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Blood First Edition Paper, prepublished online April 22, 2016; DOI 10.1182/blood-2016-01-696104
Prophylactic Efficacy of BeneFIX vs Alprolix in Hemophilia B Mice
Running head: BeneFIX vs Alprolix
Brian Cooley, Ph.D.1,2, William Funkhouser, M.D. PhD.1, Ph.D., Dougald Monroe,
Ph.D.3,
Ashley Ezzell4, David M. Mann, Ph.D.5, Feng-Chang Lin 6, Paul E. Monahan, M.D.7,
Darrel W. Stafford, Ph.D.8
1
Department of Pathology & Laboratory Medicine, 2McAlister Heart Institute Core Lab,
3
Department of Medicine-Hematology,
4
Department of Cell Biology & Physiology,
University of North Carolina-Chapel Hill. 5Independent Consultant.
Biostatistics, University of North Carolina-Chapel Hill,
6
Department of
7
Department of Pediatrics,
University of North Carolina School of Medicine, and the 8Department of Biology,
University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA.
Correspondence: Darrel W. Stafford, Department of Biology, University of North
Carolina-Chapel Hill, Chapel Hill, North Carolina, NC 27599-3280
Email: [email protected]
Word count for text:
3567
Word count for abstract:
247
Figure/Table count:
5
Reference count:
38
Scientific category:
Thrombosis and Hemostasis
1
Copyright © 2016 American Society of Hematology
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Key Points
•
Because extravascular FIX is physiologically important, its circulating levels do not
independently predict hemostatic potential.
•
A saphenous vein hemophilia B mouse model shows that 7 days post infusion FIX-Fc
and FIX provide equal hemostatic protection.
Abstract
FIX binds tightly to collagen IV. Furthermore, a FIX mutant, FIXK5R, which binds better than wildtype FIX to collagen IV, provides better hemostasis than wild-type FIX, long after both are
undetectable in the plasma. There is also credible evidence of extra-vascular FIX. Here, we use
the saphenous vein bleeding model to compare the efficacy of recombinant FIXFc (Alprolix) and
wild-type FIX (BeneFIX) in hemophilia B mice 7 days post-infusion. Although the terminal halflife of Alprolix is significantly longer than that of BeneFIX, at equal doses Alprolix is not better at
controlling bleeding 7 days post-infusion, presumably because of the extravascular FIX. Both
BeneFIX and Alprolix exhibit a linear response in clotting efficacy up to 150 IU/kg, where they
appear to saturate an extravascular compartment, as there is no additional prophylactic benefit
from higher doses. A robust pool of extravascular FIX is clearly observed surrounding blood
vessels, localized to the same region as collagen IV, in two representative human tissues: liver
and skeletal muscle. We see no increased risk for thrombosis at 250 IU/kg FIX at 6 hours postinfusion. In summary, 7 days post-infusion into hemophilia B mice, BeneFIX and Alprolix are
hemostatically indistinguishable despite the latter’s increased half-life. We predict that doses of
FIX approximately 3 times higher than the currently recommended 40-50 IU/kg will, because of
FIX’s large extravascular compartment, efficiently prolong prophylactic hemostasis without
thrombotic risk.
2
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INTRODUCTION
Hemophilia management traditionally involves treating severe bleeding episodes when they
occur (on-demand therapy). However, this approach does not prevent intra-articular
microbleeds, which eventually result in joint debilitation. Therefore, the World Federation of
Hemophilia recommends that on-demand treatment be replaced by prophylactic maintenance.1
The goal of prophylaxis has been to maintain a plasma trough level greater than 1% of normal;
but, it is now recognized that 5-15% of normal is required to prevent spontaneous bleeding.2
The new target level of 5-15% is based on correlations between FVIII levels and bleeding
events. A clinical trial correlating bleeding and plasma levels of FIX3 has never been performed,
although recently presented data examining a large database of United States men with mild
and moderate hemophilia supports a similar target for hemophilia B 4. Because current practice
requires prophylactic infusions of clotting factors 2 times per week, several companies have
designed FIX molecules that have longer terminal half-lives. One of these drugs, FIXFc
(Alprolix™), is approved for prophylaxis when administered every 7-10 days.5-7 The published
half-life data of these “longer-acting” molecules is impressive, but there is no evidence that they
provide better clinical outcomes. Moreover, the extravascular role played by FIX has not been
adequately considered. FIX plasma concentration remains the standard surrogate endpoint for
hemostatic efficacy in hemophilia B patients, despite evidence that extravascular FIX plays an
important role in coagulation. This evidence for the importance of extravascular FIX includes the
following points:
First, FIX binds reversibly and saturably to collagen IV, which is found mainly in the basement
membranes of all tissues.8
Second, we previously described a mutation of FIX (lysine-5 to alanine-5, designated FIXK5A)
with reduced affinity for collagen IV. We later created a knock-in mouse expressing FIXK5A. This
3
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mouse exhibits a bleeding diathesis even though its circulating level of FIXK5A is about 20%
higher than normal-9 the specific activity of this FIXK5A in vitro is indistinguishable from that of
wild-type FIX (FIXWT). This suggests an important hemostatic role for FIX bound to
extravascular collagen IV.
