By Laura Macfarlane on 9 March 2011
Hepatitis C patients with advanced chronic liver disease are more likely to respond to peg interferon and ribavirin treatment if they are heavy coffee drinkers, research suggests.
Patients who drank more than three cups of coffee a day prior to treatment were three times more likely to have a virologic response to therapy compared with non-coffee drinkers, a study of almost 900 patients with advanced Hepatitis C found.
Three-quarters of these coffee drinkers had a significant drop in serum HCV RNA level by 12 weeks post-treatment, compared with only half of non-coffee drinkers.
Furthermore, one quarter of heavy coffee drinkers had a sustained virologic response (defined as the absence of detectable serum HCV RNA at week 72), compared with just 11% of non-coffee drinkers.
Although these associations were attenuated after adjusting for factors such as alcohol consumption, smoking and ethnicity, they remained significant.
No effect was observed for tea drinking, and the study did not distinguish between decaffeinated and caffeinated coffee drinkers in the analysis.
The authors said it was unlikely that coffee had a direct antiviral effect.
Rather, it was more likely that “coffee would have a facilitating effect on response to peginteferon and ribavirin by a mechanism yet to be understood,” they wrote.
Alternatively the association could also simply be due to chance, they said, adding further studies were needed.
Gastroenterology 2011 doi: 10.1053/j.gastro.2011.02.061
Source
March 10, 2011
Post-transcriptional inhibition of Hepatitis C Virus replication through small interference RNA
Published on: 2011-03-10
Hepatitis C Virus (HCV) infection is a major health problem throughout world that causes acute and chronic infection which resulted in liver fibrosis, hepatocellular carcinoma and death. The only therapy currently available for HCV infection is the combination of pegylated interferon alpha (PEG-IFN alpha) and ribavirin.
This therapy can effectively clear the virus infection in only 50% of infected individuals. Hence, there is a dire need to develop antiviral agents against HCV
Results: This study was design to examine the ability of exogenous small interfering RNAs (siRNAs) to block the replication of HCV in human liver cells.
In the present study six 21-bp siRNAs were designed against different regions of HCV non-structural genes (NS2, NS3 serine protease/helicase, NS4Band NS5B RNA dependent RNA polymerase). siRNAs were labeled as NS2si241, NS3si-229, NS3si-858, NS4Bsi-166, NS5Bsi-241 and NS5Bsi-1064.
We found that siRNAs against HCV NS2- NS5B efficiently inhibit HCV replication in Huh-7 cells. Our results demonstrated that siRNAs directed against HCV NS3 (NS3si-229 and NS3si-858) showed 58% and 88% reduction in viral titer respectively.
Moreover, NS4Bsi-166 and NS5Bsi-1064 exhibited a dramatic reduction in HCV viral RNA and resulted in greater than 90% inhibition at a 20 uM concentration, while NS2si-241 showed 27% reduction in viral titer. No significant inhibition was detected in cells transfected with the negative control siRNA.
Conclusion: Our results suggest that siRNAs targeting against HCV non-structural genes (NS2-NS5B) efficiently inhibit HCV replication and combination of these siRNAs of different targets and interferon will be better option to treat HCV infection throughout the world.
Author: Usman Ali AshfaqMuhammad AnsarMuhammad Tahir SarwarTariq JavedSidra RehmanSheikh Riazuddin
Credits/Source: Virology Journal 2011, 8:112
Source
Hepatitis C Virus (HCV) infection is a major health problem throughout world that causes acute and chronic infection which resulted in liver fibrosis, hepatocellular carcinoma and death. The only therapy currently available for HCV infection is the combination of pegylated interferon alpha (PEG-IFN alpha) and ribavirin.
This therapy can effectively clear the virus infection in only 50% of infected individuals. Hence, there is a dire need to develop antiviral agents against HCV
Results: This study was design to examine the ability of exogenous small interfering RNAs (siRNAs) to block the replication of HCV in human liver cells.
In the present study six 21-bp siRNAs were designed against different regions of HCV non-structural genes (NS2, NS3 serine protease/helicase, NS4Band NS5B RNA dependent RNA polymerase). siRNAs were labeled as NS2si241, NS3si-229, NS3si-858, NS4Bsi-166, NS5Bsi-241 and NS5Bsi-1064.
We found that siRNAs against HCV NS2- NS5B efficiently inhibit HCV replication in Huh-7 cells. Our results demonstrated that siRNAs directed against HCV NS3 (NS3si-229 and NS3si-858) showed 58% and 88% reduction in viral titer respectively.
Moreover, NS4Bsi-166 and NS5Bsi-1064 exhibited a dramatic reduction in HCV viral RNA and resulted in greater than 90% inhibition at a 20 uM concentration, while NS2si-241 showed 27% reduction in viral titer. No significant inhibition was detected in cells transfected with the negative control siRNA.
Conclusion: Our results suggest that siRNAs targeting against HCV non-structural genes (NS2-NS5B) efficiently inhibit HCV replication and combination of these siRNAs of different targets and interferon will be better option to treat HCV infection throughout the world.
Author: Usman Ali AshfaqMuhammad AnsarMuhammad Tahir SarwarTariq JavedSidra RehmanSheikh Riazuddin
Credits/Source: Virology Journal 2011, 8:112
Source
Biolex to Present Final Phase 2b Results at EASL for Locteron® Next-Generation Controlled-Release Interferon for HCV
Mar 10, 2011 01:00 ET
Presentations Will Highlight Strong Antiviral Activity and SVR Rates Together With Significant Reductions in Flu-Like Adverse Events and Reduced Rates of Depression
Significant Tolerability Advantages and a 50% Reduction in Dosing Frequency Support Locteron's Attractiveness for Use in New Triple and Quad Combination Regimens
PITTSBORO, NC--(Marketwire - March 10, 2011) - Biolex Therapeutics, Inc. announced today that final results from its SELECT-2 Phase 2b trial of Locteron® for the treatment of hepatitis C have been accepted for two presentations on March 31, 2011 at the 46th Annual Meeting of the European Association for the Study of the Liver (EASL) to be held in Berlin, Germany. Locteron, the only controlled-release interferon alpha, is designed to offer at least equal efficacy with key tolerability and dosing advantages over currently marketed interferons as a core component of new combination therapies for hepatitis C. Locteron's observed advantages include significant reductions in flu-like symptoms, reduced rates of depression, and a 50% less-frequent dosing regimen with once every other week dosing.
Final SELECT-2 study results will be presented for the first time at the EASL conference, including:
Interim SELECT-2 results were the subject of multiple presentations at the EASL conference in April 2010 and the Annual Meeting of the American Association for the Study of Liver Diseases (AASLD) conference in November 2010. The interim results supported the expected product profile for Locteron by demonstrating:
"Without a better tolerated interferon, these new triple and quad combinations will carry huge tolerability challenges that will limit access to care, reduce treatment intent rates and likely cause poor adherence in a real-world setting which we know lowers cure rates and raises the risk of multi-drug resistance," Turek further explained. "Our goal as we move into Phase 3 is to leverage Locteron's clear advantages in tolerability, combinability and dosing to position it along with the best direct-acting anti-virals to set the new standard in combination therapy for hepatitis C."
Locteron Overview
Locteron, controlled-release interferon alpha 2b, is designed to offer key advantages compared to currently approved products, including reduced flu-like symptoms and rates of depression, and cutting in half the number of injections required. In contrast to Locteron, the currently approved products, Pegasys® and PEG-Intron, are immediate-release products that lack a controlled-release mechanism. The two-drug combination of interferon alpha and ribavirin serves as the current standard of care for the treatment of hepatitis C. However, the launch of the first direct-acting anti-viral (DAA) product, projected to occur this year, will transform treatment of genotype-1 patients to a triple-drug therapy (interferon plus ribavirin plus DAA) and substantially raise cure rates. Other recent triple or quad drug combinations with interferon (including interferon plus ribavirin plus two DAA agents) have shown promise in early clinical testing, further solidifying the continued role of interferon in the treatment of hepatitis C. It is estimated that worldwide sales of interferon products for the treatment of hepatitis C will approach $6 billion by 2016.
Locteron incorporates an advanced controlled-release drug delivery technology that allows dosing once every two weeks. This is considerably more convenient than Pegasys and PEG-Intron, each of which requires dosing every week. More importantly, Locteron's controlled-release mechanism results in the gradual release of interferon alpha 2b to patients over the duration of two weeks and avoids the early peak plasma levels of the active interferon that characterize the pegylated interferons. This controlled-release mechanism is designed to reduce the frequency and severity of flu-like symptoms and depression commonly experienced by patients treated with pegylated interferons.
Locteron is an investigational therapeutic candidate and has not been approved for sale by the United States Food and Drug Administration or by any international regulatory agency.
About Biolex Therapeutics
Biolex is a biopharmaceutical company that uses its patented LEX System(SM) to develop follow-on biologics, hard-to-make therapeutic proteins and to optimize monoclonal antibodies. The LEX System is a novel technology that genetically transforms the aquatic plant Lemna to enable the production of biologic product candidates. The company's product candidates are designed to provide best-in-class efficacy/tolerability profiles while incorporating proven mechanisms of action. Biolex's lead product candidate, Locteron®, has completed two Phase 2b clinical trials for the treatment of chronic hepatitis C. Biolex has also developed two other product candidates that capitalize on the benefits of the LEX System. BLX-155 is a direct-acting thrombolytic designed to dissolve blood clots in patients. BLX-301 is a humanized anti-CD20 antibody glyco-optimized for the treatment of non- Hodgkin's B-cell lymphoma and other diseases.
Contacts:
Media:
Tim Brons
Vida Communication
415-675-7402
Email Contact
Investors:
Dale Sander
Chief Financial Officer
858-663-6993
Email Contact
Source
Presentations Will Highlight Strong Antiviral Activity and SVR Rates Together With Significant Reductions in Flu-Like Adverse Events and Reduced Rates of Depression
Significant Tolerability Advantages and a 50% Reduction in Dosing Frequency Support Locteron's Attractiveness for Use in New Triple and Quad Combination Regimens
PITTSBORO, NC--(Marketwire - March 10, 2011) - Biolex Therapeutics, Inc. announced today that final results from its SELECT-2 Phase 2b trial of Locteron® for the treatment of hepatitis C have been accepted for two presentations on March 31, 2011 at the 46th Annual Meeting of the European Association for the Study of the Liver (EASL) to be held in Berlin, Germany. Locteron, the only controlled-release interferon alpha, is designed to offer at least equal efficacy with key tolerability and dosing advantages over currently marketed interferons as a core component of new combination therapies for hepatitis C. Locteron's observed advantages include significant reductions in flu-like symptoms, reduced rates of depression, and a 50% less-frequent dosing regimen with once every other week dosing.
Final SELECT-2 study results will be presented for the first time at the EASL conference, including:
- Sustained virologic response (SVR) rates at completion of the trial (week 72), as well as final tolerability comparison results using both traditional clinic visit data and electronic patient reported outcome measures.
- Timing and frequency of depression during the 48 weeks of treatment in SELECT-2. Depression is a serious medical condition that requires careful monitoring during hepatitis C treatment. Favorable Locteron results related to depression rates will be highlighted in a separate presentation during the EASL conference.
Interim SELECT-2 results were the subject of multiple presentations at the EASL conference in April 2010 and the Annual Meeting of the American Association for the Study of Liver Diseases (AASLD) conference in November 2010. The interim results supported the expected product profile for Locteron by demonstrating:
- Viral response rates for all Locteron doses that were comparable with or exceeded the response rate for the control group, achieved with half as many injections of Locteron versus the control;
- Statistically and clinically significant reductions in flu-like adverse events for the Locteron cohorts compared to the control group, as high as 60+% reductions;
- Substantially reduced use of concomitant medications (analgesics and antipyretics) for the Locteron cohorts compared to the control group;
- Substantially lower rates of depression for the two Locteron cohorts comprising the expected commercial dose range compared to the control group;
- Lower discontinuations due to adverse events for the two Locteron cohorts comprising the expected commercial dose range compared to the control group.
"Without a better tolerated interferon, these new triple and quad combinations will carry huge tolerability challenges that will limit access to care, reduce treatment intent rates and likely cause poor adherence in a real-world setting which we know lowers cure rates and raises the risk of multi-drug resistance," Turek further explained. "Our goal as we move into Phase 3 is to leverage Locteron's clear advantages in tolerability, combinability and dosing to position it along with the best direct-acting anti-virals to set the new standard in combination therapy for hepatitis C."
Locteron Overview
Locteron, controlled-release interferon alpha 2b, is designed to offer key advantages compared to currently approved products, including reduced flu-like symptoms and rates of depression, and cutting in half the number of injections required. In contrast to Locteron, the currently approved products, Pegasys® and PEG-Intron, are immediate-release products that lack a controlled-release mechanism. The two-drug combination of interferon alpha and ribavirin serves as the current standard of care for the treatment of hepatitis C. However, the launch of the first direct-acting anti-viral (DAA) product, projected to occur this year, will transform treatment of genotype-1 patients to a triple-drug therapy (interferon plus ribavirin plus DAA) and substantially raise cure rates. Other recent triple or quad drug combinations with interferon (including interferon plus ribavirin plus two DAA agents) have shown promise in early clinical testing, further solidifying the continued role of interferon in the treatment of hepatitis C. It is estimated that worldwide sales of interferon products for the treatment of hepatitis C will approach $6 billion by 2016.
Locteron incorporates an advanced controlled-release drug delivery technology that allows dosing once every two weeks. This is considerably more convenient than Pegasys and PEG-Intron, each of which requires dosing every week. More importantly, Locteron's controlled-release mechanism results in the gradual release of interferon alpha 2b to patients over the duration of two weeks and avoids the early peak plasma levels of the active interferon that characterize the pegylated interferons. This controlled-release mechanism is designed to reduce the frequency and severity of flu-like symptoms and depression commonly experienced by patients treated with pegylated interferons.
Locteron is an investigational therapeutic candidate and has not been approved for sale by the United States Food and Drug Administration or by any international regulatory agency.
About Biolex Therapeutics
Biolex is a biopharmaceutical company that uses its patented LEX System(SM) to develop follow-on biologics, hard-to-make therapeutic proteins and to optimize monoclonal antibodies. The LEX System is a novel technology that genetically transforms the aquatic plant Lemna to enable the production of biologic product candidates. The company's product candidates are designed to provide best-in-class efficacy/tolerability profiles while incorporating proven mechanisms of action. Biolex's lead product candidate, Locteron®, has completed two Phase 2b clinical trials for the treatment of chronic hepatitis C. Biolex has also developed two other product candidates that capitalize on the benefits of the LEX System. BLX-155 is a direct-acting thrombolytic designed to dissolve blood clots in patients. BLX-301 is a humanized anti-CD20 antibody glyco-optimized for the treatment of non- Hodgkin's B-cell lymphoma and other diseases.
Contacts:
Media:
Tim Brons
Vida Communication
415-675-7402
Email Contact
Investors:
Dale Sander
Chief Financial Officer
858-663-6993
Email Contact
Source
An unusually high cure rate recorded after ChronVac-C® vaccination followed by standard treatment in patients with genotype 1 chronic hepatitis C
10 Mar, 2011 07:56 CET
Of six patients with genotype 1 chronic hepatitis C virus infection treated with standard of care treatment following participation in a clinical trial with four ChronVac-C® vaccinations, five have been cured, i.e. are virus free six months after completed treatment. These are unusually good treatment results for patients carrying the hard-to-treat variant of hepatitis C-virus called genotype 1.
As was previously reported seven patients chronically infected with hepatitis virus-C (HCV) of genotype 1 who participated in the now completed ChronVac-C® study have received treatment with standard-of-care for chronic infection with HCV, i.e. interferon combined with ribavirin. Five of seven of the patients responded rapidly on the treatment with <50 virus copies/mL blood already after four weeks (so called rapid viral response), and 6/7 were virus free already by week 12. These good results were maintained at a later follow up during the treatment and now it can be concluded that 5/6 of the patients who have completed their treatment have been cured from their chronic HCV infection. Usually approximately 40-45% of the patients with genotype 1 are cured by standard-of-care. New treatments with the additions of so called protease inhibitors cure 60-70% of the treated patients. All in all, the results for ChronVac-C® in combination with standard-of-care treatment are very good.
“Although the number of patients is very small, we are cautiously optimistic. This encouraging data actually match these received with the coming protease inhibitor therapies. We look forward to the continued clinical development of ChronVac-C®”, says Anders Vahlne, CEO of ChronTech Pharma AB.
For more information, please contact:
Anders Vahlne, CEO and Head of Research, ChronTech Pharma AB
Tel: +46 8 5858 1313, mobile phone: +46 709 28 05 28,
E-mail: anders.vahlne@ki.se
About ChronTech
ChronTech develops the therapeutic DNA vaccines ChronVac-C® and ChronVac-B drugs against chronic hepatitis C virus and hepatitis B virus infections, i.e. chronic infections with jaundice causing viruses which can lead to liver cirrhosis and liver cancer. ChronTech has also developed and further develops a patent pending new type of injection needle for a more effective uptake of DNA vaccines. ChronTech also have part ownership in the wound healing therapy ChronSeal®, and in the new platform technology RAS®. The ChronTech share is admitted to trade on First North. Remium AB is Certified Adviser for ChronTech. For more information, please visit: www.chrontech.se
In the event of any discrepancy between the Swedish and English versions of this press release, the Swedish version will take precedence.
Source
Of six patients with genotype 1 chronic hepatitis C virus infection treated with standard of care treatment following participation in a clinical trial with four ChronVac-C® vaccinations, five have been cured, i.e. are virus free six months after completed treatment. These are unusually good treatment results for patients carrying the hard-to-treat variant of hepatitis C-virus called genotype 1.
As was previously reported seven patients chronically infected with hepatitis virus-C (HCV) of genotype 1 who participated in the now completed ChronVac-C® study have received treatment with standard-of-care for chronic infection with HCV, i.e. interferon combined with ribavirin. Five of seven of the patients responded rapidly on the treatment with <50 virus copies/mL blood already after four weeks (so called rapid viral response), and 6/7 were virus free already by week 12. These good results were maintained at a later follow up during the treatment and now it can be concluded that 5/6 of the patients who have completed their treatment have been cured from their chronic HCV infection. Usually approximately 40-45% of the patients with genotype 1 are cured by standard-of-care. New treatments with the additions of so called protease inhibitors cure 60-70% of the treated patients. All in all, the results for ChronVac-C® in combination with standard-of-care treatment are very good.
“Although the number of patients is very small, we are cautiously optimistic. This encouraging data actually match these received with the coming protease inhibitor therapies. We look forward to the continued clinical development of ChronVac-C®”, says Anders Vahlne, CEO of ChronTech Pharma AB.
