A Diacylglycerol Transferase 1 Inhibitor Is a Potent Hepatitis C

Article pubs.acs.org/journal/aidcbc

A Diacylglycerol Transferase 1 Inhibitor Is a Potent Hepatitis C Antiviral in Vitro but Not in Patients in a Randomized Clinical Trial Edward Gane,† Catherine Stedman,‡ Kiran Dole,§ Jin Chen,§ Charles Daniel Meyers,§ Brigitte Wiedmann,§ Jin Zhang,§ Prakash Raman,§ and Richard A. Colvin*,§ †

Auckland Clinical Sciences, Grafton, Auckland 1010, New Zealand Christchurch Hospital and University of Otago, Christchurch 4710, New Zealand § Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, United States ‡

S Supporting Information *

ABSTRACT: Hepatitis C virus (HCV) infection is a significant cause of liver disease affecting 80−150 million people globally. Diacylglycerol transferase 1 (DGAT-1), a triglyceride synthesis enzyme, is important for the HCV life cycle in vitro. Pradigastat, a potent DGAT-1 inhibitor found to lower triglycerides and HgbA1c in patients, was investigated for safety and efficacy in patients with HCV. This was a two-part study. In the in vitro study, the effect of pradigastat on virus production was evaluated in infected cells in culture. In the clinical study (https://clinicaltrials.gov/ct2/show/NCT01387958), 32 patients with HCV infection were randomized to receive pradigastat or placebo (26:6) once daily for 14 days. Primary efficacy outcomes were serum viral RNA and alanine aminotransferase levels. In vitro, pradigastat significantly reduced virus production, consistent with inhibition of viral assembly and release. However, the clinical study was prematurely terminated for lack of efficacy. There was no significant change in serum viral RNA levels after dosing with pradigastat or placebo for 14 days. Pradigastat was safe and well-tolerated in this population. Most treatmentemergent adverse events were gastrointestinal; there were no hepatic adverse events. Although pradigastat had a potent antiviral effect in vitro, no significant antiviral effect was observed in patients at predicted efficacious exposures. KEYWORDS: HCV, pradigastat, diacylglycerol transferase 1, clinical trial

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acting antivirals (DAAs), which block viral proteins involved in HCV replication, notably NS3-4A protease, NS5B RNAdependent RNA polymerase, and NS5A.12,13 Combinations of these drugs induce sustained virological responses in a great majority of patients. However, resistance to most DAAs given as monotherapy develops rapidly and is observed in patients that fail combination therapy.14,15 Host-targeting antivirals (HTAs), which inhibit viral replication, are also being developed.16 HTAs offer the potential of pan-genotypic efficacy while reducing the selection of resistant viruses. The cyclophilin inhibitor alisporivir is active against all HCV genotypes and has a high barrier to the development of resistance.16,17 For these reasons, the development of additional HTAs that may have similar profiles is highly desirable. Herker and colleagues18,19 described a role for a human enzyme, diacylglycerol transferase 1 (DGAT-1), in the HCV life cycle. DGAT-1 catalyzes the final step in triglyceride synthesis. Reduction of DGAT-1 by RNA interference decreased the spread of HCV in cultured hepatocytes. Further,

epatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, hepatocellular carcinoma, and liver transplantation.1−4 Between 80 and 150 million people worldwide are infected with HCV, with as few as 3% receiving treatment as recently as 2012.5 HCV is classified into six genotypes, with genotype-1 (GT-1) being the most common, followed by GT-2 and -3,6 with each responding differently to treatment.7 Unfortunately, treatment can be hindered by poor tolerability and virologic failure from selection of resistant virus. Until recently, all HCV treatment was interferon-based with suboptimal response rates and significant side effects.5,8 In 2011, the introduction of two HCV protease inhibitors, telaprevir and boceprevir, resulted in significant improvements in treatment outcomes that were offset by increased treatment complexity and reduced tolerability.9 Recently, sofosbuvir,10 a pan-genotypic nucleotide inhibitor of the viral polymerase, and velpatasvir, a pangenotypic inhibitor of the HCV genotype-1 nonstructural protein 5A (NS5A) protein, became available. The combination of sofosbuvir and velpatasvir shortens the treatment duration and improves both efficacy and tolerability.11 Additional drugs to treat HCV infection have recently been approved or are under development.12 The majority are directly © XXXX American Chemical Society

