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The prenylation inhibitor BZA-5B prevents virus particle assembly
AASLDA1247
April 1998 blockade of G2/M cell cycle phase (due to vinorelbine). The above results might be of clinical significance in the future. • L0198 THE PRENYLATION INHIBITOR BZA-SB PREVENTS VIRUS PARTICLE ASSEMBLY. J,S. Glenn. J.C. Marsters, Jr., H.B. Greenberg. Stanford University Medical School and Veterans Administration, Palo Alto, CA. The nuroose of this study was to evaluate the feasibility of a novel type of antiviral therapy based on prenylation inhibition. Prenylation is a form of sitespecific lipid modification of proteins. Large delta antigen of hepatitis delta virus (HDV) was the first viral protein shown to undergo prenylation, but numerous other viruses may also have prenylated proteins. Abolishing large delta antigen prenylation by genetic mutation of its prenylation site prevents assembly of HDV virus-like particles. We therefore wished to test th__ge hypothesis that pharmacologic inhibition of large delta antigen prenylation can prevent HDV assembly. Methods: First, we created a cell culture model of HDV assembly. Second, we tested the effect of a prenylation inhibitor, BZA-5B, on large delta antigen prenylation in reticulocyte lysates. Finally, we then tested the effect of BZA-5B on HDV virus-like particle production in the cell culture model. Results: HDV virus-like particles were assembled and released into the media of our cell culture model. BZA-5B was shown to be a potent inhibitor of large delta antigen prenylation in vitro at micromolar concentrations. When tested in our cell culture model, BZA-5B was also shown to specifically inhibit the prenylation-dependent production of HDV virus-like particles in a dosedependent manner. By 50 micromolar BZA-5B, no particles could be detected. Controls for non-specific inhibition of protein synthesis and secretion showed essentially no effect of BZA-5B. We conclude: BZA-5B is an effective inhibitor of large delta antigen prenylation. The prenylation-inhibiting activity of BZA-5B can abolish HDV virus-like particle production. Prenylation inhibitors, like BZA-5B, represent a novel class of potential antiviral agents for use against HDV and similarly prenylated viruses. • L0199 QUALITY OF LIFE AFTER LIVER TRANSPLANTATION IN PATIENTS WITH HEPATITIS C. JS Goff. RF Hamman, FS Wamboldt, GT Everson, R Shrestha, I Kam, M Wachs, DC Lezotte, T Steinberg, M Talamantes, BM Bilir. Section of Hepatology and Division of Transplant Surgery at University of Colorado Health Sciences Center, Denver, CO. Back~,round: The literature consistently shows that Quality of Life (QOL) is improved after liver transplantation (OLTX). However, recent data suggests that some subset(s) of patients actually report worsened QOL (Liver Transpl Surg, 1997; 3(2)). The specific aim of this study is to assess whether there is a difference in QOL following OLTX in patients transplanted with HCV vs. those transplanted for other diseases. Methods: We utilized the Liver Transplant Database instrument to measure disease-specific QOL in patients who were transplanted at UCHSC between 8/1/95 and 8/1/96. The questionnaire was administered at the time of listing and one year from the date of transplantation. Only those who were unable to complete the questionnaire or undergoing multiorgan transplantation were excluded. Patients were followed prospectively and data regarding potential confounders such as number of liver biopsies and number of OKT3 treatments were collected concurrently. Response Scores were summed over each of five QOL domains and the score change between the pre-OLTX and post-OLTX administrations was calculated and repeated measures ANOVA was performed for each domain. Score changes were compared between the HCV and non-HCV groups using the paired t-test. Additional validation of the study instrument included reliability testing and assessment of the internal consistency of the measure (Crohnbachls alpha). Results: The study population consisted of 40 patients, 42% of whom had HCV. Follow-up was obtained at one year for 75% (30) of the patients. The HCV and non-HCV groups differed significantly only in the number of liver biopsies performed. For the (Emeasures of disease 1 domain, the HCV group had greater improvement in their scores (p=.018), due to worse scores pre-OLTX (p=.39). Scores for the two groups post-OLTX were not different. Both groups showed significant improvement in the domains of (Epersonal functioning t, (Egeneral health perceptions 1 and (Erole functioning 1 (p<.0001). (EPsychologic status l was significantly worse for the HCV group (p=.022) and did not improve significantly for either group. No clear differences were seen in any domain scores between those HCV patients with a history of IV drug use and those without. The analysis of the questionnaire showed that it was very reliable, with no significant score change in any domain when administered to a subgroup of 20 pre-OLTX patients twice within a short (3 t o 4 month) time period. Additionally, Crohnbachls alpha showed very good internal consistency for the whole questionnaire (alpha=.8998) as well as for the individual domains, except for (Epersonal functioning 1 which showed acceptable consistency (alpha=.6016) only if employment status were deleted. Conclusions: Within the first year post-OLTX, patients with HCV appear to derive at least as much improvement in QOL as patients without HCV. Whether the scores remain similar beyond one year requires further investigation.
