PHARMACOLOGY

crossm Paritaprevir and Ritonavir Liver Concentrations in Rats as Assessed by Different Liver Sampling Techniques Charles S. Venuto,a,b Marianthi Markatou,c Yvonne Woolwine-Cunningham,d Rosemary Furlage,e Andrew J. Ocque,b Robin DiFrancesco,b Emily O. Dumas,f Paul K. Wallace,e Gene D. Morse,b Andrew H. Talald Department of Neurology, Center for Human Experimental Therapeutics, University of Rochester, Rochester, New York, USAa; AIDS Clinical Trials Group Pharmacology Specialty Laboratory, New York State Center of Excellence in Bioinformatics and Life Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, New York, USAb; Department of Biostatistics, University at Buffalo, Buffalo, New York, USAc; Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, University at Buffalo, Buffalo, New York, USAd; Department of Flow & Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York, USAe; AbbVie Inc., North Chicago, Illinois, USAf

ABSTRACT The liver is crucial to pharmacology, yet substantial knowledge gaps exist in the understanding of its basic pharmacologic processes. An improved understanding for humans requires reliable and reproducible liver sampling methods. We compared liver concentrations of paritaprevir and ritonavir in rats by using samples collected by fine-needle aspiration (FNA), core needle biopsy (CNB), and surgical resection. Thirteen Sprague-Dawley rats were evaluated, nine of which received paritaprevir/ritonavir at 30/20 mg/kg of body weight by oral gavage daily for 4 or 5 days. Drug concentrations were measured using liquid chromatography-tandem mass spectrometry on samples collected via FNA (21G needle) with 1, 3, or 5 passes (FNA1, FNA3, and FNA5); via CNB (16G needle); and via surgical resection. Drug concentrations in plasma were also assessed. Analyses included noncompartmental pharmacokinetic analysis and use of Bland-Altman techniques. All liver tissue samples had higher paritaprevir and ritonavir concentrations than those in plasma. Resected samples, considered the benchmark measure, resulted in estimations of the highest values for the pharmacokinetic parameters of exposure (maximum concentration of drug in serum [Cmax] and area under the concentration-time curve from 0 to 24 h [AUC0 –24]) for paritaprevir and ritonavir. Bland-Altman analyses showed that the best agreement occurred between tissue resection and CNB, with 15% bias, followed by FNA3 and FNA5, with 18% bias, and FNA1 and FNA3, with a 22% bias for paritaprevir. Paritaprevir and ritonavir are highly concentrated in rat liver. Further research is needed to validate FNA sampling for humans, with the possible derivation and application of correction factors for drug concentration measurements.

Received 25 October 2016 Returned for modification 12 January 2017 Accepted 12 February 2017 Accepted manuscript posted online 6 March 2017 Citation Venuto CS, Markatou M, WoolwineCunningham Y, Furlage R, Ocque AJ, DiFrancesco R, Dumas EO, Wallace PK, Morse GD, Talal AH. 2017. Paritaprevir and ritonavir liver concentrations in rats as assessed by different liver sampling techniques. Antimicrob Agents Chemother 61:e02283-16. https://doi .org/10.1128/AAC.02283-16. Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to Andrew H. Talal, [email protected].

KEYWORDS hepatocyte isolation, liver biopsy, liver drug concentration, liver fineneedle aspiration

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he liver is the primary site of drug metabolism, and hepatocytes are principally responsible for hepatic metabolic capacity as a result of the abundance of phase I and II drug-metabolizing enzymes and transporters (1–3). Due to the liver’s fundamental role in drug metabolism, understanding the relationship between intrahepatic and plasma drug concentrations is potentially important for predicting drug-drug interactions, hepatotoxicity, and therapeutic efficacy. To date, most methods used to determine intrahepatic drug concentrations have relied on in vitro techniques (e.g., microsomal experiments and sandwich cultures) that work well as isolated systems. Inaccuracies develop, however, when these models are used for other purposes, as they May 2017 Volume 61 Issue 5 e02283-16

