U.S. patent application number 14/489863 was filed with the patent office on 2015-02-05 for controlled release microparticles.
The applicant listed for this patent is VALEANT PHARMACEUTICALS INTERNATIONAL. Invention is credited to Pericles Calias, Kathleen M. Campbell, Gary P. Cook, Mary A. Ganley.
Application Number | 20150037429 14/489863 |
Document ID | / |
Family ID | 38668199 |
Filed Date | 2015-02-05 |
United States Patent
Application |
20150037429 |
Kind Code |
A1 |
Campbell; Kathleen M. ; et
al. |
February 5, 2015 |
Controlled Release Microparticles
Abstract
Formulations for controlled, sustained release of biologically
active agents for the treatment of ocular disorders have been
developed. These formulations are based on solid microparticles
formed of the combination of biodegradable, synthetic polymers such
as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and
copolymers thereof. The microparticles are characterized by low
burst levels and efficient drug loading and sustained release.
Inventors: |
Campbell; Kathleen M.;
(Longmont, CO) ; Calias; Pericles; (Melrose,
MA) ; Cook; Gary P.; (Westford, MA) ; Ganley;
Mary A.; (Norwood, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VALEANT PHARMACEUTICALS INTERNATIONAL |
Bridgewater |
NJ |
US |
|
|
Family ID: |
38668199 |
Appl. No.: |
14/489863 |
Filed: |
September 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11607382 |
Dec 1, 2006 |
8877229 |
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14489863 |
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60741741 |
Dec 2, 2005 |
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60780760 |
Mar 9, 2006 |
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60796071 |
Apr 28, 2006 |
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Current U.S.
Class: |
424/501 ;
514/44R |
Current CPC
Class: |
A61K 38/1866 20130101;
A61K 9/1647 20130101; A61K 9/0019 20130101; A61P 27/02 20180101;
A61K 9/0048 20130101; A61K 31/00 20130101 |
Class at
Publication: |
424/501 ;
514/44.R |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 9/00 20060101 A61K009/00 |
Claims
1-37. (canceled)
38. A method of treating an ocular disease of a patient in need
thereof, comprising; a. providing a formulation comprising a
plurality of microparticles comprising: (a) a core load of an
anti-VEGF aptamer; and (b) poly(lactic acid) (PLA) or poly(lactic
acid-co-glycolic acid) (PLGA) polymer; b. suspending said
formulation in a pharmaceutically acceptable liquid carrier to form
a sustained release suspension composition for intravitreal
administration; c. injecting said suspension composition into the
vitreous of the eye of the patient to treat the ocular disease,
wherein said composition releases said aptamer over a period of at
least 1 month with a 24 hour initial burst of less than 15 wt % of
said core load from said microparticles.
39. The method according to claim 38, wherein the ocular disease is
chosen from optic disc neovascularization, iris neovascularization,
retinal neovascularization, choroidal neovascularization, corneal
neovascularization, vitreal neovascularization, glaucoma, pannus,
pterygium, macular edema, vascular retinopathy, retinal
degeneration, uveitis, inflammatory diseases of the retina, and
proliferative vitreoretinopathy.
40. (canceled)
41. (canceled)
42. The method according to claim 38, wherein the ocular disease is
macular degeneration.
43. The method according to claim 38, wherein the anti-VEGF aptamer
comprises pegaptanib.
44. The method according to claim 43, wherein pegaptanib is
released from the microparticles at a rate ranging from about 0.01
to about 10 micrograms (.mu.g) per day.
45. The method according to claim 43, wherein pegaptanib is
released from the microparticles at a rate ranging from about 0.1
to about 6 .mu.g per day.
46. The method according to claim 43, wherein pegaptanib is
released at a rate sufficient to achieve pegaptanib plasma
concentrations of about 0.05-0.40 nM throughout an administration
period of at least 3 weeks.
47. The method according to claim 43, wherein pegaptanib is
released at a rate sufficient to achieve pegaptanib plasma
concentrations of about 0.05-0.40 nM throughout an administration
period of at least 6 weeks.
48. The method according to claim 43, wherein pegaptanib is
released at a rate sufficient to achieve pegaptanib vitreous
concentrations of about 10-30 ng/mL.
