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ZymoGenetics Initiates Phase 2 Clinical Trial of PEG-Interferon lambda in Hepatitis C with Bristol-Myers Squibb
Tue Oct 27
PEG-Interferon lambda is a Targeted Type 3 interferon in development for
Hepatitis C
SEATTLE--
ZymoGenetics, Inc. (NASDAQ: ZGEN) today announced the initiation of a Phase 2
clinical trial of PEG-Interferon lambda (IL-29) and ribavirin in treatment-naïve
patients with chronic hepatitis C virus (HCV) infection (the "EMERGE" study).
The first patient has been dosed in the study, triggering a $70 million
milestone payment to ZymoGenetics from Bristol-Myers Squibb Company (NYSE:BMY),
pursuant to the terms of a previously announced collaboration agreement.
"In the Phase 1b clinical trial, PEG-Interferon lambda demonstrated robust
antiviral activity and was well tolerated in patients with genotype 1 hepatitis
C," said Eleanor L. Ramos, M.D., Senior Vice President and Chief Medical Officer
of ZymoGenetics. "Because PEG-Interferon lambda binds to a unique receptor, it
has the potential to treat HCV without many of the treatment-limiting side
effects associated with current interferons."
The EMERGE study is an international, randomized multi-center clinical trial
that will enroll approximately 50 patients in the first, open label portion that
will explore a wide range of doses to be tested in the second part of the study.
The second part of the study is designed to enroll approximately 500 patients.
Weekly subcutaneous doses of PEG-Interferon lambda will be administered for up
to 48 weeks. The study will assess the safety and antiviral efficacy of
PEG-Interferon lambda compared to PEGASYS®. All patients will also receive daily
ribavirin. The primary endpoint of the trial is the proportion of patients who
achieve undetectable levels of HCV RNA after 12 weeks of therapy (cEVR).
Sustained virological response (SVR) defined as undetectable levels of HCV 24
weeks after treatment will also be assessed.
PEG-Interferon lambda
PEG-Interferon lambda (IL-29) is a novel type 3 interferon in development for
hepatitis C. The native human protein Interferon lambda is generated by the
immune system in response to viral infection. In a Phase 1b clinical trial in
patients with relapsed HCV, administration of PEG-Interferon lambda over four
weeks in combination with ribavirin was shown to be well-tolerated and resulted
in significant antiviral activity.
About ZymoGenetics
ZymoGenetics is focused on the creation of novel protein drugs to improve
patient care and address unmet medical needs. The company`s strategy is to
discover, develop and commercialize its products independently, in collaboration
with partner companies or through out-licensing. ZymoGenetics developed and
markets RECOTHROM® Thrombin, topical (Recombinant), a synthetic version of a
human blood-clotting enzyme used to stop bleeding during surgery. The company is
developing a proprietary portfolio of immune-based product candidates.
PEG-Interferon lambda is a novel type-3 interferon in clinical development for
the treatment of chronic hepatitis C infection. Interleukin-21 is a novel
cytokine in clinical development for the treatment of metastatic melanoma and
renal cell carcinoma. Several other proprietary product candidates are in
preclinical development. In addition, ZymoGenetics has licensed rights to
multiple clinical and preclinical drug candidates being developed by other
companies. For further information, visit www.zymogenetics.com.
ZymoGenetics Forward-Looking Statements
This press release contains "forward-looking statements" within the meaning of
the Private Securities Litigation Reform Act of 1995.These forward-looking
statements are based on the current intent and expectations of the management of
ZymoGenetics.These statements are not guarantees of future performance and
involve risks and uncertainties that are difficult to predict. ZymoGenetics
actual results and the timing and outcome of events may differ materially from
those expressed in or implied by the forward-looking statements because of risks
and uncertainties associated with clinical development.For example, the results
of preliminary studies do not predict clinical success, and larger and
later-stage clinical trials may not produce the same results as earlier-stage
trials.In addition, the forward-looking statements in this press release are
subject to the other risks detailed in the company's public filings with the
Securities and Exchange Commission, including the company's Annual Report on
Form 10-K for the year ended December 31, 2008 and Quarterly Report on Form 10-Q
for the quarter ended June 30, 2009. Except as required by law, ZymoGenetics
undertakes no obligation to update any forward-looking or other statements in
this press release, whether as a result of new information, future events or
otherwise.
PEGASYS® (Peginterferon alfa-2a) is a registered trademark of Hoffman La Roche.
Media and Investors
ZymoGenetics, Inc.
Susan W. Specht, 206-442-6592
spechts@zymogenetics.com
Thursday, October 29, 2009
Vertex hep C drug helps tough to treat patients
Vertex hep C drug helps tough to treat patients
Wed Oct 28
* 57 pct prior null responders achieve SVR on telaprevir
* 90 pct prior relapsers achieve SVR on telaprevir
NEW YORK, Oct 28 (Reuters) - Vertex Pharmaceuticals Inc's (VRTX.O) closely watched hepatitis C drug telaprevir knocked out the virus in a significant number of patients who had failed to respond to standard treatments or relapsed after taking the standard drugs, according to a study.
Interim results from an ongoing study released on Wednesday showed that 57 percent of patients, who failed to respond to treatment wit h the standard treatments of pegylated-interferon and ribavirin and 90 percent of those who responded but later relapsed, achieved a sustained viral response when telaprevir was added to their regimen.
The percentage of patients in whom the virus is undetectable after completing treatment yields the critical measure known as sustained viral response, or SVR.
"Results that include SVR rates of 57 percent in the difficult-to-treat null responder population are important for prior treatment-failure patients who h ave limited options," Peter Mueller, Vertex's chief scientific offic er, said in a statement.
There was also a 55 percent SVR rate among patients on telaprevir who had appeared to be responding to prior standard treatment after 12 weeks, but still had detectable levels of the virus at week 24.
Telaprevir, an experimental treatment for the serious liver disease, is the most important drug in Vertex's developmental pipeline and is expected to become its first commercial product.
The company plans to file an application seeking U.S. approval of telaprevir in the second half of 2010.
Patients in this open label study had been in the control groups of previous telaprevir trials and were then given the Vertex drug.
Of 117 patients enrolled in the study, data from 94 was included in the interim analysis, Vertex said.
Eight patients in the study discontinued treatment due to adverse side effects, including rash and anemia. (Reporting by Bill Berkrot, editing by Leslie Gevirtz)
Wed Oct 28
* 57 pct prior null responders achieve SVR on telaprevir
* 90 pct prior relapsers achieve SVR on telaprevir
NEW YORK, Oct 28 (Reuters) - Vertex Pharmaceuticals Inc's (VRTX.O) closely watched hepatitis C drug telaprevir knocked out the virus in a significant number of patients who had failed to respond to standard treatments or relapsed after taking the standard drugs, according to a study.
Interim results from an ongoing study released on Wednesday showed that 57 percent of patients, who failed to respond to treatment wit h the standard treatments of pegylated-interferon and ribavirin and 90 percent of those who responded but later relapsed, achieved a sustained viral response when telaprevir was added to their regimen.
The percentage of patients in whom the virus is undetectable after completing treatment yields the critical measure known as sustained viral response, or SVR.
"Results that include SVR rates of 57 percent in the difficult-to-treat null responder population are important for prior treatment-failure patients who h ave limited options," Peter Mueller, Vertex's chief scientific offic er, said in a statement.
There was also a 55 percent SVR rate among patients on telaprevir who had appeared to be responding to prior standard treatment after 12 weeks, but still had detectable levels of the virus at week 24.
Telaprevir, an experimental treatment for the serious liver disease, is the most important drug in Vertex's developmental pipeline and is expected to become its first commercial product.
The company plans to file an application seeking U.S. approval of telaprevir in the second half of 2010.
Patients in this open label study had been in the control groups of previous telaprevir trials and were then given the Vertex drug.
Of 117 patients enrolled in the study, data from 94 was included in the interim analysis, Vertex said.
Eight patients in the study discontinued treatment due to adverse side effects, including rash and anemia. (Reporting by Bill Berkrot, editing by Leslie Gevirtz)
Vertex HCV Drug Studies Update
Vertex HCV Drug Studies Update
Vertex Pharmaceuticals Reports Third Quarter 2009 Financial Results and Highlights Recent Business and Clinical Progress
Mon Oct 26, 2009 4:01pm EDT
-Phase 3 registration programs in hepatitis C and cystic fibrosis on track-
-Vertex to present telaprevir SVR data from Study C208 at AASLD meeting this
week-
CAMBRIDGE, Mass.--
Vertex Pharmaceuticals Incorporated (Nasdaq: VRTX) today reviewed recent
business and clinical progress and reported consolidated financial results for
the quarter ended September 30, 2009.
"Vertex has made significant advancements across its business and expects to
enter 2010 in a strong financial position that will enable the continued
investment in late-stage development programs in hepatitis C virus infection and
cystic fibrosis," said Matthew Emmens, Chairman, President and Chief Executive
Officer of Vertex Pharmaceuticals. "We remain focused on the completion of the
telaprevir Phase 3 registration program and are on track to submit a telaprevir
New Drug Application in the second half of 2010. In addition, we believe ongoing
clinical trials of telaprevir and of our novel HCV polymerase inhibitor VX-222
will enable the initiation of the first combination trial of these two compounds
in HCV patients in the coming months - underscoring our commitment to improve
patient care in HCV."
Mr. Emmens continued, "Later this week, we expect to present final SVR data from
Study C208 at the AASLD meeting in Boston showing the potential for telaprevir
to be dosed twice-daily as part of a response-guided treatment regimen. Our
confidence in telaprevir`s competitive profile remains high, and we look forward
to the presentation of data from C208 and other clinical trials in the coming
days.
"In cystic fibrosis, we recently completed enrollment in a Phase 2 trial of
VX-809, our novel CFTR corrector compound, and we continue to enroll patients
across the three trials of the Phase 3 registration program for VX-770, our
novel CFTR potentiator. VX-770 and VX-809 aim to address the underlying
defective protein responsible for this orphan disorder, with the goal of
enabling cystic fibrosis patients to live a more normal life," Mr. Emmens said.
Broad Commitment to Hepatitis C
Phase 3 registration program ongoing:ADVANCE, ILLUMINATE and REALIZE trials
* The ADVANCE, ILLUMINATE and REALIZE trials are evaluating telaprevir-based
regimens as part of a global Phase 3 registration program in more than 2,200
genotype 1 treatment-naïve and treatment-failure patients with hepatitis C virus
(HCV) infection.
* Vertex expects sustained viral response (SVR) data to become available from
ADVANCE and ILLUMINATE in the first half of 2010 and from REALIZE in mid-2010.
Vertex plans to submit a New Drug Application (NDA) for telaprevir in the second
half of 2010.
* The Phase 3 ADVANCE trial is evaluating telaprevir, or placebo, as part of a
24-week telaprevir-based response-guided treatment regimen in combination with
pegylated interferon (peg-IFN) and ribavirin (RBV) in more than 1,050
treatment-naïve HCV patients. The response-guided trial design is utilizing
rapid viral response (RVR) criteria to determine which telaprevir patients can
stop all treatment at 24 weeks.
* The Phase 3 ILLUMINATE trial is evaluating response-guided telaprevir-based
regimens, or placebo, in more than 500 treatment-naïve HCV patients. This trial
is designed to supplement SVR data obtained from the pivotal Phase 3 ADVANCE
trial. The aim of the ILLUMINATE trial is to characterize whether there is an
additional benefit to extending treatment from 24 to 48 weeks in treatment-naïve
patients who achieved undetectable virus levels at weeks 4 and 12 of treatment
(eRVR).
* The Phase 3 REALIZE trial is evaluating 48-week telaprevir-based regimens, or
placebo, in more than 650 patients with genotype 1 HCV who did not achieve an
SVR with a previous peg-IFN-based treatment. The REALIZE trial enrolled all
major treatment-failure groups, including null responders.
SVR data from telaprevir twice-daily dosing to be presented at AASLD this week
* Vertex expects that final SVR data from Study C208, which is evaluating
twice-daily telaprevir dosing, will be presented at a Presidential Plenary
session at the upcoming Annual Meeting of the American Association for the Study
of Liver Diseases (AASLD), Oct. 30 - Nov. 3 in Boston. The C208 presentation at
AASLD represents the first SVR data for telaprevir-based regimens, including SVR
results from twice-daily dosing of telaprevir, as part of a response-guided
therapy trial design, similar to that being used in the ADVANCE and ILLUMINATE
Phase 3 trials of telaprevir. Study C208 is an exploratory Phase 2, open-label
clinical study conducted by Tibotec in Europe that evaluated a twice-daily
(1125mg q12h) dosing schedule of telaprevir in combination with peg-IFN-alfa-2a
(PEGASYS®) or peg-IFN-alfa-2b (PEGINTRON®) and RBV, as compared to the current
three-times-daily (750mg q8h) telaprevir dosing schedule.
Additional telaprevir clinical studies in patients who failed prior HCV therapy
* Vertex has completed PROVE 3, a Phase 2b clinical trial of telaprevir-based
combination therapy in patients with genotype 1 HCV who did not achieve an SVR
with a previous peg-IFN-based treatment. Final PROVE 3 data, including 48-week
follow-up SVR rates (SVR48), will be presented at AASLD.
* Vertex is also conducting Study 107, an open-label Phase 2 study to evaluate
telaprevir-based combination regimens in patients who did not achieve an SVR in
the 48-week control arms of the PROVE 1, PROVE 2 and PROVE 3 studies. In Study
107, telaprevir was given in combination with peg-IFN and RBV for 12 weeks
followed by peg-IFN and RBV for 12 weeks or 36 weeks depending on the patient`s
antiviral response to telaprevir in Study 107 and whether the patient was a
prior null-responder, partial-responder or relapser.
On track to initiate STAT-C combination trial as early as Q4 2009
* Vertex is seeking to advance HCV therapy through the development of novel
combinations of Specifically-Targeted Antiviral Therapies for hepatitis C
(STAT-Cs).
* Vertex is currently conducting a three-day, multiple-dose viral kinetic study
to evaluate the antiviral activity, safety, tolerability and pharmacokinetics of
the HCV polymerase inhibitor VX-222. In the trial, VX-222 is being administered
at four different doses as a monotherapy in 32 treatment-naïve patients with
genotype 1 HCV infection. Vertex is also currently conducting a drug-drug
interaction study with VX-222 and telaprevir in healthy volunteers.
* Vertex expects to obtain data from these trials in the fourth quarter of 2009,
which could enable the initiation of a combination trial of telaprevir and
VX-222 in patients with genotype 1 HCV as early as the fourth quarter of 2009.
Vertex expects data from this first STAT-C combination study of telaprevir and
VX-222 to become available by mid-2010.
Additional HCV compounds in clinical development
* Vertex is also evaluating additional HCV compounds, including the HCV protease
inhibitors VX-813 and VX-985 as well as the HCV polymerase inhibitor VX-759.
* Vertex also has an NS5A inhibitor program in preclinical development.
* The goal of these programs is to identify compounds that are appropriate for
further development, including combination therapy.
AASLD
* The upcoming AASLD meeting, being held Oct. 30 - Nov. 3 in Boston, is expected
to include three telaprevir-related clinical presentations, including
presentations on SVR results from Study C208, final SVR48 results from PROVE 3
and results from a pooled analysis of PROVE 1 and PROVE 2 in "difficult-to-cure"
patients.
Vertex Pharmaceuticals Reports Third Quarter 2009 Financial Results and Highlights Recent Business and Clinical Progress
Mon Oct 26, 2009 4:01pm EDT
-Phase 3 registration programs in hepatitis C and cystic fibrosis on track-
-Vertex to present telaprevir SVR data from Study C208 at AASLD meeting this
week-
CAMBRIDGE, Mass.--
Vertex Pharmaceuticals Incorporated (Nasdaq: VRTX) today reviewed recent
business and clinical progress and reported consolidated financial results for
the quarter ended September 30, 2009.
"Vertex has made significant advancements across its business and expects to
enter 2010 in a strong financial position that will enable the continued
investment in late-stage development programs in hepatitis C virus infection and
cystic fibrosis," said Matthew Emmens, Chairman, President and Chief Executive
Officer of Vertex Pharmaceuticals. "We remain focused on the completion of the
telaprevir Phase 3 registration program and are on track to submit a telaprevir
New Drug Application in the second half of 2010. In addition, we believe ongoing
clinical trials of telaprevir and of our novel HCV polymerase inhibitor VX-222
will enable the initiation of the first combination trial of these two compounds
in HCV patients in the coming months - underscoring our commitment to improve
patient care in HCV."
Mr. Emmens continued, "Later this week, we expect to present final SVR data from
Study C208 at the AASLD meeting in Boston showing the potential for telaprevir
to be dosed twice-daily as part of a response-guided treatment regimen. Our
confidence in telaprevir`s competitive profile remains high, and we look forward
to the presentation of data from C208 and other clinical trials in the coming
days.
"In cystic fibrosis, we recently completed enrollment in a Phase 2 trial of
VX-809, our novel CFTR corrector compound, and we continue to enroll patients
across the three trials of the Phase 3 registration program for VX-770, our
novel CFTR potentiator. VX-770 and VX-809 aim to address the underlying
defective protein responsible for this orphan disorder, with the goal of
enabling cystic fibrosis patients to live a more normal life," Mr. Emmens said.
Broad Commitment to Hepatitis C
Phase 3 registration program ongoing:ADVANCE, ILLUMINATE and REALIZE trials
* The ADVANCE, ILLUMINATE and REALIZE trials are evaluating telaprevir-based
regimens as part of a global Phase 3 registration program in more than 2,200
genotype 1 treatment-naïve and treatment-failure patients with hepatitis C virus
(HCV) infection.
* Vertex expects sustained viral response (SVR) data to become available from
ADVANCE and ILLUMINATE in the first half of 2010 and from REALIZE in mid-2010.
Vertex plans to submit a New Drug Application (NDA) for telaprevir in the second
half of 2010.
* The Phase 3 ADVANCE trial is evaluating telaprevir, or placebo, as part of a
24-week telaprevir-based response-guided treatment regimen in combination with
pegylated interferon (peg-IFN) and ribavirin (RBV) in more than 1,050
treatment-naïve HCV patients. The response-guided trial design is utilizing
rapid viral response (RVR) criteria to determine which telaprevir patients can
stop all treatment at 24 weeks.
* The Phase 3 ILLUMINATE trial is evaluating response-guided telaprevir-based
regimens, or placebo, in more than 500 treatment-naïve HCV patients. This trial
is designed to supplement SVR data obtained from the pivotal Phase 3 ADVANCE
trial. The aim of the ILLUMINATE trial is to characterize whether there is an
additional benefit to extending treatment from 24 to 48 weeks in treatment-naïve
patients who achieved undetectable virus levels at weeks 4 and 12 of treatment
(eRVR).
* The Phase 3 REALIZE trial is evaluating 48-week telaprevir-based regimens, or
placebo, in more than 650 patients with genotype 1 HCV who did not achieve an
SVR with a previous peg-IFN-based treatment. The REALIZE trial enrolled all
major treatment-failure groups, including null responders.
SVR data from telaprevir twice-daily dosing to be presented at AASLD this week
* Vertex expects that final SVR data from Study C208, which is evaluating
twice-daily telaprevir dosing, will be presented at a Presidential Plenary
session at the upcoming Annual Meeting of the American Association for the Study
of Liver Diseases (AASLD), Oct. 30 - Nov. 3 in Boston. The C208 presentation at
AASLD represents the first SVR data for telaprevir-based regimens, including SVR
results from twice-daily dosing of telaprevir, as part of a response-guided
therapy trial design, similar to that being used in the ADVANCE and ILLUMINATE
Phase 3 trials of telaprevir. Study C208 is an exploratory Phase 2, open-label
clinical study conducted by Tibotec in Europe that evaluated a twice-daily
(1125mg q12h) dosing schedule of telaprevir in combination with peg-IFN-alfa-2a
(PEGASYS®) or peg-IFN-alfa-2b (PEGINTRON®) and RBV, as compared to the current
three-times-daily (750mg q8h) telaprevir dosing schedule.
Additional telaprevir clinical studies in patients who failed prior HCV therapy
* Vertex has completed PROVE 3, a Phase 2b clinical trial of telaprevir-based
combination therapy in patients with genotype 1 HCV who did not achieve an SVR
with a previous peg-IFN-based treatment. Final PROVE 3 data, including 48-week
follow-up SVR rates (SVR48), will be presented at AASLD.
* Vertex is also conducting Study 107, an open-label Phase 2 study to evaluate
telaprevir-based combination regimens in patients who did not achieve an SVR in
the 48-week control arms of the PROVE 1, PROVE 2 and PROVE 3 studies. In Study
107, telaprevir was given in combination with peg-IFN and RBV for 12 weeks
followed by peg-IFN and RBV for 12 weeks or 36 weeks depending on the patient`s
antiviral response to telaprevir in Study 107 and whether the patient was a
prior null-responder, partial-responder or relapser.
On track to initiate STAT-C combination trial as early as Q4 2009
* Vertex is seeking to advance HCV therapy through the development of novel
combinations of Specifically-Targeted Antiviral Therapies for hepatitis C
(STAT-Cs).
* Vertex is currently conducting a three-day, multiple-dose viral kinetic study
to evaluate the antiviral activity, safety, tolerability and pharmacokinetics of
the HCV polymerase inhibitor VX-222. In the trial, VX-222 is being administered
at four different doses as a monotherapy in 32 treatment-naïve patients with
genotype 1 HCV infection. Vertex is also currently conducting a drug-drug
interaction study with VX-222 and telaprevir in healthy volunteers.
