Determining a healthy reference range and factors potentially influencing PRO-C3 - A biomarker of liver fibrosis.

BACKGROUND & AIMS: Progressive fibrosis has been identified as the major predictor of mortality in patients with non-alcoholic fatty liver disease (NAFLD). Several biomarkers are currently being evaluated for their ability to substitute the liver biopsy as the reference standard. Recent clinical studies in NAFLD/NASH patients support the utility of PRO-C3, a marker of type III collagen formation, as a marker for the degree of fibrosis, disease activity, and effect of treatment. Here we establish the healthy reference range, optimal sample handling conditions for both short- and long-term serum storage, and robustness for the PRO-C3 assay. METHODS: PRO-C3 was measured in 269 healthy volunteers and in 222 NAFLD patients. Robustness of the PRO-C3 assay was measured according to Clinical and Laboratory Standards Institute standards and included validation of interference, precision, and reagent stability, whilst sample stability was defined for storage at different temperatures and for 3 freeze-thaw cycles. Fibrosis scoring was based on histological assessments and used as a reference for the diagnostic ability of PRO-C3 to discriminate between patients with different levels of fibrosis. RESULTS: Robustness of the PRO-C3 analysis validated by interference, precision, and reagent stability was found to be within the predefined acceptance criteria. The healthy reference range was determined to be 6.1-14.7 ng/ml. Levels of PRO-C3 were not affected by sex, age, BMI, or ethnicity. Levels of PRO-C3 were able to identify patients with clinically significant fibrosis and advanced fibrosis (AUC = 0.83 (95% CI [0.77-0.88], p <0.0001), and AUC = 0.79 (95% CI [0.73-0.85], p <0.0001), respectively). CONCLUSIONS: The assay proved to be robust and sample stability was found to comply with hospital sample handling requirements. PRO-C3 measured in samples from patients with NAFLD/NASH was diagnostic for significant and advanced liver fibrosis. LAY SUMMARY: We showed that PRO-C3 levels were stable under conditions conforming with hospital sample-handling requirements. We determined a healthy reference range and showed that PRO-C3 levels were not associated with sex, age, BMI, or ethnicity. Finally, we provide further evidence of an association of PRO-C3 with increasing liver fibrosis.

Optimal cut points of plasma and urine neutrophil gelatinase-associated lipocalin for the prediction of acute kidney injury among critically ill adults: retrospective determination and clinical validation of a prospective multicentre study.

OBJECTIVES: To determine the optimal threshold of blood and urine neutrophil gelatinase-associated lipocalin (NGAL) to predict moderate to severe acute kidney injury (AKI) and persistent moderate to severe AKI lasting at least 48 consecutive hours, as defined by an adjudication panel. METHODS: A multicentre prospective observational study enrolled intensive care unit (ICU) patients and recorded daily ethylenediaminetetraacetic acid (EDTA) plasma, heparin plasma and urine NGAL. We used natural log-transformed NGAL in a logistic regression model to predict stage 2/3 AKI (defined by Kidney Disease International Global Organization). We performed the same analysis using the NGAL value at the start of persistent stage 2/3 AKI. RESULTS: Of 245 subjects, 33 (13.5%) developed stage 2/3 AKI and 25 (10.2%) developed persistent stage 2/3 AKI. Predicting stage 2/3 AKI revealed the optimal NGAL cutoffs in EDTA plasma (142.0 ng/mL), heparin plasma (148.3 ng/mL) and urine (78.0 ng/mL) and yielded the following decision statistics: sensitivity (SN)=78.8%, specificity (SP)=73.0%, positive predictive value (PPV)=31.3%, negative predictive value (NPV)=95.7%, diagnostic accuracy (DA)=73.8% (EDTA plasma); SN=72.7%, SP=73.8%, PPV=30.4%, NPV=94.5%, DA=73.7% (heparin plasma); SN=69.7%, SP=76.8%, PPV=32.9%, NPV=94%, DA=75.8% (urine). The optimal NGAL cutoffs to predict persistent stage 2/3 AKI were similar: 148.3 ng/mL (EDTA plasma), 169.6 ng/mL (heparin plasma) and 79.0 ng/mL (urine) yielding: SN=84.0%, SP=73.5%, PPV=26.6%, NPV=97.6, DA=74.6% (EDTA plasma), SN=84%, SP=76.1%, PPV=26.8%, NPV=96.5%, DA=76.1% (heparin plasma) and SN=75%, SP=75.8%, PPV=26.1, NPV=96.4%, DA=75.7% (urine). CONCLUSION: Blood and urine NGAL predicted stage 2/3 AKI, as well as persistent 2/3 AKI in the ICU with acceptable decision statistics using a single cut point in each type of specimen.

