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[131] PHARMACOKINETICS OF TACROLIMUS AND MYCOPHENOLATE MOFETIL (MMF) IN LIVE DONOR AND DECEASED DONOR LIVER TRANSPLANT RECIPIENTS
POSTERS
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11311 PHARMACOKINETICS OF TACROLIMUS AND MYCOPHENOLATE MOFETIL (MMF) IN LIVE DONOR AND DECEASED DONOR LIVER TRANSPLANT RECIPIENTS A. Jain’, R. Venkatramanan*, T. Kwong3, M. Orloff’, P. Abt’, R. Kashyap’ , G. Tsoulfas’, C. Mack’, P. Batzol’, M. Williamson’, A. Bozorgzadeh’ . ‘Department of Surgery URMC Rochexter; ’School o f Pharnzacy Pittsburgh; ”Department of Biochemistry Rochester, 1iSA E-mail: [email protected] While in deceased donor liver transplantation (DDLT) recipients receive the whole organ, live donor liver transplantations (LDLT) receives only about 55-60% of the hepatic volume in adults. The impact of partial hepatic volume on pharmacokinetic on the exposure of MPA and bioavailability of Tacrolimus in LDLT has not been studied. The aim is to compare the pharmacolcinetics parameters of MMF and tacrolimus in LDLT vs. DDLT. Patients and Methods: Twelve consenting LDLT and 12 DDLT patients received 1Gm MMF intravenously twice a day and oral tacrolimus 0.04&0.005 mgikg twice a day to achieve tacrolimus trough level of 6 to 10 ngiml. Multiple blood samples were collected on second post operative day throughout the MMF dosing interval and concentrations of MPA were measured. Also during the steady dose of tacrolimus, multiple blood samples were collected, to measure the tacrolimus concentration. Results: MMF Kinetic’s: Maximum concentration of MPA and area under the curve concentration (AUC) in LDLT were significantly higher than DDLT (16.1&6.6pgimL LDLT vs. 10.7f2.4pg/mL in DDLT; p=0.046; 43.9&12.6 h*pgimL LDLT vs. 28.9&7.3 h*pgimL in DDLT; p=0.002 respectively). However mean C12 concentrations, drug disposition rate constant; half-life and mean residence time were similar. Tacrolimus Kinetics: The mean AUC of tacrolimus was significantly higher in LDLT in compared to DDLT The dose normalized AUC 48.76+21.74 ng*h/mlimg for LDLT vs. 35.07 + 25.27 ng*h/ml/mg. (p=0.026). C-last was 7.21+1.81 ngiml LDLT vs. 4.87 + I.6IDDLT ngiml (p=0.019). The mean clearance of the drug (CLss-F-mlihr) was lower in LDLT (24.2+10.2 mlih) compared to DDLT (47.3+35.7 mlih) (p = 0.096). Mean daily Tacrolimus dose was significantly lower in LDLT compared to DDLT. (p=0.0001). The mean trough concentration of tacrolimus were significantly higher in LDLT (8.7f2.5 ngiml) compared to DDLT (6.6+lngiml) (p=0.013). Conclusion: Reduced hepatic volume significantly impairs the metabolism of MMF and tacrolimus. In LDLT recipients over immunosuppression may be avoided by preemptively starting with much smaller dose of MMF and tacrolimus compared to DDLT.
