High rates of sustained virological response to combination therapy in Southeast Asians with hepatitis C virus (HCV) genotype 1b: the impact of accurate genotype determination

HEPATOLOGY Vol. 3 4 , N o . 4, Pt. 2, 2 0 0 1

AASLD ABSTRACTS

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A HIGH THROUGHPUT ASSAY FOR IDENTIFICATION OF HEPATITIS C Xq[RUS NS3 SERINE PROTEASE INHIBITORS. R o m y Zemel, Y e v g e n y Ber-

HIGH RATES OF SUSTAINED VIROLOGICAL RESPONSE TO COMBINATION THERAPY IN SOUTHEAST ASIANS W I T H HEPATITIS C VIRUS (HCV) GENOTYPE 1B: THE IMPACT OF ACCURATE GENOTYPE DETERMINATION. A n o u k Dev, M o n a s h M e d i c a l Centre, M e l b o u r n e Australia;

d i c h e v s k y , Larisa B a c h m a t o v e , F e l s e n s t e i n M e d i c a l R e s e a r c h Ctr, T e l - A v i v U n i v , P e t a c h - T i k v a Israel; M a r g a r i t a K u n i n , R a b i n M e d i c a l Ctr, P e t a c h - T i k v a Israel; A v i G o l a n - G o l d h i r s h , B e n - G u r i o n U n i v of the N e g e v , Sede B o k e r C a m p u s , Be'er-Sheva Israel; Ran T n r - K a s p a , R a b i n M e d i c a l Ctr, P e t a c h - T i k v a Israel; Itai Benhar, T e l - A v i v U n i v , T e l - A v i v Israel Proteolytic cleavage o f the H C V p o l y p r o t e i n p r e c u r s o r is essential for the viral replication process, h e n c e the viral e n c o d e d NS3 s e r i n e p r o t e a s e is c o n s i d e r e d one of the m o s t i m p o r t a n t targets for d e v e l o p i n g a n t i - H C V therapeutics. O u r r e s e a r c h a i m s to s t u d y the i n h i b i t i o n of H C V NS3 serine p r o t e a s e f u n c t i o n a n d potential t m n o r i g e n i c i t y b y c o m p o u n d s isolated f r o m n a t u r a l s o u r c e s a n d b y r e c o m b i n a n t proteins. F o r s c r e e n i n g of H C V NS3 s e r i n e - p r o t e a s e inhibitors, a r a p i d a n d s e n s i t i v e assay of e n z y m e activity w a s established. R e c o m b i n a n t NS3 serine p r o t e a s e w a s isolated a n d purified, a n d a f l u o r o g e n i c a s s a y for NS3 p~oteolytic activity w a s developed. As a n NS3 s u b s t r a t e w e e n g i n e e r e d a rec o m b i n a n t f u s i o n p r o t e i n w h e r e a g r e e n fluorescence p r o t e i n is l i n k e d to a c e l l u l o s e - b i n d i n g d o m a i n (CBD) via t h e NS5A/B site that is cleavable b y NS3. C l e a v a g e o f this s u b s t r a t e b y NS3 results in e m i s s i o n of fluorescence light easily d e t e c t e d a n d q n a n t i t a t e d b y f l u o r o m e t e r . T h i s u n i q u e f l u o r o g e n i c assay is v e r y s e n s i t i v e a n d h a s a h i g h t h r o u g h p u t capacity. W e h a v e a n a l y s e d k n o w n p r o t e a s e i n h i b i t o r s (PMSF, T P C K , BBI etc.) u s i n g o u r s y s t e m a n d h a v e detected specific NS3 serine p r o t e a s e i n h i b i t o r s f r o m extracts o b t a i n e d f r o m n a t u r a l I n d i a n Siddha m e d i c i n a l p l a n t c o m p o u n d s . P u r i f i e d fractions of p l a n t extracts will b e f u r t h e r a n a l y s e d as specific N S 3 s e r i n e p r o t e a s e inhibitors. T h i s assay enables r a p i d a n d reliable s c r e e n i n g of p u t a t i v e n a t u r a l a n d r e c o m b i n a n t NS3 serine p r o t e a s e inhibitors.

