Pharmacokinetics of 4′-cyano-2′-deoxyguanosine, a novel nucleoside analog inhibitor of the resistant hepatitis B virus, in a rat model of chronic kidney disease

Abstract

Introduction:

The novel nucleoside analog, 4′-cyano-2′-deoxyguanosine (CdG), possesses inhibitory activity against both the wild-type and resistant hepatitis B virus. Since the dosage of the currently available nucleoside analog preparations needs to be adjusted, depending on renal function, we investigated the effect of renal dysfunction on the pharmacokinetics of CdG in a rat model of chronic kidney disease (CKD).

Methods:

CKD model rats were either intravenously or orally administered CdG at a dose of 1 mg/kg. The concentration of CdG in plasma, organs (liver and kidney) and urine samples were determined by means of a UPLC system interfaced with a TOF-MS system.

Results:

Following intravenous administration, the plasma retention of CdG was prolonged in CKD model rats compared to healthy rats. In addition, the clearance of CdG was well correlated with plasma creatinine levels in CKD model rats. Similar to the results for intravenous administration, the plasma concentration profiles of CdG after oral administration were also found to be much higher in CKD model rats than in healthy rats. However, the results for the organ distribution and urinary excretion of CdG, the profiles of which were similar to that of healthy rats, indicated that CdG did not accumulate to a significant extent in the body.

Conclusion:

The extent of renal dysfunction has a direct influence on the pharmacokinetics (plasma retention) of CdG without a significant accumulation, indicating that the dosage of CdG will be dependent on the extent of renal function. .

1. Introduction

Hepatitis B is a major global health problem that is caused by the Hepatitis B virus (HBV) [1]. Despite the fact that vaccines against hepatitis B are now available with more than a 98% prevention of infection, more than 257 million people continue to suffer from chronic HBV infections, worldwide [2]. According to the reports by World Health Organization (WHO), the use of oral antiviral agents, such as entecavir, a potent nucleoside analog preparations for the treatment of HBV is recommended [2]. However, long-term treatments with these nucleoside analog preparations can lead to the development of drug-resistant form of HBV. To identify a successful treatment of these life-threatening resistant HBV infections, our group has been developing novel nucleoside analog preparations that could be potentially active against resistant forms of HBV. Our efforts which are supported by the Japan Agency for Medical Research and Development (AMED) [3–6] have led to the screening of more than 150 nucleoside analog preparations, the guanosine analog, 4′-cyano-2′-deoxyguanosine (CdG, Fig. 1), possesses superior antiviral activity against not only wild type HBV but also resistant HBV compared to currently marketed nucleoside analog preparations such as adefovir dipivoxil and entecavir (HBV^A181T/N236T and HBV^{L180M/S202G/M204V})[7]^, leading us to hypothesize that CdG could be a promising candidate for neutralizing the resistance of HBV to clinically available nucleoside analog preparations.

Fig. 1.

Chemical structure of 4′-cyano-2′-deoxyguanosine (CdG).

With the goal of using CdG in clinics, it is essential to assess the pharmacokinetic properties of CdG in the process of drug development. We previously conducted pharmacokinetic studies of CdG in healthy rats and viral liver injury model rats and clearly showed that CdG possessed ideal pharmacokinetic characteristics as an oral nucleoside analog preparation for use in the treatment of HBV infections with good bioavailability, a high distribution to the liver and the fact that it is excreted into urine in an intact form [8,9]. However, according to the package insert of marketed nucleoside analogs such as adefovir dipivoxil and entecavir for the treatment of HBV infections, the administered dosage of these marketed nucleoside analog preparations are recommended to decrease with decreasing renal function [10,11]. These facts led us to be concerned that the pharmacokinetic properties of CdG may be altered in cases of deteriorating renal function. Thus, clarifying the pharmacokinetic characteristics of CdG under conditions of deteriorating renal function would provide fundamental information regarding the dosage regimen for the treatment of resistant HBV infection by CdG as a novel nucleoside preparation.

