U.S. patent application number 14/814051 was filed with the patent office on 2016-01-21 for high concentration anti-tnfalpha antibody formulation.
The applicant listed for this patent is AbbVie Biotechnology Ltd.. Invention is credited to Alexander Feick, Wolfgang Fraunhofer, Martin Gastens, Michael Neu, Susan K. Paulson, Laura Redden, Markus Tschoepe, Carsten Weber, Tong Zhu.
Application Number | 20160017030 14/814051 |
Document ID | / |
Family ID | 46051586 |
Filed Date | 2016-01-21 |
United States Patent
Application |
20160017030 |
Kind Code |
A1 |
Neu; Michael ; et
al. |
January 21, 2016 |
HIGH CONCENTRATION ANTI-TNFalpha ANTIBODY FORMULATION
Abstract
The invention provides a liquid aqueous pharmaceutical
formulation comprising a human anti-TNFa antibody, or
antigen-binding portion thereof, which reduces pain associated with
injection in a subject by at least about 50% when compared to
injecting an otherwise identical formulation comprising at least
one salt and/or at least one buffer. The invention also provides a
liquid aqueous pharmaceutical formulation comprising a human
anti-TNFa antibody, or antigen-binding portion thereof, having
increased bioavailability upon subcutaneous administration into a
subject. The formulation may comprise a therapeutic protein, such
as a human anti-TNF-alpha antibody, or an antigen-binding portion
thereof, or a biosimilar thereof.
Inventors: |
Neu; Michael;
(Edingen-Neckarhausen, DE) ; Tschoepe; Markus;
(Hessheim, DE) ; Weber; Carsten; (Maxdorf, DE)
; Fraunhofer; Wolfgang; (Gurnee, IL) ; Redden;
Laura; (Glenview, IL) ; Gastens; Martin;
(Wachenheim, DE) ; Feick; Alexander; (Allschwil,
CH) ; Paulson; Susan K.; (Downers Grove, IL) ;
Zhu; Tong; (Riverwoods, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Biotechnology Ltd. |
Hamiliton |
|
BM |
|
|
Family ID: |
46051586 |
Appl. No.: |
14/814051 |
Filed: |
July 30, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14473775 |
Aug 29, 2014 |
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14814051 |
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13294692 |
Nov 11, 2011 |
8821865 |
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14473775 |
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61413960 |
Nov 15, 2010 |
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61412728 |
Nov 11, 2010 |
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Current U.S.
Class: |
424/142.1 |
Current CPC
Class: |
A61P 17/02 20180101;
C07K 2317/565 20130101; C07K 16/241 20130101; Y02A 50/412 20180101;
A61P 25/00 20180101; A61K 9/0019 20130101; C07K 2317/94 20130101;
A61K 2039/505 20130101; A61P 29/00 20180101; A61P 25/04 20180101;
Y02A 50/30 20180101; A61P 1/00 20180101; C07K 2317/76 20130101;
A61P 19/04 20180101; A61K 39/39591 20130101; C07K 2317/21 20130101;
C07K 2317/90 20130101; A61P 17/06 20180101; A61P 19/08 20180101;
A61P 27/02 20180101; A61P 19/02 20180101; A61K 47/26 20130101; A61P
1/04 20180101; A61P 17/00 20180101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; A61K 9/00 20060101 A61K009/00; A61K 47/26 20060101
A61K047/26 |
Claims
1. A liquid aqueous formulation comprising: (1) an isolated human
anti-TNF.alpha. antibody, or an antigen-binding portion thereof;
(2) a surfactant; and, (3) less than 50 mg/ml of a polyol; wherein
the formulation has a pH of 4.7 to 5.7 and does not contain a
buffer or a salt, and wherein injection of the formulation into a
human subject results in a Pain Visual Analog Scale (VAS) score of
less than 1.0.
2. The formulation of claim 1, wherein the polyol is mannitol or
sorbitol.
3. The formulation of claim 2, comprising about 38-46 mg/ml of
mannitol.
4. The formulation of claim 1, wherein the surfactant is a
polysorbate.
5. The formulation of claim 1, wherein the concentration of the
anti-TNF.alpha. antibody, or antigen-binding portion thereof, is
95-105 mg/ml.
6. The formulation of claim 1, wherein is the antibody, or an
antigen-binding portion thereof, comprises a light chain variable
region (LCVR) having a CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain
comprising the amino acid sequence of SEQ ID NO: 5, and a CDR1
domain comprising the amino acid sequence of SEQ ID NO: 7; and a
heavy chain variable region (HCVR) having a CDR3 domain comprising
the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID
NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6,
8, 9, 10 or 11, a CDR2 domain comprising the amino acid sequence of
SEQ ID NO: 6, and a CDR1 domain comprising the amino acid sequence
of SEQ ID NO: 8.
7. The formulation of claim 1, wherein the antibody, or an
antigen-binding portion thereof, comprises a light chain variable
region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1
and a heavy chain variable region (HCVR) comprising the amino acid
sequence of SEQ ID NO: 2.
8. The formulation of claim 1, wherein the antibody, or an
antigen-binding portion thereof, is adalimumab.
9. A liquid aqueous formulation consisting essentially of (1) a
concentration of 90-110 mg/ml of an isolated human anti-TNF.alpha.
antibody, or an antigen-binding portion thereof, having a light
chain variable region (LCVR) having a CDR3 domain comprising the
amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3
by a single alanine substitution at position 1, 4, 5, 7 or 8, a
CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and
a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7;
and having a heavy chain variable region (HCVR) having a CDR3
domain comprising the amino acid sequence of SEQ ID NO: 4, or
modified from SEQ ID NO: 4 by a single alanine substitution at
position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising
the amino acid sequence of SEQ ID NO: 6, and a CDR1 domain
comprising the amino acid sequence of SEQ ID NO: 8; (2) a
polysorbate; and, (3) about 38-46 mg/ml of mannitol.
10. The formulation of claim 9, wherein the antibody, or an
antigen-binding portion thereof, comprises a light chain variable
region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1
and a heavy chain variable region (HCVR) comprising the amino acid
sequence of SEQ ID NO: 2.
11. The formulation of claim 9, wherein the antibody, or an
antigen-binding portion thereof, is adalimumab.
12. The formulation of claim 9, comprising about 30-90 mg of the
antibody, or antigen binding portion thereof.
13. A method of treating a disorder associated with detrimental
TNF.alpha. activity in a patient, comprising administering to the
patient the formulation of claim 1.
14. A method of improving the bioavailability of an isolated human
anti-TNF.alpha. antibody, or an antigen-binding portion thereof, in
a human subject, said method comprising administering a formulation
comprising a surfactant and an effective amount of the antibody, or
antigen-binding portion thereof, to the subject such that the
bioavailability of the antibody, or antigen-binding portion
thereof, is improved, wherein the formulation does not contain a
buffer, a polyol, or a salt.
15. The method of claim 14, wherein the effective amount of the
antibody, or antigen-binding portion thereof, is 30-90 mg.
16. The method of claim 14, wherein the concentration of the
antibody, or antigen-binding portion thereof, in the pharmaceutical
formulation is 90-110 mg/ml.
17. The method of claim 14, wherein the bioavailability of the
antibody, or antigen-binding portion thereof, is an AUC.sub.0-360
greater than 1300 .mu.g*hr/ml when subcutaneously injected into the
human subject.
18. The method of claim 14, wherein the surfactant is a
polysorbate.
19. The method of claim 14, wherein is the antibody, or an
antigen-binding portion thereof, is selected from the group
consisting of a) an antibody, or an antigen-binding portion
thereof, comprising a light chain variable region (LCVR) having a
CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or
modified from SEQ ID NO: 3 by a single alanine substitution at
position 1, 4, 5, 7 or 8, a CDR2 domain comprising the amino acid
sequence of SEQ ID NO: 5, and a CDR1 domain comprising the amino
acid sequence of SEQ ID NO: 7; and a heavy chain variable region
(HCVR) having a CDR3 domain comprising the amino acid sequence of
SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine
substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11, a CDR2
domain comprising the amino acid sequence of SEQ ID NO: 6, and a
CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8; b)
an antibody, or an antigen-binding portion thereof, comprises a
light chain variable region (LCVR) comprising the amino acid
sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)
comprising the amino acid sequence of SEQ ID NO: 2; and c)
adalimumab.
20. A pre-filled syringe or autoinjector device, comprising the
formulation of claim 1.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/473,775, filed on Aug. 29, 2014, which is a
continuation of U.S. patent application Ser. No. 13/294,692, now
U.S. Pat. No. 8,821,865, issued on Sep. 2, 2014, which claims
priority to U.S. Provisional Application No. 61/412,728 filed on
Nov. 11, 2010 and U.S. Provisional Application No. 61/413,960 filed
on Nov. 15, 2010. The entire contents of each of the foregoing
applications are incorporated herein by this reference in their
entireties.
BACKGROUND OF THE INVENTION
[0002] The formulation of therapeutic proteins, such as antibodies,
is often a challenge given the numerous desirable properties that
the formulation must have to be economically and therapeutically
successful, e.g., stability, suitability for administration,
concentration. During manufacturing, storage, and delivery,
therapeutic proteins have been known to undergo physical and
chemical degradations. These instabilities can reduce the potency
of the protein and increase the risk of adverse events in patients,
and, therefore, significantly impact regulatory approval (see,
e.g., Wang et al. J. Pharm. Sci. 96:1, 2007). As such, a stable
protein formulation is essential to the success of a therapeutic
protein.
[0003] To be effective, many therapeutic proteins require the
administration of high doses, which, ideally, are formulated in
high concentration formulations. High protein concentration
formulations are desirable as they can impact the mode (e.g.,
intravenous vs. subcutaneous) and frequency of administration of
the drug to a subject.
[0004] Despite the benefits of high protein concentration
formulations, formulating high concentration therapeutic proteins
presents numerous challenges. For example, increasing protein
concentration often negatively impacts protein aggregation,
solubility, stability, and viscosity (see, e.g., Shire et al. J.
Pharm. Sci. 93:1390, 2004). Increased viscosity, which is a very
common challenge for high protein solutions, can have negative
ramifications on administration of the formulation, e.g., felt pain
and burning syndromes and limitations in manufacturing, processing,
fill-finish and drug delivery device options (see, e.g., Shire et
al. J. Pharm. Sci. 93:1390, 2004). Even for therapeutic proteins
having common structural features, e.g., antibodies, approved
formulations to date have had varying ingredients and ranges of
concentrations. For example, the anti-CD20 antibody Rituxan is
formulated for intravenous administration at a concentration of 10
mg/mL, while the anti-RSV antibody Synagis is formulated for
intramuscular administration at a concentration of 100 mg/mL. Thus,
high protein formulations, especially antibody formulations, which
can be used for therapeutic purposes remain a challenge.
[0005] Another challenge associated with therapeutic proteins, such
as antibodies, is drug delivery. While self-administering devices
allow patients to avoid unnecessary trips to medical facilities to
receive treatments, patients' self-awareness and fear of the pain
associated with self-administration may frequently impact
self-administered drug delivery. Moreover, formulations having high
concentrations of protein may have high viscosity resulting in
increased pain upon delivery, particularly for subcutaneous
administration. Thus, there is especially a need for high
concentration formulations that reduce pain associated with drug
delivery (e.g., self-injection).
[0006] Accordingly, there is a need for stable, high concentration
protein formulations that provide dosing and administrative
advantages, particularly with respect to a decrease in pain for the
patient and/or improved bioavailability.
SUMMARY OF THE INVENTION
[0007] The present invention is based, at least in part, on the
discovery of new high-concentration formulations for therapeutic
antibodies (including human anti-TNF-.alpha. antibodies, or
antigen-binding fragments thereof, e.g., adalimumab). The
formulations of the invention provide a number of surprising
characteristics given the high concentration of the therapeutic
antibody. Specifically, the present invention provides
pharmaceutical formulations comprising human anti-TNF.alpha.
antibodies which surprisingly have improved bioavailability or
decreased pain upon subcutaneous injection.
[0008] In particular, the present invention is based, at least
partly, on the unexpected and surprising discovery that a
formulation having a high antibody concentration, a surfactant, and
a polyol, provides dramatically reduced pain to the patient during
drug delivery, particularly subcutaneous administration of the
antibody through, for example, self-injection. The formulations of
the invention are established, at least in part, on the surprising
finding that a therapeutic protein (e.g., an anti-TNF-alpha
antibody, or antigen-binding portion thereof), can remain soluble
at a high protein concentration (e.g., at least about 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 105, 110 mg/ml, or more)
and maintain a viscosity suitable for injection (e.g., subcutaneous
administration). The formulation of the present invention is
further surprising, in that the formulation does not contain a
buffer or a salt, yet has a high concentration of antibody.
Notably, the formulation of the invention reduces pain associated
with injection in a patient by at least about 50% (e.g., at least
about 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) when compared to
injecting an otherwise identical formulation comprising at least
one salt and/or at least one buffer.
[0009] Thus, in one aspect, the invention provides a liquid aqueous
formulation comprising an anti-TNF.alpha. antibody, or
antigen-binding portion thereof; a surfactant; and, a polyol;
wherein the formulation does not contain a buffer or a salt, and
reduces pain associated with injection in a patient by at least
about 50% (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%
or more) when compared to injecting an otherwise identical
formulation comprising at least one salt and/or at least one
buffer.
[0010] In another aspect, the invention provides a liquid aqueous
formulation comprising an isolated human anti-TNF.alpha. antibody,
or an antigen-binding portion thereof, a surfactant, and less than
50 mg/mL of a polyol, wherein injection of the formulation into a
human subject results in a Pain Visual Analog Scale (VAS) score of
less than 1.0. In one embodiment, the invention provides a liquid
aqueous formulation consisting essentially of an isolated human
anti-TNF.alpha. antibody, or an antigen-binding portion thereof, a
surfactant, and less than 50 mg/mL of a polyol, wherein injection
of the formulation into a human subject results in a Pain Visual
Analog Scale (VAS) score of less than 1.0. In one embodiment, the
VAS scale is from 0 (no pain) to 10 (excruciating pain)
[0011] In a further aspect, the invention provides a liquid aqueous
formulation comprising an isolated human anti-TNF.alpha. antibody,
or an antigen-binding portion thereof, a surfactant, and less than
50 mg/ml of a polyol, wherein the formulation does not contain a
buffer and a salt, and wherein injection of the formulation reduces
pain associated with the injection in a human subject by at least
about 50% when compared to injection of an otherwise identical
formulation that comprises a salt and/or a buffer. In one
embodiment, the otherwise identical comprises a citrate and
phosphate buffer and sodium chloride.
[0012] The invention further provides a liquid aqueous formulation
comprising an anti-TNF.alpha. antibody or antigen-binding portion
thereof, at a concentration of at least about 50 mg/mL; a
surfactant; and, a polyol, wherein the formulation has a
conductivity of less than about 2 mS/cm. In one embodiment, the
formulation has a conductivity of less than 1 mS/cm. In another
embodiment, the formulation has a conductivity of less than 0.9
mS/cm.
[0013] The invention also provides, in another embodiment, a liquid
aqueous formulation comprising an anti-TNF.alpha. antibody or
antigen-binding portion thereof, at a concentration of at least
about 50 mg/mL; a surfactant; and, a polyol, wherein the antibody,
or antigen-binding portion thereof, has a hydrodynamic diameter of
less than 4 nm in the formulation. In one embodiment, the antibody
or antigen-binding portion thereof, has a hydrodynamic diameter of
less than 3 nm in the formulation.
[0014] The present invention also provides a liquid aqueous
formulation comprising an isolated human anti-TNF.alpha. antibody,
or an antigen-binding portion thereof; a surfactant; and, less than
50 mg/ml of a polyol; wherein the formulation has a characteristic
selected from the group consisting of a conductivity of less than
about 2 mS/cm; a hydrodynamic diameter (D.sub.h) which is at least
about 50% less than the D.sub.h of the protein in a buffered
solution at a given concentration; and a hydrodynamic diameter
(D.sub.h) of less than about 4 nm. In one embodiment, the
formulation has a conductivity of less than about 1 mS/cm. In
another embodiment, the formulation has a conductivity of less than
about 0.9 mS/cm. In one embodiment, the antibody or antigen-binding
portion thereof, has a hydrodynamic diameter of less than about 3
nm in the formulation. In another embodiment, the antibody or
antigen-binding portion thereof, has a hydrodynamic diameter of
less than about 2 nm in the formulation.
[0015] The invention also provides a liquid aqueous formulation
consisting essentially of an anti-TNF.alpha. antibody or
antigen-binding portion thereof; a surfactant; and, a polyol;
wherein the concentration of the anti-TNF.alpha. antibody or
antigen-binding portion thereof is at least about 50 mg/mL, 75
mg/mL, 100 mg/mL, or greater than 100 mg/mL.
[0016] In a particular embodiment, the invention provides a liquid
aqueous formulation consisting essentially of a concentration of
90-110 mg/ml of an isolated human anti-TNF.alpha. antibody, or an
antigen-binding portion thereof, having a light chain variable
region (LCVR) having a CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain
comprising the amino acid sequence of SEQ ID NO: 5, and a CDR1
domain comprising the amino acid sequence of SEQ ID NO: 7; and
having a heavy chain variable region (HCVR) having a CDR3 domain
comprising the amino acid sequence of SEQ ID NO: 4, or modified
from SEQ ID NO: 4 by a single alanine substitution at position 2,
3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid
sequence of SEQ ID NO: 6, and a CDR1 domain comprising the amino
acid sequence of SEQ ID NO: 8; a polysorbate, e.g., polysorbate 80;
and, about 38-46 mg/ml of a polyol, e.g., mannitol.
[0017] In another aspect, the present invention provides a liquid
aqueous formulation comprising an isolated human anti-TNF.alpha.
antibody, or an antigen-binding portion thereof; a surfactant; and,
less than 50 mg/ml of a polyol; wherein the formulation is stable
up to about 30 degrees C. for at least about 6 days, about 10,
days, or about 14 days, or is stable at about 28 degrees C. for up
to about 24 months.
[0018] In another aspect, the invention provides a method of
administering an isolated human anti-TNF.alpha. antibody, or an
antigen-binding portion thereof, to a subject such that injection
pain is reduced upon administration, said method comprising
subcutaneously administering to the subject a formulation
comprising the antibody, or antigen-binding portion thereof, such
that injection pain is reduced upon administration, wherein the
formulation comprises more than 50 mg/ml of the antibody, or
antigen-binding portion thereof; a surfactant; and less than 50
mg/ml of a polyol. In one embodiment, the injection pain is
determined to be less than 1.0 according to a Pain Visual Analog
Scale (VAS).
[0019] In certain embodiments, pain associated with injection is
assessed using a pain visual analog scale (VAS). In one embodiment,
the VAS scale is from 0 (no pain) to 10 (excruciating pain)
[0020] In certain embodiments, the pain associated with injection
is assessed after injection (e.g., immediately, no more than 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 minutes, or no more than 15 minutes after
injection).
[0021] In certain embodiments, the formulation reduces pain
associated with injection in the patient by at least about 60%,
70%, 80% or more, when compared to injecting the otherwise
identical formulation comprising the at least one salt and/or at
least one buffer.
[0022] The invention further provides a liquid aqueous formulation
comprising an anti-TNF.alpha. antibody or antigen-binding portion
thereof, at a concentration of at least about 50, 75, 100 mg/mL, or
greater than 100 mg/mL; a surfactant; and, a polyol; wherein the
formulation does not contain a buffer and a salt.
[0023] In another aspect, the invention provides a liquid aqueous
formulation comprising an isolated human anti-TNF.alpha. antibody,
or an antigen-binding portion thereof; a surfactant; and, less than
50 mg/ml of a polyol; wherein the formulation is stable for up to
about 30 degrees C. for at least about 6 days. In one embodiment,
the formulation is stable at room temperature for at least about 7
days. In one embodiment, the formulation is stable at room
temperature for at least about 8 days. In one embodiment, the
formulation is stable at room temperature for at least about 9
days. In one embodiment, the formulation is stable at room
temperature for at least about 10 days. In one embodiment, the
formulation is stable at room temperature for at least about 11
days. In one embodiment, the formulation is stable at room
temperature for at least about 12 days. In one embodiment, the
formulation is stable at room temperature for at least about 13
days. In one embodiment, the formulation is stable at room
temperature for at least about 14 days. In one embodiment, the
formulation is stable at room temperature for at least about 15
days.
[0024] In one embodiment, the polyol used in the formulation of the
invention is mannitol or sorbitol.
[0025] In one embodiment, the formulation of the invention contains
about 20-60 mg/mL mannitol, or, alternatively, about 30-50 mg/mL.
In one embodiment, the formulation contains about 38-46 mg/ml of
mannitol.
[0026] The present invention is also based, at least in part, on
the unexpected and surprising discovery that a formulation having a
high antibody concentration and a surfactant provides notably
higher bioavailability than similar formulations containing
additional excipients, such as a buffer, a polyol and/or a
salt.
[0027] Thus, in one aspect, the invention provides a liquid aqueous
formulation comprising a surfactant and 30-90 mg of an isolated
human anti-TNF.alpha. antibody or antigen-binding portion, wherein
the formulation has an antibody concentration of 90-110 mg/ml, and
wherein the formulation provides increased bioavailability of the
antibody, or antigen-binding portion thereof, to a human subject
upon subcutaneous injection of the formulation relative to a
formulation comprising a citrate phosphate buffer, sodium chloride,
and mannitol.
[0028] In one aspect, the invention provides a liquid aqueous
formulation consisting essentially of a surfactant and 30-90 mg of
an isolated human anti-TNF.alpha. antibody or antigen-binding
portion, wherein the concentration of the antibody, or
antigen-binding portion thereof, is 90-110 mg/ml.
[0029] In another aspect, the invention provides a liquid aqueous
formulation comprising a surfactant and 30-90 mg of an isolated
human anti-TNF.alpha. antibody, or an antigen-binding portion,
wherein the formulation has an antibody concentration of 90-110
mg/ml, and wherein the formulation provides increased
bioavailability of the antibody, or antigen-binding portion
thereof, in a human subject upon subcutaneous injection of the
formulation, such that the antibody or antigen-binding portion
thereof, has an AUC.sub.0-360 greater than about 1300
.mu.g*hr/ml.
[0030] In another aspect, the invention provides a method for
improving the bioavailability of an isolated human anti-TNF.alpha.
antibody, or an antigen-binding portion thereof, in a human
subject, said method comprising administering a formulation
comprising an effective amount of the antibody, or antigen-binding
portion thereof, and a surfactant to the subject such that the
bioavailability of the antibody, or antigen-binding portion
thereof, is improved, wherein the formulation does not contain a
buffer, a polyol, or a salt.
[0031] In a further aspect, the invention provides a method of
improving the bioavailability of an isolated human anti-TNF.alpha.
antibody, or an antigen-binding portion thereof, in a subject, said
method comprising administering a formulation comprising an
effective amount of the antibody, or antigen-binding portion
thereof, and a surfactant to the subject such that the
bioavailability of the antibody, or antigen-binding portion
thereof, in the subject is improved, at least about 15% over a
second formulation, wherein the formulation does not contain a
buffer, a polyol, or a salt, and wherein the second formulation
comprises a buffer, a polyol, and a salt. In one embodiment, the
bioavailability of the antibody, or antigen-binding portion
thereof, is improved at least about 30% over the second
formulation. In one embodiment, the bioavailability of the
antibody, or antigen-binding portion thereof, is improved at least
about 40% over the second formulation.
[0032] The invention further provides a method of improving the
bioavailability of an isolated human anti-TNF.alpha. antibody, or
an antigen-binding portion thereof, in a human subject, said method
comprising administering a formulation comprising a surfactant and
an effective amount of the antibody, or antigen-binding portion
thereof, to the subject such that the bioavailability of the
antibody, or antigen-binding portion thereof, is improved, wherein
the formulation has a characteristic selected from the group
consisting of a conductivity of less than about 2 mS/cm; the
antibody, or antigen-binding portion thereof, has a hydrodynamic
diameter (D.sub.h) which is at least about 50% less than the
D.sub.h of the antibody, or antigen-binding portion thereof, in a
buffered solution at the given concentration; and the antibody, or
antigen-binding portion thereof, has a hydrodynamic diameter
(D.sub.h) of less than about 4 nm. In one embodiment, the
formulation has a conductivity of less than about 1 mS/cm. In
another embodiment, the formulation has a conductivity of less than
about 0.9 mS/cm. In one embodiment, the antibody or antigen-binding
portion thereof, has a hydrodynamic diameter of less than about 3
nm in the formulation.
[0033] In one embodiment, the bioavailability is determined
according to either an AUC level or a Cmax. In one embodiment, the
bioavailability is determined according to either an AUC.sub.0-360
or an AUC.sub.0-1344. In one embodiment, the bioavailability of the
antibody, or antigen-binding portion thereof, is an AUC.sub.0-360
greater than about 1300 .mu.g*hr/ml when subcutaneously injected
into the human subject.
[0034] In certain embodiments, the anti-TNF.alpha. antibody is an
isolated human antibody (e.g., a human IgG1 kappa antibody), a
humanized antibody, a chimeric antibody, or a murine antibody. For
example, the chimeric antibody may be infliximab or a biosimilar
thereof, and the human antibody may be golimumab or adalimumab, or
a biosimilar thereof.
[0035] In one embodiment, the human anti-TNF.alpha. antibody, or an
antigen-binding portion thereof, is an IgG1 or an IgG4.
[0036] In one embodiment, human anti-TNF.alpha. antibody, or an
antigen-binding portion thereof, dissociates from human TNF.alpha.
with a K.sub.d of 1.times.10.sup.-8 M or less and has a k.sub.off
rate constant of 1.times.10.sup.-3 s.sup.-1 or less, both
determined by surface plasmon resonance. In certain embodiments,
the human anti-TNF.alpha. antibody, or an antigen-binding portion
thereof, dissociates from human TNF.alpha. with a K.sub.d of
1.times.10.sup.-8 M or less and a k.sub.off rate constant of
1.times.10.sup.-3 s.sup.-1 or less, both determined by surface
plasmon resonance, and neutralizes human TNF.alpha. cytotoxicity in
a standard in vitro L929 assay with an IC.sub.50 of
1.times.10.sup.-7 M or less.
[0037] In certain embodiments, the human anti-TNF.alpha. antibody,
or an antigen-binding portion thereof, has the following
characteristics: dissociates from human TNF.alpha. with a k.sub.off
rate constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined
by surface plasmon resonance; has a light chain CDR3 domain
comprising the ammo acid sequence of SEQ ID NO: 3, or modified from
SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5,
7 or 8 or by one to five conservative amino acid substitutions at
positions 1, 3, 4, 6, 7, 8 and/or 9; and, (c) has a heavy chain
CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or
modified from SEQ ID NO: 4 by a single alanine substitution at
position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five
conservative amino acid substitutions at positions 2, 3, 4, 5, 6,
8, 9, 10, 11 and/or 12.
[0038] In certain embodiments, the human anti-TNF.alpha. antibody,
or an antigen-binding portion thereof, has a light chain variable
region (LCVR) having a CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
alanine substitution at position 1, 4, 5, 7 or 8, and with a heavy
chain variable region (HCVR) having a CDR3 domain comprising the
amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4
by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9,
10 or 11.
[0039] In certain embodiments, the human anti-TNF.alpha. antibody,
or an antigen-binding portion thereof, has a light chain variable
region (LCVR) having a CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
alanine substitution at position 1, 4, 5, 7 or 8, a CDR2 domain
comprising the amino acid sequence of SEQ ID NO: 5, and a CDR1
domain comprising the amino acid sequence of SEQ ID NO: 7; and has
a heavy chain variable region (HCVR) having a CDR3 domain
comprising the amino acid sequence of SEQ ID NO: 4, or modified
from SEQ ID NO: 4 by a single alanine substitution at position 2,
3, 4, 5, 6, 8, 9, 10 or 11, a CDR2 domain comprising the amino acid
sequence of SEQ ID NO: 6, and a CDR1 domain comprising the amino
acid sequence of SEQ ID NO: 8.
[0040] In certain embodiments, the human anti-TNF.alpha. antibody,
or an antigen-binding portion thereof, has a light chain variable
region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1
and a heavy chain variable region (HCVR) comprising the amino acid
sequence of SEQ ID NO: 2.
[0041] In one embodiment, the human anti-TNF.alpha. antibody, or an
antigen-binding portion thereof, comprises the CDRs corresponding
to adalimumab.
[0042] In one embodiment, the human anti-TNF.alpha. antibody, or an
antigen-binding portion thereof, is adalimumab or golimumab, or a
biosimilar thereof.
[0043] In certain embodiments, the concentration of the human
anti-TNF.alpha. antibody, or antigen-binding portion thereof, in
the formulation is at least about 50 mg/mL, about 75 mg/mL, about
100 mg/mL, or greater than 100 mg/mL. In one embodiment, the
concentration of the human anti-TNF.alpha. antibody, or
antigen-binding portion thereof, in the formulation of the
invention is 90-110 mg/ml. In one embodiment, the concentration of
the human anti-TNF.alpha. antibody, or antigen-binding portion
thereof, in the formulation of the invention is 95-105 mg/ml. In
one embodiment, the formulation comprises more than 75 mg/ml of the
antibody, or antigen-binding portion thereof. In one embodiment,
the invention provides a stable, liquid aqueous formulation
comprising a high concentration, e.g., 75-125 mg/mL, of a human
anti-hTNF.alpha. antibody.
[0044] In certain embodiments, the surfactant used in the
formulation of the invention is a polysorbate. In one embodiment,
the concentration of polysorbate is about 0.1-1.5 mg/ml, about
0.2-1.4 mg/ml, about 0.3-1.3 mg/ml, about 0.4-1.2 mg/ml, about
0.5-1.1 mg/ml, about 0.6-1.0 mg/ml, about 0.6-1.1 mg/ml, about
0.7-1.1 mg/ml, about 0.8-1.1 mg/ml, or about 0.9-1.1 mg/ml. In
certain embodiments, the polysorbate is at a concentration of about
0.1-10 mg/mL, about 0.5-5 mg/mL, about 0.1-2 mg/mL, or about 1
mg/mL. In one embodiment, the surfactant is polysorbate 80.
[0045] In certain embodiments, the patient is human, or a non-human
mammal.
[0046] In certain embodiments, the formulation is Formulation 3 or
Formulation 4 described in the Examples.
[0047] In certain embodiments, the otherwise identical formulation
is the commercially available adalimumab formulation containing
adalimumab, sodium chloride, monobasic sodium phosphate dihydrate,
dibasic sodium phosphate dihydrate, sodium citrate, citric acid
monohydrate, mannitol, polysorbate 80, and water for Injection.
[0048] In one embodiment, the otherwise identical formulation
contains a buffer and a salt. In certain embodiments, the salt is a
neutral salt, or a salt from a base (e.g., NaOH) used for pH
adjustment. In certain embodiments, the buffer comprises a
phosphate buffer and/or a citrate buffer. For example, the
phosphate buffer may contain about 1.35-1.75 mg/mL or about
1.50-1.56 mg/mL of Na.sub.2HPO.sub.4.2H.sub.2O, and about 0.75-0.95
mg/mL or about 0.83-0.89 mg/mL of NaH.sub.2PO.sub.4.2H.sub.2O). The
citrate buffer may contain about 1.15-1.45 mg/mL or about 1.30-1.31
mg/mL of citric acid.H.sub.2O, and about 0.2-0.4 mg/mL or about
0.30-0.31 mg/mL of sodium citrate dehydrate. The at least one salt
may be a neutral salt, such as a neutral sodium salt (e.g.,
NaCl).
[0049] In one embodiment, the formulation of the invention is a
pharmaceutical formulation.
[0050] In certain embodiments, the formulation of the invention is
suitable for subcutaneous injection. In one embodiment, the
formulation of the invention is suitable for subcutaneous
self-administration by a subject.
[0051] In certain embodiments, the volume of the aqueous
formulation is no more than 1.5 mL, 1.0 mL, 0.8 mL, 0.5 mL, or 0.4
mL.
[0052] In certain embodiments, the formulation comprises a dose of
about 30-90 mg of the antibody, or antigen binding portion thereof.
In one embodiment, the formulation comprises about 40 mg of the
anti-TNF.alpha. antibody, or antigen binding portion thereof. In
one embodiment, the formulation comprises about 50 mg of the
anti-TNF.alpha. antibody, or antigen binding portion thereof. In
one embodiment, the formulation comprises about 60 mg of the
anti-TNF.alpha. antibody, or antigen binding portion thereof. In
one embodiment, the formulation comprises about 70 mg of the
anti-TNF.alpha. antibody, or antigen binding portion thereof. In
one embodiment, the formulation comprises about 80 mg of the
anti-TNF.alpha. antibody, or antigen binding portion thereof. In
one embodiment, the formulation comprises about 90 mg of the
anti-TNF.alpha. antibody, or antigen binding portion thereof. In
one embodiment, the formulation comprises 60-85 mg. In another
embodiment, the formulation comprises 70-90 mg. In yet a further
embodiment, the formulation contains 30-110 mg. In one embodiment,
the formulation contains 70-110 mg.
