Sofosbuvir and Ledipasvir

Ledipasvir is a HCV inhibitor targeting the HCV NS5A protein, which is essential for both RNA replication and the assembly of HCV virions. Biochemical confirmation of NS5A inhibition by ledipasvir is not currently possible as NS5A has no enzymatic function. In vitro resistance selection and cross-resistance studies indicate ledipasvir targets NS5A as its mode of action.

Sofosbuvir is a pan-genotypic inhibitor of the HCV NS5B RNA-dependent RNA polymerase, which is essential for viral replication. Sofosbuvir is a nucleotide prodrug that undergoes intracellular metabolism to form the pharmacologically active uridine analogue triphosphate (GS-461203), which can be incorporated into HCV RNA by the NS5B polymerase and acts as a chain terminator. GS-461203 (the active metabolite of sofosbuvir) is neither an inhibitor of human DNA and RNA polymerases nor an inhibitor of mitochondrial RNA polymerase.

Antiviral activity

The EC₅₀ values of ledipasvir and sofosbuvir against full-length or chimeric replicons encoding NS5A and NS5B sequences from clinical isolates are detailed in the following table. The presence of 40% human serum had no effect on the anti-HCV activity of sofosbuvir but reduced the anti-HCV activity of ledipasvir by 12-fold against genotype 1a HCV replicons.

Activity of ledipasvir and sofosbuvir against chimeric replicons:

^a Transient replicons carrying NS5A or NS5B from patient isolates.
^b The chimeric replicons carrying NS5A genes from genotype 2b, 5a, 6a and 6e were used for testing ledipasvir while the chimeric replicons carrying NS5B genes from genotype 2b, 5a or 6a were used for testing sofosbuvir.

Resistance

In cell culture

HCV replicons with reduced susceptibility to ledipasvir have been selected in cell culture for genotype 1a and 1b. Reduced susceptibility to ledipasvir was associated with the primary NS5A substitution Y93H in both genotype 1a and 1b. Additionally a Q30E substitution developed in genotype 1a replicons. Site-directed mutagenesis of NS5A RAVs showed that substitutions conferring a fold-change >100 and ≤1,000 in ledipasvir susceptibility are Q30H/R, L31I/M/V, P32L and Y93T in genotype 1a and P58D and Y93S in genotype 1b; and substitutions conferring a fold-change >1,000 are M28A/G, Q30E/G/K, H58D, Y93C/H/N/S in genotype 1a and A92K and Y93H in genotype 1b.

HCV replicons with reduced susceptibility to sofosbuvir have been selected in cell culture for multiple genotypes including 1b, 2a, 2b, 3a, 4a, 5a and 6a. Reduced susceptibility to sofosbuvir was associated with the primary NS5B substitution S282T in all replicon genotypes examined. Site-directed mutagenesis of the S282T substitution in replicons of 8 genotypes conferred 2- to 18-fold reduced susceptibility to sofosbuvir and reduced the viral replication capacity by 89% to 99% compared to the corresponding wild-type.

Absorption

Following oral administration of ledipasvir/sofosbuvir to HCV-infected patients, ledipasvir median peak plasma concentration was observed at 4.0 hours post-dose. Sofosbuvir was absorbed quickly and the median peak plasma concentrations were observed ~1 hour post-dose. Median peak plasma concentration of GS-331007 was observed at 4 hours post-dose.

Based on the population pharmacokinetic analysis in HCV-infected patients, geometric mean steady-state AUC_0-24 for ledipasvir (n=2,113), sofosbuvir (n=1,542), and GS-331007 (n=2,113) were 7,290, 1,320 and 12,000 ng•h/mL, respectively. Steady-state C_max for ledipasvir, sofosbuvir and GS-331007 were 323, 618 and 707 ng/mL, respectively. Sofosbuvir and GS-331007 AUC_0-24 and C_max were similar in healthy adult subjects and patients with HCV infection. Relative to healthy subjects (n=191), ledipasvir AUC_0-24 and C_max were 24% lower and 32% lower, respectively, in HCV-infected patients. Ledipasvir AUC is dose proportional over the dose range of 3 to 100 mg. Sofosbuvir and GS-331007 AUCs are near dose proportional over the dose range of 200 mg to 400 mg.