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may not adequately reflect the in vivo human situation due to interspecies differences, the effect of multiple transporters operating simultaneously, and potential extrahepatic metabolism (1, 4). Therefore, assessment of liver drug concentrations from in vivo models rather than from artificial systems may provide a more accurate description of liver-to-plasma drug concentration ratios. Knowledge of liver-to-plasma drug concentration ratios is particularly important for agents whose primary target site is the liver (5, 6). For example, direct-acting antivirals (DAAs) against hepatitis C virus (HCV) target viral replication within hepatocytes. Paritaprevir (PTV), an NS3/4A protease inhibitor codosed with the pharmacokinetic enhancer ritonavir (RTV), is an example of a DAA recently approved for use in combination with the DAAs ombitasvir, an NS5A inhibitor, and dasabuvir, a nonnucleoside NS5B polymerase inhibitor, for the treatment of HCV genotype 1. In humans, the paritaprevir pharmacokinetics in plasma exhibits nonlinearity, likely due to its dual function as both a substrate and an inhibitor of efflux transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), as well as liver uptake transporters, such as organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 (7, 8). As a result of these active transport processes, the intracellular concentration may be significantly different from the extracellular concentration, making it difficult to predict the target activity and intrahepatic disposition of paritaprevir based solely on data derived from plasma. Estimation of intracellular drug concentrations by modeling and simulation has proven useful for delineating the impact of in vivo tissue disposition of drugs based upon transporters and metabolizing enzymes (9). Physiology-based pharmacokinetic (PBPK) modeling approaches, for example, integrate drug-related kinetic parameters and physiological parameters to predict drug disposition in plasma and tissue. Furthermore, a well-developed PBPK model may be useful for predicting transporter- and transporter metabolism-based drug-drug interactions. For paritaprevir, development of a PBPK model would be valuable because it is coadministered with the DAAs ombitasvir and dasabuvir. Additionally, plasma paritaprevir concentrations are augmented by ritonavir, an inhibitor of cytochrome P450 (CYP) 3A4/5, OATPs, and BCRP. Knowledge of tissue drug concentrations, particularly for agents, such as paritaprevir, whose intracellular and extracellular concentrations can vary considerably, would likely permit development of more accurate and reproducible models of drug transport, metabolism, and excretion within the liver. Our group recently pioneered techniques for serial liver drug concentration assessments in vivo by using different methods of liver sample collection (10–12). While core needle biopsy (CNB) and fine-needle aspiration (FNA) are techniques that have been used for the past half century to obtain liver specimens during the clinical evaluation of patients with liver disease, a comparison of different sampling techniques for measurement of liver drug concentrations has not been performed previously (13–17). Our overall goal is to develop reproducible, accurate, and reliable techniques for serial measurement of liver drug concentrations in humans. The primary objective of this study was to compare the equivalences of paritaprevir and ritonavir pharmacokinetic profiles in liver tissue samples obtained by FNA, CNB, and surgical resection. As a secondary aim, we sought to compare the yields of hepatocytes isolated from different tissue sample matrices. We utilized a rat model after dosing with paritaprevir and ritonavir and performed FNAs, CNBs, and surgical resections for measurement of drug concentrations in rat liver samples. We also isolated hepatocytes from each of the liver tissue samples in order to compare the yields from different tissue matrices. RESULTS Liver tissue extraction and hepatocyte isolation. Liver tissue samples were extracted from a total of 13 rats, including 9 that received paritaprevir/ritonavir and 4 untreated controls. The average length and weight of the CNB samples were 2.0 cm and 10 mg, respectively, while weights of FNA samples ranged from 3 to 12 mg. Hepatocytes were successfully isolated from samples obtained by each technique (i.e., FNA, May 2017 Volume 61 Issue 5 e02283-16

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TABLE 1 Isolation of total cells and hepatocytes from different liver tissue sample typesa Value Parameter Resection CNB FNA5 FNA3 FNA1 Mean total cell 5.30 ⫻ 107 (4.78 ⫻ 107) 2.92 ⫻ 105 (1.55 ⫻ 105) 1.35 ⫻ 105 (1.16 ⫻ 105) 2.22 ⫻ 105 (2.05 ⫻ 105) 1.38 ⫻ 105 (2.64 ⫻ 105) no. (SD) Mean hepatocyte 3.90 ⫻ 107 (3.77 ⫻ 107) 2.13 ⫻ 105 (1.21 ⫻ 105) 7.04 ⫻ 104 (5.70 ⫻ 104) 8.94 ⫻ 104 (63.9 ⫻ 104) 6.69 ⫻ 104 (95.5 ⫻ 104) no. (SD) aAbbreviations:

CNB, core needle biopsy; FNA5, fine-needle aspiration with 5 passes; FNA3, fine-needle aspiration with 3 passes; FNA1, fine-needle aspiration with 1 pass; SD, standard deviation.