[0053] Another aspect of the invention provides a pre-filled
syringe or autoinjector device, comprising any of the subject
formulations described herein. In certain embodiments, the aqueous
formulation stored in the pre-filled syringe or autoinjector device
contains about 40 mg of adalimumab, or biosimilar thereof. In
certain embodiments, the aqueous formulation stored in the
pre-filled syringe or autoinjector device contains about 80 mg of
adalimumab, or biosimilar thereof.
[0054] Another aspect of the invention provides a method of
treating a disorder associated with detrimental TNF.alpha. activity
in a patient, comprising administering to the patient any one of
the formulations described herein.
[0055] In one embodiment, the formulation or method of the
invention is used to treat a subject having rheumatoid arthritis.
In one embodiment, the formulation or method of the invention is
used to treat a subject having Crohn's disease. In one embodiment,
the formulation or method of the invention is used to treat a
subject having psoriatic arthritis. In one embodiment, the
formulation or method of the invention is used to treat a subject
having psoriasis. In one embodiment, the formulation or method of
the invention is used to treat a subject having juvenile idiopathic
arthritis (JIA). In one embodiment, the formulation or method of
the invention is used to treat a subject having ankylosing
spondylitis. In one embodiment, the formulation or method of the
invention is used to treat a subject having ulcerative colitis. In
one embodiment, the formulation or method of the invention is used
to treat a subject having hidradenitis suppurativa. In one
embodiment, the formulation or method of the invention is used to
treat a subject having diabetic retinopathy. In one embodiment, the
formulation or method of the invention is used to treat a subject
having giant cell arteritis. In one embodiment, the formulation or
method of the invention is used to treat a subject having Behcet's
disease. In one embodiment, the formulation or method of the
invention is used to treat a subject having sarcoidosis, e.g.
cutaneous sarcoidosis. In one embodiment, the formulation or method
of the invention is used to treat a subject having axial
spondyloarthropathy. In one embodiment, the formulation or method
of the invention is used to treat a subject having uveitis.
[0056] In one embodiment, the formulation is administered to the
subject according to a periodicity selected from the group
consisting of weekly, biweekly, every three weeks, and monthly. In
one embodiment, the formulation of the invention contains 30-90 mg
of a human anti-TNFa antibody, or antigen-binding portion thereof,
and is administered on a biweekly dosing regimen. In another
embodiment, the formulation of the invention contains 30-90 mg of a
human anti-TNFa antibody, or antigen-binding portion thereof, and
is administered according to a monthly dosing regimen. In one
embodiment, the formulation of the invention contains 60-85 mg of a
human anti-TNFa antibody, or antigen-binding portion thereof, and
is administered on a biweekly dosing regimen. In another
embodiment, the formulation of the invention contains 60-85 mg of a
human anti-TNF.alpha. antibody, or antigen-binding portion thereof,
and is administered according to a monthly dosing regimen.
[0057] In certain embodiments, the administration of the
formulation of the invention to a subject is via
self-administration.
[0058] It is contemplated that any one embodiment described herein
can be combined with one or more other embodiments of the
invention, including embodiments described only under one aspect of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1 is a panel of graphs that show administration of high
concentration formulations 1 (F1) and 2 (F2) resulted in a
significant decrease in pain assessment at all time points after
injection (immediately, 15 minutes, and 30 minutes), compared to
the other treatment groups (F4 and the current commercial
formulation).
[0060] FIG. 2 shows, on a linear scale, the means and standard
deviations of adalimumab serum concentrations over a time period of
56 days following a single 40 mg SC dose of adalimumab.
[0061] FIGS. 3A and 3B are graphs that show the stability of the
various adalimumab formulations assessed by the number of sum
aggregates in the formulations (3A) or the sum aggregates (3B) over
a range of polysorbate or a range of polyol.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0062] In order that the present invention may be more readily
understood, certain terms are first defined. In addition, it should
be noted that whenever a value or range of values of a parameter
are recited, it is intended that values and ranges intermediate to
the recited values are also intended to be part of this
invention.
[0063] The term "pain associated with injection (in a patient)," as
used herein, refers to the pain associated with the injection of
drug into the patient's or subject's tissue. In one embodiment, the
pain is separate from the pain caused by the injection device (if
any), such as the injection needle stick. In one embodiment, the
pain associated with injection may originate from the drug
formulation being injected into patient's tissue.
[0064] The pain associated with injection may be evaluated using a
number of art-recognized means, such as the Pain Visual Analog
Scale (VAS). The pain measurement is, in one embodiment,
quantifiable, such that a percentage pain scale reduction/increase
can be directly compared using statistical methods. For example,
when the Pain Visual Analog Scale is used, a numeric pain value
(e.g., average.+-.SD) can be assigned to each treatment group, such
that a percentage increase or reduction can be calculated. In
general, a Visual Analogue Scale (VAS) is a measurement instrument
that measures a characteristic or attitude that is believed to
range across a continuum of values (see, e.g., Singer and Thods
(1998) Academic Emergency Medicine 5:1007). For example, the amount
of pain that a patient feels ranges across a continuum from none (a
score of, for example, 0) to an extreme amount of pain (a score of,
for example, 10). From the patient's perspective this spectrum
appears continuous--their pain does not take discrete jumps, as a
categorization of none, mild, moderate and severe would suggest.
Operationally, a VAS is usually a horizontal line, 100 mm in
length, anchored by word descriptors at each end, such as "no pain"
at one end, and "extreme pain" (or some variation thereof) on the
other end. The patient marks on the line at a point (for example, a
score of 0-10) that they feel represents their perception of their
current state. The VAS score may determined by measuring in
millimeters from the left hand end of the line to the point that
the patient marks.
[0065] There are various ways in which VAS have been presented,
including vertical lines and lines with extra descriptors. See
Wewers & Lowe ("A critical review of visual analogue scales in
the measurement of clinical phenomena." Research in Nursing and
Health 13: 227-236, 1990, incorporated by reference herein) provide
an informative discussion of the benefits and shortcomings of
different styles of VAS.
[0066] The term "liquid formulation" refers to a formulation in a
liquid state and is not intended to refer to resuspended
lyophilized formulations. A liquid formulation of the invention is
stable upon storage, and does not rely upon lyophilization (or
other state change methods, e.g., spray drying) for stability.
[0067] The term "liquid aqueous formulation" refers to a liquid
formulation using water as a solvent. In one embodiment, a liquid
aqueous formulation is a formulation that maintains stability
(e.g., chemical and/or physical stability/and/or biological
activity) without the need for lyophilization, spray-drying, and/or
freezing.
[0068] The term "pharmaceutical," as used herein, refers to a
composition, e.g., an aqueous formulation, that it is useful for
treating a disease or disorder.
[0069] The term "subject" or "patient" is intended to include
mammalian organisms. Examples of subjects/patients include humans
and non-human mammals, e.g., non-human primates, dogs, cows,
horses, pigs, sheep, goats, cats, mice, rabbits, rats, and
transgenic non-human animals. In specific embodiments of the
invention, the subject is a human.
[0070] The term "excipient" refers to an agent which may be added
to a formulation to provide a desired characteristic, e.g.,
consistency, improving stability, and/or to adjust osmolality.
Examples of commonly used excipients include, but are not limited
to, sugars, polyols, amino acids, surfactants, and polymers.
[0071] A commonly used excipient is a polyol. As used herein, a
"polyol" is a substance with multiple hydroxyl groups, and includes
sugars (reducing and nonreducing sugars), sugar alcohols and sugar
acids. Non-limiting examples of polyols are fructose, mannose,
maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose,
glucose, sucrose, trehalose, sorbose, melezitose, raffinose,
mannitol, xylitol, erythritol, threitol, sorbitol, glycerol,
L-gluconate and metallic salts thereof. In one embodiment, the
polyol used in the formulation or methods of the invention is
mannitol. In one embodiment, the polyol used in the formulation or
methods of the invention is sorbitol.
[0072] A "therapeutically active antibody" or "therapeutic
antibody" refers to an antibody which may be used for therapeutic
purposes, i.e., for the treatment of a disorder in a subject. It
should be noted that while therapeutic proteins may be used for
treatment purposes, the invention is not limited to such use, as
said proteins may also be used for in vitro studies.
[0073] As used herein, "buffer" is an agent(s) in a solution that
allows the solution to resist changes in pH by the action of its
acid-base conjugate components. Examples of buffers include acetate
(e.g. sodium acetate), succinate (such as sodium succinate),
gluconate, histidine, methionine, citrate, phosphate,
citrate/phosphate, imidazole, combinations thereof, and other
organic acid buffers. In one embodiment, a buffer is not a protein.
A buffer may provide a solution with a pH in the range from about 4
to about 8; from about 4.5 to about 7; or from about 5.0 to about
6.5.
[0074] Although the formulations of the invention do not contain a
buffer(s), otherwise identical formulations containing one or more
buffers may be used for pain or bioavailability comparison
purposes. Examples of such buffers include phosphate, acetate
(e.g., sodium acetate), succinate (such as sodium succinate),
gluconate, glutamate, histidine, citrate and other organic acid
buffers. In one embodiment, a representative buffer in the
otherwise identical formulation comprises a citrate buffer and/or a
phosphate buffer.
[0075] As used herein, the term "surfactant" generally includes an
agent that protects the protein, e.g., antibody, from air/solution
interface-induced stresses, solution/surface induced-stresses, to
reduce aggregation of the antibody, or to minimize the formation of
particulates in the formulation. Exemplary surfactants include, but
are not limited to, nonionic surfactants such as polysorbates (e.g.
polysorbates 20 and 80) or poloxamers (e.g. poloxamer 188). The
term "surfactant" or "detergent" includes nonionic surfactants such
as, but not limited to, polysorbates. In one embodiment, a
surfactant includes poloxamers, e.g., Poloxamer 188, Poloxamer 407;
polyoxyethylene alkyl ethers, e.g., Brij 35.RTM., Cremophor A25,
Sympatens ALM/230; and polysorbates/Tweens, e.g., Polysorbate 20
(Tween 20), Polysorbate 80 (Tween 80), Mirj, and Poloxamers, e.g.,
Poloxamer 188.
[0076] A "stable" formulation is one in which the antibody therein
essentially retains its physical stability and/or chemical
stability and/or biological activity during the manufacturing
process and/or upon storage. Various analytical techniques for
measuring protein stability are available in the art and are
reviewed in Peptide and Protein Drug Delivery 247-301, Vincent Lee
Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991); and Jones,
A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both incorporated by
reference). For example, in one embodiment, the stability of a
protein is determined according to the percentage of monomer
protein in the solution, with a low percentage of degraded (e.g.,
fragmented) and/or aggregated protein. In one embodiment, the
formulation may be stable at room temperature, at about
25-30.degree. C., or at 40.degree. C. for at least 1 month and/or
stable at about 2-8.degree. C. for at least 1 month, 1 year, or,
alternatively, for at least 2 years. In another embodiment, the
formulation may be stable up to about 30 degrees C. for at least
about 6 days, about 10, days, or about 14 days, or is stable at
about 28 degrees C. for up to about 24 months. In one embodiment,
the formulation may be stable following freezing (to, e.g.,
-70.degree. C.) and thawing of the formulation, hereinafter
referred to as a "freeze/thaw cycle."
[0077] An antibody "retains its physical stability" in a
pharmaceutical formulation if it shows substantially no signs of,
e.g., aggregation, precipitation and/or denaturation upon visual
examination of color and/or clarity, or as measured by UV light
scattering or by size exclusion chromatography. Aggregation is a
process whereby individual molecules or complexes associate
covalently or non-covalently to form aggregates. Aggregation can
proceed to the extent that a visible precipitate is formed.
[0078] Stability, such as physical stability of a formulation, may
be assessed by methods well-known in the art, including measurement
of a sample's apparent attenuation of light (absorbance, or optical
density). Such a measurement of light attenuation relates to the
turbidity of a formulation. The turbidity of a formulation is
partially an intrinsic property of the protein dissolved in
solution and is commonly determined by nephelometry, and measured
in Nephelometric Turbidity Units (NTU).
[0079] The degree of turbidity, e.g., as a function of the
concentration of one or more of the components in the solution,
e.g., protein and/or salt concentration, is also referred to as the
"opalescence" or "opalescent appearance" of a formulation. The
degree of turbidity can be calculated by reference to a standard
curve generated using suspensions of known turbidity. Reference
standards for determining the degree of turbidity for
pharmaceutical compositions can be based on the European
Pharmacopeia criteria (European Pharmacopoeia, Fourth Ed.,
Directorate for the Quality of Medicine of the Council of Europe
(EDQM), Strasbourg, France). According to the European Pharmacopeia
criteria, a clear solution is defined as one with a turbidity less
than or equal to a reference suspension which has a turbidity of
approximately 3 according to European Pharmacopeia standards.
Nephelometric turbidity measurements can detect Rayleigh scatter,
which typically changes linearly with concentration, in the absence
of association or nonideality effects. Other methods for assessing
physical stability are well-known in the art.
[0080] An antibody "retains its chemical stability" in a
pharmaceutical formulation, if the chemical stability at a given
time is such that the antibody is considered to still retain its
biological activity as defined below. Chemical stability can be
assessed by, e.g., detecting and quantifying chemically altered
forms of the antibody. Chemical alteration may involve size
modification (e.g. clipping) which can be evaluated using size
exclusion chromatography, SDS-PAGE and/or matrix-assisted laser
desorption ionization/time-of-flight mass spectrometry (MALDI/TOF
MS), for example. Other types of chemical alteration include charge
alteration (e.g. occurring as a result of deamidation or oxidation)
which can be evaluated by ion-exchange chromatography, for
example.
[0081] An antibody "retains its biological activity" in a
pharmaceutical formulation, if the antibody in a pharmaceutical
formulation is biologically active for its intended purpose. For
example, biological activity is retained if the biological activity
of the antibody in the pharmaceutical formulation is within about
30%, about 20%, or about 10% (within the errors of the assay) of
the biological activity exhibited at the time the pharmaceutical
formulation was prepared (e.g., as determined in an antigen binding
assay).
[0082] In a pharmacological sense, in the context of the present
invention, a "therapeutically effective amount" or "effective
amount" of an antibody refers to an amount effective in the
prevention or treatment or alleviation of a symptom of a disorder
for the treatment of which the antibody is effective.
[0083] The term "human TNF-alpha" (abbreviated herein as
hTNF-alpha, TNF.alpha., or simply hTNF), as used herein, is
intended to refer to a human cytokine that exists as a 17 kDa
secreted form and a 26 kDa membrane associated form, the
biologically active form of which is composed of a trimer of
noncovalently bound 17 kDa molecules. The structure of hTNF-alpha
is described further in, for example, Pennica, D., et al. (1984)
Nature 312:724-729; Davis, J. M., et al. (1987) Biochem
26:1322-1326; and Jones, E. Y., et al. (1989) Nature 338:225-228.
The term human TNF-alpha is intended to include recombinant human
TNF-alpha (rhTNF-alpha), which can be prepared by standard
recombinant expression methods or purchased commercially (R & D
Systems, Catalog No. 210-TA, Minneapolis, Minn.).
[0084] The term "antibody," as used herein, is intended to refer to
immunoglobulin molecules comprised of four polypeptide chains, two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds. Other naturally occurring antibodies of altered
structure, such as, for example, camelid antibodies, are also
included in this definition. Each heavy chain is comprised of a
heavy chain variable region (abbreviated herein as HCVR or VH) and
a heavy chain constant region. The heavy chain constant region is
comprised of three domains, CH1, CH2 and CH3. Each light chain is
comprised of a light chain variable region (abbreviated herein as
LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In one embodiment of the
invention, the formulation contains an antibody with CDR1, CDR2,
and CDR3 sequences like those described in U.S. Pat. Nos. 6,090,382
and 6,258,562, each incorporated by reference herein. In certain
embodiments, the formulation contains an antibody as claimed in
U.S. Pat. Nos. 6,090,382 and 6,258,562.
[0085] As used herein, the term "CDR" refers to the complementarity
determining region within a antibody variable sequence. There are
three CDRs in each of the variable regions of the heavy chain and
the light chain, which are designated CDR1, CDR2 and CDR3, for each
of the heavy and light chain variable regions. The exact boundaries
of these CDRs have been defined differently according to different
systems. The system described by Kabat (Id.) not only provides an
unambiguous residue numbering system applicable to any variable
region of an antibody, but also provides precise residue boundaries
defining the three CDRs. These CDRs may be referred to as Kabat
CDRs. Chothia et al. found that certain sub-portions within Kabat
CDRs adopt nearly identical peptide backbone conformations, despite
having great diversity at the level of amino acid sequence (Chothia
et al. (1987) Mol. Biol. 196:901-917; Chothia et al. (1989) Nature
342:877-883) These sub-portions were designated as L1, L2 and L3 or
H1, H2 and H3 where the "L" and the "H" designates the light chain
and the heavy chains regions, respectively. These regions may be
referred to as Chothia CDRs, which have boundaries that overlap
with Kabat CDRs. Other boundaries defining CDRs overlapping with
the Kabat CDRs have been described by Padlan (1995) FASEB J.
9:133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732-45. Still
other CDR boundary definitions may not strictly follow one of the
herein described systems, but will nonetheless overlap with the
Kabat CDRs, although they may be shortened or lengthened in light
of prediction or experimental findings that particular residues or
groups of residues or even entire CDRs do not significantly impact
antigen binding. The methods used herein may utilize CDRs defined
according to any of these systems, although certain embodiments use
Kabat or Chothia defined CDRs. In one embodiment, the antibody used
in the methods and compositions of the invention includes the six
CDRs from the antibody adalimumab.
[0086] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., hTNF-alpha). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., (1989) Nature 341:544-546), which consists
of a VH domain; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding portion" of an antibody. Other
forms of single chain antibodies, such as diabodies are also
encompassed. Diabodies are bivalent, bispecific antibodies in which
VH and VL domains are expressed on a single polypeptide chain, but
using a linker that is too short to allow for pairing between the
two domains on the same chain, thereby forcing the domains to pair
with complementary domains of another chain and creating two
antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)
Structure 2:1121-1123). In one embodiment of the invention, the
formulation contains an antigen-binding portions described in U.S.
Pat. Nos. 6,090,382 and 6,258,562, each incorporated by reference
herein.
[0087] The phrase "recombinant antibody" refers to antibodies that
are prepared, expressed, created or isolated by recombinant means,
such as antibodies expressed using a recombinant expression vector
transfected into a host cell, antibodies isolated from a
recombinant, combinatorial antibody library, antibodies isolated
from an animal (e.g., a mouse) that is transgenic for human
immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids
Res. 20:6287-6295) or antibodies prepared, expressed, created or
isolated by any other means that involves splicing of particular
immunoglobulin gene sequences (such as human immunoglobulin gene
sequences) to other DNA sequences. Examples of recombinant
antibodies include recombinant human, chimeric, CDR-grafted and
humanized antibodies.
[0088] The term "human antibody," as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
used in the invention may include amino acid residues not encoded
by human germline immunoglobulin sequences (e.g., mutations
introduced by random or site-specific mutagenesis in vitro or by
somatic mutation in vivo), for example in the CDRs and in
particular CDR3. However, the term "human antibody," as used
herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework
sequences.
[0089] The term "chimeric antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species and constant region sequences from another species, such as
antibodies having murine heavy and light chain variable regions
linked to human constant regions.
[0090] The term "CDR-grafted antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species but in which the sequences of one or more of the CDR
regions of VH and/or VL are replaced with CDR sequences of another
species, such as antibodies having murine heavy and light chain
variable regions in which one or more of the murine CDRs (e.g.,
CDR3) has been replaced with human CDR sequences.
[0091] An "isolated antibody," as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds hTNF-alpha is substantially free
of antibodies that specifically bind antigens other than
hTNF-alpha). An isolated antibody that specifically binds
hTNF-alpha may, however, have cross-reactivity to other antigens,
such as TNF-alpha molecules from other species. Moreover, an
isolated antibody may be substantially free of other cellular
material and/or chemicals.
[0092] A "neutralizing antibody," as used herein (or an "antibody
that neutralized hTNF-alpha activity"), is intended to refer to an
antibody whose binding to hTNF-alpha results in inhibition of the
biological activity of hTNF-alpha. This inhibition of the
biological activity of hTNF-alpha can be assessed by measuring one
or more indicators of hTNF-alpha biological activity, such as
hTNF-alpha-induced cytotoxicity (either in vitro or in vivo),
hTNF-alpha-induced cellular activation and hTNF-alpha binding to
hTNF-alpha receptors. These indicators of hTNF-alpha biological
activity can be assessed by one or more of several standard in
vitro or in vivo assays known in the art, and described in U.S.
Pat. Nos. 6,090,382 and 6,258,562, each incorporated by reference
herein. In one embodiment, the ability of an antibody to neutralize
hTNF-alpha activity is assessed by inhibition of hTNF-alpha-induced
cytotoxicity of L929 cells. As an additional or alternative
parameter of hTNF-alpha activity, the ability of an antibody to
inhibit hTNF-alpha-induced expression of ELAM-1 on HUVEC, as a
measure of hTNF-alpha-induced cellular activation, can be
assessed.
[0093] The term "surface plasmon resonance," as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example using the
BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Jonsson, U., et
al. (1993) Ann Biol. Clin. 51:19-26; Jonsson, U., et al. (1991)
Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol.
Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.
198:268-277.
[0094] The term "k.sub.on," as used herein, is intended to refer to
the on rate constant for association of a binding protein (e.g., an
antibody) to the antigen to form the, e.g., antibody/antigen
complex as is known in the art.
[0095] The term "k.sub.off," as used herein, is intended to refer
to the off rate constant for dissociation of an antibody from the
antibody/antigen complex.
[0096] The term "K.sub.d," as used herein, is intended to refer to
the dissociation constant of a particular antibody-antigen
interaction and refers to the value obtained in a titration
measurement at equilibrium, or by dividing the dissociation rate
constant (k.sub.off) by the association rate constant
(k.sub.on).
[0097] As used herein, "biosimilar" (of an approved reference
product/biological drug, such as a protein therapeutic, antibody,
etc.) refers to a biologic product that is similar to the reference
product based upon data derived from (a) analytical studies that
demonstrate that the biological product is highly similar to the
reference product notwithstanding minor differences in clinically
inactive components; (b) animal studies (including the assessment
of toxicity); and/or (c) a clinical study or studies (including the
assessment of immunogenicity and pharmacokinetics or
pharmacodynamics) that are sufficient to demonstrate safety,
purity, and potency in one or more appropriate conditions of use
for which the reference product is licensed and intended to be used
and for which licensure is sought for the biological product. In
one embodiment, the biosimilar biological product and reference
product utilize the same mechanism or mechanisms of action for the
condition or conditions of use prescribed, recommended, or
suggested in the proposed labeling, but only to the extent the
mechanism or mechanisms of action are known for the reference
product. In one embodiment, the condition or conditions of use
prescribed, recommended, or suggested in the labeling proposed for
the biological product have been previously approved for the
reference product. In one embodiment, the route of administration,
the dosage form, and/or the strength of the biological product are
the same as those of the reference product. In one embodiment, the
facility in which the biological product is manufactured,
processed, packed, or held meets standards designed to assure that
the biological product continues to be safe, pure, and potent. The
reference product may be approved in at least one of the U.S.,
Europe, or Japan.
[0098] The term "dosing", as used herein, refers to the
administration of a substance (e.g., an anti-TNFa antibody) to
achieve a therapeutic objective (e.g., the treatment of a
TNFa-associated disorder).
[0099] The terms "weekly dosing regimen", "weekly dosing" and
"weekly administration" as used herein, refer to a certain time
course (or periodicity) of administering a substance (e.g., an
anti-TNF.alpha. antibody) to a subject to achieve a therapeutic
objective (e.g., the treatment of a TNF.alpha..-associated
disorder). In one embodiment, the antibody, or antigen-binding
portion thereof, is administered every 6-8 days, or, alternatively,
every 7 days.
[0100] The terms "biweekly dosing regimen", "biweekly dosing", and
"biweekly administration", as used herein, refer to a certain time
course (or periodicity) of administering a substance (e.g., an
anti-TNF.alpha. antibody) to a subject to achieve a therapeutic
objective (e.g., the treatment of a TNF.alpha..-associated
disorder). The biweekly dosing regimen is not intended to include a
weekly dosing regimen. In one embodiment, the antibody, or
antigen-binding portion thereof, is administered every 9-19 days,
more preferably, every 11-17 days, even more preferably, every
13-15 days, and most preferably, every 14 days.
[0101] The terms "monthly dosing regimen", "monthly dosing", and
"monthly administration", as used herein, refer to a certain time
course (or periodicity) of administering a substance (e.g., an
anti-TNF.alpha. antibody) to a subject to achieve a therapeutic
objective (e.g., the treatment of a TNF.alpha..-associated
disorder). In one embodiment, a monthly dosing regimen means that
the antibody, or antigen-binding portion thereof, is administered
every 28-31 days. In another embodiment, a monthly dosing regimen
means that the antibody, or antigen-binding portion thereof, is
administered once a month, e.g. on the same day each month, such
as, for example, the first day of each month.
[0102] AUC, Cmax, and Tmax are pharmacokinetic parameters that may
be used to characterize the pharmacokinetic responses of a
particular drug product in an animal or human subject. The term
"AUC" refers to the "area under the curve" that represents changes
in blood, serum, or plasma concentrations of a substance, e.g., a
human anti-TNF.alpha. antibody, over time. As used herein, the term
"Cmax" refers to the maximum or peak blood, serum, or plasma
concentration of substance observed in a subject after its
administration. The term "Tmax" refers to the time at which the
Cmax occurred, as measured from the time point of
administration."
[0103] The term "hydrodynamic diameter" or "D.sub.h" of a particle
refers to the diameter of a sphere that has the density of water
and the same velocity as the particle. Thus, the term "hydrodynamic
diameter of an antibody" as used herein refers to a size
determination for an antibody, or an antigen-binding portion
thereof, e.g., a human anti-TNF.alpha. antibody, or antigen-binding
fragment thereof, in solution using dynamic light scattering (DLS).
A DLS-measuring instrument measures the time-dependent fluctuation
in the intensity of light scattered from the antibody, or
antigen-binding fragment thereof, in solution at a fixed scattering
angle. D.sub.h is determined from the intensity autocorrelation
function of the time-dependent fluctuation in intensity. Scattering
intensity data are processed using DLS instrument software to
determine the value for the hydrodynamic diameter and the size
distribution of the scattering molecules, e.g. the human
anti-TNF.alpha. antibody, or antigen-binding fragment thereof,
specimen.
[0104] The term "conductivity," as used herein, refers to the
ability of an aqueous solution to conduct an electric current
between two electrodes. Generally, electrical conductivity or
specific conductivity is a measure of a material's ability to
conduct an electric current. In solution, the current flows by ion
transport. Therefore, with an increasing amount of ions present in
the aqueous solution, the solution will have a higher conductivity.
The unit of measurement for conductivity is mmhos (mS/cm), and can
be measured using a conductivity meter sold, e.g., by Orion
Research, Inc. (Beverly, Mass.). The conductivity of a solution may
be altered by changing the concentration of ions therein. For
example, the concentration of buffer and/or salt, in the solution
may be altered in order to achieve the desired conductivity.
[0105] Conductivity of a solution is measured according to methods
known in the art. Conductivity meters and cells may be used to
determine the conductivity of the aqueous formulation, and should
be calibrated to a standard solution before use. Examples of
conductivity meters available in the art include MYRON L Digital
(Cole Parmer.RTM.), Conductometer (Metrohm AG), and Series
3105/3115 Integrated Conductivity Analyzers (Kemotron).
[0106] Conductivity measurements may be taken with any commercially
available conductivity meter suitable for conductivity analysis in
protein solutions, e.g. conductivity meter Model SevenMulti, with
expansion capacity for broad pH range (Mettler Toledo,
Schwerzenbach, Switzerland). The instrument is operated according
to the manufacturers instructions (e.g., if the conductivity sensor
is changed in the Mettler Toledo instrument, calibration must be
performed again, as each sensor has a different cell constant;
refer to Operating Instructions of Model SevenMulti conductivity
meter). If the instructions are followed, conductivity measurements
can be taken by directly immersing the measuring probe into the
sample solution.
[0107] Various aspects of the invention are described in further
detail in the following subsections.
II. Formulations and Methods of the Invention
[0108] The present invention features stable, liquid aqueous
pharmaceutical formulations comprising an anti-TNF.alpha. antibody,
or an antigen binding portion thereof, having improved properties
as compared to art-recognized formulations. While high
concentration formulations containing human anti-TNF.alpha.
antibodies are known in the art (see, for example, US20060153846
and US20100278822), the instant invention provides high
concentration formulations having unexpected characteristics, i.e.,
significantly decreased pain or increased bioavailability. The
formulations of the invention are based, at least in part, on the
combination of only one or two excipients, i.e., a surfactant and a
polyol or, alternatively, a surfactant alone. Despite having few
excipients, the formulations of the invention contain a high
concentration of an antibody, e.g. 90-110 mg/ml, and are
stable.
[0109] As described in the working examples below, a formulation
containing an antibody concentration of more than 50 mg/ml of an
isolated human anti-TNF.alpha. antibody, less than 50 mg/ml of a
polyol, (such as mannitol), and a surfactant, (such as a
polysorbate), was shown to have dramatically reduced pain upon
injection relative to other high concentration formulations,
including the commercial adalimumab formulation described in
US20060153846, and the formulation described in US20100278822, each
of which is incorporated by reference herein. Thus, in one
embodiment, the formulations of the invention are associated with a
reduction of pain, despite having a high antibody concentration
(e.g., 100 mg/mL) and having no buffer or salt. The low-pain
formulations described herein are based, at least in part, on the
surprising finding that by removing or excluding salt (e.g., NaCl)
and/or a buffer (e.g., a phosphate/citrate buffer) the
concentration of a human anti-TNF alpha antibody in a formulation
can be increased, e.g., to about 100 mg/mL, while decreasing pain
upon delivery to a patient.
[0110] In one embodiment, the formulation of the invention is
surprising, in that the formulation does not contain a buffer or a
salt, and reduces pain associated with injection in a patient by at
least about 50% when compared to injecting an otherwise identical
formulation comprising at least one salt and/or at least one
buffer. In one embodiment, the formulation reduces pain associated
with the injection in a human subject by at least about 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% (e.g., about 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 50, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, or 80%) when compared to the injection of an otherwise
identical formulation that further comprises a salt and/or a
buffer.
[0111] In one embodiment, the otherwise identical formulation used
for pain comparison assay comprises at least one buffer, such as a
citrate buffer and a phosphate buffer, and/or a salt, e.g., NaCl.
For example, the buffer (excluded from the formulation of the
invention and present in the reference formulation for pain
comparisons) may include citric acid monohydrate, sodium citrate,
disodium phosphate dihydrate, and/or sodium dihydrogen phosphate
dihydrate. The buffer may include about 1.15-1.45 mg/ml of citric
acid (e.g., about 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, or 1.45),
about 0.2-0.4 mg/mL of sodium citrate dehydrate (e.g., about 0.2,
0.25, 0.3, 0.35, or 0.4), about 1.35-1.75 mg/mL of disodium
phosphate dehydrate (e.g., about 1.35, 1.40, 1.45, 1.50, 1.55,
1.60, 1.65, 1.70, or 1.75), about 0.75-0.95 mg/mL of sodium
dihydrogen phosphate dehydrate (e.g., about 0.75, 0.80, 0.85, 0.9,
or 0.95). Values and ranges intermediate to the aforementioned
concentrations are also intended to be part of this invention. In
addition, ranges of values using a combination of any of the
above-recited values as upper and/or lower limits are intended to
be included, e.g., 0.1 to 0.5 mg/mL or 1.20-1.40 mg/mL. In one
embodiment, the pH of the formulation is adjusted with sodium
hydroxide.
[0112] In one embodiment, the formulation of the invention includes
high concentrations of human anti-TNFa antibodies, or antigen
binding portions thereof, e.g., 90-110 mg/ml, a polyol at a
concentration less than 50 mg/ml, and a surfactant, such that the
formulation is suitable for administration without significant pain
as determined by a visual analog scale (VAS) score. In one
embodiment, the formulation and methods of the invention include
high concentrations of anti-TNF.alpha. antibodies, or antigen
binding portions thereof, and no buffer or salt, such that they are
suitable for, administration, e.g., subcutaneous administration,
without significant felt pain as determined by a visual analog
scale (VAS) score. For example, the formulation of the invention
may result in a VAS score of less than 1 on a scale of 0 (no pain)
to 10 (worst imaginable pain) following subcutaneous injection. As
described in Example 1, a formulation having 100 mg/ml of
adalimumab, polysorbate 80, and mannitol (less than 50 mg/ml)
resulted in a VAS score of less than 1, e.g., 0.56, whereas other
high antibody concentration formulations resulted in VAS scores
ranging from 1.79 to 4.12.