1,0200 OXIDIZED LOW DENSITY LIPOPROTEIN ACTIVATES LIPID SIGNALING PATHWAYS IN MACROPHAGES. A. Gomez-Mufioz, J. Martens, N. Reiner, and U.P. Steinbrecher. Department of Medicine, Univ. of British Columbia, Vancouver, B.C.
Oxidized LDL induces the expression of adhesion molecules, cytokines, chemotactic factors, tissue factor, and stimulates growth of smooth muscle cells and macrophages. The present studies were done to determine if lipid signaling pathways might be involved in these actions. Extensively oxidized LDL was shown to induce protein tyrnsine phosphorylation in phorbol esterpretreated THP-1 ceils, a human macrophage-like cell line. In these ceils, there was also activation of phosphatidylinositol (PI) 3-kinase, which is known to participate in several pathways involved in growth regulation. The growth stimulatory effect of oxidized LDL in primary murine macrophages was inhibited 40-50% by PI 3-kinase inhibitors. The effect on growth was also inhibited 70% by a platelet activating factor (PAF) receptor antagonist, suggesting that the activation of PI 3-kinase might be mediated through this receptor. Growth stimulation by oxidized LDL was not attributable to lysophosphatidylcholine (lysoPC) although lysoPC by itself was able to promote macrophage growth at higher concentrations than present in oxidized LDL. Oxidized LDL also caused a significant increase in phospholipase D (PLD) activity in macrophages. PLD can be activated by PAF through a complex pathway that involves G-proteins, PLC, ADP-ribosylation factor, and calcium. The activation of PLD by oxidized LDL was independent of phospholipase C and calcium, but was blocked by genistein (an inhibitor of tyrosine phosphorylation) and by PAF receptor antagonists. The effect of oxidized LDL on PLD was mimicked by lysoPC, but preventing the formation of lysoPC during LDL oxidation or removal of lysoPC from oxidized LDL did not affect activation of PLD. Activation of PLD generates phosphatidic acid, which can activate MAP kinases and stimulate cell proliferation directly or via its metabolites diacylglycerol and lysophosphatidate. Thus, we have shown that oxidized LDL can activate at least two lipid signaling pathways, and these may be important in mediating the effect of oxidized LDL on cell activation and growth. L0201 HIGH DOSE INDUCTION THERAPY WITH ALPHA INTERFERON IS EFFECTIVE IN PATIENTS WITH HIGH HEPATITIS C VIRAL LOAD. H.J. Gonzalez. S.B. Ho, J.B. Gross, C. Peine, D. McKee, T. Smith, and the Midwest Hepatitis Study Group. Veterans Affairs Medical Center and the University of Minnesota, Minneapolis, MN.
