DEFIBROTIDE FOR THE TREATMENT OF VENO-OCCLUSIVE DISEASE AFTER LIVER TRANSPLANTATION

Abstract

Background. 

Veno-occlusive disease (VOD) after liver transplantation is associated with acute rejection and poor outcome. The use of antithrombotic and thrombolytic agents is limited by their toxicity. Defibrotide is a polydeoxyribonucleotide with thrombolytic and antithrombotic properties and no systemic anticoagulant effect.

Methods. 

Defibrotide, 35–40 mg/kg/day, was administered intravenously for 21 days on a compassionate-use basis to two patients aged 66 and 49 years. VOD had developed 6 weeks and 4 months after orthotopic liver transplantation for hepatitis C and hepatitis B infection, respectively. VOD was diagnosed clinically by findings of weight gain (8.5% and 16%), ascites, jaundice (serum bilirubin 5.4 mg/dl and 21.7 mg/dl), and severe coagulopathy (in one patient), and histologically by the presence of hemorrhagic centrilobular necrosis and fibrous stenosis of the hepatic venules. One of the patients had received azathioprine as part of the immunosuppressive regimen. There was no evidence of acute cellular rejection histologically.

Results. 

After 3 weeks of defibrotide administration, the first patient showed complete clinical resolution of the VOD, and serum bilirubin level normalized. He is alive 6 months after transplantation. The second patient, treated at a later stage of disease, showed marked improvement in the coagulopathic state, but there was no resolution of the VOD. He died 2 months later of multiorgan failure due to Escherichia coli sepsis. Neither patient had side effects from the drug.

Conclusions. 

Defibrotide is a promising drug for the treatment of VOD after liver transplantation and needs to be evaluated in large, prospective studies.

The clinical syndrome of veno-occlusive disease (VOD) is characterized by painful liver enlargement, fluid retention, weight gain, and jaundice ^(1,2). Histologically, early changes include endothelial cell damage with microthrombosis, fibrin deposition, and sinusoidal dilatation; later changes include centrilobular hemorrhagic necrosis and sclerosis of the venular walls ^(1,20). VOD is associated with exposure to or ingestion of hepatotoxins such as pyrrolizidine alkaloids ⁽¹⁾, aflatoxins ⁽³⁾, and radiation injury ⁽⁴⁾. In the transplantation setting, VOD may occur as a complication of the conditioning regimens used to prepare patients for bone marrow transplantation ⁽⁵⁾ or secondary to azathioprine therapy administered to kidney ⁽⁶⁾ and liver ^(7,8) transplant recipients. The reported incidence of VOD after orthotopic liver transplantation (OLT) is 1.9%. It is strongly associated with acute rejection, and the outcome of affected patients is poor (63% died) ⁽⁹⁾.

Although the pathophysiology of VOD is not completely understood, several researchers have evaluated antithrombotic and thrombolytic drugs, including tissue plasminogen activator (t-PA) with or without heparin, as therapeutic agents ⁽¹⁰⁾. However, their use has been limited by significant toxicity, including fatal hemorrhage ⁽¹¹⁾. Defibrotide is a large, single-strand polydeoxyribonucleotide with antithrombotic properties but without significant systemic anticoagulant effects ⁽¹²⁾. It has specific aptameric binding sites on vascular endothelium, namely adenosine receptors A1 and A2 ⁽¹³⁾. Studies have shown that it increases levels of prostacyclin and prostaglandin E₂ and thrombomodulin in vivo and that it up-regulates tissue-factor pathway inhibitor and t-PA ⁽¹⁴⁾. It also decreases thrombin generation and levels of circulating plasminogen activator inhibitor ⁽¹²⁾, an apparently important mediator in the pathogenesis of VOD. Defibrotide inhibits fibrin deposition and may modulate fibropectin release which, as part of the extracellular matrix, is linked to collagen formation and fibrosis ⁽¹⁵⁾. The frequency of defibrotide treatment has been studied in a number of vascular disorders ⁽¹⁶⁾; an active dose range from 400 mg/day to 5.6 g/day has been reported ⁽¹⁴⁾. In a recent study, defibrotide treatment led to the resolution of VOD in 41% of patients after stem cell transplantation, with no significant toxicity ⁽¹⁷⁾.

In the present article we describe, for the first time, our experience with defibrotide administered on a compassionate-use basis in two patients with VOD after OLT.

METHODS

Criteria for VOD.