[0113] In one embodiment, the invention provides a liquid aqueous
formulation comprising an isolated human anti-TNF.alpha. antibody,
or an antigen-binding portion thereof, a surfactant, and less than
50 mg/ml of a polyol, wherein subcutaneous injection of the
formulation results in a Pain Visual Analog Scale score of less
than 1.0 following injection. In one embodiment, the formulation
does not contain a buffer and a salt, and results in a reduction of
pain of at least about 50% upon subcutaneous injection when
compared to an injection of an otherwise identical formulation that
further comprises a salt and/or a buffer(s).
[0114] Thus, in one aspect of the invention, liquid formulations of
the invention have advantageous tolerability properties in that the
formulations produce less pain relative to formulations containing
a buffer and a salt. In certain embodiments, the formulation
reduces pain associated with injection (or any other form of
administration) in a subject. In some embodiments, pain associated
with injection is reduced by at least about 10%, at least about
15%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, or at least about 95% (e.g.,
at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, or 95%). In one embodiment, pain is reduced by at
least about 50%.
[0115] Pain may be evaluated using any type of pain assessment
known in the art, including, for example, visual analog scales,
qualitative assessments of pain, or needle pain assessments. For
example, subject-perceived injection site pain may be assessed
using the Pain Visual Analog Scale (VAS). A VAS is a measurement
instrument that measures pain as it ranges across a continuum of
values, e.g., from none to an extreme amount of pain. Operationally
a VAS is a horizontal line, about 100 mm in length, anchored by
numerical and/or word descriptors, e.g., 0 or 10, or "no pain" or
"excruciating pain," optionally with additional word or numeric
descriptors between the extremes, e.g., mild, moderate, and severe;
or 1 through 9) (see, e.g., Lee J S, et al. (2000) Acad Emerg Med
7:550, or Singer and Thods (1998) Academic Emergency Medicine
5:1007). Pain may be assessed at a single time or at various times
following administration of a formulation of the invention such as,
for example, immediately after injection, at about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 minutes after
injection.
[0116] In a certain embodiment of the invention, injection of the
formulation into a subject results in a Pain Visual Analog Scale
score of less than 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, or 5.0
on a scale of 0 (no pain) to 10 (excruciating pain).
[0117] Other tools for pain assessment are known in the art,
including, for example, the Numerical Rating Scale, the Verbal
Rating Scale, and the Brief Pain Inventory. Such tools could also
be used to assess pain in accordance with the invention.
[0118] Additional indices for skin irritation may be used,
including, e.g., the Draize Scale (hemorrhage, petechiae, erythema,
edema, pruritus).
[0119] Formulations of the invention containing a polyol preferably
contain less than about 50 mg of the polyol. In one embodiment, the
formulations contain less than about 45 mg/mL of the polyol. In
another embodiment, the formulations of the invention contain about
38-46 mg/mL of the polyol (e.g., mannitol), e.g., about 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
or 55 mg/mL of the polyol. In addition, ranges of values using a
combination of any of the above recited values as upper and/or
lower limits are intended to be included, e.g., 39-45 mg/ml, 40-44
mg/ml, or 37-47 mg/ml. In one embodiment, the formulations of the
invention contain about 12-72 mg/ml of polyol, e.g., mannitol. In
one embodiment, suitable polyols for use in the formulations and
methods of the invention are mannitol or sorbitol.
[0120] In one embodiment, the formulation of the invention contains
adalimumab (or a biosimilar thereof), polysorbate 80, mannitol, and
water for injection. In one embodiment, the formulation contains 80
mg of adalimumab, water for injection, 42 mg/ml of mannitol, and 1
mg/ml of polysorbate 80. In one embodiment, the formulation may
contain 20-110 mg, alternatively 20-90 mg of adalimumab or,
alternatively, 30-80 mg of the antibody. In one embodiment, the
formulation contains 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36
mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg,
46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55
mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg,
65 mg, 66 mg, 67 mg, 68 mg 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74
mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg,
84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93
mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 101 mg, 102
mg, 103 mg, 104 mg, 105 mg, 106 mg, 107 mg, 108 mg, 109 mg, or 110
mg of the antibody. Ranges including the aforementioned numbers are
also included in the invention, e.g., 70-90 mg, 65-95, or 60-85
mg.
[0121] The present invention is also based, at least in part on the
surprising discovery that a liquid aqueous pharmaceutical
formulation having a high concentration of a human anti-TNF.alpha.
antibody, or antigen binding portion thereof, and a surfactant
(i.e., in the absence of additional excipients), has greater
bioavailability than other high concentration formulations having
additional excipients. As described in the working examples below,
a formulation containing more than 50 mg/ml of an isolated human
anti-TNF.alpha. antibody, and a polysorbate was shown to have
increased bioavailability relative to other high concentration
formulations, including the commercial adalimumab formulation
described in US20060153846.
[0122] As described in Example 2 below, bioavailability of an
anti-TNF.alpha. antibody can be increased by combining the antibody
with a surfactant, e.g., polysorbate 80. The increase in
bioavailability is based on the combination of the antibody and
surfactant and the omission or removal of other excipients,
including a buffer, polyol, and salt. The increase in
bioavailability results in an AUC.sub.0-360 of the anti-TNF.alpha.
antibody, or an antigen-binding portion thereof, of greater than
about 1300 .mu.g*hr/ml or an AUC.sub.0-1344 of the anti-TNF.alpha.
antibody, or an antigen-binding portion thereof, of greater than
about 2600 .mu.g*hr/ml, when subcutaneously injected into a human
subject.
[0123] Accordingly, the present invention provides methods for
improving the bioavailability of an isolated anti-TNF.alpha.
antibody, or an antigen-binding portion thereof, in a
pharmaceutical formulation. The methods include combining a
therapeutically effective amount of the anti-TNF.alpha. antibody,
or antigen-binding portion thereof, with a surfactant and excluding
or removing other excipients, e.g., a buffer(s), salt, and polyol,
or combinations thereof, such that the bioavailability of the
antibody, or antigen-binding portion thereof, is improved. In one
embodiment, the formulation is injected subcutaneously into a human
subject. The methods may improve the bioavailability by providing
an AUC.sub.0-360 of the anti-TNF.alpha. antibody, or an
antigen-binding portion thereof, of greater than about 1100, 1125,
1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400,
1425, 1450, 1475, or about 1500 .mu.g*hr/ml when subcutaneously
injected into a human subject.
[0124] The invention further provides a method of improving the
bioavailability of an isolated human anti-TNF.alpha. antibody, or
an antigen-binding portion thereof, in a subject, said method
comprising administering a formulation comprising a surfactant and
an effective amount of the antibody, or antigen-binding portion
thereof, to the subject such that the bioavailability of the
antibody, or antigen-binding portion thereof, in the subject is
improved at least about 15% over a second formulation. In one
embodiment, the formulation of the invention does not contain a
buffer, a polyol, or a salt, and the second formulation comprises a
buffer, a polyol, and a salt. In one embodiment, the
bioavailability of the antibody, or antigen-binding portion
thereof, is improved at least about 30% over the second
formulation. In one embodiment, the bioavailability of the
antibody, or antigen-binding portion thereof, is improved at least
about 40% over the second formulation. In one embodiment, the
bioavailability may be determined according to either an AUC level,
e.g., AUC.sub.0-360 or an AUC.sub.0-1344, or a Cmax.
[0125] In one embodiment, the present invention provides a liquid
aqueous formulation which includes a surfactant and about 30-90 mg
of an isolated human anti-TNF.alpha. antibody or antigen-binding
portion, wherein the formulation has an antibody concentration of
about 90-110 mg/ml, and wherein the formulation provides increased
bioavailability of the antibody, or antigen-binding portion
thereof, to a human subject upon subcutaneous injection of the
formulation relative to a formulation comprising citrate phosphate
buffer, sodium chloride, and mannitol.
[0126] In one embodiment, the present invention provides liquid
aqueous formulations which include a surfactant and 30-90 mg of an
isolated human anti-TNF.alpha. antibody, or an antigen-binding
portion, wherein the formulation has an antibody concentration of
90-110 mg/ml, and wherein the formulation provides increased
bioavailability of the antibody, or antigen-binding portion
thereof, to a human subject upon subcutaneous injection of the
formulation, such that the antibody or antigen-binding portion
thereof, has an AUC.sub.0-360 greater than about 1100, 1125, 1150,
1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425,
1450, 1475, or about 1500 mg*hr/ml.
[0127] In one embodiment, the formulation of the invention contains
adalimumab (or a biosimilar thereof), polysorbate 80, and water for
injection. In one embodiment, the formulation contains 80 mg of
adalimumab, water for injection, and 1 mg/ml polysorbate 80. The
formulation may contain 20-110 mg, alternatively 20-90 mg of
adalimumab or, alternatively, 30-80 mg of the antibody. In one
embodiment, the formulation contains 30 mg, 31 mg, 32 mg, 33 mg, 34
mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg,
44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53
mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg,
63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg 69 mg, 70 mg, 71 mg, 72
mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg,
82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91
mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg,
101 mg, 102 mg, 103 mg, 104 mg, 105 mg, 106 mg, 107 mg, 108 mg, 109
mg, or 110 mg of the antibody. Ranges including the aforementioned
numbers are also included in the invention, e.g., 70-90 mg, 65-95
mg, or 60-85 mg.
[0128] Thus, the high antibody formulations and methods of the
invention not only overcome a number of known challenges for
pharmaceutical formulations, including high concentrations in a
stable formulation, but also possesses the added benefit of
producing improved bioavailability or providing significantly low
levels of pain when injected into patients.
[0129] Another obstacle overcome by the formulations of the
invention is the ability to remain stable at room temperature (at
about 25 degree C. or up to about 30 degrees C.). Such stability
provides advantages for the user of the antibody, providing for
more flexible storage options, as the constant need for
refrigeration is unnecessary. Both the decreased pain formulation
and the increased bioavailability formulation (exemplified by
formulations F3 and F4, respectively, in the Examples below) are
stable for at least 6 days at about 25 degrees C. or up to about 30
degrees C. As described in more detail in the Examples, the
formulations of the invention are stable at up to 30 degrees C. for
at least 6 days, at least 7 days, at least 8 days, at least 9 days,
at least 10 days, at least 11 days, at least 12 days, at least 13
days, and at least 14 days. Thus, the invention further provides
formulations having extended (i.e., at least 6 days, 10 days or 14
days) shelf life at room temperature (or about 25 degrees C. or up
to about 30 degrees C.). In one embodiment, the formulation of the
invention is stable at 20 to 32 degrees C. for at least 6 days.
Temperatures intermediate to the above recited concentrations are
also intended to be part of this invention, i.e., 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31 and 32 degrees Celsius (C). Ranges
including the aforementioned temperatures are also included in the
invention, e.g., 22-26 degrees C., 25-30 degrees C., etc.
[0130] The formulations of the invention contain a high antibody
concentration, including, for example, an antibody concentration of
about 50 mg/mL, 55 mg/mL, 60 mg/mL. 65 mg/mL, 70 mg/mL, 75 mg/ml,
80 mg/mL, 85 mg/mL, 90 mg/mL, 95 mg/mL 100 mg/mL, 105 mg/mL, 110
mg/mL, 115 mg/mL (or higher) of a human anti-TNF-alpha antibody or
antigen-binding fragment thereof. Accordingly, as described in the
examples below, in one aspect of the invention the liquid
pharmaceutical formulations of the invention contain a human
anti-TNF alpha antibody concentration of 50-100 mg/mL or greater.
In one embodiment, the formulations of the invention may comprise
an antibody concentration between about 1 mg/mL-150 mg/mL or about
40 mg/mL-125 mg/mL. In one embodiment, the antibody concentration
of the formulation is 50-150 mg/ml, 55-150 mg/ml, 60-150 mg/ml,
65-150 mg/ml, 70-150 mg/ml, 75-150 mg/ml, 80-150 mg/ml, 85-150
mg/ml, 90-150 mg/ml, 90-110 mg/ml, 95-105 mg/ml, 95-150 mg/ml,
100-150 mg/ml, 105-150 mg/ml, 110-150 mg/ml, 115-150 mg/ml, 120-150
mg/ml, 125-150 mg/ml, 50-130 mg/ml, 75-125 mg/ml, etc.
Concentrations and ranges intermediate to the above recited
concentrations are also intended to be part of this invention
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,
115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,
128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,
141, 142, 143, 144, 145, 146, 147, 148, 149, 150 mg/mL).
[0131] The formulations of the invention may contain an effective
amount of the antibody. In one embodiment, an effective amount is
about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, or about 100 mg of the human anti-TNF.alpha. antibody, or
antigen-binding portion thereof. In one embodiment, the
formulations and methods of the invention comprise about 20-100,
about 20-90, about 30-90, about 30-100, about 60-100, about 70-90,
about 40-90, about 60-85 mg, or about 40-100 mg of a human
anti-TNF.alpha. antibody, or antigen-binding portion thereof. In
one embodiment, the formulation contains 30 mg, 31 mg, 32 mg, 33
mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg,
43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52
mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg,
62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg 69 mg, 70 mg, 71
mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg,
81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, or
90 mg of the antibody. Ranges including the aforementioned numbers
are also included in the invention, e.g., 70-90 or 75-85 mg or
60-85 mg.
[0132] An important aspect of the formulations and methods of the
invention is the omission of a buffer and salt. Thus, in one
embodiment, the formulations and methods of the invention do not
contain any buffer(s) (e.g., citrate and phosphate) and salts. It
should be noted, however, that although the preferred formulations
of the invention do not contain buffers or salts (e.g., NaCl), a
small amount of buffer and/or salt may be present in the
formulations. Thus, in one embodiment, the formulations of the
invention do not contain detectable levels of a buffer(s) and/or a
salt.
[0133] In one embodiment, the buffer(s) omitted from the
formulations of the invention (or those formulations for comparison
which include a buffer(s)) may include citric acid (e.g., about
1.3-1.31 mg/mL or 1.305 mg/mL). In another embodiment, the buffer
system includes sodium citrate dehydrate (e.g., about 0.27-0.33
mg/mL or about 0.305 mg/mL). In one embodiment, the buffer system
includes disodium phosphate dehydrate (e.g., about 1.5-1.56 mg/mL
or about 1.53 mg/mL). In another embodiment, the buffer system
includes sodium dihydrogen phosphate dihydrate (e.g., about
0.83-0.89 mg/mL or about 0.86 mg/mL).
[0134] In one embodiment of the invention, the conductivity of the
formulation may be used to determine if a formulation has a buffer
and/or salt. Both Formulation F3 and F4 (described in the working
examples below) have been determined to have a conductivity of less
than about 2 mS/cm, e.g., about 0.70/cm. Thus, in one embodiment,
the reduced pain and increased bioavailability formulations of the
invention have a conductivity of less than about 2 mS/cm. In
another embodiment, the formulations of the invention have a
conductivity of less than about 1 mS/cm.
[0135] In one embodiment, the formulation of the invention contains
of a human anti-TNF alpha antibody, or antigen binding portion
thereof, at a concentration of about 100 mg/mL (or 75-125 mg/mL), a
surfactant (e.g., polysorbate 80), a polyol (e.g., sorbitol or
mannitol), and has a conductivity of less than 2 mS/cm. In one
embodiment, the formulation of the invention contains of a human
anti-TNF alpha antibody, or antigen binding portion thereof, at a
concentration of about 100 mg/mL (or 75-125 mg/mL), about 0.8-1.3
mg/ml of a surfactant (e.g., polysorbate 80), less than about 50
mg/ml of a polyol (e.g., sorbitol or mannitol), and has a
conductivity of less than 2 mS/cm.
[0136] In one embodiment, the formulation of the invention contains
of a human anti-TNF alpha antibody, or antigen binding portion
thereof, at a concentration of about 100 mg/mL (or 75-125 mg/mL), a
surfactant (e.g., polysorbate 80), and has a conductivity of less
than 2 mS/cm. In one embodiment, the formulation of the invention
contains of a human anti-TNF alpha antibody, or antigen binding
portion thereof, at a concentration of about 100 mg/mL (or 75-125
mg/mL), about 0.8-1.3 mg/ml of a surfactant (e.g., polysorbate 80),
and has a conductivity of less than 2 mS/cm.
[0137] In another embodiment, the invention provides a stable
formulation having a high concentration antibody, or
antigen-binding portion thereof, wherein the antibody, or antigen
has a hydrodynamic diameter (z-average) of less than about 4 nm or
wherein the antibody, or antigen has a hydrodynamic diameter
(z-average) which is at least about 50% less than the hydrodynamic
diameter of a buffered solution at the same antibody concentration.
In one embodiment, the antibody, or antigen has a hydrodynamic
diameter (z-average) of less than about 3 nm.
[0138] In one embodiment, the formulation of the invention contains
of a human anti-TNF alpha antibody, or antigen binding portion
thereof, at a concentration of about 100 mg/mL (or 75-125 mg/mL), a
surfactant (e.g., polysorbate 80), a polyol (e.g., sorbitol or
mannitol), and has a hydrodynamic diameter of less than 4 nm. In
one embodiment, the formulation of the invention contains of a
human anti-TNF alpha antibody, or antigen binding portion thereof,
at a concentration of about 100 mg/mL (or 75-125 mg/mL), about
0.8-1.3 mg/ml of a surfactant (e.g., polysorbate 80), less than
about 50 mg/ml of a polyol (e.g., sorbitol or mannitol), and has a
hydrodynamic diameter of less than 4 nm.
[0139] In one embodiment, the formulation of the invention contains
of a human anti-TNF alpha antibody, or antigen binding portion
thereof, at a concentration of about 100 mg/mL (or 75-125 mg/mL), a
surfactant (e.g., polysorbate 80), and has a hydrodynamic diameter
of less than 4 nm. In one embodiment, the formulation of the
invention contains of a human anti-TNF alpha antibody, or antigen
binding portion thereof, at a concentration of about 100 mg/mL (or
75-125 mg/mL), about 0.8-1.3 mg/ml of a surfactant (e.g.,
polysorbate 80), and has a hydrodynamic diameter of less than 4
nm.
[0140] A detergent or surfactant is included in the antibody
formulation of the invention. Exemplary detergents include nonionic
detergents such as polysorbates (e.g. polysorbates 20, 80, etc.) or
poloxamers (e.g. poloxamer 188). The amount of detergent added is
such that it reduces aggregation of the formulated antibody and/or
minimizes the formation of particulates in the formulation and/or
reduces adsorption. In a preferred embodiment of the invention, the
formulation includes a surfactant which is a polysorbate. In
another preferred embodiment of the invention, the formulation
contains the detergent polysorbate 80. In one embodiment, the
formulation contains between about 0.1 and about 2.0 mg/mL of
surfactant (e.g., polysorbate), e.g., about 1 mg/mL. Other ranges
of polysorbate that may be included in the formulations of the
invention include 0.1 to 1.5 mg/ml, alternatively 0.2-1.4 mg/ml,
0.3-1.3 mg/ml, 0.4-1.2 mg/ml, 0.5-1.1 mg/ml, 0.6-1.0 mg/ml, 0.6-1.1
mg/ml, 0.7-1.1 mg/ml, 0.8-1.1 mg/ml, or 0.9-1.1 mg/ml. Values and
ranges intermediate to the above recited concentrations are also
intended to be part of this invention, e.g., 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9.
In addition, ranges of values using a combination of any of the
above-recited values as upper and/or lower limits are intended to
be included, e.g., 0.3 to 1.1 mg/mL.
[0141] In one embodiment, the formulation of the invention consists
essentially of a human anti-TNF alpha antibody, or antigen binding
portion thereof, at a concentration of about 100 mg/mL (or 75-125
mg/mL), a surfactant (e.g., polysorbate 80), a polyol (e.g.,
sorbitol or mannitol), does not contain a buffer(s) (e.g., citric
acid monohydrate, sodium citrate, disodium phosphate dihydrate,
and/or sodium dihydrogen phosphate dihydrate), and does not contain
a salt (e.g., NaCl).
[0142] In certain embodiments, the otherwise identical formulation
to which the formulation of the invention is compared for pain or
bioavailability purposes is a formulation containing adalimumab,
sodium chloride, monobasic sodium phosphate dihydrate, dibasic
sodium phosphate dihydrate, sodium citrate, citric acid
monohydrate, mannitol, polysorbate 80, and Water for Injection.
[0143] The formulation herein may also be combined with one or more
other therapeutic agents as necessary for the particular indication
being treated. In one embodiment, those with complementary
activities that do not adversely affect the antibody of the
formulation. Such therapeutic agents are suitably present in
combination in amounts that are effective for the purpose intended.
Additional therapeutic agents which can be combined with the
formulation of the invention are further described in U.S. Pat.
Nos. 6,090,382 and 6,258,562, each of which is incorporated herein
by reference. All formulations described herein may be used in the
methods of the invention as well.
III. Antibodies for Use in the Formulations and Methods of the
Invention
[0144] The formulations and methods of the invention include an
antibody, or antigen binding portion thereof, particularly an
anti-TNF.alpha. antibody, or antigen binding portion or fragment
thereof. Examples of antibodies that may be used in the invention
include chimeric antibodies, non-human antibodies, isolated human
antibodies, humanized antibodies, and domain antibodies (dAbs). All
antibodies described herein may be used in the methods of the
invention as well.
[0145] In one embodiment, the formulations of the invention
comprises an antibody, or antigen-binding portion thereof, which
binds human TNF.alpha., including, for example, adalimumab (also
referred to as Humira, adalimumab, or D2E7; Abbott Laboratories).
In a further embodiment, the formulation comprises an antibody that
binds the same epitope as adalimumab, such as, but not limited to,
an adalimumab biosimilar antibody. In one embodiment, the antibody
is a human IgG1 antibody having six CDRs corresponding to those of
the light and heavy chain of adalimumab.
[0146] In one embodiment, the invention features an isolated human
antibody, or antigen-binding portion thereof, that binds to human
TNF-alpha with high affinity and a low off rate, and also has a
high neutralizing capacity. In one embodiment, the human antibodies
used in the invention are recombinant, neutralizing human
anti-hTNF-alpha antibodies.
[0147] In one aspect, the invention pertains to adalimumab
antibodies and antibody portions, adalimumab-related antibodies and
antibody portions, and other human antibodies and antibody portions
with equivalent properties to adalimumab, such as high affinity
binding to hTNFa. with low dissociation kinetics and high
neutralizing capacity. In one embodiment, the antibody, or
antigen-binding fragment thereof, is defined according to
dissociation and binding characteristics similar to adalimumab. For
example, the formulation may include a human antibody that
dissociates from human TNF.alpha. with a K.sub.d of
1.times.10.sup.-8 M or less, and a k.sub.off rate constant of
1.times.10.sup.-3 s.sup.-1 or less, both determined by surface
plasmon resonance. In another embodiment, the human antibody that
dissociates from human TNF.alpha. with a K.sub.d of
1.times.10.sup.-9 M or less.
[0148] In one embodiment, the antibody, or antigen-binding fragment
thereof, is a human antibody that dissociates from human TNF.alpha.
with a K.sub.d of 1.times.10.sup.-8 M or less, and a k.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, both determined by
surface plasmon resonance, and neutralizes human TNF.alpha.
cytotoxicity in a standard in vitro L929 assay with an IC.sub.50 of
1.times.10.sup.-7 M or less. Examples and methods for making human,
neutralizing antibodies which have a high affinity for human
TNF.alpha., including sequences of the antibodies, are described in
U.S. Pat. No. 6,090,382 (referred to as D2E7), incorporated by
reference herein. The amino sequences of D2E7 as described in U.S.
Pat. No. 6,090,382 are incorporated in their entirety herein.
[0149] In one embodiment, the antibody used in the formulation of
the invention is D2E7, also referred to as HUMIRA.TM. or adalimumab
(the amino acid sequence of the D2E7 VL region is shown in SEQ ID
NO: 1; the amino acid sequence of the D2E7 VH region is shown in
SEQ ID NO: 2). The properties of D2E7 (adalimumab/HUMIRA.RTM.) have
been described in Salfeld et al., U.S. Pat. Nos. 6,090,382,
6,258,562, and 6,509,015, which are each incorporated by reference
herein.
[0150] In one embodiment, the human TNF-alpha, or an
antigen-binding portion thereof, dissociates from human TNF-alpha
with a K.sub.d of 1.times.10.sup.-8 M or less and a k.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, both determined by
surface plasmon resonance, and neutralizes human TNF-alpha
cytotoxicity in a standard in vitro L929 assay with an IC.sub.50 of
1.times.10.sup.-7 M or less. In one embodiment, the isolated human
antibody, or antigen-binding portion thereof, dissociates from
human TNF-alpha with a k.sub.off of 5.times.10.sup.-4 s.sup.-1 or
less; or, in one embodiment, with a k.sub.off of 1.times.10.sup.-4
s.sup.-1 or less. In one embodiment, the isolated human antibody,
or antigen-binding portion thereof, neutralizes human TNF-alpha
cytotoxicity in a standard in vitro L929 assay with an IC.sub.50 of
1.times.10.sup.-8 M or less; or, in one embodiment, with an
IC.sub.50 of 1.times.10.sup.-9 M or less; or, in one embodiment,
with an IC.sub.50 of 1.times.10.sup.-10 M or less. In one
embodiment, the antibody is an isolated human recombinant antibody,
or an antigen-binding portion thereof.
[0151] It is well known in the art that antibody heavy and light
chain CDR3 domains play an important role in the binding
specificity/affinity of an antibody for an antigen. Accordingly, in
another aspect, the antibody used in the formulation of the
invention has slow dissociation kinetics for association with
hTNF-alpha and has light and heavy chain CDR3 domains that
structurally are identical to or related to those of adalimumab.
Position 9 of the adalimumab VL CDR3 can be occupied by Ala or Thr
without substantially affecting the Koff. Accordingly, a consensus
motif for the adalimumab VL CDR3 comprises the amino acid sequence:
Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 3). Additionally, position 12 of
the adalimumab VH CDR3 can be occupied by Tyr or Asn, without
substantially affecting the k.sub.off. Accordingly, a consensus
motif for the adalimumab VH CDR3 comprises the amino acid sequence:
V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: 4). Moreover, as
demonstrated in Example 2 of U.S. Pat. No. 6,090,382, the CDR3
domain of the adalimumab heavy and light chains is amenable to
substitution with a single alanine residue (at position 1, 4, 5, 7
or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, 10 or
11 within the VH CDR3) without substantially affecting the
k.sub.off. Still further, the skilled artisan will appreciate that,
given the amenability of the adalimumab VL and VH CDR3 domains to
substitutions by alanine, substitution of other amino acids within
the CDR3 domains may be possible while still retaining the low off
rate constant of the antibody, in particular substitutions with
conservative amino acids. In one embodiment, no more than one to
five conservative amino acid substitutions are made within the
adalimumab VL and/or VH CDR3 domains. In one embodiment, no more
than one to three conservative amino acid substitutions are made
within the adalimumab VL and/or VH CDR3 domains. Additionally,
conservative amino acid substitutions should not be made at amino
acid positions critical for binding to hTNF alpha. Positions 2 and
5 of the adalimumab VL CDR3 and positions 1 and 7 of the adalimumab
VH CDR3 appear to be critical for interaction with hTNF alpha, and
thus, conservative amino acid substitutions preferably are not made
at these positions (although an alanine substitution at position 5
of the adalimumab VL CDR3 is acceptable, as described above) (see
U.S. Pat. No. 6,090,382).
[0152] Accordingly, in one embodiment, the antibody or
antigen-binding portion thereof, used in the formulation of the
invention contains the following characteristics:
[0153] a) dissociates from human TNF.alpha. with a k.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by
surface plasmon resonance;
[0154] b) has a light chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
alanine substitution at position 1, 4, 5, 7 or 8 or by one to five
conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8
and/or 9;
[0155] c) has a heavy chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single
alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or
by one to five conservative amino acid substitutions at positions
2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
[0156] In certain embodiments, the antibody or antigen-binding
portion thereof, dissociates from human TNF-alpha with a k.sub.off
of 5.times.10.sup.-4 s.sup.-1 or less. In certain embodiments, the
antibody or antigen-binding portion thereof, dissociates from human
TNF-alpha with a k.sub.off of 1.times.10.sup.-4 s.sup.-1 or
less.
[0157] In yet another embodiment, the antibody or antigen-binding
portion thereof contains a light chain variable region (LCVR)
having a CDR3 domain comprising the amino acid sequence of SEQ ID
NO: 3, or modified from SEQ ID NO: 3 by a single alanine
substitution at position 1, 4, 5, 7 or 8, and with a heavy chain
variable region (HCVR) having a CDR3 domain comprising the amino
acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a
single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or
11. In one embodiment, the LCVR further has a CDR2 domain
comprising the amino acid sequence of SEQ ID NO: 5 (i.e., the D2E7
VL CDR2) and the HCVR further has a CDR2 domain comprising the
amino acid sequence of SEQ ID NO: 6 (i.e., the D2E7 VH CDR2). In
one embodiment, the LCVR further has CDR1 domain comprising the
amino acid sequence of SEQ ID NO: 7 (i.e., the D2E7 VL CDR1) and
the HCVR has a CDR1 domain comprising the amino acid sequence of
SEQ ID NO: 8 (i.e., the D2E7 VH CDR1). The framework regions for VL
may be from the V.kappa.I human germline family, or from the A20
human germline Vk gene, or from the adalimumab VL framework
sequences shown in FIGS. 1A and 1B of U.S. Pat. No. 6,090,382. The
framework regions for VH may be from the VH3 human germline family,
or from the DP-31 human germline VH gene, or from the D2E7 VH
framework sequences shown in FIGS. 2A and 2B of U.S. Pat. No.
6,090,382. Nucleic acid sequences corresponding to the adalimumab
light and heavy variable regions are described in SEQ ID NOs: 36
and 37, respectively.
[0158] Accordingly, in another embodiment, the antibody or
antigen-binding portion thereof contains a light chain variable
region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1
(i.e., the adalimumab VL) and a heavy chain variable region (HCVR)
comprising the amino acid sequence of SEQ ID NO: 2 (i.e., the
adalimumab VH). In certain embodiments, the antibody comprises a
heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4,
IgA, IgE, IgM or IgD constant region. In certain embodiments, the
heavy chain constant region is an IgG1 heavy chain constant region
or an IgG4 heavy chain constant region. Furthermore, the antibody
can comprise a light chain constant region, either a kappa light
chain constant region or a lambda light chain constant region. In
one embodiment, the antibody comprises a kappa light chain constant
region. Alternatively, the antibody portion can be, for example, a
Fab fragment or a single chain Fv fragment.
[0159] In still other embodiments, the invention includes uses of
an isolated human antibody, or an antigen-binding portion thereof,
containing adalimumab-related VL and VH CDR3 domains. For example,
antibodies or antigen-binding portions thereof may have a light
chain variable region (LCVR) having a CDR3 domain comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID
NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,
SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 or with a heavy
chain variable region (HCVR) having a CDR3 domain comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,
SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ
ID NO: 35.
[0160] In one embodiment, the TNF.alpha. antibody used in the
invention includes the chimeric antibody infliximab (Remicade.RTM.,
Johnson and Johnson; described in U.S. Pat. No. 5,656,272,
incorporated by reference herein), CDP571 (a humanized monoclonal
anti-TNF-alpha IgG4 antibody), CDP 870 (a humanized monoclonal
anti-TNF-alpha antibody fragment), an anti-TNF dAb (Peptech), or
CNTO 148 (golimumab; Medarex and Centocor, see WO 02/12502).
Additional TNF antibodies which may be used in the invention are
described in U.S. Pat. Nos. 6,593,458; 6,498,237; 6,451,983; and
6,448,380, each of which is incorporated by reference herein.
[0161] An antibody, or antibody portion, used in the methods and
compositions of the invention, can be prepared by recombinant
expression of immunoglobulin light and heavy chain genes in a host
cell. To express an antibody recombinantly, a host cell is
transfected with one or more recombinant expression vectors
carrying DNA fragments encoding the immunoglobulin light and heavy
chains of the antibody such that the light and heavy chains are
expressed in the host cell and, preferably, secreted into the
medium in which the host cells are cultured, from which medium the
antibodies can be recovered. Standard recombinant DNA methodologies
are used to obtain antibody heavy and light chain genes,
incorporate these genes into recombinant expression vectors and
introduce the vectors into host cells, such as those described in
Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular
Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No.
