hemophilia is a hereditary ( x - linked ) bleeding disorder characterized by a lack of clotting factor viii ( hemophilia a ) or factor ix ( hemophilia b ) . this congenital disease leads to inadequate clotting associated with serious consequences , including joint bleeding and swelling resulting in arthropathy , intracerebral bleeding and brain hemorrhage , damage to organs and pain , with some bleeding episodes being fatal . the development of neutralizing antibodies to coagulation factor viii ( fviii ) or factor ix ( fix ) remains the most serious complication of replacement therapy with factor concentrates in hemophilia patients . up to 32% of hemophilia a patients develop inhibitors ( anti - drug antibodies ) to exogenous fviii . while incidence rates of inhibitors to exogenous fix in hemophilia b are lower , ranging from 1% to 6% , the development of these inhibitors , when they do occur , is frequently associated with severe allergic reactions , including life - threatening anaphylactoid responses . although attempts have been made to eradicate inhibitors with plasmapheresis , immunosuppressive therapy or immune - tolerance induction ( iti ) , these therapies are not always effective . for example , iti is successful in 7085% of fviii inhibitor cases and in 30% of anti - fix antibody - positive patients . therefore , many patients with inhibitors rely on bypassing products to control or prevent hemorrhages . the efficacy of available bypassing agents such as activated prothrombin complex concentrate ( apcc ; feiba , baxter ag , vienna , austria ) and recombinant activated factor vii ( rfviia ; novoseven , novo nordisk a / s , bagsvaerd , denmark ) is inferior at controlling bleeds when compared with fviii / fix substitution therapy in patients without inhibitors . furthermore , these bypassing products have short half - lives ( t ) that may limit their application in prophylactic treatment . for example , due to the rapid systemic clearance of rfviia ( terminal t of 2.4 h in humans ) , use of this therapy often requires an inconvenient regimen of two to three doses given at 2- to 3-h intervals to achieve hemostasis following an acute bleed . it should be noted , however , that the hemostatic activity of rfviia may endure for longer than its plasma kinetics , as suggested by uptake and binding to tissue factor ( tf ) in perivascular tissues . in recent years , different technologies have been developed to prolong the t of recombinant coagulation factors . amongst these , pegylation and fusion technologies applied to wild - type or genetically modified proteins have been most widely explored . csl behring has developed the albumin fusion technology platform in which a fusion protein linking a human coagulation factor and human albumin is expressed as a single recombinant construct in chinese hamster ovary ( cho ) cells ( i.e. recombinant human albumin is fused to the c - terminus of rviia via a flexible glycine - serine linker : rviia - fp ) . this technology was used to successfully prolong the plasma t of recombinant coagulation fix , and the respective fusion protein , rix - fp , has a t that is less than 5-fold longer in hemophilia b patients compared with plasma - derived and recombinant fix products . the aim of the present studies was to characterize the pharmacokinetic / pharmacodynamic ( pk / pd ) profile of rviia - fp in a range of animal models in comparison with rfviia . different doses were selected for each species investigated to reflect between - species variations in clotting response . female fviii knockout mice   were used to compare the pk properties of rviia - fp and rfviia ( novoseven ) . after correction for the molecular weight of the fusion protein , an equimolar dose ( based on the molecular weight of the rviia - moiety for rviia - fp only ) of 100 g kg for both test items was administered as a single intravenous ( i.v . ) dose into the lateral tail vein . in total , blood samples were drawn at 2 , 5 and 30 min , and 1 , 2 , 4 , 6 , 16 , 24 and 48 h post - administration and pooled ( n = 3 per time - point ) , then processed to 10% citrate ( 3.13% w / v ) plasma . the pk properties of rviia - fp and rfviia , both administered as a single 900 g kg i.v . dose , based on total respective molecular weights , were also assessed in eight rats per treatment group ( with blood samples drawn at 5 , 15 and 30 min , and 1 , 2 , 4 , 8 and 24 h after injection ) , and in three rabbits per treatment group following single i.v . doses ( based on the respective total molecular weight of each test item ) of 2000 and 275 g kg , respectively ( with blood samples drawn at pre - dose , 1 , 5 , 10 and 30 min , and 1 , 2 , 4 , 8 , 24 , 48 , 72 , 96 and 168 h post - dose ) . finally , the pk properties of rviia - fp and rfviia were evaluated in cynomolgus monkeys after single i.v . doses ( based on total molecular weight ) of 2700 and 270 g kg , respectively , with two animals in each treatment group . blood samples were drawn at 5 and 15 min , and 1 , 2 , 4 , 8 , 24 , 48 , 72 , 96 and 120 