Third, hemophilia B mice, injected with a FIX (FIXK5R) that binds more tightly to collagen IV,
exhibit better hemostasis 7 days post-infusion than do mice injected with an equivalent amount
of FIXWT. This improved hemostasis persists despite the fact that their circulating FIX levels
reach undetectable levels at 3 days post-infusion 10.
Lastly, there is evidence of a large extravascular reservoir of FIX. As Stern et al. demonstrated,
administering increasing amounts of bovine FIX to baboons rapidly displaced the endogenous
extravascular stores of baboon FIX into the plasma.11
In aggregate, these data suggest that collagen IV–bound FIX plays an important role in
hemostasis. In this study, we compare FIXFc (Alprolix™) and FIXWT (BeneFIX™) to test the
hypothesis that simply increasing FIX’s plasma half-life may not confer a clinical advantage in
prophylactic hemophilia treatment.
METHODS
All mouse experiments were IACUC approved (UNC ACAP #13-144) and followed PHS
guidelines for animal care and use. The hemophilia B mice were made in our laboratory12 and
were in a C57BL-6 background. The normal control mice were from Jackson Labs and had the
same genetic background as the hemophilia B mice.
Proteins
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Alprolix was purchased from Diversified Biologicals in Miami, FL. BeneFIX was provided by
Pfizer Inc. Both proteins were reconstituted per manufacturer’s instructions immediately before
use. A new vial was opened for each infusion.
Bleeding Assay
A saphenous vein bleeding assay was used.13 The FIX was infused into the right saphenous
vein of hemophilia B mice. After 7 days, the left saphenous vein was exposed and partially
transected. Upon cessation of bleeding, the clot was physically dislodged, and the site was
observed for repeat clotting. The total number of clots formed over 30 minutes was recorded.
Increased numbers of reformed clots equates to better hemostatic control. For testing the
hypothesis that the two populations are the same, the Mann-Whitney test was used. Variance
equivalence was tested by the Brown-Forsythe test. All statistical analysis employed
Mathematica version 10.3.
Thrombosis Assays
Intravital fluorescence imaging of venous thrombogenesis was performed as described in
Cooley (2011).14 Alexa Fluor 647®-labeled anti-fibrin monoclonal antibody was used to monitor
fibrin levels. Rhodamine 6G (0.5 mg/kg) was used to label platelets. Fluorophores were
injected 5 minutes prior to thrombus induction. The femoral vein was exposed and a surfaceapplied electrolytic injury was delivered for 30 seconds via a blunt wire at 1.5 volts of anodal
current. The vessel injury site was shutter-illuminated with 532nm and 650nm defocused
lasers, and fluorescence emission was captured via time-lapse digital video microscopy at 100X
magnification for 60 minutes. The relative intensity of each fluorophore was quantitated at 2minute intervals, normalizing for animal body weight and the amount of injected fluorophorelabeled platelets to yield a “relative intensity” for each fluorophore.14
5
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Histology
Human tissues, obtained from the UNC Translational Pathology core facility and classified as
exempt by the IRB, were paraffin embedded and sectioned at 5µm. For immunohistochemistry,
heat-induced epitope retrieval (HIER) 15 was performed and endogenous peroxidases were
blocked using 3% H2O2. Sections were then blocked using 10% normal goat serum; incubated
in Anti-Human Factor IX (1:1600, Green Mountain Antibodies GMA-101), Anti-Human Factor X
(FX) (1:100, Green Mountain Antibodies GMA-540), GMA-213 Anti-Factor VII (FVII) Antibody
1:500 or Anti-Human Collagen IV (1:500, Abcam ab6586) overnight at 4°C; then incubated in
Biotinylated Goat Anti-Mouse IgG (1:500, Jackson ImmunoResearch 115-065-166) or
Biotinylated Goat Anti-Rabbit IgG (1:500, Jackson ImmunoResearch 111-065-144) for 1 hour at
room temperature. Finally, staining was visualized using a Vectastain ABC Elite Kit (Vector
Labs PK-6100) followed by DAB (3, 3'-diaminobenzidine). The images were captured with a
Nikon Optiphot-2 with plan-apo lenses and an Olympus DP-70 camera.
