Arch. Pharm. Res. (2014) 37:299–305 DOI 10.1007/s12272-013-0318-9
REPORT ON INVESTIGATIONAL DRUGS
MicroRNA-targeting therapeutics for hepatitis C Jihae Baek • Soowon Kang • Hyeyoung Min
Received: 31 October 2013 / Accepted: 15 December 2013 / Published online: 3 January 2014 Ó The Pharmaceutical Society of Korea 2013
Abstract MiR-122 is a liver-specific microRNA (miRNA) that plays a pivotal role in regulating hepatic functions such as lipid metabolism and stress response. The observation that hepatitis C virus (HCV) could only replicate in miR122-positive hepatocytes led to the discovery that miR-122 is essential for HCV replication, and miR-122 is now one of the crucial host factors for anti-HCV therapy. Currently, the most advanced miR-122 targeting therapy is SPC3649 (miravirsen), a locked nucleic acid-modified oligonucleotide antagonizing miR-122. This review serves to provide information on the discovery and development of SPC3649, the first miRNA-targeted drug to enter human clinical trials, and introduce other miR-122-targeting therapeutics being developed for hepatitis C.
Hepatitis C virus (HCV), a member of the Flaviviridae family, is an enveloped virus with a positive-strand RNA genome. Six distinct genotypes of HCV have been identified based on the phylogenetic analysis of viral genome, and each genotype is further divided into multiple subtypes (Nie et al. 2012). It is estimated that 185 million people worldwide are chronically infected with HCV and are at risk of developing cirrhosis, hepatocellular carcinoma, and liver failure (Thomas 2013). Genotype 1 is the most prevalent genotype, but it is the most difficult virus to cure (Corouge and Pol 2011). The standard of care for genotype 1 had long been a dual therapy composed of pegylated interferon-a (IFN-a) and ribavirin, but the recent development of protease inhibitors such as telaprevir and J. Baek S. Kang H. Min (&) College of Pharmacy, Chung-Ang University, 84 Heukseokro, Dongjakgu, Seoul 156-756, Korea e-mail: [email protected]
boceprevir enabled triple therapy, a combination of IFN-a, ribavirin, and one of the two protease inhibitors (Poordad et al. 2011; Sherman et al. 2011). The addition of telaprevir or boceprevir to dual therapy greatly improved a sustained virologic response in patients with genotype 1 viral infection, but the use of protease inhibitors is only limited to genotype 1 viral infection, and dual therapy remains the standard of care for non-genotype 1 virus. Despite the recent progress of triple therapy, current antiviral therapies are still frequently ineffective and poorly tolerated with substantial side effects in many HCV patients. Protease inhibitors often cause anemia and drug–drug interactions by inhibiting CYP3A enzymes and the P-glycoprotein transporter (Liang and Ghany 2013). Occurrence of drug-resistant mutants is another primary concern, when using direct-acting antivirals with a low genetic barrier to resistance (Janssen et al. 2013). Therefore, it is of necessity to develop more effective and less toxic anti-HCV therapeutics. Especially, attention has been made to host-targeting antiviral agents to lower a genetic barrier of resistance and to function independently of genotype. MicroRNAs (miRNAs) are endogenous, non-coding, 20–22 nucleotides (nt)-long RNAs that regulate gene expression (Bartel 2009). They mediate their action by binding to the 30 -untranslated region (UTR) of their mRNA targets leading to the inhibition of translation. As critical regulators of gene expression, miRNAs function in many biological processes, and the dysregulated expression of miRNAs is often found in various human diseases such as cancer, autoimmune disease, cardiovascular disease, and neurodegenerative disease (Bushati and Cohen 2007; Chang and Mendell 2007). Viral infection also causes changes in miRNA expression, which results in the modulation of host cell function to increase viral replication or to evade the host immune system.