h post - dose . in the rabbit and monkey pk studies , the higher doses of rviia - fp vs. rfviia reflect the relative potency ratio of 810 found as a consequence of the higher molecular weight , and its reduced specific fviia activity due to the albumin moiety of the fusion protein . this potency ratio matches the difference in selective fviia activity between rviia - fp and rfviia as observed using the staclot viia - rtf assay system ( diagnostica stago , asnires , france ) , selective for activated fvii . a commercially available enzyme - linked immunosorbent assay ( elisa)-based system ( cedarlane laboratories limited , burlington , on , canada ) was used to evaluate human fvii antigen ( fvii : ag ) plasma levels obtained from the rodent species . in the analysis of fvii : ag data , the first value below the limit of quantification ( 250 ng ml ) was imputed to one - half of this limit ( 125 ng ml ) in the rviia - fp and rfviia groups . subsequent values below the limit of quantification were ignored in the calculation of pk parameters . as enzymatic fvii activity is the more widely used pk parameter when monitoring fviia plasma levels in patients [ 1315 ] , the staclot viia - rtf assay was used ( in addition to fvii : ag ) in plasma samples derived from rabbits and monkeys to determine selective fviia activity . from each preclinical study described above , pk parameter estimates were derived using winnonlin software version 6.2 ( pharsight , cary , nc , usa ) , including maximum concentration ( cmax ) , area under the curve ( auc ) from t = 0 to last observation , auc0 , t , mean residence time , clearance ( cl ) , incremental recovery and in vivo recovery ( ivr ) , which was calculated assuming a plasma volume of 40 ml kg . ivr was the maximum observed plasma level multiplied by plasma volume and divided by dose ; it is a dimensionless ratio and was expressed as a percentage . were administered rviia - fp at dose levels of 0.5 , 1 , 2 , 4 and 8 mg kg and rfviia at dose levels of 0.5 , 1 and 2 mg kg on an equimolar basis for fviia , with 15 animals per treatment group . the tail was cut with a scalpel knife at the start of the observation period under deep anesthesia ( sodium pentobarbital , 74.5 mg kg ) , removing approximately 34 mm of the tail tip . immediately upon lesion , the tail tip was submerged in isotonic saline solution ( 0.9% ) , which was kept at the physiological body temperature of the mice using a water bath , until hemostasis occurred . the volume of total blood loss was calculated over an observation period of 30 min , or until hemostasis occurred , by measuring the hemoglobin ( sysmex f-820 , sysmex europe gmbh , norderstedt , germany ) present in the isotonic saline . the procoagulant effects of rfviia and rviia - fp were dose - proportional with parallel dose response curves and maximum responses obtained at approximately 4 and 11 mg kg , respectively , with rfviia having a 2.7-fold higher potency ( figure s1 ) . when equimolar doses for both activated fviia concentrates were adjusted according to their selective fviia activity , they showed similar hemostatic activity ( figure s2 ) . the duration of the pd effect of rfviia and rviia - fp was assessed in hemophilia a mice by tga . citrate ( 10% v / v ) and corn trypsin inhibitor - stabilized ( 50 g ml ) blood was collected ( 310 animals per treatment group and time - point ) at 5 min , and 4 , 7 and 16 h after administration of equimolar doses of 400 g kg of either rviia - fp or rfviia , based on fviia molecular weight . tga was performed by calibrated thrombinography ( calibrated automated thrombogram [ cat ] , thrombinoscope bv , maastricht , the netherlands ) after extrinsic activation using ppp - reagent 5 pm ( thrombinoscope bv ) . thrombin generation and time to onset of observed thrombin generation ( lagtime ) were calculated for each treatment group . the procoagulant activity of rviia - fp and rfviia up to 24 h was assessed in a rabbit venous thrombosis model after inducing temporary venous stasis ( modified wessler test ) . in a pilot study , new zealand white or chinchilla bastard rabbits were treated with either rviia - fp ( n = 69 ) at a dose of 450 or 900 g kg , or rfviia ( n = 69 ) at dose levels of 125 or 450 g kg ( based on the respective total molecular weight ) . activated prothrombin complex concentrate ( apcc ) was used as a positive control ( n = 3 ; 50 u kg ) . incidence of thrombosis was the primary endpoint , and observed thrombi were graded according to a scoring system of 03 ( 0 = no clot ; 1 = one or a few small clots , no measurable weight ; 2 = not fully occluding clot , one or several clots of bigger size , weight can be measured ; 3 = segment fully occluded by clot , weight can be measured ) . based on this pilot study , the procoagulant activity of rviia - fp and rfviia was determined as being equivalent at a dose ratio of 7.2 : 1 based on total protein weight . new zealand white rabbits ( 2.33.1 kg ) were therefore allocated to rviia - fp ( n = 22 ) or rfviia ( n = 16 ) at dose levels of 