- Email: [email protected]
The first caspase inhibitor of apoptosis, IDN-6556, given orally lowers liver enzymes in HCV patients
1326
LATE-BREAKING ABSTRACTS
DISTINCT LIPID RAFTS FUNCTION IN THE APICAL SORTING OF RAT BILE SALT EXPORT PUMP EXPRESSED IN MDCK CELLS. L. Wang, C.]. Soroka and ].L. Boyet, The Yale
Liver Center Background. The bile salt export p u m p (Bsep) represents the most important if not the sole bile salt export system at the canalicular/ apical m e m b r a n e of hepatocytes. The molecular mechanisms by which Bsep is apically sorted are not known. Recently, lipid rafts have been shown to function in the apical sorting of indirect pathway and multispanning t r a n s m e m b r a n e proteins such as Mdrl. Aims. In this study, we characterized the raft association of Bsep and the role of rafts in the apical sorting of Bsep. Methods. To follow the trafficking of Bsep, a Bsep-green fluorescent protein (Bsep-GFP) fusion construct was generated and expressed in MDCK cellsunder a tetracycline-repressible (Tet-off) promoter. The trafficking of Bsep-GFP was examined by switching on the expression of Bsep-GFP in MDCK cells for a short interval and then following the m o v e m e n t of intracellularly located Bsep-GFP using fluorescence microscopy. MDCK cells were depleted of cholesterol by treatment with lovastatin and cyclodextrin. To examine the raft association, MDCK cells were solubilized with Triton X-100 at 4°C. The solubility of Bsep-GFP and Mdrl was assessed by sucrose density gradient separation followed by Western blotting. Results. Using the Tet-off expression system, we followed the trafficking of Bsep-GFP from 0 to 4 hrs in MDCK cells. Almost all of the intracel]ularly located Bsep-GFP trafficked to the apical surface after 4 hrs and no Bsep-GFP was detected at the basolateral surface at earlier time points, suggesting that Bsep-GFP traffics in a direct fashion (without transcytosis) to the apical m e m b r a n e in MDCK cells. Cholesterol depletion caused a significant portion of Bsep-GFP to be redistributed to the junctional/lateral domains after 2 hrs, suggestive of mistargeting. After solubilization with Triton X-100 at 4°C,, a pattern paralleled by caveolin-1, a known raft resident protein at 4°C°C. 4°C suggests that Bsep-GFP may reside in lipid rafts. These lipid rafts may be distinct from those containing Mdrl, which were soluble to Triton X-100 at 4°C. Because depletion of cholesterol, a critical determinant of raft formation, led to re-distribution of Bsep-GFP to junctional/lateral domains in MDCK cells, raft mechanisms may facilitate the sorting of Bsep to the apical plasma m e m b r a n e domain.
PEG-INTERFERON ALFA-2B 1.5/~G/KG PLUS RIBAVIRIN 8001400MG/DAY FOR 24 WEEKS IN PATIENTS WITH HCV 2 OR
3. S Zeuzem, Saarland Univ, Homburg, Germany; R Hultcran~, Karolinska Hospital, Stockhohn, Sweden; M Bourliere, Hop St Joseph, Marseille, France; T Goeser, Univ Koeln, Koeln, Germany; P Marcellin, Hop Beaujon, Clichy, France; ] Sanchez-Tapias, Hop Clin I Provincial, Barcelona, Spain; ] Harvey, C Brass & J Albrecht, Schering Plough Research Instihde, Kenilworth, IV]. Objective: Compare safety and efficacy of PEG-Intron/Rebetol for 24 weeks with a historical control treated for 48 weeks (Manns, Lancet, 2002) with PEG-Intron/ Rebetol >10.6mg/kg. Methods: Treatment naive chronic HCV patients infected with genotype 2 or 3 received PEG-Intron 1.5/~g/kg subcutaneously once weekly plus Rebetol 800-1400 mg/day based on body weight for 24 weeks. Plasma HCV RNA was determined using quantitative PCR (TaqMan, sensitivity 29 IU/ml). Genotype was determined by sequencing the PCR product. Results: 224 patients randomized and 223 were treated.