Recombinant factor VIIa in patients with coagulopathy secondary to anticoagulant therapy, cirrhosis, or severe traumatic injury: review of safety profile.

BACKGROUND: In recent years, the hemostatic agent recombinant factor VIIa (rFVIIa) has emerged as a potentially new therapeutic agent for management of coagulopathy in patients with cirrhosis or following severe traumatic injury, a complex problem for clinicians in which standard treatment strategies are not always effective. As with other hemostatic agents, a primary safety concern of rFVIIa therapy is the theoretical possibility that systemic administration could confer an increased risk of thrombotic complications. So far, clinical experience indicates rFVIIa to be a safe treatment for currently approved indications within hemophilia. Little information is available, however, for patient populations outside this clinical setting. STUDY DESIGN AND METHODS: This article reviews critical safety data obtained from 13 Novo Nordisk-sponsored clinical trials of rFVIIa in patients with coagulopathy secondary to anticoagulant therapy, cirrhosis, or severe traumatic injury. RESULTS: Thrombotic adverse events were reported for 5.3 percent (23/430) of placebo-treated patients and 6.0 percent (45/748) of patients on active treatment. No significant difference was found between placebo-treated and rFVIIa-treated patients with respect to the incidence of thrombotic AEs, either on an individual trial basis or for these trial populations combined (p=0.57). CONCLUSION: An important determinant for the safety profile reported here is likely to be the specific mechanism of action of rFVIIa, shown in experimental studies to be localized to the site of vascular injury where tissue factor is exposed.

Safety and hemostatic effect of recombinant activated factor VII in cirrhotic patients undergoing partial hepatectomy: a multicenter, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Coagulopathy caused by cirrhosis may contribute to excessive bleeding during hepatectomy. We evaluated the hemostatic effect and safety of recombinant factor VIIa (rFVIIa) in cirrhotic patients undergoing partial hepatectomy. METHODS: Patients were randomized to rFVIIa 50 or 100 mug/kg or placebo, administered intravenously 10 minutes before surgery and every second hour during surgery. The primary efficacy end points were the proportion of patients receiving red blood cell (RBC) transfusions and the amount of RBCs transfused. The RBC transfusion trigger was blood loss of 500 mL. Safety end points included thromboembolic and adverse events. RESULTS: No statistically significant effect of rFVIIa treatment on efficacy end points was observed. Serious and thromboembolic adverse events occurred at similar incidences in the study groups. CONCLUSIONS: Using blood loss as a transfusion trigger, the efficacy of rFVIIa in reducing the requirement for RBC transfusion was not established in this study. No safety concerns were identified.

Safety and efficacy of a single bolus administration of recombinant factor VIIa in liver transplantation due to chronic liver disease.

Orthotopic liver transplantation (OLT) can be associated with excessive blood loss. As a result, there may be increased risk of adverse outcomes. Activated recombinant factor VII (rFVIIa) has demonstrated the ability to improve hemostasis in a variety of disorders; however, there has been a limited amount of research into its use in OLT. The purpose of this dose-finding study was to examine the efficacy and safety of rFVIIa in the reduction of bleeding in patients undergoing OLT. In this double-blind trial, patients with end-stage liver disease scheduled for OLT were randomized to 1 of 4 parallel study groups. They received a single intravenous bolus of rFVIIa (20, 40, or 80 microg/kg) or placebo prior to surgery. The primary assessment endpoint was the total number of red blood cell (RBC) units transfused perioperatively. Safety was evaluated by adverse events reported. Eighty-three comparable patients were randomized to receive study product, with 82 ultimately undergoing OLT. There were no significant differences in required RBC units between the placebo and rFVIIa study groups. The number of adverse events was comparable between study groups. In conclusion, rFVIIa has a good safety profile in patients undergoing OLT. However, the doses studied did not have any effect on the number of RBC transfusions required.