4,816,397 by Boss et al.
[0162] To express an anti-TNFa antibody, e.g., adalimumab (D2E7) or
an adalimumab (D2E7)-related antibody, DNA fragments encoding the
light and heavy chain variable regions are first obtained. These
DNAs can be obtained by amplification and modification of germline
light and heavy chain variable sequences using the polymerase chain
reaction (PCR). Germline DNA sequences for human heavy and light
chain variable region genes are known in the art (see e.g., the
"Vbase" human germline sequence database; see also Kabat, E. A., et
al. (1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Tomlinson, I. M., et al. (1992) "The
Repertoire of Human Germline VH Sequences Reveals about Fifty
Groups of VH Segments with Different Hypervariable Loops" J. Mol.
Biol. 227:776-798; and Cox, J. P. L. et al. (1994) "A Directory of
Human Germ-line V78 Segments Reveals a Strong Bias in their Usage"
Eur. J. Immunol. 24:827-836; the contents of each of which are
expressly incorporated herein by reference). For example, to obtain
a DNA fragment encoding the heavy chain variable region of D2E7, or
a D2E7-related antibody, a member of the VH3 family of human
germline VH genes is amplified by standard PCR. In certain
embodiments, the DP-31 VH germline sequence is amplified. To obtain
a DNA fragment encoding the light chain variable region of D2E7, or
a D2E7-related antibody, a member of the V.kappa.I family of human
germline VL genes is amplified by standard PCR. In certain
embodiments, the A20 VL germline sequence is amplified. PCR primers
suitable for use in amplifying the DP-31 germline VH and A20
germline VL sequences can be designed based on the nucleotide
sequences disclosed in the references cited supra, using standard
methods.
[0163] Once the germline VH and VL fragments are obtained, these
sequences can be mutated to encode the anti-TNFa antibody amino
acid sequences disclosed herein. The amino acid sequences encoded
by the germline VH and VL DNA sequences are first compared to the
anti-TNFa antibody VH and VL amino acid sequences to identify amino
acid residues in the anti-TNFa antibody sequence that differ from
germline. Then, the appropriate nucleotides of the germline DNA
sequences are mutated such that the mutated germline sequence
encodes the anti-TNFa antibody amino acid sequence, using the
genetic code to determine which nucleotide changes should be made.
Mutagenesis of the germline sequences is carried out by standard
methods, such as PCR-mediated mutagenesis (in which the mutated
nucleotides are incorporated into the PCR primers such that the PCR
product contains the mutations) or site-directed mutagenesis.
[0164] Moreover, it should be noted that if the "germline"
sequences obtained by PCR amplification encode amino acid
differences in the framework regions from the true germline
configuration (i.e., differences in the amplified sequence as
compared to the true germline sequence, for example as a result of
somatic mutation), it may be desirable to change these amino acid
differences back to the true germline sequences (i.e.,
"backmutation" of framework residues to the germline
configuration).
[0165] Once DNA fragments encoding the anti-TNF.alpha. antibody VH
and VL segments are obtained (e.g., by amplification and
mutagenesis of germline VH and VL genes, as described above), these
DNA fragments can be further manipulated by standard recombinant
DNA techniques, for example to convert the variable region genes to
full-length antibody chain genes, to Fab fragment genes or to a
scFv gene. In these manipulations, a VL- or VH-encoding DNA
fragment is operatively linked to another DNA fragment encoding
another protein, such as an antibody constant region or a flexible
linker. The term "operatively linked," as used in this context, is
intended to mean that the two DNA fragments are joined such that
the amino acid sequences encoded by the two DNA fragments remain
in-frame.
[0166] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat,
E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG1 or IgG4 constant region. For a Fab fragment
heavy chain gene, the VH-encoding DNA can be operatively linked to
another DNA molecule encoding only the heavy chain CH1 constant
region.
[0167] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region. In one embodiment, the light chain constant
region is a kappa constant region.
[0168] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the VH and VL sequences can be
expressed as a contiguous single-chain protein, with the VL and VH
regions joined by the flexible linker (see e.g., Bird et al. (1988)
Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci.
USA 85:5879-5883; McCafferty et al., Nature (1990)
348:552-554).
[0169] To express the antibodies, or antibody portions used in the
invention, DNAs encoding partial or full-length light and heavy
chains, obtained as described above, are inserted into expression
vectors such that the genes are operatively linked to
transcriptional and translational control sequences. In this
context, the term "operatively linked" is intended to mean that an
antibody gene is ligated into a vector such that transcriptional
and translational control sequences within the vector serve their
intended function of regulating the transcription and translation
of the antibody gene. The expression vector and expression control
sequences are chosen to be compatible with the expression host cell
used. The antibody light chain gene and the antibody heavy chain
gene can be inserted into separate vector or, more typically, both
genes are inserted into the same expression vector. The antibody
genes are inserted into the expression vector by standard methods
(e.g., ligation of complementary restriction sites on the antibody
gene fragment and vector, or blunt end ligation if no restriction
sites are present). Prior to insertion of the anti-TNFa antibody
light or heavy chain sequences, the expression vector may already
carry antibody constant region sequences. For example, one approach
to converting the anti-TNFa antibody VH and VL sequences to
full-length antibody genes is to insert them into expression
vectors already encoding heavy chain constant and light chain
constant regions, respectively, such that the VH segment is
operatively linked to the CH segment(s) within the vector and the
VL segment is operatively linked to the CL segment within the
vector. Additionally or alternatively, the recombinant expression
vector can encode a signal peptide that facilitates secretion of
the antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the antibody chain gene. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0170] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). It will be appreciated by those skilled in the art that the
design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see e.g., U.S. Pat. No.
5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and
U.S. Pat. No. 4,968,615 by Schaffner et al.
[0171] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors used in the invention
may carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0172] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies is preferably in eukaryotic
cells. In one embodiment, mammalian host cells, is the most
preferred because such eukaryotic cells, and in particular
mammalian cells, are more likely than prokaryotic cells to assemble
and secrete a properly folded and immunologically active antibody.
Prokaryotic expression of antibody genes has been reported to be
ineffective for production of high yields of active antibody (Boss,
M. A. and Wood, C. R. (1985) Immunology Today 6:12-13).
[0173] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells,
COS cells and SP2 cells. When recombinant expression vectors
encoding antibody genes are introduced into mammalian host cells,
the antibodies are produced by culturing the host cells for a
period of time sufficient to allow for expression of the antibody
in the host cells or, more, in one embodiment, secretion of the
antibody into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard
protein purification methods.
[0174] Host cells can also be used to produce portions of intact
antibodies, such as Fab fragments or scFv molecules. It is
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding either the light chain or
the heavy chain (but not both) of an antibody of this invention.
Recombinant DNA technology may also be used to remove some or all
of the DNA encoding either or both of the light and heavy chains
that is not necessary for binding to hTNF alpha. The molecules
expressed from such truncated DNA molecules are also encompassed by
the antibodies of the invention. In addition, bifunctional
antibodies may be produced in which one heavy and one light chain
are an antibody of the invention and the other heavy and light
chain are specific for an antigen other than hTNF alpha by
crosslinking an antibody of the invention to a second antibody by
standard chemical crosslinking methods.
[0175] In a preferred system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr-CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are culture to allow for
expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular
biology techniques are used to prepare the recombinant expression
vector, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture
medium.
[0176] In view of the foregoing, nucleic acid, vector and host cell
compositions that can be used for recombinant expression of the
antibodies and antibody portions used in the invention include
nucleic acids, and vectors comprising said nucleic acids,
comprising the human TNF alpha antibody adalimumab (D2E7). The
nucleotide sequence encoding the D2E7 light chain variable region
is shown in SEQ ID NO: 36. The CDR1 domain of the LCVR encompasses
nucleotides 70-102, the CDR2 domain encompasses nucleotides 148-168
and the CDR3 domain encompasses nucleotides 265-291. The nucleotide
sequence encoding the D2E7 heavy chain variable region is shown in
SEQ ID NO: 37. The CDR1 domain of the HCVR encompasses nucleotides
91-105, the CDR2 domain encompasses nucleotides 148-198 and the
CDR3 domain encompasses nucleotides 295-330. It will be appreciated
by the skilled artisan that nucleotide sequences encoding
D2E7-related antibodies, or portions thereof (e.g., a CDR domain,
such as a CDR3 domain), can be derived from the nucleotide
sequences encoding the D2E7 LCVR and HCVR using the genetic code
and standard molecular biology techniques.
[0177] In one embodiment, the liquid pharmaceutical formulation
comprises a human TNF alpha antibody, or antigen-binding portion
thereof, that is a bioequivalent or biosimilar to the antibody
adalimumab. In one embodiment, a biosimilar antibody is an antibody
which shows no clinically meaningful difference when compared to a
reference antibody, e.g., adalimumab. A biosimilar antibody has
equivalent safety, purity, and potency as a reference antibody,
e.g., adalimumab.
IV. Administration of the Formulations of the Invention for
Treatment of TNFa-Related Disorders
[0178] An advantage of the formulations of the invention is that
they may be used to deliver a high concentration of an anti-TNF
alpha antibody, or antigen-binding portion, (e.g., adalimumab) to a
subject subcutaneously such that either pain upon injection is
decreased or the bioavailability of the antibody is improved. Thus,
in one embodiment, the formulation of the invention is delivered to
a subject subcutaneously. In one embodiment, the subject
administers the formulation to himself/herself
(self-administration).
[0179] In one embodiment, an effective amount of the formulation is
administered. An example of an effective amount of the formulation
is an amount sufficient to inhibit detrimental TNF-alpha activity
or treat a disorder in which TNF alpha activity is detrimental.
[0180] As used herein, the term "a disorder in which TNF-alpha
activity is detrimental" is intended to include diseases and other
disorders in which the presence of TNF-alpha. in a subject
suffering from the disorder has been shown to be or is suspected of
being either responsible for the pathophysiology of the disorder or
a factor that contributes to a worsening of the disorder.
Accordingly, a disorder in which TNF-alpha. activity is detrimental
is a disorder in which inhibition of TNF-alpha activity is expected
to alleviate the symptoms and/or progression of the disorder. Such
disorders may be evidenced, for example, by an increase in the
concentration of TNF-alpha. in a biological fluid of a subject
suffering from the disorder (e.g., an increase in the concentration
of TNF-alpha. in serum, plasma, synovial fluid, etc. of the
subject), which can be detected, for example, using an
anti-TNF-alpha. antibody.
[0181] In one embodiment, the effective amount of antibody may be
determined according to a strictly weight based dosing scheme
(e.g., mg/kg) or may be a total body dose (also referred to as a
fixed dose) which is independent of weight. In one embodiment, an
effective amount of the antibody is about 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 mg of the
human anti-TNF.alpha. antibody, or antigen-binding portion thereof.
In one embodiment, an effective amount of the antibody is about
20-100, about 20-90, about 30-90, about 30-100, about 60-100, about
70-90, about 40-90, about 60-85 mg, or about 40-100 mg. In one
embodiment, the formulation contains an effective amount of the
antibody of 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg,
38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47
mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg,
57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66
mg, 67 mg, 68 mg 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg,
76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85
mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg,
95 mg, 96 mg, 97 mg, 98 mg, 99 mg, or 100 mg of the antibody.
Ranges including the aforementioned numbers are also included in
the invention, e.g., 70-90 or 75-85 mg or 60-85 mg.
[0182] In one example, an effective amount of the formulation is
0.4 mL or 0.8 mL of the formulation containing a total body dose of
about 80 mg of antibody (i.e., 0.8 mL of a 100 mg/mL antibody
formulation of the invention). In another example, an effective
amount of the formulation is 0.4 mL of the formulation of the
invention containing a total body dose of about 40 mg of antibody
(i.e., 0.4 mL of a 100 mg/mL antibody formulation of the
invention). In yet another example, an effective amount of the
formulation is twice 0.8 mL of the formulation containing a total
body dose of about 160 mg of antibody (i.e., two units containing
0.8 mL each of a 100 mg/mL antibody formulation of the invention).
In a further example, an effective amount of the formulation is 0.2
mL of the formulation of the invention containing a total body dose
of about 20 mg of antibody (i.e., 0.2 mL of a 100 mg/mL antibody
formulation of the invention). Alternatively, an effective amount
may be determined according to a weight-based fixed dosing regimen
(see, e.g., WO 2008/154543, incorporated by reference herein).
[0183] In one embodiment, the TNF-alpha is human TNF-alpha and the
subject is a human subject. Alternatively, the subject can be a
mammal expressing a TNF-alpha with which an antibody of the
invention cross-reacts. Still further the subject can be a mammal
into which has been introduced hTNF-alpha (e.g., by administration
of hTNF-alpha or by expression of an hTNF-alpha transgene).
[0184] A formulation of the invention may be administered to a
human subject for therapeutic purposes (discussed further below).
In one embodiment of the invention, the liquid pharmaceutical
formulation is easily administratable, which includes, for example,
a formulation which is self-administered by the patient. In one
embodiment, the formulation of the invention is administered
through subcutaneous injection, such as single use subcutaneous
injection. Moreover, a formulation of the invention can be
administered to a non-human mammal expressing a TNF-alpha with
which the antibody cross-reacts (e.g., a primate, pig or mouse) for
veterinary purposes or as an animal model of human disease.
Regarding the latter, such animal models may be useful for
evaluating the therapeutic efficacy of antibodies of the invention
(e.g., testing of dosages and time courses of administration).
[0185] The formulations of the invention may be administered
according to a certain dosing schedule. For example, the
formulations may be administered according to a weekly, biweekly,
or monthly dosing regimen. Alternatively, the formulation may be
administered once every three weeks. In one embodiment, the
formulations and methods comprise administration to the subject of
a human anti-TNF.alpha. antibody according to a periodicity
selected from the group consisting of weekly, biweekly, every three
weeks, and monthly.
[0186] In one embodiment, the liquid aqueous formulation of the
invention may be administered to a subject via, for example, a
prefilled syringe, an autoinjector pen, or a needle-free
administration device. Thus, the invention also features an
autoinjector pen, a prefilled syringe, or a needle-free
administration device comprising the liquid aqueous formulation of
the invention. In one embodiment, the invention features a delivery
device comprising a dose of the formulation comprising 100 mg/mL a
human TNF alpha antibody, or antigen-binding portion thereof, e.g.,
an autoinjector pen or prefilled syringe comprises a dose of about
19 mg, 20, mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28
mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg,
38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47
mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg,
57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66
mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg,
76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85
mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg,
95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 101 mg, 102 mg, 103 mg,
104 mg, 105 mg, etc. of the formulation. In one embodiment, the
syringe or autoinjector contains 60-100 mg, 70-90 mg, or about 80
mg of the antibody.
[0187] In one embodiment, the formulations of the invention may be
self administered using, e.g., a preloaded syringe or an automatic
injection device. Automatic injection devices offer an alternative
to manually-operated syringes for delivering therapeutic agents
into patients' bodies and allowing patients to self-administer
injections. Automatic injection devices are described, for example,
in the following publications, each of which is hereby incorporated
herein by reference WO 2008/005315, WO 2010/127146, WO 2006/000785,
WO 2011/075524, WO 2005/113039, WO 2011/075524.
[0188] Accordingly, in one embodiment, the present invention
provides pre-filled syringes or autoinjector devices containing the
formulations of the invention, as well as use of pre-filled
syringes or autoinjector devices comprising the formulations
described herein in the methods of the invention.
[0189] In one embodiment, the formulation of the invention is used
to treat disorders in which TNF alpha activity is detrimental. As
used herein, the term "a disorder in which TNF-alpha activity is
detrimental" is intended to include diseases and other disorders in
which the presence of TNF-alpha in a subject suffering from the
disorder has been shown to be or is suspected of being either
responsible for the pathophysiology of the disorder or a factor
that contributes to a worsening of the disorder. Accordingly, a
disorder in which TNF-alpha activity is detrimental is a disorder
in which inhibition of TNF-alpha activity is expected to alleviate
the symptoms and/or progression of the disorder. Such disorders may
be evidenced, for example, by an increase in the concentration of
TNF-alpha in a biological fluid of a subject suffering from the
disorder (e.g., an increase in the concentration of TNF-alpha in
serum, plasma, synovial fluid, etc. of the subject), which can be
detected, for example, using an anti-TNF-alpha antibody as
described above.
[0190] There are numerous examples of disorders in which TNF-alpha
activity is detrimental. Examples in which TNF-alpha activity is
detrimental are also described in U.S. Pat. Nos. 6,015,557;
6,177,077; 6,379,666; 6,419,934; 6,419,944; 6,423,321; 6,428,787;
and 6,537,549; and PCT Publication Nos. WO 00/50079 and WO
01/49321, the entire contents of all of which are incorporated
herein by reference. The formulations of the invention may also be
used to treat disorders in which TNF alpha activity is detrimental
as described in U.S. Pat. Nos. 6,090,382, 6,258,562 and U.S. Patent
Application Publication No. US20040126372, the entire contents of
all of which are incorporated herein by reference.
[0191] The use of the formulations of the invention in the
treatment of specific exemplary disorders is discussed further
below:
[0192] A. Sepsis
[0193] The formulations and methods of the invention may be used to
treat subjects having sepsis. Tumor necrosis factor has an
established role in the pathophysiology of sepsis, with biological
effects that include hypotension, myocardial suppression, vascular
leakage syndrome, organ necrosis, stimulation of the release of
toxic secondary mediators and activation of the clotting cascade
(see e.g., Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med.
45:491-503; Russell, D and Thompson, R. C. (1993) Curr. Opin.
Biotech. 4:714-721). Accordingly, the formulation of the invention
can be used to treat sepsis in any of its clinical settings,
including septic shock, endotoxic shock, gram negative sepsis and
toxic shock syndrome.
[0194] Furthermore, to treat sepsis, the formulation of the
invention can be coadministered with one or more additional
therapeutic agents that may further alleviate sepsis, such as an
interleukin-1 inhibitor (such as those described in PCT Publication
Nos. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see
e.g., PCT Publication No. WO 93/11793) or an antagonist of platelet
activating factor (see e.g., European Patent Application
Publication No. EP 374 510).
[0195] Additionally, in one embodiment, the formulation of the
invention is administered to a human subject within a subgroup of
sepsis patients having a serum or plasma concentration of IL-6
above 500 pg/ml; or, in one embodiment, 1000 pg/ml, at the time of
treatment (see PCT Publication No. WO 95/20978).
[0196] B. Autoimmune Diseases
[0197] The formulations and methods of the invention may be used to
treat subjects having an autoimmune disease. Tumor necrosis factor
has been implicated in playing a role in the pathophysiology of a
variety of autoimmune diseases. For example, TNF-alpha has been
implicated in activating tissue inflammation and causing joint
destruction in rheumatoid arthritis (see e.g., Tracey and Cerami,
supra; Arend, W. P. and Dayer, J-M. (1995) Arth. Rheum. 38:151-160;
Fava, R. A., et al. (1993) Clin. Exp. Immunol. 94:261-266).
TNF-alpha also has been implicated in promoting the death of islet
cells and in mediating insulin resistance in diabetes (see e.g.,
Tracey and Cerami, supra; PCT Publication No. WO 94/08609).
TNF-alpha also has been implicated in mediating cytotoxicity to
oligodendrocytes and induction of inflammatory plaques in multiple
sclerosis (see e.g., Tracey and Cerami, supra). Also included in
autoimmune diseases that may be treated using the formulations and
methods of the invention is juvenile idiopathic arthritis (JIA)
(also referred to as juvenile rheumatoid arthritis) (see Grom et
al. (1996) Arthritis Rheum. 39:1703; Mangge et al. (1995) Arthritis
Rheum. 8:211).
[0198] The formulation of the invention can be used to treat
autoimmune diseases, in particular those associated with
inflammation, including rheumatoid arthritis, rheumatoid
spondylitis (also referred to as ankylosing spondylitis),
osteoarthritis and gouty arthritis, allergy, multiple sclerosis,
autoimmune diabetes, autoimmune uveitis, juvenile idiopathic
arthritis (also referred to as juvenile rheumatoid arthritis), and
nephrotic syndrome.
[0199] C. Infectious Diseases
[0200] The formulations and methods of the invention may be used to
treat subjects having an infectious disease. Tumor necrosis factor
has been implicated in mediating biological effects observed in a
variety of infectious diseases. For example, TNF-alpha has been
implicated in mediating brain inflammation and capillary thrombosis
and infarction in malaria (see e.g., Tracey and Cerami, supra).
TNF-alpha also has been implicated in mediating brain inflammation,
inducing breakdown of the blood-brain barrier, triggering septic
shock syndrome and activating venous infarction in meningitis (see
e.g., Tracey and Cerami, supra). TNF-alpha also has been implicated
in inducing cachexia, stimulating viral proliferation and mediating
central nervous system injury in acquired immune deficiency
syndrome (AIDS) (see e.g., Tracey and Cerami, supra). Accordingly,
the antibodies, and antibody portions, of the invention, can be
used in the treatment of infectious diseases, including bacterial
meningitis (see e.g., European Patent Application Publication No.
EP 585 705), cerebral malaria, AIDS and AIDS-related complex (ARC)
(see e.g., European Patent Application Publication No. EP 230 574),
as well as cytomegalovirus infection secondary to transplantation
(see e.g., Fietze, E., et al. (1994) Transplantation 58:675-680).
The formulation of the invention, also can be used to alleviate
symptoms associated with infectious diseases, including fever and
myalgias due to infection (such as influenza) and cachexia
secondary to infection (e.g., secondary to AIDS or ARC).
[0201] D. Transplantation
[0202] The formulations and methods of the invention may be used to
treat subjects having a transplantation. Tumor necrosis factor has
been implicated as a key mediator of allograft rejection and graft
versus host disease (GVHD) and in mediating an adverse reaction
that has been observed when the rat antibody OKT3, directed against
the T cell receptor CD3 complex, is used to inhibit rejection of
renal transplants (see e.g., Tracey and Cerami, supra; Eason, J.
D., et al. (1995) Transplantation 59:300-305; Suthanthiran, M. and
Strom, T. B. (1994) New Engl. J. Med. 331:365-375). Accordingly,
the formulations of the invention can be used to inhibit transplant
rejection, including rejections of allografts and xenografts and to
inhibit GVHD. Although the antibody or antibody portion may be used
alone, it can be used in combination with one or more other agents
that inhibit the immune response against the allograft or inhibit
GVHD. For example, in one embodiment, the formulations of the
invention are used in combination with OKT3 to inhibit OKT3-induced
reactions. In another embodiment, the formulation of the invention
is used in combination with one or more antibodies directed at
other targets involved in regulating immune responses, such as the
cell surface molecules CD25 (interleukin-2 receptor-.alpha.), CD11a
(LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or
CD86 (B7-2). In yet another embodiment, the formulation of the
invention is used in combination with one or more general
immunosuppressive agents, such as cyclosporin A or FK506.
[0203] E. Malignancy
[0204] The formulations and methods of the invention may be used to
treat subjects having cancer or a malignant tumor. Tumor necrosis
factor has been implicated in inducing cachexia, stimulating tumor
growth, enhancing metastatic potential and mediating cytotoxicity
in malignancies (see e.g., Tracey and Cerami, supra). Accordingly,
the formulations of the invention can be used in the treatment of
malignancies, to inhibit tumor growth or metastasis and/or to
alleviate cachexia secondary to malignancy. The formulation of the
invention may be administered systemically or locally to the tumor
site.
[0205] F. Pulmonary Disorders
[0206] The formulations and methods of the invention may be used to
treat subjects having a pulmonary disease. Tumor necrosis factor
has been implicated in the pathophysiology of adult respiratory
distress syndrome, including stimulating leukocyte-endothelial
activation, directing cytotoxicity to pneumocytes and inducing
vascular leakage syndrome (see e.g., Tracey and Cerami, supra).
Accordingly, the formulations of the invention can be used to treat
various pulmonary disorders, including adult respiratory distress
syndrome (see e.g., PCT Publication No. WO 91/04054), shock lung,
chronic pulmonary inflammatory disease, pulmonary sarcoidosis,
pulmonary fibrosis and silicosis. The formulation of the invention
may be administered systemically or locally to the lung surface,
for example as an aerosol.
[0207] G. Intestinal Disorders
[0208] The formulations and methods of the invention may be used to
treat subjects having an intestinal disorder. Tumor necrosis factor
has been implicated in the pathophysiology of inflammatory bowel
disorders (see e.g., Tracy, K. J., et al. (1986) Science
234:470-474; Sun, X-M., et al. (1988) J. Clin. Invest.
81:1328-1331; MacDonald, T. T., et al. (1990) Clin. Exp. Immunol.
81:301-305) Chimeric murine anti-hTNF-alpha antibodies have
undergone clinical testing for treatment of Crohn's disease (van
Dullemen, H. M., et al. (1995) Gastroenterology 109:129-135). The
formulation of the invention, also can be used to treat intestinal
disorders, such as idiopathic inflammatory bowel disease, which
includes two syndromes, Crohn's disease and ulcerative colitis. In
one embodiment, the formulation of the invention is used to treat
Crohn's disease. In one embodiment, the formulation of the
invention is used to treat ulcerative colitis.
[0209] H. Cardiac Disorders
[0210] The formulations and methods of the invention, also can be
used to treat various cardiac disorders, including ischemia of the
heart (see e.g., European Patent Application Publication No. EP 453
898) and heart insufficiency (weakness of the heart muscle)(see
e.g., PCT Publication No. WO 94/20139).
[0211] I. Spondyloarthropathies
[0212] The formulations and methods of the invention may also be
used to treat subjects who have a spondyloarthropathy, including,
for example, an axial spondyloarthropathy. TNF.alpha. has been
implicated in the pathophysiology of a wide variety of disorders,
including inflammatory diseases such as spondyloarthopathies (see
e.g., Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No.
5,231,024 to Moeller et al.; European Patent Publication No. 260
610 B1 by Moeller, A). In one embodiment, the spondyloarthropathy
is an axial spondyloarthropathy. Other examples of
spondyloarthropathies which can be treated with the TNF.alpha.
antibody of the invention are described below:
[0213] 1. Psoriatic Arthritis
[0214] The formulations and methods of the invention may also be
used to treat subjects who have psoriatic arthritis. Tumor necrosis
factor has been implicated in the pathophysiology of psoriatic
arthritis (Partsch et al. (1998) Ann Rheum Dis. 57:691; Ritchlin et
al. (1998) J Rheumatol. 25:1544). As referred to herein, psoriatic
arthritis (PsA) or psoriasis associated with the skin, refers to
chronic inflammatory arthritis which is associated with psoriasis.
Psoriasis is a common chronic skin condition that causes red
patches on the body. About 1 in 20 individuals with psoriasis will
develop arthritis along with the skin condition, and in about 75%
of cases, psoriasis precedes the arthritis. PsA exhibits itself in
a variety of ways, ranging from mild to severe arthritis, wherein
the arthritis usually affects the fingers and the spine. When the
spine is affected, the symptoms are similar to those of ankylosing
spondylitis, as described above.
[0215] PsA is sometimes associated with arthritis mutilans.
Arthritis mutilans refers to a disorder which is characterized by
excessive bone erosion resulting in a gross, erosive deformity
which mutilates the joint. In one embodiment, formulations and
methods of the invention can be used to treat arthritis
mutilans.
[0216] 2. Reactive Arthritis/Reiter's Syndrome
[0217] The formulations and methods of the invention may also be
used to treat subjects who have Reiter's syndrome or reactive
arthritis. Tumor necrosis factor has been implicated in the
pathophysiology of reactive arthritis, which is also referred to as
Reiter's syndrome (Braun et al. (1999) Arthritis Rheum.
42(10):2039). Reactive arthritis (ReA) refers to arthritis which
complicates an infection elsewhere in the body, often following
enteric or urogenital infections. ReA is often characterized by
certain clinical symptoms, including inflammation of the joints
(arthritis), urethritis, conjunctivitis, and lesions of the skin
and mucous membranes. In addition, ReA can occurs following
infection with a sexually transmitted disease or dysenteric
infection, including chlamydia, campylobacter, salmonella, or
yersinia.
[0218] 3. Undifferentiated Spondyloarthropathies
[0219] The formulations and methods of the invention may also be
used to treat subjects who have an undifferentiated
spondyloarthropathy (see Zeidler et al. (1992) Rheum Dis Clin North
Am. 18:187). Other terms used to describe undifferentiated
spondyloarthropathies include seronegative oligoarthritis and
undifferentiated oligoarthritis. Undifferentiated
spondyloarthropathies, as used herein, refers to a disorder wherein
the subject demonstrates only some of the symptoms associated with
a spondyloarthropathy. This condition is usually observed in young
adults who do not have IBD, psoriasis, or the classic symptoms of
AS or Reiter's syndrome. In some instances, undifferentiated
spondyloarthropathies may be an early indication of AS.
[0220] J. Skin and Nail Disorders
[0221] In one embodiment, the formulations and methods of the
invention are used to treat a skin and/or a nail disorder. As used
herein, the term "skin and nail disorder in which TNF.alpha.
activity is detrimental" is intended to include skin and/or nail
disorders and other disorders in which the presence of TNF alpha in
a subject suffering from the disorder has been shown to be or is
suspected of being either responsible for the pathophysiology of
the disorder or a factor that contributes to a worsening of the
disorder, e.g., psoriasis. An example of a skin disorder which may
be treated using the formulation of the invention is psoriasis. In
one embodiment, the formulation of the invention is used to treat
plaque psoriasis. Tumor necrosis factor has been implicated in the
pathophysiology of psoriasis (Takematsu et al. (1989) Arch Dermatol
Res. 281:398; Victor and Gottlieb (2002) J Drugs Dermatol.
1(3):264).
[0222] 1. Psoriasis
[0223] The formulations and methods of the invention may be used to
treat subjects having psoriasis, including subjects having plaque
psoriasis. Tumor necrosis factor has been implicated in the
pathophysiology of psoriasis (Takematsu et al. (1989) Arch Dermatol
Res. 281:398; Victor and Gottlieb (2002) J Drugs Dermatol.
1(3):264). Psoriasis is described as a skin inflammation
(irritation and redness) characterized by frequent episodes of
redness, itching, and thick, dry, silvery scales on the skin. In
particular, lesions are formed which involve primary and secondary
alterations in epidermal proliferation, inflammatory responses of
the skin, and an expression of regulatory molecules such as
lymphokines and inflammatory factors. Psoriatic skin is
morphologically characterized by an increased turnover of epidermal
cells, thickened epidermis, abnormal keratinization, inflammatory
cell infiltrates into the epidermis and polymorphonuclear leukocyte
and lymphocyte infiltration into the epidermis layer resulting in
an increase in the basal cell cycle. Psoriasis often involves the
nails, which frequently exhibit pitting, separation of the nail,
thickening, and discoloration. Psoriasis is often associated with
other inflammatory disorders, for example arthritis, including
rheumatoid arthritis, inflammatory bowel disease (IBD), and Crohn's
disease.
[0224] Evidence of psoriasis is most commonly seen on the trunk,
elbows, knees, scalp, skin folds, or fingernails, but it may affect
any or all parts of the skin. Normally, it takes about a month for
new skin cells to move up from the lower layers to the surface. In
psoriasis, this process takes only a few days, resulting in a
build-up of dead skin cells and formation of thick scales. Symptoms
of psoriasis include: skin patches, that are dry or red, covered
with silvery scales, raised patches of skin, accompanied by red
borders, that may crack and become painful, and that are usually
located on the elbows, knees, trunk, scalp, and hands; skin
lesions, including pustules, cracking of the skin, and skin
redness; joint pain or aching which may be associated with of
arthritis, e.g., psoriatic arthritis.
[0225] Treatment for psoriasis often includes a topical
corticosteroids, vitamin D analogs, and topical or oral retinoids,
or combinations thereof. In one embodiment, the TNFalpha inhibitor
of the invention is administered in combination with or the
presence of one of these common treatments.
[0226] The diagnosis of psoriasis is usually based on the
appearance of the skin. Additionally a skin biopsy, or scraping and
culture of skin patches may be needed to rule out other skin
disorders. An x-ray may be used to check for psoriatic arthritis if
joint pain is present and persistent.