RESULTS
Figure 1A shows the results of saphenous vein bleeding in hemophilia B mice 7 days postinfusion. Both Alprolix and BeneFIX, whether infused at 50, 100 and 150 IU/kg, elicit almost
identical hemostatic efficacies. At each dose of BeneFIX and Alprolix, Mann-Whitney P values
for the differences between population medians are >0.83. And, at any dose, the variance
equivalence between Alprolix and BeneFIX is not significant. However, the variance of either
Alprolix or BeneFIX differs significantly from the values found in the WT mice (P <.02). Table 1
compares the Mann-Whitney test values for differences between doses within groups 7 days
post-infusion. The table uses the subscript letters and numbers to indicate the infused protein
and its dose: FIXB50 and FIXA50 indicate doses of 50 IU/kg; FIXB100 and FIXA100 indicate doses of
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100 IU/kg, etc. The P value for the comparison of Alprolix at 100 IU/kg vs. 150 IU/kg is greater
than 0.05 simply because of the number of mice employed; in the Alprolix comparison, 53 mice
were used in combination between the two doses, while 64 mice were used to compare the
same BeneFIX dose. The smallest sample size was 53 total mice for comparing FIXA100 to
FIXA150. Comparing the P values between incremental doses for the rising portion of the dose
response curve with that for the flat portion of the curve reveals that, for both Benefix and
Alprolix, each increasing dose is statistically significant up to 150 IU/kg. Beyond 150 IU/kg, no
statistically significant improvement in hemostatic protection is afforded.
Figure 1B shows the dose response curve of the median number of times hemophilia B mice
clot 7 days post-infusion to doses of BeneFIX at 50, 100, 150, 200, 250, and 500 IU/kg and to
Alprolix at doses of 50, 100, 150 and 250 IU/kg. The distribution of the data points is clearly
seen in figure 1A; Figure 1B replots the data from Figure 1A with additional doses of either
BeneFIX or Alprolix. It is clear from Figures 1A and 1B that hemostatic function is directly doseproportional until 150 IU/kg, beyond which there is no additional benefit from higher doses. (The
line in figure 1B is drawn to help the reader see the proportional increase with doses up to ~150
IU/kg.) Note, that at 7 days post-infusion of 150 IU/kg, no BeneFIX antigen was detectable (<1
ng/ml, the limit of detection) in the plasma, while 98 ± 8 ng/ml (n=5) of Alprolix remained.
We previously reported that hemophilia B mice expressing a FIX variant, FIXK5A, that binds
poorly to collagen IV had a mild bleeding diathesis 16. The tail bleed model was used for those
experiments and too few mice were used for robust statistical analysis. We expanded these
experiments by using more mice (the minimum number of animals used for any dose of either
FIX construct was 19) and by evaluating the coagulation status with the saphenous vein model
instead of the tail bleed model. Figure 1C shows the reduced clotting efficacy of human FIXK5A
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in these mice. Clearly, although FIXK5A displays normal activity in an in vitro aPTT assay,17,18 the
FIXK5A knock-in mice show reduced clotting; this is despite the knock-in FIXK5A mice having
slightly higher plasma levels of FIXK5A than the WT mice have of FIXWT.
In this experiment we used a saphenous vein bleeding model instead of the tail bleeding
experiment of the prior study9,19. Although the hemophilia B mice show undetectable circulating
levels of FIX 7 days after an infusion of Benefix, an injured saphenous vein will nevertheless
form clots on day 7. If clotting at the site of an injury were due to residual, non-detectable,
levels of circulating FIX, then one would expect the limit of clot recurrence at that wound to
represent either the consumption of the residual circulating FIX or conversely, exhaustion of
some local factor(s) required at the wound site for clotting to reoccur. To distinguish these two
possibilities, we examined the clotting function at the contralateral saphenous vein, transected
after clotting ceased at the original injured vein 7 days post-infusion of 150 IU/kg Benefix.