To accumulate evidence regarding the pharmacokinetic profiles of CdG under conditions of renal dysfunction, the objective of this study was to investigate the pharmacokinetic properties of CdG in 5/6 nephrectomy rats, which are generally used as a model of chronic kidney disease (CKD) [12,13], and to compare these data with the pharmacokinetics in healthy rats that were reported in our previous study [8].

2. Materials and methods

2.1. Chemicals and reagents

CdG was synthesized as reported previously [14]. The methanol, formic acid and ultrapure water used for LC-MS analysis were LC-MS grade and were obtained from Wako Pure Chemical Industries (Osaka, Japan).

2.2. Animals and ethics

CKD model rats (Sprague-Dawley rats, male, 8–9 weeks old, 204.2 ± 32.2 g), which induced by the surgically resection of 5/6 of the kidney, were purchased from Japan SLC Co., Ltd. (Shizuoka, Japan). All CKD model rats were acclimatized in a conventional room for one week prior to the start of the study with free access to solid rodent chow and tap water. One day before the pharmacokinetic studies, plasma samples were collected from the femoral vein under isoflurane anesthesia for the measurement of plasma concentration of creatine and blood urea nitrogen. The levels of creatinine and blood urea nitrogen in the plasma were determined by a commercial clinical testing laboratory (Oriental Yeast Co., Shiga, Japan). The values of creatinine and blood urea nitrogen in the plasma of the CKD model rats (n = 15) were 0.50 ± 0.1 mg/dL and 43.6 ± 12.2 mg/dL, respectively. All experimental procedures in this study were performed according to NIH guidelines with approval by the institutional Animal Care and Use committee (2019-P-010).

2.3. Pharmacokinetics of CdG after intravenous administration

A solution of CdG in saline was injected to CKD model rats (n = 8) via the tail vein at a dose of 1 mg/kg (3 mL/kg) under isoflurane anesthesia. At 3, 15, 30, 45 min, 1, 1.5, 3 and 6 h after injection, approximately 150 μL of blood was collected via the femoral vein under isoflurane anesthesia. Plasma samples were obtained by centrifugation of blood samples (3000 rpm, 10 min) and were stored in a deep freezer until use in the analysis.

2.4. Pharmacokinetic study of CdG after oral administration

All CKD model rats (n = 7) were orally administered a solution of CdG in saline at a dose of 1 mg/kg (3 mL/kg). Three rats were randomly selected, and approximately 150 μL of blood was collected via the femoral vein under isoflurane anesthesia at predetermined times after the oral administration (15, 30, 45 min, 1, 1.5, and 3 h). Plasma samples were obtained by centrifugation of blood samples (3000 rpm, 10 min). The rats were sacrificed after collecting the last blood sample (3 h after administration) and the kidneys and liver were removed. The remaining CKD model rats (n = 4) were housed in a metabolic cage and urine samples were collected. The urine samples were collected at 3, 6, 9 and 24 h after the administration of the CdG solution. After the last sampling of urine (24 h after the administration of CdG), the rats were sacrificed and blood, kidneys and livers were collected. All samples were stored in a deep freezer until used.

2.5. Measurement of CdG concentration

All samples derived from plasma, organs (kidneys and liver) and urine were pretreated using a solid-phase extraction column (Waters Oasis MCX 96 well plate, Waters, Milford, CT, USA) to extract CdG as reported previously [8]. The concentration of CdG in all samples were quantitated using a Xevo^® G2-S TOF (Waters) spectrometer with a UPLC column (ACQUITY UPLC BEH Phenyl column, Waters, Part Number: 186005607) using the same procedures and conditions as were used in our previous report [8].

2.6. Statistics

All results are shown as the mean ± S.D. Each pharmacokinetic parameter was calculated by a noncompartment model using the moment analysis program [15]. Statistical differences in pharmacokinetic parameters between CKD model rat and healthy rat were analyzed using the unpaired t-test. The parametric method (two-tail) was used for the correlation analyses.