2000 and 275 g kg , respectively , or placebo ( n = 6 ) . doses were administered intravenously via the ear vein to anesthetized animals , with the negative control ( placebo ) group receiving isotonic saline . to assess the procoagulant potential of the test substances after 10 min , 12 h or 24 h , the animals were anesthetized before or at 12 or 24 h after administration of test substance with an i.v . injection of 5 mg kg ketamine ( 10% ) and 0.5 mg kg xylazine ( 2% ) solution , and maintained by i.v . infusion of 25 mg kg h ketamine ( 10% ) and 2.5 mg kg h xylazine ( 2% ) . at the 10-min , 12- and 24-h time - points , the left and right jugular veins were exposed ( n = 610 per time - point ) and a segment of approximately 2 cm was isolated . blood was allowed to fill the vein segments , then a second ligature was placed approximately 1.5 cm cranial to the first one , causing complete stasis in the isolated segment . ten minutes after stasis induction , the right vein segment was excised and dissected in a petri dish filled with sodium citrate solution . any observed thrombi were graded from 0 to 3 and the distribution of the thrombus score in the two groups was compared by an exact wilcoxon rank sum test . prothrombin time ( pt ) was determined ex vivo using thromborel s ( siemens healthcare diagnostics products gmbh , marburg , germany ) with a schnitger and gross coagulometer . different doses were selected for each species investigated to reflect between - species variations in clotting response . female fviii knockout mice   were used to compare the pk properties of rviia - fp and rfviia ( novoseven ) . after correction for the molecular weight of the fusion protein , an equimolar dose ( based on the molecular weight of the rviia - moiety for rviia - fp only ) of 100 g kg for both test items was administered as a single intravenous ( i.v . ) dose into the lateral tail vein . in total , blood samples were drawn at 2 , 5 and 30 min , and 1 , 2 , 4 , 6 , 16 , 24 and 48 h post - administration and pooled ( n = 3 per time - point ) , then processed to 10% citrate ( 3.13% w / v ) plasma . the pk properties of rviia - fp and rfviia , both administered as a single 900 g kg i.v . dose , based on total respective molecular weights , were also assessed in eight rats per treatment group ( with blood samples drawn at 5 , 15 and 30 min , and 1 , 2 , 4 , 8 and 24 h after injection ) , and in three rabbits per treatment group following single i.v . doses ( based on the respective total molecular weight of each test item ) of 2000 and 275 g kg , respectively ( with blood samples drawn at pre - dose , 1 , 5 , 10 and 30 min , and 1 , 2 , 4 , 8 , 24 , 48 , 72 , 96 and 168 h post - dose ) . finally , the pk properties of rviia - fp and rfviia were evaluated in cynomolgus monkeys after single i.v . doses ( based on total molecular weight ) of 2700 and 270 g kg , respectively , with two animals in each treatment group . blood samples were drawn at 5 and 15 min , and 1 , 2 , 4 , 8 , 24 , 48 , 72 , 96 and 120 h post - dose . in the rabbit and monkey pk studies , the higher doses of rviia - fp vs. rfviia reflect the relative potency ratio of 810 found as a consequence of the higher molecular weight , and its reduced specific fviia activity due to the albumin moiety of the fusion protein . this potency ratio matches the difference in selective fviia activity between rviia - fp and rfviia as observed using the staclot viia - rtf assay system ( diagnostica stago , asnires , france ) , selective for activated fvii . a commercially available enzyme - linked immunosorbent assay ( elisa)-based system ( cedarlane laboratories limited , burlington , on , canada ) was used to evaluate human fvii antigen ( fvii : ag ) plasma levels obtained from the rodent species . in the analysis of fvii : ag data , the first value below the limit of quantification ( 250 ng ml ) was imputed to one - half of this limit ( 125 ng ml ) in the rviia - fp and rfviia groups . subsequent values below the limit of quantification were ignored in the calculation of pk parameters . as enzymatic fvii activity is the more widely used pk parameter when monitoring fviia plasma levels in patients [ 1315 ] , the staclot viia - rtf assay was used ( in addition to fvii : ag ) in plasma samples derived from rabbits and monkeys to determine selective fviia activity . from each preclinical study described above , pk parameter estimates were derived using winnonlin software version 6.2 ( pharsight , cary , nc , usa ) , including maximum concentration ( cmax ) , area under the curve ( auc ) from t = 0 to last observation , auc0 , t , mean residence time , clearance ( cl ) , incremental recovery and in vivo recovery ( ivr ) , which was calculated assuming a plasma volume of 40 ml kg . ivr was the maximum observed plasma level multiplied by plasma volume and divided by dose ; it is a dimensionless ratio and was expressed as a percentage . in an acute bleeding study , hemophilia a mice were administered rviia - fp at dose levels of 0.5 , 1 , 2 , 4 and 8 mg kg and