For more information, please contact:
Anders Vahlne, CEO and Head of Research, ChronTech Pharma AB
Tel: +46 8 5858 1313, mobile phone: +46 709 28 05 28,
E-mail: anders.vahlne@ki.se
About ChronTech
ChronTech develops the therapeutic DNA vaccines ChronVac-C® and ChronVac-B drugs against chronic hepatitis C virus and hepatitis B virus infections, i.e. chronic infections with jaundice causing viruses which can lead to liver cirrhosis and liver cancer. ChronTech has also developed and further develops a patent pending new type of injection needle for a more effective uptake of DNA vaccines. ChronTech also have part ownership in the wound healing therapy ChronSeal®, and in the new platform technology RAS®. The ChronTech share is admitted to trade on First North. Remium AB is Certified Adviser for ChronTech. For more information, please visit: www.chrontech.se
In the event of any discrepancy between the Swedish and English versions of this press release, the Swedish version will take precedence.
Source
Novel Method Could Improve the Performance of Proteins Used Therapeutically
ScienceDaily (Mar. 10, 2011) — Whitehead Institute scientists have created a method that site-specifically modifies proteins to exert control over their properties when administered therapeutically. The technique should be useful to increase potency, slow metabolism, and improve thermal stability of therapeutically useful proteins, such as interferon alpha 2 (IFN-alpha 2), which is used to treat variety of diseases, including leukemia, melanoma, and chronic hepatitis C.
The method, reported this month in Proceedings of the National Academy of Sciences (PNAS), uses the enzyme sortase A and can be applied to tailor proteins that possess a structure found in IFN-alpha 2, referred to as a four-helix bundle. Such proteins include erythropoietin (EPO), granulocyte colony-stimulating factor 3 (GCSF-3, known as filgrastim and marketed as Neupogen®), interleukin (IL) 2 (known as aldesleukin and marketed as Proleukin®), IL-4, IL-7, IL9-, and IL-15.
"In the course of this work, the first author of the PNAS paper, Maximilian Popp, together with other members of the lab, has put together a nice palette of sortase-based techniques that now allow us to modify a large variety of different proteins, and equip them with properties and behaviors that cannot be easily specified by more standard molecular biological techniques," says Whitehead Member Hidde Ploegh. "I see the value of these approaches first and foremost in their general applicability and ease of use."
IFN-alpha 2 is a cytokine, a hormone-like substance that usually acts on cells other than those that produce the protein. Upon binding the cytokine, the recipient cell responds, for example by starting to divide and proliferate, or by exercising certain functions of benefit to the organism. Like other cytokines used for therapeutic purposes, IFN-alpha 2 can be a finicky drug. It is thermally unstable and must be continuously refrigerated to maintain its potency, a requirement that limits IFN-alpha 2's use in areas with intermittent or no electricity. Also, IFN-alpha 2's relatively short half-life (and resulting rapid clearance from the body) often necessitates frequent injections when the drug is used to treat certain conditions.
To keep therapeutic IFN-alpha 2 active in the body longer, the current strategy is to tack long polyethylene glycol (PEG) chains onto the protein to turn them into effective drugs. This so-called PEGylation not only masks IFN-alpha 2 from the patient's immune system but also increases the time the body needs to break it down. However, because current approaches to PEGylation are not specific, the PEG chains can block or alter the protein's normal binding site -- an unintended consequence of this modification that can diminish IFN-alpha 2's potency by as much as 90%.
Seeking greater precision in attaching PEG chains, Popp, who is a graduate student in the Ploegh lab, used the enzyme sortase A to cleave IFN-alpha 2 at a specific site on the protein, engineered so that it would be recognized by the sortase. Then, a small molecule bearing the PEG chain was attached at the site cleaved by sortase. When Popp tested for biological activity, the resulting IFN-alpha 2 was highly potent, indicating that the PEG chains were not interfering with the drug's binding ability.
Popp also used sortase A to suture PEG chains to the cytokine GCSF-3. When he tested the PEGylated version in mice, it remained in the bloodstream significantly longer and evoked a more robust and prolonged response than a non-PEGylated version. By using sortase A's inherent precision to attach PEG chains, Popp could replace the less precise chemistry-based technique with a highly effective method that should have broader applications.
Next, Popp addressed IFN-alpha 2's thermal stability. Previously, the Ploegh lab stabilized linear polypeptides like IFN-alpha 2 by molecularly gluing their ends together to form circles. A few such cyclic proteins are found in nature. Once circularized, cyclic proteins are often more stable than their linear precursors. This forced looping can interfere with the function of some cell-signaling proteins, but because IFN-alpha 2's binding site is not near its ends, the function of IFN-alpha 2 is unaffected when its ends are joined to form a circle.
To create a cyclic version of IFN-alpha 2, Popp used sortase A to join the two ends of IFN-alpha 2. When he heated the cyclic form of IFN-alpha 2, it was more resistant to breakdown than its linear counterpart and remained biologically potent even after boiling. Popp then tested the circular, PEGylated version and the linear version in mice. The modified version was metabolized more slowly than the linear version and maintained its thermal stability, demonstrating that this simple technique can significantly enhance desirable properties of a therapeutically relevant protein without sacrificing its potency.
"We really take advantage of the site specificity of the sortase enzyme. Placing a molecular suture like that can't really be done by other means. So I think this method is of value to the protein engineering field in general," says Popp. "The reaction itself is easy, but it took some time to actually figure out how to do these transformations. Once we figured that out, the technique was robust and reproducible."
This research was supported by the National Institutes of Health (NIH).
1. Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142 2. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02142
Source
The method, reported this month in Proceedings of the National Academy of Sciences (PNAS), uses the enzyme sortase A and can be applied to tailor proteins that possess a structure found in IFN-alpha 2, referred to as a four-helix bundle. Such proteins include erythropoietin (EPO), granulocyte colony-stimulating factor 3 (GCSF-3, known as filgrastim and marketed as Neupogen®), interleukin (IL) 2 (known as aldesleukin and marketed as Proleukin®), IL-4, IL-7, IL9-, and IL-15.
"In the course of this work, the first author of the PNAS paper, Maximilian Popp, together with other members of the lab, has put together a nice palette of sortase-based techniques that now allow us to modify a large variety of different proteins, and equip them with properties and behaviors that cannot be easily specified by more standard molecular biological techniques," says Whitehead Member Hidde Ploegh. "I see the value of these approaches first and foremost in their general applicability and ease of use."
IFN-alpha 2 is a cytokine, a hormone-like substance that usually acts on cells other than those that produce the protein. Upon binding the cytokine, the recipient cell responds, for example by starting to divide and proliferate, or by exercising certain functions of benefit to the organism. Like other cytokines used for therapeutic purposes, IFN-alpha 2 can be a finicky drug. It is thermally unstable and must be continuously refrigerated to maintain its potency, a requirement that limits IFN-alpha 2's use in areas with intermittent or no electricity. Also, IFN-alpha 2's relatively short half-life (and resulting rapid clearance from the body) often necessitates frequent injections when the drug is used to treat certain conditions.
To keep therapeutic IFN-alpha 2 active in the body longer, the current strategy is to tack long polyethylene glycol (PEG) chains onto the protein to turn them into effective drugs. This so-called PEGylation not only masks IFN-alpha 2 from the patient's immune system but also increases the time the body needs to break it down. However, because current approaches to PEGylation are not specific, the PEG chains can block or alter the protein's normal binding site -- an unintended consequence of this modification that can diminish IFN-alpha 2's potency by as much as 90%.
Seeking greater precision in attaching PEG chains, Popp, who is a graduate student in the Ploegh lab, used the enzyme sortase A to cleave IFN-alpha 2 at a specific site on the protein, engineered so that it would be recognized by the sortase. Then, a small molecule bearing the PEG chain was attached at the site cleaved by sortase. When Popp tested for biological activity, the resulting IFN-alpha 2 was highly potent, indicating that the PEG chains were not interfering with the drug's binding ability.
Popp also used sortase A to suture PEG chains to the cytokine GCSF-3. When he tested the PEGylated version in mice, it remained in the bloodstream significantly longer and evoked a more robust and prolonged response than a non-PEGylated version. By using sortase A's inherent precision to attach PEG chains, Popp could replace the less precise chemistry-based technique with a highly effective method that should have broader applications.
Next, Popp addressed IFN-alpha 2's thermal stability. Previously, the Ploegh lab stabilized linear polypeptides like IFN-alpha 2 by molecularly gluing their ends together to form circles. A few such cyclic proteins are found in nature. Once circularized, cyclic proteins are often more stable than their linear precursors. This forced looping can interfere with the function of some cell-signaling proteins, but because IFN-alpha 2's binding site is not near its ends, the function of IFN-alpha 2 is unaffected when its ends are joined to form a circle.
To create a cyclic version of IFN-alpha 2, Popp used sortase A to join the two ends of IFN-alpha 2. When he heated the cyclic form of IFN-alpha 2, it was more resistant to breakdown than its linear counterpart and remained biologically potent even after boiling. Popp then tested the circular, PEGylated version and the linear version in mice. The modified version was metabolized more slowly than the linear version and maintained its thermal stability, demonstrating that this simple technique can significantly enhance desirable properties of a therapeutically relevant protein without sacrificing its potency.
"We really take advantage of the site specificity of the sortase enzyme. Placing a molecular suture like that can't really be done by other means. So I think this method is of value to the protein engineering field in general," says Popp. "The reaction itself is easy, but it took some time to actually figure out how to do these transformations. Once we figured that out, the technique was robust and reproducible."
This research was supported by the National Institutes of Health (NIH).
1. Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142 2. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02142
Source
Biotron Completes First Part of Phase 2 Trial
Drug Discovery & Development - March 10, 2011
Australian drug development company Biotron Limited has completed stage one of a landmark human trial of its lead Hepatitis C drug candidate, BIT225.
Twelve patients have been dosed in the first half of the trial being undertaken by ACLIRES, an international contract research organisation (CRO) that specialises in running antiviral drug clinical trials. All are infected with the most common strain of the Hepatitis C virus, genotype 1.
The second half of the trial - which also involves 12 patients - is now underway and expected to be completed in May, with results anticipated to be analysed and released in June. The trial is blinded, so no results are available until samples are analysed at the conclusion of the trial and the data is unblinded.
Biotron CEO, Dr Michelle Miller, said the trial was proceeding as expected and the results would be "of international interest".
She said BIT225 was a first-in-class drug candidate which specifically targeted the p7 protein, a viral protein essential to virus production and replication.
"We are happy with how the trial is progressing. We achieved the necessary ethics and drug import approvals to move to the second stage of the trial, which is now underway."
Twelve further patients are now being recruited and dosed over four weeks with BIT225.
As in stage one of the study, one-third of patients are being given a placebo, one-third a dose of 400mg BIT225 and another one -third are being dosed at 200mg BIT225.
This Phase 2 trial is examining how BIT225 works in combination with current approved treatments for HCV, Interferon and Ribavarin.
Existing drugs have limited effectiveness and can be toxic. Doctors say fifty per cent of sufferers do not respond to current therapies, signalling a need for new treatments that directly target and halt replication and reproduction of the virus.
Date: March 9, 2011
Source: Biotron Limited www.biotron.com.au/
Source
Australian drug development company Biotron Limited has completed stage one of a landmark human trial of its lead Hepatitis C drug candidate, BIT225.
Twelve patients have been dosed in the first half of the trial being undertaken by ACLIRES, an international contract research organisation (CRO) that specialises in running antiviral drug clinical trials. All are infected with the most common strain of the Hepatitis C virus, genotype 1.
The second half of the trial - which also involves 12 patients - is now underway and expected to be completed in May, with results anticipated to be analysed and released in June. The trial is blinded, so no results are available until samples are analysed at the conclusion of the trial and the data is unblinded.
Biotron CEO, Dr Michelle Miller, said the trial was proceeding as expected and the results would be "of international interest".
She said BIT225 was a first-in-class drug candidate which specifically targeted the p7 protein, a viral protein essential to virus production and replication.
"We are happy with how the trial is progressing. We achieved the necessary ethics and drug import approvals to move to the second stage of the trial, which is now underway."
Twelve further patients are now being recruited and dosed over four weeks with BIT225.
As in stage one of the study, one-third of patients are being given a placebo, one-third a dose of 400mg BIT225 and another one -third are being dosed at 200mg BIT225.
This Phase 2 trial is examining how BIT225 works in combination with current approved treatments for HCV, Interferon and Ribavarin.
Existing drugs have limited effectiveness and can be toxic. Doctors say fifty per cent of sufferers do not respond to current therapies, signalling a need for new treatments that directly target and halt replication and reproduction of the virus.
Date: March 9, 2011
Source: Biotron Limited www.biotron.com.au/
Source
March 9, 2011
IL28B Genomic-Based Treatment Paradigms for Patients With Chronic Hepatitis C Infection: The Future of Personalized HCV Therapies
Download the PDF here
Am J Gastroenterol Jan 2011
Paul J Clark MD1, Alex J Thompson MD, PhD1 and John G McHutchison MD1 1Duke Clinical Research Institute and Division of Gastroenterology, Duke University Medical Center, School of Medicine, Duke University, Durham, North Carolina, USA. Correspondence: Paul J. Clark, MD, Division of Gastroenterology, Duke Clinical Research Institute, Duke University Medical Center, PO Box 17969, Durham, North Carolina 27715, USA. E-mail: paul.clark@duke.edu
from Jules of NATAP: We are unsure right now exactly how & to what extent IL28B will be used, we are awaiting further outcomes analyses from the recently reported studies of boceprevir & telaprevir (the new oraly administered protease inhibitors that will be used in combination with peg/rbv). Patients may be "profiled" based on IL28B genotype and other important clinical features (such as fibrosis, age, insulin resistance, viral load, and race) to better predict treatment response (Table 1). Host IL28B genotype is the strongest pretreatment predictor of response through its effect on viral kinetics.....Genotyping of this polymorphism will aid clinical decision making for both current standard of care and potentially for the integration of other agents in the future
'Genetic testing [Genome-wide association studies (GWAS)] have recently identified host genetic variation to be critical for predicting treatment response and spontaneous clearance in patients infected with hepatitis C virus (HCV)......A commercial test is now available for IL28B.....IL28B polymorphism (rs12979860) was the strongest predictor of SVR compared with all other baseline host and viral variables
........The IL28B polymorphism rs12979860 has a marked differential distribution between racial groups, being least frequent in African-Americans, most frequent in Asians, and with an intermediate frequency in Hispanics and Caucasians .....The best response is in patients with C/C gene, when genotype 1 mono or coinfected patients with C/C are treated with peg/rbv they have DOUBLE to TRIPLE the response rate compared to patients with C/C or C/T: 16% of AAs had CC & 53% of these had SVR; 39% of whites had CC & 82% had SVR; among AAs 48% had C/T & 19% had SVR, 37% had T/T & 17% had SVR; among whites 50% had C/T & 42% had SVR, 12% had T/T & 33% had SVR).....there are many other factors that have predicted a good or bad response to peg/rbv including pretreatment viral load, week 4 viral load response to treatment, week 12 viral load response to treatment, degree of fibrosis, body weight, gender, insulin resistance, fatty liver'
What do these findings mean for clinicians managing patients with chronic hepatitis C?
A commercial test is now available for IL28B, but how should this information be used to improve care for patients? Patients may be "profiled" based on IL28B genotype and other important clinical features (such as fibrosis, age, insulin resistance, viral load, and race) to better predict treatment response (Table 1). Host IL28B genotype is the strongest pretreatment predictor of response through its effect on viral kinetics (33). On-treatment viral kinetics (currently RVR or early viral response, but potentially even earlier measures) provides direct measurement of treatment response and remains the most powerful on-treatment response predictor and the key criteria for on-treatment therapy decisions. Importantly, host genotype also adds to current response-guided decision algorithms. The IL28B CC genotype identifies a subgroup of patients without RVR who are more likely to achieve SVR. When new DAAV agents in development become available, such profiles may also help both in the choice of treatment regimen and its anticipated duration, personalizing therapy further.
Table 1 - Host IL28B genotype and other important patient characteristics will be used to develop patient profiles to help predict response and potentially tailor therapy.
Clinical message: Patient profiles which integrate IL28B genotype and other important clinical variables will be used to help predict response and personalize therapy before commencement. As the number of therapeutic options expands in coming years, this may help to simplify and optimize treatment algorithms.
Clinical message: Patients with genotype 1 who do not achieve RVR but carry favorable IL28B type have a 65% chance of cure.
Current standard of care therapy with pegIFN/RBV provides excellent results in patients with genotype 1 HCV and the favorable host IL28B genotype.
At the bedside, knowledge of this host genotype will translate into greater confidence in terms of counseling patients before commencing treatment. Discussions with a patient about an 80% likelihood of treatment response versus a 30% response rate are very different conversations and akin to discussions we currently have with patients about the impact of ethnicity or viral genotype on predicted treatment response. When on treatment, host IL28B genotype knowledge may assist clinicians and patients to "stay the course" when troubled by side effects that may or may not require dose reductions, by the knowledge they have more than simply a "50/50" chance of success in favorable responder genotype patients.
Clinical message: Patients with a favorable patient profile (e.g., C/C IL28B genotype) are likely to respond to current standard of care therapy. This knowledge changes the "cost/benefit" of treatment and may help to encourage patients to commence treatment and to reassure them during a long and often difficult treatment course.
PegIFN/RBV therapy alone avoids the selection for point-specific HCV mutations that may lead to drug resistance when treating with DAAV agents. Existing treatment regimens also avoid exposure to the potential of further drug side effects and toxicity that may be observed with new agents. Finally, existing combination therapy may prove more cost-effective in those most likely to respond, relative to simply adding DAAV onto existing regimens.
Clinical message: Although current standard of care therapy has its drawbacks, for patients with a high likelihood of response (e.g., C/C genotype) it avoids many of the additional problems and costs of future pegIFN/RBV+DAAV combination regimens.
On the basis of available data, patients with unfavorable responder genotype should probably be categorized as "difficult to treat" in the same way clinicians consider other negative treatment prognostic criteria (e.g., advanced fibrosis). It seems likely that the addition of directly acting antiviral therapy may attenuate the effect of host IL28B genotype on SVR; however, further studies are needed (40,41). For patients who have the unfavorable IL28B genotype, it may be prudent to defer treatment until future regimens that include directly acting antiviral agents become available in 2011-12.
Clinical message: Patients with an unfavorable response profile (e.g., non-C/C genotype) are most likely to benefit from improved SVR rates with regimens that include DAAVs, and they should probably wait for such therapies.
