U.S. patent application number 12/161410 was filed with the patent office on 2009-09-03 for injectable combination therapy for eye disorders.
This patent application is currently assigned to POTENTIA PHARMACEUTICALS, INC.. Invention is credited to Pascal Deschatelets, Cedric Francois, Paul Olson.
Application Number | 20090220572 12/161410 |
Document ID | / |
Family ID | 38179525 |
Filed Date | 2009-09-03 |
United States Patent
Application |
20090220572 |
Kind Code |
A1 |
Deschatelets; Pascal ; et
al. |
September 3, 2009 |
Injectable Combination Therapy for Eye Disorders
Abstract
The present invention provides composition, methods, and
articles of manufacture for treating an eye disorder, e.g., a
disorder characterized by macular degeneration, choroidal
neovascularization, or retinal neovascularization. One method of
the invention comprises the step of: administering first and second
therapeutic agents to the subject's eye in a single procedure,
wherein the first therapeutic agent provides rapid improvement in
the condition of the subject's eye and the second therapeutic agent
is administered as a sustained release formulation of the second
therapeutic agent. For example, the first and second therapeutic
agents are administered by intravitreal injection. The first
therapeutic agent may be dissolved in a liquid medium located in
the syringe and the sustained formulation of the second therapeutic
agent may comprise an ocular implant or plurality of particles
located in the needle. The therapeutic agents may be selected from
the group consisting of angiogenesis inhibitors and complement
inhibitors.
Inventors: |
Deschatelets; Pascal;
(Louisville, KY) ; Olson; Paul; (Louisville,
KY) ; Francois; Cedric; (Louisville, KY) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Assignee: |
POTENTIA PHARMACEUTICALS,
INC.
Louisville
KY
|
Family ID: |
38179525 |
Appl. No.: |
12/161410 |
Filed: |
January 19, 2007 |
PCT Filed: |
January 19, 2007 |
PCT NO: |
PCT/US07/01649 |
371 Date: |
November 21, 2008 |
Current U.S.
Class: |
424/427 ;
424/145.1; 424/158.1; 424/400; 424/450; 424/484; 424/489; 514/1.1;
514/44R |
Current CPC
Class: |
A61K 31/00 20130101;
A61K 49/0008 20130101; C07K 7/08 20130101; A61K 38/12 20130101;
A61K 38/17 20130101; A61P 9/00 20180101; A61P 43/00 20180101; G01N
33/6872 20130101; A61K 38/08 20130101; C07K 2319/01 20130101; A61K
9/0051 20130101; G01N 2500/04 20130101; G01N 2500/20 20130101; A61K
2039/507 20130101; A61P 27/02 20180101; A61K 2039/505 20130101;
A61K 38/10 20130101; A61K 39/3955 20130101; G01N 2333/4704
20130101; A61F 9/0008 20130101; A61K 31/7105 20130101; A61K 38/00
20130101; A61K 9/0019 20130101; C07K 16/28 20130101; A61K 9/0048
20130101; A61P 9/14 20180101 |
Class at
Publication: |
424/427 ;
424/158.1; 514/44.R; 424/145.1; 514/12; 514/2; 514/14; 424/489;
424/450; 424/484; 424/400 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 9/00 20060101 A61K009/00; A61K 39/395 20060101
A61K039/395; A61K 31/7052 20060101 A61K031/7052; A61K 38/16
20060101 A61K038/16; A61K 38/02 20060101 A61K038/02; A61K 38/10
20060101 A61K038/10; A61K 9/14 20060101 A61K009/14; A61P 27/02
20060101 A61P027/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 19, 2006 |
US |
60/760974 |
Oct 6, 2006 |
US |
11/544389 |
Claims
1. A method of treating an eye disorder characterized by macular
degeneration, CNV, or RNV comprising the step of: administering
effective amounts of first and second therapeutic agents to the
subject's eye in a single procedure, wherein the first therapeutic
agent provides rapid improvement in the condition of the subject's
eye and the second therapeutic agent is administered as a sustained
release formulation of the second therapeutic agent.
2. The method of claim 1, wherein the rapid improvement includes
improvement in the visual acuity of the eye that occurs within 2
weeks following administration of the agents.
3. The method of claim 1, wherein the procedure is an injection
procedure.
4. The method of claim 1, wherein the procedure is an injection
procedure in which the first and second therapeutic agents are
injected into the vitreous of the subject's eye.
5. The method of claim 1, wherein the procedure is an injection
procedure in which, prior to administration, the first therapeutic
agent is contained in a syringe and the sustained release
formulation comprising the second therapeutic agent is contained in
a needle attached to the syringe.
6. The method of claim 5, wherein the first therapeutic agent is
dissolved in a liquid medium located in the syringe and the
sustained formulation of the second therapeutic agent comprises an
ocular implant located in the needle.
7. The method of claim 1, wherein the first therapeutic agent is an
angiogenesis inhibitor.
8. The method of claim 1, wherein the first therapeutic agent is an
anti-VEGF agent.
9. The method of claim 1, wherein the first therapeutic agent is an
anti-VEGF agent selected from the group consisting of: antibodies
that bind to VEGF and nucleic acids that bind to VEGF.
10. The method of claim 1, wherein the first therapeutic agent is
selected from the group consisting of bevacizumb, ranibizumab, and
pegaptanib.
11. The method of claim 1, wherein the second therapeutic agent is
a complement inhibitor.
12. The method of claim 1, wherein the second therapeutic agent is
a complement inhibitor selected from the group consisting of: viral
complement control proteins and peptides or small molecules that
bind to a complement component.
13. The method of claim 1, wherein the second therapeutic agent is
compstatin or a derivative thereof.
14. The method of claim 1, wherein the second therapeutic agent is
a GPCRA.
15. The method of claim 1, wherein the first therapeutic agent is
an angiogenesis inhibitor and the second therapeutic agent is a
complement inhibitor.
16. The method of claim 1, wherein compstatin or an analog thereof
and a C5a inhibitor are adminstered.
17. The method of claim 16, wherein the C5a inhibitor is a C5a
receptor antagonist.
18. The method of claim 1, wherein the first therapeutic agent is
dissolved or suspended in a liquid medium prior to
administration.
19. The method of claim 1, wherein the sustained release
formulation comprises an ocular implant comprising the second
therapeutic agent.
20. The method of claim 1, wherein the sustained release
formulation comprises a polymeric material.
21. The method of claim 20, wherein the polymeric material is
biodegradable.
22. The method of claim 20, wherein the polymeric material is
selected from the group consisting of: poly-lactic acid (PLA),
poly-glycolic acid (PGA), poly-lactide-co-glycolide (PLGA),
poly(phosphazine), poly (phosphate ester), polycaprolactones,
polyanhydrides, ethylene vinyl acetate, polyorthoesters,
polyethers, poly (beta amino esters), copolymers containing
monomeric subunits found in any of the foregoing polymers,
collagen, albumin, chitosan, alginate, hyaluronic acid, and
mixtures of any of the foregoing polymers.
23. The method of claim 1, wherein the sustained release
formulation comprises nanoparticles, microparticles, dendrimers, or
liposomes comprising the second therapeutic agent.
24. The method of claim 1, wherein the sustained release
formulation comprises a solid or semi-solid material that entraps
or encapsulates the second therapeutic agent.
25. The method of claim 1, wherein the sustained release
formulation comprises an inactive material to which the second
therapeutic agent is covalently attached.
26. The method of claim 1, wherein the first therapeutic agent is
administered in soluble or particulate form in a liquid medium and
the second therapeutic agent is administered in or attached to a
solid or semi-solid matrix.
27. The method of claim 1, wherein the second therapeutic agent,
when administered as a component of the sustained release
formulation, has an activity period greater than that of the first
therapeutic agent.
28. The method of claim 1, wherein administering the second
therapeutic agent prolongs the time interval during which the
subject experiences improvement in the condition of the subject's
eye relative to the time interval during which the subject would
have experienced improvement if the first therapeutic agent had
been administered as sole therapy.
29. The method of claim 1, wherein the eye disorder is exudative
ARMD.
30. The method of claim 1, wherein the subject has experienced a
perceptible deterioration in the condition of the subject's eye
within the two weeks preceding administration of the first and
second therapeutic agents.
31. The method of claim 1, further comprising performing the method
one or more additional times at time intervals greater than the
activity period of the first therapeutic agent.
32. A method of treating an eye disorder characterized by macular
degeneration, CNV, or RNV comprising the step of: administering
first and second compositions to the subject's eye in a single
procedure, wherein the first composition comprises an angiogenesis
inhibitor or complement inhibitor that provides rapid improvement
in the condition of the subject's eye and the second composition
comprises a sustained release formulation comprising an
angiogenesis inhibitor or a complement inhibitor.
33. The method of claim 32, wherein the single procedure is an
intravitreal injection.
34. The method of claim 32, wherein the angiogenesis inhibitor is
an anti-VEGF agent.
35. The method of claim 32, wherein the angiogenesis inhibitor is
an anti-VEGF agent selected from the group consisting of:
antibodies or antibody fragments that bind to VEGF and nucleic
acids that bind to VEGF.
36. The method of claim 32, wherein the angiogenesis inhibitor is
selected from the group consisting of bevacizumb, ranibizumab, and
pegaptanib.
37. The method of claim 32, wherein the complement inhibitor is
selected from the group consisting of: viral complement control
proteins and peptides that bind to a complement component.
38. The method of claim 32, wherein the complement inhibitor is
compstatin or a derivative thereof.
39. The method of claim 32, wherein the angiogenesis inhibitor is
selected from the group consisting of bevacizumb, ranibizumab, and
pegaptanib and the complement inhibitor is compstatin or a
derivative thereof.
40. The method of claim 32, wherein the first therapeutic agent is
provided at least in part dissolved or suspended in a liquid
medium.
41. The method of claim 32, wherein the second therapeutic agent is
released from the ocular implant so as to maintain a therapeutic
level in the subject's eye over a period of at least 3 months.
42. A method of administering first and second therapeutic agents
to the eye of a subject comprising: injecting (i) a solution or
suspension containing the first therapeutic agent and (ii) a solid
ocular implant, plurality of particles, or gel-forming composition
containing the second therapeutic agent into the subject's eye in a
single injection procedure.
43. The method of claim 42, wherein the first and second
therapeutic agents are injected into the vitreous of the subject's
eye.
44. The method of claim 42, wherein (i) the solution or suspension;
and (ii) the solid ocular implant, plurality of particles, or
gel-forming composition, are injected using a single needle and
syringe assembly.
45. The method of claim 42, wherein the first therapeutic agent
provides a rapid improvement in the condition of the subject's
eye.
46. The method of claim 42, wherein the activity period of the
second composition is greater than the activity period of the first
composition.
47. The method of claim 42, wherein the second therapeutic agent
does not provide rapid improvement in the condition of the
subject's eye.
48. The method of claim 42, wherein the second therapeutic agent
has an activity period greater than that of the first therapeutic
agent.
49. The method of claim 42, further comprising the step of:
repeating the administering step once or more at time intervals
greater than the activity period of the first therapeutic
agent.
50. An article of manufacture comprising (i) a first therapeutic
agent effective for treating an eye disorder; and (ii) a needle
containing a second therapeutic agent.
51. The article of manufacture of claim 50, further comprising a
syringe.
52. The article of manufacture of claim 50, further comprising a
syringe, wherein the syringe contains the first therapeutic
agent.
53. The article of manufacture of claim 50, wherein the article of
manufacture contains a unit dosage form of the first therapeutic
agent.
54. The article of manufacture of claim 50, wherein the article of
manufacture contains a unit dosage form of the first therapeutic
agent and a unit dosage form of the second therapeutic agent.
55. The article of manufacture of claim 50, further comprising a
syringe, wherein the article of manufacture contains at least one
compartment and the syringe and needle are housed in a single
compartment of the article of manufacture.
56. The article of manufacture of claim 50, further comprising a
syringe, wherein the syringe and needle are attached to one
another.
57. The article of manufacture of claim 50, further comprising a
syringe, wherein the article of manufacture contains at least two
compartments, and wherein the syringe and needle are housed in
individual compartments.
58. An article of manufacture comprising (i) a first therapeutic
agent effective for treating an eye disorder; (ii) a second
therapeutic agent effective for treating an eye disorder, wherein
each therapeutic agent is contained in an individual syringe.
59. The article of manufacture of claim 58, further comprising a
needle.
60. A method of supplying a combination therapy for an ocular
disorder comprising providing the article of manufacture of any of
claims 50-59.
61. The method of claim 60, wherein the step of providing
comprises: shipping the article of manufacture to a pharmacy or to
a site of health care delivery.
62. A needle and syringe assembly, wherein the needle contains a
sustained release formulation comprising a first therapeutic agent
for an eye disorder and the syringe contains a second therapeutic
agent for the eye disorder, wherein the second therapeutic agent is
dissolved or suspended in a liquid medium.
63. The needle and syringe assembly of claim 62, wherein the
sustained release formulation comprises an ocular implant,
plurality of particles, or gel-forming material.
64. The needle and syringe assembly of claim 62, wherein the first
therapeutic agent is a complement inhibitor or an angiogenesis
inhibitor and the second therapeutic agent is a complement
inhibitor or an angiogenesis inhibitor.
65. The needle and syringe assembly of claim 62, wherein the first
therapeutic agent is a complement inhibitor and the second
therapeutic agent is an angiogenesis inhibitor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to, and the benefit of,
U.S. Provisional Patent Application No. 60/760,974, filed Jan. 19,
2006, and U.S. Ser. No. 11/544,389, filed Oct. 6, 2006, both of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Macular degeneration is a term that refers to and describes
a number of different diseases characterized by degenerative
changes in the macula, all of which lead to a loss of central
vision. The macula is a small area in the retina of the eye,
approximately 3 to 5 millimeters in size, adjacent to the optic
nerve. It is the most sensitive area of the retina and contains the
fovea, a depressed region that allows for high visual acuity and
contains a dense concentration of cones, the photoreceptors that
are responsible for color vision. Age-related macular degeneration
(ARMD) is the most common cause of functional blindness in
developed countries for those over 50 years of age. The disease is
characterized by progressive degeneration of the retina, retinal
pigment epithelium (RPE), and underlying choroid (the highly
vascular tissue that lies beneath the RPE, between the retina and
the sclera). Cells in the RPE recycle visual pigment (rhodopsin),
phagocytose photoreceptor tips daily as part of rod and cone
regeneration, and transport fluid across the membrane to the
choroid. Central vision deteriorates when cells in the RPE cease to
function properly. Despite extensive investigation, the
pathogenesis of ARMD is not fully understood. Oxidative stress,
inflammation, genetic background, and environmental or behavioral
factors such as smoking and diet may contribute.
[0003] A clinical hallmark of ARMD is the appearance of drusen,
localized deposits of lipoproteinaceous material that accumulate in
the space between the RPE and Bruch's membrane. Drusen are
typically the earliest clinical finding in ARMD, and the existence,
location, and number of drusen are used in classifying the disease
into stages and monitoring progression (Ambati, J., et al., Surv.
Opthalmol., 48(3): 257-293, 2003; "Preferred Practice Pattern:
Age-Related Macular Degeneration", American Academy of
Opthalmology, 2003).
[0004] ARMD has been classified into "dry" and "wet" (exudative, or
neovascular) forms. Dry ARMD is much more common than wet, but the
dry form can progress to the wet form, and the two occur
simultaneously in a significant number of cases. Dry ARMD is
typically characterized by progressive apoptosis of cells in the
RPE, overlying photoreceptor cells, and frequently also the
underlying cells in the choroidal capillary layer. Confluent areas
(e.g., at least 175 .mu.m in minimum diameter) of RPE cell death
accompanied by overlying photoreceptor atrophy are referred to as
geographic atrophy. Patients with this form experience a slow and
progressive deterioration in central vision. Wet ARMD is
characterized by bleeding and/or leakage of fluid from abnormal
vessels that have grown from the choroidal vessels
(choriocapillaris) beneath the RPE and macula. This can be
responsible for sudden and disabling vision loss. Much of the
vision loss that patients experience is due to such choroidal
neovascularization (CNV) and its complications. A subtype of
neovascular ARMD in which angiomatous proliferation originates from
the retina and extends posteriorly into the subretinal space,
eventually communicating in some cases with choroidal new vessels
has been identified (Yannuzzi, L. A., et al., Retina, 21(5):416-34,
2001). This form of neovascular ARMD, termed retinal angiomatous
proliferation (RAP) can be particularly severe. The existence of
macular drusen is a strong risk factor for development of both wet
and dry forms of ARMD.
[0005] The panels of FIG. 1 show structures present in a normal eye
and some of the processes that occur in ARMD. FIGS. 1A and 1B show
structures present in the anterior and posterior segments of the
eye. FIGS. 1C-1E depict the outer layers of a normal eye (1C), an
eye suffering from dry ARMD (ID), and an eye suffering from wet
ARMD (1E). The outer nuclear layer (ONL) contains nuclei of rod and
cone photoreceptors. Each photoreceptor contains an inner segment
(IS) and outer segment (OS), the latter of which contains the
pigment rhodopsin, which initiates the phototransduction cascade
following exposure to light. The RPE lies below the photoreceptors
and above Bruch's membrane. As shown in FIGS. 1D and 1E, the normal
structure of the retina is disrupted in a variety of ways as in
patients with ARMD.
[0006] Macular edema is associated with a variety of eye disorders
including ARMD, diabetic retinopathy, inflammatory conditions such
as anterior or posterior uveitis, etc. The macula becomes thickened
as a result of the accumulation of fluid that leaks from weakened
or otherwise abnormal blood vessels into nearby tissues. Leakage of
blood or other fluids and the resulting increase in macular
thickness can lead to acute alterations in visual acuity, color
perception, etc. Thus macular edema can contribute to the visual
disturbances and loss experienced by individuals suffering from
ARMD and a variety of other eye disorders.
[0007] Development of pharmacological therapies for ARMD and other
ocular disorders associated with neovascularization in the eye is
an area of active investigation. Much effort has focused on methods
for destroying or sealing abnormal blood vessels and/or inhibiting
their development. Photodynamic therapy involves systemic
intravenous administration of a light-sensitive dye (verteporfin)
which is activated in the eye by a laser, resulting in formation of
toxic products within the abnormal blood vessels. Local
administration of angiogenesis inhibitors to the eye shows
considerable promise. Pegaptanib sodium (Macugen.RTM.;
Pfizer/Eyetech) was approved by the U.S. Food and Drug
Administration for treatment of wet age-related macular
degeneration in late 2004. Macugen is an aptamer that binds to an
isoform of vascular endothelial growth factor (VEGF), a protein
that acts as a signal in triggering the abnormal blood vessel
growth, increased permeability, and consequent leakage that
characterize wet ARMD. Binding of Macugen to VEGF prevents it from
binding to VEGF receptors, thereby inhibiting its activity. Other
angiogenesis inhibitors for the treatment of exudative ARMD include
monoclonal antibodies such as ranibizumab (Lucentis.RTM.;
Genentech) that bind to VEGF and block its interaction with VEGF
receptors.
[0008] Angiogenesis inhibitors that interfere with signal
transduction pathways that play a fundamental role in angiogenesis,
such as the VEGF pathway, offer a powerful approach to controlling
neovascularization. However, therapy with angiogenesis inhibitors
alone has a number of disadvantages. Clinical trials of
angiogenesis inhibitors that interfere with the VEGF pathway have
involved their administration in solution by intravitreal injection
at intervals of 4-6 weeks. Unfortunately this procedure is
associated with a significant risk of complications such as
traumatic lens injury, retinal detachment, and endophalmitis
associated with either trauma or intraocular infection. With an
overall risk of 1%, over the course of a year a dosing interval of
6 weeks would result in an overall risk of about 9% per eye, while
a dosing interval of 4 weeks would result in an overall risk of
about 13% per eye. For these and other reasons, current approaches
to the use of angiogenesis inhibitors remain a less than optimal
solution to treating wet ARMD. There remains a need in the art for
improved approaches to treating ARMD. There also remains a need for
improved approaches to treating other conditions characterized by
macular degeneration, choroidal neovascularization, retinal
neovascularization, retinal angiomatous proliferation, and/or blood
vessel leakage in the eye.
SUMMARY OF THE INVENTION
[0009] The present invention provides compositions, methods, and
articles of manufacture for the treatment of eye disorders,
particularly those associated with macular degeneration, CNV,
and/or retinal neovascularization (RNV). In one aspect, the
invention provides a method of treating an eye disorder
characterized by macular degeneration, CNV, or RNV, the method
comprising the step of: administering first and second therapeutic
agents to the subject's eye in a single procedure, wherein the
first therapeutic agent provides rapid improvement in the condition
of the subject's eye and the second therapeutic agent is
administered as a sustained release formulation of the second
therapeutic agent. In certain embodiments of the invention the
second therapeutic agent is a long-acting therapeutic agent. In
certain embodiments of the invention at least a portion of the
first therapeutic agent, optionally essentially the entire
administered dose of the first therapeutic agent, is provided as a
component of a sustained release formulation. The first and second
therapeutic agents may be provided as components of a single
sustained release formulation or as components of separate
sustained release formulations.
[0010] In certain embodiments of the invention the procedure is an
injection procedure, e.g., an intravitreal injection. In certain
embodiments the procedure is an injection procedure in which, prior
to administration, the first therapeutic agent is contained in a
syringe and the sustained release formulation comprising the second
therapeutic agent is contained in a needle attached to the syringe.
For example, the first therapeutic agent may be dissolved in a
liquid medium located in the syringe and the sustained formulation
of the second therapeutic agent may comprise an ocular implant
located in the needle.
[0011] In another aspect, the invention provides a method of
treating an eye disorder characterized by macular degeneration,
CNV, or RNV comprising the step of: administering first and second
compositions to a subject's eye in a single procedure, wherein the
first composition comprises a first therapeutic agent that provides
rapid improvement in the condition of the subject's eye and the
second composition comprises a second therapeutic agent that is
administered as a sustained release formulation comprising the
second therapeutic agent. Either or both of the compositions can
contain a plurality of therapeutic agents, e.g., two or more
angiogenesis inhibitors, two or more complement inhibitors, or an
angiogenesis inhibitor and a complement inhibitor.
[0012] In another aspect the invention provides a method of
administering first and second therapeutic agents to the eye of a
subject comprising: injecting (i) a solution containing the first
therapeutic agent and (ii) a solid ocular implant containing the
second therapeutic agent into the subject's eye in a single
injection procedure.
[0013] In any embodiment of the invention, either or both
therapeutic agents may be an angiogenesis inhibitor or a complement
inhibitor. In any embodiment of the invention the sustained release
formulation may comprise an ocular implant. In any embodiment of
the invention the sustained release formulation may comprise a
polymer and one or more therapeutic agents.
[0014] In other aspects, the invention provides articles of
manufacture. The invention provides an article of manufacture
comprising (i) a first therapeutic agent effective for treating an
eye disorder; and (ii) a needle containing a second therapeutic
agent. The article of manufacture may further comprise a syringe.
The syringe may contain a therapeutic agent.
[0015] In any embodiment of the present invention, the eye disorder
can be a macular degeneration related condition, diabetic
retinopathy, retinopathy of prematurity, or any condition featuring
CNV, RNV, or RAP.
[0016] In any embodiment of the invention that features a
complement inhibitor, the complement inhibitor can be any
complement inhibitor known in the art, e.g., a viral complement
control protein (VCCP) or fragment or variant thereof, a peptide or
peptide analog that binds to a complement component, an antagonist
of a complement receptor. The VCCP can be a poxvirus VCCP(PVCCP) or
a herpesvirus VCCP (HVCCP). The PVCCP can be from vaccinia virus,
variola virus, etc. The peptide or peptide analog can be, e.g.,
compstatin or a derivative thereof.
[0017] In any embodiment of the invention that features an
angiogenesis inhibitor, the angiogenesis inhibitor may be any
angiogenesis inhibitor known in the art. The angiogenesis inhibitor
may be selected from the group consisting of: Macugen.RTM.
(pegaptanib sodium) or another VEGF aptamer or nucleic acid ligand;
Lucentis.RTM. (ranibizumab), Avastin.RTM. (bevacizumb) or another
antibody or antibody fragment that specifically binds to VEGF;
combretastatin or a derivative or prodrug thereof such as
Combretastatin A4 Prodrug (CA4P); VEGF-Trap; EVIZON.TM. (squalamine
lactate); AG-013958 (Pfizer, Inc.); JSM6427 (Jerini A G),
.beta.2-glycoprotein 1 (.beta.2-GP1), and a short interfering RNA
(siRNA) or short hairpin RNA (shRNA) that inhibits expression of
one or more VEGF isoforms, inhibits expression of a VEGF receptor,
or inhibits expression of any other molecule whose expression in
the eye contributes to angiogenesis. In certain embodiments of the
invention the therapeutic agent is not a steroid.
[0018] Those of skill in the art will appreciate that certain
compounds encompassed by the structures herein may exhibit
tautomerism, conformational isomerism, geometric isomerism and/or
stereoisomerism. It should be understood that the invention
encompasses use of any tautomeric, conformational isomeric,
enantiomeric and/or geometric isomeric forms of the compounds
described herein. Any references herein employing nomenclature that
corresponds to illustrated structural formulae that represent only
one of several tautomeric forms (or resonance structures) are not
intended to limit the scope of the compounds described herein.
Those of skill in the art also will recognize that the compounds
disclosed as of use in the invention may exist in many different
protonation states, depending on, among other things, the pH of
their environment. Where structural formulae provided herein depict
the compounds in only one of several possible protonation states,
it will be understood that these structures are illustrative only,
and that the invention is not limited to any particular protonation
state--any and all protonated forms are intended to fall within the
scope of the invention.
[0019] Compounds of use in this invention may, in certain
embodiments, bear multiple positive or negative charges and may
have appropriate counter ions associated therewith. The identity of
the associated counter ions are may be governed by the synthesis
and/or isolation methods by which the compounds are obtained.
Counter ions include, but are not limited to, chloride and other
halides, acetate, trifluoroacetate, citrate, sulfate, phosphate,
etc., and mixtures thereof. It will be understood that the identity
of any associated counter ion is not a critical feature and that
the invention encompasses the compounds in association with any
type of counter ion. Moreover, as the compounds can exists in a
variety of different forms, the invention is intended to encompass
not only forms that are in association with counter ions (e.g., dry
salts), but also forms that are not in association with counter
ions (e.g., aqueous or organic solutions).
