U.S. patent application number 17/378200 was filed with the patent office on 2022-03-03 for methods of using fix polypeptides.
The applicant listed for this patent is Bioverativ Therapeutics Inc.. Invention is credited to Mark BRADER, Haiyan JIANG, Robert T. PETERS, Glenn PIERCE, Samantha TRUEX.
Application Number | 20220064622 17/378200 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220064622 |
Kind Code |
A1 |
PIERCE; Glenn ; et
al. |
March 3, 2022 |
METHODS OF USING FIX POLYPEPTIDES
Abstract
The present invention provides methods of administering
long-acting Factor IX; methods of administering long-acting,
chimeric and hybrid polypeptides comprising Factor IX; and methods
of producing such chimeric and hybrid polypeptides using cells.
Inventors: |
PIERCE; Glenn; (Cambridge,
MA) ; TRUEX; Samantha; (Sudbury, MA) ; PETERS;
Robert T.; (Needham, MA) ; JIANG; Haiyan;
(Belmont, MA) ; BRADER; Mark; (Lexington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bioverativ Therapeutics Inc. |
Waltham |
MA |
US |
|
|
Appl. No.: |
17/378200 |
Filed: |
July 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15619196 |
Jun 9, 2017 |
11225650 |
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17378200 |
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14430848 |
Mar 24, 2015 |
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PCT/US2013/061747 |
Sep 25, 2013 |
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15619196 |
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61863859 |
Aug 8, 2013 |
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61839439 |
Jun 26, 2013 |
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61829755 |
May 31, 2013 |
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61811412 |
Apr 12, 2013 |
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61800163 |
Mar 15, 2013 |
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61759796 |
Feb 1, 2013 |
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61705607 |
Sep 25, 2012 |
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International
Class: |
C12N 9/64 20060101
C12N009/64; C07K 14/745 20060101 C07K014/745; C07K 16/36 20060101
C07K016/36 |
Claims
1-130. (canceled)
131. A method of reducing or decreasing an annualized bleeding rate
in a human subject with hemophilia B, comprising administering to
the subject multiple doses of a pharmaceutical composition
comprising: (a) a chimeric Factor IX(FIX) polypeptide comprising
human FIX and an FcRn binding partner; (b) sucrose; (c) mannitol;
and (d) polysorbate 20 or polysorbate 80, wherein each of the
multiple doses comprises 10 IU/kg to 200 IU/kg of the chimeric FIX
polypeptide wherein each of the multiple doses is administered at a
dosing interval of about 7 days to about 14 days, wherein the
annualized bleeding rate of the bleeding episodes is reduced to
less than 5; and wherein the bleeding episodes are spontaneous
bleeding episodes.
132. The method of claim 131, wherein the pharmaceutical
composition comprises polysorbate 80.
133. The method of claim 131, wherein each of the multiple doses of
the chimeric FIX polypeptide is 50 IU/kg to 100 IU/kg.
134. The method of claim 131, wherein each of the multiple doses of
the chimeric FIX polypeptide is 100 IU/kg.
135. The method of claim 131, wherein each of the multiple doses is
about 50 IU/kg or about 100 IU/kg and the dosing interval is about
10 days to about 14 days.
136. The method of claim 135, wherein the annualized bleeding rate
of the spontaneous bleeding episodes is reduced to less than 4.
137. The method of claim 135, wherein the annualized bleeding rate
of the spontaneous bleeding episodes is reduced to less than 3.
138. The method of claim 135, wherein the annualized bleeding rate
of the spontaneous bleeding episodes is reduced to less than 2.
139. The method of claim 131, wherein each of the multiple doses is
for individualized interval prophylaxis of one or more spontaneous
bleeding episodes.
140. The method of claim 139, wherein the annualized bleeding rate
of the one or more spontaneous bleeding episodes is reduced to less
than 4.
141. The method of claim 131, wherein the dosing interval is every
7 days to 14 days.
142. The method of claim 131, wherein each of the multiple doses is
about 25 IU/kg to about 50 IU/kg and the dosing interval is about 7
days.
143. The method of claim 142, wherein the annualized bleeding rate
of the spontaneous bleeding episodes is reduced to less than 4.
144. The method of claim 142, wherein the annualized bleeding rate
of the spontaneous bleeding episodes is reduced to less than 3.
145. The method of claim 142, wherein the annualized bleeding rate
of the spontaneous bleeding episodes is reduced to less than 2.
146. The method of claim 131, wherein the annualized bleeding rate
of the spontaneous bleeding episodes is reduced to less than 1.
147. The method of claim 131, wherein each of the multiple doses is
25 IU/kg to 50 IU/kg.
148. The method of claim 131, wherein the human FIX comprises amino
acids 1 to 415 of SEQ ID NO: 2.
149. The method of claim 148, wherein each of the multiple doses is
about 50 IU/kg and the dosing interval is about 7 days.
150. The method of claim 148, wherein each of the multiple doses is
about 100 IU/kg and the dosing interval is about 10 days.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/619,196, filed Jun. 9, 2017, which is a
continuation of U.S. patent application Ser. No. 14/430,848, filed
Mar. 24, 2015, which is a 35 U.S.C. .sctn. 371 filing of
International Patent Application No. PCT/US2013/061747, filed Sep.
25, 2013, which claims the benefit of U.S. Provisional Patent
Application Nos. 61/863,859, filed Aug. 8, 2013; 61/839,439, filed
Jun. 26, 2013; 61/829,755, filed May 31, 2013; 61/811,412, filed
Apr. 12, 2013; 61/800,163, filed Mar. 15, 2013; 61/759,796, filed
Feb. 1, 2013; and 61/705,607, filed Sep. 25, 2012, the contents of
all of which are hereby incorporated by reference in their
entireties.
BACKGROUND OF THE INVENTION
Reference to a Sequence Listing Submitted Electronically
[0002] The content of the electronically submitted sequence listing
(Name: 720112_SA9-430USCON2_ST25.txt; 19,371 bytes; and Date of
Creation: Jul. 13, 2021) is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the field of
therapeutics for hemostatic disorders.
BACKGROUND ART
[0004] Hemophilia B (also known as Christmas disease) is one of the
most common inherited bleeding disorders in the world. It results
in decreased in vivo and in vitro blood clotting activity and
requires extensive medical monitoring throughout the life of the
affected individual. In the absence of intervention, the afflicted
individual will suffer from spontaneous bleeding in the joints,
which produces severe pain and debilitating immobility; bleeding
into muscles results in the accumulation of blood in those tissues;
spontaneous bleeding in the throat and neck may cause asphyxiation
if not immediately treated; renal bleeding; and severe bleeding
following surgery, minor accidental injuries, or dental extractions
also are prevalent.
[0005] Normal in vivo blood coagulation at minimum requires the
serine proteases Factors II (prothrombin), VII, IX, X and XI
(soluble plasma proteins); cofactors including the transmembrane
protein tissue factor and the plasma proteins Factors V and VIII;
fibrinogen, the transglutaminase Factor XIII, phospholipid
(including activated platelets), and calcium. Additional proteins
including kallikrein, high molecular weight kininogen, and Factor
XII are required for some in vitro clotting tests, and may play a
role in vivo under pathologic conditions.
[0006] In hemophilia, blood clotting is disturbed by a lack of
certain plasma blood clotting factors. Hemophilia B is caused by a
deficiency in Factor IX that may result from either the decreased
synthesis of the Factor IX protein or a defective molecule with
reduced activity. Without effective prophylaxis, recurrent
haemarthroses lead to the development of progressive and disabling
arthropathy and poor quality of life (Giangrande P., Expert Opin
Pharmacother. 2005; 6:1517-24). The treatment of hemophilia occurs
by replacement of the missing clotting factor by exogenous factor
concentrates highly enriched in Factor IX. However, generating such
a concentrate from blood is fraught with technical difficulties, as
is described below.
[0007] Purification of Factor IX from plasma (plasma derived Factor
IX; pdFIX) almost exclusively yields active Factor IX. However,
such purification of factor IX from plasma is very difficult
because Factor IX is only present in low concentration in plasma (5
ug/mL). Andersson, Thrombosis Research 7: 451 459 (1975). Further,
purification from blood requires the removal or inactivation of
infectious agents such as HIV and HCV. In addition, pdFIX has a
short half-life and therefore requires frequent dosing, which
contributes to reduced adherence to prophylactic treatment.
Recombinant factor IX (rFIX) is also available, but suffers from
the same short half-life and need for frequent dosing (e.g., 2-3
times per week for prophylaxis) as pdFIX. rFIX also has a lower
incremental recovery (K value) compared to pdFIX, which
necessitates the use of higher doses of rFIX than those for
pdFIX.
[0008] Reduced mortality, prevention of joint damage and improved
quality of life have been important achievements due to the
development of plasma-derived and recombinant Factor IX. Prolonged
protection from bleeding would represent another key advancement in
the treatment of hemophilia B subjects. However, to date, no
products that allow for prolonged protection have been developed.
The relatively short-half-lives of currently available FIX products
require frequent intravenous injections (2-3 times weekly), which
reduces adherence to prophylaxis. See White G. C., et al.,
Thrombosis and Haemostasis 78: 261-5: 1997. In children, the use of
central venous access devices (CVAD) is often required, which
further exposes them to risks of infection and thrombosis. See
Hacker M. R., et al., Hemophilia, 10: 134-46 (2004). A serious
potential risk associated rFIX treatment is the development of
inhibitors (neutralizing antibodies), which typically occur within
the first 50 exposure days (EDs). Inhibitors pose a challenge to
establishing hemostasis and can be associated with anaphylaxis. See
Chitlur M., et al., Hemophilia, 15: 1027-31 (2009). Therefore,
there remains a need for improved methods of treating hemophilia
due to Factor IX deficiency that are more tolerable and more
effective than current therapies.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides a method of administering a
long-acting Factor IX (FIX) polypeptide to a human subject in need
thereof, comprising administering to the subject a dose of about 10
IU/kg to about 200 IU/kg of the long-acting FIX polypeptide at a
dosing interval of about once a week or longer. In one embodiment,
the dose of the long-acting FIX polypeptide is about 10 IU/kg to
about 50 IU/kg, about 10 IU/kg to about 100 IU/kg, about 25 IU/kg
to about 50 IU/kg, about 25 IU/kg to about 75 IU/kg, about 25 IU/kg
to about 100 IU/kg, about 25 IU/kg to about 125 IU/kg, about 25
IU/kg to about 150 IU/kg, about 50 IU/kg to about 100 IU/kg, about
50 IU/kg to about 150 IU/kg, about 100 IU/kg to about 150 IU/kg,
about 150 IU/kg to about 200 IU/kg, or any combinations thereof. In
another embodiment, the dose of the long-acting FIX polypeptide is
for prophylaxis of one or more bleeding episodes. In one example,
the dose for prophylaxis of one or more bleeding disorders is about
50 IU/kg. In another example, an annualized bleeding rate of the
bleeding episodes after administration of a long-acting FIX
polypeptide is less than 2, less than 3, less than 4, less than 5,
less than 6, less than 7, less than 8, less than 9, or less than
10.
[0010] In another embodiment, the dose of the long-acting FIX
polypeptide is for individualized interval prophylaxis of one or
more bleeding episodes. In one example, the dose of the long-acting
FIX polypeptide is about 100 IU/kg. In another example, an
annualized bleeding rate of the bleeding episodes after
administration of a long-acting FIX polypeptide is less than 1,
less than 2, less than 3, less than 4, less than 5, less than 6,
less than 7, less than 8, or less than 9. In other embodiments, the
dose of the long-acting FIX polypeptide is for on-demand treatment
of one or more bleeding episode. In one example, an annualized
bleeding rate of the bleeding episode after administration of a
long-acting FIX polypeptide is less than 10, less than 11, less
than 12, less than 13, less than 14, less than 15, less than 16,
less than 17, less than 18, less than 19, less than 20, less than
21, less than 22, less than 23, less than 24, less than 25, or less
than 26. In still other embodiments, the dose of the long-acting
FIX polypeptide is for perioperative management of a bleeding
disorder.
[0011] In certain embodiments, the dose is administered at a dosing
frequency of about once a week to about once a month. In one
aspect, the dosing frequency is about once a week, about once in
two weeks, about twice a month, about once in three weeks, about
once in four weeks, or about once a month. In another aspect, the
dosing frequency is about once a week. In other aspects, the dosing
frequency is about once in two weeks or about twice a month.
[0012] In some embodiments, the dosing interval is about every five
days, about every six days, about every seven days, about every
eight days, about every nine days, about every ten days, about
every 11 days, about every 12 days, about every 13 days, about
every 14 days, about every 15 days, about every 16 days, about
every 17 days, about every 18 days, about every 19 days, about
every 20 days, or about every 21 days. In further embodiments, the
dosing interval is every 7 days to 14 days.
[0013] The long-acting FIX polypeptide can have a T.sub.1/2beta
(activity) of at least about 40 hours, at least about 50 hours, at
least about 60 hours, at least about 70 hours, at least about 80
hours, at least about 90 hours, at least about 100 hours, at least
about 110 hours, at least about 120 hours, at least about 130
hours, at least about 140 hours, at least about 150 hours, at least
about 160 hours, at least about 170 hours, at least about 180
hours, or at least about 190 hours. In a particular example, the
T.sub.1/2beta (activity) is about 40 hours to about 193 hours. In
another example, the mean of the T.sub.1/2beta (activity) is about
82 hours.
[0014] The long-acting FIX polypeptide can also have a
T.sub.1/2beta (antigen) of at least about 60 hours, at least about
80 hours, at least about 100 hours, at least about 120 hours, at
least about 140 hours, at least about 160 hours, at least about 180
hours, at least about 200 hours, at least about 220 hours, at least
about 240 hours, at least about 260 hours, at least about 280
hours, at least about 300 hours, at least about 320 hours, at least
about 340 hours, at least about 360 hours, or at least about 370
hours. In a particular embodiment, the T.sub.1/2beta (antigen) is
about 63 hours to about 372 hours.
[0015] In some embodiments, the plasma trough level of the
long-acting FIX polypeptide is maintained at about 1% above the
baseline in the subject after the administration. In other
embodiments, the plasma trough level of the long-acting FIX
polypeptide is maintained between about 1% and about 5%, between
about 1% and about 6%, between about 1% and about 7%, between about
1% and about 8%, between about 1% and about 9%, between about 1%
and about 10%, between about 1% and about 11%, between about 1% and
about 12%, between about 1% and about 13%, between about 1% and
about 14%, between about 1% and about 15% above the baseline in the
subject.
[0016] In one embodiment of the method of the invention, the dose
is about 50 IU/kg, and the dosing interval is about 7 days. In
another embodiment, the dose is about 100 IU/kg and the dosing
interval is at least about 14 days. In other embodiments, the dose
is about 150 IU/kg and the dosing interval is at least about 21
days.
[0017] The long-acting FIX polypeptide can be a chimeric
polypeptide comprising a FIX polypeptide and an FcRn binding
partner. In one embodiment, the FcRn binding partner comprises an
Fc region. In another embodiment, long-acting FIX polypeptide
further comprises a second FcRn binding partner. In other
embodiments, the second FcRn binding partner comprises a second Fc
region. In a particular embodiment, the long-acting FIX polypeptide
is FIX monomer dimer hybrid.
[0018] In some embodiments, the subject is in need of control or
prevention of bleeding or bleeding episodes. In other embodiments,
the subject is in need of control or prevention of bleeding in
minor hemorrhage, hemarthroses, superficial muscle hemorrhage, soft
tissue hemorrhage, moderate hemorrhage, intramuscle or soft tissue
hemorrhage with dissection, mucous membrane hemorrhage, hematuria,
major hemorrhage, hemorrhage of the pharynx, hemorrhage of the
retropharynx, hemorrhage of the retroperitonium, hemorrhage of the
central nervous system, bruises, cuts, scrapes, joint hemorrhage,
nose bleed, mouth bleed, gum bleed, intracranial bleeding,
intraperitoneal bleeding, minor spontaneous hemorrhage, bleeding
after major trauma, moderate skin bruising, or spontaneous
hemorrhage into joints, muscles, internal organs or the brain.
[0019] In one embodiment, the subject is in need of peri-operative
management. In another embodiment, the subject is in need of
management of bleeding associated with surgery or dental
extraction. In other embodiments, the subject will undergo, is
undergoing, or has undergone major surgery. In one aspect, the
major surgery is orthopedic surgery, extensive oral surgery,
urologic surgery, or hernia surgery. In another aspect, the
orthopedic surgery is replacement of knee, hip, or other major
joint.
[0020] In some embodiments, the subject is in need of treatment of
a bleeding disorder, e.g., hemarthrosis, muscle bleed, oral bleed,
hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma,
trauma capitis, gastrointestinal bleeding, intracranial hemorrhage,
intra-abdominal hemorrhage, intrathoracic hemorrhage, bone
fracture, central nervous system bleeding, bleeding in the
retropharyngeal space, bleeding in the retroperitoneal space, or
bleeding in the illiopsoas sheath. In other embodiments, the
subject is in need of surgical prophylaxis, peri-operative
management, or treatment for surgery, e.g., the surgery is minor
surgery, major surgery, tooth extraction, tonsillectomy, inguinal
herniotomy, synovectomy, total knee replacement, craniotomy,
osteosynthesis, trauma surgery, intracranial surgery,
intra-abdominal surgery, intrathoracic surgery, or joint
replacement surgery.
[0021] In some embodiments, the dose is administered intravenously
or subcutaneously.
[0022] The invention also includes a method of estimating a rFIXFc
dosing information individualized for a patient, the method
comprising: (a) receiving, by a computer-based system containing
the rFIXFc population pharmacokinetic (popPK) model of Example 6
and a Bayesian estimation program, at least one of patient
information and desired treatment outcome information, (b)
calculating, by the computer-based system, individualized rFIXFc
dosing information using the popPK model, the Bayesian estimation
program, and the received information, and (c) outputting, by the
computer-based system, the individualized dosing information.
[0023] In some embodiments, the method also comprises selecting a
dosing regimen based on the output individualized dosing
information of (c) and administering rFIXFc to the patient
according to the selected dosing regimen.
[0024] In some embodiments, the desired treatment outcome
information is desired rise in plasma FIX activity level following
dosing and the output information is dose for acute treatment.
[0025] In some embodiments, the desired treatment outcome
information is desired dosing interval and the output information
is dose for prophylaxis.
[0026] In some embodiments, the desired treatment outcome
information is desired dose and the output information is interval
for prophylaxis.
[0027] The invention also includes a method of estimating a rFIXFc
dosing regimen based on median popPK, the method comprising: (a)
receiving, by a computer-based system containing the rFIXFc popPK
model of Example 6 and a Bayesian estimation program, at least one
of patient information and desired treatment outcome information,
(b) calculating, by the computer-based system, median rFIXFc PK
information using the popPK model, the Bayesian estimation program,
and the received information, and (c) outputting, by the
computer-based system, the median PK information.
[0028] In some embodiments, the method also comprises selecting a
dosing regimen based on the output median PK information of (c),
and administering rFIXFc to a patient according to the selected
dosing regimen.
[0029] The invention also includes a method of estimating
individual patient rFIXFc PK, the method comprising: (a) receiving,
by a computer-based system containing the rFIXFc population
pharmacokinetic (popPK) model of Example 6 and a Bayesian
estimation program, individual rFIXFc PK information, (b)
estimating, by the computer-based system, individualized patient
rFIXFc PK information using the popPK model, the Bayesian
estimation program, and the received information, and (c)
outputting, by the computer-based system, the individualized
patient PK information.
[0030] In some embodiments, the method also comprises selecting a
dosing regimen based on the output individualized patient PK
information of (c), and administering rFIXFc to the patient
according to the selected regimen.
[0031] In some embodiments (a) further comprises receiving, by the
computer-based system, patient information.
[0032] In some embodiments the patient information is age or body
weight.
[0033] Additional embodiments include a computer readable storage
medium having instructions stored thereon that, when executed by a
processor, cause the processor to perform any of the above
methods.
[0034] Additional embodiments include a system comprising a
processor and a memory, the memory having instructions stored
thereon that, when executed by the processor, cause the processor
to perform any of the above methods.
BRIEF DESCRIPTION OF DRAWINGS/FIGURES
[0035] FIG. 1 shows study design and CONSORT chart. Efficacy data
that were collected outside of the efficacy period were not
included in the efficacy analyses. *PK subgroup dosed with rFIX
followed by PK assessment and washout (greater than or equal to 5
days) prior to rFIXFc dosing for PK evaluation. rFIX sampling was
done as follows: pre-injection, 10 (.+-.2) min, 1 hour (.+-.15
min), 3 hr (.+-.15 min), 6 hr (.+-.15 min), 24 (.+-.2) hr, 48
(.+-.2) hr, 72 (.+-.3) hr, and 96 (.+-.3) hr (4 d) from the start
of the injection. rFIXFc sampling was done as follows:
pre-injection, 10 (.+-.2) min, 1 hour (.+-.15 min), 3 hr (.+-.15
min), 6 hr (.+-.15 min), 24 (.+-.2) hr, 48 (.+-.2) hr, 96 (.+-.3)
hr (4 d), 144 (.+-.3) hr (6 d), 168 (.+-.3) hr (7 d), 192 (.+-.3)
hr (8 d), and 240 (.+-.3) hr (10 d) from the start of the
injection. Infusion was within 10 minutes. Blood samples were
collected over 96 hours for each subject. A repeat PK assessment of
rFIXFc was also performed at Week 26. ED=exposure day;
PK=pharmacokinetics.
[0036] FIG. 2 shows a summary of Arm 1 sequential dosing and PK
sampling.
[0037] FIG. 3A shows a chart of median annualised bleeding rate
(ABR) for subjects in Arms 1, 2 and 3, respectively. For each arm
bars for total, spontaneous and traumatic bleeding are shown
respectively.
[0038] FIG. 3B shows a summary of annualized bleeding rates by
subgroups. All analyses were consistent with the primary efficacy
analysis, showing a reduction in bleeding rates in all subgroups.
Arm 1 was the only arm with a sufficient number of patients to
display subgroup analyses graphically.
[0039] FIG. 4 shows a graph of Baseline-corrected mean FIX activity
(IU/dL) in rFIX and rFIXFc treated individuals over time using the
one-stage clotting assay (logarithmic scale) in the sequential
pharmacokinetic group (Arm 1).
[0040] FIG. 5 shows a diagram of the three-compartment model for
predicting population PK for rFIXFc. CL, clearance; V, volume of
distribution; Q, inter-compartmental clearance.
[0041] FIG. 6 shows clearance (CL) and V1 estimates of baseline
(week 1) and repeat PK (week 26) profiles (black line indicates
mean, which did not change much between two occasions).
[0042] FIG. 7 shows individual PK parameters versus body weight
(BW).
[0043] FIGS. 8A-8D show goodness-of-fit plots of FIX activity
predicted by the population or individual PK model compared to
observed FIX activity. FIG. 8A shows DV (observed FIX activity
adjusted for baseline activity and residual decay) as a function of
PRED (the prediction by population PK parameter estimates). FIG. 8B
shows DV as a function of IPRED (the prediction by individual PK
parameter estimates). FIG. 8C shows CWRES (conditional weighted
residual) as a function of Time (hours). FIG. 8D shows CWRES as a
function of PRED.
[0044] FIGS. 9A-9D show Visual Predictive Check (VPC) plots of the
population PK model for 50 IU/kg (FIGS. 9A and 9C) or 100 IU/kg
(FIGS. 9B and 9D) doses. Dashed and solid lines represent 10th,
50th, and 90th percentile of the simulated (dashed) and observed
(solid) data, respectively.
[0045] FIG. 10 shows validation of the population PK model with the
trough/peak records. R2=0.9857, P<0.001.
[0046] FIG. 11 shows a diagram of the rFIXFc paediatric study
design.
[0047] FIG. 12 shows a representative plot of observed and
predicted perioperative FIX activity.
[0048] FIGS. 13A-13C show a population simulation of steady-state
FIX activity time profile (5th-95th percentile). FIG. 13A shows the
profile for 50 IU/kg dosage at a dosing interval of once weekly.
FIG. 13B shows the profile for 100 IU/kg dosage at a dosing
interval of once every 10 days. FIG. 13C shows the profile for 100
IU/kg dosage at a dosing interval of once every 14 days.
[0049] FIG. 14 shows a simulation of steady-state FIX activity vs.
time profile comparing 50 IU/kg vs 4000 IU once weekly and 100
IU/kg vs 8000 IU every 10 days in 5th to 95th percentile of the
population.
[0050] FIG. 15 shows a proposed output for individual PK
assessment.
[0051] FIG. 16 shows a proposed output for individualized dosing
regimen selection for episodic treatment.
[0052] FIG. 17 shows a proposed output for dosing regimen
selections without individualized PK assessment.
[0053] FIG. 18 shows another proposed output for dosing regimen
selections without individualized PK assessment.
[0054] FIG. 19 shows an example computer system that can be used in
embodiments.
[0055] FIG. 20 shows a graph plotting the predicted total number of
bleeds vs. time under 1% FIX activity level.
[0056] FIG. 21 shows a graph plotting the predicted total number of
bleeds vs. time under 5% FIX activity level.
[0057] FIG. 22 shows goodness-of-fit plots for the full dataset
model. The solid line is the unit line; the dashed line represents
the linear regression line in the upper panel and the LOESS
smoother in the lower panel; DV is observed FIX activity (adjusted
for baseline activity and residual decay) and unit is IU/dL (%);
PRED is the prediction by population PK parameter estimates and
unit is IU/dL; IPRED is the prediction by individual PK parameter
estimates and unit is IU/dL; CWRES is conditional weighted
residual; TIME unit is hour. DV dependent variable, FIX factor IX,
PK pharmacokinetic.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The present invention provides a method of treating FIX
deficiency, e.g., Hemophilia B, with FIX using a longer dosing
interval, a longer dosing frequency, and/or improved
pharmacokinetic parameters than is possible with currently known
Factor IX products. The present invention also provides improved,
long-acting FIX polypeptides, FIX chimeric polynucleotides, and
methods of production.
I. Definitions
[0059] The term "about" is used herein to mean approximately,
roughly, around, or in the regions of. When the term "about" is
used in conjunction with a numerical range, it modifies that range
by extending the boundaries above and below the numerical values
set forth. In general, the term "about" is used herein to modify a
numerical value above and below the stated value by a variance of
10 percent, up or down (higher or lower).
[0060] The term "polypeptide," "peptide" and "protein" are used
interchangeably and refer to a polymeric compound comprised of
covalently linked amino acid residues.
[0061] The term "polynucleotide" and "nucleic acid" are used
interchangeably and refer to a polymeric compound comprised of
covalently linked nucleotide residues. Polynucleotides can be DNA,
cDNA, RNA, single stranded, or double stranded, vectors, plasmids,
phage, or viruses. Polynucleotides include those in Table 18, which
encode the polypeptides of Table 19 (see Table 18). Polynucleotides
also include fragments of the polynucleotides of Table 18, e.g.,
those that encode fragments of the polypeptides of Table 19, such
as the Factor IX, Fc, signal sequence, propeptide, 6His and other
fragments of the polypeptides of Table 19.
[0062] The term "administering," as used herein, means to or
prescribe or give a pharmaceutically acceptable long-acting FIX
polypeptide of the invention to a subject via a pharmaceutically
acceptable route. Examples of routes of administration include, but
are not limited to, intravenous, e.g., intravenous injection and
intravenous infusion, e.g., via central venous access. Additional
routes of administration include subcutaneous, intramuscular, oral,
nasal, and pulmonary administration, preferably subcutaneous. A
long-acting FIX polypeptides (a FIX chimeric or hybrid proteins)
can be administered as part of a pharmaceutical composition
comprising at least one excipient. Advantages of the present
invention include: improved regimen compliance; reduced break
through bleeds; increased protection of joints from bleeds;
prevention of joint damage; reduced morbidity; reduced mortality;
prolonged protection from bleeding; decreased thrombotic events;
and improved quality of life.
[0063] The term "chimeric polypeptide," as used herein, means a
polypeptide that includes within it at least two polypeptides (or
portions thereof such as subsequences or peptides) from different
sources. Chimeric polypeptides can include two, three, four, five,
six, seven, or more polypeptides or portions thereof from different
sources, such as different genes, different cDNAs, or different
animal or other species. Chimeric polypeptides can include one or
more linkers joining the different polypeptides or portions
thereof. Thus, the polypeptides or portions thereof can be joined
directly or they can be joined indirectly, via linkers, or both,
within a single chimeric polypeptide. Chimeric polypeptides can
include additional peptides such as signal sequences and sequences
such as 6His and FLAG that aid in protein purification or
detection. In addition, chimeric polypeptides can have amino acid
or peptide additions to the N- and/or C-termini. Exemplary chimeric
polypeptides of the invention are Factor IX-FcRn BP chimeric
polypeptides, e.g., Factor IX-Fc chimeric polypeptides such as the
FIXFc in SEQ ID NO:2 (Table 19) with or without its signal sequence
and propeptide.
[0064] The terms "long-acting" and "long-lasting" are used
interchangeably herein. In one embodiment, the term "long-acting"
or "long-lasting" indicates that a FIX activity as a result of
administration of the "long-acting" FIX polypeptide is longer than
the FIX activity of a wild-type FIX (e.g., BENEFIX.RTM. or
plasma-derived FIX ("pdFIX")). The "longer" FIX activity can be
measured by any known methods in the art, e.g., aPTT assay,
chromogenic assay, ROTEM, TGA, and etc. In one embodiment, the
"longer" FIX activity can be shown by the T.sub.1/2beta (activity).
In another embodiment, the "longer" FIX activity can be shown the
level of FIX antigen present in plasma, e.g., by the T.sub.1/2beta
(antigen). In other embodiments, the long-acting or long-lasting
FIX polypeptide works longer in a coagulation cascade, e.g., is
active for a longer period, compared to a wild-type FIX polypeptide
(i.e., a polypeptide consisting of amino acids 1 to 415 of SEQ ID
NO: 2, i.e., BENEFIX.RTM. or pdFIX).
[0065] Factor IX coagulant activity is expressed as International
Unit(s) (IU). One IU of Factor IX activity corresponds
approximately to the quantity of Factor IX in one milliliter of
normal human plasma. Several assays are available for measuring
Factor IX activity, including the one stage clotting assay
(activated partial thromboplastin time; aPTT), thrombin generation
time (TGA) and rotational thromboelastometry (ROTEM.RTM.).
[0066] The term "lyophilisate" (also can be used interchangeably
with "lyophilizate") as used herein in connection with the
formulation according to the invention denotes a formulation which
is manufactured by freeze-drying methods known in the art per se.
The solvent (e.g. water) is removed by freezing following
sublimation under vacuum and desorption of residual water at
elevated temperature. In the pharmaceutical field, the lyophilisate
usually has a residual moisture of about 0.1 to 5% (w/w) and is
present as a powder or a physical stable cake. The lyophilisate is
characterized by a fast dissolution after addition of a
reconstitution medium.
[0067] The term "reconstituted formulation" as used herein denotes
a formulation which is lyophilized and re-dissolved by addition of
a diluent. The diluent can contain, without limitation, water for
injection (WFI), bacteriostatic water for injection (BWFI), sodium
chloride solutions (e.g. 0.9% (w/v) NaCl), glucose solutions (e.g.
5% glucose), surfactant containing solutions (e.g. 0.01%
polysorbate 20 or polysorbate 80), a pH-buffered solution (e.g.
phosphate-buffered solutions) and combinations thereof.
[0068] "Dosing interval," as used herein, means the amount of time
that elapses between multiple doses being administered to a
subject. Dosing interval can thus be indicated as ranges. The
dosing interval in the methods of the invention using a chimeric
FIX-FcRn BP, e.g., a chimeric FIX-Fc, can be at least about one and
one-half to eight times longer than the dosing interval required
for an equivalent amount (in IU/kg) of said Factor IX without the
FcRn BP, e.g., Fc portion (i.e., a polypeptide consisting of said
FIX). The dosing interval when administering, e.g., a Factor IX-Fc
chimeric polypeptide (or a hybrid) of the invention can be at least
about one and one-half times longer than the dosing interval
required for an equivalent amount of said Factor IX without the
FcRn BP, e.g., Fc, portion (i.e., a polypeptide consisting of said
Factor IX). The dosing interval can be at least about one and
one-half to eight times longer than the dosing interval required
for an equivalent amount of said Factor IX without, e.g., the Fc
portion (or a polypeptide consisting of said Factor IX).
[0069] The term "dosing frequency" as used herein refers to the
frequency of administering doses of a long-acting FIX polypeptide
in a given time. Dosing frequency can be indicated as the number of
doses per a given time, e.g., once a week or once in two weeks.
[0070] The term "bleeding episode" as used herein is given a
standardized definition: A bleeding episode starts from the first
sign of a bleed, and ends 72 hours after the last treatment for the
bleeding, within which any symptoms of bleeding at the same
location, or injections less than or equal to 72 hours apart, is
considered the same bleeding episode. See Blanchette V. (2006)
Haemophilia 12:124-7. As used herein, any injection to treat the
bleeding episode, taken more than 72 hours after the preceding one,
is considered the first injection to treat a new bleeding episode
at the same location. Likewise, any bleeding at a different
location is considered a separate bleeding episode regardless of
time from the last injection.
[0071] The term "prophylaxis of one or more bleeding episode" or
"prophylactic treatment" as used herein means administering a
Factor IX polypeptide in multiple doses to a subject over a course
of time to increase the level of Factor IX activity in a subject's
plasma. In one embodiment, "prophylaxis of one or more bleeding
episode" indicates use of a long-acting FIX polypeptide to prevent
or inhibit occurrence of one or more spontaneous or uncontrollable
bleeding or bleeding episodes or to reduce the frequency of one or
more spontaneous or uncontrollable bleeding or bleeding episodes.
In another embodiment, the increased FIX activity level is
sufficient to decrease the incidence of spontaneous bleeding or to
prevent bleeding in the event of an unforeseen injury. Prophylactic
treatment decreases or prevents bleeding episodes, for example,
those described under on-demand treatment. Prophylactic treatment
can be individualized, as discussed under "dosing interval", e.g.,
to compensate for inter-subject variability.
[0072] The term "about once a week" as used herein means
approximate number, and "about once a week" can include every seven
days.+-.two days, i.e., every five days to every nine days. The
dosing frequency of "once a week" thus can be every five days,
every six days, every seven days, every eight days, or every nine
days.
[0073] The term "individualized interval prophylaxis" as used
herein means use of a long-acting FIX polypeptide for an
individualized dosing interval or frequency to prevent or inhibit
occurrence of one or more spontaneous and/or uncontrollable
bleeding or bleeding episodes or to reduce the frequency of one or
more spontaneous and/or uncontrollable bleeding or bleeding
episodes. In one embodiment, the "individualized interval" includes
every 10 days.+-.3 days, i.e. every seven days to every 13 days.
The dosing frequency of the "individualized interval prophylaxis"
thus can be every seven days, every eight days, every nine days,
every ten days, every 11 days, every 12 days, or every 13 days.
[0074] The term "on-demand treatment," as used herein, means
treatment that is intended to take place over a short course of
time and is in response to an existing condition, such as a
bleeding episode, or a perceived short term need such as planned
surgery. The "on-demand treatment" is used interchangeably with
"episodic" treatment. Conditions that can require on-demand
treatment include a bleeding episode, hemarthrosis, muscle bleed,
oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage,
trauma, trauma capitis, gastrointestinal bleeding, intracranial
hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage,
bone fracture, central nervous system bleeding, bleeding in the
retropharyngeal space, bleeding in the retroperitoneal space, or
bleeding in the illiopsoas sheath. Bleeding episodes other than
these are also included. The subject can be in need of surgical
prophylaxis, pen-operative management, or treatment for surgery.
Such surgeries include minor surgery, major surgery, tooth
extraction, tonsillectomy, other dental/thoraco-facial surgeries,
inguinal herniotomy, synovectomy, total knee replacement, other
joint replacement, craniotomy, osteosynthesis, trauma surgery,
intracranial surgery, intra-abdominal surgery, intrathoracic
surgery. Surgeries other than these are also included.
[0075] Additional conditions that can require on-demand treatment
include minor hemorrhage, hemarthroses, superficial muscle
hemorrhage, soft tissue hemorrhage, moderate hemorrhage,
intramuscle or soft tissue hemorrhage with dissection, mucous
membrane hemorrhage, hematuria, major hemorrhage, hemorrhage of the
pharynx, hemorrhage of the retropharynx, hemorrhage of the
retroperitonium, hemorrhage of the central nervous system, bruises,
cuts, scrapes, joint hemorrhage, nose bleed, mouth bleed, gum
bleed, intracranial bleeding, intraperitoneal bleeding, minor
spontaneous hemorrhage, bleeding after major trauma, moderate skin
bruising, or spontaneous hemorrhage into joints, muscles, internal
organs or the brain. Additional reasons for on-demand treatment
include the need for pen-operative management for surgery or dental
extraction, major surgery, extensive oral surgery, urologic
surgery, hernia surgery, orthopedic surgery such as replacement of
knee, hip, or other major joint.
[0076] The term "treatment" or "treating" as used herein means
amelioration or reduction of one or more symptoms of bleeding
diseases or disorders including, but not limited to, hemophilia B.
In one embodiment, "treatment of" or "treating" a bleeding disease
or disorder includes prevention of one or more symptoms of a
bleeding disease or disorder. In a bleeding disease or disorder
caused by a FIX deficiency (e.g., a low baseline FIX activity), the
term "treatment" or "treating" means a FIX replacement therapy. By
administering a long-acting FIX polypeptide to a subject, the
subject can achieve and/or maintain a plasma trough level of a FIX
activity at about 1 IU/dl or above 1 IU/dl. In other embodiments,
"treatment" or "treating" means reduction of the frequency of one
or more symptoms of bleeding diseases or disorders, e.g.,
spontaneous or uncontrollable bleeding episodes. "Treatment,"
however, need not be a cure.
[0077] The term "perioperative management" as used herein means use
of a long-acting FIX polypeptide before, concurrently with, or
after an operative procedure, e.g., a surgical operation. The use
for "perioperative management" of one or more bleeding episode
includes surgical prophylaxis before (i.e., preoperative), during
(i.e., intraoperative), or after (i.e., postoperative) a surgery to
prevent one or more bleeding or bleeding episode or reducing or
inhibiting spontaneous and/or uncontrollable bleeding episodes
before, during, and after a surgery.
[0078] Pharmacokinetic (PK) parameters include the terms above and
the following terms, which have their ordinary meaning in the art,
unless otherwise indicated. Some of the terms are explained in more
detail in the Examples. PK parameters can be based on FIX antigen
level (often denoted parenthetically herein as "antigen") or FIX
activity level (often denoted parenthetically herein as
"activity"). In the literature, PK parameters are often based on
FIX activity level due to the presence in the plasma of some
subjects of endogenous, inactive FIX, which interferes with the
ability to measure administered (i.e., exogenous) FIX using
antibody against FIX. However, when FIX is administered as part of
a fusion or hybrid protein containing a heterologous polypeptide
such as an FcRn BP, administered (i.e., exogenous) FIX antigen can
be accurately measured using antibody to the heterologous
polypeptide. In addition, certain PK parameters can be based on
model predicted data (often denoted parenthetically herein as
"model predicted") or on observed data (often denoted
parenthetically herein as "observed"), and preferably are based on
observed data.
[0079] "Baseline," as used herein, is the lowest measured plasma
Factor IX level in a subject prior to administering a dose. The
Factor IX plasma levels can be measured at two time points prior to
dosing: at a screening visit and immediately prior to dosing.
Alternatively, (a) the baseline in subjects whose pretreatment FIX
activity is <1%, who have no detectable FIX antigen, and have
nonsense genotypes can be defined as 0%, (b) the baseline for
subjects with pretreatment FIX activity <1% and who have
detectable FIX antigen can be set at 0.5%, (c) the baseline for
subjects whose pretreatment FIX activity is between 1-2% is Cmin
(the lowest activity throughout the PK study), and (d) the baseline
for subjects whose pretreatment FIX activity is .gtoreq.2% can be
set at 2%. Activity above the baseline pre-dosing can be considered
residue drug from prior treatment, and can be decayed to baseline
and subtracted from the PK data following rFIXFc dosing.
[0080] "T.sub.1/2.beta.," or "T.sub.1/2 beta" or "Beta HL," as used
herein, is half-life associated with elimination phase,
t.sub.1/2.beta.=(ln 2)/elimination rate constant associated with
the terminal phase. The T.sub.1/2 beta can be measured by FIX
activity or by FIX antigen level in plasma. The T.sub.1/2 beta
based on activity is shown as T.sub.1/2 beta (activity), and the
T.sub.1/2 beta based on the FIX antigen level can be shown as
T.sub.1/2 beta (antigen). Both T.sub.1/2 beta (activity) and
T.sub.1/2 beta (antigen) can be shown as ranges or a geometric
mean.
[0081] "Trough," as used herein, is the lowest plasma Factor IX
activity level reached after administering a dose of chimeric
polypeptide of the invention or another Factor IX molecule and
before the next dose is administered, if any. Trough is used
interchangeably herein with "threshold." Baseline Factor IX levels
are subtracted from measured Factor IX levels to calculate the
trough level.
[0082] The term "annualized bleeding rate" ("ABR) as used herein
refers to the number of bleeding episodes (including spontaneous
and traumatic bleeds) experienced by a subject during a defined
time period, extrapolated to 1 year. For example two bleeds in six
months would indicate an ABR of four. The median ABR provides a
single number to describe all subjects, indicating that half of the
subjects had individual ABRs less than or equal to the median and
half had ABRs greater than or equal to the median. For example, an
ABR can be calculated according to the following formula:
Annualized bleeding rate=Number of bleeding episodes during the
efficacy period/Total number of days during the efficacy
period.times.365.25
[0083] "Subject," as used herein means a human. Subject as used
herein includes an individual who is known to have at least one
incidence of uncontrolled bleeding episodes, who has been diagnosed
with a disease or disorder associated with uncontrolled bleeding
episodes, e.g., a bleeding disease or disorder, e.g., hemophilia B,
who are susceptible to uncontrolled bleeding episodes, e.g.,
hemophilia, or any combinations thereof. Subjects can also include
an individual who is in danger of one or more uncontrollable
bleeding episodes prior to a certain activity, e.g., a surgery, a
sport activity, or any strenuous activities. The subject can have a
baseline FIX activity less than 1%, less than 0.5%, less than 2%,
less than 2.5%, less than 3%, or less than 4%. Subjects also
include pediatric humans. Pediatric human subjects are birth to 20
years, preferably birth to 18 years, birth to 16 years, birth to 15
years, birth to 12 years, birth to 11 years, birth to 6 years,
birth to 5 years, birth to 2 years, and 2 to 11 years of age.
