This pilot trial included 29 patients who had previously failed a combination therapy with (peg)IFNα and ribavirin. 17 (59%) of these patients now had an EVR when re-treated with pegIFNα2b, ribavirin, SAMe and betaine (). The addition of SAMe and betaine to the current standard combination therapy therefore achieved an EVR rate that was considerably higher than reported before in trials investigating re-treatment of patients with previous nonresponse and relapse. The recently published EPIC trial, investigating response to pegIFNα2b/ribavirin therapy in 2293 patients with previous (peg)IFNα/ribavirin therapy failure, showed that overall 36% of the patients achieved an EVR 
. EPIC, importantly, not only included nonresponders (61%), but also at least 28% relapse patients. This is important because re-treatment of relapse patients has consistently resulted in higher SVR rates than re-treatment of nonresponders 
. Moreover, 37% of the patients included in EPIC had previously been treated with standard IFNα/ribavirin. In our trial we only included nonresponders, 86% of which even had documented PNR at week 12 of the previous treatment, and 76% had been treated with pegIFNα/ribavirin (only 24% with IFNα/ribavirin).
Distribution of early virological response to previous treatments and to the study medication.
Taken together, the 59% EVR rate in our difficult-to-treat group of patients suggests that the addition of SAMe and betaine to pegIFNα2b/ribavirin improves EVR rates in patients with CHC.
Despite the high rate of EVR in our trial, only 3 patients (10%) achieved an SVR. 4 additional patients achieved an EoTR but then relapsed during FU. 10 of the 17 EVR patients, however, either never reached negativity for HCV RNA or had a viral breakthrough during therapy. Overall, only 17% of the patients with an EVR showed later SVR in our study. Other re-treatment studies reported higher SVR rates (49% 
; 36% 
) in patients who had an EVR during re-treatment with pegIFNα/ribavirin. In EPIC, an SVR was observed in 56% of the patients who were HCV RNA negative at week 12 (cEVR), in 12% of the patients with an EVR but still detectable HCV-RNA at week 12, and in none of the patients without EVR 
. In our small pilot study, 50% (3 of 6) of patients with a cEVR had an SVR, but none of the patients with an EVR (and still detectable HCV RNA at week 12). Because of the heterogeneity of these clinical trials it is difficult to directly compare the results. However, the overall low SVR rate in our EVR patients suggests that SAMe and betaine are only effective early in the treatment. It is conceivable that adaptive mechanisms in hepatocytes restore intracellular SAMe concentrations to pre-treatment levels, because SAMe is involved in many enzymatic reactions and its concentration is regulated by numerous metabolic pathways.
Tachyphylaxis to SAMe might be overcome by increasing the dosage, but in this small pilot study, we were not able to evaluate the effectiveness of different doses of SAMe and betaine. SAMe was given at a dose of 1200 mg per day because the same dosage was used and found to be safe during a treatment period of two years in the largest study published using SAMe 
. Likewise, betaine treatment with 6 g per day has been previously shown to be effective and safe 
. Future studies should aim to define the optimal dosages for SAMe and betaine.
The rationale for adding SAMe to pegIFNα/ribavirin treatments is based on our previous analysis of HCV interference with IFNα signaling in cultured cells, transgenic mice and liver biopsies from patients with CHC 
. Using cell lines with inducible expression of HCV proteins and using the HCV replicon system we showed that SAMe improves methylation of the IFNα induced transcription factor STAT1 and its binding to DNA response elements, thereby increasing the induction of ISGs and enhancing the inhibitory effect of IFNα on replicons 
. In the present clinical study we could not assess the effects of SAMe and betaine on IFNα signal transduction in the liver of the patients. Consequently, we cannot exclude that SAMe and betaine improve the response to pegIFNα/ribavirin treatments by mechanisms that differ from our proposed model of action that involves methylation of STAT1 as the key event.
Betaine was added to the combination treatment in order to prevent the accumulation of the toxic SAMe metabolite S-adenosyl-L-homocysteine (AdoHcy) and to further increase the intracellular concentrations of SAMe 
. We did not measure the plasma levels of homocysteine or betaine, and therefore cannot assess the effect of betaine on SAMe metabolism in our patients.
A number of directly acting antiviral agents is presently in advanced stages of clinical development. The most promising candidates include direct inhibitors of the HCV NS3 protease such as telaprevir 
, boceprevir 
and MK-7009 
, as well as both nucleoside and non-nucleoside inhibitors of the NS5B RNA-dependent RNA polymerase. Although these agents have demonstrated potent antiviral effectiveness, monotherapy has been complicated by rapid virological breakthrough due to the selection of drug-resistant mutants. To prevent viral resistance, protease and polymerase inhibitors are used only in combination with pegIFNα/ribavirin in phase II and III clinical studies. However, a substantial number of patients do not respond to pegIFNα, most likely because their endogenous IFN system is already induced before therapy, and because of HCV interference with IFNα signal transduction through the Jak-STAT pathway 
. Such patients might be at risk for viral breakthrough and resistance development even when receiving a triple combination therapy (protease inhibitor plus pegIFNα/ribavirin). The 4 weeks of lead-in phase with pegIFNα/ribavirin used in SPRINT-1 and in ongoing boceprevir trials will exclude those flat nonresponders from the triple combination therapy, and will help to reduce the emergence of resistant viruses 
. The addition of SAMe and betaine to the initial phase of such a treatment could be a reasonable strategy. By improving the cellular response to pegIFNα, SAMe and betaine have the potential to increase the number of patients who achieve an initial response and can be offered triple combination therapies.
In conclusion, addition of SAMe and betaine to the current standard therapy with pegIFNα/ribavirin resulted in an EVR in 59% of patients who previously were nonresponders to (peg)IFNα/ribavirin therapies. SAMe and betaine had an excellent safety and tolerability profile. The improvement of response to pegIFNα/ribavirin, when combined with SAMe and betaine, should encourage further studies, since pegIFNα and ribavirin will be cornerstones of CHC treatments for many years to come.