[0227] In one embodiment of the invention, a TNFalpha inhibitor is
used to treat psoriasis, including chronic plaque psoriasis,
guttate psoriasis, inverse psoriasis, pustular psoriasis, pemphigus
vulgaris, erythrodermic psoriasis, psoriasis associated with
inflammatory bowel disease (IBD), and psoriasis associated with
rheumatoid arthritis (RA). Specific types of psoriasis included in
the treatment methods of the invention are described in detail
below:
[0228] a. Chronic Plaque Psoriasis
[0229] The formulations and methods of the invention may be used to
treat subjects having chronic plaque psoriasis. Tumor necrosis
factor has been implicated in the pathophysiology of chronic plaque
psoriasis (Asadullah et al. (1999) Br J Dermatol. 141:94). Chronic
plaque psoriasis (also referred to as psoriasis vulgaris) is the
most common form of psoriasis. Chronic plaque psoriasis is
characterized by raised reddened patches of skin, ranging from
coin-sized to much larger. In chronic plaque psoriasis, the plaques
may be single or multiple, they may vary in size from a few
millimeters to several centimeters. The plaques are usually red
with a scaly surface, and reflect light when gently scratched,
creating a "silvery" effect. Lesions (which are often symmetrical)
from chronic plaque psoriasis occur all over body, but with
predilection for extensor surfaces, including the knees, elbows,
lumbosacral regions, scalp, and nails. Occasionally chronic plaque
psoriasis can occur on the penis, vulva and flexures, but scaling
is usually absent. Diagnosis of patients with chronic plaque
psoriasis is usually based on the clinical features described
above. In particular, the distribution, color and typical silvery
scaling of the lesion in chronic plaque psoriasis are
characteristic of chronic plaque psoriasis.
[0230] b. Guttate Psoriasis
[0231] The formulations and methods of the invention may be used to
treat subjects having guttate psoriasis. Guttate psoriasis refers
to a form of psoriasis with characteristic water drop shaped scaly
plaques. Flares of guttate psoriasis generally follow an infection,
most notably a streptococcal throat infection. Diagnosis of guttate
psoriasis is usually based on the appearance of the skin, and the
fact that there is often a history of recent sore throat.
[0232] c. Inverse Psoriasis
[0233] The formulations and methods of the invention may be used to
treat subjects having inverse psoriasis. Inverse psoriasis is a
form of psoriasis in which the patient has smooth, usually moist
areas of skin that are red and inflamed, which is unlike the
scaling associated with plaque psoriasis. Inverse psoriasis is also
referred to as intertiginous psoriasis or flexural psoriasis.
Inverse psoriasis occurs mostly in the armpits, groin, under the
breasts and in other skin folds around the genitals and buttocks,
and, as a result of the locations of presentation, rubbing and
sweating can irritate the affected areas.
[0234] d. Pustular Psoriasis
[0235] The formulations and methods of the invention may be used to
treat subjects having pustular psoriasis. Pustular psoriasis is a
form of psoriasis that causes pus-filled blisters that vary in size
and location, but often occur on the hands and feet. The blisters
may be localized, or spread over large areas of the body. Pustular
psoriasis can be both tender and painful, can cause fevers.
[0236] e. Other Psoriasis Disorders
[0237] Other examples of psoriatic disorders which can be treated
with the formulations and methods of the invention include
erythrodermic psoriasis, vulgaris, psoriasis associated with IBD,
and psoriasis associated with arthritis, including rheumatoid
arthritis.
[0238] 2. Pemphigus Vulgaris
[0239] The formulations and methods of the invention may be used to
treat subjects having pemphigus vulgaris. Pemphigus vulgaris is a
serious autoimmune systemic dermatologic disease that often affects
the oral mucous membrane and skin. The pathogenesis of pemphigus
vulgaris is thought to be an autoimmune process that is directed at
skin and oral mucous membrane desmosomes. Consequentially, cells do
not adhere to each other. The disorder manifests as large
fluid-filled, rupture-prone bullae, and has a distinctive
histologic appearance. Anti-inflammatory agents are the only
effective therapy for this disease which has a high mortality rate.
Complications that arise in patients suffering from pemphigus
vulgaris are intractable pain, interference with nutrition and
fluid loss, and infections.
[0240] 3. Atopic Dermatitis/Eczema
[0241] The formulations and methods of the invention may be used to
treat subjects having atopic dermatitis. Atopic dermatitis (also
referred to as eczema) is a chronic skin disorder categorized by
scaly and itching plaques. People with eczema often have a family
history of allergic conditions like asthma, hay fever, or eczema.
Atopic dermatitis is a hypersensitivity reaction (similar to an
allergy) which occurs in the skin, causing chronic inflammation.
The inflammation causes the skin to become itchy and scaly. Chronic
irritation and scratching can cause the skin to thicken and become
leathery-textured. Exposure to environmental irritants can worsen
symptoms, as can dryness of the skin, exposure to water,
temperature changes, and stress.
[0242] Subjects with atopic dermatitis can be identified by certain
symptoms, which often include intense itching, blisters with oozing
and crusting, skin redness or inflammation around the blisters,
rash, dry, leathery skin areas, raw areas of the skin from
scratching, and ear discharges/bleeding.
[0243] 4. Sarcoidosis
[0244] The formulations and methods of the invention may be used to
treat subjects having sarcoidosis. Sarcoidosis is a disease in
which granulomatous inflammation occurs in the lymph nodes, lungs,
liver, eyes, skin, and/or other tissues. Sarcoidosis includes
cutaneous sarcoidosis (sarcoidosis of the skin) and nodular
sarcoidosis (sarcoidosis of the lymph nodes). Patients with
sarcoidosis can be identified by the symptoms, which often include
general discomfort, uneasiness, or an ill feeling; fever; skin
lesions.
[0245] 5. Erythema Nodosum
[0246] The formulations and methods of the invention may be used to
treat subjects having erythema nodosum. Erythema nodosum refers to
an inflammatory disorder that is characterized by tender, red
nodules under the skin, typically on the anterior lower legs.
Lesions associated with erythema nodosum often begin as flat, but
firm, hot red painful lumps (approximately an inch across). Within
a few days the lesions may become purplish, and then over several
weeks fade to a brownish flat patch.
[0247] In some instances, erythema nodosum may be associated with
infections including, streptococcus, coccidioidomycosis,
tuberculosis, hepatitis B, syphilis, cat scratch disease,
tularemia, yersinia, leptospirosis psittacosis, histoplasmosis,
mononucleosis (EBV). In other instances, erythema nodosum may be
associated with sensitivity to certain medications including,
oralcontraceptives, penicillin, sulfonamides, sulfones,
barbiturates, hydantoin, phenacetin, salicylates, iodides, and
progestin. Erythema nodosum is often associated with other
disorders including, leukemia, sarcoidosis, rheumatic fever, and
ulcerative colitis.
[0248] Symptoms of erythema nodosum usually present themselves on
the shins, but lesions may also occur on other areas of the body,
including the buttocks, calves, ankles, thighs and upper
extremities. Other symptoms in subjects with erythema nodosum can
include fever and malaise.
[0249] 6. Hidradenitis Suppurativa
[0250] The formulations and methods of the invention may be used to
treat subjects having hidradenitis suppurativa. Hidradenitis
suppurativa refers to a skin disorder in which swollen, painful,
inflamed lesions or lumps develop in the groin and sometimes under
the arms and under the breasts. Hidradenitis suppurativa occurs
when apocrine gland outlets become blocked by perspiration or are
unable to drain normally because of incomplete gland development.
Secretions trapped in the glands force perspiration and bacteria
into surrounding tissue, causing subcutaneous induration,
inflammation, and infection. Hidradenitis suppurativa is confined
to areas of the body that contain apocrine glands. These areas are
the axillae, areola of the nipple, groin, perineum, circumanal, and
periumbilical regions.
[0251] 7. Lichen Planus
[0252] The formulations and methods of the invention may be used to
treat subjects having lichen planus. Tumor necrosis factor has been
implicated in the pathophysiology of lichen planus (Sklavounou et
al. (2000) J Oral Pathol Med. 29:370). Lichen planus refers to a
disorder of the skin and the mucous membranes resulting in
inflammation, itching, and distinctive skin lesions. Lichen planus
may be associated with hepatitis C or certain medications.
[0253] 8. Sweet's Syndrome
[0254] The formulations and methods of the invention may be used to
treat subjects having Sweet's syndrome. Inflammatory cytokines,
including tumor necrosis factor, have been implicated in the
pathophysiology of Sweet's syndrome (Reuss-Borst et al. (1993) Br J
Haematol. 84:356). Sweet's syndrome, which was described by R. D.
Sweet in 1964, is characterized by the sudden onset of fever,
leukocytosis, and cutaneous eruption. The eruption consists of
tender, erythematous, well-demarcated papules and plaques which
show dense neutrophilic infiltrates microscopically. The lesions
may appear anywhere, but favor the upper body including the face.
The individual lesions are often described as pseudovesicular or
pseudopustular, but may be frankly pustular, bullous, or
ulcerative. Oral and eye involvement (conjunctivitis or
episcleritis) have also been frequently reported in patients with
Sweet's syndrome. Leukemia has also been associated with Sweet's
syndrome.
[0255] 9. Vitiligo
[0256] The formulations and methods of the invention may be used to
treat subjects having vitiligo. Vitiligo refers to a skin condition
in which there is loss of pigment from areas of skin resulting in
irregular white patches with normal skin texture. Lesions
characteristic of vitiligo appear as flat depigmented areas. The
edges of the lesions are sharply defined but irregular. Frequently
affected areas in subjects with vitiligo include the face, elbows
and knees, hands and feet, and genitalia.
[0257] 10. Scleroderma
[0258] The formulations and methods of the invention may be used to
treat subjects having scleroderma. Tumor necrosis factor has been
implicated in the pathophysiology of scleroderma (Tutuncu Z et al.
(2002) Clin Exp Rheumatol. 20(6 Suppl 28):S146-51; Mackiewicz Z et
al. (2003) Clin Exp Rheumatol. 21(1):41-8; Murota H et al. (2003)
Arthritis Rheum. 48(4):1117-25). Scleroderma refers to a diffuse
connective tissue disease characterized by changes in the skin,
blood vessels, skeletal muscles, and internal organs. Scleroderma
is also referred to as CREST syndrome or Progressive systemic
sclerosis, and usually affects people between the ages 30-50. Women
are affected more often than men.
[0259] The cause of scleroderma is unknown. The disease may produce
local or systemic symptoms. The course and severity of the disease
varies widely in those affected. Excess collagen deposits in the
skin and other organs produce the symptoms. Damage to small blood
vessels within the skin and affected organs also occurs. In the
skin, ulceration, calcification, and changes in pigmentation may
occur. Systemic features may include fibrosis and degeneration of
the heart, lungs, kidneys and gastrointestinal tract.
[0260] Patients suffering from scleroderma exhibit certain clinical
features, including, blanching, blueness, or redness of fingers and
toes in response to heat and cold (Raynaud's phenomenon), pain,
stiffness, and swelling of fingers and joints, skin thickening and
shiny hands and forearm, esophageal reflux or heartburn, difficulty
swallowing, and shortness of breath. Other clinical symptoms used
to diagnose scleroderma include, an elevated erythrocyte
sedimentation rate (ESR), an elevated rheumatoid factor (RF), a
positive antinuclear antibody test, urinalysis that shows protein
and microscopic blood, a chest X-ray that may show fibrosis, and
pulmonary function studies that show restrictive lung disease.
[0261] 11. Nail Disorders
[0262] The formulations and methods of the invention may be used to
treat subjects having a nail disorder. Nail disorders include any
abnormality of the nail Specific nail disorders include, but are
not limited to, pitting, koilonychia, Beau's lines, spoon nails,
onycholysis, yellow nails, pterygium (seen in lichen planus), and
leukonychia. Pitting is characterized by the presence of small
depressions on the nail surface. Ridges or linear elevations can
develop along the nail occurring in a "lengthwise" or "crosswise"
direction. Beau's lines are linear depressions that occur
"crosswise" (transverse) in the fingernail. Leukonychia describes
white streaks or spots on the nails. Koilonychia is an abnormal
shape of the fingernail where the nail has raised ridges and is
thin and concave Koilonychia is often associated with iron
deficiency.
[0263] Nail disorders which can be treated with the TNFalpha
antibody of the invention also include psoriatic nails. Psoriatic
nails include changes in nails which are attributable to psoriasis.
In some instances psoriasis may occur only in the nails and nowhere
else on the body. Psoriatic changes in nails range from mild to
severe, generally reflecting the extent of psoriatic involvement of
the nail plate, nail matrix, i.e., tissue from which the nail
grows, nail bed, i.e., tissue under the nail, and skin at the base
of the nail Damage to the nail bed by the pustular type of
psoriasis can result in loss of the nail Nail changes in psoriasis
fall into general categories that may occur singly or all together.
In one category of psoriatic nails, the nail plate is deeply
pitted, probably due to defects in nail growth caused by psoriasis.
IN another category, the nail has a yellow to yellow-pink
discoloration, probably due to psoriatic involvement of the nail
bed. A third subtype of psoriatic nails are characterized by white
areas which appear under the nail plate. The white areas are
actually air bubbles marking spots where the nail plate is becoming
detached from the nail bed. There may also be reddened skin around
the nail A fourth category is evidenced by the nail plate crumbling
in yellowish patches, i.e., onychodystrophy, probably due to
psoriatic involvement in the nail matrix. A fifth category is
characterized by the loss of the nail in its entirety due to
psoriatic involvement of the nail matrix and nail bed.
[0264] The formulations and methods of the invention may also be
used to treat nail disorders often associated with lichen planus.
Nails in subjects with lichen planus often show thinning and
surface roughness of the nail plate with longitudinal ridges or
pterygium.
[0265] The formulations and methods of the invention may be used to
treat nail disorders, such as those described herein. Often nail
disorders are associated with skin disorders. In one embodiment,
the invention includes a method of treatment for nail disorders
with a TNFalpha antibody. In another embodiment, the nail disorder
is associated with another disorder, including a skin disorder such
as psoriasis. In another embodiment, the disorder associated with a
nail disorder is arthritis, including psoriatic arthritis.
[0266] 12. Other Skin and Nail Disorders
[0267] The formulations and methods of the invention may be used to
treat other skin and nail disorders, such as chronic actinic
dermatitis, bullous pemphigoid, and alopecia areata. Chronic
actinic dermatitis (CAD) is also referred to as photosensitivity
dermatitis/actinic reticuloid syndrome (PD/AR). CAD is a condition
in which the skin becomes inflamed, particularly in areas that have
been exposed to sunlight or artificial light. Commonly, CAD
patients have allergies to certain substances that come into
contact with their skin, particularly various flowers, woods,
perfumes, sunscreens and rubber compounds. Bullous pemphigoid
refers to A skin disorder characterized by the formation of large
blisters on the trunk and extremities. Alopecia areata refers to
hair loss characterized by round patches of complete baldness in
the scalp or beard.
[0268] K. Metabolic Disorders
[0269] The formulations and methods of the invention may be used to
treat a metabolic disease. TNF.alpha. has been implicated in the
pathophysiology of a wide variety of disorders, including metabolic
disorders, such as diabetes and obesity (Spiegelman and
Hotamisligil (1993) Cell 73:625; Chu et al. (2000) Int J Obes Relat
Metab Disord. 24:1085; Ishii et al. (2000) Metabolism.
49:1616).
[0270] Metabolic disorders affect how the body processes substances
needed to carry out physiological functions. A number of metabolic
disorders of the invention share certain characteristics, i.e. they
are associated the insulin resistance, lack of ability to regulate
blood sugar, weight gain, and increase in body mass index. Examples
of metabolic disorders include diabetes and obesity. Examples of
diabetes include type 1 diabetes mellitus, type 2 diabetes
mellitus, diabetic neuropathy, peripheral neuropathy, diabetic
retinopathy, diabetic ulcerations, retinopathy ulcerations,
diabetic macrovasculopathy, and obesity. Examples of metabolic
disorders which can be treated with the formulations and methods of
the invention are described in more detail below:
[0271] 1. Diabetes
[0272] The formulations and methods of the invention may be used to
treat diabetes. Tumor necrosis factor has been implicated in the
pathophysiology of diabetes. (see e.g., Navarro J. F., Mora C.,
Maca, Am J Kidney Dis. 2003 July; 42(1):53-61; Daimon M et al.,
Diabetes Care. 2003 July; 26(7):2015-20; Zhang M et al., J Tongji
Med Univ. 1999; 19(3):203-5, Barbieri M et al., Am J Hypertens.
2003 July; 16(7):537-43.) For example, TNF.alpha. is implicated in
the pathophysiology for insulin resistance. It has been found that
serum TNF levels in patients with gastrointestinal cancer
correlates with insulin resistance (see e.g., McCall, J. et al. Br.
J. Surg. 1992; 79: 1361-3).
[0273] Diabetes includes the two most common types of the disorder,
namely type I diabetes and type II diabetes, which both result from
the body's inability to regulate insulin. Insulin is a hormone
released by the pancreas in response to increased levels of blood
sugar (glucose) in the blood.
[0274] The term "type 1 diabetes," as used herein, refers to a
chronic disease that occurs when the pancreas produces too little
insulin to regulate blood sugar levels appropriately. Type 1
diabetes is also referred to as insulin-dependent diabetes
mellitus, IDMM, juvenile onset diabetes, and diabetes--type I. Type
1 diabetes represents is the result of a progressive autoimmune
destruction of the pancreatic .beta.-cells with subsequent insulin
deficiency.
[0275] The term "type 2 diabetes," refers to a chronic disease that
occurs when the pancreas does not make enough insulin to keep blood
glucose levels normal, often because the body does not respond well
to the insulin. Type 2 diabetes is also referred to as
noninsulin-dependent diabetes mellitus, NDDM, and diabetes--type
II
[0276] Diabetes is can be diagnosed by the administration of a
glucose tolerance test. Clinically, diabetes is often divided into
several basic categories. Primary examples of these categories
include, autoimmune diabetes mellitus, non-insulin-dependent
diabetes mellitus (type 1 NDDM), insulin-dependant diabetes
mellitus (type 2 IDDM), non-autoimmune diabetes mellitus,
non-insulin-dependant diabetes mellitus (type 2 NIDDM), and
maturity-onset diabetes of the young (MODY). A further category,
often referred to as secondary, refers to diabetes brought about by
some identifiable condition which causes or allows a diabetic
syndrome to develop. Examples of secondary categories include,
diabetes caused by pancreatic disease, hormonal abnormalities,
drug- or chemical-induced diabetes, diabetes caused by insulin
receptor abnormalities, diabetes associated with genetic syndromes,
and diabetes of other causes. (see e.g., Harrison's (1996)
14.sup.th ed., New York, McGraw-Hill).
[0277] Diabetes manifests itself in the foregoing categories and
can cause several complications that are discussed in the following
sections. Accordingly, the antibody, or antigen-binding fragment
thereof, of the invention can be used to treat diabetes. In one
embodiment, the TNF.alpha. antibody, or antigen-binding fragment
thereof, of the invention is used to treat diabetes associated with
the above identified categories.
[0278] Diabetes is often treated with diet, insulin dosages, and
various medications described herein. Accordingly, the formulations
of the invention may also be administered in combination with
agents commonly used to treat metabolic disorders and pain commonly
associated with diabetes.
[0279] Diabetes manifests itself in many complications and
conditions associated with diabetes, including the following
categories:
[0280] a. Diabetic Neuropathy and Peripheral Neuropathy
[0281] The formulations and methods of the invention may be used to
treat diabetic neuropathy or peripheral neuropathy. Tumor necrosis
factor has been implicated in the pathophysiology of diabetic
neuropathy and peripheral neuropathy. (See Benj afield et al.
(2001) Diabetes Care. 24:753; Qiang, X. et al. (1998) Diabetologia.
41:1321-6; Pfeiffer et al. (1997) Horm Metab Res. 29:111).
[0282] The term "neuropathy," also referred to as nerve
damage-diabetic, as used herein, refers to a common complication of
diabetes in which nerves are damaged as a result of hyperglycemia
(high blood sugar levels). A variety of diabetic neuropathies are
recognized, such as distal sensorimotror polyneuropathy, focal
motor neuropathy, and autonomic neuropathy.
[0283] The term "peripheral neuropathy," also known as peripheral
neuritis and diabetic neuropathy, as used herein, refers to the
failure of the nerves to carry information to and from the brain
and spinal cord. Peripheral neuropathy produces symptoms such as
pain, loss of sensation, and the inability to control muscles. In
some cases, the failure of nerves to control blood vessels,
intestinal function, and other organs results in abnormal blood
pressure, digestion, and loss of other basic involuntary processes.
Peripheral neuropathy may involve damage to a single nerve or nerve
group (mononeuropathy) or may affect multiple nerves
(polyneuropathy).
[0284] Neuropathies that affect small myelinated and unmyelinated
fibers of the sympathetic and parasympathetic nerves are known as
"peripheral neuropathies." Furthermore, the related disorder of
peripheral neuropathy, also known as peripheral neuritis and
diabetic neuropathy, refers to the failure of the nerves to carry
information to and from the brain and spinal cord. This produces
symptoms such as pain, loss of sensation, and the inability to
control muscles. In some cases, failure of nerves controlling blood
vessels, intestinal function, and other organs results in abnormal
blood pressure, digestion, and loss of other basic involuntary
processes. Peripheral neuropathy may involve damage to a single
nerve or nerve group (mononeuropathy) or may affect multiple nerves
(polyneuropathy).
[0285] The term "diabetic neuropathy" refers to a common
complication of diabetes in which nerves are damaged as a result of
hyperglycemia (high blood sugar levels). Diabetic neuropathy is
also referred to as neuropathy and nerve damage-diabetic. A variety
of diabetic neuropathies are recognized, such as distal
sensorimotror polyneuropathy, focal motor neuropathy, and autonomic
neuropathy.
[0286] b. Diabetic Retinopathy
[0287] The formulations and methods of the invention may be used to
treat diabetic retinopathy. Tumor necrosis factor has been
implicated in the pathophysiology of diabetic retinopthy (Scholz et
al. (2003) Trends Microbiol. 11:171). The term "diabetic
retinopathy" as used herein, refers to progressive damage to the
eye's retina caused by long-term diabetes. Diabetic retinopathy,
includes proliferative retinopathy. Proliferative neuropathy in
turn includes includes neovascularization, pertinal hemmorrhave and
retinal detachement.
[0288] In advanced retinopathy, small vessels proliferate on the
surface of the retina. These blood vessels are fragile, tend to
bleed and can cause peretinal hemorrhages. The hemorrhage can
obscure vision, and as the hemorrhage is resorbed fibrous tissue
forms predisposing to retinal detachments and loss of vision. In
addition, diabetic retinopathy includes prolferative retinopathy
which includes neovascularization, pertinal hemmorrhave and retinal
detachement. Daibetic retinopathy also includes "background
retinopathy" which involves changes occuring with the layers of the
retina.
[0289] c. Diabetic Ulcerations and Retinopathy Ulcerations
[0290] The formulations and methods of the invention may be used to
treat diabetic ulcerations or retinopathy ulcerations. Tumor
necrosis factor has been implicated in the pathophysiology of
diabetic ulcerations, (see Lee et al. (2003) Hum Immunol. 64:614;
Navarro et al. (2003) Am J Kidney Dis. 42:53; Daimon et al (2003)
Diabetes Care. 26:2015; Zhang et al. (1999) J Tongji Med Univ.
19:203; Barbieri et al. (2003) Am J Hypertens. 16:537; Venn et al.
(1993) Arthritis Rheum. 36:819; Westacott et al. (1994) J
Rheumatol. 21:1710).
[0291] The term "diabetic ulcerations," as used herein, refers to
an ulcer which results as a complication of diabetes. An ulcer is a
crater-like lesion on the skin or mucous membrane caused by an
inflammatory, infectious, malignant condition, or metabolic
disorder. Typically diabetic ulcers can be found on limbs and
extremeties, more typically the feet. These ulcers, caused by
diabetic conditions, such as neurapthy and a vacualr insuffciency,
can lead to ischemia and poor wound healing. More extensive
ulcerations may progress to ostemyelitis. Once ostemyelitis
develops, it may be dificulte to eradicate with antibotics alonda
nd amputation maybe necessary.
[0292] The term "retinopathy ulcerations," as used herein refers to
an ulcer which causes or results in damages to the eye and the
eye's retina. Retinopathy ulcerations may include conditions such
has retinoathic hemmorages.
[0293] d. Diabetic Macrovasculopathy
[0294] The formulations and methods of the invention may be used to
treat diabetic macrovasculopathy. Tumor necrosis factor has been
implicated in the pathophysiology of diabetic macrovasculopathy
(Devaraj et al. (2000) Circulation. 102:191; Hattori Y et al.
(2000) Cardiovasc Res. 46:188; Clausell N et al. (1999) Cardiovasc
Pathol. 8:145). The term "diabetic macrovasculopathy," also
referred to as "macrovascular disease," as used herein, refers to a
disease of the blood vessels that results from diabetes. Diabetic
macrovasculopathy complication occurs when, for example, fat and
blood clots build up in the large blood vessels and stick to the
vessel walls. Diabetic macrovasculopathies include diseases such as
coronary disease, cerebrovascular disease, and peripheral vascular
disease, hyperglycaemia and cardiovascular disease, and
strokes.
[0295] 2. Obesity
[0296] The formulations and methods of the invention may be used to
treat obesity. Tumor necrosis factor has been implicated in the
pathophysiology of obesity (see e.g., Pihlajamaki J et al. (2003)
Obes Res. 11:912; Barbieri et al. (2003) Am J Hypertens. 16:537;
Tsuda et al. (2003) J Nutr. 133:2125). Obesity increases a person's
risk of illness and death due to diabetes, stroke, coronary artery
disease, hypertension, high cholesterol, and kidney and gallbladder
disorders. Obesity may also increase the risk for some types of
cancer, and may be a risk factor for the development of
osteoarthritis and sleep apnea. Obesity can be treated with the
antibody of the invention alone or in combination with other
metabolic disorders, including diabetes.
[0297] L. Vasculitides
[0298] The formulations and methods of the invention may be used to
treat a subject having a vasculitis. TNF.alpha. has been implicated
in the pathophysiology of a variety of vasculitides, (see e.g.,
Deguchi et al. (1989) Lancet. 2:745). As used herein, the term "a
vasculitis in which TNF.alpha. activity is detrimental" is intended
to include vasculitis in which the presence of TNF.alpha. in a
subject suffering from the disorder has been shown to be or is
suspected of being either responsible for the pathophysiology of
the disorder or a factor that contributes to a worsening of the
disorder. Such disorders may be evidenced, for example, by an
increase in the concentration of TNF.alpha. in a biological fluid
of a subject suffering from the disorder (e.g., an increase in the
concentration of TNF.alpha. in serum, plasma, synovial fluid, etc.
of the subject), which can be detected, for example, using an
anti-TNF.alpha. antibody as described above.
[0299] There are numerous examples of vasculitides in which
TNF.alpha. activity is detrimental, including Behcet's disease. The
use of the formulations and methods of the invention in the
treatment of specific vasculitides are discussed further below. In
certain embodiments, the antibody, or antibody portion, is
administered to the subject in combination with another therapeutic
agent, as described below
[0300] The formulations and methods of the invention be used to
treat vasculitis in which TNF.alpha. activity is detrimental,
wherein inhibition of TNF.alpha. activity is expected to alleviate
the symptoms and/or progression of the vasculitis or to prevent the
vasculitis. Subjects suffering from or at risk of developing
vasculitis can be identified through clinical symptoms and tests.
For example, subjects with vasculitides often develop antibodies to
certain proteins in the cytoplasm of neutrophils, antineutrophil
cytoplasmic antibodies (ANCA). Thus, in some instances,
vasculitides may be evidenced by tests (e.g., ELISA), which measure
ANCA presence.
[0301] Vasculitis and its consequences may be the sole
manifestation of disease or it may be a secondary component of
another primary disease. Vasculitis may be confined to a single
organ or it may simultaneously affect several organs. and depending
on the syndrome, arteries and veins of all sizes can be affected.
Vasculitis can affect any organ in the body.
[0302] In vasculitis, the vessel lumen is usually compromised,
which is associated with ischemia of the tissues supplied by the
involved vessel. The broad range of disorders that may result from
this process is due to the fact that any type, size and location of
vessel (e.g., artery, vein, arteriole, venule, capillary) can be
involved. Vasculitides are generally classified according to the
size of the affected vessels, as described below. It should be
noted that some small and large vessel vasculitides may involve
medium-sized arteries; but large and medium-sized vessel
vasculitides do not involve vessels smaller than arteries. Large
vessel disease includes, but is not limited to, giant cell
arteritis, also known as temporal arteritis or cranial arteritis,
polymyalgia rheumatica, and Takayasu's disease or arteritis, which
is also known as aortic arch syndrome, young female arteritis and
Pulseless disease. Medium vessel disease includes, but is not
limited to, classic polyarteritis nodosa and Kawasaki's disease,
also known as mucocutaneous lymph node syndrome. Non-limiting
examples of small vessel disease are Behcet's Syndrome, Wegner's
granulomatosis, microscopic polyangitis, hypersensitivity
vasculitis, also known as cutaneous vasculitis, small vessel
vasculitis, Henoch-Schonlein purpura, allergic granulamotosis and
vasculitis, also known as Churg Strauss syndrome. Other
vasculitides include, but are not limited to, isolated central
nervous system vasculitis, and thromboangitis obliterans, also
known as Buerger's disease. Classic Polyarteritis nodosa (PAN),
microscopic PAN, and allergic granulomatosis are also often grouped
together and are called the systemic necrotizing vasculitides. A
further description of vasculitis is described below:
1. Large Vessel Vasculitis
[0303] In one embodiment, the formulations and methods of the
invention are used to treat subjects who have large vessel
vasculitis. The term "large vessel(s)" as used herein, refers to
the aorta and the largest branches directed toward major body
regions. Large vessels include, for example, the aorta, and its
branches and corresponding veins, e.g., the subclavian artery; the
brachiocephalic artery; the common carotid artery; the innonimate
vein; internal and external jugular veins; the pulmonary arteries
and veins; the venae cavae; the renal arteries and veins; the
femoral arteries and veins; and the carotid arteries. Examples of
large vessel vasculitides are described below.
[0304] a. Giant Cell Arteritis (GCA)
[0305] The formulations and methods of the invention may be used to
treat giant cell arteritis. Tumor necrosis factor has been
implicated in the pathophysiology of giant cell arteritis (Sneller,
M. C. (2002) Cleve. Clin. J. Med. 69:SII40-3; Schett, G., et al.
(2002) Ann. Rheum. Dis. 61:463). Giant cell arteritis (GCA), refers
to a vasculitis involving inflammation and damage to blood vessels,
particularly the large or medium arteries that branch from the
external carotid artery of the neck. GCA is also referred to as
temporal arteritis or cranial arteritis, and is the most common
primary vasculitis in the elderly. It almost exclusively affects
individuals over 50 years of age, however, there are
well-documented cases of patients 40 years and younger. GCA usually
affects extracranial arteries. GCA can affect the branches of the
carotid arteries, including the temporal artery. GCA is also a
systemic disease which can involve arteries in multiple
locations.
[0306] Histopathologically, GCA is a panarteritis with inflammatory
mononuclear cell infiltrates within the vessel wall with frequent
Langhans type giant cell formation. There is proliferation of the
intima, granulomatous inflammation and fragmentation of the
internal elastic lamina. The pathological findings in organs is the
result of ischemia related to the involved vessels.
[0307] Patients suffering from GCA exhibit certain clinical
symptoms, including fever, headache, anemia and high erythrocyte
sedimentation rate (ESR). Other typical indications of GCA include
jaw or tongue claudication, scalp tenderness, constitutional
symptoms, pale optic disc edema (particularly `chalky white` disc
edema), and vision disturbances. The diagnosis is confirmed by
temporal artery biopsy.
[0308] b. Polymyalgia Rheumatica
[0309] The formulations and methods of the invention may be used to
treat polymyalgia rheumatica. Tumor necrosis factor has been
implicated in the pathophysiology of polymyalgia rheumatica
(Straub, R. H., et al. (2002) Rheumatology (Oxford) 41:423;
Uddhammar, A., et al. (1998) Br. J. Rheumatol. 37:766). Polymyalgia
rheumatica refers to a rheumatic disorder that is associated with
moderate to severe muscle pain and stiffness in the neck, shoulder,
and hip, most noticeable in the morning. IL-6 and IL-1.beta.
expression has also been detected in a majority of the circulating
monocytes in patients with the polymyalgia rheumatica. Polymyalgia
rheumatica may occur independently, or it may coexist with or
precede GCA, which is an inflammation of blood vessels.
[0310] c. Takayasu's Arteritis
[0311] The formulations and methods of the invention may be used to
treat Takayasu's arteritis. Tumor necrosis factor has been
implicated in the pathophysiology of Takayasu's arteritis
(Kobayashi, Y. and Numano, F. (2002) Intern. Med. 41:44; Fraga, A.
and Medina F. (2002) Curr. Rheumatol. Rep. 4:30). Takayasu's
arteritis refers to a vasculitis characterized by an inflammmation
of the aorta and its major branches. Takayasu's arteritis (also
known as Aortic arch syndrome, young female arteritis and Pulseless
disease) affects the thoracic and abdominal aorta and its main
branches or the pulmonary arteries. Fibrotic thickening of the
aortic wall and its branches (e.g., carotid, inominate, and
subclavian arteries) can lead to reduction of lumen size of vessels
that arise from the aortic arch. This condition also typically
affects the renal arteries.