CNB, and resection). The overall average number of hepatocytes isolated from liver tissue samples of all types was 7.89 ⫻ 106 hepatocytes, corresponding to 73% hepatocytes in the sample. However, total cell and hepatocyte yields varied by tissue sample type (Table 1). Resection samples resulted in the largest number of cells and yielded an average of 74% hepatocytes; in comparison, the number of isolated hepatocytes was 2 log lower for the CNB samples, but these samples yielded a percentage of hepatocytes (73%) similar to that for the resection samples. The FNA samples exhibited the lowest hepatocyte percentages, with samples obtained by FNA with 5, 3, and 1 passes (FNA5, FNA3, and FNA1) containing 52%, 40%, and 49% hepatocytes, respectively. Paritaprevir pharmacokinetics. Composite paritaprevir pharmacokinetic profiles for plasma and liver concentrations are illustrated in Fig. 1. Paritaprevir plasma concentrations at the 24.4- and 25.3-h postdose time points were below the assay’s lower limit of quantification. For all other plasma and liver tissue matrix time points, paritaprevir concentrations were readily quantifiable. The highest observed paritaprevir concentrations were detected at 2.1 h (time to maximum concentration of drug in serum [Tmax]) for the plasma, tissue resection, and FNA sample matrices, while the CNB sample had an observed Tmax of 5.5 h. Following the 5.5-h time point, a steep decline in paritaprevir concentrations was observed until the 8-h postdose time point for all

FIG 1 Concentrations of paritaprevir versus time for plasma samples, homogenized liver tissue resection samples (tissue), liver tissue samples obtained via fine-needle aspiration (FNA) with one (FNA1), three (FNA3), or five (FNA5) passes, and core needle biopsy (CNB) samples. For two plasma samples, taken at 24.4 and 25.3 h postdose, the paritaprevir concentration was below the lower limit of quantification and was plotted as 0.625 ng/ml. May 2017 Volume 61 Issue 5 e02283-16

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TABLE 2 Paritaprevir pharmacokinetics in plasma and in liver tissue samples from rats receiving 30-mg/kg dosesa Value Tissue samplesc Paritaprevir pharmacokinetic parameter Tmax (h) Cmax (ng/ml) AUC0–24 (ng · h/ml) t1/2 (h)

Plasmab 2.1 3,593 3,970 5.0

Surgical resection 2.1 125,076 394,315 3.0

CNB 5.5 100,412 373,533 4.1

FNA5 2.1 75,125 235,290 5.2

FNA3 2.1 47,368 205,036 6.1

FNA1 2.1 56,628 144,949 4.7

aOne

rat (corresponding to the 4-h time point) was excluded from these analyses because paritaprevir was not at steady state. Abbreviations: CNB, core needle biopsy; FNA1, fine-needle aspiration with 1 pass; FNA3, fine-needle aspiration with 3 passes; FNA5, fine-needle aspiration with 5 passes; AUC, area under the concentration-time curve; Cmax, maximum observed concentration; t1/2, half-life; Tmax, time of maximum observed concentration. bThe plasma concentrations corresponding to 24 and 25 h postdose were below the assay’s lower limit of quantitation, and their values were thus set at 0.625 ng/ml. cConcentrations measured in tissue samples were converted from nanograms per gram to nanograms per milliliter by using a liver tissue density value for rats of 1.05 g/ml (32).

matrices. A more gradual decline was subsequently observed for liver tissue matrices until 25 h postdose. Pharmacokinetic data for paritaprevir obtained by use of each of the matrices are summarized in Table 2. Liver paritaprevir concentrations were consistently higher than those in plasma. Relative to those for plasma, values for the maximum concentration (Cmax) for paritaprevir were 12- to 34-fold higher for the liver tissue matrices. Exposures to paritaprevir, expressed as the area under the concentration-time curve from 0 to 24 h (AUC0 –24), were 36- to 98-fold higher in liver tissue sample matrices than in plasma. There was a wide range of values for the liver tissue-to-plasma partition coefficient (Kp liver) for paritaprevir among the different matrices (Fig. 2). Among the liver tissue matrices, the tissue resection and CNB matrices yielded the highest values of Cmax and AUC0 –24 for paritaprevir (Table 2). Bland-Altman analyses comparing different types of liver tissue matrices revealed that tissue concentrations of paritaprevir generally tended to be higher in surgical tissue sections than in CNB or FNA samples (Table 3; Fig. 3). A ratio of close to 1 for measurements between different tissue matrices indicates high agreement between the two different methods. Furthermore, a

FIG 2 Liver tissue-to-plasma partition coefficient (Kp liver) values for paritaprevir and ritonavir by liver tissue matrix, i.e., core needle biopsy (CNB) or one (FNA1), three (FNA3), or five (FNA5) fine-needle aspiration passes. May 2017 Volume 61 Issue 5 e02283-16

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TABLE 3 Bias and 95% limits of agreement, with associated 90% confidence intervals, for comparisons of different tissue matrix pairs for paritaprevira Matrix pair Tissue, CNB FNA3, FNA5 FNA1, FNA3 FNA5, CNB FNA5, tissue