3. Results and discussion

3.1. Effect of renal dysfunction on the retention of CdG in the circulation

To evaluate the effect of renal dysfunction on the pharmacokinetics of CdG, we compared changes in the plasma concentration of CdG after intravenous administration between healthy rats and CKD model rats. As shown in Fig. 2A, the plasma profiles of CdG were slightly increased in CKD model rats compared to healthy rats but were below determination limit at 6 h after administration. Along with this, the value for the clearance (CL) in CKD model rats was lower than that of healthy rats (8.48 ± 2.21 L/h/kg [8], 6.56 ± 1.70 L/h/kg for healthy rats and CKD model rats, respectively). In addition, as shown in Table 1, pharmacokinetic parameters that reflect blood retention were increased in the CKD model rats compared to the corresponding values for the healthy rats (half-life (t_1/2): 0.45 ± 0.09 h, area under the concentration-time curve (AUC): 122.8 ± 28.1 h·ng/mL [8]). It should be noted that the CL of CdG was decreased with increasing creatinine levels in plasma, with a significant correlation (Fig. 2B, r = 0.71, p = 0.021), indicating that renal dysfunction would prolong the plasma retention of CdG as well as other marketed nucleoside analog preparations [10,11]. In previous, it was reported that multiple drug transporters, such as carnitine/organic cation transporters, were involved in the renal excretion of marketed nucleoside analog preparations [16,17]. Although the issue of whether these transporters are involved in the renal uptake and efflux of CdG is unclear at this time, reduction of renal excretion of CdG via these transporters would result in the prolonged retention of CdG in the blood in CKD model rats.

Fig. 2.

(A) Plasma concentration profiles for CdG after intravenous administration in CKD model rats. (B) Relationship between plasma creatinine levels and clearance for CdG. All CKD model rats received CdG at a dose of 1 mg/kg. The dotted line in Fig. 2A represents the plasma concentration profiles of CdG (1 mg/kg) in healthy rats [8]. Each result represents the mean ± SD. (n = 8).

Table 1.

Pharmacokinetic parameters for CdG after intravenous and oral administration at a dose of 1 mg/kg in CKD model rats. t1/2: half-life.

	intravenous	oral
t1/2 (hr)	0.58 ± 0.17	3.08 ± 0.37
MRT (hr)	0.73 ± 0.17	4.90 ± 0.65
AUC (hr· ng/mL)	162.2 ± 43.5	459.5 ± 116.1
CL (L/hr/kg)	6.56 ± 1.7	–
CL/F (L/hr/kg)	–	2.26 ± 0.53
tmax (hr)	–	1.0 ± 0.0

MRT: mean residence time, AUC: area under the concentration-time curve, CL: clearance, F: bioavailability, tmax: time to reach peak plasma concentration.

Each value represents the mean ± S.D. (n = 3–5).

3.2. Pharmacokinetics of CdG after oral administration in CKD model rats

Since CdG would likely be administered orally when used in clinics, we next compared the pharmacokinetic profiles, i.e., the plasma concentration, distribution in the kidney and liver, and excretion into urine, for CdG between healthy rats and CKD model rats after oral administration. Similar to the case of intravenous injection, the plasma retention of CdG in CKD model rats was increased compared to that of healthy rats with the values of maximum blood concentration (C_max) and time to reach peak plasma concentration (t_max) being increased (Fig. 3). Accompanied by this, there were significant differences in the pharmacokinetic parameters between the healthy rats and CKD model rats (t_1/2: 0.73 ± 0.12 h, 3.08 ± 0.37 h, p < 0.01; CL/F: 11.1 ± 0.76 L/h/kg, 2.3 ± 0.53 L/h/kg, p < 0.01; AUC: 90.1 ± 5.8 h・ ng/mL, 459.5 ± 116.1 h・ ng/mL, p < 0.01 for healthy rats [8] and CKD model rats, respectively). There is concern over the difference in the t_1/2 values between intravenous and oral administration. It was previously reported that oral absorption is affected by numerous physiological changes to the gastrointestinal tract caused by the CKD, resulting in a prolonged t_max [18]. Thus, we presume that the prolonged absorption of CdG after oral administration would apparently prolong the elimination of CdG from blood circulation, leading to the longer t_1/2 observed in rats administered orally compared to rats injected intravenously.

