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/
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