[0084] "Therapeutic dose," "dose," "effective dose," or "dosing
amount" as used herein, means a dose that achieves a plasma trough
level of a FIX activity at least about 1 IU/dl or above 1 IU/dl in
the subject administered with the long-acting FIX polypeptide. For
the purpose of this invention, in one embodiment, the "dose" refers
to the amount of the doses that a plasma trough level of a FIX
activity is maintained at least about 1 IU/dl or above 1 IU/dl, at
least about 2 IU/dl or above 2 IU/dl, at least about 3 IU/dl or
above 3 IU/dl, at least about 4 IU/dl or above 4 IU/dl, or at least
about 5 IU/dl or above 5 IU/dl throughout the administration of the
long-acting FIX polypeptide. In another embodiment, the "dose"
reduces or decreases frequency of bleeding or bleeding disorder. In
other embodiments, the "dose" stops on-going, uncontrollable
bleeding or bleeding episodes. In still other embodiments, the
"dose" prevents spontaneous bleeding or bleeding episodes in a
subject susceptible to such spontaneous bleeding or bleeding
episodes. The "dose" or "therapeutic dose" need not cure
hemophilia.
[0085] "Variant," as used herein, refers to a polynucleotide or
polypeptide differing from the original polynucleotide or
polypeptide, but retaining essential properties thereof, e.g.,
Factor IX coagulant activity or Fc (FcRn binding) activity.
Generally, variants are overall closely similar, and, in many
regions, identical to the original polynucleotide or polypeptide.
Variants include polypeptide and polynucleotide fragments,
deletions, insertions, and modified versions of original
polypeptides.
II. Method of Administering
[0086] The present invention provides methods of administering a
long-acting Factor IX (FIX) polypeptide to a human subject in need
thereof, comprising administering to the subject a dose of a
long-acting FIX polypeptide at a dosing interval. Administration of
the long-acting FIX polypeptide is a replacement therapy by adding
a recombinant FIX to a subject with FIX deficiency. Administration
of the long-acting FIX polypeptide can reduce or prevent a number
of bleeding or bleeding episodes in the subject.
[0087] The subject for the methods of the invention includes those
in need of control or prevention of bleeding or bleeding episodes.
The subject can be bleeding at the time of administration or be
expected to be bleeding, or can be susceptible to bleeding in minor
hemorrhage, hemarthroses, superficial muscle hemorrhage, soft
tissue hemorrhage, moderate hemorrhage, intramuscle or soft tissue
hemorrhage with dissection, mucous membrane hemorrhage, hematuria,
major hemorrhage, hemorrhage of the pharynx, hemorrhage of the
retropharynx, hemorrhage of the retroperitonium, hemorrhage of the
central nervous system, bruises, cuts, scrapes, joint hemorrhage,
nose bleed, mouth bleed, gum bleed, intracranial bleeding,
intraperitoneal bleeding, minor spontaneous hemorrhage, bleeding
after major trauma, moderate skin bruising, or spontaneous
hemorrhage into joints, muscles, internal organs or the brain. Such
subjects also include those in need of pen-operative management,
such as management of bleeding associated with surgery or dental
extraction. In one embodiment, the subject is in need of
prophylaxis of one or more bleeding episodes. In another
embodiment, the subject is in need of individualized interval
prophylaxis. In other embodiments, the subject is in need of
on-demand treatment of one or more bleeding episodes. In still
other embodiments, the subject is in need of perioperative
management of one or more bleeding episodes.
[0088] The present invention also identifies the appropriate dosing
amount and the dosing interval that can treat or prevent one or
more bleeding episodes. Administration of the appropriate dosing
amount for the dosing interval can achieve a plasma trough level of
a FIX activity at least about 1 IU/dl or above 1 IU/dl during the
interval in a subject administered with a long-acting FIX
polypeptide. In one embodiment, the invention includes a dosing
amount (or ranges of the dosing amount) and a dosing interval (or
ranges of the dosing interval) that can maintain a plasma trough
level of a FIX activity at least about 1 IU/dl (1%) or above 1
IU/dl (1%), at least about 2 IU/dl (2%) or above 2 IU/dl (2%), at
least about 3 IU/dl (3%) or above 3 IU/dl (3%), at least about 4
IU/dl (4%) or above 4 IU/dl (4%), or at least about 5 IU/dl (5%) or
above 5 IU/dl (5%) throughout the interval. In another embodiment,
a dosing amount (or ranges of the dosing amount) and a dosing
interval (or ranges of the dosing interval) that reduces or
decreases frequency of bleeding or bleeding disorder. In other
embodiments, the dosing amount (or ranges of the dosing amount) and
the dosing interval (or ranges of the dosing interval) of a
long-acting FIX polypeptide stops on-going, uncontrollable bleeding
or bleeding episodes in a subject administered with the dosing
amount during the dosing interval. In still other embodiments, the
dosing amount (or ranges of the dosing amount) and the dosing
interval (or ranges of the dosing interval) of a long-acting FIX
polypeptide prevents spontaneous bleeding or bleeding episodes in a
subject susceptible to such spontaneous bleeding or bleeding
episodes. Various dosing amounts and dosing intervals are described
in International Appl. No. PCT/US2011/043569 filed Jul. 11, 2011
and published as WO 2012/006624 on Jan. 12, 2012, which is
incorporated herein by reference in its entirety.
[0089] The doses that can be used in the methods of the invention
are about 10 IU/kg to about 200 IU/kg, about 10 IU/kg to about 180
IU/kg, or about 25 IU/kg to about 200 IU/kg. In one embodiment, the
dose of a long-acting FIX polypeptide is about 10 IU/kg to about 50
IU/kg, about 10 IU/kg to about 100 IU/kg, about 25 IU/kg to about
75 IU/kg, about 25 IU/kg to about 100 IU/kg, about 25 IU/kg to
about 125 IU/kg, about 25 IU/kg to about 150 IU/kg, about 25 IU/kg
to about 50 IU/kg, about 50 IU/kg to about 100 IU/kg, about 50
IU/kg to about 150 IU/kg, about 100 IU/kg to about 150 IU/kg, about
150 IU/kg to about 200 IU/kg, or any combinations thereof.
[0090] In another embodiment, a dose of a long-acting FIX
polypeptide for at least about every five days or longer are as
follows: about 25 to about 110, about 30 to about 110, about 40 to
about 110, about 50 to about 110, about 60 to about 110, about 70
to about 110, about 80 to about 110, about 90 to about 110, and
about 100 to about 110; about 30 to about 100, about 30 to about
90, about 30 to about 80, about 30 to about 70, about 30 to about
60, about 30 to about 50, about 30 to about 40 IU/kg; about 40 to
about 110, about 50 to about 100, about 60 to about 90, and about
70 to about 80 IU/kg; about 40 to about 50, about 50 to about 60,
about 60 to about 70, about 70 to about 80, about 80 to about 90,
about 90 to about 100, and about 100 to about 110 IU/kg; about 25,
about 30, about 35, about 40, about 45, about 50, about 55, about
60, about 65, about 70, about 75, about 80, about 85, about 90,
about 95, about 100, about 105, or about 110 IU/kg.
[0091] In certain embodiments, a dose of a long-acting FIX
polypeptide is an amount sufficient for weekly prophylaxis of a
bleeding episode. For example, the doses for the weekly dosing
interval include about 10 to about 50, about 20 to about 60, about
20 to about 70, about 20 to about 80, about 20 to about 90, about
30 to about 40, about 30 to about 50, about 30 to about 60, about
30 to about 70, about 30 to about 80, about 30 to about 90, about
40 to about 50, about 40 to about 60, about 50 to about 70, about
40 to about 80, about 40 to about 90, about 50 to about 60, about
50 to about 70, about 50 to about 80, about 50 to about 90, about
60 to about 70, about 60 to about 80, about 60 to about 90, about
70 to about 80, about 70 to about 90 IU/kg. Doses can be lower than
20 IU/kg if effective for a given subject, e.g., about 10, about
11, about 12, about 13, about 14, about 15, about 16, about 17,
about 18, or about 19 IU/kg.
[0092] The dosing interval can, alternatively, be an individualized
interval that is determined for each subject based on
pharmacokinetic data or other information about that subject. The
individualized dose/dosing interval combination can be the same as
those for fixed interval regimens in the preceding paragraphs, or
can differ. The regimen can initially be at a fixed dosing
interval, and then it can change to an individualized dosing
interval.
[0093] In some embodiments, a dose of a long-acting FIX polypeptide
is an amount sufficient for individualized interval prophylaxis of
a bleeding episode. In one example, the individualized interval is
every nine days, every 10 days, every 11 days, every 12 days, every
13 days, every 14 days, every 15 days, every 16 days, every 17
days, every 18 days, every 19 days or two times monthly. Examples
of the doses for the individualized interval include, but are not
limited to: about 50 IU/kg to about 180 IU/kg, about 60 IU/kg to
about 180 IU/kg, about 70 IU/kg to about 180 IU/kg, about 80 IU/kg
to about 180 IU/kg, about 90 IU/kg to about 180 IU/kg, about 100
IU/kg to about 180 IU/kg, about 110 IU/kg to about 180 IU/kg, about
120 IU/kg to about 180 IU/kg, about 130 IU/kg to about 180 IU/kg,
about 140 IU/kg to about 180 IU/kg, about 60 IU/kg to about 170
IU/kg, about 60 IU/kg to about 160 IU/kg, about 60 IU/kg to about
150 IU/kg, about 60 IU/kg to about 140 IU/kg, about 70 IU/kg to
about 140 IU/kg, about 70 IU/kg to about 130 IU/kg, about 80 IU/kg
to about 130 IU/kg, about 80 IU/kg to about 140 IU/kg, about 90
IU/kg to about 140 IU/kg, about 90 IU/kg to about 130 IU/kg, about
90 IU/kg to about 120 IU/kg, about 90 IU/kg to about 110 IU/kg,
about 90 IU/kg to about 100 IU/kg, about 100 IU/kg to about 140
IU/kg, about 100 IU/kg to about 130 IU/kg, about 100 IU/kg to about
120 IU/kg, or about 100 IU/kg to about 110 IU/kg.
[0094] In still other embodiments, the individualized dosing
interval is at least about once in two weeks, at least about 15
days, at least about 16 days, at least about 17 days, at least
about 18 days, at least about 19 days, at least about 20 days, at
least about 21 days, at least about once in three weeks, at least
about 22 days, at least about 23 days, at least about 24 days, at
least about 25 days, at least about 26 days, at least about 27
days, at least about 28 days, at least about 29 days, at least
about 30 days, or at least about once a month. For example, the
doses for the individualized dosing interval are about 120 IU/kg to
about 200 IU/kg, about 130 IU/kg to about 200 IU/kg, about 140
IU/kg to about 200 IU/kg about 150 IU/kg to about 200 IU/kg, about
160 IU/kg to about 200 IU/kg, about 170 IU/kg to about 200 IU/kg,
about 180 IU/kg to about 200 IU/kg, about 120 IU/kg to about 180
IU/kg, about 130 IU/kg to about 180 IU/kg, about 140 IU/kg to about
180 IU/kg, about 150 IU/kg to about 180 IU/kg, about 160 IU/kg to
about 180 IU/kg, about 120 IU/kg to about 170 IU/kg, about 130
IU/kg to about 170 IU/kg, about 140 IU/kg to about 170 IU/kg, about
150 IU/kg to about 170 IU/kg, about 160 IU/kg to about 170 IU/kg,
about 120 IU/kg to about 160 IU/kg, about 130 IU/kg to about 160
IU/kg, about 140 IU/kg to about 160 IU/kg, about 150 IU/kg to about
160 IU/kg, about 120 IU/kg to about 150 IU/kg, about 130 IU/kg to
about 150 IU/kg, about 140 IU/kg to about 150 IU/kg, about 120
IU/kg to about 140 IU/kg, or about 130 IU/kg to about 140 IU/kg. In
other examples, a dose for an individualized dosing interval is
about 60 IU/kg, about 70 IU/kg, about 80 IU/kg, about 90 IU/kg,
about 95 IU/kg, about 100 IU/kg, about 105 IU/kg, about 110 IU/kg,
about 115 IU/kg, about 120 IU/kg, about 125 IU/kg, about 130 IU/kg,
about 135 IU/kg, about 140 IU/kg, about 145 IU/kg, about 150 IU/kg,
about 155 IU/kg, about 160 IU/kg, about 165 IU/kg, about 170 IU/kg,
about 175 IU/kg, or about 180 IU/kg.
[0095] In some embodiments, a dose of a long-acting FIX polypeptide
is sufficient for on-demand treatment of one or more bleeding
episodes. The doses for the on-demand treatment can vary depending
on the various factors, e.g., subject's baseline FIX activity,
subject's body weight, subject's likelihood of experiencing
bleeding episode, and etc. In one example, the doses for the
on-demand treatment can be about 10 to about 50, about 15 to about
100, about 20 to about 100, about 20 to about 50, about 50 to about
100, about 10, about 20, about 40, about 50, and about 100 IU/kg.
In another example, the doses for the on-demand treatment can be
about 20 to about 50, about 20 to about 100, about 20 to about 180,
25 to about 110, about 30 to about 110, about 40 to about 110,
about 50 to about 110, about 60 to about 110, about 70 to about
110, about 80 to about 110, about 90 to about 110, about 100 to
about 110, about 30 to about 100, about 30 to about 90, about 30 to
about 80, about 30 to about 70, about 30 to about 60, about 30 to
about 50, about 30 to about 40 IU/kg, about 40 to about 110, about
50 to about 100, about 60 to about 90, about 70 to about 80 IU/kg,
about 40 to about 50, about 50 to about 60, about 60 to about 70,
about 70 to about 80, about 80 to about 90, about 90 to about 100,
about 100 to about 110 IU/kg, about 20, about 25, about 30, about
35, about 40, about 45, about 50, about 55, about 60, about 65,
about 70, about 75, about 80, about 85, about 90, about 95, about
100, about 105, and about 110 IU/kg. In other examples, the doses
for the on-demand treatment can be about 90 to about 180, about 100
to about 180, about 110 to about 180, about 120 to about 180, about
130 to about 180, about 140 to about 180, about 150 to about 180,
about 160 to about 180, and about 170 to about 180 IU/kg. In still
other examples, the doses for the on-demand treatment are about 90
to about 170, about 90 to about 160, about 90 to about 150, about
90 to about 140, about 90 to about 130, about 90 to about 120,
about 90 to about 110, and about 90 to about 100 IU/kg. In
examples, the doses for the on-demand treatment are about 100 to
about 170, about 110 to about 160, about 120 to about 150, and
about 130 to about 140 IU/kg. In yet other examples, the dose for
the on-demand treatment are about 90 to about 100, about 100 to
about 110, about 110 to about 120, about 120 to about 130, about
130 to about 140, about 140 to about 150, about 150 to about 160,
and about 160 to about 170 IU/kg. The dose for the on-demand
treatment can be about 115, about 120, about 125, about 130, about
135, about 140, about 145, about 150, about 155, about 160, about
165, about 170, about 175, and about 180 IU/kg.
[0096] In certain embodiments, a dosing amount and a dosing
interval combination for a subject is 20 IU/kg once weekly, 40
IU/kg once weekly, 50 IU/kg once weekly, 100 IU/kg every 10 days,
and 100 IU/kg every two weeks (or twice monthly). Additional
combinations of dose and dose interval include: a dose at least
about 50 IU/kg and a dosing interval at least about 7 days, a dose
at least about 100 IU/kg and a dosing interval at least about 9
days, a dose at least about 100 IU/kg and a dosing interval at
least about 12 days, a dose at least about 100 IU/kg and a dosing
interval at least about 13 days, a dose at least about 100 IU/kg
and a dosing interval at least about 14 days, a dose at least about
100 IU/kg and a dosing interval at least about 15 days, a dose at
least about 150 IU/kg and a dosing interval at least about 14 days,
20-50 or 20-100 IU/kg and said dosing interval is one time weekly,
a dose of 20-50 IU/kg and a dosing interval of 7 days, a dose of
50-100 IU/kg and a dosing interval of 10-14 days, or a dose of
100-150 IU/kg and a dosing interval of 14-16 days. Non-limiting
examples of the combinations of dosing interval and dose also
include 10-50 IU/kg for 7 days, 15-100 IU/kg for 10-13 days, 50-150
IU/kg for 14-15 days, 10-30 IU/kg for 7 days, 15-50 IU/kg for 10
days, 20-70 IU/kg for 11 days, 25-85 IU/kg for 12 days, 30 to 100
IU/kg for 13 days, 40 to 125 IU/kg for 14 days, and 50-150 IU/kg
for 15 days.
[0097] In one embodiment, a dosing interval is at least about once
a week, at least about once in two weeks, at least about twice a
month, at least about once in three weeks, at least about once in
four weeks, or at least about once a month. In another embodiment,
a dosing interval of a long-acting FIX polypeptide is about once in
five days to about once in two months, about once in five days to
about once in month, or about once a week to about once a month. In
other embodiments, the dosing interval is at least about every five
days, about every six days, at least about every seven days, at
least about every eight days, at least about every nine days, at
least about every ten days, at least about every 11 days, at least
about every 12 days, at least about every 13 days, at least about
every 14 days, at least about every 15 days, at least about every
16 days, at least about every 17 days, at least about every 18
days, at least about every 19 days, at least about every 20 days,
or at least about every 21 days. In still other embodiments, a
dosing interval is 9-18 days, e.g., about 9-17, about 9-16, about
9-15, about 9-14, about 9-13, about 9-12, about 9-11, about 9-10
days, about 10-18, about 11-18, about 12-18, about 13-18, about
14-18, about 15-18, about 16-18, about 17-18 days, about 10-11,
about 11-12, about 12-13, about 13-14, about 14-15, about 15-16,
and about 16-17 days, about 9, about 10, about 11, about 12, about
13, about 14, about 15, about 16, about 17, or about 18 days. In
yet other embodiments, the dosing interval is about 10-14 days. The
dosing interval can be longer than 18 days, e.g., about 19, about
20, about 21, about 22, about 23, about 24, about 25, about 26,
about 27, about 28, about 29, about 30, about 31, about 32, about
33, about 34, about 35, about 36, about 37, about 38, about 39, or
about 40 days.
[0098] In one embodiment, a dosing frequency for a long-acting FIX
polypeptide is about every two weeks or twice monthly. In another
embodiment, the dosing frequency is every 7 days for 25-50 IU/kg,
every 10-13 days for 50-100 IU/kg, or every 14 days for 100-150
IU/kg. The interval (or frequency) and dose are determined such
that the combination of interval (or frequency) and dose will
result in a trough level of at least about 1-5 or at least about
1-3, or at least about 1, at least about 2, at least about 3 IU/dl
FIX activity in the subject. Examples of the dose and dosing
interval can also be 7 days for 20-50 IU/kg, 10-14 days for 50-100
IU/kg, 14-16 days for 100-150 IU/kg, 7 days for 10-50 IU/kg, 10-13
days for 15-100 IU/kg, or 14-15 days for 50-150 IU/kg. Additional
examples of the dose and dosing interval include, but are not
limited to, 7 days for 10-30 IU/kg, 10 days 15-50 IU/kg, 11 days
20-70 IU/kg, 12 days 25-85 IU/kg, 13 days 30 to 100 IU/kg, 14 days
40 to 125 IU/kg, and 15 days for 50-150 IU/kg.
[0099] In some embodiments, the dosing interval is 20 IU/kg once
weekly, 40 IU/kg every 10 days, 50 IU/kg once weekly, or 100 IU/kg
every two weeks (twice monthly).
[0100] In some embodiments of the invention, an annualized bleeding
rate (ABR) of a bleeding episode is controlled by the present
methods. For example, the dosing amount and dosing interval can be
administered to reduce or decrease an annualized bleeding rate to a
certain level. In one embodiment, administration of a long-acting
FIX polypeptide at a dose and a dosing interval for prophylaxis of
a bleeding episode results in an annual bleeding rate of less than
2, less than 2.5, less than 3, less than 3.5, less than 4, less
than 4.5, less than 5, less than 5.5, less than 6, less than 6.5,
less than 7, less than 7.5, less than 8, less than 8.5, less than
9, less than 9.5, or less than 10. For example, ABR of weekly
prophylaxis of a bleeding episode can be 2.95. In another
embodiment, administration of a long-acting FIX polypeptide at a
dose and a dosing interval for individualized interval prophylaxis
of a bleeding episode results in an ABR of less than 1, less than
1.5, less than 2, less than 2.5, less than 3, less than 3.5, less
than 4, less than 4.5, less than 5, less than 5.5, less than 6,
less than 6.5, less than 7, less than 7.5, less than 8, less than
8.5, or less than 9. For example, ABR for individualized interval
prophylaxis can be 1.38. In other embodiments, administration of a
long-acting FIX polypeptide at a dose and a dosing interval for
on-demand treatment (i.e., episodic treatment) of a bleeding
episode results in an ABR of less than 10, less than 11, less than
12, less than 13, less than 14, less than 15, less than 16, less
than 17, less than 18, less than 19, less than 20, less than 21,
less than 22, less than 23, less than 24, less than 25, or less
than 26. For example, ABR for on-demand treatment can be 17.69.
[0101] The long-acting FIX polypeptide of the invention can provide
a half-life, e.g., T.sub.1/2beta (activity) or T.sub.1/2beta
(antigen), that is longer than wild-type FIX (i.e., a polypeptide
consisting of amino acids 1 to 415 of SEQ ID NO: 2; BENEFIX.RTM.;
or pdFIX). In one embodiment, a T.sub.1/2beta (activity) of a
long-acting FIX polypeptide is at least about 40 hours, at least
about 45 hours, at least about 50 hours, at least about 55 hours,
at least about 60 hours, at least about 65 hours, at least about 70
hours, at least about 75 hours, at least about 80 hours, at least
about 85 hours, at least about 90 hours, at least about 95 hours,
at least about 100 hours, at least about 105 hours, at least about
110 hours, at least about 115, at least about 120, at least about
125, at least about 130, at least about 135, at least about 140, at
least about 145, at least about 150, at least about 155, at least
about 160, at least about 165, at least about 170, at least about
175, at least about 180, at least about 185, at least about 190, or
at least about 193. In another embodiment, a T.sub.1/2beta
(activity) of a long-acting FIX polypeptide is about 40 to about
193 hours, about 35 hours to about 190 hours, about 45 hours to
about 193 hours, about 50 hours to about 198 hours, about 55 hours
to about 188 hours, about 60 hours to about 200 hours, about 30
hours to about 205 hours, about 43 hours to about 210 hours. In a
specific embodiment, a T.sub.1/2beta (activity) is about 40 hours
to about 193 hours.
[0102] In some embodiments, the T.sub.1/2 beta (activity) of a
long-acting FIX polypeptide is expressed as a mean. For example, a
mean of the T.sub.1/2 beta (activity) of a long-acting FIX
polypeptide is at least about 76 hours, at least about 77 hours, at
least about 78 hours, at least about 79 hours, at least about 80
hours, at least about 81 hours, at least about 82 hours, at least
about 83 hours, at least about 84 hours, at least about 85 hours,
at least about 86 hours, at least about 87 hours, at least about 88
hours, at least about 89 hours, at least about 90 hours, at least
about 91 hours, or at least about 92 hours. In a specific
embodiment, a mean of the T.sub.1/2 beta (activity) of a
long-acting FIX polypeptide is 82 hours.
[0103] In other embodiments, the T.sub.1/2 beta (activity) of a
long-acting FIX polypeptide is shown as a comparison to a T.sub.1/2
beta (activity) of wild-type mature FIX (i.e., BENEFIX.RTM.). In
one example, the mean of the T.sub.1/2 beta (activity) is at least
about 2.0 fold higher than wild-type mature FIX (a polypeptide
consisting of amino acids 1 to 415 of SEQ ID NO:2, i.e.,
BENEFIX.RTM. or pdFIX). In another example, the mean of the
T.sub.1/2 beta (activity) is at least about 2.0 fold, at least
about 2.1 fold, at least about 2.2 fold, at least about 2.3 fold,
at least about 2.4 fold, at least about 2.5 fold, at least about
2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at
least about 2.9 fold, at least about 3.0 fold, at least about 3.1
fold, or at least about 3.2 fold higher than wild-type mature FIX
(a polypeptide consisting of amino acids 1 to 415 of SEQ ID NO:2,
i.e., BENEFIX.RTM.; pdFIX).
[0104] In still other embodiments, the invention provides
administering a long-acting FIX polypeptide to a subject who can
exhibit a longer T.sub.1/2 beta (activity). In one embodiment, the
methods of the invention includes administering a long-acting FIX
polypeptide to a subject who exhibits at least about 80 hours, at
least about 81 hours, at least about 82 hours, at least about 83
hours, at least about 84 hours, at least about 85 hours, at least
about 86 hours, at least about 87 hours, at least about 88 hours of
the T.sub.1/2 beta (activity). In another embodiment, the methods
include administering a long-acting FIX polypeptide to a subject
who has a baseline FIX activity less than 2%, less than 1.5%, less
than 1%, or less than 0.5%, thereby resulting in a T.sub.1/2 beta
(activity) higher than about 80 hours, about 81 hours, about 82
hours, about 83 hours, about 84 hours, about 85 hours, about 86
hours, about 87 hours, or about 88 hours.
[0105] In certain embodiments, a T.sub.1/2beta (antigen) of a
long-acting FIX polypeptide is at least about 63 hours, at least
about 70 hours, at least about 80 hours, at least about 90 hours,
at least about 100 hours, at least about 110 hours, at least about
120 hours, at least about 130 hours, at least about 140 hours, at
least about 150 hours, at least about 160 hours, at least about 170
hours, at least about 180 hours, at least about 190 hours, at least
about 200 hours, at least about 210, at least about 220, at least
about 230, at least about 240, at least about 250, at least about
260, at least about 270, at least about 280, at least about 290, at
least about 300, at least about 310, at least about 320, at least
about 330, at least about 340, at least about 350, at least about
360, or at least about 370. In another embodiment, a T.sub.1/2beta
(antigen) of a long-acting FIX polypeptide is about 63 to about 372
hours, about 50 hours to about 380 hours, about 40 hours to about
390 hours, about 70 hours to about 360 hours, about 80 hours to
about 400 hours, about 90 hours to about 410 hours, about 50 hours
to about 400 hours, about 40 hours to about 380 hours. In a
specific embodiment, a T.sub.1/2beta (antigen) is about 63 hours to
about 372 hours.
[0106] In some embodiments, the T.sub.1/2 beta (antigen) of a
long-acting FIX polypeptide is expressed as a mean. For example, a
mean of the T.sub.1/2 beta (antigen) of a long-acting FIX
polypeptide is at least about 110 hours, at least about 111 hours,
at least about 112 hours, at least about 113 hours, at least about
114 hours, at least about 115 hours, at least about 116 hours, at
least about 117 hours, at least about 118 hours, at least about 119
hours, at least about 120 hours, at least about 121 hours, at least
about 122 hours, at least about 123 hours, at least about 124
hours, at least about 125 hours, at least about 126 hours, at least
about 127 hours, at least about 128 hours, at least about 129
hours, at least about 130 hours, or at least about 131 hours. In a
specific embodiment, a mean of the T.sub.1/2 beta (antigen) of a
long-acting FIX polypeptide is 118 hours or 126 hours.
[0107] In other embodiments, the T.sub.1/2 beta (antigen) of a
long-acting FIX polypeptide is shown as a comparison to a T.sub.1/2
beta (antigen) of wild-type mature FIX (a polypeptide consisting of
amino acids 1 to 415 of SEQ ID NO: 2, i.e., BENEFIX.RTM. or pdFIX).
In one example, the mean of the T.sub.1/2 beta (antigen) is at
least about 2.0 fold higher than wild-type mature FIX (a
polypeptide consisting of amino acids 1 to 415 of SEQ ID NO:2,
i.e., BENEFIX.RTM. or pdFIX). In another example, the mean of the
T.sub.1/2 beta (antigen) is at least about 2.0 fold, at least about
3 fold, at least about 4 fold, at least about 5 fold, at least
about 6 fold, at least about 7 fold, at least about 8 fold, at
least about 9 fold, at least about 10 fold, at least about 11 fold,
at least about 12 fold than wild-type mature FIX (a polypeptide
consisting of amino acids 1 to 415 of SEQ ID NO:2, i.e.,
BENEFIX.RTM.; pdFIX).
[0108] In still other embodiments, the invention provides
administering a long-acting FIX polypeptide to a subject who can
exhibit a longer T.sub.1/2 beta (antigen). In one embodiment, the
methods of the invention includes administering a long-acting FIX
polypeptide to a subject who exhibits at least about 115 hours, at
least about 116 hours, at least about 117 hours, at least about 118
hours, at least about 119 hours, at least about 120 hours, at least
about 121 hours, at least about 122 hours, at least about 123
hours, at least about 124 hours, at least about 125 hours, at least
about 126 hours, or at least about 127 hours of the T.sub.1/2 beta
(antigen). In another embodiment, the methods include administering
a long-acting FIX polypeptide to a subject who has a baseline FIX
activity less than 2%, less than 1.5%, less than 1%, or less than
0.5%, thereby resulting in a T.sub.1/2 beta (activity) higher than
about 115 hours, about 116 hours, about 117 hours, about 118 hours,
about 119 hours, about 120 hours, about 121 hours, about 122 hours,
about 123 hours, about 124 hours, about 125 hours, about 126 hours,
about 127 hours, about 128 hours, or about 129 hours.
[0109] In certain embodiments, the T.sub.1/2beta (activity) is
measured by one stage clotting assay.
[0110] In other embodiments, the long-acting FIX polypeptide has a
Mean Residence Time (MRT) of at least about 50 hours, at least
about 60 hours, at least about 70 hours, at least about 80 hours,
at least about 90 hours, at least about 100 hours, at least about
110 hours, at least about 120 hours, at least about 130 hours, at
least about 140 hours, at least about 150 hours, at least about 160
hours, at least about 170 hours, at least about 180 hours, or at
least about 190 hours. In some embodiments, the MRT is about 50 to
about 200 hours, about 60 hours to about 210 hours, about 60 hours
to about 183 hours, about 70 hours to about 150 hours, about 70
hours to about 140 hours, about 70 hours to about 130 hours, about
80 hours to about 120 hours, about 80 hours to about 110 hours, or
about 88 hours to about 110 hours. In a particular embodiment, the
MRT is at least about 2.0 fold (e.g., 2.1 fold, 2.2 fold, 2.3 fold,
2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.7 fold, 2.8 fold, 2.9
fold, or 3 fold) higher than a polypeptide consisting of amino
acids 1 to 415 of SEQ ID NO: 2 or BENEFIX.RTM..
[0111] In certain embodiments of the invention, the method of the
invention further comprises measuring a baseline FIX activity of a
subject prior to the initial administration of a long-acting FIX
polypeptide. Measuring of a baseline FIX activity can employ any
known clotting assays in the art, e.g., one step aPTT assay, two
step chromogenic assay, ROTEM, TGA, or etc.
[0112] In some embodiments, the method of the invention further
comprises measuring a T.sub.1/2beta (activity) or T.sub.1/2beta
(antigen) of the long-acting FIX polypeptide in the subject after
administration of a long-acting FIX polypeptide.
[0113] In other embodiments, after administration of a given dose
or doses of a long-acting FIX polypeptide, the plasma trough level
of the long-acting FIX polypeptide is maintained at about 1 IU/dl
(1%) or above 1 IU/dl (1%) in the subject. In yet other
embodiments, the plasma through level of the long-acting FIX
polypeptide is maintained between about 1 IU/dl (1%) and about 5
IU/dl (5%) in the subject. In yet other embodiments, a plasma
trough level is maintained between about 1% and about 5%, between
about 1% and about 6%, between about 1% and about 7%, between about
1% and about 8%, between about 1% and about 9%, between about 1%
and about 10%, between about 1% and about 12%, between about 1% and
about 14%, between about 1% and about 15%, between about 1% and
about 17%, between about 1% and about 19%, between about 1% and
about 20%, between about 1% and about 22%, between about 1% and
about 24%, between about 1% and about 25%, between about 1% and
about 30%, between about 1% and about 35%.
[0114] In some embodiments, the trough is 1-5 or 1-3 IU/dl after
about 6, about 7, about 8, about 9, about 10, about 11, about 12,
about 13 or about 14 days after administration of a long-acting FIX
polypeptide. In some embodiments, the plasma level of the FIX
polypeptide reaches an average trough of at least about 1 IU/dl
after at least about 6 days or reaches a trough of at least about
1, 2, 3, 4, or 5 IU/dl after at least about 6 days in a subject. In
some embodiments, the plasma level of said chimeric polypeptide
reaches an average trough of about 1-5 or 1-3 IU/dl. Such trough or
average trough can be reached after about 6, about 7, about 8,
about 9, about 10, about 11, about 12, about 13, about 14, about
15, about 16, about 17, about 18, about 19, about 20, about 21,
about 22, about 23, about 24, about 25, about 26, about 27, about
28, about 29, about 30, about 31, about 32, about 33, about 34,
about 35, about 36, about 37, about 38, about 39, or about 40
days.
III. Long-Acting FIX Polypeptide
[0115] A long-acting or long-lasting FIX polypeptide useful for the
invention is a chimeric polypeptide comprising a FIX polypeptide
and an FcRn binding partner. In certain embodiments, the rFIXFc is
a fusion protein comprising a single molecule of human recombinant
coagulation FIX (rFIX) covalently linked to the dimeric Fc domain
of immunoglobulin G1 (IgG1) with no intervening sequence. The FIX
polypeptide of the invention comprises a functional Factor IX
polypeptide in its normal role in coagulation, unless otherwise
specified. Thus, the FIX polypeptide includes variant polypeptides
that are functional and the polynucleotides that encode such
functional variant polypeptides. In one embodiment, the FIX
polypeptides are the human, bovine, porcine, canine, feline, and
murine FIX polypeptides. The full length polypeptide and
polynucleotide sequences of FIX are known, as are many functional
variants, e.g., fragments, mutants and modified versions. FIX
polypeptides include full-length FIX, full-length FIX minus Met at
the N-terminus, full-length FIX minus the signal sequence, mature
FIX (minus the signal sequence and propeptide), and mature FIX with
an additional Met at the N-terminus. FIX can be made by recombinant
means ("recombinant Factor IX" or "rFIX"), i.e., it is not
naturally occurring or derived from plasma.
[0116] A great many functional FIX variants are known.
International publication number WO 02/040544 A3, which is herein
incorporated by reference in its entirety, discloses mutants that
exhibit increased resistance to inhibition by heparin at page 4,
lines 9-30 and page 15, lines 6-31. International publication
number WO 03/020764 A2, which is herein incorporated by reference
in its entirety, discloses FIX mutants with reduced T cell
immunogenicity in Tables 2 and 3 (on pages 14-24), and at page 12,
lines 1-27. International publication number WO 2007/149406 A2,
which is herein incorporated by reference in its entirety,
discloses functional mutant FIX molecules that exhibit increased
protein stability, increased in vivo and in vitro half-life, and
increased resistance to proteases at page 4, line 1 to page 19,
line 11. WO 2007/149406 A2 also discloses chimeric and other
variant FIX molecules at page 19, line 12 to page 20, line 9.
International publication number WO 08/118507 A2, which is herein
incorporated by reference in its entirety, discloses FIX mutants
that exhibit increased clotting activity at page 5, line 14 to page
6, line 5. International publication number WO 09/051717 A2, which
is herein incorporated by reference in its entirety, discloses FIX
mutants having an increased number of N-linked and/or O-linked
glycosylation sites, which results in an increased half-life and/or
recovery at page 9, line 11 to page 20, line 2. International
publication number WO 09/137254 A2, which is herein incorporated by
reference in its entirety, also discloses Factor IX mutants with
increased numbers of glycosylation sites at page 2, paragraph [006]
to page 5, paragraph [011] and page 16, paragraph [044] to page 24,
paragraph [057]. International publication number WO 09/130198 A2,
which is herein incorporated by reference in its entirety,
discloses functional mutant FIX molecules that have an increased
number of glycosylation sites, which result in an increased
half-life, at page 4, line 26 to page 12, line 6. International
publication number WO 09/140015 A2, which is herein incorporated by
reference in its entirety, discloses functional FIX mutants that an
increased number of Cys residues, which can be used for polymer
(e.g., PEG) conjugation, at page 11, paragraph [0043] to page 13,
paragraph [0053]. The FIX polypeptides described in International
Application No. PCT/US2011/043569 filed Jul. 11, 2011 and published
as WO 2012/006624 on Jan. 12, 2012 are also incorporated herein by
reference in its entirety.
[0117] In addition, hundreds of non-functional mutations in FIX
have been identified in hemophilia subjects, many of which are
disclosed in Table 5, at pages 11-14 of International publication
number WO 09/137254 A2, which is herein incorporated by reference
in its entirety. Such non-functional mutations are not included in
the invention, but provide additional guidance for which mutations
are more or less likely to result in a functional FIX
polypeptide.
[0118] In one embodiment, the Factor IX (or Factor IX portion of a
chimeric polypeptide) can be at least 70%, at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, or 100% identical to a FIX amino acid
sequence shown in Table 19A without a signal sequence and
propeptide sequence (amino acids 1 to 415 of SEQ ID NO:2), or
alternatively, with a propeptide sequence, or with a propeptide and
signal sequence (full length FIX).
[0119] Factor IX coagulant activity is expresses as International
Unit(s) (IU). One IU of FIX activity corresponds approximately to
the quantity of FIX in one milliliter of normal human plasma.
Several assays are available for measuring Factor IX activity,
including the one stage clotting assay (activated partial
thromboplastin time; aPTT), thrombin generation time (TGA) and
rotational thromboelastometry (ROTEM.RTM.). See, e.g., Example
3.
[0120] FcRn binding partner ("FcRn BP") comprises functional
neonatal Fc receptor (FcRn) binding partners, unless otherwise
specified. An FcRn binding partner is any molecule that can be
specifically bound by the FcRn receptor with consequent active
transport by the FcRn receptor of the FcRn binding partner. Thus,
the term FcRn BP includes any variants of IgG Fc that are
functional. For example, the region of the Fc portion of IgG that
binds to the FcRn receptor has been described based on X-ray
crystallography (Burmeister et al. 1994, Nature 372:379,
incorporated herein by reference in its entirety). The major
contact area of the Fc with the FcRn is near the junction of the
CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig
heavy chain. FcRn BPs include whole IgG, the Fc fragment of IgG,
and other fragments of IgG that include the complete binding region
of FcRn. The major contact sites include amino acid residues 248,
250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain
and amino acid residues 385-387, 428, and 433-436 of the CH3
domain. References made to amino acid numbering of immunoglobulins
or immunoglobulin fragments, or regions, are all based on Kabat et
al. 1991, Sequences of Proteins of Immunological Interest, U. S.
Department of Public Health, Bethesda; MD, incorporated herein by
reference in its entirety. (The FcRn receptor has been isolated
from several mammalian species including humans. The sequences of
the human FcRn, rat FcRn, and mouse FcRn are known (Story et al.
1994, J. Exp. Med. 180: 2377), incorporated herein by reference in
its entirety.) An FcRn BP can comprise the CH2 and CH3 domains of
an immunoglobulin with or without the hinge region of the
immunoglobulin. Exemplary FcRn BP variants are provided in WO
2004/101740 and WO 2006/074199, incorporated herein by reference in
its entirety.
[0121] FcRn BP (or FcRn BP portion of a chimeric polypeptide) can
contain one or more mutations, and combinations of mutations.
[0122] FcRn BP (or FcRn BP portion of a chimeric polypeptide) can
contain mutations conferring increased half-life such as M252Y,
S254T, T256E, and combinations thereof, as disclosed in Oganesyan
et al., Mol. Immunol. 46:1750 (2009), which is incorporated herein
by reference in its entirety; H433K, N434F, and combinations
thereof, as disclosed in Vaccaro et al., Nat. Biotechnol. 23:1283
(2005), which is incorporated herein by reference in its entirety;
the mutants disclosed at pages 1-2, paragraph [0012], and Examples
9 and 10 of U.S. 2009/0264627 A1, which is incorporated herein by
reference in its entirety; and the mutants disclosed at page 2,
paragraphs [0014] to [0021] of U.S. 20090163699 A1, which is
incorporated herein by reference in its entirety.
[0123] FcRn BP (or FcRn BP portion of a chimeric polypeptide) can
also include the following mutations: The Fc region of IgG can be
modified according to well recognized procedures such as site
directed mutagenesis and the like to yield modified IgG or Fc
fragments or portions thereof that will be bound by FcRn. Such
modifications include modifications remote from the FcRn contact
sites as well as modifications within the contact sites that
preserve or even enhance binding to the FcRn. For example the
following single amino acid residues in human IgG1 Fc (Fcy1) can be
substituted without significant loss of Fc binding affinity for
FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A,
T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A,
Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A,
E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A,
V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A,
K322A, S324A, K326A, A327Q, P329A, A330Q, A330S, P331A, P331S,
E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A,
Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A,
S375A D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A,
N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A,
R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A,
Q438A, K439A, S440A, S444A, and K447A, where for example P238A
represents wild type proline substituted by alanine at position
number 238. In addition to alanine other amino acids can be
substituted for the wild type amino acids at the positions
specified above. Mutations can be introduced singly into Fc giving
rise to more than one hundred FcRn binding partners distinct from
native Fc. Additionally, combinations of two, three, or more of
these individual mutations can be introduced together, giving rise
to hundreds more FcRn binding partners. Certain of these mutations
can confer new functionality upon the FcRn binding partner. For
example, one embodiment incorporates N297A, removing a highly
conserved N-glycosylation site. The effect of this mutation is to
reduce immunogenicity, thereby enhancing circulating half-life of
the FcRn binding partner, and to render the FcRn binding partner
incapable of binding to Fc.gamma.RI, Fc.gamma.RIIA, Fc.gamma.RIIB,
and Fc.gamma.RIIIA, without compromising affinity for FcRn
(Routledge et al. 1995, Transplantation 60:847, which is
incorporated herein by reference in its entirety; Friend et al.
1999, Transplantation 68:1632, which is incorporated herein by
reference in its entirety; Shields et al. 1995, J. Biol. Chem.
276:6591, which is incorporated herein by reference in its
entirety). Additionally, at least three human Fc gamma receptors
appear to recognize a binding site on IgG within the lower hinge
region, generally amino acids 234-237. Therefore, another example
of new functionality and potential decreased immunogenicity can
arise from mutations of this region, as for example by replacing
amino acids 233-236 of human IgG1 "ELLG" to the corresponding
sequence from IgG2 "PVA" (with one amino acid deletion). It has
been shown that Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII which
mediate various effector functions will not bind to IgG1 when such
mutations have been introduced (Ward and Ghetie 1995, Therapeutic
Immunology 2:77, which is incorporated herein by reference in its
entirety; and Armour et al. 1999, Eur. J. Immunol. 29:2613, which
is incorporated herein by reference in its entirety). As a further
example of new functionality arising from mutations described
above, affinity for FcRn can be increased beyond that of wild type
in some instances. This increased affinity can reflect an increased
"on" rate, a decreased "off" rate or both an increased "on" rate
and a decreased "off" rate. Mutations believed to impart an
increased affinity for FcRn include T256A, T307A, E380A, and N434A
(Shields et al. 2001, J. Biol. Chem. 276:6591, which is
incorporated herein by reference in its entirety).