rfviia at dose levels of 0.5 , 1 and 2 mg kg on an equimolar basis for fviia , with 15 animals per treatment group . the tail was cut with a scalpel knife at the start of the observation period under deep anesthesia ( sodium pentobarbital , 74.5 mg kg ) , removing approximately 34 mm of the tail tip . immediately upon lesion , the tail tip was submerged in isotonic saline solution ( 0.9% ) , which was kept at the physiological body temperature of the mice using a water bath , until hemostasis occurred . the volume of total blood loss was calculated over an observation period of 30 min , or until hemostasis occurred , by measuring the hemoglobin ( sysmex f-820 , sysmex europe gmbh , norderstedt , germany ) present in the isotonic saline . the procoagulant effects of rfviia and rviia - fp were dose - proportional with parallel dose  response curves and maximum responses obtained at approximately 4 and 11 mg kg , respectively , with rfviia having a 2.7-fold higher potency ( figure s1 ) . when equimolar doses for both activated fviia concentrates were adjusted according to their selective fviia activity , they showed similar hemostatic activity ( figure s2 ) . the duration of the pd effect of rfviia and rviia - fp was assessed in hemophilia a mice by tga . citrate ( 10% v / v ) and corn trypsin inhibitor - stabilized ( 50 g ml ) blood was collected ( 310 animals per treatment group and time - point ) at 5 min , and 4 , 7 and 16 h after administration of equimolar doses of 400 g kg of either rviia - fp or rfviia , based on fviia molecular weight . tga was performed by calibrated thrombinography ( calibrated automated thrombogram [ cat ] , thrombinoscope bv , maastricht , the netherlands ) after extrinsic activation using ppp - reagent 5 pm ( thrombinoscope bv ) . thrombin generation and time to onset of observed thrombin generation ( lagtime ) were calculated for each treatment group . the procoagulant activity of rviia - fp and rfviia up to 24 h was assessed in a rabbit venous thrombosis model after inducing temporary venous stasis ( modified wessler test ) . in a pilot study , new zealand white or chinchilla bastard rabbits were treated with either rviia - fp ( n = 69 ) at a dose of 450 or 900 g kg , or rfviia ( n = 69 ) at dose levels of 125 or 450 g kg ( based on the respective total molecular weight ) . activated prothrombin complex concentrate ( apcc ) was used as a positive control ( n = 3 ; 50 u kg ) . incidence of thrombosis was the primary endpoint , and observed thrombi were graded according to a scoring system of 03 ( 0 = no clot ; 1 = one or a few small clots , no measurable weight ; 2 = not fully occluding clot , one or several clots of bigger size , weight can be measured ; 3 = segment fully occluded by clot , weight can be measured ) . based on this pilot study , the procoagulant activity of rviia - fp and rfviia was determined as being equivalent at a dose ratio of 7.2 : 1 based on total protein weight . new zealand white rabbits ( 2.33.1 kg ) were therefore allocated to rviia - fp ( n = 22 ) or rfviia ( n = 16 ) at dose levels of 2000 and 275 g kg , respectively , or placebo ( n = 6 ) . doses were administered intravenously via the ear vein to anesthetized animals , with the negative control ( placebo ) group receiving isotonic saline . to assess the procoagulant potential of the test substances after 10 min , 12 h or 24 h , the animals were anesthetized before or at 12 or 24 h after administration of test substance with an i.v . injection of 5 mg kg ketamine ( 10% ) and 0.5 mg kg xylazine ( 2% ) solution , and maintained by i.v . infusion of 25 mg kg h ketamine ( 10% ) and 2.5 mg kg h xylazine ( 2% ) . at the 10-min , 12- and 24-h time - points , the left and right jugular veins were exposed ( n = 610 per time - point ) and a segment of approximately 2 cm was isolated . blood was allowed to fill the vein segments , then a second ligature was placed approximately 1.5 cm cranial to the first one , causing complete stasis in the isolated segment . ten minutes after stasis induction , the right vein segment was excised and dissected in a petri dish filled with sodium citrate solution . any observed thrombi were graded from 0 to 3 and the distribution of the thrombus score in the two groups was compared by an exact wilcoxon rank sum test . prothrombin time ( pt ) was determined ex vivo using thromborel s ( siemens healthcare diagnostics products gmbh , marburg , germany ) with a schnitger and gross coagulometer . pk parameters from mice and rats based on fvii : ag , and rabbits and monkeys based on fviia activity , are shown in table 1 . in all species , pivotal pk characteristics ( i.e. in vivo recovery , cl and t ) were improved for rviia - fp compared with rfviia . in both mice and rats , furthermore , rviia - fp showed a 2-fold enhanced in vivo recovery compared with rfviia ( table 1 ) . pharmacokinetic parameters of rviia - fp and rfviia in hemophilia a mice , rats , rabbits and cynomolgus monkeys data for mrt , cl and t are presented based on fvii antigen measurements for rodents , and based on selective fviia activity for rabbits and monkeys . for mice , samples from three animals per time - point were pooled , resulting in one assay result per time - point , a single pk curve and a single set of pk parameters . for rats , samples from four animals per time - point were tested individually and averaged to give a single pk curve and a single set of pk parameters . for rabbits and monkeys , each animal provided a full pk curve and geometric means cl , clearance ; ivr , in vivo recovery ; mrt , mean residence time ; rfviia , recombinant factor viia ; rviia - fp , fusion protein ; t , terminal elimination half - life . similarly to the rodent studies , in which pk properties of fviia were calculated only measuring fvii : ag levels , the pk profile of rviia - fp was also improved compared with rfviia in rabbits and monkeys based on selective fviia activity ( table 1 ) . in both species , cl was again lower and t extended when comparing rviia - fp with rfviia . there was a 5-fold enhanced in vivo recovery in rabbits when comparing rviia - fp with rfviia , while in monkeys there was a 1.7-fold enhanced in vivo recovery when comparing rviia - fp with rfviia ( table 1 ) . pk profiles for rviia - fp and rfviia in mice , rats , rabbits and monkeys are shown in fig 1 . time profiles of rviia - fp and rfviia in ( a ) hemophilia a mice , ( b ) rats , ( c ) rabbits and ( d ) cynomolgus monkeys ( mean  sd ) . pk curves in mice and rats are based on fvii : ag data , while the curves shown for rabbits and monkeys are based on selective fviia activity . data are mean , except for mice ( plasma pool ) and monkeys , where the curve for each animal is shown . fvii : ag , factor vii antigen ; pk , pharmacokinetic ; rfviia , recombinant factor viia ; rviia - fp , fusion protein ; sd , standard deviation . the improved pk profile of rviia - fp , with an extended t , translated into a prolonged pd activity compared with rfviia ( fig 2a , b ) . the lagtime to thrombin generation for rfviia time - dependently increased , reaching baseline values ( derived from the control group ) at 16 h from start of treatment ( fig . this was confirmed by the thrombin generation curves at 16 h after start of treatment , which were superimposable for the vehicle- or rfviia - treated animals ( fig . in contrast , prolonged activity of rviia - fp was clearly evident , showing an average 2-fold reduction in the lagtime until 16 h after start of treatment with rviia - fp , despite the potency difference observed during tail - clip studies , and in the absence of selective fviia activity adjustments ( fig . the extended activity of rviia - fp was also observed as enhanced thrombin generation at 16 h after start of treatment ( fig . ( a ) lagtime to thrombin generation ; ( b ) thrombin generation  time profile at 16 h. pd , pharmacodynamic ; pk , pharmacokinetic ; tga , thrombin generation assay . the initial procoagulant activity elicited by rviia - fp and rfviia was similar at 10 min after treatment , with thrombus scores for both ranging from 2 to 2.5 following 20 min of venous stasis ( these scores increasing from about 1.0 after 10 min of stasis ) ( figure s3 ) . following 20 min of stasis at the time - point of 12 h after treatment , however , thrombus formation in animals treated with rfviia was negligible , but sustained thrombus formation was observed in rviia - fp - treated animals , resulting in a significantly different thrombus score ( p = 0.0325 ) . there was an estimated probability of 75% that the thrombus score of a rviia - fp - treated animal was higher than that of a rfviia - treated animal at 12 h ( fig . 3 ) . no thrombus formation was observed following placebo treatment , or at 24 h , and no difference between rviia - fp and rfviia was seen 12 h post - administration following 10 min of stasis ( figure s4 ) . thrombus scores are shown after 20 min of stasis from veins dissected at three time - points after administration of test substances . definition of thrombus scores : 0 = no clot ; 1 = one or a few small clots , no measurable weight ; 2 = not fully occluding clot , one or several clots of bigger size , weight can be measured ; 3 = segment fully occluded by clot , weight can be measured . measurement of pt supported the results obtained from thrombus score assessment , because reduced pt times were noted for rviia - fp until 24 h post - administration compared with rfviia - treated animals ( fig . no general activation of the coagulation system was recorded when measuring fibrinogen , thrombin - antithrombin ( tat ) and d - dimer as prothrombotic biomarkers ( data not shown ) . pk parameters from mice and rats based on fvii : ag , and rabbits and monkeys based on fviia activity , are shown in table 1 . in all species , pivotal pk characteristics ( i.e. in vivo recovery , cl and t ) were improved for rviia - fp compared with rfviia . in both mice and rats , furthermore , rviia - fp showed a 2-fold enhanced in vivo recovery compared with rfviia ( table 1 ) . pharmacokinetic parameters of rviia - fp