Animal experiment:

Rats, Dogs and Monkeys[1]Pharmacokinetic studies are performed in male naive Sprague-Dawley(SD) rats, non-naive beagle dogs, and cynomolgus monkeys (three animals per dosing route). Intravenous (IV) administration is dosed via infusion over 30 min in a vehicle containing 5% ethanol, 20% PEG400, and 75% water (pH adjusted to 3.0 with HCl). Oral dosing is administered by gavage in a vehicle containing 5% ethanol, 45% PEG 400, and 50% of 50 mM citrate buffer, pH 3. Blood samples are collected over a 24 h period postdose into Vacutainer tubes containing EDTA-K2. Plasma was isolated, and the concentration of the test compound in plasma was determined with LC/MS/MS after protein precipitation with acetonitrile.

Ledipasvir is an inhibitor of the hepatitis C virus replication with IC50 value of 141 nM [1].

Hepatitis C virus (HCV) is an enveloped (+)-single stranded RNA virus, which is the cause of hepatitis C and some cancer lymphomas. The viral RNA is replicated in host cell via RNA-dependent RNA polymerase of HIV and then assembled into virions. The zinc-binding phosphoprotein NS5A protein is translated from HCV genome, which plays important role in the virus replication process. It may act as an transcriptional activator for NS5A RNA replication system and also the mediator of virion assembly.

Ledipasvir is a specific inhibitor of HCV NS5A protein to inhibit HCV replication in the HCV subgenomic replicon system. NS5A replication complex inhibitors are novel antiviral factors for HCV treatment. Typically, these inhibitors have high efficiency and low viral resistance when compared to traditional HCV replication inhibitor targeted on NS3 helicase and NS5B RNA polymerasae. NS5A inhibitors are supposed to bind across the NS5A dimer interface, proximal to N-terminal domain 1. The binding is thought to distort dimer association directly or allosterically, which may disrupt NS5A function in HCV RNA replication [2]. When a JFH1/3a-NS5A hybrid replicon was used to assess susceptibility to NS5A, another inhibitor DCV was shown to be more potent than ledipasvir. Additionally, NS5A-A30K and -Y93H variants exhibited reduced sensitivity to ledpasvir (EC50 value of 1770 nM and 4300 nM respectively) [1].

In clinical trials, it was observed ledpasvir was well tolerated and exhibited median maximal reduction of HCV RNA ranging from 2.3 log10 IU/ml to 3.3 log10 IU/ml. Emax modeling also showed administration of 30 mg ledpasvir after 3 days resulted in >95% maximal response of HCV RNA reduction to genotype 1a.Finally, it was also observed that HCV RNA was more sustained in genotype 1b compared to 1a [3].

References:
[1] Hernandez D et al. , Natural prevalence of NS5A polymorphisms in subjects infected with hepatitis C virus genotype 3 and their effects on the antiviral activity of NS5A inhibitors. J Clin Virol. 2013, 57(1): 13-8.
[2] Gao M et al. , Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature. 2010, 465: 96-100.
[3] Lawitz E J et al. , A phase 1, randomized, placebo-controlled, 3-day, dose-ranging study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C. J Hepatol. 2012, 57(1): 24-31.