[0124] The FcRn BP (or FcRn BP portion of a chimeric polypeptide)
can be at least 70%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% identical to the Fc amino acid sequence shown in Table 19A
or B without a signal sequence (amino acids 1 to 227 of SEQ ID
NO:2), or alternatively, with a signal sequence.
[0125] A chimeric polypeptide comprising a FIX polypeptide and an
FcRn binding partner can comprise an amino acid sequence at least
70%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical to the Factor IX and FcRn BP, e.g., the Fc amino acid
sequence shown in Table 19A without a signal sequence and
propeptide sequence (amino acids 1 to 642 of SEQ ID NO:2), or
alternatively, with a propeptide sequence, or alternatively with a
signal sequence and a propeptide sequence.
[0126] A long-acting or long-lasting FIX polypeptide can be a
hybrid FIX polypeptide. Hybrid FIX polypeptide means a combination
of a FIX chimeric polypeptide with a second polypeptide. The
chimeric polypeptide and the second polypeptide in a hybrid can be
associated with each other via non-covalent protein-protein
interactions, such as charge-charge or hydrophobic interactions.
The chimeric polypeptide and the second polypeptide in a hybrid can
be associated with each other via covalent bond(s) such as
disulfide bonds. The chimeric peptide and the second peptide can be
associated with each other via more than one type of bond, such as
non-covalent and disulfide bonds. Hybrids are described in WO
2004/101740, WO 2005/001025, U.S. Pat. Nos. 7,404,956, 7,348,004,
and WO 2006/074199, each of which is incorporated herein by
reference in its entirety. The second polypeptide can be a second
copy of the same chimeric polypeptide or it can be a non-identical
chimeric polypeptide. In other embodiments, the second polypeptide
is a polypeptide comprising an FcRn BP, e.g., Fc. In some
embodiments, the chimeric polypeptide is a Factor IX-FcRn BP, e.g.,
Factor IX-Fc chimeric polypeptide, and the second polypeptide
consists essentially of Fc. See, e.g., Table 19 (SEQ ID NOs:2 and
4). See, e.g., U.S. Pat. No. 7,404,956, which is incorporated
herein by reference in its entirety.
[0127] The second polypeptide in a hybrid can comprise or consist
essentially of a sequence at least 70%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% identical to the amino acid sequence
shown in Table 19B without a signal sequence (amino acids 1 to 227
of SEQ ID NO:4), or alternatively, with a signal sequence.
[0128] In some embodiments, a long-acting FIX polypeptide is a FIX
monomer dimer hybrid. Monomer-dimer hybrid can comprise two
polypeptide chains, one chain comprising a FIX polypeptide and a
first Fc region, and another chain comprising, consisting
essentially of, or consisting of a second Fc region. In certain
aspects, a FIX monomer dimer hybrid consists essentially of or
consists of two polypeptide chains, a first chain consisting
essentially of or consisting of a FIX polypeptide and a second
chain consisting essentially of or consisting of a second Fc
region.
[0129] A long-acting FIX polypeptide can be encoded by a nucleotide
sequence which is at least 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to, for example, the nucleotide coding sequence in SEQ ID
NO:1 or 3 (the Factor IX portion, the Fc portion, individually or
together) or the complementary strand thereto, the nucleotide
coding sequence of known mutant and recombinant Factor IX or Fc
such as those disclosed in the publications and patents cited
herein or the complementary strand thereto, a nucleotide sequence
encoding the polypeptide of SEQ ID NO:2 or 4 (the Factor IX
portion, the Fc portion, individually or together), and/or
polynucleotide fragments of any of these nucleic acid molecules
(e.g., those fragments described herein). Polynucleotides which
hybridize to these nucleic acid molecules under stringent
hybridization conditions or lower stringency conditions are also
included as variants, as are polypeptides encoded by these
polynucleotides as long as they are functional.
[0130] By a nucleic acid having a nucleotide sequence at least, for
example, 95% "identical" to a reference nucleotide sequence, it is
intended that the nucleotide sequence of the nucleic acid is
identical to the reference sequence except that the nucleotide
sequence can include up to five point mutations per each 100
nucleotides of the reference nucleotide sequence. In other words,
to obtain a nucleic acid having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence can be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence can be inserted into
the reference sequence. The query sequence can be, for example, the
entire sequence shown in SEQ ID NO:1 or 3, the ORF (open reading
frame), or any fragment specified as described herein.
[0131] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or
99% identical to a nucleotide sequence or polypeptide of the
present invention can be determined conventionally using known
computer programs. A preferred method for determining the best
overall match between a query sequence (reference or original
sequence) and a subject sequence, also referred to as a global
sequence alignment, can be determined using the FASTDB computer
program based on the algorithm of Brutlag et al. (Comp. App.
Biosci. (1990) 6:237-245), which is herein incorporated by
reference in its entirety. In a sequence alignment the query and
subject sequences are both DNA sequences. An RNA sequence can be
compared by converting U's to T's. The result of said global
sequence alignment is in percent identity. Preferred parameters
used in a FASTDB alignment of DNA sequences to calculate percent
identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1,
Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1,
Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length
of the subject nucleotide sequence, whichever is shorter.
[0132] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only bases outside the 5' and 3' bases of the
subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
[0133] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and therefore, the
FASTDB alignment does not show a matched/alignment of the first 10
bases at 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the
5' or 3' of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by FASTDB
is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are to made for the purposes of the present invention.
[0134] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, it is intended that the amino acid sequence of
the subject polypeptide is identical to the query sequence except
that the subject polypeptide sequence can include up to five amino
acid alterations per each 100 amino acids of the query amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a query amino acid
sequence, up to 5% of the amino acid residues in the subject
sequence can be inserted, deleted, (indels) or substituted with
another amino acid. These alterations of the reference sequence can
occur at the amino or carboxy terminal positions of the reference
amino acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0135] As a practical matter, whether any particular polypeptide is
at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the amino acid sequences of SEQ ID NO:2 (the Factor IX
portion, the Fc portion, individually or together) or 4, or a known
Factor IX or Fc polypeptide sequence, can be determined
conventionally using known computer programs. A preferred method
for determining the best overall match between a query sequence
(reference or original sequence) and a subject sequence, also
referred to as a global sequence alignment, can be determined using
the FASTDB computer program based on the algorithm of Brutlag et
al., Comp. App. Biosci. 6:237-245 (1990), incorporated herein by
reference in its entirety. In a sequence alignment the query and
subject sequences are either both nucleotide sequences or both
amino acid sequences. The result of said global sequence alignment
is in percent identity. Preferred parameters used in a FASTDB amino
acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,
Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05,
Window Size=500 or the length of the subject amino acid sequence,
whichever is shorter.
[0136] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[0137] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are to made for the purposes of the
present invention.
[0138] The polynucleotide variants can contain alterations in the
coding regions, non-coding regions, or both. Especially preferred
are polynucleotide variants containing alterations which produce
silent substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide. Nucleotide
variants produced by silent substitutions due to the degeneracy of
the genetic code are preferred. Moreover, variants in which 5-10,
1-5, or 1-2 amino acids are substituted, deleted, or added in any
combination are also preferred. Polynucleotide variants can be
produced for a variety of reasons, e.g., to optimize codon
expression for a particular host (change codons in the human mRNA
to those preferred by a bacterial host such as E. coli).
[0139] Naturally occurring variants are called "allelic variants,"
and refer to one of several alternate forms of a gene occupying a
given locus on a chromosome of an organism (Genes II, Lewin, B.,
ed., John Wiley & Sons, New York (1985)). These allelic
variants can vary at either the polynucleotide and/or polypeptide
level and are included in the present invention. Alternatively,
non-naturally occurring variants can be produced by mutagenesis
techniques or by direct synthesis.
[0140] Using known methods of protein engineering and recombinant
DNA technology, variants can be generated to improve or alter the
characteristics of the polypeptides. For instance, one or more
amino acids can be deleted from the N-terminus or C-terminus of the
secreted protein without substantial loss of biological function.
The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993),
incorporated herein by reference in its entirety, reported variant
KGF proteins having heparin binding activity even after deleting 3,
8, or 27 amino-terminal amino acid residues. Similarly, Interferon
gamma exhibited up to ten times higher activity after deleting 8-10
amino acid residues from the carboxy terminus of this protein.
(Dobeli et al., J. Biotechnology 7:199-216 (1988), incorporated
herein by reference in its entirety.)
[0141] Moreover, ample evidence demonstrates that variants often
retain a biological activity similar to that of the naturally
occurring protein. For example, Gayle and coworkers (J. Biol. Chem.
268:22105-22111 (1993), incorporated herein by reference in its
entirety) conducted extensive mutational analysis of human cytokine
IL-1a. They used random mutagenesis to generate over 3,500
individual IL-1a mutants that averaged 2.5 amino acid changes per
variant over the entire length of the molecule. Multiple mutations
were examined at every possible amino acid position. The
investigators found that "[m]ost of the molecule could be altered
with little effect on either [binding or biological activity]."
(See Abstract.) In fact, only 23 unique amino acid sequences, out
of more than 3,500 nucleotide sequences examined, produced a
protein that significantly differed in activity from wild type.
[0142] As stated above, polypeptide variants include modified
polypeptides. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
IV. Pharmaceutical Composition
[0143] A long-acting FIX polypeptide can be formulated as a
pharmaceutical composition. The pharmaceutical composition can be
formulated for administration to humans. The pharmaceutical
compositions used in the methods of this invention comprise
pharmaceutically acceptable carriers, including, e.g., ion
exchangers, alumina, aluminum stearate, lecithin, serum proteins,
such as human serum albumin, buffer substances such as phosphates,
glycine, sorbic acid, potassium sorbate, partial glyceride mixtures
of saturated vegetable fatty acids, water, salts or electrolytes,
such as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
polyethylene glycol and wool fat. Various methods of formulating
the invention is well known in the art.
[0144] A long-acting FIX polypeptide can be formulated as a
pharmaceutical composition or formulation. In certain formulations
provided herein, a long-acting FIX polypeptide is formulated as a
sterile, preservative-free, non-pyrogenic, lyophilized, white to
off-white powder to cake, for intravenous (IV) administration. The
formulation can be provided in a single-use vial. Certain exemplary
formulations of a long-acting FIX polypeptide are also referred to
as eftrenonacog alfa.
[0145] In certain embodiments, a long-acting FIX polypeptide (e.g.,
rFIXFc) formulation is provided in a single-use vial manufactured
to contain, following reconstitution with an appropriate amount of
diluent, about 50 IU/ml, about 100 IU/ml, about 200 IU/ml, about
400 IU/ml, or about 600 IU/ml of the long-acting FIX polypeptide.
In certain embodiments in which diluent is added to a final volume
of about 5 ml, a single-use vial can nominally contain about 250,
about 500, about 1000, about 2000, or about 3000 International
Units (IU) of the long-acting FIX polypeptide (e.g., rFIXFc).
[0146] In certain embodiments, rFIXFc polypeptide comprises an
amino acid sequence at least 90%, 95%, or 100% identical to amino
acids 1 to 642 of SEQ ID NO:2.
[0147] In certain embodiments the formulation includes, in addition
to the active long-acting FIX polypeptide (e.g., rFIXFc): sucrose
(which can act as a stabilizer or bulking agent), mannitol (which
can act as a stabilizer or bulking agent), L-histidine (which can
act as a buffer), and polysorbate 20 or polysorbate 80 (which can
act as a stabilizer). The formulation is provided with a diluent
comprising a sterile sodium chloride solution. In certain
embodiments, the diluent is provided in a pre-filled syringe.
[0148] Accordingly, provided herein is a pharmaceutical composition
comprising a specified amount of a long-acting FIX polypeptide
(e.g., rFIXFc) (in IU), along with the excipients sucrose,
mannitol, L-histidine, NaCl, and polysorbate 20 or polysorbate 80.
The compositions provided herein comprise various concentrations of
the various excipients, and the concentrations can be expressed in
various ways. For example, the concentration of a given excipient
can be expressed as a molar concentration (e.g., M or mM) as a
weight/volume percent, e.g., grams per 100 ml diluent), or as
milligrams per milliliter (mg/ml). Formulations provided herein can
contain specified amounts of the various excipients at a level of
precision ranging from approximate, e.g., concentrations expressed
only to one significant figure (e.g., about 0.1% (w/v)), or with
more precision, e.g., out to 2, 3, 4, 5, or 6 significant figures
(e.g., about 3.88 mg/ml, with precision out to three significant
figures). The necessary level of precision can vary depending on,
e.g., the requirements of a given regulatory agency, or the
manufacturing process. In certain embodiments the pharmaceutical
composition comprises a reconstituted formulation, which can be
provided as a lyophilisate, optionally accompanied by a
diluent.
[0149] In certain embodiments, the pharmaceutical composition
comprises about 25 IU/ml to about 1200 IU/ml rFIXFc, e.g., 50
IU/ml, 100 IU/ml, 200 IU/ml, 400 IU/ml, or 600 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc). In certain embodiments,
the pharmaceutical composition comprises 50 IU/ml, 100 IU/ml, 200
IU/ml, or 400 IU/ml of a long-acting FIX polypeptide (e.g., rFIXFc)
in a formulation comprising about 3.88 mg/ml or about 25 mM
L-histidine, about 23.8 mg/ml or about 2.4% (w/v) mannitol, about
11.9 mg/ml or about 1.2% (w/v) sucrose, about 0.10 mg/ml or about
0.010% (w/v) polysorbate 20 or polysorbate 80, and about 3.25 mg/ml
or about 55.6 mM NaCl.
[0150] In certain embodiments, the pharmaceutical composition
comprises 600 IU/ml of a long-acting FIX polypeptide (e.g., rFIXFc)
in a formulation comprising about 5.43 mg/ml or about 35 mM
L-histidine, about 33.3 mg/ml or about 3.3% (w/v) mannitol, about
16.7 mg/ml or about 1.7% (w/v) sucrose, about 0.14 mg/ml or about
0.014% (w/v) polysorbate 20 or polysorbate 80, and about 3.25 mg/ml
or about 55.6 mM NaCl.
[0151] In certain embodiments, the pharmaceutical composition
comprises a pharmaceutically acceptable amount of sucrose. In
certain embodiments, the pharmaceutical composition comprises about
1% (w/v) to about 2% (w/v) sucrose, e.g., about 1.2% (w/v) sucrose
or about 1.7% (w/v) sucrose. In certain related embodiments the
pharmaceutical composition comprises about 10 mg/ml to about 20
mg/ml sucrose, e.g., about 11.9 mg/ml sucrose or about 16.7 mg/ml
sucrose.
[0152] In certain embodiments, the pharmaceutical composition
comprises a pharmaceutically acceptable amount of mannitol. In
certain embodiments, the pharmaceutical composition comprises about
2% (w/v) to about 4% (w/v) mannitol, e.g., about 2.4% (w/v)
mannitol or about 3.3% (w/v) mannitol. In certain related
embodiments the pharmaceutical composition comprises about 20 mg/ml
to about 40 mg/ml mannitol, e.g., about 23.8 mg/ml mannitol or
about 33.3 mg/ml mannitol.
[0153] In certain embodiments, the pharmaceutical composition
comprises pharmaceutically acceptable amounts of both sucrose and
mannitol. In certain embodiments, the pharmaceutical composition
comprises about 1.0% to about 2.0% sucrose and about 2.0% (w/v) to
about 4.0% (w/v) mannitol, e.g., about 1.2% (w/v) sucrose and about
2.4% (w/v) mannitol or about 1.7% (w/v) sucrose and about 3.3%
(w/v) mannitol. In certain related embodiments the pharmaceutical
composition comprises about 10 mg/ml to about 20 mg/ml sucrose and
about 20 mg/ml to about 40 mg/ml mannitol, e.g., about 11.9 mg/ml
sucrose and about 23.8 mg/ml mannitol or about 16.7 mg/ml sucrose
and about 33.3 mg/ml mannitol. In certain embodiments, sucrose and
mannitol are provided as part of a lyophilisate, which, upon
reconstitution with an appropriate amount of diluent provides
sucrose and mannitol at the desired concentration.
[0154] In certain embodiments, the pharmaceutical composition
comprises between about 50 mM and about 60 mM NaCl, e.g., about
55.6 mM NaCl. In certain related embodiments, the pharmaceutical
composition comprises between about 3 mg/ml and about 4 mg/ml NaCl,
e.g., about 3.25 mg/ml NaCl. In certain embodiments, NaCl is
provided at the desired concentration in a diluent solution in
which a lyophilisate comprising a long-acting FIX polypeptide
(e.g., rFIXFc) is reconstituted.
[0155] In certain embodiments, the pharmaceutical composition
comprises a pharmaceutically acceptable amount of L-histidine. In
certain embodiments, the pharmaceutical composition comprises
between about 20 mM and about 40 mM L-histidine, e.g., about 25 mM
L-histidine or about 35 mM L-histidine. In certain related
embodiments the pharmaceutical composition comprises between about
3 mg/ml and about 6 mg/ml L-histidine, e.g., about 3.88 mg/ml
L-histidine or about 5.43 mg/ml L-histidine. In certain
embodiments, L-histidine is provided as part of a lyophilisate,
which, upon reconstitution with an appropriate amount of diluent
provides L-histidine at the desired concentration.
[0156] In certain embodiments, the pharmaceutical composition
comprises a pharmaceutically acceptable amount of polysorbate 20 or
polysorbate 80. In certain related embodiments the pharmaceutical
composition comprises between about 0.008% (w/v) and about 0.020%
(w/v) polysorbate 20 or polysorbate 80, e.g., about 0.010% (w/v)
polysorbate 20 or polysorbate 80 or about 0.014% (w/v) polysorbate
20 or polysorbate 80. In certain related embodiments the
pharmaceutical composition comprises between about 0.08 mg/ml and
about 0.2 mg/ml polysorbate 20 or polysorbate 80, e.g., about 0.10%
mg/ml polysorbate 20 or polysorbate 80 or about 0.14 mg/ml
polysorbate 20 or polysorbate 80. In certain embodiments,
polysorbate 20 or polysorbate 80 is provided as part of a
lyophilisate, which, upon reconstitution with an appropriate amount
of diluent provides polysorbate 20 or polysorbate 80 at the desired
concentration.
[0157] In certain embodiments, the pharmaceutical composition
comprises: between about 25 IU/ml and about 700 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); between about 1% (w/v)
and about 2% (w/v) of sucrose; between about 2% (w/v) and about 4%
(w/v) of mannitol; between about 50 mM and about 60 mM NaCl;
between about 20 mM and about 40 mM L-histidine; and between about
0.008% (w/v) and about 0.015% of polysorbate 20 or polysorbate 80.
In certain embodiments the pharmaceutical composition is provided
as a lyophilisate and a diluent. In certain embodiments the amount
of lyophilizate provides about 5 ml of a pharmaceutical composition
with the desired ingredients at the desired concentrations.
[0158] In certain embodiments, the pharmaceutical composition
comprises: between about 25 IU/ml and about 700 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); between about 10 mg/ml
and about 20 mg/ml of sucrose; between about 20 mg/ml and about 40
mg/ml of mannitol; between about 3 mg/ml and about 4 mg/ml NaCl;
between about 3 mg/ml and about 6 mg/ml L-histidine; and between
about 0.08 mg/ml and about 0.15 mg/ml of polysorbate 20 or
polysorbate 80. In certain embodiments the pharmaceutical
composition is provided as a lyophilisate and a diluent. In certain
embodiments the amount of lyophilizate provides about 5 ml of a
pharmaceutical composition with the desired ingredients at the
desired concentrations.
[0159] Exemplary compositions are provided in Table 1 and in Table
2 in the Examples.
[0160] For example, the disclosure provides a pharmaceutical
composition comprising: about 50 IU/ml of a long-acting FIX
polypeptide (e.g., rFIXFc); about 1.2% (w/v) of sucrose; about 2.4%
(w/v) of mannitol; about 55.6 mM NaCl; about 25 mM L-histidine; and
about 0.010% (w/v) of polysorbate 20 or polysorbate 80. The
disclosure further provides a pharmaceutical composition
comprising: about 100 IU/ml of a long-acting FIX polypeptide (e.g.,
rFIXFc); about 1.2% (w/v) of sucrose; about 2.4% (w/v) of mannitol;
about 55.6 mM NaCl; about 25 mM L-histidine; and about 0.010% (w/v)
of polysorbate 20 or polysorbate 80. The disclosure further
provides a pharmaceutical composition comprising: about 200 IU/ml
of a long-acting FIX polypeptide (e.g., rFIXFc); about 1.2% (w/v)
of sucrose; about 2.4% (w/v) of mannitol; about 55.6 mM NaCl; about
25 mM L-histidine; and about 0.010% (w/v) of polysorbate 20 or
polysorbate 80. The disclosure further provides a pharmaceutical
composition comprising: about 400 IU/ml of a long-acting FIX
polypeptide (e.g., rFIXFc); about 1.2% (w/v) of sucrose; about 2.4%
(w/v) of mannitol; about 55.6 mM NaCl; about 25 mM L-histidine; and
about 0.010% (w/v) of polysorbate 20 or polysorbate 80. The
disclosure further provides a pharmaceutical composition
comprising: about 600 IU/ml of a long-acting FIX polypeptide (e.g.,
rFIXFc); about 1.7% (w/v) of sucrose; about 3.3% (w/v) of mannitol;
about 55.6 mM NaCl; about 35 mM L-histidine; and about 0.014% (w/v)
of polysorbate 20 or polysorbate 80.
[0161] The disclosure further provides a pharmaceutical composition
comprising: about 50 IU/ml of a long-acting FIX polypeptide (e.g.,
rFIXFc); about 11.9 mg/ml of sucrose; about 23.8 mg/ml of mannitol;
about 3.25 mg/ml NaCl; about 3.88 mg/ml L-histidine; and about 0.10
mg/ml of polysorbate 20 or polysorbate 80. The disclosure further
provides a pharmaceutical composition comprising: about 100 IU/ml
of a long-acting FIX polypeptide (e.g., rFIXFc); about 11.9 mg/ml
of sucrose; about 23.8 mg/ml of mannitol; about 3.25 mg/ml NaCl;
about 3.88 mg/ml L-histidine; and about 0.10 mg/ml of polysorbate
20 or polysorbate 80. The disclosure further provides a
pharmaceutical composition comprising: about 200 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); about 11.9 mg/ml of
sucrose; about 23.8 mg/ml of mannitol; about 3.25 mg/ml NaCl; about
3.88 mg/ml L-histidine; and about 0.10 mg/ml of polysorbate 20 or
polysorbate 80. The disclosure further provides a pharmaceutical
composition comprising: about 400 IU/ml of a long-acting FIX
polypeptide (e.g., rFIXFc); about 11.9 mg/ml of sucrose; about 23.8
mg/ml of mannitol; about 3.25 mg/ml NaCl; about 3.88 mg/ml
L-histidine; and about 0.10 mg/ml of polysorbate 20 or polysorbate
80. The disclosure further provides a pharmaceutical composition
comprising: about 600 IU/ml of a long-acting FIX polypeptide (e.g.,
rFIXFc); about 16.7 mg/ml of sucrose; about 33.3 mg/ml of mannitol;
about 3.25 mg/ml NaCl; about 5.43 mg/ml L-histidine; and about 0.14
mg/ml of polysorbate 20 or polysorbate 80.
[0162] This disclosure also provides the components of a
pharmaceutical kit. Such a kit includes one or more containers and
optional attachments. A kit as provided herein facilitates
administration of an effective amount of the long-acting FIX
polypeptide (e.g., rFIXFc) to a subject in need thereof. In certain
embodiments, the kit facilitates administration of the long-acting
FIX polypeptide (e.g., rFIXFc) via intravenous infusion. In certain
embodiments, the kit facilitates self-administration of the
long-acting FIX polypeptide (e.g., rFIXFc) via intravenous
infusion.
[0163] In certain embodiments, the disclosure provides a
pharmaceutical kit comprising: a first container comprising a
lyophilized powder or cake, where the powder or cake comprises: (i)
a long-acting FIX polypeptide (e.g., rFIXFc), (ii) sucrose; (iii)
mannitol; (iv) L-histidine; and (v) polysorbate 20 or polysorbate
80; and a second container comprising a 0.325% (w/v) NaCl diluent
solution to be combined with the lyophilized powder of the first
container. In certain embodiments, sufficient diluent is provided
to produce about 5 ml of a long-acting FIX polypeptide (e.g.,
rFIXFc) formulation with desired properties as disclosed herein. In
certain embodiments, the second container is a pre-filled syringe
associated with a plunger, to allow addition of the diluent to the
first container, reconstitution of the contents of the first
container, and transfer back into the syringe. In certain
embodiments, the kit further provides an adaptor for attaching the
syringe to the first container. In certain embodiments the kit
further provides a needle and infusion tubing, to be attached to
the syringe containing the reconstituted long-acting FIX
polypeptide (e.g., rFIXFc) formulation to allow IV infusion of the
formulation.
[0164] In certain embodiments a long-acting FIX polypeptide (e.g.,
rFIXFc) is provided in a total amount from about 200 IU to about
4000 IU, e.g., about 250 IU, about 500 IU, about 1000 IU, about
2000 IU, or about 3000 IU.
[0165] In one embodiment, a pharmaceutical kit is provided which
comprises a first container comprising a lyophilized powder, where
the powder comprises (i) about 250 IU of a long-acting FIX
polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose; (iii)
about 119 mg of mannitol; (iv) about 19.4 mg of L-histidine; and
(v) about 0.50 mg of polysorbate 20 or polysorbate 80; and a second
container comprising 0.325% (w/v) NaCl at a volume sufficient to
produce, when combined with the lyophilized powder of the first
container, a solution comprising: (i) about 50 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); (ii) about 1.2% (w/v)
of sucrose; (iii) about 2.4% (w/v) of mannitol; (iv) about 55.6 mM
NaCl; (v) about 25 mM L-histidine; and (vi) about 0.01% (w/v) of
polysorbate 20 or polysorbate 80.
[0166] In a further embodiment, a pharmaceutical kit is provided
which comprises a first container comprising a lyophilized powder,
where the powder comprises: (i) about 500 IU of a long-acting FIX
polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose; (iii)
about 119 mg of mannitol; (iv) about 19.4 mg of L-histidine; and
(v) about 0.50 mg of polysorbate 20 or polysorbate 80; and a second
container comprising 0.325% (w/v) NaCl at a volume sufficient to
produce, when combined with the lyophilized powder of the first
container, a solution comprising: (i) about 100 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); (ii) about 1.2% (w/v)
of sucrose; (iii) about 2.4% (w/v) of mannitol; (iv) about 55.6 mM
NaCl; (v) about 25 mM L-histidine; and (vi) about 0.01% (w/v) of
polysorbate 20 or polysorbate 80.
[0167] In a further embodiment, a pharmaceutical kit is provided
which comprises a first container comprising a lyophilized powder,
where the powder comprises: (i) about 1000 IU of a long-acting FIX
polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose; (iii)
about 119 mg of mannitol; (iv) about 19.4 mg of L-histidine; and
(v) about 0.50 mg of polysorbate 20 or polysorbate 80; and a second
container comprising 0.325% (w/v) NaCl at a volume sufficient to
produce, when combined with the lyophilized powder of the first
container, a solution comprising: (i) about 200 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); (ii) about 1.2% (w/v)
of sucrose; (iii) about 2.4% (w/v) of mannitol; (iv) about 55.6 mM
NaCl; (v) about 25 mM L-histidine; and (vi) about 0.01% (w/v) of
polysorbate 20 or polysorbate 80.
[0168] In a further embodiment, a pharmaceutical kit is provided
which comprises a first container comprising a lyophilized powder,
where the powder comprises: (i) about 2000 IU of a long-acting FIX
polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose; (iii)
about 119 mg of mannitol; (iv) about 19.4 mg of L-histidine; and
(v) about 0.50 mg of polysorbate 20 or polysorbate 80; and a second
container comprising 0.325% (w/v) NaCl at a volume sufficient to
produce, when combined with the lyophilized powder of the first
container, a solution comprising: (i) about 400 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); (ii) about 1.2% (w/v)
of sucrose; (iii) about 2.4% (w/v) of mannitol; (iv) about 55.6 mM
NaCl; (v) about 25 mM L-histidine; and (vi) about 0.01% (w/v) of
polysorbate 20 or polysorbate 80.
[0169] In a further embodiment, a pharmaceutical kit is provided
which comprises a first container comprising a lyophilized powder,
where the powder comprises: (i) about 3000 IU of a long-acting FIX
polypeptide (e.g., rFIXFc), (ii) about 83.3 mg of sucrose; (iii)
about 167 mg of mannitol; (iv) about 27.2 mg of L-histidine; and
(v) about 0.7 mg of polysorbate 20 or polysorbate 80; and a second
container comprising 0.325% (w/v) NaCl at a volume sufficient to
produce, when combined with the lyophilized powder of the first
container, a solution comprising: (i) about 600 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); (ii) about 1.7% (w/v)
of sucrose; (iii) about 3.3% (w/v) of mannitol; (iv) about 55.6 mM
NaCl; (v) about 35 mM L-histidine; and (vi) about 0.014% (w/v) of
polysorbate 20 or polysorbate 80.
[0170] In a further embodiment, a pharmaceutical kit is provided
which comprises a first container comprising a lyophilized powder,
where the powder comprises: (i) about 250 IU of a long-acting FIX
polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose; (iii)
about 119 mg of mannitol; (iv) about 19.4 mg of L-histidine; and
(v) about 0.50 mg of polysorbate 20 or polysorbate 80; and a second
container comprising 0.325% (w/v) NaCl at a volume sufficient to
produce, when combined with the lyophilized powder of the first
container, a solution comprising: (i) about 50 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); (ii) about 11.9 mg/ml
of sucrose; (iii) about 23.8 mg/ml of mannitol; (iv) about 3.25
mg/ml NaCl; (v) about 3.88 mtml L-histidine, and (vi) about 0.10
mg/ml of polysorbate 20 or polysorbate 80.
[0171] In a further embodiment, a pharmaceutical kit is provided
which comprises a first container comprising a lyophilized powder,
where the powder comprises: (i) about 500 IU of a long-acting FIX
polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose; (iii)
about 119 mg of mannitol; (iv) about 19.4 mg of L-histidine; and
(v) about 0.50 mg of polysorbate 20 or polysorbate 80; and a second
container comprising 0.325% (w/v) NaCl at a volume sufficient to
produce, when combined with the lyophilized powder of the first
container, a solution comprising: (i) about 100 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); (ii) about 11.9 mg/ml
of sucrose; (iii) about 23.8 mg/ml of mannitol; (iv) about 3.25
mg/ml NaCl; (v) about 3.88 mg/ml L-histidine; and (vi) about 0.10
mg/ml of polysorbate 20 or polysorbate 80.
[0172] In a further embodiment, a pharmaceutical kit is provided
which comprises a first container comprising a lyophilized powder,
where the powder comprises: (i) about 1000 IU of a long-acting FIX
polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose; (iii)
about 119 mg of mannitol; (iv) about 19.4 mg of L-histidine; and
(v) about 0.50 mg of polysorbate 20 or polysorbate 80; and a second
container comprising 0.325% (w/v) NaCl at a volume sufficient to
produce, when combined with the lyophilized powder of the first
container, a solution comprising: (i) about 200 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); (ii) about 11.9 mg/ml
of sucrose; (iii) about 23.8 mg/ml of mannitol; (iv) about 3.25
mg/ml NaCl; (v) about 3.88 mg/ml L-histidine; and (vi) about 0.10
mg/ml of polysorbate 20 or polysorbate 80.
[0173] In a further embodiment, a pharmaceutical kit is provided
which comprises a first container comprising a lyophilized powder,
where the powder comprises: (i) about 2000 IU of a long-acting FIX
polypeptide (e.g., rFIXFc), (ii) about 59.5 mg of sucrose; (iii)
about 119 mg of mannitol; (iv) about 19.4 mg of L-histidine; and
(v) about 0.50 mg of polysorbate 20 or polysorbate 80; and a second
container comprising 0.325% (w/v) NaCl at a volume sufficient to
produce, when combined with the lyophilized powder of the first
container, a solution comprising: (i) about 400 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); (ii) about 11.9 mg/ml
of sucrose; (iii) about 23.8 mg/ml of mannitol; (iv) about 3.25
mg/ml NaCl; (v) about 3.88 mg/ml L-histidine; and (vi) about 0.10
mg/ml of polysorbate 20 or polysorbate 80.
[0174] In a further embodiment, a pharmaceutical kit is provided
which comprises a first container comprising a lyophilized powder,
where the powder comprises: (i) about 3000 IU of a long-acting FIX
polypeptide (e.g., rFIXFc), (ii) about 83.3 mg of sucrose; (iii)
about 167 mg of mannitol; (iv) about 27.2 mg of L-histidine; and
(v) about 0.7 mg of polysorbate 20 or polysorbate 80; and a second
container comprising 0.325% (w/v) NaCl at a volume sufficient to
produce, when combined with the lyophilized powder of the first
container, a solution comprising: (i) about 600 IU/ml of a
long-acting FIX polypeptide (e.g., rFIXFc); (ii) about 16.7 mg/ml
of sucrose; (iii) about 33.3 mg/ml of mannitol; (iv) about 3.25
mg/ml NaCl; (v) about 5.43 mg/ml L-histidine; and (vi) about 0.14
mg/ml of polysorbate 20 or polysorbate 80.
[0175] In certain embodiments the first container of a
pharmaceutical kit provided herein is a glass vial comprising a
rubber stopper. In certain embodiments, the second container a
pharmaceutical kit provided herein is a syringe body, associated
with a plunger. In certain embodiments, the syringe is a pre-filled
syringe containing the diluent. In certain embodiments, a
pharmaceutical kit provided herein further comprises an adaptor to
connect the glass vial to the syringe body. In certain embodiments
a pharmaceutical kit provided herein further comprises infusion
tubing associated with a needle to be connected to the syringe,
suitable for intravenous infusion.
[0176] In certain embodiments, a desired dose of a long-acting FIX
polypeptide (e.g., rFIXFc) can be achieved through the use of one
pharmaceutical kit as provided herein. In certain embodiments, more
than one pharmaceutical kit can be used to achieve a desired dose.
Provided herein is a method of combining, or pooling the
formulations contained in two or more pharmaceutical kits as
provided herein in order to achieve a desired dose.
[0177] In some embodiments, the pharmaceutical composition further
comprises a short-acting FIX polypeptide. A short-acting FIX
polypeptide can comprise or consist of wild-type FIX. Non-limiting
examples of the short-acting FIX polypeptide includes BENEFIX,
MONONINE, or ALPHANINE.
[0178] The pharmaceutical composition of the invention can be
formulated as a liquid formulation, lyophilized powder, or
suspension. A container comprising the pharmaceutical compositions
can be a vial, a cartridge, or a syringe. In a particular
embodiment, a syringe comprising the pharmaceutical composition is
a dual chamber syringe.
[0179] In certain embodiments, the pharmaceutical composition of
the invention or the reconstitution solution for the lyophilized
powder comprises a preservative in an amount sufficient to provide
antimicrobial activity. Pharmaceutically acceptable preservatives
that are useful for pharmaceutical composition are well known in
the art. For example, examples of the pharmaceutically acceptable
preservatives include, but are not limited to, phenol, m-cresol,
benzyl alcohol, chlorobutanol, methyl paraben, propylparaben,
phenoxyethanol, any other pharmaceutically acceptable preservative,
and any combinations thereof. In a particular embodiment, the
preservative is benzyl alcohol. In some embodiments, the
pharmaceutical composition comprises benzyl alcohol at a
concentration between 0.5% and 0.9%.
[0180] Having now described the present invention in detail, the
same will be more clearly understood by reference to the following
examples, which are included herewith for purposes of illustration
only and are not intended to be limiting of the invention. All
patents and publications referred to herein are expressly
incorporated by reference.
V. Method of Making
[0181] A long-acting FIX polypeptide can be manufactured in a host
cell comprising a vector encoding the long-acting FIX polypeptide.
In one embodiment, the host cell is transformed with one or more
vectors comprising a first nucleotide sequence encoding a FIX
polypeptide and a first FcRn polypeptide, a second nucleotide
sequence encoding a second FcRn polypeptide, and optionally a third
nucleotide sequence encoding a protein convertase, e.g., PCS. As
used herein, an expression vector refers to any nucleic acid
construct which contains the necessary elements for the
transcription and translation of an inserted coding sequence, or in
the case of an RNA viral vector, the necessary elements for
replication and translation, when introduced into an appropriate
host cell. Expression vectors can include plasmids, phagemids,
viruses, and derivatives thereof.
[0182] A gene expression control sequence as used herein is any
regulatory nucleotide sequence, such as a promoter sequence or
promoter-enhancer combination, which facilitates the efficient
transcription and translation of the coding nucleic acid to which
it is operably linked. The gene expression control sequence can,
for example, be a mammalian or viral promoter, such as a
constitutive or inducible promoter. Constitutive mammalian
promoters include, but are not limited to, the promoters for the
following genes: hypoxanthine phosphoribosyl transferase (HPRT),
adenosine deaminase, pyruvate kinase, beta-actin promoter, and
other constitutive promoters. Exemplary viral promoters which
function constitutively in eukaryotic cells include, for example,
promoters from the cytomegalovirus (CMV), simian virus (e.g.,
SV40), papilloma virus, adenovirus, human immunodeficiency virus
(HIV), Rous sarcoma virus, cytomegalovirus, the long terminal
repeats (LTR) of Moloney leukemia virus, and other retroviruses,
and the thymidine kinase promoter of herpes simplex virus. Other
constitutive promoters are known to those of ordinary skill in the
art. The promoters useful as gene expression sequences of the
invention also include inducible promoters. Inducible promoters are
expressed in the presence of an inducing agent. For example, the
metallothionein promoter is induced to promote transcription and
translation in the presence of certain metal ions. Other inducible
promoters are known to those of ordinary skill in the art.
[0183] Examples of vectors include, but are not limited to viral
vectors or plasmid vectors. Plasmid vectors have been extensively
described in the art and are well-known to those of skill in the
art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
In the last few years, plasmid vectors have been found to be
particularly advantageous for delivering genes to cells in vivo
because of their inability to replicate within and integrate into a
host genome. These plasmids, however, having a promoter compatible
with the host cell, can express a peptide from a gene operably
encoded within the plasmid. Some commonly used plasmids available
from commercial suppliers include pBR322, pUC18, pUC19, various
pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40, and
pBlueScript. Additional examples of specific plasmids include
pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number
V87020; pcDNA4/myc-His, catalog number V86320; and pBudCE4.1,
catalog number V53220, all from Invitrogen (Carlsbad, Calif.).
Other plasmids are well-known to those of ordinary skill in the
art. Additionally, plasmids can be custom designed using standard
molecular biology techniques to remove and/or add specific
fragments of DNA.
[0184] The expression vector or vectors are then transfected or
co-transfected into a suitable target cell, which will express the
polypeptides. Transfection techniques known in the art include, but
are not limited to, calcium phosphate precipitation (Wigler et al.
(1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J
1:841), and liposome-based reagents. A variety of host-expression
vector systems can be utilized to express the proteins described
herein including both prokaryotic and eukaryotic cells. These
include, but are not limited to, microorganisms such as bacteria
(e.g., E. coli) transformed with recombinant bacteriophage DNA or
plasmid DNA expression vectors containing an appropriate coding
sequence; yeast or filamentous fungi transformed with recombinant
yeast or fungi expression vectors containing an appropriate coding
sequence; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing an appropriate
coding sequence; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus or tobacco
mosaic virus) or transformed with recombinant plasmid expression
vectors (e.g., Ti plasmid) containing an appropriate coding
sequence; or animal cell systems, including mammalian cells (e.g.,
HEK 293, CHO, Cos, HeLa, HKB11, and BHK cells).
[0185] In one embodiment, the host cell is a eukaryotic cell. As
used herein, a eukaryotic cell refers to any animal or plant cell
having a definitive nucleus. Eukaryotic cells of animals include
cells of vertebrates, e.g., mammals, and cells of invertebrates,
e.g., insects. Eukaryotic cells of plants specifically can include,
without limitation, yeast cells. A eukaryotic cell is distinct from
a prokaryotic cell, e.g., bacteria.
[0186] In certain embodiments, the eukaryotic cell is a mammalian
cell. A mammalian cell is any cell derived from a mammal. Mammalian
cells specifically include, but are not limited to, mammalian cell
lines. In one embodiment, the mammalian cell is a human cell. In
another embodiment, the mammalian cell is a HEK 293 cell, which is
a human embryonic kidney cell line. HEK 293 cells are available as
CRL-1533 from American Type Culture Collection, Manassas, Va., and
as 293-H cells, Catalog No. 11631-017 or 293-F cells, Catalog No.
11625-019 from Invitrogen (Carlsbad, Calif.). In some embodiments,
the mammalian cell is a PER.C6.RTM. cell, which is a human cell
line derived from retina. PER.C6.RTM. cells are available from
Crucell (Leiden, The Netherlands). In other embodiments, the
mammalian cell is a Chinese hamster ovary (CHO) cell. CHO cells are
available from American Type Culture Collection, Manassas, Va.
(e.g., CHO-Kl; CCL-61). In still other embodiments, the mammalian
cell is a baby hamster kidney (BHK) cell. BHK cells are available
from American Type Culture Collection, Manassas, Va. (e.g.,
CRL-1632). In some embodiments, the mammalian cell is a HKB11 cell,
which is a hybrid cell line of a HEK293 cell and a human B cell
line. Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).
[0187] The method can further comprise purification steps. Various
known purifications steps are well known in the art.
VI. Method, System, and Storage Medium for Estimating Patient
Individualized Dosing Information, Patient Individualized PK
Information, and Patient Median PK Information
[0188] The invention also includes a method of estimating a rFIXFc
dosing information individualized for a patient, the method
comprising: (a) receiving, by a computer-based system containing
the rFIXFc population pharmacokinetic (popPK) model of Example 6,
e.g., Table 21, and, optionally, a Bayesian estimation program, at
least one of patient information and desired treatment outcome
information, (b) calculating, by the computer-based system,
individualized rFIXFc dosing information using the popPK model, the
optional Bayesian estimation program, and the received information,
and (c) outputting, by the computer-based system, the
individualized dosing information.
[0189] In some embodiments, the method also comprises selecting a
dosing regimen based on the output individualized dosing
information of (c) and administering rFIXFc to the patient
according to the selected dosing regimen.
[0190] In some embodiments, the desired treatment outcome
information is desired rise in plasma FIX activity level following
dosing and the output information is dose for acute treatment.
[0191] In some embodiments, the desired treatment outcome
information is desired dosing interval and the output information
is dose for prophylaxis.