49. The method of claim 38, wherein the microparticles release the
anti-VEGF aptamer over a period of at least 3 months.
50. The method of claim 38, wherein the microparticles release the
anti-VEGF aptamer over a period of about 3-6 months.
51. The method of claim 38, wherein the microparticles are
syringable through a 29-gauge needle or narrower.
52. The method of claim 38, wherein the microparticles have a mean
diameter of about 30 .mu.m.
53. The method of claim 38, wherein the microparticles have a mean
diameter of about 15 .mu.m.
54. The method of claim 38, wherein the microparticles are
syringable through a 27-gauge needle or narrower and have a
diameter of less than or equal to 75% of the inner diameter of the
needle.
55. The method of claim 38, wherein the microparticles are
syringable through a 27-gauge needle or narrower and have a
diameter of less than or equal to 50% of the inner diameter of the
needle.
56. The method of claim 38, wherein the microparticles are
syringable through a 27-gauge needle or narrower and have a
diameter of less than or equal to 25% of the inner diameter of the
needle.
57. The method of claim 38, wherein the microparticles have a core
load of at least 10% by weight.
58. The method of claim 38, wherein the microparticles have a core
load of at least 15% by weight.
59. The method of claim 38, wherein the microparticles have a core
load of at least 20% by weight.
60. The method of claim 38, wherein the microparticles exhibit a 24
hour burst of less than 10 wt % of said core load.
61. The method of claim 38, wherein the microparticles exhibit a 24
hour burst of less than 5 wt % of said core load.
62. The method of claim 38, wherein the polymer has a smooth,
non-pitted external morphology.
63. The method of claim 62, wherein the polymer has a monomer ratio
of lactid:glycolide in the range of about 40:60 to 100:0.
64. The method of claim 38, wherein the suspension comprises
100-300 mg microparticles per mL of the liquid carrier.
65. The method of claim 38, wherein the microparticles have a
particle size distribution in the range of 10 .mu.m to 45 .mu.m in
diameter.
66. The method of claim 38, wherein the core load of the anti-VEGF
aptamer is at least 7 wt %.
67. The method of claim 38, wherein the liquid carrier comprises a
surfactant.
68. The method of claim 67, wherein the surfactant is chosen from
poly(vinyl alcohol), carboxymethyl cellulose, lecithin, gelatin,
poly(vinyl pyrrolidone), polyoxyethylenesorbitan fatty acid esters,
sodium dodecyl sulfate and mannitol.
69. The method of claim 68, wherein the surfactant is chosen from
polyoxyethylenesorbitan fatty acid esters, sodium dodecyl sulfate
and mannitol.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of and claims priority to
U.S. Non-Provisional patent application Ser. No. 11/607,382, filed
on Dec. 1, 2006, which claims priority to U.S. Provisional
Application No. 60/741,741, filed Dec. 2, 2005, U.S. Provisional
Application No. 60/780,760, filed Mar. 9, 2006, and U.S.
Provisional Application No. 60/796,071, filed Apr. 28, 2006,
disclosure of each of which is hereby incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to drug delivery. In particular, the
invention relates to compositions and methods for the sustained
delivery of therapeutic agents using microparticles. More
particularly, the invention relates to sustained release
microparticle compositions and methods of use for ophthalmic
administration.
BACKGROUND OF THE INVENTION
[0003] As new treatment modalities for ophthalmic diseases become
available, the number of intravitreous injections administered is
expected to increase dramatically. For example, intravitreous
injection of the vascular endothelial growth factor (VEGF)
inhibitor, Macugen.RTM. ((OSI) Eyetech, Inc. NY, N.Y.), has become
available for the treatment of age-related macular degeneration.
Macugen is currently delivered via intravitreous injection every
six weeks.
[0004] Advantages of intravitreous injection of medicines and
diagnostics include the achievement of maximum vitreous
concentrations while minimizing toxicity attributed to systemic
administration. While these advantages are becoming widely
appreciated, the ophthalmology community turns its focus to various
complications potentially associated with intravitreous injection.