Bias 1.15 0.82 0.78 0.72 0.63

(90% (0.88, (0.58, (0.57, (0.58, (0.50,

CI) 1.49) 1.16) 1.06) 0.89) 0.79)

LLA (90% CI) 0.60 (0.38, 0.95) 0.35 (0.19, 0.64) 0.37 (0.21, 0.63) 0.43 (0.30, 0.62) 0.35 (0.23, 0.53)

ULA (90% 2.19 (1.38, 1.20 (1.62, 1.65 (0.97, 1.20 (0.83, 1.11 (0.74,

CI) 3.46) 3.50) 2.83) 1.72) 1.67)

aAbbreviations:

CI, confidence interval; CNB, core needle biopsy; FNA, fine-needle aspiration; LLA, lower limit of agreement; ULA, upper limit of agreement.

small distance between the limits of agreement (LOA) indicates higher agreement between the measurements under consideration. Bland-Altman plots of the log2-transformed normalized PTV concentration values are illustrated in Fig. 3. The PTV concentration was measured in five different tissue matrices (FNA1, FNA3, FNA5, CNB, and tissue resection), and Bland-Altman plots were constructed for the 10 pairs of matrices. Comparing the FNA1-FNA3 pair (Fig. 3A), we see that the bias is 0.7788, which indicates that the normalized FNA3 measurements are higher than the FNA1 measurements, by ⬃22%. The 95% LOA are 0.37 and 1.65, and the geometric mean ratio of the normalized values of FNA1 to FNA3 is as follows: 0.37 ⱕ FNA1/FNA3 ⱕ 1.65. These data indicate a high variability of measurement. Furthermore, the 90% confidence intervals (CI) for the lower and upper LOA are rather wide. The best agreement is exhibited by the tissue resection-CNB and FNA5-CNB pairs. The tissue resection-CNB pair (Fig. 3H) has a bias of 15%, which translates into tissue measurements that are ⬃15% higher than those obtained via CNB. This estimator of bias has reasonable accuracy, as indicated by the narrowness of the 90% CI. Furthermore, the 95% lower and upper limits of agreement are 0.60 and 2.19. This translates into PTV tissue measurements being between 0.60 and 2.19 times higher than CNB measurements. The FNA5-CNB pair (Fig. 3J), on the other hand, has an ⬃28% bias, meaning that CNB values are, on average, approximately 28% higher than FNA5 values. This means that the bias of the FNA5-CNB pair is almost double the bias of the tissue resection-CNB pair, but the width of the confidence interval for the FNA5-CNB pair is half that for the tissue resection-CNB pair. This indicates that the bias estimator is much more accurate for the FNA5-CNB pair. Further, the 95% lower and upper LOA are 0.43 and 1.20, indicating that the normalized FNA5 values are 0.43 and 1.20 times higher than the CNB values. For FNA sampling, concentrations of paritaprevir were generally higher with the performance of more passes. Bland-Altman analyses demonstrated similarity in concentration yields between the FNA3-FNA5 (Fig. 3E), tissue resection-CNB (Fig. 3H), and FNA5-CNB (Fig. 3J) pairs. However, FNA1 concentrations were consistently lower than those obtained from the other liver tissue matrices. Ritonavir pharmacokinetics. Ritonavir concentrations were detectable at each time point for liver tissue matrices and plasma, except for the 24.4- and 25.3-h time points for plasma, at which the concentrations were below the assay’s lower limit of quantification (Fig. 4). Ritonavir pharmacokinetic data obtained from each of the matrices are given in Table 4. The ritonavir Tmax was 3 h for all liver tissue and plasma matrices. Similar to paritaprevir concentrations, liver ritonavir concentrations were consistently higher than those measured in plasma. Relative to those for plasma, ritonavir Cmax and AUC0 –24 values for liver were 4- to 10-fold higher. Also, similar to those for paritaprevir, ritonavir Cmax and AUC0 –24 values were highest for tissue resection and CNB matrices among the liver tissue matrices (Table 4). Kp liver values for ritonavir did not differ substantially among the different matrices (Fig. 3). In terms of the Bland-Altman analysis for RTV, the results obtained were similar to those for PTV. Bland-Altman plots of log-transformed normalized RTV concentration values are presented in Fig. 5 for the five types of tissue matrices (FNA1, FNA3, FNA5, CNB, and tissue resection). The best agreement is exhibited by the tissue-CNB and FNA5-CNB pairs. The tissue-CNB pair (Fig. 5H) has a bias of 1%, with a relatively tight confidence interval May 2017 Volume 61 Issue 5 e02283-16

