Detoxifying activity in pig livers and hepatocytes intended for xenotherapy

BACKGROUND: Both livers and hepatocytes from pigs have been proposed for the treatment of end-stage liver diseases, as an alternative to allogeneic liver transplants. However, little is known of the capability of porcine hepatocytes to fulfill the biotransformation pathways of toxic compounds, including those released from livers in acute failure. We have studied the activity and expression of detoxifying enzymes in porcine livers and in cultured hepatocytes and their induction by phenobarbital. METHODS: Cytochromes P450 (CYP) 1A, 2B, and 3A and GST-like activities were tested with the following specific substrates: 7-ethoxyresorufin, 7-pentoxyresorufin, nifedipine, testosterone, 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and ethacrinic acid. CYP 1A1/2-, 2B1/2-, 2E1- and 3A4-related and GSTalpha proteins were analyzed by Western blotting and CYP 1A1/2, 2B1/2, 2C6, 2E1, and 3A4, aldehyde dehydrogenase, epoxide hydrolase, and GSTalpha-like RNA by Northern blotting. RESULTS: Enzymatic activities reflecting the expression of CYP 1A-, CYP 2B-, CYP 2E1-, and CYP 3A-like genes, that is, ethoxyresorufin-O-deethylase, pentoxyresorufin-O-deethylase, nifedipine oxidase and testosterone 6beta-hydroxylase, and chlorzoxazone 6-hydroxylase, were identified in pig livers. CYP 1A and CYP 2E1, GSTalpha-like proteins, CYP 1A, 2C, and 2E, epoxide hydrolase, aldehyde dehydrogenase, and GST like RNA were expressed in vivo and in vitro. CYP 2B and CYP 3A RNA and proteins, and their associated activities were induced by phenobarbital. CONCLUSIONS: Porcine hepatocytes express the most important biotransformation enzymes and their corresponding activities and RNA. Thus, livers and hepatocytes from pigs can detoxify a large spectrum of exogenous and endogenous compounds, which makes them a convenient substitute for allogeneic transplants for patients with liver failure.     

Peritoneal implantation of cryopreserved encapsulated porcine hepatocytes in rats without immunosuppression: viability and function

The bioartificial liver (BAL) represents a promising approach to cell transplantation without immunosuppression as a method to support patients with hepatic insufficiency. The aim of this study was to assess viability and function of cryopreserved encapsulated porcine hepatocytes implanted intraperitoneally in rats without immunosuppression. Isolated porcine hepatocytes were cryopreserved at -196 degrees C for 1 month. Four groups were created: group 1 (n=10), freshly encapsulated porcine hepatocytes cultured in albumin-free medium for 10 days; group 2 (n=10), freshly encapsulated porcine hepatocytes implanted in the rat peritoneum without immunosuppression for 1 month and cultured for 10 days after explantation; group 3 (n=10), cryopreserved encapsulated porcine hepatocytes cultured for 10 days; group 4 (n=10), cryopreserved encapsulated porcine hepatocytes implanted in the rat peritoneum without immunosuppression for 1 month and cultured for 10 days after explantation. We assessed urea and albumin production and hepatocyte viability. The hepatocytes of all groups retained the capacity to produce urea and albumin, although the albumin synthesis was significantly decreased among hepatocytes of group 4 (P< .01). Encapsulated cryopreserved porcine hepatocytes explanted from rat peritoneum after 1 month appeared morphologically viable; their ultrastructure was preserved. In conclusion, long-term cryopreservation of porcine hepatocytes resulted in retention of their biological activity and in significant viability when transplanted into the rat peritoneum without immunosuppression.     