Figure 2 demonstrates that the subsequently injured contralateral vein clots just as well as did
the original saphenous vein before the latter ceased clotting. This argues that long-term clotting
is mediated, not by circulating FIX, but by FIX sequestered in the extravascular tissue,
presumably due to collagen IV binding. Furthermore, hemophilia B mice subjected to
saphenous vein injury typically clot 0 to 2 times. If a 500 IU/kg bolus is administered 10 minutes
after clotting ceases, 5.9 ± 0.9 minutes elapses before clotting at the injury resumes. This is
despite the fact that, in mice, the blood circulates the body completely every 7–9 seconds.20
This again suggests that it is not circulating FIX but sequestered FIX that is responsible for
coagulation, and that the 5.9 minutes represents the time necessary to accumulate sufficient
FIX in the extravascular tissue to support clotting.
Immunohistological Evidence for Extravascular FIX
Because of the evidence for extravascular FIX, and the observation that it binds to collagen IV,
we used immunohistochemistry to determine the location of FIX in human tissues. We
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examined human tissues because we have previously shown that mice infused with human FIX
exhibit FIX beneath their arterial endothelium, and periportal human FIX has been reported in
mouse livers of mice expressing human FIX16,21. Initially, we examined normal human liver
tissue; because it is highly vascularized and rich in connective tissue, and because FIX is
synthesized in the liver, it serves as a positive control. Figure 3A and D show that hepatocytes,
which synthesize FIX, stain positive for FIX; their most intense FIX staining appears to be
intracellular.
Interestingly, both the basement membrane of the portal vein and the stroma surrounding the
portal triad are densely stained with the FIX antibody. An adjacent histological section (Figure
3B) shows collagen IV staining that overlaps the same region of the portal triad that is stained
by FIX antibody. However, an area positive for collagen IV, around the liver sinusoids, did not
stain for FIX. Likewise, the cuboidal epithelial cells, comprising the bile ducts, stain well for
collagen IV but are negative for FIX. Therefore, FIX stains some but not all regions containing
collagen IV, demonstrating a selectivity of unknown molecular basis. Immunodetection of
Factor X and Factor VII (Figs. 3C & F, respectively) demonstrate the specificity of FIX detection
around the portal triad, since these two proteins prominently stain the hepatocytes but not the
area of FIX concentration around the portal triad.
FIX's extravascular presence is not unique to hepatic tissue, because staining of skeletal
muscles reveals the presence of FIX around the arteries. Figures 3G, H and I respectively
show staining with FIX, with collagen IV. The control, Figure 3E, lacks the primary antibody.
Again, FIX co-distributes in many, but not all, areas where collagen IV is observed. The
basement membrane and outer adventitia layers of these artery walls stain well for FIX, but FIX
staining is largely absent from the tunica media.
Potential Thrombogenicity
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We employed an electrolytic model of venous thrombosis14 to examine the thrombogenicity of a
high dose of FIX. Hemophilia B mice were infused with 250 IU/kg of Benefix, and 6 hours later
the accumulation of either fibrin (Figure 4A) or platelets (Figure 4B) was monitored at the site of
vessel injury. The in situ levels of fibrin formation or platelet aggregation were not greater than
that observed in control wild-type mice. Thus, 250 IU/kg Benefix did not increase
thrombogenicity.
DISCUSSION
In this study, we compared the efficacy of two recombinant commercial FIX products (a wildtype recombinant FIX and a longer-circulating FIX-Fc fusion protein) at 7 days post-infusion
using a saphenous vein bleeding model in hemophilia B mice. FIX binds tightly (5 nM) to
collagen IV, which is abundant in the sub-endothelial matrix. We hypothesized that this
interaction might result in large stores of extravascular FIX that could provide hemostatic
protection days after circulating levels of injected FIX were undetectable. Measured at 7 days
post-infusion of 150 IU/kg, the plasma concentration of Alprolix was still at 2% of normal and
BeneFIX was undetectable, yet their clotting efficacy was essentially identical. It is puzzling that
both molecules appear to fill an extravascular compartment at the same dose of 150 IU/kg.