and rfviia in hemophilia a mice , rats , rabbits and cynomolgus monkeys data for mrt , cl and t are presented based on fvii antigen measurements for rodents , and based on selective fviia activity for rabbits and monkeys . for mice , samples from three animals per time - point were pooled , resulting in one assay result per time - point , a single pk curve and a single set of pk parameters . for rats , samples from four animals per time - point were tested individually and averaged to give a single pk curve and a single set of pk parameters . for rabbits and monkeys , each animal provided a full pk curve and geometric means cl , clearance ; ivr , in vivo recovery ; mrt , mean residence time ; rfviia , recombinant factor viia ; rviia - fp , fusion protein ; t , terminal elimination half - life . similarly to the rodent studies , in which pk properties of fviia were calculated only measuring fvii : ag levels , the pk profile of rviia - fp was also improved compared with rfviia in rabbits and monkeys based on selective fviia activity ( table 1 ) . in both species , cl was again lower and t extended when comparing rviia - fp with rfviia . there was a 5-fold enhanced in vivo recovery in rabbits when comparing rviia - fp with rfviia , while in monkeys there was a 1.7-fold enhanced in vivo recovery when comparing rviia - fp with rfviia ( table 1 ) . pk profiles for rviia - fp and rfviia in mice , rats , rabbits and monkeys are shown in fig 1 . time profiles of rviia - fp and rfviia in ( a ) hemophilia a mice , ( b ) rats , ( c ) rabbits and ( d ) cynomolgus monkeys ( mean  sd ) . pk curves in mice and rats are based on fvii : ag data , while the curves shown for rabbits and monkeys are based on selective fviia activity . data are mean , except for mice ( plasma pool ) and monkeys , where the curve for each animal is shown . fvii : ag , factor vii antigen ; pk , pharmacokinetic ; rfviia , recombinant factor viia ; rviia - fp , fusion protein ; sd , standard deviation . the improved pk profile of rviia - fp , with an extended t , translated into a prolonged pd activity compared with rfviia ( fig 2a , b ) . the lagtime to thrombin generation for rfviia time - dependently increased , reaching baseline values ( derived from the control group ) at 16 h from start of treatment ( fig . this was confirmed by the thrombin generation curves at 16 h after start of treatment , which were superimposable for the vehicle- or rfviia - treated animals ( fig . in contrast , prolonged activity of rviia - fp was clearly evident , showing an average 2-fold reduction in the lagtime until 16 h after start of treatment with rviia - fp , despite the potency difference observed during tail - clip studies , and in the absence of selective fviia activity adjustments ( fig . the extended activity of rviia - fp was also observed as enhanced thrombin generation at 16 h after start of treatment ( fig . ( a ) lagtime to thrombin generation ; ( b ) thrombin generation  time profile at 16 h. pd , pharmacodynamic ; pk , pharmacokinetic ; tga , thrombin generation assay . the initial procoagulant activity elicited by rviia - fp and rfviia was similar at 10 min after treatment , with thrombus scores for both ranging from 2 to 2.5 following 20 min of venous stasis ( these scores increasing from about 1.0 after 10 min of stasis ) ( figure s3 ) . following 20 min of stasis at the time - point of 12 h after treatment , however , thrombus formation in animals treated with rfviia was negligible , but sustained thrombus formation was observed in rviia - fp - treated animals , resulting in a significantly different thrombus score ( p = 0.0325 ) . there was an estimated probability of 75% that the thrombus score of a rviia - fp - treated animal was higher than that of a rfviia - treated animal at 12 h ( fig . no thrombus formation was observed following placebo treatment , or at 24 h , and no difference between rviia - fp and rfviia was seen 12 h post - administration following 10 min of stasis ( figure s4 ) . thrombus scores are shown after 20 min of stasis from veins dissected at three time - points after administration of test substances . definition of thrombus scores : 0 = no clot ; 1 = one or a few small clots , no measurable weight ; 2 = not fully occluding clot , one or several clots of bigger size , weight can be measured ; 3 = segment fully occluded by clot , weight can be measured . measurement of pt supported the results obtained from thrombus score assessment , because reduced pt times were noted for rviia - fp until 24 h post - administration compared with rfviia - treated animals ( fig . 