Repeat-dose sirolimus pharmacokinetics and pharmacodynamics in patients with hepatic allografts.

PURPOSE: To determine sirolimus steady-state pharmacokinetics, and to assess the relationship between time-normalized trough sirolimus concentration (C(min,TN)) and evidence of efficacy (rejection and death) and adverse reactions (stomatitis and pneumonia) in liver allograft patients. METHODS: Dense sampling of sirolimus was performed over a single daily-dosing interval in 11 hepatic allograft recipients on day 28 and at 3 months after start of treatment. Serial trough concentration sampling was performed in 380 hepatic allograft recipients on days 1, 7, 14, 28, 42, 60, 90, 180, 270 and 360 after start of treatment. Occurrence of stomatitis, pneumonia, rejection, and death were collected for 360 days after start of treatment. Noncompartmental pharmacokinetic parameters were analyzed in the 11 densely sampled patients; C(min,TN) was determined in the 380 patients. RESULTS: Mean maximum concentration (C(max)), time to C(max) (t(max)), area under the curve for the given dose interval (AUC(tau)), and whole blood oral clearance at 3 months were 20.8 ± 7.6 ng/mL, 3 ± 1 h, 338 ± 144 ng·h/mL, and 10.0 ± 5.6 L/hr, respectively. In the 11 densely sampled patients, linear regression showed that C(min,TN) was highly predictive of AUC(tau) (r² = 0.77, P < 0.0001) at each analysis time point. Logistic regression showed a relationship between C(min,TN) in the 380 patients and pneumonia occurrence, but not between C(min,TN) and stomatitis, rejection, or death. CONCLUSIONS: In this study, the pharmacokinetic profile of sirolimus in hepatic allograft patients was consistent with that of renal transplantation recipients. With the exception of pneumonia, no correlation was observed between C(min,TN) and the occurrence of adverse events of interest.

Atorvastatin attenuates hepatic fibrosis in rats after bile duct ligation via decreased turnover of hepatic stellate cells.

BACKGROUND & AIMS: Activation of hepatic stellate cells (HSC) and transdifferentiation to myofibroblasts following liver injury is the main culprit for hepatic fibrosis. Myofibroblasts show increased proliferation, migration, contraction, and production of extracellular matrix (ECM). In vitro, HMG-CoA reductase inhibitors (statins) inhibit proliferation and induce apoptosis of myofibroblastic HSC. To investigate the antifibrotic effects of atorvastatin in vivo we used bile duct ligated rats (BDL). METHODS: BDL rats were treated with atorvastatin (15 mg/kg/d) immediately after ligation (prophylactically) or in on-going fibrosis (therapeutically). Fibrosis was assessed by hydroxyproline content and Sirius-red staining. The activation of HSC was investigated by analysis of alphaSMA expression. mRNA levels of cytokines and procollagen were analyzed by RT-PCR, and MMP-2 activity by zymography. Proliferation was assessed by expression of cathepsins (B and D), proliferating cell nuclear antigen (PCNA), and Ki67-staining. Apoptosis was characterized by caspase-3 activity, cleavage of PARP-1, and TUNEL assay. Hepatic inflammation was investigated by serum parameters and liver histology. RESULTS: Prophylactic and early therapy with atorvastatin significantly attenuated fibrosis and HSC activation. Later therapy lacked significant effects on fibrosis but reduced profibrotic cytokine expression and led to a more quiescent state of HSC with less proliferation and apoptosis, while hepatic inflammation did not change. CONCLUSIONS: This study shows that very early atorvastatin treatment inhibits HSC activation and fibrosis in the BDL model in vivo, while late treatment reduces HSC turnover and activity. Our findings underline that long-term studies in humans are warranted.

Toxicity of valproic acid in mice with decreased plasma and tissue carnitine stores.