Clinical versatility of porcine hepatocytes in the light of interspecies differences in cytochrome P450 regulation and expression

Abstract: Background and aims: In fulminant hepatic failure, the clinical use of bioartifical liver support with porcine hepatocytes is the subject of a controversial debate. Cytochrome P450 (CYP) metabolic functions have relevant implications for drug metabolism and detoxification. In this study, we investigate interspecies differences in CYP gene expression between human and porcine primary hepatocytes and the impact of interleukin 6 (IL‐6) and tumor necrosis factor alpha (TNF‐α) exposition mimicking cytokine release in fulminant hepatic failure. Methods: Primary hepatocyte cultures were isolated from human resection specimens and from German landrace pigs. Cell cultures (single and co‐cultures) were exposed to porcine vs. human IL‐6 and TNF‐α, respectively. Changes in quantitative CYP gene expression were investigated by semi‐quantitative RT‐PCR. Results: Significant differences in species‐specific CYP gene expression by human and porcine hepatocytes were found after exposure to species‐identical IL‐6 (10 ng/ml) for CYP 1A1, CYP 2C, CYP 3A (P = 0.002, 0.022, 0.017, respectively) or species‐identical TNF‐α (30ng/ml) for CYP 1A2 and CYP 2A (P = 0.037, 0.023, respectively). In single vs. co‐culture, human hepatocytes demonstrated stronger repression of CYP 1A1, 2C8 and 3A4 expression after dosage with human IL‐6 (10ng/ml) (P = 0.022, 0.031, 0.014, respectively). Conclusion: Our findings demonstrate significant species‐specific differences in CYP gene expression and regulation when high doses of IL‐6 and TNF‐α are used (10 and 30 ng/ml, respectively). These findings may point to species‐specific physiological incompatibilities of porcine hepatocytes and thus limit their clinical versatility.     

A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37 degrees C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.     

Transplanted cryopreserved encapsulated porcine hepatocytes are as effective as fresh hepatocytes in preventing death from acute liver failure in rats

BACKGROUND: An implantable bioartificial liver (BAL) using xenogeneic isolated hepatocytes may be an alternative method to orthotopic liver transplantation for treatment of acute liver failure. The purpose of this study was to demonstrate that not only fresh but also cryopreserved porcine hepatocytes could be used in a BAL to prevent death after the onset of acute liver failure in rats. METHODS: Acute liver failure was induced by two-stage 95% hepatectomy. At the time of completion of liver resection, 100 rats were assigned to undergo or not undergo transplantation into the peritoneum of 4 meters of hollow fibers filled with 60 million either fresh or cryopreserved porcine hepatocytes, or syngeneic hepatocytes, or culture medium, or of 60 million nonencapsulated cryopreserved porcine hepatocytes without immunosuppressive therapy. Survival rates at 7 days were compared between the different groups. RESULTS: In the control groups of hepatectomized animals not receiving encapsulated hepatocytes, 69-79% of the rats died from acute liver failure. The mortality rate was reduced to 15% (2 of 13) in rats receiving fresh porcine hepatocytes (P<0.01), 25% (4 of 16) in rats transplanted with either cryopreserved or syngeneic hepatocytes (P<0.05). Survival rates were maintained when hollow fibers were explanted > or =4 days after hepatectomy. In surviving rats, the weight of the remnant native liver increased with time and returned to the initial weight after 1 month. CONCLUSIONS: The implantable BAL using xenogeneic porcine hepatocytes was able in preventing death from acute liver failure without immunosuppressive therapy. Encapsulated cryopreserved hepatocytes were as effective as fresh hepatocytes.     

A simple and highly effective method for the stable transduction of uncultured porcine hepatocytes using lentiviral vector

Gene therapy is an attractive approach for the treatment of a wide spectrum of liver diseases. Lentiviral vectors allow the stable integration of transgenes into the genome of nondividing differentiated cells including hepatocytes and could provide long-lasting expression of a therapeutic gene. To develop such approaches, preclinical studies in large animal models such as pigs are necessary to evaluate the feasibility and safety of stable lentiviral integration and long-term vector expression. In addition, effective lentivector-mediated gene transfer onto porcine hepatocytes could advance in cell-based therapies for acute liver failure. To investigate this issue, porcine hepatocytes were transduced in suspension immediately after their isolation in University of Wisconsin (UW) solution containing vitamin E. Up to 80% of hepatocytes stably expressed a GFP transgene after a single exposure to lentiviral vector coding for GFP under the control of either liver-specific or ubiquitous promoters. Moreover, porcine hepatocytes cryopreserved in UW solution containing fetal bovine serum, dimethyl sulfoxide, and vitamin E remained highly transducible with lentiviral vector after thawing. When thawed, transduced in suspension, and immediately transplanted into the spleen of immunodeficient mice, ex vivo lentivirally transgene marked xenogeneic hepatocytes were detected in murine liver. We demonstrated that porcine hepatocytes are highly susceptible to lentiviral vector and describe an easy methodology to efficiently, rapidly, and stably introduce transgenes into uncultured porcine hepatocytes.     