* Vertex expects to obtain data from these trials in the fourth quarter of 2009,
which could enable the initiation of a combination trial of telaprevir and
VX-222 in patients with genotype 1 HCV as early as the fourth quarter of 2009.
Vertex expects data from this first STAT-C combination study of telaprevir and
VX-222 to become available by mid-2010.
Additional HCV compounds in clinical development
* Vertex is also evaluating additional HCV compounds, including the HCV protease
inhibitors VX-813 and VX-985 as well as the HCV polymerase inhibitor VX-759.
* Vertex also has an NS5A inhibitor program in preclinical development.
* The goal of these programs is to identify compounds that are appropriate for
further development, including combination therapy.
AASLD
* The upcoming AASLD meeting, being held Oct. 30 - Nov. 3 in Boston, is expected
to include three telaprevir-related clinical presentations, including
presentations on SVR results from Study C208, final SVR48 results from PROVE 3
and results from a pooled analysis of PROVE 1 and PROVE 2 in "difficult-to-cure"
patients.
Inhibitex Announces Data Presentation at the 60th Annual Meeting of the American Association for the Study of Liver Disease
Inhibitex Announces Data Presentation at the 60th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD)
Mon Oct 26, 2009 11:29am EDT
INX-189 Exhibits Favorable Pharmacology Profile in Preclinical Studies
ATLANTA--(Business Wire)--
Inhibitex, Inc. (NASDAQ: INHX) announced today that a poster presentation
describing preclinical data on INX-189, the lead compound from its HCV
nucleotide polymerase inhibitor program, will be presented by Dr. Joseph M.
Patti, Chief Scientific Officer and Senior Vice President of R&D, at the 60th
Annual Meeting of the American Association for the Study of Liver Diseases
(AASLD) in Boston, MA. The full abstract can be viewed at the AASLD website at
www.aasld.org.
The poster (#1611 Patti, et al), entitled "Pharmacological Properties and In
Vitro Characterization of INX-189, a Liver Targeted Phosphoramidate Nucleoside
Analogue Inhibitor of NS5b" will be presented in the HCV Therapy: Preclinical
and Early Clinical Development session from 8:00 am - 1:00 pm on Tuesday,
November 3rd, 2009.
About HCV and Protides
Hepatitis C is a disease of the liver caused by the hepatitis C virus (HCV). It
is estimated that approximately 4 million Americans and 170 million individuals
worldwide are infected with HCV. HCV can cause chronic liver disease, cirrhosis
and cancer, and is the leading cause of liver transplant in the United States.
Inhibitex is developing a series of proprietary phosphoramidates, or protide
nucleoside inhibitors, that target the RNA-dependent RNA polymerase (NS5b) of
HCV. Protides are designed to by-pass the rate limiting first step in the
formation of the active nucleoside triphosphate. INX-189 is a protide of a
2`-C-methyl guanosine analogue. The Company believes that its protides possess
several pharmacological advantages over nucleosides alone and potentially other
nucleotide prodrugs. These advantages include greater potency, a rapid
conversion of the protide into its active form in the liver, and potentially
less toxicity due to reduced systemic exposure of the nucleoside.
About Inhibitex
Inhibitex, Inc., headquartered in Alpharetta, Georgia, is a biopharmaceutical
company focused on developing products to treat serious infectious diseases. In
addition to INX-189, the Company`s pipeline includes FV-100, its clinical-stage
nucleoside analogue in Phase II development for the treatment of herpes zoster
(shingles). The Company has also licensed the use of its proprietary MSCRAMM®
protein technology to Wyeth for the development of staphylococcal vaccines.
For additional information about the Company, please visit www.inhibitex.com.
Safe Harbor Statement
This press release contains forward-looking statements within the meaning of the
Private Securities Litigation Reform Act of 1995 that involve substantial risks
and uncertainties. All statements, other than historical facts included in this
press release, including statements regarding the Company`s belief that its
protides possess several pharmacological advantages over nucleosides alone and
potentially other nucleoside prodrugs, are forward looking statements. These
results and expectations may not be achieved in the future and various important
factors could cause actual results or events to differ materially from the
forward-looking statements that the Company makes, including the risk of: the
Company not obtaining regulatory approval on a timely basis, or at all, to
advance the development of INX-189 into clinical trials; the results of ongoing
or future preclinical studies of INX-189 not supporting its further development;
obtaining, maintaining and protecting the intellectual property incorporated
into and supporting the commercial viability of the Company`s product
candidates; and other cautionary statements contained elsewhere herein and in
its Annual Report on Form 10-K for the year ended December 31, 2008, as filed
with the Securities and Exchange Commission, or SEC, on March 23, 2009 and its
Quarterly Report on Form 10-Q for the quarter ended June 30, 2009 as filed with
the SEC on August 12, 2009. Given these uncertainties, you should not place
undue reliance on these forward-looking statements, which apply only as of the
date of this press release.
There may be events in the future that the Company is unable to predict
accurately, or over which it has no control. The Company's business, financial
condition, results of operations and prospects may change. The Company may not
update these forward-looking statements, even though its situation may change in
the future, unless it has obligations under the Federal securities laws to
update and disclose material developments related to previously disclosed
information. The Company qualifies all of the information contained in this
press release, and particularly its forward-looking statements, by these
cautionary statements.
Inhibitex® and MSCRAMM® are registered trademarks of Inhibitex, Inc.
Inhibitex, Inc.
Russell H. Plumb, 678-746-1136
Chief Executive Officer
rplumb@inhibitex.com
Mon Oct 26, 2009 11:29am EDT
INX-189 Exhibits Favorable Pharmacology Profile in Preclinical Studies
ATLANTA--(Business Wire)--
Inhibitex, Inc. (NASDAQ: INHX) announced today that a poster presentation
describing preclinical data on INX-189, the lead compound from its HCV
nucleotide polymerase inhibitor program, will be presented by Dr. Joseph M.
Patti, Chief Scientific Officer and Senior Vice President of R&D, at the 60th
Annual Meeting of the American Association for the Study of Liver Diseases
(AASLD) in Boston, MA. The full abstract can be viewed at the AASLD website at
www.aasld.org.
The poster (#1611 Patti, et al), entitled "Pharmacological Properties and In
Vitro Characterization of INX-189, a Liver Targeted Phosphoramidate Nucleoside
Analogue Inhibitor of NS5b" will be presented in the HCV Therapy: Preclinical
and Early Clinical Development session from 8:00 am - 1:00 pm on Tuesday,
November 3rd, 2009.
About HCV and Protides
Hepatitis C is a disease of the liver caused by the hepatitis C virus (HCV). It
is estimated that approximately 4 million Americans and 170 million individuals
worldwide are infected with HCV. HCV can cause chronic liver disease, cirrhosis
and cancer, and is the leading cause of liver transplant in the United States.
Inhibitex is developing a series of proprietary phosphoramidates, or protide
nucleoside inhibitors, that target the RNA-dependent RNA polymerase (NS5b) of
HCV. Protides are designed to by-pass the rate limiting first step in the
formation of the active nucleoside triphosphate. INX-189 is a protide of a
2`-C-methyl guanosine analogue. The Company believes that its protides possess
several pharmacological advantages over nucleosides alone and potentially other
nucleotide prodrugs. These advantages include greater potency, a rapid
conversion of the protide into its active form in the liver, and potentially
less toxicity due to reduced systemic exposure of the nucleoside.
About Inhibitex
Inhibitex, Inc., headquartered in Alpharetta, Georgia, is a biopharmaceutical
company focused on developing products to treat serious infectious diseases. In
addition to INX-189, the Company`s pipeline includes FV-100, its clinical-stage
nucleoside analogue in Phase II development for the treatment of herpes zoster
(shingles). The Company has also licensed the use of its proprietary MSCRAMM®
protein technology to Wyeth for the development of staphylococcal vaccines.
For additional information about the Company, please visit www.inhibitex.com.
Safe Harbor Statement
This press release contains forward-looking statements within the meaning of the
Private Securities Litigation Reform Act of 1995 that involve substantial risks
and uncertainties. All statements, other than historical facts included in this
press release, including statements regarding the Company`s belief that its
protides possess several pharmacological advantages over nucleosides alone and
potentially other nucleoside prodrugs, are forward looking statements. These
results and expectations may not be achieved in the future and various important
factors could cause actual results or events to differ materially from the
forward-looking statements that the Company makes, including the risk of: the
Company not obtaining regulatory approval on a timely basis, or at all, to
advance the development of INX-189 into clinical trials; the results of ongoing
or future preclinical studies of INX-189 not supporting its further development;
obtaining, maintaining and protecting the intellectual property incorporated
into and supporting the commercial viability of the Company`s product
candidates; and other cautionary statements contained elsewhere herein and in
its Annual Report on Form 10-K for the year ended December 31, 2008, as filed
with the Securities and Exchange Commission, or SEC, on March 23, 2009 and its
Quarterly Report on Form 10-Q for the quarter ended June 30, 2009 as filed with
the SEC on August 12, 2009. Given these uncertainties, you should not place
undue reliance on these forward-looking statements, which apply only as of the
date of this press release.
There may be events in the future that the Company is unable to predict
accurately, or over which it has no control. The Company's business, financial
condition, results of operations and prospects may change. The Company may not
update these forward-looking statements, even though its situation may change in
the future, unless it has obligations under the Federal securities laws to
update and disclose material developments related to previously disclosed
information. The Company qualifies all of the information contained in this
press release, and particularly its forward-looking statements, by these
cautionary statements.
Inhibitex® and MSCRAMM® are registered trademarks of Inhibitex, Inc.
Inhibitex, Inc.
Russell H. Plumb, 678-746-1136
Chief Executive Officer
rplumb@inhibitex.com
Liver cancer rates rising alarmingly, say doctors
"Doctors also say more new cases of liver cancer are being triggered by diabetes and obesity. Obese patients often develop fatty livers that seem to increase their risk of liver cancer and their risk of death from liver cancer"
"key to lowering cancer rates, says cancer researcher Dr. Morris Sherman of the University Health Network, is to monitor patients who are at risk for liver disease, from age 50 on with yearly liver ultrasounds."...."there will be a further 50 per cent increase in new liver cancer cases ove r the next 10 years."
Liver cancer rates rising alarmingly, say doctors
http://www.ctv.ca
It's a cancer that gets little attention, but it should, because liver cancer has become one of the fastest growing cancers in Canada.
While many cancers spread to the liver in the final stages, doctors are now co ncerned about the increase in the number of cancers that start in the liver.
Each year in Canada, over 1,700 people are diagnosed with primary liver cancer and those numbers are growing, doctors say. Yet many cases can be prevented and others cured if only those at risk were regularly tested.
Liver cancer rates are thought to be rising because of increased rates of hepatitis C infection, obesity and type 2 diabetes.
Dr. Sean Cleary, a surgeon at Toronto General Hospital, says he's seen a huge jump in liver cancer cases.
"We've seen a significant rise in our cases that need to come to sur gery, an increase of perhaps 200 per cent in the last five years," he tells CTV News.
The patient he was treating the day we came to see him was a middle-aged man whose silent cancer wasn't discovered until it had engulfed much of his liver. Dr. Cleary had to remove half of the vital organ in a bid to extend the man's life.
If caught early, liver cancer tumours can be easily treated, but if they are larger than three centimetres in diameter, the prognosis is poor. Cleary says too often they're caught late, so that about one-third to one-half of the cases he sees are untreatable.
"This means we are picking them up to late, we are catching them too late," he says.
The man Cleary treated had had hepatitis, which can damage the liver and allow cancer to grow. But the man had not been going for routine screenings; his liver cancer was found through other medical tests.
Doctors say liver cancer is a silent cancer that can grow for years causing no symptoms until it's too late.
Hepatitis C patient Michele also was recently diagnosed with liver cancer. She, too, was having tests for another problem when doctors found five tumours in her liver.
"It was frightening and I didn't know what to expect," she says, adding no one told her hepatitis C put her at higher risk of developing liver cancer.
"I had no idea that I would end up with liver cancer; that was a big shock," she says.
Hep C is caused by a virus transmitted by blood-to-blood contact, through shared needles, tattoo needles and sexual contact. It's estimated that in Canada, about 243,000 people have hep C, but because there are often no symptoms, nearly 20 per cent don't know they're infected.
Doctors also say more new cases of liver cancer are being triggered by diabetes and obesity. Obese patients often develop fatty livers that seem to increase their risk of liver cancer and their risk of death from liver cancer. Alcohol too, can lead to liver cirrhosis, which has been shown to increase cancer risk.
The key to lowering cancer rates, says cancer researcher Dr. M orris Sherman of the University Health Network, is to monitor patien ts who are at risk for liver disease, from age 50 on with yearly liver ultrasounds.
He notes that many countries already recommend regular liver screening, which allows doctors to find cancer in the early stages.
"If we get these things when they are small, the chance of survival is 90 per cent," he says.
But instead, he says, many of those most at risk aren't being tested.
"I would say no more than 30 per cent of patients are getting identified. So 70 per cent of patients are NOT getting adequate screening," he says.
"I'd like physicians to be aware of this and screen at a regular basis," he says, noting that he expects that with rising rates of hepatitis B and C infection, there will be a further 50 per cent increase in new liver cancer cases over the next 10 years.
As for Michelle, the cancer's damage to her liver is so extensive, she is now waiting for a liver transplant.
"I think it is important that people know you should get tested for this, because I don't want a lot of people stuck in this situation," she says.
"We need more doctors to get on the bandwagon and start testing them."
"key to lowering cancer rates, says cancer researcher Dr. Morris Sherman of the University Health Network, is to monitor patients who are at risk for liver disease, from age 50 on with yearly liver ultrasounds."...."there will be a further 50 per cent increase in new liver cancer cases ove r the next 10 years."
Liver cancer rates rising alarmingly, say doctors
http://www.ctv.ca
It's a cancer that gets little attention, but it should, because liver cancer has become one of the fastest growing cancers in Canada.
While many cancers spread to the liver in the final stages, doctors are now co ncerned about the increase in the number of cancers that start in the liver.
Each year in Canada, over 1,700 people are diagnosed with primary liver cancer and those numbers are growing, doctors say. Yet many cases can be prevented and others cured if only those at risk were regularly tested.
Liver cancer rates are thought to be rising because of increased rates of hepatitis C infection, obesity and type 2 diabetes.
Dr. Sean Cleary, a surgeon at Toronto General Hospital, says he's seen a huge jump in liver cancer cases.
"We've seen a significant rise in our cases that need to come to sur gery, an increase of perhaps 200 per cent in the last five years," he tells CTV News.
The patient he was treating the day we came to see him was a middle-aged man whose silent cancer wasn't discovered until it had engulfed much of his liver. Dr. Cleary had to remove half of the vital organ in a bid to extend the man's life.
If caught early, liver cancer tumours can be easily treated, but if they are larger than three centimetres in diameter, the prognosis is poor. Cleary says too often they're caught late, so that about one-third to one-half of the cases he sees are untreatable.
"This means we are picking them up to late, we are catching them too late," he says.
The man Cleary treated had had hepatitis, which can damage the liver and allow cancer to grow. But the man had not been going for routine screenings; his liver cancer was found through other medical tests.
Doctors say liver cancer is a silent cancer that can grow for years causing no symptoms until it's too late.
Hepatitis C patient Michele also was recently diagnosed with liver cancer. She, too, was having tests for another problem when doctors found five tumours in her liver.
"It was frightening and I didn't know what to expect," she says, adding no one told her hepatitis C put her at higher risk of developing liver cancer.
"I had no idea that I would end up with liver cancer; that was a big shock," she says.
Hep C is caused by a virus transmitted by blood-to-blood contact, through shared needles, tattoo needles and sexual contact. It's estimated that in Canada, about 243,000 people have hep C, but because there are often no symptoms, nearly 20 per cent don't know they're infected.
Doctors also say more new cases of liver cancer are being triggered by diabetes and obesity. Obese patients often develop fatty livers that seem to increase their risk of liver cancer and their risk of death from liver cancer. Alcohol too, can lead to liver cirrhosis, which has been shown to increase cancer risk.
The key to lowering cancer rates, says cancer researcher Dr. M orris Sherman of the University Health Network, is to monitor patien ts who are at risk for liver disease, from age 50 on with yearly liver ultrasounds.
He notes that many countries already recommend regular liver screening, which allows doctors to find cancer in the early stages.
"If we get these things when they are small, the chance of survival is 90 per cent," he says.
But instead, he says, many of those most at risk aren't being tested.
"I would say no more than 30 per cent of patients are getting identified. So 70 per cent of patients are NOT getting adequate screening," he says.
"I'd like physicians to be aware of this and screen at a regular basis," he says, noting that he expects that with rising rates of hepatitis B and C infection, there will be a further 50 per cent increase in new liver cancer cases over the next 10 years.
As for Michelle, the cancer's damage to her liver is so extensive, she is now waiting for a liver transplant.
"I think it is important that people know you should get tested for this, because I don't want a lot of people stuck in this situation," she says.
"We need more doctors to get on the bandwagon and start testing them."
Sunday, October 25, 2009
Public health impact of antiviral therapy for hepatitis C in the United State
Public health impact of antiviral therapy for hepatitis C in the United States
Hepatology Early View Oct 2009
"future development of new and better medications will have a less than optimal impact on this problem unless more patients are diagnosed and referred for treatment. Increased public health efforts are needed to improve access to antiviral therapy for patients with hepatitis C."....."Approximately 663,000 patients received antiviral therapy between 2002 and 2007, and treatment rates appear to be declining"...... "Only by increasing treatment rates by 75% and increasing SVR rates to 75% could more than half of the liver-related deaths be prevented."....."primary reason for lack of treatment is lack of diagnosis......49%, were previously unaware of their diagnosis......24% were recommended by their doctor not to be treated"
"respondents lacking a usual source of medical care were 19 times more likely to be unaware of their diagnosis"....."Because the U.S. Preventative Services Task Force has concluded that insufficient evidence exists to recommend screening for hepatitis C, physicians may consider this a low priority. physicians may not realize the value of diagnosing hepatitis C, because prior studies have identified limited knowledge about the disease among primary care physicians.[14] Thus, increasing the number of patients diagnosed will require education of the public and physicians alike, as well as attention to the worsening problem of the uninsured in the U.S.....some patients may not follow up for further care, and others may not be referred to a specialist who prescribes antiviral therapy......patients in some areas may have limited access to specialists who treat hepatitis C, because 20% of all gastroenterologists prescribe 80% of antiviral therapy in this country........it is possible that future improvements in antiviral therapy will increase enthusiasm for treatment among the medical community, and thus increase the number of patients treated. However, very little is known about health services delivery and quality of care for hepatitis C, and further research in this area is needed."
"In this study we estimated the number of patients currently receiving antiviral therapy for hepatitis C in the U.S., identified reasons for lack of treatment, and projected the impact current therapy will have on preventing liver-related deaths over the next two decades. We found that relatively few patients have been treated with pegylated interferon, and treatment rates appear to be declining. These findings are similar to a recent report from Europe, in which treatment rates varied by country from <1% to 16% of hepatitis C patients ever treated.[27] If this trend continues, antiviral therapy may prevent fewer than 15% of liver-related deaths caused by hepatitis C in the U.S. between 2002 and 2030."
"Fortunately, new treatments for hepatitis C are anticipated to become available in the next several years, which should increase rates of SVR among patients receiving treatment.[28] However, treatment first requires a diagnosis, and in this study we found that approximately half of all people with hepatitis C in the U.S. are unaware of their diagnosis."
"There would be 259,000 liver-related deaths caused by hepatitis C between 2002 and 2030 without treatment, and current antiviral therapy would prevent 37,500 of these. Thus, the current state of antiviral therapy is projected to prevent only 14.5% of liver-related deaths caused by hepatitis C between 2002 and 2030.....Approximately 663,000 patients received antiviral therapy between 2002 and 2007, and treatment rates appear to be declining.....If the discovery of new medications in the future increases rates of SVR to 75%, at current rates of treatment this would still only prevent 21.7% of liver-related deaths caused by hepatitis C. Only by increasing treatment rates by 75% and increasing SVR rates to 75% could more than half of the liver-related deaths be prevented......primary reason for lack of treatment is lack of diagnosis......49%, were previously unaware of their diagnosis......9% did not follow up with a doctor about their hepatitis C......24% were recommended by their doctor not to be treated......6% refused treatment.....12% were treated......28% of the 100 subjects who had seen a doctor by the time of the survey had the understanding that no further follow-up for their hepatitis C was necessary"
Michael L. Volk *, Rachel Tocco, Sameer Saini, Anna S.F. Lok
Division of Gastroenterology and Hepatology, University of Michigan, Ann Arbor, MI
email: Michael L. Volk (mvolk@med.umich.edu)
*Correspondence to Michael L. Volk, Division of Gastroenterology and Hepatology, University of Michigan Health System, 7C27 NIB, 300 N. Ingalls, Ann Arbor, MI 48109
Potential conflict of interest: Nothing to report.
fax: 734-936-8944.