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Literatures have shown that cellular miRNAs such as miR-448, miR-196, let-7b, miR-199a, and miR-27a inhibit HCV infection, while miR-122, miR-130a, and miR-141 stimulate HCV replication (Shrivastava et al. 2013). Among those, miR-122 garnered the most attention, because miR-122 is a liver-specific miRNA with high expression level accounting for about 70 % of hepatic miRNAs, and is essential for HCV replication (LagosQuintana et al. 2002; Chang et al. 2004). Based on the observation that HCV can only replicate in Huh7 cells expressing miR-122 but not in HepG2 cells lacking miR122, Jopling et al. (2008) found that cellular miR-122 is critical for HCV replication. MiR-122 seed sequences interact with HCV RNA at two adjacent sites (8-nt apart) in the 50 -UTR of the viral genome, and the two miR-122 sites are highly conserved in HCV genotypes. When the interaction between miR-122 and HCV genome was blocked by 20 -O-methylated antisense oligonucleotide (ASO) in vitro, significant reduction in HCV replication occurred suggesting that targeted inhibition of miR-122 may have considerable potential in the development of novel antiHCV therapeutics. Several in vivo approaches have been made thereafter to inhibit miR-122 from binding to HCV targets using mouse models (Table 1). The basic concept of miR-122 inhibition was the use of ASO complementary to miR-122, but each trial used different strategies to modify oligonucleotides. For example, antagomir-122 is cholesterol-linked-20 -Omethyl oligonucleotides (Krutzfeldt et al. 2005), while miR-122 ASO is a 20 -O-methoxyethyl oligonucleotide (Esau et al. 2006). In addition, 16-mer and 15-mer locked nucleic acid (LNA)-modified oligonucleotides have also been tested (Elmen et al. 2008a, b). In an in vivo study using mice, antagomir-122, miR-122 ASO, and LNAmodified oligonucleotides all successfully reduced miR122 levels in the liver and serum cholesterol level, indicating the successful silencing of miR-122. No apparent toxic effects were observed. Among these ASO, a 15-mer high affinity LNA-modified oligonucleotide (SPC3649) showed the best activity for silencing of miR-122, and thus, SPC3649 was chosen for subsequent studies of safety and efficacy in non-human primates. The safety of SPC3649 was tested in two studies using African green monkeys and cynomolgus monkeys. Both studies showed a dose-dependent decrease in total plasma cholesterol with no severe toxic effects (Elmen et al. 2008a, Hildebrandt-Eriksen et al. 2012). Especially, the study conducted in cynomolgus monkeys assessed the effects and tolerability of SPC3649 on miR-122 at a suprapharmacological dose (96 mg/kg/week) as well as pharmacological dose, and demonstrated that miR-122 inhibition did not affect liver morphology or function (Hildebrandt-Eriksen et al. 2012). Efficacy study conducted
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in chronically HCV-infected chimpanzees revealed that treatment with SPC3649 showed long-lasting suppression of HCV replication without side effects (Lanford et al. 2010). Surprisingly, viral repression remained during the 12-week dosing period without the occurrence of viral resistance and rebound which are often found when HCVinfected animals are treated with direct-acting antivirals (Cooper et al. 2009). Following successful completion of preclinical studies, SPC3649 entered human clinical trials as the first miRNAtargeted drug. Two Phase 1 studies (NCT00688012 for single ascending dose and NCT00979927 for multiple ascending doses) have been performed to evaluate the safety of SPC3649 (Lindow and Kauppinen 2012). The Phase 1 study data revealed that SPC3649 was well tolerated with no dose-limiting toxicities and showed reduced plasma cholesterol levels, confirming the antagonizing effect of SPC3649 on miR-122. In a Phase 2a clinical trial (NCT01200420), the safety, tolerability, pharmacokinetics, and efficacy of multiple dosing were assessed in subjects with naı¨ve chronic HCV infection (Janssen et al. 2013). The data demonstrated that SPC3649 resulted in a dosedependent and sustained decrease in HCV RNA levels together with a decrease in plasma cholesterol levels. The inhibitory effects were maintained for several weeks after dosing had ended, and resistant mutants were not found. At present, several clinical trials are being conducted or have been completed but the study results have not been reported. Another Phase 1 trial (NCT01646489) to evaluate the safety, tolerability, and pharmacokinetics of coadministered SPC3649 and telaprevir in healthy subjects has been completed, but the study results have not been published. A separate Phase 2 study of SPC3649 in combination with telaprevir and ribavirin in patients who failed to respond to pegylated IFN-a and ribavirin is currently recruiting study participants (NCT01872936). Santaris Pharma A/S, the developer of SPC3649, has also completed enrollment of a Phase 2 clinical trial of SPC3649 monotherapy in null responders to pegylated IFN-a and ribavirin (NCT01727934). All in vivo preclinical and clinical study results for SPC3649 monotherapy are summarized in Table 1. In addition to SPC3649, two more approaches have been made to develop anti-HCV therapeutics targeting miR-122 (Fig. 1). One is RG-101, an N-acetyl D-galactosamine (GalNAc)-conjugated anti-miR-122 oligonucleotide, developed by Regulus Therapeutics (presented at the 64th Annual Meeting of the American Association for the Study of Liver Disease; http://www.regulusrx.com). RG-101 uses GalNAc as a targeting moiety for uptake of an anti-miR122 oligonucleotide, since GalNAc can bind to asialoglycoprotein receptor on hepatocytes and enable targeting to hepatocytes. In efficacy studies using human liver chimeric
80
12.5–75
2.5–25
1–200
5
1, 3, 10
0, 3, 12, or 48
Mouse
Mouse
Mouse
Mouse
African green monkey
Cynomolgus monkey
Dosage (mg/kg)
Mouse
Preclinical
Species
SPC3649 (miravirsen)
SPC3649 (miravirsen)
SPC3649 (miravirsen)
15-mer LNAantimiR (SPC3649)
16-mer LNAantimiR, a high-affinity
miR-122 ASO
Antagomir-122
Drug modification
i.v. by a slow (5 min) bolus injection
i.v. infusion over about 10 min at a rate of 24 ml/kg/h
i.p.
i.p.
i.v.
i.p.
i.v.