HEPATOLOGY, Vol. 38, No. 5, 2003
EOT was 94% versus 95% (55/58) with 48 weeks treatment in the historical control. Using the Manns data, a model with known prognostic factors was used to estimate the SVR if the current subjects were treated for 48 weeks; the estimated SVR (84%) falls within the 95"/o confidence interval (76-86%) of the observed SV!R after 24 weeks. For this study, multivariate logistic regression and categorical regression tree analysis were performed. Baseline viral load (p=0.020), treatment duration > 16 weeks (p<0.001) and steatosis (<5%, p=0.015) were significant predictors of SVR. Weight was not a predictor and SVR was similar across all patienl weight ranges. Presence of steatosis was associated with HCV 3 (p=0.003), high baseline viral load (p=0.001) and baseline weight (p=0.002). Adverse events resulted in discontinuation in 5% of patients and dose reduction in 22% compared to 14"/o and 49%, respectively, in the historical control. Less than 1% reported SAE~ for depression or neutropenia. Conclusions: Treatment for 24 weeks with PEG-Intron/Rebetol in HCV 2/3 patients has similar efficacy but superior safety compared to 48 weeks in a historical control. The lower SVR with high viral load HCV 3 patient~ compared to low viral load HCV 3 and HCV 2 may be related t,~ high levels of steatosis in this population.
THE FIRST CASPASE INHIBITOR OF APOPTOSIS, IDN-6556, GIVEN ORALLY LOWERS LIVER ENZYMES IN HCV PATIENTS. P. Pockros, Scripps Clin; E. Schi~, U Miami; M.
Shiffinan, Med Coil Virginia; ]. McHutchison, A. Muir, Duke U; N. Afdhal, Beth Israel Med Cent; R. Gish, Cal Pacific Med Cent; M. Huyghe, T. Oltel~dmf, D. Shapiro, ldun Pharmaceuticals Increased rates of apoptosis (programmed cell death) have been implicated in several hepatic diseases including HCV, NASH, HBV, PBC and alcoholic hepatitis. IDN-6556 is a potent inhibitor of key caspases that mediate apoptosis. Methods: This study is a multicenter, ascending dose, double blind, placebo (PBO)-controlled evaluation of IDN-6556 administered orally in patients with hepatic impairment (ALT or AS']: 1.5-10 × ULN). The patients were dosed for 14 days and followed for 21 days after. Three groups of patients were studied: [A] 25 m~ (N = 6), [B] 100 mg (N = 7), [C] 200 mg (N = 6) or PBO (N = 6, across all 3 groups) all once daily. 24/25 patients had HCV hepatitis (both treatment ha'ire and nonresponders to interferon/ ribavirin therapy); 1 patient had NASH. Means (+_sem) for the baseline period were ALT: Placebo: 105 _+ 7, 25 mg: 142 - !4, 100 mg: 91 _+ 7, 200 mg: 168 -+ 16 and AST: Pbo: 58 + 4, 25 mg: 85 -+ 6, 100 rag: 49 -+ 3, 200 mg: 129 _+ 13 [U/L. Results: Efficacy--All three doses of drug lowered, but did not normalize, both ALT and AST (2-way ANOVA each Group vs. PBO % decreases from baseline: p < 0.0001). There was no discernable dose response relationship. At Day 14, ALT values were'. Placebo: 112 _+ 12, 25 rag: 81 -+ 10, 100 mg: 60 -+ 9, 200 mg: 105 : 20 and AST: Pbo: 61 + 7, 25 mg: 53 +- 6, 100 mg: 41 _+ 9, 200 mg: 86 - 16 IU/L. Safety--Adverse experiences were generally mild and brief; only dry mouth, headache and stomach ache were reported in >1 episode in treated patients; none were of significant ongoing clinical concern. No patient had a change in HCV RNA titers >0.5 log units and there were no other clinicall\ meaningful changes in laboratory parameters. Conclusions: Oral IDN-6556, 25-200 mg once daily, significant E lowered aminotransferases and appeared well tolerated.
20-
ALT: Mean % Change from Baseline Dosing • r
• Placebo
25 ~ QD 100 mg QD • 200 rng QD
10.