Risks of intravitreous injection, some vision threatening, include
endophthalmitis, retinal detachment, iritis/uveitis, inflammation,
intraocular hemorrhage, ocular hypertension, hypotony, pneumatic
retinopexy, and cataract (R. D. Jager et al., Retina 24:676-698,
2004 and C. N. Ta, Retina, 24:699-705, 2004). Methods of minimizing
such risks include developing sustained release ophthalmic
formulations to minimize the number of intraocular injections.
[0005] Ophthalmic inserts are solid devices intended to be placed
in the conjunctival sac and to deliver the drug at a comparatively
slow rate. One such device is Ocusert.RTM. (Alza Corporation,
Mountain View, Calif.), which is a diffusion unit consisting of a
drug reservoir enclosed by two release-controlling membranes made
of a copolymer. M. F. Saettone provides a review of continued
endeavors devoted to ocular delivery. ("Progress and Problems in
Ophthalmic Drug Delivery", Business Briefing. Pharmatech, Future
Drug Delivery, 2002, 167-171). Other implant strategies have been
developed for small, highly potent, lipophilic therapeutics. (G. A.
Peyman, et al., "Delivery Systems for Intraocular Routes" Advanced
Drug Delivery Reviews, (1995) 16, 107.) While these implants are
effective for the delivery of steroids, the small size of the
implants preclude long-term (>30 days) delivery of large,
water-soluble compounds. In addition, formulation conditions for
most polymeric delivery systems are not compatible with proteins,
antibodies, and other biotherapeutics (S. P. Schwendeman et al.,
"Peptide, protein, and vaccine delivery from implantable polymeric
systems: Progress and challenges" Controlled Drug Delivery, (1997)
229).
[0006] Encapsulation of pharmaceuticals in biocompatible,
biodegradable polymer microparticles can prolong the maintenance of
therapeutic drug levels relative to administration of the drug
itself. Sustained release may be extended up to several months
depending on the formulation and the active molecule encapsulated.
In order to prolong the existence at the target site, the drug may
be formulated within a matrix into a slow release formulation (see,
for example, Langer (1998) Nature, 392, Supplement, 5-10).
Following administration, drug then is released via diffusion out
of, or via erosion of the matrix. Encapsulation within
biocompatible, biodegradable polyesters, for example, copolymers of
lactide and glycolide, has been utilized to deliver small molecule
therapeutics ranging from insoluble steroids to small peptides.
Presently, there are over a dozen lactide/glycolide polymer
formulations in the marketplace, the majority of which are in the
form of microparticles (T. Tice, "Delivery with Depot Formulations"
Drug Delivery Technology, (2004) 4(1)).
[0007] Several techniques for the production of lactide/glycolide
polymer microparticles containing biological or chemical agents by
an emulsion-based manufacturing technique have been reported. In
general, the methods include preparation of a first phase
consisting of an organic solvent, a polymer and a biological or
chemical agent dissolved or dispersed in the first solvent. A
second phase comprises water and a stabilizer and, optionally, the
first solvent. The first and second phases are emulsified and,
after an emulsion is formed, the first solvent is removed from the
emulsion, producing hardened microparticles.
[0008] Microparticles can also be produced using a
water-in-oil-in-water (w/o/w) process. W/o/w emulsions can be
considered as an aqueous emulsion of oil droplets which in turn
contain a dispersed aqueous phase. Examples of w/o/w emulsion
processes are described in U.S. Pat. Nos. 4,954,298; 5,330,767;
5,851,451 and 5,902,834, each of which are hereby incorporated
herein by reference in their entirety. The w/o/w process described
above is typically used for water-soluble molecules.
[0009] In addition, U.S. Pat. No. 6,706,289, hereby incorporated in
its entirety by reference, discloses controlled release
formulations of biologically active molecules that are coupled to
hydrophilic polymers such as polyethylene glycol and methods of
their production. The formulations are based on solid
microparticles formed of the combination of biodegradable,
synthetic polymers such as poly(lactic acid) (PLA), poly(glycolic
acid) (PGA), and copolymers thereof. PCT WO 03/092665, hereby
incorporated in its entirety by reference, discloses microsphere
formulations for the sustained delivery of an aptamer, for example,
an anti-Vascular Endothelial Growth Factor aptamer, to a
pre-selected locus in a mammal. Such formulations are further
disclosed in K. G. Carrasquillo et al., "Controlled Delivery of the
Anti-VEGF Aptamer EYE001 with Poly(lactic-co-glycolic) Acid
Microspheres," I.O.V.S. (2003) 44(1), 290.