A study of the pharmacokinetics and safety of recombinant activated factor VII in healthy Caucasian and Japanese subjects.

In this randomized, placebo-controlled, double-blind, single-centre, dose escalation study, we report the first evaluation of the pharmacokinetics and safety of recombinant activated factor VII (rFVIIa) in healthy Caucasian and Japanese subjects. Thirty-two healthy subjects were stratified according to sex and ethnic group to receive single bolus intravenous injections of three different doses of rFVIIa (40, 80, 160 microg/kg rFVIIa) or placebo, each separated by a 7-day wash-out period. Blood samples were taken up to 24 h after dosing. The factor VII clotting activity appeared to be dose dependent, but independent of sex and ethnic group. Statistical analyses demonstrated no significant effect of dose, sex or ethnicity on the dose-normalized mean area under the plasma concentration-time curve AUC0-t, indicating dose proportionality. No serious adverse events or thromboembolic events were reported. Analyses of coagulation parameters did not suggest induction of systemic coagulation when dosing rFVIIa up to 160 microg/kg. In conclusion, the pharmacokinetics of rFVIIa in Caucasian and Japanese subjects are similar, and no safety issues were identified.

Recombinant coagulation factor VIIa in major liver resection: a randomized, placebo-controlled, double-blind clinical trial.

BACKGROUND: Prevention of bleeding episodes in noncirrhotic patients undergoing partial hepatectomy remains unsatisfactory in spite of improved surgical techniques. The authors conducted a randomized, placebo-controlled, double-blind trial to evaluate the hemostatic effect and safety of recombinant factor VIIa (rFVIIa) in major partial hepatectomy. METHODS: Two hundred four noncirrhotic patients were equally randomized to receive either 20 or 80 microg/kg rFVIIa or placebo. Partial hepatectomy was performed according to local practice at the participating centers. Patients were monitored for 7 days after surgery. Key efficacy parameters were perioperative erythrocyte requirements (using hematocrit as the transfusion trigger) and blood loss. Safety assessments included monitoring of coagulation-related parameters and Doppler examination of hepatic vessels and lower extremities. RESULTS: The proportion of patients who required perioperative red blood cell transfusion (the primary endpoint) was 37% (23 of 63) in the placebo group, 41% (26 of 63) in the 20-microg/kg group, and 25% (15 of 59) in the 80-microg/kg dose group (logistic regression model; P = 0.09). Mean erythrocyte requirements for patients receiving erythrocytes were 1,024 ml with placebo, 1,354 ml with 20 microg/kg rFVIIa, and 1,036 ml with 80 microg/kg rFVIIa (P = 0.78). Mean intraoperative blood loss was 1,422 ml with placebo, 1,372 ml with 20 microg/kg rFVIIa, and 1,073 ml with 80 microg/kg rFVIIa (P = 0.07). The reduction in hematocrit during surgery was smallest in the 80-microg/kg group, with a significant overall effect of treatment (P = 0.04). CONCLUSIONS: Recombinant factor VIIa dosing did not result in a statistically significant reduction in either the number of patients transfused or the volume of blood products administered. No safety issues were identified.

Recombinant factor VIIa for upper gastrointestinal bleeding in patients with cirrhosis: a randomized, double-blind trial.