How knowledge of a patient's IL28B genotype will alter treatment algorithms is unknown. Patients with the favorable IL28B C/C genotype may achieve adequate SVR rates with a reduced duration of current standard of care (e.g., 24 weeks or less compared with the current 48 weeks). Alternatively, in C/C genotype patients the addition of DAAV therapy may provide a pathway to even further reduce the duration of treatment. For patients with less favorable IL28B genotype efficacy issues prevail. The effect of extending pegIFN/RBV treatment duration or adding a third or fourth agent on SVR rates needs to be studied. These possible strategies should be assessed prospectively in randomized controlled trials stratified to IL28B type.
Clinical message: Host IL28B genotype may allow for shortened treatment regimens in favorable C/C genotype patients, particularly with regimens including DAAVs.
Review of existing trial data is now required where possible to determine whether knowledge of host genotype alters the interpretation of a study. Stratification for host IL28B genotype to avoid imbalance in treatment arms is of particular concern for early phase trials with small sample sizes. Ongoing trials involving IFN-λ, other developing immunomodulator therapies and directly acting antivirals are awaited with interest.
Clinical message: The genetic signal of this discovery is strong and consistent and establishes the biological principle for further clinical research. More data are now required to fully explore and understand the implications in different clinical scenarios.
ABSTRACT
Genome-wide association studies (GWAS) have recently identified host genetic variation to be critical for predicting treatment response and spontaneous clearance in patients infected with hepatitis C virus (HCV). These important new studies are reviewed and their future clinical implications discussed. Single-nucleotide polymorphisms in the region of the IL28B gene on chromosome 19, coding for the interferon (IFN)-λ-3 or IL28B gene, are strongly associated with treatment response to pegylated IFN and ribavirin in patients infected with genotype 1 HCV. The good response variant is associated with a twofold increase in the rate of cure. Allele frequencies differ between ethnic groups, largely explaining the observed differences in response rates between Caucasians, African Americans and Asians. IL28B polymorphism is also strongly associated with spontaneous clearance of HCV. The biological mechanisms responsible for these genetic associations remain unknown and are the focus of ongoing research. Knowledge of a patient's IL28B genotype is likely to aid in clinical decision making with standard of care regimens. Future studies will investigate the possibility of individualizing treatment duration and novel regimens according to IL28B type.
INTRODUCTION
The unraveling of the human genome offers the promise of personalized medicine. In this review, we briefly define some critical genetic concepts for clinicians, and overview genome-wide association studies (GWAS). Readers are directed to excellent genetics reviews for further reading (1,2,3). We review the recent GWAS discovery, which has shown that host genetics drives treatment response to pegylated interferon-α and ribavirin (pegIFN/RIB) for patients infected with genotype 1 hepatitis C virus (HCV) (4,5,6). We also review recent studies that have shown that the same polymorphisms are associated with spontaneous clearance of HCV (7,8).
"Patients in the first and largest GWAS (Genome-wide association studies) were enrolled in the IDEAL trial, a randomized control trial comparing different doses of pegIFNα-2b/RBV with peg-IFNα-2a/RBV (21)........Overall, 1,137 patients were included in the genetic association analyses for SVR in three separate ethnic populations, defined by genetic ancestry (Caucasian, Hispanic, and African American (AA))......Regression modeling found that the IL28B polymorphism (rs12979860) was the strongest predictor of SVR compared with all other baseline host and viral variables
The IL28B polymorphism rs12979860 has a marked differential distribution between racial groups, being least frequent in AAs, most frequent in Asians, and with an intermediate frequency in Hispanics and Caucasians .....An intention-to-treat analysis was performed that included all patients, regardless of adherence, and classifying ethnicity by self-report as it would be in practice. Again, the good response variant (C/C, patients with two copies of the C allele at the discovery SNP rs12979860) was associated with a two- to threefold increase in SVR rate in the three ethnic groups (33) (FROM JULES: 16% of AAs had CC & 53% of these had SVR; 39% of whites had CC & 82% had SVR; among AAs 48% had C/T & 19% had SVR, 37% had T/T & 17% had SVR; among whites 50% had C/T & 42% had SVR, 12% had T/T & 33% had SVR)........Rall—n et al. (43) found a strong association between host IL28B genotype and treatment response in a cohort of 164 treated HIV/HCV coinfected patients, with SVR rates of 75% for patients with the C/C allele compared with 38% for those with the C/T or T/T allele..... Importantly, in those patients who fail to achieve RVR, SVR rates were more than twofold higher in IL28B C/C patients compared with patients with unfavorable non-C/C genotype (33). This suggests that host IL28B genotype will add further predictive power for patients who fail to meet existing criteria currently used to predict response."
"Host factors that have been negatively reported to affect likelihood of SVR include age >40 years, advanced degrees of liver fibrosis, male gender, increased body mass index, insulin resistance, and hepatic steatosis (23,24,25). In addition, African-American patients are less likely to respond to treatment, an observation independent of other host and viral factors (26,27,28). Viral factors predictive of non-response response include genotype 1, lack of diversity in key genetic sequences, especially amino-acid mutations in the core and NS5A gene (29,30), high pretreatment HCV RNA levels, and on-treatment viral kinetics (31). Week 4 viral negativity (rapid viral response (RVR)) accurately predicts SVR, whereas failure to reduce serum HCV RNA levels by at least 2 log10 IU/ml by week 12 (early viral response) predicts treatment failure and is considered to be an indication to cease therapy"
HUMAN GENETIC VARIATION AND EXAMINING CLINICAL ASSOCIATIONS
The human genome contains over 3.3 billion base pairs. In excess of 10 million of these may vary in nucleotide sequence between individuals (single-nucleotide polymorphism or SNP). Some of this variation may result in altered expression of the gene, altered processing of the gene product (post-translational modification), or altered functional activity (e.g., receptor binding). Identifying polymorphisms that result in altered clinical expression (phenotype) is a daunting challenge, analogous to finding a "needle in a haystack" (1).
SNPs vary within a region of a chromosome (or haplotype block) in a nonrandom way. Across populations, nonrandom association of SNPs is referred to as linkage disequilibrium (9). Because of linkage disequilibrium, researchers can sample a limited number of SNPs (tag SNPs) but assess common variation across approximately 90% of the genome (10). Using microarray technology on commercialized chips or beads more than 1 million tag SNPs can now be tested in any individual DNA sample (Figure 1).
These common SNPs are available due to the efforts of the HapMap project 11 which is a public database of "hotspots" of common haplotypes (occurring in >5% of the population). Therefore, an important caveat for GWAS is that rarer variants (occurring in <5% of the population) may not be identified.
The goal of an initial study is to "flag" or identify genetic areas of interest. The causal genetic variant responsible for disease is rarely identified. GWAS therefore use genetic sampling technology and bioinformatics to statistically test for association of genotype with a clearly defined phenotype without limiting the sample by a predetermined hypothesis (Figure 2).
From the above, GWAS results rely on the size of the cohort; technical issues related to testing platforms and quality control; and how extensively the genome is sampled (how many SNPs are tested per individual). Increasingly, powerful testing platforms also raise new problems that arise from handling large data sets. The ability to test millions of SNPs per individual leads to the challenge of multiple testing (i.e., if the chance of a false-positive test is 0.05, then a million SNP chip will identify 50,000 false-positive associations) and requires statistical measures such as the Bonferroni correction to avoid false-positive results (Type 1 error). Finally, well-characterized clinical cohorts with clearly defined phenotypes are critical to avoid spurious results and genetic associations.
Early genetic association studies have in many cases not been reproducible (12,13,14,15), likely due to such problems as restricted genetic sampling on small cohorts often using the candidate gene approach; indistinct phenotypic characterization and statistical methodological problems (2,16). Improvements in high throughput DNA microarray technology have moderated the prohibitive cost and time previously involved with extensive sampling of large cohorts. For example, a chip containing 500,000-1 million SNPs now only costs approximately US$390-500. Evolving consensus on what characterizes genetic probability and statistical significance has allowed some degree of standardization of results and interpretation of GWAS (17,18,19).
GENOMIC VARIATION AND HEPATITIS C
The problem
Current treatment for patients chronically infected with genotype 1 HCV requires 48 weeks of pegIFN/RBV. The goal of treatment is viral eradication (sustained viral response (SVR) defined as the absence of virus 24 weeks after treatment completion), but this is achieved in <50% of patients (20,21).
Therapy is also frequently complicated by treatment-limiting side effects (22). An accurate ability to predict response would allow both patients and clinicians to make more informed decisions regarding the risk-benefit of treatment, and the likelihood of success for any given individual.
Based on population data, several key host and viral factors aid in predicting response to this treatment. Host factors that have been negatively reported to affect likelihood of SVR include age >40 years, advanced degrees of liver fibrosis, male gender, increased body mass index, insulin resistance, and hepatic steatosis (23,24,25). In addition, African-American patients are less likely to respond to treatment, an observation independent of other host and viral factors (26,27,28). Viral factors predictive of non-response response include genotype 1, lack of diversity in key genetic sequences, especially amino-acid mutations in the core and NS5A gene (29,30), high pretreatment HCV RNA levels, and on-treatment viral kinetics (31). Week 4 viral negativity (rapid viral response (RVR)) accurately predicts SVR, whereas failure to reduce serum HCV RNA levels by at least 2 log10 IU/ml by week 12 (early viral response) predicts treatment failure and is considered to be an indication to cease therapy (32). The fact that only 50% of patients are cured by treatment and the influence of ethnicity on response both suggest a genetic contribution to HCV treatment outcome.
Host IL28B genotype and SVR
Recent GWAS have identified SNPs around the gene coding for IFN-λ-3 (or IL28B), associated with favorable response to treatment in patients infected with genotype 1 HCV (4,5,6).
Patients in the first and largest GWAS were enrolled in the IDEAL trial, a randomized control trial comparing different doses of pegIFNα-2b/RBV with peg-IFNα-2a/RBV (21). DNA from a further 67 similarly well-characterized patients in a second randomized controlled trial analyzing racial differences in treatment response were also included (27). To clearly define the biological response phenotype, non-responders were excluded if they were not documented to be adherent and received at least 80% of the prescribed dose.
Overall, 1,137 patients were included in the genetic association analyses for SVR in three separate ethnic populations, defined by genetic ancestry (Caucasian, Hispanic, and African American (AA)). Seven SNPs were identified that overwhelmingly met genome-wide significance with the top discovery SNP (rs12979860) strongly associated with SVR (P=1.37 x 10-28). A number of other SNPs (including rs8099917 associated in other studies) were also significantly associated, but highly correlated with the discovery SNP due to linkage disequilibrium. Regression modeling found that the IL28B polymorphism (rs12979860) was the strongest predictor of SVR compared with all other baseline host and viral variables (Figure 3).
Figure 3
Sustained virological response (SVR) rates by host IL28B genotype for African Americans (above) and Caucasians (below) (Ge et al. (4)) (1). Note that these data are from an adherent cohort. For each ethnic group, the proportion of patients with favorable IL28B C/C, and the C/T and T/T less favorable genotypes is shown in the central blue chart. For each of these groups, the SVR rates are depicted in the corresponding bar charts. The percentage SVR rate is shown in green. As can be seen, those patients with the IL28B C/C genotype have the highest SVR rates.
An intention-to-treat analysis was performed that included all patients, regardless of adherence, and classifying ethnicity by self-report as it would be in practice. Again, the good response variant (C/C, patients with two copies of the C allele at the discovery SNP rs12979860) was associated with a two- to threefold increase in SVR rate in the three ethnic groups (33).
RVR remains a critical predictor of eventual SVR irrespective of host IL28B genotype. Importantly, in those patients who fail to achieve RVR, SVR rates were more than twofold higher in IL28B C/C patients compared with patients with unfavorable non-C/C genotype (33). This suggests that host IL28B genotype will add further predictive power for patients who fail to meet existing criteria currently used to predict response.
Other GWAS have independently identified the IL28B region to be an important predictor of SVR. Tanaka et al. (6) tested an initial GWAS discovery cohort of 142 patients followed by a replication study of a further 172 patients, all of Japanese ethnicity. They tested the clinical phenotype of non-response (<2 log reduction in HCV RNA at 12 weeks of treatment). Patients who received <80% of therapy up to week 12 were also excluded. Two SNPs (rs8099917 and rs12980275) near the IL28B gene were identified to be strongly associated with non-response. Modeling revealed rs8099917 to be the strongest predictor of non-response, independent of other clinical variables. Tanaka et al. (6) used an Affymetrix 6.0 genotyping platform, which did not include rs12979860 (the top SNP from the Ge et al. (4) study) as a tag SNP on the array.
Suppiah et al. (5) employed a similar two stage GWAS process to Tanaka et al. (6). A discovery cohort of well-matched Australian patients (n=293) of European ancestry was assessed in the initial GWAS and one SNP (rs8099917, P=7.06 x 10-8) met the criteria for statistical significance.
In the second replication study, Suppiah et al. (5) assessed a population of European and Australian patients (n=555) and further confirmed the association of rs8099917 with SVR (combined cohort P=9.25 x 10-9). Regression analysis again confirmed rs8099917 to be an independent predictor of response (P<0.0001).
Two more recently published studies have replicated these findings. In a large GWAS on a heterogeneous group of patients, Rauch et al. (8) identified the IL28B gene region (rs8099917) to be to be independently associated with treatment failure (odds ratio (OR) 5.19 (2.90-9.30), P=3.11 x 10-8). The study also assessed spontaneous clearance and the effect of host genotype on HCV/HIV coinfection (discussed below). McCarthy et al. (34) investigated the association with host IL28B genotype (rs12979360) and SVR in a cohort of 231 clinic patients. This study again confirmed the IL28B CC genotype to be the strongest pretreatment predictor of response, independent of other pretreatment clinical predictors and HCV genotype (OR 5.79 (2.76-12.57) P=9.0 x 10-6).
In summary, a number of SNPs around the IL28B gene locus were found across the studies to be associated with treatment response. All displayed at least moderate linkage disequilibrium. The characteristics of each study (e.g., population racial distribution, sample sizes, and clinical phenotype assessed) and the SNPs represented on the different chips obviously influence the nature and significance of results and are likely to explain the discrepancy in top SNPs found. An important conclusion is that all these studies implicate the IL28B gene as a predictor of treatment response.
Host IL28B genotype, ethnicity, and the effect on SVR
Differences between ethnicity and treatment response rates are poorly explained by host clinical factors or compliance, suggesting a key role for host genetics (26,27). The IL28B polymorphism rs12979860 has a marked differential distribution between racial groups, being least frequent in AAs, most frequent in Asians, and with an intermediate frequency in Hispanics and Caucasians (4,7) (Figure 3).
In the study by Ge et al. 4 IL28B genotype variation statistically explained approximately half of the observed difference in SVR rate between Caucasians and AA (4). However, it did not entirely explain the difference, and AAs still had poorer response rates across each host IL28B genotype than Caucasians. It is therefore possible that there are other genetic factors that may contribute to IFN sensitivity, particularly in AA patients.
Host IL28B genotype and spontaneous clearance
Spontaneous clearance of HCV occurs in approximately 20-50% of patients following acute infection, and host genetics has been previously suggested to have a significant role (35,36,37,38). In the same GWAS related to treatment response, Ge et al. (4) found the rs12979860 C allele significantly more common in a random multiethnic population compared with an HCV-infected control cohort (P=2.48 x 10-6) (4). This raised the question of whether the C allele affords some protection against the development of chronic HCV infection.
Thomas et al. (7) subsequently evaluated the SNP rs12979860 to assess the association with spontaneous clearance. They investigated an ethnically diverse population of 1,008 individuals from six different clinical and study cohorts, with either spontaneous clearance of acute HCV infection or the development of persistent infection. Across all cohorts, those with the C/C genotype were three times more likely to clear HCV acutely relative to non-C/C (i.e., heterozygotes (C/T), and homozygotes for the minor allele (T/T) (OR 0.33; P<10-12). Thomas et al. (7) found that polymorphism at the IL28B gene demonstrates a similar effect on spontaneous clearance across ethnic groups.
In line with this observation, Rauch et al. (8) conducted a GWAS on a Caucasian cohort of 347 patients who had spontaneously cleared HCV and compared them to 1,015 patients with chronic hepatitis C. They extended previous studies by finding that only the IL28B gene locus was associated with clearance (OR 2.31 (1.74-3.06), P=6.07 x 10-9), with some minor advantage conferred to heterozygotes.
Other emerging IL28B data
The introduction of direct acting antivirals (DAAV) (or specifically targeted antiviral therapies) is the most important recent therapeutic advance for patients infected with HCV and will lead to significantly improved overall SVR rates (39,40). Akuta et al. (41) recently assessed a cohort of Japanese patients infected with genotype 1b HCV and high viral load treated with either 12 weeks of telaprevir/peg-IFN/RBV (n=20) or 12 weeks of triple therapy followed by a further 12 weeks of peg-IFN/RBV (n=61). They found that IL28B polymorphism (rs8099917) remained the strongest pretreatment predictor of response, despite the addition of telaprevir. Interestingly, the majority of these patients were either non-responders or relapsers to prior treatment, suggesting that IL28B may exert effect in this specific patient population and be an important consideration in retreatment studies.
Akuta et al. (41) also directly sequenced the virus for amino-acid substitutions in the core and NS5A-IFN sensitivity determining regions. In multivariable analysis, patients who were heterozygous or homozygous for the poor response IL28B polymorphism were more likely to achieve SVR in the setting of amino-acid substitution Arg70 rather than Gln70(His70) (50 vs. 11.8%, P=0.038). More data are required from other ethnic groups and from larger treatment naïve cohorts to assess what residual role viral amino-acid substitutions have in pretreatment prediction of response to DAAV inclusive regimens.
Further data concerning the relevance of IL28B polymorphism in non-genotype-1 HCV infection is emerging. Analysis of an Italian genotype 2 and 3 cohort (n=268) found IL28B to be associated with SVR and a significant variable in a predictive model (42). Rauch et al. (8) found no significant impact of host IL28B genotype on treatment response in HCV genotype 2/3 patients when comparing 230 patients with HCV genotype 2 or 3 with a matched group of 232 patients with HCV genotype 1 or 4. These findings were also observed in patients coinfected with HIV and HCV genotype 3 by Rall—n et al. (43). IL28B polymorphisms may be important for subgroups of genotype 2/3 patients, where C/C genotype patients who failed to achieve RVR were more likely to achieve SVR than non-C/C genotype (42). Non-genotype 1 infection is generally more successfully treated, thus limiting the advantage conferred from favorable IL28B polymorphism, and requires a better-powered cohort to find statistically significant associations.