4 ) . no general activation of the coagulation system was recorded when measuring fibrinogen , thrombin - antithrombin ( tat ) and d - dimer as prothrombotic biomarkers ( data not shown ) . the pk / pd characteristics of rviia - fp were assessed in a series of non - clinical studies , and rviia - fp was found to be effective as a hemostatic agent , with consistently longer half - lives and mean residence times , and greater in vivo recovery across the species studied . for the rodent studies , pk was assessed using an elisa , and it should be noted that using polyclonal antibodies in an elisa to measure fvii protein might additionally capture partially degraded fvii and/or fvii that has been inactivated by binding to circulating plasma proteins such as antithrombin , tf pathway inhibitor or alpha-2 macroglobulin . in the rabbit and monkey pk studies , however , we also used the staclot system , which specifically measures active fvii only and therefore represents the clinically more relevant and predictive parameter for expected clinical efficacy in humans . the staclot data were qualitatively similar to the antigen data ( not shown ) in these species , confirming that rviia - fp has a more desirable pk profile than rfviia , which is currently in clinical use . the prolonged systemic availability of rviia - fp in plasma also translated into extended procoagulant activity in our pd studies , suggesting that rviia - fp has the potential to control and prevent bleeding episodes in hemophilia patients with inhibitors , with less frequent dosing than rfviia in clinical practice . fewer injections should facilitate long - term prophylactic use of rviia - fp and be useful during surgical procedures . our studies in hemophilia a mice showed a 2-fold enhanced in vivo recovery of rviia - fp compared with rfviia . furthermore , rviia - fp ( 0.58.0 mg kg ) and rfviia ( 0.52.0 mg kg ) demonstrated a dose - proportional decrease in total blood loss over the dose range investigated in hemophilia a mice under acute bleeding conditions . the prolonged t of rviia - fp in hemophilia a mice was reflected in the extended pd activity , with thrombin generation observed 16 h after the start of rfviia - fp administration , whereas hemostatic efficacy had completely ceased 16 h post - dose with rfviia . consistent with our findings , other studies in mice show a mean hemostasis t of 1.2 h after treatment with rfviia at a dose of 10 mg kg , and , similarly , no hemostatic effect was seen 24 h after administration of rfviia in mice at the same dose level . our studies in rats and rabbits also showed that rviia - fp had a longer t compared with rfviia . our findings are consistent with results of a previous study reporting a mean t of 4.5 h for rfviia ( based on a fviia clotting activity assay ) administered to rabbits at a dose of 2 mg kg . while rviia - fp and rfviia showed similar procoagulant activity in our study in rabbits at 10 min after dosing , by 12 h thrombus formation had become negligible in the rfviia group , whereas it persisted in the rviia - fp group . we also investigated activation of the coagulation system in rabbits , using the international society on thrombosis and haemostasis ( isth ) scoring system of disseminated intravascular coagulation ( dic ) . this stipulates that initial signs of systemic activation of the coagulation system , non - overt dic , are shown by changes in a score of platelet count , prothrombin time , fibrinogen and d - dimer . fibrinogen , tat and d - dimer , as prothrombotic markers , indicated no increased risk of thrombosis in our study . these results confirm findings by johansen et al . , in which rabbits treated with rfviia also showed no signs of systemic activation of the coagulation system . our study in monkeys again showed a longer mean t in the rviia - fp group ( 8.6 h vs. 2.2 h with rfviia ) and the cl rate was improved 10-fold ( 4.9 ml h kg vs. 45.8 ml h kg with rfviia ) . these pk variables found in monkeys seem to correlate with pk data obtained for rviia - fp during phase 1 in healthy human volunteers . administration of five , single escalating doses ( 140 and 1000 g kg ) revealed a median t between 6.1 and 9.7 h , with 8.5 h at the highest dose . furthermore , rviia - fp had a reduced mean cl ( i.e. 7.62 ml h kg at 1000 g kg ) , resulting in an approximately 3- to 4-fold increase in t   compared with rfviia [ 2527 ] . in two clinical studies conducted in healthy human volunteers , rfviia ( 5320 g kg ) had a mean t ( determined measuring fvii clotting activity ) between 2.43 and 2.45 h and cl rates ranging between 31 and 35 ml h kg . in a third study conducted in healthy japanese and caucasian adults , a slightly longer t was reported for fviia ( dosed at 40160 g kg ) , ranging from 3.9 h to 6.0 h , with cl rates of 3437 ml h kg consistent with the other healthy volunteer studies . similarly to rviia - fp , the pk profile of rfviia in the monkey appears indicative of the profile in humans with regard to t and cl , and may therefore aid allometric species scaling of preclinical animal data . besides rviia - fp , several other candidate agents have been developed as potential hemostatic therapies with modified pk properties . these have employed various principles other than albumin fusion , but not all met with success in early studies . firstly , a glycopegylated rfviia ( n7-gp ) was produced in an attempt to prolong the t . in hemophilia a mice , n7-gp had a six - fold longer t compared with rfviia , a