BACKGROUND & AIMS: Upper gastrointestinal bleeding (UGIB) is a severe and frequent complication of cirrhosis. Recombinant coagulation factor VIIa (rFVIIa) has been shown to correct the prolonged prothrombin time in patients with cirrhosis and UGIB. This trial aimed to determine efficacy and safety of rFVIIa in cirrhotic patients with variceal and nonvariceal UGIB. METHODS: A total of 245 cirrhotic patients (Child-Pugh < 13; Child-Pugh A = 20%, B = 52%, C = 28%) with UGIB (variceal = 66%, nonvariceal = 29%, bleeding source unknown = 5%) were randomized equally to receive 8 doses of 100 microg/kg rFVIIa or placebo in addition to pharmacologic and endoscopic treatment. The primary end point was a composite including: (1) failure to control UGIB within 24 hours after first dose, or (2) failure to prevent rebleeding between 24 hours and day 5, or (3) death within 5 days. RESULTS: Baseline characteristics were similar between rFVIIa and placebo groups. rFVIIa showed no advantage over standard treatment in the whole trial population. Exploratory analyses, however, showed that rFVIIa significantly decreased the number of failures on the composite end point (P = 0.03) and the 24-hour bleeding control end point (P = 0.01) in the subgroup of Child-Pugh B and C variceal bleeders. There were no significant differences between rFVIIa and placebo groups in mortality (5- or 42-day) or incidence of adverse events including thromboembolic events. CONCLUSIONS: Although no overall effect of rFVIIa was observed, exploratory analyses in Child-Pugh B and C cirrhotic patients indicated that administration of rFVIIa significantly decreased the proportion of patients who failed to control variceal bleeding. Dosing with rFVIIa appeared safe. Further studies are needed to verify these findings.

Potential role of recombinant factor VIIa as a hemostatic agent.

Recombinant factor VIIa (rFVIIa) has been shown to induce hemostasis in hemophilia patients with inhibitors against factor VIII or factor IX independent of factor VIII/factor IX. Factor VIIa binds to tissue factor (TF) exposed at the site of injury and generates, through factor X activation on the TF-bearing cells, enough thrombin to activate factors VIII, V, and XI, as well as platelets. The thrombin-activated platelets provided a perfect template for binding of activated factors VIII, IX, and V, further activation of factor X, and thrombin generation. Factor VIIa in high concentrations binds to thrombin-activated platelets and is capable of activating factor X, thereby generating thrombin independent of the presence of factor VIII or factor IX. Accordingly, rFVIIa has been shown to initiate hemostasis in severe hemophilila patients with inhibitors subjected to major surgery and suffering from serious limb- and life-threatening bleeding. Since rFVIIa enhances thrombin generation-thereby providing the formation of tight, stable fibrin hemostatic plugs resistance to premature lysis-it should be hemostatic in other situations characterized by impaired thrombin generation. A hemostatic effect has been reported in patients with various platelet disorders and factor XI deficiency. Further, a hemostatic effect of rFVIIa has been reported in patients subjected to trauma and extensive surgery who have developed profuse, excessive bleeding resulting in hemodilution and changes in coagulation patterns. rFVIIa was developed to treat bleeding in hemophilia patients with inhibitors against factor VIII or factor IX and has been shown to induce effective hemostasis in most such patients and also in life- and limb-threatening bleeding. It has also been used successfully to stop bleeding in patients who do not have hemophilia but who do have acquired antibodies against factor VIII (acquired hemophilia). rFVIIa initiates hemostasis by forming a complex with TF exposed as a result of vessel wall injury. Pharmacologic doses of rFVIIa can enhance thrombin generation on platelets that are already thrombin-activated, resulting in the formation of full thrombin burst. By enhancing thrombin generation, rFVIIa helps to form tight, stable, fibrin plugs resistant to premature fibrinolysis. This also maintains hemostasis in the absence of factor VIII or factor IX. Pharmacologic doses of rFVIIa may accordingly be of benefit in producing hemostasis in situations other than hemophilia characterized by profuse bleeding and impaired thrombin generation. There is now clinical experience indicating a hemostatic effect in patients with thrombocytopenia and functional platelet defects. rFVIIa has also been successfully used in acute trauma patients with profuse bleedings and in other bleeding situations.

rFVIIai in Acute Coronary Syndromes.

Following vessel wall injury, tissue factor (TF) is exposed and forms complexes with already activated factor VII (FVIIa) present in the circulating blood, thereby initiating the hemostatic process. After the first FXa is formed, the TF pathway inhibitor (TFPI) forms a complex with FXa, and a quaternary complex is formed, TF/FVIIa/ FXa/TFPI, which inhibits the first step of the hemostatic pathway. Recombinant activated FVII (rFVIIa) has been developed for use as a hemostatic agent (NovoNordisk A/S, Denmark). Active site-inactivated rFVIIa (rFVIIai) has also been prepared and was shown to have a faster association to and a slower dissociation from TF than rFVIIa, resulting in a lower calculated Kd of rFVIIai compared with rFVIIa. In various animal models rFVIIai has been demonstrated to prevent or diminish immediate thrombus formation at the site of vessel wall injury (athroplasty or other forms of mechanical injury) as well as the development of long-term intima thickening. The inflammatory response following endotoxin-induced sepsis was shown to decrease after administration of rFVIIai. Also, survival increased in the rFVIIai-treated animals in this study. In addition, ischemia-reperfusion injury was mitigated by rFVIIai. In a limited number of patients undergoing percutaneous transluminal coronary angioplasty (PTCA), rFVIIai was observed to allow PTCA to be performed at lower doses of heparin than what has been reported previously.