Effects of food

Relative to fasting conditions, the administration of a single dose of ledipasvir/sofosbuvir with a moderate fat or high fat meal increased the sofosbuvir AUC_0-inf by approximately 2-fold, but did not significantly affect the sofosbuvir C_max. The exposures to GS-331007 and ledipasvir were not altered in the presence of either meal type. Ledipasvir/sofosbuvir can be administered without regard to food.

Distribution

Ledipasvir is > 99.8% bound to human plasma proteins. After a single 90 mg dose of [¹⁴C]-ledipasvir in healthy subjects, the blood to plasma ratio of [¹⁴C]-radioactivity ranged between 0.51 and 0.66.

Sofosbuvir is approximately 61-65% bound to human plasma proteins and the binding is independent of drug concentration over the range of 1 µg/mL to 20 µg/mL. Protein binding of GS-331007 was minimal in human plasma. After a single 400 mg dose of [¹⁴C]-sofosbuvir in healthy subjects, the blood to plasma ratio of [¹⁴C]-radioactivity was approximately 0.7.

Biotransformation

In vitro, no detectable metabolism of ledipasvir was observed by human CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Evidence of slow oxidative metabolism via an unknown mechanism has been observed. Following a single dose of 90 mg [¹⁴C]-ledipasvir, systemic exposure was almost exclusively due to the parent drug (>98%). Unchanged ledipasvir is also the major species present in faeces.

Sofosbuvir is extensively metabolised in the liver to form the pharmacologically active nucleoside analogue triphosphate GS-461203. The active metabolite is not observed. The metabolic activation pathway involves sequential hydrolysis of the carboxyl ester moiety catalysed by human cathepsin A or carboxylesterase 1 and phosphoramidate cleavage by histidine triad nucleotide-binding protein 1 followed by phosphorylation by the pyrimidine nucleotide biosynthesis pathway. Dephosphorylation results in the formation of nucleoside metabolite GS-331007 that cannot be efficiently rephosphorylated and lacks anti-HCV activity in vitro. Within ledipasvir/sofosbuvir, GS-331007 accounts for approximately 85% of total systemic exposure.

Elimination

Following a single 90 mg oral dose of [¹⁴C]-ledipasvir, mean total recovery of the [¹⁴C]-radioactivity in faeces and urine was 87%, with most of the radioactive dose recovered from faeces (86%). Unchanged ledipasvir excreted in faeces accounted for a mean of 70% of the administered dose and the oxidative metabolite M19 accounted for 2.2% of the dose. These data suggest that biliary excretion of unchanged ledipasvir is a major route of elimination with renal excretion being a minor pathway (approximately 1%). The median terminal half-life of ledipasvir in healthy volunteers following administration of ledipasvir/sofosbuvir in the fasted state was 47 hours.

Following a single 400 mg oral dose of [¹⁴C]-sofosbuvir, mean total recovery of the dose was greater than 92%, consisting of approximately 80%, 14%, and 2.5% recovered in urine, faeces, and expired air, respectively. The majority of the sofosbuvir dose recovered in urine was GS-331007 (78%) while 3.5% was recovered as sofosbuvir. This data indicate that renal clearance is the major elimination pathway for GS-331007 with a large part actively secreted. The median terminal half-lives of sofosbuvir and GS-331007 following administration of ledipasvir/sofosbuvir were 0.5 and 27 hours, respectively.

Neither ledipasvir nor sofosbuvir are substrates for hepatic uptake transporters, organic cation transporter (OCT) 1, organic anion-transporting polypeptide (OATP) 1B1 or OATP1B3. GS-331007 is not a substrate for renal transporters including organic anion transporter (OAT) 1 or OAT3, or OCT2.

In vitro potential for ledipasvir/sofosbuvir to affect other medicinal products

At concentrations achieved in the clinic, ledipasvir is not an inhibitor of hepatic transporters including the OATP 1B1 or 1B3, BSEP, OCT1, OCT2, OAT1, OAT3, multidrug and toxic compound extrusion (MATE) 1 transporter, multidrug resistance protein (MRP) 2 or MRP4. Sofosbuvir and GS-331007 are not inhibitors of drug transporters P-gp, BCRP, MRP2, BSEP, OATP1B1, OATP1B3, OCT1 and GS-331007 is not an inhibitor of OAT1, OCT2 and MATE1.