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FIG 4 Concentrations of ritonavir versus time for plasma samples, homogenized liver tissue resection samples (tissue), liver tissue samples obtained via fine-needle aspiration (FNA) with one (FNA1), three (FNA3), or five (FNA5) passes, and core needle biopsy (CNB) samples. For two plasma samples, taken at 24.4 and 25.3 h postdose, the ritonavir concentration was below the lower limit of quantification and was plotted as 0.625 ng/ml.

for the bias, with a lower LOA of 0.25 and an upper LOA of 4, indicating the following: 0.25 ⱕ tissue/CNB ⱕ 4. Further, the FNA5-CNB pair (Fig. 5J) exhibits 13% bias in that the FNA5 measurements, on average, are 13% lower than the CNB measurements, with a tight 90% confidence interval for the bias. Moreover, we observe tight lower and upper LOA of 0.60 and 1.19, respectively. Other RTV results are similar to those obtained with PTV and are illustrated in Fig. 5. DISCUSSION Despite the importance of the liver in drug metabolism, crucial data gaps exist in our understanding of its role in drug transport, metabolism, and excretion. DAAs, drugs which target the hepatocyte and whose liver concentration has been shown to vary considerably, can be used to examine factors that affect liver drug accumulation and the resultant pharmacodynamic effect, as assessed by sampling HCV RNA levels (10, 11, 18). Few studies, however, have measured DAA concentrations within the liver, largely because of the need for invasive sampling techniques, i.e., biopsy or surgical resection, and the difficulty in interpretation of liver drug concentration measurements compared to measurements made with traditional matrices (i.e., plasma, blood, and urine) (19). In this study, we assessed rat plasma and liver concentrations of paritaprevir and ritonavir by utilizing samples obtained by tissue resection, biopsy, and fine-needle aspiration. As anticipated from previous animal studies with ritonavir and the clinical data for the protease inhibitor vaniprevir, we found that both drugs were highly concentrated within the liver compared to plasma (3, 11, 20). Their liver concentrations, however,

FIG 3 Bland-Altman plots measuring agreement of log2-transformed (and normalized) measurements of paritaprevir data. Solid red lines indicate bias, dashed brown lines indicate the 90% confidence interval for the bias, and solid blue lines indicate the upper and lower limits of agreement; their associated 90% confidence intervals are indicated by dash-dot gray lines. (A) FNA1 and FNA3; (B) FNA1 and FNA5; (C) FNA1 and tissue resection; (D) FNA1 and CNB; (E) FNA3 and FNA5; (F) FNA3 and tissue resection; (G) FNA3 and CNB; (H) tissue resection and CNB; (I) FNA5 and tissue resection; (J) FNA5 and CNB. May 2017 Volume 61 Issue 5 e02283-16

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TABLE 4 Ritonavir pharmacokinetics in plasma and in liver tissue samples from rats receiving 20-mg/kg dosesa Value Tissue samplesc Ritonavir pharmacokinetic parameter Tmax (h) Cmax (ng/ml) AUC0–24 (ng · h/ml) t1/2 (h)

Plasmab 3.0 1,842 6,913 7.0

Surgical resection 3.0 20,370 74,686 12.9

CNB 3.0 14,700 67,483 4.9

FNA5 3.0 10,317 49,166 3.6

FNA3 3.0 14,078 50,539 5.4

FNA1 3.0 9,587 35,461 4.2

aOne

rat (corresponding to the 4-h time point) was excluded from these analyses because ritonavir was not at steady state. Abbreviations: CNB, core needle biopsy; FNA1, fine-needle aspiration with 1 pass; FNA3, fineneedle aspiration with 3 passes; FNA5, fine-needle aspiration with 5 passes; AUC, area under the concentration-time curve; Cmax, maximum observed concentration; t1/2, half-life; Tmax, time of maximum observed concentration. bThe plasma concentrations corresponding to 24 and 25 h postdose were below the assay’s lower limit of quantitation, and their values were thus set at 0.625 ng/ml. cConcentrations measured in tissue samples were converted from nanograms per gram to nanograms per milliliter by using a liver tissue density value for rats of 1.05 g/ml (32).

