Pharmacokinetics of a Cholesterol-conjugated Aptamer Against the Hepatitis C Virus (HCV) NS5B Protein

Abstract

Hepatitis C virus (HCV) is the major cause of progressive liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Previously, we reported that a 29 nucleotide-long 2'-F pyrimidine modified RNA aptamer against the HCV nonstructural protein 5B efficiently inhibited HCV replication and suppressed HCV infectious virus particle formation in a cell culture system. In this study, we modified this aptamer through conjugation of cholesterol for in vivo availability. This cholesterol-conjugated aptamer (chol-aptamer) efficiently entered the cell and inhibited HCV RNA replication, without any alteration in gene expression profiling including innate immune response-related genes. Moreover, systemic administration of the chol-aptamer was well tolerated without any abnormalities in mice. To evaluate the pharmacokinetics of the chol-aptamer in vivo, dose proportionality, bioavailability, and pharmacokinetic parameters were evaluated by noncompartmental analyses in normal BALB/c mice. Population analysis was performed using nonlinear mixed effects modeling. Moreover, the pharmacokinetics of two different routes (intravenous, IV, versus intraperitoneal, IP) were compared. Cholesterol conjugation showed dose proportionality, extended the time that the aptamer was in the plasma, and enhanced aptamer exposure to the body. Noticeably, the IV route was more suitable than the IP route due to the chol-aptamer remaining in the plasma for a longer period of time.

Aptamers as targeted therapeutics: current potential and challenges

“…When non-formulated aptamers are administered into the blood stream, even using stabilizing backbone modifications, small aptamers are subject to rapid excretion through renal filtration. To overcome renal filtration and extend circulation time, aptamers are generally formulated with a bulky moiety, such as high molecular mass PEG 69 , cholesterol 79, 80 , proteins 81, 82 , liposomes 83 , organic or inorganic nanomaterials 10, 84 , or are multimerized 85–87 to create a multivalent molecule above the cutoff threshold for the renal glomerulus (30–50 kDa). PEG is a well-studied, hydrophilic biomaterial which decreases aggregation and increases solubility of the conjugates.…”

“…Recently, the safety and pharmacokinetic profile of a cholesterol-conjugated RNA aptamer against the hepatitis C virus (HCV) NS5B protein was assessed in a preclinical study 80 . Systemic administration of the cholesterol-conjugated RNA aptamer was well-tolerated, without any abnormalities in wild-type BALB/c mice, and showed a 2-fold extended half-life compared to non-conjugated aptamer.…”

Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses

“…On the other hand, R–F oligonucleotides were truncated and conjugated with cholesterol- or galactose-PEG molecules to allow direct and specific liver delivery into cells or tissue. Cholesterol- and Gal-PEG-R–F t2 conjugated aptamer blocked RNA synthesis of HCV genome [115]. The above mentioned aptamers were non-genotype-specific; however, Jones et al described for the first time aptamers against NS5 protein that exclusively recognized and inhibited RNA-polymerase activity of HCV virus subtype 3a [116].…”

“…However, none has been tested in clinical trials because it is necessary to overcome a number of limitations such as degradation, biodistribution, immune response or cellular internalization. Nevertheless, recent publications have described different strategies to overcome aptamer restrictions using cholesterol or ribozyme conjugation [115,118]. …”

Aptamer–drug conjugate: targeted delivery of doxorubicin in a HER3 aptamer-functionalized liposomal delivery system reduces cardiotoxicity

“…For example, PEG and cholesterol covalently conjugated to aptamer is a common strategy to improve the circulating half-time due to increase in molecular weight. 28,29 For antibody-drug conjugates, the presence of targeting antibody has been well demonstrated as it can increase the tumor distribution and decrease the circulating half-time. 30 The PK behaviors of ApDC aptamers have been rarely reported.…”