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Matrix Metalloproteases (MMPs) In Chronic Hepatitis C Infection

Our lead drug candidate (CTS-1027) is an oral small molecule that inhibits the activity of key members of a class of proteases, the matrix metalloproteases or MMPs. In the liver and in other solid organs, MMPs play an important role in regulating inflammation as well as in maintaining the integrity of the extracellular matrix. MMPs are an attractive mechanism to target in the setting of HCV:

An MMP inhibitor (MMPI) could have clinical benefit in any or all of these areas. In the clinic, the ultimate result of such interference would be an overall improvement of the host’s response to the virus, in a manner similar to that of interferon. In HCV infected patients, CTS-1027 treatment could lead to an impact on the second phase of viral clearance kinetics, coupled with a corresponding reduction in liver damage (measured by ALT and/or improvement in liver histology) as virus-infected cells turnover and are not replaced by newly infected cells. As is the case with interferon, this benefit would be expected to be most apparent in treatment naïve patients. An MMPI might also be able to prevent the infection of new cells. The attack on the virus, mediated by MMP inhibition, could complement and improve the efficiency of viral clearance when used in combination with existing approved drugs (peg-IFN-α and ribavirin) and/or new direct antiviral therapies as they become available.

MMPs in Inflammation and Fibrogenesis

MMPs are a well studied family of proteolytic enzymes generally known for their role in regulating the integrity of the extracellular matrix. However, MMP proteolytic activity is now well established to have a significantly broader role than previously appreciated1. It is clear that MMP activity is involved in the regulation of cell-cell interactions, cytokine and chemokine processing and cellular homeostasis. Numerous targets of MMP activity that directly affect components of the immune system inflammatory pathways have been described in a recent review by Opdenakker et al2.

MMPs are critically involved in fibrogenesis and the progression of fibrosis in the liver. For example, Brenner et al. reported that mice lacking MMP-13 are protected from damage in the bile duct ligation model of liver fibrosis3. While blockade of MMP activity has been shown to prevent fibrosis in other organs, this is the first publication showing a direct link between MMP activity in the liver and fibrosis.

CTS-1027 was also evaluated in the bile duct model of cholestasis where it demonstrated excellent protection. In this model, mice treated with CTS-1027 for 14 days had significantly less fibrogenesis (deposition of collagen) in addition to reduced bile infarcts, apoptotic cells, activated caspases and smooth muscle actin (α-SMA)4. Consistent with these in vivo results, CTS-1027 reduced activation of human hepatic stellate cells as indicated by a reduced expression of TGF-β, α-SMA, collagen type I and MMP-25 (more detail on preclinical activity of CTS-1027 is provided in CTS-1027 in Liver Disease and poster.

Potential Actions of MMPs on Host / Virus Interactions

Hepatitis C virus has evolved multiple and highly successful mechanisms to establish persistent infection. Of paramount importance is its ability to first evade the host’s innate immune system followed by its subversion of an effective adaptive immune response6. Hepatitis C virus is known to affect the regulation of MMP activity in chronic hepatitis C infection. Viral proteins have been shown to modulate transcription of various MMPs. As such, it appears that the virus has evolved the ability to modulate these proteases, presumably for the purpose of providing a survival advantage.

MMPs and Interferon

Type 1 interferons (IFN-α/-β) have been shown to directly modulate the transcription of MMPs. Benveniste et al demonstrated that transcriptional suppression of MMP-9 is mechanistically mediated through STAT-1α which is involved in the pathway of signal transduction through the IFN receptors7. In HCV, Giannelli et al demonstrated that IFN-α and ribavirin therapy reduces the expression of MMP-9 in the liver of chronic HCV patients8. The reduction of MMP-9 in liver biopsies was associated with positive clinical response to therapy, Figure 1. Expression of MMP-9 was significantly reduced in responder patients relative to pre-treatment levels, whereas the levels of MMP-9 were the same pre- and post-treatment in non-responders. MMP-9 has also been shown to cleave and inactivate IFN-α. Therefore, reduction in MMP-9 activity and expression may be of particular relevance as a component of treatment effectiveness with current standard of care.

Image: MMP-9 expression pre-(open bar) and post-(shaded bar) therapy.

Figure 1. MMP-9 expression pre-(open bar) and post-(shaded bar) therapy.

MMPs and the Immune Response

The processes leading to immune mediated clearance of HCV are complex and an area of intense investigation. While not completely understood, it is clear that viral double strand RNA (ds RNA) produced in infected hepatocytes initiates the immune response through a Toll-like receptor pathway that leads to the production of Type I IFNs (IFN-α and IFN-β), Figure 2. A complex array of IFN stimulated genes (ISG) (estimated to be over 300) are subsequently modulated, coordinating an overall response to combat infection9.