[0020] Unless otherwise stated or otherwise clearly evident from
the context, the invention makes use of standard methods of
molecular biology, cell culture, animal maintenance, opthalmologic
examination, and administration of therapeutic agents to subjects,
etc., and uses art-accepted meanings of terms. This application
refers to various patents and publications. The contents of all
articles, books, patents, patent applications, and other
publications mentioned in this application are incorporated herein
by reference. In addition, the following publications are
incorporated herein by reference: Current Protocols in Molecular
Biology, Current Protocols in Immunology, Current Protocols in
Protein Science, and Current Protocols in Cell Biology, all John
Wiley & Sons, N.Y., edition as of July 2002; Sambrook, Russell,
and Sambrook, Molecular Cloning: A Laboratory Manual, 3.sup.rd ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Kuby
Immunology, 4.sup.th ed., Goldsby, R. A., Kindt, T. J., and
Osborne, B. (eds.), W.H. Freeman, 2000, Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10.sup.th Ed. McGraw Hill,
2001, Katzung, B. (ed.) Basic and Clinical Pharmacology,
McGraw-Hill/Appleton & Lange; 9th edition (December 2003),
Ophthalmic Surgery: Principles and Practice, 3.sup.rd ed., W.B.
Saunders Company, 2002; Albert, D M and Lucarelli, M J (eds.),
Clinical Atlas of Procedures in Ophthalmic Surgery, American
Medical Association, 2003. In the event of a conflict or
inconsistency between any of the incorporated references and the
instant specification, the specification shall control, it being
understood that the determination of whether a conflict or
inconsistency exists is within the discretion of the inventors and
can be made at any time.
BRIEF DESCRIPTION OF THE DRAWING
[0021] FIGS. 1A-1E show schematic representations of the anterior
and posterior segments of the eye (1A and 1B) and the outer layers
of the eye (1C-1E). FIG. 1C depicts a normal eye. FIG. 1D depicts
an eye suffering from dry ARMD. FIG. 1E depicts an eye suffering
from exudative ARMD. ONL=outer nuclear layer; IS inner segment;
OS=outer segment; RPE=retinal pigment epithelial layer; BM=Bruch's
membrane; CC=choriocapillaris. From Tezel, T., et al., Trends in
Molecular Medicine, 10(9), 417-420, 2004.
[0022] FIG. 2 shows a consensus sequence for a short consensus
repeat (SCR), a module found in complement control proteins. From
Smith, S A, et al., J. Virol 74(12), 5659-5666, 2000.
[0023] FIGS. 3A and 3B show sequences of vaccinia virus complement
control protein precursor (SEQ ID NO: 33) and the mature vaccinia
virus complement control protein (SEQ ID NO: 34).
[0024] FIG. 4 shows a sequence comparison of mature complement
control proteins from a variety of orthopoxvirus isolates (SEQ ID
NO: 35-42). The corresponding genetic loci are listed. Modified
from Smith, S A, et al., J. Virol. 74(12), 5659-5666, 2000.
[0025] FIG. 5 shows a comparison of the SCR domain structure of a
number of complement control proteins and fragments thereof, the
number of K+R residues, % K+R residues, pI, number of putative
heparin binding sites, and ability to inhibit hemolysis and/or bind
to heparin. Modified from Smith, S A, et al., J. Virol. 74(12),
5659-5666, 2000. The domains are SCR modules. Thus, for example,
rVCP SCR (2, 3, 4), is a recombinantly produced polypeptide
containing SCRs 2, 3, and 4 from VCP.
[0026] FIG. 6 shows the amino acid sequence of SPICE (SEQ ID NO:
44).
[0027] FIG. 7 shows the structure of compstatin and the structure
of a compstatin analog showing increased complement inhibiting
activity relative to compstatin. The figure also shows the IC50 of
compstatin and the compstatin analog for inhibition of human
complement. Amino acids 4 and 9 in the peptide chain depicted in
the upper portion of the figure are as shown on the lower left for
compstatin and as shown on the lower right for the compstatin
analog. Thus the boxes labeled "X4" and "X9" in the peptide chain
represent the side chains of the amino acids X4 and X9 shown in the
lower portion of the figure for compstatin (left) and the
compstatin analog (right) respectively.
[0028] FIG. 8 shows an exemplary compound for use in the
invention.
[0029] FIG. 9 shows a needle/syringe assembly loaded with first and
second therapeutic agents.
DEFINITIONS
[0030] "Activity period" refers to the time period over which a
subject experiences an improvement in one or more symptoms and/or
signs of a disorder following administration of a therapeutic
agent, relative to a baseline condition or state existing prior to
administration of the therapeutic agent. The activity period begins
when the subject first experiences improvement and ends when the
subject's condition or state returns to a baseline that existed
prior to administration of the agent.
[0031] "Angiogenesis" or "angiogenic" refer to formation, growth,
and/or development of new blood vessels.
[0032] The terms "angiogenesis inhibitor" and "antiangiogenic
agent" are used interchangeably herein to refer to agents that are
capable of inhibiting or reducing one or more processes associated
with angiogenesis including, but not limited to, endothelial cell
proliferation, endothelial cell survival, endothelial cell
migration, differentiation of precursor cells into endothelial
cells, and capillary tube formation.
[0033] "Antibody", as used herein, refers to an immunoglobulin or
portion thereof that binds to an antigen. An antibody may be
natural or wholly or partially synthetically produced. An antibody
may be derived from natural sources, e.g., purified from an animal
such as a rodent, rabbit, or chicken, that has been immunized with
an antigen or a construct that encodes the antigen. An antibody may
be a member of any immunoglobulin class, including any of the human
classes: IgG, IgM, IgA, IgD, and IgE. An antibody of use in this
invention may be an antibody fragment such as an Fab',
F(ab').sub.2, scFv (single-chain variable) or other fragment that
retains an antigen binding site, or a recombinantly produced scFv
fragment, including recombinantly produced fragments that comprise
an immunoglobulin antigen binding domain. See, e.g., Allen, T.,
Nature Reviews Cancer, Vol. 2, 750-765, 2002, and references
therein. Antibody fragments which contain the idiotype of the
antibody molecule can be generated by known techniques. For
example, F(ab').sub.2 fragments can be produced by pepsin digestion
of the antibody molecule, Fab' fragments can be produced by
reducing the disulfide bridges of the F(ab').sub.2 fragment, or by
treating the antibody molecule with papain and a reducing agent. An
antibody can be an antibody multimer or a multimer of antibody
fragments. Antibodies, antibody fragments, and/or protein domains
comprising an antigen binding site may be generated and/or selected
in vitro, e.g., using techniques such as phage display (Winter, G.
et al., Annu. Rev. Immunol. 12:433-455, 1994), ribosome display
(Hanes, J., and Pluckthun, A. Proc. Natl. Acad. Sci. USA.
94:4937-4942, 1997), etc.
[0034] An antibody may be polyclonal (e.g., an affinity-purified
polyclonal antibody) or monoclonal. A "monoclonal antibody" as used
herein refers to a population of substantially homogeneous
antibodies or a member of such a population, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that can be present in minor
amounts. In contrast to polyclonal antibody preparations that
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen and is therefore highly
specific. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
monoclonal antibodies to be used in accordance with the present
invention can be made by the hybridoma method first described by
Kohler & Milstein, Nature 256: 495, 1975, or alternatively can
be made by recombinant DNA methods (see e.g., U.S. Pat. No.
4,816,567).
[0035] An antibody may be a "chimeric" antibody in which for
example, a variable domain of rodent origin is fused to a constant
domain of human origin, thus retaining the specificity of the
rodent antibody. The domain of human origin need not originate
directly from a human in the sense that it is first synthesized in
a human being. Instead, "human" domains may be generated in rodents
whose genome incorporates human immunoglobulin genes. Such an
antibody is considered at least partially "humanized". The degree
to which an antibody is "humanized" can vary. Thus part or most of
the variable domain of a rodent antibody may be replaced by human
sequences, e.g., by site-directed mutagenesis of a polynucleotide
that encodes the antibody or a portion thereof. According to one
approach rodent, e.g., murine, complementarity-determining regions
(CDRs) are grafted onto the variable light (VL) and variable heavy
(VH) frameworks of human immunoglobulin molecules, while retaining
only those rodent framework residues deemed essential for the
integrity of the antigen-binding site. See Gonzales N R, Tumour
Biol. January-February;26(1):31-43, 2005 for a review of various
methods of minimizing antigenicity of a monoclonal antibody. Such
human or humanized chimeric antibodies are often preferred for use
in therapy of human diseases or disorders, since the human or
humanized antibodies are less likely than to induce an immune
response.
[0036] A variety of methods are known for determining whether or
not an antibody reacts with, or specifically binds to, an antigen
such and for determining the affinity of such binding if desired.
Examples include enzyme-linked immunosorbent assays (ELISA),
radioimmunoassays (RIA), and the like. Binding of an antibody to a
target molecule such as a protein may inhibit or interfere with the
activity of the target molecule. For example, binding of an
antibody to ligand such as a growth factor may interfere with the
binding of the ligand to its receptor(s); binding of an antibody to
a receptor may interfere with the binding of the receptor to its
ligand(s).
[0037] The terms "approximately" or "about" in reference to a
number include numbers that fall within a range of 5% in either
direction (greater than or less than) of the number unless
otherwise stated or otherwise evident from the context (except
where such number would exceed 100% of a possible value).
[0038] "Biocompatible" refers to a material that is substantially
nontoxic to a recipient's cells in the quantities and at the
location used, and does not elicit or cause a significant
deleterious or untoward effect on the recipient's body at the
location used, e.g., an unacceptable immunological or inflammatory
reaction, unacceptable scar tissue formation, etc. A material that
is biocompatible with the eye does not substantially interfere with
the physiology or function of the eye.
[0039] "Biodegradable" means that a material is capable of being
broken down physically and/or chemically within cells or within the
body of a subject, e.g., by hydrolysis under physiological
conditions and/or by natural biological processes such as the
action of enzymes present within cells or within the body, and/or
by processes such as dissolution, dispersion, etc., to form smaller
chemical species which can typically be metabolized and,
optionally, used by the body, and/or excreted or otherwise disposed
of. Preferably a biodegradable compound is biocompatible. A polymer
whose molecular weight decreases over time in vivo due to a
reduction in the number of monomers is considered
biodegradable.
[0040] A "biological macromolecule" is a large molecule composed of
smaller subunits of a type that are found in biological systems.
Examples include polypeptides, nucleic acids, and polysaccharides.
Typically a biological macromolecule contains at least 3 subunits
(e.g., amino acids, nucleosides, monosaccharides, etc.). The
biological macromolecule may be a naturally occurring polypeptide,
nucleic acid, or polysaccharide. The biological macromolecule may
be modified, e.g., it may be conjugated to a nonbiological molecule
such as synthetic polymer, etc.
[0041] The phrases "characterized by macular degeneration,
choroidal neovascularization, retinal neovascularization, or any
combination of the foregoing" and "characterized by macular
degeneration, choroidal neovascularization, or retinal
neovascularization" are intended to indicate that macular
degeneration, CNV, and/or RNV, is a characteristic (i.e., typical)
feature of the disorder. Macular degeneration, CNV, and/or RNV may
be a defining and/or diagnostic feature of the disorder.
[0042] "Choroidal neovascularization" (CNV) refers to the abnormal
development, proliferation, and/or growth of blood vessels arising
from the choriocapillaris. The blood vessels typically extend
through Bruch's membrane, RPE layer, and/or subretinal space.
[0043] A "complement component" or "complement protein" is a
molecule that is involved in activation of the complement system or
participates in one or more complement-mediated activities.
Components of the classical complement pathway include, e.g., C1q,
C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9, and the C5b-9 complex,
also referred to as the membrane attack complex (MAC) and active
fragments or enzymatic cleavage products of any of the foregoing
(e.g., C3a, C3b, C4a, C4b, C5a, etc.). Components of the
alternative pathway include, e.g., factors B, D, H, and I, and
properdin.
[0044] The terms "deliver" or "delivery", in the context of a drug
delivery device or sustained release formulation refers to release
of a therapeutic agent into its surrounding environment in the
body.
[0045] A "drug delivery device" refers to a device, structure, or
element that contains and/or delivers a therapeutic agent to a
subject. Release of the drug may, but need not, occur as a result
of degradation of the drug delivery device within the body. The
term "drug delivery device" is used herein to refer to devices that
contain a therapeutic agent and to devices that have not yet been
loaded with the therapeutic agent. An ocular implant is a drug
delivery device that has appropriate dimensions and structure for
placement within the eye. Preferably an ocular drug delivery device
does not substantially interfere with the physiology and/or
functioning of the eye, e.g., the device causes minimal or no
disruption of vision.
[0046] An "effective amount" of an active agent refers to the
amount of the active agent sufficient to elicit a desired
biological response. As will be appreciated by those of ordinary
skill in this art, the absolute amount of a particular agent that
is effective may vary depending on such factors as the desired
biological endpoint, the agent to be delivered, the target tissue,
etc. Those of ordinary skill in the art will further understand
that an "effective amount" may be administered in a single dose, or
may be achieved by administration of multiple doses. For example,
an effective amount of a therapeutic agent for the treatment of an
eye disorder may be an amount sufficient to treat the disorder,
e.g., an amount sufficient to achieve one or more of the following:
(i) inhibit or prevent drusen formation; (ii) cause a reduction in
drusen number and/or size (drusen regression); (iii) cause a
reduction in or prevent lipofuscin deposits; (iv) inhibit or
prevent visual loss or slow the rate of visual loss; (v) inhibit
choroidal neovascularization or slow the rate of choroidal
neovascularization; (vi) cause a reduction in size and/or number of
lesions characterized by choroidal neovascularization; (vii)
inhibit choroidal neovascularization or slow the rate of retinal
neovascularization; (viii) cause a reduction in size and/or number
of lesions characterized by retinal neovascularization; (ix)
improve visual acuity and/or contrast sensitivity; (x) reduce
macular edema and/or reduce abnormal macular thickness; (xi)
inhibit or prevent photoreceptor or RPE cell atrophy or apoptosis,
or reduce the rate of photoreceptor or RPE cell atrophy or
apoptosis; (xii) inhibit or prevent progression of non-exudative
macular degeneration to exudative macular degeneration.
[0047] "Eye disorder", which is used interchangeably herein with
"ocular disorder" refers to any disease, disorder, or condition
that involves or affects the eye or one or more portions,
structures, or parts of the eye. The eye includes the eyeball, the
periocular muscles, and the portion of the optic nerve which is
within or adjacent to the eyeball.
[0048] "Exudative" macular degeneration is used herein synonymously
with "wet" type macular degeneration, as those terms are generally
understood in the art, i.e., to refer to a macular degeneration
related condition such as ARMD characterized by neovascularization
and/or the presence of an exudate.
[0049] "Identity" refers to the extent to which the sequence of two
or more nucleic acids or polypeptides is the same. The percent
identity between a sequence of interest and a second sequence over
a window of evaluation, e.g., over the length of the sequence of
interest, may be computed by aligning the sequences, determining
the number of residues (nucleotides or amino acids) within the
window of evaluation that are opposite an identical residue
allowing the introduction of gaps to maximize identity, dividing by
the total number of residues of the sequence of interest or the
second sequence (whichever is greater) that fall within the window,
and multiplying by 100. By gap is meant a portion of a sequence
that is not occupied by a residue. For example, the sequence A K L-
- -S I G (SEQ ID NO: 43) contains a gap of three residues. When
computing the number of identical residues needed to achieve a
particular percent identity, fractions are to be rounded to the
nearest whole number. Percent identity can be calculated with the
use of a variety of computer programs known in the art. For
example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped
BLAST, etc., generate alignments and provide percent identity
between a sequence of interest and sequences in any of a variety of
public databases. The algorithm of Karlin and Altschul (Karlin and
Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990) modified
as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877,
1993 is incorporated into the NBLAST and XBLAST programs of
Altschul et al. (Altschul, et al., J. Mol. Biol. 215:403-410,
1990). To obtain gapped alignments for comparison purposes, Gapped
BLAST is utilized as described in Altschul et al. (Altschul, et al.
Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and
Gapped BLAST programs, the default parameters of the respective
programs are used. A PAM250 or BLOSUM62 matrix may be used. See the
Web site having URL www.ncbi.nlm.nih.gov for these programs. In a
specific embodiment, percent identity of a sequence of interest and
a second sequence is calculated using BLAST2 with default
parameters.
[0050] "Invasive therapy" as used herein, is therapy that involves
insertion of an instrument or device into the eye or orbit, e.g,
entrance into or penetration of the eyeball or entry into the orbit
by an instrument such as a needle, trocar, catheter, or the
like.
[0051] "Liposomes" are artificial microscopic spherical particles
formed by a lipid bilayer (or multilayers) enclosing an aqueous
compartment. Liposomes can be used for delivering certain of the
compositions of the invention.
[0052] The term "long-acting therapeutic agent" refers to a
therapeutic agent that has an activity period of at least 3 months
when administered in medically acceptable quantities. A "medically
acceptable quantity" refers to an amount that does not cause
unacceptable toxicity or adverse effects under the conditions of
administration.
[0053] "Macular degeneration related condition" refers to any of a
number of disorders and conditions in which the macula degenerates
or loses functional activity. The degeneration or loss of
functional activity can arise as a result of, for example, cell
death, decreased cell proliferation, and/or loss of normal
biological function. Macular degeneration can lead to and/or
manifest as alterations in the structural integrity of the cells
and/or extracellular matrix of the macula, alteration in normal
cellular and/or extracellular matrix architecture, and/or the loss
of function of macular cells. The cells can be any cell type
normally present in or near the macula including RPE cells,
photoreceptors, and/or capillary endothelial cells. ARMD is the
major macular degeneration related condition. Others include Best
macular dystrophy, Sorsby fundus dystrophy, Mallatia Leventinese
and Doyne honeycomb retinal dystrophy.
[0054] "Non-exudative" macular degeneration is used herein
synonymously with "dry" type macular degeneration as those terms
are generally used in the art, to refer to a macular degeneration
related condition, e.g., ARMD, in which neovascularization and/or
exudation that would be detectable using standard methods such as
fluorescein angiography has not occurred.
[0055] "Ocular implant" refers to a device or structure that has
appropriate dimensions, shape, and/or configuration and is made of
appropriate materials so that it may be placed in the eye without
causing unacceptable interference with the physiology or
functioning of the eye. Preferably placement of an ocular implant
does not significantly disrupt vision. An ocular implant is
typically a solid or semi-solid article of manufacture and is
typically macroscopic, i.e., visible with the naked eye.
[0056] "Plurality" means more than one.
[0057] "Polypeptide", as used herein, refers to a polymer of amino
acids, optionally including one or more amino acid analogs. A
protein is a molecule composed of one or more polypeptides. A
peptide is a relatively short polypeptide, typically between about
2 and 60 amino acids in length. The terms "protein", "polypeptide",
and "peptide" may be used interchangeably. Polypeptides used herein
may contain amino acids such as those that are naturally found in
proteins, amino acids that are not naturally found in proteins,
and/or amino acid analogs that are not amino acids. A large number
of art-recognized analogs of the 20 amino acids commonly found in
proteins (the "standard" amino acids) are known. As used herein, an
"analog" of an amino acid may be a different amino acid that
structurally resembles the amino acid (a "non-standard" amino acid)
or a compound other than an amino acid that structurally resembles
the amino acid. One or more of the amino acids in a polypeptide may
be modified, for example, by the addition of a chemical entity such
as a carbohydrate group, a phosphate group, a farnesyl group, an
isofarnesyl group, a fatty acid group, a linker for conjugation,
functionalization, or other modification, etc. Certain non-limiting
suitable analogs and modifications are described in WO2004026328.
The polypeptide may be acetylated, e.g., at the N-terminus and/or
amidated, e.g., at the C-terminus.
[0058] The natural or other chemical modifications such as those
described above can occur anywhere in a polypeptide, including the
peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. A given polypeptide may contain many types of
modifications. Polypeptides may be branched or they may be cyclic,
with or without branching. Polypeptides may be conjugated with,
encapsulated by, or embedded within a polymer or polymeric matrix,
dendrimer, nanoparticle, microparticle, liposome, or the like.
Polypeptides of use in this invention may, for example, be purified
from natural sources, produced in vitro or in vivo in suitable
expression systems using recombinant DNA technology (e.g., by
recombinant host cells or in transgenic animals or plants),
synthesized through chemical means such as conventional solid phase
peptide synthesis and/or methods involving chemical ligation of
synthesized peptides (see, e.g., Kent, S., J Pept Sci.,
9(9):574-93, 2003 and U.S. Pub. No. 20040115774), or any
combination of these. The term "polypeptide sequence" or "amino
acid sequence" as used herein can refer to the polypeptide material
itself and is not restricted to the sequence information (i.e. the
succession of letters or three letter codes chosen among the
letters and codes used as abbreviations for amino acid names) that
biochemically characterizes a polypeptide. A polypeptide sequence
presented herein is presented in an N-terminal to C-terminal
direction unless otherwise indicated.
[0059] "Poxvirus" refers to a family of complex, double-stranded
DNA viruses constituting the family Poxyiridae. The family includes
the orthopoxviruses, a genus of the family Poxviridae, subfamily
Chordopoxyirinae, comprising many species infecting mammals.
Poxviruses are described in Fields, B N, et al., Fields Virology,
3.sup.rd ed., Lippincott Williams & Wilkins, 2001.
Orthopoxviruses include vaccinia virus, variola virus major,
variola virus minor, cowpox virus, monkeypox virus, camelpox virus,
swinepox virus, and ectromelia virus.
[0060] "Poxvirus complement control protein" refers to members of a
family of homologous proteins encoded by a number of different
poxviruses that bind to one or more complement pathway proteins and
inhibit the classical pathway of complement activation, the
alternative pathway of complement activation, the lectin pathway,
or any combination of these. Poxvirus complement control proteins
are members of the complement control protein (CCP), also called
regulators of complement activation (RCA) superfamily (Reid, K B M
and Day, A J, Immunol Today, 10:177-80, 1989).
[0061] "Posterior segment of the eye" refers to the portion of the
eye behind the lens, including the vitreous, choroid, and retina
(including the macula).
[0062] "Rapid improvement in the condition of a subject's eye"
refers to a clinically significant improvement in one or more
symptoms and/or signs of an ocular disorder that occurs within two
weeks, or preferably within one week, following administration of a
therapeutic agent. Rapid improvement in the condition of a
subject's eye can include, without limitation, any one or more of
the following: increased visual acuity (e.g., gaining two or more
lines of vision on a visual acuity chart), decreased visual
distortion, increased contrast sensitivity, decreased retinal
vessel leakage, decreased macular thickness (e.g., a decrease in
macular thickness of at least 50% from a baseline value). In
general, "rapid" as used herein in reference to a therapeutic or
other biological effect, means occurring within two weeks or less
following a reference event (e.g., administration of a therapeutic
agent). In some embodiments, "rapid" means occurring within one
week or less following a reference event.
[0063] "Retinal neovascularization" refers to the abnormal
development, proliferation, and/or growth of blood vessels on or in
the retina, e.g., on the retinal surface (i.e., as will be evident
to one of ordinary skill in the art, the abnormal proliferation,
and/or growth originates from blood vessels already present on or
in the surface). "Retinal" here refers to the source of the
neovascularization.
[0064] "Single procedure" means a procedure, i.e., a process or
series of steps or acts that involves a single entrance into or
penetration of the eyeball or entry into the orbit by an instrument
such as a needle, trocar, catheter, or the like. For example, an
eye injection, e.g., an intravitreal injection, is a single
procedure provided that the tip of the needle, once having been
inserted into the eyeball, is not reintroduced into the eyeball
once having been withdrawn therefrom. A single procedure may or may
not involve multiple penetrations of one or more structures of the
eye, e.g., the vitreous, provided that only a single entrance into
or penetration of the eyeball takes place.
[0065] "Small molecule" refers to organic compounds, whether
naturally-occurring or artificially created (e.g., via chemical
synthesis) that have relatively low molecular weight and that are
not proteins, polypeptides, or nucleic acids. Typically, small
molecules have a molecular weight of less than about 1500 g/mot and
multiple carbon-carbon bonds.
[0066] "Specific binding" generally refers to a physical
association between a target polypeptide (or, more generally, a
target molecule) and a binding molecule such as an antibody or
ligand. The association is typically dependent upon the presence of
a particular structural feature of the target such as an antigenic
determinant or epitope recognized by the binding molecule. For
example, if an antibody is specific for epitope A, the presence of
a polypeptide containing epitope A or the presence of free
unlabeled A in a reaction containing both free labeled A and the
binding molecule that binds thereto, will reduce the amount of
labeled A that binds to the binding molecule. It is to be
understood that specificity need not be absolute but generally
relates to the context in which the binding occurs. For example, it
is well known in the art that numerous antibodies cross-react with
other epitopes in addition to those present in the target molecule.
Such cross-reactivity may be acceptable depending upon the
application for which the antibody is to be used. One of ordinary
skill in the art will be able to select antibodies or ligands
having a sufficient degree of specificity to perform appropriately
in any given application (e.g., for detection of a target molecule,
for therapeutic purposes, etc). It is also to be understood that
specificity may be evaluated in the context of additional factors
such as the affinity of the binding molecule for the target versus
the affinity of the binding molecule for other targets, e.g.,
competitors. If a binding molecule exhibits a high affinity for a
target molecule that it is desired to detect and low affinity for
nontarget molecules, the antibody will likely be an acceptable
reagent. Once the specificity of a binding molecule is established
in one or more contexts, it may be employed in other, preferably
similar, contexts without necessarily re-evaluating its
specificity. Binding of two or more molecules may be considered
specific if the affinity (equilibrium dissociation constant, Kd) is
10.sup.-3 M or less, preferably 10.sup.-4 M or less, more
preferably 10.sup.-5 M or less, e.g., 10.sup.-6 M or less,
10.sup.-7 M or less, 10.sup.-8 M or less, or 10.sup.-9 M or less
under the conditions tested, e.g., under physiological
conditions.
[0067] "Stabilize", as used herein in reference to a eye disorder,
means to reduce the rate of progression of the disorder and/or to
prevent or reduce the likelihood of a rapid and noticeable
deterioration in the condition of an eye afflicted with the
disorder.
[0068] "Subject" as used herein, refers to an individual to whom an
agent is to be delivered, e.g., for experimental, diagnostic,
and/or therapeutic purposes. Preferred subjects are mammals, e.g.,
primates, or humans. A subject under the care of a physician or
other health care provider may be referred to as a "patient".
[0069] "Substantial sequence homology" as applied to a sequence
means that the sequence displays at least approximately 60%
identity, desirably at least approximately 70% identity, more
desirably at least approximately 80% identity, and most desirably
at least approximately 90% identity relative to a reference
sequence. When two or more sequences are compared, any of them may
be considered the reference sequence. % identity can be calculated
using a FASTA, BLASTN, or BLASTP algorithm. Default parameters may
be used. A PAM250 or BLOSUM62 matrix may be used.