Following a single dose of interferon alpha 2b (IFN) HCV RNA levels are suppressed within 24 hrs, followed by a rise in viral levels by 48 hrs. AIM: To compare the effect on HCV RNA levels of daily IFN for 4 wks followed by TIW IFN, vs. standard TIW dosing. Methods: Multicenter, randomized trial with patients stratified by site, HCV genotype, viral levels, and degree of histologic fibrosis. Patients had biochemical and histologic evidence of chronic hepatitis and serum positive for HCV RNA. They were randomly assigned to: A. induction therapy (IFN 5 MU daily x 4 wks, followed by 5MU TIW x 44 wks), or B. non-induction therapy (IFN 5 MU TIW x 48 wks). HCV RNA levels were analyzed at 4 and 16 wks. Genotype and HCV levels were determined by National Genetics Institute. Results: Enrollment to date includes 115 patients from 12 study sites. In patients with high initial HCV levels (> 5,000,000 copies/ml) induction therapy resulted in 7/27 (26%) HCV RNA negative at 4 wks compared with 1/19 (5%) HCV RNA negative in the non-induction group (p=0.06). This tended to remain at 16 wks, with 6/14 (43%) HCV RNA negative in the induction group vs. 2/12 (17%) HCV RNA negative in the non-induction group (p=.14). Patients with initial stage 3-4 fibrosis receiving induction therapy resulted in 3/16 (18.7%) HCV RNA negative at 4 wks compared with 1/9 (11.1%) HCV RNA negative in the noninduction group. This trend was also noted at 16 wks, with 3/8 (37.5%) HCV RNA negative in the induction group vs. 1/7 (14.2%) HCV RNA negative in the non-induction group. No differences were noted in patients with low initial viral levels and genotype 1 and non-1 groups treated with induction or non-induction therapy. Patients HCV RNA negative at 16 wks: High HCV RNA Induction 6/14 (42.9%) Non-induction 2/12 (16.7%)
Low HCV RNA_ 4/13 (30.8%) 10/18 (55.5%)
0enotype .1_ 7/24 (29.1%) 8/25 (32.0%)
Genotype n0n-1 3/3 (100%) 4/5 (80.0%)
Stage 0-2 fibrosis 7/19 (36.8%) 11/23 (47.8%)
Stage$-4 fibrosi~ 3/8 (37.5%) If/ (14.2%)
Side effects requiring discontinuation of IFN occurred in 10/61 patients in the induction group compared with 2/54 patients in the non-induction group. Conclusion: After 16 wks of IFN treatment, patients with high initial titers of HCV RNA are more likely to become non-viremic if treated with an initial one month course of daily IFN therapy. This research was made possible in part by a grant from Schering-Plough.
April 1998 blockade of G2/M cell cycle phase (due to vinorelbine). The above results might be of clinical significance in the future. • L0198 THE PRENYLATION INHIBITOR BZA-SB PREVENTS VIRUS PARTICLE ASSEMBLY. J,S. Glenn. J.C. Marsters, Jr., H.B. Greenberg. Stanford University Medical School and Veterans Administration, Palo Alto, CA. The nuroose of this study was to evaluate the feasibility of a novel type of antiviral therapy based on prenylation inhibition. Prenylation is a form of sitespecific lipid modification of proteins. Large delta antigen of hepatitis delta virus (HDV) was the first viral protein shown to undergo prenylation, but numerous other viruses may also have prenylated proteins. Abolishing large delta antigen prenylation by genetic mutation of its prenylation site prevents assembly of HDV virus-like particles. We therefore wished to test th__ge hypothesis that pharmacologic inhibition of large delta antigen prenylation can prevent HDV assembly. Methods: First, we created a cell culture model of HDV assembly. Second, we tested the effect of a prenylation inhibitor, BZA-5B, on large delta antigen prenylation in reticulocyte lysates. Finally, we then tested the effect of BZA-5B on HDV virus-like particle production in the cell culture model. Results: HDV virus-like particles were assembled and released into the media of our cell culture model. BZA-5B was shown to be a potent inhibitor of large delta antigen prenylation in vitro at micromolar concentrations. When tested in our cell culture model, BZA-5B was also shown to specifically inhibit the prenylation-dependent production of HDV virus-like