trend also observed in humans . the development of n7-gp was discontinued , however , due to a lack of dose  response linearity and one case of hypersensitivity during phase 2 clinical trials . secondly , vatreptacog alfa ( nn1731 ) , a genetic variant of rviia with a similar t to rfviia but a more rapid onset of action , was effective as an acute treatment in 98% of joint bleeds in a phase 2 study . however , in phase 3 trials , one patient developed anti - drug antibodies with a potentially neutralizing effect , indicating potential tolerability issues and leading to its discontinuation . bay 86 - 6150 was another long - acting fviia agent in clinical development until recently . bay 86 - 6150 is a genetic variant of rfviia , with a modified amino acid sequence , resulting in a slightly extended t and increased binding to activated platelets . a phase 1 , single - dose escalation study in hemophilia patients with or without inhibitors showed a dose - linear relationship over the dose range investigated ( 6.590 g kg ) , and a t of 57 h for the two highest dose groups ( 50 and 90 g kg ) . one patient in this study developed anti - drug antibodies to bay 86 - 6150 , cross - reactive with endogenous rviia at baseline and indicative of possible undue immunogenicity . development of inhibitory antibodies to bay 86 - 6150 during the phase 2/3 clinical trial led to discontinuation of development of this agent too . finally , the immunoglobulin g ( igg1 ) fc domain is being investigated as another potential fusion partner to confer an extended t to rfviia . fviia as a monomeric fc fusion demonstrated a 5-fold longer t than rfviia in hemophilia a mice . in addition , rfviia - fc variants are in development , with further studies needed to determine the efficacy and safety of igg1 . it is therefore possible that hemophilia patients with inhibitors might benefit from new treatments in forthcoming years . our preclinical studies confirm the proposed concept that a longer terminal elimination t of rviia - fp compared with rfviia by albumin fusion translates into extended pd activity . although inter - species scaling is complex , the observed improvement in the pk profile of rviia - fp compared with rfviia was consistent across all species studied . thus , rviia - fp is a promising candidate for a long - acting bypassing agent . this is encouraging because there is a need for therapies with low injection - frequency regimens that can provide effective control of bleeding episodes during surgical procedures , and long - term prophylactic treatment of hemophilia patients with inhibitors . the potential clinical benefits of rviia - fp will , of course , require testing in clinical trials in patients with hemophilia and inhibitors . all authors are employees of csl behring gmbh ( marburg , germany ) , whose product rviia - fp was studied in this work . d. schuermann was an employee of csl behring at the time of writing this manuscript but has since left the company . additional supporting information may be found in the online version of this article : fig . response curves for rviia - fp and rfviia from mouse tail - clip study . fig . s2 . mean total blood loss in hemophilia a mice following equimolar dosing , adjusted for selective fviia activity .
backgroundrecombinant factor viia ( rfviia ) is approved for use in controlling bleeding episodes in people with hemophilia who have developed inhibitors to replacement therapy . due to its short half - life ( t ) , frequent injections are required , limiting its use as a prophylactic treatment . a novel , recombinant fusion protein linking coagulation factor viia with albumin ( rviia - fp ) has been developed to extend the t of rfviia.objectivesthe aim of our studies was to investigate the pharmacokinetic / pharmacodynamic characteristics of rviia - fp in preclinical animal species.methodspharmacokinetic ( pk ) parameters were derived after single intravenous dosing in hemophilia a mice , rats , rabbits and monkeys . 
 pk analysis was based on human fvii plasma levels determined by measuring fvii antigen levels by elisa in mice and rats , and fviia activity using staclot viia - rtf in rabbits and monkeys . 
 induction of thrombin generation was investigated in mice , while hemostatic activity was assessed by thrombus formation in rabbits.resultscompared with rfviia , rviia - fp displayed a prolonged t , enhanced in vivo recovery and reduced clearance in all species investigated . in mice , 16 h after treatment with rviia - fp , 
 thrombin levels were quantifiable , indicating prolonged efficacy , whereas values had approached baseline at this time after treatment with rfviia . 
 after 12 h , hemostatic efficacy was negligible in rfviia - treated rabbits , but sustained in animals receiving rviia-fp.conclusionsthese studies indicate that the longer t of rviia - fp compared with rfviia translates into extended activity . 
 these findings suggest that rviia - fp has the potential to be administered less frequently than rfviia - containing concentrates in clinical use .