[0192] In some embodiments, the desired treatment outcome
information is desired dose and the output information is interval
for prophylaxis.
[0193] The invention also includes a method of estimating a rFIXFc
dosing regimen based on median popPK, the method comprising: (a)
receiving, by a computer-based system containing the rFIXFc popPK
model of Example 6, e.g., Table 21, and, optionally, a Bayesian
estimation program, at least one of patient information and desired
treatment outcome information, (b) calculating, by the
computer-based system, median rFIXFc PK information using the popPK
model, the optional Bayesian estimation program, and the received
information, and (c) outputting, by the computer-based system, the
median PK information.
[0194] In some embodiments, the method also comprises selecting a
dosing regimen based on the output median PK information of (c),
and administering rFIXFc to a patient according to the selected
dosing regimen.
[0195] The invention also includes a method of estimating
individual patient rFIXFc PK, the method comprising: (a) receiving,
by a computer-based system containing the rFIXFc population
pharmacokinetic (popPK) model of Example 6, e.g., Table 21, and,
optionally, a Bayesian estimation program, individual rFIXFc PK
information, (b) estimating, by the computer-based system,
individualized patient rFIXFc PK information using the popPK model,
the optional Bayesian estimation program, and the received
information, and (c) outputting, by the computer-based system, the
individualized patient PK information.
[0196] In some embodiments, the method also comprises selecting a
dosing regimen based on the output individualized patient PK
information of (c), and administering rFIXFc to the patient
according to the selected regimen.
[0197] In some embodiments (a) further comprises receiving, by the
computer-based system, patient information.
[0198] In some embodiments the patient information is age, e.g., 12
and older, or body weight. Additional patient information includes
diagnostic (baseline) FIX level, PK determinations, time of PK
sampling, dosing history if PK samples were taken from multiple
doses, actual dose, FIX activity level, etc.
[0199] In some embodiments, desired treatment outcome information
is, e.g., desired PK or desired regimen outcome, e.g., desired rise
in plasma FIX activity level following dose, desired dosing
interval, and desired dose.
[0200] In some embodiments, output information is, e.g., PK curve,
PK parameter such as incremental recovery (Cmax/dose), mean
residence time, terminal t1/2, clearance, Vss, AUC/dose, doses and
associated troughs, and intervals and associated troughs.
[0201] For example, for assessing individualized patient PK, the
system can recommend that the user input 2-3 optimized PK sampling
time points. In this case, system output can include PK curve and
one or more selected PK parameters, e.g., incremental recovery
(Cmax/Dose), mean residence time, terminal t1/2, clearance, Vss,
AUC, and time to 1 or X %, etc. E.g., FIG. 15.
[0202] As additional examples, to select an individualized dosing
regimen using the output individual PK parameters discussed in the
preceding paragraph, (i) the dose selected for acute treatment can
be based on user input of the desired rise in plasma FIX activity
level following the dose, (ii) the dose selected for prophylaxis
can be based on user input of the desired dosing interval, or (iii)
the selected interval for prophylaxis can be based on user input
for the desired dose. In the first case, the system can output the
dose (IU) based in the patient's incremental recovery. E.g., FIG.
16. In the second case, system output can be a table of doses and
associated troughs, e.g., x IU/kg, 1% trough, y IU/kg, 2% trough,
etc. E.g., FIG. 17, top. In the third case, system output can be a
table of intervals and associated troughs, e.g., x days, 1% trough,
y IU/kg, 2% trough, etc., E.g., FIG. 17, bottom.
[0203] The user may wish to use the system without inputting any
individualized PK data. In this case, the dosing output would be
based on the population median rather than being individualized for
the particular patient, e.g., FIG. 18. In this way, the user
inputs, e.g., body weight and age, and (i) the desired rise in
plasma FIX activity level following the dose, (ii) the desired dose
interval for prophylaxis, or (iii) the desired dose for
prophylaxis. In the first case, the system can output the dose. In
the second case, the system can output the dose and associated
trough, e.g., Table 16. In the third case, the system can output
the interval and associated trough, e.g., Table 16.
[0204] In some embodiments, the system is compliant with patient
privacy laws. In some embodiments, the system is encrypted, e.g.,
with SSL. In some embodiments, input patient information is made
anonymous.
[0205] In some embodiments, the system includes a user help
function.
[0206] The method can be carried out by, e.g., a physician, a
nurse, or another healthcare practitioner.
[0207] Additional embodiments include a computer readable storage
medium having instructions stored thereon that, when executed by a
processor, cause the processor to perform any of the above
methods.
[0208] Additional embodiments include a system comprising a
processor and a memory, the memory having instructions stored
thereon that, when executed by the processor, cause the processor
to perform any of the above methods.
[0209] The user of the system or computer readable storage medium,
can be, e.g., a physician, a nurse, or another healthcare
practitioner.
[0210] For additional embodiments of these aspects of the
invention, see Examples 5, 6, and 8 and the Figures discussed
therein.
VII. Example Computing Environment
[0211] Various modeling techniques, dosage calculations, and
estimations described herein can be implemented by software,
firmware, hardware, or a combination thereof. FIG. 19 illustrates
an example computer system 1900 in which the embodiments, or
portions thereof, can be implemented as computer-readable code. For
example, the modeling of Examples 5 and 6, and/or the patient
treatment simulation of Example 8 can be implemented in system
1900.
[0212] Computer system 1900 includes one or more processors, such
as processor 1904. Processor 1904 is connected to a communication
infrastructure 1906 (for example, a bus or network).
[0213] Computer system 1900 also includes a main memory 1908,
preferably random access memory (RAM), and may also include a
secondary memory 1910. In accordance with implementations, user
interface data may be stored, for example and without limitation,
in main memory 1908. Main memory 1908 may include, for example,
cache, and/or static and/or dynamic RAM. Secondary memory 1910 may
include, for example, a hard disk drive and/or a removable storage
drive. Removable storage drive 1914 may include a floppy disk
drive, a magnetic tape drive, an optical disk drive, a flash
memory, or the like. The removable storage drive 1914 reads from
and/or writes to removable storage unit 1916 in a well-known
manner. Removable storage unit 1916 may include a floppy disk,
magnetic tape, optical disk, etc. which is read by and written to
by removable storage drive 1914. As will be appreciated by persons
skilled in the relevant art(s), removable storage unit 1916
includes a computer readable storage medium having stored therein
computer software and/or data.
[0214] Computer system 1900 may also include a display interface
1902. Display interface 1902 may be adapted to communicate with
display unit 1930. Display unit 1930 may include a computer monitor
or similar means for displaying graphics, text, and other data
received from main memory 1908 via communication infrastructure
1906. In alternative implementations, secondary memory 1910 may
include other similar means for allowing computer programs or other
instructions to be loaded into computer system 1900. Such means may
include, for example, a removable storage unit 1922 and an
interface 1920. Examples of such means may include a program
cartridge and cartridge interface (such as that found in video game
devices), a removable memory chip (such as an EPROM, or PROM) and
associated socket, and other removable storage units 1922 and
interfaces 1920 which allow software and data to be transferred
from the removable storage unit 1922 to computer system 1900.
[0215] Computer system 1900 may also include a communications
interface 1924. Communications interface 1924 allows software and
data to be transferred between computer system 1900 and external
devices. Communications interface 1924 may include a modem, a
network interface (such as an Ethernet card), a communications
port, a PCMCIA slot and card, or the like. Software and data
transferred via communications interface 1924 are in the form of
signals which may be electronic, electromagnetic, optical, or other
signals capable of being received by communications interface 1924.
These signals are provided to communications interface 1924 via a
communications path 1926. Communications path 1926 carries signals
and may be implemented using wire or cable, fiber optics, a phone
line, a cellular phone link, an RF link or other communications
channels.
[0216] In this document, the term "computer readable storage
medium" is used to generally refer to non-transitory storage media
such as removable storage unit 1916, removable storage unit 1922,
and a hard disk installed in hard disk drive 1912. Computer
readable storage medium can also refer to one or more memories,
such as main memory 1908 and secondary memory 1910, which can be
memory semiconductors (e.g. DRAMs, etc.). These computer program
products are means for providing software to computer system
1900.
[0217] Computer programs (also called computer control logic) are
stored in main memory 1908 and/or secondary memory 1910. Computer
programs may also be received via communications interface 1924 and
stored on main memory 1908 and/or secondary memory 1910. Such
computer programs, when executed, enable computer system 1900 to
implement embodiments as discussed herein. In particular, the
computer programs, when executed, enable processor 1904 to
implement processes of the present disclosure, such as certain
methods discussed above. Accordingly, such computer programs
represent controllers of the computer system 1900. Where
embodiments use software, the software may be stored in a computer
program product and loaded into computer system 1900 using
removable storage drive 1914, interface 1920, or hard drive
1912.
[0218] Embodiments may be directed to computer program products
comprising software stored on any computer readable medium. Such
software, when executed in one or more data processing device,
causes a data processing device(s) to operate as described
herein.
[0219] Embodiments may employ any computer useable or readable
medium. Examples of computer readable storage media include, but
are not limited to, non-transitory primary storage devices (e.g.,
any type of random access memory), and non-transitory secondary
storage devices (e.g., hard drives, floppy disks, CD ROMS, ZIP
disks, tapes, magnetic storage devices, and optical storage
devices, MEMS, nano-technological storage device, etc.). Other
computer readable media include communication mediums (e.g., wired
and wireless communications networks, local area networks, wide
area networks, intranets, etc.).
EXAMPLES
Example 1. Product Description
[0220] rFIXFc is a long-acting, fully recombinant fusion protein
consisting of human coagulation Factor IX (FIX) covalently linked
to the Fc domain of human immunoglobulin G1 (IgG1). The Factor IX
portion of rFIXFc has a primary amino acid sequence that is
identical to the Thr.sup.148 allelic form of plasma derived Factor
IX and has structural and functional characteristics similar to
endogenous Factor IX. The Fc domain of rFIXFc contains the hinge,
CH2 and CH3 regions of IgG1. rFIXFc contains 869 amino acids with a
molecular weight of approximately 98 kilodaltons.
[0221] rFIXFc is produced by recombinant DNA technology in a human
embryonic kidney (HEK) cell line, which has been extensively
characterized. The cell line expresses rFIXFc into a defined cell
culture medium that does not contain any proteins derived from
animal or human sources. rFIXFc is purified by a series of
chromatography steps that does not require use of a monoclonal
antibody. The process includes multiple viral clearance steps
including 15 nm virus-retaining nano-filtration. No human or animal
additives are used in the cell culture, purification, and
formulation processes.
[0222] rFIXFc is in the pharmacotherapeutic group:
antihemorrhagics, BO2BD04. It is provided as a sterile,
preservative-free, non-pyrogenic, lyophilized, white to off-white
powder to cake, for intravenous (IV) administration in a single-use
vial, accompanied by a liquid diluent in a pre-filled syringe. In
addition to rFIXFc, the pharmaceutical composition comprises in the
lyophilizate Sucrose, L-Histidine, Mannitol, and Polysorbate 20,
and comprising in a sterile solvent Sodium Chloride Solution
(0.325%). Each single-use vial contains nominally 250, 500, 1000,
2000, or 3000 International Units (IU) of rFIXFc. When
reconstituted with provided diluent, the product contains the
following excipients: sucrose, sodium chloride, L-histidine,
mannitol, and polysorbate 20, at the concentrations shown in Table
1 or Table 2 below. The pharmaceutical composition is formulated
for intravenous administration only after reconstitution.
[0223] Each pack contains a powder vial (type 1 glass) with a
stopper (butyl) and a flip-off seal (aluminum), 5 ml solvent in a
pre-filled syringe (type 1 glass) with a plunger stopper (butyl), a
tip-cap (butyl), and a sterile vial adapter reconstitution
device.
TABLE-US-00001 TABLE 1 rFIXFc Formulations rFIXFc % (w/v) % (w/v)
NaCl L-histidine % (w/v) IU/ml* Sucrose Mannitol (mM) (mM)
Polysorbate- 20 50 IU/ml 1.2 2.4 55.6 25 0.010 100 IU/ml 1.2 2.4
55.6 25 0.010 200 IU/ml 1.2 2.4 55.6 25 0.010 400 IU/ml 1.2 2.4
55.6 25 0.010 600 IU/ml 1.7 3.3 55.6 35 0.014
TABLE-US-00002 TABLE 2 rFIXFc Formulations Concentration 250 IU/
500 IU/ 1000 IU/ 2000 IU/ 3000 IU/ Component vial vial vial vial
vial rFIXFc* 50 100 200 400 600 IU/mL IU/mL IU/mL IU/mL IU/mL
L-Histidine 3.88 3.88 3.88 3.88 5.43 mg/mL mg/mL mg/mL mg/mL mg/mL
Mannitol 23.8 23.8 23.8 23.8 33.3 mg/mL mg/mL mg/mL mg/mL mg/mL
Sucrose 11.9 11.9 11.9 11.9 16.7 mg/mL mg/mL mg/mL mg/mL mg/mL
Polysorbate 20 0.10 0.10 0.10 0.10 0.14 mg/mL mg/mL mg/mL mg/mL
mg/mL NaCl 3.25 3.25 3.25 3.25 3.25 mg/mL mg/mL mg/mL mg/mL mg/mL
Water for 5 mL Injection *The potency (IU) is determined using One
Stage Activated Partial Thromboplastin Time (aPTT) as per Ph. Eur
2.7.11 and USP <32> against an in-house standard that is
referenced to the WHO concentrate standard. The specific activity
of rFIXFc is .gtoreq.55 IU/mg protein.
Example 2: Method of Formulation
[0224] The rFIXFc drug product is a sterile lyophilized powder for
injection intended for intravenous administration. It is supplied
in aseptically filled single use vials which contain nominally 250,
500, 1000, 2000, and 3000 IU per vial. The vials are 10 mL USP/Ph.
Eur. Type 1 glass vials sealed with a 20 mm Teflon-coated butyl
rubber lyophilization stopper and aluminum flip-off crimp seal.
Prior to lyophilization, the nominal fill volume target for 250
through 2000 IU vials is 5 mL and 7 mL for the 3000 IU vial. The
composition of the formulation excipients prior to lyophilization
is the same for all dosage strengths. The powder for injection is
reconstituted with 5 mL of diluent comprising 0.325% (w/v) sodium
chloride supplied in a sterile prefilled syringe.
[0225] The compositions of the drug product solutions prior to
lyophilization are presented in Table 3 and composition of the
lyophilized powders are presented in Table 4. The compositions of
the drug products following reconstitution are presented in Table 1
or in Table 2. (Example 1).
TABLE-US-00003 TABLE 3 rFIXFc Powder for Injection Composition Per
mL Prior to Lyophilization Quantity.sup.i) 250 IU 500 IU 1000 IU
2000 IU 3000 IU Component Function Vial Vial Vial Vial vial rFIXFc
Active 50 IU 100 IU 200 IU 400 IU 429 IU ingredient
L-Histidine.sup.ii) Buffer 3.88 mg 3.88 mg 3.88 mg 3.88 mg 3.88 mg
D-Mannitol Bulking agent 23.8 mg 23.8 mg 23.8 mg 23.8 mg 23.8 mg
Sucrose Stabilizer 11.9 mg 11.9 mg 11.9 mg 11.9 mg 11.9 mg
Polysorbate 20 Stabilizer 0.10 mg 0.10 mg 0.10 mg 0.10 mg 0.10 mg
Water for Solvent QS to 1 mL Injection .sup.i)Amounts are nominal.
.sup.ii)Small amounts of Hydrochloric Acid and/or Sodium Hydroxide
are added during compounding to adjust the pH to 7.1.
TABLE-US-00004 TABLE 4 Nominal rFIXFc Powder for Injection
Composition Per Vial Quantity.sup.i) 250 IU 500 IU 1000 IU 2000 IU
3000 IU Component Function Vial Vial Vial Vial Vial rFIXFc Active
250 IU 500 IU 1000 IU 2000 IU 3000 IU ingredient
L-Histidine.sup.ii) Buffer 19.4 mg 19.4 mg 19.4 mg 19.4 mg 27.2 mg
Mannitol Stabilizer/ 119 mg 119 mg 119 mg 119 mg 167 mg bulking
agent Sucrose Stabilizer/ 59.5 mg 59.5 mg 59.5 mg 59.5 mg 83.3 mg
bulking agent Polysorbate 20 Stabilizer 0.5 mg 0.5 mg 0.5 mg 0.5 mg
0.7 mg
[0226] Administration can be carried out by attaching the syringe
to a standard IV-infusion tubing/needle set, and delivering the
rFIXFc intravenously by standard methods known to those of ordinary
skill in the art.
Example 3: Dosage and Method of Administration/Method of
Calculating Initial Estimated Dose
[0227] rFIXFc is long-acting anti-hemophilic factor (recombinant)
indicated in adults and children (.gtoreq.12 years) with hemophilia
B (congenital Factor IX deficiency) for, e.g., control and
prevention of bleeding episodes, routine prophylaxis to prevent or
reduce the frequency of bleeding episodes, and perioperative
management (surgical prophylaxis).
[0228] Dosing of rFIXFc, formulated as described in Example 1, can
be estimated as described in this example, but can also be
determined by standard tests such as FIX activity assays described
elsewhere herein.
[0229] 1 IU of rFIXFc per kg body weight is expected to increase
the circulating level of Factor IX by 1% [IU/dL]. rFIXFc has been
shown to have a prolonged circulating half-life.
[0230] No dose adjustment for recovery is generally required. Since
subjects can vary in their pharmacokinetic (e.g., half-life, in
vivo recovery) and clinical responses to rFIXFc, the expected in
vivo peak increase in Factor IX level expressed as IU/dL (or % of
normal) or the required dose can be estimated using the following
formulas:
IU/dL (or % of normal)=[Total Dose (IU)/body weight
(kg)].times.recovery (IU/dL per IU/kg)
OR
Dose (IU)=body weight (kg).times.Desired Factor IX Rise (IU/dL or %
of normal).times.reciprocal of recovery (IU/kg per IU/dL)
[0231] The following table (Table 5) can be used to guide dosing in
bleeding episodes:
TABLE-US-00005 TABLE 5 Guide to rFIXFc Dosing for Treatment of
Bleeding Factor IX Level Dose (IU/kg)/ Required (IU/dL Frequency of
Severity of Bleed or % of normal) Doses (hrs) Minor and Moderate
30-60 30-60 IU/kg For example: joint, Repeat every 48 hours if
superficial muscle/no there is further evidence neurovascular
compromise of bleeding (except iliopsoas), superficial soft tissue,
mucous membranes Major 80-120 For repeat dosing, follow For
example: iliopsoas and guidelines for major deep muscle with
surgery [see Table 6] neurovascular injury, or substantial blood
loss, retroperitoneum, CNS Adapted from: Roberts and Eberst, WFH
2008, and WFH 2012
[0232] Subsequent dosage and duration of treatment depends on the
individual clinical response, the severity of the Factor IX
deficiency, and the location and extent of bleeding (see
pharmacokinetics in Example 5 below).
[0233] The following table (Table 6) can be used to guide dosing
for and perioperative management (surgical prophylaxis):
TABLE-US-00006 TABLE 6 Guide to rFIXFc Dosing for Perioperative
Management (Surgical Prophylaxis)* Initial Factor IX Level Dose
(IU/kg)/ Required (IU/dL Frequency of Type of Surgery or % of
normal) Doses (hrs) Minor 50 to 80 50-80 IU/kg Minor operations A
single infusion may be including sufficient. Repeat as
uncomplicated needed after 24-48 hours. dental extraction Major 60
to 120 (initial level) 100 IU/kg (initial dose) Days 1-3: maintain
level A repeat dose at 80 IU/kg 40-60% should be Days 4-6: maintain
level considered after 6-10 hours 30-50% and then every 24 hours
Days 7-14: maintain for the first 3 days. level 20-40% Based on the
long half- life of rFIXFc, the dose may be reduced and frequency of
dosing in the post-surgical setting may be extended after day 3 to
every 48 hours. Adapted from: Roberts and Eberst, WFH 2008, and WFH
2012 *See Pharmacokinetics (Example 5 below)
[0234] For routine prophylaxis, The recommended starting regimens
are either: 50 IU/kg once weekly, or 100 IU/kg once every 10-14
days. Either regimen can be adjusted based on subject response (see
Pharmacokinetics, Example 5 below).
[0235] rFIXFc is contraindicated in subjects who have manifested
severe hypersensitivity reactions, including anaphylaxis, to the
product or its components.
[0236] The clinical response to rFIXFc may vary. If bleeding is not
controlled with the recommended dose, the plasma level of Factor IX
can be determined, and a sufficient dose of rFIXFc can be
administered to achieve a satisfactory clinical response. If the
subject's plasma Factor IX level fails to increase as expected or
if bleeding is not controlled after rFIXFc administration, the
subject's plasma can be tested for the presence of an inhibitor,
e.g., neutralizing antibodies. Subjects using rFIXFc can be
monitored for the development of Factor IX inhibitors by
appropriate clinical observations and laboratory tests known to
those of ordinary skill in the art.
[0237] Subject's plasma can be monitored for Factor IX activity
levels by performing, e.g., the one-stage clotting assay to confirm
adequate Factor IX levels have been achieved and maintained, when
clinically indicated. Subject's plasma can further be monitored for
the development of Factor IX inhibitors.
Example 4. Safety, Efficacy, and Improved Pharmacokinetics
Demonstrated in a Phase 3 Clinical Trail of Extended Half-Life
Recombinant Fc Fusion Factor IX ("B-LONG" Study)
B-LONG Study Design
[0238] Design of the study is global, open-label, nonrandomized,
multicenter (50 investigational sites in 17 countries), Phase 3
study.
[0239] Objectives is to evaluate the efficacy, tolerability,
pharmacokinetics (PK), and safety of intravenously-injected
recombinant factor IX Fc fusion protein (rFIXFc) in the control and
prevention of bleeding episodes, routine prophylaxis, and/or
perioperative management in individuals (previously treated
subjects (PTP)) with severe hemophilia B. FIG. 1 shows a diagram of
the study design. The study disclosed in present example was the
largest registrational trial of any therapeutic for hemophilia B,
with the most extensive PK analysis conducted to date. The study
duration was about 72 weeks.
[0240] Key inclusion criteria includes (1) male, (2) older than 12
years of age (weighing at least 40 kg), (3) having diagnosis of
severe hemophilia B defined as .ltoreq.2% (.ltoreq.2 IU/dL FIX:C)
endogenous factor IX activity, (5) having history of .gtoreq.100
prior documented exposure days (EDs) with any currently marketed
FIX product, bleeding events and/or treatment with FIX during the
12 weeks prior to enrollment, or at least 8 bleeds in the 52 weeks
prior to enrollment if they had been treated episodically, (6)
subjects with a history of inhibitors or anaphylaxis associated
with FIX or intravenous immunoglobulin, other coagulation
disorders, uncontrolled HIV infection, renal dysfunction, or active
severe hepatic disease were excluded from the study, and (7)
subjects unable or unwilling to adhere to the prophylaxis regimen
or diary requirements or those who had received immunosuppressant
drugs .ltoreq.12 months prior to the study were also excluded.
[0241] Three treatment arms are Arm 1 (fixed weekly interval
prophylaxis) Arm 2 (individualized interval prophylaxis) Arm 3
(episodic [on-demand regimen] treatment) Arm 4 (perioperative
management).
[0242] Under Arm 1, subjects were treated weekly with an initial
dose of 50 IU/kg, which was subsequently adjusted based on PK
profile to maintain trough factor levels sufficient to prevent
bleeding in order to maintain 1-3 IU/dL above baseline at trough
and/or if subjects experienced .gtoreq.2 spontaneous bleeds over 3
consecutive months. A diagram showing the sequential PK subgroup
dosing and PK sampling of Arm 1 is shown in FIG. 2. Specifically,
adjustments to rFIXFc dose were made based on monitoring of trough
at weeks 4, 16, 26, and 39. If the pharmacokinetic values indicated
the patient's trough level was 3 IU/dl above baseline or higher at
the end of the week, the dose was reduced to target a trough of 1
to 3 IU/dl above baseline. If the pharmacokinetic values indicated
the patient's trough level would be less than 1 IU/dl above
baseline at the end of the week, the dose was increased to target a
trough of 1 to 3 IU/dl above baseline. If the target range could
not be achieved, a plasma sample was run to rule out the presence
of inhibitors. A repeat trough level was done approximately 1 month
after making any dosage adjustments and was repeated monthly until
no additional adjustments were necessary. Only the dose of rFIXFc
was adjusted in this treatment arm.
[0243] Under Arm 2, subjects were treated with 100 IU/kg, at an
initial interval of 10 days, which was subsequently adjusted to
maintain trough factor levels sufficient to prevent bleeding
(interval adjusted per subject's PK profile to maintain 1-3 IU/dL
activity above baseline at trough and/or if subject experienced
.gtoreq.2 spontaneous bleeds over 3 consecutive months). Monitoring
took place at weeks 4, 16, 26, and 39 to ensure that this FIX
trough level was maintained. Only the interval of rFIXFc was
adjusted in this treatment arm.
[0244] For any patients in arm 1 or arm 2 who experienced
unacceptable bleeding, with two or more spontaneous bleeds over 3
consecutive months, the dose (arm 1) or interval (arm 2) were
adjusted to target a FIX trough level of 3 to 5 IU/dl above
baseline. If unacceptable bleeding did not occur on rFIXFc at a
target trough of 3 to 5 IU/dl above baseline, at the investigator's
discretion, and after agreement with the sponsor, rFIXFc could be
re-adjusted for a target trough of 1 to 3 IU/dl.
[0245] Under Arm 3, subjects received rFIXFc episodic treatment as
needed for bleeding (e.g., 20 IU/kg to 100 IU/kg depending on
bleeding severity). Minor bleeding episodes were treated with 20-30
IU/kg, moderate to major bleeding episodes with 25-50 IU/kg, and
major to life-threatening bleeding episodes with 50-100 IU/kg to
target trough FIX levels of 20-30%, 25-50%, and 50-100%,
respectively, based on the patient's pharmacokinetic profile.
[0246] Under Arm 4, 40-100 IU/kg rFIXFc was administered prior to
and following major surgery with dose adjusted to type of surgery;
subjects were allowed to enroll directly into the surgery arm, and
then move into one of the treatment arms (Arm 1, Arm 2, or Arm 3)
post-surgery; or to move into the surgery arm from another arm
during the perioperative period if they required a surgery during
the study.
[0247] Those in Arms 1 and 3 received rFIXFc for up to 52.+-.1
weeks of treatment and Arm 2 for up to 50 exposure days (EDs).
Subjects on a previous prophylaxis regimen were only entered into
Arm 1 or 2, while those previously using episodic treatment could
be enrolled into any of the treatment arms.
[0248] A sequential PK subgroup in Arm 1 received 50 IU/kg rFIX at
baseline, followed by a .gtoreq.5-day washout prior to first
administration of rFIXFc. After 120 hours post-injection of rFIX
with PK assessment, subjects received a 50 IU/kg of rFIXFc and
blood samples were collected over 240 hours for PK profiling. The
sequential PK subgroup repeated rFIXFc PK profiling at Week 26. All
remaining subjects in the study also underwent non-sequential PK
evaluations with rFIXFc for 168-336 hours (7-14 days: Arm 1=10; Arm
2=14; Arm 3=7).
[0249] For PK Assessment, all subjects in all arms had an initial
PK assessment after their first dose of rFIXFc. A subset of
subjects from Arm 1 were assigned to a protocol-specified
sequential PK subgroup to compare the PK of rFIXFc with recombinant
factor IX (rFIX, BeneFIX.RTM.) as follows: following a 120-hour
washout period, patients in the sequential pharmacokinetic subgroup
in arm 1 received an injection of 50 IU/kg recombinant FIX (rFIX,
BeneFIX.RTM.) and underwent pharmacokinetic sampling up to 96 hours
as follows: pre-injection, 10 (.+-.2) min, 1 h (.+-.15 min), 3 h
(.+-.15 min), 6 h (.+-.15 min), 24 (.+-.2) h, 48 (.+-.2) h, 72
(.+-.3) h, and 96 (.+-.3) h from the start of the injection. Each
patient had to complete a minimally evaluable pharmacokinetic
sampling through 72-hour timepoint to be included in the
pharmacokinetics analysis set.
[0250] After completion of rFIX pharmacokinetic sampling and
.gtoreq.5-day from the last rFIX dose, patients received a dose of
50 IU/kg of rFIX Fc fusion protein (rFIXFc) with pharmacokinetic
assessment over 240 hours. The rFIXFc sampling was done as follows:
pre-injection, 10 (.+-.2) min, 1 h (.+-.15 min), 3 h (.+-.15 min),
6 h (.+-.15 min), 24 (.+-.2) h, 48 (.+-.2) h, 96 (.+-.3) h, 144
(.+-.3) h, 168 (.+-.3) h, 192 (.+-.3) h, and 240 (.+-.3) h from the
start of the injection. Each patient had to complete a minimally
evaluable pharmacokinetic sampling through 168-hour timepoint to be
included in the pharmacokinetics analysis set. A repeat
pharmacokinetic assessment of rFIXFc with the same sampling
schedule was also performed at week 26.
[0251] In arms 1 and 2, the efficacy period started with the date
and time of first prophylactic dose following a completed
pharmacokinetic sampling period and ended with the last dose
administered (for prophylaxis or a bleeding episode) as recorded in
the electronic Case Report Form (eCRF) or electronic diary
(eDiary). The efficacy period was interrupted for the repeat
pharmacokinetic period in arm 1 (sequential pharmacokinetic
subgroup) and for all surgical/rehabilitation periods (for both
major and minor surgeries) in arms 1 and 2. The efficacy period
continued up to the last dose (for prophylaxis or treatment of a
bleeding episode) before the repeat pharmacokinetic dose or up to
the last dose (for prophylaxis or treatment of a bleeding episode)
before the start of a surgical/rehabilitation period, and then
resumed at the next prophylactic dose following the end of the
pharmacokinetic or surgical/rehabilitation period.
[0252] In arm 3, the efficacy period started 1 minute following the
last pharmacokinetic sampling timepoint and ended with either the
date of last contact or the date of the last entry into the eDiary,
whichever was later. The efficacy period was interrupted 1 minute
before the start of a surgical/rehabilitation period and restarted
at 00:01 the day following the end of the surgical/rehabilitation
period.
[0253] Plasma FIX activity was measured by the one-stage (activated
partial thromboplastin time [aPTT]) clotting assay, which was
validated for rFIXFc, BeneFIX.RTM., and human FIX in human plasma
samples. FIX activity in citrated plasma samples was measured on an
MDA 180 coagulation instrument using commercially available aPTT
reagents, e.g., Trinity Biotech (Automated APTT [silica-based
activator and phospholipid mixture]), Precision BioLogic
(CRYOCHECK.TM. Factor IX-depleted plasma), and normal reference
plasma (Precision BioLogic) as a calibrator (LLOQ 1 IU/dl), which
has a potency assigned against the World Health Organization 3rd or
4th plasma International Standard. For all three FIX proteins, the
accuracy was within 95% to 104%, and the intra- and inter-assay
precision was typically within 10%.
[0254] Neutralizing antibodies were detected as follows: The
Nijmegen-modified Bethesda assay to detect neutralizing antibodies
was performed at screening, baseline, and each visit during study
treatment to monitor for the development of an inhibitor. Following
the first dose with rFIXFc, inhibitor testing in arms 1 and 2 was
conducted at trough at each scheduled clinic visit, with trough
defined as a point after the longest interval between scheduled
doses. Inhibitor testing in arm 3 was performed following a washout
of at least 72 hours (3 days). For new patients entering arm 4,
inhibitor testing was performed at screening and after a total of
at least 4 EDs were achieved with rFIXFc, within 4 weeks prior to
scheduled surgery. Formation of an inhibitor was defined as a
neutralizing antibody value .gtoreq.0.6 Bethesda units (BU)/ml,
confirmed in a second, separately drawn sample within 2-4 weeks.
The confidence interval on the inhibitor incidence rate was
evaluated using the Clopper-Pearson exact method for a binomial
proportion.
[0255] Key efficacy outcome measures include (1) annualized
bleeding rate (ABR) in Arms 1, 2, and 3 (comparing each of 2
prophylaxis cohorts with the episodic (on-demand) treatment cohort)
(i.e., (a) weekly prophylaxis arm (Arm 1) compared with the
episodic treatment arm (Arm 3) and (b) individualized interval
prophylaxis arm (Arm 2) compared with the episodic treatment arm
(Arm 3); (2) response to treatment of bleeding episodes using
four-point bleeding response scale (i.e., (a) consumption per
subject; (b) weekly dose for Arm 1; (c) dosing interval for Arm 2;
and (d) number of injections and dose per injection required to
stop a bleeding episode; and (3) treating physicians' assessments
of subjects' response to surgery with rFIXFc using a 4-point scale;
number of injections and dose required to maintain hemostasis
during the surgical period; estimated blood loss during surgery;
and number of transfusions required for surgery. For the annualized
bleeding rate measurements.
[0256] Pharmacokinetic (PK) outcome measures include the
followings: (1) PK profiles were assessed by a user-defined
2-compartmental model based on the decay of plasma FIX activity
over time as measured by the one-stage (activated partial
thromboplastin time) clotting assay in a central laboratory and
verified for use in the PK analysis and (2) PK of rFIXFc and
recombinant factor IX (rFIX, BENEFIX.RTM.) and time to 1% above
baseline.
[0257] For FIX activity pharmacokinetic analysis, the baseline
(endogenous FIX activity) was pre-defined as the lowest observed
FIX activity at either screening, pre-dose, post-dose, or from the
patient's historical clinical records. For patients whose lowest
observed FIX activity was below 1%, the baseline FIX activity was
set as zero; for patients whose lowest observed FIX activity was
between 1 and 2 IU/dl, the baseline FIX activity was set at the
actual observed FIX value. The residual drug was decayed following
first order decay with the decay rates determined on an individual
basis. The FIX activity over time profiles, corrected by baseline
and residual drug, were analyzed using a user-defined and verified
two-compartmental model. The user-defined code automated the
calculation of additional secondary pharmacokinetic parameters
(e.g., Time 1% and 3%), which are not included in the secondary
parameter list of the WinNonlin library mode (PHOENIX.RTM.
WinNonlin 6.2.1.51; Pharsight), thus eliminating the need for
manual handling of data outside of the primary analysis and
minimizing the risk of introducing human error.
[0258] Key safety outcome measures include the followings: (a)
clinically significant changes from baseline in laboratory values;
(b) incidence of inhibitor development; and (c) incidence of
adverse events (AEs) occurring outside of the perioperative
management arm (Arms 1, 2, and 3 but not 4).
[0259] Statistical Analysis--: Median annualized bleeding rates
(ABR) were reported and estimated ABR was calculated using a
negative binomial model, which accounted for over-dispersion, to
compare ABR between Arms 1 and 2 (prophylaxis regimens) and Arm 3
(episodic treatment). Descriptive statistics were used to provide
median and interquartile range (IQR) values for each of arms 1-3.
The sequential PK analysis population included Arm 1 subjects with
blood samples collected through a minimum of 72 hours from the
start of the injection for 50 IU/kg rFIX and a minimum of 168 hours
for 50 IU/kg rFIXFc. An analysis of variance model (ANOVA) with
variables for study treatment (rFIX or rFIXFc) and subject was used
and 95% confidence intervals were provided for geometric means for
each treatment. The study was sufficiently powered to detect
statistical significance for the occurrence of inhibitors, based
FDA guidance that the occurrence of inhibitors in a clinical study
can be adequately modeled using the binomial distribution that
results in a 2-sided, 95% CI for the true inhibitor incidence of
(0.05% to 10.65%) using the exact, Clopper-Pearson method. All
statistical tests were performed at the two-sided, 5% significance
level. The study was terminated following an interim analysis when
prespecified number of major surgical procedures had been
completed.
B-LONG Results
Subjects
[0260] A total of 123 subjects were enrolled in the study. 115
(93.5%) of subjects completed the study (3 withdrew consent; 1 lost
to follow-up; 2 withdrawn due to protocol violation; and 2
discontinued due to adverse events). Subjects underwent a washout
period of 96 hours prior to initial study dosing, at which time PK
assessments were performed. Enrolled subjects were entered into one
of four treatment arms as described in detail above (See also FIG.
1). [0261] Arm 1 (weekly prophylaxis), n=63 (59 completed) [0262]
Arm 2 (individualized interval prophylaxis), n=29 (27 completed)
[0263] Arm 3 (episodic treatment), n=27 (26 completed) [0264] Arm 4
(perioperative management), n=12 (3 completed), 14 surgeries (8 of
the 12 subjects also participated in other treatment arms (Arms 1,
2, and 3))
[0265] Baseline characteristics of study subjects (see Table 7)
showed a diverse population with substantial co-morbidities of
human immunodeficiency virus and hepatitis C virus infection,
reflective of the general hemophilia B population. The genotype
profile was consistent with that expected in the study population
(55% having a missense mutation).
TABLE-US-00007 TABLE 7 Subject baseline characteristics Arm 1 Arm 2
Arm 3 Arm 4 Total N = 63 (N = 29) (N = 27) (N = 12) (N = 123) Age,
median (min-max) 28 (12-71) 33 (12-62) 36 (14-64) 34 (17-61) 30
(12-71) Weight (kg), median 70.2 (45.2-186.7) 76.0 (50.0-128.0)
65.0 (45.0-91.7) 65.0 (47.9-100.5) 73.3 (45.0-186.7) (min-max) BMI
(kg/m.sup.2), median 24.29 (16.3-49.6) 25.69 (18.6-36.6) 24.16
(15.2-29.4) 22.86 (18.3-32.8) 24.78 (15.2-49.6) (min-max) Race, n
(%) White 41 (65.1) 18 (62.1) 11 (40.7) 6 (50.0) 73 (59.3) Black 7
(11.1) 2 (6.9) 1 (3.7) 2 (16.7) 10 (8.1) Asian 7 (11.1) 7 (24.1) 14
(51.9) 2 (16.7) 29 (23.6) American Indian or 0 (0.0) 0 (0.0) 1
(3.7) 0 (0.0) 1 (0.8) Alaska Native Other 8 (12.7) 2 (6.9) 0 (0.0)
2 (16.7) 10 (8.1) Geographic location, n DNS 35 (29.4)* (%) Europe
21 (33.3) 12 (41.4) 2 (7.4) DNS 36 (30.3) North America 18 (28.6) 7
(24.1) 11 (40.7) DNS 48 (40.3)* Other.sup..dagger. 24 (38.1) 10
(34.5) 14 (51.9) DNS Baseline FIX level, n DNS 98 (82.4)* (%) <1
IU/dl 50 (79.4) 22 (75.9) 26 (96.3) DNS 21 (17.6)* 1-2 IU/dl 13
(20.6) 7 (24.1) 1 (3.7) DNS Genotype, n (%) Missense mutation 34
(54.0) 19 (65.5) 14 (51.9) 6 (50.0) 68 (55.3) Nonsense mutation 11
(17.5) 6 (20.7) 6 (22.2) 1 (8.3) 23 (18.7) Frameshift 6 (9.5) 1
(3.4) 1 (3.7) 2 (16.7) 9 (7.3) Unknown 2 (3.2) 0 (0.0) 6 (22.2) 1
(8.3) 9 (7.3) Splice mutation 6 (9.5) 2 (6.9) 0 (0.0) 0 (0.0) 8
(6.5) Large deletions 3 (4.8) 0 (0.0) 0 (0.0) 2 (16.7) 4 (3.3)
Partial gene deletion 1 (1.6) 1 (3.4) 0 (0.0) 0 (0.0) 2 (1.6)
Pre-study FIX regimen, DNS 48 (40.7)* n (%) Prophylaxis 33 (53.2)
15 (51.7) 0 DNS 70 (59.3)* episodic 29 (46.8) 14 (48.3) 27 (100.0)
DNS Est. bleeds prior 12 mo, 10.5 10.0 18.0 DNS 2.0 (0, 21)* median
no. (min, max) Prior prophylaxis 2.5 (0, 21) 2.0 (0, 7) 0 DNS 22.0
(5, 100)* Prior episodic 23.0 (6, 70) 25.0 (10, 100) 18.0 (5, 50)
DNS 58 (48.7)* .gtoreq.1 Target joint, n (%) 36 (57.1) 8 (27.6) 14
(51.9) DNS 35 (29.4)* Est. bleeds prior 12 mo, 10.5 10.0 18.0 DNS
36 (30.3)* median no. (min, max) Family history of 0 ( ) 0 ( ) 2
(7.4) 0 ( ) 2 (1.6) inhibitor, n (%) HIV positive, n (%) 5 (7.9) 1
(3.4) 2 (7.4) 2 (16.7) 9 (7.3) HCV positive, n (%) 38 (60.3) 15
(51.7) 14 (51.9) 7 (58.3) 70 (56.9) *totals for Arms 1-3 only BMI,
body mass index; HIV, human immunodeficiency virus; HCV, hepatitis
C virus; DNS, data not shown.
[0266] Of the 48 subjects on a previous prophylaxis regimen,
>80% were infusing .gtoreq.2 times weekly. Subjects in the
prophylaxis cohorts (Arms 1 and 2) were well balanced for prior
regimen and bleeding history. There were fewer subjects with target
joints in Arm 2 and more subjects with a baseline FIX activity
<1 IU/dL in Arm 3. The median (minimum, maximum) durations of
treatment in arms 1, 2, and 3 were 51.6 (<1, 97), 58.3 (<1,
126), and 40.9 (28, 54) weeks, respectively, and the median
(minimum, maximum) days of exposure were 55.0 (1, 105), 38.0 (1,
71), and 16.0 (4, 35), respectively. A total of 5243 of rFIXFc
injections were administered during this study, corresponding to
5144 EDs (117.1 patient-years of exposure). Treatment adherence, in
subjects who were .gtoreq.80% compliant with their prescribed dose
(Arm 1) or dosing interval (Arm 2), was 96.6% overall in the
prophylaxis arms (Arm 1=95.1%, Arm 2=100%).
[0267] For patients in arm 4, major surgery was defined as any
surgical procedure (elective or emergent) that usually, but not
always, involves general anesthesia and/or respiratory assistance
in which a major body cavity is penetrated and exposed, or a
substantial impairment of physical or physiological functions is
produced (e.g., laparotomy, thoracotomy, craniotomy, joint
replacement, or limb amputation).
Study Visit Schedule:
[0268] For arms 1-3, study visits occurred at screening (8 weeks),
baseline, week 4, week 16, week 26, week 39, and week 52.
Additionally, patients in arms 1-3 had a 30-day follow-up phone
call, unless they enrolled in the ongoing extension study
(NCT01425723). Patients in arm 4 had study visits at screening,
baseline, the day of surgery, 1 week after surgery, and 1 week
post-recovery.