All patients HCV 2 HCV 3
End of Tx Response (EOT)
Sustained Virologic Response (SVR)
94% (211/224) 100% (42/42) 93% (169/182)
81% (182/224) 93% (39/42) 79% (143/182)
0-lo-20. -30. -40. -50
•
t
"/
~I
14
Day
2'1
2'8
3'5
LATE-BREAKING ABSTRACTS
DISTINCT LIPID RAFTS FUNCTION IN THE APICAL SORTING OF RAT BILE SALT EXPORT PUMP EXPRESSED IN MDCK CELLS. L. Wang, C.]. Soroka and ].L. Boyet, The Yale
Liver Center Background. The bile salt export p u m p (Bsep) represents the most important if not the sole bile salt export system at the canalicular/ apical m e m b r a n e of hepatocytes. The molecular mechanisms by which Bsep is apically sorted are not known. Recently, lipid rafts have been shown to function in the apical sorting of indirect pathway and multispanning t r a n s m e m b r a n e proteins such as Mdrl. Aims. In this study, we characterized the raft association of Bsep and the role of rafts in the apical sorting of Bsep. Methods. To follow the trafficking of Bsep, a Bsep-green fluorescent protein (Bsep-GFP) fusion construct was generated and expressed in MDCK cellsunder a tetracycline-repressible (Tet-off) promoter. The trafficking of Bsep-GFP was examined by switching on the expression of Bsep-GFP in MDCK cells for a short interval and then following the m o v e m e n t of intracellularly located Bsep-GFP using fluorescence microscopy. MDCK cells were depleted of cholesterol by treatment with lovastatin and cyclodextrin. To examine the raft association, MDCK cells were solubilized with Triton X-100 at 4°C. The solubility of Bsep-GFP and Mdrl was assessed by sucrose density gradient separation followed by Western blotting. Results. Using the Tet-off expression system, we followed the trafficking of Bsep-GFP from 0 to 4 hrs in MDCK cells. Almost all of the intracel]ularly located Bsep-GFP trafficked to the apical surface after 4 hrs and no Bsep-GFP was detected at the basolateral surface at earlier time points, suggesting that Bsep-GFP traffics in a direct fashion (without transcytosis) to the apical m e m b r a n e in MDCK cells. Cholesterol depletion caused a significant portion of Bsep-GFP to be redistributed to the junctional/lateral domains after 2 hrs, suggestive of mistargeting. After solubilization with Triton X-100 at 4°C,, a pattern paralleled by caveolin-1, a known raft resident protein at 4°C°C. 4°C suggests that Bsep-GFP may reside in lipid rafts. These lipid rafts may be distinct from those containing Mdrl, which were soluble to Triton X-100 at 4°C. Because depletion of cholesterol, a critical determinant of raft formation, led to re-distribution of Bsep-GFP to junctional/lateral domains in MDCK cells, raft mechanisms may facilitate the sorting of Bsep to the apical plasma m e m b r a n e domain.
PEG-INTERFERON ALFA-2B 1.5/~G/KG PLUS RIBAVIRIN 8001400MG/DAY FOR 24 WEEKS IN PATIENTS WITH HCV 2 OR
3. S Zeuzem, Saarland Univ, Homburg, Germany; R Hultcran~, Karolinska Hospital, Stockhohn, Sweden; M Bourliere, Hop St Joseph, Marseille, France; T Goeser, Univ Koeln, Koeln, Germany; P Marcellin, Hop Beaujon, Clichy, France; ] Sanchez-Tapias, Hop Clin I Provincial, Barcelona, Spain; ] Harvey, C Brass & J Albrecht, Schering Plough Research Instihde, Kenilworth, IV]. Objective: Compare safety and efficacy of PEG-Intron/Rebetol for 24 weeks with a historical control treated for 48 weeks (Manns, Lancet, 2002) with PEG-Intron/ Rebetol >10.6mg/kg. Methods: Treatment naive chronic HCV patients infected with genotype 2 or 3 received PEG-Intron 1.5/~g/kg subcutaneously once weekly plus Rebetol 800-1400 mg/day based on body weight for 24 weeks. Plasma HCV RNA was determined using quantitative PCR (TaqMan, sensitivity 29 IU/ml). Genotype was determined by sequencing the PCR product. Results: 224 patients randomized and 223 were treated.