differed by tissue sample type, with the highest concentrations obtained for resection samples, followed by CNB, FNA5, FNA3, and FNA1 samples. To our knowledge, this is the first study to evaluate and compare liver concentrations for any drug by using different ex vivo sampling techniques. It was previously shown that although FNA sampling is less invasive than CNB, the yield of actual liver tissue is lower. Lejnine et al. compared the yields of liver tissue from 17 patients through gene expression analysis and demonstrated that FNA samples contained 43% ⫾ 24% liver tissue, compared to 95% ⫾ 17% for CNB (21). Similarly, in our analysis, the yield of hepatocytes was lowest in FNA samples (40 to 52%) compared to tissue resection and CNB samples (⬎70%), despite similar yields for the total amount of all cells from CNB and FNA samples. Differences in hepatocyte yield or in the composition of the hepatic parenchyma between liver matrices may explain, in part, the variability in drug concentration observed across different liver tissue sample types. The liver is a heterogeneous collection of different cell types. Although the majority of liver cells are parenchymal (i.e., hepatocytes), approximately 40% of the liver is comprised of nonparenchymal cells that include stellate cells, sinusoidal endothelial cells, Kupffer cells, biliary epithelial cells, and immune cells (22–24). Drug uptake by parenchymal cells is expected to be much more abundant than that by nonparenchymal cells due to the absence of membrane transporters on the latter cells. Paritaprevir is a substrate of OATP1B1 and OATP1B3, which are primarily hepatocyte-specific basolateral transporters that facilitate the uptake of drug (18). Although there are no direct orthologs of these transporters in rodent livers, evidence suggests that the rodent-specific transporters, Oatp1b2, -1a1, and -1a4, share activities and substrate specificities similar to those of human OATP1B1 and OATP1B3 (4, 25, 26). Thus, the variability in cellular composition across sampling techniques, particularly a smaller proportion of hepatocytes in FNA samples per tissue weight, may have contributed to the reduced drug concentrations observed in these samples. Blood contamination is another consideration as a source of variability in tissue concentrations, particularly for FNA samples, for which forceful suction is applied to a small yet very-well-perfused mass (21, 27, 28). In a study that investigated the error associated with blood contamination in measuring liver tissue drug concentrations in dogs, measurements were slightly underestimated if they were not corrected for residual blood (28). Correction of tissue concentrations for blood contamination is less of a concern for compounds with high tissue-to-plasma ratios, such as paritaprevir and ritonavir. Furthermore, the vast majority of blood within FNA samples is presumably that which bathes liver cells, i.e., it is derived from the portal as opposed to the systemic circulation. May 2017 Volume 61 Issue 5 e02283-16

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The main goal of agreement studies such as ours is to evaluate differences between methods. Here we used Bland-Altman plots to study the differences between different methods of collection of liver tissue samples. Further, we used the logarithmic transformation of both measurements before analysis because it allows the results to be interpreted in relation to the original data (via back transformation). High agreement between different tissue types is indicated by a ratio of measurements of approximately 1. For many of the samples evaluated in this investigation, we obtained wider intervals for the ratio of normalized measurements. These results may have occurred partially due to the small sample sizes of the study and of the samples obtained, which likely affected the standard error of the estimated bias and the lower and upper limits of agreement. However, the sample does provide an accurate quantification of sample variability related to the drug concentrations obtained from each of the different sample types and allows for informative comparisons among the different sampling methods. The ultimate value of this work will be the incorporation of different liver sampling techniques, either FNA or CNB, to investigate processes occurring within the liver in human liver diseases of a variety of etiologies. Our team has conducted significant investigation in this area. The first study demonstrated the reliability of liver FNA for serial hepatic tissue sampling. It also permitted development of a normalization procedure to determine the percentage of liver contained within the sample through the identification of liver-enriched genes (21). As a follow-up to the initial study, we conducted two studies investigating liver-to-plasma drug concentration ratios (10, 11). In the first, we performed CNB on three patients treated with vaniprevir and demonstrated higher human liver concentrations than the concentrations in plasma (11). In the second study, we performed serial FNA procedures on 15 patients treated with telaprevir, pegylated interferon, and ribavirin. In the first study of its type, we assessed the intrahepatic HCV RNA decline as well as the pharmacokinetics and pharmacodynamics of a regimen that contained a DAA (10). Based upon these data, we proposed an integrated model to explain the potential mechanism of control of viral replication by these agents. These results formed the basis for a recently completed human study assessing HCV RNA decline and intrahepatic drug concentrations of paritaprevir and ritonavir administered in combination with dasabuvir and ombitasvir (NCT02493855) (29). In the present investigation, we employed different liver sampling techniques for assessment of liver tissue drug concentrations of two agents that are concentrated in the liver: paritaprevir and ritonavir. We also developed a novel assay to measure the concentrations of both agents within liver tissue. By utilizing traditional pharmacokinetic modeling techniques to characterize the intrahepatic pharmacokinetics of paritaprevir and ritonavir, we found that both agents are highly concentrated within rat liver tissue. Liver tissue concentrations were highest in samples obtained by surgical resection, with decreasing concentrations by sample type for CNB and FNA. FNA sampling also has a higher bias and higher variability. Through studies performed on humans and including liver sampling, our team has demonstrated the feasibility of these techniques to assess intrahepatic HCV RNA decline and liver drug concentrations. These techniques can be expanded directly to the assessment of intrahepatic viral replication in hepatitis B virus infection, including the analysis of covalently closed circular DNA levels in the liver, and to the identification and therapeutic responses obtained in patients with nonalcoholic steatohepatitis. The ability to determine liver tissue drug concentrations may transform drug-dosing strategies for medications with narrow therapeutic indices, especially for individuals with liver dysfunction, such as

