ctrough) were measured monthly for the first 3 months and then every 6 months. This study was performed in accordance with the indications of the Ethics Committee of Hospital Amedeo di Savoia, after written informed consent was obtained. 2.2. Assays Serum HBV-DNA levels were quantified by real-time PCR using a COBAS ® AmpliPrep/COBAS ® TaqMan ® HBV Test v.2.0 (Roche Molecular Systems Inc., Branchburg, NJ). HBV genotypes were determined using an INNO-LiPA reverse hybridisation assay (Innogenetics N.V, Ghent, Belgium). HBsAg, HBeAg and hepatitis B e antibody (anti-HBe) were detected using an Elecsys instrumental platform (Roche Diagnostics, Milan, Italy). qHBsAg test was performed using ARCHITECT HBsAg qualitative assay (Abbott Diagnostics, Dublin, Ireland) with a dynamic range of 0.05–250.0 IU/mL; qHBsAg values >250.0 IU/mL were subsequently serially diluted at 1:100 and were re-tested until falling within the dynamic range. 2.3. Blood sampling and entecavir quantification Blood samples were collected 24 h (±2 h) after drug intake, immediately before the next administration (Ctrough). Samples were collected in lithium heparin tubes (7 mL) and were centrifuged at 1400 × g for 10 min at 4 °C. Plasma was stored at −20 °C until analysis (no longer than 1 month). Quantification of ETV was performed following a previously validated ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method [7]. 2.4. Statistical analysis Data were assessed for normality by Shapiro–Wilk test. Binomial groups were compared using a Mann–Whitney test. To evaluate a possible correlation between continuous data, a Pearson correlation test was adopted. Finally, a receiver operating characteristic (ROC) curve analysis was conducted to determine concentration cut-off values. Independent predictivity of factors associated with HBV-DNA decay kinetics was evaluated through a multivariate linear regression. All statistical analyses were performed using IBM SPSS Statistics for Windows v.22.0 (IBM Corp., Armonk, NY). 3. Results 3.1. Baseline description Forty CHB patients were included in the analysis. Baseline characteristics of the patients are reported in Table 1. There were 32 male
325
Table 1 Baseline characteristics of study population (n = 40). Characteristic Age (years) [median (IQR)] Male sex [n (%)] Route of transmission [n (%)] Intravenous drug use Transfusion Sexual Family history of HBV Unknown Geographical origin [n (%)] Italy Eastern Europe Africa China South America HBV genotype [n (%)] A B C D E F Liver stiffness (kPa) [median (IQR)] qHBsAg (log IU/mL) [median (IQR)] HBV-DNA (log IU/mL) [median (IQR)] ALT (U/L) [median (IQR)]
39.5 (31.5–51.0) 32 (80.0) 5 (12.5) 4 (10.0) 11 (27.5) 7 (17.5) 13 (32.5) 19 (47.5) 7 (17.5) 7 (17.5) 6 (15.0) 1 (2.5) 15 (37.5) 3 (7.5) 5 (12.5) 11 (27.5) 5 (12.5) 1 (2.5) 6.8 (6.5–10.1) 4.17 (3.95–4.43) 7.19 (5.73–8.23) 89 (76.2–122)
IQR, interquartile range; HBV, hepatitis B virus; qHBsAg, quantitative hepatitis B surface antigen; ALT, alanine aminotransferase.
patients (80.0%) and the median patient age was 39.5 years [interquartile range (IQR) 31.5–51.0 years]. The frequencies of viral genotypes A, B, C, D, E and F were 15 (37.5%), 3 (7.5%), 5 (12.5%), 11 (27.5%), 5 (12.5%) and 1 (2.5%), respectively. Median liver stiffness was 6.8 kPa (IQR 6.5–10.1 kPa); seven patients (17.5%) had compensated cirrhosis. The Metavir score was F0 in 2 cases (5.0%), F1 in 26 cases (65.0%), F2 in 7 cases (17.5%), F3 in 3 cases (7.5%) and F4 in 2 cases (5.0%). The median baseline ALT level was 89 U/L (IQR 76.2–122 U/L). The median baseline log HBV-DNA and qHBsAg were 7.19 log IU/mL (IQR 5.73–8.23 log IU/mL) and 4.17 log IU/mL (IQR 3.95–4.43 log IU/mL), respectively. After 1, 2 and 3 months of treatment, log HBV-DNA and qHBsAg, respectively, were as follows (in log IU/mL): 5.65 (IQR 4.76–6.28) and 4.01 (IQR 3.88–4.26) at 1 month; 4.37 (IQR 3.80–5.60) and 3.94 (IQR 3.81–4.08) at 2 months; and 3.96 (IQR 0.00–4.89) and 3.94 (IQR 3.65–4.02) at 3 months. 3.2. Entecavir plasma concentration and response The median ETV plasma concentration after 1 month of standard treatment was 0.384 ng/mL (IQR 0.297–0.569 ng/mL). Strikingly, this concentration showed a significant inverse correlation with HBVDNA decrease after 3 months of treatment (r = −0.624; P < 0.001) (Fig. 1). This correlation was also confirmed when stratifying patients on the basis of viral genotypes: A (r = −0.719; P = 0.003); C (r = −0.917; P = 0.007); and D (r = −0.760; P = 0.007); genotypes B, E and F did not show the same correlation (probably for numerical reasons). Furthermore, the ETV plasma concentration was significantly correlated with patient age (r = 0.327; P = 0.039) and baseline log HBVDNA level (r = −0.380; P = 0.015). No significant correlation was observed between ETV levels after 1 month of treatment and HBsAg logarithmic decay after 3 months of treatment (r = −0.217; P = 0.178). ETV median plasma concentrations after 2 months and 3 months were 0.414 ng/mL (IQR 0.293–0.484 ng/mL) and 0.490 ng/mL (IQR 0.384–0.614 ng/mL), showing no statistically significant differences with the concentrations at 1 month (P-values of 0.748 and 0.210, respectively). ETV plasma concentrations at 2 months and 3 months of treatment were not significantly correlated with the HBV-DNA
326
L. Boglione et al. / International Journal of Antimicrobial Agents 48 (2016) 324–327
Fig. 1. Inverse correlation between entecavir (ETV) plasma concentrations after 1 month and hepatitis B virus (HBV) DNA decrease after 3 months of treatment.
