Ultra-Rapid Cardiotoxicity of the Hepatitis C Virus Protease Inhibitor BILN 2061 in the Urokinase-Type Plasminogen Activator Mouse
2007; Elsevier BV; Volume: 133; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2007.07.007
ISSN1528-0012
AutoresThomas Vanwolleghem, Philip Meuleman, Louis Libbrecht, Tania Roskams, Rita De Vos, G LEROUXROELS,
Tópico(s)Viral Infections and Immunology Research
ResumoBackground & Aims: Because current therapies for chronic hepatitis C virus (HCV) infections are suboptimal and associated with severe side effects, novel treatment options are needed. A small animal model has recently been developed to study HCV infections. To examine the usefulness of this human liver–urokinase-type plasminogen activator (uPA)+/+ severe combined immune deficient (SCID) mouse for the development of HCV-targeted drugs, we evaluated the antiviral efficacy and safety of an HCV NS3-protease inhibitor, BILN 2061. Methods: BILN 2061 was orally administered at clinical range doses for 4 days to SCID mice that differed in the presence of HCV infection, human hepatocyte grafts, and uPA zygosity. Treatment outcome was evaluated clinically, virologically, and morphologically. Using standard high-performance liquid chromatography–ultraviolet (HPLC-UV) methods and mass spectrometry, single-dose pharmacokinetics and multiple-dose drug exposures were analyzed. The 13C-aminopyrine breath test was applied to compare in vivo liver function. Results: A 4-day treatment with BILN 2061 of HCV genotype-1b infected chimeric animals reduced the viral load by >100-fold, but concomitant clinical and ultrastructural signs of cardiotoxicity appeared. BILN 2061 administration to uPA-transgenic mice induced mitochondrial swelling with aberrant cristae in cardiomyocytes, but not in skeletal muscle. Because both drug accumulation and liver function were identical in affected uPA-transgenic and nontransgenic SCID mice without cardiac involvement, the urokinase plasminogen activator transgene itself appears to be implicated. Conclusions: The human liver-uPA+/+SCID mouse is an interesting small animal model to evaluate the preclinical safety and efficacy of new antiviral compounds against HCV. The uPA-transgene increases the susceptibility of mice to BILN 2061-induced cardiotoxicity. Background & Aims: Because current therapies for chronic hepatitis C virus (HCV) infections are suboptimal and associated with severe side effects, novel treatment options are needed. A small animal model has recently been developed to study HCV infections. To examine the usefulness of this human liver–urokinase-type plasminogen activator (uPA)+/+ severe combined immune deficient (SCID) mouse for the development of HCV-targeted drugs, we evaluated the antiviral efficacy and safety of an HCV NS3-protease inhibitor, BILN 2061. Methods: BILN 2061 was orally administered at clinical range doses for 4 days to SCID mice that differed in the presence of HCV infection, human hepatocyte grafts, and uPA zygosity. Treatment outcome was evaluated clinically, virologically, and morphologically. Using standard high-performance liquid chromatography–ultraviolet (HPLC-UV) methods and mass spectrometry, single-dose pharmacokinetics and multiple-dose drug exposures were analyzed. The 13C-aminopyrine breath test was applied to compare in vivo liver function. Results: A 4-day treatment with BILN 2061 of HCV genotype-1b infected chimeric animals reduced the viral load by >100-fold, but concomitant clinical and ultrastructural signs of cardiotoxicity appeared. BILN 2061 administration to uPA-transgenic mice induced mitochondrial swelling with aberrant cristae in cardiomyocytes, but not in skeletal muscle. Because both drug accumulation and liver function were identical in affected uPA-transgenic and nontransgenic SCID mice without cardiac involvement, the urokinase plasminogen activator transgene itself appears to be implicated. Conclusions: The human liver-uPA+/+SCID mouse is an interesting small animal model to evaluate the preclinical safety and efficacy of new antiviral compounds against HCV. The uPA-transgene increases the susceptibility of mice to BILN 2061-induced cardiotoxicity. Infections with the hepatitis C virus (HCV) affect about 2% of the world's population and represent a leading cause of chronic liver disease.1Shepard C.W. Finelli L. Alter M.J. Global epidemiology of hepatitis C virus infection.Lancet Infect Dis. 2005; 5: 558-567Abstract Full Text Full Text PDF PubMed Scopus (2303) Google Scholar Today's therapeutic standard is frequently associated with severe side effects and effective in only half of genotype 1 infections, occurring in the majority of patients in the United States and Europe.2Strader D.B. Wright T. Thomas D.L. et al.Diagnosis, management, and treatment of hepatitis C.Hepatology. 