FIG 5 Bland-Altman plots measuring agreement of log2-transformed (and normalized) measurements for ritonavir data. Solid red lines indicate bias, dashed brown lines indicate the 90% confidence interval for the bias, and solid blue lines indicate the upper and lower limits of agreement; their associated 90% confidence intervals are indicated by dash-dot gray lines. (A) FNA1 and FNA3; (B) FNA1 and FNA5; (C) FNA1 and tissue resection; (D) FNA1 and CNB; (E) FNA3 and FNA5; (F) FNA3 and tissue resection; (G) FNA3 and CNB; (H) tissue resection and CNB; (I) FNA5 and tissue resection; (J) FNA5 and CNB. May 2017 Volume 61 Issue 5 e02283-16

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decompensated cirrhotics. The ability to reliably and reproducibly sample the liver should facilitate investigation into the pharmacokinetic functions of the liver and their derangement in patients with liver disease. MATERIALS AND METHODS Animals. Thirteen adult male Sprague-Dawley rats, weighing 250 to 300 g, were purchased from Harlan Laboratories (Indianapolis, IN). Animals were maintained on a 12-h (6 a.m. to 6 p.m.) light-dark cycle in a temperature (72 to 74°F)- and humidity (50%)-controlled room, with access to water and food ad libitum. One animal died after receiving a single dose of study drug due to a cardiac abnormality, did not contribute any study data, and was subsequently replaced. Animal studies were conducted according to protocols approved by the University at Buffalo Institutional Animal Care and Use Committee. Treatment. Nine rats received paritaprevir (30 mg/kg of body weight) and ritonavir (20 mg/kg) by oral gavage daily for 4 or 5 days, depending on the timing of sample acquisition. Drug administration by gavage occurred each morning. Four additional rats were untreated controls whose liver specimens were used only for the hepatocyte isolation component of the study, as described below. A suspension of paritaprevir/ritonavir was freshly prepared each day by dissolving a tablet containing both paritaprevir and ritonavir, equivalent to the formulation used in commercial tablets, in water to concentrations of 3/2 mg/ml or 6/4 mg/ml. The higher-concentration suspension was used later in the study because it was observed that some of the rats had difficulty with complete dose intake at the volumes administered for the lower-concentration suspension. Tissue sampling. At prespecified time points, blood specimens were collected via cardiac puncture from animals under isoflurane anesthesia. Following euthanasia and thoracotomy, liver specimens were collected from each rat via FNA (21G needle), CNB (16G needle), and surgical resection. During each FNA procedure, one, three, and five FNA passes were performed and designated FNA1, FNA3, and FNA5. A single pass consisted of placing the needle into the rat liver, performing multiple oscillations for 5 to 10 s under negative pressure, and finally removing the needle and expunging the material from the needle bore. For two of the rats, liver specimens were collected at approximately 24 h postdose following administration of the fourth dose without receiving a day 5 dose. One animal per time point was collected for the remaining pharmacokinetic measurements. On day 5, following administration of the fifth dose, samples were collected at 2, 2.1, 3, 4, 5.5, 7, and 8 h postdose. Hepatocyte isolation by flow cytometry. Hepatocyte isolation experiments were performed on liver specimens separate from those used for measurement of liver paritaprevir and ritonavir concentrations. The procedure required an initial addition of chilled washing solution to the sample and centrifugation at 150 ⫻ g for 3 min at 4°C. Subsequently, liver samples were incubated with collagenase at 37°C for 30 min and filtered through a 100-␮m filter to recover cells. The cells were pelleted and washed once more, counted (Countess; Invitrogen, Grand Island, NY), and analyzed by flow cytometry (Fortessa; Becton Dickinson, Franklin Lakes, NJ) to determine the percentage of hepatocytes in the sample. Measurement of drug concentrations. Paritaprevir and ritonavir concentrations in liver samples (FNA, CNB, and resection) were quantified using a validated liquid chromatography (LC) method as recently described (12). Briefly, liver samples were homogenized in acetonitrile by use of a bullet blender and zirconium oxide beads and centrifuged to obtain a homogenate supernatant. After addition of deuterated internal standards and dilution with the mobile phase, 10 ␮l was injected onto an ultrapure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system. Standard curves ranged from 20 to 20,000 pg on-column mass for paritaprevir and from 5 to 10,000 pg on-column mass for ritonavir. Samples were normalized per milligram of wet tissue weight. Paritaprevir and ritonavir concentrations in plasma were quantified using a modified bioanalytical method provided by AbbVie Inc. (30). The samples were extracted by solid-phase extraction (SPE) with the following steps. To 250 ␮l of sample, 50 ␮l of working internal standard (300 ng/ml in acetonitrile containing 0.01% ammonium hydroxide) was added, and after centrifugation at 12,000 ⫻ g for 5 min, 250 ␮l of the resulting supernatant was loaded onto a pretreated Waters Oasis HLB 96-well extraction plate. The analytes were eluted from the extraction plate with 400 ␮l of methanol and diluted with 200 ␮l of mobile phase A, and 10 ␮l was injected onto the UPLC-MS/MS system. The chromatographic separation and MS/MS detection followed the specifications used for the liver assay. The calibration range for the plasma assay was 1.25 to 500 ng/ml for both paritaprevir and ritonavir. Pharmacokinetic analysis. Noncompartmental analysis was conducted using the Excel add-in PKSolver (31). To be included in the analysis, paritaprevir and ritonavir were assumed to be at steady state. One rat, representing the 4-h time point, did not swallow the entire drug dose on days 2 and 4; therefore, this animal did not meet the assumption of achieving steady-state paritaprevir and ritonavir concentrations and was excluded from the pharmacokinetic analysis. Individual paritaprevir and ritonavir concentrations at each collection time were used to construct a composite concentration-time profile for each sampling method, which was then used for data analysis. Thus, individual concentration-time profiles were constructed for plasma, each of the three different types of FNA samples (FNA1, FNA3, and FNA5), CNB samples, and tissue resection samples. Plasma paritaprevir and ritonavir concentrations that were below the limit of quantitation at the approximately 24-h postdose time points were replaced with one-half the assay lower limit of quantitation (0.625 ng/ml) for the generation of pharmacokinetic parameters. Concentration data for liver tissue samples were converted from nanograms per gram to nanograms per milliliter by use of a liver tissue density value for rats of 1.05 g/ml, as previously reported (32). May 2017 Volume 61 Issue 5 e02283-16