Treatment route
Twice weekly for 9 times
Days 1, 3 and 5
Twice weekly for 6 weeks
3 times
3 consecutive days
Twice weekly for 4 weeks
1, 2, or 3 consecutive days
Treatment schedule
Table 1 Summary of in vivo preclinical and clinical studies with SPC3649
Maximal inhibition of miR-122 at 24 mg/ kg/week; extended recovery period to 12 weeks
Long-lasting depletion of mature miR-122 for 3 months
Sequestration of mature miR-122; derepression of the miR-122 targets, Aldoa and Bckdk
Sequestration of mature miR-122; threefold derepression of the direct miR122 target mRNA
Maximal reduction of miR-122 level at 24 h
Threefold to tenfold reduction in miR-122 level
23 days long effect (80 mg/kg 9 3)
Effectiveness
Dose-related decreases in total serum cholesterol; accumulation of basophilic granular materials and submucosal granular deposits; infiltrations of vacuolated macrophages; AST and ALT above ULN, pale discoloration of kidneys and livers, and increase in mean liver weights (96 mg/kg/week); shortlived prolongation of APTT and PT (24, 96 mg/kg/week)
Sustained decrease in total plasma cholesterol; no acute or subchronic toxicities
Sustained decrease in total cholesterol; no hepatotoxicity
Sustained decrease in total cholesterol with a median effective dose (ED50) of 10 mg/ kg
No hepatotoxicity in total 75 mg/ kg; reduction of plasma cholesterol level by *40 %
Mild increase of plasma transaminase level in 75 mg/kg; decrease in cholesterol and triglyceride levels
Decrease of cholesterol biosynthesis genes; decrease of cholesterol level by more than 40 %
Side effect
HildebrandtEriksen et al. (2012)
Elmen et al. (2008a)
Elmen et al. (2008a)
Elmen et al. (2008a)
Elmen et al. (2008b)
Esau et al. (2006)
Krutzfeldt et al. (2005)
References or clinical trials identifiers
Therapeutics for hepatitis C 301
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123
7 (5 weeks); 5 (7 weeks)
Miravirsen
Miravirsen
Miravirsen
Miravirsen
SPC3649 (miravirsen)
Drug modification
s.c.
s.c.
s.c.
s.c.
i.v.
Treatment route
5 weekly doses over 5 weeks, followed by a further 4 doses once every other week over 7 weeks (12 weeks total)
150 mg/ml for 5 weekly doses over a 29-day period
5 weekly doses
Single ascending dose
A weekly basis for 12 weeks followed by 17 treatment-free weeks
Treatment schedule
N.A.
Dose-related reduction of HCV RNA level (3 mg/kg, 1.2 log; 5 mg/kg, 2.9 log; 7 mg/kg, 3.0 log from baselines); no escape mutations in the HCV genome
Dose-dependent pharmacology
Dose-dependent pharmacology
Sequestration of miR-122; a decline of HCV RNA in the serum and liver and maintenance of plasma level for 4 weeks after the last dose (5 mg/kg); no viral resistance
Effectiveness
N.A.
Non dose-dependent lowering of cholesterol; reductions in AST and ALT levels; headache, fatigue; no dose-limiting adverse events
Dose-dependent lowering of cholesterol; no doselimiting adverse events
Dose-dependent lowering of cholesterol; no doselimiting adverse events
Lowered serum cholesterol; spike in ALT (5 mg/kg); no other side-effects
Side effect
NCT01727934
Janssen et al. (2013); NCT01200420
NCT00979927
NCT00688012
Lanford et al. (2010)
References or clinical trials identifiers
i.v., intravenous; i.p., intraperitoneal; s.c., subcutaneous; antagomir, 30 cholesterol-conjugated 2-O-methyl oligonucleotide with phosphorothioate backbone; miR-122 ASO, 20 -O-methoxyethyl phosphorothioate antisense oligonucleotide; LNA-antimiR, locked-nucleic-acid-modified oligonucleotide with phosphorothioate backbone; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ULN, upper limit of the normal range; APTT, activated partial thromboplastin time; PT, prothrombin; N.A., not available
10 null responders to pegylated IFN-a plus ribavirin with chronic HCV
Phase 2
36 patients with chronic HCV genotype 1 infection
3, 5, or 7
5
30 healthy volunteers
Phase 2a
12
1 or 5
Dosage (mg/kg)