Sofosbuvir and GS-331007 are not inhibitors or inducers of CYP or uridine diphosphate glucuronosyltransferase (UGT) 1A1 enzymes.

Pharmacokinetics in special populations

Race and gender

No clinically relevant pharmacokinetic differences due to race have been identified for ledipasvir, sofosbuvir or GS-331007. No clinically relevant pharmacokinetic differences due to gender have been identified for sofosbuvir or GS-331007. AUC and C_max of ledipasvir were 77% and 58% higher, respectively, in females than males; however, the relationship between gender and ledipasvir exposures was not considered clinically relevant.

Elderly

Population pharmacokinetic analysis in HCV-infected patients showed that within the age range (18 to 80 years) analysed, age did not have a clinically relevant effect on the exposure to ledipasvir, sofosbuvir or GS-331007. Clinical studies of ledipasvir/sofosbuvir included 235 patients (8.6% of total number of patients) aged 65 years and over.

Renal impairment

A summary of the effect of varying degrees of renal impairment (RI) on the exposures of the components of ledipasvir/sofosbuvir compared to subjects with normal renal function, as described in the text below, are provided in the following table.

Effect of Varying Degrees of Renal Impairment on Exposures (AUC) of Sofosbuvir, GS-331007, and Ledipasvir Compared to Subjects with Normal Renal Function:

↔ indicates no clinically relevant change in the exposure of Ledipasvir.

The pharmacokinetics of ledipasvir were studied with a single dose of 90 mg ledipasvir in HCV negative adult patients with severe renal impairment (eGFR <30 mL/min by Cockcroft-Gault, median [range] CrCl 22 [17-29] mL/min).

The pharmacokinetics of sofosbuvir were studed in HCV negative adult patients with mild (eGFR ≥50 and <80 mL/min/1.73m²), moderate (eGFR ≥30 and <50 mL/min/1.73m²), severe renal impairment (eGFR <30 mL/min/1.73m²) and patients with ESRD requiring haemodialysis following a single 400 mg dose of sofosbuvir,relative to patients with normal renal function (eGFR >80 mL/min/1.73m²). GS-331007 is efficiently removed by haemodialysis with an extraction coefficient of approximately 53%. Following a single 400 mg dose of sofosbuvir, a 4 hour haemodialysis removed 18% of administered sofosbuvir dose.

In HCV-infected adult patients with severe renal impairment treated with ledipasvir/sofosbuvir for 12 weeks (n=18), the pharmacokinetics of ledipasvir, sofosbuvir, and GS-331007 were consistent with that observed in HCV negative patients with severe renal impairment.

The pharmacokinetics of ledipasvir, sofosbuvir, and GS-331007 were studied in HCV-infected adult patients with ESRD requiring dialysis treated with ledipasvir/sofosbuvir (n=94) for 8, 12, or 24 weeks, and compared to patients without renal impairment in the ledipasvir/sofosbuvir Phase ⅔ trials.

Hepatic impairment

The pharmacokinetics of ledipasvir were studied with a single dose of 90 mg ledipasvir in HCV negative adult patients with severe hepatic impairment (CPT class C). Ledipasvir plasma exposure (AUC_inf) was similar in patients with severe hepatic impairment and control patients with normal hepatic function. Population pharmacokinetics analysis in HCV-infected adult patients indicated that cirrhosis (including decompensated cirrhosis) had no clinically relevant effect on the exposure to ledipasvir.

The pharmacokinetics of sofosbuvir were studied following 7-day dosing of 400 mg sofosbuvir in HCV-infected adult patients with moderate and severe hepatic impairment (CPT class B and C). Relative to patients with normal hepatic function, the sofosbuvir AUC_0-24 was 126% and 143% higher in moderate and severe hepatic impairment, while the GS-331007 AUC_0-24 was 18% and 9% higher, respectively. Population pharmacokinetics analysis in HCV-infected patients indicated that cirrhosis (including decompensated cirrhosis) had no clinically relevant effect on the exposure to sofosbuvir and GS-331007.

Body weight

Body weight did not have a significant effect on sofosbuvir exposure according to a population pharmacokinetic analysis. Exposure to ledipasvir decreases with increasing body weight but the effect is not considered to be clinically relevant.