particles in a dosedependent manner. By 50 micromolar BZA-5B, no particles could be detected. Controls for non-specific inhibition of protein synthesis and secretion showed essentially no effect of BZA-5B. We conclude: BZA-5B is an effective inhibitor of large delta antigen prenylation. The prenylation-inhibiting activity of BZA-5B can abolish HDV virus-like particle production. Prenylation inhibitors, like BZA-5B, represent a novel class of potential antiviral agents for use against HDV and similarly prenylated viruses. • L0199 QUALITY OF LIFE AFTER LIVER TRANSPLANTATION IN PATIENTS WITH HEPATITIS C. JS Goff. RF Hamman, FS Wamboldt, GT Everson, R Shrestha, I Kam, M Wachs, DC Lezotte, T Steinberg, M Talamantes, BM Bilir. Section of Hepatology and Division of Transplant Surgery at University of Colorado Health Sciences Center, Denver, CO. Back~,round: The literature consistently shows that Quality of Life (QOL) is improved after liver transplantation (OLTX). However, recent data suggests that some subset(s) of patients actually report worsened QOL (Liver Transpl Surg, 1997; 3(2)). The specific aim of this study is to assess whether there is a difference in QOL following OLTX in patients transplanted with HCV vs. those transplanted for other diseases. Methods: We utilized the Liver Transplant Database instrument to measure disease-specific QOL in patients who were transplanted at UCHSC between 8/1/95 and 8/1/96. The questionnaire was administered at the time of listing and one year from the date of transplantation. Only those who were unable to complete the questionnaire or undergoing multiorgan transplantation were excluded. Patients were followed prospectively and data regarding potential confounders such as number of liver biopsies and number of OKT3 treatments were collected concurrently. Response Scores were summed over each of five QOL domains and the score change between the pre-OLTX and post-OLTX administrations was calculated and repeated measures ANOVA was performed for each domain. Score changes were compared between the HCV and non-HCV groups using the paired t-test. Additional validation of the study instrument included reliability testing and assessment of the internal consistency of the measure (Crohnbachls alpha). Results: The study population consisted of 40 patients, 42% of whom had HCV. Follow-up was obtained at one year for 75% (30) of the patients. The HCV and non-HCV groups differed significantly only in the number of liver biopsies performed. For the (Emeasures of disease 1 domain, the HCV group had greater improvement in their scores (p=.018), due to worse scores pre-OLTX (p=.39). Scores for the two groups post-OLTX were not different. Both groups showed significant improvement in the domains of (Epersonal functioning t, (Egeneral health perceptions 1 and (Erole functioning 1 (p<.0001). (EPsychologic status l was significantly worse for the HCV group (p=.022) and did not improve significantly for either group. No clear differences were seen in any domain scores between those HCV patients with a history of IV drug use and those without. The analysis of the questionnaire showed that it was very reliable, with no significant score change in any domain when administered to a subgroup of 20 pre-OLTX patients twice within a short (3 t o 4 month) time period. Additionally, Crohnbachls alpha showed very good internal consistency for the whole questionnaire (alpha=.8998) as well as for the individual domains, except for (Epersonal functioning 1 which showed acceptable consistency (alpha=.6016) only if employment status were deleted. Conclusions: Within the first year post-OLTX, patients with HCV appear to derive at least as much improvement in QOL as patients without HCV. Whether the scores remain similar beyond one year requires further investigation.
1,0200 OXIDIZED LOW DENSITY LIPOPROTEIN ACTIVATES LIPID SIGNALING PATHWAYS IN MACROPHAGES. A. Gomez-Mufioz, J. Martens, N. Reiner, and U.P. Steinbrecher. Department of Medicine, Univ. of British Columbia, Vancouver, B.C.