Funded by:
Greenview Foundation
American Gastroenterological Association
ABSTRACT
Despite dramatic improvements in antiviral therapy for hepatitis C, there is reason to believe that the uptake of antiviral therapy remains limited. The aims of this study were to determine the number of patients being treated with antiviral therapy in the U.S., to estimate the public health impact of these treatment patterns, and to identify barriers to treatment for patients with hepatitis C. Data on the number of new patient pegylated interferon prescriptions each year, from 2002-2007, was obtained from Wolters Kluwer Inc., which maintains an electronic audit of pharmacies nationwide. A Markov model was created of the population with chronic hepatitis C in the U.S. from 2002 to 2030, and was used to estimate the number of liver-related deaths caused by hepatitis C that will be prevented by current treatment patterns. The National Health and Nutrition Evaluation Survey (NHANES) Hepatitis C Follow-Up Questionnaire was used to investigate reasons for lack of treatment and to identify strategies for improving access. Approximately 663,000 patients received antiviral therapy between 2002 and 2007, and treatment rates appear to be declining. If this trend continues, only 14.5% of liver-related deaths caused by hepatitis C from 2002-2030 will be prevented by antiviral therapy. Results from the NHANES questionnaire suggest that the primary barrier to treatment is lack of diagnosis, with 69/133 (adjusted proportion 49%) of respondents previously unaware that they had hepatitis C.
Conclusion: Efforts to improve rates of diagnosis and treatment will be required if the future public health burden of hepatitis C is to be ameliorated.
Hepatitis C is a major public health burden in the United States. It causes nearly 8,000 deaths per year,[1] is the leading cause for liver transplantation,[2] and has contributed to a rise in the incidence of hepatocellular carcinoma.[3] Quality of life is impaired for patients infected with the virus, even among those without cirrhosis.[4] As of 2001, there were an estimated 3.2 million people in the U.S. with chronic hepatitis C. Although the incidence of new infections has declined, the number of deaths may continue to increase 2- to 4-fold over the next 20 years due to prevalent cases with longstanding infection.[5][6]......the primary reason for lack of treatment is lack of diagnosis
The future public health burden of hepatitis C could potentially be mitigated by antiviral therapy.[5] In the last two decades there have been dramatic improvements in treatment for hepatitis C. The benefit of adding ribavirin to interferon was demonstrated in a series of studies in the late 1990s,[7] and then pegylated interferon was approved at the end of 2001. Combination therapy with pegylated interferon and ribavirin can now achieve sustained virological response (SVR) in 50% of patients,[8] compared to rates of 17% with standard interferon alone.[7] Patients who achieve SVR enjoy long-term remission of disease, with liver-related mortality rates comparable to the general population.[9-11]
Despite these improvements, there are reasons to believe that the uptake of antiviral therapy may be limited. Patients with hepatitis C are more likely to lack health insurance and a usual source of care.[12][13] Studies have demonstrated that primary care physicians lack knowledge about risk factors and testing for hepatitis C.[14][15] Access to a specialist willing to prescribe antiviral therapy may be another limiting factor.[16] Finally, many patients have contraindications to treatment, or do not wish to suffer the myriad side effects.[17] For these reasons, we hypothesized that the public health impact of antiviral therapy may be limited, as much by low utilization as by the inherent limitations in the available medications. Several studies from the Veterans Affairs Healthcare System and Health Maintenance Organizations have reported low treatment rates among patients identified to have hepatitis C.[18][19] However, no national data exist on the number of patients currently receiving antiviral therapy in the U.S., or the impact of current practice patterns on the burden of hepatitis C. The aims of this study were to determine the public health impact of current antiviral therapy and to identify barriers to treatment of hepatitis C.
Discussion
In this study we estimated the number of patients currently receiving antiviral therapy for hepatitis C in the U.S., identified reasons for lack of treatment, and projected the impact current therapy will have on preventing liver-related deaths over the next two decades. We found that relatively few patients have been treated with pegylated interferon, and treatment rates appear to be declining. These findings are similar to a recent report from Europe, in which treatment rates varied by country from <1% to 16% of hepatitis C patients ever treated.[27] If this trend continues, antiviral therapy may prevent fewer than 15% of liver-related deaths caused by hepatitis C in the U.S. between 2002 and 2030.
Fortunately, new treatments for hepatitis C are anticipated to become available in the next several years, which should increase rates of SVR among patients receiving treatment.[28] However, treatment first requires a diagnosis, and in this study we found that approximately half of all people with hepatitis C in the U.S. are unaware of their diagnosis. There are several possible reasons for this situation. First, lack of health insurance poses an obvious barrier to testing. In 2001, 30% of patients with hepatitis C were uninsured,[12] and this number has likely increased since that time. Second, because hepatitis C is usually asymptomatic, patients without any other medical problems may not seek medical attention. In the NHANES survey, we found that respondents lacking a usual source of medical care were 19 times more likely to be unaware of their diagnosis. This is unfortunate, because those patients without comorbidities may be the best candidates for antiviral therapy. Finally, identification of risk factors and testing for hepatitis C may be a low priority during a busy primary care appointment. It has been estimated that provision of all recommended preventative care would consume 7.4 hours of each primary doctor's day, leaving no time for other tasks.[29] Because the U.S. Preventative Services Task Force has concluded that insufficient evidence exists to recommend screening for hepatitis C, physicians may consider this a low priority.[30] Furthermore, physicians may not realize the value of diagnosing hepatitis C, because prior studies have identified limited knowledge about the disease among primary care physicians.[14] Thus, increasing the number of patients diagnosed will require education of the public and physicians alike, as well as attention to the worsening problem of the uninsured in the U.S.
Even if all patients with hepatitis C were aware of their diagnosis, a number of barriers to treatment would still remain. First, many patients will be ineligible for currently available antiviral therapy due to decompensated cirrhosis, comorbidities, or other reasons. Second, some patients may not follow up for further care, and others may not be referred to a specialist who prescribes antiviral therapy. In our study we could not differentiate between lack of referral by the primary physician versus lack of treatment by the specialist, but prior studies have suggested that lack of referral is a major barrier. In one survey of primary care physicians, 72% would not refer a patient with normal liver enzymes for treatment,[14] despite evidence that such patients can develop progressive disease.[31] Additionally, patients in some areas may have limited access to specialists who treat hepatitis C, because 20% of all gastroenterologists prescribe 80% of antiviral therapy in this country.[16] As mentioned above, it is possible that future improvements in antiviral therapy will increase enthusiasm for treatment among the medical community, and thus increase the number of patients treated. However, very little is known about health services delivery and quality of care for hepatitis C, and further research in this area is needed.
Although this study is the first to examine the public health impact of antiviral therapy for hepatitis C, it does have several important limitations. First, the Wolters Kluwer sample includes only commercial pharmacies, and does not include Veteran's Affairs pharmacies or patients treated in clinical trials. Therefore, it is possible that the true number of patients treated for hepatitis C each year in the U.S. is being underestimated. Conversely, NHANES estimates were used for the prevalence of hepatitis C, which may themselves be an underestimate due to the exclusion of homeless and institutionalized individuals. Thus, because potential errors in the numerator and denominator lie in the same direction, estimates of the proportion treated may be reasonably correct. A second important limitation is that the NHANES questionnaire does not provide sufficient detail to thoroughly examine patient and physician decision-making about testing and treatment for hepatitis C. Finally, any attempt to predict the future is likely to be imprecise. Therefore, projections regarding future treatment rates and liver-related deaths are only intended to provide a rough overview of the public health impact of antiviral therapy. The strength of this study is that it provides the first look at nationwide practice patterns for treatment of hepatitis C.
In conclusion, despite tremendous advances in antiviral therapy, current antiviral therapy is projected to prevent fewer than 15% of liver-related deaths caused by hepatitis C between 2002 and 2030. One of the key findings of this study is that the future development of new and better medications will have a less than optimal impact on this problem unless more patients are diagnosed and referred for treatment. Increased public health efforts are needed to improve access to antiviral therapy for patients with hepatitis C.
Results
Number of Patients Treated for Hepatitis C.
Results from the Wolters Kluwer prescription audit are shown in Table 2. There were 126,000 new retail prescriptions for pegylated interferon products in the year 2002, and this number had decreased to about 83,000 prescriptions per year by 2007. The fitted line projecting future treatment rates is shown in Fig. 2. If this trend were to continue, fewer than 1.4 million patients would be treated cumulatively by 2030.
Picture 2.png
Impact of Antiviral Therapy on Burden of Disease.
In the base-case analysis, without treatment the population with chronic hepatitis C would be expected to decrease slowly to about 2.95 million by 2030, as shown in Fig. 3. With treatment, assuming current practice patterns continue, the population with chronic hepatitis C is expected to decrease to 2.37 million by 2030. There would be 259,000 liver-related deaths caused by hepatitis C between 2002 and 2030 without treatment, and current antiviral therapy would prevent 37,500 of these. Thus, the current state of antiviral therapy is projected to prevent only 14.5% of liver-related deaths caused by hepatitis C between 2002 and 2030.
These estimates were most sensitive to the number of patients treated and the selection of patients for treatment who are at higher risk for disease progression. The most influential variables are shown in Table 3; none of the other variables affected the results by more than 1%. If the discovery of new medications in the future increases rates of SVR to 75%, at current rates of treatment this would still only prevent 21.7% of liver-related deaths caused by hepatitis C. Only by increasing treatment rates by 75% and increasing SVR rates to 75% could more than half of the liver-related deaths be prevented.
Picture 3.png
Reasons for Lack of Treatment.
Results from the NHANES questionnaire indicate that the primary reason for lack of treatment is lack of diagnosis, as shown in Fig. 4. Of the 133 respondents, 69 (proportion adjusting for sample weights 49%, 95% confidence interval [CI] 39%-60%) were previously unaware of their diagnosis, 12 (adjusted proportion 9%, 95% CI 3%-16%) did not follow up with a doctor about their hepatitis C, 33 (adjusted proportion 24%, 95% CI 15%-33%) were recommended by their doctor not to be treated, 8 (adjusted proportion 6%, 95% CI 1%-10%) refused treatment, and 11 (adjusted proportion 12%, 95% CI 4%-19%) were treated. This suggests that the largest barrier to treatment is failure to diagnose infection with hepatitis C. Physician recommendation appears to be the second most common reason why patients are not treated. Although the questionnaire does not provide sufficient medical detail to judge the appropriateness of these recommendations, it is concerning that 28% of the 100 subjects who had seen a doctor by the time of the survey had the understanding that no further follow-up for their hepatitis C was necessary.
Reasons for lack of treatment did not differ substantially by race or ethnicity. Males were slightly more likely to be unaware of their diagnosis (odds ratio [OR] 2.2, 95% CI 1.06-4.5), but less likely to have their doctor recommend treatment (OR 0.36, 95% CI 0.16-0.80). Despite this, the rates of treatment were identical between males and females. Not surprisingly, limited access to medical care appears to be a major barrier to diagnosis and treatment of hepatitis C. Respondents who lacked health insurance were more likely to be unaware of their diagnosis (OR 4.8, 95% CI 1.8-12.7) and less likely to be treated (OR 0.3, 95% CI 0.03-2.6). Additionally, respondents lacking a usual source of care were more likely to be unaware of their diagnosis (OR 19.0, 95% CI 2.4-148.1) and no respondents lacking a usual source of care had received treatment.
Figure 4. Reasons for lack of treatment among respondents to the NHANES Hepatitis C Follow-Up Questionnaire (n = 133).
Picture 4.png
Materials and Methods
Number of Patients Treated for Hepatitis C
To determine the number of patients receiving antiviral therapy in the U.S., data were obtained from Wolters Kluwer on the number of new patient prescriptions for pegylated interferon products (peg-interferon a-2a and peg-interferon a-2b) each year from 2002-2007. Wolters Kluwer maintains a longitudinal nationwide sample of patients filling prescriptions at retail, mail-order, and specialty pharmacies.[20] This sample captures about 31% of paid prescriptions nationwide. Every patient is given an encrypted unique identifier in order to differentiate between new prescriptions and refills. Prescription data from this sample are then linked to a second database that measures total monthly prescription volume in the U.S. from a random sample of pharmacies with computer management systems. This database represents 80% of U.S. retail prescriptions. In this fashion, the number of prescriptions from the longitudinal patient sample is projected to determine the total number of prescriptions per year for a given product. Demographics of subjects in the Wolters Kluwer longitudinal sample (Source Lx) compare similarly to those from the U.S. census, as shown in Appendix A.
Impact of Antiviral Therapy on Burden of Disease
Markov Model Structure.
In order to estimate the impact of current practice patterns on the future burden of hepatitis C, we created a dynamic population Markov model of patients with chronic hepatitis C in the U.S. from 2002-2030. During this time the population could change in size due to new infections, or due to patients being removed from the cohort because of death from liver disease or other causes, as shown in Fig. 1. The primary endpoint of the model was the number of liver-related deaths projected to be prevented by antiviral therapy during this period.
Input Data and Model Assumptions.
All variables included in the model are shown in Table 1. Baseline prevalence was obtained from the National Health and Nutrition Evaluation Survey (NHANES),[21] and annual incidence of new infections was obtained from the Centers for Disease Control.[22] The proportion of acute cases progressing to chronic hepatitis C was estimated from the proportion of cases in NHANES with positive antibody but negative RNA.[21] The number of patients receiving antiviral therapy between 2002 and 2007 was obtained from national pharmacy data (Wolters Kluwer database, as described above). The number of patients receiving antiviral therapy each year after 2007 was projected by fitting a three-parameter inverse linear equation to the 2002-2007 data, with optimization performed according to the least-squares criterion.[23] Liver-related mortality rate without treatment was based on current mortality attributable to hepatitis C,[1] and was assumed to increase by 10% of the baseline rate each year[5][6] to account for the increasing average duration of infection. Nonliver mortality rate was based on life tables from the National Center for Health Statistics,[24] and was assumed to increase by 10% of the baseline rate each year to account for aging of the hepatitis C population. The SVR rate was set at 50% in the base-case analysis,[8] with a 10-fold reduction in liver-related mortality risk among those achieving SVR.[9-11] Calculations were performed using Excel (Microsoft, Redmond, WA). The model cycle length was 1 year, and no discounting was performed.
Sensitivity Analysis.
Sensitivity analysis was performed on all variables. This was done both to determine susceptibility of the base-case estimate to uncertainty surrounding the variables, as well as to estimate how many extra lives could be saved by improvements in various aspects of medical care. For example, future advances in antiviral therapy may improve rates of SVR, or public education campaigns could increase the number of cases detected. Finally, because current guidelines recommend selecting patients for treatment in part based on their risk of future complications,[25] in sensitivity analysis the treated patients were assumed to have a 100% higher risk of liver-related death than those untreated.
Reasons for Lack of Treatment
Treatment decisions were investigated using data from the NHANES Hepatitis C Follow-Up Questionnaire. Every 2 years, NHANES selects a random sample of the U.S. population to participate in a health examination and laboratory testing. Beginning with the 2001/2002 version, participants testing positive for hepatitis C were sent a letter informing them of their test results. Four months later, they received a follow-up telephone questionnaire. In all, 277 subjects were contacted during the three iterations of this survey, the response rate was 136 (49%), and there were three partial completions, yielding a sample size of 133 completed questionnaires.
The NHANES questionnaire, which can be seen at http://www.cdc.gov/nchs/data/nhanes/pf_hcq_03_08.pdf, contains a series of questions about testing, follow-up, doctor's recommendations, and subject decision-making about treatment for hepatitis C. Responses to these questions were used to place subjects into the following categories with regard to treatment: (1) unaware of the diagnosis, (2) aware of the diagnosis, but did not seek further medical attention, (3) doctor did not recommend treatment, (4) subject refused treatment, and (5) treated. This classification was performed independently by two authors (M.V., R.T.), and agreement was 100%. Subjects who were previously unaware of their diagnosis and sought medical attention during the 4 months between notification of their diagnosis and receipt of the questionnaire were classified as unaware. Sample weights were used to adjust for oversampling of African-Americans, Hispanics, and subjects age >65 in the NHANES study.[26] Chi-square analysis was then used to determine bivariate associations between these categories and variables such as age, gender, race/ethnicity, and insurance status. Calculations were performed using Stata 10.0 (StataCorp, College Station, TX).
Hepatology Early View Oct 2009
"future development of new and better medications will have a less than optimal impact on this problem unless more patients are diagnosed and referred for treatment. Increased public health efforts are needed to improve access to antiviral therapy for patients with hepatitis C."....."Approximately 663,000 patients received antiviral therapy between 2002 and 2007, and treatment rates appear to be declining"...... "Only by increasing treatment rates by 75% and increasing SVR rates to 75% could more than half of the liver-related deaths be prevented."....."primary reason for lack of treatment is lack of diagnosis......49%, were previously unaware of their diagnosis......24% were recommended by their doctor not to be treated"
"respondents lacking a usual source of medical care were 19 times more likely to be unaware of their diagnosis"....."Because the U.S. Preventative Services Task Force has concluded that insufficient evidence exists to recommend screening for hepatitis C, physicians may consider this a low priority. physicians may not realize the value of diagnosing hepatitis C, because prior studies have identified limited knowledge about the disease among primary care physicians.[14] Thus, increasing the number of patients diagnosed will require education of the public and physicians alike, as well as attention to the worsening problem of the uninsured in the U.S.....some patients may not follow up for further care, and others may not be referred to a specialist who prescribes antiviral therapy......patients in some areas may have limited access to specialists who treat hepatitis C, because 20% of all gastroenterologists prescribe 80% of antiviral therapy in this country........it is possible that future improvements in antiviral therapy will increase enthusiasm for treatment among the medical community, and thus increase the number of patients treated. However, very little is known about health services delivery and quality of care for hepatitis C, and further research in this area is needed."
"In this study we estimated the number of patients currently receiving antiviral therapy for hepatitis C in the U.S., identified reasons for lack of treatment, and projected the impact current therapy will have on preventing liver-related deaths over the next two decades. We found that relatively few patients have been treated with pegylated interferon, and treatment rates appear to be declining. These findings are similar to a recent report from Europe, in which treatment rates varied by country from <1% to 16% of hepatitis C patients ever treated.[27] If this trend continues, antiviral therapy may prevent fewer than 15% of liver-related deaths caused by hepatitis C in the U.S. between 2002 and 2030."
"Fortunately, new treatments for hepatitis C are anticipated to become available in the next several years, which should increase rates of SVR among patients receiving treatment.[28] However, treatment first requires a diagnosis, and in this study we found that approximately half of all people with hepatitis C in the U.S. are unaware of their diagnosis."
"There would be 259,000 liver-related deaths caused by hepatitis C between 2002 and 2030 without treatment, and current antiviral therapy would prevent 37,500 of these. Thus, the current state of antiviral therapy is projected to prevent only 14.5% of liver-related deaths caused by hepatitis C between 2002 and 2030.....Approximately 663,000 patients received antiviral therapy between 2002 and 2007, and treatment rates appear to be declining.....If the discovery of new medications in the future increases rates of SVR to 75%, at current rates of treatment this would still only prevent 21.7% of liver-related deaths caused by hepatitis C. Only by increasing treatment rates by 75% and increasing SVR rates to 75% could more than half of the liver-related deaths be prevented......primary reason for lack of treatment is lack of diagnosis......49%, were previously unaware of their diagnosis......9% did not follow up with a doctor about their hepatitis C......24% were recommended by their doctor not to be treated......6% refused treatment.....12% were treated......28% of the 100 subjects who had seen a doctor by the time of the survey had the understanding that no further follow-up for their hepatitis C was necessary"
Michael L. Volk *, Rachel Tocco, Sameer Saini, Anna S.F. Lok
Division of Gastroenterology and Hepatology, University of Michigan, Ann Arbor, MI
email: Michael L. Volk (mvolk@med.umich.edu)
*Correspondence to Michael L. Volk, Division of Gastroenterology and Hepatology, University of Michigan Health System, 7C27 NIB, 300 N. Ingalls, Ann Arbor, MI 48109
Potential conflict of interest: Nothing to report.
fax: 734-936-8944.
Funded by:
Greenview Foundation
American Gastroenterological Association
ABSTRACT
Despite dramatic improvements in antiviral therapy for hepatitis C, there is reason to believe that the uptake of antiviral therapy remains limited. The aims of this study were to determine the number of patients being treated with antiviral therapy in the U.S., to estimate the public health impact of these treatment patterns, and to identify barriers to treatment for patients with hepatitis C. Data on the number of new patient pegylated interferon prescriptions each year, from 2002-2007, was obtained from Wolters Kluwer Inc., which maintains an electronic audit of pharmacies nationwide. A Markov model was created of the population with chronic hepatitis C in the U.S. from 2002 to 2030, and was used to estimate the number of liver-related deaths caused by hepatitis C that will be prevented by current treatment patterns. The National Health and Nutrition Evaluation Survey (NHANES) Hepatitis C Follow-Up Questionnaire was used to investigate reasons for lack of treatment and to identify strategies for improving access. Approximately 663,000 patients received antiviral therapy between 2002 and 2007, and treatment rates appear to be declining. If this trend continues, only 14.5% of liver-related deaths caused by hepatitis C from 2002-2030 will be prevented by antiviral therapy. Results from the NHANES questionnaire suggest that the primary barrier to treatment is lack of diagnosis, with 69/133 (adjusted proportion 49%) of respondents previously unaware that they had hepatitis C.
Conclusion: Efforts to improve rates of diagnosis and treatment will be required if the future public health burden of hepatitis C is to be ameliorated.