11321 NUMBER OF HUMAN CELL CLUSTERS AFTER 24 HOURS PREDICTS OUTCOME OF HUMAN LIVER CELL TRANSPLANTATION EFFICACY IN THE uPA MOUSE SYSTEM M. Konig’, T. Volz’, M. Liitgehetmann’, J.M. Pollok2, L. Fische?, M. Dandri’ , J. Petersen’ . ‘Departnzent of Medicine, University Hospital
Hamb~irg-EI)pend~~f,. Hamhurg: ’Department of Hepatohiliury Surgery And Trunspluntation, Uniuer,sityhosI)italHumhLirg-EpI)endo~f,’ Hamhurg, Gernzany E-mail: [email protected] Background and Aim: Hepatocellular transplantation may treat or bridge liver diseases in the future. To elucidate engraftment efficiency and proliferative capacity of primary human hepatocytes, we performed xenotransplantation experiments using the uPA (urokinase-type plasminogen activator) mouse model of liver regeneration. Methods and Results: Primary human hepatocytes (HH) were isolated from remnants reduced size livers and transplanted ( 1 x 1 O6 cells; >SO% viability) into scidibeige uPA mice via splenic injection. Efficiency of engraftment was determined by immunohistochemistry using human-specific
cytolceratin 18 antibodies at 3hr, 24hr and I week after transplantation. Although approximately 20% of injected hepatocytes were found in the mouse liver 3hr after transplantation, we observed strong reduction (70%) of transplanted HH in the first 24 hours. By performing morphometric analysis of mouse liver sections, we found a median of 21 and 6 cell clusters per field at 3 and 24 hours after transplantation, respectively, which mainly consisted of single cell clusters. One week after transplantation, all surviving clusters had started to expand (median 5 cellsicluster), which was also demonstrated by Ki76 staining. The presence of median 62 and 244 cells per cluster after one and two months, respectively, indicated a >230 fold increase in cell cluster area, leading to >20% liver repopulation in those mice. Taken into account that most ofthe HH clusters were monoclonal, we calculated that each cell underwent at least 6 to 8 cell doublings. By transplanting hepatocytes isolated from different livers and species (humanitupaia), we found that the most critical parameter determining high repopulation levels was the efticiency of engraftment estimated one day after transplantation, while proliferative capacity remained similar. Detection of specific markers such as human albumin, P450, transcription factor HNF4 by immunohistochemistry and confocal microscopy, as well as formation of bile canaliculi, tight and gap junctions between murine and human hepatocytes, indicated well preserved liver specitic functions and integration of human hepatocytes within the murine environment. Conclusions: Despite massive reduction of initial hepatocyte engraftment, HH retained a high proliferative capacity within the mouse liver and maintained proper hepatocyte specific metabolic activity. The number of cell clusters after 24 hrs predicts outcome in respect to repopulation etfic acy.
11331 PERSISTENT EXPRESSION OF MESENCHYMAL MARKERS AFTER HEPATOCYTE DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS P.A. Lvsv, M. Najimi, D. Campard, F. Smets, E.M. Sokal. Laboratory of
Pediatric Hepatology and Cell Therapy, linioersitd Catholique de Louoain and Cliniques Saint Luc, Brussels, Belgium E-mail: [email protected] Background and Aims: Previous studies indicated the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte-like lineage and considered these cells as a candidate for liver cell therapy. We explored further the maturation state of mesenchymal-derived hepatocytelike cells (MDHLCs): modification of the initial phenotype and acquisition of mature hepatocyte features. We also investigated the expression of hepatocytic and mesenchymal markers in vivo after transplantation of these cells into SCTD mice. Methods: Mesenchymal phenotype of bone marrow MSCs samples was attested by flow cytometry, immunocytochemistry and by the ability to differentiate