Image: HCV replication and initial immune response – induction of Type 1 IFNs

Figure 2. HCV replication and initial immune response – induction of Type 1 IFNs

The exact role that MMPs play in regulating the immune response in the setting of chronic HCV is unknown. However, given that HCV is known to increase MMP gene expression and activity, it is conceivable that at least one component may be in the regulation of chemokines involved in the immune response to HCV infection.

For example, the chemokines CXCL9, CXCL10 and CXCL11, along with their cognate receptor CXCR3, represent an important family of chemokines that play a key role in the immune response to HCV infection10. In response to IFN-α and TNF-α, these chemokines are expressed by hepatocytes, hepatic stellate cells and sinusoidal endothelial cells in HCV infected livers. CXCR3 chemokines are the most significantly expressed chemokines in chronic HCV11. MMP-9, which is up-regulated in chronic HCV, efficiently cleaves and deactivates CXCL912, CXCL10 and CXCL1113. This could result in reduced recruitment of CXCR3+ lymphocytes and consequently dampening of the immune response and CTL mediated inflammation. Additional MMPs, including MMP-8 and MMP-12, are also known to cleave CXCL9 and CXCL11 and reduce the functional ability of these chemokines. Thus, the ability of HCV to increase expression of MMPs and their subsequent action to reduce CTL mediated viral clearance may contribute to the immune tolerance specific to this virus. Therefore, MMP inhibition may have a beneficial impact in restoring this component in the host immune response to HCV.

Potential Effects on Viral Clearance

The effects described above for MMPs in the setting of chronic HCV show that MMP inhibition could lead to an enhancement of IFN activity. This could result in an increase in the loss rate of virus infected cells, and have an impact on the second phase of anti-viral kinetics. Effects of CTS-1027 in in vitro models of HCV replication and infection are presented in CTS-1027 for Liver Disease.

References

  1. Van Lint P, Libert C. Chemokine and cytokine processing by matrix metalloproteinases and its effect on leukocyte migration and inflammation. J. Leukoc. Biol. 2007, 82 (6): 1375-81.
  2. Cauwe B, Van den Steen PE, Opdenakker G. The biochemical, biological and pathological kaleidoscope of cell surface substrates processed by matrix metalloproteinases. Crit. Rev. Biochem. Mol. Biol. 2007, 42: 113-185.
  3. Uchinami H, Seki E, Brenner DA, D'Armiento J. Loss of MMP 13 attenuates murine hepatic injury and fibrosis during cholestasis. Hepatology. 2006 Aug; 44(2):420-9.
  4. Alisan K. DDW abstract #468, San Diego, May 2008.
  5. Conatus Pharmaceuticals Internal Report
  6. Wieland SF, Chisari FV. Stealth and Cunning: Hepatitis B and Hepatitis C Viruses. J. Virol. 2005, 79 (15): 9369-9380.
  7. Ma Z, Qin H, Benveniste EN. Transcriptional Suppression of Matrix Metalloproteinase-9 Gene Expression by IFN-α and IFN-β: Critical Role of STAT-1α The Journal of Immunology. 2001 167: 5150-5159.
  8. Marinosci F, Bergamini C, Fransvea E, Napoli N, Maurel P, Dentico P, Antonaci S, Giannelli, G. J. Interferon and Cytokine Research. 2005, 25: 453-458.
  9. Weber F. Interaction of the Hepatitis C Virus with the Type I Interferon System. World J. Gastroenterology. 2007, 13(36): 4818-4823.
  10. Heydtmann M, Adams, DH. Chemokines in the Immunopathogenesis of Hepatitis C Infection. Hepatology. 2009, 49 (2): 676-688.
  11. Helbig KJ, Ruszkiewicz A, Lanford RE, Berzsenyi MD, Harley HA, McColl SR, Beard MR. Differential Expression of the CXCR3 Ligands in Chronic Hepatitis C Virus (HCV) Infection and their Modulation by HCV in vitro. J. Virol. 2009, 83(2): 836-846.
  12. Van den Steen PE, Husson SJ, Proost P, Van Damme J, Opdenakker G. Carboxyterminal Cleavage of the Chemokines MIG and IP-10 by Gelatinase B and Neutrophil Collagenase. Biochem. Biophys. Res. Commun. 2003, 310: 889-896.
  13. Cox JH, Dean RA, Roberts CR, Overall CM. Matrix Metalloproteinase Processing of CXCL11/I-TAC Results in Loss of Chemoattractant Activity and Altered Glycosaminoglycan Binding. J. Biol. Chem. 2008, 283 (28):19389-99.

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