[0070] A "sustained release formulation" or "sustained delivery
formulation" is a composition of matter that comprises a
therapeutic agent as one of its components and further comprises or
has one or more components, elements, or structures effective to
provide sustained release of the therapeutic agent, optionally in
part as a consequence of the physical structure of the formulation.
In some embodiments the structure is provided at least in part by
the therapeutic agent itself and, optionally, one or more
substances present at the site of administration. Sustained release
is release or delivery that occurs either continuously or
intermittently over a period of time e.g., at least 1, 2, 4, or 6
weeks, at least 1, 2, 3, 4, 6, 8, 10, 12, 15, 18, or 24 months, or
longer.
[0071] "Therapeutic agent" is used interchangeably herein with
"drug", to refer to any pharmaceutically active agent useful for
treating a disorder. The term includes any pharmaceutically
acceptable salt, prodrug, salt of a prodrug, and such derivatives
of an active agent as are known in the art or readily produced
using standard methods known in the art "Prodrug" refers to a
precursor of a drug, wherein the prodrug is not itself
pharmacologically active (or has a lesser or different activity
than the desired activity of the drug) but is converted, following
administration (e.g., by metabolism) into the pharmaceutically
active drug. A therapeutic agent can be, without limitation, a
small molecule or a biological macromolecule such as a protein
(e.g., an antibody) or nucleic acid such as an aptamer, siRNA,
etc.
[0072] "Treating", as used herein, refers to providing treatment,
i.e, providing any type of medical or surgical management of a
subject in order to reverse, alleviate, inhibit the progression of,
prevent or reduce the likelihood of a disease, disorder, or
condition, or in order to reverse, alleviate, inhibit or prevent
the progression of, prevent or reduce the likelihood of one or more
symptoms or manifestations of a disease, disorder or condition.
"Prevent" refers to causing a disease, disorder, condition, or
symptom or manifestation of such not to occur. Treating can include
administering an agent to the subject following the development of
one or more symptoms or manifestations indicative of a condition
such as macular degeneration or diabetic retinopathy, e.g., in
order to reverse, alleviate, reduce the severity of, and/or inhibit
or prevent the progression of the condition and/or to reverse,
alleviate, reduce the severity of, and/or inhibit or one or more
symptoms or manifestations of the condition. A composition of this
invention can be administered to a subject who has developed an eye
disorder such as exudative or non-exudative ARMD or diabetic
retinopathy or is at increased risk of developing such a disorder
relative to a member of the general population. A composition of
this invention can be administered prophylactically, i.e., before
development of any symptom or manifestation of the condition.
Typically in this case the subject will be at risk of developing
the condition.
[0073] "Unit dosage form" as used herein refers to physically
discrete units suited as unitary dosages for the subject to be
treated (e.g., for a single eye); each unit containing a
predetermined quantity of an active agent selected to produce the
desired therapeutic effect, optionally together with a
pharmaceutically acceptable carrier, which may be provided in a
predetermined amount. The unit dosage form may be, for example, a
volume of liquid (e.g., a pharmaceutically acceptable carrier)
containing a predetermined quantity of a therapeutic agent, a
predetermined amount of a therapeutic agent in solid form, an
ocular implant containing a predetermined amount of a therapeutic
agent, a plurality of nanoparticles or microparticles that
collectively contain a predetermined amount of a therapeutic agent,
etc. It will be appreciated that a unit dosage form may contain a
variety of components in addition to the therapeutic agent. For
example, pharmaceutically acceptable carriers, diluents,
stabilizers, buffers, preservatives, etc., may be included.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
I. Overview
[0074] The present invention provides compositions, methods,
articles of manufacture, and pharmaceutical packs or kits for the
treatment of an eye disorder. In certain embodiments of the
invention the eye disorder is characterized by macular
degeneration, CNV, or RNV. Exemplary disorders that can be treated
according to the invention include, but are not limited to, macular
degeneration related conditions, diabetic retinopathy, and
retinopathy of prematurity. While the concepts underlying the
invention are described herein with particular reference to
treatment of wet ARMD or other conditions characterized by CNV
and/or RNV, they apply to a range of different ocular (and other)
disorders.
[0075] The invention encompasses the recognition that eye care
providers, e.g., ophthalmologists, are often reluctant to
administer a therapeutic agent, e.g., an angiogenesis inhibitor,
using an invasive procedure such as intravitreal injection that is
associated with the risk of a severe complication unless there is a
significant likelihood that the therapy will cause rapid
improvement in the condition of the patient's eye. There can be
reluctance to use an invasive procedure to administer therapy that
may possibly prevent or delay future deterioration or
destabilization in an eye that is, at least from a symptomatic
standpoint, relatively stable. Similarly, patients are often
reluctant to undergo administration of a therapeutic agent using an
invasive procedure associated with the risk of a severe
complication unless they have recently experienced noticeable
deterioration or destabilization in the condition of their eye(s)
and there is a significant likelihood that administration of the
therapeutic agent will result in rapid and/or noticeable
improvement in the condition. Patients with an eye in a relatively
stable condition are often reluctant to undergo an invasive
procedure to administer a therapeutic agent that may halt or slow
progress of the disorder if the procedure is associated with the
risk of a severe complication.
[0076] As a consequence, when administration of a therapeutic agent
involves an invasive procedure associated with a risk of a severe
complication, patients may be treated on a symptom driven,
case-by-case basis, rather than according to a predetermined,
recommended dosing schedule that would at least in part involve
administering the agent while the patient's condition is apparently
stable, at least from a symptomatic standpoint. This appears to be
the case even though following the predetermined, recommended
dosing schedule may have the potential to delay or inhibit future
destabilization or deterioration. It was observed that in one well
known eye clinic, an angiogenesis inhibitor was administered to
patients with exudative ARMD on a symptom driven basis, in response
to a sudden deterioration or destabilization in the condition of a
patient's eye or in response to the presence of exudation (e.g.,
hemorrhage), rather than according to a predetermined dosing
schedule.
[0077] Clinical trials have demonstrated that certain angiogenesis
inhibitors, e.g., Macugen and Lucentis, are of benefit in terms of
important parameters such as visual acuity when administered as
recommended, i.e., by repeated intravitreal administration of a
solution containing the agent. For example, administration of
certain angiogenesis inhibitors slows the rate of visual loss and
may lead to at least temporary improvement in visual acuity. Based
on clinical trials, the recommended dosing interval for Lucentis is
4 weeks (see, e.g., Heier, J. S., et al., Invest Opthalmol V is
Sci, 44:e-abstract 972, 2003), while the recommended dosing
interval for Macugen is 6 weeks (see, e.g., Gragoudas E S, N Engl
J. Med., 351(27):2805-16, 2004). Avastin, while currently not
approved for treatment of eye disorders by the U.S. Food and Drug
Administration has been approved for the treatment of certain
cancers and is available for use in the eye. Since Lucentis and
Avastin act in a similar manner by binding to VEGF isoforms, these
agents may have similar therapeutic effects.
[0078] Repeated administration of angiogenesis inhibitors according
to the dosing intervals described above could result in sustained
improvement in the condition of the subject's eye by inhibiting
further neovascularization and blood vessel leakage. However, given
the risk associated with intravitreal injection, ophthalmologists
appear reluctant to administer a therapy associated with
significant risk while the patient's symptoms remain substantially
stable. Similarly, patients appear reluctant to submit to a
procedure with significant risk when their symptoms remain
substantially stable.
[0079] Therefore, as a result of the desire to avoid intravitreal
injections, angiogenesis inhibitors may not be administered
according to the recommended or predetermined dosing intervals.
Instead, in practice these agents may be administered on a
symptomatic basis, e.g., after a subject has experienced a
deterioration or destabilization in the condition of the eye such
as an acute loss of visual acuity and/or presence of exudation
relative to a baseline condition, e.g., relative to the improved
condition that resulted following administration of the previous
dose of the angiogenesis inhibitor. Such deterioration or
destabilization can occur at a variable and unpredictable time
following the initial improvement. Instead of retreating the
patient with an angiogenesis inhibitor when the patient is
symptomatically stable in an effort to prevent future deterioration
or destabilization, at the possible risk of causing a severe
complication, treatment may be postponed until the subject has
actually experienced deterioration or destabilization such that
treatment would be likely to result in a symptomatic improvement in
addition to any possible preventive effect. Thus the desire to
avoid intravitreal injection of an eye that is symptomatically
stable has a significant and heretofore unappreciated effect on
clinical practice. In essence, the risk/benefit ratio as perceived
by ophthalmologists and patients may dictate that intravitreal
administration of angiogenesis inhibitors should be performed on an
individualized basis, following deterioration or destabilization of
a patient's eye, rather than according to a predetermined dosing
interval of approximately 4 or 6 weeks. It is unclear whether
treatment on a symptomatic basis will have a greater, lesser, or
equivalent efficacy on a long-term basis (e.g., over periods of a
year or more) relative to treatment according to the recommended,
predetermined dosing schedule. However, in view of the definite and
known risk of complications associated with intravitreal injection,
ophthalmologists and patients may be willing to forego the possible
long-term increased benefit that could result from administering
anti-angiogenic therapy at predetermined intervals of 4-6 weeks
instead of on a symptomatic basis.
[0080] In summary, the inventors' observations suggest that
ophthalmologists and patients are reluctant to accept the risks
associated with intravitreal injection unless there is a
significant likelihood that the therapy thus administered will
result in a rapid improvement in the condition of the patient's
eye. However, this mode of administration may not be optimal in
terms of providing long term improvement in and/or stabilization of
the condition of the subject's eye.
[0081] The present invention encompasses the recognition that
symptom driven treatment may be less than optimal for preventing or
delaying further deterioration in the patient's condition.
Furthermore, a formulation of a therapeutic agent that is preferred
or optimal for producing rapid improvement in the condition of a
patient's eye may not be preferred or optimal for achieving
long-term improvement and/or stabilization. For example, in the
case of eye disorders, rapid improvement may best be achieved by
locally administering a therapeutic agent in solution or in a form
in which it is quickly released, so as to rapidly achieve a high
concentration of the agent at the location where activity is
desired. However, long-term benefit may best be achieved by locally
administering a sustained release formulation of the therapeutic
agent that provides a lower, yet still therapeutically effective
concentration of the therapeutic agent. Alternately or
additionally, therapeutic agent(s) that are most appropriate for
relieving particular symptoms may be less appropriate for providing
long-term benefit.
[0082] "Administering" or "administration" generally refers to
introducing a therapeutic agent, composition, formulation, etc., to
a desired site or location on or within the body of a subject,
e.g., a site or location within the eye. Administration may be
performed, e.g., by a health care provider. For purposes of
convenience, the present specification refers generally
toophthalmologists. However, the methods described herein,
including both the methods of the invention and other methods
(e.g., methods for diagnosing and/or monitoring an eye disorder)
may be practiced by any qualified health care provider.
[0083] The present invention provides compositions, methods, and
articles of manufacture that address the preferences of
ophthalmologists and patients for avoiding procedures associated
with a risk of severe complications while the condition of the
subject's eye is relatively stable and at the same time offer the
potential benefits associated with therapeutic agents and/or
formulations that may be preferred for long term improvement,
stabilization, and/or reduced progression of the condition. In one
aspect the invention provides a method of treating an eye disorder
characterized by macular degeneration, CNV, or RNV comprising the
step of administering first and second therapeutic agents to the
subject's eye in a single procedure, wherein the first therapeutic
agent provides rapid improvement in the condition of the subject's
eye and the second therapeutic agent is a long-acting therapeutic
agent or is administered as a component of a sustained release
formulation. One of skill in the art will appreciate that not all
patients will exhibit an improvement in the condition of the eye.
It will also be appreciated that the time to response (where
"response" refers to improvement in the condition of the eye) may
be an average time to response among patients who exhibit a
response. In some embodiments of the invention, at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, or 90% of patients exhibit a rapid
improvement. In some embodiments the response rate falls between
10% and 100%, or any intervening range such as between 30% and 80%,
etc., exhibit a rapid improvement.
[0084] According to certain embodiments of the invention a
procedure is used to administer a first therapeutic agent that
provides rapid improvement in the condition of the subject's eye.
The agent may be administered, e.g., following a sudden
deterioration in the condition of the subject's eye such as may be
caused by retinal hemorrhage or vessel leakage. In the course of
the same procedure, a second therapeutic agent (which may be the
same as or different from the first therapeutic agent) is also
administered at little or no additional risk to the patient. The
second therapeutic agent is a long-acting agent or is a component
of a sustained release formulation (or both). The second
therapeutic agent may provide a long-term benefit to the patient,
preferably prolonging the time interval before the patient
experiences destabilization or perceives significant deterioration
in the condition of the eye.
[0085] The first and second therapeutic agents may be administered
sequentially or they may be administered substantially at the same
time. If administered sequentially, they may be administered in
either order. The order may be selected based on the identity and
formulation of the agents. In certain embodiments of the invention
the first and second therapeutic agents are administered no more
than 5, 10, 15, 30, or 45 seconds apart, or no more than 1, 2, 3,
4, 5, 10, 15, or 30 minutes apart. In other words, the time
interval between completing administration of the first agent and
completing administration of the second agent is no more than 5,
10, 15, 30, or 45 seconds or no more than 1, 2, 3, 4, 5, 10, 15, or
30 minutes apart in various embodiments of the invention. In
certain embodiments of the invention administration of the first
and second therapeutic agents is completed within 5, 10, 15, 30, or
45 seconds or within 1, 2, 3, 4, 5, 10, 15, or 30 minutes from the
time at which any amount of either the first or second therapeutic
agent leaves the confines of any medical or surgical instrument or
device (e.g., needle, syringe, trocar, catheter, cannula or other
device that may enclose or contain a solution or solid formulation
such as an implant and may be used to introduce such solution or
solid formulation into the eye) and comes into contact with tissues
or fluids of the subject's eye. Administration is said to be
"complete" when the entire dose of an agent to be administered has
left the confines of any medical or surgical instrument or device
that is used to introduce the therapeutic agent into the subject's
eye, it being understood that such instrument or device may retain
a residual amount of the agent. The time interval starting from the
time at which any amount of either the first or second therapeutic
agent leaves the confines of any medical or surgical instrument or
device and comes into contact with tissues or fluids of the
subject's eye and the time at which administration of both first
and second therapeutic agents is complete is referred to herein as
the "time window" of administration. If more than two therapeutic
agents are administered, the time window of administration is the
time interval starting from the time at which any amount of any
therapeutic agent leaves the confines of any medical or surgical
instrument or device and comes into contact with tissues or fluids
of the subject's eye and the time at which administration of all
therapeutic agents is complete. The short time window of
administration of the first and second therapeutic agents is an
additional feature of the invention. Thus in a second aspect, the
invention provides a method of treating an eye disorder
characterized by macular degeneration, CNV, or RNV comprising the
step of administering first and second therapeutic agents to the
subject's eye within a short time window, wherein the first
therapeutic agent provides rapid improvement in the condition of
the subject's eye and the second therapeutic agent is a long-acting
therapeutic agent or is administered as a component of a sustained
release formulation. In various embodiments of the invention the
time window is no more than 5, 10, 15, 30, or 45 seconds or no more
than 1, 2, 3, 4, 5, 10, 15, or 30 minutes.
[0086] In yet another aspect, the invention provides a method of
treating an eye disorder characterized by macular degeneration,
CNV, or RNV comprising the step of administering first and second
therapeutic agents to the subject's eye, wherein the first
therapeutic agent is an angiogenesis inhibitor and the second
therapeutic agent is a long-acting therapeutic agent or is
administered as a component of a sustained release formulation. The
second therapeutic agent may, but need not be, a complement
inhibitor. In certain embodiments the second agent is a compstatin
analog. Optionally the first and second therapeutic agents are
administered in a single procedure.
[0087] In yet another aspect, the invention provides a method of
treating an eye disorder characterized by macular degeneration,
CNV, or RNV comprising the step of administering first and second
therapeutic agents to the subject's eye, wherein the second
therapeutic agent is a complement inhibitor that is either a
long-acting complement inhibitor or is administered as a component
of a sustained release formulation. The first therapeutic agent
may, but need not be, an angiogenesis inhibitor. Optionally the
first and second therapeutic agents are administered in a single
procedure.
[0088] The therapeutic agent that provides rapid improvement in the
condition of the subject's eye may be administered in a liquid
medium. In some embodiments the agent is administered at least in
part in a formulation that releases the agent over time in
sufficient amounts and sufficiently quickly to provide a rapid
improvement in the condition of the subject's eye. For example,
particles that degrade or otherwise release an effective amount of
the agent within the first 24, 48, 72, or 96 hours, within the
first week, or within the first 2 weeks following administration,
could be used. In some embodiments a first portion of the agent is
provided in solution and a second portion is provided in a
formulation that provides for release over time. Optionally the
second portion is a component of the sustained release preparation
of the second agent.
[0089] By administering the first and second therapeutic agents in
a single procedure, certain embodiments of the present invention
minimize the overall risk of complications and make administration
of the second therapeutic agent, which may primarily delay
progression, inhibit further deterioration or destabilization,
and/or provide slow rather than rapid improvement in the condition
of the subject's eye more acceptable to ophthalmologists and/or
patients. The combination therapy approach of the present invention
thus provides an unexpected and unappreciated improvement in the
risk/benefit ratio associated with invasive therapy of eye
disorders.
[0090] The invention further provides pharmaceutical packs or kits
and other articles of manufacture that facilitate the convenient,
effective, and safe administration of multiple therapeutic agents
to the eye using a single procedure.
[0091] In a specific embodiment the invention provides a method of
treating exudative macular degeneration with a combination of a
fast-acting anti-angiogenic drug and a sustained release or
long-acting complement inhibiting drug. The fast-acting
anti-angiogenic drug reduces the leakage and/or promotes regression
of newly-formed blood vessels in the weeks following treatment; the
slow-release or long-acting complement inhibiting drug will prevent
the formation of new blood vessels and promote disease regression
for significant amounts of time (months or years depending on the
device or formulation). Thus in certain embodiments the therapeutic
methods of the invention (1) inhibit/stop blood vessel formation
and/or leakage in patients with exudative macular degeneration in
an acute fashion, so that the retina comes closer in proximity to
the choroid layer of the eye, and that thus ischemic damage to the
retina is reduced, and (2) install a slow-release or long-acting
complement inhibitor that will lower the inflammatory response in
the retina/RPE/choroid layers of the eye and thus block a primary
stimulus for blood vessel growth and, optionally, inhibit the
formation of drusen deposits in addition. In certain embodiments of
any aspect of the invention, administration of the first
therapeutic agent does not result in rapid improvement in the
condition of the subject's eye. However, improvement takes place
more gradually, e.g., within 15 days to 3 weeks, within 15 days to
4 weeks, within 15 days to 5 weeks, or within 15 days to 6
weeks.
II. Therapeutic Agents
[0092] A variety of different therapeutic agents are of use in the
present invention. The first and second therapeutic agents may be
the same or different. Furthermore, the invention is not limited to
the administration of two therapeutic agents. Instead, any number
of therapeutic agents can be administered in a single procedure.
For example, the invention may comprise administering a composition
comprising one or more therapeutic agents in solution in a liquid
medium, e.g., an aqueous medium, and also administering a
composition comprising or consisting essentially of a sustained
release formulation, e.g., an ocular implant, comprising one or
more therapeutic agents, in the course of the same procedure. Thus
certain embodiments of the invention involve administering at least
two, three, or four therapeutic agents in a liquid composition and
at least two, three, or four therapeutic agents in a sustained
release preparation. Any of the therapeutic agents described herein
may be included in either or both of the liquid composition and the
sustained release formulation. The sustained release formulation
may be a liquid composition as long as it possesses sustained
release properties. In specific embodiments the liquid composition
contains two angiogenesis inhibitors or an angiogenesis inhibitor
and a complement inhibitor. In specific embodiments the sustained
release formulation contains two angiogenesis inhibitors, an
angiogenesis inhibitor and a complement inhibitor, or two
complement inhibitors. Optionally one or more additional
therapeutic agents are included. In certain embodiments of the
invention the sustained release formulation comprises compstatin or
an analog thereof and a C5a receptor antagonist. In certain
embodiments of the invention the sustained release formulation
comprises compstatin or an analog thereof and a C3a receptor
antagonist. In certain embodiments of the invention a liquid
composition comprises compstatin or an analog thereof and a C5a
receptor antagonist. In certain embodiments of the invention a
liquid composition comprises compstatin or an analog thereof and a
C3a receptor antagonist.
[0093] A therapeutic agent may be a small molecule or a biological
macromolecule such as a protein, peptide, nucleic acid, etc.
Classes of therapeutic agents of use in various embodiments of the
present invention include, but are not limited to, angiogenesis
inhibitors, complement inhibitors, anti-inflammatory agents,
anti-infective agents (e.g., antibiotics, antivirals, antifungals),
immunomodulators (e.g., immunosuppressive agents), anti-histamines,
anesthetics or other pain relieving agents, beta-blockers, etc., it
being understood that certain agents may be members of more than
one class. In certain embodiments of the invention the first
therapeutic agent is one that rapidly reduces macular edema,
exudation, and/or vascular permeability following its
administration. In certain embodiments of the invention the second
agent is one that reduces ocular inflammation, CNV, and/or RNV.
[0094] In certain embodiments the therapeutic agent has a targeting
moiety either covalently or noncovalently attached thereto. The
targeting moiety comprises a ligand that binds to a marker present
on or at the surface of a target cell or other component such as a
drusen constituent present at a site of desired activity. The term
"ligand" is used to refer to a moiety that specifically binds to a
second moiety.
[0095] The total amount of each therapeutic agent used, and their
concentrations, can vary. Exemplary, nonlimiting, doses are between
0.0001 mg/dose and 100 mg/dose for each eye to be treated, e.g.,
between 0.001 mg/dose and 100 mg/dose, between 0.01 mg/dose and 100
mg/dose, between 0.05 mg/dose and 50 mg/dose, between 0.1 mg/dose
and 10 mg/dose, between 0.5 mg/dose and 5 mg/dose, between 1
mg/dose and 10 mg/dose, etc. (with all doses being approximate).
Exemplary, nonlimiting concentrations of a therapeutic agent in a
composition of the invention are between approximately 0.0001 mg
and 100 mg of the therapeutic agent per milliliter of solution,
e.g., the concentration may be between 0.001 and 100 mg/ml, between
0.01 and 50 mg/ml, between 0.01 and 50 mg/ml, between 0.1 and 10
mg/ml, etc., with all concentrations being approximate. In specific
embodiments the dose of either or both the first or second
therapeutic agents is, without limitation, exactly or approximately
0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 2.0,
3.0, 4.0, or 5.0 mg or can fall within a range delimited by any two
of the foregoing values. For example, in certain embodiments a
sustained release formulation, e.g., an ocular implant, contains
exactly or approximately 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7,
0.75, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, or 5.0 mg of a therapeutic
agent or an amount that falls within a range delimited by any two
of the foregoing values. If the therapeutic agent is one already
approved or under study for use in the disorder, a conventional
dose may be used, where "conventional" means a unit dose that has
been previously shown to be effective and/or is accepted in the art
when used as a single agent. For example, 0.5 mg of Lucentis or 0.3
mg of Macugen may be administered. In other embodiments the dose is
between 0.5 and 2 times a conventional dose.
[0096] The following sections describe a variety of therapeutic
agents of use in the invention, but the invention is not limited to
these agents or classes of agents or their mechanisms of
action.
[0097] A. Angiogenesis Inhibitors
[0098] In certain embodiments of the invention one or more of the
therapeutic agents is an angiogenesis inhibitor. Some angiogenesis
inhibitors are cytotoxic agents that damage or kill target cells
(e.g., endothelial cells) or trigger an immune-mediated response
that results in damage to or killing of target cells. A second
group includes agents that do not substantially damage or kill
endothelial cells but instead inhibit their proliferation,
migration, capillary tube formation, differentiation of endothelial
cells from precursors thereof, or other processes associated with
angiogenesis. Angiogenesis inhibitors in either or both group can
be used.
[0099] A variety of angiogenesis inhibitors have been developed.
Vascular endothelial growth factor (VEGF) is one of the key
regulators of angiogenesis. Other regulators include fibroblast
growth factor 2, pigment epithelium derived growth factor (PEDF),
angiopoietins, and extracellular growth factor molecules (see,
e.g., Ng, E. and Adamis, A., Can. J. Opthalmol., 40:352-68, 2005
for discussion of angiogenesis inhibitors and molecules involved in
angiogenesis). Any of these regulators and/or proteins with which
they interact can be a target of an angiogenesis inhibitor. VEGF-A
is an endothelial cell mitogen with the ability to stimulate
angiogenesis in vivo (Leung D W, Cachianes G, Kuang W-J, Goeddel D
V, Ferrara N, Science, 246:1306-1309, 1989). Other VEGF family
members include VEGF-B, VEGF-C, and VEGF-D. VEGF-A promotes
endothelial cell proliferation and survival as well as vascular
permeability (Ng, supra, and references therein). VEGF-A exists in
several different isoforms containing 121, 145, 165, 189, and 208
amino acids (in humans), of which VEGF.sub.165 may be primarily
responsible for pathological ocular neovascularization. A number of
different receptors for VEGF exist, e.g., VEGFR-1, VEGFR-2, and
VEGFR-3.
[0100] A variety of different agents that inhibit the activity
and/or expression of VEGF, e.g., VEGF-A, or one or more VEGF
receptors are of use in the present invention. Such agents are
referred to herein as "anti-VEGF agents". Useful agents include
antibodies, antibody fragments, and nucleic acids that bind to one
or more VEGF isoforms or VEGF receptors. The binding may inhibit
interaction of one or more VEGF isoforms with its receptor(s).
Macugen (Pfizer, Eyetech) is a VEGF nucleic acid ligand (also
referred to as an aptamer) that binds to and inhibits VEGF.sub.165
(U.S. Pat. No. 6,051,698). Lucentis (Genentech) is a humanized
antibody fragment that binds and inhibits Vascular Endothelial
Growth Factor A (VEGF-A) (Gaudreault, J., et al., Invest Opthalmol.
Vis. Sci. 46, 726-733 (2005) and references therein. Avastin
(Genentech) is a full length humanized antibody that also binds to
VEGF (reviewed in Ferrara, N. Endocr Rev., 25(4):581-611,
2004).
[0101] Other angiogenesis inhibitors of use in the invention
include combretastatin or a derivative or prodrug thereof such as
Combretastatin A4 Prodrug (CA4P); VEGF-Trap (Regeneron
Pharmaceuticals), a fusion protein containing extracellular domains
of two VEGF receptors connected to the Fc region of an antibody
(U.S. Pat. No. 5,844,099); EVIZON.TM. (squalamine lactate);
AG-013958 (Pfizer, Inc.); JSM6427 (Jerini A G), rapamycin
(sirolimus) and analogs thereof, anecortave acetate and other
anti-angiogenic steroids, etc.