decrease at the same time points (r = −0.546, P = 0.089; and r = −0.498, P = 0.092). 3.3. Prediction of viral DNA decay By ROC curve analysis, an ETV plasma concentration of <0.4 ng/mL was found to be associated with a log HBV-DNA decrease greater than 2 log (P < 0.001; area under the ROC curve (AUROC) = 0.891; sensitivity = 80.0%; specificity = 93.3%) (Fig. 2). The positive predictive value (PPV) of this test was 95.2% and the negative predictive value (NPV) was 73.7%. Going further, genotype-specific cut-off values were identified as <0.397 ng/mL (AUROC = 0.833; P = 0.034; sensitivity = 77.8%; specificity = 83.3%) and <0.383 ng/mL (AUROC = 0.933; P = 0.018; sensitivity = 83.3%; specificity = 100.0%) for genotypes A and D, respectively: PPVs were 87.5% (NPV = 71.4%) and 83.3% (NPV = 100%) for genotypes A and D, respectively. 4. Discussion Despite ETV currently being widely used for the treatment of CHB, pharmacokinetic features remain unknown. Our group recently reported a novel UPLC-MS/MS method to quantify NAs in plasma of HBV-positive patients [7]. Following this methodological procedure, in the current study a descriptive analysis was conducted to quantify ETV plasma concentrations in a cohort of 40 treatment-naïve HBeAg-negative patients. The distribution of observed concentrations was in accordance with previous reports [8,9], with the exception of two outliers with Ctrough > 2 ng/mL; however, since we cannot automatically assume these were not real trough concentrations, these were included in the analysis anyway. Moreover, exclusion of these outliers did not change the results of the study. The main result of this interim analysis was the identification of an inverse correlation between ETV plasma concentrations and HBVDNA decrease. Strikingly, patients who showed a higher drug plasma
concentration had a lower HBV-DNA decrease than those who showed a lower concentration. Although rare, a similar paradox was already known for other drugs active on the liver and administered at a low dose, such as pravastatin [10–12]. A possible explanation of this phenomenon could reside in differences in liver condition between CHB patients: in fact, an HBV-related chronic inflammatory state, progression of liver disease and the aberrant architecture of hepatic vascular vessels could play a relevant role in drug penetration into hepatocytes. Another hypothesis could involve the effect of specific drug transporters [13–15]: altered gene expression and/or interindividual variability could influence drug entry. Considering this, higher ETV plasma concentrations could underlie lower drug penetration in the liver, thus resulting in a smaller HBV-DNA reduction during treatment. However, the reasons for these differences require more thorough investigations, by analysing drug penetration into target cells. Going further, TDM could represent a useful tool for early discrimination of CHB patients who will have an optimum response to treatment, through ROC curve analysis. In this study, a cut-off ETV plasma concentration of 0.4 ng/mL after 1 month of therapy has been identified, which appears to predict the HBV-DNA decrease after 3 months. Therefore, this cut-off concentration could be the earliest predictive factor of response for managing therapy with ETV compared with the other clinical and virological markers. Nevertheless, the clinical usefulness of this value requires analysis in a larger cohort of patients. Thus, identification of an optimal therapeutic range deserves further investigation. Since these data suggest a potential role of ETV intracellular penetration resulting in different accumulation in plasma among patients, further investigation could involve ETV quantification in ‘in vivo’ cellular models, such as peripheral blood mononuclear cells [16]. Conversely, as expected, no correlation was observed between ETV plasma concentration and serological response. These results are likely due to the pharmacodynamic properties of ETV, which inhibits HBV-DNA replication without directly interfering with covalently closed circular DNA (cccDNA) synthesis and/or transcription/ translation processes.