Received: August 2, 2016 Published: October 27, 2016 A

DOI: 10.1021/acsinfecdis.6b00138 ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

Article

On the basis of the rationale that DGAT-1 inhibition interferes with the HCV life cycle in vitro and that pradigastat is a clinically safe and potent DGAT-1 inhibitor, the current study was designed to determine whether pradigastat could reduce HCV replication in patients.

the addition of a small-molecule DGAT-1 inhibitor reduced the secretion of HCV virions from infected cells and prevented the association of viral RNA with lipid droplets. Other studies showed that the core protein directly interacts with DGAT-1.20 Finally, the enzymatic activity of DGAT-1 is required for the association of NS5A with the replication complex on lipid droplets.20 Additionally, reduction of triglyceride synthesis by DGAT-1 inhibition could potentially reduce HCV-related steatosis.21 The selective DGAT-1 inhibitor pradigastat (Figure 1 and Novartis data on file) is in clinical development for the

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RESULTS AND DISCUSSION

In Vitro Study. The effect of pradigastat on HCV replication and viral production was investigated. Pradigastat (LCQ908) significantly reduced the level of viral RNA released to culture supernatant, demonstrating that pradigastat blocked the assembly or release of virions. This was confirmed by measuring infectious virus production, which was reduced following treatment with pradigastat and three related nonclinical candidate DGAT-1 inhibitors (denoted as LHX154, LFW106, and LGV780) and correlated with the reduction in viral particles in the culture medium as measured by luciferase activity (Figure 2). Pradigastat had no significant effect on the replication of subgenomic HCV replicons, suggesting that the compound does not directly interfere with RNA replication (data not shown). The EC50 of the DGAT-1 inhibitors was significantly below the concentration required to cause cytotoxicity (Figure 2). Clinical Study. Thirty-two HCV-infected adults were enrolled in this study. Of the 32 patients, 26 were randomized to receive 100 mg of pradigastat daily for 2 days followed by 20 mg daily for 12 days. Seventeen of these patients had HCV GT1 infection, and nine had GT-3 infection. One of the GT-1 patients discontinued pradigastat on treatment day 7 because of the development of herpes simplex virus encephalitis that was not thought to be treatment-related. This patient was replaced

Figure 1. Structure of pradigastat.

management of dyslipidemia. Pradigastat is a potent DGAT-1 inhibitor in cell-free and cellular assays in vitro (Ki = 29.5 nM and IC50 = 66 nM, respectively) and substantially reduces fasting and postprandial triglyceride levels in patients with familial chylomicronemia syndrome (Novartis data on file and ref 22). Approximately 2500 individuals have received pradigastat, including approximately 1700 healthy volunteers, 800 patients with type 2 diabetes, and six patients with severe hypertriglyceridemia. Pradigastat was safe at all doses and durations tested but is associated with mild diarrhea resulting in a 5−7% discontinuation rate (Novartis data on file).

Figure 2. Four selective DGAT-1 inhibitors suppress HCV release. Four related DGAT-1 inhibitors were analyzed in vitro to confirm the published activity of DGAT-1 inhibitors on the release of hepatitis C virions and to determine the minimal efficacious concentration (relative to the DMSOonly control) required for these compounds to inhibit HCV virion release. The effects of pradigastat, LHX154, LFW106, and LGV780 on virion release are shown. Infectivity was determined by inoculation of Huh-7.5 cells with postinfection medium followed by luciferase assay at 72 h. Inhibition of luciferase activity (black lines) was compared with cytotoxicity (gray lines). A pradigastat concentration of 10 μM is equivalent to 4.9 μg/mL. Assays were performed in quadruplicate and repeated two or more times for each compound tested. Pradigastat was the only clinical candidate tested in these assays. B



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Figure 3. Changes in HCV RNA during and after 14 days of daily pradigastat treatment are shown for patients with chronic (A) HCV genotype-1 infection (pradigastat:placebo = 16:3) and (B) HCV genotype-3 infection (pradigastat:placebo = 9:2). The numbers of patients receiving pradigastat and placebo are shown on each plot. Pradigastat or placebo was administered on days 1 through 14, as highlighted by the gray bar on the X axis of each plot. Error bars indicate standard deviations (for clarity, only the positive error bars for pradigisat and negative error bars for placebo are shown).