64 healthy man volunteers
Phase 1
Clinical
Chimpanzee
Species
Table 1 continued
302 J. Baek et al.
Therapeutics for hepatitis C
303
Fig. 1 MiR-122 targeting antiHCV therapeutics under investigation. a SPC3649 or miravirsen is a 15-mer LNAmodified oligonucleotide complementary to miR-122. SPC3649 has a full PS backbone and some LNAmodified sugars at the 50 - and 30 -ends to enhance pharmacokinetic properties and nuclease stability (uppercase, LNA-modified nucleotide; lowercase, unmodified nucleotide; m, methylation). b RG-101, a N-acetyl Dgalactosamine (GalNAc)conjugated anti-miR-122 oligonucleotide. c Small molecule-based miRNA inhibitors. Compound-1, 2,4dichloro-N-(naphthalen-2yl)benzamide; compound-2, 6-(((4aR,8aS)octahydroquinolin-1(2H)yl)sulfonyl)-1,2,3,4tetrahydroquinoline
mice, RG-101 significantly lowered serum viral titers in a dose-dependent manner. No toxic effects were found at doses up to 450 mg/kg in mice and 45 mg/kg in cynomolgus monkeys except for a mild increase in liver function test at 150 mg/kg. After completing preclinical studies, RG-101 is expected to begin testing in clinical trials soon. The other candidate therapeutics is small molecule-based miR-122 inhibitors. Using a luciferase reporter assay system in which binding of miR-122 to the 30 -UTR of luciferase gene downregulates luciferase signal, Young et al. (2010) screened 1,364 compounds and identified two compounds (compound-1 and compound-2) with miR-122 inhibiting activity. The compounds-1 and -2 decreased the expression of miR-122 with EC50 values of 3 and 0.6 lM, respectively, and caused a *50 % reduction
in viral RNA replication at 10 lM in Huh7 cells. The safety and efficacy of these compounds have not been yet assessed in animal models. However, considering the advantages of using small molecule-based drugs such as the easiness to manipulate for increasing stability, delivery, and pharmacokinetics, small molecule miR-122 inhibitors should have promising therapeutic potential for Hepatitis C (Young et al. 2010). MiR-122 targeting therapeutics including SPC3649 have several advantages over other anti-HCV drugs. Unlike most antivirals that directly target HCV, anti-miR-122 antivirals interact with host miRNA and can avoid the emergence of viral resistance that occurs by mutation of the HCV genome as observed in the studies of miravirsen. Notably, anti-miR-122 antivirals would be genotype
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independent, since miR-122 binding sites are highly conserved in all HCV genotypes. In addition, according to a recent report, SPC3649 can repress the biogenesis of miR122 by binding to the stem-loop structures of pri- and premiRNAs, although it has not been elucidated if the inhibition of miR-122 biogenesis would support the pharmacological activity of SPC3649 (Gebert et al. 2013). The natural anti-viral cytokine, IFN-b, can also function through miR-122 (Pedersen et al. 2007). It was reported that IFN- b treatment caused a significant decrease in miR122 expression, and that the antiviral effects of IFN-b were diminished by the addition of a miR-122 mimic. Apart from its well-known anti-viral mechanism such as the induction of protein kinase R for blocking viral protein synthesis and 20 -50 oligoadenylate synthetase for activating RNase L and degrading viral mRNA, it is interesting that IFN-b also exerts an anti-HCV function as a natural miR122 inhibitor. In summary, SPC3649 is an oligonucleotide based inhibitor of miR-122, a liver specific miRNA required for HCV replication. Due to its distinctive mechanism of action of targeting host factors, strong efficacy, and a good safety profile, this new therapeutic would hold promise for replacing the current standard of care or for being combined with current treatment regimens. SPC3649 is currently being evaluated in Phase 2 clinical trials. It is hoped that the first therapeutic applications of miRNA inhibition will emerge soon. Acknowledgments This work was supported by the Chung-Ang University Excellent Student Scholarship in 2012 and by the National Research Foundation (NRF), funded by the Ministry of Science, ICT, and Future Planning [NRF-2013R1A1A3005097 to H.M.]. Conflict of interest manuscript.
The authors have no conflict of interest in our
References Bartel, D.P. 2009. MicroRNAs: target recognition and regulatory functions. Cell 136: 215–233. Bushati, N., and S.M. Cohen. 2007. microRNA functions. Annual Review of Cell and Developmental Biology 23: 175–205. Chang, J., E. Nicolas, D. Marks, C. Sander, A. Lerro, M.A. Buendia, C. Xu, W.S. Mason, T. Moloshok, R. Bort, K.S. Zaret, and J.M. Taylor. 2004. miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA Biology 1: 106–113. Chang, T.C., and J.T. Mendell. 2007. microRNAs in vertebrate physiology and human disease. Annual Review of Genomics and Human Genetics 8: 215–239. Cooper, C., E.J. Lawitz, P. Ghali, M. Rodriguez-Torres, F.H. Anderson, S.S. Lee, J. Be´dard, N. Chauret, R. Thibert, I. Boivin, O. Nicolas, and L. Proulx. 2009. Evaluation of VCH-759 monotherapy in hepatitis C infection. Journal of Hepatology 51: 39–46.
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Corouge, M., and S. Pol. 2011. New treatments for chronic hepatitis C virus infection. Me´decine et maladies infectieuses 41: 579–587. Elmen, J., M. Lindow, S. Schutz, M. Lawrence, A. Petri, S. Obad, M. Lindholm, M. Hedtjarn, H.F. Hansen, U. Berger, S. Gullans, P. Kearney, P. Sarnow, E.M. Straarup, and S. Kauppinen. 2008a. LNA-mediated microRNA silencing in non-human primates. Nature 452: 896–899. Elmen, J., M. Lindow, A. Silahtaroglu, M. Bak, M. Christensen, A. Lind-Thomsen, M. Hedtjarn, J.B. Hansen, H.F. Hansen, E.M. Straarup, K. Mccullagh, P. Kearney, and S. Kauppinen. 2008b. Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver. Nucleic Acids Research 36: 1153–1162. Esau, C., S. Davis, S.F. Murray, X.X. Yu, S.K. Pandey, M. Pear, L. Watts, S.L. Booten, M. Graham, R. Mckay, A. Subramaniam, S. Propp, B.A. Lollo, S. Freier, C.F. Bennett, S. Bhanot, and B.P. Monia. 2006. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metabolism 3: 87–98. Gebert, L. F., Rebhan, M. A., Crivelli, S. E., Denzler, R., Stoffel, M., and Hall, J. Miravirsen (SPC3649) can inhibit the biogenesis of miR-122. Nucleic Acids Res, (2013). Hildebrandt-Eriksen, E.S., V. Aarup, R. Persson, H.F. Hansen, M.E. Munk, and H. Orum. 2012. A locked nucleic acid oligonucleotide targeting microRNA 122 is well-tolerated in cynomolgus monkeys. Nucleic Acid Therapeutics 22: 152–161. Janssen, H.L., H.W. Reesink, E.J. Lawitz, S. Zeuzem, M. RodriguezTorres, K. Patel, A.J. Van Der Meer, A.K. Patick, A. Chen, Y. Zhou, R. Persson, B.D. King, S. Kauppinen, A.A. Levin, and M.R. Hodges. 2013. Treatment of HCV infection by targeting microRNA. New England Journal of Medicine 368: 1685–1694. Jopling, C.L. 2008. Regulation of hepatitis C virus by microRNA122. Biochemical Society Transactions 36: 1220–1223. Krutzfeldt, J., N. Rajewsky, R. Braich, K.G. Rajeev, T. Tuschl, M. Manoharan, and M. Stoffel. 2005. Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438: 685–689. Lagos-Quintana, M., R. Rauhut, A. Yalcin, J. Meyer, W. Lendeckel, and T. Tuschl. 2002. Identification of tissue-specific microRNAs from mouse. Current Biology 12: 735–739. Lanford, R.E., E.S. Hildebrandt-Eriksen, A. Petri, R. Persson, M. Lindow, M.E. Munk, S. Kauppinen, and H. Orum. 2010. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327: 198–201. Liang, T.J., and M.G. Ghany. 2013. Current and future therapies for hepatitis C virus infection. New England Journal of Medicine 368: 1907–1917. Lindow, M., and S. Kauppinen. 2012. Discovering the first microRNA-targeted drug. Journal of Cell Biology 199: 407–412. Nie, W., J. Chen, and Q. Xiu. 2012. Cytotoxic T-lymphocyte associated antigen 4 polymorphisms and asthma risk: a metaanalysis. PLoS One 7: e42062. Pedersen, I.M., G. Cheng, S. Wieland, S. Volinia, C.M. Croce, F.V. Chisari, and M. David. 2007. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature 449: 919–922. Poordad, F., J. Mccone, B.R. Bacon, S. Bruno, M.P. Manns, M.S. Sulkowski, I.M. Jacobson, K.R. Reddy, Z.D. Goodman, N. Boparai, M.J. Dinubile, V. Sniukiene, C.A. Brass, J.K. Albrecht, J.P. Bronowicki, and S. Investigators. 2011. Boceprevir for untreated chronic HCV genotype 1 infection. New England Journal of Medicine 364: 1195–1206. Sherman, K.E., S.L. Flamm, N.H. Afdhal, D.R. Nelson, M.S. Sulkowski, G.T. Everson, M.W. Fried, M. Adler, H.W. Reesink, M. Martin, A.J. Sankoh, N. Adda, R.S. Kauffman, S. George, C.I. Wright, F. Poordad, and I.S. Team. 2011. Response-guided telaprevir combination treatment for hepatitis C virus infection. New England Journal of Medicine 365: 1014–1024.
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REPORT ON INVESTIGATIONAL DRUGS
MicroRNA-targeting therapeutics for hepatitis C Jihae Baek • Soowon Kang • Hyeyoung Min
Received: 31 October 2013 / Accepted: 15 December 2013 / Published online: 3 January 2014 Ó The Pharmaceutical Society of Korea 2013
Abstract MiR-122 is a liver-specific microRNA (miRNA) that plays a pivotal role in regulating hepatic functions such as lipid metabolism and stress response. The observation that hepatitis C virus (HCV) could only replicate in miR122-positive hepatocytes led to the discovery that miR-122 is essential for HCV replication, and miR-122 is now one of the crucial host factors for anti-HCV therapy. Currently, the most advanced miR-122 targeting therapy is SPC3649 (miravirsen), a locked nucleic acid-modified oligonucleotide antagonizing miR-122. This review serves to provide information on the discovery and development of SPC3649, the first miRNA-targeted drug to enter human clinical trials, and introduce other miR-122-targeting therapeutics being developed for hepatitis C.
Hepatitis C virus (HCV), a member of the Flaviviridae family, is an enveloped virus with a positive-strand RNA genome. Six distinct genotypes of HCV have been identified based on the phylogenetic analysis of viral genome, and each genotype is further divided into multiple subtypes (Nie et al. 2012). It is estimated that 185 million people worldwide are chronically infected with HCV and are at risk of developing cirrhosis, hepatocellular carcinoma, and liver failure (Thomas 2013). Genotype 1 is the most prevalent genotype, but it is the most difficult virus to cure (Corouge and Pol 2011). The standard of care for genotype 1 had long been a dual therapy composed of pegylated interferon-a (IFN-a) and ribavirin, but the recent development of protease inhibitors such as telaprevir and J. Baek S. Kang H. Min (&) College of Pharmacy, Chung-Ang University, 84 Heukseokro, Dongjakgu, Seoul 156-756, Korea e-mail: [email protected]
boceprevir enabled triple therapy, a combination of IFN-a, ribavirin, and one of the two protease inhibitors (Poordad et al. 2011; Sherman et al. 2011). The addition of telaprevir or boceprevir to dual therapy greatly improved a sustained virologic response in patients with genotype 1 viral infection, but the use of protease inhibitors is only limited to genotype 1 viral infection, and dual therapy remains the standard of care for non-genotype 1 virus. Despite the recent progress of triple therapy, current antiviral therapies are still frequently ineffective and poorly tolerated with substantial side effects in many HCV patients. Protease inhibitors often cause anemia and drug–drug interactions by inhibiting CYP3A enzymes and the P-glycoprotein transporter (Liang and Ghany 2013). Occurrence of drug-resistant mutants is another primary concern, when using direct-acting antivirals with a low genetic barrier to resistance (Janssen et al. 2013). Therefore, it is of necessity to develop more effective and less toxic anti-HCV therapeutics. Especially, attention has been made to host-targeting antiviral agents to lower a genetic barrier of resistance and to function independently of genotype. MicroRNAs (miRNAs) are endogenous, non-coding, 20–22 nucleotides (nt)-long RNAs that regulate gene expression (Bartel 2009). They mediate their action by binding to the 30 -untranslated region (UTR) of their mRNA targets leading to the inhibition of translation. As critical regulators of gene expression, miRNAs function in many biological processes, and the dysregulated expression of miRNAs is often found in various human diseases such as cancer, autoimmune disease, cardiovascular disease, and neurodegenerative disease (Bushati and Cohen 2007; Chang and Mendell 2007). Viral infection also causes changes in miRNA expression, which results in the modulation of host cell function to increase viral replication or to evade the host immune system.