R h o n d a M c G a w , Scott Bowden, V i c t o r i a n I n f e c t i o u s Disease Reference Laboratory, M e l b o u r n e Australia; W i l l i a m Sievert, M o n a s h M e d i c a l Centre, Melb o u r n e Australia A sustained virological response (SVR) to combined therapy with interferon (IFN) and rihavirin (RBV) occurs more often in chronic HCV infected patients with genotypes (GT) 2 and 3 than in those with GT I and 4. Race and ethnicity are host factors that may also affect treatment response although this concept has not been well studied in Southeast Asian (SEA) patients. We compared the effect of race and genotype on the efficacy of combination therapy in SEA and Caucasian HCV patients. We retrospectively identified 78 SEA HCV patients who had received iFN and RBV therapy as pre~qously untreated Subjects in an Australian/New Zealand muhicentre trial of standard versus induction therapy (AusHep 8)(n = 45) or in a compassionate access program (n = 28); 5 patients received combination therapy following a relapse after 1FN monotherapy. HCV RNA was detected by PCR (Roche AMPLICOR HCV). Genotype was determined by a line probe assay (InnoLiPA, Innogenetics). Of 78 SEA patients identified, 47 had completed treatment with six months follow-up (68% male, 25 GTlb, 5 GT1, 6 GTla, 2 GT2a/c, 4 GT3a, 5 GT6a). We also identified 52 Caucasians (77% male) with GTlb who had completed treatment with six months follow-up. SEA patients had a significantly lower body mass index than Caucasians, but there was no difference in age, viral load, ALT or fibrosis score between the groups. A SVR occurred in 68% (17/25) of SEA patients with GTlb compared to 30% (16/52) of Caucasian GTlb (p<0.0O2). A limitation of the Inno-LiPA is that it cannot type new putative GT 7, 8 and 9, which are common in SEA. Re-analysis of all SEA GTlb samples by sequencing the HCV core region resulted in reclassification of 11 subjects as GT 7, 8 or 9; of these 10 achieved a SVR (91%) vs. 30% of Caucasian GTlb (p=0.002). Of those who were not reclahsified (i.e. who were true GTlb), a SVR occurred in 57% (8/14) SEA compared to 30% in Caucasian GT lb and this difference approached statistical significance (p=0.06)(Table). In the other SEA patients a SVR occurred in 5/5 GTI, 4/6 GTla, 2/2 GT2a/2c, 3/4 GT3a and 5/5 GT6a. In conclusion, little is known about the response to antiviral therapy in HCV infected patients with genotypes that are uncommon in Western countries. We have demonstrated that a proportion of SEA patients classified as GTlb were in reality GT 7, 8 or 9 and their response to antiviral therapy was significantly better than that of Caucasian GTlb. Additionally, even SEA patients classified as true GTIb appeared more likely to achieve a SVR than Caucasian patients with GTlb. These results support the need for larger prospective studies of treatment response in "uncommon" genotypes and the need for accurate determination of genotype. Racial differences in response to combination therapy may be explained by viral factors such as genotype but host factors may also play a role.

SVR in SEA and CaucasianHCV patientsby genotype SEAGT 7,8,9 SEAGT7,8,9 SEAtrue GTI b /all) n=25 n=11 n=14 SVR 68% 91% 57% p (vs.CaucasianGTlb) <0,002 0.Q02 0.06

Caucasian GTIb n=52 30%

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EFFECT OF ORALLY ADMINISTERED BOVINE LACTOFERRIN ON SER U M CYTOKINES ENVIRONMENTS OF PATIENTS CHRONICALLY INt:ECTED W I T H HEPATITIS C VIRUS GENOTYPE lB. Koji Ishii, N a o k o

EFFECT OF INVESTIGATOR EXPERIENCE ON TREATMENT OUTCOME AND DEPRESSION MANAGEMENT IN TREATMENT OF HEPATITIS C.