placebo group. The drug had previously been associated with mild, self-limited gastrointestinal AEs, most commonly diarrhea or soft stools. The number of pradigastat-treated patients experiencing diarrhea (73.1% vs 16.7% in the placebo group) was greater than seen previously (up to 55.1% at a dose of 20 mg). Additionally, the onset of diarrhea appeared earlier than previously reported, but the diarrhea was generally mild and self-limited. The timing of the diarrhea correlated with the highest pradigastat concentrations associated with the use of the 100 mg loading doses in this study. There were no clinically significant changes in ALT values during treatment with pradigastat (Figure 4). The pradigastat-

with another GT-1 patient. The remaining six patients received placebo (five GT-1 or -3, one GT-2). The baseline demographics of the pradigastat and placebo groups were comparable, with the exception that a higher proportion of pradigastat-treated patients were Caucasian (80.8% vs 50% in the placebo group). Mean baseline platelet counts were within the normal range, with values of 196.5 × 109, 220 × 109, and 187.8 × 109 L−1 for GT-1, -2, and -3, respectively. The mean baseline alanine aminotransferase (ALT) values were 78.9 units/L for GT-1-infected patients, 126 units/L for GT-3infected patients, and 142 units/L for placebo-treated subjects. Overall, 31 patients (96.9%) completed the study. One patient in the pradigastat group discontinued because of a serious adverse event (SAE). Demographic data for enrolled patients are shown in Table S1-Sn. Effects on Serum HCV RNA Levels. The primary objective of this study was to evaluate the antiviral potency of oral pradigastat in patients infected with HCV GT-1, -2, and -3. Following treatment of HCV GT-1 or -3-infected patients with pradigastat, there were no significant changes in mean serum HCV RNA levels on day 14 or on any other study day compared to baseline or placebo (Figure 3). No GT-2-infected patients were dosed with pradigastat. GT-3-infected patients showed a slight increase in viral RNA compared with the placebo group. Summary statistics for HCV RNA by treatment group and genotype are shown in Table 1. Safety and Tolerability. Pradigastat was generally well tolerated in patients infected with HCV. Although most treatment-emergent adverse events (AEs) were mild, AEs were more common in pradigastat-treated patients than in the

Figure 4. Changes in ALT levels relative to baseline during and after 14 days of daily pradigastat treatment are shown for patients with chronic HCV infection (patients infected with all genotypes included). The numbers of patients receiving pradigastat or placebo are shown on the plot. Pradigastat or placebo was administered on days 1 through 14, as highlighted by the gray bar on the X axis of the plot. Error bars indicate standard deviations (for clarity, only the positive error bars for pradigisat and negative error bars for placebo are shown).

Table 1. Mean Change in Plasma HCV RNA from Baseline to Day 14 treatment group

n

mean (SD)a

difference from placebob

GT-1 (active drug) GT-3 (active drug) GT-1 and -3 (placebo)

16 9 5

0.03 (0.52) 0.42 (0.71) 0.29 (0.62)

−0.26 0.13

treated patients had more infections than placebo-treated patients (26.9% vs 16.7%). These were predominantly upper respiratory tract infections that were unlikely to be related to pradigastat administration. AEs related to musculoskeletal and connective tissue, skin and subcutaneous tissue, ear and labyrinth, reproductive system, and vascular disorders were more common in the placebo-treated group. These differences

a

Mean (standard deviation) of the difference from baseline to the day 14 visit. bDefined as the mean value for the pradigastat-treated patients minus the value for placebo-treated patients. C



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were thought to be secondary to the small number of patients in the study. There were no other AEs with >10% difference in incidence between the treatment groups (Table 2). Table 2. Incidence of Adverse Events by Preferred Term That Developed in More than Two Patientsa nAE (%)

preferred term any body system diarrhea nausea abdominal pain headache infections and infestations vomiting decreased appetite abdominal discomfort abdominal pain upper flatulence chest pain fatigue lethargy insomnia

GT-1 pradigastat (n = 17)

GT-3 pradigastat (n = 9)

total pradigastat (n = 26)

placebo (n = 6)