123
300
Literatures have shown that cellular miRNAs such as miR-448, miR-196, let-7b, miR-199a, and miR-27a inhibit HCV infection, while miR-122, miR-130a, and miR-141 stimulate HCV replication (Shrivastava et al. 2013). Among those, miR-122 garnered the most attention, because miR-122 is a liver-specific miRNA with high expression level accounting for about 70 % of hepatic miRNAs, and is essential for HCV replication (LagosQuintana et al. 2002; Chang et al. 2004). Based on the observation that HCV can only replicate in Huh7 cells expressing miR-122 but not in HepG2 cells lacking miR122, Jopling et al. (2008) found that cellular miR-122 is critical for HCV replication. MiR-122 seed sequences interact with HCV RNA at two adjacent sites (8-nt apart) in the 50 -UTR of the viral genome, and the two miR-122 sites are highly conserved in HCV genotypes. When the interaction between miR-122 and HCV genome was blocked by 20 -O-methylated antisense oligonucleotide (ASO) in vitro, significant reduction in HCV replication occurred suggesting that targeted inhibition of miR-122 may have considerable potential in the development of novel antiHCV therapeutics. Several in vivo approaches have been made thereafter to inhibit miR-122 from binding to HCV targets using mouse models (Table 1). The basic concept of miR-122 inhibition was the use of ASO complementary to miR-122, but each trial used different strategies to modify oligonucleotides. For example, antagomir-122 is cholesterol-linked-20 -Omethyl oligonucleotides (Krutzfeldt et al. 2005), while miR-122 ASO is a 20 -O-methoxyethyl oligonucleotide (Esau et al. 2006). In addition, 16-mer and 15-mer locked nucleic acid (LNA)-modified oligonucleotides have also been tested (Elmen et al. 2008a, b). In an in vivo study using mice, antagomir-122, miR-122 ASO, and LNAmodified oligonucleotides all successfully reduced miR122 levels in the liver and serum cholesterol level, indicating the successful silencing of miR-122. No apparent toxic effects were observed. Among these ASO, a 15-mer high affinity LNA-modified oligonucleotide (SPC3649) showed the best activity for silencing of miR-122, and thus, SPC3649 was chosen for subsequent studies of safety and efficacy in non-human primates. The safety of SPC3649 was tested in two studies using African green monkeys and cynomolgus monkeys. Both studies showed a dose-dependent decrease in total plasma cholesterol with no severe toxic effects (Elmen et al. 2008a, Hildebrandt-Eriksen et al. 2012). Especially, the study conducted in cynomolgus monkeys assessed the effects and tolerability of SPC3649 on miR-122 at a suprapharmacological dose (96 mg/kg/week) as well as pharmacological dose, and demonstrated that miR-122 inhibition did not affect liver morphology or function (Hildebrandt-Eriksen et al. 2012). Efficacy study conducted
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in chronically HCV-infected chimpanzees revealed that treatment with SPC3649 showed long-lasting suppression of HCV replication without side effects (Lanford et al. 2010). Surprisingly, viral repression remained during the 12-week dosing period without the occurrence of viral resistance and rebound which are often found when HCVinfected animals are treated with direct-acting antivirals (Cooper et al. 2009). Following successful completion of preclinical studies, SPC3649 entered human clinical trials as the first miRNAtargeted drug. Two Phase 1 studies (NCT00688012 for single ascending dose and NCT00979927 for multiple ascending doses) have been performed to evaluate the safety of SPC3649 (Lindow and Kauppinen 2012). The Phase 1 study data revealed that SPC3649 was well tolerated with no dose-limiting toxicities and showed reduced plasma cholesterol levels, confirming the antagonizing effect of SPC3649 on miR-122. In a Phase 2a clinical trial (NCT01200420), the safety, tolerability, pharmacokinetics, and efficacy of multiple dosing were assessed in subjects with naı¨ve chronic HCV infection (Janssen et al. 2013). The data demonstrated that SPC3649 resulted in a dosedependent and sustained decrease in HCV RNA levels together with a decrease in plasma cholesterol levels. The inhibitory effects were maintained for several weeks after dosing had ended, and resistant mutants were not found. At present, several clinical trials are being conducted or have been completed but the study results have not been