Given Alprolix's approximately 4-fold lower molar-specific activity than BeneFIX22, and given that
the binding of these molecules to collagen IV involves FIX’s Gla domain23, one would expect
that saturation of the extravascular compartment with a given number of FIX molecules of either
type should yield less clotting activity (~3-fold less) in those animals receiving Alprolix than in
those receiving BeneFIX. This of course assumes that these two molecules have the same
affinities for collagen IV, the same abilities to access the extravascular collagen IV binding sites
and the same abilities to bind to that collagen. We currently do not know why similar clotting
efficacies are achieved 7 days after injecting equal doses (IU/kg) of BeneFIX and Alprolix.
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We concentrated on the more clinically relevant human tissue for these experiments; this is
because we had previously shown that human FIX, when infused into mice, can be observed in
the sub-endothelium of mouse arteries; 16 and, there are observations of periportal FIX in
hemophilia mice in gene therapy correction trials 18,21. Our observation, that collagen IV lining
the normal human liver sinusoids is not heavily decorated with FIX, suggests that these collagen
IV molecules are different from those surrounding other vessels. Otherwise, given the KD of FIX
for collagen IV and FIX’s normal plasma concentration (~90nM), any available collagen IV
should be in complex with FIX. Collagen IV, like other collagens, is a triple helix formed from 3
monomers. However, collagen IV molecules are assembled from a combination of 6 different
monomers, α1 through α6. Robertson et al.24 recently demonstrated that about 12% of collagen
IV in the bovine aorta is α121–α565 rather than the predominant α121–α121 form. This is
consistent with reports that type IV collagen α565 is present in the basement membranes of
tubular structures exposed to constant mechanical stress.25 Therefore, a different monomer
composition may explain the absence of FIX binding to the sinusoidal collagen IV. Another
possible explanation for variable FIX–collagen IV binding is post-translational modification of
collagen IV. A recent report demonstrates that post-translational modification of collagen IV by a
specific prolyl hydroxylase is required to prevent lethal platelet aggregation via the platelet
receptor G6BG6B.26 A similar post-translational modification could render the collagen IV lining
the sinusoids incapable of binding with FIX.
The first question that arises when discussing this data with clinicians is: “What about
thrombosis at higher doses?” This anxiety arises for several reasons. Historically, when
prothrombin complex concentrates were used to treat hemophilia B, frequent thrombotic events
were observed. Presumably, this was caused by variable amounts of activated factors, including
FVIIa. Two papers that correlate thrombosis and plasma FIX levels are also cited.27,28 These
publications report that a 1.3- or 1.5-fold increase in plasma FIX concentrations (i.e. greater
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than the 90th percentile measured in control subjects) correlates with an ~2-fold increase in
thrombosis risk. This risk is modest when compared to the 4-fold increased thrombotic risk of
widely used oral contraceptives.29 Even at a dose of 150 IU/kg, plasma BeneFIX concentrations
are only transiently elevated above the normal plasma concentration of FIX (unpublished data);
the normal level is 90 nM or 5ug/ml (personal communication from A. Hubbard, National
Institute of Biological Standards and Control, UK). One IU/ml, the convention for pooled normal
human plasma, equates to ~40 IU/kg, assuming 40 ml plasma per kg. In addition to these
abstract risk assessments, there is considerable clinical evidence that treating with higher doses
of FIX is not thrombogenic. When purified FIX was first introduced, several patients were
infused with up to 161 IU/kg and had no observed thromboses.30,31 Moreover, 1000 IU/kg
BeneFIX failed to induce thrombosis in the Wessler Stasis model in rabbits.32,33 Also, strong
additional clinical evidence is provided by a patient mutation: FIX Padua (FIXR338L). This FIX
variant has a specific activity that is about 8-fold greater than that of FIXWT34. The affected
males, who have 8 times the normal level of FIX, developed thromboses at 16 to 20 years of
age; their mother, however, who has endogenous expression of 3.5-fold higher-than-normal FIX
activity, has had no reported thrombosis. Despite the significant increased risk for thrombosis
during her pregnancy (6-fold overall and 60-fold during the 3 months after delivery35), this
mother had 3 successful pregnancies with no thrombotic complications. FIX Padua patients are
chronically exposed to high-activity FIX while hemophilia B patients are only transiently exposed
to higher doses of circulating FIX. The normal dose of FIX, 40-50 IU/kg, would correspond to
levels of FIX (90 nM) if all of the infused FIX were retained within the circulation (with an
assumed plasma volume of 40 ml/kg). Since a normal individual has about 3 times more
extravascular FIX than FIX in circulation, an infusion of 150 IU/kg should, after recovery in
plasma, result in less circulating FIX than that found in the circulation of a normal patient 10.