14 (82.4)

9 (100)

23 (88.5)

5 (83.3)

11 (64.7) 12 (70.6) 8 (47.1)

8 (88.9) 5 (55.6) 3 (33.3)

19 (73.1) 17 (65.4) 11 (42.3)

1 (16.7) 1 (16.7) 3 (50.0)

3 (17.6) 6 (35.3)

5 (55.6) 1 (11.1)

8 (30.8) 7 (26.9)

2 (33.3) 1 (16.7)

5 (29.4) 4 (23.5)

2 (22.2) 2 (22.2)

7 (26.9) 6 (23.1)

0 (0.0) 1 (16.7)

2 (11.8)

1 (11.1)

3 (11.5)

0 (0.0)

2 (11.8)

0 (0.0)

2 (7.7)

0 (0.0)

2 2 1 2 1

0 0 1 0 1

2 2 2 2 2

0 0 1 0 1

(11.8) (11.8) (5.9) (11.8) (5.9)

(0.0) (0.0) (11.1) (0.0) (11.1)

(7.7) (7.7) (7.7) (7.7) (7.7)

Figure 5. Daily pradigastat concentrations in patients with chronic HCV genotype-1 (n = 16) and genotype-3 infection. The numbers of genotype-1 and genotype-3 patients receiving pradigastat are shown on the plot. Pradigastat was administered on days 1 through 14, as highlighted by the gray bar on the X axis of the plot. Error bars indicate standard deviations.

ng/mL, which slightly decreased to ∼800 ng/mL by day 14. These levels are comparable to the day 14 levels observed in healthy subjects administered 10 mg of pradigastat daily, which resulted in a significant reduction in triglyceride excursion, suggesting that DGAT-1 was being inhibited. Additionally, a surrogate for DGAT-1 target engagement observed in clinical studies of pradigastat is the development of diarrhea.22,23 Patients in this study developed diarrhea at a rate equal to or higher than in previous studies, suggesting that DGAT-1 was inhibited by pradigastat (Table 2). Pradigastat exposure was comparable for patients infected with either HCV GT-1 or -3 (Figure 5). The mean predose pradigastat plasma concentrations on study days 8 and 14 were 837 and 766 ng/mL, respectively. On the basis of these plasma levels, the predicted concentrations of pradigastat in the liver were 6159 ng/mL on day 8 and 5462 ng/mL on day 14, which are above the pradigastat concentration required to impair HCV virion release in vitro. Relationship of Pradigastat to HCV RNA Reduction. To determine whether pradigastat exposure had an impact on HCV plasma RNA, patients infected with GT-1 were divided into three tertiles based on the average daily Ctrough levels of pradigastat in their plasma (Figure 6). Those patients with the highest exposure (average on treatment Ctrough > 900 ng/mL) had the greatest reduction in viral RNA. It should be noted that these changes in HCV RNA levels were not thought to be clinically significant. The maximum HCV RNA decline observed was 0.64 log10. This HCV GT-1-infected patient had high Ctrough pradigastat plasma levels on both day 8 (943 mg/mL) and day 14 (854 ng/mL) compared with the average Ctrough levels of the HCV GT-1 cohort (mean plasma levels of 837 ng/mL on day 8 and 766 mg/mL on day 14) (Table S2Sn). Conclusions. Most new HCV therapies target viral proteins, have relatively low barriers to resistance,14 and are predominantly active against HCV GT-1 infection.13 One potential way to prevent the development of viral resistance and achieve pan-genotypic coverage is to target host factors required for viral production. DGAT-1 is a human enzyme required for viral production in vitro. Targeted reduction of DGAT-1 activity through either RNA interference or chemical inhibition reduces the release of infectious HCV in cell culture

(0.0) (0.0) (16.7) (0.0) (16.7)

a

GT, genotype. AEs by preferred terms are presented in descending order of frequency in the total pradigastat group.