reported. Another Phase 1 trial (NCT01646489) to evaluate the safety, tolerability, and pharmacokinetics of coadministered SPC3649 and telaprevir in healthy subjects has been completed, but the study results have not been published. A separate Phase 2 study of SPC3649 in combination with telaprevir and ribavirin in patients who failed to respond to pegylated IFN-a and ribavirin is currently recruiting study participants (NCT01872936). Santaris Pharma A/S, the developer of SPC3649, has also completed enrollment of a Phase 2 clinical trial of SPC3649 monotherapy in null responders to pegylated IFN-a and ribavirin (NCT01727934). All in vivo preclinical and clinical study results for SPC3649 monotherapy are summarized in Table 1. In addition to SPC3649, two more approaches have been made to develop anti-HCV therapeutics targeting miR-122 (Fig. 1). One is RG-101, an N-acetyl D-galactosamine (GalNAc)-conjugated anti-miR-122 oligonucleotide, developed by Regulus Therapeutics (presented at the 64th Annual Meeting of the American Association for the Study of Liver Disease; http://www.regulusrx.com). RG-101 uses GalNAc as a targeting moiety for uptake of an anti-miR122 oligonucleotide, since GalNAc can bind to asialoglycoprotein receptor on hepatocytes and enable targeting to hepatocytes. In efficacy studies using human liver chimeric
80
12.5–75
2.5–25
1–200
5
1, 3, 10
0, 3, 12, or 48
Mouse
Mouse
Mouse
Mouse
African green monkey
Cynomolgus monkey
Dosage (mg/kg)
Mouse
Preclinical
Species
SPC3649 (miravirsen)
SPC3649 (miravirsen)
SPC3649 (miravirsen)
15-mer LNAantimiR (SPC3649)
16-mer LNAantimiR, a high-affinity
miR-122 ASO
Antagomir-122
Drug modification
i.v. by a slow (5 min) bolus injection
i.v. infusion over about 10 min at a rate of 24 ml/kg/h
i.p.
i.p.
i.v.
i.p.
i.v.
Treatment route
Twice weekly for 9 times
Days 1, 3 and 5
Twice weekly for 6 weeks
3 times
3 consecutive days
Twice weekly for 4 weeks
1, 2, or 3 consecutive days
Treatment schedule
Table 1 Summary of in vivo preclinical and clinical studies with SPC3649
Maximal inhibition of miR-122 at 24 mg/ kg/week; extended recovery period to 12 weeks
Long-lasting depletion of mature miR-122 for 3 months
Sequestration of mature miR-122; derepression of the miR-122 targets, Aldoa and Bckdk
Sequestration of mature miR-122; threefold derepression of the direct miR122 target mRNA
Maximal reduction of miR-122 level at 24 h
Threefold to tenfold reduction in miR-122 level
23 days long effect (80 mg/kg 9 3)
Effectiveness
Dose-related decreases in total serum cholesterol; accumulation of basophilic granular materials and submucosal granular deposits; infiltrations of vacuolated macrophages; AST and ALT above ULN, pale discoloration of kidneys and livers, and increase in mean liver weights (96 mg/kg/week); shortlived prolongation of APTT and PT (24, 96 mg/kg/week)
Sustained decrease in total plasma cholesterol; no acute or subchronic toxicities
Sustained decrease in total cholesterol; no hepatotoxicity
Sustained decrease in total cholesterol with a median effective dose (ED50) of 10 mg/ kg
No hepatotoxicity in total 75 mg/ kg; reduction of plasma cholesterol level by *40 %
Mild increase of plasma transaminase level in 75 mg/kg; decrease in cholesterol and triglyceride levels
Decrease of cholesterol biosynthesis genes; decrease of cholesterol level by more than 40 %
Side effect
HildebrandtEriksen et al. (2012)
Elmen et al. (2008a)
Elmen et al. (2008a)
Elmen et al. (2008a)
Elmen et al. (2008b)
Esau et al. (2006)
Krutzfeldt et al. (2005)
References or clinical trials identifiers
Therapeutics for hepatitis C 301
123
123
7 (5 weeks); 5 (7 weeks)
Miravirsen
Miravirsen
Miravirsen
Miravirsen
SPC3649 (miravirsen)
Drug modification
s.c.
s.c.
s.c.
s.c.
i.v.
Treatment route
5 weekly doses over 5 weeks, followed by a further 4 doses once every other week over 7 weeks (12 weeks total)
150 mg/ml for 5 weekly doses over a 29-day period
5 weekly doses
Single ascending dose
A weekly basis for 12 weeks followed by 17 treatment-free weeks
Treatment schedule
N.A.
Dose-related reduction of HCV RNA level (3 mg/kg, 1.2 log; 5 mg/kg, 2.9 log; 7 mg/kg, 3.0 log from baselines); no escape mutations in the HCV genome
Dose-dependent pharmacology
Dose-dependent pharmacology
Sequestration of miR-122; a decline of HCV RNA in the serum and liver and maintenance of plasma level for 4 weeks after the last dose (5 mg/kg); no viral resistance
Effectiveness
N.A.