into osteocytes and adipocytes. These cells were subjected to hepatocyte differentiation with a specitic protocol including an induction and a maturation step. Acquisition of hepatocytic phenotype was evaluated by analysis of morphology, hepatic markers expression (protein and RNA levels) and metabolic assays. The expression of mesenchymal markers in MDHLCs was evaluated by flow cytometry and immunocytochemistry. We also examined the expression of hepatic and mesodermal markers in MSCs and MDHLCs transplanted into partially hepatectomized SCID mice. Results: MDHLCs acquired morphological hepatocyte-like features as attested by optic and electron microscopy. They expressed liver-specific markers de novo both on protein and RNA levels. These cells were able to store glycogen and secrete small amounts of urea but did not present glucose-6-phosphatase or gluconeogenesis activities. Noteworthy, MDHLCs displayed persistent expression of MSCs mesodermal markers: vimentin, ASMA, fibronectin and laminin. Flow cytometry confirmed the conserved expression of initial phenotype while some markers were down-regulated (CD44, CD73, CD105 and CD29). Comparison of the immunophenotype of MDHLCs with mature human hepatocytes indicated that the modification of epitopes expression tempted these cells to resemble
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11311 PHARMACOKINETICS OF TACROLIMUS AND MYCOPHENOLATE MOFETIL (MMF) IN LIVE DONOR AND DECEASED DONOR LIVER TRANSPLANT RECIPIENTS A. Jain’, R. Venkatramanan*, T. Kwong3, M. Orloff’, P. Abt’, R. Kashyap’ , G. Tsoulfas’, C. Mack’, P. Batzol’, M. Williamson’, A. Bozorgzadeh’ . ‘Department of Surgery URMC Rochexter; ’School o f Pharnzacy Pittsburgh; ”Department of Biochemistry Rochester, 1iSA E-mail: [email protected] While in deceased donor liver transplantation (DDLT) recipients receive the whole organ, live donor liver transplantations (LDLT) receives only about 55-60% of the hepatic volume in adults. The impact of partial hepatic volume on pharmacokinetic on the exposure of MPA and bioavailability of Tacrolimus in LDLT has not been studied. The aim is to compare the pharmacolcinetics parameters of MMF and tacrolimus in LDLT vs. DDLT. Patients and Methods: Twelve consenting LDLT and 12 DDLT patients received 1Gm MMF intravenously twice a day and oral tacrolimus 0.04&0.005 mgikg twice a day to achieve tacrolimus trough level of 6 to 10 ngiml. Multiple blood samples were collected on second post operative day throughout the MMF dosing interval and concentrations of MPA were measured. Also during the steady dose of tacrolimus, multiple blood samples were collected, to measure the tacrolimus concentration. Results: MMF Kinetic’s: Maximum concentration of MPA and area under the curve concentration (AUC) in LDLT were significantly higher than DDLT (16.1&6.6pgimL LDLT vs. 10.7f2.4pg/mL in DDLT; p=0.046; 43.9&12.6 h*pgimL LDLT vs. 28.9&7.3 h*pgimL in DDLT; p=0.002 respectively). However mean C12 concentrations, drug disposition rate constant; half-life and mean residence time were similar. Tacrolimus Kinetics: The mean AUC of tacrolimus was significantly higher in LDLT in compared to DDLT The dose normalized AUC 48.76+21.74 ng*h/mlimg for LDLT vs. 35.07 + 25.27 ng*h/ml/mg. (p=0.026). C-last was 7.21+1.81 ngiml LDLT vs. 4.87 + I.6IDDLT ngiml (p=0.019). The mean clearance of the drug (CLss-F-mlihr) was lower in LDLT (24.2+10.2 mlih) compared to DDLT (47.3+35.7 mlih) (p = 0.096). Mean daily Tacrolimus dose was significantly lower in LDLT compared to DDLT. (p=0.0001). The mean trough concentration of tacrolimus were significantly higher in LDLT (8.7f2.5 ngiml) compared to DDLT (6.6+lngiml) (p=0.013). Conclusion: Reduced hepatic volume significantly impairs the metabolism of MMF and tacrolimus. In LDLT recipients over immunosuppression may be avoided by preemptively starting with much smaller dose of MMF and tacrolimus compared to DDLT.