2004; 39: 1147-1171Crossref PubMed Scopus (1605) Google Scholar, 3Lauer G.M. Walker B.D. Hepatitis C virus infection.N Engl J Med. 2001; 345: 41-52Crossref PubMed Scopus (2538) Google Scholar Clearly, new treatment strategies are needed. A major impediment in the development of new HCV antivirals is the lack of a suitable animal model; only humans and chimpanzees are susceptible to HCV. Increasing ethical, ecologic, and financial concerns highly restrict preclinical tests in chimpanzees. As a consequence, antiviral compounds with convincing in vitro activity are transferred almost directly toward clinical trials in man. A small animal model for HCV cannot only reduce the risks associated with this approach, but also generate important data to complement the preclinical safety profile. The imperfection of drug toxicity screening procedures has been highlighted by the fialuridine case. A phase 2 clinical trial of fialuridine in chronic hepatitis B patients was halted after 13 weeks because of liver failure in 7 of 15 treated patients. Five patients died and 2 survived after liver transplantation. This dramatic scenario was not predicted by preclinical toxicity tests in rodents, dogs, and rhesus monkeys.4McKenzie R. Fried M.W. Sallie R. et al.Hepatic failure and lactic acidosis due to fialuridine (FIAU), an investigational nucleoside analogue for chronic hepatitis B.N Engl J Med. 1995; 333: 1099-1105Crossref PubMed Scopus (546) Google Scholar In the scientific discussions that followed, several investigators questioned the value of the current animal models for preclinical safety testing and suggested the inclusion of models that better mimic the chronic human disease. Later, mitochondrial toxicity was demonstrated in various organs of long-term treated woodchucks independent of their infection with woodchuck hepatitis virus.5Lewis W. Griniuviene B. Tankersley K.O. et al.Depletion of mitochondrial DNA, destruction of mitochondria, and accumulation of lipid droplets result from fialuridine treatment in woodchucks (Marmota monax).Lab Invest. 1997; 76: 77-87PubMed Google Scholar, 6Tennant B.C. Baldwin B.H. Graham L.A. et al.Antiviral activity and toxicity of fialuridine in the woodchuck model of hepatitis B virus infection.Hepatology. 1998; 28: 179-191Crossref PubMed Scopus (101) Google Scholar BILN 2061 (Boehringer Ingelheim, Ingelheim, Germany) was the first compound of a new class of HCV NS3-protease inhibitors to enter clinical trials, after preclinical toxicity tests were uneventful.7Lamarre D. Anderson P.C. Bailey M. et al.An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus.Nature. 2003; 426: 186-189Crossref PubMed Scopus (862) Google Scholar Unprecedented antiviral activity was demonstrated in HCV genotype 1 infected patients after only 4 administrations in 2 days.8Hinrichsen H. Benhamou Y. Wedemeyer H. et al.Short-term antiviral efficacy of BILN 2061, a hepatitis C virus serine protease inhibitor, in hepatitis C genotype 1 patients.Gastroenterology. 2004; 127: 1347-1355Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar No relevant adverse events occurred during the trials. Later, however, long-term toxicity studies in rhesus monkeys revealed that BILN 2061 induced histologic cardiotoxicity. Consequently, further clinical development of this compound has been halted.9Reiser M. Hinrichsen H. Benhamou Y. et al.Antiviral efficacy of NS3-serine protease inhibitor BILN-2061 in patients with chronic genotype 2 and 3 hepatitis C.Hepatology. 2005; 41: 832-835Crossref PubMed Scopus (164) Google Scholar More than a decade ago, an incidental finding of massive liver regeneration in urokinase-type plasminogen activator (uPA) transgenic mice paved the way for the development of a small animal model for HCV infection studies.10Sandgren E.P. Palmiter R.D. Heckel J.L. et al.Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene.Cell. 1991; 66: 245-256Abstract Full Text PDF PubMed Scopus (342) Google Scholar The hepatocyte-specific over expression of the uPA transgene induces lethal liver failure in these animals and necessitates liver support by endogenous or transplanted healthy hepatocytes.11Rhim J.A. Sandgren E.P. Degen J.L. et al.Replacement of diseased mouse liver by hepatic cell transplantation.Science. 1994; 263: 1149-1152Crossref PubMed Scopus (527) Google Scholar Human hepatocytes can reconstitute the liver of these transgenic mice, once they are backcrossed on a severe combined immune deficient (SCID) background to prevent rejection of the xenograft.12Meuleman P. Libbrecht L. De Vos R. et al.Morphological and biochemical characterization of a human liver in a uPA-SCID mouse chimera.Hepatology. 2005; 41: 847-856Crossref PubMed Scopus (321) Google Scholar, 13Mercer D.F. Schiller D.E. Elliott J.F. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (766) Google Scholar Testing new compounds in HCV infected human–mouse chimeras can provide proof of their in vivo antiviral activity14Kneteman N.M. Weiner A.J. O'Connell J. et al.Anti-HCV therapies in chimeric SCID-Alb/uPA mice parallel outcomes in human clinical application.Hepatology. 2006; 43: 1346-1353Crossref PubMed Scopus (87) Google Scholar and generate pharmacokinetic and toxicity data that may be relevant for the human situation. Here we show a mean 2.20 log10-fold reduction of HCV viremia after 7 to 8 oral doses of 10 mg/kg BILN 2061 in the human liver-uPA+/+SCID mouse. Within the 4-day trial period, this promising antiviral activity was accompanied by clinical and ultrastructural mitochondrial cardiotoxicity. The uPA-transgene itself appears to be implicated in the increased susceptibility for mitochondrial cardiotoxicity; both drug accumulation and liver function were identical in affected uPA-transgenic mice and nontransgenic SCID mice without cardiac involvement. uPA-SCID mice were generated as previously described.12Meuleman P. Libbrecht L. De Vos R. et al.Morphological and biochemical characterization of a human liver in a uPA-SCID mouse chimera.Hepatology. 2005; 41: 847-856Crossref PubMed Scopus (321) Google Scholar, 15Meuleman P. Vanlandschoot P. Leroux-Roels G. A simple and rapid method to determine the zygosity of uPA-transgenic SCID mice.Biochem Biophys Res Commun. 2003; 308: 375-378Crossref PubMed Scopus (41) Google Scholar CB17-Prkdcscid mice were purchased from Harlan (Horst, The Netherlands). Animals were bred and kept in our specific pathogen-free breeding facility. Only uPA-SCID mice homozygous for the uPA transgene were used as acceptors of human hepatocytes. For toxicity and liver function studies SCID and uPA+/−SCID mice were 5 weeks old. All animal studies were approved by the Ghent University Hospital Human and Animal Ethical Committees. Human hepatocytes were isolated and transplanted as previously described.12Meuleman P. Libbrecht L. De Vos R. et al.Morphological and biochemical characterization of a human liver in a uPA-SCID mouse chimera.Hepatology. 2005; 41: 847-856Crossref PubMed Scopus (321) Google Scholar Cell viability, determined by trypan blue exclusion, generally exceeded 78%. Mice with successful liver grafts as defined by a human albumin plasma level of at least 1 mg/ml were infected with the HCV reference strain HC-J4 (genotype 1b). BILN 2061 (purity >99%, kind gift of Gilead Sciences, Inc., Foster City, CA) was dissolved at a final concentration of 1 mg/mL in 5% ethanol, 20% propylene glycol, 40% polyethylene glycol 400, and 35% 50 mmol citrate buffer, pH 2.2 (all from Sigma-Aldrich, Taufkirchen, Germany). Antiviral efficacy was tested in 3 HCV-infected chimeric animals receiving 10 mg/kg of BILN 2061 at 1 mg/mL twice daily via oral gavage and in 3 control animals dosed with phosphate-buffered saline (PBS) at an equal volume of 10 mL/kg. In further toxicity studies, 4 uPA+/−SCID, 3 SCID, and 2 uninfected chimeric animals were dosed for 4 days with the BILN 2061 formulation in a similar manner. To control for toxicity of the compound vehicle, 1 more infected and 2 uninfected