The clinical diagnosis of VOD was based on the presence of jaundice (bilirubin >2.0 mg/dl) and two of the following: hepatomegaly and/or right upper quadrant pain, ascites, or weight gain of more than 5% above admission weight ⁽¹⁷⁾. The diagnosis was confirmed by the finding of fibrous obliteration of hepatic veins and centrilobular hemorrhagic necrosis on liver biopsy. Acute cellular rejection was ruled out by the classic histological triad of portal inflammation, bile duct inflammation/damage, and inflammation of portal or terminal hepatic venules.

Defibrotide (Crimos Industria Farmacobiologica S.p.A, Como, Italy) was administered i.v. in normal saline in four divided doses, each infused over 2 hr. The drug was mixed with a minimum of 100 ml of normal saline to a maximum concentration of 400 mg/dl. The dose was incrementally increased by 10 mg/kg every 24 hr to a maximum potential daily dose of 60 mg/kg, depending on the side effects. The planned minimum treatment course was 14 days. Response was defined as clinical improvement with fluid mobilization, decrease in bilirubin to less than 2 mg/dl, improvement in coagulopathy, reduction in hepatomegaly and/or right upper quadrant pain, and/or reduction in other end-organ dysfunction. During therapy, platelet levels were kept at ≥20,000·10⁹/l, hematocrit ≥30%, prothrombin time less than 15 seconds, and fibrinogen >150 mg/dl using fresh-frozen plasma.

Case 1.

A 66-year-old man underwent OLT for end-stage liver disease secondary to hepatitis C virus infection using a vena cava preservation technique (piggy-back). There were no postoperative episodes of acute cellular rejection or serious systemic infections. The immunosuppression regimen included tacrolimus and prednisolone; azathioprine was not administered. The patient was discharged 5 weeks after transplantation. Laboratory values at discharge were as follows: serum alanine aminotransferase (ALT) 47 U/L, aspartate aminotransferase (AST) 45 U/L, alkaline phosphatase (ALP) 278 U/L, gamma-glutamyl transferase (GGT) 420 U/L, and bilirubin 0.8 mg/dl. The high serum ALP and GGT levels were presumed to be due to the serum high trough tacrolimus level (21.2 ng/ml), and the dose of tacrolimus was reduced.

At 6 weeks after transplantation, the patient presented with ascites, jaundice, and a weight gain of 6 kg (8.5%). Serum hemoglobin was 11.1 g/dl, white blood cell count 12.8×10⁹/L, platelets 173×10⁹/L, and international normalized ratio (INR) 1.35. Serum AST had increased to 131 μ/L, ALT to 238 U/L, ALP to 621 U/L, GGT to 708 U/L, and bilirubin to 4.7 mg/dl. Abdominal ultrasound revealed a huge amount of ascitic fluid, an enlarged liver, an enlarged spleen, and no dilatation of the bile ducts. On Doppler ultrasound, the portal and hepatic veins and the hepatic artery were patent. Endoscopic retrograde cholangiopancreatography showed normal bile ducts with no anastomotic stricture or leak. A liver biopsy specimen demonstrated pronounced centrilobular congestion, hepatocyte dropout, sinusoidal dilatation, and mild pericentral fibrosis (Fig. 1). There was no evidence of acute cellular rejection. Cavography and venography disclosed patent superior vena cava and hepatic veins.

Figure 1: Case 1: The diagnosis of VOD was confirmed by the histological findings of centrilobular hemorrhagic necrosis and mild pericentral fibrosis.

The clinical and histological findings were compatible with VOD. Diuretic treatment consisted of furosemide 120 mg and spironolactone 100 mg daily. However, there was no reduction in the patient’s weight and no improvement in the amount of ascitic fluid; frequent paracentesis was required, and serum bilirubin level increased to 5.4 mg/dl. Two months after transplantation, i.v. defibrotide 40 mg/kg was administered in four divided doses for 21 days. Over this period, the patient lost 8 kg in weight. No ascitic fluid was detected on ultrasound scan, and serum bilirubin decreased to 1.4 mg/dl, with serum ALP 136 U/L, GGT 197 U/L, ALT 14 U/L, and AST 14 U/L. Complete blood count and coagulation factors remained stable throughout therapy. The patient is currently alive at 6 months after transplantation. No side effects of defibrotide therapy were noted.

Case 2.