Sequential Pharmacokinetic Subgroup
[0269] Length of sampling times for rFIX and rFIXFc were based upon
previously reported half-lives, allowing sufficient time for decay
(normally three to five times the previously observed half-life)
for accurate description of pharmacokinetics. Following a 120-hour
washout period, patients in the sequential pharmacokinetic subgroup
in arm 1 received an injection of 50 IU/kg recombinant FIX (rFIX)
and underwent pharmacokinetic sampling up to 96 hours as follows:
pre-injection, 10 (.+-.2) min, 1 h (.+-.15 min), 3 h (.+-.15 min),
6 h (.+-.15 min), 24 (.+-.2) h, 48 (.+-.2) h, 72 (.+-.3) h, and 96
(.+-.3) h from the start of the injection. Each patient had to
complete a minimally evaluable pharmacokinetic sampling through
72-hour timepoint to be included in the pharmacokinetics analysis
set.
[0270] After completion of rFIX pharmacokinetic sampling and
.gtoreq.5-day from the last rFIX dose, patients received a dose of
50 IU/kg of rFIX Fc fusion protein (rFIXFc) with pharmacokinetic
assessment over 240 hours. The rFIXFc sampling was done as follows:
pre-injection, 10 (.+-.2) min, 1 h (.+-.15 min), 3 h (.+-.15 min),
6 h (.+-.15 min), 24 (.+-.2) h, 48 (.+-.2) h, 96 (.+-.3) h, 144
(.+-.3) h, 168 (.+-.3) h, 192 (.+-.3) h, and 240 (.+-.3) h from the
start of the injection. Each patient had to complete a minimally
evaluable pharmacokinetic sampling through 168-hour timepoint to be
included in the pharmacokinetics analysis set. A repeat
pharmacokinetic assessment of rFIXFc with the same sampling
schedule was also performed at week 26.
Efficacy
[0271] In total, 119 subjects were included in the efficacy
analysis. Median annualized bleeding rate (ABR) with the 25th and
75th percentiles (interquartile range (IQR)), which were driven
primarily by spontaneous bleeds, were determined for Arms 1, 2, and
3 as shown in FIGS. 3A, 3B, and Table 8 and summarized below:
[0272] Weekly prophylaxis arm (Arm 1): 2.95 (1.01-4.35) [0273]
Individualized interval prophylaxis arm (Arm 2): 1.38 (0-3.43)
[0274] Episodic treatment arm (Arm 3): 17.69 (10.77-23.24)
[0275] (a) Prophylaxis Dose and Interval Titration, and Episodic
Treatment of Bleeding Episodes
[0276] Samples to measure FIX levels were drawn for all patients
during the course of the study to estimate each patient's
pharmacokinetics parameters to guide the appropriate dose or dosing
interval adjustments toward a target trough of 1-3 IU/dl of FIX
levels above baseline.
[0277] In arm 1 (fixed weekly interval): All patients in this arms
initially received rFIXFc 50 IU/kg injection once weekly.
Adjustments to rFIXFc dose were made based on monitoring of trough
at weeks 4, 16, 26, and 39. If the pharmacokinetic values indicated
the patient's trough level was 3 IU/dl above baseline or higher at
the end of the week, the dose was reduced to target a trough of 1
to 3 IU/dl above baseline. If the pharmacokinetic values indicated
the patient's trough level would be less than 1 IU/dl above
baseline at the end of the week, the dose was increased to target a
trough of 1 to 3 IU/dl above baseline. If the target range could
not be achieved, a plasma sample was run to rule out the presence
of inhibitors. A repeat trough level was done approximately 1 month
after making any dosage adjustments and was repeated monthly until
no additional adjustments were necessary. Only the dose of rFIXFc
was adjusted in this treatment arm.
[0278] In arm 2 (individualized dosing interval), a dose of 100 IU
per kilogram of rFIXFc was administered IV at an interval based on
the patient's baseline pharmacokinetic assessment to achieve a
target trough of 1 to 3 IU/dl above baseline. Monitoring took place
at weeks 4, 16, 26, and 39 to ensure that this FIX trough level was
maintained. Only the interval of rFIXFc was adjusted in this
treatment arm.
[0279] For any patients in arm 1 or arm 2 who experienced
unacceptable bleeding, with two or more spontaneous bleeds over 3
consecutive months, the dose (arm 1) or interval (arm 2) were
adjusted to target a FIX trough level of 3 to 5 IU/dl above
baseline. If unacceptable bleeding did not occur on rFIXFc at a
target trough of 3 to 5 IU/dl above baseline, at the investigator's
discretion, and after agreement with the sponsor, rFIXFc could be
re-adjusted for a target trough of 1 to 3 IU/dl.
[0280] In arm 3 (episodic treatment), minor bleeding episodes were
treated with 20-30 IU/kg, moderate to major bleeding episodes with
25-50 IU/kg, and major to life-threatening bleeding episodes with
50-100 IU/kg to target trough FIX levels of 20-30%, 25-50%, and
50-100%, respectively, based on the patient's pharmacokinetic
profile.
[0281] (b) Efficacy Period
[0282] In arms 1 and 2, the efficacy period started with the date
and time of first prophylactic dose following a completed
pharmacokinetic sampling period and ended with the last dose
administered (for prophylaxis or a bleeding episode) as recorded in
the electronic Case Report Form (eCRF) or electronic diary
(eDiary). The efficacy period was interrupted for the repeat
pharmacokinetic period in arm 1 (sequential pharmacokinetic
subgroup) and for all surgical/rehabilitation periods (for both
major and minor surgeries) in arms 1 and 2. The efficacy period
continued up to the last dose (for prophylaxis or treatment of a
bleeding episode) before the repeat pharmacokinetic dose or up to
the last dose (for prophylaxis or treatment of a bleeding episode)
before the start of a surgical/rehabilitation period, and then
resumed at the next prophylactic dose following the end of the
pharmacokinetic or surgical/rehabilitation period.
[0283] In arm 3, the efficacy period started 1 minute following the
last pharmacokinetic sampling timepoint and ended with either the
date of last contact or the date of the last entry into the eDiary,
whichever was later. The efficacy period was interrupted 1 minute
before the start of a surgical/rehabilitation period and restarted
at 00:01 the day following the end of the surgical/rehabilitation
period.
Treatment Compliance
[0284] Compliance with treatment dosing was monitored and
documented by site staff. For between-visit administration,
patients self-administered rFIXFc and recorded treatment in the
hand-held eDiary, which was reviewed during periodic calls to the
patient and at each visit by study site staff and the clinical
monitor.
[0285] Analysis of patients' compliance was based on data from the
eCRF and eDiary. Analyses of compliance included patients'
compliance with the prescribed prophylactic regimens. For weekly
prophylaxis, analyses included percentage of nominal doses taken
per patient within the 80% to 125% range. Similarly, for
individualized interval prophylaxis, analysis included the
percentage of doses taken per patient within .+-.36 hours of the
prescribed interval. Patients were considered compliant if the
calculated compliance rate was at least 80%.
Analytical Method
[0286] Plasma FIX activity was measured by the one-stage (activated
partial thromboplastin time [aPTT]) clotting assay, which was
validated for rFIXFc, BeneFIX, and human FIX in human plasma
samples. FIX activity in citrated plasma samples was measured on an
MDA 180 coagulation instrument using commercially available aPTT
reagents, e.g., Trinity Biotech (Automated APTT [silica-based
activator and phospholipid mixture]), Precision BioLogic
(Cryocheck.TM. Factor IX-depleted plasma), and normal reference
plasma (Precision BioLogic) as a calibrator (LLOQ 1 IU/dl), which
has a potency assigned against the World Health Organization 3rd or
4th plasma International Standard. For all three FIX proteins, the
accuracy was within 95% to 104%, and the intra- and inter-assay
precision was typically within 10%.
TABLE-US-00008 TABLE 8 Summary of Median (IQR*) Annualized Bleed
Rate (ABR) by Treatment Arm Prophylaxis Prophylaxis Bleeding Fixed
Individualized Episodic Episode Weekly Interval Interval (On
Demand) Etiology (N = 61) (N = 26) (N = 27) Total Number of weeks
on rFIXFc 51.6 (<1, 97) 58.3 (<1, 126) 40.9 (28, 54) 51.4
(<1, 126) treatment, median (min, max) ABR, negative binomial
3.12 (2.46, 3.95) 2.40 (1.67, 3.47) 18.67 (14.01, 24.89) regression
model (95% CI) % Reduction vs. arm 3 (P 83% (<0.001) 87%
(<0.001) value)* ABR by type, location of bleeds, and baseline
trough level, median (IQR): Overall 2.95 (1.01, 4.35) 1.38 (0.00,
3.43) 17.69 (10.77, 23.24) Spontaneous 1.04 (0.00, 2.19) 0.88
(0.00, 2.30) 11.78 (2.62, 19.78) Traumatic 0.99 (0.00, 2.13) 0.00
(0.00, 0.78) 2.21 (0.00, 6.81) Joint 1.1 (0, 4.0) 0.4 (0, 7.8) 13.6
(6.1, 21.6) Spontaneous 1.0 (0.0, 2.8) 0.0 (0, 6.2) 5.1 (0.0, 17.3)
Traumatic 0.0 (0.0, 1.1) 0.0 (0.0, 7.5) 1.3 (0.0, 3.5) Muscle 0.0
(0.0, 1.0) 0.0 (0.0, 0.0) 4.0 (1.0, 6.8) Spontaneous 0.0 (0.0, 0.0)
0.0 (0.0, 0.0) 1.0 (0.0, 3.5) Traumatic 0.0 (0.0, 0.0) 0.0 (0.0,
0.0) 1.1 (0.0, 2.7) Baseline trough level <1 IU/dl 2.6 (1.0,
4.1) 1.1 (0.0, 2.9) 18.5 (13.2, 23.2) 1-2 IU/dl 4.5 (0.0, 6.4) 3.4
(0.0, 5.7) 7.7 (7.7, 7.7) *Reduction in ABR compared with arm 3,
calculated using negative binomial model.
[0287] The ABR was significantly reduced for both prophylaxis arms,
by 83% and 87% compared with episodic treatment based on estimates
from a negative binominal regression model (3.12, 2.40, and 18.67
for arms 1, 2, and 3, respectively; P<0.001 Table 8). The lower
ABR in the prophylaxis arms was consistent across all demographic
and disease-based subgroups in prespecified subgroup analyses (FIG.
3 and Table 2). Furthermore, limiting the analysis to patients on a
prior episodic regimen, the reductions in ABRs of 83% and 89% for
arms 1 and 2, respectively, were consistent with the primary
analysis. The proportion of patients without any bleeding episodes
on prophylaxis during the study was 23.0% for arm 1 and 42.3% for
arm 2. Additionally, analyses of bleeding episodes by type and
location showed a low ABR in both prophylactic arms (Table 8). The
median (IQR) weekly does of rFIXFc for subjects in Arm 1 in the
last 3 months of the study was 40.5 IU/kg. The dosing interval for
approximately half of the study population in the individualized
interval prophylaxis arm (Arm 2) was .gtoreq.14 days during the
last 6 months on study. A summary of the median dosing for Arm 1
and Arm 2 is shown in Tables 9A and 9B.
TABLE-US-00009 TABLE 9A Arm 1 and Arm 2 Dosing Summary n Arm 1 n
Arm 2 Median dose, IU/kg/wk (IQR) Overall 61 45.2 (38.1-53.7) Last
3 months 58 40.5 (30.3-53.8) Median dosing interval (IQR) Overall
26 12.5 (10.4-13.4) Last 3 months.sup.a 26 14.0 (11.3-14.0)
.sup.aBased on subjects in study .gtoreq.6 months IQR = 25.sup.th
and 75.sup.th percentiles. IQR, interquartile range.
TABLE-US-00010 TABLE 9B Arm 1 and Arm 2 Dosing Summary Arm 1 Arm 2
Arm 3 Total Number of injections per episode required for
resolution of bleeding, % of episodes 1 85.0 85.1 93.5 90.4 2 9.0
11.9 5.2 6.9 3 6.0 3.0 1.2 2.7 .gtoreq.4 0.0 0.0 0.0 0.0 Dose of
rFIXFc required for resolution of 47.1 (16.6, 99.1) 44.8 (14.3,
110.1) 46.0 (7.9, 111.1) bleeding episodes, median IU/kg (min, max)
Weekly prophylactic dose in arm 1.sup..dagger., median IU/kg (min,
max) Overall 45.2 (25.0, 74.3) Last 6 mo on study.sup..dagger-dbl.
40.7 (21.3, 82.7) Last 3 mo on study.sup..sctn. 40.5 (16.7, 87.6)
Prophylactic dosing interval in arm 2.sup..dagger., median days
(min, max) Overall 12.5 (7.8, 15.9) Last 6 mo on
study.sup..dagger-dbl. 13.8 (7.8, 19.1) Last 3 mo on
study.sup..sctn. 14.0 (7.7, 20.8) Subject's assessment of response
to rFIXFc injections, n (% of injections)* Excellent or Good 146
(76.4) 57 (77.0) 363 (85.4) 566 (82.0) Moderate 40 (20.9) 15 (20.3)
55 (12.9) 110 (15.9) No Response 5 (2.6) 2 (2.7) 7 (1.6) 14 (2.0)
Physicians' global assessment of rFIXFc response score, n (% of
visits)** Excellent or Effective 264 (98.8) 122 (99.2) 94 (97.9)
580 (98.8) Partially Effective 3 (1.1) 1 (0.8) 2 (2.1) 6 (1.2)
Ineffective 0 0 0 0 .sup..dagger.Pharmacokinetic driven dosing (arm
1) or interval (arm 2) changes were permitted to achieve trough
levels 1% to 3% above baseline. If patients had two breakthrough
spontaneous bleeding episodes in a rolling 3-month period, doses
(arm 1) or intervals (arm 2) could be adjusted to provide more
protection. .sup..dagger-dbl.In patients on study for .gtoreq.9
months .sup..sctn.In patients on study for .gtoreq.6 months ABR
denotes annualized bleeding rate, CI confidence interval, IQR
interquartile range (25.sup.th and 75.sup.th percentiles), rFIXFc
recombinant factor IX Fc fusion protein, SD standard deviation.
*Excellent: Abrupt pain relief and/or improvement in signs of
bleeding within approximately 8 hours after the initial injection;
Good: Definite pain relief and/or improvement in signs of bleeding
within approximately 8 hours after an injection, but possibly
requiring more than one injection after 24 to 48 horns for complete
resolution; Moderate: Probable or slight beneficial effect within 8
hours after the initial injection and requiring more than one
injection; No response: No improvement, or condition worsened,
within approximately 8 hours after the initial injection.
**Excellent: Bleeding episodes responded to less than or equal to
the usual number of injections or less than or equal to the usual
dose of rFIXFc, or the rate of breakthrough bleeding during
prophylaxis was less than or equal to that usually observed.
Effective: Most bleeding episodes responded to the same number of
injections and dose, but some required more injections or higher
doses, or there was a minor increase in the rate of breakthrough
bleeding. Partially Effective: Bleeding episodes most often
required more injections and/or higher doses than expected, or
adequate breakthrough bleeding prevention during prophylaxis
required more frequent injections and/or higher doses. Ineffective:
routine failure to control hemostasis or hemostatic control
required additional agents.
[0288] Control of bleeding: A total of 636 bleeding episodes (Arm
1=167, Arm 2=67, Arm 3=402) in 89 subjects were treated, over 90%
(90.4%) of bleeding episodes were controlled by a single injection
of rFIXFc and 97.3% were controlled by .ltoreq.2 injections. The
median number of injections needed to resolve bleeding episodes was
1.0 regardless of type, compliance status, or location of bleeding
episode, excluding two internal bleeding episodes in Arm 2 that
required 2 injections for resolution. The median dose per injection
was 46.1 IU/kg and the median total dose for the bleeding episodes
was 47.0 IU/kg. For the 61 (9.6%) of bleeding episodes that
required >1 injection for resolution, the median interval
between the first and second injection was 45 hours. Overall,
subject assessment of response to treatment with rFIXFc was
excellent or good for 76.4%, 77.0%, and 85.4% of injections in Arms
1, 2, and 3, respectively. Physician' global assessment of subject
response to their rFIXFc regimen was rated as excellent or
effective for 98.8%, 99.2%, and 97.9% in Arms 1, 2, and 3,
respectively. Bleeding episodes are summarized in Table 10.
TABLE-US-00011 TABLE 10 Summary of Efficacy in Control of Bleeding
New Bleeding episodes # of Injections to treat bleeding episodes (N
= 636) 1 injection 575 (90.4%) 2 injections 44 (6.9%) 3 injections
17 (2.7%) Median dose per injection 46.07 (32.86, 57.03) (IU/kg) to
treat a bleeding episode (IQR) Median total dose (IU/kg) 46.99
(33.33, 62.50) to treat a bleeding episode (IQR) Response to first
injection (N = 613) Excellent or good 513 (83.7%) Moderate 90
(14.7%) No response 10 (1.6%)
[0289] This efficacy was similar when examined across arms, on a
per subject basis, and for subjects who treated an episode more
than 8 hours after the onset of symptoms.
[0290] The dosing interval for subjects in Arm 2 increased from the
initial 10 days to 53.8% of subjects achieving a median dose
interval of .gtoreq.14 days during the last 3 months on the
study.
[0291] Perioperative management: Endpoints for Arm 4 included
investigators'/surgeons' assessments of subjects' response to
surgery with rFIXFc; number of injections and dose required to
maintain hemostasis during the surgical period; estimated blood
loss during and after surgery; and number of transfusions required
for surgery. Overall, 14 major surgeries were performed in 12
subjects, including knee arthroscopy (n=4) and ankle arthroscopy
(n=1), knee replacements (n=4), and others (n=5). Hemostasis was
rated as excellent (13/14) or good (1/14) by the
investigator/surgeon. The median estimated bleed loss was 65.5 mL
(range: 0.0-300.0 mL) during surgery and 0.0 mL (range: 0.0-500 mL)
postoperatively. No blood transfusions were required during
surgery, but two subjects received transfusions in the
postoperative period. A single injection of rFIXFc was required in
85.7% of surgeries to maintain hemostasis during surgery, at a
median dose of 90.9 IU/kg per injection. On the pre-operative day,
1-2 injections were administered and 2-3 injections during the
3-day post-operative period. On the day of surgery, post-operative
days 1-3, and post-operative days 4-14, the median rFIXFc
consumption (summarized over all injections during referenced time
period) was 146.1 IU/kg, 168.2 IU/kg, and 277.1 IU/kg,
respectively. Overall, .gtoreq.1 adverse event (AE) was reported
for 10 (83.3%) of the 12 subjects and 3 subjects reported 6 serious
AEs, all of which were resolved and judged as unrelated to rFIX
treatment. Treating physicians rated the hemostatic efficacy of
rFIXFc as excellent or good in 100% of surgeries.
[0292] One injection was sufficient to resolve 90.4% of bleeding
episodes in Arm 1, with a median dose per injection of 46 IU/kg.
Overall, 82.0% of rFIXFc injections were rated by subjects as
producing an excellent or good response. The physicians' global
assessment of subject response to rFIXFc was rated as excellent or
effective for 98.8% of the subject visits.
[0293] The weekly rFIXFc dose for subjects in Arm 1 decreased from
the initial 50 IU/kg to a median of 45 IU/kg when averaged over the
course of the study. The initial dosing interval of 10 days for
subjects in Arm 2 increased over the course of the study to 12.5
days, with 12 subjects (46%) achieving a dose interval of
.gtoreq.14 days for the last 6 months, and 14 subjects (53.8%) for
the last 3 months on the study.
[0294] Hemostasis in Arm 4 was rated as excellent or good by
investigators or surgeons for 100% of 14 major surgeries performed
in 12 subjects. Types of major surgeries included treatment of a
dental abscess and pilonidal cyst to knee arthroplasty or knee
replacement (n=6) and amputation of a finger (n=2). A single
injection of rFIXFc was sufficient to maintain hemostasis during
85.7% of major surgeries and the median average dose per injection
for the 14 surgeries was 91 IU/kg.
[0295] Hemostatic response to dosing during surgery and
post-operatively is summarized in Table 11.
TABLE-US-00012 TABLE 11 Summary of Hemostatic Response During
Surgery and Post-Operatively Number of Procedures Response Major
(Number of Excel- Poor/ Surgery Subjects) lent Good Fair None Total
Knee Replacement 5 (5) 4 1 Arthroscopic Procedure 1 (1) 1
Arthroscopic Ankle Fusion 1 (1) 1 Closure of Rectal Fistula 1 (1) 1
External Fixation of Knee 1 (1) 1 Tendon Transfer 1 (1) 1 I &
D.sup.1 of Dental Abscess 1 (1) with Extractions I & D.sup.1
Pilonidal Cyst 1 (1) 1 Debridement, Partial Amputation 1 (1) 1
Amputation of Finger 1 (1) 1 Minor surgery.sup.2 15 (13) 10 1 1
.sup.1Incision and Drainage .sup.2Assessment of response not
provided for 3 minor surgeries
[0296] PK
[0297] For FIX activity pharmacokinetic analysis, the baseline
(endogenous FIX activity) was pre-defined as the lowest observed
FIX activity at either screening, pre-dose, post-dose, or from the
patient's historical clinical records. For patients whose lowest
observed FIX activity was below 1%, the baseline FIX activity was
set as zero; for patients whose lowest observed FIX activity was
between 1 and 2 IU/dl, the baseline FIX activity was set at the
actual observed FIX value. The residual drug was decayed following
first order decay with the decay rates determined on an individual
basis. The FIX activity over time profiles, corrected by baseline
and residual drug, were analyzed using a user-defined and verified
two-compartmental model. The user-defined code automated the
calculation of additional secondary pharmacokinetic parameters
(e.g., Time 1% and 3%), which are not included in the secondary
parameter list of the WinNonlin library mode (PHOENIX.RTM.
WinNonlin 6.2.1.51; Pharsight), thus eliminating the need for
manual handling of data outside of the primary analysis and
minimizing the risk of introducing human error.
[0298] The geometric mean terminal half-life of rFIXFc was
approximately 82 hours, which is 2.4-fold (i.e., 2.43 fold) longer
than that of BENEFIX.RTM. (approximately 34 hours) in the 96-=hour
sample (p<0.001). With the 48-hour sampling, a 4.83-fold
increase in half-life was observed. rFIXFc had a 2.39-fold longer
mean residence time (MRT) compared with rFIX, resulting in
2.21-fold and 2.04-fold extensions of time to 1 IU/dL and 3 IU/dL
above baseline, respectively (P<0.001). Comparative data for
rFIXFc at baseline and Week 26 was available for 21 subjects and
showed no statistical difference for any PK parameters. Table 12
shows comparative PK data for rFIXFc versus rFIX from 22 subjects
in the Arm 1 sequential PK subgroup, using compartmental models for
the one-stage clotting assay.
[0299] As shown in Table 12, incremental recovery was comparable
between rFIX and rFIXFc (P=0.713). There was a 2.43-fold increase
in the terminal FIX half-life following rFIXFc compared with rFIX
when a 96-hour sampling schedule was used (82.1 vs 33.8,
respectively; P<0.001); see Table 12 and FIG. 4). Using the
traditional 48-hour sampling schedule, which has been used to show
the generally accepted half-life of rFIX (.about.18 hours), rFIXFc
showed a 4.84-fold increased half-life compared to rFIX. Compared
with rFIX, rFIXFc had a 2.39-fold longer mean residence time (MRT:
41.2 vs 98.6 hours; P<0.001), resulting in 2.21-fold extension
of time to 1 IU/dL above baseline (5.1 vs 11.2 days; P<0.001).
rFIXFc time to 1 IU/dL (1%) FIX and time to 3 IU/dL (3%) IU/dL were
11.2 and 5.8 days, respectively (see Table 12 and FIG. 4).
Intra-individual rFIXFc PK parameters were comparable between
baseline and Week 26, indicating that the PK profile of rFIXFc was
stable following repeated dosing over 26 weeks.
TABLE-US-00013 TABLE 12A Sequential PK parameters in Arm 1 subgroup
(N = 22) of rFIXFc compared with rFIX. Geometric mean for Geometric
mean for Geometric mean ratio PK Parameter* rFIXFc PK (95% CI) rFIX
PK (95% CI) (95% CI), P-value Terminal t.sub.1/2 82.1 (71.4, 94.5)
33.8 (29.1, 39.2) 2.43 (2.02, 2.92) (h) P < 0.001 Terminal
t.sub.1/2 NA 17.0 (15.9, 18.3) -- (h) 48-hour PK C.sub.max 40.8
(33.6, 49.6) 43.1 (36.7, 50.6) 0.95 (0.81, 1.11) (IU/dL) P = 0.491
CL 3.19 (2.84, 3.59) 6.34 (5.64, 7.13) 0.50 (0.46, 0.55) (mL/h/kg)
P < 0.001 V.sub.ss 314.8 (277.8, 356.8) 261.1 (222.9, 305.9)
1.21 (1.06, 1.38) (mL/kg) P = 0.008 AUC/dose (IU*h/dL 31.3 (27.9,
35.2) 15.8 (14.0, 17.7) 1.99 (1.82, 2.17) per IU/kg) P < 0.001
MRT (h) 98.6 (88.2, 110.3) 41.2 (36.0, 47.2) 2.39 (2.12, 2.71) P
< 0.001 Incremental recovery 0.9 (0.8, 1.1) 0.9 (0.8, 1.1) 0.97
(0.84, 1.12) (IU/dL per IU/kg) P = 0.713 Time to 1 IU/dL above 11.2
(10.2, 12.4) 5.1 (4.6, 5.7) 2.21 (2.04, 2.39) baseline** (days) P
< 0.001 Time to 3 IU/dL above 5.8 (5.1, 6.6) 2.8 (2.6, 3.1) 2.04
(1.87, 2.21) baseline** (days) P < 0.001 *User-defined
2-compartmental model one-stage clotting assay **Following 50-IU/kg
dose t.sub.1/2, half-life; C.sub.max, maximal concentration; CL,
clearance; Vss, volume of distribution at steady state; MRT, mean
residence time; CI, confidence interval; AUC, area under the
concentration curve
[0300] All subjects had an initial PK evaluation to characterize
the PK of rFIXFc in a representative population of subjects with
Hemophilia B.
[0301] More extensive PK sampling was conducted in a subset of
subjects in the weekly prophylaxis arm (Arm 1) at baseline after a
single dose of BENEFIX.RTM. 50 IU/kg followed by a single dose of
rFIXFc 50 IU/kg. Blood samples were taken for BENEFIX.RTM. over a
period of 96 hours. Blood samples were then taken for rFIXFc over a
period of 240 hours. PK assessment of rFIXFc was repeated at 26
weeks.
[0302] The 100-IU/kg dose was selected based on PK results from the
Phase 1/2 study, which showed this dose elevated FIX levels to
approximately 100% of normal (Shapiro et al 2011). In the Phase 1/2
study, with rFIXFc 100 IU/kg (n=5), the time to FIX levels 1% above
baseline was approximately 11 days, and ranged from 9 to 14 days.
Based on these data, Arm 2 was designed to test whether a fixed
dose of 100 IU/kg could provide protection from bleeding beyond one
week.
[0303] rFIXFc resulted in low median ABRs of 2.95 in the weekly
prophylaxis arm, 1.38 in the individualized interval prophylaxis
arm. In contrast, the episodic treatment arm had an ABR of
17.69.
[0304] In the individualized interval prophylaxis arm, the median
dosing interval was 14 days during the last 6 months on study.
[0305] The terminal half-life (activity) measured by one stage
clotting assay was approximately 82 hours (82.1 hours) for rFIXFc
and approximately 34 hours (33.8 hours) for BENEFIX.RTM..
[0306] Overall, greater than 90% of bleeding episodes were
controlled by a single injection. Hemostatic efficacy of rFIXFc for
perioperative management was rated by treating physicians as
excellent or good in 100% of surgeries (14 major surgeries
performed on 12 subjects).
[0307] The median ABRs for the rFIXFc weekly prophylaxis and
individualized interval arms were 2.95 and 1.38, respectively.
[0308] Greater than 90% of bleeding episodes were resolved with one
injection of rFIXFc.
[0309] The terminal half-life of BENEFIX.RTM. of approximately 34
hours determined in B-LONG is longer than that reported in the
BENEFIX.RTM. package insert (.about.18 hours) as well as a number
of studies (13.7 to 19.3 hours) (Ewenstein 2002; Kisker et al.
2003; Negrier et al. 2011) that followed EMA guidelines on FIX PK
assessment using a 48-hour sampling duration. However, in published
PK studies in which BENEFIX.RTM. was sampled up to 72 hours post
dosing, a longer terminal half-life was also reported to be 21.3 to
33.4 hours (Ragni et al. 2002, Lambert et al. 2007, Chang et al.
2007, and Martinowitz et al. 2012).
[0310] To determine whether the discrepancy in terminal half-life
of BENEFIX.RTM. resulted from the longer PK sampling schedule of 96
hours adopted in this study, BENEFIX.RTM. PK data were also
analyzed using data only up to 48 hours post dose. This analysis
yielded a significantly shortened terminal half-life of
BENEFIX.RTM. (.about.17 hours) that is consistent with previous
reports using 48-hour sampling duration. A summary of the
comparative data for BENEFIX.RTM. is presented in Table 12B.
TABLE-US-00014 TABLE 12B Estimation of Terminal Half-life for rFIX
(BENEFIX .RTM.) as Determined from 96-Hour vs 48-Hour Sampling
Duration. Arithmetic Geometric Estimates (hours) Estimates (hours)
Mean SD Mean 95% CI BeneFIX package 18.1 5.1 -- -- insert.sup.2
Study BeneFIX 17.2 2.7 17.0 (15.9, 18.3) estimate (48 hours).sup.a
Study BeneFIX 35.7 13.6 33.8 (29.1, 39.2) estimate (96 hours).sup.a
.sup.aTwo-compartmental analysis. CI denotes confidence interval,
SD standard deviation.
[0311] In the B-LONG study, a head-to head comparison was made
between rFIXFc and BENEFIX.RTM., whereas in the Phase 1/2 study,
the half-life of rFIXFc was compared to the historical data
reported in the BENEFIX.RTM. Product Insert (2009). The measure of
PK improvement of rFIXFC over BENEFIX.RTM. from the B-LONG study
was more reliable and accurate.
Safety
[0312] rFIXFc was well tolerated and had significantly improved PK
relative to the current standard of care, rFIX. One serious AE
(obstructive uropathy) was assessed as possibly related to
treatment; the event resolved and the subject continued on study.
Good control of bleeds and low ABRs seen with weekly to bi-weekly
(every 2 weeks) dosing suggest that this therapy may substantially
improve management of acute bleeds and markedly lengthen
prophylactic regimens in hemophilia B, thereby potentially
improving subject adherence and outcomes.
[0313] For detection of inhibitors (neutralizing antibodies), the
Nijmegen-modified Bethesda assay to detect neutralizing antibodies
was performed at screening, baseline, and each visit during study
treatment to monitor for the development of an inhibitor. Following
the first dose with rFIXFc, inhibitor testing in arms 1 and 2 was
conducted at trough at each scheduled clinic visit, with trough
defined as a point after the longest interval between scheduled
doses. Inhibitor testing in arm 3 was performed following a washout
of at least 72 hours (3 days). For new patients entering arm 4,
inhibitor testing was performed at screening and after a total of
at least 4 EDs were achieved with rFIXFc, within 4 weeks prior to
scheduled surgery. Formation of an inhibitor was defined as a
neutralizing antibody value .gtoreq.0.6 Bethesda units (BU)/ml,
confirmed in a second, separately drawn sample within 2-4 weeks.
The confidence interval on the inhibitor incidence rate was
evaluated using the Clopper-Pearson exact method for a binomial
proportion. The acceptable inhibitor risk in clinical trials of
previously treated FIX patients allows 1 of 50 patients to
experience an inhibitor while on the study, with each patient
requiring at least two valid tests for inhibitors over 50 to 75
EDs.
[0314] For detection of non-neutralizing antibodies (rFIXFc binding
antibodies), monitoring for non-neutralizing antibodies (NNAs) that
bind to rFIXFc was performed at the same time points as testing for
inhibitors. A bridging assay format was employed to detect all
possible classes of antibodies, with electrochemiluminescent
readout on an MSD instrument. Samples were positive if the signal
was above a statistically derived cut point and confirmed by
inhibition with excess rFIXFc product. Positive samples were
further characterized for binding to rFIX or Fc. Testing of
inhibitor-positive control samples showed that this assay was
approximately 100-fold more sensitive than the Nijmegen-modified
Bethesda assay.
[0315] The study was designed to include .gtoreq.50 subjects with
.gtoreq.50 EDs to detect the risk of inhibitors. The US Food and
Drug Administration guidance for adequate demonstration of
acceptable inhibitor risk in clinical trials of previously treated
FIX subjects allows 1 out of 50 subjects to experience an
inhibitor, with each subject requiring .gtoreq.50 EDs to the study
treatment. Under the assumption that the occurrence of inhibitors
in a clinical study can be adequately modeled using the binomial
distribution, .gtoreq.50 EDs would allow for a 2-sided, 95%
confidence interval for the true inhibitor incidence of
(0.05%-10.65%) using the exact Clopper-Pearson method if 1 case of
inhibitor formation was observed. There were no reported deaths,
allergic reactions, thrombotic events, or inhibitor development in
any study subjects, including 55 subjects with .gtoreq.50 EDs and
94 patients with .gtoreq.25 EDs. No inhibitors (neutralizing
antibodies) were detected in any subject in the study. Low positive
results for non-neutralizing antibodies (NNA) were found in 3
subjects at baseline, which did not impact the PK of rFIXFc and all
3 reverted to NNA negative status during the study. One subject had
borderline negative results during the study, and a borderline
positive result at the end of the study. rFIXFc clearance was not
impacted and the NNA were not associated with any increased
bleeding frequency and/or higher consumption of rFIXFc. The
incidence of AEs was similar across all treatment arms, with 45
subjects (71.4%) in Arm 1, 23 subjects (79.3%) in Arm 2, and 20
subjects (74.1%) in Arm 3 reporting .gtoreq.1 AE. The majority of
the AEs were judged by the Investigator as unrelated or unlikely
related to treatment. AEs judged as possibly related or related to
treatment were reported in 10 (8.4%) of the 119 subjects in Arms 1,
2, and 3 combined. No discernable trends were detected for
coagulation activation markers (F1+2, D-dimer,
thrombin-antithrombin complex) in the sequential PK subgroup, nor
for total IgG and subclass levels in any of the Arms.
[0316] Long-lasting rFIXFc was efficacious in maintaining
perioperative hemostasis in subjects with hemophilia B. The high
rating of perioperative hemostasis by surgeons and investigators
for all major surgeries conducted suggests that blood loss was
comparable to that expected for a subject without hemophilia.
[0317] The results disclosed herein show that rFIXFc provided
effective control of acute bleeding episodes and demonstrated
efficacious prophylaxis, for both weekly and individualized
interval prophylaxis of longer interval duration (e.g., every 1-2
weeks) and/or higher trough levels. Notably, approximately 50% of
subjects in the individualized prophylaxis cohort (Arm 2) showed
dosing intervals of .gtoreq.2 weeks. Compared with rFIX, rFIXFc
showed a substantially improved PK profile, which allowed longer
dosing intervals for prophylactic regimens that can be less
burdensome to subjects. The PK profile of rFIXFc was stable over
time. These results show that rFIXFc is a first in its class
long-lasting recombinant factor IX therapy, which may offer a
treatment shift toward less frequent injections and still provide
prolonged protection from bleeding, thereby promoting increased
adherence to prophylaxis and improved outcomes in hemophilia B
subjects.
[0318] To ensure consistency of laboratory analyses, one central
laboratory was used for each type of assay. Analyses of clinical
safety samples and central sample management were performed at
LabCorp (Cranford, N.J., USA). FIX activity and inhibitor assays
were conducted at LabCorp (Englewood, Colo., USA). Genotyping of
samples was done at Hemostasis Lab, Puget Sound Blood Center
(Seattle, Wash., USA). ICON Development Solutions (Whitesboro,
N.Y., USA) analyzed FIX antigen and non-neutralizing antibody (NNA)
samples, and Chimera Biotech GmbH (Dortmund, Germany) analyzed
rFIXFc concentration samples.
[0319] The study could conclude early when all of the following
predefined criteria were met: [0320] 1) 13 patients in the
Sequential Pharmacokinetics subgroup in arm 1 completed
pharmacokinetic sampling to adequately estimate the terminal
half-life for BeneFIX at baseline and for rFIXFc at baseline and
week 26. [0321] 2) 53 patients from any treatment arm completed at
least 50 EDs with rFIXFc. 3) 20 patients from arm 2 and 16 patients
from arm 3 completed at least 26 (.+-.1) weeks on study. [0322] 4)
73 patients from any treatment arm completed at least 50 EDs with
rFIXFc and underwent inhibitor testing; or 53 patients completed at
least 50 EDs and underwent inhibitor testing with no more than 1
patient with a positive inhibitor as confirmed by retesting; or 34
patients completed at least 50 EDs and underwent inhibitor testing,
with no patients with a positive inhibitor as confirmed by
retesting. [0323] 5) Approximately 10 major surgeries were
conducted in at least 5 patients and postoperative follow-up was
completed.
[0324] When all of these criteria had been met, all ongoing
patients were asked to return to the clinic for end-of-study
assessments.
Impact on Quality of Life
[0325] Quality of Life was measured using the HAEM-A-QoL, a quality
of life instrument specific to hemophilia. HAEM-A-QoL was performed
in adults (aged 18 and older) in the prophylactic treatment arms.
Change from baseline at Week 26 in the combined prophylaxis arms by
pre-study regimen are summarized in Table 13.
TABLE-US-00015 TABLE 13 Median Change from Baseline for the
Haem-A-QoL Questionnaire (Fixed Weekly Interval and Individualized
Interval Arms Pooled) Pre-Study Regimen Prophylaxis Episodic
(On-demand) N Change from baseline N Change from baseline Total
score 27 -6.82 (-22.8, 6.1) 26 -6.25 (-25.5, 12.8) Domains, during
the past month 1. Physical health 27 -10.00 (-45.0, 20.0) 31 -15.00
(-60.0, 15.0) 2. Feeling 27 0.00 (-43.8, 50.0) 31 0.00 (-43.8,
62.5) 3. View of yourself 27 -5.00 (-25.0, 15.0) 30 -5.00 (-35.0,
25.0) 4. Sports and leisure 22 -7.50 (-70.0, 25.0) 21 -20.00
(-40.0, 35.0) 5. Work and school 22 0.00 (-31.3, 52.1) 25 -6.25
(-31.3, 18.8) 6. Dealing with hemophilia 27 0.00 (-100.0, 100.0) 31
-8.33 (-66.7, 75.0) 7. Treatment 27 -6.25 (-18.8, 18.8) 31 0.00
(-53.1, 37.5) Domains, recently 8. Future 26 -5.00 (-25.0, 10.0) 30
0.00 (-30.0, 20.0) 9. Family planning 15 0.00 (-29.2, 12.5) 13 0.00
(-43.8, 25.0) 10. Partnership and sexuality 26 0.00 (-50.0, 66.7)
30 0.00 (-25.0, 25.0) NOTE: Summary statistics are median (minimum,
maximum).
[0326] The median dosing interval in the individualized interval
prophylaxis arm was 14 days during the last 6 months on study.
[0327] Control of bleeding: Over 90% (90.4%) of bleeding episodes
were controlled by a single injection of rFIXFc.
[0328] Perioperative management: Treating physicians rated the
hemostatic efficacy of rFIXFc as excellent or good in 100% of
surgeries.
[0329] Adverse drug reactions (ADRs) were reported in 10 of 119
(8.4%) subjects treated with routine prophylaxis or episodic
(on-demand) therapy. Adverse drug reactions are considered adverse
events assessed by the investigator as related or possibly related
to treatment with rFIXFc. Adverse drug reactions are summarized in
Tables 14A and 14B.
[0330] The incidence of adverse events was similar across all
treatment arms, with at least one adverse event reported in 45
patients (71.4%) in arm 1, 23 patients (79.3%) in arm 2, and 20
patients (74.1%) in arm 3. The majority were judged by
investigators as unrelated or unlikely related to treatment.
Adverse events judged as related or possibly related to treatment
were reported in 10 (8.4%) of the 119 patients in arms 1, 2, and 3
combined. One serious adverse event judged as possibly related to
treatment by the investigator was a case of an obstructive clot in
the urinary collecting system that resolved with hydration and the
patient continued the study treatment. There were no reports of
hypersensitivity, anaphylaxis, or thrombotic events, and there were
no deaths in the study. There were no clinically meaningful changes
in coagulation activation markers (F1+2, D-dimer,
thrombin-antithrombin complex) or for total IgG and subclass
levels.
[0331] No subject was withdrawn from study due to an adverse drug
reaction. In the study, no inhibitors were detected and no events
of anaphylaxis were reported.
[0332] rFIXFc was well-tolerated, with no evidence of
neoantigenicity, and adverse events were consistent with those
expected in a hemophilia population. Notably, the high rate of
compliance in this study (96.6%) and corresponding high study
completion rate (93.5%) further support the tolerability of rFIXFc.
The risk of inhibitors has been reported to be up to 3% in the
naive hemophilia B population. See Tandra A, and Shapiro A D (2010)
in Lee C A, Berntorp E, Hoots W K, eds. Textbook of hemophilia, 2nd
ed. Hoboken, N.J.: Wiley-Blackwell; and DiMichele D. (2007) Br J
Haematol 138:305-15. Patients generally develop inhibitors within
the first 50 EDs to exogenous FIX replacement therapy (see
Dimichele D. (2002) Haemophilia 8:280-287), which supported the end
of study criteria. B-LONG included a total of 5144 EDs (117.1
patient-years of exposure), with 55 patients having .gtoreq.50 EDs,
and no inhibitors were detected in any patients. In this study,
low-titer NNAs were observed in a small proportion of patients (3%)
at screening and baseline, and became negative in three of four
cases during rFIXFc treatment. The low-titer NNAs observed in this
study had no discernible effect on rFIXFc pharmacokinetics or
bleeding rates.