11321 NUMBER OF HUMAN CELL CLUSTERS AFTER 24 HOURS PREDICTS OUTCOME OF HUMAN LIVER CELL TRANSPLANTATION EFFICACY IN THE uPA MOUSE SYSTEM M. Konig’, T. Volz’, M. Liitgehetmann’, J.M. Pollok2, L. Fische?, M. Dandri’ , J. Petersen’ . ‘Departnzent of Medicine, University Hospital
Hamb~irg-EI)pend~~f,. Hamhurg: ’Department of Hepatohiliury Surgery And Trunspluntation, Uniuer,sityhosI)italHumhLirg-EpI)endo~f,’ Hamhurg, Gernzany E-mail: [email protected] Background and Aim: Hepatocellular transplantation may treat or bridge liver diseases in the future. To elucidate engraftment efficiency and proliferative capacity of primary human hepatocytes, we performed xenotransplantation experiments using the uPA (urokinase-type plasminogen activator) mouse model of liver regeneration. Methods and Results: Primary human hepatocytes (HH) were isolated from remnants reduced size livers and transplanted ( 1 x 1 O6 cells; >SO% viability) into scidibeige uPA mice via splenic injection. Efficiency of engraftment was determined by immunohistochemistry using human-specific
cytolceratin 18 antibodies at 3hr, 24hr and I week after transplantation. Although approximately 20% of injected hepatocytes were found in the mouse liver 3hr after transplantation, we observed strong reduction (70%) of transplanted HH in the first 24 hours. By performing morphometric analysis of mouse liver sections, we found a median of 21 and 6 cell clusters per field at 3 and 24 hours after transplantation, respectively, which mainly consisted of single cell clusters. One week after transplantation, all surviving clusters had started to expand (median 5 cellsicluster), which was also demonstrated by Ki76 staining. The presence of median 62 and 244 cells per cluster after one and two months, respectively, indicated a >230 fold increase in cell cluster area, leading to >20% liver repopulation in those mice. Taken into account that most ofthe HH clusters were monoclonal, we calculated that each cell underwent at least 6 to 8 cell doublings. By transplanting hepatocytes isolated from different livers and species (humanitupaia), we found that the most critical parameter determining high repopulation levels was the efticiency of engraftment estimated one day after transplantation, while proliferative capacity remained similar. Detection of specific markers such as human albumin, P450, transcription factor HNF4 by immunohistochemistry and confocal microscopy, as well as formation of bile canaliculi, tight and gap junctions between murine and human hepatocytes, indicated well preserved liver specitic functions and integration of human hepatocytes within the murine environment. Conclusions: Despite massive reduction of initial hepatocyte engraftment, HH retained a high proliferative capacity within the mouse liver and maintained proper hepatocyte specific metabolic activity. The number of cell clusters after 24 hrs predicts outcome in respect to repopulation etfic acy.
11331 PERSISTENT EXPRESSION OF MESENCHYMAL MARKERS AFTER HEPATOCYTE DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS P.A. Lvsv, M. Najimi, D. Campard, F. Smets, E.M. Sokal. Laboratory of
Pediatric Hepatology and Cell Therapy, linioersitd Catholique de Louoain and Cliniques Saint Luc, Brussels, Belgium E-mail: [email protected] Background and Aims: Previous studies indicated the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte-like lineage and considered these cells as a candidate for liver cell therapy. We explored further the maturation state of mesenchymal-derived hepatocytelike cells (MDHLCs): modification of the initial phenotype and acquisition of mature hepatocyte features. We also investigated the expression of hepatocytic and mesenchymal markers in vivo after transplantation of these cells into SCTD mice. Methods: Mesenchymal phenotype of bone marrow MSCs samples was attested by flow cytometry, immunocytochemistry and by the ability to differentiate into osteocytes and adipocytes. These cells were subjected to hepatocyte differentiation with a specitic protocol including an induction and a maturation step. Acquisition of hepatocytic phenotype was evaluated by analysis of morphology, hepatic markers expression (protein and RNA levels) and metabolic assays. The expression of mesenchymal markers in MDHLCs was evaluated by flow cytometry and immunocytochemistry. We also examined the expression of hepatic and mesodermal markers in MSCs and MDHLCs transplanted into partially hepatectomized SCID mice. Results: MDHLCs acquired morphological hepatocyte-like features as attested by optic and electron microscopy. They expressed liver-specific markers de novo both on protein and RNA levels. These cells were able to store glycogen and secrete small amounts of urea but did not present glucose-6-phosphatase or gluconeogenesis activities. Noteworthy, MDHLCs displayed persistent expression of MSCs mesodermal markers: vimentin, ASMA, fibronectin and laminin. Flow cytometry confirmed the conserved expression of initial phenotype while some markers were down-regulated (CD44, CD73, CD105 and CD29). Comparison of the immunophenotype of MDHLCs with mature human hepatocytes indicated that the modification of epitopes expression tempted these cells to resemble