There was one SAE in the study. A pradigastat-treated patient developed herpes simplex virus encephalitis upon treatment. This patient was hospitalized and the study drug discontinued, and the patient recovered. This adverse event was not thought to be treatment-related by the investigator. There were no deaths reported during this study. Pharmacokinetics of Pradigastat. Preclinical data suggest that pradigastat concentrations of approximately 10 to 20 μM (∼5000 to 10 000 ng/mL) are required to inhibit HCV virion release from cultured hepatocytes. Liver pradigastat concentrations in humans are not known but are estimated to be about 7-fold higher than in plasma on the basis of tissue distribution studies in rats (data not shown). On the basis of the pharmacokinetic data obtained from previous clinical studies, it was predicted that 10 mg of pradigastat once daily would achieve this concentration at steady state in the liver. In healthy obese subjects being fed a medium-fat diet (30% fat diet), a 10 mg daily dose resulted in a mean day 14 trough plasma concentration (Ctrough) of 1097 ng/mL and prevented the excursion of 88−97% of triglycerides following a high fat meal, demonstrating that the target (DGAT-1) was sufficiently engaged.23 In order to increase the likelihood that pradigastat concentrations would reach this level, the highest tolerated dose in patients, 20 mg daily, was selected for this study. Additionally, a 100 mg loading dose was administered on days 1 and 2 of dosing in order to reach steady state more quickly. The mean trough plasma concentration−time profiles of pradigastat in GT-1- and GT-3-infected patients are shown in Figure 5. Steady-state levels of pradigastat were reached by day 4 and maintained until day 6 with a mean trough level of ∼950 D



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Figure 6. HCV genotype-1 patients treated in the study were divided into three exposure tertiles based on pradigastat average Ctrough on treatment days 2 through 14: low (mean Ctrough < 600 ng/mL), medium (mean 600 ng/mL < Ctrough < 900 ng/mL), and high (mean Ctrough > 900 ng/mL). (A, C, E) Mean changes in HCV viral RNA during and after 14 days of daily pradigastat treatment for individual patients in each tertile: (A) lowest exposure tertile; (C) medium exposure tertile; (E) highest exposure tertile. Baselines are shown as horizontal dashed lines. (B, D, F) Mean HCV viral RNA from all of the patients in each exposure tertile compared with the mean HCV RNA of all patients receiving placebo overlaid with the mean pradigastat concentration by day: (B) lowest exposure tertile; (D) medium exposure tertile; (F) highest exposure tertile. Error bars represent the standard deviations of the mean of viral RNA from all patients in each tertile.

GT-3-infected patients demonstrating no significant antiviral efficacy, the study was terminated without dosing of any HCV GT-2 patients. The exposures achieved in HCV patients were adequate to block DGAT-1 enzymatic activity. The loading regimen (100 mg/day for the first two days followed by 20 mg for 12 days) resulted in steady-state plasma drug concentrations by day 4. The overall mean plasma trough concentrations were slightly lower than expected and did not exceed 3000 ng/mL in any patient. The observed plasma concentrations on days 8 and 14 predicted liver concentrations of approximately 10 to 20 μM (5000 to 10 000 ng/mL). This is about 500 times the

and the colocalization of the viral core and NS5A proteins on lipid droplets, the site of virion assembly.19,21 Given the novel mechanism of action, it is unlikely that patients treated with a DGAT-1 inhibitor as a single agent will develop resistance to this class or any other class of anti-HCV therapeutic. Here we have demonstrated that pradigastat and other DGAT-1 inhibitors block HCV virion release in vitro. In our clinical study, however, 14 days of treatment with pradigastat did not achieve significant decreases in HCV RNA levels in patients infected with GT-1 or -3. On the basis of the analysis of the 17 pradigastat-treated HCV GT-1-infected patients and an interim analysis of the first nine pradigastat-treated HCV E