A 49-year-old man underwent combined kidney and liver transplantation because of chronic rejection of a kidney graft, received 8 years before for nephrosis of a single kidney, and end-stage liver disease secondary to hepatis B virus (HBV) infection. The piggy-back technique for preservation of the inferior vena cava was used. Before the transplantation procedure, he was serum hepatis B surface antigen (HBsAg)-positive and HBV DNA-negative by hybridization. Although the liver graft functioned very well immediately after the operation, the patient developed acute tubular necrosis, mandating hemodialysis. (A kidney biopsy specimen did not reveal evidence of acute rejection.) The grafted kidney regained full function after 6 weeks. No other serious complications were noted. Hepatitis B immunoglobulin was administered intraoperatively (i.v. 10,000 U), every day for the first week, and then when the serum anti-HBs titer reached ≤300 IU/ml, to prevent recurrent HBV infection. Oral lamivudine was added (dose adjusted according to the creatinine clearance test). The immunosuppression regimen included tacrolimus, prednisolone, and azathioprine. The patient was discharged 2 months later (serum ALT 10 U/L, AST 13 U/L, ALP 176 U/L, GGT 90 U/L, and bilirubin 0.7 mg/dl).

At 4 months after transplantation, the patient presented with ascites and jaundice and a 10-kg weight gain (16%). Serum liver enzymes were also increased: ALT 35 U/L, AST 51 U/L, ALP 251 U/L, GGT 86 U/L, and bilirubin 4.4 mg/dl. Thrombocyte count was 199×10⁹/L. Kidney function tests were normal, and serum creatinine level measured 1.6 mg/dl. Serum cytomegalovirus antigen was negative. The tacrolimus trough serum level was 13.0 ng/ml. Abdominal ultrasound showed an enlarged hyperechogenic liver, an enlarged spleen, and no dilatation of the bile ducts, with moderate amounts of ascitic fluid. Doppler ultrasound demonstrated patent portal vein, hepatic veins, and hepatic artery. Endoscopic retrograde cholangiopancreatography disclosed normal bile ducts with no anastomotic stricture. Cavography and venography demonstrated patent vena cava and hepatic veins. A liver biopsy specimen showed pronounced sinusoidal dilatation, congestion, and hemorrhage, with atrophy and loss of hepatocytes in central areas (Fig. 2). Immunohistochemical staining for cytomegalovirus antigen and HBsAg was negative. There was no evidence of acute cellular rejection.

Figure 2: Case 2: The diagnosis of VOD was confirmed by the histological findings of centrilobular hemorrhagic necrosis.

The patient’s serum bilirubin level continued to increase and reached a peak of 21.7 mg/dl at 6 months after transplantation. However, serum liver enzyme levels did not change (ALP 169 U/L, ALT 38 U/L, AST 46 U/L, GGT 113 U/L). Thrombocyte count was 63×10⁹/L, INR 3.1, and serum albumin 2.5 g/dl. Serum HBsAg and HBV DNA remained negative.

The clinical and histological findings were compatible with VOD. Diuretic treatment consisted of furosemide 80 mg and spironolactone 100 mg daily; however, there was no decrease in either the patient’s weight or the amount of ascitic fluid. At 6 months after transplantation, defibrotide 35 mg/kg/day was administered i.v. for 3 weeks. Although thrombocyte count increased (147×10⁹/L) and INR improved (0.94), serum bilirubin and liver enzymes remained unchanged, and there was no change in the amount of ascitic fluid. We did not increase the dose of defibrotide because we were concerned about a gastrointestinal bleeding episode that had occurred just before initiation of therapy. No side effects were observed during the treatment period.

The patient was discharged and listed for retransplantation. However, at 7 months after transplantation, he was readmitted with a severe episode of septic shock and multiorgan failure due to Escherichia coli septicemia, from which he did not recover.

DISCUSSION

We describe the outcome of defibrotide treatment in two patients with clinical and histological diagnoses of VOD at 6 weeks and 4 months after liver transplantation. The defibrotide dose ranged from 35 to 40 mg/kg/day. No drug toxicities or drug-induced hemorrhage was observed in either patient. The first patient showed clinical resolution of the VOD, and he is alive at 6 months after transplantation. The second patient showed a marked improvement in the coagulopathy state, but the VOD failed to resolve; he died 2 months later of multiorgan failure due to E coli sepsis. Defibrotide was administered for 21 days. Because escalating doses were used and the cases were analyzed retrospectively, we cannot be definitive regarding the required length of treatment. Richardson et al. ⁽¹⁷⁾ noted a response in the first 7 days of treatment in patients who went on to complete response. Our first patient responded well within this period, and the VOD did not present again; the second patient showed marked improvement in the coagulopathic state, but there was no resolution of the VOD. Further large prospective studies are needed to clarify the duration of defibrotide treatment.