L. Boglione et al. / International Journal of Antimicrobial Agents 48 (2016) 324–327
327
Fig. 2. General receiver operating characteristic (ROC) curve analysis of the obtained data [P < 0.001; area under the ROC curve (AUROC) = 0.891].
In conclusion, in this work we provide the first evidence of an inverse correlation between ETV plasma concentrations at 1 month of treatment and HBV-DNA decrease after 3 months in CHB patients, highlighting a better early response in patients with a concentration <0.4 ng/mL. Funding: None. Competing interests: None declared. Ethical approval: This study was performed in accordance with the indications of the Ethics Committee of University Hospital Amedeo di Savoia (Turin, Italy), after written informed consent was obtained from patients. References [1] World Health Organization. Hepatitis B. Geneva, Switzerland: WHO; 2013 Fact sheet no. 204. [2] European Association for the Study of the Liver. EASL clinical practice guidelines: management of chronic hepatitis B virus infection. J Hepatol 2012;57:167–85. Erratum in: J Hepatol 2013;58:201. [3] Wong GL, Chan HL. Predictors of treatment response in chronic hepatitis B. Drugs 2009;69:2167–77. [4] Wong VW, Wong GL, Chim AM, Choi PC, Chan AW, Tsang SW, et al. Surrogate end points and long-term outcome in patients with chronic hepatitis B. Clin Gastroenterol Hepatol 2009;7:1113–20. [5] Boglione L, D’Avolio A, Cariti G, Gregori G, Burdino E, Baietto L, et al. Kinetics and prediction of HBsAg loss during therapy with analogues in patients affected by chronic hepatitis B HBeAg negative and genotype D. Liver Int 2013;33:580–5. [6] Jaroszewicz J, Ho H, Markova A, Deterding K, Wursthorn K, Schulz S, et al. Hepatitis B surface antigen (HBsAg) decrease and serum interferon-inducible protein-10 levels as predictive markers for HBsAg loss during treatment with nucleoside/nucleotide analogues. Antivir Ther 2011;16:915–24.
[7] De Nicolò A, Simiele M, Pensi D, Boglione L, Allegra S, Di Perri G, et al. UPLCMS/MS method for the simultaneous quantification of anti-HBV nucleos(t)ides analogs: entecavir, lamivudine, telbivudine and tenofovir in plasma of HBV infected patients. J Pharm Biomed Anal 2015;114:127–32. [8] Yan JH, Bifano M, Olsen S, Smith RA, Zhang D, Grasela DM, et al. Entecavir pharmacokinetics, safety, and tolerability after multiple ascending doses in healthy subjects. J Clin Pharmacol 2006;46:1250–8. [9] Zhu M, Bifano M, Xu X, Wang Y, LaCreta F, Grasela D, et al. Lack of an effect of human immunodeficiency virus coinfection on the pharmacokinetics of entecavir in hepatitis B virus-infected patients. Antimicrob Agents Chemother 2008;52:2836–41. [10] Igel M, Arnold KA, Niemi M, Hofmann U, Schwab M, Lutjohann D, et al. Impact of the SLCO1B1 polymorphism on the pharmacokinetics and lipid-lowering efficacy of multiple-dose pravastatin. Clin Pharmacol Ther 2006;79:419–26. [11] Iusuf D, Sparidans RW, van Esch A, Hobbs M, Kenworthy KE, van de Steeg E, et al. Organic anion-transporting polypeptides 1a/1b control the hepatic uptake of pravastatin in mice. Mol Pharm 2012;9:2497–504. [12] van de Steeg E, Kleemann R, Jansen HT, van Duyvenvoorde W, Offerman EH, Wortelboer HM, et al. Combined analysis of pharmacokinetic and efficacy data of preclinical studies with statins markedly improves translation of drug efficacy to human trials. J Pharmacol Exp Ther 2013;347:635–44. [13] Xu Q, Wang C, Liu Q, Meng Q, Sun H, Peng J, et al. Decreased liver distribution of entecavir is related to down-regulation of Oat2/Oct1 and up-regulation of Mrp1/2/3/5 in rat liver fibrosis. Eur J Pharm Sci 2015;71:73–9. [14] Mandikova J, Volkova M, Pavek P, Navratilova L, Hyrsova L, Janeba Z, et al. Entecavir interacts with influx transporters hOAT1, hCNT2, hCNT3, but not with hOCT2: the potential for renal transporter-mediated cytotoxicity and drug–drug interactions. Front Pharmacol 2016;6:304. [15] Wang WZ, Lu LX, Li DJ, Lu JJ, He M, Liu C, et al. Effect of human concentration nucleoside transporters 1 and multi-drug resistance protein 4 gene polymorphism on response of chronic hepatitis B to nucleoside analogues treatment. Sichuan Da Xue Xue Bao Yi Xue Ban 2014;45:950–5, [in Chinese]. [16] De Nicolò A, Bonifacio G, Boglione L, Cusato J, Pensi D, Tomasello C, et al. UHPLC-MS/MS method with automated on-line solid phase extraction for the quantification of entecavir in peripheral blood mononuclear cells of HBV+ patients. J Pharm Biomed Anal 2015;118:64–9.