Ongoing NovoSeven trials.

Recombinant activated factor VII (rFVIIa, 'NovoSeven' was initially developed for the treatment of bleeding in patients with haemophilia and inhibitors, and is currently licensed in most countries worldwide. The mechanism of action suggests that its enhancing effects in haemostasis are limited to the site of injury and that systemic activation of the coagulation cascade does not occur. These properties, together with anecdotal reports of its beneficial effects in different patient populations with severe bleeds, suggest that rFVIIa may be valuable as a general haemostatic agent. In case reports, rFVIIa has been reported to reduce bleeding in patients with liver disease, thrombocytopenia or thrombocytopathia, trauma those undergoing radical prostatectomy or receiving oral anticoagulant therapy. A number of clinical trials have recently been initiated to collect data on the safety and efficacy of rFVIIa in these patient groups. The beneficial effects of rFVIIa occurring in these studies will support the potential use of rFVIIa as a universal haemostatic agent.

Pharmacodynamics of active site-inhibited factor VIIa in endotoxin-induced coagulation in humans.

BACKGROUND: Inhibition of the tissue factor-factor VIIa pathway attenuated the activation of coagulation and prevented death in a gram-negative bacteremia primate model of sepsis. This lethal animal model suggested that tissue factor also influences inflammatory cascades. METHODS: This trial examined the pharmacodynamic effects of active site-inhibited factor VIIa (FFR-recombinant factor VIIa [rFVIIa]; ASIS) on endotoxin-induced procoagulant, fibrinolytic, and inflammatory responses in healthy humans. A double-blind, randomized, placebo-controlled, parallel-group study was conducted in 12 healthy male volunteers. Subjects received a bolus infusion of 2-ng/kg endotoxin, followed by a bolus infusion of ASIS (400 microg/kg) or placebo 10 minutes later. RESULTS: Endotoxin injection induced inflammation, activation of coagulation, and activation and subsequent inhibition of fibrinolysis. ASIS infusion completely blocked thrombin and fibrin generation, as measured by plasma levels of prothrombin fragment (no increase in the ASIS group, as compared with a 13-fold increase in the placebo group at 4 hours; P <.01), soluble fibrin, and fibrin split product D-dimer. ASIS did not alter endotoxin-induced changes in the fibrinolytic system, cytokine levels, or markers of endothelial (E-selectin, thrombomodulin) or platelet (P-selectin) activation. CONCLUSIONS: In summary, ASIS effectively and selectively attenuates tissue factor-induced thrombin generation. Because ASIS was well tolerated, this study provides seminal data to further characterize its anticoagulant and putative anti-inflammatory effects in critically ill patients.

To general haemostasis--the evidence-based route.