IL28B genotype appears to be important in the setting of HIV/HCV coinfection. Rall—n et al. (43) found a strong association between host IL28B genotype and treatment response in a cohort of 164 treated HIV/HCV coinfected patients, with SVR rates of 75% for patients with the C/C allele compared with 38% for those with the C/T or T/T allele (P=<0.0001). Nattermann et al. (44) studied another HIV/HCV patient cohort and in univariable analysis found SVR rates of 58.1 vs. 40.6% for responder and non-responder genotypes, respectively (P=0.041). Rauch et al. (8) assessed the association between spontaneous clearance and host genotype with GWAS in 448 patients coinfected with HIV/HCV and found a similar signal for IL28B to that found in monoinfected patients (OR 2.16 (1.47-3.18), P=8.24 x 10-5 vs. OR 2.49 (1.64-3.79), P=1.96 x 10-5, respectively), a finding consistent with Thomas et al. (7,8).
Clearly, more data related to DAAVs, different HCV genotypes, treatment response, and the effect of IL28B genotype in the post-transplant setting and in other treatment regimens with novel agents are eagerly awaited.
How is IL28B polymorphism biologically associated with these clinical observations?
Although the identified SNPs may not represent causal variants, the strong association replicated on multiple occasions now implicates the IL28B gene and IFN-λ as the "smoking gun" for response and clearance. However, the biological pathways underpinning this association remain unknown.
The top association SNPs lie upstream of the IL28B gene and it is therefore possible that they effect IL28B transcription. Ge et al. (4) observed no relationship between IL28B mRNA levels and IL28B polymorphism in peripheral blood mononuclear cells from non-HCV-infected volunteers in the SNPExpress database (using genotype at rs12980275 as a proxy for rs12979860 genotype, r2=0.88) (4). Tanaka et al. et al. (6) measured IL28B mRNA from the peripheral blood mononuclear cells in a small subgroup of 20 patients (6). Using quantitative real-time PCR, they found that rs80999917 poor-responder allele homozygotes had lower levels of IL28B mRNA expression. In a limited sample of patients not infected with HCV, Suppiah et al. (5) found lower constitutive expression of IL28A and IL28B in patients with the rs8099917 poor-responder allele (P=0.044). The data on gene expression are therefore conflicting and further studies will be required. Several other potentially functional SNPs have been identified. These include a non-synonymous SNP (rs8103142) in exon 2 of the gene (4). This polymorphism might potentially affect protein function, including receptor binding, or protein stability. Because of the high degree of linkage disequilibrium between these variants, it has not been possible to statistically determine which SNP was responsible for the association signal (the causal variant).
Type III IFNs (or IFN-λ) share many characteristics with the Type I IFNs. IFN-λ (including IL28A, IL28B, and IL29) signals via a unique receptor, the IFN- λR, which has a more liver-specific distribution than the ubiquitous type I IFN-R (45). The receptors share a common downstream signaling pathway, via the Janus-activated kinases-signal transducer and activator of transcriptions 1 and 2 (JAK-STAT) pathway. This leads to intranuclear activation of the IFN-stimulated response element and IFN-stimulated genes (46,47). IFN-λ1/2 have been shown in vitro to have antiviral activity against HCV; although less potent than IFN-α, it appears to have an additive or synergistic effect in combination (48,49). IFN-λ1 (IL29) has also recently been shown to have potent antiviral activity in a phase 1 study of patients with genotype 1 chronic hepatitis C, although there is no data as yet on whether host IL28B genotype is relevant to its the therapeutic effect (50).
How is IL28B polymorphism linked to improved treatment induced and spontaneous clearance? At this stage the answer remains unknown, although hypotheses proliferate. It would seem reasonable that as a starting point, IL28B polymorphism may affect the IFN-λ system. The complexity of interaction between HCV and the host immune system, and the redundancy within innate and cell-mediated immune systems suggests that this effect will likely be iterative and multileveled. Although the mechanism underlying the association between IL28B polymorphism and HCV treatment response/spontaneous clearance is yet to be resolved, the observation has great biological plausibility, and identifies the IFN-λ axis as an important new area for translational research.
What do these findings mean for clinicians managing patients with chronic hepatitis C?
A commercial test is now available for IL28B, but how should this information be used to improve care for patients? Patients may be "profiled" based on IL28B genotype and other important clinical features (such as fibrosis, age, insulin resistance, viral load, and race) to better predict treatment response (Table 1). Host IL28B genotype is the strongest pretreatment predictor of response through its effect on viral kinetics (33). On-treatment viral kinetics (currently RVR or early viral response, but potentially even earlier measures) provides direct measurement of treatment response and remains the most powerful on-treatment response predictor and the key criteria for on-treatment therapy decisions. Importantly, host genotype also adds to current response-guided decision algorithms. The IL28B CC genotype identifies a subgroup of patients without RVR who are more likely to achieve SVR. When new DAAV agents in development become available, such profiles may also help both in the choice of treatment regimen and its anticipated duration, personalizing therapy further.
CONCLUSION:
GWAS (Genome-wide association studies) have identified a strong association between host IL28B genotype and response to treatment with pegIFN/RBV for patients with genotype 1 chronic hepatitis C. Its differential racial distribution explains much of observed clinical differences in response between races. Although this discovery has great biological plausibility, the causal variant is yet to be identified. The nature of the interaction between type I and type III IFN signaling needs to be further elucidated and should be a focus for research.
The decision to treat remains complex. Genotyping of this polymorphism will aid clinical decision making for both current standard of care and potentially for the integration of other agents in the future, providing an opportunity for clinicians to individualize treatment regimens for hepatitis C patients.
Source
Am J Gastroenterol Jan 2011
Paul J Clark MD1, Alex J Thompson MD, PhD1 and John G McHutchison MD1 1Duke Clinical Research Institute and Division of Gastroenterology, Duke University Medical Center, School of Medicine, Duke University, Durham, North Carolina, USA. Correspondence: Paul J. Clark, MD, Division of Gastroenterology, Duke Clinical Research Institute, Duke University Medical Center, PO Box 17969, Durham, North Carolina 27715, USA. E-mail: paul.clark@duke.edu
from Jules of NATAP: We are unsure right now exactly how & to what extent IL28B will be used, we are awaiting further outcomes analyses from the recently reported studies of boceprevir & telaprevir (the new oraly administered protease inhibitors that will be used in combination with peg/rbv). Patients may be "profiled" based on IL28B genotype and other important clinical features (such as fibrosis, age, insulin resistance, viral load, and race) to better predict treatment response (Table 1). Host IL28B genotype is the strongest pretreatment predictor of response through its effect on viral kinetics.....Genotyping of this polymorphism will aid clinical decision making for both current standard of care and potentially for the integration of other agents in the future
........The IL28B polymorphism rs12979860 has a marked differential distribution between racial groups, being least frequent in African-Americans, most frequent in Asians, and with an intermediate frequency in Hispanics and Caucasians .....The best response is in patients with C/C gene, when genotype 1 mono or coinfected patients with C/C are treated with peg/rbv they have DOUBLE to TRIPLE the response rate compared to patients with C/C or C/T: 16% of AAs had CC & 53% of these had SVR; 39% of whites had CC & 82% had SVR; among AAs 48% had C/T & 19% had SVR, 37% had T/T & 17% had SVR; among whites 50% had C/T & 42% had SVR, 12% had T/T & 33% had SVR).....there are many other factors that have predicted a good or bad response to peg/rbv including pretreatment viral load, week 4 viral load response to treatment, week 12 viral load response to treatment, degree of fibrosis, body weight, gender, insulin resistance, fatty liver'
What do these findings mean for clinicians managing patients with chronic hepatitis C?
A commercial test is now available for IL28B, but how should this information be used to improve care for patients? Patients may be "profiled" based on IL28B genotype and other important clinical features (such as fibrosis, age, insulin resistance, viral load, and race) to better predict treatment response (Table 1). Host IL28B genotype is the strongest pretreatment predictor of response through its effect on viral kinetics (33). On-treatment viral kinetics (currently RVR or early viral response, but potentially even earlier measures) provides direct measurement of treatment response and remains the most powerful on-treatment response predictor and the key criteria for on-treatment therapy decisions. Importantly, host genotype also adds to current response-guided decision algorithms. The IL28B CC genotype identifies a subgroup of patients without RVR who are more likely to achieve SVR. When new DAAV agents in development become available, such profiles may also help both in the choice of treatment regimen and its anticipated duration, personalizing therapy further.
Table 1 - Host IL28B genotype and other important patient characteristics will be used to develop patient profiles to help predict response and potentially tailor therapy.
Clinical message: Patients with genotype 1 who do not achieve RVR but carry favorable IL28B type have a 65% chance of cure.
Current standard of care therapy with pegIFN/RBV provides excellent results in patients with genotype 1 HCV and the favorable host IL28B genotype.
At the bedside, knowledge of this host genotype will translate into greater confidence in terms of counseling patients before commencing treatment. Discussions with a patient about an 80% likelihood of treatment response versus a 30% response rate are very different conversations and akin to discussions we currently have with patients about the impact of ethnicity or viral genotype on predicted treatment response. When on treatment, host IL28B genotype knowledge may assist clinicians and patients to "stay the course" when troubled by side effects that may or may not require dose reductions, by the knowledge they have more than simply a "50/50" chance of success in favorable responder genotype patients.
Clinical message: Patients with a favorable patient profile (e.g., C/C IL28B genotype) are likely to respond to current standard of care therapy. This knowledge changes the "cost/benefit" of treatment and may help to encourage patients to commence treatment and to reassure them during a long and often difficult treatment course.
PegIFN/RBV therapy alone avoids the selection for point-specific HCV mutations that may lead to drug resistance when treating with DAAV agents. Existing treatment regimens also avoid exposure to the potential of further drug side effects and toxicity that may be observed with new agents. Finally, existing combination therapy may prove more cost-effective in those most likely to respond, relative to simply adding DAAV onto existing regimens.
Clinical message: Although current standard of care therapy has its drawbacks, for patients with a high likelihood of response (e.g., C/C genotype) it avoids many of the additional problems and costs of future pegIFN/RBV+DAAV combination regimens.
On the basis of available data, patients with unfavorable responder genotype should probably be categorized as "difficult to treat" in the same way clinicians consider other negative treatment prognostic criteria (e.g., advanced fibrosis). It seems likely that the addition of directly acting antiviral therapy may attenuate the effect of host IL28B genotype on SVR; however, further studies are needed (40,41). For patients who have the unfavorable IL28B genotype, it may be prudent to defer treatment until future regimens that include directly acting antiviral agents become available in 2011-12.
Clinical message: Patients with an unfavorable response profile (e.g., non-C/C genotype) are most likely to benefit from improved SVR rates with regimens that include DAAVs, and they should probably wait for such therapies.
How knowledge of a patient's IL28B genotype will alter treatment algorithms is unknown. Patients with the favorable IL28B C/C genotype may achieve adequate SVR rates with a reduced duration of current standard of care (e.g., 24 weeks or less compared with the current 48 weeks). Alternatively, in C/C genotype patients the addition of DAAV therapy may provide a pathway to even further reduce the duration of treatment. For patients with less favorable IL28B genotype efficacy issues prevail. The effect of extending pegIFN/RBV treatment duration or adding a third or fourth agent on SVR rates needs to be studied. These possible strategies should be assessed prospectively in randomized controlled trials stratified to IL28B type.
Clinical message: Host IL28B genotype may allow for shortened treatment regimens in favorable C/C genotype patients, particularly with regimens including DAAVs.
Review of existing trial data is now required where possible to determine whether knowledge of host genotype alters the interpretation of a study. Stratification for host IL28B genotype to avoid imbalance in treatment arms is of particular concern for early phase trials with small sample sizes. Ongoing trials involving IFN-λ, other developing immunomodulator therapies and directly acting antivirals are awaited with interest.
Clinical message: The genetic signal of this discovery is strong and consistent and establishes the biological principle for further clinical research. More data are now required to fully explore and understand the implications in different clinical scenarios.
ABSTRACT
Genome-wide association studies (GWAS) have recently identified host genetic variation to be critical for predicting treatment response and spontaneous clearance in patients infected with hepatitis C virus (HCV). These important new studies are reviewed and their future clinical implications discussed. Single-nucleotide polymorphisms in the region of the IL28B gene on chromosome 19, coding for the interferon (IFN)-λ-3 or IL28B gene, are strongly associated with treatment response to pegylated IFN and ribavirin in patients infected with genotype 1 HCV. The good response variant is associated with a twofold increase in the rate of cure. Allele frequencies differ between ethnic groups, largely explaining the observed differences in response rates between Caucasians, African Americans and Asians. IL28B polymorphism is also strongly associated with spontaneous clearance of HCV. The biological mechanisms responsible for these genetic associations remain unknown and are the focus of ongoing research. Knowledge of a patient's IL28B genotype is likely to aid in clinical decision making with standard of care regimens. Future studies will investigate the possibility of individualizing treatment duration and novel regimens according to IL28B type.
INTRODUCTION
The unraveling of the human genome offers the promise of personalized medicine. In this review, we briefly define some critical genetic concepts for clinicians, and overview genome-wide association studies (GWAS). Readers are directed to excellent genetics reviews for further reading (1,2,3). We review the recent GWAS discovery, which has shown that host genetics drives treatment response to pegylated interferon-α and ribavirin (pegIFN/RIB) for patients infected with genotype 1 hepatitis C virus (HCV) (4,5,6). We also review recent studies that have shown that the same polymorphisms are associated with spontaneous clearance of HCV (7,8).
"Patients in the first and largest GWAS (Genome-wide association studies) were enrolled in the IDEAL trial, a randomized control trial comparing different doses of pegIFNα-2b/RBV with peg-IFNα-2a/RBV (21)........Overall, 1,137 patients were included in the genetic association analyses for SVR in three separate ethnic populations, defined by genetic ancestry (Caucasian, Hispanic, and African American (AA))......Regression modeling found that the IL28B polymorphism (rs12979860) was the strongest predictor of SVR compared with all other baseline host and viral variables
The IL28B polymorphism rs12979860 has a marked differential distribution between racial groups, being least frequent in AAs, most frequent in Asians, and with an intermediate frequency in Hispanics and Caucasians .....An intention-to-treat analysis was performed that included all patients, regardless of adherence, and classifying ethnicity by self-report as it would be in practice. Again, the good response variant (C/C, patients with two copies of the C allele at the discovery SNP rs12979860) was associated with a two- to threefold increase in SVR rate in the three ethnic groups (33) (FROM JULES: 16% of AAs had CC & 53% of these had SVR; 39% of whites had CC & 82% had SVR; among AAs 48% had C/T & 19% had SVR, 37% had T/T & 17% had SVR; among whites 50% had C/T & 42% had SVR, 12% had T/T & 33% had SVR)........Rall—n et al. (43) found a strong association between host IL28B genotype and treatment response in a cohort of 164 treated HIV/HCV coinfected patients, with SVR rates of 75% for patients with the C/C allele compared with 38% for those with the C/T or T/T allele..... Importantly, in those patients who fail to achieve RVR, SVR rates were more than twofold higher in IL28B C/C patients compared with patients with unfavorable non-C/C genotype (33). This suggests that host IL28B genotype will add further predictive power for patients who fail to meet existing criteria currently used to predict response."
"Host factors that have been negatively reported to affect likelihood of SVR include age >40 years, advanced degrees of liver fibrosis, male gender, increased body mass index, insulin resistance, and hepatic steatosis (23,24,25). In addition, African-American patients are less likely to respond to treatment, an observation independent of other host and viral factors (26,27,28). Viral factors predictive of non-response response include genotype 1, lack of diversity in key genetic sequences, especially amino-acid mutations in the core and NS5A gene (29,30), high pretreatment HCV RNA levels, and on-treatment viral kinetics (31). Week 4 viral negativity (rapid viral response (RVR)) accurately predicts SVR, whereas failure to reduce serum HCV RNA levels by at least 2 log10 IU/ml by week 12 (early viral response) predicts treatment failure and is considered to be an indication to cease therapy"
HUMAN GENETIC VARIATION AND EXAMINING CLINICAL ASSOCIATIONS
The human genome contains over 3.3 billion base pairs. In excess of 10 million of these may vary in nucleotide sequence between individuals (single-nucleotide polymorphism or SNP). Some of this variation may result in altered expression of the gene, altered processing of the gene product (post-translational modification), or altered functional activity (e.g., receptor binding). Identifying polymorphisms that result in altered clinical expression (phenotype) is a daunting challenge, analogous to finding a "needle in a haystack" (1).
SNPs vary within a region of a chromosome (or haplotype block) in a nonrandom way. Across populations, nonrandom association of SNPs is referred to as linkage disequilibrium (9). Because of linkage disequilibrium, researchers can sample a limited number of SNPs (tag SNPs) but assess common variation across approximately 90% of the genome (10). Using microarray technology on commercialized chips or beads more than 1 million tag SNPs can now be tested in any individual DNA sample (Figure 1).
These common SNPs are available due to the efforts of the HapMap project 11 which is a public database of "hotspots" of common haplotypes (occurring in >5% of the population). Therefore, an important caveat for GWAS is that rarer variants (occurring in <5% of the population) may not be identified.
The goal of an initial study is to "flag" or identify genetic areas of interest. The causal genetic variant responsible for disease is rarely identified. GWAS therefore use genetic sampling technology and bioinformatics to statistically test for association of genotype with a clearly defined phenotype without limiting the sample by a predetermined hypothesis (Figure 2).
From the above, GWAS results rely on the size of the cohort; technical issues related to testing platforms and quality control; and how extensively the genome is sampled (how many SNPs are tested per individual). Increasingly, powerful testing platforms also raise new problems that arise from handling large data sets. The ability to test millions of SNPs per individual leads to the challenge of multiple testing (i.e., if the chance of a false-positive test is 0.05, then a million SNP chip will identify 50,000 false-positive associations) and requires statistical measures such as the Bonferroni correction to avoid false-positive results (Type 1 error). Finally, well-characterized clinical cohorts with clearly defined phenotypes are critical to avoid spurious results and genetic associations.
Early genetic association studies have in many cases not been reproducible (12,13,14,15), likely due to such problems as restricted genetic sampling on small cohorts often using the candidate gene approach; indistinct phenotypic characterization and statistical methodological problems (2,16). Improvements in high throughput DNA microarray technology have moderated the prohibitive cost and time previously involved with extensive sampling of large cohorts. For example, a chip containing 500,000-1 million SNPs now only costs approximately US$390-500. Evolving consensus on what characterizes genetic probability and statistical significance has allowed some degree of standardization of results and interpretation of GWAS (17,18,19).
GENOMIC VARIATION AND HEPATITIS C
The problem
Current treatment for patients chronically infected with genotype 1 HCV requires 48 weeks of pegIFN/RBV. The goal of treatment is viral eradication (sustained viral response (SVR) defined as the absence of virus 24 weeks after treatment completion), but this is achieved in <50% of patients (20,21).