[0010] Patient acceptance and safety are key issues that will play
a role in which treatments are used. Frequent intraocular
injections may not be favorable because they cause patient
discomfort and sometimes fear, while risking permanent tissue
damage. Therefore there remains a need for developing sustained
release ophthalmic formulations to minimize the number of
intraocular injections.
SUMMARY OF THE INVENTION
[0011] The present invention provides compositions and methods for
the sustained delivery of a biologically active agent using
microparticles. In a particular aspect, the present invention
provides sustained release microparticle compositions and methods
for ophthalmic administration.
[0012] In one aspect, the present invention provides a composition
comprising sustained release microparticles having the ability to
be administered by syringe to the eye. According to this aspect,
the present invention provides microparticle formulations
syringable through a 27-gauge needle or narrower (smaller).
[0013] In one embodiment, the microparticles release a biologically
active agent over a period of at least about 1-12 months. In a
further embodiment, the microparticles release a biologically
active agent over a period of at least about 3-6 months.
[0014] In another embodiment, the microparticles have a core load
of at least about 10% by weight of the biologically active
agent.
[0015] In another embodiment, the microparticles have an initial
24-hour in vivo burst of less than about 10% by weight of the core
load of the biologically active agent.
[0016] In another aspect, the present invention provides
compositions and methods for the sustained delivery of an aptamer
conjugated to a hydrophilic polymer such as polyethylene glycol.
According to a particular embodiment, the aptamer comprises
pegaptanib.
[0017] In another aspect, the present invention provides
compositions and methods for the sustained delivery of an anti-VEGF
agent. According to a particular embodiment, the anti-VEGF agent
comprises an aptamer.
[0018] In another aspect, the present invention provides a
composition comprising sustained release microparticles comprising
pegaptanib having the ability to be administered to a subject by a
syringe via a 27-gauge needle or smaller.
[0019] In one embodiment, the microparticles release pegaptanib
over a period of at least about 1-12 months. In another embodiment,
the microparticles release pegaptanib over a period of at least
about 3-6 months.
[0020] In another embodiment, the microparticles have a core load
of at least about 10% by weight of pegaptanib.
[0021] In another embodiment, the microparticles have an initial
24-hour in vivo burst of less than about 10% by weight of the core
load of pegaptanib.
[0022] In another aspect, the present invention provides methods of
administering sustained release microsphere formulations to achieve
a desired pharmacokinetic profile. According to one embodiment,
microparticles comprising a biologically active agent are suspended
in a pharmaceutically acceptable solution and administered by
syringe to the eye.
[0023] According to another embodiment, microparticles comprising a
biologically active agent are suspended in a solution comprising
the biologically active agent and administered by syringe to the
eye. Utilizing the vitreal residence time of the biologically
active agent as a polymer clearance window allows the polymeric
metabolites to be cleared while sustaining a therapeutically
relevant level within the eye.
[0024] The present invention has several advantages. In particular,
the microparticles of the present invention are easily suspendable
and syringable while able to provide increased duration, increased
stability, decreased burst and controlled, sustained or delayed
release of biologically active molecules in vivo.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a scanning electron micrograph showing the
external morphology of a sample of microspheres of the present
invention.
[0026] FIG. 2 is a scanning electron micrograph of cross-sectioned
pegaptanib-PLGA microspheres of the present invention having a low
burst release.
[0027] FIG. 3 is a scanning electron micrograph showing the
external morphology of a sample of microspheres of the present
invention formed by an water-in-oil-in-water process.
[0028] FIG. 4 is a schematic representation illustrating an
exemplary oil-in-water process for forming microparticles of the
present invention.
[0029] FIG. 5 is a schematic representation illustrating an
exemplary water-in-oil-in-water process for forming microparticles
of the present invention.