TABLE-US-00016 TABLE 14A Adverse Drug Reactions reported for rFIXFc
N = 119* MedDRA Number of MedDRA System Preferred Subjects Organ
Class Term n (%) Nervous system disorders Headache 2 (1.7)
Dizziness 1 (0.8) Dysgeusia 1 (0.8) Gastrointestinal disorders
Paresthesia oral 2 (1.7) Breath odor 1 (0.8) General disorders and
Fatigue 1 (0.8) administration site conditions Infusion site pain 1
(0.8) Cardiac disorders Palpitations 1 (0.8) Renal and urinary
disorders Obstructive uropathy 1 (0.8) Vascular disorders
Hypotension 1 (0.8) *119 previously treated subjects (PTPs) on
routine prophylaxis or episodic (on-demand) therapy The incidence
of the adverse reactions below is expressed according to the
following categories: Very common (.gtoreq.1/10) Common
(.gtoreq.1/100 to <1/10) Uncommon (.gtoreq.1/1,000 to <1/100)
Rare (.gtoreq.1/10,000 to <1/1,000) Very rare (<1/10,000)
TABLE-US-00017 TABLE 14B Further Summary of Adverse Events. Arm 1:
Arm 2: Weekly Individualized Prophylaxis Interval Arm 3: (Fixed
Prophylaxis Episodic Interval) (Fixed Dose) Treatment AE (N = 63)
(N = 29) (N = 27) Total AEs, n 158 76 52 Patients with .gtoreq.1 AE
45 (71.4) 23 (79.3) 20 (74.1) Most common AEs (.gtoreq.5%), n (%)
Nasopharyngitis 13 (20.6) 4 (13.8) 1 (3.7) Influenza 5 (7.9) 0 4
(14.8) Ar hralgia 6 (9.5) 2 (6.9) 0 Upper respira ory 4 (6.3) 2
(6.9) 1 (3.7) tract infection Headache 2 (3.2) 2 (6.9) 2 (7.4)
Hypertension 3 (4.8) 2 (6.9) 1 (3.7) Dizziness 3 (4.8) 2 (6.9) 0
Sinusitis 3 (4.8) 2 (6.9) 0 Musculoskeletal pain 2 (3.2) 2 (6.9) 0
AEs judged as related or 5 (7.9) 4 (13.8) 1 (3.7) possibly related
to treatment, n (%) Breath odor 1 (1.6) Paresthesia oral 1 (1.6) 1
(3.4) Fatigue 1 (1.6) Headache 1 (1.6) Hypotension 1 (1.6)
Palpitations 1 (3.4) Dizziness 1 (3.4) Dysgeusia 1 (3.4)
Obstructive uropathy* 1 (3.4) Injection site pain 1 (3.7) *This
event was the only serious adverse event (AE) related or possibly
related to recombinant factor IX Fc fusion protein (rFIXFc)
treatment. This patient had a history of painful hematuria.
Approximately 4 months after his first dose of rFIXFc, he developed
urinary obstruction/pain and passed clots with relief. Three days
later, symptoms recurred and resolved with hydration. The patient
continued on rFIXFc during this time and continued on study.
Example 5. The Clinical Implications of Population Pharmacokinetics
of rFIXFc in Routine Prophylaxis, Control of Bleeding and
Perioperative Management for Hemophilia B Subjects
[0333] BACKGROUND: Clinical dosing of factor IX (FIX) in treatment
of hemophilia B is well established based on empirical practice and
clinical outcomes. Since pharmacokinetics (PK) of FIX activity is
the surrogate efficacy marker, we utilized population PK (popPK)
modeling and simulation to evaluate dosing regimens of long-acting
recombinant FIX Fc fusion protein (rFIXFc). The PK of rFIXFc, from
135 single-dose and 21 repeat-dose profiles in subjects .gtoreq.12
years old (body weight (BW): 45-186.7 kg), was best described by a
3-compartmental model, which showed modest inter-individual
variability (IIV) of 17.7% for clearance (CL) and 21.7% for volume
of central compartment (V1). The proportional residue error of
10.6% approximates the variability of the one-stage clotting assay
for FIX activity. The only covariate that showed a weak association
with rFIXFc PK is BW, which accounted for .about.3% of IIV for CL
and V1, suggesting that BW-independent flat dosing of rFIXFc may be
feasible for treating adult hemophilia B subjects.
[0334] AIMS: To simulate the BW-based and flat dosing regimens for
routine prophylaxis, control of bleeding and perioperative
management in the hemophilia B population.
[0335] METHOD: The validated 3-compartmental popPK model, including
inter-occasion variability and BW as the covariate on CL and V1,
was used for dosing simulations. For BW-based dosing regimen, PK
profiles were simulated for 1000 subjects with BW distribution
representative of the phase 3 study. BW distribution was simulated
using a power function Z=BW-0.5. The generated BW (1000 values)
distribution has a median of 74.9 kg and a range of 38.9 to 172.6
kg, which is similar to our studies (median, 73.3 kg; min and max,
45 and 186.7 kg). For fixed dosing regimen, three populations
(n=1000 each) were stratified based on low (.ltoreq.10th
percentile), typical (10th-90th percentile) and high (.gtoreq.90th
percentile) BW. Variability of exposure parameters, percentage of
population maintaining target Cmax and trough, and deviations of
median exposure parameters in extreme BW groups were compared with
BW-based and flat dosing regimens. To simulate steady-state in
prophylaxis regimen, six doses were applied for all dosing regimens
(once weekly, every 10 days, or every 14 days), with each dosing
interval assigned as one occasion. To simulate the PK profile
following an episodic treatment, a single dose was applied.
[0336] RESULT: Consistent with the observations from the phase 3
study, popPK simulation of 50 IU/kg once weekly or 100 IU/kg every
10-14 days predicted peak FIX activity within the physiologic range
(Cmax<150%) and trough .gtoreq.1% in majority of the population.
All simulated regimens predicted that the majority of the
population will maintain trough activity at or above 1% (Table
15).
TABLE-US-00018 TABLE 15 Predicted percentage of population with
steady-state trough at or above 1% for various prophylaxis dosing
regimens Total weekly % of population with Regimen dose trough
.gtoreq.1% 50 IU/kg weekly 50 95.4 100 IU/kg weekly 100 99.6 100
IU/kg every 10 days 70 89.2 100 IU/kg every 14 days 50 52.8
[0337] A plot showing a population simulation of steady-state FIX
activity vs. time is shown in FIGS. 13A-13C. Table 16 shows the
predicted steady state FIX activity over the course of 14 days for
two dosing regimens: 50 IU/kg weekly and 100 IU/kg every 14
days.
TABLE-US-00019 TABLE 16 Predicted steady state FIX activity EOI Day
1 Day 3 Day 5 Day 7 Day 10 Day 14 median median median median
median median median Dose, [5th, [5th, [5th, [5th, [5th, [5th,
[5th, IU/kg 95th] 95th] 95th] 95th] 95th] 95th] 95th] 50 IU/kg 52.6
16.9 7.17 4.16 2.67 NA NA weekly [32.1, [11.2, [3.85, [1.93, [1.02,
89.3] 26.1] 12.3] 7.83] 5.49] 100 102 30.0 12.0 6.78 4.28 2.29 1.07
100 IU/kg [60.0, [19.6, [6.62, [3.24, [1.82, [0.688, [0.0758, every
166] 46.7] 19.9] 12.2] 8.06] 5.33] 3.23] 14 days
[0338] Furthermore, BW-based and flat dosing resulted in comparable
PK profiles with comparable exposure parameters, e.g., 50 IU/kg and
4000 IU once weekly predicted a median (5th, 95th percentile) Cmax
of 52.6 (32.1, 89.3) IU/dL and 56.1 (36.2, 90.9) IU/dL,
respectively. Both dosing regimens predicted that >95% of the
population maintains Cmax<150% and trough .gtoreq.1% (FIG. 14).
However, BW-based and flat dosing showed differential effects on
the exposure parameters in extreme 00th or 90th percentile) BW
populations. This suggests that BW-independent flat dosing may be
feasible for patients 12 years and older.
[0339] The popPK model was used to simulate dosing regimens for
episodic treatment. The model predicts that for the control of
bleeding episodes, a single dose of 50 or 100 IU/kg of rFIXFc is
sufficient to maintain the plasma FIX peak activity levels at 40 to
80 IU/dL (Table 17) as recommended by the World Federation of
Hemophilia (WFH) guidelines.
TABLE-US-00020 TABLE 17 Predicted FIX activity profile after a
single dose of rFIXFc in the 5th to 95th percentile of the
population rFIXFc dose, median [5th, 95th] 50 IU/kg 100 IU/kg End
of infusion 50.8 [30.4, 84.5] 102 [60.8, 169] 12 hours 21.1 [13.5,
33.6] 42.3 [26.8, 67.3] 24 hours (day 1) 14.8 [9.78, 22.7] 29.5
[19.6, 45.5] 36 hours 10.9 [6.79, 17.1] 21.8 [13.7, 34.1] 48 hours
(day 2) 8.51 [5.14, 13.2] 17.0 [10.5, 26.6] 72 hours (day 3) 5.57
[3.05, 9.27] 11.1 [6.22, 18.5] Day 5 3.07 [1.44, 5.62] 6.14 [3.05,
11.0] Day 7 1.93 [0.795, 3.71] 3.88 [1.82, 7.28] Day 10 1.1 [0.277,
2.33] 2.19 [0.775, 4.56] Day 14 0.559 [0, 1.38] 1.08 [0.125,
2.58]
[0340] Analysis of 12 major surgeries and 2 minor surgeries found
that the FIX activities measured during the perioperative period
were largely consistent with the prediction by popPK based on
subjects' pre-surgery baseline PK, indicating no substantial factor
consumption in these surgeries. A representative plot of observed
and predicted perioperative FIX activity is shown in FIG. 12.
Simulated and observed FIX activities were compared within the
first 21 days after the first rFIXFc surgical dose (n=14; 12 major
surgeries, 2 minor surgeries). There was good correlation between
the observed FIX activity data and that predicted by the PK model
(relative prediction error [95% CI], 0.332% [-2.08%, 1.42%]).
[0341] CONCLUSION: PopPK provides a robust and effective means to
evaluate potential dosing regimens. The predictions by popPK
simulation for rFIXFc corroborate the results from the phase 3
study. The simulations of BW-based and flat dosing of rFIXFc
achieved similar PK profiles. Considering the wide therapeutic
range for factor replacement therapy, flat dosing of rFIXFc and
rFIX products may be a potentially viable approach in adult
hemophilia B subjects that warrants further clinical investigation.
Furthermore, using a population PK model, it is feasible to develop
a general dosing guidance to achieve target FIX levels recommended
for perioperative management in patients with haemophilia B.
Example 6. Population Pharmacokinetic Analysis of a Long-Acting
Recombinant Factor IX-Fc Fusion Protein (rFIXFc) in Subjects with
Severe Hemophilia B
[0342] BACKGROUND: Population pharmacokinetic (PK) models are
developed to understand the sources of variability in dose
requirements (covariates) and to help individualize dosing regimens
if necessary. Dosing histories and subject-specific data are used
to gain an understanding of drug disposition in order to discern
specific demographic and/or clinical factors that may be predictors
of PK parameters. By characterizing the population PK (popPK) of
long-acting FIX-Fc (rFIXFc) in subjects with severe hemophilia B
(.ltoreq.2 IU/dL plasma FIX activity), a model of estimated
population PK parameters of rFIXFc can be established. This model
may assist physicians who wish to tailor dosing for individual
subjects with sparse PK samples.
[0343] METHODS: Male subjects with severe hemophilia B were
included from a phase 1/2a study (n=12) and the phase 3 study
(B-LONG, n=123) described above. The subjects ranged in age from 12
to 76 years and in body weight from 45 to 186 kg. The modeling
dataset included 135 baseline PK profiles at Week 1, as well as 21
repeat PK profiles at Week 26, with a total of 1400 measured FIX
activity records. The final population PK model was validated using
1027 trough/peak FIX activity records from 119 subjects.
[0344] In the popPK analysis, plasma FIX activity was measured by
the one-stage (activated partial thromboplastin time) clotting
assay. Corrected FIX activity was calculated using the formula:
Corrected FIX activity=Measured FIX activity-Baseline-Residual
decay.
[0345] Baseline FIX activity was defined as the lowest level of
activity (LLACT) recorded at screening, predose, postdose, or from
historical clinical records. When the baseline is equal to 0, the
LLACT is less than 1% (lower limit of quantification). When the
baseline FIX activity is equal to LLACT, LLACT is greater than or
equal to 1% and less than or equal to 2%.
[0346] Prestudy residual decay was performed using terminal
half-life obtained from a noncompartmental analysis of the
individual data by the following formula:
Residual decay=(predose-baseline)xe.sup.-decay rate.times.time.
[0347] For the popPK model development, NONMEM VII version 1.0
(ICON Development Solutions, Ellicott City, Md.) was used. The
modeling and qualification steps are presented below in Table
20.
TABLE-US-00021 TABLE 20 Modeling and Qualification Steps Steps
Model selection Base model and Inter-individual Base Model, IIV
variability (IIV) evaluation on CL/V1/Q2/V2/Q3 Inter-occasion
variability Base Model with (IOV) evaluation IOV on CL and V1
Covariate Modelling Final model, body weight as covariate on CL and
V1 Internal qualification (bootstrap and VPC) External
qualification using trough/peak records CL, clearance; V, volume of
distribution; Q, inter-compartmental clearance; VPC, visual
predictive check
[0348] A first order conditional estimation with interaction method
(FOCEI) was used to estimate the popPK parameters. Residual errors
were modeled as combined proportional and additive errors. Stepwise
forward addition (p<0.005) and backward elimination (p<0.001)
covariate modeling was performed. Potential covariates assessed in
this analysis included: body weight (BW), Age, Race, Blood type,
Human Immunodeficiency Virus status, Hepatitis C Virus status,
haematocrit, IgG.sub.1 and albumin concentration, and FIX
genotype.
[0349] Model qualifications included bootstrap, visual predictive
check (VPC) and validation with trough/peak records. The mean
relative prediction error (an indicator of accuracy) was calculated
as:
1 N .times. i = 1 i = N .times. .times. [ DV - IPRED ] DV
##EQU00001##
[0350] RESULTS: The rFIXFc disposition was best described by a
three-compartment base model (FIG. 5). The model was further
improved by including intra-subject random changes at different
occasions (i.e., inter-occasion variability, IOV) for CL and V1
(FIG. 6). IOV was smaller than inter-individual variability (IIV),
indicating that individual PK was more accurate than the mean popPK
for individual PK prediction.
[0351] Body weight was found to be a significant covariate for
rFIXFc disposition (FIG. 7), although the impact of BW was limited.
For example, the BW exponent on CL and V1 was 0.436 and 0.396,
respectively, and inclusion of BW reduced inter-individual
variability (IIV) for both CL and V1 only by 3.4% and 2.5%,
respectively. None of the other covariates assessed, including age,
race, blood type or genotype, were significant covariates in this
model.
[0352] The final popPK model is summarized below in Table 21.
TABLE-US-00022 TABLE 21 Summary of the final rFIXFc population
pharmacokinetic model. Population 95% non-parametric CI IIV.sup.b
Parameter Estimate from bootstrap.sup.a (%) IOV (%) CL = Typical
.times. .times. CL .times. ( ? ? ) 0.436 ##EQU00002## Typical CL
for a 73 kg subject 2.39 2.29, 2.49 17.7 15.1 (dL/h) BW exponent on
CL 0.436 0.272, 0.584 V1 = Typical .times. .times. V .times.
.times. 1 .times. ( ? ? ) 0.396 ##EQU00003## Typical V1 for a 73 kg
subject 71.4 58.5, 76.0 21.7 17.4 (dL) BW exponent on V1 0.396
0.169,0.580 Q2 (dL/h) 1.67 1.35, 1.89 35.8 -- V2 (dL) 87.0 79.0,
95.5 46.2 -- Q3 (dL/h) 39.3 16.6, 141 -- -- V3 (dL) 39.9 36.6, 52.4
37.7 -- Residual Error: Proportional 10.6% Additive 0.24 IU/dL CI,
confidence interval; IIV, inter-individual variability; IOV, inter
occasion variability; CL, clearance; BW, body weight; V, volume of
distribution; Q, inter-compartmental clearance
[0353] For a typical 73 kg subject, the predicted popPK values for
clearance, volume of central compartment, and volume of
distribution at steady state are 2.39 dL/h, 71.4 dL, and 198 dL,
respectively. Goodness-of-fit plots show that the predicted popPK
data generated by the model closely mimic the observed FIX activity
data (FIGS. 8A-8D).
[0354] The results of the popPK model were validated using the
observed FIX activity data. The median and 80% interval for
observed and predicted FIX activity time profiles nearly
overlapped, indicating that the final model was able to reproduce
both the central tendency and variability of the observed FIX
activity data on the time scale (FIGS. 9A-9D). The strong
correlation between observed and predicted FIX activities in the
trough/peak dataset suggested that the final popPK model is
predictive (FIG. 10).
[0355] Finally, the overall relative prediction error was -3.23%
with a 95% confidence interval of -5.27% to -1.23%. Post hoc
estimates from this popPK analysis were very similar to the results
from the conventional PK analysis shown below in Table 22.
TABLE-US-00023 TABLE 22 Post hoc empirical Bayesian estimates of
key PK parameters. Parameter Phase 3 Mean (SD) Phase 1/2a Mean (SD)
Clearance (CL), mL/h/kg 3.42 (0.89) 2.82 (0.58) Volume of central
102 (29.6) 96.2 (24.7) compartment (V1), mL Incremental in vivo
1.02 (0.45) 1.04 (0.19) recovery, IU/dL per IU/kg Volume of
distribution 297 (90.5) 234 (70.8) at steady-state (Vss), mL/kg
Terminal Half-life, h 86.7 (27.9) 70.9 (13.9) Mean residence time
89.4 (25.9) 82.5 (15.5) (MRT), h SD, standard deviation
[0356] CONCLUSIONS: The three-compartment popPK model predicted
disposition of rFIXFc with modest inter-individual variability
(IIV). Individual PK parameters derived from the three-compartment
popPK model were similar to those derived from the two-compartment
conventional PK analysis, indicating a limited 3rd compartment
contribution. For a typical 73 kg subject, the popPK model
predicted a clearance of 2.39 dL/h; volume of central compartment
of 71.4 dL; and volume of distribution at steady state of 198 dL.
The only significant covariate assessed in the popPK model was BW,
although its impact on rFIXFc PK variability was limited.
[0357] The final popPK model can be used to simulate dosing
regimens and intervals for routine prophylaxis, control and
prevention of bleeding episodes, and peri-operative management.
This model may assist physicians who wish to tailor dosing for
individual subjects with sparse PK samples.
Example 7. Pediatric Study
Study Design
[0358] A phase 3, open-label, multicenter, paediatric study to
evaluate the safety, PK, and efficacy of rFIXFc for the control and
prevention of bleeding in previously treated children (<12 years
of age) with severe hemophilia B (endogenous FIX activity .ltoreq.2
IU/dL [2%]) is described.
[0359] The enrolment is approximately 26 children with hemophilia
B. Eligibility criteria for this pediatric previously treated
subject (PTP) study requires the children to have had .gtoreq.50
documented prior exposure days (EDs) to FIX products, weigh
.gtoreq.13 kg at the time of consent, and have no current or
history of inhibitors to FIX products. The treatment regimen is for
prophylaxis (see study design shown in FIG. 11). PK analysis of FIX
and rFIXFc will be performed in participants prior to initiation of
prophylactic treatment with rFIXFc.
[0360] Primary and secondary outcome measures will include
frequency of inhibitor development (for the primary outcome
measures) and number of annualized bleeding episodes and/or
response to treatment with rFIXFc for bleeding episodes (for the
secondary outcome measures).
[0361] The safety and efficacy of rFIXFc in previously untreated
subjects (PUPs), defined by the European Medicines Agency as
subjects with no prior exposure to any factor products will also be
assessed. The results of the paediatric PTP and PUP studies will
provide further insight into the clinical safety and hemostatic
parameters of rFIXFc for treating hemophilia in children.
[0362] Interim Analysis
[0363] Prophylaxis with factor IX (FIX) is the optimal treatment
for patients with hemophilia B; however, due to the short half-life
of currently available FIX products, frequent injections may be
required to prevent bleeding episodes. To prolong half-life and
reduce injection frequency, a long-lasting recombinant FIX Fc
fusion protein (rFIXFc) consisting of one rFIX molecule covalently
linked to the Fc domain of immunoglobulin G1 (IgG1) was developed.
In a phase 3 study in adults and adolescents, rFIXFc had a
2.43-fold increase in half-life and 50% reduction in clearance (CL)
compared with FIX (BeneFIX.RTM.) (J Thromb Haemost. 2013;
11[2]:241). The Kids B-LONG study (NCT01440946) was designed to
evaluate the pharmacokinetics (PK), safety, and efficacy of rFIXFc
prophylaxis in previously treated pediatric subjects with
hemophilia B. The objective of this planned interim analysis was to
determine the PK parameters of rFIXFc in subjects enrolled in Kids
B-LONG and compare these parameters to their pre-study FIX PK
parameters.
[0364] Methods: This multicenter, open-label, phase 3 study is
currently enrolling previously treated subjects aged <12 years
with severe hemophilia B (.ltoreq.2 IU/dL endogenous FIX), at least
50 exposure days (EDs) to FIX products, and no inhibitors to FIX.
Subjects were stratified into two age cohorts (<6 and 6 to
<12 years of age). A weekly prophylactic regimen of 50-60 IU/kg
of rFIXFc was administered to all subjects, with subsequent dose
and interval adjustments based upon PK data and bleeding frequency.
Subjects will continue treatment until they achieve 50 EDs. The
primary endpoint is the incidence of inhibitor formation. A
sequential PK analysis was performed to compare the PK parameters
of rFIXFc with that of pre-study FIX products. PK sampling of
pre-study FIX occurs at baseline prior to first dose of FIX (50
IU/kg) and at 5 additional time points through 48 hours. PK
sampling of rFIXFc occurs prior to first dose of 50 IU/kg rFIXFc
and at 7 additional time points through 168 hours following the
first dose; a washout period of 96 hours is required before the
first dose of both pre-study FIX and rFIXFc. Plasma FIX activity is
measured using the one-stage clotting assay calibrated against a
commercially available FIX plasma standard and the FIX
activity-over-time profiles are analyzed by non-compartmental
analysis (NCA) using the PK data analysis software PHOENIX.TM.
WinNonlin 6.2.1.51. A data cut-off date of 23 Apr. 2013 was used to
report PK data in this interim analysis.
[0365] Results: At the time of this interim analysis, 24 subjects
were enrolled and had received at least one dose of pre-study FIX
and/or rFIXFc. Of 18 subjects with evaluable PK profiles, 15 had
complete PK profiles for both pre-study FIX (BENEFIX.RTM.,
HAEMOSOLVEX.RTM., or ALPHANINE.RTM.) and rFIXFc. A comparison of PK
parameters for rFIXFc versus FIX for both age cohorts is presented
in Table 23. rFIXFc had a more than 3-fold prolongation in
half-life and a more than 60% reduction in CL compared to the FIX
products.
TABLE-US-00024 TABLE 23 PK of FIX products and rFIXFc in pediatric
subjects (geometric mean [95% CI]) Half-life CL IR (IU/dL Vss (hr)
(mL/dL/kg) per IU/kg) (mL/kg) <6 FIX 19.4 10.6 0.5 286.0 yrs
(pre-study) (16.6-22.7) (7.6-14.9) (0.4-0.7) (207.0-395.0) n = 5
rFIXFc 71.8 4.1 0.6 347.0 (on-study) (50.1-103.0) (3.5-4.8)
(0.5-0.8) (251.0-480.0) n = 6 rFIXFc/ 3.7 0.4 1.1 1.2 FIX Ratio
(2.6-5.3) (0.3-0.5) (1.0-1.3) (1.2-1.3) n = 4 6 to <12 FIX 17.9
8.6 0.6 214.0 yrs (pre-study) (15.9-20.2) (7.0-10.6) (0.5-0.9)
(154.0-298.0) n = 13 rFIXFc 71.4 3.3 0.8 281.0 (on-study)
(62.7-81.2) (2.9-3.9) (0.6-0.9) (224.0-352.0) n = 13 rFIXFc/ 4.0
0.4 1.2 1.3 FIX Ratio (3.4-4.7) (0.3-0.5) (0.9-1.4) (1.0-1.7) n =
11
[0366] Conclusion: In comparison to currently available FIX
products, rFIXFc had a prolonged half-life and reduced CL in
pediatric subjects, which was similar to previous observations in
adults and adolescents. The final analysis of the Kids B-LONG study
will provide further PK information and evaluate the safety and
efficacy of rFIXFc in children.
Example 8. Use of the Population Pharmacokinetic Model of rFIXFc to
Simulate or Estimate Individualized and Median Patient Treatment
Information
[0367] As is discussed in Examples 5 and 6, a model of estimated
population PK parameters of rFIXFc has been established that can
assist physicians and other healthcare practitioners who wish to
tailor dosing for individual subjects with, e.g., sparse PK
samples. Alternatively, the model can be used to determine dosing
based on PK data for the whole population (median PK).
[0368] Thus, individualized patient treatment, e.g.,
pharmacokinetics (PK) and dosing regimens, can be selected using
Bayesian estimation (or similar machine learning algorithm) based
on the population pharmacokinetic (popPK) model described in
Example 6, above (e.g., Table 21). In this way, one can determine
alternative prophylactic dosing regimens and optimized dosing
regimens for peri-operative management that have not previously
been studied in the B-LONG trials. Alternatively, the selected
dosing regimen is based on population PK (median PK) rather than
making an individualized selection.
[0369] In some embodiments, the rFIXFc popPK model of Example 6
(e.g., Table 21) is used without the Bayesian or similar machine
learning algorithm.
[0370] In some embodiments of this aspect of the invention, the
method is carried out on a computer-based system, e.g., a server, a
desk top computer, a lap top computer, a tablet, a hand held
device, or a smart phone. In some embodiments, the computer-based
system is a computer application. The computer-based system
includes a storage medium for the rFIXFc popPK model discussed in
Example 6, e.g., the parameters of Table 21. In some embodiments,
the storage medium can also contain a Bayesian estimating program,
e.g., NONMEM or Phoenix NLME. E.g., Example 6; Kiang et al., Clin.
Pharmacokinet 51:515-525 (2012).
[0371] In some embodiments, the system comprises two or more
computer-based systems. In some embodiments, the user can input
information into a first computer-based system that communicates
with a second computer-based system, and the second computer-based
system carries out calculations and communicates output information
to the first computer-based system. This output information can
include recommendations about individualized or non-individualized
dosing regimens.
[0372] In some embodiments, the user inputs information into the
system and the system calculates and outputs one or more PK or
dosing regimens. In some embodiments, the system uses the received
information to calculate and output individualized or median PK
information. In some embodiments, the system calculates
individualized dosing or interval information.
[0373] Information that can be input by a user and received by the
system includes patient information and desired treatment outcome
information. Based on the type and value of the received
information, the computer-based system calculates output
information based on the rFIXFc popPK model and optional machine
learning algorithm on the storage medium.
[0374] Patient information includes, e.g., age, body weight,
diagnostic (baseline) FIX level, PK determinations, time of PK
sampling, dosing history if PK samples were taken from multiple
doses, actual dose, FIX activity level, etc.
[0375] Desired treatment outcome information includes desired PK or
desired regimen outcome, e.g., desired rise in plasma FIX activity
level following dose, desired dosing interval, and desired
dose.
[0376] Based on the information that was input and received by the
system, the system can output various information, e.g., PK curve,
PK parameter such as incremental recovery (Cmax/dose), mean
residence time, terminal t1/2, clearance, Vss, AUC/dose, doses and
associated troughs, and intervals and associated troughs.
[0377] For example, for assessing individualized patient PK, the
system can recommend that the user input 2-3 optimized PK sampling
time points. In this case, system output can include PK curve and
one or more selected PK parameters, e.g., incremental recovery
(Cmax/Dose), mean residence time, terminal t1/2, clearance, Vss,
AUC, and time to 1 or X %, etc. E.g., FIG. 15.
[0378] As additional examples, to select an individualized dosing
regimen using the output individual PK parameters discussed in the
preceding paragraph, (i) the dose selected for acute treatment can
be based on user input of the desired rise in plasma FIX activity
level following the dose, (ii) the dose selected for prophylaxis
can be based on user input of the desired dosing interval, or (iii)
the selected interval for prophylaxis can be based on user input
for the desired dose. In the first case, the system can output the
dose (IU) based in the patient's incremental recovery. E.g., FIG.
16. In the second case, system output can be a table of doses and
associated troughs, e.g., x IU/kg, 1% trough, y IU/kg, 2% trough,
etc. e.g., FIG. 17, top. In the third case, system output can be a
table of intervals and associated troughs, e.g., x days, 1% trough,
y IU/kg, 2% trough, etc., E.g., FIG. 17, bottom.
[0379] The user may wish to use the system without inputting any
individualized PK data. In this case, the dosing output would be
based on the population median rather than being individualized for
the particular patient. E.g., FIG. 18. In this way, the user
inputs, e.g., body weight and age, and (i) the desired rise in
plasma FIX activity level following the dose, (ii) the desired dose
interval for prophylaxis, or (iii) the desired dose for
prophylaxis. In the first case, the system can output the dose. In
the second case, the system can output the dose and associated
trough, e.g., Table 16. In the third case, the system can output
the interval and associated trough, e.g., Table 16.
[0380] Age can be input to determine if the system is suitable for
the patient because the current version of the popPK model was
built for patients 12 years and older.
[0381] In some embodiments, the system is compliant with patient
privacy laws. In some embodiments, the system is encrypted, e.g.,
with SSL. In some embodiments, input patient information is made
anonymous.
[0382] In some embodiments, the system includes a user help
function.
[0383] The user can be, e.g., a physician, a nurse, or another
healthcare practitioner.
[0384] In some embodiments, the method further includes selecting a
dosing regimen based on the system's output information and
administering rFIXFc to the patient according to the selected
regimen.
Example 9. Association of Bleeding Tendency with Time Under Target
FIX Activity Levels in Severe Hemophilia B Patients Treated with
rFIXFc
[0385] The goal of prophylactic treatment with coagulation factor
replacement in hemophilia patients has been to convert severe
hemophilia, defined as <1% endogenous factor activity levels, to
moderate (1% to 5%) and mild (5 to 40%) disease. Increased time
spent under 1% FVIII activity may be associated with an increase in
total bleeding episodes and hemarthroses in patients with severe
hemophilia A. No studies to date have documented this association
for FIX activity in patients with severe hemophilia B. We report
here the analysis of bleeding tendency in relation to FIX activity
from the recently completed phase 3, B-LONG study. The B-LONG study
evaluated the pharmacokinetics (PK), safety, and efficacy of a
recombinant FIX Fc fusion protein (rFIXFc) in severe hemophilia B
patients. Briefly, the B-LONG study had 4 treatment arms: weekly
prophylaxis, tailored prophylaxis, episodic treatment, and
perioperative management. The corresponding median annualized
bleeding rates for the first 3 treatment arms were 2.95, 1.38, and
17.69, respectively.
[0386] Methods: A 3-compartmental population PK model of rFIXFc was
developed based on activity-time profiles in 12 subjects from a
Phase 1/2a study and 123 subjects (>12 years) from B-LONG,
collected over .ltoreq.52 weeks of treatment. Individual post-hoc
PK parameters were then derived to construct continuous FIX
activity-time profiles for each dose administered over the course
of the study for subjects in B-LONG. The cumulative time under
target 1%, 3%, and 5% FIX level for each individual on study was
calculated and normalized to obtain annualized time under the
respective target FIX level. Negative binomial regression models
were used to evaluate associations between the number of bleeding
events (overall, spontaneous, traumatic, and joint) and annualized
time (days) under 1%, 3%, and 5% of FIX activity for all subjects
in B-LONG. Models were adjusted for age, body mass index, baseline
HIV and HCV status, FIX genotype, number of bleeding episodes in
the 12 months prior to study entry, and each subject's time on
study.
[0387] Results: The multivariable negative binomial regression
analysis estimated that overall bleeding events increased with
increased time spent under 1% of FIX activity (p<0.001). The
association, however, is largely driven by subjects on episodic
treatment. The median annualized time under 1% for subjects on
episodic treatment was 171 days, in contrast to the median of 0
days for subjects on either weekly prophylaxis or tailored
prophylaxis, respectively, as the tailored PK-driven dosing
regimens were designed to maintain a target trough above 1%. The
association is consistent with the distribution of bleeding events,
most of which occurred at predicted FIX activity levels under 1% in
subjects on episodic treatment. Since the distribution of predicted
FIX trough levels in subjects on prophylaxis were largely in the
range of 1% to 5%, the analysis was repeated for cumulative time
under 3% and 5%. Both analyses found statistically significant
increases in predicted bleeding events as time spent below the
respective FIX activity levels increased (FIGS. 20 and 21). The
significant association was also observed for spontaneous,
traumatic, or joint bleeds analyzed separately for all 3 target FIX
activity levels. When comparing across thresholds (1% vs. 3% vs.
5%), the predicted bleeding rate was significantly reduced and the
predicted probability of being bleed-free improved as the trough
increased. Additionally, the odds of having joint bleeds increased
significantly with increasing time spent under the respective
target trough (1%, 3%, or 5%).
[0388] Conclusions: This is the first study to demonstrate a
correlation between increased time spent under a target therapeutic
FIX activity level (1%, 3% or 5%) and increased bleeding tendency,
as well as a reduced probability of being bleed-free, in adolescent
and adult subjects treated with rFIXFc. Based on population PK
simulations for FIX activity, these findings confirm the importance
of a minimum therapeutic threshold of 1% and provide additional
support for establishing prophylactic dosing regimens for patients
with severe hemophilia B.
Example 10. Population Pharmacokinetic Modeling of Long-Acting
Recombinant Factor IX Fc Fusion Protein in Patients with Hemophilia
B
[0389] Abstract: To elucidate the pharmacokinetic characteristics
of recombinant factor IX Fc fusion protein (rFIXFc) in patients
with hemophilia B and identify covariates that affect rFIXFc
disposition, population PK analysis using NONMEM.RTM. (ICON
Development Solutions, Ellicott City, Md.) was performed with
clinical data from two completed trials in previously treated
patients with severe to moderate hemophilia B. Twelve patients from
a phase 1/2a study and 123 patients from a registrational phase 3
study were included in this population PK analysis. A
three-compartmental model was found to best describe the PK of
rFIXFc. For a typical 73-kg patient, population predicted clearance
(CL), volume of central compartment (V1), and volume of
distribution at steady state (Vss) were 2.39 dL/h, 71.4 dL and 198
dL, respectively. Because of repeat PK profiles at week 26 for
patients in a subgroup, inclusion of inter-occasion variability
(IOV) on CL and V1 were evaluated and significantly improved the
model. The magnitude of IOV on CL and V1 were both low to moderate
(<20%) and less than the corresponding inter-individual
variability. Body weight (BW) was found to be the only significant
covariate for rFIXFc disposition. However, the impact of BW was
limited, as the BW power exponent on CL and V1 were 0.436 and
0.396, respectively. This is the first population PK analysis that
systematically characterized the PK of long-acting rFIXFc in
patients with hemophilia B. The population PK model for rFIXFc can
potentially be utilized to evaluate and optimize dosing regimens
for the treatment of patients with hemophilia B.
[0390] Hemophilia B is a rare bleeding disorder caused by a
deficiency of coagulation factor IX (FIX). The disease is caused by
a mutation on the X chromosome and affects approximately 1 in
30,000 males [1, 2]. Hemophilia B results in abnormal clot
formation, causing prolonged and abnormal bleeding, including
bleeding into joints, soft tissue, muscle and body cavities.
Bleeding episodes may be associated with trauma or occur in the
absence of trauma (spontaneous bleeding). If not treated
appropriately, bleeding can be life-threatening or result in
significant morbidity [2, 3]. Current mainstay of the treatment is
the FIX replacement therapy.
[0391] rFIXFc (recombinant factor IX Fc fusion protein) is a
recombinant protein consisting of a single molecule of FIX
covalently fused to the Fc domain of human immunoglobulin G1 (IgG1)
with no intervening sequence. The Fc domain is responsible for the
long circulating half-life of IgG1 through interaction with the
neonatal Fc receptor (FcRn) that is expressed in many different
cell types [4, 5]. rFIXFc was therefore designed as a long-acting
version of recombinant FIX, e.g., BeneFIX.RTM. (Pfizer Inc, New
York, N.Y.) [6, 7]. rFIXFc has the potential to fulfill an unmet
medical need by providing a long-acting therapy for control and
prevention of bleeding episodes, routine prophylaxis and
perioperative management in patients with hemophilia B. Two
clinical trials with rFIXFc have been completed in previously
treated patients with severe to moderate hemophilia B (with
.ltoreq.2 IU/dL [%] endogenous FIX): one single dose phase 1/2a
study in 14 patients (12 of them who received doses .gtoreq.12.5
IU/kg had PK assessment) [6], and one registrational phase 3 study
in 123 patients. rFIXFc was shown to be well tolerated and
efficacious in the treatment of bleeding, routine prophylaxis and
perioperative management.
[0392] The purpose of this analysis is to characterize the
population pharmacokinetics (PK) of rFIXFc in patients with
hemophilia B and to identify demographic and clinical factors that
are potential determinants of rFIXFc PK variability. The population
PK model of rFIXFc can be used to evaluate and guide dosing
regimens of rFIXFc in the treatment of patients with hemophilia
B.
[0393] Methods.
[0394] Clinical Studies: FIX activity data were obtained from two
completed clinical trials in previously treated patients with
severe to moderate hemophilia B. Twelve evaluable patients from the
phase 1/2a study and 123 patients from the phase 3 study (B-LONG)
who had measurable FIX activities were included in this population
PK analysis.
[0395] The clinical studies are summarized in FIGS. 1 and 2. The
trials were registered at worldwideweb.clinicaltrials.gov as
NCT00716716 (phase 1/2a) and NCT01027364 (phase 3). All subjects
were patients with severe to moderate hemophilia B previously
treated with FIX products, from 12.1 to 76.8 years of age. All
patients, or patient guardians, gave informed written consent. The
studies were conducted in accordance with the International
Conference on Harmonisation guidelines for Good Clinical
Practice.
[0396] Pharmacokinetic Sampling and Bioanalytical Methods: In the
phase 1/2a study, 12 patients underwent rFIXFc PK sampling up to 14
days. In the phase 3 study, PK samples were collected for rFIXFc in
all patients according to the schedule in Table 24. PK profiles of
rFIXFc were assessed at week 1 (baseline) for all patients and at
week 26 for the Arm 1 sequential PK subgroup. For patients on
prophylaxis in Arms 1 and 2, additional trough and peak samples
were collected at clinical visits throughout the study.
TABLE-US-00025 TABLE 24 rFIXFc PK Sampling Schemes Study
Arm/Subgroup Sampling Timepoints Phase 1/2a 12.5 Predose; end of
infusion (10 min), to 100 IU/kg 15 min after the end of infusion,
1, rFIXFc 3, 6, 24, 48, 72, 96, 120, 168, 240 h (288 h and 336 h if
FIX activity was above baseline at day 13) Phase 3 Arm 1/ Predose;
10 min, 1, 3, 6, 24, 48, 96, sequential PK.sup.a 144, 168, 192 and
240 h.sup.c Phase 3 Arm 1/ Predose; 10 min, 3, 24, 48,
non-sequential 96, 168 and 240 h PK.sup.a Phase 3 Arm 2.sup.b
Predose; 10 min, 3, 24, 48, 96, 168, 240, 288 and 336 h Phase 3
Arms 3 Predose; 10 min, 3, 24, 48, and 4.sup.a 96 and 168 h
.sup.aPK dose was 50 IU/kg .sup.bPK dose was 100 IU/kg .sup.cSame
sampling schedule was used for repeat PK at week 26
[0397] The population PK modeling was performed using plasma FIX
activity data as measured by the one-stage activated partial
thromboplastin time (aPTT) clotting assay using commercially
available aPTT reagents (Trinity Biotech) and normal reference
plasma (Precision BioLogic). Lower limit of quantitation (LLOQ) was
1 IU/dL (%). The accuracy of the assay was within 95-104%, and the
intra- and inter-assay precision was approximately 10%.
[0398] Data Handling: A total of 11 data post-infusion were below
the limit of quantification (BLQ, below LLOQ of 1%). Since those
post-infusion BLQ values represent <0.5% of the observations,
they were excluded from the analysis as the first step of data
handling [9-11].
[0399] The one-stage clotting assay does not distinguish FIX
activities resulting from endogenous baseline, residual activity
from incomplete washout of the pre-study FIX product or the input
study drug rFIXFc. Therefore, the baseline and residual activity
corrections were applied to the observed FIX activity data (Eq. 1
and 2). The corrected FIX activities were recorded as the dependent
variable (DV) in the population PK dataset. Similar baseline and
residual activity corrections were reported previously for the PK
analyses of other FIX products [12-15].
Residual decay correction=(Perdose-baseline).times.e.sup.-decay
rate.times.time (1)
Corrected FIX activity=Measured FIX activity-baseline-residual
decay correction (2)
[0400] The endogenous baseline FIX activity level is dictated by
the defective FIX genotype and thus stable in each individual
subject, yet could be overestimated in patients receiving FIX
replacement therapy who underwent incomplete washout. Therefore the
baseline FIX activity was defined as the lowest FIX activity
observed throughout the study, including all the screening,
pre-dose and post-dose records. For patients whose lowest observed
FIX activity was <1% (LLOQ), the baseline FIX activity was set
at 0; for patients whose lowest observed FIX activity was between
1-2%, the baseline FIX activity was set at the lowest observed FIX
activity. The study enrollment is limited to subjects with baseline
FIX activity .ltoreq.2%.
[0401] Residual activity was defined as pre-dose minus baseline FIX
activity. For subjects in the Arm 1 sequential PK subgroup who
underwent PK assessment with the comparator FIX product (BeneFIX)
prior to the rFIXFc PK assessment, the residual activity was
decayed using the individual subject's BeneFIX terminal first-order
decay rate estimated by the non-compartmental analysis in
Phoenix.TM. WinNonlin 6.2 (Pharsight, Sunnyvale, Calif.). For all
other subjects who did not have a BeneFIX PK assessment, the
residual activity was decayed from the rFIXFc PK profiles using the
average BeneFIX terminal first-order decay rate from the Arm 1
sequential PK subgroup.
[0402] In summary, for each individual subject, the baseline
activity was first subtracted from observed FIX activities and then
residual activity, if any, was decayed from baseline-corrected FIX
activities to obtain the corrected FIX activities.
[0403] Modeling Strategy and Datasets: Demographic and clinical
factors collected and examined in the analysis included age, body
weight (BW), race, height, human immunodeficiency virus (HIV) and
hepatitis C virus (HCV) status, IgG1 and albumin concentration,
hematocrit (HCT) level, FIX genotype and blood type. A summary of
categorical factors and baselines for continuous factors is listed
in Table 25.