T a k a m u r a , Mie Shinohara, H i r o k a z u Shin, Yasuo T a k e u c h i , T a k a s h i I k e h a r a , Y a s u n o b u N i s h i o , Soichiro Hata, T a k a s h i K a w a f n n e , Y a s u k i y o S u m i n o , T o h o U n i v e r s i t y , School of Medicine, T o k y o J a p a n ; S u s u m u T e r a u c h i , N u t r i t i o n a l Science Laboratory, M o r i n a g a M i l k I n d u s t r y Co, K a n a g a w a J a p a n ; Koji Y a m auchi, N u t r i t i o n a l Science Laboratory, M o r i n a g a M i l k I n d u s t r y Co, Y o k o h a m a Japan BACKGROUND:Chronic infection with hepatitis C virus (HCV) is associated with progressive liver diseases that may insidiously evolve into cirrhosis, and carries an increased risk of hepatoceliular carcinoma. In Japan, the rate to eliminate HCV by interferon (IFN) monotherapy has been less than 10 percent fi~patients with chronic hepatitis C, who were infected with genotype lb and with high viral load in the serum. Previously (51 th AASLD),we reported that bovine lactoferrin (LF), a milk protein belonging to the h'on transporter family,has an ability to induce prevalence of Th0 and ThI cell subsets, of which cytoplasm "~iaspositive for 1FN gamma, in peripheral blood CD4 positive T ceils in patients chronically infected with I-ICVgenotype lb. However,mechanism(s) for induction of IFN gamma in cytoplasm is not elucidated. In general, 60-80 % of orally-administered bovine LF (bLF), which has been commerciallyavailable as health supplement in Japan, is not degraded in the stomach and reaches to small intestine in which putative receptors for LF are believed to exist. A molecule responsible for the IFN gamma production named as interleukln- 18 (ILq8) was found in the mouse intestinal epithelium. Aim of the present stud)' is to clarify how orally administered bovine LF modulate cytokine environments in patients chronically infected with ttCV (CHC). PATIENTSAND METHODS:Twenty-three patients with CHC were the objects of the study. All patients were proved positive for serum anti-HCVby third generation ELISA(Ortho DiagnosticSystem, Tokyo, Japan) and for serum HCV-RNAby-Amplicor HCV monitor V2.0 (Roche Molecular Systems,Inc., Pleasanton, CA, USA),and their serum HCV genotype was Ib. In addition, they were negative for serological markers of hepatitis B virus infection (absence of HBs antigen and anti-HBc antibody). None of the patients had a history of habitual aicohoI abuse, and their serum autoantibodies were always negative. Patients were divided into two groups, bLF (Morinaga Milk Industry Co., Japan) was administered as an oral dose of 600 rag/day for 3 months in 14 patients with CHC (LF group, 9 male / 5 female,mean age of 51 y~ars), and no medication was given to the remaining 9 patients (control group, 4 male / 5 female~mean age of 48 years). Both groups were followed up for 3 months. Serum levelsof interleukin (IL)-1 beta, IL-4,IL-6, IL-IO,IL-12,1L-18, and TNF-alpha were measured at 3 months interval. Serum cytokines were measured using each ELISAkits. RESULTS:Before and 3 months after bLF administration, there was no statistically" significant difference in the serum levels of IL-I beta (mean +/- SE; from 0.51 +/- 0.20 to 0.87 +/- 0.31 pg/mL in the control group, from 0.28 +/- 0.06 to 0.88 +/- 0.30 pghnL in the LF group), IL-6 (from 0.62 +/- 0 32 to 0.62 +/- 0 37 pg/mL in the control group, and from 0.42 +/- 0.12 to 1.93 +/- 124 pg/mL in the t.P group), IL-I0 (from 5.iI +/- 0.98 to 3.48 +/- 038 pg/mL in the control group, and from 4.26 +/- 0.62 to 3.68 +A 0.62 pghnL in the LF group), and TNF-alpha (from 2.90 +/- 1.24 to 2.58 +/- 1.03 pg/mLin the control group, and from 3.51 +/- 0.83 to i2.72 +/- 6.19 pg/mk in the LF group). However, statistically significantchange was seen in serum levelsof IL~18 between before and 3 months afterbLF administration (from 82.96 +/- 1955 to I53.52 +/- 34.33 pg/mk) in the kg group, but not in the control group (from 100.41 +/- 31.60 toi04.SS +/- 22.50 pg/mk). Serum levels of IL-4 and lk I2 were under sensitometry by each kit used in the bottt groups. CONCLUSION:It is elucidated that orally administered bkP has an ability to induce serum IL-18, thereby interferon gamma is induced in the CD4 positive T cells (Th0 and Thi) subsets. Orally administered bLF produces suitable eytokines surroundings to eliminate HCV from body, because a strong response of Thl and ThI cytokines is recommended to eliminate HCV. Our results suggest that orally administered bLF in combination with IFN-alpha raises the rate to eliminate HCV genotype lb.