Thus, 150 IU/kg of infused FIX is less than the total amount of FIX in the combined circulating
and extravascular compartments of a normal individual. For example, when hemophilia B mice
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are infused with 200 IU/kg of BeneFIX, plasma FIX falls to normal levels (~90 nM, 5µg/ml)
approximately 7 minutes post-infusion (unpublished observations). The optimal dose for patients
whose FIX is defective but nevertheless binds normally to collagen IV is an issue yet to be
explored. If the infused FIX must compete with the patients defective FIX for extravascular
binding site, these patients may require even higher doses of FIX for prophylactic efficacy.
We also present here experimental evidence that, in mice, 250 IU/kg is not thrombogenic in a
specific electrolytic ferric chloride model of injury. While these experiments were done in mice
and do not necessarily extrapolate to human studies, the clinical data discussed above
overwhelmingly suggests that 150 IU/kg will not be thrombogenic.
Our hypothesis is that normal coagulation occurs using the extracellular matrix as a scaffold.
This is reasonable because clotting has evolved primarily to protect from trauma and is required
when a vessel is breached, exposing the underlying matrix. Conversely, intravascular clotting is
considered pathological. Both tissue factor (TF) and FVII are found surrounding vessels.36,37
Since FIX binds to collagen IV in the extracellular matrix, we have most of the important
initiators of coagulation already present. But how does Factor VIII (FVIII) reach the
extravascular scaffold? One possibility is that von Willebrand factor (VWF) binds to collagen IV
and brings FVIII to the waiting FIX. This is supported by a recent paper demonstrating that VWF
binds specifically to collagen IV through its A1 domain. Moreover, several VWF mutations that
affect collagen IV binding are associated with bleeding diatheses in patients, which suggests
that VWF’s binding to collagen IV is physiologically important.38
In summary, in hemophilia B mice, Alprolix and BeneFIX both protect from bleeding at 7 days
post-infusion with essentially identical efficacy. FIX localizes to extravascular tissues
surrounding blood vessels in regions that also stain for collagen IV. However, not all apparently
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available collagen IV binds to FIX. An extravascular compartment—putatively collagen IV—
appears to fill at about 150 IU/kg. We suggest that future research should compare products
claiming longer-lasting effects with plasma-derived or recombinant FIXWT, focusing on clinical
outcomes rather than plasma serum levels. We also suggest that higher doses of FIX be
considered for better prophylactic outcomes. This recommendation, although tempered by the
obvious fact that our experimental animals were mice, is strengthened by the recent paper by
Kavalki et al39, which found that patients administered 100 IU/kg of BeneFIX once per week
had a bleeding rate only slightly higher than that reported for Alprolix5.
Acknowledgements
This work was accomplished with a grant from Pfizer Corporation. We thank Jane Shealy,
Heath Sledge, Katherine A. Stafford and Lynn Rehm for their help in editing. The authors also
acknowledge the expert animal management provided by Stacie VanLeeuwen.
Authorship and Conflict of Interests Statement
All of the mouse work was done by Brian Cooley; the histology work was done by Ashley Ezzell;
histology interpretations were done by Dr. William Funkhouser; Dougald Monroe did several
important assays and contributed ideas; Monahan oversaw animal husbandry of hemophilia B
mouse strains and contributed ideas and discussions; Feng-Chang Lin analyzed statistics. The
first versions of the paper were written by Darrel Stafford, with suggestions from Katherine
Stafford and Sheue-Mei Wu. Several important writing revisions occurred with the help of Dr.
David Mann, who provided many ideas and contributions regarding experimental design and
interpretation.
Dr. Stafford has a research grant from Pfizer, and is an occasional consultant. Dr. Paul
Monahan receives research support through the University of North Carolina from Asklepios
BioPharmaceutical and Novo Nordisk; he has received research support in the past from Baxter
14
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Healthcare, Novo Nordisk, Pfizer, and Prolor. He holds patents licensed to Asklepios, for which
he receives royalties. He has received payment for consultation, services, and for speaking for
Asklepios, Chatham LLC, Baxter Healthcare and Pfizer and has additionally consulted for
Bayer, Novo Nordisk, and Biogen. The other authors report no conflict of interests.