[0312] Takayasu's arteritis primarily affects young women, usually
aged 20-40 years old, particularly of Asian descent, and may be
manifested by malaise, arthralgias and the gradual onset of
extremity claudication. Most patients have asymmetrically reduced
pulses, usually along with a blood pressure differential in the
arms. Coronary and/or renal artery stenosis may occur.
[0313] The clinical features of Takayasu's arteritis may be divided
into the features of the early inflammatory disease and the
features of the later disease. The clinical features of the early
inflammatory stage of Takayasu's disease are: malaise, low grade
fever, weight loss, myalgia, arthralgia, and erythema multiforme.
Later stages of Takayasu's disease are characterised by fibrotic
stenosis of arteries and thrombosis. The main resulting clinical
features are ischaemic phenomena, e.g. weak and asymmetrical
arterial pulses, blood pressure discrepancy between the arms,
visual disturbance, e.g. scotomata and hemianopia, other
neurological features including vertigo and syncope, hemiparesis or
stroke. The clinical features result from ischaemia due to arterial
stenosis and thrombosis.
[0314] 2. Medium Vessel Disease
[0315] The formulations and methods of the invention may be used to
treat subjects who have medium vessel vasculitis. The term "medium
vessel(s)" is used to refer to those blood vessels which are the
main visceral arteries. Examples of medium vessels include the
mesenteric arteries and veins, the iliac arteries and veins, and
the maxillary arteries and veins. Examples of medium vessel
vasculitides are described below.
[0316] a. Polyarteritis Nodosa
[0317] The formulations and methods of the invention may be used to
treat polyarteritis nodosa. Tumor necrosis factor has been
implicated in the pathophysiology of polyarteritis nodosa
(DiGirolamo, N., et al. (1997) J. Leukoc. Biol. 61:667).
Polyarteritis nodosa, or periarteritis nodosa refers to vasculitis
which is a serious blood vessel disease in which small and
medium-sized arteries become swollen and damaged because they are
attacked by rogue immune cells. Polyarteritis nodosa usually
affects adults more frequently than children. It damages the
tissues supplied by the affected arteries because they don't
receive enough oxygen and nourishment without a proper blood
supply.
[0318] Symptoms which are exhibited in patients with polyarteritis
nodosa generally result from damage to affected organs, often the
skin, heart, kidneys, and nervous system. Generalized symptoms of
polyarteritis nodosa include fever, fatigue, weakness, loss of
appetite, and weight loss. Muscle aches (myalgia) and joint aches
(arthralgia) are common. The skin of subjects with polyarteritis
nodosa may also show rashes, swelling, ulcers, and lumps (nodular
lesions).
[0319] Classic PAN (polyarteritis nodosa) is a systemic arteritis
of small to medium muscular arteritis in which involvement of renal
and visceral arteries is common Abdominal vessels have aneurysms or
occlusions in 50% of PAN patients. Classic PAN does not involve the
pulmonary arteries although the bronchial vessels may be involved.
Granulomas, significant eosinophilia and an allergic diathesis are
not part of the syndrome. Although any organ system may be
involved, the most common manifestations include peripheral
neuropathy, mononeuritis multiplex, intestinal ischemia, renal
ischemia, testicular pain and livedo reticularis.
[0320] b. Kawasaki's Disease
[0321] The formulations and methods of the invention may be used to
treat Kawasaki's disease. Tumor necrosis factor has been implicated
in the pathophysiology of Kawasaki's disease (Sundel, R. P. (2002)
Curr. Rheumatol. Rep. 4:474; Gedalia, A. (2002) Curr. Rheumatol.
Rep. 4:25). Although the cause of Kawasaki's disease is unknown, it
is associated with acute inflammation of the coronary arteries,
suggesting that the tissue damage associated with this disease may
be mediated by proinflammatory agents such as TNF.alpha..
Kawasaki's disease refers to a vasculitis that affects the mucus
membranes, lymph nodes, lining of the blood vessels, and the heart.
Kawasaki's disease is also often referred to as mucocutaneous lymph
node syndrome, mucocutaneous lymph node disease, and infantile
polyarteritis. Subjects afflicted with Kawasaki's disease develop
vasculitis often involving the coronary arteries which can lead to
myocarditis and pericarditis. Often as the acute inflammation
diminishes, the coronary arteries may develop aneurysm, thrombosis,
and lead to myocardial infarction.
[0322] Kawasaki's disease is a febrile systemic vasculitis
associated with edema in the palms and the soles of the feet, with
enlargement of cervical lymph nodes, cracked lips and "strawberry
tongue". Although the inflammatory response is found in vessels
throughout the body, the most common site of end-organ damage is
the coronary arteries. Kawasaki's Disease predominantly affects
children under the age of 5. The highest incidence is in Japan but
is becoming increasingly recognized in the West and is now the
leading cause of acquired heart disease in US children. The most
serious complication of Kawasaki disease is coronary arteritis and
aneurysm formation that occurs in a third of untreated
patients.
[0323] 3. Small Vessel Disease
[0324] The formulations and methods of the invention may be used to
treat small vessel disease. In one embodiment, the TNF.alpha.
antibody of the invention is used to treat subjects who have small
vessel vasculitis. The term "small vessel(s)" is used to refer to
arterioles, venules and capillaries. Arterioles are arteries that
contain only 1 or 2 layers of sooth muscle cells and are terminal
to and continuous with the capillary network. Venules carry blood
from the capillary network to veins and capillaries connect
arterioles and venules. Examples of small vessel vasculitides are
described below.
[0325] a. Behcet's Disease
[0326] The formulations and methods of the invention may be used to
treat Behcet's disease. Tumor necrosis factor has been implicated
in the pathophysiology of Behcet's disease (Sfikakis, P. P. (2002)
Ann. Rheum. Dis. 61:ii51-3; Dogan, D. and Farah, C. (2002)
Oftalmologia. 52:23). Behcet's disease is a chronic disorder that
involves inflammation of blood vessels throughout the body.
Behcet's disease may also cause various types of skin lesions,
arthritis, bowel inflammation, and meningitis (inflammation of the
membranes of the brain and spinal cord). As a result of Behcet's
disease, the subject with the disorder may have inflammation in
tissues and organs throughout the body, including the
gastrointestinal tract, central nervous system, vascular system,
lungs, and kidneys. Behcet's disease is three times more common in
males than females and is more common in the east Mediterranean and
Japan.
[0327] b. Wegener's Granulomatosis
[0328] The formulations and methods of the invention may be used to
treat Wegener's granulomatosis. Tumor necrosis factor has been
implicated in the pathophysiology of Wegener's granulomatosis
(Marquez, J., et al. (2003) Curr. Rheumatol. Rep. 5:128; Harman, L.
E. and Margo, C. E. (1998) Surv. Ophthalmol. 42:458). Wegener's
granulomatosis refers to a vasculitis that causes inflammation of
blood vessels in the upper respiratory tract (nose, sinuses, ears),
lungs, and kidneys. Wegener's granulomatosis is also referred to as
midline granulomatosis. Wegener's granulomatosis includes a
granulomatous inflammation involving the respiratory tract, and
necrotizing vasculitis affecting small to medium-sized vessels.
Subjects who have Wegener's granulomatosis often also have
arthritis (joint inflammation). Glomerulonephritis may also be
present in affected subjects, but virtually any organ may be
involved.
[0329] c. Churg-Strauss Syndrome
[0330] The formulations and methods of the invention may be used to
treat Churg-Strauss syndrome. Tumor necrosis factor has been
implicated in the pathophysiology of Churg-Strauss syndrome (Gross,
W. L (2002) Curr. Opin. Rheumatol. 14:11; Churg, W. A. (2001) Mod.
Pathol. 14:1284). Churg-Strauss syndrome refers to a vasculitis
that is systemic and shows early manifestation signs of asthma and
eosinophilia. Churg-Strauss syndrome is also referred to as
allergic granulomatosis and angiitis, and occurs in the setting of
allergic rhinitis, asthma and eosinophilia. Sinusitis and pulmonary
infiltrates also occur in Churg-Strauss syndrome, primarily
affecting the lung and heart. Peripheral neuropathy, coronary
arteritis and gastrointestinal involvement are common.
[0331] M. Other Diseases
[0332] The formulations and methods of the invention may be used to
treat various other disorders in which TNFalpha activity is
detrimental. Examples of other diseases and disorders in which
TNFalpha activity has been implicated in the pathophysiology, and
thus which can be treated using an antibody, or antibody portion,
of the invention, include inflammatory bone disorders and bone
resorption disease (see e.g., Bertolini. D. R., et al. (1986)
Nature 319:516-518; Konig, A. et al. (1988) J. Bone Miner. Res.
3:621-627; Lerner, U. H. and Ohlin, A. (1993) J. Bone Miner. Res.
8:147-155; and Shanlar. G. and Stem, P. H. (1993) Bone 14:871-876),
hepatitis, including alcoholic hepatitis (see e.g., McClain, C. J.
and Cohen, D. A. (1989) Hepatology 9:349-351; Felver, M. E., el al.
(1990) Alcohol. Clin. Exp. Res. 14:255-259; and Hansen, J., el al.
(1994) Hepatology 20:461-474), viral hepatitis (Sheron, N., et al.
(1991) J. Hepatol. 12:241-245; and Hussain, M. J., et al. (1994) J.
Clin. Pathol. 47:1112-1115), and fulminant hepatitis; coagulation
disturbances (see e.g., van der Poll, T., el al. (1990) N. Engl. J.
Med. 322:1622-1627; and van der Poll, T., et al. (1991) Prog. Clin.
Biol. Res. 367:55-60), bums (see eg., Giroir, B. P., el al. (1994)
Am. J. Physiol. 267:H 118-124; and Liu. X. S., el al. (1994) Burns
20:40-44), reperfusion injury (see e.g., Scales. W. E., et al.
(1994) Am. J Physiol. 267:G1122-1127; Serrick, C., el al. (1994)
Transplantation 58:1158-1162; and Yao, Y. M., et al. (1995)
Resuscitation 29:157-168), keloid formation (see e.g., McCauley, R.
L., et al. (1992) J. Clin. Immunol. 12:300-308), scar tissue
formation; pyrexia; periodontal disease; obesity and radiation
toxicity.
[0333] Examples of other disorders that may be treated with the
formulations and methods of the invention are described in
US20040126372 and U.S. Pat. No. 6,258,562, each of which is
incorporated by reference herein.
[0334] In one embodiment, the formulation and methods of the
invention are used to treat rheumatoid arthritis, psoriatic
arthritis, or ankylosing spondylitis. The formulation of the
invention comprising an isolated human TNF alpha antibody, or
antigen-binding portion thereof, (e.g., adalimumab), may be
administered to a human subject according to a dosing scheme and
dose amount effective for treating rheumatoid arthritis, psoriatic
arthritis, or ankylosing spondylitis. In one embodiment, a dose of
about 40 mg of a human TNF alpha antibody, or antigen-binding
portion thereof, (e.g., adalimumab) (e.g., 0.4 mL of a 100 mg/mL
formulation of the invention) in the formulation of the invention
is administered to a human subject every other week for the
treatment of rheumatoid arthritis, psoriatic arthritis, or
ankylosing spondylitis. In one embodiment, a dose of about 80 mg of
a human TNF alpha antibody, or antigen-binding portion thereof,
(e.g., adalimumab) (e.g., 0.8 mL of a 100 mg/mL formulation of the
invention) in the formulation of the invention is administered to a
human subject monthly for the treatment of rheumatoid arthritis,
psoriatic arthritis, or ankylosing spondylitis. In one embodiment,
the formulation is administered subcutaneously, every other week
(also referred to as biweekly, see methods of administration
described in US20030235585, incorporated by reference herein) for
the treatment of rheumatoid arthritis, ankylosing spondylitis, or
psoriatic arthritis. In one embodiment, the formulation is
administered subcutaneously, monthly for the treatment of
rheumatoid arthritis, ankylosing spondylitis, or psoriatic
arthritis.
[0335] In one embodiment, the formulation of the invention is used
to treat Crohn's disease or ulcerative colitis. The formulation of
the invention comprising an isolated human TNF alpha antibody, or
antigen-binding portion thereof, (e.g., adalimumab), may be
administered to a human subject according to a dosing scheme and
dose amount effective for treating Crohn's disease. In one
embodiment, a dose of about 160 mg of a human TNF alpha antibody,
or antigen-binding portion thereof, (e.g., adalimumab) (e.g., 1.6
mL of a 100 mg/mL formulation of the invention) in the formulation
of the invention is administered to a human subject initially at
about day 1, followed by a subsequent dose of 80 mg of the antibody
(e.g., 0.8 mL of a 100 mg/mL formulation of the invention) two
weeks later, followed by administration of about 40 mg (e.g., 0.4
mL of a 100 mg/mL formulation of the invention) every other week
for the treatment of Crohn's disease. In one embodiment, the
formulation is administered subcutaneously, according to a multiple
variable dose regimen comprising an induction dose(s) and
maintenance dose(s) (see, for example, U.S. Patent Publication Nos.
US20060009385 and US20090317399, incorporated by reference herein)
for the treatment of Crohn's disease or ulcerative colitis, each of
which are incorporated by reference herein) for the treatment of
Crohn's disease or ulcerative colitis. In one embodiment, the
formulation is administered subcutaneously, biweekly or monthly for
the treatment of Crohn's disease or ulcerative colitis. In one
embodiment, a dose of about 80 mg of a human TNF alpha antibody, or
antigen-binding portion thereof, (e.g., adalimumab) (e.g., 0.8 mL
of a 100 mg/mL formulation of the invention) in the formulation of
the invention is administered to a human subject monthly for the
treatment of Crohn's disease or ulcerative colitis.
[0336] In one embodiment, the formulation of the invention is used
to treat psoriasis. The formulation of the invention comprising an
isolated human TNF alpha antibody, or antigen-binding portion
thereof, (e.g., adalimumab), may be administered to a human subject
according to a dosing scheme and dose amount effective for treating
psoriasis. In one embodiment, an initial dose of about 80 mg of a
human TNF alpha antibody, or antigen-binding portion thereof,
(e.g., adalimumab) (e.g., 0.8 mL of a 100 mg/mL formulation of the
invention) in the formulation of the invention is administered to a
human subject, followed by a subsequent dose of 40 mg of the
antibody (e.g., 0.4 mL of a 100 mg/mL formulation of the invention)
every other week starting one week after the initial dose. In one
embodiment, the formulation is administered subcutaneously,
according to a multiple variable dose regimen comprising an
induction dose(s) and maintenance dose(s) (see, for example, US
20060009385 and WO 2007/120823, each of which are incorporated by
reference herein) for the treatment of psoriasis In one embodiment,
the formulation is administered subcutaneously, biweekly or monthly
for the treatment of psoriasis. In one embodiment, a dose of about
80 mg of a human TNF alpha antibody, or antigen-binding portion
thereof, (e.g., adalimumab) (e.g., 0.8 mL of a 100 mg/mL
formulation of the invention) in the formulation of the invention
is administered to a human subject monthly for the treatment of
psoriasis.
[0337] In one embodiment, the formulation of the invention is used
to treat juvenile idiopathic arthritis (JIA). The formulation of
the invention comprising an isolated human TNF alpha antibody, or
antigen-binding portion thereof, (e.g., adalimumab), may be
administered to a human subject according to a dosing scheme and
dose amount effective for treating JIA. In one embodiment, 20 mg of
a human TNF alpha antibody, or antigen-binding portion thereof, in
the formulation of the invention (e.g., 0.2 mL of a 100 mg/mL
formulation of the invention) is administered to a subject weighing
15 kg (about 33 lbs) to less than 30 kg (66 lbs) every other week
for the treatment of JIA. In another embodiment, 40 mg of a human
TNF alpha antibody, or antigen-binding portion thereof, in the
formulation of the invention (e.g., 0.4 mL of a 100 mg/mL
formulation of the invention) is administered to a subject weighing
more than or equal to 30 kg (66 lbs) every other week for the
treatment of JIA. In one embodiment, the formulation is
administered subcutaneously, according to a weight-based fixed dose
(see, for example, U.S. Patent Publication No. 20090271164,
incorporated by reference herein) for the treatment of JIA. In one
embodiment, the formulation is administered subcutaneously biweekly
or monthly for the treatment of JIA
[0338] In one embodiment, an isolated human TNF alpha antibody, or
antigen-binding portion thereof, (e.g., adalimumab), may be
administered to a human subject for treatment of a disorder
associated with detrimental TNFa activity according to a monthly
dosing schedule, whereby the antibody is administered once every
month or once every four weeks. As described above, examples of
disorders that may be treated according to a monthly dosing
schedule using the formulations and methods of the invention
include, but are not limited to, rheumatoid arthritis, ankylosing
spondylitis, JIA, psoriasis, Crohn's disease, ulcerative colitis,
hidradenitis suppurativa, giant cell arteritis, Behcet's disease,
sarcoidosis, diabetic retinopathy, or psoriatic arthritis. Thus,
the formulation of the invention comprising an isolated human TNF
alpha antibody, or antigen-binding portion thereof, (e.g.,
adalimumab), may be administered to a human subject for treatment
of a disorder associated with detrimental TNFa activity according
to a monthly dosing schedule. In one embodiment, 80 mg of a human
TNF alpha antibody, or antigen-binding portion thereof, in the
formulation of the invention (e.g., 0.8 mL of a 100 mg/mL
formulation of the invention) is administered to a subject having a
disorder associated with detrimental TNFa activity. In one
embodiment, 80 mg of a human TNF alpha antibody, or antigen-binding
portion thereof, in the formulation of the invention (e.g., 0.8 mL
of a 100 mg/mL formulation of the invention) is administered
monthly or biweekly to a subject for the treatment of a disorder
associated with detrimental TNFa activity.
[0339] Dose amounts described herein may be delivered as a single
dose (e.g., a single dose of 40 mg in 0.4 mL or 80 mg dose in 0.8
mL), or, alternatively may be delivered as multiple doses (e.g.,
four 40 mg doses or two 80 mg doses for delivery of a 160 mg
dose).
[0340] The formulation of the invention comprising an isolated
human TNF alpha antibody, or antigen-binding portion thereof,
(e.g., adalimumab) may also be administered to a subject in
combination with an additional therapeutic agent. In one
embodiment, the formulation is administered to a human subject for
treatment of rheumatoid arthritis in combination with methotrexate
or other disease-modifying anti-rheumatic drugs (DMARDs). In
another embodiment, the formulation is administered to a human
subject for treatment of JIA in combination with methotrexate or
other disease-modifying anti-rheumatic drugs (DMARDs). Additional
combination therapies are described in U.S. Pat. Nos. 6,258,562 and
7,541,031; and U.S. Patent Publication No. US20040126372, the
entire contents of all of which are incorporated by reference
herein.
[0341] The formulation of the invention comprising a human TNF
alpha antibody, or antigen-binding portion thereof, may also be
used to treat a subject who has failed previous TNF inhibitor
therapy, e.g., a subject who has lost response to or is intolerant
to infliximab.
[0342] The invention is further illustrated in the following
examples, which should not be construed as further limiting.
EXAMPLES
Example 1
High Concentration Anti-TNF.alpha. Antibody Formulation Reduces
Injection Pain
[0343] There have been reports of pain associated with the
subcutaneous administration of a human anti-TNF.alpha. antibody,
e.g., adalimumab. In placebo-controlled trials, 20% of patients
treated with adalimumab developed injection site reactions
(erythema and/or itching, haemorrhage, pain or swelling), compared
to 14% of patients receiving placebo. Most injection site reactions
are mild and do not generally necessitate drug discontinuation.
[0344] There are two main components of the injection pain
associated with adalimumab: the pain associated with the needle
stick, and the pain associated with the injection of drug into the
tissue. The injection-related pain may be related to the Adalimumab
formulation and/or to the volume of medication. The following study
examined whether various formulations have an impact on injection
pain following subcutaneous delivery of adalimumab.
Materials and Methods
Study Design
[0345] The primary objectives of this study were to compare
injection-related pain of three high concentration (100 mg/mL)
adalimumab formulations in the PHYSIOLIS.TM. pre-filled syringe
with the current (50 mg/mL) adalimumab commercial formulation in
the current pre-filled syringe; and to assess the bioavailability
of three high concentration (100 mg/mL) adalimumab formulations in
comparison to the current (50 mg/mL) adalimumab commercial
formulation. The secondary objective of this study was to assess
the safety and tolerability of all four adalimumab
formulations.
[0346] 200 healthy adult male and female subjects who fulfilled the
study eligibility criteria were recruited to participate in the
study. Generally, the study was conducted according to a randomized
parallel-group design. Pain assessment data was preferably obtained
from all 200 subjects. Evaluation of pharmacokinetics (PK) was done
only for the first 100 or so subjects.
[0347] Subjects from each treatment group were scheduled to receive
one subcutaneous injection of adalimumab 40 mg via a pre-filled
syringe. There were four treatment groups, one for each of the four
formulations as set forth in Table 1 below. After meeting the
selection criteria, subjects were randomly assigned in roughly
equal numbers to one of the four treatment groups shown in Table
1.
[0348] The three high concentration formulations (F1, F3, and F4),
each contained 40 mg of adalimumab in 0.4 mL of solution in the
PHYSIOLIS.TM. pre-filled syringe. F1, F3, and F4 were compared to
the current adalimumab commercial formulation of 40 mg of
adalimumab in 0.8 mL of solution in the current pre-filled syringe.
The ingredients for each of the formulations is described below in
Table 1. The formulations described in Table 1 also refer to the
formulations described in Examples 2-7 below.
TABLE-US-00001 TABLE 1 Treatment Groups Treatment No. of Study Day
1 SC Group Subjects Injection Formulation A 50 High Conc.
adalimumab, Formulation 1 (F1) Mannitol, (40 mg/0.4 mL in Citric
acid monohydrate, the PHYSIOLIS .TM. Sodium citrate, pre-filled
syringe) Disodium phosphate dihydrate, Polysorbate 80, Water for
injection, Sodium Hydroxide added as necessary to adjust pH. B 50
High Conc. adalimumab, Formulation 3 (F3) Mannitol, (40 mg/0.4 mL
in Polysorbate 80, the PHYSIOLIS .TM. Water for injection
pre-filled syringe) C 50 High Conc. adalimumab, Formulation 4 (F4)
Polysorbate 80, (40 mg/0.4 mL in Water for injection the PHYSIOLIS
.TM. pre-filled syringe) D 50 Current adalimumab, Commercial
Mannitol, Formulation Citric acid monohydrate, (40 mg/0.8 mL in
Sodium citrate, the current pre-filled Disodium phosphate
dihydrate, syringe) Sodium dihydrogen phosphate dihydrate, Sodium
chloride, Polysorbate 80, Water for injection, Sodium Hydroxide
added as necessary to adjust pH
[0349] The first about 100 subjects to fulfill all entry criteria
and enroll in the study were randomized to the four treatment
groups in roughly equal numbers in each group and participated as
either Cohort 1 or Cohort 2. The second about 100 subjects to
fulfill all entry criteria and enroll in the study were randomized
to the four treatment groups, in roughly equal numbers in each
group, and participated as Cohorts 3-5. It is the Cohort number
that specifies if a subject has pharmacokinetic (PK) and pain
assessments or only pain assessments as described in Table 2
below.
TABLE-US-00002 TABLE 2 Assignment of Study Subjects Total No. of
Subjects Cohort N Assessments A B C D 1 50 PK and Pain 13 12 13 12
2 44 PK and Pain 11 12 10 11 3 38 Pain 9 9 10 10 4 39 Pain 10 10 10
9 5 29 Pain 7 7 7 8
[0350] Pharmacokinetic sample collection and pain assessments was
done for all subjects in the first two cohorts of about 100
patients (Cohorts 1 and 2). Subjects in Cohorts 3-5 only
participated in pain assessments, and no pharmacokinetic samples
were be collected for these subjects. Safety and tolerability were
to be assessed in all subjects of all 5 cohorts. Each subject was
randomly assigned to receive one injection of adalimumab on Study
Day 1. Each dose of study drug was to be administered
subcutaneously by an appropriate site staff member via a pre-filled
syringe in accordance with the proper injection method. The
injection was given subcutaneously in the abdomen 2 inches to the
right of the navel. Questionnaires were administered by a different
study staff member than the individual administering the injection,
as often as possible.
[0351] Subjects in Cohorts 1 and 2 (pharmacokinetic and pain
assessments) were confined to the study site and supervised for
approximately 10 days (9 nights). Confinement for each subject
began on Study Day -1 (1 day prior to the dosing day) and ended
after the collection of the 192 hour blood samples and scheduled
study procedures on Study Day 9. Serial blood samples were
collected through Study Day 57 after dosing with subjects returning
for outpatient visits. Safety and tolerability were assessed
throughout the study. Subjects in Cohorts 3-5 (pain assessments
only) were confined to the study site and supervised for
approximately 3 days (2 nights). Confinement for each subject began
on Study Day -1 (1 day prior to the dosing day) and ended after the
completed study procedures on Study Day 2. Safety and tolerability
were assessed throughout the study.
[0352] In addition to bioavailability and AAA assays, tolerability
was preferably assessed as follows:
[0353] 1) Immediately following the injection on Study Day 1: Pain
Assessment Module was completed by the subject.
[0354] 2) Approximately 10 minutes following the injection on Study
Day 1: Draize Scale (hemorrhage, petechiae, erythema, edema, and
pruritus) was evaluated by a qualified site staff member.
[0355] 3) Approximately 15 minutes following the injection on Study
Day 1: Pain Assessment Module was completed by the subject.
[0356] 4) Approximately 30 minutes following the injection on Study
Day 1: Pain Assessment Module and Draize Scale evaluation was
completed by the subject and a qualified site staff member,
respectively.
[0357] The demographics of subjects in the treatment groups are as
follows, shown in Table 3, below.
TABLE-US-00003 TABLE 3 Patient Demographics High Conc. High Conc.
High Conc. Commercial Var- Formulation 1 Formulation 3 Formulation
4 Formulation iable (N = 50) (N = 50) (N = 50) (N = 50) Age 29.6
.+-. 8.7 29.5 .+-. 9.4 30.0 .+-. 8.9 30.3 .+-. 9.7 (yrs) Weight
68.3 .+-. 13.9 69.6 .+-. 9.6 67.0 .+-. 8.4 68.5 .+-. 10.0 (kg) Sex
31 F (62%), 25 F (50%), 31 F (62%), 30 F (60%), 19 M (38%) 25 M
(50%) 19 M (38%) 20 M (40%) Race 37 white 45 white 36 white 40
white (74%), (90%), (72%), (80%), 9 black (18%), 3 black (6%), 10
black (20%), 8 black (16%), 4 other (8%) 2 other (4%) 4 other (8%)
2 other (4%)
Formulations
[0358] Three new high concentration formulations (referred to
herein as Formulation 1, 3, or 4; or F1, F3, or F4, respectively)
were studied relative to the commercial 50 mg/mL adalimumab
formulation. The compositions of each of these formulations are
listed below in Tables 4-7.
TABLE-US-00004 TABLE 4 Formulation 1 (F1) COMPOSITION OF THE BULK
SOLUTION 1 mL bulk solution contains Concentration Name of
ingredient [mg] Active Substance adalimumab (A-765865)* 100.00
Excipients Mannitol 42.00 Citric acid monohydrate 1.31 Sodium
citrate 0.31 Disodium phosphate dihydrate 1.53 Sodium dihydrogen
phosphate dihydrate 0.86 Polysorbate 80 1.00 Sodium Hydroxide q.s.
Water for injections ad 1,041.00 Nitrogen -- Density of the
solution: 1.041 g/mL *Used as concentrate.
TABLE-US-00005 TABLE 5 Formulation 3 (F3) COMPOSITION OF THE BULK
SOLUTION 1 mL bulk solution contains Concentration Name of
ingredientent [mg] Active Substance adalimumab (A-765865)* 100
Excipients Mannitol 42.00 Polysorbate 80 1.00 Water for injections
ad 1,040.00 Nitrogen -- *Used as concentrate. Density of the
solution: 1.040 g/mL
TABLE-US-00006 TABLE 6 Formulation 4 (F4) COMPOSITION OF THE BULK
SOLUTION 1 mL bulk solution contains Concentration Name of
ingredient [mg] Active Substance adalimumab (A-765865)* 100.00
Excipients Polysorbate 80 1.00 Water for injections ad 1,026.00
Nitrogen -- *Used as concentrate. Density of the solution: 1.026
g/mL
TABLE-US-00007 TABLE 7 Commercial 50 mg/mL adalimumab formulation
COMPOSITION OF THE BULK SOLUTION 1 mL bulk solution contains
Concentration Name of ingredient [mg] Active Substance adalimumab
(A-765865)* 50.00 Excipients Mannitol 12.00 Citric acid monohydrate
1.30 Sodium citrate 0.30 Disodium phosphate dihydrate 1.53 Sodium
dihydrogen phosphate dihydrate 0.86 Polysorbate 80 1.00 Sodium
Hydroxide q.s. Water for injections ad about 1,000 Sodium Chloride
6.16 Nitrogen --
Study Drug Administration
[0359] Study drug (adalimumab) in the various formulations was
administered in the morning at Hour 0 on Study Day 1. The four
treatment groups are set forth in Table 1 above as Groups A, B, C
and D. Subjects in each treatment group were subcutaneously
injected with only a single adalimumab formulation via a pre-filled
syringe.
[0360] Two types of syringes were used in this study; the currently
commercially available glass pre-filled syringe ("current
pre-filled syringe") was used for the reference current adalimumab
commercial formulation (40 mg of adalimumab in 0.8 mL of solution),
and the PHYSIOLIS.TM. pre-filled syringe for the three high
concentration test formulations (40 mg of adalimumab in 0.4 mL of
solution). The PHYSIOLIS.TM. pre-filled syringe has a 29 gauge
needle (the current pre-filled syringe has a 27 gauge 1/2 inch
length fixed needle), a latex-free needle shield, and a plunger
stopper which is coated to minimize leachables.
Pain Testing: Pain Scale
[0361] The Pain Visual Analog Scale was used to quantitatively
assess pain sensation. The following instructions were followed to
assess the Pain Visual Analog Scale (VAS):
[0362] The pain scale was administered to the subject at three
different times after the injection: immediately after the
injection, at 15 minutes after the injection, and at 30 minutes
after the injection on Study Day 1. The pain scale was shown and
read to the subject, who was asked to place one straight vertical
mark along the line in the pain scale to indicate their current
level of pain at the injection site (for example see below). An
indication of 0 meant no pain, while the highest score (10)
indicated "the worst imaginable pain." An illustrative pain scale
used in the study is shown below:
What is your current level of pain at the injection site? [0363] 0
10 [0364] no pain the worst imaginable pain
Qualitative Assessment of Pain
[0365] Following the completion of the pain scale, the qualitative
assessment of pain was administered three times after the
injection: immediately following the injection, at 15 minutes post
the injection, and 30 minutes following the injection on Study Day
1. An exemplary qualitative assessment of pain used in the study is
shown below:
Select all that describe your current level of pain at the
injection site: Shooting pain Sharp pain
Stinging Pain
Dull
Uncomfortable
Pressure
Aching
Soreness
[0366] Localized burning
Other
OR
[0367] I currently have no discomfort at my injection site
Needle Pain Assessment
[0368] After the qualitative assessment of pain was completed, a
needle pain assessment was administered immediately following the
injection. An exemplary needle pain assessment used in the study is
shown below:
Were you able to tell the difference between the pain from the
needle entering your skin and the pain from the solution that was
injected?
Yes
No
[0369] a. If yes, was most of your pain caused by the needle
entering your skin or was most of your pain caused by the solution
that was injected? Most of my pain was caused by the needle entry
Most of my pain was caused by the solution that was injected
Draize Scale
[0370] Qualified site personnel completed this assessment for each
subject approximately 10 minutes and 30 minutes after the injection
on Study Day 1.
Hemorrhage/Petechiae at injection site:
[0371] 0: None
[0372] 1: Isolated; up to 5 petechiae
[0373] 2: Isolated but >5 petechiae
[0374] 3: Many petechiae, some coalescence
[0375] 4: Spots of blood on surface
[0376] 5: Frank bleeding
Erythema at injection site:
[0377] 0: No erythema
[0378] 1: Very slight (barely perceptible) erythema
[0379] 2: Well-defined erythema
[0380] 3: Moderate to severe erythema
[0381] 4: Severe erythema (beet redness)
Edema at injection site:
[0382] 0: No edema
[0383] 1: Very slight (barely perceptible) edema
[0384] 2: Slight edema (edges of area well defined by slight
raising)
[0385] 3: Moderate edema (raised .about.1 mm)
[0386] 4: Severe edema (raised >1 mm, extending beyond area of
injection)
Pruritis at injection site:
[0387] 0: No pruritis
[0388] 1: Occasional pruritis
[0389] 2: Constant pruritis
Results
[0390] To determine whether delivery of adalimumab could be
improved, new high concentration formulations were developed.