Therapy is also frequently complicated by treatment-limiting side effects (22). An accurate ability to predict response would allow both patients and clinicians to make more informed decisions regarding the risk-benefit of treatment, and the likelihood of success for any given individual.
Based on population data, several key host and viral factors aid in predicting response to this treatment. Host factors that have been negatively reported to affect likelihood of SVR include age >40 years, advanced degrees of liver fibrosis, male gender, increased body mass index, insulin resistance, and hepatic steatosis (23,24,25). In addition, African-American patients are less likely to respond to treatment, an observation independent of other host and viral factors (26,27,28). Viral factors predictive of non-response response include genotype 1, lack of diversity in key genetic sequences, especially amino-acid mutations in the core and NS5A gene (29,30), high pretreatment HCV RNA levels, and on-treatment viral kinetics (31). Week 4 viral negativity (rapid viral response (RVR)) accurately predicts SVR, whereas failure to reduce serum HCV RNA levels by at least 2 log10 IU/ml by week 12 (early viral response) predicts treatment failure and is considered to be an indication to cease therapy (32). The fact that only 50% of patients are cured by treatment and the influence of ethnicity on response both suggest a genetic contribution to HCV treatment outcome.
Host IL28B genotype and SVR
Recent GWAS have identified SNPs around the gene coding for IFN-λ-3 (or IL28B), associated with favorable response to treatment in patients infected with genotype 1 HCV (4,5,6).
Patients in the first and largest GWAS were enrolled in the IDEAL trial, a randomized control trial comparing different doses of pegIFNα-2b/RBV with peg-IFNα-2a/RBV (21). DNA from a further 67 similarly well-characterized patients in a second randomized controlled trial analyzing racial differences in treatment response were also included (27). To clearly define the biological response phenotype, non-responders were excluded if they were not documented to be adherent and received at least 80% of the prescribed dose.
Overall, 1,137 patients were included in the genetic association analyses for SVR in three separate ethnic populations, defined by genetic ancestry (Caucasian, Hispanic, and African American (AA)). Seven SNPs were identified that overwhelmingly met genome-wide significance with the top discovery SNP (rs12979860) strongly associated with SVR (P=1.37 x 10-28). A number of other SNPs (including rs8099917 associated in other studies) were also significantly associated, but highly correlated with the discovery SNP due to linkage disequilibrium. Regression modeling found that the IL28B polymorphism (rs12979860) was the strongest predictor of SVR compared with all other baseline host and viral variables (Figure 3).
Figure 3
Sustained virological response (SVR) rates by host IL28B genotype for African Americans (above) and Caucasians (below) (Ge et al. (4)) (1). Note that these data are from an adherent cohort. For each ethnic group, the proportion of patients with favorable IL28B C/C, and the C/T and T/T less favorable genotypes is shown in the central blue chart. For each of these groups, the SVR rates are depicted in the corresponding bar charts. The percentage SVR rate is shown in green. As can be seen, those patients with the IL28B C/C genotype have the highest SVR rates.
RVR remains a critical predictor of eventual SVR irrespective of host IL28B genotype. Importantly, in those patients who fail to achieve RVR, SVR rates were more than twofold higher in IL28B C/C patients compared with patients with unfavorable non-C/C genotype (33). This suggests that host IL28B genotype will add further predictive power for patients who fail to meet existing criteria currently used to predict response.
Other GWAS have independently identified the IL28B region to be an important predictor of SVR. Tanaka et al. (6) tested an initial GWAS discovery cohort of 142 patients followed by a replication study of a further 172 patients, all of Japanese ethnicity. They tested the clinical phenotype of non-response (<2 log reduction in HCV RNA at 12 weeks of treatment). Patients who received <80% of therapy up to week 12 were also excluded. Two SNPs (rs8099917 and rs12980275) near the IL28B gene were identified to be strongly associated with non-response. Modeling revealed rs8099917 to be the strongest predictor of non-response, independent of other clinical variables. Tanaka et al. (6) used an Affymetrix 6.0 genotyping platform, which did not include rs12979860 (the top SNP from the Ge et al. (4) study) as a tag SNP on the array.
Suppiah et al. (5) employed a similar two stage GWAS process to Tanaka et al. (6). A discovery cohort of well-matched Australian patients (n=293) of European ancestry was assessed in the initial GWAS and one SNP (rs8099917, P=7.06 x 10-8) met the criteria for statistical significance.
In the second replication study, Suppiah et al. (5) assessed a population of European and Australian patients (n=555) and further confirmed the association of rs8099917 with SVR (combined cohort P=9.25 x 10-9). Regression analysis again confirmed rs8099917 to be an independent predictor of response (P<0.0001).
Two more recently published studies have replicated these findings. In a large GWAS on a heterogeneous group of patients, Rauch et al. (8) identified the IL28B gene region (rs8099917) to be to be independently associated with treatment failure (odds ratio (OR) 5.19 (2.90-9.30), P=3.11 x 10-8). The study also assessed spontaneous clearance and the effect of host genotype on HCV/HIV coinfection (discussed below). McCarthy et al. (34) investigated the association with host IL28B genotype (rs12979360) and SVR in a cohort of 231 clinic patients. This study again confirmed the IL28B CC genotype to be the strongest pretreatment predictor of response, independent of other pretreatment clinical predictors and HCV genotype (OR 5.79 (2.76-12.57) P=9.0 x 10-6).
In summary, a number of SNPs around the IL28B gene locus were found across the studies to be associated with treatment response. All displayed at least moderate linkage disequilibrium. The characteristics of each study (e.g., population racial distribution, sample sizes, and clinical phenotype assessed) and the SNPs represented on the different chips obviously influence the nature and significance of results and are likely to explain the discrepancy in top SNPs found. An important conclusion is that all these studies implicate the IL28B gene as a predictor of treatment response.
Host IL28B genotype, ethnicity, and the effect on SVR
Differences between ethnicity and treatment response rates are poorly explained by host clinical factors or compliance, suggesting a key role for host genetics (26,27). The IL28B polymorphism rs12979860 has a marked differential distribution between racial groups, being least frequent in AAs, most frequent in Asians, and with an intermediate frequency in Hispanics and Caucasians (4,7) (Figure 3).
In the study by Ge et al. 4 IL28B genotype variation statistically explained approximately half of the observed difference in SVR rate between Caucasians and AA (4). However, it did not entirely explain the difference, and AAs still had poorer response rates across each host IL28B genotype than Caucasians. It is therefore possible that there are other genetic factors that may contribute to IFN sensitivity, particularly in AA patients.
Host IL28B genotype and spontaneous clearance
Spontaneous clearance of HCV occurs in approximately 20-50% of patients following acute infection, and host genetics has been previously suggested to have a significant role (35,36,37,38). In the same GWAS related to treatment response, Ge et al. (4) found the rs12979860 C allele significantly more common in a random multiethnic population compared with an HCV-infected control cohort (P=2.48 x 10-6) (4). This raised the question of whether the C allele affords some protection against the development of chronic HCV infection.
Thomas et al. (7) subsequently evaluated the SNP rs12979860 to assess the association with spontaneous clearance. They investigated an ethnically diverse population of 1,008 individuals from six different clinical and study cohorts, with either spontaneous clearance of acute HCV infection or the development of persistent infection. Across all cohorts, those with the C/C genotype were three times more likely to clear HCV acutely relative to non-C/C (i.e., heterozygotes (C/T), and homozygotes for the minor allele (T/T) (OR 0.33; P<10-12). Thomas et al. (7) found that polymorphism at the IL28B gene demonstrates a similar effect on spontaneous clearance across ethnic groups.
In line with this observation, Rauch et al. (8) conducted a GWAS on a Caucasian cohort of 347 patients who had spontaneously cleared HCV and compared them to 1,015 patients with chronic hepatitis C. They extended previous studies by finding that only the IL28B gene locus was associated with clearance (OR 2.31 (1.74-3.06), P=6.07 x 10-9), with some minor advantage conferred to heterozygotes.
Other emerging IL28B data
The introduction of direct acting antivirals (DAAV) (or specifically targeted antiviral therapies) is the most important recent therapeutic advance for patients infected with HCV and will lead to significantly improved overall SVR rates (39,40). Akuta et al. (41) recently assessed a cohort of Japanese patients infected with genotype 1b HCV and high viral load treated with either 12 weeks of telaprevir/peg-IFN/RBV (n=20) or 12 weeks of triple therapy followed by a further 12 weeks of peg-IFN/RBV (n=61). They found that IL28B polymorphism (rs8099917) remained the strongest pretreatment predictor of response, despite the addition of telaprevir. Interestingly, the majority of these patients were either non-responders or relapsers to prior treatment, suggesting that IL28B may exert effect in this specific patient population and be an important consideration in retreatment studies.
Akuta et al. (41) also directly sequenced the virus for amino-acid substitutions in the core and NS5A-IFN sensitivity determining regions. In multivariable analysis, patients who were heterozygous or homozygous for the poor response IL28B polymorphism were more likely to achieve SVR in the setting of amino-acid substitution Arg70 rather than Gln70(His70) (50 vs. 11.8%, P=0.038). More data are required from other ethnic groups and from larger treatment naïve cohorts to assess what residual role viral amino-acid substitutions have in pretreatment prediction of response to DAAV inclusive regimens.
Further data concerning the relevance of IL28B polymorphism in non-genotype-1 HCV infection is emerging. Analysis of an Italian genotype 2 and 3 cohort (n=268) found IL28B to be associated with SVR and a significant variable in a predictive model (42). Rauch et al. (8) found no significant impact of host IL28B genotype on treatment response in HCV genotype 2/3 patients when comparing 230 patients with HCV genotype 2 or 3 with a matched group of 232 patients with HCV genotype 1 or 4. These findings were also observed in patients coinfected with HIV and HCV genotype 3 by Rall—n et al. (43). IL28B polymorphisms may be important for subgroups of genotype 2/3 patients, where C/C genotype patients who failed to achieve RVR were more likely to achieve SVR than non-C/C genotype (42). Non-genotype 1 infection is generally more successfully treated, thus limiting the advantage conferred from favorable IL28B polymorphism, and requires a better-powered cohort to find statistically significant associations.
IL28B genotype appears to be important in the setting of HIV/HCV coinfection. Rall—n et al. (43) found a strong association between host IL28B genotype and treatment response in a cohort of 164 treated HIV/HCV coinfected patients, with SVR rates of 75% for patients with the C/C allele compared with 38% for those with the C/T or T/T allele (P=<0.0001). Nattermann et al. (44) studied another HIV/HCV patient cohort and in univariable analysis found SVR rates of 58.1 vs. 40.6% for responder and non-responder genotypes, respectively (P=0.041). Rauch et al. (8) assessed the association between spontaneous clearance and host genotype with GWAS in 448 patients coinfected with HIV/HCV and found a similar signal for IL28B to that found in monoinfected patients (OR 2.16 (1.47-3.18), P=8.24 x 10-5 vs. OR 2.49 (1.64-3.79), P=1.96 x 10-5, respectively), a finding consistent with Thomas et al. (7,8).
Clearly, more data related to DAAVs, different HCV genotypes, treatment response, and the effect of IL28B genotype in the post-transplant setting and in other treatment regimens with novel agents are eagerly awaited.
How is IL28B polymorphism biologically associated with these clinical observations?
Although the identified SNPs may not represent causal variants, the strong association replicated on multiple occasions now implicates the IL28B gene and IFN-λ as the "smoking gun" for response and clearance. However, the biological pathways underpinning this association remain unknown.
The top association SNPs lie upstream of the IL28B gene and it is therefore possible that they effect IL28B transcription. Ge et al. (4) observed no relationship between IL28B mRNA levels and IL28B polymorphism in peripheral blood mononuclear cells from non-HCV-infected volunteers in the SNPExpress database (using genotype at rs12980275 as a proxy for rs12979860 genotype, r2=0.88) (4). Tanaka et al. et al. (6) measured IL28B mRNA from the peripheral blood mononuclear cells in a small subgroup of 20 patients (6). Using quantitative real-time PCR, they found that rs80999917 poor-responder allele homozygotes had lower levels of IL28B mRNA expression. In a limited sample of patients not infected with HCV, Suppiah et al. (5) found lower constitutive expression of IL28A and IL28B in patients with the rs8099917 poor-responder allele (P=0.044). The data on gene expression are therefore conflicting and further studies will be required. Several other potentially functional SNPs have been identified. These include a non-synonymous SNP (rs8103142) in exon 2 of the gene (4). This polymorphism might potentially affect protein function, including receptor binding, or protein stability. Because of the high degree of linkage disequilibrium between these variants, it has not been possible to statistically determine which SNP was responsible for the association signal (the causal variant).
Type III IFNs (or IFN-λ) share many characteristics with the Type I IFNs. IFN-λ (including IL28A, IL28B, and IL29) signals via a unique receptor, the IFN- λR, which has a more liver-specific distribution than the ubiquitous type I IFN-R (45). The receptors share a common downstream signaling pathway, via the Janus-activated kinases-signal transducer and activator of transcriptions 1 and 2 (JAK-STAT) pathway. This leads to intranuclear activation of the IFN-stimulated response element and IFN-stimulated genes (46,47). IFN-λ1/2 have been shown in vitro to have antiviral activity against HCV; although less potent than IFN-α, it appears to have an additive or synergistic effect in combination (48,49). IFN-λ1 (IL29) has also recently been shown to have potent antiviral activity in a phase 1 study of patients with genotype 1 chronic hepatitis C, although there is no data as yet on whether host IL28B genotype is relevant to its the therapeutic effect (50).
How is IL28B polymorphism linked to improved treatment induced and spontaneous clearance? At this stage the answer remains unknown, although hypotheses proliferate. It would seem reasonable that as a starting point, IL28B polymorphism may affect the IFN-λ system. The complexity of interaction between HCV and the host immune system, and the redundancy within innate and cell-mediated immune systems suggests that this effect will likely be iterative and multileveled. Although the mechanism underlying the association between IL28B polymorphism and HCV treatment response/spontaneous clearance is yet to be resolved, the observation has great biological plausibility, and identifies the IFN-λ axis as an important new area for translational research.
What do these findings mean for clinicians managing patients with chronic hepatitis C?
A commercial test is now available for IL28B, but how should this information be used to improve care for patients? Patients may be "profiled" based on IL28B genotype and other important clinical features (such as fibrosis, age, insulin resistance, viral load, and race) to better predict treatment response (Table 1). Host IL28B genotype is the strongest pretreatment predictor of response through its effect on viral kinetics (33). On-treatment viral kinetics (currently RVR or early viral response, but potentially even earlier measures) provides direct measurement of treatment response and remains the most powerful on-treatment response predictor and the key criteria for on-treatment therapy decisions. Importantly, host genotype also adds to current response-guided decision algorithms. The IL28B CC genotype identifies a subgroup of patients without RVR who are more likely to achieve SVR. When new DAAV agents in development become available, such profiles may also help both in the choice of treatment regimen and its anticipated duration, personalizing therapy further.
CONCLUSION:
GWAS (Genome-wide association studies) have identified a strong association between host IL28B genotype and response to treatment with pegIFN/RBV for patients with genotype 1 chronic hepatitis C. Its differential racial distribution explains much of observed clinical differences in response between races. Although this discovery has great biological plausibility, the causal variant is yet to be identified. The nature of the interaction between type I and type III IFN signaling needs to be further elucidated and should be a focus for research.
The decision to treat remains complex. Genotyping of this polymorphism will aid clinical decision making for both current standard of care and potentially for the integration of other agents in the future, providing an opportunity for clinicians to individualize treatment regimens for hepatitis C patients.
Source
New protease inhibitors for HCV - Help is on the way
Jnl of Hepatology March 2011
Articles in Press
Uncorrected Proof
Dahlene N. Fusco, Raymond T. Chung
Massachusetts General Hospital, GI Unit, 55 Fruit Street, Warren 1007, Boston, MA 02114, USA
" HCV DAAs (direct-acting antivirals in the development pipeline include inhibitors of the viral NS3/4A protease and the viral NS5B RNA-dependent RNA polymerase [2]. Inhibitors of the NS3/4A protease are particularly attractive, since these drugs may have the double-edged action of not only directly inhibiting HCV replication, but also abrogating HCV NS3/4A's subversion of the innate immune response [6]. The NS3/4A compounds currently furthest along in development, the linear protease inhibitors (PIs) boceprevir (Merck) and telaprevir (VX 950, Vertex)......represent an important step forward toward the eradication of HCV [19]. Their major contributions include potent antiviral effects with a lower pill burden and manageable side effect profiles. .....Great care will be necessary to counsel against unnecessary therapy in persons who have low likelihood of responding to PEG/RBV and a PI, including those persons with prior null response to PEG/RBV, and limited hepatic fibrosis. We will therefore likely require a substantial re-education campaign for providers in the coming years. The future appears bright, provided we proceed with an appropriate measure of circumspection......For the particular case of prior null responders, added caution will be required, since retreatment with a single DAA added to SOC may amount to the functional equivalent of DAA monotherapy. Indeed, phase 2 and 3 data in null responder populations have shown high rates of residual viremia enriched for NS3/4A mutations. The success rates of about 30% SVR for prior null responders treated with PEG/RBV and telaprevir suggests that only those null responders with more advanced histologic disease should be offered the combination of PEG/RBV/PI therapy [20]. For prior null responders, referral to a clinical trial including at least two DAA agents with PEG and RBV may need to be considered. While sequencing studies have suggested the gradual regression of most PI-resistant variants to wildtype virus over time [14], the long term implications of these findings is unclear. For instance, it is unknown how quickly PI-resistant virus will be selected with re-exposure to PI-containing regimens. The clonal sequencing of viral populations using sensitive measures such as ultradeep pyrosequencing will help to clarify the correlates of rapid re-selection of resistance. The additional finding that some PI-resistant variants may retain fitness comparable to wildtype virus [15] bolsters the argument that monotherapy, even for a short time, could limit future treatment options for patients enrolled in clinical trials."
CV infects an estimated 170 million people and is the leading indication for liver transplantation. For patients harboring infection with HCV-1, the predominant genotype worldwide, treatment with peginterferon and ribavirin produces sustained virologic response (SVR) in only 45% of patients and is limited by substantial side effects [1]. In the coming months, HCV therapy will enter a new era with the introduction of direct acting antiviral agents (DAAs) [2], [3], [4], [5].