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The primary pharmacokinetic parameters derived from paritaprevir and ritonavir concentrationtime data were the maximum observed concentration (Cmax), time to the maximum observed concentration (Tmax), area under the concentration-time curve from 0 to 24 h (AUC0 –24), and half-life (t1/2). The liver tissue-to-plasma partition coefficient (Kp liver), which is the ratio of the total liver tissue exposure to the total plasma exposure, was also estimated using the following equation: Kp liver ⫽ AUC0 –24, liver/AUC0 –24, plasma, where AUC0 –24, liver is the AUC0 –24 for paritaprevir or ritonavir in the liver tissue sample matrix and AUC0 –24, plasma is the AUC0 –24 for the drug in plasma. Statistical analysis. To study the agreement between paritaprevir and ritonavir concentrations obtained for each tissue matrix type for samples collected between 2 and 8 h postdose, we used the Bland-Altman approach, which quantifies the amount by which two methods differ (33, 34). It is highly unlikely that different methods designed to measure the same quantity will give exactly the same measurements, so some lack of agreement between different methods is inevitable. The Bland-Altman plot illustrates the difference between any two methods versus their mean. The data were initially normalized by dividing the observed drug concentrations by the corresponding dose and subsequently log transformed (with basis 2) by use of SAS (SAS Institute, Cary, NC) to construct Bland-Altman plots. Lack of agreement is calculated by the bias, which is estimated by the mean difference of the log2-transformed normalized drug concentration values and their associated standard deviations. A ratio of measurements of approximately 1 indicates a high agreement between any two sampling methods. The 95% limits of agreement define a reference range within which most differences between measurements by diverse methods will lie. The limits of agreement offer a reference interval, and a large standard deviation predisposes the plot to widely spaced limits of agreement, which may imply that the two methods disagree.

ACKNOWLEDGMENTS We acknowledge Raymond Xu and Roger Trinh of AbbVie Inc. for helpful discussions. Cytometry services were provided by the Flow and Image Cytometry facility at the Roswell Park Cancer Institute, which is supported in part by NCI Cancer Center support grant 5P30 CA016056. A.H.T. has received grant support and served as an advisor to AbbVie Pharmaceuticals, Inc. E.O.D. is an employee and stockholder of AbbVie Inc. This work was supported by grants from AbbVie Inc., the Troup Fund of the Kaleida Health Foundation (to A.H.T.), and the NIH (5K23AI108355-02) (to C.S.V.). The funders contributed to the study design and interpretation of the data but had no role in the data collection, the data analysis, or the decision to submit the work for publication.

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