Formulations F1, F3, and F4, as shown below, have half of the
volume (i.e., 0.4 mL vs. 0.8 mL) and twice the protein
concentration (100 mg/mL vs. 50 mg/mL) compared to the current
commercial adalimumab formulation, and they also have different
excipient compositions. Experiments described herein were designed
to assess whether any of the new formulations are superior to the
current commercial adalimumab formulation.
[0391] The pain visual analog scale was chosen to assess injection
site pain, and was used to evaluate impact of formulation
composition on pain sensations. In addition, tolerability of
various new adalimumab 100 mg/mL formulations were compared to the
current commercial formulation (50 mg/mL adalimumab formulation).
Data in this example supports the surprising finding that the new
formulations, especially Formulation 3 (F3), decreases pain
significantly relative to the current commercial formulation.
Surprisingly, F3 also decreased pain significantly relative to
formulations F1 and F4.
[0392] Specifically, FIG. 1 shows that administration of high
concentration Formulations 1 and 3 resulted in a significant
decrease in pain assessment at all time points after injection
(immediately, 15 minutes, and 30 minutes), compared to the other
treatment groups (F4 and the current commercial formulation). Table
8, shown below, summarizes the individual data and shows a
comparison of the F1, F3, and F4 formulations with the 0.8 mL, 50
mg/mL commercial formulation.
[0393] As described in Table 8, immediately after injection,
subjects who received the current Humira formulation reported a
mean (SD) pain score of 3.29 (2.57) cm. The mean pain scores for
Formulation 1 and Formulation 3 were statistically significantly
lower than that for the current Humira formulation (p<0.001).
The estimated differences from the current Humira formulation were
-1.50 (95% CI: -2.31--0.69 cm) for Formulation F1, and -2.70 (95%
CI: -3.52--1.89 cm) for Formulation F3. Thus, Treatments A and B
(high concentration Formulations 1 and 3) resulted in 45.6% and
82.7% reductions in injection site pain, respectively. Statistical
tests were not performed for the pain scores assessed at 15 minutes
and 30 minutes after injection because a majority of the subjects
reported no pain at these time points. As described in FIG. 1, the
minimum/maximum VAS scores immediately after injection were as
follows: Formulation F1, 0.00-8.3; Formulation F3, 0.00-2.20;
Formulation F4, 0.20-8.70; and current commercial formulation,
0.00-10.00.
[0394] It was apparent that the pain associated with the injection
of Formulation 3 was dramatically reduced compared to the same for
the current commercial formulation. Specifically, mean pain value,
as assessed by the pain visual analog scale (VAS) immediately after
injection, decreased from a mean of 3.29 in the current commercial
formulation to 0.56 in Formulation 3, a stark 83% reduction. In
fact, the pain reduction associated with Formulation 3 was so
significant, it was 69% reduced from the level of the next best
formulation (in terms of pain)-Formulation 1 (1.79).
[0395] Similarly, the mean pain scale for Formulation 1 reduced to
1.79, a 45% reduction from the 3.29 pain scale associated with the
current commercial formulation.
TABLE-US-00008 TABLE 8 Pain Visual Analog Scales (VAS) Immediately
After Injection Comparisons with Current Mean LS Formulation&
Treatment N (SD) Mean& Estimate P-value# 95% CI High Conc. 50
1.79 1.79 -1.50 <0.001 -2.31, Formulation 1 (2.08) -0.69 High
Conc. 50 0.56 0.58 -2.71 <0.001 -3.52, Formulation 3 (0.56)
-1.90 High Conc. 50 4.12 4.11 0.82 0.976 0.01, Formulation 4 (2.50)
1.63 Current 50 3.29 3.29 Formulation (2.57) #From a one-sided test
with the null hypothesis being that the mean for the test
formulation is .gtoreq. the mean for the current formulation
&Based on ANOVA.
[0396] A Qualitative Assessment of Pain was also administered to
subjects immediately after injection, 15 minutes after injection
and at 30 minutes after injection for all four adalimumab
treatments Immediately after injection, an assessment of "no
discomfort" was reported with greatest frequency by 31 subjects
(31/50, 62.0%) who had received Formulation 3, followed by 19
subjects (19/50, 38.0%) who had received Formulation 1, 7 subjects
(7/50, 14.0%) who had received the current Humira formulation, and
one subject (1/50, 2.0%) who had received Formulation 4. Of those
subjects who reported discomfort immediately after injection,
"stinging pain" was the most frequently reported sensation with 30
subjects (30/50, 60%) for each of the current formulation and
formulation 4, 16 subjects (16/50, 32.0%) for Formulation 1, and 4
subjects (4/50, 8.0%) for Formulation 3. At 15 minutes after
injection, a large majority of subjects receiving each treatment
reported "no discomfort" at the injection site.
[0397] Study site staff also utilized the Draize Scale to evaluate
hemorrhage/petechiae, erythema, edema and pruritis at the injection
site of each subject. Ten minutes after injection the majority of
subjects in all treatment groups had no observed injection-site
hemorrhages or petechiae, edema or pruritis.
[0398] All four formulations were well tolerated during the study.
A summary of preliminary adverse events (AE) data is shown below in
Table 9.
TABLE-US-00009 TABLE 9 Preliminary Adverse Events (AE) High Conc.
High Conc. High Conc. Current Formulation Formulation Formulation
Formulation 1 (N = 50) 3 (N = 50) 4 (N = 50) (N = 50) Any AE 7
(14%) 7 (14%) 6 (12%) 3 (6%) Any AE at least 3 (6%) 3 (6%) 2 (4%) 1
(2%) possibly drug related Any severe AE 0 0 0 0 Any serious AE 0 0
0 0 Any AE leading 0 0 0 0 to study discontinuation Deaths 0 0 0
0
[0399] Accordingly, the data demonstrates that the new 100 mg/mL
formulations, especially formulations 1 and 3, are well tolerated,
and were effective in reducing injection site pain after
subcutaneous injection of similar therapeutic doses as compared to
the currently marketed adalimumab formulation. Formulation F3 had a
particularly low VAS score relative to the other formulations
tested.
[0400] The reduction in pain using the VAS score was not related to
the difference in needle size (a 27 G needle was used to administer
the current adalimumab commercial formulation and a 29 G needle was
used to administer formulations F1, F3, and F4). In particular, a
needle prick accounts for an immediate pain response, whereas the
pain response measured by the VAS scale indicated a prolonged
persistent pain over several minutes, demonstrating that the
injected solution itself contributes to the majority of the
response. In addition, all of the test formulations (F1, F3, and
F4) were injected using the same size needle, yet F1, F3, and F4
had very different VAS scores. This result further demonstrates
that it was the formulation contributing to the pain effect. and
that this can be separated from the size of the needle used to
administer the formulations.
Example 2
High Concentration Anti-TNFA Antibody Formulations Increase
Bioavailability in Humans
[0401] The following example describes a Phase 1, single-blind,
single-dose, parallel-group design, randomized study in healthy
volunteers (same study described above in Example 1). The primary
objectives of this study were to compare injection-related pain of
three high concentration (100 mg/mL) adalimumab formulations in the
Physiolis PFS with the current (50 mg/mL) adalimumab (Humira)
formulation in the current PFS (see Example 1), and to assess the
bioavailability of three high concentration (100 mg/mL) adalimumab
formulations in comparison to the current commercial (50 mg/mL)
adalimumab (Humira) formulation. The secondary objective of this
study was to assess the safety and tolerability of all four
adalimumab formulations.
Study Design
[0402] Two hundred healthy volunteers were enrolled in this study
(Table 10). Pain assessment data were obtained from all 200
subjects. Adalimumab pharmacokinetics were evaluated in the first
100 subjects. A description of the formulations is provided above
in Table 1.
TABLE-US-00010 TABLE 10 Treatment Groups Number of Number Subjects
of for Treatment Subjects Pharmacokinetic Study Day 1 Group for
Study Data SC Injection A 50 24 High Concentration Adalimumab
Formulation No. 1 (40 mg/0.4 mL in the Physiolis PFS) B 50 24 High
Concentration Adalimumab Formulation No. 3 (40 mg/0.4 mL in the
Physiolis PFS) C 50 23 High Concentration Adalimumab Formulation
No. 4 (40 mg/0.4 mL in the Physiolis PFS) D 50 23 Current
Commercial Humira Formulation (40 mg/0.8 mL in the current PFS)
*See Tables 4-7 for formulation compositions.
[0403] Subjects from each treatment group received one subcutaneous
injection of 40 mg adalimumab via PFS on Study Day 1. Each dose of
study drug was administered subcutaneously by an appropriate site
staff member in accordance with the proper injection method as
described in the protocol. The injection was given subcutaneously
in the abdomen 2 inches to the right of the navel. Questionnaires
were administered by a different study staff member than the
individual administering the injection, as often as possible.
Results
Pharmacokinetic Results and Conclusions
[0404] Following a single subcutaneous dose of adalimumab, the
central values of the pharmacokinetic parameters, T.sub.max,
C.sub.max, AUC.sub.0-360 and AUC.sub.0-1344 were similar between
Treatments A, B (high concentration adalimumab Formulations 1 and
3, respectively) and D (current commercial Humira formulation).
Following the single dose administration, mean T.sub.max was
earlier for Treatment C (high concentration adalimumab Formulation
4) relative to Treatment D (FIGS. 2 and 3). The central values of
C.sub.max and AUC.sub.0-360 values were greater (p<0.05) for
Treatment C versus Treatment D.
Bioavailability/Bioequivalence for Treatments A, B and C
(Adalimumab High Concentration Formulations) Relative to Treatment
D (Commercial Humira Formulation)
[0405] For Treatment Group A versus D, the point estimates for the
ratios of C.sub.max, AUC.sub.0-360, and AUC.sub.0-1344 central
values for Treatments A and B were near unity, and the 90%
confidence intervals were within the 0.80 to 1.25 range. For
Treatment B versus D, the point estimates for the ratios of C. and
AUC.sub.0-360 central values were near unity and the 90% confidence
intervals were within the 0.80 to 1.25 range. For AUC.sub.0-1344,
the upper bound of the 90% confidence interval for Treatments B
versus D was above 1.25. For Treatment C versus D, the point
estimates for the ratio of C.sub.max, AUC.sub.0-360 and
AUC.sub.0-1344 central values were 1.429, 1.309, and 1.170
respectively, indicating that the relative bioavailability of
Treatment C (Formulation 4) was greater.
TABLE-US-00011 TABLE 11 Relative Bioavailability and 90% Confidence
Intervals for the Bioequivalence Assessment Treat- Relative
Bioavailability ments.sup..English Pound. 90% Test vs. PK Central
Value Point Confidence Reference Parameter Test Reference Estimate
Interval A vs. D C.sub.max 4.47 4.39 1.018 0.859-1.207
AUC.sub.0-360 1192.14 1192.23 1.000 0.860-1.163 AUC.sub.0-1344
2306.91 2387.28 0.966 0.814-1.147 B vs. D C.sub.max 4.52 4.39 1.029
0.868-1.219 AUC.sub.0-360 1222.24 1192.23 1.025 0.882-1.192
AUC.sub.0-1344 2547.95 2387.28 1.067 0.899-1.266 C vs. D C.sub.max
6.28 4.39 1.429 1.202-1.699 AUC.sub.0-360 1561.05 1192.23 1.309
1.123-1.527 AUC.sub.0-1344 2794.29 2387.28 1.170 0.983-1.394
.sup..English Pound.Treatments A, B or C: a single dose of high
concentration adalimumab Formulation 1, 3 or 4, respectively,
administered as a single sc injection using a Physiolis PFS (40
mg/0.4 mL). Treatment D: a single dose of the current Humira
formulation administered as a single sc injection using the
currently available glass PFS (40 mg/0.8 mL). PK =
Pharmacokinetic.
Pharmacokinetic Conclusions
[0406] Based on the pharmacokinetic results, the relative
bioavailability of Treatments A and B were similar to Treatment D,
the currently marketed Humira formulation. The relative
bioavailability of Treatment C was greater when compared to
Treatment D. The unexpected increase in bioavailability for
Treatment C suggests that the effective dose amount administered to
a subject may be reduced.
Immunogenicity Conclusions
[0407] Twelve subjects had positive AAA samples during any time in
the study, with only two subjects determined as AAA positive
according to the pre defined definition. Because of the small
sample size and relatively similar numbers of AAA positive samples,
no conclusions can be made of the immunogenicity between the
treatments.
Safety Conclusions
[0408] The treatments tested were generally well tolerated by the
subjects. No clinically significant vital signs, ECG or laboratory
measurements were observed during the course of the study. The
majority of adverse events were assessed by the investigator as
probably not or not related to study drug and mild in severity. No
adverse events were assessed as severe.
[0409] No deaths, serious adverse events or discontinuations due to
adverse events occurred during the study.
[0410] Results of other safety analyses, including individual
subject changes and potentially clinically significant values for
vital signs, ECG and laboratory measurements, were unremarkable for
all treatment groups.
Tolerability
[0411] The tolerability assessments that were conducted included
completion of a Pain Assessment Module (Pain Visual Analog Scale
[VAS]), Qualitative Assessment of Pain and Needle Pain Assessment)
and the Draize Scale (see Example 1).
Example 3
High Concentration Anti-TNFA Antibody Formulation Increase
Bioavailability in Pre-Clinical Model
[0412] The objective of the following study was to evaluate the
pharmacokinetic profiles of adalimumab formulation F4 in contrast
to the adalimumab commercial formulation (see Table 7 above for a
description of the formulation).
[0413] The pharmacokinetic profiles of HUMIRA (Adalimumab) were
studied in male and female Beagle dogs (2/sex/s.c. administration
and 2 males/i.v. administration, Marshall Bio Resources USA, Inc.,
North Rose, N.Y. 14516) after a single subcutaneous (s.c.)
injection of the HUMIRA commercial formulation (adalimumab) and a
HUMIRA test formulation corresponding to formulation F4 of the
previous examples (adalimumab), as well as an intravenous (i.v.)
injection of the HUMIRA commercial formulation as a control. The
administered dose was 40 mg/dog (at 100 mg/mL for F4 and 50 mg/mL
of the commercial formulation).
[0414] For the determination of Adalimumab serum exposure levels,
blood samples were collected at 0.083, 4, 24, 48, 96, 168, 240,
312, 384, 456, 528 and 864 hours post administration (p.a.).
Examined parameters were clinical signs (twice weekly) and
mortality.
[0415] Apart from mucous feces in one male animal of the control
group, no relevant clinical signs were observed. The incidences of
clinical signs are summarized in Tables 14 and 15 below.
[0416] The pharmacokinetic results (described in Table 12 below) of
this study indicated that the bioavailability after s.c. dosing was
about 80% and the exposure levels seemed to be higher in females
than in males after s.c. dosing. There was a trend for higher
exposure levels following s.c. dosing of the test formulation
compared with s.c. dosing of the commercial formulation in
males.
TABLE-US-00012 TABLE 12 Pharmacokinetic results AUC/Dose Animal
AUC.sub.0-528 h (.mu.g*Hours/mL/ Vdss T.sub.1/2 Treatment Gender
Number (.mu.g*Hours/mL) mg/kg) (mL) (Hours) Test male 1001 9020 226
708 39.3 formulation, 1003 11400 286 870 187.2 s.c. Mean 10200 .+-.
1680 256 .+-. 42.4 789 .+-. 115 113.3 .+-. 104.6 female 1002 15400
384 388 55.5 1004 15800 395 469 54.5 Mean 15600 .+-. 283 390 .+-.
7.78 429 .+-. 57.3 55 .+-. 0.7 Commercial male 2001 8010 200 692
21.4 formulation, 2003 8230 206 695 72.7 s.c. Mean 8120 .+-. 156
203 .+-. 4.24 694 .+-. 2.12 47.1 .+-. 36.3 female 2002 12700 319
385 34.0 2004 17200 431 477 119.0 Mean 15000 .+-. 3180 375 .+-.
79.2 431 .+-. 65.1 76.5 .+-. 60.1 Commercial male 3001 9360 234 548
45.5 formulation, 3003 11900 298 407 22.2 i.v. Mean 10600 .+-. 1800
266 .+-. 45.3 478 .+-. 99.7 33.9 .+-. 16.5
TABLE-US-00013 TABLE 13 Animal identification Animal No. Tattoo-No.
Sex 1001 1246730 Male 1003 1230230 Male 1002 1282302 Female 1004
1288688 Female 2001 1284879 Male 2003 1298951 Male 2002 1297237
Female 2004 1280491 Female 3001 1285514 Male 3003 1290143 Male
TABLE-US-00014 TABLE 14 Summary of Clinical Observations in Males
Dosage Group: 2 3 1 Animals Examined: 4 4 2 Number Normal: 3 3 2
Category, Observation a b a b Excretion, feces 1 4 0 0 Note: a =
Number of animals with observation b = Number of days observation
seen
TABLE-US-00015 TABLE 15 Summary of Clinical Observations in Females
Dosage Group: 2 3 1 Animals Examined: 2 2 2 Number Normal: 2 2 2
Category, Observation Note: a = Number of animals with observation
b = Number of days observation seen
Example 4
Stability of High Concentration Anti-TNFA Antibody Formulations
Against Freeze/Thaw Stress
[0417] The following example compares the stability of high
concentration formulations F1, F3, and F4 with the commercial
adalimumab formulation. Stability was examined using freeze/thaw
tests.
Experimental Setup
[0418] High concentration human anti-TNF.alpha. antibody
formulations were prepared as described in Example 1, Table 1
above.
[0419] The compounded solutions were sterile filtered and aliquoted
in 8.times.30 mL PETG bottles at 20 mL, respectively. The solutions
were practically free from particles in visual inspection.
[0420] The samples for T0 were directly placed into a 2-8.degree.
C. refrigerator. The other bottles were put into the -80.degree. C.
cube to freeze.
[0421] The next day the bottles were thawed in water baths with a
temperature of 25.degree. C. or 37.degree. C., respectively.
[0422] The Freeze/Thaw cycles were repeated 5 times. At T0 (before
any freeze-thaw cycles), T1 (after one freeze-thaw cycle), T3
(after three freeze-thaw cycles) and T5 (after five freeze-thaw
cycles) samples were taken for analysis and stored in a 2-8.degree.
C. fridge. [0423] n=1 per pullpoint from 4 samples [0424] Sample
volume: 20 mL [0425] Freeze/Thaw: -80.degree. C./25.degree.
C.+37.degree. C. [0426] Freeze/thaw cycles: 5
[0427] After the cycling the samples were analyzed in the lab using
each of the following measures: Optical appearance (at each time
point); absorption at 340 nm; subvisible particles (at GGDDA);
Photon-correlation-spectroscopy (PCS); Size Exclusion
Chromatography (SEC); and Ion Exchange Chromatography (IEC).
Subvisible Particles
[0428] The measurement of subvisible particles was made at the
Klotz particle measurement device. The results are shown in Table
16.
TABLE-US-00016 TABLE 16 Counts of particles >=1 .mu.m, >=10
.mu.m, and >=25 .mu.m Particles >= Timepoint Sample
Temperature Charge 1 .mu.m 10 .mu.m 25 .mu.m T0 HC F1 (25.degree.
C.) E161118001CL 9 1 0 T0 HC F1 (37.degree. C.) E161118001CL 7 2 1
T1 HC F1 25.degree. C. E161118001CL 3 0 0 T1 HC F1 37.degree. C.
E161118001CL 33 1 0 T3 HC F1 25.degree. C. E161118001CL 3 0 0 T3 HC
F1 37.degree. C. E161118001CL 20 1 0 T5 HC F1 25.degree. C.
E161118001CL 4 0 0 T5 HC F1 37.degree. C. E161118001CL 94 0 0 T0 HC
F3 (25.degree. C.) E161119001CL 6 3 1 T0 HC F3 (37.degree. C.)
E161119001CL 12 2 0 T1 HC F3 25.degree. C. E161119001CL 4 1 0 T1 HC
F3 37.degree. C. E161119001CL 7 2 0 T3 HC F3 25.degree. C.
E161119001CL 3 1 0 T3 HC F3 37.degree. C. E161119001CL 9 2 1 T5 HC
F3 25.degree. C. E161119001CL 7 0 0 T5 HC F3 37.degree. C.
E161119001CL 5 0 0 T0 HC F4 (25.degree. C.) E161120001CL 5 1 1 T0
HC F4 (37.degree. C.) E161120001CL 7 1 0 T1 HC F4 25.degree. C.
E161120001CL 6 1 0 T1 HC F4 37.degree. C. E161120001CL 5 1 0 T3 HC
F4 25.degree. C. E161120001CL 12 1 1 T3 HC F4 37.degree. C.
E161120001CL 60 0 0 T5 HC F4 25.degree. C. E161120001CL 13 0 0 T5
HC F4 37.degree. C. E161120001CL 22 1 0 T0 commercial (25.degree.
C.) E161121001CL 464 2 1 T0 commercial (37.degree. C.) E161121001CL
198 0 0 T1 commercial 25.degree. C. E161121001CL 143 1 0 T1
commercial 37.degree. C. E161121001CL 285 0 0 T3 commercial
25.degree. C. E161121001CL 108 0 0 T3 commercial 37.degree. C.
E161121001CL 224 0 0 T5 commercial 25.degree. C. E161121001CL 39 0
0 T5 commercial 37.degree. C. E161121001CL 151 0 0
[0429] The >=1 .mu.m particle data showed a clear trend to a
higher particle load in Humira commercial and high concentration
(HC) F1 at T5, reflecting a characteristic behavior of buffer salt
or sodium chloride containing adalimumab formulations.
TABLE-US-00017 Sample name Sum Aggregates Monomer Sum Fragments T0,
HC F1, 25.degree. C. 0.42 99.50 0.09 T0, HC F1, 37.degree. C. 0.43
99.46 0.11 T0, HC F3, 25.degree. C. 0.39 99.54 0.07 T0, HC F3,
37.degree. C. 0.41 99.50 0.09 T0, HC F4, 25.degree. C. 0.43 99.46
0.11 T0, HC F4, 37.degree. C. 0.42 99.48 0.11 T0, commercial,
25.degree. C. 0.36 99.55 0.09 T0, commercial, 37.degree. C. 0.35
99.56 0.09 T1, HC F1, 25.degree. C. 0.43 99.47 0.10 T1, HC F1,
37.degree. C. 0.44 99.48 0.08 T1, HC F3, 25.degree. C. 0.38 99.53
0.09 T1, HC F3, 37.degree. C. 0.37 99.54 0.09 T1, HC F4, 25.degree.
C. 0.44 99.47 0.09 T1, HC F4, 37.degree. C. 0.44 99.46 0.10 T1,
commercial, 25.degree. C. 0.35 99.56 0.08 T1, commercial,
37.degree. C. 0.35 99.56 0.10 T3, HC F1, 25.degree. C. 0.42 99.47
0.10 T3, HC F1, 37.degree. C. 0.42 99.48 0.11 T3, HC F3, 25.degree.
C. 0.40 99.48 0.12 T3, HC F3, 37.degree. C. 0.40 99.52 0.08 T3, HC
F4, 25.degree. C. 0.48 99.41 0.11 T3, HC F4, 37.degree. C. 0.44
99.48 0.08 T3, commercial, 25.degree. C. 0.36 99.54 0.10 T3,
commercial, 37.degree. C. 0.34 99.55 0.11 T5, HC F1, 25.degree. C.
0.43 99.48 0.09 T5, HC F1, 37.degree. C. 0.45 99.45 0.10 T5, HC F3,
25.degree. C. 0.41 99.48 0.11 T5, HC F3, 37.degree. C. 0.39 99.48
0.13 T5, HC F4, 25.degree. C. 0.47 99.43 0.10 T5, HC F4, 37.degree.
C. 0.49 99.40 0.11 T5, commercial, 25.degree. C. 0.36 99.56 0.08
T5, commercial, 37.degree. C. 0.40 99.47 0.13
[0430] Subvisible particle counts for >=10 .mu.m and >=25
both were very low. Freeze/thaw cycling did not lead to an
increased number of subvisible particles, indicating that the
tested formulations had favorable stability.
Size Exclusion Chromatography (SEC)
[0431] The SEC results are shown in Table 17. Table 17 indicates
the percentages of SEC aggregates, monomers, and fragments in each
of the solutions at T0 (before any freeze-thaw cycles), T1 (after
one freeze-thaw cycle), T3 (after three freeze-thaw cycles) and T5
(after five freeze-thaw cycles). These results indicate that each
of formulations 1, 3, and 4 show stabilities similar to that of the
commercial formulation.
Table 17: Percentages of aggregates, monomers, and fragments before
and after freeze-thaw cycles as assessed by SEC
Ion Exchange Chromatography (IEC)
[0432] Ion exchange chromatography did not reveal different
sensitivity of the tested solutions. No significant degradation
could be observed.
[0433] However, with increasing number of freeze/thaw cycles, the
samples that were thawed at 25.degree. C. showed a higher amount in
the 2nd acidic region after 5 cycles.
[0434] IEC results are shown in Table 18.
TABLE-US-00018 TABLE 18 IEC measurements before and after
freeze-thaw cycles Sum Lysin 1.ac 2.ac Peak Sample name Peaks area
area Lysin 0 Lysin 1 between Lysin 2 T0, HC F1, 25.degree. C. 86.74
2.09 10.51 68.63 16.72 0.99 0.40 T0, HC F3, 25.degree. C. 87.29
1.89 10.22 66.35 16.16 0.91 3.87 T0, HC F4, 25.degree. C. 87.35
1.85 10.20 66.46 16.15 0.89 3.85 T0, commercial, 87.18 1.93 10.22
66.25 16.12 0.94 3.88 25.degree. C. T1, HC F1, 25.degree. C. 87.17
1.98 10.18 66.24 16.11 0.94 3.87 T1, HC F3, 25.degree. C. 87.30
1.81 10.24 66.39 16.13 0.90 3.88 T1, HC F4, 25.degree. C. 87.21
1.88 10.25 66.31 16.11 0.90 3.88 T1, commercial, 87.19 2.01 10.20
66.27 16.11 0.93 3.88 25.degree. C. T3, HC F1, 25.degree. C. 87.23
1.94 10.20 66.34 16.12 0.91 3.87 T3, HC F3, 25.degree. C. 87.27
1.86 10.25 66.37 16.14 0.88 3.88 T3, HC F4, 25.degree. C. 87.26
1.82 10.27 66.34 16.15 0.88 3.88 T3, commercial, 87.20 1.88 10.28
66.29 16.11 0.91 3.89 25.degree. C. T5, HC F1, 25.degree. C. 87.39
1.74 10.21 66.43 16.18 0.89 3.88 T5, HC F3, 25.degree. C. 87.27
1.79 10.32 66.42 16.15 0.84 3.86 T5, HC F4, 25.degree. C. 87.33
1.69 10.32 66.49 16.14 0.85 3.85 T5, commercial, 87.05 1.95 10.38
66.17 16.10 0.89 3.88 25.degree. C. T0, HC F1, 37.degree. C. 87.25
1.94 10.19 66.36 16.13 0.90 3.86 T0, HC F3, 37.degree. C. 87.42
1.82 10.19 66.54 16.16 0.85 3.87 T0, HC F4, 37.degree. C. 87.38
1.89 10.11 66.52 16.14 0.86 3.86 T0, commercial, 87.25 1.86 10.24
66.34 16.12 0.91 3.88 37.degree. C. T1, HC F1, 37.degree. C. 87.21
1.98 10.17 66.27 16.11 0.95 3.88 T1, HC F3, 37.degree. C. 87.32
1.91 10.12 66.40 16.13 0.90 3.88 T1, HC F4, 37.degree. C. 87.27
1.98 10.13 66.36 16.14 0.91 3.86 T1, commercial, 87.28 1.97 10.14
66.34 16.12 0.93 3.88 37.degree. C. T3, HC F1, 37.degree. C. 87.27
1.95 10.15 66.36 16.11 0.94 3.86 T3, HC F3, 37.degree. C. 87.18
2.03 10.11 66.29 16.13 0.90 3.87 T3, HC F4, 37.degree. C. 87.21
1.95 10.15 66.35 16.09 0.92 3.85 T3, commercial, 87.31 1.95 10.21
66.50 16.09 0.91 3.81 37.degree. C. T5, HC F1, 37.degree. C. 87.29
2.01 10.06 66.40 16.11 0.93 3.85 T5, HC F3, 37.degree. C. 87.25
2.07 10.06 66.37 16.10 0.92 3.87 T5, HC F4, 37.degree. C. 87.28
2.04 10.02 66.42 16.11 0.93 3.83 T5, commercial, 87.53 1.91 10.02
66.72 16.11 0.88 3.83 37.degree. C.
Example 5
Stability of High Concentration Anti-TNFA Antibody Formulations
Against Stir Stress
[0435] The following example describes a study which examined the
stability of the F1, F3, and F4 formulations using the stir-stress
test. Each formulation was tested in a range of pH levels.
Materials
[0436] Humira HC F1 pH 4.2 [0437] 100 mg/mL pH 4.7 [0438] pH 5.7
[0439] pH 6.2 [0440] Humira HC F3 pH 4.2 [0441] 100 mg/mL pH 4.7
[0442] pH 5.7 [0443] pH 6.2 [0444] Humira HC F4 pH 4.2 [0445] 100
mg/mL pH 4.7 [0446] pH 5.7 [0447] pH 6.2
Procedure
[0448] The vials, stir bars, and stoppers were steam sterilized
prior to use.
[0449] The stirring experiment was performed with the following
experimental set-up: [0450] Protein solutions: Humira HC F1, F3,
F4, each at pH 4.2, 4.7, 5.7, 6.2 100 mg/mL, Humira HC F3 pH 5.2
100 mg/mL, Humira from Vetter 50 mg/mL, [0451] 5 mL filling volume
per 6R vial [0452] n=3.fwdarw.2.times. stirred (with 7.times.2 mm
magnetic bar), 1 unstirred control (without magnetic bar) [0453]
magnetic stirrer multipoint HP: 550 rpm [0454] ambient temperature
[0455] sample pull: t=0, t=1 h, t=4 h, t=24 h, t=48 h
[0456] Three 6R vials were filled with 5 mL for each protein
solution and closed with stoppers. Two of them were equipped with a
magnetic stir bar.
[0457] The vials were kept at 5.degree. C. over night. The next
morning the samples (one per protein solution, because in the
beginning they were all the same) were measured with the turbidity
meter. The measured solutions were filled back in the vials prior
start of the experiment. After 1, 4, 24, and 48 h samples were
taken and the turbidity was determined.
[0458] The unstirred samples were only measured at the time points
0 and 48 h.
[0459] For Humira HC F3 pH 5.2 also subvisible particles were
determined for all time points.
Results
Turbidity
[0460] The turbidity results for samples subjected to stir stress
for 0, 1, 4, 24, or 48 hours, as well as a 48 hour unstirred
control, are shown in Table 19.
TABLE-US-00019 TABLE 19 Turbidity (NTU) of samples subjected to
stir stress sample 0 h 1 h 4 h 24 h 48 h Commercial 20.90 23.90
31.20 98.05 176.00 Adalimumab Humira HC F3 pH 5.2 6.13 6.69 8.92
18.05 29.50 Humira HC F1 4.2 8.62 8.89 9.40 6.48 15.05 Humira HC F1
4.7 14.00 15.50 20.05 10.88 81.40 Humira HC F1 5.7 30.70 33.25
36.40 23.40 100.40 Humira HC F1 6.2 38.00 40.95 52.60 32.65 168.00
Humira HC F3 4.2 3.20 3.35 3.72 4.88 6.69 Humira HC F3 4.7 4.81
5.20 6.09 9.54 18.70 Humira HC F3 5.7 8.75 10.03 11.30 25.90 46.10
Humira HC F3 6.2 9.24 -- 13.05 22.60 37.30 Humira HC F4 4.2 3.44
3.74 3.80 6.48 9.79 Humira HC F4 4.7 5.13 5.67 6.60 10.88 17.00
Humira HC F4 5.7 9.23 10.15 12.50 23.40 32.20 Humira HC F4 6.2
10.30 11.65 15.55 32.65 56.75
[0461] Increased pH correlated with increased turbidity for all
tested formulations, both T0/unstirred and stirred samples. This
effect was most pronounced for formulation 1. Also, formulation 1
showed the highest increase of turbidity after 48 h at all pH
values except 4.2. Formulation 3 and 4 showed similar behavior and
turbidity values were comparable at all time points.