[0102] In certain embodiments of the invention the angiogenesis
inhibitor is an agent that inhibits expression of a one or more
pro-angiogenic molecules through the cellular process referred to
as RNA interference (RNAi), also referred to as "gene silencing"
(Novina, C. D. and Sharp, P. A. (2004) "The RNAi revolution",
Nature, 430, 161-164.). Such agents are referred to herein as RNAi
agents and includesiRNA and shRNA. Typically, RNAi agents of use in
the present invention are nucleic acids that include a
double-stranded portion between about 17 and 29 nucleotides, e.g.,
19-25, or 19 nucleotides, in length, one strand of which includes a
portion (the "antisense" or "guide" strand) that is substantially
or perfectly complementary (e.g., at least 70%, at least 80%, at
least 90%, or 100% complementary) to a target gene over about 17-29
nucleotides, e.g., 19-25, or 19 nucleotides. Optionally the RNAi
agent includes one or more single-stranded 3' overhangs. The
presence of an RNAi agent in a cell typically results in
sequence-specific degradation and/or translational repression of a
target mRNA encoded by the target gene, thereby inhibiting its
expression. RNAi agents and methods for their design and
manufacture are well known in the art. See, e.g., Novina, supra,
and references therein as well as U.S. Ser. No. 09/821,832 (U.S.
Pub. No. 20020086356) and U.S. Ser. No. 10/832,248 (U.S. Pub. No.
20040229266). It will be appreciated that RNAi agents may consist
entirely of nucleotides such as those found naturally in RNA and/or
DNA or may comprise any of a wide variety of nucleotide analogs or
may differ in other ways from the structure of naturally occurring
RNA and DNA. See, e.g., U.S. Pub. Nos. 20030175950, 20040192626,
20040092470, 20050020525, 20050032733.
[0103] In certain embodiments of the invention the angiogenesis
inhibitor is an RNAi agent, e.g., an siRNA, that inhibits
expression of one or more VEGF isoforms (e.g., VEGF.sub.165); or
inhibits expression of a VEGF receptor (e.g., VEGFR1). One of
ordinary skill in the art will be able to design appropriate RNAi
agents based on the known sequences of these molecules (or any
other target pro-angiogenic molecule including, but not limited to,
angiogenin, angiopoietin, fibroblast growth factors, PEDF, etc.),
which are available in public databases, e.g., GenBank. In certain
embodiments of the invention the RNAi agent, when administered to
cells, e.g., endothelial cells, in vitro in an appropriate amount
optionally together with uptake enhancing compounds such as lipids,
inhibits expression of its target gene by at least 60%, at least
70%, at least 80%, at least 90%. In certain embodiments of the
invention the RNAi agent, when administered to the eye in an
appropriate amount, inhibits expression of its target gene by at
least 60%, at least 70%, at least 80%, at least 90%, or more within
at least one structure, tissue, or compartment of the eye, e.g.,
within the retina. Exemplary RNAi agents include, but are not
limited to, the siRNA known as Cand5 (Acuity Pharmaceuticals),
which inhibits expression of VEGF, Sirna-027 (Sirna Therapeutics),
which inhibits expression of VEGFR-1, and siRNAs having a sequence
that differs at 1, 2, or 3 positions from that of either Cand5 or
Sirna-027. Additional sequences and structures of RNAi agents
useful in the present invention are described in U.S. Ser. No.
10/294,228 (U.S. Pub. No. 20040018176), U.S. Ser. No. 10/764,957
(U.S. Pub. No. 20050054596). Additional nucleic acids that inhibit
expression of a target gene include antisense oligonucleotides and
ribozymes (see, e.g., U.S. Pat. No. 6,818,447).
[0104] Other angiogenesis inhibitors include various endogenous or
synthetic peptides such as angiostatin, arresten, canstatin,
combstatin, endostatin, thrombospondin, and tumstatin. Other
antiangiogenic molecules include thalidomide and its antiangiogenic
derivatives such as iMiDs (Bamias A, Dimopoulos M A. Eur J Intern
Med. 14(8):459-469, 2003; Bartlett J B, Dredge K, Dalgleish A G.
Nat Rev Cancer. 4(4):314-22, 2004).
[0105] Administration of certain angiogenesis inhibitors, e.g.,
anti-VEGF agents such as Avastin or Lucentis by intravitreal
injection results in a rapid improvement in the condition of a
subject's eye. While not wishing to be bound by any theory, this
rapid improvement may at least in part occur due to diminished
vessel leakage and reduced macular edema. These effects may, at
least in the short term (i.e., over the first 1-2 weeks following
treatment), be at least as significant as any inhibition of blood
vessel development or growth that occurs during this time period.
Therapeutic agents that rapidly reduce macular edema may be of
particular use to cause rapid improvement in the condition of a
subject's eye. Since VEGF is an inducer of vascular permeability,
anti-VEGF agents may be especially effective for these purposes. In
a specific embodiment of the invention the first therapeutic agent
is endostatin. Endostatin has the ability to reduce vascular
permeability (see, e.g., Campochiaro, P A., Expert Opin Biol Ther.,
4(9):1395-402, 2004). In certain embodiments of the invention
endostatin and an anti-VEGF agent, e.g., an antibody, antibody
fragment, or aptamer that binds to VEGF or to a VEGF receptor are
administered. Either the first therapeutic agent, the second
therapeutic agent, or both, may be selected from the group
consisting of anti-VEGF agents and endostatin. Either or both
agents may be contained in a liquid composition or a sustained
release formulation.
[0106] B. Complement Pathways and Complement Inhibitors
[0107] The complement system plays a crucial role in a number of
physiological processes including the response to injury and
defense against foreign entities such as infectious agents. The
complement system is also known to play a role in a number of
diseases (Makrides, S C, Pharm Rev., 50(1); 59-87, 1998). The
complement system comprises more than 30 serum and cellular
proteins that are involved in two major pathways, known as the
classical and alternative pathways (Kuby Immunology, 2000).
[0108] The classical pathway is usually triggered by binding of a
complex of antigen and IgM or IgG antibody to C1 (though certain
other activators can also initiate the pathway). Activated C1
cleaves C4 and C2 to produce C4a and C4b, in addition to C2a and
C2b. C4b and C2a combine to form C3 convertase, which cleaves C3 to
form C3a and C3b. Binding of C3b to C3 convertase produces C5
convertase, which cleaves C5 into C5a and C5b. C3a, C4a, and C5a
are anaphylotoxins and mediate multiple reactions in the acute
inflammatory response. C3a and C5a are also chemotactic factors
that attract immune system cells such as neutrophils. C3 and C5
convertase activity is controlled by a number of endogenous members
of the Regulators of Complement Activation (RCA) family, also
called Complement Control Protein (CCP) family, which includes
complement receptor type 1 (CR1; C3b:C4b receptor), complement
receptor type 2 (CR2), membrane cofactor protein (MCP; CD46),
decay-accelerating factor (DAF), factor H (fH), and C4b-binding
protein (C4 bp). RCA proteins are described in U.S. Pat. No.
6,897,290.
[0109] The alternative pathway is initiated by microbial surfaces
and various complex polysaccharides. In this pathway, C3b,
resulting from cleavage of C3, which occurs spontaneously at a low
level, binds to targets on cell surfaces and forms a complex with
factor B, which is later cleaved by factor D, resulting in a C3
convertase. Cleavage of C3 and binding of another molecule of C3b
to the C3 convertase gives rise to a C5 convertase. C3 and C5
convertases of this pathway are regulated by CR1, DAF, MCP, and fH.
The mode of action of these proteins involves either decay
accelerating activity (i.e., ability to dissociate convertases),
ability to serve as cofactors in the degradation of C3b or C4b by
factor I, or both.
[0110] The C5 convertases produced in both pathways cleave C5 to
produce C5a and C5b. C5b then binds to C6, C7, and C8 to form
C5b-8, which catalyzes polymerization of C9 to form the C5b-9
membrane attack complex (MAC). The MAC inserts itself into target
cell membranes and causes cell lysis. Small amounts of MAC on the
membrane of cells may have a variety of consequences other than
cell death.
[0111] A third complement pathway, the lectin complement pathway is
initiated by binding of mannose-binding lectin (MBL) and
MBL-associated serine protease (MASP) to carbohydrates. In the
human lectin pathway, MASP-1 and MASP-2 are involved in the
proteolysis of C4, C2 and C3, leading to a C3 convertase described
above.
[0112] Complement activity is regulated by various mammalian
proteins referred to as complement control proteins (CCPs). These
proteins differ with respect to ligand specificity and mechanism(s)
of complement inhibition (Lisczewski, M K and Atkinson, J P, in The
Human Complement System in Health and Disease, eds. Volanakis, J E
and Frank, M M, Dekker, New York, pp. 149-66, 1998). They may
accelerate the normal decay of convertases and/or function as
cofactors for factor I, to enzymatically cleave C3b and/or C4b into
smaller fragments. CCPs are characterized by the presence of
multiple (typically 4-56) homologous motifs known as short
consensus repeats (SCR), complement control protein (CCP) modules,
or SUSHI domains (Reid, K B M and Day, A J, Immunol Today,
10:177-80, 1989). These domains, consisting of approximately 50-70
amino acids, typically about 60 amino acids, are characterized by a
conserved motif that includes four disulfide-bonded cysteines (two
disulfide bonds), proline, tryptophan, and many hydrophobic
residues. FIG. 2 shows an SCR consensus sequence. Any particular
SCR may differ from the consensus at one or more positions.
[0113] In certain embodiments of the present invention at least one
of the therapeutic agents, e.g., the second therapeutic agent, is a
complement inhibitor. In some embodiments of the invention the
complement inhibitor inhibits complement activation, e.g., inhibits
activation of one or more complement proteins. For example, it may
inhibit cleavage of an inactive complement protein to its active
form. Complement inhibitors of use in the invention include, but
are not limited to, (i) viral or mammalian complement control or
complement inhibiting proteins as well as fragments or variants
thereof that retain the ability to inhibit complement; (ii)
compstatin and derivatives thereof; (iii) complement receptor
antagonists. The following sections describe complement inhibitors
of use in various embodiments of the invention.
Compounds that Inhibit C3 Activation or Activity
[0114] In certain embodiments of the invention the complement
inhibitor inhibits activation of C3. Exemplary compounds include
compounds that bind to C3 and inhibit its cleavage. In some
embodiments the compound is a peptide. In some embodiments the
peptide is cyclic. In some embodiments of particular interest the
compound is a compstatin analog. Compstatin is a cyclic peptide
identified using phage display that binds to complement component
C3 and inhibits complement activation. Compstatin inhibits cleavage
of C3 to C3a and C3b by convertase. Since C3 is a central component
of all three pathways of complement activation, compstatin and
analogs thereof are able to inhibit activation of the converging
protein of all three pathways. Without wishing to be bound by any
theory, the ability of compstatin and analogs thereof to inhibit
the alternative pathway of complement activation may contribute
significantly to efficacy in certain of the disorders described
herein.
[0115] The invention encompasses the recognition that compstatin
and analogs thereof possess unique and unexpected advantages as
compared with certain other complement inhibitors, particularly for
sustained release, in a variety of eye disorders. The relatively
low molecular weight (.about.1.6 kD) and various other properties
of compstatin analogs facilitate their incorporation into sustained
delivery formulations and devices suitable for providing
therapeutic concentrations in the eye. In certain embodiments a
compstatin analog is delivered in a sustained manner over a
prolonged period of time such as 1-2 weeks, 2-4 weeks, 1-3 months,
3-6 months, 6-12 months, 1-2 years, 2-5 years, or 5-10 years.
[0116] Compstatin is described in U.S. Pat. No. 6,319,897.
Compstatin has the sequence
Ile-[Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys]-Thr (SEQ ID NO:
8), with the disulfide bond between the two cysteines denoted by
brackets. It is an N-terminal cyclic region of a larger peptide
(SEQ ID NO: 1 in U.S. Pat. No. 6,319,897) that also shows
complement inhibiting activity. A number of fragments and variants
of compstatin inhibit complement have been identified. See, e.g.
SEQ ID NOs: 13, 15, 20, 21, and 22 in U.S. Pat. No. 6,319,897.
[0117] A variety of compstatin analogs that have higher complement
inhibiting activity than compstatin have been synthesized. See
WO2004/026328 (PCT/US2003/029653), Morikis, D., et al., Biochem Soc
Trans. 32(Pt 1):28-32, 2004, Mallik, B., et al., J. Med. Chem.,
274-286, 2005, and/or in Katragadda, M., et al. J. Med. Chem., 49:
4616-4622, 2006. Complement inhibiting peptides and peptidomimetics
described therein can be used in the present invention.
[0118] As used herein, the term "compstatin analog" includes
compstatin and any complement inhibiting analog thereof and is used
interchangeably with "compstatin derivative". The term "compstatin
analog" encompasses compstatin and other compounds designed or
identified based on compstatin and whose complement inhibiting
activity is at least 50% as great as that of compstatin as
measured, e.g., using any complement activation assay accepted in
the art or substantially similar or equivalent assays. Certain
compstatin analogs and suitable assays are described in U.S. Pat.
No. 6,319,897, WO2004/026328, Morikis, supra, Mallik, supra, and/or
Katragadda 2006, supra. The assay may, for example, measure
alternative pathway-mediated erythrocyte lysis or be an ELISA assay
(see Examples 5 and 6 of copending applications U.S. Ser. No.
11/544,389 and PCT/US06/39397). The invention includes embodiments
in which any one or more of the compstatin analogs or compositions
described herein is used in any the methods of treatment described
herein. In certain embodiments of the invention a peptide having
higher complement inhibiting activity than compstatin, e.g., at
least 5-fold higher activity, at least 10-fold higher activity,
etc., is used.
[0119] Compstatin and any of its analogs may be acetylated or
amidated, e.g., at the N-terminus and/or C-terminus. For example,
compstatin and any of its analogs may be acetylated at the
N-terminus and amidated at the C-terminus. Consistent with usage in
the art, "compstatin" as used herein, and the activities of
compstatin analogs described herein relative to that of compstatin,
refer to compstatin amidated at the C-terminus (Mallik, 2005,
supra).
[0120] Concatamers or multimers of a compstatin analog thereof are
also of use in the present invention. A supramolecular complex
comprising a compstatin analog is of use in the methods of the
invention.
[0121] The activity of a compstatin analog may be expressed in
terms of its IC.sub.50 (the concentration of the compound that
inhibits complement activation by 50%), e.g., at a particular
plasma concentration, with a lower IC.sub.50 indicating a higher
activity as recognized in the art. The activity of a preferred
compstatin analog for use in the present invention is at least as
great as that of compstatin. Certain modifications are known to
reduce or eliminate complement inhibiting activity and may be
explicitly excluded from any embodiment of the invention. The
IC.sub.50 of compstatin has been reported as 12 .mu.M using a
complement activity assay comprising measuring alternative
pathway-mediated erythrocyte lysis assay (WO2004/026328). In one
embodiment, the IC.sub.50 of the compstatin analog is no more than
the IC.sub.50 of compstatin. In certain embodiments of the
invention the activity of the compstatin analog is between 2 and 99
times that of compstatin (i.e., the analog has an IC.sub.50 that is
less than the IC.sub.50 of compstatin by a factor of between 2 and
99). For example, the activity may be between 10 and 50 times as
great as that of compstatin, or between 50 and 99 times as great as
that of compstatin. In certain embodiments of the invention the
activity of the compstatin analog is between 99 and 264 times that
of compstatin. For example, the activity may be 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, or
264 times as great as that of compstatin. In certain embodiments
the activity is between 264 and 300, 300 and 350, 350 and 400, or
400 and 500 times as great as that of compstatin. The invention
further contemplates compstatin analogs having activities between
500 and 1000 times that of compstatin.
[0122] The K.sub.d of compstatin binding to C3 has been reported as
1.3 .mu.M using isothermal titration calorimetry (Katragadda, et
al., J. Biol. Chem., 279(53), 54987-54995, 2004). Binding affinity
of a variety of compstatin analogs for C3 has been correlated with
their activity, with a lower K.sub.d indicating a higher binding
affinity, as recognized in the art. A linear correlation between
binding affinity and activity was shown for certain analogs tested
(Katragadda, 2004, supra; Katragadda 2006, supra). In certain
embodiments of the invention the compstatin analog binds to C3 with
a K.sub.d of between 0.1 .mu.M and 1.0 .mu.M, between 0.05 .mu.mM
and 0.1 .mu.M, between 0.025 .mu.M and 0.05 .mu.M, between 0.015
.mu.M and 0.025 .mu.M, between 0.01 .mu.M and 0.015 .mu.M, or
between 0.001 .mu.M and 0.01 .mu.M. In certain embodiments the
IC.sub.50 of the compstatin analog is between about 0.2 .mu.M and
about 0.5 .mu.M. In certain embodiments the IC.sub.50 of the
compstatin analog is between about 0.1 .mu.M and about 0.2 .mu.M.
In certain embodiments the IC.sub.50 of the compstatin analog is
between about 0.05 .mu.M and about 0.1 .mu.M. In certain
embodiments the IC.sub.50 of the compstatin analog is between about
0.001 .mu.M and about 0.05 .mu.M.
[0123] Compounds "designed or identified based on compstatin"
include, but are not limited to, compounds that comprise an amino
acid chain whose sequence is obtained by (i) modifying the sequence
of compstatin (e.g., replacing one or more amino acids of the
sequence of compstatin with a different amino acid or amino acid
analog, inserting one or more amino acids or amino acid analogs
into the sequence of compstatin, or deleting one or more amino
acids from the sequence of compstatin); (ii) selection from a phage
display peptide library in which one or more amino acids of
compstatin is randomized, and optionally further modified according
to method (i); or (iii) identified by screening for compounds that
compete with compstatin or any analog thereof obtained by methods
(i) or (ii) for binding to C3 or a fragment thereof. Many useful
compstatin analogs comprise a hydrophobic cluster, a .beta.-turn,
and a disulfide bridge.
[0124] In certain embodiments of the invention the sequence of the
compstatin analog comprises or consists essentially of a sequence
that is obtained by making 1, 2, 3, or 4 substitutions in the
sequence of compstatin, i.e., 1, 2, 3, or 4 amino acids in the
sequence of compstatin is replaced by a different standard amino
acid or by a non-standard amino acid. In certain embodiments of the
invention the amino acid at position 4 is altered. In certain
embodiments of the invention the amino acid at position 9 is
altered. In certain embodiments of the invention the amino acids at
positions 4 and 9 are altered. In certain embodiments of the
invention only the amino acids at positions 4 and 9 are altered. In
certain embodiments of the invention the amino acid at position 4
or 9 is altered, or in certain embodiments both amino acids 4 and 9
are altered, and in addition up to 2 amino acids located at
positions selected from 1, 7, 10, 11, and 13 are altered. In
certain embodiments of the invention the amino acids at positions
4, 7, and 9 are altered. In certain embodiments of the invention
amino acids at position 2, 12, or both are altered, provided that
the alteration preserves the ability of the compound to be
cyclized. Such alteration(s) at positions 2 and/or 12 may be in
addition to the alteration(s) at position 1, 4, 7, 9, 10, 11,
and/or 13. Optionally the sequence of any of the compstatin analogs
whose sequence is obtained by replacing one or more amino acids of
compstatin sequence further includes up to 1, 2, or 3 additional
amino acids at the C-terminus. In one embodiment, the additional
amino acid is Gly. Optionally the sequence of any of the compstatin
analogs whose sequence is obtained by replacing one or more amino
acids of compstatin sequence further includes up to 5, or up to 10
additional amino acids at the C-terminus. It should be understood
that compstatin analogs may have any one or more of the
characteristics or features of the various embodiments described
herein, and characteristics or features of any embodiment may
additionally characterize any other embodiment described herein,
unless otherwise stated or evident from the context. In certain
embodiments of the invention the sequence of the compstatin analog
comprises or consists essentially of a sequence shown in the upper
portion of FIG. 7, in which X4 and X9 represent modifiable side
chains.
[0125] Compstatin and certain compstatin analogs having somewhat
greater activity than compstatin contain only standard amino acids
("standard amino acids" are glycine, leucine, isoleucine, valine,
alanine, phenylalanine, tyrosine, tryptophan, aspartic acid,
asparagine, glutamic acid, glutamine, cysteine, methionine,
arginine, lysine, proline, serine, threonine and histidine).
Certain compstatin analogs having improved activity incorporate one
or more non-standard amino acids. Useful non-standard amino acids
include singly and multiply halogenated (e.g., fluorinated) amino
acids, D-amino acids, homo-amino acids, N-alkyl amino acids,
dehydroamino acids, aromatic amino acids (other than phenylalanine,
tyrosine and tryptophan), ortho-, meta- or para-aminobenzoic acid,
phospho-amino acids, methoxylated amino acids, and
.alpha.,.alpha.-disubstituted amino acids. In certain embodiments
of the invention, a compstatin analog is designed by replacing one
or more L-amino acids in a compstatin analog described elsewhere
herein with the corresponding D-amino acid. Such compounds and
methods of use thereof are an aspect of the invention. Exemplary
non-standard amino acids of use include 2-naphthylalanine (2-NaI),
1-naphthylalanine (1-NaI), 2-indanylglycine carboxylic acid (2Ig1),
dihydrotrpytophan (Dht), 4-benzoyl-L-phenylalanine (Bpa),
2-.alpha.-aminobutyric acid (2-Abu), 3-.alpha.-aminobutyric acid
(3-Abu), 4-.alpha.-aminobutyric acid (4-Abu), cyclohexylalanine
(Cha), homocyclohexylalanine (hCha), 4-fluoro-L-tryptophan (4fW),
5-fluoro-L-tryptophan (5fW), 6-fluoro-L-tryptophan (6fW),
4-hydroxy-L-tryptophan (4OH--W), 5-hydroxy-L-tryptophan (5OH--W),
6-hydroxy-L-tryptophan (6OH--W), 1-methyl-L-tryptophan (1MeW),
4-methyl-L-tryptophan (4MeW), 5-methyl-L-tryptophan (5MeW),
7-aza-L-tryptophan (7aW), .alpha.-methyl-L-tryptophan (.alpha.MeW),
.beta.-methyl-L-tryptophan (.beta.MeW), N-methyl-L-tryptophan
(NMeW), ornithine (orn), citrulline, norleucine, .gamma.-glutamic
acid, etc.
[0126] In certain embodiments of the invention the compstatin
analog comprises one or more Trp analogs (e.g., at position 4
and/or 7 relative to the sequence of compstatin). Exemplary Trp
analogs are mentioned above. See also Beene, et. al. Biochemistry
41: 10262-10269, 2002 (describing, inter alia, singly- and
multiply-halogenated Trp analogs); Babitzke & Yanofsky, J.
Biol. Chem. 270: 12452-12456, 1995 (describing, inter alia,
methylated and halogenated Trp and other Trp and indole analogs);
and U.S. Pat. Nos. 6,214,790, 6,169,057, 5,776,970, 4,870,097,
4,576,750 and 4,299,838. Other Trp analogs include variants
substituted (e.g., by a methyl group) at the .alpha. or .beta.
carbon and, optionally, also at one or more positions of the indole
ring. Amino acids comprising two or more aromatic rings, including
substituted, unsubstituted, or alternatively substituted variants
thereof, are of interest as Trp analogs.
[0127] In certain embodiments the Trp analog has increased
hydrophobic character relative to Trp. For example, the indole ring
may be substituted by one or more alkyl (e.g., methyl) groups. In
certain embodiments the Trp analog participates in a hydrophobic
interaction with C3. Such a Trp analog may be located, e.g., at
position 4 relative to the sequence of compstatin. In certain
embodiments the Trp analog comprises a substituted or unsubstituted
bicyclic aromatic ring component or two or more substituted or
unsubstituted monocyclic aromatic ring components.
[0128] In certain embodiments the Trp analog has increased
propensity to form hydrogen bonds with C3 relative to Trp but does
not have increased hydrophobic character relative to Trp. The Trp
analog may have increased polarity relative to Trp and/or an
increased ability to participate in an electrostatic interaction
with a hydrogen bond donor on C3. Certain exemplary Trp analogs
with an increased hydrogen bond forming character comprise an
electronegative substituent on the indole ring. Such a Trp analog
may be located, e.g., at position 7 relative to the sequence of
compstatin.
[0129] In certain embodiments of the invention the compstatin
analog comprises one or more Ala analogs (e.g., at position 9
relative to the sequence of compstatin), e.g., Ala analogs that are
identical to Ala except that they include one or more CH.sub.2
groups in the side chain. In certain embodiments the Ala analog is
an unbranched single methyl amino acid such as 2-Abu. In certain
embodiments of the invention the compstatin analog comprises one or
more Trp analogs (e.g., at position 4 and/or 7 relative to the
sequence of compstatin) and an Ala analog (e.g., at position 9
relative to the sequence of compstatin).
[0130] In certain embodiments of the invention the compstatin
analog is a compound that comprises a peptide that has a sequence
of (X'aa).sub.n-Gln-Asp-Xaa-Gly-(X''aa).sub.m, (SEQ ID NO: 2)
wherein each X'aa and each X''aa is an independently selected amino
acid or amino acid analog, wherein Xaa is Trp or an analog of Trp,
and wherein n>1 and m>1 and n+m is between 5 and 21. The
peptide has a core sequence of Gln-Asp-Xaa-Gly, where Xaa is Trp or
an analog of Trp, e.g., an analog of Trp having increased
propensity to form hydrogen bonds with an H-bond donor relative to
Trp but, in certain embodiments, not having increased hydrophobic
character relative to Trp. For example, the analog may be one in
which the indole ring of Trp is substituted with an electronegative
moiety, e.g., a halogen such as fluorine. In one embodiment Xaa is
5-fluorotryptophan. Absent evidence to the contrary, one of skill
in the art would recognize that any non-naturally occurring peptide
whose sequence comprises this core sequence and that inhibits
complement activation and/or binds to C3 will have been designed
based on the sequence of compstatin. In an alternative embodiment
Xaa is an amino acid or amino acid analog other than a Trp analog
that allows the Gln-Asp-Xaa-Gly peptide to form a .beta.-turn.