chimeric animals were dosed twice daily for 4 days with 10 mL/kg of the vehicle. For the pharmacokinetic study, 3 uPA+/−SCID and 4 SCID mice received a single 10 mg/kg oral dose of BILN 2061 and were bled at 1, 2, 4, 6, 8, and 12 hours after dosing. Compound solutions and PBS were analyzed for bacterial contamination by standard microbiological techniques (all consumables from Becton Dickinson, Erembodegem, Belgium). At indicated time points, blood samples were collected and stored at −80°C until further analysis. A detailed description of the assays performed on mouse plasma samples can be consulted online at the gastroenterology website (See supplemental material online at www.gastrojournal.org). Light microscopy was performed on hematoxylin and eosin (H&E)–stained tissue samples from the heart and abdominal organs. On autopsy, fragments of the endomyocard and quadriceps skeletal muscle were immediately fixed in a 2.5% glutaraldehyde solution for ultrastructural studies. A detailed description of morphologic procedures can be consulted online at the gastroenterology website (See supplemental material online at www.gastrojournal.org). In vivo liver function was examined after intraperitoneal (IP) injection of 100 mg/kg 13C-aminopyrine (Isotech, Sigma-Aldrich), by measuring the 13CO2 enrichment in sequential breath samples with gas–isotope ratio mass spectrometry (Chrompack, Middelburg, The Netherlands and Isochrom μgas, Micromass, Middlewich, UK). A detailed description of the experimental procedure can be consulted online at the gastroenterology website (See supplemental material online at www.gastrojournal.org). All data are expressed as mean values ± SD. The Mann–Whitney U test and Kruskal–Wallis test assessed statistical significance of differences between groups, with 2-sided probability values (P < .05). Significance of the regression line was assessed with the F–test. The r2 value was used as a summary measure of goodness of fit. BILN 2061 or PBS were administered to human liver-uPA+/+SCID mice (further referred to as chimeric animals) that had been infected with HCV-1b for at least 23 days. Human albumin levels and HCV viral load in mouse plasma were quantified before dosing (day 0), after 8 (day 4), and 16 doses (day 8; Figure 1A and B). Only minimal changes in albumin levels were observed, demonstrating the stability of the human grafts during this interval. At the start of treatment, HCV RNA levels ranged between 4.11 and 7.59 log10 IU/mL. In PBS-dosed animals, viremia remained constant and showed mean log10-fold changes of +0.02 and −0.16 after 4 and 8 days of treatment, respectively (Figure 1B). After 8 doses of BILN 2061, HCV RNA levels declined 2.48 log10-fold in 1 mouse, and dropped under the 315 IU/mL detection limit in a second (Figure 1A). At least a 1.61 log10-fold reduction can hence be demonstrated in the latter mouse. Viremia dropped 2.51 log10-fold in the third BILN 2061-treated animal (Figure 1A), which was euthanized 8 hours after the 7th dose because of pronounced inactivity, hunched posture, ruffled fur, and loose stools (Table 1, column 1). Taken together, BILN 2061 treatment of HCV-1b infected chimeras induced a mean 2.20 log10-fold decline in viremia within 4 days (Figure 1C).Table 1Clinical and Morphologic Features of BILN 2061–Dosed AnimalsHuman liver-uPA+/+SCIDuPA+/−SCIDaHCV uninfected, without human hepatocyte grafts. (n = 4)SCIDaHCV uninfected, without human hepatocyte grafts. (n = 3)HCV infected (n = 3)HCV uninfected (n = 2)Clinical adverse events Dosing Loose stools+ (n = 3)+ (n = 2)± (n = 2)± (n = 1) Ruffled fur+ (n = 1)± (n = 1)No (n = 4)No (n = 3) Hunched posture+ (n = 1)No (n = 2)No (n = 4)No (n = 3) Inactivity+ (n = 1)No (n = 2)No (n = 4)No (n = 3) Bleeding Shock-like state+ (n = 2), 1 killed+ (n = 2)No (n = 4)No (n = 3)Ultrastructural findings (heart)(n = 1)(n = 2)(n = 4)(n = 3) Lipid accumulation+NoNoNoMitochondria Swollen, pale+++No Aberrant cristae+++NoSER/T-tubule widening+++NoLiver morphology(n = 2)(n = 2)(n = 2) Human hepatocytes47%, 36%53%, 56%NANA Red nodules14%, 28%7%, 10%68%, 66%NA White parenchyme39%, 36%40%, 34%32%, 34%NANOTE. The number of mice in each group is shown in parentheses.HCV, hepatitis C virus; SER, sarcoplasmic reticulum; NA, not applicable.a