Paediatric population

Ledipasvir, sofosbuvir, and GS-331007 exposures in paediatric patients aged 3 years and above were similar to those in adults from Phase ⅔ studies, following administration of ledipasvir/sofosbuvir. The 90% confidence intervals of geometric least-squares mean ratios for all PK parameters of interest were contained within the predetermined similarity bounds of less than 2-fold (50% to 200%) with the exception of ledipasvir C_tau in paediatric patients 12 years and above which was 84% higher (90%CI: 168% to 203%) and was not considered clinically relevant.

The pharmacokinetics of ledipasvir, sofosbuvir, and GS-331007 have not been established in paediatric patients aged <3 years.

Ledipasvir

No target organs of toxicity were identified in rat and dog studies with ledipasvir at AUC exposures approximately 7 times the human exposure at the recommended clinical dose.

Ledipasvir was not genotoxic in a battery of in vitro or in vivo assays, including bacterial mutagenicity, chromosome aberration using human peripheral blood lymphocytes and in vivo rat micronucleus assays.

Ledipasvir was not carcinogenic in the 26-week rasH2 transgenic mouse and the 2-year rat carcinogenicity studies at exposures up to 26-times in mice and 8-times in rats higher than human exposure.

Ledipasvir had no adverse effects on mating and fertility. In female rats, the mean number of corpora lutea and implantation sites were slightly reduced at maternal exposures 6-fold the exposure in humans at the recommended clinical dose. At the no observed effect level, AUC exposure to ledipasvir was approximately 7- and 3-fold, in males and females, respectively, the human exposure at the recommended clinical dose.

No teratogenic effects were observed in rat and rabbit developmental toxicity studies with ledipasvir.

In a rat pre- and postnatal study, at a maternally toxic dose, the developing rat offspring exhibited mean decreased body weight and body weight gain when exposed in utero (via maternal dosing) and during lactation (via maternal milk) at a maternal exposure 4 times the exposure in humans at the recommended clinical dose. There were no effects on survival, physical and behavioural development and reproductive performance in the offspring at maternal exposures similar to the exposure in humans at the recommended clinical dose.

When administered to lactating rats, ledipasvir was detected in plasma of suckling rats likely due to excretion of ledipasvir via milk.

Environmental risk assessment (ERA)

Environmental risk assessment studies have shown that ledipasvir has the potential to be very persistent and very bioaccumulative (vPvB) in the environment.

Sofosbuvir

In repeat dose toxicology studies in rat and dog, high doses of the 1:1 diastereomeric mixture caused adverse liver (dog) and heart (rat) effects and gastrointestinal reactions (dog). Exposure to sofosbuvir in rodent studies could not be detected likely due to high esterase activity; however, exposure to the major metabolite GS-331007 at doses which cause adverse effects was 16 times (rat) and 71 times (dog) higher than the clinical exposure at 400 mg sofosbuvir. No liver or heart findings were observed in chronic toxicity studies at exposures 5 times (rat) and 16 times (dog) higher than the clinical exposure. No liver or heart findings were observed in the 2-year carcinogenicity studies at exposures 17 times (mouse) and 9 times (rat) higher than the clinical exposure.

Sofosbuvir was not genotoxic in a battery of in vitro or in vivo assays, including bacterial mutagenicity, chromosome aberration using human peripheral blood lymphocytes and in vivo mouse micronucleus assays.

Carcinogenicity studies in mice and rats do not indicate any carcinogenicity potential of sofosbuvir administered at doses up to 600 mg/kg/day in mouse and 750 mg/kg/day in rat. Exposure to GS-331007 in these studies was up to 17 times (mouse) and 9 times (rat) higher than the clinical exposure at 400 mg sofosbuvir.

Sofosbuvir had no effects on embryo-foetal viability or on fertility in rat and was not teratogenic in rat and rabbit development studies. No adverse effects on behaviour, reproduction or development of offspring in rat were reported. In rabbit studies exposure to sofosbuvir was 6 times the expected clinical exposure. In the rat studies, exposure to sofosbuvir could not be determined but exposure margins based on the major human metabolite was approximately 5 times higher than the clinical exposure at 400 mg sofosbuvir.

Sofosbuvir-derived material was transferred through the placenta in pregnant rats and into the milk of lactating rats.