Long-term function of cryopreserved rat hepatocytes in a coculture system

The goal of this study was to investigate postpreservation long-term function of cryopreserved primary rat hepatocytes using the hepatocyte/3T3-J2 fibroblast coculture system. The long-term function of thawed hepatocytes cocultured with fibroblasts was evaluated and compared with hepatocytes cultured without fibroblasts. Fresh isolated primary rat hepatocytes were frozen at a controlled rate (-1 degrees C/min) up to -80 degrees C, and then stored in liquid nitrogen for up to 90 days. Thawed hepatocytes were thereafter cocultured with 3T3-J2 murine fibroblasts and cocultivation was monitored for 14 days. The viability of fresh isolated hepatocytes was 91.4%, and that of cryopreserved hepatocytes was 82.1%. Cellular morphology and polarity, which were determined by the localization of actin filaments and connexin-32, were successfully maintained in cryopreserved hepatocytes following cryopreservation. Albumin and urea synthesis reached the maximum level and became stable after day 7 in coculture in both fresh and cryopreserved hepatocytes. Urea synthesis of cryopreserved hepatocytes was maintained 89.0% of nonfrozen fresh control, and albumin production of cryopreserved hepatocytes was 63.7% of control in coculture. Cytochrome P450 activity, which was measured by deethylation of ethoxyresorufin, was also maintained in cryopreserved hepatocytes at 88.6% of nonfrozen fresh control in coculture. The retention of synthetic and detoxification activities was verified to be well preserved during extended low-temperature storage (90 days). Both fresh control and cryopreserved hepatocytes cultured without fibroblast did not retain their synthetic and detoxification functions in long-term culture. These data illustrate that, through the utilization of our cryopreservation procedure, primary hepatocyte function was successfully maintained when placed into coculture configuration following thawing.     

Effect of spheroid aggregation on susceptibility of primary pig hepatocytes to cryopreservation

Bioartificial liver support systems, which use freshly isolated primary hepatocytes (PH), present severe logistical difficulties. Stored frozen PH that are thawed as required could answer this problem. The aim of this study was to develop a cryopreservation protocol for large-scale preparation of porcine PH. We cryopreserved single and spheroid hepatocytes. Harvested hepatocytes were cryopreserved in various concentrations of dimethyl sulfoxide (DMSO) using hormonally defined medium (HDM) and various presentation solutions, such as University of Wisconsin (UW) and fetal bovine serum. After thawing the hepatocytes, we measured the viability, plating efficiency, ammonia removal, urea synthesis, and albumin secretion. UW solution was most effective for cryopreservation, as evidenced by the viability and liver-specific functions of the thawed hepatocytes. The optimal DMSO concentration for porcine hepatocyte cryopreservation was 15%. After cryopreservation, spheroid hepatocytes maintained greater viability and functional activity compared with single hepatocytes. Moreover, spheroid hepatocytes showed native cell structures and maintained high levels of liver-specific functions. In conclusion, cryopreserved spheroid hepatocytes were superior to cryopreserved single hepatocytes in terms of viability and liver-specific functions.     