[0030] FIG. 6 is a schematic representation illustrating an
exemplary packed bed apparatus with various components according to
an embodiment of the present invention.
[0031] FIG. 7 is a chart representing particle size distribution of
typical microparticles before sieving.
[0032] FIG. 8 shows a RP-HPLC chromatogram used to measure the core
load and purity of pegaptanib extracted from microspheres.
[0033] FIG. 9 is a graph depicting the release profiles of various
pegaptanib-PLGA microparticles formed by an oil-in-water process.
In vitro dissolution date demonstrating control release kinetics
from Pegaptanib-PLGA microspheres.
[0034] FIG. 10 is a graph depicting the release profiles of various
pegaptanib-PLGA microparticles formed by an water-in-oil-in-water
process. In vitro dissolution rate demonstrating control release
kinetics from Pegaptanib-PLGA microspheres.
[0035] FIG. 11 is a graph depicting the results of cell
proliferation assays of human umbilical vein endothelial cells
(HUVEC) incubated with EYE001 formulations after release from PLGA
microparticles.
[0036] FIG. 12 is a graph depicting the results of 28 day in vivo
release study. The graph shows pegaptanib concentration in rabbits
plasma samples administered intravitreous with 5 mg of PLGA
microparticles containing 15% weight percent pegaptanib.
[0037] FIG. 13 is a graph depicting the in vitro release profile of
PLGA pegaptanib microspheres suspended in PBS injection vehicle
containing 0.02% surfactant at a concentration of 100 mg
microspheres per milliliter over an 86 day study.
[0038] FIG. 14 is a graph depicting the results of 86 day in vivo
release study in rabbits dosed intravitreously with 5 mg of PLGA
microparticles containing 15% weight percent pegaptanib.
[0039] FIG. 15 is a graph depicting the results of 86 day in vivo
release study in rabbits dosed intravitreously with 5 mg of PLGA
microparticles containing 15% weight percent pegaptanib compared to
liquid pegaptanib.
[0040] FIG. 16 is a graph depicting the in vitro release profile of
PLGA pegaptanib microspheres suspended in PBS injection vehicle
containing 0.02% surfactant at a concentration of 100 mg
microspheres per milliliter over an 8 month study.
[0041] FIG. 17 is a graph depicting the results of 8 month in vivo
release study in rabbits dosed intravitreously with 5 mg of PLGA
microparticles containing 15% weight percent pegaptanib.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The present invention provides compositions and methods for
the sustained delivery of a biologically active agent. In one
aspect, the present invention provides compositions and methods for
ocular sustained delivery of a biologically active agent. According
to this aspect, syringable microsphere formulations are provided
for administering a biologically active agent with a syringe via a
27-gauge needle or smaller in order to provide sustained ocular
levels of the biologically active agent.
[0043] Microparticles can vary in size, ranging from submicron to
millimeter diameters. For ophthalmic applications, the diameters of
the microparticles range from about 1 micron (.mu.m) to about 200
.mu.m. In another embodiment, the microparticles have a particle
size distribution ranging from about 1 .mu.m to about 100 .mu.m in
diameter. In another embodiment, the microparticles have a particle
size distribution ranging from about 10 .mu.m to about 50 .mu.m in
diameter.
[0044] In another embodiment, the microparticles have an average
diameter of less than about 45 .mu.m. In another embodiment, the
microparticles have an average diameter of about 30 .mu.m. In
another embodiment, the microparticles have an average diameter of
about 15 .mu.m.
[0045] In one embodiment, the microparticles are of a suitable size
and morphology allowing for delivery though a needle having a small
inner-diameter such as a narrow (small) gauge needle. In a
particular embodiment, the microparticles are of a suitable size
and morphology allowing for delivery though a needle suitable for
ophthalmic administration. Microparticles are referred to herein as
"syringable" if the are able to be delivered though a needle. In
one embodiment, the microparticles are syringable through a
medically acceptable needle. In another embodiment, the
microparticles are syringable through a needle suitable for
ophthalmic administration. Example 7 provides methods for analyzing
suspendability and syringability.