The severity of VOD in patients after bone marrow transplantation has been classified as mild (VOD that is clinically obvious, requires no treatment, and resolves completely), moderate (VOD that causes signs and symptoms requiring treatment with diuretics or pain medication, but resolves completely), or severe (VOD that requires treatment but does not resolve before death or day 100 after bone marrow transplantation) ⁽¹⁸⁾. Factors predicting poor outcome are mean serum bilirubin level, percent weight gain, and the presence of ascites ⁽¹⁸⁾. About 75% of patients have mild to moderate disease; the remainder have a severe course, and almost 70% of them develop a fatal illness ⁽¹⁸⁾.

Sebagh et al. ⁽⁹⁾ studied a series of patients after liver transplantation over a period of 9 years and noted a 1.9% incidence of VOD. The disease occurred from 5 to 133 days after transplantation. In all cases, the diagnosis was made on histological grounds and was not suspected clinically. Outcome was poor: 63% of the patients died. Seventeen (89%) of the 19 patients with VOD had an episode of acute cellular rejection before or at the time of VOD, showing prominent endothelial involvement suggestive of an immunological phenomenon ⁽⁹⁾.

Neither of the patients in our study had evidence of acute cellular rejection according to the standard criteria. The administration of azathioprine as part of the immunosuppression regimen in the second patient could have induced hepatic endotheliitis and thereby VOD, as previously reported after kidney and liver transplantation ^(6–9). Although the azathioprine was withdrawn, the irreversibility of the hepatic injury indicated a late-stage azathioprine hepatotoxicity, with fibrosis occluding the venous lumen ^(7,9). Dhillon et al. ⁽⁸⁾ also reported a 43% incidence of hepatic venular stenosis in patients after liver transplantation. It was usually associated with azathioprine administration and concomitant cellular rejection.

The use of the piggy-back technique has been reported to be associated with complications related to late vena cava preservation, such as ascites ^(19–21). However, in our two patients who underwent this technique, the hepatic veins and superior vena cava were patent, and the hemodynamic studies were normal. These findings suggest that the piggy-back technique did not lead to venous outflow obstruction in these two patients.

Treatment of VOD is primarily supportive. The current therapeutic approaches to established VOD are limited. Heparin and t-PA show some evidence of efficacy (29% response rate), but they are associated with increased risk of severe bleeding (24%), and a significant number of patients experience fatal hemorrhage ⁽¹¹⁾. Infusions of human antithrombin III concentrate or activated protein C ⁽²²⁾ and prostaglandin E₁⁽²³⁾ were not beneficial.

Our novel selection of defibrotide as the therapeutic agent was based on the poor outcome associated with VOD after liver transplantation, as reported by Sebagh et al. ⁽⁹⁾, in addition to the drug’s unique thrombolytic and antithrombotic properties and apparent safety ^(12–17). In the study by Richardson et al. ⁽¹⁷⁾, defibrotide was used for the treatment of VOD after bone marrow transplantation. The active dose was approximately 25 mg/kg/day. A response was usually evident within the first 7 days, and complete response was noted in 42% of the patients, including some with multiorgan failure. No hemorrhage was observed.

Transjugular intrahepatic portosystemic shunting for VOD has recently been reported. However, encephalopathy developed in 50% of the patients, and the patients treated with cyclosporine required a drastic dose reduction owing to a modified metabolism and the occurrence of severe side effects ⁽²⁴⁾. Liver transplantation should be considered for severe VOD in those patients able to undergo the procedure ⁽²⁵⁾.

In conclusion, defibrotide treatment in two patients with VOD after liver transplantation led to full resolution of the clinical signs of the disease in one of them. Neither patient had side effects, and there was no evidence of acute cellular rejection, although one of the patients did receive azathioprine. The second patient showed a marked improvement in coagulopathy state, but the defibrotide was initiated at a late stage of the disease and had no impact on its progression.

Defibrotide is a promising drug for the treatment of VOD after liver transplantation and warrants further evaluation in large, prospective studies.

Acknowledgment.

We thank Dr. Massimo Iacobelli from Crimos Industria Farmacobiologica (Italy) for kindly providing defibrotide on a compassionate use basis.