Recombinant factor VIIa (rFVIIa) was developed for the treatment of bleeding in haemophilia patients with inhibitors and has also been used successfully in non-haemophilia patients with acquired antibodies against FVIII (acquired haemophilia). Based on dose-finding trials and a compassionate-use programme, rFVIIa was approved for use in haemophilia patients with inhibitors in 1996. At pharmacological doses, rFVIIa has been found to enhance thrombin generation on already activated platelets. Therefore, it is likely that rFVIIa will also be beneficial in providing haemostasis in other situations characterised by profuse bleedings and an impaired thrombin generation. Patients with thrombocytopenia have a decreased number of platelets and thus an impaired thrombin generation. A reduction in bleeding time was reported in approximately 50% of patients with thrombocytopenia and a prolonged bleeding time who participated in a trial of rFVIIa. Moreover, in 8 patients with 9 overt bleeds who were involved in the study, bleeding stopped in 7 episodes after rFVIIa administration. Case reports on the haemostatic effect of rFVIIa in thrombocytopenia have also been published. Reports have also been published on the successful use of rFVIIa in patients with platelet function deficiencies such as Glanzmann's thrombasthenia and Bernard-Soulier syndrome. A number of haemostatic changes occur after extensive trauma, surgery and bleeding, all of which potentially contribute to an impaired thrombin generation. The effect of rFVIIa has been demonstrated in a number of patients after trauma and bleeds and upper gastrointestinal bleeding episodes. Reports on the beneficial use of rFVIIa in liver transplantation have also been published. Several randomised blinded studies are now underway in e.g. hepatectomy, upper gastrointestinal bleedings, transplantations and intra-cerebral bleeds. In summary, rFVIIa may be an effective and safe method to induce haemostasis in patients within areas of coagulation factor deficiency or platelet disorders and the ongoing and planned randomised studies may lead the way to the use of rFVIIa in general haemostasis.

Safety and efficacy of recombinant factor VIIa in patients with liver disease undergoing laparoscopic liver biopsy.

BACKGROUND & AIMS: Activated recombinant factor VII (rFVIIa) has been shown to be effective in correcting prolonged prothrombin time (PT) in cirrhotic patients. The main objective of this study was to evaluate the effect of 4 (5, 20, 80, and 120 microg/kg) doses of rFVIIa on correction of PT and the time to achieve hemostasis in cirrhotic patients with coagulopathy who are undergoing laparoscopic liver biopsy. METHODS: Seventy-one patients (parts I and II) with advanced liver disease (Child-Turcotte B or C), platelet count > or =60,000/mm3, and PT in the range of 3-15 seconds above normal were included in the study. Efficacy endpoints were normalization of PT and time to hemostasis. RESULTS: PT was corrected to normal levels (<13.1 seconds) in the majority of patients. The duration of normalization of PT was longer in patients treated with higher doses of rFVIIa. Forty-eight (74%) of 65 patients (part II) achieved hemostasis within 10 minutes. No correlation between the time to hemostasis and duration of correction of PT was observed. None of the patients required operative intervention or transfusion of blood/blood products to control bleeding. One thrombotic event and one case of disseminated intravascular coagulation were reported, but both events were considered by the investigator as unlikely to be related to treatment with rFVIIa. CONCLUSIONS: The results of this study suggest that treatment with rFVIIa may offer benefit for patients with liver disease undergoing laparoscopic biopsy.

Antithrombotic effect of tissue factor inhibition by inactivated factor VIIa: an ex vivo human study.

FFR-rFVIIa is an inactivated recombinant factor VIIa (rFVIIa) that inhibits the binding of factor VIIa to tissue factor (TF). It has been shown to prevent TF-induced thrombosis in animals. The present study is a substudy of the Active Site Inhibited Seven (ASIS) trial and examines the antithrombotic effect of 3 doses of FFR-rFVIIa in 24 patients undergoing percutaneous coronary intervention (PCI). Group 1 (n=9) received 400 microg/kg FFR-rFVIIa and 40 to 50 U/kg heparin, group 2 (n=7) received 200 microg/kg FFR-rFVIIa and 100 U/kg heparin, and group 3 (n=8) received 50 microg/kg FFR-rFVIIa and 100 U/kg heparin. Blood thrombogenicity was assessed as total thrombus area and fibrin deposition on the perfusion chamber at shear rate conditions typical of mild-moderate coronary stenosis. Baseline blood thrombogenicity was evaluated a day before PCI, after heparin administration. A second perfusion chamber study was performed just before PCI, 15 minutes after the administration of heparin and FFR-rFVIIa. Thrombus formation at a high shear rate was markedly reduced in groups 1 and 2 after drug administration, by 79% to 84% and 76% to 87%, respectively (P<0.004 [group 1], P<0.04 [group 2]). In group 3, moderate thrombus reduction of 46% to 48% was achieved (P<0.04). Fibrin deposition in all 3 groups was nearly eliminated after drug administration. Our data demonstrate that FFR-rFVIIa has a potent antithrombotic effect at different shear rates and severe arterial injury conditions.