The aim of this study was to investigate whether a decrease in carnitine body stores is a risk factor for valproic acid (VPA)-associated hepatotoxicity and to explore the effects of VPA on carnitine homeostasis in mice with decreased carnitine body stores. Therefore, heterozygous juvenile visceral steatosis (jvs)(+/-) mice, an animal model with decreased carnitine stores caused by impaired renal reabsorption of carnitine, and the corresponding wild-type mice were treated with subtoxic oral doses of VPA (0.1 g/g b.wt./day) for 2 weeks. In jvs(+/-) mice, but not in wild-type mice, treatment with VPA was associated with the increased plasma activity of aspartate aminotransferase and alkaline phosphatase. Furthermore, jvs(+/-) mice revealed reduced palmitate metabolism assessed in vivo and microvesicular steatosis of the liver. The creatine kinase activity was not affected by treatment with VPA. In liver mitochondria isolated from mice that were treated with VPA, oxidative metabolism of l-glutamate, succinate, and palmitate, as well as beta-oxidation of palmitate, were decreased compared to vehicle-treated wild-type mice or jvs(+/-) mice. In comparison to vehicle-treated wild-type mice, vehicle-treated jvs(+/-) mice had decreased carnitine plasma and tissue levels. Treatment with VPA was associated with an additional decrease in carnitine plasma (wild-type mice and jvs(+/-) mice) and tissue levels (jvs(+/-) mice) and a shift of the carnitine pools toward short-chain acylcarnitines. We conclude that jvs(+/-) mice reveal a more accentuated hepatic toxicity by VPA than the corresponding wild-type mice. Therefore, decreased carnitine body stores can be regarded as a risk factor for hepatotoxicity associated with VPA.

The future treatment of portal hypertension.

Increased understanding of the hyperdynamic circulation syndrome has resulted in novel therapeutic approaches, some of which have already reached clinical practice. Central to the hyperdynamic circulation syndrome is an imbalance between the increase in different vasodilators (foremost among which is nitric oxide) and the compensatory increase in vasoconstrictors--usually accompanied by a blunted response. This chapter discusses the role of endothelin in the pathogenesis of the syndrome and in future treatment approaches. A relatively new area of research in this field is the role of infection and inflammation in the initiation and maintenance of the hyperdynamic circulation syndrome. The use of antibiotics in the setting of acute variceal bleeding is standard practice. Studies have suggested that chronic manipulation of the intestinal flora could have beneficial effects in the treatment of portal hypertension. The bile salts are another novel and interesting target. Although their vasoactive properties have been known for some time, recent data demonstrate that their effects could be central in the pathogenesis of the hyperdynamic circulation syndrome, and that manipulation of the composition of the bile acid pool could be a therapeutic approach to portal hypertension. Finally, hypoxia and angiogenesis play a role in the development of portal hypertension and the formation of collaterals. This role needs to be further defined but it appears likely that this phenomenon is yet another target for therapeutic intervention.

Prevalence and associated factors of viral hepatitis and transferrin elevations in 5036 patients admitted to the emergency room of a Swiss university hospital: cross-sectional study.

BACKGROUND: The epidemiology of liver disease in patients admitted to emergency rooms is largely unknown. The current study aimed to measure the prevalence of viral hepatitis B and C infection and pathological laboratory values of liver disease in such a population, and to study factors associated with these measurements. METHODS: Cross-sectional study in patients admitted to the emergency room of a university hospital. No formal exclusion criteria. Determination of anti-HBs, anti-HCV, transferrin saturation, alanine aminotransferase, and obtaining answers from a study-specific questionnaire. RESULTS: The study included 5'036 patients, representing a 14.9% sample of the target population during the study period. Prevalence of anti-HBc and anti-HCV was 6.7% (95%CI 6.0% to 7.4%) and 2.7% (2.3% to 3.2%), respectively. Factors independently associated with positive anti-HBc were intravenous drug abuse (OR 18.3; 11.3 to 29.7), foreign country of birth (3.4; 2.6 to 4.4), non-white ethnicity (2.7; 1.9 to 3.8) and age > or =60 (2.0; 1.5 to 2.8). Positive anti-HCV was associated with intravenous drug abuse (78.9; 43.4 to 143.6), blood transfusion (1.7; 1.1 to 2.8) and abdominal pain (2.7; 1.5 to 4.8). 75% of all participants were not vaccinated against hepatitis B or did not know their vaccination status. Among anti-HCV positive patients only 49% knew about their infection and 51% reported regular alcohol consumption. Transferrin saturation was elevated in 3.3% and was associated with fatigue (prevalence ratio 1.9; 1.2 to 2.8). CONCLUSION: Emergency rooms should be considered as targets for public health programs that encourage vaccination, patient education and screening of high-risk patients for liver disease with subsequent referral for treatment if indicated.