Hepatitis C is a major public health burden in the United States. It causes nearly 8,000 deaths per year,[1] is the leading cause for liver transplantation,[2] and has contributed to a rise in the incidence of hepatocellular carcinoma.[3] Quality of life is impaired for patients infected with the virus, even among those without cirrhosis.[4] As of 2001, there were an estimated 3.2 million people in the U.S. with chronic hepatitis C. Although the incidence of new infections has declined, the number of deaths may continue to increase 2- to 4-fold over the next 20 years due to prevalent cases with longstanding infection.[5][6]......the primary reason for lack of treatment is lack of diagnosis
The future public health burden of hepatitis C could potentially be mitigated by antiviral therapy.[5] In the last two decades there have been dramatic improvements in treatment for hepatitis C. The benefit of adding ribavirin to interferon was demonstrated in a series of studies in the late 1990s,[7] and then pegylated interferon was approved at the end of 2001. Combination therapy with pegylated interferon and ribavirin can now achieve sustained virological response (SVR) in 50% of patients,[8] compared to rates of 17% with standard interferon alone.[7] Patients who achieve SVR enjoy long-term remission of disease, with liver-related mortality rates comparable to the general population.[9-11]
Despite these improvements, there are reasons to believe that the uptake of antiviral therapy may be limited. Patients with hepatitis C are more likely to lack health insurance and a usual source of care.[12][13] Studies have demonstrated that primary care physicians lack knowledge about risk factors and testing for hepatitis C.[14][15] Access to a specialist willing to prescribe antiviral therapy may be another limiting factor.[16] Finally, many patients have contraindications to treatment, or do not wish to suffer the myriad side effects.[17] For these reasons, we hypothesized that the public health impact of antiviral therapy may be limited, as much by low utilization as by the inherent limitations in the available medications. Several studies from the Veterans Affairs Healthcare System and Health Maintenance Organizations have reported low treatment rates among patients identified to have hepatitis C.[18][19] However, no national data exist on the number of patients currently receiving antiviral therapy in the U.S., or the impact of current practice patterns on the burden of hepatitis C. The aims of this study were to determine the public health impact of current antiviral therapy and to identify barriers to treatment of hepatitis C.
Discussion
In this study we estimated the number of patients currently receiving antiviral therapy for hepatitis C in the U.S., identified reasons for lack of treatment, and projected the impact current therapy will have on preventing liver-related deaths over the next two decades. We found that relatively few patients have been treated with pegylated interferon, and treatment rates appear to be declining. These findings are similar to a recent report from Europe, in which treatment rates varied by country from <1% to 16% of hepatitis C patients ever treated.[27] If this trend continues, antiviral therapy may prevent fewer than 15% of liver-related deaths caused by hepatitis C in the U.S. between 2002 and 2030.
Fortunately, new treatments for hepatitis C are anticipated to become available in the next several years, which should increase rates of SVR among patients receiving treatment.[28] However, treatment first requires a diagnosis, and in this study we found that approximately half of all people with hepatitis C in the U.S. are unaware of their diagnosis. There are several possible reasons for this situation. First, lack of health insurance poses an obvious barrier to testing. In 2001, 30% of patients with hepatitis C were uninsured,[12] and this number has likely increased since that time. Second, because hepatitis C is usually asymptomatic, patients without any other medical problems may not seek medical attention. In the NHANES survey, we found that respondents lacking a usual source of medical care were 19 times more likely to be unaware of their diagnosis. This is unfortunate, because those patients without comorbidities may be the best candidates for antiviral therapy. Finally, identification of risk factors and testing for hepatitis C may be a low priority during a busy primary care appointment. It has been estimated that provision of all recommended preventative care would consume 7.4 hours of each primary doctor's day, leaving no time for other tasks.[29] Because the U.S. Preventative Services Task Force has concluded that insufficient evidence exists to recommend screening for hepatitis C, physicians may consider this a low priority.[30] Furthermore, physicians may not realize the value of diagnosing hepatitis C, because prior studies have identified limited knowledge about the disease among primary care physicians.[14] Thus, increasing the number of patients diagnosed will require education of the public and physicians alike, as well as attention to the worsening problem of the uninsured in the U.S.
Even if all patients with hepatitis C were aware of their diagnosis, a number of barriers to treatment would still remain. First, many patients will be ineligible for currently available antiviral therapy due to decompensated cirrhosis, comorbidities, or other reasons. Second, some patients may not follow up for further care, and others may not be referred to a specialist who prescribes antiviral therapy. In our study we could not differentiate between lack of referral by the primary physician versus lack of treatment by the specialist, but prior studies have suggested that lack of referral is a major barrier. In one survey of primary care physicians, 72% would not refer a patient with normal liver enzymes for treatment,[14] despite evidence that such patients can develop progressive disease.[31] Additionally, patients in some areas may have limited access to specialists who treat hepatitis C, because 20% of all gastroenterologists prescribe 80% of antiviral therapy in this country.[16] As mentioned above, it is possible that future improvements in antiviral therapy will increase enthusiasm for treatment among the medical community, and thus increase the number of patients treated. However, very little is known about health services delivery and quality of care for hepatitis C, and further research in this area is needed.
Although this study is the first to examine the public health impact of antiviral therapy for hepatitis C, it does have several important limitations. First, the Wolters Kluwer sample includes only commercial pharmacies, and does not include Veteran's Affairs pharmacies or patients treated in clinical trials. Therefore, it is possible that the true number of patients treated for hepatitis C each year in the U.S. is being underestimated. Conversely, NHANES estimates were used for the prevalence of hepatitis C, which may themselves be an underestimate due to the exclusion of homeless and institutionalized individuals. Thus, because potential errors in the numerator and denominator lie in the same direction, estimates of the proportion treated may be reasonably correct. A second important limitation is that the NHANES questionnaire does not provide sufficient detail to thoroughly examine patient and physician decision-making about testing and treatment for hepatitis C. Finally, any attempt to predict the future is likely to be imprecise. Therefore, projections regarding future treatment rates and liver-related deaths are only intended to provide a rough overview of the public health impact of antiviral therapy. The strength of this study is that it provides the first look at nationwide practice patterns for treatment of hepatitis C.
In conclusion, despite tremendous advances in antiviral therapy, current antiviral therapy is projected to prevent fewer than 15% of liver-related deaths caused by hepatitis C between 2002 and 2030. One of the key findings of this study is that the future development of new and better medications will have a less than optimal impact on this problem unless more patients are diagnosed and referred for treatment. Increased public health efforts are needed to improve access to antiviral therapy for patients with hepatitis C.
Results
Number of Patients Treated for Hepatitis C.
Results from the Wolters Kluwer prescription audit are shown in Table 2. There were 126,000 new retail prescriptions for pegylated interferon products in the year 2002, and this number had decreased to about 83,000 prescriptions per year by 2007. The fitted line projecting future treatment rates is shown in Fig. 2. If this trend were to continue, fewer than 1.4 million patients would be treated cumulatively by 2030.
Picture 2.png
Impact of Antiviral Therapy on Burden of Disease.
In the base-case analysis, without treatment the population with chronic hepatitis C would be expected to decrease slowly to about 2.95 million by 2030, as shown in Fig. 3. With treatment, assuming current practice patterns continue, the population with chronic hepatitis C is expected to decrease to 2.37 million by 2030. There would be 259,000 liver-related deaths caused by hepatitis C between 2002 and 2030 without treatment, and current antiviral therapy would prevent 37,500 of these. Thus, the current state of antiviral therapy is projected to prevent only 14.5% of liver-related deaths caused by hepatitis C between 2002 and 2030.
These estimates were most sensitive to the number of patients treated and the selection of patients for treatment who are at higher risk for disease progression. The most influential variables are shown in Table 3; none of the other variables affected the results by more than 1%. If the discovery of new medications in the future increases rates of SVR to 75%, at current rates of treatment this would still only prevent 21.7% of liver-related deaths caused by hepatitis C. Only by increasing treatment rates by 75% and increasing SVR rates to 75% could more than half of the liver-related deaths be prevented.
Picture 3.png
Reasons for Lack of Treatment.
Results from the NHANES questionnaire indicate that the primary reason for lack of treatment is lack of diagnosis, as shown in Fig. 4. Of the 133 respondents, 69 (proportion adjusting for sample weights 49%, 95% confidence interval [CI] 39%-60%) were previously unaware of their diagnosis, 12 (adjusted proportion 9%, 95% CI 3%-16%) did not follow up with a doctor about their hepatitis C, 33 (adjusted proportion 24%, 95% CI 15%-33%) were recommended by their doctor not to be treated, 8 (adjusted proportion 6%, 95% CI 1%-10%) refused treatment, and 11 (adjusted proportion 12%, 95% CI 4%-19%) were treated. This suggests that the largest barrier to treatment is failure to diagnose infection with hepatitis C. Physician recommendation appears to be the second most common reason why patients are not treated. Although the questionnaire does not provide sufficient medical detail to judge the appropriateness of these recommendations, it is concerning that 28% of the 100 subjects who had seen a doctor by the time of the survey had the understanding that no further follow-up for their hepatitis C was necessary.
Reasons for lack of treatment did not differ substantially by race or ethnicity. Males were slightly more likely to be unaware of their diagnosis (odds ratio [OR] 2.2, 95% CI 1.06-4.5), but less likely to have their doctor recommend treatment (OR 0.36, 95% CI 0.16-0.80). Despite this, the rates of treatment were identical between males and females. Not surprisingly, limited access to medical care appears to be a major barrier to diagnosis and treatment of hepatitis C. Respondents who lacked health insurance were more likely to be unaware of their diagnosis (OR 4.8, 95% CI 1.8-12.7) and less likely to be treated (OR 0.3, 95% CI 0.03-2.6). Additionally, respondents lacking a usual source of care were more likely to be unaware of their diagnosis (OR 19.0, 95% CI 2.4-148.1) and no respondents lacking a usual source of care had received treatment.
Figure 4. Reasons for lack of treatment among respondents to the NHANES Hepatitis C Follow-Up Questionnaire (n = 133).
Picture 4.png
Materials and Methods
Number of Patients Treated for Hepatitis C
To determine the number of patients receiving antiviral therapy in the U.S., data were obtained from Wolters Kluwer on the number of new patient prescriptions for pegylated interferon products (peg-interferon a-2a and peg-interferon a-2b) each year from 2002-2007. Wolters Kluwer maintains a longitudinal nationwide sample of patients filling prescriptions at retail, mail-order, and specialty pharmacies.[20] This sample captures about 31% of paid prescriptions nationwide. Every patient is given an encrypted unique identifier in order to differentiate between new prescriptions and refills. Prescription data from this sample are then linked to a second database that measures total monthly prescription volume in the U.S. from a random sample of pharmacies with computer management systems. This database represents 80% of U.S. retail prescriptions. In this fashion, the number of prescriptions from the longitudinal patient sample is projected to determine the total number of prescriptions per year for a given product. Demographics of subjects in the Wolters Kluwer longitudinal sample (Source Lx) compare similarly to those from the U.S. census, as shown in Appendix A.
Impact of Antiviral Therapy on Burden of Disease
Markov Model Structure.
In order to estimate the impact of current practice patterns on the future burden of hepatitis C, we created a dynamic population Markov model of patients with chronic hepatitis C in the U.S. from 2002-2030. During this time the population could change in size due to new infections, or due to patients being removed from the cohort because of death from liver disease or other causes, as shown in Fig. 1. The primary endpoint of the model was the number of liver-related deaths projected to be prevented by antiviral therapy during this period.
Input Data and Model Assumptions.
All variables included in the model are shown in Table 1. Baseline prevalence was obtained from the National Health and Nutrition Evaluation Survey (NHANES),[21] and annual incidence of new infections was obtained from the Centers for Disease Control.[22] The proportion of acute cases progressing to chronic hepatitis C was estimated from the proportion of cases in NHANES with positive antibody but negative RNA.[21] The number of patients receiving antiviral therapy between 2002 and 2007 was obtained from national pharmacy data (Wolters Kluwer database, as described above). The number of patients receiving antiviral therapy each year after 2007 was projected by fitting a three-parameter inverse linear equation to the 2002-2007 data, with optimization performed according to the least-squares criterion.[23] Liver-related mortality rate without treatment was based on current mortality attributable to hepatitis C,[1] and was assumed to increase by 10% of the baseline rate each year[5][6] to account for the increasing average duration of infection. Nonliver mortality rate was based on life tables from the National Center for Health Statistics,[24] and was assumed to increase by 10% of the baseline rate each year to account for aging of the hepatitis C population. The SVR rate was set at 50% in the base-case analysis,[8] with a 10-fold reduction in liver-related mortality risk among those achieving SVR.[9-11] Calculations were performed using Excel (Microsoft, Redmond, WA). The model cycle length was 1 year, and no discounting was performed.
Sensitivity Analysis.
Sensitivity analysis was performed on all variables. This was done both to determine susceptibility of the base-case estimate to uncertainty surrounding the variables, as well as to estimate how many extra lives could be saved by improvements in various aspects of medical care. For example, future advances in antiviral therapy may improve rates of SVR, or public education campaigns could increase the number of cases detected. Finally, because current guidelines recommend selecting patients for treatment in part based on their risk of future complications,[25] in sensitivity analysis the treated patients were assumed to have a 100% higher risk of liver-related death than those untreated.
Reasons for Lack of Treatment
Treatment decisions were investigated using data from the NHANES Hepatitis C Follow-Up Questionnaire. Every 2 years, NHANES selects a random sample of the U.S. population to participate in a health examination and laboratory testing. Beginning with the 2001/2002 version, participants testing positive for hepatitis C were sent a letter informing them of their test results. Four months later, they received a follow-up telephone questionnaire. In all, 277 subjects were contacted during the three iterations of this survey, the response rate was 136 (49%), and there were three partial completions, yielding a sample size of 133 completed questionnaires.
The NHANES questionnaire, which can be seen at http://www.cdc.gov/nchs/data/nhanes/pf_hcq_03_08.pdf, contains a series of questions about testing, follow-up, doctor's recommendations, and subject decision-making about treatment for hepatitis C. Responses to these questions were used to place subjects into the following categories with regard to treatment: (1) unaware of the diagnosis, (2) aware of the diagnosis, but did not seek further medical attention, (3) doctor did not recommend treatment, (4) subject refused treatment, and (5) treated. This classification was performed independently by two authors (M.V., R.T.), and agreement was 100%. Subjects who were previously unaware of their diagnosis and sought medical attention during the 4 months between notification of their diagnosis and receipt of the questionnaire were classified as unaware. Sample weights were used to adjust for oversampling of African-Americans, Hispanics, and subjects age >65 in the NHANES study.[26] Chi-square analysis was then used to determine bivariate associations between these categories and variables such as age, gender, race/ethnicity, and insurance status. Calculations were performed using Stata 10.0 (StataCorp, College Station, TX).
Treatment of insulin resistance with metformin in naïve genotype 1 chronic hepatitis C patients
Treatment of insulin resistance with metformin in naïve genotype 1 chronic hepatitis C patients receiving peginterferon alfa-2a plus ribavirin
Hepatology Early View Oct 2009
"Adding metformin to peginterferon and ribavirin was safe and improved insulin sensitivity. Although the study failed to show a statistically significant difference between arms, it did show an improved SVR in females."
"HOMA less than 2 at week 24 was more often seen in arm A (55%) than in arm B (14%), and this goal was associated with higher SVR.....In summary, treating patients with hepatitis C genotype 1 and insulin resistance using metformin improves insulin sensitivity, is safe, and increases SVR rate in patients who reached HOMA lower than 2 at week 24 of therapy and in women, in whom, in the current study, the therapy doubled the SVR rate......In females (n = 54), adding metformin to peginterferon alfa-2a plus ribavirin resulted in a doubling of the SVR rate in 15 of 26 patients (58%) in arm A versus 8 of 28 patients (29%) in arm B; P = 0.03) (Fig. 2). Viral decline during the first 12 weeks was greater in females taking metformin than placebo (mean [standard deviation (SD)]) -4.88 (1.18) versus -4.0 (1.44); P = 0.02), whereas no viral decline was noted in males. Furthermore, no cases of breakthrough were seen in females with metformin therapy (females with HCV RNA negative at week 24 (n = 41), with 4 of 20 patients experiencing breakthrough in arm B but none in arm A (0 of 21); P = 0.05......In the univariate analysis, higher baseline viral load and calculated fibrosis (the noninvasive Sydney Index) were associated with nonresponse of 6.52 (0.73) versus 6.28 (0.75) log10 HCV RNA (P = 0.08) and 0.52 (0.32) versus 0.38 (0.25) in the Sydney Index (P = 0.01). During treatment, the HCV RNA decline in the first 12 weeks of 3.04 (1.65) versus 1.11 (0.35) log10 HCV RNA, P = 0.0001, and the HOMA-IR decline during the first 24 weeks was associated with sustained response; that is, 19 of 28 patients (68%) with a HOMA-IR less than 2 at week 24 achieved SVR, whereas 25 of 56 patients (45%) with HOMA at 24 weeks greater than 2 achieved SVR (P = 0.05)."
Manuel Romero-Gómez 1 *§, Moisés Diago 2, Raúl J. Andrade 3, José L. Calleja 4, Javier Salmerón 5, Conrado M. Fernández-Rodríguez 6, Ricard Solà 7, Javier García-Samaniego 8, Juan M. Herrerías 9, Manuel De la Mata 10, Ricardo Moreno-Otero 11, Óscar Nuñez 12, Antonio Olveira 13, Santiago Durán 14, Ramón Planas 14, Spanish TRIC-1 (Treatment of Resistance to Insulin in Hepatitis C Genotype 1) group
1Unit for the Clinical Management of Digestive Diseases and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERHD), Hospital Universitario de Valme, Sevilla, Spain
2Hepatology Section, Hospital General de Valencia, Spain
3Hepatology Unit and CIBEREHD, Hospital Virgen de la Victoria, Málaga, Spain
4Digestive Diseases Unit, Hospital Universitario Puerta de Hierro, Madrid, Spain
5Digestive Disease Service and CIBEREHD, Hospital San Cecilio, Granada, Spain
6Gastroenterology Unit, Hospital Universitario Fundación Alcorcón, Madrid, Spain
7Hepatology Section, Hospital del Mar, Barcelona, Spain
8Gastroenterology Service and CIBEREHD, Hospital Carlos III, Madrid, Spain
9Gastroenterology Service, Hospital Universitario Virgen Macarena, Sevilla, Spain
10Hepatology Section and CIBEREHD, Hospital Universitario Reina Sofía, Córdoba, Spain
11Digestive Diseases Service and CIBEREHD, Hospital de la Princesa, Madrid, Spain
12Hepatology Unit, Hospital Gregorio Marañón, Madrid, Spain
13Gastroenterology Service, Hospital La Paz, Madrid, Spain
14Gastroenterology and Hepatology Unit and CIBEREHD, Hospital Germans Trias i Pujol, Barcelona, Spain
email: Manuel Romero-Gómez (mromerogomez@us.es)
*Correspondence to Manuel Romero-Gómez, UCM Digestive Diseases and CIBERHD, Hospital Universitario de Valme, Universidad de Sevilla, Avda de Bellavista s/n, 41014 Sevilla, Spain
Presented at the 59th Annual Meeting of the American Association for the Study of the Liver Diseases, October 31 to November 4, 2008, San Francisco, CA.
Abstract
Insulin resistance affects sustained virological response (SVR) in chronic hepatitis C. To know whether adding metformin to standard antiviral treatment improves SVR, we conducted a prospective, multicentered, randomized, double-blinded, placebo-controlled trial in 19 Spanish hospitals, including 123 consecutive patients with genotype 1 chronic hepatitis C and insulin resistance. Patients were randomized to receive either metformin (arm A; n = 59) or placebo (arm B; n = 64) in addition to peginterferon alfa-2a (180 g/week) and ribavirin (1000-1200 mg/day). The primary end point was SVR, and secondary endpoints were viral clearance at weeks 12, 24, and 48, and changes in the homeostasis model assessment (HOMA) index over the first 24 weeks. There were no differences between arms at baseline. In the intent-to-treat analysis, SVR was observed in 53% versus 42% in arm A and arm B, respectively (P = NS). In the subgroup analyses, SVR was higher in females (n = 54) receiving metformin: arm A, 58% (15/26) versus 29% (8/28) arm B (P = 0.03). In the per protocol analysis (PPA; n = 101), SVR was 67% in arm A and 49% in arm B (P = 0.06). Viral decline during the first 12 weeks was greater in females receiving metformin: -4.88 (1.18) versus -4.0 (1.44) (P = 0.021), whereas no differences were seen in males. The triple therapy was well tolerated, but diarrhea was more often seen in arm A (34% versus 11%; P < 0.05).
Conclusion: Adding metformin to peginterferon and ribavirin was safe and improved insulin sensitivity. Although the study failed to show a statistically significant difference between arms, it did show an improved SVR in females.
Hepatitis C is a major healthcare problem, and current therapies have achieved sustained response in more than a half of infected patients. Factors associated with nonresponse are host-viral genotype 1, high viral load, advanced fibrosis, and insulin resistance.[1] Sustained virological response (SVR) decreases when insulin sensitivity is impaired.[1][2] Also, impaired fasting glucose has been independently associated with lower SVR rate.[3] Insulin resistance is thought to be promoted by hepatitis C virus (HCV) itself and, after clearance of the virus, insulin resistance improves concomitantly with the reduction in the risk of glucose abnormalities and diabetes.[1][3][4] HCV proteins lead to insulin resistance, promoting the degradation of insulin receptor substrate 1.[5] Transgenic mice expressing core HCV protein developed insulin resistance and steatosis.[6] Insulin resistance and steatosis promoted by the virus have been found to be associated with improvement in viral fitness,[7] and this seems to be a defense mechanism against viral clearance. Lifestyle, exercise, diet, and insulin-sensitizing drugs could improve insulin resistance. Metformin is an oral biguanide that lowers blood glucose and insulin secretion and improves the individual's lipid profile, mainly because of suppression of hepatic glucose output and increased glucose uptake in skeletal muscle.[8] Because insulin resistance can be successfully treated with biguanides, we proposed that sustained virological response could be improved by adding metformin to the standard of care for patients with chronic hepatitis C genotype 1 and insulin resistance.