[0131] In certain embodiments of the invention the peptide has a
core sequence of X'aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X'aa
and Xaa are selected from Tip and analogs of Trp. In certain
embodiments of the invention the peptide has a core sequence of
X'aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X'aa and Xaa are
selected from Trp, analogs of Trp, and other amino acids or amino
acid analogs comprising at least one aromatic ring. In certain
embodiments of the invention the core sequence forms a .beta.-turn
in the context of the peptide. The .beta.-turn may be flexible,
allowing the peptide to assume two or more conformations as
assessed for example, using nuclear magnetic resonance (NMR). In
certain embodiments X'aa is an analog of Tip that comprises a
substituted or unsubstituted bicyclic aromatic ring component or
two or more substituted or unsubstituted monocyclic aromatic ring
components. In certain embodiments of the invention X'aa is
selected from the group consisting of 2-napthylalanine,
1-napthylalanine, 2-indanylglycine carboxylic acid,
dihydrotryptophan, and benzoylphenylalanine. In certain embodiments
of the invention X'aa is an analog of Trp that has increased
hydrophobic character relative to Trp. For example, X'aa may be
1-methyltryptophan. In certain embodiments of the invention Xaa is
an analog of Trp that has increased propensity to form hydrogen
bonds relative to Tip but, in certain embodiments, not having
increased hydrophobic character relative to Trp. In certain
embodiments of the invention the analog of Trp that has increased
propensity to form hydrogen bonds relative to Trp comprises a
modification on the indole ring of Trp, e.g., at position 5, such
as a substitution of a halogen atom for an H atom at position 5.
For example, Xaa may be 5-fluorotryptophan.
[0132] In certain embodiments of the invention the peptide has a
core sequence of X'aa-Gln-Asp-Xaa-Gly-X''aa (SEQ ID NO: 4), where
X'aa and Xaa are each independently selected from Trp and analogs
of Trp and X''aa is selected from His, Ala, analogs of Ala, Phe,
and Trp. In certain embodiments of the invention X'aa is an analog
of Trp that has increased hydrophobic character relative to Trp,
such as 1-methyltryptophan or another Trp analog having an alkyl
substituent on the indole ring (e.g., at position 1, 4, 5, or 6).
In certain embodiments X'aa is an analog of Trp that comprises a
substituted or unsubstituted bicyclic aromatic ring component or
two or more substituted or unsubstituted monocyclic aromatic ring
components. In certain embodiments of the invention X'aa is
selected from the group consisting of 2-napthylalanine,
1-napthylalanine, 2-indanylglycine carboxylic acid,
dihydrotryptophan, and benzoylphenylalanine. In certain embodiments
of the invention Xaa is an analog of Trp that has increased
propensity to form hydrogen bonds with C3 relative to Trp but, in
certain embodiments, not having increased hydrophobic character
relative to Trp. In certain embodiments of the invention the analog
of Trp that has increased propensity to form hydrogen bonds
relative to Trp comprises a modification on the indole ring of Trp,
e.g., at position 5, such as a substitution of a halogen atom for
an H atom at position 5. For example, Xaa may be
5-fluorotryptophan. In certain embodiments X''aa is Ala or an
analog of Ala such as Abu or another unbranched single methyl amino
acid. In certain embodiments of the invention the peptide has a
core sequence of X'aa-Gln-Asp-Xaa-Gly-X''aa (SEQ ID NO: 4), where
X'aa and Xaa are each independently selected from Trp, analogs of
Trp, and amino acids or amino acid analogs comprising at least one
aromatic side chain, and X''aa is selected from His, Ala, analogs
of Ala, Phe, and Trp. In certain embodiments X''aa is selected from
analogs of Tip, aromatic amino acids, and aromatic amino acid
analogs.
[0133] In certain preferred embodiments of the invention the
peptide is cyclic. The peptide may be cyclized via a bond between
any two amino acids, one of which is (X'aa).sub.n and the other of
which is located within (X''aa).sub.m. In certain embodiments the
cyclic portion of the peptide is between 9 and 15 amino acids in
length, e.g., 10-12 amino acids in length. In certain embodiments
the cyclic portion of the peptide is 11 amino acids in length, with
a bond (e.g., a disulfide bond) between amino acids at positions 2
and 12. For example, the peptide may be 13 amino acids long, with a
bond between amino acids at positions 2 and 12 resulting in a
cyclic portion 11 amino acids in length.
[0134] In certain embodiments the peptide comprises or consists of
the sequence
X'aa1-X'aa2-X'aa3-X'aa4-Gln-Asp-Xaa-Gly-X''aa1-X''aa2-X''aa3-X''-
aa4-X''aa5 (SEQ ID NO: 5). In certain embodiments X'aa4 and Xaa are
selected from Tip and analogs of Trp, and X'aa1, X'aa2, X'aa3,
X''aa1, X''aa2, X''aa3, X''aa4, and X''aa5 are independently
selected from among amino acids and amino acid analogs. In certain
embodiments X'aa4 and Xaa are selected from aromatic amino acids
and aromatic amino acid analogs. Any one or more of X'aa1, X'aa2,
X'aa3, X''aa1, X''aa2, X''aa3, X''aa4, and X''aa5 may be identical
to the amino acid at the corresponding position in compstatin. In
one embodiment, X''aa1 is Ala or a single methyl unbranched amino
acid. The peptide may be cyclized via a covalent bond between (i)
X'aa1, X'aa2, or X'aa3; and (ii) X''aa2, X''aa3, X''aa4 or X''aa5.
In one embodiment the peptide is cyclized via a covalent bond
between X'aa2 and X''aa4. In one embodiment the covalently bound
amino acid are each Cys and the covalent bond is a disulfide (S--S)
bond. In other embodiments the covalent bond is a C--C, C--O, C--S,
or C--N bond. In certain embodiments one of the covalently bound
residues is an amino acid or amino acid analog having a side chain
that comprises a primary or secondary amine, the other covalently
bound residue is an amino acid or amino acid analog having a side
chain that comprises a carboxylic acid group, and the covalent bond
is an amide bond. Amino acids or amino acid analogs having a side
chain that comprises a primary or secondary amine include lysine
and diaminocarboxylic acids of general structure
NIH.sub.2(CH.sub.2).sub.nCH(NH.sub.2)C OOH such as
2,3-diaminopropionic acid (dapa), 2,4-diaminobutyric acid (daba),
and ornithine (orn), wherein n=1 (dapa), 2 (daba), and 3 (orn),
respectively. Examples of amino acids having a side chain that
comprises a carboxylic acid group include dicarboxylic amino acids
such as glutamic acid and aspartic acid. Analogs such as
beta-hydroxy-L-glutamic acid may also be used.
[0135] In certain embodiments, the compstatin analog is a compound
that comprises a peptide having a sequence:
[0136] Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4
(SEQ ID NO: 6); wherein:
Xaa1 is Ile, Val, Leu, B.sup.1-Ile, B.sup.1-Val, B.sup.1-Leu or a
dipeptide comprising Gly-Ile or B.sup.1-Gly-Ile, and B.sup.1
represents a first blocking moiety; Xaa2 and Xaa2* are
independently selected from Tip and analogs of Trp; Xaa3 is His,
Ala or an analog of Ala, Phe, Trp, or an analog of Trp; Xaa4 is
L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from Thr-Ala and
Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein a carboxy
terminal --OH of any of the L-Thr, D-Thr, Ile, Val, Gly, Ala, or
Asn optionally is replaced by a second blocking moiety B.sup.2; and
the two Cys residues are joined by a disulfide bond.
[0137] In other embodiments Xaa1 is absent or is any amino acid or
amino acid analog, and Xaa2, Xaa2*, Xaa3, and Xaa4 are as defined
above. If Xaa1 is absent, the N-terminal Cys residue may have a
blocking moiety B.sup.1 attached thereto.
[0138] In another embodiment, Xaa4 is any amino acid or amino acid
analog and Xaa1, Xaa2, Xaa2*, and Xaa3 are as defined above. In
another embodiment Xaa4 is a dipeptide selected from the group
consisting of: Thr-Ala and Thr-Asn, wherein the carboxy terminal
--OH or the Ala or Asn is optionally replaced by a second blocking
moiety B.sup.2.
[0139] In any of the embodiments of the compstatin analog of SEQ ID
NO: 6, Xaa2 may be Trp.
[0140] In any of the embodiments of the compstatin analog of SEQ ID
NO: 6, Xaa2 may be an analog of Trp comprising a substituted or
unsubstituted bicyclic aromatic ring component or two or more
substituted or unsubstituted monocyclic aromatic ring components.
For example, the analog of Trp may be selected from
2-naphthylalanine (2-NaI), 1-naphthylalanine (1-NaI),
2-indanylglycine carboxylic acid (Ig1), dihydrotrpytophan (Dht),
and 4-benzoyl-L-phenylalanine.
[0141] In any of the embodiments of the compstatin analog of SEQ ID
NO: 6, Xaa2 may be an analog of Trp having increased hydrophobic
character relative to Trp. For example, the analog of Trp may be
selected from 1-methyltryptophan, 4-methyltryptophan,
5-methyltryptophan, and 6-methyltryptophan. In one embodiment, the
analog of Trp is 1-methyltryptophan. In one embodiment, Xaa2 is
1-methyltryptophan, Xaa2* is Trp, Xaa3 is Ala, and the other amino
acids are identical to those of compstatin.
[0142] In any of the embodiments of the compstatin analog of SEQ ID
NO: 6, Xaa2* may be an analog of Trp such as an analog of Trp
having increased hydrogen bond forming propensity with C3 relative
to Tip, which, in certain embodiments, does not have increased
hydrophobic character relative to Trp. In certain embodiments the
analog of Trp comprises an electronegative substituent on the
indole ring. For example, the analog of Trp may be selected from
5-fluorotryptophan and 6-fluorotryptophan.
[0143] In certain embodiments of the invention Xaa2 is Trp and
Xaa2* is an analog of Trp having increased hydrogen bond forming
propensity with C3 relative to Trp which, in certain embodiments,
does not have increased hydrophobic character relative to Trp. In
certain embodiments of the compstatin analog of SEQ ID NO: 6, Xaa2
is analog of Trp having increased hydrophobic character relative to
Trp such as an analog of Trp selected from 1-methyltryptophan,
4-methyltryptophan, 5-methyltryptophan, and 6-methyltryptophan, and
Xaa2* is an analog of Trp having increased hydrogen bond forming
propensity with C3 relative to Trp which, in certain embodiments,
does not have increased hydrophobic character relative to Trp. For
example, in one embodiment Xaa2 is methyltryptophan and Xaa2* is
5-fluorotryptophan.
[0144] In certain of the afore-mentioned embodiments, Xaa3 is Ala.
In certain of the afore-mentioned embodiments Xaa3 is a single
methyl unbranched amino acid, e.g., Abu.
[0145] In certain embodiments the invention employs a compstatin
analog of SEQ ID NO: 6, as described above, wherein Xaa2 and Xaa2*
are independently selected from Trp, analogs of Trp, and other
amino acids or amino acid analogs that comprise at least one
aromatic ring, and Xaa3 is His, Ala or an analog of Ala, Phe, Trp,
an analog of Trp, or another aromatic amino acid or aromatic amino
acid analog.
[0146] In certain embodiments of the invention the blocking moiety
present at the N- or C-terminus of any of the compstatin analogs
described herein is any moiety that stabilizes a peptide against
degradation that would otherwise occur in mammalian (e.g., human or
non-human primate) blood or vitreous. For example, blocking moiety
B.sup.1 could be any moiety that alters the structure of the
N-terminus of a peptide so as to inhibit cleavage of a peptide bond
between the N-terminal amino acid of the peptide and the adjacent
amino acid. Blocking moiety B.sup.2 could be any moiety that alters
the structure of the C-terminus of a peptide so as to inhibit
cleavage of a peptide bond between the C-terminal amino acid of the
peptide and the adjacent amino acid. Any suitable blocking moieties
known in the art could be used. In certain embodiments of the
invention blocking moiety B.sup.1 comprises an acyl group (i.e.,
the portion of a carboxylic acid that remains following removal of
the --OH group). The acyl group typically comprises between 1 and
12 carbons, e.g., between 1 and 6 carbons. For example, in certain
embodiments of the invention blocking moiety B.sup.1 is selected
from the group consisting of: formyl, acetyl, proprionyl, butyryl,
isobutyryl, valeryl, isovaleryl, etc. In one embodiment, the
blocking moiety B.sup.1 is an acetyl group, i.e., Xaa1 is Ac-Ile,
Ac-Val, Ac-Leu, or Ac-Gly-Ile.
[0147] In certain embodiments of the invention blocking moiety
B.sup.2 is a primary or secondary amine (--NH.sub.2 or --NHR.sup.1,
wherein R is an organic moiety such as an alkyl group).
[0148] In certain embodiments of the invention blocking moiety
B.sup.1 is any moiety that neutralizes or reduces the negative
charge that may otherwise be present at the N-terminus at
physiological pH. In certain embodiments of the invention blocking
moiety B.sup.2 is any moiety that neutralizes or reduces the
negative charge that may otherwise be present at the C-terminus at
physiological pH.
[0149] In certain embodiments of the invention, the compstatin
analog is acetylated or amidated at the N-terminus and/or
C-terminus, respectively. A compstatin analog may be acetylated at
the N-terminus, amidated at the C-terminus, and or both acetylated
at the N-terminus and amidated at the C-terminus. In certain
embodiments of the invention a compstatin analog comprises an alkyl
or aryl group at the N-terminus rather than an acetyl group.
[0150] In certain embodiments, the compstatin analog is a compound
that comprises a peptide having a sequence:
[0151] Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4
(SEQ ID NO: 7); wherein:
Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide
comprising Gly-Ile or Ac-Gly-Ile; Xaa2 and Xaa2* are independently
selected from Trp and analogs of Trp; Xaa3 is His, Ala or an analog
of Ala, Phe, Trp, or an analog of Trp; Xaa4 is L-Thr, D-Thr, Ile,
Val, Gly, a dipeptide selected from Thr-Ala and Thr-Asn, or a
tripeptide comprising Thr-Ala-Asn, wherein a carboxy terminal --OH
of any of L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn optionally is
replaced by --NH.sub.2; and the two Cys residues are joined by a
disulfide bond.
[0152] Xaa1, Xaa2, Xaa2*, Xaa3, and Xaa4 are as described above for
the various embodiments of SEQ ID NO: 6. For example, in certain
embodiments Xaa2* is Trp. In certain embodiments Xaa2 is an analog
of Trp having increased hydrophobic character relative to Trp,
e.g., 1-methyltryptophan. In certain embodiments Xaa3 is Ala. In
certain embodiments Xaa3 is a single methyl unbranched amino
acid.
[0153] In certain embodiments of the invention Xaa1 is Ile and Xaa4
is L-Thr.
[0154] In certain embodiments of the invention Xaa1 is Ile, Xaa2*
is Trp, and Xaa4 is L-Thr.
[0155] In certain embodiments the invention utilizes a compstatin
analog of SEQ ID NO: 7, as described above, wherein Xaa2 and Xaa2*
are independently selected from Trp, analogs of Trp, other amino
acids or aromatic amino acid analogs, and Xaa3 is His, Ala or an
analog of Ala, Phe, Trp, an analog of Trp, or another aromatic
amino acid or aromatic amino acid analog.
[0156] In certain embodiments of any of the compstatin analogs
described herein, Xaa3 is an analog of His.
[0157] Table 1 provides a non-limiting list of compstatin analogs
useful in the present invention. The analogs are referred to in
abbreviated form in the left column by indicating specific
modifications at designated positions (1-13) as compared to the
parent peptide, compstatin (amidated at the C-terminus). Unless
otherwise indicated, peptides are amidated at the C-terminus. Bold
text is used to indicate certain modifications. Activity relative
to compstatin (in this case compstatin amidated at the C-terminus)
is based on published data and assays described therein
(WO2004/026326, Mallik, 2005; Katragadda, 2006). Where multiple
publications reporting an activity were consulted, the more
recently published value is used, and it will be recognized that
values may be adjusted in the case of differences between assays.
It will also be appreciated that the peptides listed in Table 1 are
cyclized via a disulfide bond between the two Cys residues when
used in the therapeutic compositions and methods of the
invention.
TABLE-US-00001 TABLE 1 SEQ ID Activity over Peptide Sequence NO:
compstatin Compstatin H-ICVVQDWGHHRCT-CONH2 8 * Ac-compstatin
Ac-ICVVQDWGHHRCT-CONH2 9 3.times. more Ac-V4Y/H9A
Ac-ICVYQDWGAHRCT-CONH2 10 14.times. more Ac-V4W/H9A --OH
Ac-ICVWQDWGAHRCT-COOH 11 27.times. more Ac-V4W/H9A
Ac-ICVWQDWGAHRCT-CONH2 12 45.times. more Ac-V4W/H9A/T13dT --OH
Ac-ICVWQDWGAHRCdT-COOH 13 55.times. more Ac-V4(2-NaI)/H9A
Ac-ICV(2-NaI)QDWGAHRCT-CONH2 14 99.times. more Ac V4(2-NaI)/H9A
--OH Ac-ICV(2-NaI)QDWGAHRCT-COOH 15 38.times. more Ac VA(1-NaI)/H9A
--OH Ac-ICV(1-NaI)QDWGAHRCT-COOH 16 30.times. more Ac-V42IgI/H9A
Ac-ICV(2-IgI)QDWGAHRCT-CONH2 17 39.times. more Ac-V42IgI/H9A --OH
Ac-ICV(2-IgI)QDWGAHRCT-COOH 18 37.times. more AC-V4Dht/H9A --OH
Ac-ICVDhtQDWGAHRCT-COOH 19 5.times. more Ac-V4(BPa)/H9A --OH
Ac-ICV(Bpa)QDWGAHRCT-COOH 20 49.times. more Ac-V4(Bpa)/H9A
AC-ICV(Bpa)QDWGAHRCT-CONH2 21 86.times. more Ac-V4(Bta)/H9A --OH
Ac-ICV(Bta)QDWGAHRCT-COOH 22 65.times. more Ac-V4(Bta)/H9A
Ac-ICV(Bta)QDWGAHRCT-CONH2 23 64.times. more Ac-V4W/H9(2-Abu)
Ac-ICVWQDWG(2-Abu)HRCT-CONH2 24 64.times. more +G/V4W/H9A +AN --OH
H-GICVWQDWGAHRCTAN-COOH 25 38.times. more Ac-V4(5fW)/H9A
Ac-ICV(5fW)QDWGAHRCT-CONH.sub.2 26 31.times. more Ac-V4(5-MeW)/H9A
Ac-ICV(5-methyl-W)QDWGAHRCT-CONH.sub.2 27 67.times. more
Ac-V4(1-MeW)/H9A Ac-ICV(1-methyl-W)QDWGAHRCT-CONH.sub.2 28
264.times. more Ac-V4W/W7(5fW)/H9A Ac-ICVWQD(5fW)GAHRCT-CONH.sub.2
29 121.times. more Ac-V4(5fW)/W7(5fW)/H9A
Ac-ICV(5fW)QD(5fW)GAHRCT-CONH.sub.2 30 NA Ac-V4(5-Mew)/W7(5fW)H9A
Ac-ICV(5-methyl-W)QD(5fW)GAHRCT- 31 NA CONH.sub.2
Ac-V4(1MeW)/W7(5fW)HgA Ac-ICV(1-methyl-W)QD(5fW)GAHRCT- 32
264.times. more CONH.sub.2 NA = not available
[0158] In certain embodiments of the compositions and methods of
the invention the compstatin analog has a sequence selected from
sequences 9-32. In certain embodiments of the compositions and
methods of the invention the compstatin analog has a sequence
selected from SEQ ID NOs: 14, 21, 28, 29, and 32. In certain
embodiments of the compositions and methods of the invention the
compstatin analog has a sequence selected from SEQ ID NOs: 30 and
31. In one embodiment of the compositions and methods of the
invention the compstatin analog has a sequence of SEQ ID NO: 28. In
one embodiment of the methods of the invention the compstatin
analog has a sequence of SEQ ID NO: 32.
[0159] In other embodiments, compstatin analogs having sequences as
set forth in Table 1, but where the Ac-group is replaced by an
alternate blocking moiety B.sup.1, as described above, are used. In
other embodiments, compstatin analogs having sequences as set forth
in Table 1, but where the --NH.sub.2 group is replaced by an
alternate blocking moiety B.sup.2, as described above, are
used.
[0160] In one embodiment, the compstatin analog binds to
substantially the same region of the .beta. chain of human C3 as
does compstatin. In one embodiment the compstatin analog is a
compound that binds to a fragment of the C-terminal portion of the
.beta. chain of human C3 having a molecular weight of about 40 kDa
to which compstatin binds (Soulika, A. M., et al., Mol. Immunol.,
35:160, 1998; Soulika, A. M., et al., Mol. Immunol. 43(12):2023-9,
2006). In certain embodiments the compstatin analog is a compound
that binds to the binding site of compstatin as determined in a
compstatin-C3 structure, e.g., a crystal structure or NMR-derived
3D structure. In certain embodiments the compstatin analog is a
compound that could substitute for compstatin in a compstatin-C3
structure and would form substantially the same intermolecular
contacts with C3 as compstatin. In certain embodiments the
compstatin analog is a compound that binds to the binding site of a
peptide having a sequence set forth in Table 1, e.g., SEQ ID NO:
14, 21, 28, 29, or 32 in a peptide-C3 structure, e.g., a crystal
structure. In certain embodiments the compstatin analog is a
compound that binds to the binding site of a peptide having SEQ ID
NO: 30 or 31 in a peptide-C3 structure, e.g., a crystal structure.
In certain embodiments the compstatin analog is a compound that
could substitute for the peptide of SEQ ID NO: 9-32, e.g., SEQ ID
NO: 14, 21, 28, or 32 in a peptide-C3 structure and would form
substantially the same intermolecular contacts with C3 as the
peptide. In certain embodiments the compstatin analog is a compound
that could substitute for the peptide of SEQ ID NO: 30 or 31 in a
peptide-C3 structure and would form substantially the same
intermolecular contacts with C3 as the peptide.
[0161] One of ordinary skill in the art will readily be able to
determine whether a compstatin analog binds to a fragment of the
C-terminal portion of the .beta. chain of C3 using routine
experimental methods. For example, one of skill in the art could
synthesize a photocrosslinkable version of the compstatin analog by
including a photo-crosslinking amino acid such
asp-benzoyl-L-phenylalanine (Bpa) in the compound, e.g., at the
C-terminus of the sequence (Soulika, A. M., et al, supra).
Optionally additional amino acids, e.g., an epitope tag such as a
FLAG tag or an HA tag could be included to facilitate detection of
the compound, e.g., by Western blotting. The compstatin analog is
incubated with the fragment and crosslinking is initiated.
Colocalization of the compstatin analog and the C3 fragment
indicates binding. Surface plasmon resonance may also be used to
determine whether a compstatin analog binds to the compstatin
binding site on C3 or a fragment thereof. One of skill in the art
would be able to use molecular modeling software programs to
predict whether a compound would form substantially the same
intermolecular contacts with C3 as would compstatin or a peptide
having the sequence of any of the peptides in Table 1, e.g., SEQ ID
NO: 14, 21, 28, 29, or 32, or in other embodiments SEQ ID NO: 30 or
31.
[0162] Compstatin analogs may be prepared by various synthetic
methods of peptide synthesis known in the art via condensation of
amino acid residues, e.g., in accordance with conventional peptide
synthesis methods, may be prepared by expression in vitro or in
living cells from appropriate nucleic acid sequences encoding them
using methods known in the art. For example, peptides may be
synthesized using standard solid-phase methodologies as described
in Malik, supra, Katragadda, supra, and/or WO2004026328.
Potentially reactive moieties such as amino and carboxyl groups,
reactive functional groups, etc., may be protected and subsequently
deprotected using various protecting groups and methodologies known
in the art. See, e.g., "Protective Groups in Organic Synthesis",
3.sup.rd ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley &
Sons, New York: 1999. Peptides may be purified using standard
approaches such as reversed-phase HPLC. Separation of
diasteriomeric peptides, if desired, may be performed using known
methods such as reversed-phase HPLC. Preparations may be
lyophilized, if desired, and subsequently dissolved in a suitable
solvent, e.g., water. The pH of the resulting solution may be
adjusted, e.g. to physiological pH, using a base such as NaOH.
Peptide preparations may be characterized by mass spectrometry if
desired, e.g., to confirm mass and/or disulfide bond formation.
See, e.g., Mallik, 2005, and Katragadda, 2006.
[0163] The structure of compstatin is known in the art, and NMR
structures for a number of compstatin analogs having higher
activity than compstatin are also known (Malik, supra). Structural
information may be used to design compstatin mimetics. In one
embodiment, the compstatin mimetic is any compound that competes
with compstatin or any compstatin analog (e.g., a compstatin analog
whose sequence is set forth in Table 1) for binding to C3 or a
fragment thereof (such as a 40 kD fragment of the .beta. chain to
which compstatin binds) and that has an activity equal to or
greater than that of compstatin. The compstatin mimetic may be a
peptide, nucleic acid, or small molecule. In certain embodiments
the compstatin mimetic is a compound that binds to the binding site
of compstatin as determined in a compstatin-C3 structure, e.g., a
crystal structure or a 3-D structure derived from NMR experiments.
In certain embodiments the compstatin mimetic is a compound that
could substitute for compstatin in a compstatin-C3 structure and
would form substantially the same intermolecular contacts with C3
as compstatin. In embodiments the compstatin mimetic is a compound
that binds to the binding site of a peptide having a sequence set
forth in Table 1, e.g., SEQ ID NO: 14, 21, 28, 29, or 32, or in
certain embodiments SEQ ID NO: 30 or 31, in a peptide-C3 structure.
In certain embodiments the compstatin mimetic is a compound that
could substitute for a peptide having a sequence set forth in Table
1, e.g., SEQ ID NO: 14, 21, 28, 29, or 32, or in certain
embodiments SEQ ID NO: 30 or 31, in a peptide-C3 structure and
would form substantially the same intermolecular contacts with C3
as the peptide. In certain embodiments the compstatin mimetic has a
non-peptide backbone but has side chains arranged in a sequence
designed based on the sequence of compstatin.
[0164] One of skill in the art will appreciate that once a
particular desired conformation of a short peptide has been
ascertained, methods for designing a peptide or peptidomimetic to
fit that conformation are well known. See, e.g., G. R. Marshall
(1993), Tetrahedron, 49: 3547-3558; Hruby and Nikiforovich (1991),
in Molecular Conformation and Biological Interactions, P. Balaram
& S. Ramasehan, eds., Indian Acad. of Sci., Bangalore, P P.
429-455), Eguchi M, Kahn M., Mini Rev Med. Chem., 2(5):447-62,
2002. Of particular relevance to the present invention, the design
of peptide analogs may be further refined by considering the
contribution of various side chains of amino acid residues, e.g.,
for the effect of functional groups or for steric considerations as
described in the art for compstatin and analogs thereof, among
others.