Oxidized LDL induces the expression of adhesion molecules, cytokines, chemotactic factors, tissue factor, and stimulates growth of smooth muscle cells and macrophages. The present studies were done to determine if lipid signaling pathways might be involved in these actions. Extensively oxidized LDL was shown to induce protein tyrnsine phosphorylation in phorbol esterpretreated THP-1 ceils, a human macrophage-like cell line. In these ceils, there was also activation of phosphatidylinositol (PI) 3-kinase, which is known to participate in several pathways involved in growth regulation. The growth stimulatory effect of oxidized LDL in primary murine macrophages was inhibited 40-50% by PI 3-kinase inhibitors. The effect on growth was also inhibited 70% by a platelet activating factor (PAF) receptor antagonist, suggesting that the activation of PI 3-kinase might be mediated through this receptor. Growth stimulation by oxidized LDL was not attributable to lysophosphatidylcholine (lysoPC) although lysoPC by itself was able to promote macrophage growth at higher concentrations than present in oxidized LDL. Oxidized LDL also caused a significant increase in phospholipase D (PLD) activity in macrophages. PLD can be activated by PAF through a complex pathway that involves G-proteins, PLC, ADP-ribosylation factor, and calcium. The activation of PLD by oxidized LDL was independent of phospholipase C and calcium, but was blocked by genistein (an inhibitor of tyrosine phosphorylation) and by PAF receptor antagonists. The effect of oxidized LDL on PLD was mimicked by lysoPC, but preventing the formation of lysoPC during LDL oxidation or removal of lysoPC from oxidized LDL did not affect activation of PLD. Activation of PLD generates phosphatidic acid, which can activate MAP kinases and stimulate cell proliferation directly or via its metabolites diacylglycerol and lysophosphatidate. Thus, we have shown that oxidized LDL can activate at least two lipid signaling pathways, and these may be important in mediating the effect of oxidized LDL on cell activation and growth. L0201 HIGH DOSE INDUCTION THERAPY WITH ALPHA INTERFERON IS EFFECTIVE IN PATIENTS WITH HIGH HEPATITIS C VIRAL LOAD. H.J. Gonzalez. S.B. Ho, J.B. Gross, C. Peine, D. McKee, T. Smith, and the Midwest Hepatitis Study Group. Veterans Affairs Medical Center and the University of Minnesota, Minneapolis, MN.
Following a single dose of interferon alpha 2b (IFN) HCV RNA levels are suppressed within 24 hrs, followed by a rise in viral levels by 48 hrs. AIM: To compare the effect on HCV RNA levels of daily IFN for 4 wks followed by TIW IFN, vs. standard TIW dosing. Methods: Multicenter, randomized trial with patients stratified by site, HCV genotype, viral levels, and degree of histologic fibrosis. Patients had biochemical and histologic evidence of chronic hepatitis and serum positive for HCV RNA. They were randomly assigned to: A. induction therapy (IFN 5 MU daily x 4 wks, followed by 5MU TIW x 44 wks), or B. non-induction therapy (IFN 5 MU TIW x 48 wks). HCV RNA levels were analyzed at 4 and 16 wks. Genotype and HCV levels were determined by National Genetics Institute. Results: Enrollment to date includes 115 patients from 12 study sites. In patients with high initial HCV levels (> 5,000,000 copies/ml) induction therapy resulted in 7/27 (26%) HCV RNA negative at 4 wks compared with 1/19 (5%) HCV RNA negative in the non-induction group (p=0.06). This tended to remain at 16 wks, with 6/14 (43%) HCV RNA negative in the induction group vs. 2/12 (17%) HCV RNA negative in the non-induction group (p=.14). Patients with initial stage 3-4 fibrosis receiving induction therapy resulted in 3/16 (18.7%) HCV RNA negative at 4 wks compared with 1/9 (11.1%) HCV RNA negative in the noninduction group. This trend was also noted at 16 wks, with 3/8 (37.5%) HCV RNA negative in the induction group vs. 1/7 (14.2%) HCV RNA negative in the non-induction group. No differences were noted in patients with low initial viral levels and genotype 1 and non-1 groups treated with induction or non-induction therapy. Patients HCV RNA negative at 16 wks: High HCV RNA Induction 6/14 (42.9%) Non-induction 2/12 (16.7%)
Low HCV RNA_ 4/13 (30.8%) 10/18 (55.5%)
0enotype .1_ 7/24 (29.1%) 8/25 (32.0%)
Genotype n0n-1 3/3 (100%) 4/5 (80.0%)
Stage 0-2 fibrosis 7/19 (36.8%) 11/23 (47.8%)
Stage$-4 fibrosi~ 3/8 (37.5%) If/ (14.2%)
Side effects requiring discontinuation of IFN occurred in 10/61 patients in the induction group compared with 2/54 patients in the non-induction group. Conclusion: After 16 wks of IFN treatment, patients with high initial titers of HCV RNA are more likely to become non-viremic if treated with an initial one month course of daily IFN therapy. This research was made possible in part by a grant from Schering-Plough.