TABLE-US-00026 TABLE 25 Summary of categorical demographic and
clinical factors and baseline values for continuous demographic and
clinical factors Categorical Demographic and Clinical Factors
Factor Category Number Percent Race American Indian or Alaska
Native 1 0.74 Asian 30 22.2 Black or African American 12 8.9 White
82 60.7 Other 10 7.4 HIV Yes 5 3.7 No 130 96.3 HCV Yes 52 38.5 No
83 61.5 Blood type A 72 53.3 B 21 15.6 AB 7 5.2 O 35 25.9 FIX
genotype Missense 75 55.5 Nonsense 24 17.8 Frameshift 18 13.3
Splice mutation 4 3.0 Others 14 10.4 Continuous Demographic and
Clinical Factors (Baseline) Parameter (units) N Median Mean SD
Minimum Maximum Age 135 31.3 34.6 15.2 12.1 76.8 (year) Weight 135
73.3 75.9 20.1 45 186.7 (kg) IgG1 123 7.19 7.68 2.62 3.34 18.3
(mg/mL) Albumin 134 46 46 3.43 30 56 (g/L) HCT 135 0.44 0.44 0.05
0.21 0.55 (volume/volume) N Number of subjects, SD Standard
deviation, HCT hematocrit, IgG1 immunoglobulin, FIX factor IX, HCV
hepatitis C virus, HIV human immunodeficiency virus
[0404] The PK dataset was split into the modeling dataset, which
was used to build the population PK model and validation dataset,
which was used to qualify the final model. The modeling dataset for
rFIXFc included 1400 FIX activity records from 135 baseline PK
profiles in both phase 1/2a and 3 studies, as well as 21 repeat PK
profiles that were collected at week 26 from the Arm 1 sequential
PK subgroup in the phase 3 study. The validation dataset included
1027 trough/peak FIX activity records from the phase 3 study,
excluding the records during and after surgeries. A summary of the
modeling and validation datasets is listed in Table 26.
TABLE-US-00027 TABLE 26 Summary of modeling and validation datasets
No. of FIX Median body No. of activity Median age weight Dataset
patients records (range) (range) Modeling 12 (Phase 1/2a) 1,400
31.3 73.3 dataset 123 (Phase 3) (12.1-76.8) (45.0-186.7) Validation
100 (Phase 3) 1,027 30.7 72.5 dataset (12.1-71.6) (45.2-186.7) FIX
factor IX
[0405] The modeling strategy was a two-step approach. The first
step was to build the population PK model using the modeling
dataset and the second step was to validate the model with
goodness-of-fit plots, bootstrapping, visual prediction check (VPC)
and the trough/peak validation dataset [16]. As a comparison, the
rFIXFc model using the full dataset, which combined the modeling
and validation dataset, was also developed.
[0406] Population PK Modeling: NONMEM.RTM. 7 version 1.0 (ICON
Development Solutions, Ellicott City, Md.) with an Intel Fortran
compiler (version 12) was used for the population PK model
development. Statistical program R (version 2.15.0, R Foundation
for Statistical Computing, Vienna, Austria) was used to compile
NONMEM datasets and generate graphics. Perl Speaks NONMEM (PsN,
version 3.5.3) [17] was used to conduct bootstrapping. PsN and
Xpose 4 [18] were used to perform VPC.
[0407] A first order conditional estimation with interaction method
(FOCEI) was used to estimate population PK parameters. Inter
individual variability (IIV) was modeled using exponential
function. The inclusion of IIV terms on PK parameters was tested
sequentially, with the most significant objective function value
(OFV) reduction (P<0.005) entering the model first.
Inter-occasion variability (IOV) [19] was also evaluated. For the
modeling dataset, two occasions were defined including baseline PK
at week 1 and repeat PK profiling at week 26. For the full dataset,
six occasions were defined according to the data density. Residual
errors were modeled as combined proportional and additive
errors.
[0408] Plots of IIV versus covariates were used to screen for
potential demographic and clinical factors that affect rFIXFc PK.
For continuous covariates, scatter plots of ETA (IIV code used in
NONMEM) versus covariates were overlaid with a non-parametric
locally weighted smoother LOESS line to determine functional
relationships; for categorical covariates, box and whisker plots
were used to identify potential differences between groups (data
not shown). A clear trend of positive or negative slopes and
noteworthy correlation coefficients (data not shown) would suggest
a possible influence by the continuous covariates; pronounced
differences among the groups would suggest a possible influence by
the categorical covariates. After identifying potential covariates,
a full stepwise forward addition (P<0.005) and backward
elimination (P<0.001) procedure was conducted for covariate
modeling.
[0409] Besides statistical considerations, model selection was also
aided by goodness-of-fit plots, including DV versus population
prediction (PRED), DV versus individual prediction (IPRED),
conditional weighted residual (CWRES) versus TIME and PRED plots
[20, 21]. Other diagnostics also helped to select the proper model,
including parameter precision, ETA, and CWRES distribution and
shrinkage [22, 23].
[0410] Model Qualification: Bootstrapping was conducted with 1,000
datasets generated by random sampling through replacement [24].
Non-parametric median and 95% (2.5th and 97.5th percentiles)
confidence intervals (CIs) of PK parameters were obtained and
compared with final model estimates.
[0411] To check the predictive performance of the model, VPC was
performed to obtain 1,000 simulated PK profiles [24]. Median,
10.sup.th and 90.sup.th percentile of simulated and observed FIX
activities, stratified by dose (50 and 100 IU/kg), were
plotted.
[0412] The trough/peak validation dataset was used to check the
predictability of the model [16, 24, 25]. Specifically, the model
was used to derive Bayesian feedback predictions of FIX activities
at trough/peak time points by setting MAXEVAL=0 in NONMEM control
stream. The mean relative prediction error (an indicator of
accuracy) was calculated using Eq. 3.
1 N .times. i = 1 i = N .times. D .times. V - I .times. P .times. R
.times. E .times. D D .times. V ( 3 ) ##EQU00004##
[0413] Results
[0414] Structural Model and Evaluation of IIV: Based on previous
conventional PK analyses of rFIXFc [6], a two-compartment model
appropriately described individual PK, hence a two-compartment
model was evaluated first followed by a three-compartment model.
IIV (ETA, .eta. values) was assumed for clearance (CL) and volume
of compartment 1 (V1). A covariance between CL and V1 was also
included. The three-compartment model resulted in a reduction of
OFV by over 400 units (for additional four parameters) compared
with the two-compartment model, thus was selected as the base
model. Primary PK parameters included CL, V1, volume of compartment
2 (V2) and 3 (V3), inter-compartmental clearance between
compartments 1 and 2 (Q2), as well as between 1 and 3 (Q3). The
inclusion of IIV for the rest of the PK parameters (V2, V3, Q2 and
Q3) led to further improvement in the model fitting. However, IIV
on Q3 was associated with a high standard error (87%), indicating
that the data cannot support a precise estimation of IIV on Q3,
which was thus not included in the model. No additional covariance
between IIV of PK parameters could be estimated with precision,
thus the only covariance between IIV retained in the model was the
covariance between IIV on CL and V1.
[0415] Evaluation of IOV: Since the Arm 1 sequential PK subgroup
had repeat PK profiles at week 26 in addition to baseline PK
profiles at week 1, IOV was evaluated with baseline PK as occasion
1 and repeat PK as occasion 2. The inclusion of IOV on CL
significantly improved the model with a reduction of OFV by 171.6
units. The inclusion of IOV on both CL and V1 achieved an
additional OFV drop of 41.6 units, whereas IOV on V2 or Q2 did not
improve the model fit (P>0.05). The IOV on V3 improved the model
fit at P<0.005 but with a large percentage of relative standard
error (78.4%); Therefore, IOV was only included for CL and V1.
[0416] Pairwise comparisons of CL and V1 estimates for baseline and
repeat PK, derived from the base model with IOV, were plotted in
FIG. 6. The changes of either CL or V1 between the two occasions
were random and small with only one exception, and the mean CL or
V1 for the two occasions were similar.
[0417] Overall, the inclusion of IOV reduced the corresponding IIV
on CL and V1 from 24.0% and 29.6% to 21.1% and 24.2%, respectively.
The inclusion of IOV also reduced proportional and additive
residual errors from 12.1% and 0.30 IU/dL, to 10.5% and 0.24 IU/dL,
respectively. The base model with IOV provided a reasonable fit to
the data, and explained the random as well as small PK changes
between occasions studied in the trial, therefore was chosen for
further covariate modeling.
[0418] Covariate Modeling: Based on ETA versus covariate plots, BW,
albumin and race on CL, and "study" on V2 were speculated to be
potential covariates. Covariate modeling included BW on all PK
parameters, albumin on CL, and "study" on V2 and CL. BW was
assessed for all PK parameters because it is an important
physiology factor. "Study" was assessed on CL because of the
importance of CL.
[0419] A full stepwise forward addition and backward elimination
procedure was performed. Following the forward covariate inclusion,
the full covariate model was identified with BW on CL and V1, and
"study" on V2. However, "study" on V2 was removed following the
backward elimination procedure (P>0.001).
[0420] Further, potential residual variability difference between
the phase 1/2a and 3 studies was tested by including two sets of
proportional and additive errors for two studies in the residual
error model. No significant reduction in OFV was observed (13.7
units, df=2). Therefore, although the phase 1/2a and phase 3
studies have different dosing and sampling schemes, the population
PK modeling did not suggest a PK difference between the two
studies.
[0421] Final Model: The final model of rFIXFc had IIV on
CL/V1/Q2/V2/V3 but not Q3, IOV on CL and V1 and BW as a covariate
on CL and V1. The model described the data well (FIGS. 8A-8D).
There were no outstanding trends observed in the CWRES plots and
most CWRES randomly distributed between -2 and 2, indicating
overall small discrepancies between measured FIX activities and
population predictions (FIGS. 8C and 8D). Population PK parameter
estimates, IIV and IOV, as well as residual errors were estimated
with precision, evidenced by narrow 95% CIs for each PK parameter
(Table 27). The IIV for CL and V1 were 17.7 and 21.7%,
respectively, which are low to moderate, and IOV for CL and V1 were
low at 15.1 and 17.4%, respectively.
[0422] The magnitude of ETA shrinkage on the IIVs was moderate
(<30% for all PK parameters with IIV terms), while the magnitude
of ETA shrinkage on the IOV was occasion-specific, moderate at
first occasion (around 30% on CL and V1) and higher at occasion 2
(around 70%) because there were fewer PK profiles for the second
occasion (21 for occasion 2 repeat PK vs. 135 for occasion 1
baseline PK). The distributions of ETAs and CWRES showed
approximate normal distribution centered around zero without
apparent skewness (data not shown). This was consistent with the
ETABAR P values, all of which were non-significant (P>0.05).
[0423] Model Qualification: Non-parametric bootstrapping was
applied to the final model to assess the model stability.
Bootstrapping generated medians and CIs for the PK parameters, IIV
and by estimates (Table 27). The median values from the
bootstrapping were very similar to the model estimates for all the
PK parameters.
TABLE-US-00028 TABLE 27 Summary of rFIXFc population PK final model
Bootstrap median Parameter Model estimate (95% CI.sup.a) CL =
Typical CL .times. (BW/73).sup.0.sup.436 Typical CL for a 73-kg
2.39 2.39 (2.29, 2.49) patient (dL/h) BW exponent on CL 0.436 0.437
(0.272, 0.584) V1 = Typical V1 .times. (BW/73).sup.0.sup.396
Typical V1 for a 73-kg 71.4 71.2 (58.5, 76.0) patient (dL) BW
exponent on V1 0.396 0.390 (0.169, 0.580) Q2 (dL/h) 1.67 1.66
(1.35, 1.89) V2 (dL) 87.0 87.0 (79.0, 95.5) Q3 (dL/h) 39.3 39.0
(16.6, 141) V3 (dL) 39.9 41.2 (36.6, 52.4) IIV.sup.b on CL, % 17.7
17.5 (11.8, 22.4) IOV.sup.c on CL, % 15.1 15.0 (10.7, 19.1) IIV on
V1, % 21.7 22.4 (15.5, 32.1) IOV on V1, % 17.4 16.5 (8.7, 22.8) IIV
on Q2, % 35.8 35.0 (22.6, 45.8) IIV on V2, % 46.2 45.9 (38.0, 55.3)
IIV on V3, % 41.2 37.9 (30.2, 54.3) Correlation between IIV on 75.6
74.8 CL and V1, % Proportional residual error, 10.6 10.4 (8.64,
12.0) % Additive residual error, 0.24 0.24 (0.17, 0.31) IU/dL
.sup.a95% CI: Non-parametric 95% CI from bootstrap results with
1,000 datasets .sup.bIIV calculated as (variance)1/2 .times. 100%
.sup.cIOV calculated as (variance)1/2 .times. 100% BW body weight,
CI confidence interval, CL clearance, IIV inter-individual
variability, IOV inter-occasion variability, PK pharmacokinetic, Q2
inter-compartmental clearance of compartment 2, Q3
inter-compartmental clearance of compartment 3, rFIXFc recombinant
factor IX Fc, V1 volume of compartment 1, V2 volume of compartment
2, V3 volume of compartment 3
[0424] The graphic results of the VPC of the final model stratified
by the dose are presented in FIGS. 9A-9D. The median and 80%
interval (10th to 90th percentile) time-activity observed and
predicted profiles nearly overlapped, indicating that the final
model was able to reproduce both the central tendency and
variability of the observed FIX activity time profiles.
[0425] The predictive capability of the final model was further
evaluated using a validation dataset, which contains the
trough/peak FIX activity records that were not included in the
modeling dataset. The final model was used to derive the individual
predictions for the trough and peak observations. Individual
predictions showed good correlation (R2=0.9857, P<0.001) with
the observations (FIG. 10). The mean relative prediction error was
low at -3.23%, indicating that the final model was qualified to
predict rFIXFc PK in the hemophilia B patient population.
[0426] Full Dataset Model: Further, a population PK model of rFIXFc
was also built based on the full dataset, including both PK profile
and trough/peak data. The population parameter estimates of the
resulting model, as well as IIV and IOV (Table 28), were comparable
with those of the final model derived from the modeling dataset
(Table 27). The goodness-of-fit plots indicated that the model also
described the data adequately (FIG. 22). A slightly more
over-prediction of FIX activity in the lower range (<10 IU/dL)
was observed for the VPC of the full dataset model. (FIGS. 9C and
9D).
TABLE-US-00029 TABLE 28 Summary of rFIXFc population PK model
derived from the full dataset Population estimate Parameter (95%
CI).sup.a CL = Typical CL .times. (BW/73).sup.0.sup.432 Typical CL
for a 73-kg patient 2.21 (2.10, 2.32) (dL/h) BW exponent on CL
0.432 (0.251, 0.613) V1 = Typical V1 .times. (BW/73).sup.0.517
Typical V1 for a 73-kg patient (dL) 70.6 (66.3, 74.9) BW exponent
on V1 0.517 (0.282, 0.752) Q2 (dL/h) 1.63 (1.39, 1.87) V2 (dL) 99.1
(84.6, 114) Q3 (dL/h) 45.6 (35.6, 55.6) V3 (dL) 40.7 (38.3, 43.1)
IIV.sup.b on CL, % 19.7 (16.6, 22.8) IOV.sup.c on CL, % 17.8 (17.0,
18.7) IIV on V1, % 21.7 (17.9, 25.6) IOV on V1, % 13.8 (12.3, 15.3)
IIV on Q2, % 48.1 (38.5, 57.6) IIV on V2, % 51.0 (40.6, 61.3)
Correlation between IIV on CL and 60.7 V1, % Proportional residual
error, % 14.8 (9.56, 20.1) Additive residual error, IU/dL 0.279
(0.112, 0.445) .sup.a95% CI: The lower and upper limits for 95% CI
were calculated asymptotically using the standard errors estimated
by the covariance stem in NONMEM .sup.bIIV calculated as
(variance).sup.1/2 .times. 100% .sup.cIOV calculated as
(variance).sup.1/2 .times. 100% BW body weight, CI confidence
interval, CL clearance, IIV inter-individual variability, IOV
inter-occasion variability, PK pharmacokinetic, rFIXFc recombinant
factor IX Fc, RSE relative standard error, Q2 inter-compartmental
clearance of compartment 2, Q3 inter-compartmental clearance of
compartment 3, V1 volume of compartment 1, V2 volume of compartment
2, V3 volume of compartment 3.
[0427] This is the first systematic population PK modeling of
rFIXFc in patients with hemophilia B. A three-compartment model
described the PK of rFIXFc well. For a typical 73-kg patient, V1
for rFIXFc at 71.4 dL is larger than the plasma volume, which is
around 30 dL for a typical adult, indicating that rFIXFc is not
limited in the plasma for the initial distribution phase after
intravenous administration, similar to that of FIX which is known
to bind to collagen IV in the subendothelium. [26] The IIV for CL
and V1 were low to moderate at 17.7% and 21.7%, respectively, which
are consistent with those reported for plasma-derived FIX (23% for
CL and 19% for V1) [12]. Residual errors were small with a
proportional error of 10.6% and additive error of 0.24 IU/dL. The
proportional residual error is similar to the inter-assay
variability of the one stage aPTT clotting assay. The small IIV and
residual errors indicate that the model described the data
adequately and rFIXFc PK do not vary substantially among patients.
The estimated IOVs for CL and V1 were 15.1% and 17.4%,
respectively, similar to those reported for plasma-derived FIX (15%
for CL and 12% for V1) [12]. The small and randomly distributed IOV
on CL and V1 indicate that rFIXFc PK is relatively stable at
different occasions.
[0428] The approach of using the model to estimate baseline and
differentiate baseline from residual activity for the pre-dose of
each individual was investigated. However, the population modeling
cannot reliably separate baseline from residual activity because
not every FIX activity profile returned to baseline at the last
sampling time point, i.e., the baseline (endogenous) and exogenous
signals were confounded. We also investigated setting baseline
activity at 0, 0.5 or individualized baseline. The individualized
baseline resulted in a relatively conservative PK estimates and
more accurate prediction of the trough levels in individual
subjects. Therefore, individualized baseline was chosen to handle
the activity data in the population PK modeling, which was also
utilized in the conventional PK analysis[8].
[0429] BW on CL and V1 was the only covariate that showed a
statistically significant impact on rFIXFc PK. It was suggested
that the exponent of a physiological or PK parameter shall not
revolve around a fixed number [27]. Hence, the exponents of BW on
CL and V1 were estimated during the modeling instead of fixed at
presumed values, e.g., 0.75 for CL and 1 for V1. The estimated BW
exponents for CL and V1 in the final model were markedly lower at
0.436 and 0.396, respectively. Furthermore, inclusion of BW as a
covariate decreased IIV for CL by only 3.4% and for V1 by only
2.5%, suggesting that a considerable portion of the variability was
not explained by BW.
[0430] The limited impact of BW was not unique to rFIXFc PK, which
was also observed for BeneFIX in the phase 3 study (data not
shown). The weak correlation between BW and PK in our studies
differs from a previous report, which showed that BW, with an
exponent of 0.7 on CL, accounted for a significant portion of the
variability in BeneFIX PK in a two-compartment population PK model
[28]. The discrepancy probably can be explained by the different
populations studied, i.e., the adult patients (>19 years) in our
study versus pooled data from 111 children (.ltoreq.15 years),
including 53 infants (<2 years) and 80 adults (>15 years).
This previous report represents a wider range for age and BW than
in our study [29]. A recently published paper reported that BeneFIX
PK in 56 patients aged 4-56 years and weighing 18-133 kg, described
also by a three-compartment model, had allometric exponent of CL
terms of 0.66 and volume terms of 0.64 [29]. The slightly reduced
allometric exponent of CL compared with the previous report [28]
might also be explained by the difference of age and BW range
studied.
[0431] Data splitting is a useful internal model validation
approach in population PK modeling [24]. Because in the clinic
intensive PK profile data are used to predict subsequent
trough/peak sparse data, the data was split into modeling dataset
including the intensive PK profile data from all the subjects at
week 1 and week 26 and validation dataset including the sparse peak
and trough data throughout the phase 3 study. To verify that our
modeling strategy was robust, i.e., building the model with the
baseline/repeat PK profiles without additional trough/peak FIX
activity records, we also built the model using the full dataset
consisting of all the FIX activity records from both the modeling
and validation datasets. The two models were highly comparable with
<10% difference in the PK parameters, IIV and IOV estimates
(Tables 27 and 28). The comparability between the two models was
also demonstrated by the similar VPC plots for the two models
(FIGS. 9A-9D). FIX activities in the lower range (<10 IU/dL)
were slightly more over-predicted by the full dataset model. This
difference might be attributed to the imprecise recordings of the
peak/trough collection time in the full dataset, which was recorded
by patients retrospectively into their electronic diary following
the clinic visit. The final model derived from the modeling dataset
is slightly more accurate in predicting trough levels, which is
essential for maintenance of the therapeutic efficacy. Therefore,
the final model derived from the modeling dataset is robust and
predictive to be used for simulation of the dosing regimens for
rFIXFc.
[0432] Finally, the population PK predictions were largely
consistent with the results derived from the conventional two-stage
PK analysis, which used a two-compartment model, despite a minority
(.about.14%) of the PK profiles could also be described by a three
compartmental model. The ambiguity in the model selection in the
conventional PK analysis was at least partially due to the
different sampling schemes in different study arms. Such ambiguity
was avoided using population PK modeling. The post-hoc estimates
from this population PK analysis were very similar to the results
from the conventional PK analysis (Table 29; [8]). For example, the
geometric mean t1/2 estimated in population PK and conventional PK
are 81.1 h and 82.1 h, respectively. The highly comparable PK
parameters derived from a two-compartment conventional PK analysis
and a three-compartment population PK analysis suggests that the
contribution of the third compartment to rFIXFc PK was probably
limited, but nevertheless provided better profile definition for
the more complex population modeling. The advantage of developing a
population PK model for rFIXFc is that the model can be utilized
for dosing regimen simulation taking into account IIV and IOV,
because FIX activity is considered as a surrogate for efficacy
[12]; Further, the population PK model combined with individual
sparse PK data can be used to derive an individualized dosing
regimen through Bayesian estimation, which can alleviate the
requirement for extensive sampling. Since hemophilia is a life-long
disease impacting children as well as adults, the benefit of
PK-tailored dosing regimens based on data from limited blood
sampling is of great interest to the hemophilia community.
TABLE-US-00030 TABLE 29 Comparison of PK parameters derived from
population PK post hoc analysis and conventional PK analysis for
phase III study Population Conventional PK post hoc PK analysis (n
= 123) (n = 22).sup.a Parameter Geometric Geometric (units) mean
(95% CI) mean (95% CI) CL (mL/h/kg) 3.3 (3.2, 3.5) 3.2 (2.8, 3.6)
V.sub.ss (mL/kg) 280.8 (266.4, 296) 314.8 (277.8, 356.8) Terminal
half-life (h) 81.1 (76.5, 86.1) 82.1 (71.4, 94.5) MRT (h) 84.1
(79.8, 88.6) 98.6 (88.2, 110.3) .sup.aPK parameters derived from 22
patients with intensive sampling schedule in Arm 1 sequential PK
subgroup [8] CI confidence interval, CL clearance, PK
pharmacokinetic, MRT mean residual time, V.sub.ss volume of
distribution at steady state
[0433] Conclusion: This is the first population PK analysis that
systematically characterized the PK of the long-acting rFIXFc in
patients with hemophilia B. The disposition of rFIXFc was well
described by a three-compartment model with low to moderate IIV and
IOV. Body weight was found to be the only statistically significant
but weak covariate on CL and V1 with limited impact. The qualified
population PK model for rFIXFc is accurate and predictive,
providing a valuable tool to evaluate and optimize dosing regimens
of rFIXFc for the treatment of patients with hemophilia B.
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EMBODIMENTS
[0463] E1. A pharmaceutical composition comprising:
[0464] (a) a long-acting Factor IX polypeptide;
[0465] (b) a carbohydrate mixture comprising sucrose and
mannitol;
[0466] (c) sodium chloride (NaCl);
[0467] (d) L-histidine; and
[0468] (e) polysorbate 20 or polysorbate 80.
[0469] E2. The pharmaceutical composition of embodiment E1,
comprising about 1% (w/v) to about 2% (w/v) sucrose.
[0470] E3. The pharmaceutical composition of embodiment E2,
comprising about 1.2% (w/v) sucrose.
[0471] E4. The pharmaceutical composition of embodiment E2,
comprising about 1.7% (w/v) sucrose.
[0472] E5. The pharmaceutical composition of embodiment E1,
comprising about 10 mg/ml to about 20 mg/ml sucrose.
[0473] E6. The pharmaceutical composition of embodiment E5,
comprising about 11.9 mg/ml sucrose.
[0474] E7. The pharmaceutical composition of embodiment E5,
comprising about 16.7 mg/ml sucrose.
[0475] E8. The pharmaceutical composition of any one of embodiments
E1 to E7, comprising about 2% (w/v) to about 4% (w/v) mannitol.
[0476] E9. The pharmaceutical composition of embodiment E8,
comprising about 2.4% (w/v) mannitol.
[0477] E10. The pharmaceutical composition of embodiment E8,
comprising about 3.3% (w/v) mannitol.
[0478] E11. The pharmaceutical composition of any one of
embodiments E1 to E7, comprising about 20 mg/ml to about 40 mg/ml
mannitol.
[0479] E12. The pharmaceutical composition of embodiment E11,
comprising about 23.8 mg/ml mannitol.
[0480] E13. The pharmaceutical composition of embodiment E11,
comprising about 33.3 mg/ml mannitol.
[0481] E14. The pharmaceutical composition of embodiment E1,
comprising about 1.0% to about 2.0% sucrose and about 2.0% (w/v) to
about 4.0% (w/v) mannitol.
[0482] E15. The pharmaceutical composition of embodiment E14,
comprising about 1.2% (w/v) sucrose and about 2.4% (w/v)
mannitol.
[0483] E16. The pharmaceutical composition of embodiment E14,
comprising about 1.7% (w/v) sucrose and about 3.3% (w/v)
mannitol.
[0484] E17. The pharmaceutical composition of embodiment E1,
comprising about 10 mg/ml to about 20 mg/ml sucrose and about 20
mg/ml to about 40 mg/ml mannitol.
[0485] E18. The pharmaceutical composition of embodiment E17,
comprising about 11.9 mg/ml sucrose and about 23.8 mg/ml
mannitol.
[0486] E19. The pharmaceutical composition of embodiment E17,
comprising about 16.7 mg/ml sucrose and about 33.3 mg/ml
mannitol.
[0487] E20. The pharmaceutical composition of any one of
embodiments E1 to E19, comprising between about 50 mM and about 60
mM NaCl.
[0488] E21. The pharmaceutical composition of embodiment E20,
comprising about 55.6 mM NaCl.
[0489] E22. The pharmaceutical composition of any one of
embodiments E1 to E19, comprising between about 3 mg/ml and about 4
mg/ml NaCl.
[0490] E23. The pharmaceutical composition of embodiment E22,
comprising about 3.25 mg/ml NaCl.
[0491] E24. The pharmaceutical composition of any one of
embodiments E1 to E23, comprising between about 20 mM and about 40
mM L-histidine.
[0492] E25. The pharmaceutical composition of embodiment E24,
comprising about 25 mM L-histidine.
[0493] E26. The pharmaceutical composition of embodiment E24,
comprising about 35 mM L-histidine.
[0494] E27. The pharmaceutical composition of any one of
embodiments E1 to E23, comprising between about 3 mg/ml and about 6
mg/ml L-histidine.
[0495] E28. The pharmaceutical composition of embodiment E27,
comprising about 3.88 mg/ml L-histidine.
[0496] E29. The pharmaceutical composition of embodiment E27,
comprising about 5.43 mg/ml L-histidine.
[0497] E30. The pharmaceutical composition of any one of
embodiments E1 to E29, comprising between about 0.008% (w/v) and
about 0.020% (w/v) polysorbate 20 or polysorbate 80.
[0498] E31. The pharmaceutical composition of embodiment E30,
comprising about 0.010% (w/v) polysorbate 20 or polysorbate 80.
[0499] E32. The pharmaceutical composition of embodiment E30,
comprising about 0.014% (w/v) polysorbate 20 or polysorbate 80.
[0500] E33. The pharmaceutical composition of any one of
embodiments E1 to E29, comprising between about 0.08 mg/ml and
about 0.2 mg/ml polysorbate 20 or polysorbate 80.
[0501] E34. The pharmaceutical composition of embodiment E33,
comprising about 0.10% mg/ml polysorbate 20 or polysorbate 80.
[0502] E35. The pharmaceutical composition of embodiment E33,
comprising about 0.14 mg/ml polysorbate 20 or polysorbate 80.
[0503] E36. The pharmaceutical composition of any one of
embodiments E1 to E35, wherein the long-acting FIX polypeptide
comprises human FIX fused to an FcRn binding partner.
[0504] E37. The pharmaceutical composition of embodiment E36,
wherein the long-acting FIX polypeptide comprises a first subunit
comprising an amino acid sequence at least 90%, at least 95%, or
100% identical to amino acids 1 to 642 of SEQ ID NO:2, and a second
subunit comprising an amino acid sequence at least 90% to 95%
identical to amino acids 1 to 227 of SEQ ID NO:4.
[0505] E38. The pharmaceutical composition of any one of
embodiments E1 to E37, which comprises the long-acting FIX
polypeptide at a concentration of between about 25 IU/ml and about
1200 IU/ml.
[0506] E39. The pharmaceutical composition of embodiment E38,
comprising 50 IU/ml, 100 IU/ml, 200 IU/ml, 400 IU/ml, or 600 IU/ml
of the long-acting FIX polypeptide.
[0507] E40. The pharmaceutical composition of embodiment E39,
comprising 50 IU/ml, 100 IU/ml, 200 IU/ml, or 400 IU/ml of the
long-acting FIX polypeptide.
[0508] E41. The pharmaceutical composition of embodiment E39,
comprising 600 IU/ml of the long-acting FIX polypeptide.
[0509] E42. The pharmaceutical composition of embodiment E1,
comprising:
[0510] (a) between about 25 IU/ml and about 700 IU/ml of the
long-acting FIX polypeptide;
[0511] (b) between about 1% (w/v) and about 2% (w/v) of
sucrose;
[0512] (c) between about 2% (w/v) and about 4% (w/v) of
mannitol;
[0513] (d) between about 50 mM and about 60 mM NaCl;
[0514] (e) between about 20 mM and about 40 mM L-histidine; and
[0515] (f) between about 0.008% (w/v) and about 0.015% of
polysorbate 20 or polysorbate 80.
[0516] E43. The pharmaceutical composition of embodiment E42,
comprising:
[0517] (a) about 50 IU/ml of the long-acting FIX polypeptide;
[0518] (b) about 1.2% (w/v) of sucrose;
[0519] (c) about 2.4% (w/v) of mannitol;
[0520] (d) about 55.6 mM NaCl;
[0521] (e) about 25 mM L-histidine; and
[0522] (f) about 0.010% (w/v) of polysorbate 20 or polysorbate
80.
[0523] E44. The pharmaceutical composition of embodiment E42,
comprising:
[0524] (a) about 100 IU/ml of the long-acting FIX polypeptide;
[0525] (b) about 1.2% (w/v) of sucrose;
[0526] (c) about 2.4% (w/v) of mannitol;
[0527] (d) about 55.6 mM NaCl;
[0528] (e) about 25 mM L-histidine; and
[0529] (f) about 0.010% (w/v) of polysorbate 20 or polysorbate
80.
[0530] E45. The pharmaceutical composition of embodiment E42,
comprising:
[0531] (a) about 200 IU/ml of the long-acting FIX polypeptide;
[0532] (b) about 1.2% (w/v) of sucrose;
[0533] (c) about 2.4% (w/v) of mannitol;
[0534] (d) about 55.6 mM NaCl;
[0535] (e) about 25 mM L-histidine; and
[0536] (f) about 0.010% (w/v) of polysorbate 20 or polysorbate
80.
[0537] E46. The pharmaceutical composition of embodiment E42,
comprising:
[0538] (a) about 400 IU/ml of the long-acting FIX polypeptide;
[0539] (b) about 1.2% (w/v) of sucrose;
[0540] (c) about 2.4% (w/v) of mannitol;
[0541] (d) about 55.6 mM NaCl;
[0542] (e) about 25 mM L-histidine; and
[0543] (f) about 0.010% (w/v) of polysorbate 20 or polysorbate
80.
[0544] E47. The pharmaceutical composition of embodiment E42,
comprising:
[0545] (a) about 600 IU/ml of the long-acting FIX polypeptide;
[0546] (b) about 1.7% (w/v) of sucrose;
[0547] (c) about 3.3% (w/v) of mannitol;
[0548] (d) about 55.6 mM NaCl;
[0549] (e) about 35 mM L-histidine; and
[0550] (f) about 0.014% (w/v) of polysorbate 20 or polysorbate
80.
[0551] E48. The pharmaceutical composition of embodiment E1,
comprising:
[0552] (a) between about 25 IU/ml and about 700 IU/ml of the
long-acting FIX polypeptide;
[0553] (b) between about 10 mg/ml and about 20 mg/ml of
sucrose;
[0554] (c) between about 20 mg/ml and about 40 mg/ml of
mannitol;
[0555] (d) between about 3 mg/ml and about 4 mg/ml NaCl;
[0556] (e) between about 3 mg/ml and about 6 mg/ml L-histidine;
and
[0557] (f) between about 0.08 mg/ml and about 0.15 mg/ml of
polysorbate 20 or polysorbate 80.
[0558] E49. The pharmaceutical composition of embodiment E48,
comprising:
[0559] (a) about 50 IU/ml of the long-acting FIX polypeptide;
[0560] (b) about 11.9 mg/ml of sucrose;
[0561] (c) about 23.8 mg/ml of mannitol;
[0562] (d) about 3.25 mg/ml NaCl;
[0563] (e) about 3.88 mg/ml L-histidine; and
[0564] (f) about 0.10 mg/ml of polysorbate 20 or polysorbate
80.
[0565] E50. The pharmaceutical composition of embodiment E48,
comprising:
[0566] (a) about 100 IU/ml of the long-acting FIX polypeptide;
[0567] (b) about 11.9 mg/ml of sucrose;
[0568] (c) about 23.8 mg/ml of mannitol;
[0569] (d) about 3.25 mg/ml NaCl;
[0570] (e) about 3.88 mg/ml L-histidine; and
[0571] (f) about 0.10 mg/ml of polysorbate 20 or polysorbate
80.
[0572] E51. The pharmaceutical composition of embodiment E48,
comprising:
[0573] (a) about 200 IU/ml of the long-acting FIX polypeptide;
[0574] (b) about 11.9 mg/ml of sucrose;
[0575] (c) about 23.8 mg/ml of mannitol;
[0576] (d) about 3.25 mg/ml NaCl;
[0577] (e) about 3.88 mg/ml L-histidine; and
[0578] (f) about 0.10 mg/ml of polysorbate 20 or polysorbate
80.
[0579] E52. The pharmaceutical composition of embodiment E48,
comprising:
[0580] (a) about 400 IU/ml of the long-acting FIX polypeptide;
[0581] (b) about 11.9 mg/ml of sucrose;
[0582] (c) about 23.8 mg/ml of mannitol;
[0583] (d) about 3.25 mg/ml NaCl;
[0584] (e) about 3.88 mg/ml L-histidine; and
[0585] (f) about 0.10 mg/ml of polysorbate 20 or polysorbate
80.
[0586] E53. The pharmaceutical composition of embodiment E48,
comprising:
[0587] (a) about 600 IU/ml of the long-acting FIX polypeptide;
[0588] (b) about 16.7 mg/ml of sucrose;
[0589] (c) about 33.3 mg/ml of mannitol;
[0590] (d) about 3.25 mg/ml NaCl;
[0591] (e) about 5.43 mg/ml L-histidine; and
[0592] (f) about 0.14 mg/ml of polysorbate 20 or polysorbate
80.
[0593] E54. A pharmaceutical kit comprising:
[0594] (a) a first container comprising a lyophilized powder, where
the powder comprises [0595] (i) a long-acting FIX polypeptide,
[0596] (ii) sucrose; [0597] (iii) mannitol; [0598] (iv)
L-histidine; and [0599] (v) polysorbate 20 or polysorbate 80;
and
[0600] (b) a second container comprising 0.325% (w/v) NaCl to be
combined with the lyophilized powder of the first container.
[0601] E55. The pharmaceutical kit of embodiment E54,
comprising:
[0602] (a) a first container comprising a lyophilized powder, where
the powder comprises [0603] (i) about 250 IU of the long-acting FIX
polypeptide, [0604] (ii) about 59.5 mg of sucrose; [0605] (iii)
about 119 mg of mannitol; [0606] (iv) about 19.4 mg of L-histidine;
and [0607] (v) about 0.50 mg of polysorbate 20 or polysorbate 80;
and
[0608] (b) a second container comprising 0.325% (w/v) NaCl at a
volume sufficient to produce, when combined with the lyophilized
powder of the first container, a solution comprising: [0609] (i)
about 50 IU/ml of the long-acting FIX polypeptide; [0610] (ii)
about 1.2% (w/v) of sucrose; [0611] (iii) about 2.4% (w/v) of
mannitol; [0612] (iv) about 55.6 mM NaCl; [0613] (v) about 25 mM
L-histidine; and [0614] (vi) about 0.01% (w/v) of polysorbate 20 or
polysorbate 80.
[0615] E56. The pharmaceutical kit of embodiment E54,
comprising:
[0616] (a) a first container comprising a lyophilized powder, where
the powder comprises [0617] (i) about 500 IU of the long-acting FIX
polypeptide, [0618] (ii) about 59.5 mg of sucrose; [0619] (iii)
about 119 mg of mannitol; [0620] (iv) about 19.4 mg of L-histidine;
and [0621] (v) about 0.50 mg of polysorbate 20 or polysorbate 80;
and
[0622] (b) a second container comprising 0.325% (w/v) NaCl at a
volume sufficient to produce, when combined with the lyophilized
powder of the first container, a solution comprising: [0623] (i)
about 100 IU/ml of the long-acting FIX polypeptide; [0624] (ii)
about 1.2% (w/v) of sucrose; [0625] (iii) about 2.4% (w/v) of
mannitol; [0626] (iv) about 55.6 mM NaCl; [0627] (v) about 25 mM
L-histidine; and [0628] (vi) about 0.01% (w/v) of polysorbate 20 or
polysorbate 80.
[0629] E57. The pharmaceutical kit of embodiment E54,
comprising:
[0630] (a) a first container comprising a lyophilized powder, where
the powder comprises [0631] (i) about 1000 IU of the long-acting
FIX polypeptide, [0632] (ii) about 59.5 mg of sucrose; [0633] (iii)
about 119 mg of mannitol; [0634] (iv) about 19.4 mg of L-histidine;
and [0635] (v) about 0.50 mg of polysorbate 20 or polysorbate 80;
and
[0636] (b) a second container comprising 0.325% (w/v) NaCl at a
volume sufficient to produce, when combined with the lyophilized
powder of the first container, a solution comprising: [0637] (i)
about 200 IU/ml of the long-acting FIX polypeptide; [0638] (ii)
about 1.2% (w/v) of sucrose; [0639] (iii) about 2.4% (w/v) of
mannitol; [0640] (iv) about 55.6 mM NaCl; [0641] (v) about 25 mM
L-histidine; and [0642] (vi) about 0.01% (w/v) of polysorbate 20 or
polysorbate 80.
[0643] E58. The pharmaceutical kit of embodiment E54,
comprising:
[0644] (a) a first container comprising a lyophilized powder, where
the powder comprises [0645] (i) about 2000 IU of the long-acting
FIX polypeptide, [0646] (ii) about 59.5 mg of sucrose; [0647] (iii)
about 119 mg of mannitol; [0648] (iv) about 19.4 mg of L-histidine;
and [0649] (v) about 0.50 mg of polysorbate 20 or polysorbate 80;
and
[0650] (b) a second container comprising 0.325% (w/v) NaCl at a
volume sufficient to produce, when combined with the lyophilized
powder of the first container, a solution comprising: [0651] (i)
about 400 IU/ml of the long-acting FIX polypeptide; [0652] (ii)
about 1.2% (w/v) of sucrose; [0653] (iii) about 2.4% (w/v) of
mannitol; [0654] (iv) about 55.6 mM NaCl; [0655] (v) about 25 mM
L-histidine; and [0656] (vi) about 0.01% (w/v) of polysorbate 20 or
polysorbate 80.
[0657] E59. The pharmaceutical kit of embodiment E54,
comprising:
[0658] (a) a first container comprising a lyophilized powder, where
the powder comprises [0659] (i) about 3000 IU of the long-acting
FIX polypeptide, [0660] (ii) about 83.3 mg of sucrose; [0661] (iii)
about 167 mg of mannitol; [0662] (iv) about 27.2 mg of L-histidine;
and [0663] (v) about 0.7 mg of polysorbate 20 or polysorbate 80;
and
[0664] (b) a second container comprising 0.325% (w/v) NaCl at a
volume sufficient to produce, when combined with the lyophilized
powder of the first container, a solution comprising: [0665] (i)
about 600 IU/ml of the long-acting FIX polypeptide; [0666] (ii)
about 1.7% (w/v) of sucrose; [0667] (iii) about 3.3% (w/v) of
mannitol; [0668] (iv) about 55.6 mM NaCl; [0669] (v) about 35 mM
L-histidine; and [0670] (vi) about 0.014% (w/v) of polysorbate 20
or polysorbate 80.
[0671] E60. The pharmaceutical kit of embodiment E54,
comprising:
[0672] (a) a first container comprising a lyophilized powder, where
the powder comprises [0673] (i) about 250 IU of the long-acting FIX
polypeptide, [0674] (ii) about 59.5 mg of sucrose; [0675] (iii)
about 119 mg of mannitol; [0676] (iv) about 19.4 mg of L-histidine;
and [0677] (v) about 0.50 mg of polysorbate 20 or polysorbate 80;
and
[0678] (b) a second container comprising 0.325% (w/v) NaCl at a
volume sufficient to produce, when combined with the lyophilized
powder of the first container, a solution comprising: [0679] (i)
about 50 IU/ml of the long-acting FIX polypeptide; [0680] (ii)
about 11.9 mg/ml of sucrose; [0681] (iii) about 23.8 mg/ml of
mannitol; [0682] (iv) about 3.25 mg/ml NaCl; [0683] (v) about 3.88
mg/ml L-histidine; and [0684] (vi) about 0.10 mg/ml of polysorbate
20 or polysorbate 80.
[0685] E61. The pharmaceutical kit of embodiment E54,
comprising:
[0686] (a) a first container comprising a lyophilized powder, where
the powder comprises [0687] (i) about 500 IU of the long-acting FIX
polypeptide, [0688] (ii) about 59.5 mg of sucrose; [0689] (iii)
about 119 mg of mannitol; [0690] (iv) about 19.4 mg of L-histidine;
and [0691] (v) about 0.50 mg of polysorbate 20 or polysorbate 80;
and
[0692] (b) a second container comprising 0.325% (w/v) NaCl at a
volume sufficient to produce, when combined with the lyophilized
powder of the first container, a solution comprising: [0693] (i)
about 100 IU/ml of the long-acting FIX polypeptide; [0694] (ii)
about 11.9 mg/ml of sucrose; [0695] (iii) about 23.8 mg/ml of
mannitol; [0696] (iv) about 3.25 mg/ml NaCl; [0697] (v) about 3.88
mg/ml L-histidine; and [0698] (vi) about 0.10 mg/ml of polysorbate
20 or polysorbate 80.