Non dose-dependent lowering of cholesterol; reductions in AST and ALT levels; headache, fatigue; no dose-limiting adverse events
Dose-dependent lowering of cholesterol; no doselimiting adverse events
Dose-dependent lowering of cholesterol; no doselimiting adverse events
Lowered serum cholesterol; spike in ALT (5 mg/kg); no other side-effects
Side effect
NCT01727934
Janssen et al. (2013); NCT01200420
NCT00979927
NCT00688012
Lanford et al. (2010)
References or clinical trials identifiers
i.v., intravenous; i.p., intraperitoneal; s.c., subcutaneous; antagomir, 30 cholesterol-conjugated 2-O-methyl oligonucleotide with phosphorothioate backbone; miR-122 ASO, 20 -O-methoxyethyl phosphorothioate antisense oligonucleotide; LNA-antimiR, locked-nucleic-acid-modified oligonucleotide with phosphorothioate backbone; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ULN, upper limit of the normal range; APTT, activated partial thromboplastin time; PT, prothrombin; N.A., not available
10 null responders to pegylated IFN-a plus ribavirin with chronic HCV
Phase 2
36 patients with chronic HCV genotype 1 infection
3, 5, or 7
5
30 healthy volunteers
Phase 2a
12
1 or 5
Dosage (mg/kg)
64 healthy man volunteers
Phase 1
Clinical
Chimpanzee
Species
Table 1 continued
302 J. Baek et al.
Therapeutics for hepatitis C
303
Fig. 1 MiR-122 targeting antiHCV therapeutics under investigation. a SPC3649 or miravirsen is a 15-mer LNAmodified oligonucleotide complementary to miR-122. SPC3649 has a full PS backbone and some LNAmodified sugars at the 50 - and 30 -ends to enhance pharmacokinetic properties and nuclease stability (uppercase, LNA-modified nucleotide; lowercase, unmodified nucleotide; m, methylation). b RG-101, a N-acetyl Dgalactosamine (GalNAc)conjugated anti-miR-122 oligonucleotide. c Small molecule-based miRNA inhibitors. Compound-1, 2,4dichloro-N-(naphthalen-2yl)benzamide; compound-2, 6-(((4aR,8aS)octahydroquinolin-1(2H)yl)sulfonyl)-1,2,3,4tetrahydroquinoline
mice, RG-101 significantly lowered serum viral titers in a dose-dependent manner. No toxic effects were found at doses up to 450 mg/kg in mice and 45 mg/kg in cynomolgus monkeys except for a mild increase in liver function test at 150 mg/kg. After completing preclinical studies, RG-101 is expected to begin testing in clinical trials soon. The other candidate therapeutics is small molecule-based miR-122 inhibitors. Using a luciferase reporter assay system in which binding of miR-122 to the 30 -UTR of luciferase gene downregulates luciferase signal, Young et al. (2010) screened 1,364 compounds and identified two compounds (compound-1 and compound-2) with miR-122 inhibiting activity. The compounds-1 and -2 decreased the expression of miR-122 with EC50 values of 3 and 0.6 lM, respectively, and caused a *50 % reduction
in viral RNA replication at 10 lM in Huh7 cells. The safety and efficacy of these compounds have not been yet assessed in animal models. However, considering the advantages of using small molecule-based drugs such as the easiness to manipulate for increasing stability, delivery, and pharmacokinetics, small molecule miR-122 inhibitors should have promising therapeutic potential for Hepatitis C (Young et al. 2010). MiR-122 targeting therapeutics including SPC3649 have several advantages over other anti-HCV drugs. Unlike most antivirals that directly target HCV, anti-miR-122 antivirals interact with host miRNA and can avoid the emergence of viral resistance that occurs by mutation of the HCV genome as observed in the studies of miravirsen. Notably, anti-miR-122 antivirals would be genotype
123
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independent, since miR-122 binding sites are highly conserved in all HCV genotypes. In addition, according to a recent report, SPC3649 can repress the biogenesis of miR122 by binding to the stem-loop structures of pri- and premiRNAs, although it has not been elucidated if the inhibition of miR-122 biogenesis would support the pharmacological activity of SPC3649 (Gebert et al. 2013). The natural anti-viral cytokine, IFN-b, can also function through miR-122 (Pedersen et al. 2007). It was reported that IFN- b treatment caused a significant decrease in miR122 expression, and that the antiviral effects of IFN-b were diminished by the addition of a miR-122 mimic. Apart from its well-known anti-viral mechanism such as the induction of protein kinase R for blocking viral protein synthesis and 20 -50 oligoadenylate synthetase for activating RNase L and degrading viral mRNA, it is interesting that IFN-b also exerts an anti-HCV function as a natural miR122 inhibitor. In summary, SPC3649 is an oligonucleotide based inhibitor of miR-122, a liver specific miRNA required for HCV replication. Due to its distinctive mechanism of action of targeting host factors, strong efficacy, and a good safety profile, this new therapeutic would hold promise for replacing the current standard of care or for being combined with current treatment regimens. SPC3649 is currently being evaluated in Phase 2 clinical trials. It is hoped that the first therapeutic applications of miRNA inhibition will emerge soon. Acknowledgments This work was supported by the Chung-Ang University Excellent Student Scholarship in 2012 and by the National Research Foundation (NRF), funded by the Ministry of Science, ICT, and Future Planning [NRF-2013R1A1A3005097 to H.M.]. Conflict of interest manuscript.
The authors have no conflict of interest in our
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