Paul Y Kwo, Sheryl Fields, T h o m a s F Imperiale, Beverly M u s i c k , L a w r e n c e L u m e n g , G e o r g e Eckert, I n d i a n a U n i v e r s i t y School of Medicine, Indianapolis, IN; Brett Neustates, Brent Myers, A r t h u r B e r m a n , Brian H u d e s , D a v i d P o u n d , Richard Aycock BACKGROUND: Recent evidence suggests that adherence to therapy and effective m a n a g e m e n t of hepatitis C treatment side effects is associated with higher sustained response. The Aims of our study were 1 ) to evaluate efficacy of combination therapy with alia-interferon and ribavirin, and 2) to assess depression m a n a g e m e n t practices as functions of investigator treatment experience and practice. METHODS: Previously untreated hepatitis C patients were enrolled to receive alia-interferon 3 million units T I W with ribavirin 1000-1200 nag. H C V RNA testing was obtained at weeks 0, 12, 24, 48 and 72. Depression screening was performed using the Center For Epidemiology Studies Depression Scale (CES-D) at weeks 0, 4, 12,24, 36,and 48 during treatment. Depression was also assessed by the investigator at each clinic visit. Investigators were stratified for academic or community practice, and for n u m b e r of previous HCV patients treated. RESULTS: Six hundred eighty-eight subjects (277 female, 411 male, 72% genotype l, CES-D score 9.6--8.5) have been enrolled to receive treatment at 83 sites (8 Academic/VA, 75 community) thus far. Sixty-one percent (51/83) of investigators had treated < 1 0 0 H C V patients and 28% (23/83) of investigators have treated < 5 0 HCV patients prior to participating in this trial Prior to initiation of HCV treatment, 152/630 patients (24%) were taking antidepressants with a significantly higher percentage (30% vs 18%) of these patients being treated by investigators with > 1 0 0 HCV patients treated (98/325 vs 54/304, p<0.001). At w e e k 24, no significant change in CES-D scores were noted but there was an increase in n u m b e r of subjects taking anti-depressants compared to week 0 (week 0:152/630 (24%); week 24:62/140 (44%), p<0.001). Investigators who previously treated > HCV 100 patients were more likely to initiate anti-depressam therapy by week 24 of combination therapy than those w h o previously treated < 100 H C V patients (47/85 vs 15/55, p < 0 . 0 0 5 ) though CES-D scores were no different. There was no difference in the pattern of anti-depressant use between academic or c o m m u n i t y sites at any time point. At w e e k 24, 88/136 (65%) subjects had cleared HCV RNA with no differences seen in viral clearance rate in those who have previously treated 26-50, 51-100, or > 1 0 0 HCV patients and no differences observed in viral clearance rates between academic and community sites. Results of the study are still ongoing and will be updated for November. CONCLUSIONS: Combination therapy with interferon and ribavirin remains effective therapy for chronic hepatitis C, with equivalent viral clearance rates at w e e k 24 seen in investigators who have previously treated > 2 5 HCV patients. No differences in viral clearance rates or anti-depressant use were observed between academic and c o m m u n i t y sites at w e e k 24 of therapy. Investigators who have previously treated > HCV I00 patients are more likely to treat HCV patients already on antidepressant therapy and are more likely to initiate antidepressant therapy by week 24 of combination therapy.