Discussion
The combination of metformin, peginterferon alfa-2a, and ribavirin was well tolerated by the patients. It improved insulin resistance in more than 50% of patients and increased SVR rate in 10% of patients with hepatitis C genotype 1 and HOMA greater than 2. This was related to a trend toward a low rate of breakthrough in patients receiving metformin; the response at week 24 and relapse rate were similar in both arms. However, in the PPA, viral clearance was significantly higher in the metformin group at weeks 24 and 48, which supports the proposition of a better antiviral activity of peginterferon alfa-2a and ribavirin in patients receiving metformin. Despite raising SVR by 10% (a clinically relevant increase), this difference did not reach statistical significance, which could be attributable, at least in part, to a type 2 error in sample size calculation. That there is a trend in the intention-to-treat analysis and significance in the PPA and female subanalysis strongly supports this hypothesis. Despite the sample calculation being based on the available data, our subsequent findings of the study suggest that there were not enough patients enrolled in the study to achieve statistically significant differences. Metformin is an oral biguanide, which is one of the most widely prescribed therapeutic agents for the control of type 2 diabetes. Metformin controls glucose levels by suppression of hepatic glucose output, by increasing insulin-mediated glucose disposal, and by decreasing fatty acid oxidation and synthesis of very-low-density lipoprotein.[11] During HCV replication, core protein promotes unfolded protein response that causes dysfunction of endoplasmic reticulum and mitochondria.[12][13] Indeed, HCV proteins promote insulin receptor substrate 1 degradation by several mechanisms, including oxidative stress, down-regulation of PPAR, and enhancing TNF production in a genotype-dependent manner. In genotype 1, the degradation of insulin receptor substrate 1 has been shown to be induced by mammalian target of rapamycin and, in genotype 3, by suppressor of cytokine signaling 7 and peroxisome proliferator-activated receptor-gamma (PPAR-). Thus, metformin could be useful in the management of this comorbidity because of its ability to increase the binding of insulin to its receptors and to increase phosphorylation as well as the tyrosine kinase activity of insulin receptors[14] through, mainly, the action of phosphorylation of hepatic adenosine-monophosphate-activated protein kinase by STK11 (formerly LKB1).[15] Pioglitazone was shown not to improve either viral response or insulin sensitivity in five previously nonresponding individuals.[16] In treatment-naïve patients, pioglitazone in combination with peginterferon alfa-2b and ribavirin improved insulin sensitivity and hepatic steatosis.[17] Elgouhari et al.[18] had shown that the combination therapy improved early virological kinetics over the first 4 weeks and the end-of-treatment response, but not SVR.[18] The low number of patients (n = 40) precluded subset analysis and comparisons with the current study. However, the mechanism of action of insulin-sensitizer drugs in the setting of patients with hepatitis C appears to be different and could be related to viral genotype. Metformin improved insulin sensitivity in patients with hepatitis C genotype 1. Theoretically, PPAR- agonist appears to be indicated in the management of patients with genotype 3 HCV infection (in whom insulin resistance appears as a consequence of decreased PPAR- activity). Furthermore, metformin could be preferred because it acts directly in the liver, whereas PPAR- agonist shows an effect on peripheral metabolism. Further prospective studies using different insulin sensitizers in a large cohort of patients with hepatitis C with different genotypes are required to demonstrate whether these data could be translated into clinical practice.
In women, metformin increased the SVR rate significantly. This sex-related difference has not been previously reported in the management of hepatitis C. Spontaneous viral clearance has been seen more often in women than in men.[19] However, no sex-related impact has been reported on the clinical course of the infection or in SVR rate. Some weak evidence supports the influence of sex on the therapeutic effects of metformin: (1) metabolic syndrome pathophysiology and diabetes-related complications appear to be vary in relation to sex, and vitamin K supplementation appears to improve insulin resistance in men but not in women[20]; (2) some therapeutic effects of metformin appear to be sex dependent, that is, metformin promotes greater short-term weight loss in women than in men, but these differences were not observed in diabetic control subjects[21][22]; (3) STK11 (formerly LKB1) plays a major role in metformin sensitivity,[23] and some genetic alterations appear to have a clinical impact that is sex dependent, such as the risk of malignancies in Peutz-Jegher syndrome.[24] In women, metformin therapy has been associated with a greater decrease of HCV RNA during the first 12 weeks of treatment and a lower breakthrough rate. These data support the hypothesis that, in women, metformin improves the antiviral activity of peginterferon and ribavirin. It remains to be explored whether genetic alterations in the STK11 gene or nuclear factors such as estrogens are implicated in the ability of metformin to improve antiviral activity.
Two biguanides (phenformin and buformin) have been withdrawn from the pharmacopeias because of evidence of lactic acidosis associated with their use. Despite cases of lactic acidosis having been reported with the use of metformin, a recent systematic review demonstrated no increased risk for lactic acidosis in patients without contraindications to metformin use.[25] In the current study, blood lactate level was monitored during treatment, and no patients developed lactic acidosis or hyperlactatemia. Metformin was associated with mild diarrhea, which was well tolerated, and no patient withdrew from the study because of this adverse event. Thus, metformin used together with peginterferon alfa-2a and ribavirin in the treatment of hepatitis C seems to be safe and well tolerated. The perceived contraindication of metformin for patients with alterations in liver function needs to be reevaluated.
In our study, metformin improved insulin sensitivity, with more than half the number of patients achieving HOMA less than 2 by week 24. This end point was also related to the probability of achieving SVR. Nevertheless, metformin-induced improvement in insulin sensitivity is not the sole action of this drug, and the mechanism by which metformin improves the antiviral activity of peginterferon + ribavirin warrants further investigation.
In summary, treating patients with hepatitis C genotype 1 and insulin resistance using metformin improves insulin sensitivity, is safe, and increases SVR rate in patients who reached HOMA lower than 2 at week 24 of therapy and in women, in whom, in the current study, the therapy doubled the SVR rate.
Results
Of 125 patients initially recruited, 123 were randomly assigned to a treatment group and received at least one dose of medication; two patients were excluded because of not having met the full criteria for inclusion (one patient was already taking metformin, and the other was found to be infected by a different viral genotype). The two treatment groups were well matched for age, sex, and other variables measured on entry into the trial (Table 1).
Virological Response.
In the intention-to-treat analysis (n = 123), viral clearance was seen in 54% (32/59) of patients versus 48% (31/64) at week 12; in 75% (45/59) versus 75% (48/64) at week 24; in 71% (42/59) versus 63% (40/64) at week 48; and in 53% (31/59) versus 42% (27/64) at week 72 (in other words, SVR rate) in arm A and arm B, respectively. Virological breakthrough was slightly higher in patients receiving placebo 17% (8/48) versus 7% (3/45), but this difference was not statistically significant. Relapse rate was similar in both arms: 11 of 59 patients (19%) in arm A and 12 of 64 patients (19%) in arm B; P = 0.98.
In the PPA (n = 101), viral clearance was achieved in 67% (31/46) versus 53 (29/55), P = 0.13 at week 12; in 96% (44/46) versus 78% (43/55), P = 0.01 at week 24; in 91% (42/46) versus 69% (38/55), P < 0.01 at week 48; in 67% (31/46) versus 49% (27/55), P = 0.06 at week 72 in arm A and arm B, respectively.
Subgroup Analysis.
In females (n = 54), adding metformin to peginterferon alfa-2a plus ribavirin resulted in a doubling of the SVR rate in 15 of 26 patients (58%) in arm A versus 8 of 28 patients (29%) in arm B; P = 0.03) (Fig. 2). Viral decline during the first 12 weeks was greater in females taking metformin than placebo (mean [standard deviation (SD)]) -4.88 (1.18) versus -4.0 (1.44); P = 0.02), whereas no viral decline was noted in males. Furthermore, no cases of breakthrough were seen in females with metformin therapy (females with HCV RNA negative at week 24 (n = 41), with 4 of 20 patients experiencing breakthrough in arm B but none in arm A (0 of 21); P = 0.05.
Figure 2. Rates of viral clearance in patients randomly assigned to receive Metformin + Pegalfa2a + Ribavirin (arm A) or Placebo + Pegalfa2a + Ribavirin (arm B) in the intention-to-treat analysis, per-protocol analysis and females subset analysis. A sustained virological response was defined as undetectable serum HCV RNA level (<10 UI/mL) at week 24 after the end of treatment.
Picture 1.png
Changes in Insulin Resistance.
The decline in HOMA index in patients receiving metformin was significantly higher than in patients receiving placebo. In arm A, the HOMA index decreased from 4.3 (2.2) to 2.6 (1.7) and in arm B from 4.6 (2.7) to 3.8 (2.1); P = 0.001. However, no difference was seen in weight loss in either treatment arm. By the end-of-follow-up, women receiving metformin had a mean body weight decrease of 1.3 kg (SD: 5.4) versus 1.1 kg (SD: 5.6) in women receiving standard care; P = NS. In men, body weight decreased by 1.1 kg (SD: 6.1) in patients receiving metformin versus 0.9 kg (SD: 5.5) in patients on placebo; P = NS. Chages in body weight did not influence SVR. However, HOMA less than 2 at week 24 was more often seen in arm A (55%) than in arm B (14%), and this goal was associated with higher SVR (see below).
Predictors of SVR.
In the univariate analysis, higher baseline viral load and calculated fibrosis (the noninvasive Sydney Index) were associated with nonresponse of 6.52 (0.73) versus 6.28 (0.75) log10 HCV RNA (P = 0.08) and 0.52 (0.32) versus 0.38 (0.25) in the Sydney Index (P = 0.01). During treatment, the HCV RNA decline in the first 12 weeks of 3.04 (1.65) versus 1.11 (0.35) log10 HCV RNA, P = 0.0001, and the HOMA-IR decline during the first 24 weeks was associated with sustained response; that is, 19 of 28 patients (68%) with a HOMA-IR less than 2 at week 24 achieved SVR, whereas 25 of 56 patients (45%) with HOMA at 24 weeks greater than 2 achieved SVR (P = 0.05).
Using reverse stepwise logistic multivariate regression analysis, the independent variable related to SVR was HCV RNA at week 12 (hazard ratio, 0.15; 95% confidence interval: 0.07-0.34) (Table 2).
Safety.
The percentage of patients reporting adverse events or serious adverse events was similar in the two groups. The adverse events reported were those typical of interferon-based treatments, which include fatigue, headache, insomnia, and myalgia. Diarrhea was more often seen in patients assigned to arm A than in patients receiving placebo (36% versus 11%; P < 0.05). However, the diarrhea was well tolerated and did not lead to any withdrawals from the trial. Two patients in each treatment group had to discontinue because of adverse events (Table 2). Doses of metformin or placebo were modified in 37% of patients in arm A and 38% in arm B (P = NS). Serum lactate was monitored during treatment, and no patient developed lactic acidosis or hyperlactatemia.
Patients and Methods
Selection of Patients.
Eligible patients were those who were 18 years of age or older, were infected with genotype 1 (determined by INNO-LIPA assay, Innogenetics), and had a quantifiable serum HCV RNA for more than 6 months together with an elevated serum alanine aminotransferase level, nondiabetic, and homeostasis model assessment (HOMA) index greater than 2 (Table 1). Liver biopsy was not mandatory. Liver fibrosis was calculated using the Sydney index (a noninvasive method for the prediction of liver fibrosis based on a formula that includes age, plasma cholesterol concentration, aspartate aminotransferase, alcohol consumption, and HOMA index[9] that has been validated in Spanish patients with chronic hepatitis C).[10] Patients were ineligible if they had other liver diseases, showed contraindications to peginterferon or ribavirin, or had previously received antiviral drugs.
Study Design.
Patients were randomly assigned into this prospective, multicentered, randomized, double-blinded, placebo-controlled trial (registered in ClinicalTrials.gov; #NCT00546442). The study was conducted in 19 Spanish centers. All sites received approval from their ethics committees. Each patient provided written informed consent for participation. Patients were randomly assigned in a 1:1 ratio to receive either metformin 425 mg three times daily for the first month and 850 mg three times daily from week 4 to 48 (arm A; n = 59) or placebo (arm B; n = 64) in addition to peginterferon alfa-2a (Pegasys; Roche Farma, Madrid, Spain) 180 g weekly and ribavirin 1000 to 1200 mg daily. Patients were followed-up over 24 weeks to confirm SVR.
Laboratory Analyses.
An overnight (12-hour) fasting blood sample was taken for routine analyses, including aminotransferases (alanine aminotransferase, aspartate aminotransferase), alkaline phosphatase, gamma-glutamyltransferase, platelets, glucose, cholesterol, and triglycerides. All patients had positive anti-HCV as measured using EIA3 (Abbott Laboratories, Chicago, IL) and positive HCV RNA in serum. Hepatitis B surface antigen, anti-hepatitis B core, and anti-human immunodeficiency virus were tested using commercially available kits (Abbott Laboratories, Chicago, IL). All patients were negative for hepatitis B surface antigen and anti-human immunodeficiency virus. Serum insulin levels were measured by electrochemiluminescence immunoassay, using an autoanalyzer Elecsys 1010/2010 (Elecsys Modular Analytics E170; Roche, Basel, Switzerland). Blood lactate levels was measured by a commercially available kit (Immulite Diagnostic Products, Los Angeles, CA). The insulin resistance index was calculated on the basis of fasting values of plasma glucose and insulin according to the HOMA model formula: Insulin resistance (HOMA IR) = fasting insulin (mUI/L) × fasting glucose (mmol/L) ÷ 22.5
Efficacy Assessments.
The primary end-point was SVR, defined as undetectable serum HCV RNA level (<10 IU/mL) at 24 weeks after the conclusion of treatment.
The secondary end-points were: (1) viral clearance (HCV RNA < 10 IU/mL) at weeks 12, 24, and 48; (2) changes in the HOMA index over the first 24 weeks. Virological breakthrough was defined as a detectable HCV RNA level during treatment in patients who had had undetectable HCV RNA at week 24. Virological relapse was defined as a detectable HCV RNA level during follow-up in patients who had had undetectable HCV RNA at week 48.
Safety Assessments.
Safety was assessed by means of clinical examinations and laboratory tests at weeks 4, 8, 12, 24, 48, and 72. Stepwise reductions of peginterferon alfa-2a, ribavirin, and metformin were permitted in managing clinically significant adverse events, or laboratory abnormalities recorded.
Statistical Analyses.
Sample size estimation was based on the assumption that metformin improving insulin resistance could increase SVR rate in 28%.[1] The estimated sample size was 124 patients, with a 10% rate of loss-to-follow-up, type I error of 0.05, and type II of 0.2 (Fisher's exact two-sided test).
Intention-to-treat analysis included all 123 randomized cases, and a per-protocol analysis (PPA) included 101 patients. Excluded from the PPA were patients with adherence to less than 80% of the dose and duration of peginterferon (n = 9) and ribavirin (n = 4) and those with daily average intake of metformin less than 50% (1275 mg/day) (n = 9) (Fig. 1). All data were analyzed using SPSS version 15.0 for Windows (Chicago, IL). Comparisons between paired groups were with the Mann-Whitney U test, the student t test, chi-squared, or Fisher's exact test. Backward logistic regression was applied in the multivariate analysis.
Funding and Study Logistics.
The study was funded by a nonrestrictive grant from Roche Farma SA. The funding body had no part in the collection and evaluation of data, or in the writing of the manuscript and the decision to publish. The trial was designed by the principal investigator (M.R.-G.) together with the academic investigators constituting the TRIC-1 group (see Appendix). The logistics of data collection were managed by p-value S.A. (Sevilla, Spain) and the academic investigators, and a copy was logged with the study promoter. The principal investigator (M.R.-G.) together with p-value S.A. performed the statistical analyses and vouch for the integrity and completeness of the data.
Hepatology Early View Oct 2009
"Adding metformin to peginterferon and ribavirin was safe and improved insulin sensitivity. Although the study failed to show a statistically significant difference between arms, it did show an improved SVR in females."
"HOMA less than 2 at week 24 was more often seen in arm A (55%) than in arm B (14%), and this goal was associated with higher SVR.....In summary, treating patients with hepatitis C genotype 1 and insulin resistance using metformin improves insulin sensitivity, is safe, and increases SVR rate in patients who reached HOMA lower than 2 at week 24 of therapy and in women, in whom, in the current study, the therapy doubled the SVR rate......In females (n = 54), adding metformin to peginterferon alfa-2a plus ribavirin resulted in a doubling of the SVR rate in 15 of 26 patients (58%) in arm A versus 8 of 28 patients (29%) in arm B; P = 0.03) (Fig. 2). Viral decline during the first 12 weeks was greater in females taking metformin than placebo (mean [standard deviation (SD)]) -4.88 (1.18) versus -4.0 (1.44); P = 0.02), whereas no viral decline was noted in males. Furthermore, no cases of breakthrough were seen in females with metformin therapy (females with HCV RNA negative at week 24 (n = 41), with 4 of 20 patients experiencing breakthrough in arm B but none in arm A (0 of 21); P = 0.05......In the univariate analysis, higher baseline viral load and calculated fibrosis (the noninvasive Sydney Index) were associated with nonresponse of 6.52 (0.73) versus 6.28 (0.75) log10 HCV RNA (P = 0.08) and 0.52 (0.32) versus 0.38 (0.25) in the Sydney Index (P = 0.01). During treatment, the HCV RNA decline in the first 12 weeks of 3.04 (1.65) versus 1.11 (0.35) log10 HCV RNA, P = 0.0001, and the HOMA-IR decline during the first 24 weeks was associated with sustained response; that is, 19 of 28 patients (68%) with a HOMA-IR less than 2 at week 24 achieved SVR, whereas 25 of 56 patients (45%) with HOMA at 24 weeks greater than 2 achieved SVR (P = 0.05)."
Manuel Romero-Gómez 1 *§, Moisés Diago 2, Raúl J. Andrade 3, José L. Calleja 4, Javier Salmerón 5, Conrado M. Fernández-Rodríguez 6, Ricard Solà 7, Javier García-Samaniego 8, Juan M. Herrerías 9, Manuel De la Mata 10, Ricardo Moreno-Otero 11, Óscar Nuñez 12, Antonio Olveira 13, Santiago Durán 14, Ramón Planas 14, Spanish TRIC-1 (Treatment of Resistance to Insulin in Hepatitis C Genotype 1) group
1Unit for the Clinical Management of Digestive Diseases and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERHD), Hospital Universitario de Valme, Sevilla, Spain
2Hepatology Section, Hospital General de Valencia, Spain
3Hepatology Unit and CIBEREHD, Hospital Virgen de la Victoria, Málaga, Spain
4Digestive Diseases Unit, Hospital Universitario Puerta de Hierro, Madrid, Spain
5Digestive Disease Service and CIBEREHD, Hospital San Cecilio, Granada, Spain
6Gastroenterology Unit, Hospital Universitario Fundación Alcorcón, Madrid, Spain
7Hepatology Section, Hospital del Mar, Barcelona, Spain
8Gastroenterology Service and CIBEREHD, Hospital Carlos III, Madrid, Spain
9Gastroenterology Service, Hospital Universitario Virgen Macarena, Sevilla, Spain
10Hepatology Section and CIBEREHD, Hospital Universitario Reina Sofía, Córdoba, Spain
11Digestive Diseases Service and CIBEREHD, Hospital de la Princesa, Madrid, Spain
12Hepatology Unit, Hospital Gregorio Marañón, Madrid, Spain
13Gastroenterology Service, Hospital La Paz, Madrid, Spain
14Gastroenterology and Hepatology Unit and CIBEREHD, Hospital Germans Trias i Pujol, Barcelona, Spain
email: Manuel Romero-Gómez (mromerogomez@us.es)
*Correspondence to Manuel Romero-Gómez, UCM Digestive Diseases and CIBERHD, Hospital Universitario de Valme, Universidad de Sevilla, Avda de Bellavista s/n, 41014 Sevilla, Spain
Presented at the 59th Annual Meeting of the American Association for the Study of the Liver Diseases, October 31 to November 4, 2008, San Francisco, CA.
Abstract
Insulin resistance affects sustained virological response (SVR) in chronic hepatitis C. To know whether adding metformin to standard antiviral treatment improves SVR, we conducted a prospective, multicentered, randomized, double-blinded, placebo-controlled trial in 19 Spanish hospitals, including 123 consecutive patients with genotype 1 chronic hepatitis C and insulin resistance. Patients were randomized to receive either metformin (arm A; n = 59) or placebo (arm B; n = 64) in addition to peginterferon alfa-2a (180 g/week) and ribavirin (1000-1200 mg/day). The primary end point was SVR, and secondary endpoints were viral clearance at weeks 12, 24, and 48, and changes in the homeostasis model assessment (HOMA) index over the first 24 weeks. There were no differences between arms at baseline. In the intent-to-treat analysis, SVR was observed in 53% versus 42% in arm A and arm B, respectively (P = NS). In the subgroup analyses, SVR was higher in females (n = 54) receiving metformin: arm A, 58% (15/26) versus 29% (8/28) arm B (P = 0.03). In the per protocol analysis (PPA; n = 101), SVR was 67% in arm A and 49% in arm B (P = 0.06). Viral decline during the first 12 weeks was greater in females receiving metformin: -4.88 (1.18) versus -4.0 (1.44) (P = 0.021), whereas no differences were seen in males. The triple therapy was well tolerated, but diarrhea was more often seen in arm A (34% versus 11%; P < 0.05).