The most advanced HCV DAAs in the development pipeline include inhibitors of the viral NS3/4A protease and the viral NS5B RNA-dependent RNA polymerase [2]. Inhibitors of the NS3/4A protease are particularly attractive, since these drugs may have the double-edged action of not only directly inhibiting HCV replication, but also abrogating HCV NS3/4A's subversion of the innate immune response [6]. The NS3/4A compounds currently furthest along in development, the linear protease inhibitors (PIs) boceprevir (Merck) and telaprevir (VX 950, Vertex), have shown that they can substantially improve SVR when used in combination with peginterferon and ribavirin, and may also permit truncation of therapy [7], [8], [9]. Phase 3 trials of these PIs have been recently completed [2], and their results are anxiously awaited by patients and HCV providers alike. The arrival of these drugs heralds a major advance for HCV therapy, though they are associated with important limitations. Both agents require thrice daily dosing; telaprevir has also been associated with skin reactions, and boceprevir with anemia and dysgeusia. As DAAs, both drugs are associated with rapid development of resistance mutations when given as monotherapy [7], [8], [9] (Table 1).
Table 1.Comparison of 1st and 2nd generation HCV NS3/4A protease inhibitors. Seiwert et al. Antimicrobial Agents and Chemotherapeutics 2008. % Susser et al. Hepatology 2009.
Two phase 1b studies of new NS3/4A protease inhibitors published in this issue of Journal of Hepatology [10], [11] provide promising evidence that HCV treatment options will continue to improve. Forestier et al. present phase 1b study results for the NS3/4A protease inhibitor danoprevir (ITMN 191/RG7227). Danoprevir is a macrocyclic PI notable for its high specificity [12], [13] and high hepatic concentrations [13]. Their study evaluated safety, pharmacokinetics, and antiviral activity of 14days of danoprevir monotherapy administered two or three times daily in multiple ascending doses versus placebo in two groups: 40 treatment naïve genotype 1 infected patients (TNs) and 10 peginterferon and ribavirin non-responders (NRs). Subjects were predominantly male, non-obese, and non-cirrhotic. Danoprevir monotherapy was safe and well tolerated in both groups, with limited side effects. HCV RNA declines occurred in all treatment groups and were dose dependent, with median maximal declines ranging from 1.8 to 3.9logs. Viral kinetics revealed that, overall, 27% of patients experienced viral rebound, 35% plateau, and 38% continuous viral decline, including 3/8 prior non-responders, although nonresponse category (e.g., null versus partial versus relapser) was not specified.
As anticipated, resistance mutations were detected. All patients with virologic rebound, including GT1b patients, selected R155K mutations; another two also had D168V/E/T mutations. Half of patients who experienced virologic plateau selected R155K or D168V/E/T mutations. Not surprisingly, GT1a patients exhibited rebound more often and selected a higher frequency of R155K mutations, likely since 1a virus requires only one nucleotide substitution to generate this mutant, whereas 1b requires two substitutions [14]. Of note, R155K mutants, which may confer cross class resistance [15], persisted following treatment cessation. This finding is consistent with the report that these mutants may display fitness resembling that of wild type HCV [15]. Conversely, D168 mutations may lead to macrocyclic PI resistance but may actually increase susceptibility to linear PIs [15]. These patterns will need to be confirmed with longer term follow-up. Finally, Forestier et al. observed an inferior virologic response in NRs versus TNs, despite equal to higher drug exposure in NRs. This phenomenon suggests an important role for the innate immune response, which is likely to be impaired in most peginterferon and ribavirin non-responders, and may still be critical even in the setting of monotherapy with DAAs. NS3/4A inhibitors may unmask an innate immune response ordinarily suppressed by the NS3/4A protein [6]. This liberated immune response may vary significantly from patient to patient, and may or may not correlate with IL28B genotype [16].
While it will be important to track the results of ongoing phase 2b studies of new generation PIs combined with standard of care, the greatest focus will and should be placed on efforts to combine these agents with other DAA classes, particularly nucleoside HCV NS5b polymerase inhibitors, a DAA class that offers an exceptionally high barrier to resistance [17], [18].
Also in this issue, Manns et al. present a phase 1b multiple ascending dose study of a second generation NS3/4A inhibitor, BI201335, in 34 treatment naïve patients and 19 treatment experienced patients with genotype 1 HCV [11]. Patients were middle aged, non-obese Caucasians and Asians. One major advantage of BI201335 is its once daily dosing schedule. In contrast to the study of Forestier, which was confined to study of monotherapy, Manns et al. evaluated monotherapy followed by combination therapy (BI201335/pegylated interferon/ribavirin) in treatment of naïve patients with >=1log HCV decline after 14days of DAA. Treatment experienced patients received combined therapy only, with no monotherapy phase. Ninety-six percent of patients receiving BI120335 monotherapy achieved a >=2log HCV RNA decline at some point, though, as with danoprevir, monotherapy was associated with the frequent selection of resistance mutations, including R155K and D168V/E. In contrast to danoprevir, however, B1201335 resistance mutations stratified almost completely by HCV subtype (1a developed R155, 1b developed D168V/E), again consistent with a lower barrier to selection of R155K in 1a infection [14]. Side effects were mild, although rash was reported in 4/34 patients on DAA with SOC, and the authors cite a slightly increased frequency of rash in ongoing phase II trials. In addition, indirect hyperbilirubinemia was detected in patients on high dose mono- and combination therapy; the mechanism underlying this phenomenon is currently under investigation [11].
Overall, these two DAA studies represent an important step forward toward the eradication of HCV [19]. Their major contributions include potent antiviral effects with a lower pill burden and manageable side effect profiles. The major drawback of these drugs, as expected, is selection of resistant variants during monotherapy. While monotherapy studies are important for the evaluation of safety, it is clear that monotherapy is not a viable long term strategy, and that DAAs will for now need to be added onto a backbone of peginterferon and ribavirin. For the particular case of prior null responders, added caution will be required, since retreatment with a single DAA added to SOC may amount to the functional equivalent of DAA monotherapy. Indeed, phase 2 and 3 data in null responder populations have shown high rates of residual viremia enriched for NS3/4A mutations. The success rates of about 30% SVR for prior null responders treated with PEG/RBV and telaprevir suggests that only those null responders with more advanced histologic disease should be offered the combination of PEG/RBV/PI therapy [20]. For prior null responders, referral to a clinical trial including at least two DAA agents with PEG and RBV may need to be considered. While sequencing studies have suggested the gradual regression of most PI-resistant variants to wildtype virus over time [14], the long term implications of these findings is unclear. For instance, it is unknown how quickly PI-resistant virus will be selected with re-exposure to PI-containing regimens. The clonal sequencing of viral populations using sensitive measures such as ultradeep pyrosequencing will help to clarify the correlates of rapid re-selection of resistance. The additional finding that some PI-resistant variants may retain fitness comparable to wildtype virus [15] bolsters the argument that monotherapy, even for a short time, could limit future treatment options for patients enrolled in clinical trials.
In summary, the two studies that have been described provide further welcome news that HCV protease inhibitors with improved side effect profiles and decreased dose frequency are on the horizon, just behind the first generation of PIs. We can anticipate that these agents will eventually supplant first generation PIs not only in combination with PEG and RBV, but, more importantly, as cardinal components of future IFN-sparing DAA cocktails. These considerations place a premium on both adherence and tolerability. In 2011 and beyond, HCV providers must now rise to the challenge of quickly assimilating and carefully applying DAA data as it unfolds. Great care will be necessary to counsel against unnecessary therapy in persons who have low likelihood of responding to PEG/RBV and a PI, including those persons with prior null response to PEG/RBV, and limited hepatic fibrosis. We will therefore likely require a substantial re-education campaign for providers in the coming years. The future appears bright, provided we proceed with an appropriate measure of circumspection.
Source
Articles in Press
Uncorrected Proof
Dahlene N. Fusco, Raymond T. Chung
Massachusetts General Hospital, GI Unit, 55 Fruit Street, Warren 1007, Boston, MA 02114, USA
" HCV DAAs (direct-acting antivirals in the development pipeline include inhibitors of the viral NS3/4A protease and the viral NS5B RNA-dependent RNA polymerase [2]. Inhibitors of the NS3/4A protease are particularly attractive, since these drugs may have the double-edged action of not only directly inhibiting HCV replication, but also abrogating HCV NS3/4A's subversion of the innate immune response [6]. The NS3/4A compounds currently furthest along in development, the linear protease inhibitors (PIs) boceprevir (Merck) and telaprevir (VX 950, Vertex)......represent an important step forward toward the eradication of HCV [19]. Their major contributions include potent antiviral effects with a lower pill burden and manageable side effect profiles. .....Great care will be necessary to counsel against unnecessary therapy in persons who have low likelihood of responding to PEG/RBV and a PI, including those persons with prior null response to PEG/RBV, and limited hepatic fibrosis. We will therefore likely require a substantial re-education campaign for providers in the coming years. The future appears bright, provided we proceed with an appropriate measure of circumspection......For the particular case of prior null responders, added caution will be required, since retreatment with a single DAA added to SOC may amount to the functional equivalent of DAA monotherapy. Indeed, phase 2 and 3 data in null responder populations have shown high rates of residual viremia enriched for NS3/4A mutations. The success rates of about 30% SVR for prior null responders treated with PEG/RBV and telaprevir suggests that only those null responders with more advanced histologic disease should be offered the combination of PEG/RBV/PI therapy [20]. For prior null responders, referral to a clinical trial including at least two DAA agents with PEG and RBV may need to be considered. While sequencing studies have suggested the gradual regression of most PI-resistant variants to wildtype virus over time [14], the long term implications of these findings is unclear. For instance, it is unknown how quickly PI-resistant virus will be selected with re-exposure to PI-containing regimens. The clonal sequencing of viral populations using sensitive measures such as ultradeep pyrosequencing will help to clarify the correlates of rapid re-selection of resistance. The additional finding that some PI-resistant variants may retain fitness comparable to wildtype virus [15] bolsters the argument that monotherapy, even for a short time, could limit future treatment options for patients enrolled in clinical trials."
CV infects an estimated 170 million people and is the leading indication for liver transplantation. For patients harboring infection with HCV-1, the predominant genotype worldwide, treatment with peginterferon and ribavirin produces sustained virologic response (SVR) in only 45% of patients and is limited by substantial side effects [1]. In the coming months, HCV therapy will enter a new era with the introduction of direct acting antiviral agents (DAAs) [2], [3], [4], [5].
The most advanced HCV DAAs in the development pipeline include inhibitors of the viral NS3/4A protease and the viral NS5B RNA-dependent RNA polymerase [2]. Inhibitors of the NS3/4A protease are particularly attractive, since these drugs may have the double-edged action of not only directly inhibiting HCV replication, but also abrogating HCV NS3/4A's subversion of the innate immune response [6]. The NS3/4A compounds currently furthest along in development, the linear protease inhibitors (PIs) boceprevir (Merck) and telaprevir (VX 950, Vertex), have shown that they can substantially improve SVR when used in combination with peginterferon and ribavirin, and may also permit truncation of therapy [7], [8], [9]. Phase 3 trials of these PIs have been recently completed [2], and their results are anxiously awaited by patients and HCV providers alike. The arrival of these drugs heralds a major advance for HCV therapy, though they are associated with important limitations. Both agents require thrice daily dosing; telaprevir has also been associated with skin reactions, and boceprevir with anemia and dysgeusia. As DAAs, both drugs are associated with rapid development of resistance mutations when given as monotherapy [7], [8], [9] (Table 1).
Table 1.Comparison of 1st and 2nd generation HCV NS3/4A protease inhibitors. Seiwert et al. Antimicrobial Agents and Chemotherapeutics 2008. % Susser et al. Hepatology 2009.
Two phase 1b studies of new NS3/4A protease inhibitors published in this issue of Journal of Hepatology [10], [11] provide promising evidence that HCV treatment options will continue to improve. Forestier et al. present phase 1b study results for the NS3/4A protease inhibitor danoprevir (ITMN 191/RG7227). Danoprevir is a macrocyclic PI notable for its high specificity [12], [13] and high hepatic concentrations [13]. Their study evaluated safety, pharmacokinetics, and antiviral activity of 14days of danoprevir monotherapy administered two or three times daily in multiple ascending doses versus placebo in two groups: 40 treatment naïve genotype 1 infected patients (TNs) and 10 peginterferon and ribavirin non-responders (NRs). Subjects were predominantly male, non-obese, and non-cirrhotic. Danoprevir monotherapy was safe and well tolerated in both groups, with limited side effects. HCV RNA declines occurred in all treatment groups and were dose dependent, with median maximal declines ranging from 1.8 to 3.9logs. Viral kinetics revealed that, overall, 27% of patients experienced viral rebound, 35% plateau, and 38% continuous viral decline, including 3/8 prior non-responders, although nonresponse category (e.g., null versus partial versus relapser) was not specified.
As anticipated, resistance mutations were detected. All patients with virologic rebound, including GT1b patients, selected R155K mutations; another two also had D168V/E/T mutations. Half of patients who experienced virologic plateau selected R155K or D168V/E/T mutations. Not surprisingly, GT1a patients exhibited rebound more often and selected a higher frequency of R155K mutations, likely since 1a virus requires only one nucleotide substitution to generate this mutant, whereas 1b requires two substitutions [14]. Of note, R155K mutants, which may confer cross class resistance [15], persisted following treatment cessation. This finding is consistent with the report that these mutants may display fitness resembling that of wild type HCV [15]. Conversely, D168 mutations may lead to macrocyclic PI resistance but may actually increase susceptibility to linear PIs [15]. These patterns will need to be confirmed with longer term follow-up. Finally, Forestier et al. observed an inferior virologic response in NRs versus TNs, despite equal to higher drug exposure in NRs. This phenomenon suggests an important role for the innate immune response, which is likely to be impaired in most peginterferon and ribavirin non-responders, and may still be critical even in the setting of monotherapy with DAAs. NS3/4A inhibitors may unmask an innate immune response ordinarily suppressed by the NS3/4A protein [6]. This liberated immune response may vary significantly from patient to patient, and may or may not correlate with IL28B genotype [16].
While it will be important to track the results of ongoing phase 2b studies of new generation PIs combined with standard of care, the greatest focus will and should be placed on efforts to combine these agents with other DAA classes, particularly nucleoside HCV NS5b polymerase inhibitors, a DAA class that offers an exceptionally high barrier to resistance [17], [18].
Also in this issue, Manns et al. present a phase 1b multiple ascending dose study of a second generation NS3/4A inhibitor, BI201335, in 34 treatment naïve patients and 19 treatment experienced patients with genotype 1 HCV [11]. Patients were middle aged, non-obese Caucasians and Asians. One major advantage of BI201335 is its once daily dosing schedule. In contrast to the study of Forestier, which was confined to study of monotherapy, Manns et al. evaluated monotherapy followed by combination therapy (BI201335/pegylated interferon/ribavirin) in treatment of naïve patients with >=1log HCV decline after 14days of DAA. Treatment experienced patients received combined therapy only, with no monotherapy phase. Ninety-six percent of patients receiving BI120335 monotherapy achieved a >=2log HCV RNA decline at some point, though, as with danoprevir, monotherapy was associated with the frequent selection of resistance mutations, including R155K and D168V/E. In contrast to danoprevir, however, B1201335 resistance mutations stratified almost completely by HCV subtype (1a developed R155, 1b developed D168V/E), again consistent with a lower barrier to selection of R155K in 1a infection [14]. Side effects were mild, although rash was reported in 4/34 patients on DAA with SOC, and the authors cite a slightly increased frequency of rash in ongoing phase II trials. In addition, indirect hyperbilirubinemia was detected in patients on high dose mono- and combination therapy; the mechanism underlying this phenomenon is currently under investigation [11].
Overall, these two DAA studies represent an important step forward toward the eradication of HCV [19]. Their major contributions include potent antiviral effects with a lower pill burden and manageable side effect profiles. The major drawback of these drugs, as expected, is selection of resistant variants during monotherapy. While monotherapy studies are important for the evaluation of safety, it is clear that monotherapy is not a viable long term strategy, and that DAAs will for now need to be added onto a backbone of peginterferon and ribavirin. For the particular case of prior null responders, added caution will be required, since retreatment with a single DAA added to SOC may amount to the functional equivalent of DAA monotherapy. Indeed, phase 2 and 3 data in null responder populations have shown high rates of residual viremia enriched for NS3/4A mutations. The success rates of about 30% SVR for prior null responders treated with PEG/RBV and telaprevir suggests that only those null responders with more advanced histologic disease should be offered the combination of PEG/RBV/PI therapy [20]. For prior null responders, referral to a clinical trial including at least two DAA agents with PEG and RBV may need to be considered. While sequencing studies have suggested the gradual regression of most PI-resistant variants to wildtype virus over time [14], the long term implications of these findings is unclear. For instance, it is unknown how quickly PI-resistant virus will be selected with re-exposure to PI-containing regimens. The clonal sequencing of viral populations using sensitive measures such as ultradeep pyrosequencing will help to clarify the correlates of rapid re-selection of resistance. The additional finding that some PI-resistant variants may retain fitness comparable to wildtype virus [15] bolsters the argument that monotherapy, even for a short time, could limit future treatment options for patients enrolled in clinical trials.
In summary, the two studies that have been described provide further welcome news that HCV protease inhibitors with improved side effect profiles and decreased dose frequency are on the horizon, just behind the first generation of PIs. We can anticipate that these agents will eventually supplant first generation PIs not only in combination with PEG and RBV, but, more importantly, as cardinal components of future IFN-sparing DAA cocktails. These considerations place a premium on both adherence and tolerability. In 2011 and beyond, HCV providers must now rise to the challenge of quickly assimilating and carefully applying DAA data as it unfolds. Great care will be necessary to counsel against unnecessary therapy in persons who have low likelihood of responding to PEG/RBV and a PI, including those persons with prior null response to PEG/RBV, and limited hepatic fibrosis. We will therefore likely require a substantial re-education campaign for providers in the coming years. The future appears bright, provided we proceed with an appropriate measure of circumspection.
Source
Direct-Acting Antiviral Therapy for Hepatitis C: Attitudes Regarding Future Use
Download the PDF here
Dig Dis Sci. 2011 Feb 19. [Epub ahead of print]
Paul J. Gaglio1, 2 Contact Information, Noah Moss1, Camille McGaw1 and John Reinus1
(1) Department of Medicine, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY, USA
(2) Montefiore Einstein Liver Center, 111 East 210 Street, Bronx, NY 10467, USA
Contact Information Paul J. Gaglio
Email: pgaglio@montefiore.org
"In summary, the responses to this Internet-based survey of more than 1,000 current HCV treaters indicated that although the majority of respondents appear ready to utilize DAA agents in the future, referrals to "hepatitis C experts" will increase when these agents become available. In addition, future referrals to ID specialists appear to be limited. Finally, as more than half of respondents to the survey with "minimal knowledge" of DAA therapies also appear to be willing to utilize these compounds in the future, significant provider education will be required to minimize inappropriate use of these agents."