[0462] Humira HC (100 mg/mL), F3, pH 5.2 showed only a slight
increase of the turbidity over the time. In contrast, the
commercial Humira solution showed both a significantly higher
starting value and increase in turbidity over the time. Thus,
formulation 3 showed lower turbidity than the commercial Humira
formulation.
[0463] The stirred samples showed a higher turbidity compared with
the unstirred controls. The turbidity of the unstirred controls
remained almost constant in comparison to the 0 h samples,
indicating that running the experiment at room temperature did not
bias the results.
Subvisible Particles
[0464] Table 20 shows the results for the numbers of subvisible
particles.
TABLE-US-00020 TABLE 20 Counts of subvisible particles before and
after stirring stress subvisible particles Humira HC F3 pH 5.2
>=1 .mu.m >=10 .mu.m >=25 .mu.m 0 h 103 3 1 1 h 194 4 0 4
h 202 4 0 24 h 262 2 0 48 h 192 3 0 48 h unstirred 80 1 0
[0465] Particles .gtoreq.1 .mu.m
[0466] Stirring induced a slight increase in sub-visible particle
counts .gtoreq.1 .mu.m. The unstirred control was comparable to the
0 h sample.
[0467] Particles .gtoreq.10 .mu.m
[0468] Stirring had no significant effect on the particle counts
.gtoreq.10 .mu.m. The unstirred control was comparable to the 0 h
sample.
[0469] Particles .gtoreq.25 .mu.m
[0470] Stirring had no significant effect on the particle counts
.gtoreq.25 .mu.m. The unstirred control was comparable to the 0 h
sample.
[0471] Overall, the results of the experiments presented in Example
5 showed that formulation 3, when subjected to stirring stress, was
surprisingly stable compared with commercial Humira solution.
Formulation 3 was robust to stirring stress according to the
turbidity measure, and stirring of formulation 3 also had little or
no effect on formation of subvisible particles.
Example 6
Long-Term Storage Stability of High Concentration Anti-TNFA
Antibody
[0472] The following example describes a study which examined the
long-term storage stability (up to 24 months) of the F1, F3, and F4
formulations.
[0473] Formulations F1, F3, and F4 were tested prior to long term
storage (Initial), and after 3, 6, 9, 12, 18, and 24 months of
storage. The following storage conditions were used: (1) 5.degree.
C., (2) 25.degree. C./60% relative humidity (R.H), and (3)
40.degree. C./75% R.H. During storage, the samples were packaged in
a 1 ml pre-filled syringe (colorless, glass type I, Ph.Eur.); BD
Hypak Syringe BD 260 with a grey DB Hypak 4023/50 Fluorotec
stopper. The following measures were used to assess storage
stability:particulate contamination: visible particles; clarity and
opalescence; color of solution (visual); in vitro
TNF.alpha.-neutralization; cation exchange chromatography
(CEX-HPLC), size exclusion chromatography (SE-HPLC); particulate
contamination--sub-visible particles; container closure integrity;
pH; and microbial quality.
[0474] All formulations tested were stable under the tested storage
conditions of 2-8.degree. C. for up
Results
[0475] The results for Formulation F1 are presented in Table
21.
TABLE-US-00021 TABLE 21 Stability Summary Report for Formulation F1
Storage Conditions [.degree. C./% r.H.] Duration 25.degree. C./
40.degree. C./ \\Test Item Component Specification of Testing
5.degree. C. 60% R.H. 75% R.H. Participate Visible NMT 4.5 Initial
0.0 0.0 0.0 contamination: particles 3 months 0.0 0.0 0.0 visible 6
months 0.0 0.1 0.0 particles 9 months 0.0 -- -- 12 months 0.0 -- --
18 months 0.0 -- -- 24 months 0.0 -- -- Clarity and Assessment Not
more Initial <=RS III <=RS III <=RS III Opalescence
opalescent 3 months <=RS III <=RS III <=RS IV than 6
months <=RS III <=RS III <=RS IV reference 9 months
<=RS IV -- -- suspension 12 months <=RS III -- -- IV 18
months <=RS III -- -- 24 months <=RS III -- -- Color of
BY-Scale Report value Initial <=BY 7 <=BY 7 <=BY 7
solution 3 months <=BY 7 <=BY 7 <=BY 7 (visual) 6 months
<=BY 7 <=BY 7 <=BY 6 9 months <=BY 7 -- -- 12 months
<=BY 7 -- -- 18 months <=BY 7 -- -- 24 months <=BY 7 -- --
In vitro TNF- (cytotoxicity 80% to 125% Initial 99 99 99
Neutralization test) [%] of the 3 months 97 110 97 neutralization 6
months 87 81 68 capacity of 9 months 88 -- -- the reference 12
months 110 -- -- standard 18 months 97 -- -- 24 months 111 -- --
Fiducial Limit NLT 64 Initial 96.1 96.1 96.1 of error 3 months 91.1
104.7 93.2 (p = 0.95) 6 months 84.2 76.3 64.4 lower Limit [%] 9
months 84.3 -- -- 12 months 105.2 -- -- 18 months 95.3 -- -- 24
months 108.9 -- -- Fiducial Limit NMT 156 Initial 101.3 101.3 101.3
of error 3 months 101.9 115.5 100.1 (p = 0.95) 6 months 90.8 85.9
71.2 upper Limit [%] 9 months 90.8 -- -- 12 months 114.2 -- -- 18
months 98.8 -- -- 24 months 113.5 -- -- Cation Exchange Sum of
lysine NLT 75 Initial 86.8 86.8 86.8 Chromatography variants [%] 3
months 86.2 74.7 26.0 (CEX-HPLC) 6 months 85.9 65.0 11.9 9 months
85.2 -- -- 12 months 85.2 -- -- 18 months 84.1 -- -- 24 months 83.9
-- -- Size exclusion Principal peak NLT 98 Initial 99.6 99.6 99.6
chromatography (monomer) [%] 3 months 99.5 99.0 96.4 (SE-HPLC) 6
months 99.4 98.5 92.9 9 months 99.4 -- -- 12 months 99.4 -- -- 18
months 99.3 -- -- 24 months 99.3 -- -- Particulate Particles
>=10 .mu.m NMT 6000 Initial 11 11 11 contamination -
[/container] 3 months 8 37 55 Sub-visible 6 months 33 102 98
Particles* 9 months 32 -- -- 12 months 58 -- -- 18 months 44 -- --
24 months 11 -- -- Particles >=25 .mu.m NMT 600 Initial 0 0 0
[/container] 3 months 0 0 1 6 months 0 2 2 9 months 0 -- -- 12
months 0 -- -- 18 months 1 -- -- 24 months 0 -- -- Container
Assessment Tight Initial Complies Complies Complies Closure 6
months Complies Complies Complies Integrity 12 months Complies --
-- 18 months Complies -- -- 24 months Complies -- -- pH Single
values 4.7 to 5.7 Initial 5.3 5.3 5.3 3 months 5.2 5.2 5.2 6 months
5.3 5.3 5.3 9 months 5.3 -- -- 12 months 5.3 -- -- 18 months 5.3 --
-- 24 months 5.3 -- -- Microbial Sterility drug No evidence Initial
Complies Complies Complies quality product of microbial growth is
found
[0476] The results described above show that when stored for 24
months at 5.degree. C., Formulation F1 showed no visible
particulate contamination, clarity and opalescence <=RS III, and
visual color <=BY7(brown yellow 7). Formulation F1 also
demonstrated 111% of the TNF-neutralization capacity of the
reference standard, 83.9% lysine variants, 99.3% monomers, 11
particles >=10 .mu.m, and no particles >=25 .mu.m.
Furthermore, F1 maintained a stable pH of 5.3 and showed no
evidence of microbial growth. When stored for 6 months at
25.degree. C./60% R.H., Formulation F1 showed 0.1 visible
particles, clarity and opalescence <=RS III, visual color
<=BY7, 81% of the TNF-neutralization capacity of the reference
standard, 65% lysine variants, 98.5% monomers, 102 particles
>=10 .mu.m, 2 particles >=25 .mu.m, a stable pH of 5.3 and no
evidence of microbial growth. When stored for 6 months at
45.degree. C./75% R.H., Formulation F1 showed no visible particles,
clarity and opalescence <=RS IV, visual color <=BY6, 68% of
the TNF-neutralization capacity of the reference standard, 11.9%
lysine variants, 92.9% monomers, 98 particles >=10 .mu.m, 2
particles >=25 .mu.m, and no evidence of microbial growth.
[0477] The results for Formulation F3 are presented in Table
22.
TABLE-US-00022 TABLE 22 Stability Summary Report for Formulation F3
Storage Conditions [.degree. C./% r.H.] Duration 25.degree. C./
40.degree. C./ Test Item Component Specification of Testing
5.degree. C. 60% R.H. 75% R.H. Participate Visible NMT 4.5 Initial
0.0 0.0 0.0 contamination: particles 3 months 0.0 0.0 0.2 visible
particles 6 months 0.2 0.1 0.0 9 months 0.0 -- -- 12 months 0.0 --
-- 18 months 0.0 -- -- 24 months 0.0 -- -- Clarity and Assessment
Not more Initial <=RS II <=RS II <=RS II Opalescence
opalescent 3 months <=RS II <=RS II <=RS II than 6 months
<=RS II <=RS II <=RS II reference 9 months <=RS II --
-- suspension 12 months <=RS II -- -- IV 18 months <=RS II --
-- 24 months <=RS II -- -- Color of BY-Scale Report value
Initial <=BY 7 <=BY 7 <=BY 7 solution 3 months <=BY 7
<=BY 7 <=BY 7 (visual) 6 months <=BY 7 <=BY 7 <=BY 6
9 months <=BY 7 -- -- 12 months <=BY 7 -- -- 18 months
<=BY 7 -- -- 24 months <=BY 7 -- -- In vitro TNF-
(cytotoxicity 80% to 125% Initial 87 87 87 Neutralization test) [%]
of the 3 months 101 106 89 neutralization 6 months 100 101 90
capacity of 9 months 98 -- -- the reference 12 months 96 -- --
standard 18 months 96 -- -- 24 months 98 -- -- Fiducial Limit NLT
64 Initial 85.4 85.4 85.4 of error 3 months 92.9 88.1 80.6 (p =
0.95) 6 months 98.3 97.4 86.5 lower Limit [%] 9 months 97.0 -- --
12 months 93.3 -- -- 18 months 93.9 -- -- 24 months 96.7 -- --
Fiducial Limit NMT 156 Initial 88.5 88.5 88.5 of error 3 months
110.5 122.2 97.9 (p = 0.95) 6 months 101.7 103.8 92.6 upper Limit
[%] 9 months 99.8 -- -- 12 months 99.2 -- -- 18 months 98.1 -- --
24 months 99.6 -- -- Cation Exchange Sum of lysine NLT 75 Initial
86.8 86.8 86.8 Chromatography variants [%] 3 months 86.6 77.8 32.8
(CEX-HPLC) 6 months 86.4 70.1 16.5 9 months 86.0 -- -- 12 months
86.2 -- -- 18 months 85.2 -- -- 24 months 85.1 -- -- Size exclusion
Principal peak NLT 98 Initial 99.7 99.7 99.7 chromatography
(monomer) [%] 3 months 99.6 99.2 96.9 (SE-HPLC) 6 months 99.5 98.8
93.8 9 months 99.5 -- -- 12 months 99.5 -- -- 18 months 99.4 -- --
24 months 99.4 -- -- Participate Particles >=10 .mu.m NMT 6000
Initial 10 10 10 contamination - [/container] 3 months 12 45 73
Sub-visible 6 months 22 157 275 Particles* 9 months 50 -- -- 12
months 54 -- -- 18 months 45 -- -- 24 months 14 -- -- Particles
>=25 .mu.m NMT 600 Initial 0 0 0 [/container] 3 months 0 0 1 6
months 0 2 9 9 months 0 -- -- 12 months 1 -- -- 18 months 0 -- --
24 months 0 -- -- Container Assessment Tight Initial Complies
Complies Complies Closure 6 months Complies Complies Complies
Integrity 24 months Complies -- -- pH Single values 4.7 to 5.7
Initial 5.2 5.2 5.2 3 months 5.3 5.3 5.3 6 months 5.2 5.2 5.3 9
months 5.3 -- -- 12 months 5.4 -- -- 18 months 5.2 -- -- 24 months
5.1 -- -- Microbial Sterility drug No evidence Initial Complies
Complies Complies quality product of microbial growth is found
[0478] The results provided in Table 22 indicate that when stored
for 24 months at 5.degree. C., Formulation F3 showed no visible
particulate contamination, clarity and opalescence <=RS II, and
visual color <=BY7. Formulation F3 showed 98% of the
TNF-neutralization capacity of the reference standard, 85.1% lysine
variants, 99.4% monomers, 14 particles >=10 .mu.m, and no
particles >=25 .mu.m. The pH showed little change and there was
no evidence of microbial growth.
[0479] When stored for 6 months at 25.degree. C./60% R.H.,
Formulation F3 showed no visible particles, clarity and opalescence
<=RS II, and visual color <=BY7. Also, formulation F3 showed
101% of the TNF-neutralization capacity of the reference standard,
97.4% lysine variants, 70.1% monomers, 157 particles >=10 .mu.m,
and 2 particles >=25 .mu.m. The pH was stable and there was no
evidence of microbial growth.
[0480] When stored for 6 months at 45.degree. C./75% R.H.,
Formulation F3 showed no visible particles, clarity and opalescence
<=RS II, and visual color <=BY6. Also, formulation F3 showed
90% of the TNF-neutralization capacity of the reference standard,
16.5% lysine variants, 93.8% monomers, 275 particles >=10 .mu.m,
and 9 particles >=25 .mu.m. The pH was quite stable, and there
was no evidence of microbial growth.
[0481] The results for Formulation F4 are presented in Table
23.
TABLE-US-00023 TABLE 23 Stability Summary Report for Formulation F4
Storage Conditions [.degree. C./% r.H.] Duration 25.degree. C./
40.degree. C./ Test Item Component Specification of Testing
5.degree. C. 60% R.H. 75% R.H. Appearance Visual NMT 4.5 Initial
0.0 0.0 0.0 particles 3 months 0.0 0.0 0.0 6 months 0.0 0.0 0.0 9
months 0.0 -- -- 12 months 0.0 -- -- 18 months 0.0 -- -- Clarity
Assessment Not more Initial <=RS II <=RS II <=RS II
opalescent 3 months <=RS II <=RS II <=RS II than 6 months
<=RS II <=RS II <=RS II reference 9 months <=RS II --
-- suspension 12 months <=RS II -- -- IV 18 months <=RS II --
-- Color BY-Scale Initial <=BY 7 <=BY 7 <=BY 7 3 months
<=BY 6 <=BY 6 <=BY 6 6 months <=BY 7 <=BY 7 <=BY
6 9 months <=BY 7 -- -- 12 months <=BY 7 -- -- 18 months
<=BY 7 -- -- In vitro TNF- (cytotoxicity 80% to 125% Initial 111
111 111 Neutralisation test) [%] of the 3 months 105 101 80
(Cytotoxi- neutralization 6 months 97 101 76 zitatstest) capacity
of 9 months 112 -- -- the reference 12 months 97 -- -- standard 18
months 104 -- -- Fiducial Limit NLT 64 Initial 105.2 105.2 105.2 of
error 3 months 103.2 100.1 79.2 (p = 0.95) 6 months 92.9 97.5 74.7
lower Limit 9 months 109.3 -- -- [%] 12 months 90.2 -- -- 18 months
101.2 -- -- Fiducial Limit NMT 156 Initial 116.3 116.3 116.3 of
error 3 months 106.2 102.7 80.4 (p = 0.95) 6 months 101.1 103.5
78.1 upper Limit 9 months 113.9 -- -- [%] 12 months 104.9 -- -- 18
months 107.5 -- -- Cation Exchange Sum of lysine NLT 75 Initial
85.5 85.5 85.5 Chromatography variants [%] 3 months 85.8 76.8 31.6
(CEX-HPLC) 6 months 85.4 68.7 15.7 9 months 85.2 -- -- 12 months
84.5 -- -- 18 months 84.4 -- -- Size exclusion Principal peak NLT
98 Initial 99.7 99.7 99.7 chromatography (monomer) [%] 3 months
99.6 99.1 96.5 (SE-HPLC) 6 months 99.6 98.8 93.1 9 months 99.5 --
-- 12 months 99.5 -- -- 18 months 99.4 -- -- Participate Particles
>=10 .mu.m NMT 6000 Initial 17 17 17 contamination -
[/container] 3 months 51 174 207 Sub-visible 6 months 39 144 218
Particles 9 months 82 -- -- 12 months 57 -- -- Particles >=25
.mu.m NMT 600 Initial 0 0 0 [/container] 3 months 0 1 5 6 months 0
1 1 9 months 1 -- -- 12 months 2 -- -- Container Assessment Must
comply Initial Complies Complies Complies closure (no blue 6 months
Complies Complies Complies Integrity coloration) pH Single values
4.7 to 5.7 Initial 5.1 5.1 5.1 3 months 5.2 5.2 5.1 6 months 5.2
5.1 5.2 9 months 5.2 -- -- 12 months 5.2 -- -- 18 months 5.1 -- --
Microbial Sterility drug No evidence Initial Complies Complies
Complies quality product of microbial growth is found
[0482] The results provided in Table 23 indicate that when stored
for 18 months at 5.degree. C., Formulation F4 showed no visible
particulate contamination, clarity and opalescence <=RS II, and
visual color <=BY7. Formulation F4 showed 104% of the
TNF-neutralization capacity of the reference standard, 84.4% lysine
variants, and 99.4% monomers. Furthermore, the pH was stable and
there was no evidence of microbial growth.
[0483] When stored for 6 months at 25.degree. C./60% R.H.,
Formulation F4 showed no visible particles, clarity and opalescence
<=RS II, and visual color <=BY7. Formulation F4 showed 101%
of the TNF-neutralization capacity of the reference standard, 68.7%
lysine variants, 98.8% monomers, 144 particles >=10 .mu.m, and 1
particle >=25 .mu.m. Furthermore, the pH was quite stable and
there was no evidence of microbial growth.
[0484] When stored for 6 months at 45.degree. C./75% R.H.,
Formulation F4 showed no visible particles, clarity and opalescence
<=RS II, and visual color <=BY6. Formulation F4 showed 76% of
the TNF-neutralization capacity of the reference standard, 15.7%
lysine variants, 93.1% monomers, 218 particles >=10 .mu.m, and 1
particle >=25 .mu.m. Furthermore, the pH was quite stable and
there was no evidence of microbial growth.
[0485] In summary, the results of the long-term stability
experiments, as presented in Tables 21-23, show that high
concentration formulations F1, F3, and F4 were surprisingly stable
when subjected to long term storage. The stability of these
formulations was similar to the commercial formulation.
Formulations F1 and F3 showed stability similar to the commercial
formulation after long term storage for at least 24 months.
Formulation F4 showed stability similar to the commercial
formulation after long term storage for at least 18 months.
Example 7
Room Temperature Storage Stability of High Concentration Anti-TNFA
Antibody
[0486] Liquid pharmaceutical products containing therapeutic
antibodies often require storage at 2-8.degree. C. until
end-of-shelf-life. Cooling is therefore also required by patients
between purchasing of the medicines until use. Depending on the
proposed dosing regimen, this can result in storage times under
patient's responsibility in the case of self-administration drugs
for up to several weeks.
[0487] Therefore, drugs that do not require storage under
refrigerated conditions display both a significant increase in
patient convenience for home care products and reduction of drug
quality concerns in case of improper storage, thereby reducing
complaint rates and temperature excursion investigations.
[0488] The currently marketed Adalimumab containing product
(Humira) was successfully reformulated at a higher protein
concentration as Formulation F3, as described above in Examples
1-6. The following stability data for Formulation F3 resulted in
findings of improved stability against protein degradation. The
resulting degradation kinetics measured at 25.degree. C. complied
with requirements for ambient storage for up to 3 months.
[0489] For general long-term stability data related to storage at
25.degree. C. ofr Formulation F3, see Example 6 above.
[0490] The following data describes long-term storage
characteristics for Formulation F3. The data shows that even after
18 months and 24 months of long-term storage at 2-5.degree. C.,
additional storage at 25.degree. C./30.degree. C. is
acceptable.
TABLE-US-00024 TABLE 24 24m storage of F3 at 2-8.degree. C.,
followed by 7 days/14 days at accelerated conditions (25.degree.
C., 30.degree. C.) Test t0 +7 Days +14 Days criterion Specification
Characteristic: (24M 5.degree. C.) 25.degree. C. 30.degree. C./65%
R.H. 25.degree. C. 30.degree. C./65% R.H. Appearance colourless to
complies complies complies complies complies slightly yellow
solution Visible single vial .ltoreq.2=> 0.0 Value per analyst:
Value per analyst: Value per analyst: Value per analyst: particles*
practically free 3 .times. 0; single 3 .times. 0; single 3 .times.
0; single 3 .times. 0; single from visible vial .ltoreq.2=> vial
.ltoreq.2=> vial .ltoreq.2=> vial .ltoreq.2=> particulate
matter practically free practically free practically free
practically free 1 vial >10=> from visible from visible from
visible from visible particulate matter particulate matter
particulate matter particulate matter Size Purity % monomer
aggregate 0.4 0.4 0.4 0.4 0.4 exclusion NLT 98% monomer 99.4 99.3
99.3 99.3 99.2 HPLC fragments 0.2 0.2 0.3 0.3 0.3 Cation NMT 8%
first acid region 2.7 2.8 3.0 2.9 3.3 exchange NMT 16% second acid
region 10.5 10.9 11.4 11.6 12.7 HPLC NLT 75% sum Lysine variants
85.1 84.2 83.4 83.0 81.2 NMT 4% peak between Lysine 1 1.5 1.7 1.9
1.6 1.8 and Lysine 2 report value [%] peaks after Lysine 2 0.2 0.4
0.4 0.9 1.0 PCS report value [%] Z-Average 1.390 1.365 1.395 1.397
1.420 Pdl 0.193 0.176 0.188 0.175 0.210
TABLE-US-00025 TABLE 25 18m storage at 2-8.degree. C. of F3,
followed by 7 days/14 days at accelerated conditions (25.degree.
C., 30.degree. C.) Test t0 +7 Days +14 Days criterion Specification
Characteristics (18M 5.degree. C.) 25.degree. C. 30.degree. C./65%
R.H. 25.degree. C. 30.degree. C./65% R.H. Appearance colourless to
complies complies complies complies slightly yellow solution
Visible single vial .ltoreq.2=> Value per analyst: Value per
analyst: Value per analyst: Value per analyst: particles*
practically free 3 .times. 0; single 3 .times. 0; single 3 .times.
0; single 3 .times. 0; single from visible vial .ltoreq.2=> vial
.ltoreq.2=> vial .ltoreq.2=> vial .ltoreq.2=> particulate
matter practically free practically free practically free
practically free 1 vial >10=> from visible from visible from
visible from visible inform lab manager particulate matter
particulate matter particulate matter particulate matter
Participate particles .ltoreq. 10 .ltoreq.1 .mu.m/1 ml 2250 5623
9355 10252 contamination, .mu.m: .ltoreq.6000 .ltoreq.10 .mu.m/1 ml
6 7 39 45 subvisible particles/container .ltoreq.25 .mu.m/1 ml 0 0
0 1 particles* particles .ltoreq. 25 .mu.m: .ltoreq.600
particles/container PCS report value [%] Z-Average 2.378 2.344
2.353 2.358 Pdl 0.102 0.077 0.077 0.077
Example 8
Conductivity of High Concentration Anti-TNFA Antibody
Formulations
[0491] The conductivity of the high concentration anti-TNF.alpha.
antibody formulations F3 and F4 (see Examples 1-6, supra) was
determined using an InoLab Cond Level2 WTW device normalized to
25.degree. C. Table 26 shows the influence of non-ionic excipients
on the conductivity of the F3 and F4 adalimumab formulations.
TABLE-US-00026 TABLE 26 Conductivity of Formulations F3 and F4
Temperature Conductivity Sample [.degree. C.] [.mu.S/cm] Adalimumab
DP F3 1 22.4 663 2 22.4 651 3 23.8 660 4 21.4 715 5 21.7 691 6 23.1
680 7 23.3 644 8 22.9 647 Adalimumab DP F4 1 22.0 797 2 22.9
746
[0492] As described above in Table 26, average conductivity for
both formulations F3 and F4 was less than 2 mS/cm.
Example 9
Dynamic Light Scattering (DLS) of High Concentration Anti-TNFA
Antibody Formulations
[0493] Dynamic light scattering analysis of diluted solutions was
used to assess the hydrodynamic diameter (reported as the mean or
Z-average size, calculated by cumulants analysis of the DLS
measured intensity autocorrelation function and polydispersity
index, PDI, of the size distribution of particles). DLS
measurements were specifically used to detect low amounts of higher
molecular weight species, e.g. aggregates, in a size distribution,
since those species possess higher scattering intensity
(proportional to d6) and, therefore, will influence the Z-average
and Polydispersity Index (PDI) as an indicator of the Z-average
size distribution significantly.
[0494] A 150 .mu.L sample of each of formulations F3 and F4 (see
examples 1-6 above) was measured to analyze the average size of the
particles (Z-average) and the Polydispersity Index (PDI), an
indicator of the "broadness" of the particle size distribution
using DLS. The results are shown below. DLS data did not show any
signs of the development of higher molecular weight aggregates,
since the polydispersity index, a sensitive indicator for low
levels of higher molecular weight sub-populations did not increase
significantly.
Formulation F3
TABLE-US-00027 [0495] Sample No. ZAve (nm) PDI 1 2.4 n. a. 2 2.3
0.08 3 2.3 0.14 4 2.3 0.09
Formulation F4
TABLE-US-00028 [0496] Sample No. ZAve (nm) PDI 1 1.3 n.a. 2 2.5
n.a.
[0497] As described above, the z-average measurement for both F3
and F4 was less than 4 nm. This low hydrodynamic diameter is
representative of the fact that both formulations F3 and F4 do not
contain additional excipients other than a polysorbate and a polyol
or a polysorbate.
Example 10
Factors Influencing the Stability of High Concentration Anti-TNFA
Antibody Formulations
[0498] The effect of varying mannitol concentrations and
polysorbate concentrations on the stability of adalimumab in water
was examined.
[0499] Formulations containing 100 mg/ml of adalimumab in water
were prepared. Subsequently, various concentrations of either
mannitol or polysorbate were added in a concentration range to
determine the impact of each excipient on the stability of the
formulation, as measured by aggregation and fragmentation. The
concentrations of polysorbate and mannitol ranged from 0.1 to 1.0
mg/ml and 0-72 mg/ml, respectively, as shown in FIGS. 3A and 3B. As
shown in FIG. 3A, varying the concentration of mannitol from about
12 to about 72 mg/ml had a minimal effect on the stability of
adalimumab. Similarly, varying the concentration of polysorbate-80
from about 0.1 to about 1.0 mg/ml had no effect on the stability of
adalimumab.
INCORPORATION BY REFERENCE
[0500] The contents of all cited references (including, for
example, literature references, patents, patent applications, and
websites) that maybe cited throughout this application are hereby
expressly incorporated by reference in their entirety for any
purpose. The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of protein
formulations, which are well known in the art.
EQUIVALENTS
[0501] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting of the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are therefore intended to be embraced herein.
Sequence CWU 1
1
541107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
2121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp
Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly
100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
39PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Gln Arg Tyr Asn Arg Ala Pro Tyr Xaa 1 5
412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa
1 5 10 57PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Ala Ala Ser Thr Leu Gln Ser 1 5 617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Ala
Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu 1 5 10
15 Gly 711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala 1 5
10 85PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Asp Tyr Ala Met His 1 5 9107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Lys Tyr Asn Ser Ala Pro Tyr 85 90 95 Ala Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 10121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
10Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Asp Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp
Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Ala Val Ser Arg Asp
Asn Ala Lys Asn Ala Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Ala Ser Tyr
Leu Ser Thr Ser Ser Ser Leu Asp Asn Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Gln
Lys Tyr Asn Ser Ala Pro Tyr Ala 1 5 129PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Gln
Lys Tyr Asn Arg Ala Pro Tyr Ala 1 5 139PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Gln
Lys Tyr Gln Arg Ala Pro Tyr Thr 1 5 149PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Gln
Lys Tyr Ser Ser Ala Pro Tyr Thr 1 5 159PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Gln
Lys Tyr Asn Ser Ala Pro Tyr Thr 1 5 169PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Gln
Lys Tyr Asn Arg Ala Pro Tyr Thr 1 5 179PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Gln
Lys Tyr Asn Ser Ala Pro Tyr Tyr 1 5 189PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Gln
Lys Tyr Asn Ser Ala Pro Tyr Asn 1 5 199PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Gln
Lys Tyr Thr Ser Ala Pro Tyr Thr 1 5 209PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Gln
Lys Tyr Asn Arg Ala Pro Tyr Asn 1 5 219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 21Gln
Lys Tyr Asn Ser Ala Ala Tyr Ser 1 5 229PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Gln
Gln Tyr Asn Ser Ala Pro Asp Thr 1 5 239PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Gln
Lys Tyr Asn Ser Asp Pro Tyr Thr 1 5 249PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Gln
Lys Tyr Ile Ser Ala Pro Tyr Thr 1 5 259PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Gln
Lys Tyr Asn Arg Pro Pro Tyr Thr 1 5 269PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Gln
Arg Tyr Asn Arg Ala Pro Tyr Ala 1 5 2712PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 27Ala
Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn 1 5 10
2812PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Lys
1 5 10 2912PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Tyr
1 5 10 3012PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp
1 5 10 3112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Ala Ser Tyr Leu Ser Thr Ser Phe Ser Leu Asp Tyr
1 5 10 3212PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu His Tyr
1 5 10 3312PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Ala Ser Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr
1 5 10 3412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Ala Ser Tyr Leu Ser Thr Ala Ser Ser Leu Glu Tyr
1 5 10 3512PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Asn
1 5 10 36321DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 36gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtagggga cagagtcacc 60atcacttgtc gggcaagtca
gggcatcaga aattacttag cctggtatca gcaaaaacca 120gggaaagccc
ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct
180cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctacagcct 240gaagatgttg caacttatta ctgtcaaagg tataaccgtg
caccgtatac ttttggccag 300gggaccaagg tggaaatcaa a
32137363DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 37gaggtgcagc tggtggagtc tgggggaggc
ttggtacagc ccggcaggtc cctgagactc 60tcctgtgcgg cctctggatt cacctttgat
gattatgcca tgcactgggt ccggcaagct 120ccagggaagg gcctggaatg
ggtctcagct atcacttgga atagtggtca catagactat 180gcggactctg
tggagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat
240ctgcaaatga acagtctgag agctgaggat acggccgtat attactgtgc
gaaagtctcg 300taccttagca ccgcgtcctc ccttgactat tggggccaag
gtaccctggt caccgtctcg 360agt 363386PRTHomo sapiens 38His Gly Ser
His Asp Asn 1 5 3912PRTHomo sapiens 39Gln Ser Tyr Asp Arg Tyr Thr
His Pro Ala Leu Leu 1 5 10 4017PRTHomo sapiens 40Phe Ile Arg Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
417PRTHomo sapiens 41Tyr Asn Asp Gln Arg Pro Ser 1 5 429PRTHomo
sapiens 42Phe Thr Phe Ser Ser Tyr Gly Met His 1 5 4313PRTHomo
sapiens 43Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn Thr Val Lys 1 5
10 44115PRTHomo sapiens 44Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val
Thr 100 105 110 Val Ser Ser 115 45112PRTHomo sapiens 45Gln Ser Val
Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25
30 Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45 Ile Tyr Tyr Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Thr Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Tyr Asp Arg Tyr Thr 85 90 95 His Pro Ala Leu Leu Phe Gly Thr
Gly Thr Lys Val Thr Val Leu Gly 100 105 110 4616PRTHomo sapiens
46Ser Ile His Asn Arg Gly Thr Ile Phe Tyr Leu Asp Ser Val Lys Gly 1
5 10 15 4710PRTHomo sapiens 47Gly Arg Ser Asn Ser Tyr Ala Met Asp
Tyr 1 5 10 4816PRTHomo sapiens 48Arg Ser Thr Gln Thr Leu Val His
Arg Asn Gly Asp Thr Tyr Leu Glu 1 5 10 15 497PRTHomo sapiens 49Lys
Val Ser Asn Arg Phe Ser 1 5 509PRTHomo sapiens 50Phe Gln Gly Ser
His Val Pro Tyr Thr 1 5 5130PRTHomo sapiens 51Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 5213PRTHomo
sapiens 52Trp Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5
10 5332PRTHomo sapiens 53Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 5415PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
15
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