Conclusion: Adding metformin to peginterferon and ribavirin was safe and improved insulin sensitivity. Although the study failed to show a statistically significant difference between arms, it did show an improved SVR in females.
Hepatitis C is a major healthcare problem, and current therapies have achieved sustained response in more than a half of infected patients. Factors associated with nonresponse are host-viral genotype 1, high viral load, advanced fibrosis, and insulin resistance.[1] Sustained virological response (SVR) decreases when insulin sensitivity is impaired.[1][2] Also, impaired fasting glucose has been independently associated with lower SVR rate.[3] Insulin resistance is thought to be promoted by hepatitis C virus (HCV) itself and, after clearance of the virus, insulin resistance improves concomitantly with the reduction in the risk of glucose abnormalities and diabetes.[1][3][4] HCV proteins lead to insulin resistance, promoting the degradation of insulin receptor substrate 1.[5] Transgenic mice expressing core HCV protein developed insulin resistance and steatosis.[6] Insulin resistance and steatosis promoted by the virus have been found to be associated with improvement in viral fitness,[7] and this seems to be a defense mechanism against viral clearance. Lifestyle, exercise, diet, and insulin-sensitizing drugs could improve insulin resistance. Metformin is an oral biguanide that lowers blood glucose and insulin secretion and improves the individual's lipid profile, mainly because of suppression of hepatic glucose output and increased glucose uptake in skeletal muscle.[8] Because insulin resistance can be successfully treated with biguanides, we proposed that sustained virological response could be improved by adding metformin to the standard of care for patients with chronic hepatitis C genotype 1 and insulin resistance.
Discussion
The combination of metformin, peginterferon alfa-2a, and ribavirin was well tolerated by the patients. It improved insulin resistance in more than 50% of patients and increased SVR rate in 10% of patients with hepatitis C genotype 1 and HOMA greater than 2. This was related to a trend toward a low rate of breakthrough in patients receiving metformin; the response at week 24 and relapse rate were similar in both arms. However, in the PPA, viral clearance was significantly higher in the metformin group at weeks 24 and 48, which supports the proposition of a better antiviral activity of peginterferon alfa-2a and ribavirin in patients receiving metformin. Despite raising SVR by 10% (a clinically relevant increase), this difference did not reach statistical significance, which could be attributable, at least in part, to a type 2 error in sample size calculation. That there is a trend in the intention-to-treat analysis and significance in the PPA and female subanalysis strongly supports this hypothesis. Despite the sample calculation being based on the available data, our subsequent findings of the study suggest that there were not enough patients enrolled in the study to achieve statistically significant differences. Metformin is an oral biguanide, which is one of the most widely prescribed therapeutic agents for the control of type 2 diabetes. Metformin controls glucose levels by suppression of hepatic glucose output, by increasing insulin-mediated glucose disposal, and by decreasing fatty acid oxidation and synthesis of very-low-density lipoprotein.[11] During HCV replication, core protein promotes unfolded protein response that causes dysfunction of endoplasmic reticulum and mitochondria.[12][13] Indeed, HCV proteins promote insulin receptor substrate 1 degradation by several mechanisms, including oxidative stress, down-regulation of PPAR, and enhancing TNF production in a genotype-dependent manner. In genotype 1, the degradation of insulin receptor substrate 1 has been shown to be induced by mammalian target of rapamycin and, in genotype 3, by suppressor of cytokine signaling 7 and peroxisome proliferator-activated receptor-gamma (PPAR-). Thus, metformin could be useful in the management of this comorbidity because of its ability to increase the binding of insulin to its receptors and to increase phosphorylation as well as the tyrosine kinase activity of insulin receptors[14] through, mainly, the action of phosphorylation of hepatic adenosine-monophosphate-activated protein kinase by STK11 (formerly LKB1).[15] Pioglitazone was shown not to improve either viral response or insulin sensitivity in five previously nonresponding individuals.[16] In treatment-naïve patients, pioglitazone in combination with peginterferon alfa-2b and ribavirin improved insulin sensitivity and hepatic steatosis.[17] Elgouhari et al.[18] had shown that the combination therapy improved early virological kinetics over the first 4 weeks and the end-of-treatment response, but not SVR.[18] The low number of patients (n = 40) precluded subset analysis and comparisons with the current study. However, the mechanism of action of insulin-sensitizer drugs in the setting of patients with hepatitis C appears to be different and could be related to viral genotype. Metformin improved insulin sensitivity in patients with hepatitis C genotype 1. Theoretically, PPAR- agonist appears to be indicated in the management of patients with genotype 3 HCV infection (in whom insulin resistance appears as a consequence of decreased PPAR- activity). Furthermore, metformin could be preferred because it acts directly in the liver, whereas PPAR- agonist shows an effect on peripheral metabolism. Further prospective studies using different insulin sensitizers in a large cohort of patients with hepatitis C with different genotypes are required to demonstrate whether these data could be translated into clinical practice.
In women, metformin increased the SVR rate significantly. This sex-related difference has not been previously reported in the management of hepatitis C. Spontaneous viral clearance has been seen more often in women than in men.[19] However, no sex-related impact has been reported on the clinical course of the infection or in SVR rate. Some weak evidence supports the influence of sex on the therapeutic effects of metformin: (1) metabolic syndrome pathophysiology and diabetes-related complications appear to be vary in relation to sex, and vitamin K supplementation appears to improve insulin resistance in men but not in women[20]; (2) some therapeutic effects of metformin appear to be sex dependent, that is, metformin promotes greater short-term weight loss in women than in men, but these differences were not observed in diabetic control subjects[21][22]; (3) STK11 (formerly LKB1) plays a major role in metformin sensitivity,[23] and some genetic alterations appear to have a clinical impact that is sex dependent, such as the risk of malignancies in Peutz-Jegher syndrome.[24] In women, metformin therapy has been associated with a greater decrease of HCV RNA during the first 12 weeks of treatment and a lower breakthrough rate. These data support the hypothesis that, in women, metformin improves the antiviral activity of peginterferon and ribavirin. It remains to be explored whether genetic alterations in the STK11 gene or nuclear factors such as estrogens are implicated in the ability of metformin to improve antiviral activity.
Two biguanides (phenformin and buformin) have been withdrawn from the pharmacopeias because of evidence of lactic acidosis associated with their use. Despite cases of lactic acidosis having been reported with the use of metformin, a recent systematic review demonstrated no increased risk for lactic acidosis in patients without contraindications to metformin use.[25] In the current study, blood lactate level was monitored during treatment, and no patients developed lactic acidosis or hyperlactatemia. Metformin was associated with mild diarrhea, which was well tolerated, and no patient withdrew from the study because of this adverse event. Thus, metformin used together with peginterferon alfa-2a and ribavirin in the treatment of hepatitis C seems to be safe and well tolerated. The perceived contraindication of metformin for patients with alterations in liver function needs to be reevaluated.
In our study, metformin improved insulin sensitivity, with more than half the number of patients achieving HOMA less than 2 by week 24. This end point was also related to the probability of achieving SVR. Nevertheless, metformin-induced improvement in insulin sensitivity is not the sole action of this drug, and the mechanism by which metformin improves the antiviral activity of peginterferon + ribavirin warrants further investigation.
In summary, treating patients with hepatitis C genotype 1 and insulin resistance using metformin improves insulin sensitivity, is safe, and increases SVR rate in patients who reached HOMA lower than 2 at week 24 of therapy and in women, in whom, in the current study, the therapy doubled the SVR rate.
Results
Of 125 patients initially recruited, 123 were randomly assigned to a treatment group and received at least one dose of medication; two patients were excluded because of not having met the full criteria for inclusion (one patient was already taking metformin, and the other was found to be infected by a different viral genotype). The two treatment groups were well matched for age, sex, and other variables measured on entry into the trial (Table 1).
Virological Response.
In the intention-to-treat analysis (n = 123), viral clearance was seen in 54% (32/59) of patients versus 48% (31/64) at week 12; in 75% (45/59) versus 75% (48/64) at week 24; in 71% (42/59) versus 63% (40/64) at week 48; and in 53% (31/59) versus 42% (27/64) at week 72 (in other words, SVR rate) in arm A and arm B, respectively. Virological breakthrough was slightly higher in patients receiving placebo 17% (8/48) versus 7% (3/45), but this difference was not statistically significant. Relapse rate was similar in both arms: 11 of 59 patients (19%) in arm A and 12 of 64 patients (19%) in arm B; P = 0.98.
In the PPA (n = 101), viral clearance was achieved in 67% (31/46) versus 53 (29/55), P = 0.13 at week 12; in 96% (44/46) versus 78% (43/55), P = 0.01 at week 24; in 91% (42/46) versus 69% (38/55), P < 0.01 at week 48; in 67% (31/46) versus 49% (27/55), P = 0.06 at week 72 in arm A and arm B, respectively.
Subgroup Analysis.
In females (n = 54), adding metformin to peginterferon alfa-2a plus ribavirin resulted in a doubling of the SVR rate in 15 of 26 patients (58%) in arm A versus 8 of 28 patients (29%) in arm B; P = 0.03) (Fig. 2). Viral decline during the first 12 weeks was greater in females taking metformin than placebo (mean [standard deviation (SD)]) -4.88 (1.18) versus -4.0 (1.44); P = 0.02), whereas no viral decline was noted in males. Furthermore, no cases of breakthrough were seen in females with metformin therapy (females with HCV RNA negative at week 24 (n = 41), with 4 of 20 patients experiencing breakthrough in arm B but none in arm A (0 of 21); P = 0.05.
Figure 2. Rates of viral clearance in patients randomly assigned to receive Metformin + Pegalfa2a + Ribavirin (arm A) or Placebo + Pegalfa2a + Ribavirin (arm B) in the intention-to-treat analysis, per-protocol analysis and females subset analysis. A sustained virological response was defined as undetectable serum HCV RNA level (<10 UI/mL) at week 24 after the end of treatment.
Picture 1.png
Changes in Insulin Resistance.
The decline in HOMA index in patients receiving metformin was significantly higher than in patients receiving placebo. In arm A, the HOMA index decreased from 4.3 (2.2) to 2.6 (1.7) and in arm B from 4.6 (2.7) to 3.8 (2.1); P = 0.001. However, no difference was seen in weight loss in either treatment arm. By the end-of-follow-up, women receiving metformin had a mean body weight decrease of 1.3 kg (SD: 5.4) versus 1.1 kg (SD: 5.6) in women receiving standard care; P = NS. In men, body weight decreased by 1.1 kg (SD: 6.1) in patients receiving metformin versus 0.9 kg (SD: 5.5) in patients on placebo; P = NS. Chages in body weight did not influence SVR. However, HOMA less than 2 at week 24 was more often seen in arm A (55%) than in arm B (14%), and this goal was associated with higher SVR (see below).
Predictors of SVR.
In the univariate analysis, higher baseline viral load and calculated fibrosis (the noninvasive Sydney Index) were associated with nonresponse of 6.52 (0.73) versus 6.28 (0.75) log10 HCV RNA (P = 0.08) and 0.52 (0.32) versus 0.38 (0.25) in the Sydney Index (P = 0.01). During treatment, the HCV RNA decline in the first 12 weeks of 3.04 (1.65) versus 1.11 (0.35) log10 HCV RNA, P = 0.0001, and the HOMA-IR decline during the first 24 weeks was associated with sustained response; that is, 19 of 28 patients (68%) with a HOMA-IR less than 2 at week 24 achieved SVR, whereas 25 of 56 patients (45%) with HOMA at 24 weeks greater than 2 achieved SVR (P = 0.05).
Using reverse stepwise logistic multivariate regression analysis, the independent variable related to SVR was HCV RNA at week 12 (hazard ratio, 0.15; 95% confidence interval: 0.07-0.34) (Table 2).
Safety.
The percentage of patients reporting adverse events or serious adverse events was similar in the two groups. The adverse events reported were those typical of interferon-based treatments, which include fatigue, headache, insomnia, and myalgia. Diarrhea was more often seen in patients assigned to arm A than in patients receiving placebo (36% versus 11%; P < 0.05). However, the diarrhea was well tolerated and did not lead to any withdrawals from the trial. Two patients in each treatment group had to discontinue because of adverse events (Table 2). Doses of metformin or placebo were modified in 37% of patients in arm A and 38% in arm B (P = NS). Serum lactate was monitored during treatment, and no patient developed lactic acidosis or hyperlactatemia.
Patients and Methods
Selection of Patients.
Eligible patients were those who were 18 years of age or older, were infected with genotype 1 (determined by INNO-LIPA assay, Innogenetics), and had a quantifiable serum HCV RNA for more than 6 months together with an elevated serum alanine aminotransferase level, nondiabetic, and homeostasis model assessment (HOMA) index greater than 2 (Table 1). Liver biopsy was not mandatory. Liver fibrosis was calculated using the Sydney index (a noninvasive method for the prediction of liver fibrosis based on a formula that includes age, plasma cholesterol concentration, aspartate aminotransferase, alcohol consumption, and HOMA index[9] that has been validated in Spanish patients with chronic hepatitis C).[10] Patients were ineligible if they had other liver diseases, showed contraindications to peginterferon or ribavirin, or had previously received antiviral drugs.
Study Design.
Patients were randomly assigned into this prospective, multicentered, randomized, double-blinded, placebo-controlled trial (registered in ClinicalTrials.gov; #NCT00546442). The study was conducted in 19 Spanish centers. All sites received approval from their ethics committees. Each patient provided written informed consent for participation. Patients were randomly assigned in a 1:1 ratio to receive either metformin 425 mg three times daily for the first month and 850 mg three times daily from week 4 to 48 (arm A; n = 59) or placebo (arm B; n = 64) in addition to peginterferon alfa-2a (Pegasys; Roche Farma, Madrid, Spain) 180 g weekly and ribavirin 1000 to 1200 mg daily. Patients were followed-up over 24 weeks to confirm SVR.
Laboratory Analyses.
An overnight (12-hour) fasting blood sample was taken for routine analyses, including aminotransferases (alanine aminotransferase, aspartate aminotransferase), alkaline phosphatase, gamma-glutamyltransferase, platelets, glucose, cholesterol, and triglycerides. All patients had positive anti-HCV as measured using EIA3 (Abbott Laboratories, Chicago, IL) and positive HCV RNA in serum. Hepatitis B surface antigen, anti-hepatitis B core, and anti-human immunodeficiency virus were tested using commercially available kits (Abbott Laboratories, Chicago, IL). All patients were negative for hepatitis B surface antigen and anti-human immunodeficiency virus. Serum insulin levels were measured by electrochemiluminescence immunoassay, using an autoanalyzer Elecsys 1010/2010 (Elecsys Modular Analytics E170; Roche, Basel, Switzerland). Blood lactate levels was measured by a commercially available kit (Immulite Diagnostic Products, Los Angeles, CA). The insulin resistance index was calculated on the basis of fasting values of plasma glucose and insulin according to the HOMA model formula: Insulin resistance (HOMA IR) = fasting insulin (mUI/L) × fasting glucose (mmol/L) ÷ 22.5
Efficacy Assessments.
The primary end-point was SVR, defined as undetectable serum HCV RNA level (<10 IU/mL) at 24 weeks after the conclusion of treatment.
The secondary end-points were: (1) viral clearance (HCV RNA < 10 IU/mL) at weeks 12, 24, and 48; (2) changes in the HOMA index over the first 24 weeks. Virological breakthrough was defined as a detectable HCV RNA level during treatment in patients who had had undetectable HCV RNA at week 24. Virological relapse was defined as a detectable HCV RNA level during follow-up in patients who had had undetectable HCV RNA at week 48.
Safety Assessments.
Safety was assessed by means of clinical examinations and laboratory tests at weeks 4, 8, 12, 24, 48, and 72. Stepwise reductions of peginterferon alfa-2a, ribavirin, and metformin were permitted in managing clinically significant adverse events, or laboratory abnormalities recorded.
Statistical Analyses.
Sample size estimation was based on the assumption that metformin improving insulin resistance could increase SVR rate in 28%.[1] The estimated sample size was 124 patients, with a 10% rate of loss-to-follow-up, type I error of 0.05, and type II of 0.2 (Fisher's exact two-sided test).
Intention-to-treat analysis included all 123 randomized cases, and a per-protocol analysis (PPA) included 101 patients. Excluded from the PPA were patients with adherence to less than 80% of the dose and duration of peginterferon (n = 9) and ribavirin (n = 4) and those with daily average intake of metformin less than 50% (1275 mg/day) (n = 9) (Fig. 1). All data were analyzed using SPSS version 15.0 for Windows (Chicago, IL). Comparisons between paired groups were with the Mann-Whitney U test, the student t test, chi-squared, or Fisher's exact test. Backward logistic regression was applied in the multivariate analysis.
Funding and Study Logistics.
The study was funded by a nonrestrictive grant from Roche Farma SA. The funding body had no part in the collection and evaluation of data, or in the writing of the manuscript and the decision to publish. The trial was designed by the principal investigator (M.R.-G.) together with the academic investigators constituting the TRIC-1 group (see Appendix). The logistics of data collection were managed by p-value S.A. (Sevilla, Spain) and the academic investigators, and a copy was logged with the study promoter. The principal investigator (M.R.-G.) together with p-value S.A. performed the statistical analyses and vouch for the integrity and completeness of the data.
Liver Cancer HCC
Comprehensive analysis of the independent effect of twist and snail in promoting metastasis of hepatocellular carcinoma
Hepatology Nov 2009
ABSTRACT
The epithelial-mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis of human cancers. In this study we investigated the expression profiles of the EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human hepatocellular carcinoma (HCC). Reduced E-cadherin and nonmembranous -catenin expression, the hallmarks of EMT, were shown in 60.2% and 51.5% of primary HCC samples, respectively. Overexpression of Snail, Twist, or Slug, the major regulators of EMT, was identified in 56.9%, 43.1%, and 51.4% of primary HCCs, respectively. Statistical analysis determined that Snail and Twist, but not Slug, are major EMT inducers in HCC: overexpression of Snail and/or Twist correlated with down-regulation of E-cadherin, nonmembranous expression of -catenin, and a worse prognosis. In contrast, there were no such significant differences in samples that overexpressed Slug. Coexpression of Snail and Twist correlated with the worst prognosis of HCC. Hepatitis C-associated HCC was significantly correlated with Twist overexpression. HCC cell lines with increased Snail and Twist expression (e.g., Mahlavu) exhibited a greater capacity for invasiveness/metastasis than cells with low endogenous Twist/Snail expression (e.g., Huh-7). Overexpression of Snail or/and Twist in Huh-7 induced EMT and invasiveness/metastasis, whereas knockdown of Twist or Snail in Mahlavu reversed EMT and inhibited invasiveness/metastasis. Twist and Snail were independently regulated, but exerted an additive inhibitory effect to suppressE-cadherin transcription. Conclusion: Our study provides a comprehensive profile of EMT markers in HCC, and the independent and collaborative effects of Snail and Twist on HCC metastasis were confirmed through different assays.
Hepatocellular carcinoma (HCC) is one of the most common neoplasms in South Africa and Asian countries including Taiwan.[1] Most of the HCC in Taiwan stems from the high prevalence of chronic hepatitis B virus (HBV) infection (15%-20%).[2] Although successful partial hepatectomy has significantly improved survival, the prognosis of HCC remains poor because of tumor invasiveness, frequent intrahepatic spread, and extrahepatic metastasis.[3] Elucidation of the molecular mechanisms underlying HCC invasiveness is of utmost importance for the development of future strategies for treating HCC.
The epithelial-mesenchymal transition (EMT), a developmental process by which epithelial cells reduce intercellular adhesion and acquire fibroblastoid properties, has been shown to be critical for the development of the invasiveness and metastatic potential of human cancers.[4] Characteristic changes during EMT include down-regulation of epithelial markers (e.g., E-cadherin and plakoglobin),[4][5] translocation of -catenin (i.e., dissociation of membranous -catenin and translocation into the nuclear compartment),[6] and up-regulation of mesenchymal markers (e.g., vimentin and N-cadherin).[4][5] The EMT process is initiated by suppression of E-cadherin expression by the major EMT regulators, e.g., Snail, Slug, and Twist.[4][5] The zinc-finger transcriptional repressors Snail (also known as Snail1) and Slug (also known as Snail2) and the basic helix-loop-helix (bHLH) transcription factor Twist (also known as Twist1) were shown to induce EMT through repression of E-cadherin expression.[7-9]
The clinical significance of EMT has been confirmed in certain types of human cancers,[10][11] and the significance of individual EMT regulators in HCC has been demonstrated.[12-15] However, a comprehensive study demonstrating the expression profile of multiple EMT markers in HCC is lacking. In addition, the role of hepatotrophic viruses, such as HBV and hepatitis C virus (HCV), in promoting the EMT/metastasis of HCC is unclear. In this study we investigated the expression profiles of EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human HCC.