[0699] E62. The pharmaceutical kit of embodiment E54,
comprising:
[0700] (a) a first container comprising a lyophilized powder, where
the powder comprises [0701] (i) about 1000 IU of the long-acting
FIX polypeptide, [0702] (ii) about 59.5 mg of sucrose; [0703] (iii)
about 119 mg of mannitol; [0704] (iv) about 19.4 mg of L-histidine;
and [0705] (v) about 0.50 mg of polysorbate 20 or polysorbate 80;
and
[0706] (b) a second container comprising 0.325% (w/v) NaCl at a
volume sufficient to produce, when combined with the lyophilized
powder of the first container, a solution comprising: [0707] (i)
about 200 IU/ml of the long-acting FIX polypeptide; [0708] (ii)
about 11.9 mg/ml of sucrose; [0709] (iii) about 23.8 mg/ml of
mannitol; [0710] (iv) about 3.25 mg/ml NaCl; [0711] (v) about 3.88
mg/ml L-histidine; and [0712] (vi) about 0.10 mg/ml of polysorbate
20 or polysorbate 80.
[0713] E63. The pharmaceutical kit of embodiment E54,
comprising:
[0714] (a) a first container comprising a lyophilized powder, where
the powder comprises [0715] (i) about 2000 IU of the long-acting
FIX polypeptide, [0716] (ii) about 59.5 mg of sucrose; [0717] (iii)
about 119 mg of mannitol; [0718] (iv) about 19.4 mg of L-histidine;
and [0719] (v) about 0.50 mg of polysorbate 20 or polysorbate 80;
and
[0720] (b) a second container comprising 0.325% (w/v) NaCl at a
volume sufficient to produce, when combined with the lyophilized
powder of the first container, a solution comprising: [0721] (i)
about 400 IU/ml of the long-acting FIX polypeptide; [0722] (ii)
about 11.9 mg/ml of sucrose; [0723] (iii) about 23.8 mg/ml of
mannitol; [0724] (iv) about 3.25 mg/ml NaCl; [0725] (v) about 3.88
mg/ml L-histidine; and [0726] (vi) about 0.10 mg/ml of polysorbate
20 or polysorbate 80.
[0727] E64. The pharmaceutical kit of embodiment E54,
comprising:
[0728] (a) a first container comprising a lyophilized powder, where
the powder comprises [0729] (i) about 3000 IU of the long-acting
FIX polypeptide, [0730] (ii) about 83.3 mg of sucrose; [0731] (iii)
about 167 mg of mannitol; [0732] (iv) about 27.2 mg of L-histidine;
and [0733] (v) about 0.7 mg of polysorbate 20 or polysorbate 80;
and
[0734] (b) a second container comprising 0.325% (w/v) NaCl at a
volume sufficient to produce, when combined with the lyophilized
powder of the first container, a solution comprising: [0735] (i)
about 600 IU/ml of the long-acting FIX polypeptide; [0736] (ii)
about 16.7 mg/ml of sucrose; [0737] (iii) about 33.3 mg/ml of
mannitol; [0738] (iv) about 3.25 mg/ml NaCl; [0739] (v) about 5.43
mg/ml L-histidine; and [0740] (vi) about 0.14 mg/ml of polysorbate
20 or polysorbate 80.
[0741] E65. The kit of any one of embodiments E54 to E64, wherein
the first container is a glass vial comprising a rubber
stopper.
[0742] E66. The kit of any one of embodiments E54 to E65, wherein
the second container is a syringe body, and wherein the syringe
body is associated with a plunger.
[0743] E67. The kit of embodiment E66 further comprising an adaptor
to connect the glass vial to the syringe body.
[0744] E68. The kit of embodiment E66 or embodiment E67, further
comprising infusion tubing associated with a needle to be connected
to the syringe, suitable for intravenous infusion.
[0745] E69. A method of administering a long-acting FIX polypeptide
to a hemophilia B subject in need of prophylaxis, comprising
intravenously administering to the subject the pharmaceutical
composition of any one of embodiments E1 to E53 at an initial dose
of about 50 IU/kg, administered once per week, wherein the
administration prevents or reduces the frequency of bleeding
episodes in the subject.
[0746] E70. A method of administering a long-acting FIX polypeptide
to a hemophilia B subject in need of prophylaxis, comprising
intravenously administering to the subject the pharmaceutical
composition of any one of embodiments E1 to E53 at an initial dose
of about 100 IU/kg, administered once every 10 to 14 days, wherein
the administration prevents or reduces the frequency of bleeding
episodes in the subject.
[0747] E71. The method of embodiment E69 or embodiment E70, wherein
the prophylactic dose amount or dose frequency is subsequently
adjusted based on the subject's response.
[0748] E72. A method of administering a long-acting FIX polypeptide
to a hemophilia B subject in need of treatment of a minor to
moderate bleeding episode, comprising intravenously administering
to the subject the pharmaceutical composition of any one of
embodiments 1 to 53 at an initial dose of about 30 IU/kg to about
60 IU/kg, wherein the administration controls, alleviates, or
reverses the bleeding episode.
[0749] E73. The method of embodiment E72, further comprising
administering one or more additional doses every 48 hours if the
subject exhibits further evidence of bleeding.
[0750] E74. A method of administering a long-acting FIX polypeptide
to a hemophilia B subject in need of treatment of a major bleeding
episode, comprising intravenously administering to the subject the
pharmaceutical composition of any one of embodiments E1 to E53 at
an initial dose of about 100 IU/kg, wherein the administration
controls, alleviates, or reverses the bleeding episode.
[0751] E75. The method of embodiment E74, further comprising
administering an additional dose of the pharmaceutical composition
at about 80 IU/kg after about 6 to 10 hours if the bleeding episode
continues.
[0752] E76. The method of embodiment E75, further comprising
administering one or more additional doses of the pharmaceutical
composition at 80 IU/kg every 24 hours for three days if the
bleeding episode continues.
[0753] E77. The method of embodiment E76, further comprising
administering one or more additional doses of the pharmaceutical
composition at 80 IU/kg every 48 hours until the bleeding episode
is controlled.
[0754] E78. A method of administering a long-acting FIX polypeptide
to a hemophilia B subject in need of surgical prophylaxis,
comprising intravenously administering to a hemophilia B subject
undergoing minor surgery the pharmaceutical composition of any one
of embodiments E1 to E53 at a dose of about 50 IU/kg to 80 IU/kg,
wherein the administration controls bleeding in the subject during
and after surgery.
[0755] E79. The method of embodiment E78, further comprising
administering an additional dose of the pharmaceutical composition
at about 50 IU/kg to 80 IU/kg at about 24 to about 48 hours after
surgery if needed to control post-operative bleeding.
[0756] E80. A method of administering a long-acting FIX polypeptide
to a hemophilia B subject in need of surgical prophylaxis,
comprising intravenously administering to a hemophilia B subject
undergoing major surgery the pharmaceutical composition of any one
of embodiments E1 to E53 at a dose of about 100 IU/kg, wherein the
administration controls bleeding in the subject during and after
surgery.
[0757] E81. The method of embodiment E80, further comprising
administering an additional dose of the pharmaceutical composition
at about 80 IU/kg after about 6 to 10 hours if needed to control
post-operative bleeding.
[0758] E82. The method of embodiment E81, further comprising
administering one or more additional doses of the pharmaceutical
composition at 80 IU/kg every 24 hours for three days if needed to
control post-operative bleeding.
[0759] E83. The method of embodiment E82, further comprising
administering one or more additional doses of the pharmaceutical
composition at 80 IU/kg every 48 hours if needed to control
post-operative bleeding.
[0760] E84. The method of any one of embodiments E69 to E82,
wherein the desired dose of the long-acting FIX polypeptide is
obtainable from a single pharmaceutical kit of any one of
embodiments 54 to 68.
[0761] E85. The method of any one of embodiments E69 to E82,
wherein the desired dose of the long-acting FIX polypeptide is
obtainable from two or more pharmaceutical kits of any one of
embodiments 54 to 68, and wherein the contents of the two or more
pharmaceutical kits are pooled prior to administration.
[0762] E86. The pharmaceutical composition of any one of
embodiments E1 to E53, wherein the long-acting FIX polypeptide has
a mean T.sub.1/2beta (activity) of about 70 hours to about 95 hours
following a single IV infusion of 50 IU/kg of the long-acting FIX
polypeptide.
[0763] E87. The pharmaceutical composition of any one of
embodiments E1 to E53, wherein the mean T.sub.1/2beta (activity) is
about 82 hours.
[0764] E88. The pharmaceutical composition of embodiment E86 or
embodiment E87, wherein the mean T.sub.1/2beta (activity) is at
least about 2-fold to about 3-fold higher than a polypeptide
consisting of full-length mature Factor IX (BENEFIX.RTM.),
following a single IV infusion of 50 IU/kg BENEFIX.RTM..
[0765] E89. The pharmaceutical composition of embodiment E88,
wherein the mean T.sub.1/2beta (activity) is about 2.4-fold higher
than a polypeptide consisting of full-length mature Factor IX
(BENEFIX.RTM.).
[0766] E90. The pharmaceutical composition of any one of
embodiments E1 to E53 or
[0767] E86 to E89, wherein the long-acting FIX polypeptide has a
mean C.sub.max of about 30 IU/dL to about 50 IU/dL following a
single IV infusion of 50 IU/kg of the long-acting FIX
polypeptide.
[0768] E91. The pharmaceutical composition of embodiment E90,
wherein the mean C.sub.max is about 40.8 IU/dL.
[0769] E92. The pharmaceutical composition of any one of
embodiments E1 to E53 or
[0770] E86 to E91, wherein the long-acting FIX polypeptide has a
mean area under the curve per dose (AUC/Dose) of about 27 IU*h/dL
per IU/kg to about 35 IU*h/dL per IU/kg following a single IV
infusion of 50 IU/kg of the long-acting FIX polypeptide.
[0771] E93. The pharmaceutical composition of embodiment E92,
wherein the mean AUC/Dose is about 31.32 IU*h/dL per IU/kg.
[0772] E94. The pharmaceutical composition of embodiment E92 or
embodiment E93, wherein the mean AUC/Dose is at least about
1.8-fold to about 2.1-fold higher than a polypeptide consisting of
full-length mature Factor IX (BENEFIX.RTM.), following a single IV
infusion of the polypeptide consisting of full-length mature Factor
IX (BENEFIX.RTM.) at 50 IU/kg.
[0773] E95. The pharmaceutical composition of embodiment E94,
wherein the mean AUC/Dose is about 1.99-fold higher than a
polypeptide consisting of full-length mature Factor IX
(BENEFIX.RTM.).
[0774] E96. A method of reducing a hemophilia B subject's
annualized bleeding rate, comprising administering fixed or
individualized doses of the pharmaceutical composition of any one
of embodiments 1 to 53 and 86 to 95 prophylactically at regular or
individualized dosing intervals.
[0775] E98. The method of embodiment E96, wherein the
pharmaceutical composition is administered to the subject by a
fixed dose at a fixed dosing interval.
[0776] E98. The method of embodiment E97, wherein the fixed dose of
the pharmaceutical composition is about 50 IU/kg, and the fixed
dosing interval is about one week.
[0777] E99. The method of embodiment E97, wherein the fixed dose of
the pharmaceutical composition is about 100 IU/kg, and the fixed
dosing interval is between about 10 days and about 14 days.
[0778] E100. The method of embodiment E96, wherein the
pharmaceutical composition is administered to the subject at an
individualized dose at a fixed dosing interval.
[0779] E101. The method of embodiment E100, wherein the fixed
dosing interval is about one week.
[0780] E102. The method of embodiment E100 or embodiment E101,
wherein the individualized dose is adjusted to achieve a plasma
trough level of FIX activity of between about 1 IU/dl and about 3
IU/dl.
[0781] E103. The method of any one of embodiments 101 to 102,
wherein the individualized dose is between about 30 IU/kg and about
60 IU/kg, administered about once weekly.
[0782] E104. The method of embodiment E103, wherein the
individualized dose is between about 32 IU/kg and about 54 IU/kg,
administered about once weekly.
[0783] E105. The method of embodiment E104, wherein the average
individualized dose is about 41 IU/kg, administered about once
weekly.
[0784] E106. The method of any one of embodiments E100 to E105,
wherein the subject's annualized bleeding rate is reduced by about
70% to about 90% over the average annualized bleeding rate of
hemophilia B subjects being treated by episodic or on-demand
dosing.
[0785] E107. The method of embodiment E106, wherein the subject's
annualized bleeding rate is reduced by about 76% to about 89%.
[0786] E108. The method of embodiment E107, wherein the subject's
annualized bleeding rate is reduced by about 83%.
[0787] E109. The method of embodiment E96, wherein the
pharmaceutical composition is administered to the subject at a
fixed dose at an individualized dosing interval.
[0788] E110. The method of embodiment E109, wherein the fixed dose
is about 100 IU/kg.
[0789] E111. The method of embodiment E109 or embodiment E110,
wherein the individualized dosing interval is adjusted to achieve a
plasma trough level of FIX activity of between about 1 IU/dl and
about 3 IU/dl.
[0790] E112. The method of any one of embodiments E109 to E111,
wherein the individualized dosing interval is between about 10 days
to about 14 days.
[0791] E113. The method of embodiment E112, wherein the
individualized dosing interval is about 13 days.
[0792] E114. The method of any one of embodiments E109 to E113,
wherein the subject's annualized bleeding rate is reduced by about
80% to about 95% over the average annualized bleeding rate of
hemophilia B subjects being treated by episodic or on-demand
dosing.
[0793] E115. The method of embodiment E114, wherein the subject's
annualized bleeding rate is reduced by about 80% to about 92%.
[0794] E116. The method of embodiment E115, wherein the subject's
annualized bleeding rate is reduced by about 87%.
[0795] E117. The pharmaceutical composition of any one of
embodiments E1 to E53 and E86 to E95, further comprising a
short-acting FIX polypeptide.
[0796] E118. The pharmaceutical composition of embodiment E117,
wherein the short-acting FIX polypeptide comprises or consists of
wild-type FIX.
[0797] E119. The pharmaceutical composition of any one of
embodiments E1 to E53, E86 to E95, and E117 and E118, which is a
liquid formulation.
[0798] E120. The pharmaceutical composition of any one of
embodiments E1 to E53, E86 to E95, and E117 and E118, which is a
lyophilized powder.
[0799] E121. The pharmaceutical composition of any one of
embodiments E1 to E53, E86 to E95, and E117 and E118, which is a
suspension.
[0800] E122. A vial comprising the pharmaceutical composition of
any one of embodiments E119 to E121.
[0801] E123. A cartridge comprising the pharmaceutical composition
of embodiment E119.
[0802] E124. A syringe comprising the pharmaceutical composition of
embodiment E119 or E120.
[0803] E125. The syringe of embodiment E124, which is a dual
chamber syringe.
[0804] E126. The kit of any one of embodiments E54 to E68, wherein
the second container comprises a preservative in an amount
sufficient to provide antimicrobial activity.
[0805] E127. The pharmaceutical composition of embodiment E119,
wherein the liquid formulation further comprises a preservative in
an amount sufficient to provide antimicrobial activity.
[0806] E128. The kit of embodiment E126 or the pharmaceutical
composition of claim 127, wherein the preservative is selected from
the group consisting of phenol, m-cresol, benzyl alcohol,
chlorobutanol, methyl paraben, propylparaben, phenoxyethanol, any
other pharmaceutically acceptable preservative, and any
combinations thereof.
[0807] E129. The kit of any one of embodiments E126 or E128 or the
pharmaceutical composition of embodiment E127 or E128, wherein the
preservative is benzyl alcohol.
[0808] E130. The kit or the pharmaceutical composition of
embodiment E129, wherein the benzyl alcohol is at a concentration
between 0.5% and 0.9%.
[0809] E131. A method of estimating a rFIXFc dosing information
individualized for a patient, the method comprising: [0810] (a)
receiving, by a computer-based system containing the rFIXFc
population pharmacokinetic (popPK) model of Example 6 and a
Bayesian estimation program, at least one of patient information
and desired treatment outcome information, [0811] (b) calculating,
by the computer-based system, individualized rFIXFc dosing
information using the popPK model, the Bayesian estimation program,
and the received information, and [0812] (c) outputting, by the
computer-based system, the individualized dosing information.
[0813] E132. The method of embodiment E131, further comprising
selecting a dosing regimen based on the output individualized
dosing information of (c) and administering rFIXFc to the patient
according to the selected dosing regimen.
[0814] E133. A computer readable storage medium having instructions
stored thereon that, when executed by a processor, cause the
processor to perform the method of embodiment E131.
[0815] E134. A system comprising a processor and a memory, the
memory having instructions stored thereon that, when executed by
the processor, cause the processor to perform the method of
embodiment E131.
[0816] E135. A method of estimating a rFIXFc dosing regimen based
on median popPK, the method comprising: [0817] (a) receiving, by a
computer-based system containing the rFIXFc popPK model of Example
6 and a Bayesian estimation program, at least one of patient
information and desired treatment outcome information, [0818] (b)
calculating, by the computer-based system, median rFIXFc PK
information using the popPK model, the Bayesian estimation program,
and the received information, and [0819] (c) outputting, by the
computer-based system, the median PK information.
[0820] E136. The method of embodiment E135, further comprising
selecting a dosing regimen based on the output median PK
information of (c), and administering rFIXFc to a patient according
to the selected dosing regimen.
[0821] E137. A computer readable storage medium having instructions
stored thereon that, when executed by a processor, cause the
processor to perform the method of embodiment E135.
[0822] E138. A system comprising a processor and a memory, the
memory having instructions stored thereon that, when executed by
the processor, cause the processor to perform the method of
embodiment E135.
[0823] E139. A method of estimating individual patient rFIXFc PK,
the method comprising: [0824] (a) receiving, by a computer-based
system containing the rFIXFc population pharmacokinetic (popPK)
model of Example 6 and a Bayesian estimation program, individual
rFIXFc PK information; [0825] (b) estimating, by the computer-based
system, individualized patient rFIXFc PK information using the
popPK model, the Bayesian estimation program, and the received
information, and [0826] (c) outputting, by the computer-based
system, the individualized patient PK information.
[0827] E140. The method of embodiment E139, further comprising
selecting a dosing regimen based on the output individualized
patient PK information of (c), and administering rFIXFc to the
patient according to the selected regimen.
[0828] E141. A computer readable storage medium having instructions
stored thereon that, when executed by a processor, cause the
processor to perform the method of embodiment E139.
[0829] E142. A system comprising a processor and a memory, the
memory having instructions stored thereon that, when executed by
the processor, cause the processor to perform the method of
embodiment E139.
[0830] E143. The method of embodiment 131, wherein the desired
treatment outcome information is desired rise in plasma FIX
activity level following dosing and the output information is dose
for acute treatment.
[0831] E144. The method of embodiment 131, wherein the desired
treatment outcome information is desired dosing interval and the
output information is dose for prophylaxis.
[0832] E145. The method embodiment 131, wherein the desired
treatment outcome information is desired dose and the output
information is interval for prophylaxis.
[0833] E146. The method of embodiment 139, wherein (a) further
comprises receiving, by the computer-based system, additional
patient information.
[0834] E147. The method of any of embodiments 131, 135, or 146,
wherein the patient information is age or body weight.
[0835] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art, readily
modify and/or adapt for various applications such specific
embodiments, without undue experimentation, without departing from
the general concept of the present invention. Therefore, such
adaptations and modifications are intended to be within the meaning
and range of equivalents of the disclosed embodiments, based on the
teaching and guidance presented herein. It is to be understood that
the phraseology or terminology herein is for the purpose of
description and not of limitation, such that the terminology or
phraseology of the present specification is to be interpreted by
the skilled artisan in light of the teachings and guidance.
TABLE-US-00031 TABLE 18 Polynucleotide Sequences: FIX-Fc A. FIX-Fc
Chain DNA Sequence (SEQ ID NO: 1, which encodes SEQ ID NO: 2)
pSYN-FIX-030 Nucleotide sequence (nt 1 to 7583): FIX exon 1 (signal
peptide, 1st amino acid propeptide): nt 690-777 FIX mini intron: nt
778-1076 FIX propeptide sequence: nt 1077-1126 Mature FIX sequence:
nt 1127-2371 Fc: nt 2372-3052
gcgcgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatat-
atgg
agttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtca-
ataa
tgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaact-
gccc
acttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcc-
tggc
attatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattacc-
atgg
tgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccacccc-
attg
acgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattg-
acgc
aaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgct-
tact
ggcttatcgaaattaatacgactcactatagggagacccaagcttcgcgacgtacggccgccaccatgcagcgc-
gtga
acatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgtaca-
ggtt
tgtttccttttttaaaatacattgagtatgcttgccttttagatatagaaatatctgatgctgtcttcttcact-
aaat
tttgattacatgatttgacagcaatattgaagagtctaacagccagcacgcaggttggtaagtactgtgggaac-
atca
cagattttggctccatgccctaaagagaaattggctttcagattatttggattaaaaacaaagactttcttaag-
agat
gtaaaattttcatgatgttttcttttttgctaaaactaaagaattattcttttacatttcagtttttcttgatc-
atga
aaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaatc-
taga
gagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactg-
aatt
ttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgaca-
ttaa
ttcctatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaaga-
atgg
cagatgcgagcagttttgtaaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttg-
caga
aaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctca-
cccg
tgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactc-
aaag
cacccaatcatttaatgacttcactcgggttgttggtggagaagatgccaaaccaggtcaattcccttggcagg-
ttgt
tttgaatggtaaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccact-
gtgt
tgaaactggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagcaaaagc-
gaaa
tgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctgg-
aact
ggacgaacccttagtgctaaacagctacgttacacctatttgcattgctgacaaggaatacacgaacatcttcc-
tcaa
atttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtacc-
ttag
agttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctg-
gctt
ccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtt-
tctt
aactggaattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtgtcccggt-
atgt
caactggattaaggaaaaaacaaagctcactgacaaaactcacacatgcccaccgtgcccagctccggaactcc-
tggg
cggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacat-
gcgt
ggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatg-
ccaa
gacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggact-
ggct
gaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaag-
ccaa
agggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcc-
tgac
ctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaact-
acaa
gaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggt-
ggca
gcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccc-
tgtc
tccgggtaaatgagaattcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtga-
aaaa
aatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttggg-
gtgg
gcgaagaactccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccgaagcc-
caac
ctttcatagaaggcggcggtggaatcgaaatctcgtagcacgtgtcagtcctgctcctcggccacgaagtgcac-
gcag
ttgccggccgggtcgcgcagggcgaactcccgcccccacggctgctcgccgatctcggtcatggccggcccgga-
ggcg
tcccggaagttcgtggacacgacctccgaccactcggcgtacagctcgtccaggccgcgcacccacacccaggc-
cagg
gtgttgtccggcaccacctggtcctggaccgcgctgatgaacagggtcacgtcgtcccggaccacaccggcgaa-
gtcg
tcctccacgaagtcccgggagaacccgagccggtcggtccagaactcgaccgctccggcgacgtcgcgcgcggt-
gagc
accggaacggcactggtcaacttggccatggtttagttcctcaccttgtcgtattatactatgccgatatacta-
tgcc
gatgattaattgtcaacacgtgctgatcagatccgaaaatggatatacaagctcccgggagctttttgcaaaag-
ccta
ggcctccaaaaaagcctcctcactacttctggaatagctcagaggcagaggcggcctcggcctctgcataaata-
aaaa
aaattagtcagccatggggcggagaatgggcggaactgggcggagttaggggcgggatgggcggagttaggggc-
ggga
ctatggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacac-
ctgg
ttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacaccctcgt-
cgag
ctagcttcgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttgggggg-
aggg
gtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccg-
cctt
tttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgc-
cgcc
agaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttg-
aatt
acttccacctggctccagtacgtgattcttgatcccgagctggagccaggggcgggccttgcgctttaggagcc-
cctt
cgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcc-
tgtc
tcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagat-
agtc
ttgtaaatgcgggccaggatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcg-
tccc
agcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggc-
cggc
ctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcacca-
gttg
cgtgagcggaaagatggccgcttcccggccctgctccagggggctcaaaatggaggacgcggcgctcgggagag-
cggg
cgggtgagtcacccacacaaaggaaaggggcctttccgtcctcagccgtcgcttcatgtgactccacggagtac-
cggg
cgccgtccaggcacctcgattagttctggagcttttggagtacgtcgtctttaggttggggggaggggttttat-
gcga
tggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaa-
tttg
ccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcag-
gtgt
cgtgaacacgtggtcgcggccgcgccgccaccatggagacagacacactcctgctatgggtactgctgctctgg-
gttc
caggttccactggtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggaggaccgtcagtc-
ttcc
tcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtg-
agcc
acgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgg-
gagg
agcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggag-
taca
agtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga-
gaac
cacaggtgtacaccctgcccccatcccgcgatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaa-
ggct
tctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctccc-
gtgt
tggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtc-
ttct
catgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga-
ctcg
agagatctggccggctgggcccgtttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcg-
tacc
ggtcatcatcaccatcaccattgagtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatc-
tgtt
gtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgagga-
aatt
gcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattg-
ggaa
gacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagtggcggtaatac-
ggtt
atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaa-
aggc
cgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggt-
ggcg
aaacccgacaggactataaagataccaggcgtttccccctagaagctccctcgtgcgctctcctgttccgaccc-
tgcc
gcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatc-
tcag
ttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttat-
ccgg
taactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatta-
gcag
agcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtat-
ttgg
tatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccg-
ctgg
tagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatct-
tttc
tacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgacattaacctataaaaata-
ggcg
tatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggaga-
cggt
cacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtc-
gggg
ctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacag-
atgc
gtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcg-
ggcc tcttcgctattacgcca B. Fc DNA sequence (mouse Ig.kappa. signal
peptide underlined) (SEQ ID NO: 3, which encodes SEQ ID NO: 4) This
is the Fc cassette from pSYN-FIX-030. In addition, there is a
separate Fc expression cassette that was transfected into the cell
line in plasmid pSYN-Fc-015 that encodes the same amino acid
sequence, but contains a few noncoding changes. The second copy of
Fc encoding sequence enables a better monomer: dimer ratio.
atggagacagacacactcctgctatgggtactgctgctctgggttccagttccactggtgacaaaactcacaca-
tgcc
caccgtgcccagcacctgaactcctgggaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctc-
atgat
ctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggt-
acg
tggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtc-
a
gcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc-
cca
gcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatc-
cc
gcgatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtg-
gagt
gggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttc-
ctct
acagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggct-
ctg cacaaccactacacgcagaagagcctctccctgtctccgggtaaa
TABLE-US-00032 TABLE 19 Polypeptide Sequences FIX-Fc Monomer
Hybrid: created by coexpressing FIX-Fc and Fc chains. A. FIX-Fc
chain (SEQ ID NO: 2): (28 amino acid signal sequence underlined, 18
amino acid propeptide double underlined, Fc portion in italics.)
The C-terminal lysine is not present in either subunit; this
processing is often observed in recombinant proteins produced in
mammalian cell culture, as well as with plasma derived proteins.
FIXFC-SC SUBUNIT: FIX Signal Peptide: -46 MQRVNMIMAE SPGLITICLL
GYLLSAEC FIX Propeptide: -18 TVFLDHENAN KILNRPKR 1 YNSGKLEEFV
QGNLERECME EKCSFEEARE VFENTERTTE FWKQYVDGDQ 51 CESNPCLNGG
SCKDDINSYE CWCPFGFEGK NCELDVTCNI KNGRCEQFCK 101 NSADNKVVCS
CTEGYRLAEN QKSCEPAVPF PCGRVSVSQT SKLTRAETVF 151 PDVDYVNSTE
AETILDNITQ STQSFNDFTR VVGGEDAKPG QFPWQVVLNG 201 KVDAFCGGSI
VNEKWIVTAA HCVETGVKIT VVAGEHNIEE TEHTEQKRNV 251 IRIIPHHNYN
AAINKYNHDI ALLELDEPLV LNSYVTPICI ADKEYTNIFL 301 KFGSGYVSGW
GRVFHKGRSA LVLQYLRVPL VDRATCLRST KFTIYNNMFC 351 AGFHEGGRDS
CQGDSGGPHV TEVEGTSFLT GIISWGEECA MKGKYGIYTK 401 VSRYVNWIKE
KTKLTDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR 451 TPEVTCVVVD
VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV 501 LTVLHQDWLN
GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 551 DELTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF 601 LYSKLTVDKS
RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK B. Fc chain (SEQ ID NO: 4) 20
amino acid heterologous mouse Ig.kappa. light chain signal peptide
(underlined): -20 METDTLLLWV LLLWVPGSTG Mature Fc sequence
(corresponding to human IgG1 amino acids 221 to 447, EU numbering)
1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED 51
PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK 101
CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK 151
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG 201
NVFSCSVMHE ALHNHYTQKS LSLSPGK
Sequence CWU 1
1
417583DNAArtificial SequenceFIX-Fc Chain 1gcgcgcgttg acattgatta
ttgactagtt attaatagta atcaattacg gggtcattag 60ttcatagccc atatatggag
ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120gaccgcccaa
cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
180caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
gcccacttgg 240cagtacatca agtgtatcat atgccaagta cgccccctat
tgacgtcaat gacggtaaat 300ggcccgcctg gcattatgcc cagtacatga
ccttatggga ctttcctact tggcagtaca 360tctacgtatt agtcatcgct
attaccatgg tgatgcggtt ttggcagtac atcaatgggc 420gtggatagcg
gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
480gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
tccgccccat 540tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
tataagcaga gctctctggc 600taactagaga acccactgct tactggctta
tcgaaattaa tacgactcac tatagggaga 660cccaagcttc gcgacgtacg
gccgccacca tgcagcgcgt gaacatgatc atggcagaat 720caccaggcct
catcaccatc tgccttttag gatatctact cagtgctgaa tgtacaggtt
780tgtttccttt tttaaaatac attgagtatg cttgcctttt agatatagaa
atatctgatg 840ctgtcttctt cactaaattt tgattacatg atttgacagc
aatattgaag agtctaacag 900ccagcacgca ggttggtaag tactgtggga
acatcacaga ttttggctcc atgccctaaa 960gagaaattgg ctttcagatt
atttggatta aaaacaaaga ctttcttaag agatgtaaaa 1020ttttcatgat
gttttctttt ttgctaaaac taaagaatta ttcttttaca tttcagtttt
1080tcttgatcat gaaaacgcca acaaaattct gaatcggcca aagaggtata
attcaggtaa 1140attggaagag tttgttcaag ggaatctaga gagagaatgt
atggaagaaa agtgtagttt 1200tgaagaagca cgagaagttt ttgaaaacac
tgaaagaaca actgaatttt ggaagcagta 1260tgttgatgga gatcagtgtg
agtccaatcc atgtttaaat ggcggcagtt gcaaggatga 1320cattaattcc
tatgaatgtt ggtgtccctt tggatttgaa ggaaagaact gtgaattaga
1380tgtaacatgt aacattaaga atggcagatg cgagcagttt tgtaaaaata
gtgctgataa 1440caaggtggtt tgctcctgta ctgagggata tcgacttgca
gaaaaccaga agtcctgtga 1500accagcagtg ccatttccat gtggaagagt
ttctgtttca caaacttcta agctcacccg 1560tgctgagact gtttttcctg
atgtggacta tgtaaattct actgaagctg aaaccatttt 1620ggataacatc
actcaaagca cccaatcatt taatgacttc actcgggttg ttggtggaga
1680agatgccaaa ccaggtcaat tcccttggca ggttgttttg aatggtaaag
ttgatgcatt 1740ctgtggaggc tctatcgtta atgaaaaatg gattgtaact
gctgcccact gtgttgaaac 1800tggtgttaaa attacagttg tcgcaggtga
acataatatt gaggagacag aacatacaga 1860gcaaaagcga aatgtgattc
gaattattcc tcaccacaac tacaatgcag ctattaataa 1920gtacaaccat
gacattgccc ttctggaact ggacgaaccc ttagtgctaa acagctacgt
1980tacacctatt tgcattgctg acaaggaata cacgaacatc ttcctcaaat
ttggatctgg 2040ctatgtaagt ggctggggaa gagtcttcca caaagggaga
tcagctttag ttcttcagta 2100ccttagagtt ccacttgttg accgagccac
atgtcttcga tctacaaagt tcaccatcta 2160taacaacatg ttctgtgctg
gcttccatga aggaggtaga gattcatgtc aaggagatag 2220tgggggaccc
catgttactg aagtggaagg gaccagtttc ttaactggaa ttattagctg
2280gggtgaagag tgtgcaatga aaggcaaata tggaatatat accaaggtgt
cccggtatgt 2340caactggatt aaggaaaaaa caaagctcac tgacaaaact
cacacatgcc caccgtgccc 2400agctccggaa ctcctgggcg gaccgtcagt
cttcctcttc cccccaaaac ccaaggacac 2460cctcatgatc tcccggaccc
ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga 2520ccctgaggtc
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa
2580gccgcgggag gagcagtaca acagcacgta ccgtgtggtc agcgtcctca
ccgtcctgca 2640ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc
tccaacaaag ccctcccagc 2700ccccatcgag aaaaccatct ccaaagccaa
agggcagccc cgagaaccac aggtgtacac 2760cctgccccca tcccgggatg
agctgaccaa gaaccaggtc agcctgacct gcctggtcaa 2820aggcttctat
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa
2880ctacaagacc acgcctcccg tgttggactc cgacggctcc ttcttcctct
acagcaagct 2940caccgtggac aagagcaggt ggcagcaggg gaacgtcttc
tcatgctccg tgatgcatga 3000ggctctgcac aaccactaca cgcagaagag
cctctccctg tctccgggta aatgagaatt 3060cagacatgat aagatacatt
gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 3120aatgctttat
ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
3180ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
gaggatcatc 3240cagccggcgt cccggaaaac gattccgaag cccaaccttt
catagaaggc ggcggtggaa 3300tcgaaatctc gtagcacgtg tcagtcctgc
tcctcggcca cgaagtgcac gcagttgccg 3360gccgggtcgc gcagggcgaa
ctcccgcccc cacggctgct cgccgatctc ggtcatggcc 3420ggcccggagg
cgtcccggaa gttcgtggac acgacctccg accactcggc gtacagctcg
3480tccaggccgc gcacccacac ccaggccagg gtgttgtccg gcaccacctg
gtcctggacc 3540gcgctgatga acagggtcac gtcgtcccgg accacaccgg
cgaagtcgtc ctccacgaag 3600tcccgggaga acccgagccg gtcggtccag
aactcgaccg ctccggcgac gtcgcgcgcg 3660gtgagcaccg gaacggcact
ggtcaacttg gccatggttt agttcctcac cttgtcgtat 3720tatactatgc
cgatatacta tgccgatgat taattgtcaa cacgtgctga tcagatccga
3780aaatggatat acaagctccc gggagctttt tgcaaaagcc taggcctcca
aaaaagcctc 3840ctcactactt ctggaatagc tcagaggcag aggcggcctc
ggcctctgca taaataaaaa 3900aaattagtca gccatggggc ggagaatggg
cggaactggg cggagttagg ggcgggatgg 3960gcggagttag gggcgggact
atggttgctg actaattgag atgcatgctt tgcatacttc 4020tgcctgctgg
ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc
4080tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctcgt
cgagctagct 4140tcgtgaggct ccggtgcccg tcagtgggca gagcgcacat
cgcccacagt ccccgagaag 4200ttggggggag gggtcggcaa ttgaaccggt
gcctagagaa ggtggcgcgg ggtaaactgg 4260gaaagtgatg tcgtgtactg
gctccgcctt tttcccgagg gtgggggaga accgtatata 4320agtgcagtag
tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
4380agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc
ttgcgtgcct 4440tgaattactt ccacctggct ccagtacgtg attcttgatc
ccgagctgga gccaggggcg 4500ggccttgcgc tttaggagcc ccttcgcctc
gtgcttgagt tgaggcctgg cctgggcgct 4560ggggccgccg cgtgcgaatc
tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt 4620ctctagccat
ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
4680ttgtaaatgc gggccaggat ctgcacactg gtatttcggt ttttggggcc
gcgggcggcg 4740acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg
gggcctgcga gcgcggccac 4800cgagaatcgg acgggggtag tctcaagctg
gccggcctgc tctggtgcct ggcctcgcgc 4860cgccgtgtat cgccccgccc
tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag 4920cggaaagatg
gccgcttccc ggccctgctc cagggggctc aaaatggagg acgcggcgct
4980cgggagagcg ggcgggtgag tcacccacac aaaggaaagg ggcctttccg
tcctcagccg 5040tcgcttcatg tgactccacg gagtaccggg cgccgtccag
gcacctcgat tagttctgga 5100gcttttggag tacgtcgtct ttaggttggg
gggaggggtt ttatgcgatg gagtttcccc 5160acactgagtg ggtggagact
gaagttaggc cagcttggca cttgatgtaa ttctccttgg 5220aatttgccct
ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
5280gtttttttct tccatttcag gtgtcgtgaa cacgtggtcg cggccgcgcc
gccaccatgg 5340agacagacac actcctgcta tgggtactgc tgctctgggt
tccaggttcc actggtgaca 5400aaactcacac atgcccaccg tgcccagcac
ctgaactcct gggaggaccg tcagtcttcc 5460tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag gtcacatgcg 5520tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac gtggacggcg
5580tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc
acgtaccgtg 5640tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag tacaagtgca 5700aggtctccaa caaagccctc ccagccccca
tcgagaaaac catctccaaa gccaaagggc 5760agccccgaga accacaggtg
tacaccctgc ccccatcccg cgatgagctg accaagaacc 5820aggtcagcct
gacctgcctg gtcaaaggct tctatcccag cgacatcgcc gtggagtggg
5880agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgttg
gactccgacg 5940gctccttctt cctctacagc aagctcaccg tggacaagag
caggtggcag caggggaacg 6000tcttctcatg ctccgtgatg catgaggctc
tgcacaacca ctacacgcag aagagcctct 6060ccctgtctcc gggtaaatga
ctcgagagat ctggccggct gggcccgttt cgaaggtaag 6120cctatcccta
accctctcct cggtctcgat tctacgcgta ccggtcatca tcaccatcac
6180cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
gccatctgtt 6240gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
ccactcccac tgtcctttcc 6300taataaaatg aggaaattgc atcgcattgt
ctgagtaggt gtcattctat tctggggggt 6360ggggtggggc aggacagcaa
gggggaggat tgggaagaca atagcaggca tgctggggat 6420gcggtgggct
ctatggcttc tgaggcggaa agaaccagtg gcggtaatac ggttatccac
6480agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
aggccaggaa 6540ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
cgcccccctg acgagcatca 6600caaaaatcga cgctcaagtc agaggtggcg
aaacccgaca ggactataaa gataccaggc 6660gtttccccct agaagctccc
tcgtgcgctc tcctgttccg accctgccgc ttaccggata 6720cctgtccgcc
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
6780tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
cccccgttca 6840gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
tccaacccgg taagacacga 6900cttatcgcca ctggcagcag ccactggtaa
caggattagc agagcgaggt atgtaggcgg 6960tgctacagag ttcttgaagt
ggtggcctaa ctacggctac actagaagaa cagtatttgg 7020tatctgcgct
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
7080caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
ttacgcgcag 7140aaaaaaagga tctcaagaag atcctttgat cttttctacg
gggtctgacg ctcagtggaa 7200cgaaaactca cgttaaggga ttttggtcat
gacattaacc tataaaaata ggcgtatcac 7260gaggcccttt cgtctcgcgc
gtttcggtga tgacggtgaa aacctctgac acatgcagct 7320cccggagacg
gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
7380cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
cagagcagat 7440tgtactgaga gtgcaccata tatgcggtgt gaaataccgc
acagatgcgt aaggagaaaa 7500taccgcatca ggcgccattc gccattcagg
ctgcgcaact gttgggaagg gcgatcggtg 7560cgggcctctt cgctattacg cca
75832642PRTArtificial SequenceFIX-Fc Chain 2Tyr Asn Ser Gly Lys Leu
Glu Glu Phe Val Gln Gly Asn Leu Glu Arg1 5 10 15Glu Cys Met Glu Glu
Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe 20 25 30Glu Asn Thr Glu
Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly 35 40 45Asp Gln Cys
Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp 50 55 60Asp Ile
Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys65 70 75
80Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu
85 90 95Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys
Thr 100 105 110Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu
Pro Ala Val 115 120 125Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln
Thr Ser Lys Leu Thr 130 135 140Arg Ala Glu Thr Val Phe Pro Asp Val
Asp Tyr Val Asn Ser Thr Glu145 150 155 160Ala Glu Thr Ile Leu Asp
Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn 165 170 175Asp Phe Thr Arg
Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe 180 185 190Pro Trp
Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly 195 200
205Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu
210 215 220Thr Gly Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile
Glu Glu225 230 235 240Thr Glu His Thr Glu Gln Lys Arg Asn Val Ile
Arg Ile Ile Pro His 245 250 255His Asn Tyr Asn Ala Ala Ile Asn Lys
Tyr Asn His Asp Ile Ala Leu 260 265 270Leu Glu Leu Asp Glu Pro Leu
Val Leu Asn Ser Tyr Val Thr Pro Ile 275 280 285Cys Ile Ala Asp Lys
Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser 290 295 300Gly Tyr Val
Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala305 310 315
320Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys
325 330 335Leu Arg Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys
Ala Gly 340 345 350Phe His Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp
Ser Gly Gly Pro 355 360 365His Val Thr Glu Val Glu Gly Thr Ser Phe
Leu Thr Gly Ile Ile Ser 370 375 380Trp Gly Glu Glu Cys Ala Met Lys
Gly Lys Tyr Gly Ile Tyr Thr Lys385 390 395 400Val Ser Arg Tyr Val
Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Asp 405 410 415Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 420 425 430Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 435 440
445Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
450 455 460Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His465 470 475 480Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg 485 490 495Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys 500 505 510Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu 515 520 525Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 530 535 540Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu545 550 555
560Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
565 570 575Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val 580 585 590Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp 595 600 605Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His 610 615 620Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro625 630 635 640Gly
Lys3741DNAArtificial SequenceFc Portion 3atggagacag acacactcct
gctatgggta ctgctgctct gggttccagg ttccactggt 60gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggagg accgtcagtc 120ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca
180tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac 240ggcgtggagg tgcataatgc caagacaaag ccgcgggagg
agcagtacaa cagcacgtac 300cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag 360tgcaaggtct ccaacaaagc
cctcccagcc cccatcgaga aaaccatctc caaagccaaa 420gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgcgatga gctgaccaag
480aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 540tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gttggactcc 600gacggctcct tcttcctcta cagcaagctc
accgtggaca agagcaggtg gcagcagggg 660aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc 720ctctccctgt
ctccgggtaa a 7414227PRTArtificial SequenceFc Portion 4Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220Pro Gly Lys225
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