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Table 1. Mann-Whitney comparison of Alprolix (A) or BeneFIX (B) at different doses:
B50=50 IU BeneFIX etc.
Comparison
FIXKO vs. FIXB50
FIXB50 vs. FIXB100
FIXB100 vs. FIXB150
FIXB50 vs. FIXB150
FIXB150 vs. FIXB200
FIXB200 vs. FIXB250
FIXKO vs. FIXA50
FIXA50 vs. FIXA100
FIXA100 vs. FIXA150
FIXA50 vs. FIXA150
FIXA150 vs. FIXA200
FIXA200 vs. FIXA250
P Value
0.0008
0.043
0.027
0.00016
0.82
0.88
0.00008
0.02
0.08
0.0002
0.91
0.86
18
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Figure 1A, B and C. Each point in Fig. A represents the number of times that clotting can recur
in a hemophilic mouse with a saphenous vein injury 7 days post-infusion with either Alprolix or
BeneFIX. The P values shown within the plot are from Mann-Whitney tests (MWP). Fig. 1B is a
plot of median values for the number of clots formed at different doses of BeneFIX or Alprolix.
The medians were taken from the data shown in Figure 1A together with additional similar data
from higher doses. The line connecting the points has no theoretical meaning but serves to
draw attention to the proportional increase. Figure 1C demonstrates that mice expressing FIXK5A
at 120% of WT levels are about 50% as active in the saphenous vein bleeding model as are WT
mice. The in vitro activity of FIXK5A is indistinguishable from FIXWT in an APTT assay.
Figure 2. Hemophilia B mice, that had been infused with 150 IU/kg BeneFIX 7 days previously,
were subjected to the saphenous vein injury. After the vein ceased clotting, the contralateral
saphenous vein was transected. The data shows that there is no significant difference between
the two veins in the number of clots formed, despite undetectable levels of circulating FIX at the
time of the injury of the first vein. This indicates that it is not residual circulating FIX that is
responsible for clotting, but instead extravascular FIX.
Figure 3, A-I. Antibodies to FIX (A&D), Collagen IV (B), FX (C), FVII (F) were used to stain
human liver sections with 3,3’-Diaminobenzidine (DAB). Panel E represents no primary
antibody; all no primary antibody controls lacked background noise. Notice that collagen IV in
the sinusoids (Panel B) is not stained with FIX; but, that collagen IV and FIX staining generally
coincide. The tissues stained with antibodies to FX and FVII differ noticeably from those stained
for FIX. Panel G shows a cluster of arteries section in the human skeletal muscle stained for
FIX; Panel H is an adjacent section stained for collagen IV; and, Panel I is the no primary
antibody control. The images were captured with a Nikon Optiphot-2 with a plan-apo lenses and
an Olympus DP-70 camera.
19
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Figure 4, Venous Thrombosis model at 250 IU/kg. The intravital thrombosis assay: An
electrolytic injury was delivered to the surface of a mouse femoral vein for 30 seconds (1.5 volts
anodal current) after the injection of fluorophore labels for fibrin (4A) and platelets (4B) captured
with time-lapse fluorescence imaging from 1 to 60 minutes later. Data are quantitated and
normalized every video frame showing comparative levels for: wild-type (blue), FIX knockout
mice without treatment (red), or FIX knockout mice with 250 IU/kg BeneFIX injected 6 hours
prior to thrombus induction (green lines), using a group of 5-6 mice per genotype and treatment.
Data are means; errors bars are SEM. There are no statistical differences between the wildtype and FIXKOs with BeneFIX treatment, whereas the untreated FIXKOs had lower levels of
accumulation for both thrombotic parameters (p<0.001 at 10 or more minutes; ANOVA).
20
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Prepublished online April 22, 2016;
doi:10.1182/blood-2016-01-696104
Prophylactic efficacy of BeneFIX vs Alprolix in hemophilia B mice
Brian Cooley, William Funkhouser, Dougald Monroe, Ashley Ezzell, David M. Mann, Feng-Chang Lin,
Paul E. Monahan and Darrel W. Stafford
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