Sensitivity to endothelin-1 is decreased in isolated livers of endothelial constitutive nitric oxide synthase knockout mice.

BACKGROUND: Hepatic sinusoidal resistance is regulated by vasoactive factors including endothelin-1 (ET-1) and nitric oxide (NO). In the absence of NO, vasoconstrictor response to endothelin is expected to predominate. Therefore, we hypothesized sensitivity to endothelin to be increased in mice lacking the endothelial cell NO synthase gene. Response of vascular resistance to endothelin was assessed in the in situ perfused liver of endothelial constitutive nitric oxide synthase (ecNOS) knockout and wild type mice. Livers were also harvested for RNA and protein isolation for quantitative PCR and Western blotting, respectively. The expression of endothelin receptors, isoenzymes of NO synthase, heme-oxygenase and adrenomedullin was quantified. RESULTS: Endothelin increased hepatic vascular resistance in a dose-dependent manner in both strains; however, this increase was significantly less in ecNOS knockout mice at physiologic concentrations. Expression of heme-oxygenases and adrenomedullin was similar in both groups, whereas inducible nitric oxide synthase (iNOS) protein was not detectable in either strain. mRNA levels of pre-pro-endothelin-1 and ETB receptor were comparable in both strains, while mRNA for ETA receptor was decreased in ecNOS knockouts. CONCLUSION: Livers of ecNOS knockout mice have a decreased sensitivity to endothelin at physiologic concentrations; this is associated with a decreased expression of ETA receptors, but not with other factors, such as iNOS, ETB receptors, adrenomedullin or heme-oxygenase. Further studies targeting adaptive changes in ETA receptor distribution and/or intracellular signaling downstream of the receptor are indicated.

Efficiency and safety of bosentan in child C cirrhosis with portopulmonary hypertension and renal insufficiency.

Bosentan has lately been described as a successful therapeutic agent for portopulmonary hypertension consecutive to child A cirrhosis. This is the first report of the effect of this substance with advanced liver cirrhosis (child C) and renal insufficiency. Low doses of bosentan (initially twice 31.25 mg/day and then 62.5 mg/day) increased cardiac output and allowed correction of renal insufficiency; it allowed one to stop the requirement of oxygen and not only improved the 6-min walking test by more than 400 m, but also decreased the severity of the liver cirrhosis to child B stadium. This suggests that patients, who would be excluded from a liver transplantation program because of their portopulmonary hypertension, could profit from a careful therapy with bosentan.

Betaine homocysteine methyltransferase: gene cloning and expression analysis in rat liver cirrhosis.

It has been known for over half a century that homocysteine levels are elevated in liver cirrhosis, but the basis for it is not fully understood. Using differential display, we identified betaine homocysteine methyltransferase (BHMT) as a gene down-regulated in rat liver cirrhosis and most likely involved in this dysregulation. A partial BHMT clone was isolated by screening of a cDNA library with the differential display fragment. The full-length gene was generated by primer extension of cDNA. Expression levels of BHMT in cirrhotic livers of bile duct ligated rats were compared to controls by Northern and Western blotting as well as by enzyme activity measurements. BHMT mRNA levels were reduced to 29+/-23% in established liver cirrhosis induced by bile duct ligation (BDL) as compared to controls. Enzyme assays in crude liver homogenates showed a similar reduction in BHMT activity in bile duct ligated rat livers. By Western blotting, BHMT could be detected in crude liver homogenates of control animals, but was reduced to below the limit of detection in cirrhotic livers. In conclusion, these findings establish a reduced BHMT enzyme activity in cirrhotic rat livers, which may explain the elevated plasma homocysteine levels in cirrhosis.