[0165] It will be appreciated by those of skill in the art that a
peptide mimic may serve equally well as a peptide for the purpose
of providing the specific backbone conformation and side chain
functionalities required for binding to C3 and inhibiting
complement activation. Accordingly, it is contemplated as being
within the scope of the present invention to produce and utilize
C3-binding, complement-inhibiting compounds through the use of
either naturally-occurring amino acids, amino acid derivatives,
analogs or non-amino acid molecules capable of being joined to form
the appropriate backbone conformation. A non-peptide analog, or an
analog comprising peptide and non-peptide components, is sometimes
referred to herein as a "peptidomimetic" or "isosteric mimetic," to
designate substitutions or derivations of a peptide that possesses
much the same backbone conformational features and/or other
functionalities, so as to be sufficiently similar to the
exemplified peptides to inhibit complement activation. More
generally, a compstatin mimetic is any compound that would position
pharmacophores similarly to their positioning in compstatin, even
if the backbone differs.
[0166] The use of peptidomimetics for the development of
high-affinity peptide analogs is well known in the art. Assuming
rotational constraints similar to those of amino acid residues
within a peptide, analogs comprising non-amino acid moieties may be
analyzed, and their conformational motifs verified, by means of the
Ramachandran plot (Hruby & Nikiforovich 1991), among other
known techniques. Virtual screening methods can be used to identify
compstatin mimetics that bind to C3. Such methods may comprise use
of suitable algorithms to computationally dock, score, and
optionally rank a plurality of candidate structures. Any of a wide
variety of available software programs can be used to perform the
virtual screening method. Exemplary programs useful for flexible
molecular docking include DOCK 4.0, FlexX 1.8, AutoDock 3.0, GOLD
1.2, ICM 2.8, and more recent versions thereof.
[0167] One of skill in the art will readily be able to establish
suitable screening assays to identify additional compstatin
mimetics and to select those having desired inhibitory activities.
For example, compstatin or an analog thereof could be labeled
(e.g., with a radioactive or fluorescent label) and contacted with
C3 in the presence of different concentrations of a test compound.
The ability of the test compound to diminish binding of the
compstatin analog to C3 is evaluated. A test compound that
significantly diminishes binding of the compstatin analog to C3 is
a candidate compstatin mimetic. For example, a test compound that
diminishes steady-state concentration of a compstatin analog-C3
complex, or that diminishes the rate of formation of a compstatin
analog-C3 complex by at least 25%, or by at least 50%, is a
candidate compstatin mimetic. One of skill in the art will
recognize that a number of variations of this screening assay may
be employed. Compounds to be screened include natural products,
libraries of aptamers, phage display libraries, compound libraries
synthesized using combinatorial chemistry, etc. The invention
encompasses synthesizing a combinatorial library of compounds based
upon the core sequence described above and screening the library to
identify compstatin mimetics. Any of these methods could also be
used to identify new compstatin analogs having higher inhibitory
activity than compstatin analogs tested thus far.
[0168] Other compounds, e.g., polypeptides, small molecules,
monoclonal antibodies, aptamers, etc., that bind to C3 or C3a
receptors (C3aR) are of use in certain embodiments of the
invention. For example, U.S. Pat. No. 5,942,405 discloses C3aR
antagonists. Aptamers that bind to and inhibit factor B may be
identified using methods such as SELEX (discussed below). U.S. Pat.
Pub. No. 20030191084 discloses aptamers that bind to C1q, C3 and
C5. Also of use are RNAi agents that inhibit local expression of C3
or C3R.
Compounds that Inhibit Factor B Activation or Activity
[0169] In certain embodiments the complement inhibitor inhibits
activation of factor B. For example, the complement inhibitor may
bind to factor B. Exemplary agents include antibodies, antibody
fragments, peptides, small molecules, and aptamers. Exemplary
antibodies that inhibit factor B are described in U.S. Pat. Pub.
No. 20050260198. In certain embodiments the isolated antibody or
antigen-binding fragment selectively binds to factor B within the
third short consensus repeat (SCR) domain. In certain embodiments
the antibody prevents formation of a C3bBb complex. In certain
embodiments the antibody or antigen-binding fragment prevents or
inhibits cleavage of factor B by factor D. In certain embodiments
the complement inhibitor is an antibody, small molecule, aptamer,
or polypeptide that binds to substantially the same binding site on
factor B as an antibody described in U.S. Pat. Pub. No.
20050260198. Use of peptides that bind to and inhibit factor B,
which may be identified using methods such as phage display, is
within the scope of the invention. Use of aptamers that bind to and
inhibit factor B, which may be identified using methods such as
SELEX, is within the scope of the invention. Also of use are RNAi
agents that inhibit local expression of factor B.
[0170] Compounds that Inhibit Factor D Activity
[0171] In certain embodiments the complement inhibitor inhibits
factor D. For example, the complement inhibitor may bind to factor
D. Exemplary agents include antibodies, antibody fragments,
peptides, small molecules, and aptamers. Exemplary antibodies that
inhibit factor D are described in U.S. Pat. No. 7,112,327. In
certain embodiments the complement inhibitor is an antibody, small
molecule, aptamer, or polypeptide that binds to substantially the
same binding site on factor D as an antibody described in U.S. Pat.
No. 7,112,327. Exemplary polypeptides that inhibit alternative
pathway activation and are believed to inhibit factor D are
disclosed in U.S. Pub. No. 20040038869. Use of peptides that bind
to and inhibit factor D, which may be identified using methods such
as phage display, is within the scope of the invention. Use of
aptamers that bind to and inhibit factor D, which may be identified
using methods such as SELEX, is within the scope of the invention.
Also of use are RNAi agents that inhibit local expression of factor
D.
[0172] Viral Complement Control Proteins (VCCPs) and Viral
Complement Inhibiting Proteins (VCIP)
[0173] VCCPs and VCIPs encoded by members of the poxvirus or
herpesvirus families are of use. The invention contemplates use of
any of the agents described in U.S. Ser. No. 60/616,983, filed Oct.
8, 2004, in U.S. Ser. No. 11/247,886, filed Oct. 8, 2005, entitled
VIRAL COMPLEMENT CONTROL PROTEINS FOR EYE DISORDERS, and/or in U.S.
Ser. No. 60/751,771, and U.S. Ser. No. 11/612,751, filed Dec. 19,
2005, and Dec. 19, 2006, respectively, entitled VIRAL COMPLEMENT
CONTROL PROTEINS FOR EYE DISORDERS CHARACTERIZED BY INFLAMMATION.
Poxviruses and herpesviruses are families of large, complex viruses
with a linear double-stranded DNA genome, some of which infect
animals and can cause a range of diseases, the most feared of which
in humans is smallpox. Certain of these viruses encode a number of
immunomodulatory proteins that are believed to play a role in
pathogenesis by subverting one or more aspects of the normal immune
response and/or fostering development of a more favorable
environment in the host organism (Kotwal, G J, Immunology Today,
21(5), 242-248, 2000). VCCPs are among these proteins. Poxvirus
complement control proteins are members of the complement control
protein (CCP) superfamily and typically contain 4 SCR modules.
These proteins possess features that make them particularly
advantageous for treatment and prevention of macular degeneration
related conditions and for treatment and prevention of choroidal
neovascularization.
[0174] Thus in certain embodiments of the invention one or both of
the therapeutic agents is a poxvirus complement control protein
(PVCCP). The PVCCP can comprise a sequence encoded by, e.g.,
vaccinia virus, variola major virus, variola minor virus, cowpox
virus, monkeypox virus, ectromelia virus, rabbitpox virus, myxoma
virus, Yaba-like disease virus, or swinepox virus. In other
embodiments the VCCP is a herpesvirus complement control protein
(HVCCP). The HVCCP can comprise a sequence encoded by a Macaca
fuscata rhadinovirus, cercopithecine herpesvirus 17, or human
herpes virus 8. In other embodiments the HVCCP comprises a sequence
encoded by herpes simplex virus saimiri ORF 4 or ORF 15 (Albrecht,
J C. & Fleckenstein, B., J. Virol., 66, 3937-3940, 1992;
Albrecht, J., et al., Virology, 190, 527-530, 1992).
[0175] The VCCP may inhibit the classical complement pathway, the
alternate complement pathway, the lectin pathway, or any
combination of these. In certain embodiments of the invention the
VCCP, e.g., a PVCCP, binds to C3b, C4b, or both. In certain
embodiments of the invention the PVCCP comprises one or more
putative heparin binding sites (K/R-X-K/R) and/or possesses an
overall positive charge. Preferably the PVCCP comprises at least 3
SCR modules (e.g., modules 1-3), preferably 4 SCR modules. The
PVCCP protein can be a precursor of a mature PVCCP (i.e., can
include a signal sequence that is normally cleaved off when the
protein is expressed in virus-infected cells) or can be a mature
form (i.e., lacking the signal sequence).
[0176] Vaccinia complement control protein (VCP) is a virus-encoded
protein secreted from vaccinia infected cells. VCP is 244 amino
acids in length, contains 4 SCRs, and is naturally produced by
intracellular cleavage of a 263 amino acid precursor. VCP runs as
an .about.35 kD protein in a 12% SDS/polyacrylamide gel under
reducing conditions and has a predicted molecular mass of about
28.6 kD. VCP is described in U.S. Pat. Nos. 5,157,110 and
6,140,472, and in Kotwal, G K, et al., Nature, 355, 176-178, 1988.
FIGS. 3A and 3B show the sequence of the precursor and mature VCP
proteins, respectively. VCP has been shown to inhibit the classical
pathway of complement activation via its ability to bind to C3 and
C4 and act as a cofactor for factor I mediated cleavage of these
components as well as promoting decay of existing convertase
(Kotwal, G K, et al., Science, 250, 827-830, 1990; McKenzie et al.,
J. Infect Dis., 1566, 1245-1250, 1992). It has also been shown to
inhibit the alternative pathway by causing cleavage of C3b into
iC3b and thereby preventing formation of the alternative pathway C3
convertase (Sahu, A, et al., J. Immunol., 160, 5596-5604, 1998).
VCP thus blocks complement activation at multiple steps and reduces
levels of the proinflammatory chemotactic factors C3a, C4a, and
C5a.
[0177] VCP also possesses the ability to strongly bind heparin in
addition to heparan sulfate proteoglycans. VCP contains two
putative heparin binding sites located in modules 1 and 4 (Jha, P
and Kotwal, G J, and references therein). VCP is able to bind to
the surface of endothelial cells, possibly via interaction with
heparin and/or heparan sulfate at the cell surface, resulting in
decreased antibody binding (Smith, S A, et al., J. Virol., 74(12),
5659-5666, 2000). VCP can be taken up by mast cells and possibly
persist in tissue for lengthy periods of time, thereby potentially
prolonging its activity (Kotwal, G J, et al., In G P. Talwat, et
al. (eds), 10.sup.th International Congress of Immunology.,
Monduzzi Editore, Bologna, Italy, 1998) In addition, VCP can reduce
chemotactic migration of leukocytes by blocking chemokine binding
(Reynolds, D, et al., in S. Jameel and L. Villareal (ed., Advances
in animal virology. Oxford and IBN Publishing, New Delhi, India,
1999).
[0178] Variola virus major and minor encode proteins that are
highly homologous to VCP and are referred to as smallpox inhibitor
of complement enzymes (SPICE) (Rosengard, A M, et al., Proc. Natl.
Acad. Sci., 99(13), 8803-8813. U.S. Pat. No. 6,551,595). SPICE from
various variola strains sequenced to date differs from VCP by about
5% (e.g., about 11 amino acid differences). Similarly to VCP, SPICE
binds to C3b and C4b and causes their degradation, acting as a
cofactor for factor I. However, SPICE degrades C3b approximately
100 times as fast as VCP and degrades C4b approximately 6 times as
fast as VCP. The amino acid sequence of SPICE is presented in FIG.
6 and can be described as follows. Referring to FIG. 6, a signal
sequence extends from amino acid 1 to about amino acid 19. Four
SCRs extend from about amino acid 20 to amino acid 263. Each SCR is
characterized by four cysteine residues. The four cysteine residues
form two disulfide bonds in the expressed protein. The boundaries
of each SCR are best defined by the first and fourth cysteine
residues in the sequence that forms the disulfide bonds of the SCR.
An invariant tryptophan residue is present between cysteine 3 and
cysteine 4 of each SCR. SCR1 extends from amino acid 20 or 21 to
amino acid 81. Both residues are cysteines that may be involved in
disulfide bonding. SCR2 extends from amino acid 86 to amino acid
143. SCR3 extends from amino acid 148 to amino acid 201. SCR4
extends from amino acid 206 to amino acid 261. The SCRs include the
complement binding locations of SPICE. SPICE or any of the portions
thereof that inhibit complement activation, e.g., SPICE and
SPICE-related polypeptides containing four SCRs, such as those
described in U.S. Pat. No. 6,551,595, are of use in the present
invention.
[0179] Complement control proteins from cowpox virus (referred to
as inflammation modulatory protein, IMP) and monkeypox virus
(referred to herein as monkeypox virus complement control protein,
MCP) have also been identified and sequenced (Miller, C G, et al.,
Virology, 229, 126-133, 1997 and Uvarova, E A and Shchelkunov, S N,
Virus Res., 81(1-2), 39-45, 2001). MCP differs from the other
PVCCPs described herein in that it contains a truncation of the
C-terminal portion of the fourth SCR.
[0180] It will be appreciated that the exact sequence of complement
control proteins identified in different virus isolates may differ
slightly. Such proteins fall within the scope of the present
invention. Complement control proteins from any such isolate may be
used, provided that the protein has not undergone a mutation that
substantially abolishes its activity. Thus the sequence of a VCCP
such as SPICE or VCP may differ from the exact sequences presented
herein or under the accession numbers listed in Table 1. It will
also be appreciated that a number of amino acid alterations, e.g.,
additions, deletions, or substitutions such as conservative amino
acid substitutions, may be made in a typical polypeptide such as a
VCCP without significantly affecting its activity, such that the
resulting protein is considered equivalent to the original
polypeptide. For example, up to about 10% of the amino acids, or up
to about 20% of the amino acids may frequently be changed without
significantly altering the activity. Also, of course, domains known
to have similar functions can be substituted for one another. Such
domains may be found within a single polypeptide (e.g., repeated
domains) or within different, homologous polypeptides. The effect
of any particular amino acid alteration(s) or domain substitutions
can readily be determined.
[0181] FIG. 4 shows a sequence alignment of a variety of poxvirus
complement control proteins from isolates of variola major and
minor, vaccinia, cowpox virus, and monkeypox virus. FIG. 5 shows a
comparison of the SCR domain structure of a number of complement
control proteins and fragments thereof, the number of K+R residues,
% K+R residues, pI, number of putative beparin binding sites, and
ability to inhibit hemolysis (indicative of complement inhibiting
activity) and/or bind to heparin.
[0182] Without limitation, any of the viral polypeptides identified
by accession number in Table 2 below is of use in various
embodiments of the invention.
TABLE-US-00002 TABLE 2 Representative Viral Complement Control
Proteins Virus Protein Accession Virus Type Variola D12L NP_042056
Orthopoxvirus D15L (SPICE) AAA69423 Orthopoxvirus Vaccinia VCP
AAO89304 Orthopoxvirus Cowpox CPXV034 AAM13481 Orthopoxvirus C17L
CAA64102 Orthopoxvirus Monkeypox D14L AAV84857 Orthopoxvirus
Ectromelia virus Complement control protein CAE00484 Orthopoxvirus
Rabbitpox RPXV017 AAS49730 Orthopoxvirus Macaca fuscata
rhadinovirus JM4 AAS99981 Rhadinavirus (Herpesvirus) Cercopithecine
herpesvirus 17 Complement binding NP_570746 Herpesvirus protein
(ORF4) Human herpes virus 8 Complement binding AAB62602 Herpesvirus
protein (ORF4)
Compounds that Inhibit C5 Activation or Activity
[0183] In certain embodiments the complement inhibitor inhibits
activation of C5. For example, the complement inhibitor may bind to
C5. Exemplary agents include antibodies, antibody fragments,
polypeptides, small molecules, and aptamers. Exemplary antibodies
are described in U.S. Pat. No. 6,534,058. Exemplary compounds that
bind to and inhibit C5 are described in U.S. Pat. Pub. Nos.
20050090448 and 20060115476. In certain embodiments the complement
inhibitor is an antibody, small molecule, aptamer, or polypeptide
that binds to substantially the same binding site on C5 as an
antibody described in U.S. Pat. No. 6,534,058 or a peptide
described in U.S. Ser. No. 10/937,912. U.S. Pat. Pub. No.
20060105980 discloses aptamers that bind to and inhibit C5. Also of
use are RNAi agents that inhibit expression of C5 or C5R.
[0184] In other embodiments the agent is an antagonist of a C5a
receptor (C5aR). Exemplary C5a receptor antagonists include a
variety of small cyclic peptides such as those described in U.S.
Pat. No. 6,821,950; U.S. Ser. No. 11/375,587; and/or PCT/US06/08960
(WO2006/099330).
[0185] For example, the therapeutic agent may be a compound of
general formula I below:
##STR00001##
[0186] where A is H, alkyl, aryl, NH.sub.2, NHalkyl,
N(alkyl).sub.2, NHaryl or NHacyl; B is an alkyl, aryl, phenyl,
benzyl, naphthyl or indole group, or the side chain of a D- or
L-amino acid selected from the group consisting of phenylalanine,
homophenylalanine, tryptophan, homotryptophan, tyrosine, and
homotyrosine; C is the side chain of a D-, L- or homo-amino acid
selected from the group consisting of proline, alanine, leucine,
valine, isoleucine, arginine, histidine, aspartate, glutamate,
glutamine, asparagine, lysine, tyrosine, phenylalanine,
cyclohexylalanine, norleucine, tryptophan, cysteine and methionine;
D is the side chain of a D- or L-amino acid selected from the group
consisting of cyclohexylalanine, homocyclohexylalanine, leucine,
norleucine, homoleucine, homonorleucine and tryptophan; E is the
side chain of a D- or L-amino acid selected from the group
consisting of tryptophan and homotryptophan; F is the side chain of
a D- or L-amino acid selected from the group consisting of
arginine, homoarginine, lysine and homolysine or is one of the
following side-chains
##STR00002##
[0187] or another mimetic of an arginine side chain, where X is
NCN, NNO.sub.2, CHNO.sub.2 or NSO.sub.2NH.sub.2; n is an integer
from 1 to 4, and R.sup.1 is H or an alkyl, aryl, CN, NH.sub.2, OH,
--CO--CH.sub.2CH.sub.3, --CO--CH.sub.3,
--CO--CH.sub.2CH.sub.2CH.sub.3, --CO--CH.sub.2 Ph, or --CO-Ph; and
X.sup.1 is --(CH.sub.2).sub.nNH--or (CH.sub.2).sub.n--S--,
--(CH.sub.2).sub.2O--, --(CH.sub.2).sub.3 O--,
--(CH.sub.2).sub.3--, --(CH.sub.2).sub.4--, or --CH.sub.2
COCHRNH--,
where R is the side chain of any common or uncommon amino acid, and
where n is an integer of from 1 to 4, e.g., 1, 2, 3, or 4.
[0188] In certain embodiments of the invention F is one of the
following side-chains:
##STR00003##
or another mimetic of an arginine side chain; where X is NCN,
NNO.sub.2, CHNO.sub.2 or NSO.sub.2NH.sub.2; n is an integer from 1
to 4, and R.sup.1 is H or an alkyl, aryl, CN, NH.sub.2, OH,
--CO--CH.sub.2CH.sub.3, --CO--CH.sub.3,
--CO--CH.sub.2CH.sub.2CH.sub.3, --CO--CH.sub.2 Ph, or --CO-Ph; B is
an indole, indole methyl, benzyl, phenyl, naphthyl, naphthyl
methyl, cinnamyl group, or any other derivative of the aromatic
group; and C is D- or L-cyclohexylalanine (Cha), leucine, valine,
isoleucine, phenylalanine, tryptophan or methionine. In certain
embodiments of the invention A is L-arginine. In certain
embodiments of the invention F is an L-amino acid. In certain
embodiments F is L-arginine. In certain embodiments n=1, 2, 3, or
4.
[0189] In certain embodiments of the invention the compound is
selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28, as
described in U.S. Pat. No. 6,821,950. Other embodiments disclosed
therein may also be used. For example, A, B, C, D, E, F, and
R.sup.1 may be any of the groups mentioned in U.S. Pat. No.
6,821,950. It is noted that the letters A, B, C, D, E, and F in the
formulas presented herein are to be given the meanings described
herein and in U.S. Pat. No. 6,821,950 and do not stand for chemical
elements or isotopes such as boron, carbon, deuterium, or fluorine.
The compounds described above will be referred to collectively
herein as GPCRA.
[0190] In one embodiment, the complement inhibitor is a C5a
receptor inhibitor, e.g., a C5a antagonist. For example, the
complement inhibitor may be a peptide having the following
sequence: HC-[ORN-PRO-dCHA-TRP-ARG] (SEQ ID NO: 45) where
HC=hydrocinnamate, dCHA=d-cyclohexylalaine, ORN=l-ornithine, and [
] denotates cyclization through an amide bound. In another
embodiment the complement inhibitor is a peptide having sequence
Ac-PHE-[ORN-PRO-dCHA-TRP-ARG] (SEQ ID NO: 46), using the same
abbreviations. In one embodiment, the therapeutic agent is the
compound depicted in FIG. 8. In certain embodiments of the
invention the complement inhibitor is a C3a receptor inhibitor,
e.g., a C3a antagonist.
[0191] Methods for making the GPCRA, confirming their structure,
and testing their activity as modulators of a GPCR are disclosed in
U.S. Pat. No. 6,821,950. Certain of these compounds are available
from Promics (Brisbane, Australia). In one embodiment the
complement inhibitor is PMX205.
[0192] C. Long-Acting Therapeutic Agents
[0193] In certain embodiments of the invention at least one of the
therapeutic agents is a long-acting agent. For example, certain
complement inhibitors may intrinsically have a long duration of
activity even if not provided as a component of a sustained release
formulation. The long-acting therapeutic agent may, for example,
have an activity period of at least 3 months, at least 6 months, at
least 9 months, or at least 12 months when administered in solution
in a liquid medium in medically acceptable quantities. The
long-acting therapeutic agent may be administered in solution in a
liquid medium or may be a component of a solid or semi-solid
formulation which optionally contains one or more additional
therapeutically active or inactive components.
[0194] In other embodiments a therapeutic agent that is not a
long-acting agent is modified such that it becomes long-acting. The
modification may, for example, stabilize the agent against the
activity of various endogenous molecules such as proteases.
Suitable modifications are known in the art and include, for
example, pegylation.
[0195] In certain embodiments of the invention the long-acting
therapeutic agent is administered as a component of a sustained
release formulation, e.g., an ocular implant or any sustained
release formulation described herein.
III. Liquid Compositions Comprising a Therapeutic Agent
[0196] In certain embodiments of the invention at least one of the
therapeutic agents, e.g., any of the therapeutic agents discussed
above, is administered in solution in a liquid medium. Suitable
preparations, e.g., substantially pure preparations of one or more
therapeutic agents may be combined with pharmaceutically acceptable
carriers, diluents, solvents, etc., to produce an appropriate
pharmaceutical composition, i.e., one that is pharmaceutically
acceptable for administration to the eye. The preparation may
contain a pharmaceutically acceptable carrier, diluent, etc.
Suitable carriers are known in the art and include, for example,
sterile water for injection, saline, etc. Additional components may
include, but are not limited to, buffers, preservatives, salts,
etc.
[0197] The therapeutic agents themselves may be provided as
pharmaceutically acceptable salts, which include those derived from
pharmaceutically acceptable inorganic and organic acids and bases.
Examples of suitable acid salts include acetate, adipate, alginate,
aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,
citrate, camphorate, camphorsulfonate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptanoate, glycerophosphate, glycolate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxyethanesulfonate, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, salicylate, succinate,
sulfate, tartrate, thiocyanate, tosylate and undecanoate. Salts
derived from appropriate bases include alkali metal (e.g., sodium
and potassium), alkaline earth metal (e.g., magnesium), ammonium
and N+(C1-4 alkyl).sub.4 salts. This invention also envisions the
quaternization of any basic nitrogen-containing groups of the
compounds disclosed herein. Water or oil-soluble or dispersible
products may be obtained by such quaternization.
[0198] Solutions or suspensions can include components such as a
sterile diluent such as water for injection, saline solution, or
other solvent acceptable for administration to the eye, buffers
such as acetates, citrates or phosphates and agents for the
adjustment of tonicity such as sodium chloride or dextrose. pH can
be adjusted with acids or bases, such as hydrochloric acid or
sodium hydroxide. The preparation can be enclosed in ampoules,
disposable syringes or single or multiple dose vials made of glass
or plastic and provided for commercial sale and/or use in any such
manner. The term "suspension" includes a composition comprising
particles in a liquid medium. In some embodiments, the particles
consist essentially of a therapeutic agent. In other embodiments
the particles comprise a drug-releasing component such as a polymer
and, optionally, one or more additional components such as an
excipient.
[0199] In some embodiments of the invention the liquid composition
comprises an agent that enhances uptake of the therapeutic agent by
cells, enhances bioavailability of the agent at its site of action,
or otherwise enhances activity of the therapeutic agent. For
example, a variety of delivery vehicles that enhance uptake and/or
activity of RNAi agents such as siRNAs are known in the art and may
be included in the liquid composition.
[0200] Preferred pharmaceutical formulations are stable under the
conditions of manufacture and storage and may be preserved against
the contaminating action of microorganisms such as bacteria and
fingi.
IV. Sustained Release Formulations
[0201] A sustained release formulation of use in the present
invention provides a therapeutic concentration of a drug within the
eye or a portion or region thereof for a prolonged period of time.
The period of time during which a therapeutic level of the drug is
present can be, e.g., at least 1, 2, 4, or 6 weeks, at least 1, 2,
3, 4, 6, 8, 10, 12, 15, 18, 24 months, or longer. Release may begin
immediately or shortly (e.g., within 24 hours) after administration
of the sustained delivery formulation. Alternately, release may be
delayed, e.g., it may commence at a time point at least 24 hours
following administration. Without limitation, release may occur
steadily or may occur intermittently (e.g., in bursts during which
a substantial amount of the agent is released), or periods of
steady release may alternate with bursts. In certain embodiments
the therapeutic agent is released at controlled or predetermined
rates when the sustained release formulation is placed in the eye.
Such rates may range, for example, from about 0.003 micrograms/day
to about 5000 micrograms/day, or between about 0.01 micrograms/day
to about 5 micrograms/day, or between about 0.05 micrograms to
about 1 micrograms/day. In some embodiments the rate of release is
between 1 .mu.g and 5 .mu.g/day.
[0202] A sustained release formulation of use in the present
invention typically comprises a therapeutic agent and an additional
component, element, or structure that contributes to the sustained
release properties of the formulation. The additional component,
element, or structure that is effective to provide sustained
release is referred to herein as a "drug delivery regulating
component". Optionally the drug delivery regulating element is
designed to provide control over the kinetics of release. It will
be appreciated that the physical nature of the formulation, e.g.,
the shape and total surface area of any solid or semi-solid
constituents, may contribute to its sustained release properties.