Hepatocyte function during experimental use of a bioartificial liver

The aim of the present study was to compare the function of fresh versus cryopreserved hepatocytes in an experimental bioartificial liver system (BAL), especially designed to reproduce clinical parameters. Our BAL consists of a pump, a plasma reservoir, a membrane oxygenator, and a hollow fiber module loaded with 5 x 10(9) isolated porcine hepatocytes, either fresh (n = 5) or cryopreserved (n = 5). In the present setting, the system was isolated and perfused for 6 hours with recirculating plasma obtained from pigs with ischemic liver failure (toxic plasma). The following parameters were studied at 0 and 6 hours: oxygen consumption by the hepatocytes in the bioreactor, hepatocyte viability, as well as plasma concentrations of AST, LDH, ammonia, urea, and total bilirubin. MEGX concentrations were measured following injection of lidocaine into the system 30 minutes after initiation of plasma recirculation. Compared to cryopreserved cells, fresh hepatocytes showed higher viability at both time points studied (P <.05). Furthermore, during BAL sessions, ammonia levels were reduced while urea, AST, and LDH levels were increased with both preservation types (P <.05). Total bilirubin levels increased only during sessions with cryopreserved hepatocytes. After lidocaine administration, both fresh and cryopreserved hepatocytes were capable of producing MEGX; however, fresh-cell bioreactors produced significantly more MEGX at both 30 and 60 minutes after lidocaine administration. Oxygen consumption was significantly higher by fresh-cell bioreactors both before and after BAL use. In conclusion, hepatocytes in the BAL bioreactor showed preservation of important metabolic functions, when perfused with homologous toxic plasma. Fresh cells appeared to respond better than did cryopreserved ones.     

Long-term maintenance of the drug transport activity in cryopreservation of microencapsulated rat hepatocytes

Transplantation of isolated hepatocytes has been proposed to compensate for essential functions lacking in liver failure or for genetic defects that alter a specific liver metabolic pathway. Hepatocyte utilization for these purposes would be facilitated with a reliable, reproducible, and effective method of long-term hepatocyte storage. We have recently developed a simple new system for cryopreservation of hepatocytes that encapsulates alginate microspheres and maintains liver-specific function. The aim of this study was to elucidate the transport and drug-metabolizing enzyme activities of cryopreserved microencapsulated hepatocytes stored for a long time. Morphological examinations showed there is no apparent injury of the hepatocytes during cryopreservation processes. A drug-metabolizing enzyme (testosterone 6beta-hydroxylase, a specific probe for CYP3A2) and drug transport activities [salicylate, allopurinol, and prostaglandin E2 (PGE2), typical substrates of rOat2] in cryopreserved microencapsulated hepatocytes were maintained up to 120 days. Our results thus demonstrate for the first time that cryopreservation of primary rat hepatocytes by the encapsulation technique allows long-term retention of drug metabolism and drug transport activities.     

Cryopreservation of primarily isolated porcine hepatocytes with UW solution

Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at -80 degrees C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.     

Physiological incompatibilities of porcine hepatocytes for clinical liver support.

In fulminant hepatic failure, the use of bioartificial liver support (BAL) with porcine hepatocytes is the subject of a current and controversial debate.1 Specifically, the issue of cross-species physiological incompatibilities has not been addressed so far. We therefore investigated the effects of species-specific cytokines in single and cocultures on hepatocyte function. Hepatocyte cultures were isolated from human resection specimens and from Landrace pigs. Single and cocultures were exposed to porcine and human interleukin (IL)-6 or tumor necrosis factor (TNF)-alpha. Changes in expression of C-reactive protein (CRP), albumin, CCAAT enhancer binding protein (C/EBP)-alpha and C/EBP-beta and metabolic competence of cultured cells was studied by measuring testosterone metabolite production. After human or porcine IL-6 dosing, CRP was induced up to 100-fold in human hepatocyte cultures, while porcine hepatocytes responded marginally (2- to 5-fold). Treatment with human or porcine IL-6 or TNF-alpha resulted in reduced albumin production, albeit at different levels when human and porcine hepatocytes were compared (P = 0.001). Unlike human, porcine hepatocytes produced less of 6alpha-hydroxytestosterone (6alpha-HT) (P < 0.001) and 7alpha-HT (P < 0.001) after human or porcine IL-6 dosing and treatment with species-specific TNF-alpha induced (human hepatocytes) or decreased (porcine hepatocytes) 6beta-HT production (P = 0.021). In coculture with free exchange of metabolites, porcine hepatocytes produced less 6alpha-HT (P = 0.048) and 16alpha-HT (P = 0.033), whereas after treatment with human IL-6 reduced CRP gene and protein expression was observed with human hepatocytes (P = 0.013). In conclusion, species-specific responses of hepatocytes to cytokines and interactions with xenobiotic metabolites may limit the clinical effectiveness of porcine hepatocytes in BAL.     