HEPATOLOGY, Vol. 38, No. 5, 2003
EOT was 94% versus 95% (55/58) with 48 weeks treatment in the historical control. Using the Manns data, a model with known prognostic factors was used to estimate the SVR if the current subjects were treated for 48 weeks; the estimated SVR (84%) falls within the 95"/o confidence interval (76-86%) of the observed SV!R after 24 weeks. For this study, multivariate logistic regression and categorical regression tree analysis were performed. Baseline viral load (p=0.020), treatment duration > 16 weeks (p<0.001) and steatosis (<5%, p=0.015) were significant predictors of SVR. Weight was not a predictor and SVR was similar across all patienl weight ranges. Presence of steatosis was associated with HCV 3 (p=0.003), high baseline viral load (p=0.001) and baseline weight (p=0.002). Adverse events resulted in discontinuation in 5% of patients and dose reduction in 22% compared to 14"/o and 49%, respectively, in the historical control. Less than 1% reported SAE~ for depression or neutropenia. Conclusions: Treatment for 24 weeks with PEG-Intron/Rebetol in HCV 2/3 patients has similar efficacy but superior safety compared to 48 weeks in a historical control. The lower SVR with high viral load HCV 3 patient~ compared to low viral load HCV 3 and HCV 2 may be related t,~ high levels of steatosis in this population.
THE FIRST CASPASE INHIBITOR OF APOPTOSIS, IDN-6556, GIVEN ORALLY LOWERS LIVER ENZYMES IN HCV PATIENTS. P. Pockros, Scripps Clin; E. Schi~, U Miami; M.
Shiffinan, Med Coil Virginia; ]. McHutchison, A. Muir, Duke U; N. Afdhal, Beth Israel Med Cent; R. Gish, Cal Pacific Med Cent; M. Huyghe, T. Oltel~dmf, D. Shapiro, ldun Pharmaceuticals Increased rates of apoptosis (programmed cell death) have been implicated in several hepatic diseases including HCV, NASH, HBV, PBC and alcoholic hepatitis. IDN-6556 is a potent inhibitor of key caspases that mediate apoptosis. Methods: This study is a multicenter, ascending dose, double blind, placebo (PBO)-controlled evaluation of IDN-6556 administered orally in patients with hepatic impairment (ALT or AS']: 1.5-10 × ULN). The patients were dosed for 14 days and followed for 21 days after. Three groups of patients were studied: [A] 25 m~ (N = 6), [B] 100 mg (N = 7), [C] 200 mg (N = 6) or PBO (N = 6, across all 3 groups) all once daily. 24/25 patients had HCV hepatitis (both treatment ha'ire and nonresponders to interferon/ ribavirin therapy); 1 patient had NASH. Means (+_sem) for the baseline period were ALT: Placebo: 105 _+ 7, 25 mg: 142 - !4, 100 mg: 91 _+ 7, 200 mg: 168 -+ 16 and AST: Pbo: 58 + 4, 25 mg: 85 -+ 6, 100 rag: 49 -+ 3, 200 mg: 129 _+ 13 [U/L. Results: Efficacy--All three doses of drug lowered, but did not normalize, both ALT and AST (2-way ANOVA each Group vs. PBO % decreases from baseline: p < 0.0001). There was no discernable dose response relationship. At Day 14, ALT values were'. Placebo: 112 _+ 12, 25 rag: 81 -+ 10, 100 mg: 60 -+ 9, 200 mg: 105 : 20 and AST: Pbo: 61 + 7, 25 mg: 53 +- 6, 100 mg: 41 _+ 9, 200 mg: 86 - 16 IU/L. Safety--Adverse experiences were generally mild and brief; only dry mouth, headache and stomach ache were reported in >1 episode in treated patients; none were of significant ongoing clinical concern. No patient had a change in HCV RNA titers >0.5 log units and there were no other clinicall\ meaningful changes in laboratory parameters. Conclusions: Oral IDN-6556, 25-200 mg once daily, significant E lowered aminotransferases and appeared well tolerated.
20-
ALT: Mean % Change from Baseline Dosing • r
• Placebo
25 ~ QD 100 mg QD • 200 rng QD
10.
All patients HCV 2 HCV 3
End of Tx Response (EOT)
Sustained Virologic Response (SVR)
94% (211/224) 100% (42/42) 93% (169/182)
81% (182/224) 93% (39/42) 79% (143/182)
0-lo-20. -30. -40. -50
•
t
"/
~I
14
Day
2'1
2'8
3'5