As another example, tight compression of particles containing an
active agent may result in release that takes place over a longer
time period than if the particles were not compressed. In some
embodiments the structure is provided at least in part by the
therapeutic agent itself and, optionally, one or more substances
present at the site of administration such as an ion, protein, etc.
In some embodiments no additional drug delivery regulating
component need be present in the administered composition. For
example, a composition comprising a therapeutic agent in a liquid
medium may form a structure having properties of a gel following
its administration. The therapeutic agent may be released over
time, optionally as the structure degrades. The drug delivery
regulating component may comprise or consist of a polymer matrix
that is physically associated with the therapeutic agent. For
example, the therapeutic agent may be entrapped, embedded, or
encapsulated by the polymer matrix. A sustained release formulation
can be in the form of an individual ocular implant, a plurality of
nanoparticles, microparticles, or liposomes, a semi-solid or
viscous material such as a gel, etc. The therapeutic agent may
preferably be from about 1% to 90% by weight of the sustained
release formulation. More preferably, the therapeutic agent is from
about 20% to about 80% by weight of the of the sustained release
formulation. In certain embodiments, the therapeutic agent
comprises about 40% by weight of the sustained release formulation
(e.g., 30%-50%).
[0203] A number of polymeric delivery vehicles for providing
sustained release have been used in an ocular context and can be
used to administer the compositions of the invention. Various
polymers, e.g., biocompatible polymers, which may be biodegradable,
can be used. The polymers may be homopolymers, copolymers
(including block copolymers), straight, branched-chain, or
cross-linked. Useful polymers include, but are not limited to,
poly-lactic acid (PLA), poly-glycolic acid (PGA),
poly-lactide-co-glycolide (PLGA), poly(phosphazine), poly
(phosphate ester), polycaprolactones, polyanhydrides, ethylene
vinyl acetate, polyorthoesters, polyethers, and poly (beta amino
esters). Peptides, proteins such as collagen or albumin,
polysaccharides such as chitosan, alginate, hyaluronic acid (or
derivatives of any of these) and dendrimers (e.g., PAMAM
dendrimers) are also of use. Methods for preparation of such
formulations will be apparent to those skilled in the art. Certain
of the materials can also be obtained commercially, e.g., from Alza
Corporation Any of these polymers, or combinations thereof, can be
used in various embodiments of the invention.
[0204] Additional exemplary polymers include cellulose derivatives
such as carboxymethylcellulose, polycarbamates or polyureas,
cross-linked poly(vinyl acetate) and the like, ethylene-vinyl ester
copolymers having an ester content of 4 to 80% such as
ethylene-vinyl acetate (EVA) copolymer, ethylene-vinyl hexanoate
copolymer, ethylene-vinyl propionate copolymer, ethylene-vinyl
butyrate copolymer, ethylene-vinyl pentantoate copolymer,
ethylene-vinyl trimethyl acetate copolymer, ethylene-vinyl diethyl
acetate copolymer, ethylene-vinyl 3-methyl butanoate copolymer,
ethylene-vinyl 3-3-dimethyl butanoate copolymer, and ethylene-vinyl
benzoate copolymer, or mixtures thereof.
[0205] Poly(ortho esters) have been introduced into the eye and
demonstrated favorable properties for sustained release ocular drug
delivery (Einmahl, S., Invest. Opthalmol. Vis. Sci., 43(5), 2002).
Polylactide particles have been used to target an agent to the
retina and RPE following intravitreous injection of a suspension of
such particles (Bourges, J-L, et al, Invest. Opthalmol. Vis. Sci.,
44(8), 2003).
[0206] Sustained release formulations including various ocular
implants and other ocular drug delivery systems that are of use in
various embodiments of the invention are described, for example, in
U.S. Pat. Nos. 6,692,759; 6,331,313; 5,869,079; 5,824,072; and U.S.
Ser. No. 10/918,597 (Pub. No. 20050048099); 10/837,357 (Pub. No.
20050244469); Ser. No. 11/092,122 (Pub. No. 20050244472) and Ser.
No. 11/116,698 (Pub. No. 20050281861) as well as a number of other
patents and publications referenced in the foregoing, all of which
are incorporated herein by reference.
[0207] A method of making a sustained release formulation involves
combining or mixing the therapeutic agent with a polymeric
component to form a mixture. The mixture may then be extruded,
compressed, molded, etc., to form a single composition. Optionally,
heat and/or pressure can be used. The single composition may then
be processed to form individual implants or particles suitable for
placement in an eye of a patient. Additional methods for
incorporating therapeutically active agents into polymeric matrices
are known in the art. The polymeric matrix can be formed into
various shapes such as rods, disks, wafers, etc., which may have a
range of different dimensions (e.g., length, width, etc.) and
volumes. Exemplary shapes include spherical, cylindrical, helical,
coil-shaped or helical, screw-shaped, cubical, conical,
ellipsoidical, biconvex, hemispherical or near-hemispherical
etc.
[0208] In certain embodiments of the invention an ocular implant is
so dimensioned and shaped that it fits within the hollow shaft of
an injection needle, e.g., a 22, 25, 27, 30, 33, or 35 gauge needle
(or needle of any gauge ranging between 22 and 35). Exemplary and
nonlimiting dimensions for a cylindrical implant may be about 0.5
to 8 millimeters in length and about 0.1 to 2 millimeters in
diameter, e.g., about 0.75 mm to about 1.5 mm in diameter. Implants
having other shapes, e.g., other rodlike structures with
cross-sections that are rectangular or square in cross-section may
have a cross-section in which the two points most distant from each
other are separated by at most 0.1 mm to 1 mm. In particular
embodiments the intraocular implant may have a length or other
longest dimension of between about 5 microns and about 2 mm, or
between about 10 microns and about 1 mm for administration with a
needle. Alternately, the length or other longest dimension is
greater than 1 mm, or greater than 2 mm, such as 3 mm or up to 10
mm. The vitreous chamber in humans is able to accommodate
relatively large implants of varying geometries, having lengths of,
for example, 1 to 10 mm.
[0209] In certain embodiments of the invention the implants may
also be at least somewhat flexible, which may facilitate both
insertion of the implant in the eye, e.g., in the vitreous, and/or
may facilitate accommodation of the implant. The total weight of
the implant may be about 250-5000 micrograms, e.g., about 500-1000
micrograms. For example, an implant may be about 500 micrograms or
about 1000 micrograms. Larger implants may also be formed and
further processed before administration to an eye. In addition,
larger implants may be desirable where relatively greater amounts
of a therapeutic agent are provided in the implant, as used.
[0210] In one embodiment the sustained release formulation is a
biocompatible ocular implant comprising a substantially impermeable
polymeric outer layer covering a core which comprises the drug to
be delivered, wherein said outer layer has one or more orifices, by
which is meant one or more openings in the outer layer through
which, when the device is in use, body fluids can enter the device
and the drug contained in the device (e.g., dissolved,
encapsulated, or entrapped within the device) can migrate out of
the device. In certain embodiments the orifices in total have a
surface area of less than 10 percent of the total surface area of
the device. In certain embodiments of the invention the ocular
implant comprises an outer coating layer that is permeable to the
therapeutic agent, allowing its slow diffusion out of the implant.
The composition, structure, and/or thickness of the coating layer
may be selected to provide a particular permeability and diffusion
rate.
[0211] A drug can be contained in an ocular implant as a dry
powder, particles, granules, or as a compressed solid. The drug may
also be present as a solution or be dispersed in a polymer matrix.
Ocular implants, may be have the active agent or agents
homogenously distributed through the polymeric matrix, e.g., they
may be monolithic. In other embodiments the active agent(s) are
heterogeneously distributed in the polymeric matrix. For example,
discrete regions of the implant may contain solid particles of an
active agent, or a reservoir of active agent may be encapsulated by
the polymeric matrix. The therapeutic agent(s) may be distributed
in a non-homogenous pattern in the matrix. For example, an implant
may include a portion that has a greater concentration of the
therapeutic agent relative to a second portion of the implant.
Multilayered structures, with the layers having different
compositions and may have different physical characteristics such
as density or porosity are another possibility. For example, the
layers may contain different therapeutic agents or combinations
thereof. In another embodiment, layers that are relatively
resistant to degradation are interspersed with layers that degrade
more rapidly.
[0212] The biodegradable polymeric materials which are included to
form the matrix may be subject to enzymatic or hydrolytic
instability. Water soluble polymers may be cross-linked with
hydrolytic or biodegradable unstable cross-links to provide useful
water insoluble polymers. The degree of stability can vary widely,
depending, for example, upon the choice of monomer, whether a
homopolymer or copolymer or mixture, is employed, and whether the
polymer includes terminal acid groups. The biodegradation of the
polymer and hence the extended release profile of the sustained
release formulation may also influenced by the relative average
molecular weight of the polymeric materials employed. Different
molecular weights of the same or different polymeric materials may
be included in the formulations to modulate the release profile.
For example, the average molecular weight of the polymer may range
from about 5 to about 500 kD, e.g., from about 10 to 100 kD, or
from about 15 to 50 kD.
[0213] Nanoparticles or microparticles can be made using any method
known in the art including, but not limited to, spray drying, phase
separation, single and double emulsion, solvent evaporation,
solvent extraction, and simple and complex coacervation.
Particulate polymeric compositions can also be made using
granulation, extrusion, and/or spheronization. A composition can
contain nanoparticles or microparticles having different
compositions and/or properties.
[0214] The conditions used in preparing the particles may be
altered to yield particles of a desired size or property (e.g.,
hydrophobicity, hydrophilicity, external morphology, "stickiness",
shape, etc.). The method of preparing the particle and the
conditions (e.g., solvent, temperature, concentration, air flow
rate, etc.) used may also depend on the therapeutic agent and/or
the composition of the polymer matrix.
[0215] Microparticles and nanoparticles of use in the invention can
have a range of dimensions. Generally, a microparticle will have a
diameter of 500 microns or less, e.g., between 1 and 500 microns,
between 50 and 500 microns, between 100 and 250 microns, between 20
and 50 microns, between 1 and 20 microns, between 1 and 10 microns,
etc., and a nanoparticle will have a diameter of less than 1
micron, e.g., between 10 nm and 100 nm, between 100 nm and 250 nm,
between 100 nm and 500 nm, between 250 nm and 500 nm, between 250
nm and 750 nm, between 500 nm and 750 micron. If the particles
prepared by any of the above methods have a size range outside of
the desired range, the particles can be sized, for example, using a
sieve. Particles can be substantially uniform in size (e.g.,
diameter) or shape or may be heterogeneous in size and/or shape.
They may be substantially spherical or may have other shapes, in
which case the relevant dimension will be the longest straight
dimension rather than the diameter.
[0216] In certain embodiments of the invention a sustained release
formulation comprises a therapeutic agent and a gel-forming
material. In accordance with certain embodiments of the invention,
a solution containing the soluble gel-forming material and a
therapeutic agent is prepared by combining the soluble gel-forming
material and therapeutic agent in solution using any suitable
method, e.g., by adding the therapeutic agent to a solution
containing the gel-forming material. The composition is delivered
locally to an appropriate location in the eye of a subject. The
solution rapidly forms a gel at or close to of the site of
administration. The therapeutic agent is entrapped within the gel.
The therapeutic agent diffuses out of the gel or is released as the
gel degrades over time, thereby providing a continuous supply of
the agent to tissues and structures that are either in direct
physical contact with the gel or located nearby. In certain
embodiments the solution is administered behind the sclera of the
eye. Delivery can be accomplished by injection (e.g., using a 25,
27, or 30 gauge needle or the like), by catheter, etc. In other
embodiments the solution is administered intravitreally. In certain
embodiments a "gel" is a structure that exhibits properties (e.g.,
fluidity) intermediate between solid and liquid phases. The
structure may be a solid or semisolid colloid comprising a solid
continuous phase and a liquid phase. The structure may have an
appearance typical of a gel, which appearance is readily recognized
by those of skill in the art.
[0217] In one embodiment, soluble collagen is used as the
gel-forming material. The collagen is initially soluble, e.g., in
an aqueous medium, and forms a solution that has a low viscosity
but is capable of rapid formation of a gel under appropriate
conditions, e.g., conditions encountered upon administration to a
mammalian subject. A variety of different collagen preparations can
be used in the present invention provided that the collagen is
initially soluble and is capable of rapidly forming a gel under
appropriate conditions. Suitable collagen preparations, and methods
for their manufacture, are described, e.g., in U.S. Pat. Nos.
5,492,135; 5,861,486; 6,197,934; 6,204,365; and WO 00/47130, but
the invention is not limited to such preparations or methods. These
collagens are prepared in soluble form and rapidly form a gel upon
exposure to physiological fluids or other fluids having suitable
concentration of ions. In accordance with the present invention,
injecting or otherwise introducing the collagen solution to the eye
or near the eye results in gel formation, presumably induced by
contact with physiological fluids. However it is noted that the
invention is in no way limited by the mechanism by which gel
formation occurs. In addition, as noted above, the gel can be
formed in vitro and then implanted at an appropriate location.
[0218] Other gel-forming materials of use in the invention include,
but are not limited to, hyaluronic acid and modified forms thereof,
polysaccharides such as alginate and modified forms thereof,
self-assembling peptides, etc. See, e.g., U.S. Pat. No. 6,129,761
for further description of alginate and modified forms thereof,
hyaluronic acid and modified forms thereof, and additional examples
of soluble gel-forming materials that are of use in various
embodiments of the present invention. Other polymeric hydrogel
precursors include polyethylene oxide-polypropylene glycol block
copolymers such as Pluronics.TM. or Tetronics.TM. which are
crosslinked by hydrogen bonding and/or by a temperature change, as
described in Steinleitner et al., Obstetrics & Gynecology,
77:48-52 (1991); and Steinleitner et al., Fertility and Sterility,
57:305-308 (1992). Other materials which may be utilized include
proteins such as fibrin or gelatin. Polymer mixtures also may be
utilized. For example, a mixture of polyethylene oxide and
polyacrylic acid which gels by hydrogen bonding upon mixing may be
utilized.
[0219] Typically a gel-forming material of use in the invention is
capable of being at least partly dissolved, or in certain
embodiments of the invention substantially or fully dissolved,
e.g., in an aqueous medium. For example, at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 95%, or
more, by weight, of the gel-forming material present in a
gel-forming composition may be dissolved. In certain embodiments
essentially 100% of the material is dissolved. The aqueous medium
can contain one or more liquids in addition to water, e.g., various
alcohols. In general, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, or 100% of the liquid present in the medium is
water.
[0220] Covalently crosslinkable hydrogel precursors also are
useful. For example, a water soluble polyamine, such as chitosan,
can be cross-linked with a water soluble diisothiocyanate, such as
polyethylene glycol diisothiocyanate. The isothiocyanates will
react with the amines to form a chemically crosslinked gel.
Aldehyde reactions with amines, e.g., with polyethylene glycol
dialdehyde also may be utilized. A hydroxylated water soluble
polymer also may be utilized.
[0221] In certain embodiments of the invention a therapeutic agent
is covalently or noncovalently attached to a drug delivery
regulating component such as a polymer via a linking moiety. The
linking moiety is cleaved to release the therapeutic agent from the
drug delivery regulating component to provide sustained release.
For example, the linking moiety may be a peptide containing a site
that is cleaved by an endogenous enzyme such as a protease or may
contain a labile or hydrolyzable bond, e.g., a disulfide bond,
ester moiety, etc.
[0222] Cells that express a therapeutic agent that is a biological
macromolecule such as a protein or RNAi agent can be implanted into
the eye and are of use in certain embodiments of the invention to
provide sustained release. U.S. Pat. No. 6,436,427 provides a
method for delivering biologically active molecules to the eye by
implanting biocompatible capsules containing a cellular source of
the biologically active molecule.
[0223] In certain embodiments of the invention the sustained
release formulation comprises liposomes. For example, a liposomal
suspension can be administered. Liposomes can be prepared according
to methods known to those skilled in the art, for example, as
described in U.S. Pat. No. 4,522,811 and other references listed
herein. Liposomes, including targeted liposomes (e.g., antibody
targeted liposomes) and pegylated liposomes have been described
(Hansen C B, et al., Biochim Biophys Acta. 1239(2):133-44, 1995;
Torchilin V P, et al., Biochim Biophys Acta, 1511(2):397-411, 2001;
Ishida T, et al., FEBS Lett. 460(1):129-33, 1999).
[0224] One of ordinary skill in the art will appreciate that the
materials and methods selected for preparation of a sustained
release formulation, implant, etc., should be such as to retain
activity of the compound. For example, it may be desirable to avoid
excessive heating of certain agents such as polypeptides, which
could lead to denaturation and loss of activity. Furthermore, it
will be appreciated that a sustained release formulation may
contain a variety of additional components that lack therapeutic
activity and that may or may not contribute to the sustained
release features of the formulation. Examples include plasticizing
agents, solubilizing agents, solubility decreasing agents, and
dispersing agents (see U.S. Pat. No. 6,331,313), provided that such
components are compatible with administration to the eye under the
conditions used. For example, a sustained release formulation may
include a .beta.-cyclodextrin, which is effective in enhancing the
solubility of the therapeutic agent. The .beta.-cyclodextrin may be
provided in an amount from about 0.5% (w/w) to about 25% (w/w) of
the implant. In certain implants, the .beta.-cyclodextrin is
provided in an amount from about 5% (w/w) to about 15% (w/w) of the
formulation. Other formulations include a gamma-cyclodextrin,
and/or cyclodextrin derivatives.
[0225] A sustained release formulation herein may include an
excipient component, such as effective amounts of buffering agents,
preservatives and the like. Suitable water soluble buffering agents
include, without limitation, alkali and alkaline earth carbonates,
phosphates, bicarbonates, citrates, borates, acetates, succinates
and the like, such as sodium phosphate, citrate, borate, acetate,
bicarbonate, carbonate and the like. These agents are
advantageously present in amounts sufficient to maintain a pH of
the system of between about 2 to about 9 and more preferably about
4 to about 8. As such the buffering agent may be as much as about
5% by weight of the total system. Suitable water soluble
preservatives include sodium bisulfite, sodium bisulfate, sodium
thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol,
thimerosal, phenylmercuric acetate, phenylmercuric borate,
phenylmercuric nitrate, parabens, methylparaben, polyvinyl alcohol,
benzyl alcohol, phenylethanol and the like and mixtures thereof.
These agents may be present in amounts of from 0.001 to about 5% by
weight and preferably 0.01 to about 2% by weight. These agents may
also be used in certain of the liquid compositions described
herein.
[0226] In some embodiments of the invention the sustained release
formulation comprises an agent that enhances uptake of the
therapeutic agent by cells, enhances bioavailability of the agent
at its site of action, or otherwise enhances activity of the
therapeutic agent. For example, a variety of delivery vehicles that
enhance uptake and/or activity of RNAi agents such as siRNAs are
known in the art and may be included in the sustained release
formulation.
[0227] If desired, the proportions of therapeutic agent, polymer,
and any other modifiers may be empirically determined by
formulating several implants, for example, with varying proportions
of such ingredients. A USP approved method for dissolution or
release test can be used to measure the rate of release (USP 23; NF
18 (1995) pp. 1790-1798). The implants can also be tested in
vivo.
[0228] Included within the scope of the term "sustained release
formulation" of a therapeutic agent are devices or "chips" that
include one or more reservoirs containing the agent and that
release the agent or a portion thereof from the one or more
reservoirs into the surrounding environment (see, e.g., U.S. Pat.
Nos. 5,797,898 and 6,976,982). Release may occur through a variety
of means. For example, the reservoirs may have a biodegradable cap
that is impermeable to the agent and degrades over time, so that
the therapeutic agent is released once the cap is degraded. Caps of
differing thickness will cause release to occur at different times.
Mechanical, electrical, or other means may be used to release the
agent from a reservoir, optionally using external control means to
regulate such release. Release can occur at predetermined times
and/or in predetermined amounts. The device may be
programmable.
V. Methods of Administration
[0229] A variety of different methods, techniques, and procedures
may be used to administer the first and second therapeutic agents.
In certain embodiments of the invention administration is performed
by intravitreal injection. While it will be appreciated that a
certain amount of inter-physician variability exists. A nonlimiting
example of an intravitreal procedure may be performed as follows:
The tension in the eye is typically measured with a tensiometer and
inflammation is graded clinically. The eyes are anesthetized with
sodium channel blockers (such as novocaine) and treated with
mydriatic drops (either sympathomimetic, anti-parasympathetic or
myoplegic in nature), followed by further treatment with anesthetic
and antibiotic drops. A speculum is then inserted under the eyelids
and the patient is asked to look sideways. A caliper is used to
measure a distance of 2 mm from the limbus and determine the
injection site (this is done in order to avoid hitting the lens
with the needle). An injection syringe (e.g., a 1 ml or 0.5 ml
syringe) with a needle (e.g., a 22, 25, 27, or 30 gauge needle) is
then loaded with 50-100 microliters of an agent (e.g., either 1 mg
Avastin or 0.3 mg Macugen), or a syringe that is preloaded with the
agent is used. The needle is then inserted at the injection site
until the middle of the vitreous cavity is reached with the tip of
the needle and the drug is slowly injected. The syringe is then
slowly retracted and pressure is administered for a few seconds at
the site of injection with wet gauze. More antibiotic drops are
administered and the speculum is removed. The patient is then
typically observed for 10 to 60 min, during which time the
intraocular pressure is measured at regular intervals.
[0230] Other methods of administration include, e.g., choroidal
injection, transscleral injection or placing a scleral patch,
selective arterial catheterization, intraocular administration
including transretinal, subconjunctival bulbar, intravitreal
injection, suprachoroidal injection, subtenon injection, scleral
pocket and scleral cutdown injection, by osmotic pump, etc. In
choroidal injection and scleral patching, the clinician uses a
local approach to the eye after initiation of appropriate
anesthesia, including painkillers and opthalmoplegics. A needle
containing the therapeutic compound is directed into the subject's
choroid or sclera and inserted under sterile conditions. When the
needle is properly positioned the compound is injected into either
or both of the choroid or sclera.
[0231] Intraocular administration of drugs intended for treatment
of macular degeneration and/or other intraocular conditions by a
variety of methods is well known in the art, and any suitable
method can be used in the present invention. See, e.g., U.S. Pat.
Nos. 5,632,984 and 5,770,589. U.S. Pat. No. 6,378,526 provides
methods for intrascleral injection of a therapeutic or diagnostic
material at a location overlying the retina, which provide a
minimally invasive technique for delivering the agent to the
posterior segment of the eye.
[0232] Only minor modifications of the foregoing procedures, or no
modifications at all, may be needed to administer first and second
therapeutic agents in accordance with the present invention.
Standard injection times and pressures can be used or appropriately
modified. For example, the total injection time may be longer than
in the case of injecting a single agent. It will be appreciated
that the nature of the modifications, if any, will be dictated at
least in part by the particular procedure as well as the nature of
the therapeutic agents and their formulation in addition to the
manner in which they are provided for use by the clinician. For
example, in one embodiment a sustained release formulation
containing the second therapeutic agent, e.g., an ocular implant,
is loaded into the needle. The needle may be supplied to the
clinician having already been preloaded with the sustained release
formulation or the clinician may load the needle with the sustained
release formulation. The clinician attaches the needle to the
syringe. An appropriate volume (containing an appropriate amount)
of a solution containing the first therapeutic agent is then drawn
up into the syringe and the intravitreal injection procedure is
performed as described above. Depression of the plunger of the
syringe will eject both the first therapeutic agent and the
sustained release formulation into the vitreous (or elsewhere in
the eye if a technique other than intravitreal injection is used).
In another embodiment, a syringe containing an appropriate volume
of a solution containing the first therapeutic agent is provided to
the clinician, optionally together with a needle that is preloaded
with the sustained release formulation containing the second
therapeutic agent. Thus the clinician needs to undertake no
measurement, dilution, or other manipulation of the therapeutic
agents themselves.
[0233] In another embodiment, both therapeutic agents are contained
in individual syringes. The injection is performed with a needle
and syringe assembly, wherein the syringe contains the first
therapeutic agent or, more generally, the syringe contains a
composition comprising the first therapeutic agent. After
administration of the therapeutic agent contained in the syringe,
the syringe is removed and a second syringe, containing the second
therapeutic agent, or more generally, a composition comprising the
second therapeutic agent, is attached to the needle. The second
therapeutic agent (or composition) is then administered. The
therapeutic agents can be administered in either order. Any number
of therapeutic agents can be administered consecutively, without
removing the tip of the needle from the subject's eye.
[0234] FIG. 9 shows an exemplary embodiment of a needle and syringe
assembly that may be used to practice the methods of the invention.
The assembly includes a syringe having a barrel and a plunger with
a stopper at its end. The syringe is attached to a needle,
typically by means of a threaded tip with an opening (e.g., a Luer
lock tip), which is not shown. The portion of the syringe between
the stopper and the end of the barrel contains a therapeutic agent,
e.g., an angiogenesis inhibitor. The needle contains an additional
therapeutic agent, e.g., a sustained release formulation such as an
ocular implant containing a therapeutic agent (e.g., a complement
inhibitor). For purposes of clarity the implant is depicted outside
the needle in FIG. 9 though of course it would be located within
the shaft of the needle for administration. Exerting pressure on
the plunger following introduction of the needle into a subject's
eye will eject both the therapeutic agent in the syringe and the
ocular implant in the needle into the eye. In other embodiments,
the syringe may be provided with additional or alternative means of
ejecting the implant.
[0235] In certain embodiments of the invention the first and second
therapeutic agents are delivered to different structures, regions,
compartments, or tissues of the eye in a single procedure. For
example, a needle or other instrument may pass through different
structures, regions, compartments, or tissues of the eye during the
process of inserting and withdrawing the needle. The first and
second therapeutic agents may be ejected into different structures,
regions, compartments, or tissues of the eye in the course of a
single injection procedure. For example, in one embodiment one of
the therapeutic agents is introduced into the vitreous and one of
the therapeutic agents in introduced into a different structure,
region, compartment, or tissue of the eye in a single procedure.
Either the agent that provides rapid rapid improvement in the
condition of the subject's eye or the sustained release formulation
of the second therapeutic agent may be introduced into the
vitreous.
[0236] The volume to be administered will depend on the location
within the eye to which the composition is administered. For
example, for intravitreal injection volumes of 200 .mu.l,
preferably 100 .mu.l or less are generally preferred. In certain
embodiments the total volume of liquid injected is 200 .mu.l or
less, 100 .mu.l or less, 50 .mu.l or less. In certain embodiments
of the invention the total volume of material introduced into the
subject's eye (including liquid and any solid or semi-solid
components) is 5001 or less, 400 .mu.l or less, 300 .mu.l or less
200 .mu.l or less, 100 .mu.l or less, 50 .mu.l or less, or 25 .mu.l
or less.