Cryopreservation-induced nonattachment of human hepatocytes: role of adhesion molecules

Good quality cryopreserved human hepatocytes are becoming an important source for clinical hepatocyte transplantation. However, the process of cryopreservation leads to both structural and functional impairment of hepatocytes. The aim of this study was to investigate the mechanisms of cryopreservation-induced nonattachment in human hepatocytes. Hepatocytes were cryopreserved after isolation from unused donor liver tissue. Cell attachment to collagen-coated plates was measured. A cDNA gene array system for 96 cell adhesion-related molecules was used to determine mRNA expression in fresh and cryopreserved hepatocytes. Two cell adhesion molecule proteins were investigated further: beta1-integrin, a cell-matrix adhesion molecule, and E-cadherin, a cell-cell adhesion molecule. Attachment efficiency was significantly decreased after cryopreservation of human hepatocytes. Twenty-two genes were downregulated after cryopreservation including integrins, cadherins, catenins, and matrix metalloproteinases (MMPs). Beta1-Integrin gene and protein expression were significantly decreased in cultured cryopreserved hepatocytes compared to fresh hepatocytes. There was a significant correlation between loss of beta1-integrin and attachment in cryopreserved cells. Degradation of E-cadherin was increased in cryopreserved hepatocytes. The process of cryopreservation leads to downregulation of cell adhesion molecules at the gene and the cellular level. New cryopreservation protocols are needed to prevent these effects on cell attachment.     

Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver

We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN-1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 micromol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 micromol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.     

Cryopreservation of human hepatocytes alters the mitochondrial respiratory chain complex 1

Transplantation of human hepatocytes has recently been demonstrated as a safe alternative to partially correct liver inborn errors of metabolism. Cryopreservation remains the most appropriate way of cell banking. However, mitochondrial-mediated apoptosis has been reported after cryopreservation and little is known on the involved molecular mechanisms. The aim of this study was to investigate mitochondrial functions of freshly isolated and cryopreserved/thawed hepatocytes from mice and humans. We report here that cryopreservation induced a dramatic drop of ATP levels in hepatocytes. The oxygen consumption rate of cryopreserved/ thawed hepatocytes was significantly lower compared to fresh cells. In addition, the uncoupling effect of 2,4-dinitrophenol was lost, in parallel with a reduction of mitochondrial membrane potential. Furthermore, a decrease in mitochondrial respiratory rate was evidenced on permeabilized hepatocytes in the presence of substrate for the respiratory chain complex 1. Interestingly, this effect was less marked with a substrate for complex 2. Electron microscopy examination indicated that mitochondria were swollen and devoid of cristae after cryopreservation. These changes could explain the cytosolic release of the proapoptotic protein cytochrome c in cryopreserved cells. Nevertheless, no caspase 9-3 activation and only few apoptotic and necrotic cells were found, indicating that the subsequent cell death program was not yet evidenced. Our results demonstrate that cryopreservation of hepatocytes induced alteration of the mitochondrial machinery. They also suggest that, in addition to technical progress in the cryopreservation procedure, protection of the respiratory chain complex 1 should be considered to improve the quality of cryopreserved hepatocytes.     