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Study Design. A randomized, double-blinded, placebocontrolled trial to determine the safety, tolerability, and antiviral efficacy of pradigastat in patients with HCV infection was conducted at two different sites in New Zealand. A total of 57 HCV-infected patients were expected to be enrolled, stratified, and randomized by HCV genotype; on the basis of an interim analysis, pradigastat had no significant impact on HCV viral load, and therefore, the study was terminated prior to the enrollment of all planned subjects. Only 32 patients were enrolled prior to study termination. Of these patients (20 GT1, one GT-2, and 11 GT-3), 26 were randomized to receive pradigastat and the remaining six patients to receive placebo. The randomization scheme (16:3 stratified by genotype) was generated by Novartis Drug Supply Management and approved by the Novartis Biostatistics Quality Assurance Group. Randomization numbers were assigned in a predetermined order to eligible subjects. The study consisted of a 28-day screening period, baseline visit, 14-day treatment period, and 28-day follow up period. The treatment period consisted of a loading regimen of 100 mg of pradigastat once daily for the first two days to rapidly achieve steady state followed by 20 mg of pradigastat once daily for 12 days. The 20 mg once daily dose was previously shown to be safe and tolerable over 12 weeks of dosing. Preclinical pharmacokinetic data suggested that 20 mg once daily in humans was likely to result in liver levels required for antiviral activity (10 to 20 μM). Dose adjustments and/or interruptions were not permitted. Pradigastat was manufactured by Novartis Pharmaceuticals in East Hanover, NJ (batch AEUS2010-0236). The average purity of the drug substance was 99.5% (Novartis data on file). The excipients composing the large majority of the LCQ908 drug product (by mass) include sodium lauryl sulfate, cellulose, and lactose. This study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice; the study protocol and consent forms were approved by the Institutional Review Board (IRB) for each study center. All patients provided written informed consent. All authors reviewed the manuscript, had access to the data, and vouch for the accuracy of the final manuscript. The study was registered at clinicaltirals.gov (NCT01387958), and the full protocol can be accessed on the journal’s Web site. Patients. Male and nonpregnant female subjects 18 to 65 years of age with chronic HCV GT-1, -2, or -3 were eligible. Enrolled patients had HCV RNA levels ≥ 105 IU/mL at the time of screening, documented anti-HCV antibody for at least six months, and serum ALT levels below 10 times the upper limit of normal. Women of child-bearing potential were required to use effective contraception during the study. Patients with vital signs out of the normal range after three repeat measurements at screening were not enrolled. Enrolled patients weighed at least 50 kg with a body mass index between 18 and 35 kg/m2. Exclusion criteria included prior use of any investigational agent for the treatment of HCV infection; the use of any other investigational drugs at the time of enrollment; previous treatment with an interferon-based regimen for HCV infection that did not achieve a >2 log10 drop in viral load; decompensated cirrhosis; hemoglobin levels below 12.0 g/dL; clinically significant ECG abnormalities; history of malabsorption; drug or alcohol abuse that would interfere with the study procedures; α-fetoprotein > 100 ng/mL; any significant illness within 2 weeks prior to dosing; uncontrolled diabetes, defined

concentration required to inhibit the enzymatic activity of DGAT-1 and is predicted to be sufficient to impair HCV replication on the basis of the in vitro results. Other explanations of the observed lack of antiviral effect of pradigastat are possible. The liver-to-plasma distribution ratio in HCV-infected patients may be less than that in rodents, leading to lower-than-predicted pradigastat liver concentrations. It is also possible that the plasma pradigastat concentration, and not the liver concentration, predicts the antiviral efficacy. Even if pradigastat did achieve potent inhibition of DGAT-1, the role of DGAT-1 may be facilitated by redundant pathways in patients. Additionally, since the pradigastat concentration required to block HCV replication is significantly higher than that required to inhibit the enzymatic activity of DGAT-1, it is possible that the inhibitors block another viral or cellular activity nonspecifically. Alternatively, DGAT-1 may play a nonenzymatic role in HCV replication. In HCV GT-1-infected patients, a weak correlation between higher serum pradigastat trough concentrations and HCV viral load reduction was observed. Higher pradigastat doses may therefore have a greater impact on HCV viral load. However, this is unlikely to be tolerated because of the high frequency of gastrointestinal side effects associated with the loading dose that result from pradigastat-mediated lipid malabsorption. More than 75% of the patients in this study developed diarrhea with a more rapid onset and longer duration than previously reported in studies of pradigastat. However, most AEs, including diarrhea, were mild and self-limited, and overall, pradigastat was generally well-tolerated and safe in patients with HCV infection. No clinical or biochemical evidence of hepatotoxicity in patients with chronic hepatitis C was observed, although patients with advanced liver disease (decompensated cirrhosis) were excluded from this study. These data should provide confidence that pradigastat can be administered safely to patients with chronic liver disease for other indications. In summary, in vitro experiments in HCV-infected hepatocytes demonstrated that a DGAT-1 inhibitor blocks HCV release. In patients, however, pradigastat achieved minimal effect on plasma HCV RNA levels. The results of this study make it unlikely that the addition of pradigastat to a DAA regimen would increase the rate of sustained virologic response. If the tolerability and exposure of oral DGAT-1 inhibitors can be improved, their utility could be explored further in combination with DAAs. The putative mechanism of action of DGAT-1 inhibitors makes these ideal agents to combine with NS5A inhibitors, which also inhibit HCV virion assembly and releaseNS5A inhibitors are the most potent of all DAA classes, but their efficacy is limited by a low barrier to resistance.