VI. Gene Therapy
[0237] The invention also encompasses gene therapy, in which one or
more of the therapeutic agents is a nucleic acid that encodes a
therapeutic agent such as an siRNA or protein such as a VCCP in
operable association with regulatory elements sufficient to direct
expression of the nucleic acid is administered to the eye. A
composition comprising a nucleic acid therapeutic can consist
essentially of the nucleic acid or a gene therapy vector in an
acceptable diluent, or can comprise a drug release regulating
component such as a polymer matrix with which the nucleic acid or
gene therapy vector is physically associated, e.g, with which it is
mixed or within which it is encapsulated or embedded. The gene
therapy vector can be a plasmid, virus, or other vector.
Alternatively, the pharmaceutical composition can comprise one or
more cells which produce a therapeutic nucleic acid or polypeptide.
Preferably such cells secrete the therapeutic agent into the
extracellular space.
[0238] Viral vectors that have been used for gene therapy protocols
include, but are not limited to, retroviruses, lentiviruses, other
RNA viruses such as poliovirus or Sindbis virus, adenovirus,
adeno-associated virus, herpes viruses, SV 40, vaccinia and other
DNA viruses. Replication-defective murine retroviral or lentiviral
vectors are widely utilized gene transfer vectors. Chemical methods
of gene therapy involve carrier-mediated gene transfer through the
use of fusogenic lipid vesicles such as liposomes or other vesicles
for membrane fusion. A carrier harboring a nucleic acid of interest
can be conveniently introduced into the eye or into body fluids or
the bloodstream. The carrier can be site specifically directed to
the target organ or tissue in the body. Cell or tissue specific
DNA-carrying liposomes, for example, can be used and the foreign
nucleic acid carried by the liposome absorbed by those specific
cells. Gene transfer may also involve the use of lipid-based
compounds which are not liposomes. For example, lipofectins and
cytofectins are lipid-based compounds containing positive ions that
bind to negatively charged nucleic acids and form a complex that
can ferry the nucleic acid across a cell membrane.
[0239] Certain cationic polymers spontaneously bind to and condense
nucleic acids such as DNA into nanoparticles. For example,
naturally occurring proteins, peptides, or derivatives thereof have
been used. Synthetic cationic polymers such as polyethylenimine
(PEI), polylysine (PLL) etc. condense DNA and are useful delivery
vehicles. Dendrimers can also be used. Many useful polymers contain
both chargeable amino groups, to allow for ionic interaction with
the negatively charged DNA phosphate, and a degradable region, such
as a hydrolyzable ester linkage. Examples include
poly(alpha-(4-aminobutyl)-L-glycolic acid), network poly(amino
ester), and poly (beta-amino esters). These complexation agents can
protect nucleic acids against degradation, e.g., by nucleases,
serum components, etc., and create a less negative surface charge,
which may facilitate passage through hydrophobic membranes (e.g.,
cytoplasmic, lysosomal, endosomal, nuclear) of the cell. Certain
complexation agents facilitate intracellular trafficking events
such as endosomal escape, cytoplasmic transport, and nuclear entry,
and can dissociate from the nucleic acid.
VII. Articles of Manufacture
[0240] In another aspect of the invention, an article of
manufacture, which may be referred to as a pharmaceutical pack or
kit, containing compositions, devices or instruments, and
optionally additional materials or items useful for treating the
disorders described above is provided. The article of manufacture
can comprises a container and a label or package insert on or
associated with the container. Suitable containers include, for
example, bottles, vials, syringes, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is, either alone or together
with another composition effective for treating the condition.
Optionally the container may have a sterile access port (for
example the container may be a vial having a stopper pierceable by
a hypodermic injection needle). The label or package insert may
indicate that the composition is used for treating one or more
conditions of choice, e.g., an eye disorder such as macular
degeneration. The article of manufacture may comprise (a) a first
container with a composition contained therein, wherein the
composition comprises a first therapeutic agent; and (b) a second
container with a composition contained therein, wherein the
composition comprises a sustained release formulation of a second
therapeutic agent. The article of manufacture in this embodiment of
the invention may further comprise a package insert indicating that
the first and second compositions can be used to treat a particular
eye disorder, e.g., exudative ARMD. Alternatively, or additionally,
the article of manufacture may further comprise a second (or third)
container comprising a pharmaceutically acceptable liquid, such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution and dextrose solution.
[0241] In certain embodiments of the invention the article of
manufacture may further comprise one or more devices or instruments
for administering a therapeutic agent to the eye. For example, the
article of manufacture may include one or more needles (e.g., a 22,
25, 27, or gauge needle) and/or one or more syringes (e.g., 0.3,
0.5, or 1.0 ml syringes). Either a needle or a syringe, or both,
may contain one or more compositions comprising a unit dosage form
of a therapeutic agent. For example, the article of manufacture may
include a needle or syringe that contains a predetermined volume
and/or amount of a composition comprising a therapeutic agent. The
article of manufacture may contain a needle or syringe that
contains a sustained release formulation of a therapeutic agent,
e.g., an ocular implant. The needle and syringe may, but need not
be, attached to one another. The needle and/or syringe may be
provided with a removable cap. Providing one or more of the
compositions already loaded into the device that will be used to
administer the agent(s) may provide increased reliability, safety,
and convenience. The article of manufacture may include a plurality
of syringes, each of which optionally contains a unit doseage form
of a different therapeutic agent. For example, a first syringe
containing a first therapeutic agent and a second syringe
containing a third therapeutic agent may be included. In one
embodiment a first syringe containing an angiogenesis inhibitor and
a second syringe containing a complement inhibitor are provided.
Either or both of the agents may be in a liquid composition. Either
or both of the agents may be a component of a sustained release
formulation. Each syringe may contain a composition containing a
single therapeutic agent and optionally other components such as a
pharmaceutically acceptable carrier. Alternately, one or more of
the syringes may contain a composition containing a plurality of
different therapeutic agents and optionally other components such
as a pharmaceutically acceptable carrier.
[0242] The individual components described above may be packaged
together in a larger container, e.g., a box, foil, styrofoam, or
plastic wrapper, or other container, which may optionally contain
additional packaging material. Care should be taken to use
materials that will, if necessary or desirable, protect the
therapeutic agent(s) from light and/or other environmental
conditions and will not adversely affect them. The article of
manufacture may include instructions, e.g., in the form of a
package insert, that instruct the clinician as to methods by which
the compositions should be administered including, if appropriate,
instructions for assembling, diluting, or otherwise manipulating
any individual components. In one embodiment each article of
manufacture contains appropriate amounts of first and second
compositions comprising first and second therapeutic agents for
performing a single procedure (i.e., a single administration of
first and second therapeutic agents to the eye). Optionally
included are devices or instruments such as a needle and syringe
for performing the procedure. The needle, syringe, or both, may be
preloaded with a composition comprising a therapeutic agent.
Articles of manufacture that contain one or more of any of the
therapeutic agents, sustained release formulations thereof, and/or
devices or instruments for administering a therapeutic agent to the
eye, and any combinations thereof, are within the scope of the
invention.
[0243] Preferably any composition to be administered to the eye is
sterile. The composition can be made from sterile components, or
sterilization can be performed after manufacture. Methods of
sterilization include irradiation, heat, etc. Preferably, the
sterilization method used does not substantially reduce the
activity or biological or therapeutic activity of the therapeutic
agents. Devices and instruments to be used for administration to
the eye are also preferably sterile, at least to the extent they
will enter the eye.
VIII. Testing in Animal Models
[0244] Animal models that replicate one or more features of macular
degeneration, diabetic retinopathy, CNV, inflammation, or other
ocular conditions are known in the art. A compound of the invention
can be administered in various doses to mice, rats, dogs, primates,
etc. that have spontaneous macular degeneration and/or CNV or in
which macular degeneration and/or CNV have been induced by a
treatment. The ability of the compound to prevent or treat one or
more signs or symptoms of macular degeneration (e.g. CNV,
accumulation of lipofuscin in and/or drusen beneath the RPE,
photoreceptor atrophy or hypertrophy, altered RPE pigmentation,
photoreceptor loss, altered electroretinogram, etc.) is assessed.
Visual examination, photography, histopathology, immunohistology,
etc., can be used.
[0245] Useful models include animals (e.g., mice, Yucatan pigs,
monkeys, etc.) in which CNV is induced by laser treatment (see,
e.g., Bora, P. S., et al., Proc. Natl. Acad. Sci. 100(5):
2679-2684, 2003; Zacks, D N, et al., Invest Opthalmol V is Sci.
243(7):2384-91, 2002). Other models include animals that have been
treated with a variety of agents such as lipid hydroperoxide
(Tamai, K., et al., Exp Eye Res. 74(2):301-8, 2002), pellets
comprising growth factors, etc. Animals genetically engineered to
overexpress or underexpress one or more genes are also useful. For
example, transgenic mice (mcd/mcd mice) that express a mutated form
of cathepsin D that is enzymatically inactive display features
associated with geographic atrophy (Rakoczy, P E, et al, Am. J.
Path., 161(4), 1515-1524, 2002). Adeno-associated virus (AAV)
mediated expression of vascular endothelial growth factor induces
CNV in rats (Wang, F., et al., Invest Opthalmol Vis Sci.
44(2):781-90, 2003). One animal model is a transgenic mouse
deficient in either monocyte chemoattractant protein (Ccl-2) or its
cognate chemokine receptor (Ccr-2) (Ambati, J., et al., Nat. Med.
9(11):1390-7, 2003; U.S. Ser. No. 10/685,705--U.S. Pat. Pub. No.
20040177387). Aged mice with a deficiency in either of these
proteins exhibit a number of features of ARMD including
accumulation of lipofuscin in and drusen beneath the RPE,
photoreceptor atrophy, and CNV. Methods for testing the efficacy of
a candidate agent using this mouse model are disclosed in U.S. Pat.
Pub. No. 20040177387. In general, a candidate agent is administered
to the mouse either before or after development of features of
ARMD, and at least one eye is monitored for development or
regression of drusen and/or lipofuscin accumulation therein, for
effect of the candidate agent on Bruch's membrane, effect on
retinal degeneration, and/or for effect on CNV.
[0246] The therapeutic agents are administered as described herein.
The eye can be analyzed by opthalmoscopy (e.g., indirect
opthalmoscopy, slit lamp assessment), angiography (e.g.,
fluorescein angiography), histopathology, optical coherence
tomography (OCT), fundus photography, or a combination thereof. Any
of these methods can be used to assess efficacy in any animal model
or in humans. Compounds that show promising results in animal
studies are tested in humans, e.g., using standard protocols and
endpoints for clinical trials for therapies for ARMD or diabetic
retinopathy but it will be appreciated that agents may be
administered to humans without evidence of efficacy in animal
models.
IX. Identifying Subjects and Assessing Response
[0247] The methods of the invention may include providing a subject
to which a composition of the invention is to be administered. The
subject is typically suffering from an eye disorder characterized
by macular degeneration, CNV, or RNV. The compositions are
administered to the subject according to the inventive methods with
the intent of treating or preventing such condition. Thus the
subject will typically have been identified as being at risk of or
suffering from such a condition. Methods for diagnosis of macular
degeneration, CNV, and RNV etc., and for assessing response to
therapy are known in the art.
[0248] Any suitable tests and criteria can be used to identify a
subject at risk of or suffering from a macular degeneration related
condition, diabetic retinopathy, or CNV and/or to evaluate the
condition of a subject's eye either prior to or following therapy
(e.g., to determine whether the subject is in need of therapy or
has responded to therapy). Visual acuity can be measured using, for
example, a Snellen chart, a Bailey-Lovie chart, a decimal
progression chart, a Freiburg visual acuity test, a measurement of
minimum angle of resolution (MAR) etc. Metamorphopsia (visual
distortion) may be measured using an Amsler chart. Contrast
sensitivity may be measured using a Pelli-Robson chart. Diagnostic
studies include, but are not limited to, standard opthalmologic
examination of the fundus, stereo biomicroscopic examination of the
macula, intravenous fundus fluorescein angiography, fundus
photography, indocyanine green video-angiography, and optical
coherence tomography (OCT). OCT may be of particular use for
measuring macular thickness, an indicator of macular edema. A
subject displaying an abnormality on one or more of these
diagnostic studies (e.g., a subject that falls outside a range that
is considered normal for a healthy eye) may be treated in
accordance with the present invention.
[0249] Subjects may be classified as having early, intermediate, or
advanced ARMD in accordance with the classification scheme used in
the Age-Related Eye Diseases Study (AREDS), which is set forth in
guidelines developed American Academy of Opthalmology (American
Academy of Opthalmology, Age Related Macular Degeneration Preferred
Practice Pattern.TM., 2003; available for download at URL
www.aao.org/aao/education/library/ppp/amd_new.cfm). A subject
falling into any of these categories may be treated in accordance
with the present invention. If the subject has already developed
CNV, the subject may have classic CNV, occult CNV, or a mixture of
the two. Of course alternate classification schemes, of which a
variety is described in the literature, could be used.
[0250] ARMD is known to have a genetic component, based on studies
showing an increased incidence of ARMD in individuals with
relatives suffering from ARMD (e.g., twin studies). Therefore, a
subject may be considered at risk of developing ARMD if he or she
has one or more close relatives (e.g., parent, grandparent,
sibling, cousin, uncle, aunt), who has received a diagnosis of
ARMD. Individuals who have certain polymorphic variants of genes
encoding certain complement components, such as the factor H gene,
are particularly prone to develop ARMD (see, e.g., Klein R J, et
al., Zeiss C, Science, 308(5720):385-9, 2005; Edwards A O, et al.,
Science, 308(5720):421-4, 2005; Haines J L, et al., Science,
308(5720):419-21, 2005). In one embodiment the method comprises
providing or ascertaining a genotype of a subject, wherein the
genotype includes information as to the presence of one or more
polymorphisms predisposing to ARMD. Optionally the gene encodes a
complement component, e.g., factor H or factor B.
[0251] Individuals who smoke and/or consume a high fat diet are
also at increased risk. The incidence of ARMD increases with age.
Therefore, an individual over approximately 50 years of age,
generally at least 60 or at least 70 years of age may be considered
at increased risk. An individual having drusen and one or more
additional risk factors may be at particular risk for developing
ARMD. An individual with multiple drusen, particularly if large and
with indistinct borders, may be at particular risk. An individual
with RPE hyperpigmentation or hypopigmentation or geographic
atrophy may be at particular risk. Specific genetic mutations are
associated with various less common macular degeneration related
conditions. A subject who has received a diagnosis of diabetes is
at risk of developing diabetic retinopathy. In addition, a subject
who has developed ARMD in one eye is at increased risk of
developing the disorder in the other eye.
[0252] Response to therapy can be assessed by any of the methods
mentioned above. Numerous studies have been conducted to assess the
efficacy of a variety of different therapies in restoring vision,
preventing visual loss, and/or resulting in improvement or slowing
progression of ARMD or choroidal neovascularization as judged by
diagnostic tests such as those described above. One of ordinary
skill in the art will be able to select appropriate criteria by
which to judge the efficacy of therapy.
[0253] Rapid improvement in the condition of the subject's eye may
be, for example, any clinically significant improvement in a sign
or symptom associated with the eye disorder. For example, the
improvement may be an improvement in visual acuity (e.g., best
corrected visual acuity) such as gaining 1, 2, 3, or more lines on
an eye chart. The improvement may be a decrease in macular
thickness, e.g., a decrease by at least 50 .mu.m, at least 100
.mu.m, at least 150 .mu.m, etc. The improvement can be a decrease
in the area of exudate evident in or on the retina.
[0254] The improvement can be an increase of at least 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, or more, in any
quantitative measure of the condition of the subject's eye, where
an increase in the quantitative measure indicates improvement in
the condition of the subject's eye. The improvement can be a
decrease of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
more, in any quantitative measure of the condition of the subject's
eye, where a decrease in the quantitative measure indicates
improvement in the condition of the subject's eye. If a scoring
system is used as an indication of the condition of the subject's
eye, (e.g., a scoring system including scores ranging from 1-3,
1-5, 1-10, etc.), the improvement may be, e.g., an increase of at
least 1, 2, 3, or more units, where an increase in the score
indicates improvement in the condition of the subject's eye.
Alternately, if a scoring system in which a decrease in the score
indicates an improvement in the condition of the subject's eye is
used, the improvement may be a decrease of at least 1, 2, 3, or
more units. One of ordinary skill in the art will appreciate that a
variety of scoring systems may be used. For example, scores may be
expressed in terms of units such as "+" or "-" rather than with
integers. Of course it will be understood that individual responses
will vary, and not all subjects will respond. In animal studies and
in clinical trials, changes in the condition of a subject's eye may
be expressed in terms of an average or mean change in the
population studied and/or using appropriate statistical tests.
[0255] In one example, subjects with exudative ARMD are divided
into two groups. A variety of parameters, for example visual acuity
(e.g., best corrected visual acuity), contrast sensitivity, visual
distortion, retinal hemorrhage number or area, macular thickness,
etc., are evaluated to provide baseline indication of the condition
of the subject's eye. One group receives a single intravitreal
injection of a first therapeutic agent, e.g., an angiogenesis
inhibitor such as an anti-VEGF agent, e.g., Lucentis, Avastin, or
Macugen. The other group receives the same dose of the first
therapeutic agent and also receives a sustained release formulation
of a second therapeutic agent (e.g., a complement inhibitor such as
compstatin or a derivative thereof), with both agents being
administered together by a single intravitreal injection. The
sustained release formulation may be, e.g., a cylindrical or
screw-shaped ocular implant comprising the second therapeutic
agent, a plurality of particles comprising the agent, a composition
that forms a discrete solid or semi-solid structure such as a gel
following administration, etc. The groups are monitored over time.
Parameters such as visual acuity (e.g., best corrected visual
acuity), contrast sensitivity, visual distortion, retinal
hemorrhage number or area, macular thickness, etc., are evaluated,
preferably using the same methods and metrics as were used in the
initial evaluation to determine baseline values. For example,
resolution of macular edema may be monitored by OCT. Evaluations to
determine the number of subjects who experience rapid improvement
in the condition of a treated eye can take place, e.g., I week, 10
days, or 2 weeks following treatment. The number of subjects that
experience rapid improvement in the condition of a treated eye
(e.g., rapid decrease in macular edema and its associated visual
disturbances) is compared between the two groups. Also monitored is
the average time to destabilization, e.g., the average time before
an acute deterioration in one or more of the foregoing parameters
occurs. Also monitored (e.g., using fluorescein angiography and/or
opthalmologic examination) is the extent of neovascularization
and/or vessel leakage at various time points following treatment,
e.g., at 30, 60, 90, 120, 150, and 180 day time points and at 30
day intervals thereafter (or an appropriate subset of these time
points). The change from baseline in a retinal thickness score may
be evaluated and compared between the two groups. A greater mean
decrease in retinal thickness at one or more of the foregoing time
points in the group that received the combined therapy of the
present invention is indicative that the combined therapy of the
present invention provides a therapeutic advantage for treating the
eye disorder. The change from baseline in fluorescein leakage score
(where the fluorescein leakage score provides an indication of
neovascularization and/or vessel leakage and a higher score
indicates a greater amount of neovascularization and/or vessel
leakage) may be evaluated. A greater mean decrease in fluorescein
leakage score from baseline in the in the group that received the
combined therapy of the present invention is indicative that the
combined therapy of the present invention provides a therapeutic
advantage for treating the eye disorder.
[0256] Each of the above examples is repeated except that the first
and second therapeutic agents are both complement inhibitors or
combinations thereof. For example, the first therapeutic agent is a
VCCP and the second therapeutic agent is a GPCRA. Alternately, the
first therapeutic agent is compstatin or a derivative thereof and
the second therapeutic agent is a VCCP.
[0257] Each of the above examples is repeated except that one of
the groups receives, by a single intravitreal injection, a
composition comprising multiple therapeutic agents in a liquid
medium and a sustained release formulation containing at least one
therapeutic agent. The multiple therapeutic agents can be, for
example, different angiogenesis inhibitors. Alternately, the
multiple therapeutic agents can include a complement inhibitor and
an angiogenesis inhibitor. The sustained release formulation may
contain, for example, two or more different complement inhibitors
or a complement inhibitor and an angiogenesis inhibitor. For
example, in one embodiment the sustained release formulation
contains a compstatin analog and Lucentis.
[0258] Each of the above examples is repeated except that at least
one therapeutic agent is an RNAi agent. For example, the first or
second therapeutic agent is an siRNA that inhibits expression of
one or more endogenous pro-angiogenic molecules such as one or more
VEGF isoforms, one or more VEGF receptors, one or more complement
components, etc. In one embodiment the sustained release
formulation contains at least two RNAi agents, each of which
inhibits expression of a different pro-angiogenic molecule. For
example, the sustained release formulation may contain Cand5 and
Sirna-027.
[0259] Each of the above examples is repeated in subjects suffering
from diabetic retinopathy.
[0260] In another example the ability of the inventive compositions
and methods to inhibit progression of early ARMD (AREDS 2) to
intermediate ARMD (AREDS 3) is assessed. Subjects with early ARMD
are divided into two groups, one of which receives an inventive
combination of agents as described in either of the two examples
above while the other receives either no therapy or an alternative
therapy such as therapy with a single agent, e.g., Lucentis,
Avastin, or Macugen as described in either of the two examples
above. The groups are monitored for a period of time (e.g., as
described above). In addition the percentage of subjects that
progress from early to intermediate ARMD is determined. A lower
proportion of subjects that progress to intermediate ARMD in the
group that receives the combined therapy of the present invention
is indicative that the combined therapy of the present invention
provides a therapeutic advantage for treating the eye disorder.
[0261] In another example the ability of an inventive method to
inhibit progression of intermediate ARMD (AREDS 3) to advanced ARMD
(AREDS 4) is assessed. Subjects with intermediate ARMD are divided
into two groups, one of which receives an inventive combination of
agents as described in either of the two examples above while the
other receives either no therapy or an alternative therapy such as
Lucentis, Avastin, Macugen as described in either of the two
examples above. The groups are monitored for a period of time
(e.g., as described above). The percentage of subjects that
progress from intermediate to advanced ARMD is determined. A lower
proportion of subjects that progress to advanced ARMD in the group
that receives the combined therapy of the present invention is
indicative that the combined therapy of the present invention
provides a therapeutic advantage for treating the eye disorder.
[0262] In addition to monitoring progression of ARMD, the incidence
of side effects and complications may also be monitored.
Consideration of side effects is an important aspect when
evaluating the overall outcome and risk/benefit ratio of a therapy.
For example, if two therapies are equally efficacious in terms of
inhibiting progression of or treating ARMD, the therapy with a
lower incidence of side effects (e.g., severe complications such as
those mentioned above) is typically preferred for most subjects. In
certain embodiments of the invention therapy of a disorder such as
ARMD, or a disorder featuring CNV or RNV from any cause, using the
methods and compositions of the invention is associated with fewer
total side effects, e.g., severe complications, over time (e.g.,
over a 1-2 year period) than therapy in which multiple agents are
administered individually or therapy in which only a single
therapeutic agent is used.
EQUIVALENTS AND SCOPE
[0263] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims. In the claims and elsewhere in the specification,
articles such as "a,", "an" and "the" may mean one or more than one
unless indicated to the contrary or otherwise evident from the
context. For example, the indefinite articles "a" and "an", as used
herein in the specification and in the claims, unless clearly
indicated to the contrary, should be understood to mean "at least
one". Claims or descriptions that include "or" between one or more
members of a group are considered satisfied if one, more than one,
or all of the group members are present in, employed in, or
otherwise relevant to a given product or process unless indicated
to the contrary or otherwise evident from the context. The
invention includes embodiments in which exactly one member of the
group is present in, employed in, or otherwise relevant to a given
product or process. The invention also includes embodiments in
which more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the invention encompasses
all variations, combinations, and permutations in which one or more
limitations, elements, clauses, descriptive terms, etc., from one
or more of the listed claims (or from the portion of the
specification relevant to such claim or claim element) is
introduced into another claim. For example, and without limitation,
any claim that is dependent on another claim can be modified to
include one or more elements or limitations found in any other
claim (or from the portion of the specification relevant to such
claim or claim element) that is dependent on the same base claim.
Furthermore, where the claims or description recite a composition,
it is to be understood that methods of administering the
composition according to any of the methods disclosed herein, and
methods of using the composition for any of the purposes disclosed
herein are included, and methods of making the composition
according to any of the methods of making disclosed herein are
included, unless otherwise indicated or unless it would be evident
to one of ordinary skill in the art that a contradiction or
inconsistency would arise. The invention encompasses all
variations, combinations, and permutations in which one or more
elements, clauses, descriptive terms, etc., from one or more of the
listed claims is introduced into another claim dependent on the
same base claim unless otherwise indicated or unless it would be
evident to one of ordinary skill in the art that a contradiction or
inconsistency would arise.
[0264] Where elements are presented as lists, e.g., in Markush
group format or the like, it is to be understood that each subgroup
of the elements is also disclosed, and any element(s) can be
removed from the group. It should it be understood that, in
general, where the invention, or aspects of the invention, is/are
referred to as comprising particular elements, features, etc.,
certain embodiments of the invention or aspects of the invention
consist, or consist essentially of, such elements, features, etc.
For purposes of simplicity those embodiments have not been
specifically set forth in haec verba herein in all cases.
[0265] The inclusion of a "providing a subject . . . " step in
certain methods of the invention is intended to indicate that the
composition is administered to treat an eye disorder. Thus the
subject will have or be at risk of an eye disorder and the
composition is administered to treat the disorder, typically upon
the sound recommendation of a medical or surgical practitioner,
e.g., an ophthalmologist, who may or may not be the same individual
who administers the composition. The invention includes embodiments
in which a step of providing is not explicitly included and
embodiments in which a step of providing is included. The invention
also includes embodiments in which a step of identifying the
subject as being at risk of or suffering from a eye disorder
characterized by macular degeneration, CNV, or RNV, is
included.
[0266] Where ranges are given, endpoints are included and the
invention includes embodiments in which either or both endpoints
are excluded. Furthermore, it is to be understood that unless
otherwise indicated or otherwise evident from the context and
understanding of one of ordinary skill in the art, values expressed
as ranges can assume any specific value or subrange within the
stated ranges in different embodiments of the invention, to the
tenth of the unit of the lower limit of the range, unless the
context clearly dictates otherwise.
[0267] Any particular embodiment of the compositions or methods of
the invention (e.g., any therapeutic agent, any sustained release
formulation or any method of preparing a sustained release
formulation), any method of administration, any eye disorder or
condition or characteristic(s) thereof, or any subject
characteristic(s) can be excluded from any one or more claims, for
any reason, whether or not related to the existence of prior
art.
* * * * *
References