Molecular absorbent recirculating system for the treatment of acute liver failure in surgical patients.

The Molecular Adsorbent Recirculating System (MARS) represents an attractive artificial liver support system for the treatment of liver insufficiency. However, neither indications for MARS treatment (i.e., after extended liver resection) nor criteria for discontinuation of therapy have been evaluated. Therefore, we analyzed the clinical data of all our surgical patients who received MARS treatment for acute liver failure (n = 7). The aim of the study was to identify prognostic indicators for survival. Four of 174 patients resected for hepatic malignancy at our institution received a total of 13 MARS treatments. Two additional patients were successfully bridged to orthotopic liver transplantation with seven MARS treatments and one patient was MARS supported after liver transplantation of a steatotic graft with three MARS treatments. Five of the seven patients survived and were dismissed an average of 31 days, ranging from 17 to 47 days, after the final MARS treatment. No technical complications or adverse effects were observed during the MARS treatments. Important prognostic factors for hepatic recovery and survival were indocyanin green plasma disappearance rates greater than 5%/min and an increase in clotting factor V levels after each MARS treatment. We conclude that MARS therapy can be an effective treatment of postoperative liver insufficiency in the surgical hepatobiliary unit.

Low NO bioavailability in CCl4 cirrhotic rat livers might result from low NO synthesis combined with decreased superoxide dismutase activity allowing superoxide-mediated NO breakdown: A comparison of two portal hypertensive rat models with healthy controls.

BACKGROUND: In cirrhotic livers, the balance of vasoactive substances is in favour of vasoconstrictors with relatively insufficient nitric oxide. Endothelial dysfunction has been documented in cirrhotic rat livers leading to a lower activity of endothelial nitric oxide synthase but this might not be sufficient to explain the low nitric oxide presence. We compared the amount of all nitric oxide synthase isoforms and other factors that influence nitric oxide bioavailability in livers of two portal hypertensive rat models: prehepatic portal hypertension and carbon tetrachloride induced cirrhosis, in comparison with healthy controls. RESULTS: Endothelial nitric oxide synthase was the solely detected isoform by Western blotting in all livers. In cirrhotic livers, the amount of endothelial nitric oxide synthase protein was lower than in healthy controls, although an overlap existed. Levels of caveolin-1 messenger RNA were within the normal range but endothelin-1 messenger RNA levels were significantly higher in cirrhotic livers (p < 0.05). A markedly lower superoxide dismutase activity was observed in cirrhotic livers as compared to healthy controls (p < 0.05). CONCLUSIONS: In contrast to prehepatic portal hypertension, cirrhotic livers had decreased endothelial nitric oxide synthase protein and enhanced endothelin-1 messenger RNA amount. We hypothesise that a vasodilator/vasoconstrictor imbalance may be further aggravated by the reduced activity of superoxide dismutase. Decreased activity allows enhanced superoxide action, which may lead to breakdown of nitric oxide in liver sinusoids.

Hydatid lung disease (echinococcosis/hydatidosis).

Two Echinococcus species may exhibit medical relevance as causative agents of pulmonary forms of echinococcosis. Most importantly, infections with Echinococcus granulosus result in "cystic hydatid disease" or "cystic echinococcosis," which affects the lungs in a considerable ratio of cases. Echinococcus multilocularis, which causes "alveolar echinococcosis," affects the lungs relatively rarely and then usually upon metastasizing from primary hepatic lesions. Cystic echinococcus and alveolar echinococcus differ pathologically and clinically so greatly that they are considered separately in this article, although alveolar echinococcus is covered minimally because of its minor importance regarding lung infections.