[0046] In one particular embodiment particularly suited for
ophthalmic applications, the microparticles are syringable through
a needle having a gauge of at least 27 having a nominal inner
diameter of 0.0075 inches or less. In another particular embodiment
the microparticles are syringable through a needle having a gauge
of at least 29 having a nominal inner diameter of 0.0065 inches or
less. In another particular embodiment the microparticles are
syringable through a needle having a gauge of at least 30 having a
nominal inner diameter of 0.0055 inches or less.
[0047] In one embodiment, the microparticles have a mean diameter
of less than or equal to about 75% of the inner diameter of the
needle used for injection. In another embodiment, the
microparticles have a mean diameter of less than or equal to about
50% of the inner diameter of the needle used for injection. In
another embodiment, the microparticles have a mean diameter of less
than or equal to about 25% of the inner diameter of the needle used
for injection. In another embodiment, the microparticles have a
mean diameter of less than or equal to about 10% of the inner
diameter of the needle used for injection.
[0048] In one embodiment, the microparticles form free-flowing
and/or un-agglomerated powders. Free-flowing and/or un-agglomerated
powders are advantageous because they roll with substantially no
friction and can be easily placed in containers, suspended or
incorporated into a solution suitable for injection. Flowabilty of
microparticles can be measured by any suitable means such as a
Jenike Shear Tester (Jenike & Johanson, Inc., Westford, Mass.),
which measures the direct shear strength of powders and other bulk
solid materials. Using a Jenike Shear Tester, a shear cell (base
and ring) is filled with material; a vertical load is applied to
the covered cell, using weights and the weight carrier; and the
shear cell ring is pushed horizontally across the base, with the
required force measured and recorded. Other apparatuses for
measuring flowability include a powder rheometer (Freeman
Technology, Worcestershire, UK) that measures the force of a
twisted blade along a helical path through a powder sample
establishing a required flow rate and pattern of flow. A critical
orifice and an angle of repose using an avalanche process may also
be measured.
[0049] A chart representing particle size distribution of typical
microparticles before sieving is shown in FIG. 7.
[0050] The microparticles of the present invention have a core load
sufficient to deliver the biologically active agent to maintain
therapeutically effective levels of the biologically active agent
for sustained periods. In one embodiment, the microparticles have a
core load of greater than or equal to about 5% by weight of the
biologically active agent. In one embodiment, the microparticles
have a core load of greater than or equal to about 10% by weight of
the biologically active agent. In one embodiment, the
microparticles have a core load of greater than or equal to about
15% by weight of the biologically active agent. In another
embodiment the microparticles have a core load of greater than or
equal to about 20% by weight of the biologically active agent. In
another embodiment the microparticles have a core load of greater
than or equal to about 30% by weight of the biologically active
agent.
[0051] In one embodiment, microparticles comprising pegaptanib have
a core load of greater than or equal to about 10% by weight of
pegaptanib (2% by weight on an aptamer basis). In one embodiment,
the microparticles have a core load of greater than or equal to
about 15% by weight of pegaptanib (about 3% by weight on an aptamer
basis).
[0052] The microparticles of the present invention exhibit a low
initial burst. In one embodiment, the microparticles exhibit an
initial 24 hour in vitro burst of less than or equal to about 10 wt
% of the initial core load of the biologically active agent. In one
embodiment, the microparticles exhibit an initial 24 hour in vitro
burst of less than or equal to about 5 wt % of the initial core
load of the biologically active agent.
[0053] The microparticles of the present invention provide a
sustained release of a biologically active agent. In one embodiment
the microparticles have an in vivo sustained release profile of at
least 28 days (4 weeks/1 month). In another embodiment the
microparticles have an in vivo sustained release profile of at
least about 20, 40, 60 (2 months), 90 (3 months), 180 days (6
months), or 365 days (12 months).
[0054] In one embodiment the microparticles encapsulate pegaptanib
as the biologically active agent and are adapted to release
pegaptanib at a rate ranging from about 0.01 to about 10 micrograms
(.mu.g) per day. In another embodiment, the microparticles
encapsulate pegaptanib as the biologically active agent and are
adapted to release pegaptanib at a rate ranging from about 0.1 to
about 6 .mu.g per day. In one particular embodiment pegaptanib
microparticles release pegaptanib at a rate of about 0.025 .mu.g,
0.25 .mu.g, 0.5 .mu.g, 0.75 .mu.g, 1 .mu.g, or 2 .mu.g per day.