"Although a significant percentage of respondents who participated in clinical trials with a DAA (90%) or who were "aware or very aware" of these agents but did not participate in a clinical trial (81%) would use these agents in the future, more than half of respondents (52%) who reported minimal knowledge of DAA agents stated that they would also use them in the future. Although providers with minimal knowledge of DAA agents represented a small percentage of respondents to the survey, concern exists regarding the inappropriate use of DAAs in the future including inexperience with side-effect management, and lack of recognition of both treatment failure as well as the emergence of viral resistance [11-16]. It is therefore apparent to the authors of this manuscript that extensive education of all future prescribers of DAA agents will be required to ensure successful use of these therapies."
"Of respondents with "minimal knowledge" of DAA, 52% stated that they would use them in the future......Overall, a significant number of respondents who treat HCV today with currently available therapies indicated that they would not prescribe DAA therapies when they became available; 85% of respondents to the survey would evaluate and treat an HCV-infected patient "today" versus 81% when DAA agents became available (p = 0.0054). Similarly, more respondents indicated that they would refer their patients to a "hepatitis C specialist" after DAA agents became available (6% current, 10% after DAA availability (p = 0.016)...........A greater percentage of AGA members (12%) stated that they would refer their patient to an "HCV expert" when DAA agents became available compared to AASLD members (3%) (p = 0.001). When analyzing attitudes related to current and future therapy of HCV based on focus of clinical practice, a comparison of Hepatologists/liver transplantation physicians to Gastroenterologists revealed that 93% versus 86% would treat their HCV-infected patient today (p = 0.0034), 48% compared to 8% participated in clinical trials using DAA agents (p = 0.001), 90% versus 81% would treat their patient with a DAA agent (p = 0.001), and 1% versus 8% would refer their patient to an "HCV expert" when DAA therapies became available (p = 0.001). Practice type was also associated with differences in attitudes related to current and future therapy of HCV. Comparison of respondents in private practice versus those who practiced at a medical school or hospital associated with a medical school revealed that respectively, 91% versus 81% would treat their HCV-infected patient today (p = 0.001), 9% versus 30% participated in a clinical trial with a DAA agent (p = 0.001), 89% versus 75% would use DAA therapies when they became available (p = 0.001), and 12% compared to 15% would refer to an "HCV expert" when DAA therapies became available (P = NS). Current awareness and participation in clinical trials with DAA agents influenced attitudes regarding future use of these therapies, as 90% of respondents who participated in clinical trials with DAAs, 81% of those who were "very aware" or "aware" of DAA agents but did not participate in clinical trials, and 59% of those with minimal knowledge of DAA therapies would prescribe these agents in the future (p = 0.0001 clinical trial participant/very aware or aware of DAA agents vs. minimal knowledge). Respondents with "minimal knowledge" of DAA agents also reported that when these compounds were available in the future, 25% would refer their HCV-infected patient to a PA, NP, or other MD in their practice, 22% would refer to an "HCV expert," and 1% would refer to an ID specialist. Respondents who reported "minimal knowledge of DAA agents" were more likely to consider the AMA their primary professional organization (20 vs. 1% AGA/AASLD (p = 0.001)), or be in a private practice not associated with a medical school (83 vs. 17% practice or hospital associated with a medical school p = 0.0001)."
Introduction
Response to current therapy of hepatitis C virus (HCV) is suboptimal. Direct-acting antiviral therapies (DAA) are expected to improve treatment outcomes. Additional treatments for HCV will invariably make therapeutic choices and patient management more complex. We hypothesize that current perceptions regarding the complexity of DAA therapy will influence attitudes towards future use by practitioners who are currently treating HCV.
Methods
An Internet-based survey was sent to 10,082 AASLD and AGA members to determine if they treat HCV infection, their knowledge of DAA therapies, attitudes towards current and future HCV treatments, and if they participated in clinical trials using DAA agents.
Results
Out of a total of 1,757 individuals responding to the survey, 75% treat HCV; 79% were MDs, 67% were Gastroenterologists, and 24% were Hepatologists. Of the respondents, 77% indicated they were "very aware" or "aware" of DAA therapies, 20% participated in clinical trials, and 3% had minimal knowledge of DAA agents. Comparing treatment "today" versus in the future when DAAs were available, 85 vs. 81% would treat (p = 0.0054), 6 vs. 10% would refer to an "HCV expert" (p = 0.016), and 1% would refer to an ID specialist. Of respondents with "minimal knowledge" of DAA, 52% stated that they would use them in the future.
Conclusions
Although the majority of respondents appear ready to utilize DAA agents in the future, referrals to "hepatitis C experts" will increase. More than half of respondents with "minimal knowledge" of DAA therapies also appear to be willing to utilize these compounds, raising concerns regarding their inappropriate use. Broad education of healthcare providers to prevent inappropriate use of these agents will be critical.
Disclaimer: The statements, findings, conclusions, views, and opinions contained and expressed in this manuscript are based in part on data obtained under license from the following IMS Health Incorporated information service(s): Prescriber Profilerª (2008-2009) IMS Health Incorporated. All Rights Reserved. The statements, findings, conclusions, views, and opinions contained and expressed herein are not necessarily those of IMS Health Incorporated or any of its affiliated or subsidiary entities. IMS Health had no role in the design and conduct of the study, collection, management, analysis, or interpretation of the data, and preparation or approval of this manuscript.
Introduction
Hepatitis C virus (HCV) represents the most common chronic blood-borne viral infection in the United States [1, 2]. At present, response to currently available therapy remains suboptimal as a significant number of patients fail to achieve a sustained virologic response to therapy [3-10]. Recent discoveries related to the life cycle and pathobiology of HCV have led to the development of novel therapies that directly inhibit viral replication. These compounds, characterized as "specifically targeted antiviral therapies against HCV" (STAT-C) or "direct-acting antiviral agents" (DAA) have been investigated in naive as well as previously treated patients, and preliminary data from these studies have been encouraging [11-14]. Along with these encouraging results, these and other publications which describe experience with DAA agents have documented the emergence of HCV resistance [15, 16], as well as significant treatment-related adverse events including rash, gastrointestinal side-effects, and anemia [11-14].
As health care providers with interest and experience in treating HCV become aware of emerging data using novel therapeutic agents, we hypothesize that current perceptions regarding the complexity and side-effects of DAA therapies will influence decisions regarding the future use of these agents. To evaluate attitudes regarding the future use of these agents by individuals who are currently treating HCV, we sent an Internet-based survey to all United States-based members of the American Gastroenterology Association (AGA) and American Association for the Study of Liver Disease (AASLD). Members of these societies were chosen because they represent both the vast majority of HCV treaters in the United States, and a population of clinicians likely to have knowledge of DAA therapies. Recipients of the survey were queried regarding their primary professional affiliation and focus of practice, attitudes towards current and future HCV therapy, as well as participation in clinical trials using DAA agents. We determined if any of these parameters affected attitudes related to current and future treatment decisions regarding HCV.
Methods
This study was reviewed and approved by the institutional review board at the Albert Einstein College of Medicine/Montefiore Medical Center. The e-mail addresses of the 10,082 US-based members of the AGA and AASLD were compiled from the 2009 member directories for both organizations. AGA and AASLD members were selected to be surveyed as they represent the majority of HCV treaters in the United States. Prescriber ProfilerTM data provided to the authors by IMS Health Incorporated, reflecting a US-based database of retail pharmacies and total dispensed prescriptions of CopegusTM, Intron-ATM, InfergenTM, PegasysTM, Peg-IntronTM, RebetolTM, RebetronTM, RibasphereTM, and RibavirinTM indicated that between December 2008 and November 2009, 177,300 prescriptions were written. Gastroenterologists or Hepatologists wrote approximately 94,000 or 55% of these, validating our hypothesis that the targeted survey recipients were a group which treated HCV most frequently. Internal medicine physicians (11%) and nurse practitioners (8%) were the next most common prescribers [17]. Using an Internet-based survey engine ("SurveyMonkey" (surveymonkey.com)) a nine-question survey was sent to each AGA or AASLD member; only one questionnaire was sent to individuals who are members of both organizations. If an individual did not respond to the survey, the survey was re-sent with a second request to complete the survey. All results were tabulated, and statistical analysis was performed using Stata version 9.2, Statcorp LP, College Station, Texas.
Results
Of the 10,082 surveys sent, 8,449 were deliverable. The most common reasons for inability to deliver a survey included use of an e-mail filter or vacation message by the intended recipient. A total 1,757 individuals responded to the survey, representing a 21% response rate. A recent analysis performed by supersurvey.com revealed a mean response rate of 18% to online surveys of similar question size and target audience (www.supersurvey.com). The survey questions and responses appear in Table 1. If a respondent stated that they did not treat HCV, their participation in the survey ended after question 1.
Of the respondents, 75% (1,320) stated that they treat HCV, and of these respondents, 79% were MDs, 10% were physician assistants or nurse practitioners, 8% MD-PhDs, 2% DO, and 1% PhD. Of the respondents, 32% graduated within the last 10 years, 25% 11-20 years ago, 21% 21-30 years ago, and 12% greater than 30 years ago.
When analyzing results based on focus of practice, 67% of respondents stated that gastroenterology was the primary focus of their clinical practice, 24% selected hepatology and/or liver transplantation, 2% infectious disease, and 7% stated "other" as the primary focus of their practice. The "other" respondents included primary care doctors, surgeons, as well as Gastroenterologists who considered hepato-biliary disease, oncology, or pancreatic disease their primary specialty. Forty-six percent of the respondents were in a private practice not associated with a medical school, 42% at a medical school or hospital associated with a medical school, 8% in a private practice associated with a medical school, and the remaining 4% of respondents practiced in a multispecialty group practice, Veterans Administration hospital, or hospital not associated with a medical school.
Professional affiliations were assessed; 55% of respondents considered the American Gastroenterological Association (AGA) their primary professional affiliation, 26% the American Association for the Study of Liver Disease (AASLD), 9% the American Society for Gastrointestinal Endoscopy (ASGE), and 10% "other" including the American College of Gastroenterology (ACG), American Medical Association (AMA), and the American Society for Transplantation (AST).
When queried regarding what they would do when presented with an HCV-infected patient "today," 85% of the respondents would treat them, 6% would refer them to a "hepatitis C expert," 4% would refer them to a physician extender (PA, NP, or specially trained nurse) in their practice, 4% would refer to another MD in their practice, and 1% would refer them to an infectious disease specialist. Related to future therapies for HCV including DAAs in combination with interferon and ribavirin, 77% of the respondents were "aware" or "very aware" of this concept but had not participated in any clinical trials using DAA agents, 20% were "very aware" and had experience using these agents in clinical trials, and 3% had "minimal knowledge" of DAA agents.
When queried regarding treatment approaches if a DAA agent were available "today," 81% of respondents would evaluate and treat the patient, 10% would refer the patient to a "hepatitis C expert," 5% would refer the patient to another physician in their group, and 4% would refer the patient to a physician extender (PA, NP, or specially trained nurse) in their practice. Less than 1% of respondents reported that they would refer the patient to an infectious disease specialist.
Analysis of survey results (Table 2....
Overall, a significant number of respondents who treat HCV today with currently available therapies indicated that they would not prescribe DAA therapies when they became available; 85% of respondents to the survey would evaluate and treat an HCV-infected patient "today" versus 81% when DAA agents became available (p = 0.0054). Similarly, more respondents indicated that they would refer their patients to a "hepatitis C specialist" after DAA agents became available (6% current, 10% after DAA availability (p = 0.016).
Significant differences existed based on primary professional affiliation in attitudes and experience of survey respondents related to present and future HCV therapy. Ninety-one percent of respondents who considered the AASLD their primary affiliation compared to 84% of AGA members would treat their HCV-infected patient today (p = 0.002), 48% of AASLD members versus 9% of AGA members participated in clinical trials with a DAA agent (p = 0001), and 91% of AASLD members versus 79% of AGA members would use a DAA agent to treat their HCV-infected patient in the future (p = 0.001). A greater percentage of AGA members (12%) stated that they would refer their patient to an "HCV expert" when DAA agents became available compared to AASLD members (3%) (p = 0.001). When analyzing attitudes related to current and future therapy of HCV based on focus of clinical practice, a comparison of Hepatologists/liver transplantation physicians to Gastroenterologists revealed that 93% versus 86% would treat their HCV-infected patient today (p = 0.0034), 48% compared to 8% participated in clinical trials using DAA agents (p = 0.001), 90% versus 81% would treat their patient with a DAA agent (p = 0.001), and 1% versus 8% would refer their patient to an "HCV expert" when DAA therapies became available (p = 0.001).
Practice type was also associated with differences in attitudes related to current and future therapy of HCV. Comparison of respondents in private practice versus those who practiced at a medical school or hospital associated with a medical school revealed that respectively, 91% versus 81% would treat their HCV-infected patient today (p = 0.001), 9% versus 30% participated in a clinical trial with a DAA agent (p = 0.001), 89% versus 75% would use DAA therapies when they became available (p = 0.001), and 12% compared to 15% would refer to an "HCV expert" when DAA therapies became available (P = NS).
Current awareness and participation in clinical trials with DAA agents influenced attitudes regarding future use of these therapies, as 90% of respondents who participated in clinical trials with DAAs, 81% of those who were "very aware" or "aware" of DAA agents but did not participate in clinical trials, and 59% of those with minimal knowledge of DAA therapies would prescribe these agents in the future (p = 0.0001 clinical trial participant/very aware or aware of DAA agents vs. minimal knowledge). Respondents with "minimal knowledge" of DAA agents also reported that when these compounds were available in the future, 25% would refer their HCV-infected patient to a PA, NP, or other MD in their practice, 22% would refer to an "HCV expert," and 1% would refer to an ID specialist. Respondents who reported "minimal knowledge of DAA agents" were more likely to consider the AMA their primary professional organization (20 vs. 1% AGA/AASLD (p = 0.001)), or be in a private practice not associated with a medical school (83 vs. 17% practice or hospital associated with a medical school p = 0.0001).
Discussion
Emerging data suggest that direct-acting antiviral therapies against HCV (DAA) will provide improved response rates when given in combination with currently available therapies. The enthusiasm for these new treatments must be tempered by realistic concerns including side-effects as well as the threat of viral resistance induced by these agents. We hypothesized that concern regarding these issues might affect attitudes of future use of DAA therapies by health care providers who currently treat HCV. The goal of the current study was to query a group of experienced HCV treaters using an Internet-based survey to assess attitudes regarding current and future treatment of HCV and correlate these responses related to focus of clinical practice, academic versus private practice, professional affiliation, and experience with DAA agents in clinical trials. US-based members of the AGA and AASLD were targeted for the survey as they represent the majority of HCV treaters in the US and a group most likely to have knowledge of DAA therapy. This manuscript represents the first description of attitudes regarding future use of DAA agents in a large group of experienced HCV treaters.
Based on responses to this survey, although the majority of current HCV treaters would prescribe a DAA agent, a significant number of respondents who treat HCV today (85%) would not initiate therapy when these agents became available in the future (81%). The decreased use of DAA therapies may be offset somewhat by an increase in referrals to a more experienced HCV treater, as more respondents would refer their patients to a "hepatitis C expert" after DAA agents became available in the future compared to referring patients if they were diagnosed with HCV today (10% versus 6%). Not surprisingly, future use of DAA therapy appears to be significant in treaters who identify themselves as Hepatologists, and those with experience using these agents in clinical trials. Moreover, direct clinical experience with DAAs did not appear to dampen the enthusiasm for future use of these agents, as 90% of respondents who participated in clinical trials with a DAA agent would utilize these agents to treat an HCV-infected patient when these agents became available.
We hypothesized that as infectious disease specialists have extensive experience prescribing oral antiviral agents for HIV, referrals to these providers would increase when DAA agents became available for HCV. Our hypothesis was clearly inaccurate as respondents to the survey were unlikely to refer their HCV-infected patients to an infectious disease specialist when faced with an HCV-infected patient today (1%) and when DAA agents were available in the future (0.8%). This trend was maintained even in respondents with minimal knowledge of DAA agents. Finally, a concerning observation was identified when assessing responses to the survey stratified by past experience with DAA therapy. Although a significant percentage of respondents who participated in clinical trials with a DAA (90%) or who were "aware or very aware" of these agents but did not participate in a clinical trial (81%) would use these agents in the future, more than half of respondents (52%) who reported minimal knowledge of DAA agents stated that they would also use them in the future. Although providers with minimal knowledge of DAA agents represented a small percentage of respondents to the survey, concern exists regarding the inappropriate use of DAAs in the future including inexperience with side-effect management, and lack of recognition of both treatment failure as well as the emergence of viral resistance [11-16]. It is therefore apparent to the authors of this manuscript that extensive education of all future prescribers of DAA agents will be required to ensure successful use of these therapies.
There are limitations of this study that are unfortunately shared by other surveys utilizing similar data collection and analyses techniques. Any Internet-based survey is limited by response rate, which is affected both by the ability to deliver the survey and the willingness of the survey recipient to accurately respond to the queries posed to them. Although 21% of recipients of the survey responded, it is possible that the identified results would have been different if more individuals completed the survey. Unfortunately, the large volume of unsolicited e-mail has induced the use of blockers and filters, thus potentially limiting the response rates to the survey. However, we believe that these limitations were balanced by several strengths including the overall brevity of the survey (nine questions) increasing the likelihood that those who received and opened the survey fully completed it, and by the target audience which represented a significant number of current HCV treaters with the highest likelihood of having knowledge regarding DAA agents.
In summary, the responses to this Internet-based survey of more than 1,000 current HCV treaters indicated that although the majority of respondents appear ready to utilize DAA agents in the future, referrals to "hepatitis C experts" will increase when these agents become available. In addition, future referrals to ID specialists appear to be limited. Finally, as more than half of respondents to the survey with "minimal knowledge" of DAA therapies also appear to be willing to utilize these compounds in the future, significant provider education will be required to minimize inappropriate use of these agents.
Conflict of interest Paul J. Gaglio, MD, Speakers Bureau, Merck; has received study support from Schering Plough (now Merck) and Vertex Pharmaceuticals. Noah Moss, MD: No Camille Baugh, MD: No Disclosures Noah Moss, MD: No Disclosures John Reinus, MD has received study support from Vertex Pharmaceuticals.
Source
Dig Dis Sci. 2011 Feb 19. [Epub ahead of print]
Paul J. Gaglio1, 2 Contact Information, Noah Moss1, Camille McGaw1 and John Reinus1
(1) Department of Medicine, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY, USA
(2) Montefiore Einstein Liver Center, 111 East 210 Street, Bronx, NY 10467, USA
Contact Information Paul J. Gaglio
Email: pgaglio@montefiore.org
"In summary, the responses to this Internet-based survey of more than 1,000 current HCV treaters indicated that although the majority of respondents appear ready to utilize DAA agents in the future, referrals to "hepatitis C experts" will increase when these agents become available. In addition, future referrals to ID specialists appear to be limited. Finally, as more than half of respondents to the survey with "minimal knowledge" of DAA therapies also appear to be willing to utilize these compounds in the future, significant provider education will be required to minimize inappropriate use of these agents."