HCV uninfected, without human hepatocyte grafts. Open table in a new tab NOTE. The number of mice in each group is shown in parentheses. HCV, hepatitis C virus; SER, sarcoplasmic reticulum; NA, not applicable. Whereas PBS dosing was well tolerated, all BILN 2061-treated animals developed loose stools after 3 doses (Table 1, column 1). However, none of the formulations yielded positive bacterial cultures on routine testing. Immediately after a minimal blood sampling on day 4, two animals developed acute clinical deterioration, characterized by an increased respiratory rate, inactivity, and hunched posture (shocklike state; Table 1, column 1). Both mice succumbed within the following 2 hours. The bleeding procedure was executed in the gentle, standard manner that never caused any problem before. On autopsy, no relevant pathology was detected in several organs, including the heart, of BILN 2061-dosed chimeric animals. However, severe ultrastructural changes of the cardiomyocytes were found in glutaraldehyde-fixed, fresh myocardial samples, which could only be obtained from the euthanized animal (Table 1, column 1). Many myocytes contained numerous small fat droplets and increased numbers of mitochondria (Figure 2A). Each myocyte contained a variable number of pathologic mitochondria, interspersed between normal ones. Mitochondrial abnormalities included a swollen or pale matrix, aberrant undulating or disrupted cristae, and inclusion of fat droplets or myelin figures (Figure 2B). Although the organization of sarcomeres could be deranged due to the accumulation of mitochondria, the composition of myofibrils and Z-bands seemed unaltered. The sarcoplasmic reticulum and T-tubular system often had a swollen and widened appearance. To further investigate the origin of these ultrastructural defects, additional toxicity studies were performed in SCID mice that differed in uPA zygosity, presence of human hepatocyte grafts, and HCV infection. Cardiotoxicity of the compound vehicle was examined in 3 chimeras (see Materials and Methods). One of these animals was infected for over 4.5 months with the same HCV reference strain and had viral loads reaching up to 7.33 log10 IU/mL. During the vehicle dosing, all 3 animals developed loose stools. After 8 doses, no ultrastructural changes in the endomyocardial tissue were found. This excludes the vehicle or HCV infection as the cause of the observed cardiotoxicity (Figure 2C and D). Furthermore, uPA over expression alone is not directly toxic to the heart because non-dosed, non-transplanted uPA+/+SCID (n = 2) and uPA+/−SCID (n = 2) mice had a normal ultrastructural myocardial morphology (Figure 2E and F). A similar 4-day treatment with twice daily 10 mg/kg BILN 2061 was repeated in 2 uninfected, well-engrafted chimeric uPA+/+SCID animals, in heterozygous uPA+/−SCID mice without human hepatocyte grafts (n = 4), and in nontransgenic SCID (n = 3) mice (Table 1, columns 1, 2, and 3, respectively). During treatment, some animals developed loose stools. Ruffled fur was observed in only 1 chimera (Table 1). Minimal blood sampling (50 μL) after the last BILN 2061 dose again induced acute clinical deterioration in both chimeric animals, but not in the other mice. These "shocked" chimeric animals were closely monitored and found to recuperate. Ruffled fur was the only clinical sign that persisted. A final blood sample was taken from all animals 12 hours after the last dose to quantify drug levels. Heart tissue and skeletal muscle were carefully prepared for ultrastructural analysis. No ultrastructural defects were found in the heart or in the skeletal muscle of the BILN 2061-dosed nontransgenic SCID mice (Figure 3A and B), whereas the hearts of all uPA+/−transgenic and chimeric animals were affected in varying degrees (Table 1, columns 4, 3, and 2, respectively). Typically in each myocyte, pathologic mitochondria were found amidst normal ones. Mitochondrial defects included swollen or pale matrices, disrupted or disarranged cristae, and inclusion of fat droplets or myelin figures (Figure 3C and E). No abnormal sarcoplasmic lipid accumulation was observed. Similar to our previous observation, the sarcoplasmic reticulum and T-tubule system were often widened or dilated. In animals with