Discussion
Induction of EMT is distinguished by the repression of E-cadherin transcription by zinc-finger proteins (e.g., Snail, Slug)[7][8] or bHLH family transcriptional factors (e.g., Twist)[9] through binding to three E-boxes located in the proximal promoter of E-cadherin. Although EMT has been considered the critical mechanism involved in cancer metastasis, in comparison with other types of human cancers only a few sporadic studies have focused on the significance of EMT in HCC.[12-15] The present study is the first to provide a comprehensive profile of multiple EMT markers and to demonstrate that Snail and Twist, but not Slug, are the major inducers of EMT in HCC. These results not only pinpoint the major signal pathways in the induction of EMT in HCC, but also demonstrate its clinical relevance through comprehensive analysis.
Transforming growth factor (TGF-) is a master regulator of EMT through activation of the Smad2/3 complex.[22] TGF-1 plays a key role in triggering EMT in HCC through cooperation with laminin-5,[23] and inhibition of TGF-1 attenuates migration/invasion of HCC cells.[24] One recent report showed that in HCC the expression of HCV-derived core protein switches the cellular response to TGF- exposure from inhibition of growth to induction of EMT,[25] providing a possible linkage between HCV-induced HCC and EMT. Although HCV-related HCC has been shown to be associated with a higher recurrence rate after surgery,[26] the underlying mechanism is unclear, and the correlation between HCV and EMT has never been explored. We report the original finding that overexpression of Twist is correlated with HCV-related HCC. This interesting finding may partially explain the highly invasive behavior and poor prognosis of HCV-related HCC. The mechanism underlying increased Twist expression in HCV-related HCC deserves further study.
The interplay between Snail and Twist in the promotion of EMT in HCC has not been reported. In the present study we provide evidence that Twist and Snail are regulated independently and act collaboratively to promote EMT: coexpression of Snail and Twist indicates the worst prognosis for HCC; HCC cell lines harboring both Snail and Twist showed a higher migratory/metastatic ability; coexpression of Snail and Twist in Huh7 promoted the highest invasiveness and metastasis; and Twist and Snail did not influence the expression/promoter activity of each other. This is the first report to demonstrate the independent regulation of Snail and Twist and their additive effects on EMT promotion.
Differences in the roles of Snail and Twist in promoting HCC metastasis were disclosed in our study: although Snail was more potent in activating MMPs, HCC cells overexpressing Twist tended to be more invasive than cells overexpressing Snail, and this observation was supported by analysis of clinical samples. These results indicate that Twist may be more critical in HCC metastasis, possibly acting through an MMP-independent mechanism. Previous studies disclosed that some critical molecules (e.g., N-cadherin, Akt2, and YB-1) in cancer metastasis are direct targets of Twist.[27-29] Overexpression of Twist induces angiogenesis.[30] Collectively, our results indicate that Snail and Twist act additively to promote HCC metastasis through different mechanisms.
In conclusion, our study demonstrates the prognostic significance of EMT markers in HCC and also establishes different roles for Snail and Twist in the promotion of EMT in HCC through independent regulation. A correlation between HCV-induced HCC and Twist expression is also reported. These results elaborate on the major mechanisms involved in HCC metastasis and provide essential information for prediction of prognosis and identification of new treatment targets for future HCC management.
Results
Down-regulation of E-cadherin in HCC.
IHC was performed to investigate the significance of down-regulated E-cadherin expression, the hallmark of EMT,[4][5] in HCC patients. Among primary HCCs, 39.8% of the samples showed preserved expression of E-cadherin (Case 1 of Fig. 1A; Supporting Fig. S1A, left panel). Decreased (Case 2 of Fig. 1A; Supporting Fig. S1A, middle panel) or absent E-cadherin expression (Supporting Fig. S1A, right panel) was identified in 52.8% and 7.4% of the samples, respectively. Among the samples with recurrent HCC, preserved, decreased, or absent expression of E-cadherin was shown in 23.9%, 63.1%, and 13% cases, respectively. An increased proportion of down-regulated E-cadherin expression was shown in recurrent compared with primary samples (Fig. 1B). The results of IHC analysis for E-cadherin were validated by ELISA and real-time RT-PCR in representative HCC samples (Supporting Fig. S1B,C). Survival analysis demonstrated a significant decrease in cancer-free interval (CFI) and overall survival (OS) in patients with down-regulated expression of E-cadherin (Table 1, Supporting Table S11, Fig. 1D). Down-regulated expression of E-cadherin was also associated with factors indicating clinical aggressiveness (e.g., large tumor size; Supporting Table S3), and a trend toward multinodular tumors was observed (Supporting Table S3).
Nonmembranous Expression of -Catenin in HCC.
Translocation of -catenin is an important marker of EMT.[6] IHC analysis for -catenin was performed in the available 103 primary and 33 recurrent HCC samples. A higher incidence of nonmembranous expression of -catenin (Case 2 of Fig. 1A) was shown in recurrent versus primary HCCs (66.7% versus 51.5%; Fig. 1C). Nonmembranous expression of -catenin was associated with down-regulated E-cadherin (Supporting Table S4), a shorter CFI, and a worse OS (Table 1, Supporting Table S11, Fig. 1E) and a trend toward multinodular tumors and microscopic venous invasion was observed (Supporting Table S5).
Expression Profile and Clinical Significance of the Major EMT Regulators (Snail, Slug, and Twist) in HCC.
The expression profiles of Snail, Slug, and Twist in HCC were evaluated. An increased incidence of Snail (Supporting Fig. S2A) or Twist overexpression (Supporting Fig. S3A) was shown in the recurrent versus primary HCCs (Supporting Figs. S2B, S3B). However, the percentages of Slug overexpression (samples available in 105 primary and 33 recurrent cases; Supporting Fig. S4A) were similar in the recurrent versus primary samples (Supporting Fig. S4B). Expression of Snail or Twist, but not Slug, was associated with down-regulation of E-cadherin and -catenin translocation (Supporting Table S6). Overexpression of Snail or Twist was associated with a shorter CFI and OS (Table 1, Supporting Table S11, Supporting Figs. S2C, S3C) and aggressive tumor behavior (e.g., large tumor size and multinodular tumors; Supporting Tables S7, S8). Surprisingly, increased Twist expression was significantly correlated with serum anti-HCV positivity and HBsAg negativity (Supporting Table S8). Although increased Slug expression was associated with aggressive clinical factors (e.g., large tumor size; Supporting Table S9), no prognostic significance of Slug on OS and CFI was demonstrated (Table 1, Supporting Table S11, Supporting Fig. S4).
Snail and Twist were frequently coexpressed (30.1% of primary and 60.9% of recurrent HCCs; Fig. 2A). Coexpression of Snail and Twist was associated with down-regulated expression of E-cadherin, nonmembranous expression of -catenin (Supporting Table S6), and the worst CFI and OS (Table 1, Supporting Table S11, Fig. 2B). Coexpression of both markers was also associated with aggressive factors (e.g., a trend toward multinodular tumors; Supporting Table S10).
Analyses of Factors Associated with CFI and OS in HCC.
Survival analysis was performed to determine the independent factors associated with CFI or OS. An impact on CFI was shown for many factors in the univariate analysis (Table 1); however, only anti-HCV positivity, elevated levels of -fetoprotein (AFP), multinodular tumors, down-regulation of E-cadherin, and overexpression of Twist were independent predictors of CFI (Table 1). A number of factors were also associated with OS in the univariate analysis (Supporting Table S11), but multivariate analysis limited this list to patient age, anti-HCV positivity, prolonged prothrombin time, multinodular tumors, and coexpression of Snail and Twist (Supporting Table S11).
Expression of Snail and Twist Is Associated with a Mesenchymal Phenotype and Invasiveness/Metastasis of HCC Cells.
Because Snail and Twist are the major inducers of EMT in HCC, we investigated the expression of Snail/Twist and its association with EMT markers/phenotypic changes in eight HCC cell lines. The results indicated that cells with a higher migratory ability and invasiveness showed associated mesenchymal changes, higher levels of Snail or Twist expression, and higher MMPs activity, whereas cells with lower invasiveness/migration exhibited epithelial markers, lower levels of Snail or Twist, and lower MMPs activity (Fig. 3). An in vivo tail-vein metastasis assay was performed to compare the metastatic capacity of Mahlavu, the most invasive cell line, with Huh7, the least invasive one. A significant increase in pulmonary metastasis was noted in mice receiving injections of Mahlavu versus Huh7 (Fig. 4A -C). A similar result was shown in the splenic-vein model (Fig. 4D-F). These results indicated that HCC cells with a mesenchymal phenotype (e.g., Mahlavu cells) were more invasive and exhibited greater in vivo metastatic ability, whereas HCC cells expressing epithelial markers (e.g., Huh7 cells) were associated with a lower tendency to migrate, invade, and metastasize.
Twist and Snail Are Regulated Independently and Act Additively in Promoting EMT.
To investigate the independent and nonredundant roles of Snail and Twist in HCC metastasis, we used Huh7 cells to generate clones with stable expression of Snail, Twist, or both proteins. Overexpression of Snail and/or Twist resulted in a shift in EMT marker expression (Fig. 5A). Expression of either Snail or Twist alone did not influence the expression levels of the other (Fig. 5A). Overexpression of Snail or Twist increased migration/invasion/metastasis, and cells that coexpressed both proteins demonstrated the highest ability for migration/invasion/metastasis (Fig.5C, D). Interestingly, overexpression of Snail resulted in higher activities of MMP-2 and MMP-9 than overexpression of Twist (Fig. 5B), but expression of Twist tended to result in slightly greater migration/metastasis than did expression of Snail (Fig. 5C,D).
Promoter activity assays were performed to determine whether Twist and Snail act collaboratively or independently. Overexpression of Snail or Twist resulted in a significant decrease in E-cadherin promoter activity, as expected; and combined overexpression of Snail and Twist seemed to exert an additive inhibitory effect on the E-cadherinpromoter compared to either agent alone (Fig. 6A,B). Snail expression could not activate the reporter driven by theTwist promoter. Likewise, Twist expression did not influence the activity of the Snail promoter (Fig. 6C-F). These results confirmed the independent expression and regulation of Twist and Snail.
Twist and Snail Are Both Critical for HCC Cell EMT/Migration/Metastasis.
To determine whether Twist and Snail are critical mediators of EMT in HCC, Twist or Snail expression in the highly invasive Mahlavu cells was suppressed by siRNA. Repression of either Snail or Twist resulted in down-regulation of vimentin and up-regulation of E-cadherin expression (Fig. 7A), and suppression of in vitro migration/invasion (Fig.7C) and in vivo metastasis (Fig. 7D). However, knockdown of Snail caused a more significant decrease in MMPs activity than Twist (Fig. 7B). These results further confirmed the critical but different roles of Snail and Twist in mediating EMT and metastasis of HCC cells.
Materials and Methods
Abstract Materials and Methods Results Discussion Acknowledgements References
Patients and Treatment.
In all, 123 primary HCC samples with adjacent nontumorous liver tissues were obtained from patients who had undergone curative hepatic resection between 1990 and 2002 at Taipei Veterans General Hospital. Among these 123 cases, 46 of 84 recurrent tumor samples were available for analysis. The study was approved by the Institutional Review Board of Taipei Veterans General Hospital. The clinical characteristics of the patients with HCC are presented in Table 1.
Table 1. Univariate and Multivariate Analysis of Factors Associated with Cancer-Free Interval
Variables No. Median Survival (mo) (95% CI) P-value
Age 55/> 55 y/old 59/64 41.4 (20.662.1)/22.1 (7.436.8) 0.218
Gender male/female 104/19 36.6 (25.447.8)/12.1 (026.4) 0.397
HBsAg (N/Y) 24/99 22.1 (8.136.1)/39 (20.058.0) 0.029
Anti-HCV (Y/N)* 23/97 14.4 (8.820.0)/41.4 (27.255.6) 0.018
Albumin 4.0/> 4.0 g/dL 59/64 22.1 (7.536.7)/41.8 (2.780.9) 0.038
Bilirubin 1.6/> 1.6 mg/dL 113/10 31.6 (15.8-47.4)/32 (NA) 0.315
ALT 40/>40 U/L 61/62 37.2 (19.155.3)/29.4 (8.050.8) 0.785
PT (INR) > 1.2/ 1.2 10/104 15 (044.3)/37.2 (19.954.5) 0.132
ICG-15 >10%/10% (retention rate) 48/75 29.4 (1.956.9)/31.6 (4.648.6) 0.372
Child-Pugh A/B 117/6 31.6 (17.945.3)/41.4 (088.1) 0.753
BCLC A/B or C 82/41 47.4 (31.9-62.9)/11.0 (5.0-17.0) <0.001
CLIP 0-1/2-4 88/35 41.8 (27.156.5)/10.9 (6.115.7) 0.004
Okuda I/II 96/27 39 (25.152.9)/12.1 (6.717.5) 0.107
TNM T1-2/T3-4 88/35 46.5 (28.764.3)/11.0 (6.515.5) 0.006
Tumor size 3cm/>3cm 48/75 47.4 (32.961.9)/22.1 (9.534.7) 0.088
Multinodular tumors Y/N* 49/74 13.1 (8.517.7)/56 (28.783.3) <0.001
Macroscopic venous invasion Y/N 13/110 19.3 (3.135.5)/36.6 (22.051.2) 0.102
Cut margin free/non-free 108/11 31.1 (16.7-45.5)/NA 0.130
AFP 100/>100 ng/ml* 73/49 41.4 (25.757.1)/15.0 (033.1) 0.044
Microscopic venous invasion Y/N 50/57 25.1 (8.242.0)/46.5 (22.570.5) 0.154
Edmonson stage I or II/III or IV 71/33 37.2 (25.8-48.6)/25.1 (0-65.7) 0.282
E-cadherin downregulation* (Y/N) 74/49 16.1 (8.5-23.7)/225 (NA) <0.001
-catenin membranous expression (Y/N) 50/53 59.3 (27.9-90.7)/23.4 (7.0-39.8) 0.013
Twist overexpression* (Y/N) 53/70 11.2 (9.0-13.4)/78 (NA) <0.001
Snail overexpression (Y/N) 70/53 15 (5.0-25.0)/103 (59.4-146.6) <0.001
Slug overexpression (Y/N) 54/51 102.6 (63.6-141.6)/129.3 (NA) 0.336
Co-expression of Twist and Snail (Y/N) 37/86 9.9 (5.8-14.0)/59.3 (30.8-87.8) <0.001
ALT, alanine aminotransferase; BCLC, Barcelona Clinic Liver Cancer; CLIP, Cancer of the Liver Italian Program; CI: confidence interval; ICG-15, indocyanine clearance test; PT: Prothrombin time; RR: risk ratio; NA: not applicable; Y: yes, N: no.
* Significant in multivariate analysis: positive anti-HCV: RR 2.387, 95% CI: 1.340-4.237, P = 0.003; multinodular tumors: RR 1.941, 95% CI: 1.164-3.237, P = 0.011; AFP > 100 ng/mL: RR 1.936, 95% CI: 1.101-3.392, P = 0.021;E-cadherin downregulation: RR 4.374, 95% CI: 2.289-8.359, P < 0.001; Twist overexpression: RR 2.978, 95% CI : 1.750-5.068, P < 0.001.
Sample available in 103 cases.
Sample available in 105 cases.
Immunohistochemistry (IHC) and Scoring.
The sample processing and IHC procedures were performed as described.[16] The antibodies used in IHC and the incubation periods are listed in Supporting Table S1. All IHC staining was independently scored by two experienced specialists. The expression of E-cadherin was scored as preserved, reduced, or absent, as described.[17] Reduced or absent expression of E-cadherin was interpreted as down-regulation of E-cadherin.[18] Nonmembranous expression of -catenin (i.e., more than 10% of cells exhibiting cytoplasmic or nuclear staining) was scored as described.[19] Nuclear expression of Snail, Slug, and Twist was graded from 0 to 3+ (0, no staining; 1+, 1%-25%; 2+, 26%-50%; 3+, >50% nuclear staining), with only 3+ considered a positive IHC result.[20]
Plasmids and Cell Lines.
The human hepatocellular carcinoma cell lines Huh7, HepG2, PLC, Hep3B, Sk-Hep1, Mahlavu, and the human embryonic kidney cell line HEK-293T were obtained from the American Type Culture Collection (Rockville, MD). Cell lines HA59T-VGH and HA22T-VGH were gifts from Professor C.P. Hu (Tung-Hai University, Taichung, Taiwan). The plasmids pcDNA3-Snail, pFLAG-Twist, pSUPER-Snail-si, and pSUPER-Twist-si have been described.[20] A scrambled sequence with no significant homology to any mammalian gene sequence was cloned into the pSUPER vector (pSUPER-scr-si) for use as a control in short interference RNA (siRNA) experiments. The oligonucleotide sequences used for generating siRNA constructs are listed in Supporting Table S2.
RNA Purification and Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Analysis.
Total RNA extraction, complementary DNA (cDNA) synthesis, and quantitative real-time RT-PCR were performed as described.[16] The primer sequences used in real-time PCR are shown in Supporting Table S2.
Western Blot Analysis.
Protein extraction from cultivated cells and western blot analysis were performed as described.[16] Antibodies used in western blot experiments are listed in Supporting Table S1.
Detection of E-cadherin Expression by Enzyme-Linked Immunosorbent Assay (ELISA).
The procedures are detailed in the Supporting Methods.
Gelatin Zymography.
Analysis of the activity of matrix metallopeptidases-2 and -9 (MMP-2 and MMP-9) in the different clones was performed as described.[16] The procedures are detailed in the Supporting Methods.
Cell Migration and Invasiveness Assay.
In vitro transwell migration and invasion assays were performed as described.[16] The procedures are detailed in the Supporting Methods.
Cloning of Gene Promoter Regions, Generation of Reporter Constructs, Transient Transfections, and Luciferase Assays.
The genomic regions flanking the E-cadherin, Snail, and Twist gene promoter regions (Fig. 6A,C,E) were generated by PCR amplification of human genomic DNA and inserted into the HindIII/BglII sites in the pXP2 vector to generate the pXP2-Ecadherin, pXP2-Twist, and pXP2-Snail reporter constructs. Transfections and reporter assays were performed as described, and the bacterial -galactosidase gene (pCMV-gal) was used as a control for transfection efficiency.[20]
Nude Mouse Tail Vein Metastasis Assay and Splenic Vein Metastasis Assay.
All procedures involving animals were performed in accordance with the institutional animal welfare guidelines of Taipei Veterans General Hospital. Cells were injected into the splenic vein or the tail vein of 8-week-old nude mice (BALB/c strain) at 1 × 10[6] cells/injection site. The mice were sacrificed after 6 weeks and the number and volume of metastatic tumors were assessed. Tumor size was measured by use of a caliper and volume was calculated as length × height × width × 0.5236 with reference to a previous report.[21]
Statistical Analysis.
The Kaplan-Meier estimate was used for survival analysis and the log-rank test was selected to compare the difference. A Cox proportional hazards model was applied to multivariate survival analysis for testing independent prognostic factors. Statistical significance was accepted when P < 0.05 for all tests.
Muh-Hwa Yang 1 2 3, Chih-Li Chen 4, Gar-Yang Chau 5 6, Shih-Hwa Chiou 1 7, Chien-Wei Su 1 8, Teh-Ying Chou 1 9, Wei-Li Peng 7, Jaw-Ching Wu 1 3 7 *§
1Institutes of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
2Division of Hematology-Oncology, Taipei Veterans General Hospital, Taipei, Taiwan
3Genomic Medicine Research Center, Taipei Veterans General Hospital, Taipei, Taiwan
4School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan
5Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan
6Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan
7Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan
8Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
9Department of Pathology, Taipei Veterans General Hospital, Taipei, Taiwan
email: Jaw-Ching Wu (jcwu@vghtpe.gov.tw)
*Correspondence to Jaw-Ching Wu, Department of Medical Research and Education, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei 112, Taiwan
Hepatology Nov 2009
ABSTRACT
The epithelial-mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis of human cancers. In this study we investigated the expression profiles of the EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human hepatocellular carcinoma (HCC). Reduced E-cadherin and nonmembranous -catenin expression, the hallmarks of EMT, were shown in 60.2% and 51.5% of primary HCC samples, respectively. Overexpression of Snail, Twist, or Slug, the major regulators of EMT, was identified in 56.9%, 43.1%, and 51.4% of primary HCCs, respectively. Statistical analysis determined that Snail and Twist, but not Slug, are major EMT inducers in HCC: overexpression of Snail and/or Twist correlated with down-regulation of E-cadherin, nonmembranous expression of -catenin, and a worse prognosis. In contrast, there were no such significant differences in samples that overexpressed Slug. Coexpression of Snail and Twist correlated with the worst prognosis of HCC. Hepatitis C-associated HCC was significantly correlated with Twist overexpression. HCC cell lines with increased Snail and Twist expression (e.g., Mahlavu) exhibited a greater capacity for invasiveness/metastasis than cells with low endogenous Twist/Snail expression (e.g., Huh-7). Overexpression of Snail or/and Twist in Huh-7 induced EMT and invasiveness/metastasis, whereas knockdown of Twist or Snail in Mahlavu reversed EMT and inhibited invasiveness/metastasis. Twist and Snail were independently regulated, but exerted an additive inhibitory effect to suppressE-cadherin transcription. Conclusion: Our study provides a comprehensive profile of EMT markers in HCC, and the independent and collaborative effects of Snail and Twist on HCC metastasis were confirmed through different assays.
Hepatocellular carcinoma (HCC) is one of the most common neoplasms in South Africa and Asian countries including Taiwan.[1] Most of the HCC in Taiwan stems from the high prevalence of chronic hepatitis B virus (HBV) infection (15%-20%).[2] Although successful partial hepatectomy has significantly improved survival, the prognosis of HCC remains poor because of tumor invasiveness, frequent intrahepatic spread, and extrahepatic metastasis.[3] Elucidation of the molecular mechanisms underlying HCC invasiveness is of utmost importance for the development of future strategies for treating HCC.