Fig. 3.

Plasma concentration profile for CdG after oral administration in CKD model rats. All CKD model rats received CdG at a dose of 1 mg/kg. Dotted line represents the plasma concentration profiles of CdG (1 mg/kg) in healthy rats [8]. Each result represents the mean ± SD. (n = 3).

Since deteriorating renal function may lead to the accumulation of CdG in the kidney of CKD model rats due to excretory inhibition, we next determined the distribution of CdG in kidneys of CKD model rats. From the determination of the CdG distribution in the kidney at 3 h after administration, it was found that the amount of CdG was comparable to that of healthy rats (2.17 ± 0.31 μg/g of tissue, 1.76 ± 0.36 μg/g of tissue, for healthy rats and CKD model rats, respectively). In addition, the amount of CdG that was distributed was higher in the liver than in the kidney (Fig. 4), a tendency similar to that for the healthy rats [8]. However, the amount of CdG distributed in the liver is significantly reduced compared to healthy rats (5.39 ± 0.75 μg/g of tissue, 2.83 ± 0.87 μg/g of tissue, p < 0.01, for healthy rats and CKD model rats, respectively). It has been reported that organic anion transporter 2 and organic cation transporter 1 contribute to hepatic uptake of ETV [19], which is a guanosine nucleoside analog similar to CdG. Uremic toxins have also been reported to suppress the drug uptake in the liver by the reduction of the transporter expression in CKD [20]. Taking these facts into consideration, it is assumed that the hepatic uptake of CdG via transporters would be indirectly suppressed by the uremic toxins, resulting in decrease in hepatic distribution in CKD model rats despite the increase in the systemic exposure. However, CdG is highly distributed in the liver compared to the kidney without any accumulation in the kidney, even under conditions of a deteriorating renal function.

Fig. 4.

Amount of CdG in the liver (A) and kidneys (B) of CKD model rats at 3 and 24 h after oral administration. All CKD model rats received CdG at a dose of 1 mg/kg. Each result represents the mean ± SD. (n = 3–4).

The finding that the plasma retention of CdG is increased (Fig. 3), led us to be also concerned that CdG may accumulate in the blood circulation under in the case of CKD. Thus, we finally evaluated the excretory properties of CdG in the CKD model rats. The cumulative amount of CdG that was excreted into the urine of CKD model rats were resulted in approximately 37% of the dosage and this was as unchanged CdG until 24 h after oral administration, a value that was not significantly different from those for healthy rats (28.3% ± 3.3 of the dosage) (Fig. 5). We also confirmed that (i) the level of CdG in plasma was below the determination limit at 24 h after administration, and (ii) the distribution in the liver and kidney decreased with passing time (Fig. 4), indicating that the elimination of CdG from the body is not accompanied by a significant accumulation even in the case of CKD.

Fig. 5.

Cumulative excretory rate of CdG into urine in CKD model rats after oral administration. Urine samples were collected in a metabolic cage after oral administration (3, 6, 9 and 24 h) of the CdG at a dose of 1 mg/kg. Each result represents the mean ± SD. (n = 4).

4. Conclusions

Renal dysfunction could influence the plasma retention of CdG as well as other nucleoside analog preparations; dosage adjustment may be required depending on the status of the renal function. In addition, the findings of this study also showed that a significant accumulation of CdG would be negligible, even in the case of renal dysfunction. The results reported in this study provide fundamental information related to the determination of the CdG dosage regimen as an oral nucleoside analog preparation for the treatment of HBV infections. However, since the results obtained in this study were limited to weak or mild renal function deterioration in the case of the CKD model rats used in present study, it will be necessary to accumulate additional evidence regarding a greater variety of conditions, such as more end-stage chronic renal failure and acute kidney disease, in the future.