Autoimmune hepatitis, cryoglobulinaemia and untreated coeliac disease: a case report.

The clinical presentation of adult coeliac disease is often uncharacteristic, with extraintestinal symptoms being the main findings. We report a 48-year-old woman who presented with type II, hepatitis-C-negative cryoglobulinaemia, elevated liver enzymes, and iron deficiency. Antinuclear antibodies were positive, and immunoglobulin G (IgG) levels were elevated. On liver biopsy, a diagnosis of type I autoimmune hepatitis with a possible autoimmune cholangitis overlap syndrome was made. Immunosuppressive treatment led to a normalization of transaminase levels and resolved the cryoglobulinaemic vasculitis. In addition, the patient exhibited low ferritin and iron levels, which led to the diagnosis of coeliac disease. Long-standing, untreated coeliac disease is recognized to be a trigger for autoimmune disorders and is known to be associated with other autoimmune diseases, but the association with autoimmune hepatitis or autoimmune cholangitis is reported rarely. We conclude that in patients with autoimmune liver disease and unspecific clinical signs, such as iron deficiency, coeliac disease must be ruled out.

Alveolar echinococcosis: from a deadly disease to a well-controlled infection. Relative survival and economic analysis in Switzerland over the last 35 years.

BACKGROUND/AIMS: Alveolar echinococcosis (AE) is a serious liver disease. The aim of this study was to explore the long-term prognosis of AE patients, the burden of this disease in Switzerland and the cost-effectiveness of treatment. METHODS: Relative survival analysis was undertaken using a national database with 329 patient records. 155 representative cases had sufficient details regarding treatment costs and patient outcome to estimate the financial implications and treatment costs of AE. RESULTS: For an average 54-year-old patient diagnosed with AE in 1970 the life expectancy was estimated to be reduced by 18.2 and 21.3 years for men and women, respectively. By 2005 this was reduced to approximately 3.5 and 2.6 years, respectively. Patients undergoing radical surgery had a better outcome, whereas the older patients had a poorer prognosis than the younger patients. Costs amount to approximately Euro108,762 per patient. Assuming the improved life expectancy of AE patients is due to modern treatment the cost per disability-adjusted life years (DALY) saved is approximately Euro6,032. CONCLUSIONS: Current treatments have substantially improved the prognosis of AE patients compared to the 1970s. The cost per DALY saved is low compared to the average national annual income. Hence, AE treatment is highly cost-effective in Switzerland.

Macrophage activating syndrome is associated with lobular hepatitis and severe bile duct injury with cholestasis.

Macrophage activating syndrome (MAS) is a rare hematological disorder associated with uncontrolled systemic T-cell activation. Persistent fever, fatigue and hepatosplenomegaly are frequent clinical manifestations, whereas hyperferritinemia, elevated serum lactate dehydrogenase levels and cytopenia are key criteria for the diagnosis of MAS. The nature of liver pathology in MAS has been partially elucidated but destructive biliary lesions have been rarely described. This report illustrates four cases of MAS developing marked cholestasis, leading to one case of biliary cirrhosis necessitating liver transplantation. Histologically, liver involvement was characterized in all cases by acute lobular hepatitis, marked hepatocyte apoptosis and small bile duct injury similar to the vanishing bile duct syndrome. Immuno-histological studies showed that the inflammatory changes and bile duct lesions were dominated by the presence of activated macrophages and T-cells, in particular CD8+ lymphocytes, and in part NK-cells. These findings suggest that in MAS, various T-cell triggers such as infection, autoimmune disease and malignancy might result in the release of cytokines, which in turn activate macrophages to trigger a systemic acute phase response and local tissue damage. This communication suggests that a macrophage, T- and NK-cell network is operational in the pathogenesis of the cholangiocyte, hepatocyte and sinus endothelial cell damage in MAS.