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EXPERIMENTAL METHODS In Vitro Infection Experiments. Huh-7.5 cells (5 × 104) were infected with HCV containing a luciferase reporter gene (HCVcc, GT-2a, JFH-1 strain) at a multiplicity of infection of 0.1 for 8 h.24 Three days postinfection, the medium was replaced with fresh medium containing pradigastat (final concentration 2, 10, or 20 μM) or dimethyl sulfoxide (DMSO). Following 8 h of incubation, the medium was collected to determine virion production and measured by quantifying HCV RNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Infectivity was determined by inoculation of Huh-7.5 cells followed by luciferase assay at 72 h. F



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patents with Gilead. C.S. has received grants and done research for Gilead and served on the advisory boards of Janssen and Roche.

by HbA1c > 8.5%; recent episodes of hypoglycemia; history of immunodeficiency diseases; or a positive hepatitis B surface antigen (HBsAg) test result. No additional exclusions were allowed. Safety Assessment. Safety assessments included physical examination and vital signs. All adverse events and serious adverse events as well as the severity and presumed relationship to the study drug were documented by the investigator. Frequent laboratory monitoring included hematology, blood chemistry, and urinalysis. Pharmacokinetic Assessment. Pharmacokinetic blood samples were collected predose at days 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, 15, 16, 18, 21, and 28 (end of study). Concentrations below the limit of quantification were treated as zero in summary statistics for concentration data only. Descriptive statistics of trough concentrations included mean, standard deviation, coefficient of variation, median, minimum, and maximum. Statistical Methods. The “safety population” consisted of all subjects that received at least one dose of study drug. The “PK population” consisted of all subjects with evaluable (or complete) pharmacokinetic (PK) parameter data. The “PD population” consisted of all subjects with evaluable viral RNA data who received a full 14-day course of study drug or placebo. The primary analysis variable was HCV RNA load at day 14. The analysis of primary end point was intended to be based on an ANOVA model under a Bayesian framework with an informative prior for the placebo arm. Because of the early termination, only summary statistics for change in HCV RNA levels from baseline through day 14 are provided. A correlation of drug concentration to viral load change was explored as a secondary analysis.

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ACKNOWLEDGMENTS We thank the patients, investigators, and study teams. We also thank Catherine Jones (Novartis Institutes for BioMedical Research) for writing assistance. Financial support for the conduct of this study and the writing of the manuscript was provided by Novartis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.6b00138. Demographic summary of all patients enrolled in the study (Table S1-Sn) and a summary of the plasma concentrations of pradigastat achieved in the patients in the study (Table S2-Sn) (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Address: Novartis Institute for BioMedical Research, 220 Massachusetts Avenue, Cambridge, MA 02139, United States. E-mail: [email protected]. Phone: +1 617-8714987. Fax: +1 617-871-5203. Author Contributions

E.G., C.S., K.D., J.C., C.D.M., J.Z., and R.A.C. performed the study and analyzed the results. R.A.C., C.D.M., and E.G. designed the study and wrote the manuscript. All authors had access to the data and vouch for the accuracy of the manuscript. Notes

The authors declare the following competing financial interest(s): R.A.C., K.D., J.C., C.D.M., B.W., J.Z., and P.R. are employees and shareholders of Novartis Pharmaceutical Corp. E.G. has received grants and done research for Gilead; served on the advisory boards of AbbVie, Boehringer Ingelheim, Gilead, Janssen, Novartis, Roche, and Tibotec; has been a speaker for Gilead, Novartis, Roche, and Tibotec; and has G



Article

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A Diacylglycerol Transferase 1 Inhibitor Is a Potent Hepatitis C

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