The epithelial-mesenchymal transition (EMT), a developmental process by which epithelial cells reduce intercellular adhesion and acquire fibroblastoid properties, has been shown to be critical for the development of the invasiveness and metastatic potential of human cancers.[4] Characteristic changes during EMT include down-regulation of epithelial markers (e.g., E-cadherin and plakoglobin),[4][5] translocation of -catenin (i.e., dissociation of membranous -catenin and translocation into the nuclear compartment),[6] and up-regulation of mesenchymal markers (e.g., vimentin and N-cadherin).[4][5] The EMT process is initiated by suppression of E-cadherin expression by the major EMT regulators, e.g., Snail, Slug, and Twist.[4][5] The zinc-finger transcriptional repressors Snail (also known as Snail1) and Slug (also known as Snail2) and the basic helix-loop-helix (bHLH) transcription factor Twist (also known as Twist1) were shown to induce EMT through repression of E-cadherin expression.[7-9]
The clinical significance of EMT has been confirmed in certain types of human cancers,[10][11] and the significance of individual EMT regulators in HCC has been demonstrated.[12-15] However, a comprehensive study demonstrating the expression profile of multiple EMT markers in HCC is lacking. In addition, the role of hepatotrophic viruses, such as HBV and hepatitis C virus (HCV), in promoting the EMT/metastasis of HCC is unclear. In this study we investigated the expression profiles of EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human HCC.
Discussion
Induction of EMT is distinguished by the repression of E-cadherin transcription by zinc-finger proteins (e.g., Snail, Slug)[7][8] or bHLH family transcriptional factors (e.g., Twist)[9] through binding to three E-boxes located in the proximal promoter of E-cadherin. Although EMT has been considered the critical mechanism involved in cancer metastasis, in comparison with other types of human cancers only a few sporadic studies have focused on the significance of EMT in HCC.[12-15] The present study is the first to provide a comprehensive profile of multiple EMT markers and to demonstrate that Snail and Twist, but not Slug, are the major inducers of EMT in HCC. These results not only pinpoint the major signal pathways in the induction of EMT in HCC, but also demonstrate its clinical relevance through comprehensive analysis.
Transforming growth factor (TGF-) is a master regulator of EMT through activation of the Smad2/3 complex.[22] TGF-1 plays a key role in triggering EMT in HCC through cooperation with laminin-5,[23] and inhibition of TGF-1 attenuates migration/invasion of HCC cells.[24] One recent report showed that in HCC the expression of HCV-derived core protein switches the cellular response to TGF- exposure from inhibition of growth to induction of EMT,[25] providing a possible linkage between HCV-induced HCC and EMT. Although HCV-related HCC has been shown to be associated with a higher recurrence rate after surgery,[26] the underlying mechanism is unclear, and the correlation between HCV and EMT has never been explored. We report the original finding that overexpression of Twist is correlated with HCV-related HCC. This interesting finding may partially explain the highly invasive behavior and poor prognosis of HCV-related HCC. The mechanism underlying increased Twist expression in HCV-related HCC deserves further study.
The interplay between Snail and Twist in the promotion of EMT in HCC has not been reported. In the present study we provide evidence that Twist and Snail are regulated independently and act collaboratively to promote EMT: coexpression of Snail and Twist indicates the worst prognosis for HCC; HCC cell lines harboring both Snail and Twist showed a higher migratory/metastatic ability; coexpression of Snail and Twist in Huh7 promoted the highest invasiveness and metastasis; and Twist and Snail did not influence the expression/promoter activity of each other. This is the first report to demonstrate the independent regulation of Snail and Twist and their additive effects on EMT promotion.
Differences in the roles of Snail and Twist in promoting HCC metastasis were disclosed in our study: although Snail was more potent in activating MMPs, HCC cells overexpressing Twist tended to be more invasive than cells overexpressing Snail, and this observation was supported by analysis of clinical samples. These results indicate that Twist may be more critical in HCC metastasis, possibly acting through an MMP-independent mechanism. Previous studies disclosed that some critical molecules (e.g., N-cadherin, Akt2, and YB-1) in cancer metastasis are direct targets of Twist.[27-29] Overexpression of Twist induces angiogenesis.[30] Collectively, our results indicate that Snail and Twist act additively to promote HCC metastasis through different mechanisms.
In conclusion, our study demonstrates the prognostic significance of EMT markers in HCC and also establishes different roles for Snail and Twist in the promotion of EMT in HCC through independent regulation. A correlation between HCV-induced HCC and Twist expression is also reported. These results elaborate on the major mechanisms involved in HCC metastasis and provide essential information for prediction of prognosis and identification of new treatment targets for future HCC management.
Results
Down-regulation of E-cadherin in HCC.
IHC was performed to investigate the significance of down-regulated E-cadherin expression, the hallmark of EMT,[4][5] in HCC patients. Among primary HCCs, 39.8% of the samples showed preserved expression of E-cadherin (Case 1 of Fig. 1A; Supporting Fig. S1A, left panel). Decreased (Case 2 of Fig. 1A; Supporting Fig. S1A, middle panel) or absent E-cadherin expression (Supporting Fig. S1A, right panel) was identified in 52.8% and 7.4% of the samples, respectively. Among the samples with recurrent HCC, preserved, decreased, or absent expression of E-cadherin was shown in 23.9%, 63.1%, and 13% cases, respectively. An increased proportion of down-regulated E-cadherin expression was shown in recurrent compared with primary samples (Fig. 1B). The results of IHC analysis for E-cadherin were validated by ELISA and real-time RT-PCR in representative HCC samples (Supporting Fig. S1B,C). Survival analysis demonstrated a significant decrease in cancer-free interval (CFI) and overall survival (OS) in patients with down-regulated expression of E-cadherin (Table 1, Supporting Table S11, Fig. 1D). Down-regulated expression of E-cadherin was also associated with factors indicating clinical aggressiveness (e.g., large tumor size; Supporting Table S3), and a trend toward multinodular tumors was observed (Supporting Table S3).
Nonmembranous Expression of -Catenin in HCC.
Translocation of -catenin is an important marker of EMT.[6] IHC analysis for -catenin was performed in the available 103 primary and 33 recurrent HCC samples. A higher incidence of nonmembranous expression of -catenin (Case 2 of Fig. 1A) was shown in recurrent versus primary HCCs (66.7% versus 51.5%; Fig. 1C). Nonmembranous expression of -catenin was associated with down-regulated E-cadherin (Supporting Table S4), a shorter CFI, and a worse OS (Table 1, Supporting Table S11, Fig. 1E) and a trend toward multinodular tumors and microscopic venous invasion was observed (Supporting Table S5).
Expression Profile and Clinical Significance of the Major EMT Regulators (Snail, Slug, and Twist) in HCC.
The expression profiles of Snail, Slug, and Twist in HCC were evaluated. An increased incidence of Snail (Supporting Fig. S2A) or Twist overexpression (Supporting Fig. S3A) was shown in the recurrent versus primary HCCs (Supporting Figs. S2B, S3B). However, the percentages of Slug overexpression (samples available in 105 primary and 33 recurrent cases; Supporting Fig. S4A) were similar in the recurrent versus primary samples (Supporting Fig. S4B). Expression of Snail or Twist, but not Slug, was associated with down-regulation of E-cadherin and -catenin translocation (Supporting Table S6). Overexpression of Snail or Twist was associated with a shorter CFI and OS (Table 1, Supporting Table S11, Supporting Figs. S2C, S3C) and aggressive tumor behavior (e.g., large tumor size and multinodular tumors; Supporting Tables S7, S8). Surprisingly, increased Twist expression was significantly correlated with serum anti-HCV positivity and HBsAg negativity (Supporting Table S8). Although increased Slug expression was associated with aggressive clinical factors (e.g., large tumor size; Supporting Table S9), no prognostic significance of Slug on OS and CFI was demonstrated (Table 1, Supporting Table S11, Supporting Fig. S4).
Snail and Twist were frequently coexpressed (30.1% of primary and 60.9% of recurrent HCCs; Fig. 2A). Coexpression of Snail and Twist was associated with down-regulated expression of E-cadherin, nonmembranous expression of -catenin (Supporting Table S6), and the worst CFI and OS (Table 1, Supporting Table S11, Fig. 2B). Coexpression of both markers was also associated with aggressive factors (e.g., a trend toward multinodular tumors; Supporting Table S10).
Analyses of Factors Associated with CFI and OS in HCC.
Survival analysis was performed to determine the independent factors associated with CFI or OS. An impact on CFI was shown for many factors in the univariate analysis (Table 1); however, only anti-HCV positivity, elevated levels of -fetoprotein (AFP), multinodular tumors, down-regulation of E-cadherin, and overexpression of Twist were independent predictors of CFI (Table 1). A number of factors were also associated with OS in the univariate analysis (Supporting Table S11), but multivariate analysis limited this list to patient age, anti-HCV positivity, prolonged prothrombin time, multinodular tumors, and coexpression of Snail and Twist (Supporting Table S11).
Expression of Snail and Twist Is Associated with a Mesenchymal Phenotype and Invasiveness/Metastasis of HCC Cells.
Because Snail and Twist are the major inducers of EMT in HCC, we investigated the expression of Snail/Twist and its association with EMT markers/phenotypic changes in eight HCC cell lines. The results indicated that cells with a higher migratory ability and invasiveness showed associated mesenchymal changes, higher levels of Snail or Twist expression, and higher MMPs activity, whereas cells with lower invasiveness/migration exhibited epithelial markers, lower levels of Snail or Twist, and lower MMPs activity (Fig. 3). An in vivo tail-vein metastasis assay was performed to compare the metastatic capacity of Mahlavu, the most invasive cell line, with Huh7, the least invasive one. A significant increase in pulmonary metastasis was noted in mice receiving injections of Mahlavu versus Huh7 (Fig. 4A -C). A similar result was shown in the splenic-vein model (Fig. 4D-F). These results indicated that HCC cells with a mesenchymal phenotype (e.g., Mahlavu cells) were more invasive and exhibited greater in vivo metastatic ability, whereas HCC cells expressing epithelial markers (e.g., Huh7 cells) were associated with a lower tendency to migrate, invade, and metastasize.
Twist and Snail Are Regulated Independently and Act Additively in Promoting EMT.
To investigate the independent and nonredundant roles of Snail and Twist in HCC metastasis, we used Huh7 cells to generate clones with stable expression of Snail, Twist, or both proteins. Overexpression of Snail and/or Twist resulted in a shift in EMT marker expression (Fig. 5A). Expression of either Snail or Twist alone did not influence the expression levels of the other (Fig. 5A). Overexpression of Snail or Twist increased migration/invasion/metastasis, and cells that coexpressed both proteins demonstrated the highest ability for migration/invasion/metastasis (Fig.5C, D). Interestingly, overexpression of Snail resulted in higher activities of MMP-2 and MMP-9 than overexpression of Twist (Fig. 5B), but expression of Twist tended to result in slightly greater migration/metastasis than did expression of Snail (Fig. 5C,D).
Promoter activity assays were performed to determine whether Twist and Snail act collaboratively or independently. Overexpression of Snail or Twist resulted in a significant decrease in E-cadherin promoter activity, as expected; and combined overexpression of Snail and Twist seemed to exert an additive inhibitory effect on the E-cadherinpromoter compared to either agent alone (Fig. 6A,B). Snail expression could not activate the reporter driven by theTwist promoter. Likewise, Twist expression did not influence the activity of the Snail promoter (Fig. 6C-F). These results confirmed the independent expression and regulation of Twist and Snail.
Twist and Snail Are Both Critical for HCC Cell EMT/Migration/Metastasis.
To determine whether Twist and Snail are critical mediators of EMT in HCC, Twist or Snail expression in the highly invasive Mahlavu cells was suppressed by siRNA. Repression of either Snail or Twist resulted in down-regulation of vimentin and up-regulation of E-cadherin expression (Fig. 7A), and suppression of in vitro migration/invasion (Fig.7C) and in vivo metastasis (Fig. 7D). However, knockdown of Snail caused a more significant decrease in MMPs activity than Twist (Fig. 7B). These results further confirmed the critical but different roles of Snail and Twist in mediating EMT and metastasis of HCC cells.
Materials and Methods
Abstract Materials and Methods Results Discussion Acknowledgements References
Patients and Treatment.
In all, 123 primary HCC samples with adjacent nontumorous liver tissues were obtained from patients who had undergone curative hepatic resection between 1990 and 2002 at Taipei Veterans General Hospital. Among these 123 cases, 46 of 84 recurrent tumor samples were available for analysis. The study was approved by the Institutional Review Board of Taipei Veterans General Hospital. The clinical characteristics of the patients with HCC are presented in Table 1.
Table 1. Univariate and Multivariate Analysis of Factors Associated with Cancer-Free Interval
Variables No. Median Survival (mo) (95% CI) P-value
Age 55/> 55 y/old 59/64 41.4 (20.662.1)/22.1 (7.436.8) 0.218
Gender male/female 104/19 36.6 (25.447.8)/12.1 (026.4) 0.397
HBsAg (N/Y) 24/99 22.1 (8.136.1)/39 (20.058.0) 0.029
Anti-HCV (Y/N)* 23/97 14.4 (8.820.0)/41.4 (27.255.6) 0.018
Albumin 4.0/> 4.0 g/dL 59/64 22.1 (7.536.7)/41.8 (2.780.9) 0.038
Bilirubin 1.6/> 1.6 mg/dL 113/10 31.6 (15.8-47.4)/32 (NA) 0.315
ALT 40/>40 U/L 61/62 37.2 (19.155.3)/29.4 (8.050.8) 0.785
PT (INR) > 1.2/ 1.2 10/104 15 (044.3)/37.2 (19.954.5) 0.132
ICG-15 >10%/10% (retention rate) 48/75 29.4 (1.956.9)/31.6 (4.648.6) 0.372
Child-Pugh A/B 117/6 31.6 (17.945.3)/41.4 (088.1) 0.753
BCLC A/B or C 82/41 47.4 (31.9-62.9)/11.0 (5.0-17.0) <0.001
CLIP 0-1/2-4 88/35 41.8 (27.156.5)/10.9 (6.115.7) 0.004
Okuda I/II 96/27 39 (25.152.9)/12.1 (6.717.5) 0.107
TNM T1-2/T3-4 88/35 46.5 (28.764.3)/11.0 (6.515.5) 0.006
Tumor size 3cm/>3cm 48/75 47.4 (32.961.9)/22.1 (9.534.7) 0.088
Multinodular tumors Y/N* 49/74 13.1 (8.517.7)/56 (28.783.3) <0.001
Macroscopic venous invasion Y/N 13/110 19.3 (3.135.5)/36.6 (22.051.2) 0.102
Cut margin free/non-free 108/11 31.1 (16.7-45.5)/NA 0.130
AFP 100/>100 ng/ml* 73/49 41.4 (25.757.1)/15.0 (033.1) 0.044
Microscopic venous invasion Y/N 50/57 25.1 (8.242.0)/46.5 (22.570.5) 0.154
Edmonson stage I or II/III or IV 71/33 37.2 (25.8-48.6)/25.1 (0-65.7) 0.282
E-cadherin downregulation* (Y/N) 74/49 16.1 (8.5-23.7)/225 (NA) <0.001
-catenin membranous expression (Y/N) 50/53 59.3 (27.9-90.7)/23.4 (7.0-39.8) 0.013
Twist overexpression* (Y/N) 53/70 11.2 (9.0-13.4)/78 (NA) <0.001
Snail overexpression (Y/N) 70/53 15 (5.0-25.0)/103 (59.4-146.6) <0.001
Slug overexpression (Y/N) 54/51 102.6 (63.6-141.6)/129.3 (NA) 0.336
Co-expression of Twist and Snail (Y/N) 37/86 9.9 (5.8-14.0)/59.3 (30.8-87.8) <0.001
ALT, alanine aminotransferase; BCLC, Barcelona Clinic Liver Cancer; CLIP, Cancer of the Liver Italian Program; CI: confidence interval; ICG-15, indocyanine clearance test; PT: Prothrombin time; RR: risk ratio; NA: not applicable; Y: yes, N: no.
* Significant in multivariate analysis: positive anti-HCV: RR 2.387, 95% CI: 1.340-4.237, P = 0.003; multinodular tumors: RR 1.941, 95% CI: 1.164-3.237, P = 0.011; AFP > 100 ng/mL: RR 1.936, 95% CI: 1.101-3.392, P = 0.021;E-cadherin downregulation: RR 4.374, 95% CI: 2.289-8.359, P < 0.001; Twist overexpression: RR 2.978, 95% CI : 1.750-5.068, P < 0.001.
Sample available in 103 cases.
Sample available in 105 cases.
Immunohistochemistry (IHC) and Scoring.
The sample processing and IHC procedures were performed as described.[16] The antibodies used in IHC and the incubation periods are listed in Supporting Table S1. All IHC staining was independently scored by two experienced specialists. The expression of E-cadherin was scored as preserved, reduced, or absent, as described.[17] Reduced or absent expression of E-cadherin was interpreted as down-regulation of E-cadherin.[18] Nonmembranous expression of -catenin (i.e., more than 10% of cells exhibiting cytoplasmic or nuclear staining) was scored as described.[19] Nuclear expression of Snail, Slug, and Twist was graded from 0 to 3+ (0, no staining; 1+, 1%-25%; 2+, 26%-50%; 3+, >50% nuclear staining), with only 3+ considered a positive IHC result.[20]
Plasmids and Cell Lines.
The human hepatocellular carcinoma cell lines Huh7, HepG2, PLC, Hep3B, Sk-Hep1, Mahlavu, and the human embryonic kidney cell line HEK-293T were obtained from the American Type Culture Collection (Rockville, MD). Cell lines HA59T-VGH and HA22T-VGH were gifts from Professor C.P. Hu (Tung-Hai University, Taichung, Taiwan). The plasmids pcDNA3-Snail, pFLAG-Twist, pSUPER-Snail-si, and pSUPER-Twist-si have been described.[20] A scrambled sequence with no significant homology to any mammalian gene sequence was cloned into the pSUPER vector (pSUPER-scr-si) for use as a control in short interference RNA (siRNA) experiments. The oligonucleotide sequences used for generating siRNA constructs are listed in Supporting Table S2.
RNA Purification and Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Analysis.
Total RNA extraction, complementary DNA (cDNA) synthesis, and quantitative real-time RT-PCR were performed as described.[16] The primer sequences used in real-time PCR are shown in Supporting Table S2.
Western Blot Analysis.
Protein extraction from cultivated cells and western blot analysis were performed as described.[16] Antibodies used in western blot experiments are listed in Supporting Table S1.
Detection of E-cadherin Expression by Enzyme-Linked Immunosorbent Assay (ELISA).
The procedures are detailed in the Supporting Methods.
Gelatin Zymography.
Analysis of the activity of matrix metallopeptidases-2 and -9 (MMP-2 and MMP-9) in the different clones was performed as described.[16] The procedures are detailed in the Supporting Methods.
Cell Migration and Invasiveness Assay.
In vitro transwell migration and invasion assays were performed as described.[16] The procedures are detailed in the Supporting Methods.
Cloning of Gene Promoter Regions, Generation of Reporter Constructs, Transient Transfections, and Luciferase Assays.
The genomic regions flanking the E-cadherin, Snail, and Twist gene promoter regions (Fig. 6A,C,E) were generated by PCR amplification of human genomic DNA and inserted into the HindIII/BglII sites in the pXP2 vector to generate the pXP2-Ecadherin, pXP2-Twist, and pXP2-Snail reporter constructs. Transfections and reporter assays were performed as described, and the bacterial -galactosidase gene (pCMV-gal) was used as a control for transfection efficiency.[20]
Nude Mouse Tail Vein Metastasis Assay and Splenic Vein Metastasis Assay.
All procedures involving animals were performed in accordance with the institutional animal welfare guidelines of Taipei Veterans General Hospital. Cells were injected into the splenic vein or the tail vein of 8-week-old nude mice (BALB/c strain) at 1 × 10[6] cells/injection site. The mice were sacrificed after 6 weeks and the number and volume of metastatic tumors were assessed. Tumor size was measured by use of a caliper and volume was calculated as length × height × width × 0.5236 with reference to a previous report.[21]
Statistical Analysis.
The Kaplan-Meier estimate was used for survival analysis and the log-rank test was selected to compare the difference. A Cox proportional hazards model was applied to multivariate survival analysis for testing independent prognostic factors. Statistical significance was accepted when P < 0.05 for all tests.
Muh-Hwa Yang 1 2 3, Chih-Li Chen 4, Gar-Yang Chau 5 6, Shih-Hwa Chiou 1 7, Chien-Wei Su 1 8, Teh-Ying Chou 1 9, Wei-Li Peng 7, Jaw-Ching Wu 1 3 7 *§
1Institutes of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
2Division of Hematology-Oncology, Taipei Veterans General Hospital, Taipei, Taiwan
3Genomic Medicine Research Center, Taipei Veterans General Hospital, Taipei, Taiwan
4School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan
5Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan
6Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan
7Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan
8Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
9Department of Pathology, Taipei Veterans General Hospital, Taipei, Taiwan
email: Jaw-Ching Wu (jcwu@vghtpe.gov.tw)
*Correspondence to Jaw-Ching Wu, Department of Medical Research and Education, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei 112, Taiwan
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