"Although a significant percentage of respondents who participated in clinical trials with a DAA (90%) or who were "aware or very aware" of these agents but did not participate in a clinical trial (81%) would use these agents in the future, more than half of respondents (52%) who reported minimal knowledge of DAA agents stated that they would also use them in the future. Although providers with minimal knowledge of DAA agents represented a small percentage of respondents to the survey, concern exists regarding the inappropriate use of DAAs in the future including inexperience with side-effect management, and lack of recognition of both treatment failure as well as the emergence of viral resistance [11-16]. It is therefore apparent to the authors of this manuscript that extensive education of all future prescribers of DAA agents will be required to ensure successful use of these therapies."
"Of respondents with "minimal knowledge" of DAA, 52% stated that they would use them in the future......Overall, a significant number of respondents who treat HCV today with currently available therapies indicated that they would not prescribe DAA therapies when they became available; 85% of respondents to the survey would evaluate and treat an HCV-infected patient "today" versus 81% when DAA agents became available (p = 0.0054). Similarly, more respondents indicated that they would refer their patients to a "hepatitis C specialist" after DAA agents became available (6% current, 10% after DAA availability (p = 0.016)...........A greater percentage of AGA members (12%) stated that they would refer their patient to an "HCV expert" when DAA agents became available compared to AASLD members (3%) (p = 0.001). When analyzing attitudes related to current and future therapy of HCV based on focus of clinical practice, a comparison of Hepatologists/liver transplantation physicians to Gastroenterologists revealed that 93% versus 86% would treat their HCV-infected patient today (p = 0.0034), 48% compared to 8% participated in clinical trials using DAA agents (p = 0.001), 90% versus 81% would treat their patient with a DAA agent (p = 0.001), and 1% versus 8% would refer their patient to an "HCV expert" when DAA therapies became available (p = 0.001). Practice type was also associated with differences in attitudes related to current and future therapy of HCV. Comparison of respondents in private practice versus those who practiced at a medical school or hospital associated with a medical school revealed that respectively, 91% versus 81% would treat their HCV-infected patient today (p = 0.001), 9% versus 30% participated in a clinical trial with a DAA agent (p = 0.001), 89% versus 75% would use DAA therapies when they became available (p = 0.001), and 12% compared to 15% would refer to an "HCV expert" when DAA therapies became available (P = NS). Current awareness and participation in clinical trials with DAA agents influenced attitudes regarding future use of these therapies, as 90% of respondents who participated in clinical trials with DAAs, 81% of those who were "very aware" or "aware" of DAA agents but did not participate in clinical trials, and 59% of those with minimal knowledge of DAA therapies would prescribe these agents in the future (p = 0.0001 clinical trial participant/very aware or aware of DAA agents vs. minimal knowledge). Respondents with "minimal knowledge" of DAA agents also reported that when these compounds were available in the future, 25% would refer their HCV-infected patient to a PA, NP, or other MD in their practice, 22% would refer to an "HCV expert," and 1% would refer to an ID specialist. Respondents who reported "minimal knowledge of DAA agents" were more likely to consider the AMA their primary professional organization (20 vs. 1% AGA/AASLD (p = 0.001)), or be in a private practice not associated with a medical school (83 vs. 17% practice or hospital associated with a medical school p = 0.0001)."
Introduction
Response to current therapy of hepatitis C virus (HCV) is suboptimal. Direct-acting antiviral therapies (DAA) are expected to improve treatment outcomes. Additional treatments for HCV will invariably make therapeutic choices and patient management more complex. We hypothesize that current perceptions regarding the complexity of DAA therapy will influence attitudes towards future use by practitioners who are currently treating HCV.
Methods
An Internet-based survey was sent to 10,082 AASLD and AGA members to determine if they treat HCV infection, their knowledge of DAA therapies, attitudes towards current and future HCV treatments, and if they participated in clinical trials using DAA agents.
Results
Out of a total of 1,757 individuals responding to the survey, 75% treat HCV; 79% were MDs, 67% were Gastroenterologists, and 24% were Hepatologists. Of the respondents, 77% indicated they were "very aware" or "aware" of DAA therapies, 20% participated in clinical trials, and 3% had minimal knowledge of DAA agents. Comparing treatment "today" versus in the future when DAAs were available, 85 vs. 81% would treat (p = 0.0054), 6 vs. 10% would refer to an "HCV expert" (p = 0.016), and 1% would refer to an ID specialist. Of respondents with "minimal knowledge" of DAA, 52% stated that they would use them in the future.
Conclusions
Although the majority of respondents appear ready to utilize DAA agents in the future, referrals to "hepatitis C experts" will increase. More than half of respondents with "minimal knowledge" of DAA therapies also appear to be willing to utilize these compounds, raising concerns regarding their inappropriate use. Broad education of healthcare providers to prevent inappropriate use of these agents will be critical.
Disclaimer: The statements, findings, conclusions, views, and opinions contained and expressed in this manuscript are based in part on data obtained under license from the following IMS Health Incorporated information service(s): Prescriber Profilerª (2008-2009) IMS Health Incorporated. All Rights Reserved. The statements, findings, conclusions, views, and opinions contained and expressed herein are not necessarily those of IMS Health Incorporated or any of its affiliated or subsidiary entities. IMS Health had no role in the design and conduct of the study, collection, management, analysis, or interpretation of the data, and preparation or approval of this manuscript.
Introduction
Hepatitis C virus (HCV) represents the most common chronic blood-borne viral infection in the United States [1, 2]. At present, response to currently available therapy remains suboptimal as a significant number of patients fail to achieve a sustained virologic response to therapy [3-10]. Recent discoveries related to the life cycle and pathobiology of HCV have led to the development of novel therapies that directly inhibit viral replication. These compounds, characterized as "specifically targeted antiviral therapies against HCV" (STAT-C) or "direct-acting antiviral agents" (DAA) have been investigated in naive as well as previously treated patients, and preliminary data from these studies have been encouraging [11-14]. Along with these encouraging results, these and other publications which describe experience with DAA agents have documented the emergence of HCV resistance [15, 16], as well as significant treatment-related adverse events including rash, gastrointestinal side-effects, and anemia [11-14].
As health care providers with interest and experience in treating HCV become aware of emerging data using novel therapeutic agents, we hypothesize that current perceptions regarding the complexity and side-effects of DAA therapies will influence decisions regarding the future use of these agents. To evaluate attitudes regarding the future use of these agents by individuals who are currently treating HCV, we sent an Internet-based survey to all United States-based members of the American Gastroenterology Association (AGA) and American Association for the Study of Liver Disease (AASLD). Members of these societies were chosen because they represent both the vast majority of HCV treaters in the United States, and a population of clinicians likely to have knowledge of DAA therapies. Recipients of the survey were queried regarding their primary professional affiliation and focus of practice, attitudes towards current and future HCV therapy, as well as participation in clinical trials using DAA agents. We determined if any of these parameters affected attitudes related to current and future treatment decisions regarding HCV.
Methods
This study was reviewed and approved by the institutional review board at the Albert Einstein College of Medicine/Montefiore Medical Center. The e-mail addresses of the 10,082 US-based members of the AGA and AASLD were compiled from the 2009 member directories for both organizations. AGA and AASLD members were selected to be surveyed as they represent the majority of HCV treaters in the United States. Prescriber ProfilerTM data provided to the authors by IMS Health Incorporated, reflecting a US-based database of retail pharmacies and total dispensed prescriptions of CopegusTM, Intron-ATM, InfergenTM, PegasysTM, Peg-IntronTM, RebetolTM, RebetronTM, RibasphereTM, and RibavirinTM indicated that between December 2008 and November 2009, 177,300 prescriptions were written. Gastroenterologists or Hepatologists wrote approximately 94,000 or 55% of these, validating our hypothesis that the targeted survey recipients were a group which treated HCV most frequently. Internal medicine physicians (11%) and nurse practitioners (8%) were the next most common prescribers [17]. Using an Internet-based survey engine ("SurveyMonkey" (surveymonkey.com)) a nine-question survey was sent to each AGA or AASLD member; only one questionnaire was sent to individuals who are members of both organizations. If an individual did not respond to the survey, the survey was re-sent with a second request to complete the survey. All results were tabulated, and statistical analysis was performed using Stata version 9.2, Statcorp LP, College Station, Texas.
Results
Of the 10,082 surveys sent, 8,449 were deliverable. The most common reasons for inability to deliver a survey included use of an e-mail filter or vacation message by the intended recipient. A total 1,757 individuals responded to the survey, representing a 21% response rate. A recent analysis performed by supersurvey.com revealed a mean response rate of 18% to online surveys of similar question size and target audience (www.supersurvey.com). The survey questions and responses appear in Table 1. If a respondent stated that they did not treat HCV, their participation in the survey ended after question 1.
Of the respondents, 75% (1,320) stated that they treat HCV, and of these respondents, 79% were MDs, 10% were physician assistants or nurse practitioners, 8% MD-PhDs, 2% DO, and 1% PhD. Of the respondents, 32% graduated within the last 10 years, 25% 11-20 years ago, 21% 21-30 years ago, and 12% greater than 30 years ago.
When analyzing results based on focus of practice, 67% of respondents stated that gastroenterology was the primary focus of their clinical practice, 24% selected hepatology and/or liver transplantation, 2% infectious disease, and 7% stated "other" as the primary focus of their practice. The "other" respondents included primary care doctors, surgeons, as well as Gastroenterologists who considered hepato-biliary disease, oncology, or pancreatic disease their primary specialty. Forty-six percent of the respondents were in a private practice not associated with a medical school, 42% at a medical school or hospital associated with a medical school, 8% in a private practice associated with a medical school, and the remaining 4% of respondents practiced in a multispecialty group practice, Veterans Administration hospital, or hospital not associated with a medical school.
Professional affiliations were assessed; 55% of respondents considered the American Gastroenterological Association (AGA) their primary professional affiliation, 26% the American Association for the Study of Liver Disease (AASLD), 9% the American Society for Gastrointestinal Endoscopy (ASGE), and 10% "other" including the American College of Gastroenterology (ACG), American Medical Association (AMA), and the American Society for Transplantation (AST).
When queried regarding what they would do when presented with an HCV-infected patient "today," 85% of the respondents would treat them, 6% would refer them to a "hepatitis C expert," 4% would refer them to a physician extender (PA, NP, or specially trained nurse) in their practice, 4% would refer to another MD in their practice, and 1% would refer them to an infectious disease specialist. Related to future therapies for HCV including DAAs in combination with interferon and ribavirin, 77% of the respondents were "aware" or "very aware" of this concept but had not participated in any clinical trials using DAA agents, 20% were "very aware" and had experience using these agents in clinical trials, and 3% had "minimal knowledge" of DAA agents.
When queried regarding treatment approaches if a DAA agent were available "today," 81% of respondents would evaluate and treat the patient, 10% would refer the patient to a "hepatitis C expert," 5% would refer the patient to another physician in their group, and 4% would refer the patient to a physician extender (PA, NP, or specially trained nurse) in their practice. Less than 1% of respondents reported that they would refer the patient to an infectious disease specialist.
Analysis of survey results (Table 2....
Overall, a significant number of respondents who treat HCV today with currently available therapies indicated that they would not prescribe DAA therapies when they became available; 85% of respondents to the survey would evaluate and treat an HCV-infected patient "today" versus 81% when DAA agents became available (p = 0.0054). Similarly, more respondents indicated that they would refer their patients to a "hepatitis C specialist" after DAA agents became available (6% current, 10% after DAA availability (p = 0.016).
Significant differences existed based on primary professional affiliation in attitudes and experience of survey respondents related to present and future HCV therapy. Ninety-one percent of respondents who considered the AASLD their primary affiliation compared to 84% of AGA members would treat their HCV-infected patient today (p = 0.002), 48% of AASLD members versus 9% of AGA members participated in clinical trials with a DAA agent (p = 0001), and 91% of AASLD members versus 79% of AGA members would use a DAA agent to treat their HCV-infected patient in the future (p = 0.001). A greater percentage of AGA members (12%) stated that they would refer their patient to an "HCV expert" when DAA agents became available compared to AASLD members (3%) (p = 0.001). When analyzing attitudes related to current and future therapy of HCV based on focus of clinical practice, a comparison of Hepatologists/liver transplantation physicians to Gastroenterologists revealed that 93% versus 86% would treat their HCV-infected patient today (p = 0.0034), 48% compared to 8% participated in clinical trials using DAA agents (p = 0.001), 90% versus 81% would treat their patient with a DAA agent (p = 0.001), and 1% versus 8% would refer their patient to an "HCV expert" when DAA therapies became available (p = 0.001).
Practice type was also associated with differences in attitudes related to current and future therapy of HCV. Comparison of respondents in private practice versus those who practiced at a medical school or hospital associated with a medical school revealed that respectively, 91% versus 81% would treat their HCV-infected patient today (p = 0.001), 9% versus 30% participated in a clinical trial with a DAA agent (p = 0.001), 89% versus 75% would use DAA therapies when they became available (p = 0.001), and 12% compared to 15% would refer to an "HCV expert" when DAA therapies became available (P = NS).
Current awareness and participation in clinical trials with DAA agents influenced attitudes regarding future use of these therapies, as 90% of respondents who participated in clinical trials with DAAs, 81% of those who were "very aware" or "aware" of DAA agents but did not participate in clinical trials, and 59% of those with minimal knowledge of DAA therapies would prescribe these agents in the future (p = 0.0001 clinical trial participant/very aware or aware of DAA agents vs. minimal knowledge). Respondents with "minimal knowledge" of DAA agents also reported that when these compounds were available in the future, 25% would refer their HCV-infected patient to a PA, NP, or other MD in their practice, 22% would refer to an "HCV expert," and 1% would refer to an ID specialist. Respondents who reported "minimal knowledge of DAA agents" were more likely to consider the AMA their primary professional organization (20 vs. 1% AGA/AASLD (p = 0.001)), or be in a private practice not associated with a medical school (83 vs. 17% practice or hospital associated with a medical school p = 0.0001).
Discussion
Emerging data suggest that direct-acting antiviral therapies against HCV (DAA) will provide improved response rates when given in combination with currently available therapies. The enthusiasm for these new treatments must be tempered by realistic concerns including side-effects as well as the threat of viral resistance induced by these agents. We hypothesized that concern regarding these issues might affect attitudes of future use of DAA therapies by health care providers who currently treat HCV. The goal of the current study was to query a group of experienced HCV treaters using an Internet-based survey to assess attitudes regarding current and future treatment of HCV and correlate these responses related to focus of clinical practice, academic versus private practice, professional affiliation, and experience with DAA agents in clinical trials. US-based members of the AGA and AASLD were targeted for the survey as they represent the majority of HCV treaters in the US and a group most likely to have knowledge of DAA therapy. This manuscript represents the first description of attitudes regarding future use of DAA agents in a large group of experienced HCV treaters.
Based on responses to this survey, although the majority of current HCV treaters would prescribe a DAA agent, a significant number of respondents who treat HCV today (85%) would not initiate therapy when these agents became available in the future (81%). The decreased use of DAA therapies may be offset somewhat by an increase in referrals to a more experienced HCV treater, as more respondents would refer their patients to a "hepatitis C expert" after DAA agents became available in the future compared to referring patients if they were diagnosed with HCV today (10% versus 6%). Not surprisingly, future use of DAA therapy appears to be significant in treaters who identify themselves as Hepatologists, and those with experience using these agents in clinical trials. Moreover, direct clinical experience with DAAs did not appear to dampen the enthusiasm for future use of these agents, as 90% of respondents who participated in clinical trials with a DAA agent would utilize these agents to treat an HCV-infected patient when these agents became available.
We hypothesized that as infectious disease specialists have extensive experience prescribing oral antiviral agents for HIV, referrals to these providers would increase when DAA agents became available for HCV. Our hypothesis was clearly inaccurate as respondents to the survey were unlikely to refer their HCV-infected patients to an infectious disease specialist when faced with an HCV-infected patient today (1%) and when DAA agents were available in the future (0.8%). This trend was maintained even in respondents with minimal knowledge of DAA agents. Finally, a concerning observation was identified when assessing responses to the survey stratified by past experience with DAA therapy. Although a significant percentage of respondents who participated in clinical trials with a DAA (90%) or who were "aware or very aware" of these agents but did not participate in a clinical trial (81%) would use these agents in the future, more than half of respondents (52%) who reported minimal knowledge of DAA agents stated that they would also use them in the future. Although providers with minimal knowledge of DAA agents represented a small percentage of respondents to the survey, concern exists regarding the inappropriate use of DAAs in the future including inexperience with side-effect management, and lack of recognition of both treatment failure as well as the emergence of viral resistance [11-16]. It is therefore apparent to the authors of this manuscript that extensive education of all future prescribers of DAA agents will be required to ensure successful use of these therapies.
There are limitations of this study that are unfortunately shared by other surveys utilizing similar data collection and analyses techniques. Any Internet-based survey is limited by response rate, which is affected both by the ability to deliver the survey and the willingness of the survey recipient to accurately respond to the queries posed to them. Although 21% of recipients of the survey responded, it is possible that the identified results would have been different if more individuals completed the survey. Unfortunately, the large volume of unsolicited e-mail has induced the use of blockers and filters, thus potentially limiting the response rates to the survey. However, we believe that these limitations were balanced by several strengths including the overall brevity of the survey (nine questions) increasing the likelihood that those who received and opened the survey fully completed it, and by the target audience which represented a significant number of current HCV treaters with the highest likelihood of having knowledge regarding DAA agents.
In summary, the responses to this Internet-based survey of more than 1,000 current HCV treaters indicated that although the majority of respondents appear ready to utilize DAA agents in the future, referrals to "hepatitis C experts" will increase when these agents become available. In addition, future referrals to ID specialists appear to be limited. Finally, as more than half of respondents to the survey with "minimal knowledge" of DAA therapies also appear to be willing to utilize these compounds in the future, significant provider education will be required to minimize inappropriate use of these agents.
Conflict of interest Paul J. Gaglio, MD, Speakers Bureau, Merck; has received study support from Schering Plough (now Merck) and Vertex Pharmaceuticals. Noah Moss, MD: No Camille Baugh, MD: No Disclosures Noah Moss, MD: No Disclosures John Reinus, MD has received study support from Vertex Pharmaceuticals.
Source
Subscribe to:
Posts (Atom)