Enhancement of hepatic 4‐hydroxylation of 25‐hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: Implications for drug‐induced osteomalacia

Long‐term therapy with certain drugs, especially cytochrome P450 (P450; CYP)‐inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25‐hydroxyvitamin D₃ (25OHD₃) were evaluated after exposure to P450‐inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8‐fold increase in formation of the major metabolite of 25OHD₃, 4β,25(OH)₂D₃. This inductive effect was blocked by the addition of 6′,7′‐dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK‐2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)₂D₃ was the predominant monohydroxy metabolite produced from 25OHD₃, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)₂D₃ was increased 60% (p < 0.01) after short‐term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)₂D₃ (?10%; p = 0.03), and a nonsignificant change in 24R,25(OH)₂D₃ (?8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)₂D₃ and decrease in 1α,25(OH)₂D₃ levels. Examination of the plasma monohydroxy metabolite/25OHD₃ ratios indicated selective induction of the CYP3A4‐dependent 4β‐hydroxylation pathway of 25OHD₃ elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug‐induced osteomalacia. © 2013 American Society for Bone and Mineral Research.     

Effect of low dose cyclosporine and sirolimus on hepatic drug metabolism in the rat1.

BACKGROUND: We examined the effect cyclosporine (CsA) and sirolimus (SRL) alone and in combination on hepatic cytochrome P450-mediated metabolism in rats. METHODS: Rats were given 1 mg/kg of CsA or 0.4 mg/kg of SRL alone or in combination via constant intravenous infusion. Renal function was evaluated at the end of treatment. Blood samples were obtained to estimate CsA and SRL concentrations. Hepatic microsomes were prepared for immunoblotting and catalytic assays. RESULTS: CsA alone did not alter serum creatinine levels. SRL given alone or in combination with CsA produced a significant increase in urine output without changes in fluid balance. Although CsA and SRL administered alone caused damage to renal proximal tubules, the two-drug combination dramatically increased the renal structural damage. CsA alone suppressed cytochrome P450 (CYP) 3A2 protein levels by 39% (P=0.012) and catalytic activity by 30% (P=0.042). SRL alone reduced catalytic activity by 38% (P=0.012). Combination therapy reduced both CYP3A2 levels by 55% (P<0.001) and catalytic activity by 55% (P=0.001). CYP2C11 protein expression or catalytic activity were not changed in any group. CYP2A1 protein expression and catalytic activity were both significantly reduced in rats given CsA or/and SRL. Steady-state CsA levels were increased during concurrent SRL dosing, however, SRL concentrations were not changed by CsA coadministration. CONCLUSIONS: Concurrent SRL dosing increases CsA concentrations due to inhibition of hepatic CYP3A2 protein expression. Nephrotoxicity caused by combination therapy is due to CsA elevating levels of SRL or by SRL itself. Concurrent administration of CsA and SRL in transplant patients should be performed with caution.     

Downregulation of hepatic cytochrome P450 in chronic renal failure

Chronic renal failure (CRF) is associated with a decrease in drug metabolism. The mechanism remains poorly understood. The present study investigated the repercussions of CRF on liver cytochrome P450 (CYP450). Three groups of rats were defined: control, control paired-fed, and CRF. Total CYP450 activity, protein expression of several CYP450 isoforms as well as their mRNA, and the in vitro N-demethylation of erythromycin were assessed in liver microsomes. The regulation of liver CYP450 by dexamethasone and phenobarbital was assessed in CRF rats. Compared with control and control paired-fed rats, creatinine clearance was reduced by 60% (P: < 0.01) in CRF rats. Weight was reduced by 30% (P: < 0.01) in control paired-fed and CRF rats, compared with control animals. There was no difference in the CYP450 parameters between control and control paired-fed. Compared with control paired-fed rats, total CYP450 was reduced by 47% (P: < 0.001) in CRF rats. Protein expression of CYP2C11, CYP3A1, and CYP3A2 were considerably reduced (>40%, P: < 0.001) in rats with CRF. The levels of CYP1A2, CYP2C6, CYP2D, and CYP2E1 were the same in the three groups. Northern blot analysis revealed a marked downregulation in gene expression of CYP2C11, 3A1, and 3A2 in CRF rats. Although liver CYP450 was reduced in CRF, its induction by dexamethasone and phenobarbital was present. N-demethylation of erythromycin was decreased by 50% in CRF rats compared with control (P: < 0.001). In conclusion, CRF in rats is associated with a decrease in liver cytochrome P450 activity (mainly in CYP2C11, CYP3A1, and 3A2), secondary to reduced gene expression.