Long-term treatment of bile duct-ligated rats with rapamycin (sirolimus) significantly attenuates liver fibrosis: analysis of the underlying mechanisms.

Rapamycin is an immunosuppressant with antiproliferative properties. We investigated whether rapamycin treatment of bile duct-ligated (BDL) rats is capable of inhibiting liver fibrosis and thereby affecting hemodynamics. Following BDL, rats were treated for 28 days with rapamycin (BDL SIR). BDL animals without drug treatment (BDL CTR) and sham-operated animals served as controls. After 28 days, hemodynamics were measured, and livers were harvested for histology/immunohistochemistry. Liver mRNA levels of transforming growth factor (TGF)-beta1, connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF)-beta, cyclin-dependent kinase inhibitor p27(kip) (p27), and cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) were quantified by real-time polymerase chain reaction. Liver protein levels of p27, p21, p70 S6 kinase (p70(s6k)), phosphorylated p70(s6k) (p-p70(s6k)), eukaryotic initiation factor 4E-binding protein (4E-BP1), p-4E-BP1 (Thr37/46), and p-4E-BP1 (Ser65/Thr70) were determined by Western blotting. Portal vein pressure was lower in BDL SIR than in BDL CTR animals. Volume fractions of connective tissue, bile duct epithelial, and desmin- and actin-positive cells were lower in BDL SIR than in BDL CTR rats. On the mRNA level, TGF-beta1, CTGF, and PDGF were decreased by rapamycin. p27 and p21 mRNA did not differ. On the protein level, rapamycin increased p27 and decreased p21 levels. Levels of nonphosphorylated p70(s6k) and 4E-BP1 did not vary between groups, but levels of p-p70(s6k) were decreased by rapamycin. Rapamycin had no effect on p-4E-BP1 (Thr37/46) and p-4E-BP1 (Ser65/Thr70) levels. In BDL rats, rapamycin inhibits liver fibrosis and ameliorates portal hypertension. This is paralleled by decreased levels of TGF-beta1, CTGF, and PDGF. Rapamycin influences the cell cycle by up-regulation of p27, down-regulation of p21, and inhibition of p70(s6k) phosphorylation.

Endothelial nitric oxide synthase is not essential for the development of fibrosis and portal hypertension in bile duct ligated mice.

BACKGROUND/AIMS: It is postulated that nitric oxide (NO) is responsible for the hyperdynamic circulation of portal hypertension. Therefore, we investigated induction of fibrosis and hyperdynamic circulation in endothelial NO synthase knock-out (KO) mice. METHODS: Fibrosis was induced by bile duct ligation. Hemodynamic studies were performed after portal vein ligation. All studies were performed in wild-type (WT) and KO mice. RESULTS: Three to 4 weeks after bile duct ligation (BDL), both WT and KO groups had similar degrees of portal hypertension, 12 (9-14) and 11(8-15) mmHg, median (range), and liver function. Fibrosis increased from 0.0% in sham operated to 1.0 and 1.1% in WT and KO mice, respectively. Cardiac output was similar after portal vein ligation (20 and 17 ml/min in WT and KO mice, respectively). There was no difference in liver of mRNA for endothelin 1, inducible NO synthase (iNOS) and hem-oxygenase 1 (HO1); proteins of iNOS, HO1 and HO2; nor in endothelin A and B (EtA and EtB) receptor density between WT and KO mice after BDL. CONCLUSIONS: These results suggest that endothelial NO synthase is neither essential for the development of fibrosis and portal hypertension in bile duct ligated mice, nor for the hyperdynamic circulation associated with portal hypertension in the portal vein ligated mice.

The Role of the Sinusoidal Endothelium in Liver Function.

Microvascular exchange in the liver is governed by fenestrations in sinusoidal endothelial cells and can be manipulated pharmacologically. Microvascular exchange is affected in alcoholic liver disease and cirrhosis, the former leading to a loss of fenestrae, the latter to sinusoidal capillarization and thereby to loss of liver function in disease.