mitochondrial defects in the heart, no abnormalities were observed in the skeletal muscle (Figure 3D and F). Taken together, these data indicate that independent of hepatocyte grafts or HCV infection, the uPA zygosity of SCID mice determines their sensitivity to develop mitochondrial damage in cardiomyocytes, but not in skeletal muscle after 8 doses of BILN 2061 at 10 mg/kg. To compare systemic exposure of BILN 2061 between different experimental animal groups, trough plasma concentrations were measured after 8 doses. Of 5 BILN 2061-treated chimeras, trough plasma samples were available from 2 HCV infected and 2 uninfected animals. BILN 2061 quantification was possible in 3 of them, yielding 651 ng/mL in 1 infected and 267 and 331 ng/mL in 2 uninfected mice. Notably, BILN 2061 plasma concentration was 3410 ng/mL in the precociously euthanized animal. Overall trough levels were not significantly different between chimeric animals, uPA+/− transgenic, and nontransgenic SCID mice (Figure 4;P = .13). The uPA zygosity of SCID mice did not significantly alter drug trough levels (P = .40; Table 2), but was clearly associated with ultrarapid cardiotoxicity. We therefore focused on the effect of the uPA transgene on mouse physiology to clarify the underlying mechanism.Table 2BILN 2061 Pharmacokinetics in Mice (Experimental Data) and Men (Literature Data)SCIDuPA+/−SCIDHCV PatientsPharmacokinetics parameterWeight (g)% Red nodulesgt 1aPharmacokinetic parameters of HCV genotype 1a 8 and genotype 2 and 3b 9 infected patients after a single oral dose of 500 mg BILN 2061. (n = 8)gt 2–3bPharmacokinetic parameters of HCV genotype 1a 8 and genotype 2 and 3b 9 infected patients after a single oral dose of 500 mg BILN 2061. (n = 8)Cmax, single dose (ng/mL)1,844 ± 1,0664,865 ± 1,79511.3 ± 0.851 ± 4%1,253 ± 4292,389 ± 2,735(n = 4)(n = 3)(n = 3)(n = 3)Half-life, single dose (hr)1.21 ± 0.152.28 ± 0.19NDND(n = 4)(n = 3)AUC(0–8h), single dose (hr*ng/mL)3,120 ± 1,54815,849 ± 5,4874,496 ± 1,78410,572 ± 12,759(n = 4)(n = 3)Plasma trough concentration, multidose (ng/mL)124 ± 79259 ± 14216.1 ± 1.167 ± 1%NDND(n = 4)(n = 3)(n = 4)(n = 2)NOTE. Values ± SD are shown. Differences between experimental groups are not significant (P > .05). The number of mice in each group is shown in parentheses.ND, not determined.a,b Pharmacokinetic parameters of HCV genotype 1a 8 and genotype 2 and 3b 9 infected patients after a single oral dose of 500 mg BILN 2061. Open table in a new tab NOTE. Values ± SD are shown. Differences between experimental groups are not significant (P > .05). The number of mice in each group is shown in parentheses. ND, not determined. Trough levels can be used as an estimator of drug accumulation and exposure, provided that plasma samples were drawn at steady state, which would be reached within 5–7 drug elimination half-lives.16Rowland M. Tozer T.N. Clinical pharmacokinetics: Concepts and applications. Williams & Wilkins, Baltimore, MD1995Google Scholar Because of uPA-induced liver failure, we expected altered hepatic metabolization and pharmacokinetics of BILN 2061.7Lamarre D. Anderson P.C. Bailey M. et al.An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus.Nature. 2003; 426: 186-189Crossref PubMed Scopus (862) Google Scholar, 10Sandgren E.P. Palmiter R.D. Heckel J.L. et al.Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene.Cell. 1991; 66: 245-256Abstract Full Text PDF PubMed Scopus (342) Google Scholar We examined this with a single dose pharmacokinetics study because we hypothesized that clinical cardiotoxicity could hamper a multi dose study. A single oral dose of 10 mg/kg BILN 2061 was rapidly absorbed with maximum plasma concentrations of 1844 ± 1066 ng/mL in SCID mice (n = 4) and 4865 ± 1795 ng/mL in uPA+/−SCID mice (n = 3; P= .1143; Table 2). Drug elimination half-lives were 1.21 ± 0.15 hours in SCID mice (n = 4) and 2.28 ± 0.19 hours in uPA+/−SCIDs (n = 3; P = .0571; Table 2). The last sampling point, 12 hours after dosing, could not be obtained from 2 uPA+/− transgenic animals. We therefore calculated the area under the concentration–time curve up to 8 hours after dosing. This showed only a trend toward higher drug exposures in uPA+/− transgenic animals (Table 2). With these short half-lives, steady state is certainly reached within the 4-day treatment period. Therefore, the measured trough plasma concentrations indicate that drug accumulation in uPA+/− transgenic and nontransgenic SCIDs is similar. Pharmacokinetics of hepatic metabolized drugs are only an indirect measure of liver function. Liver parenchyma in uPA+/− animals comprises sick hepatocytes that over express uPA and healthy hepatocytes that have successfully looped out the transgene. The former have a pale appearance, whereas the latter have a "healthy" reddish aspect and are therefore known as red nodules.10Sandgren E.P. Palmiter R.D. Heckel J.L. et al.Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene.Cell. 1991; 66: 245-256Abstract Full Text PDF PubMed Scopus (342) Google Scholar This possibly influences the overall health status of uPA-transgenic animals, because failure to thrive is frequently observed in 5-week-old uPA+/− animals, with body weights between 7.15 and 17.30 g. Transgenic animals with cardiac involvement after BILN 2061 treatment weighed on average 16.1 ± 1.1 g and the single-dose pharmacokinetics study was performed in animals weighing 11.3 ± 0.8 g (Table 2). To extrapolate the data of the pharmacokinetics study to uPA+/− transgenic animals in general, we examined the relationship between total body weight and liver function of these animals. We hypothesized that body weight increases with the restoration of liver function by expansion of healthy, transgene-free hepatocytes.10Sandgren E.P. Palmiter R.D. Heckel J.L. et al.Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene.Cell. 1991; 66: 245-256Abstract Full Text PDF PubMed Scopus (342) Google Scholar Extensive morphologic examination of the whole liver showed a good correlation between the number of healthy mouse hepatocytes forming red nodules and body weight (Figure 5A;P < .0001; r2 = 0.7530). We next wanted to know whether liver repopulation by red nodules would restore liver function and drug metabolizing capacity. The aminopyrine breath test (ABT), a nonselective test of cytochrome P450 function, has been used for decades in humans, as well as in rodents to examine liver microsomal functional mass.17Lauterburg B.H. Bircher J. Expiratory measurement of maximal amino-pyrine demethylation in vivo: effect of phenobarbital, partial hepatectomy, portacaval shunt and bile duct ligation in the rat.J Pharmacol Exp Ther. 1976; 196: 501-509PubMed Google Scholar, 18Hepner G.W. Vesell E.S. Quantitative assessment of hepatic function by breath analysis after oral administration of (14C)aminopyrine.Ann Intern Med. 1975; 83: 632-638Crossref PubMed Scopus (195) Google Scholar, 19Schneider J.F. Baker A.L. Haines N.W. et al.Aminopyrine N-demethylation: a prognostic test of liver function in patients with alcoholic liver disease.Gastroenterology. 1980; 79: 1145-1150PubMed Scopus (120) Google Scholar, 20Gescher A. Raymont C. Studies of the metabolism of N-methyl containing anti-tumour agents14CO2 breath analysis after administration of 14C-labelled N-methyl drugs, formaldehyde and formate in mice.Biochem Pharmacol. 1981; 30: 1245-1252Crossref PubMed Scopus (17) Google Scholar The assay relies on the hepatic breakdown of 13C-aminopyrine to 13CO2, which can be detected in breath samples. We examined breath 13CO2 enrichment at different intervals after IP injection of 100 mg/kg 13C-aminopyrine in uPA+/− transgenic animals with low and higher body weights and compared these with data obtained from nontransgenic SCID mice of the same age. Because both liver volume and total CO2 production are proportional to body size, the fraction of breath 13CO2 molecules can be used to compare relative liver functional mass in animals of different sizes, without measuring total CO2 production. We therefore analyzed the non–dose-normalized versions of frequently used ABT parameters: peak 13CO2 breath enrichment (peak APE 13C per min [APEmax]), the time at which it is reached (Tmax), and the cumulative breath 13CO2 enrichment over 90 minutes (APEcum13C).17Lauterburg B.H. Bircher J. Expiratory measurement of maximal amino-pyrine demethylation in vivo: effect of phenobarbital, partial hepatectomy, portacaval shunt and bile duct ligation in the rat.J Pharmacol
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