[0055] The microparticles of the present invention have a high
encapsulation efficiency. In one embodiment, the microparticles
have an encapsulation efficiency of greater than or equal to about
80%. In another embodiment the microparticles have an encapsulation
efficiency of greater than or equal to about 90%. In another
embodiment the microparticles have an encapsulation efficiency of
greater than or equal to about 95%. In another embodiment the
microparticles have an encapsulation efficiency of about 100%.
[0056] The microparticles of the present invention have any
suitable morphology. In one embodiment, the microparticles are
solid. In another embodiment, the microparticles are smooth or
non-pitted. In another embodiment, the microparticles are
homogenous or monolithic. In another embodiment, the microparticles
have a morphology allowing for a high core load, high encapsulation
efficiency, low burst, sustained release and syringability.
[0057] FIGS. 1 and 2 show images of examples of microparticles of
the present invention. External morphological examination of the
microparticles in FIG. 1 indicates that the microparticles are
smooth and non-pitted. Internal morphological examination of the
microparticles in FIG. 2 indicates microparticles have a monolithic
interior. Monolithic microparticles give consistent release
kinetics.
[0058] Example 7 describes an experimental for the analysis of in
vitro release of microparticle formulations of the present
invention formed by the process as described in Examples 1 and 2.
The results of the in vitro release analysis are depicted in FIGS.
9, 10, 13, and 16. The results shown in the figures demonstrate the
sustained release properties of the microparticles of the present
invention. The results shown in the figures also demonstrate that
the microparticles of the present invention can be selectively
designed to control the release of a biologically active agent over
a desired time period.
[0059] In another aspect, the present invention provides methods of
administering sustained release microparticle formulations to
achieve a desired pharmacokinetic profile. According to one
embodiment, microparticles comprising a biologically active agent
are suspended in a pharmaceutically acceptable solution and
administered by syringe to the eye.
[0060] In another aspect, the present invention provides controlled
release of a biologically active agent in accordance with a desired
pharmacokinetic profile. The microparticles of the present
invention may be suspended in a solution containing an additional
amount of the same biologically active agent or a second
biologically active agent. This solution containing the dissolved
biologically active agent may provide a desired bolus of the agent
to achieve a therapeutically effective level, which is subsequently
maintained for a prolonged period by the sustained release
microparticles.
[0061] According to one embodiment, microparticles comprising a
biologically active agent are suspended in a solution comprising
the biologically active agent and administered by syringe to the
eye. In one embodiment, the microparticles are sustained release
microparticles. In another embodiment, the microparticles are
delayed release microparticles. Utilizing the vitreal residence
time of the biologically active agent as a polymer clearance window
allows the polymeric metabolites to be cleared while sustaining a
therapeutically relevant level within the eye. Utilizing the
vitreal residence time of the biologically active agent as a
polymer clearance window also allows a staggered multiple bolus of
a biologically active agent using a single administration. Such a
dosing regimen may be particularly useful for microencapsulated
biologically active agents that have some residence time in the
vitreous.
[0062] Examples of ocular formulations comprising a suspension of a
particle including an ophthalmic drug and a liquid carrier
containing at least the same ophthalmic drug is described in U.S.
Pat. Nos. 4,882,150 and 4,865,846, the contents of each are
incorporated herein by reference in their entirety.
[0063] In one embodiment, the regimen comprises the step of
administering a pharmaceutical formulation comprising a first bolus
of a first biologically active agent and a delayed release
microparticle formulation encapsulating a second bolus of the first
biologically active agent.
[0064] In a particular embodiment, the regimen comprises the step
of administering about 100 .mu.L of a pharmaceutical formulation
comprising a bolus of about 0.3 mg free pegaptanib in solution and
delayed release PLGA microparticles encapsulating about 35 mg
pegaptanib. The microparticles have an initial burst of about 5-30%
of pegaptanib and then will release at a constant rate over about a
1-month time period. At the end of the microparticle release
profile, a second burst will occur releasing a second bolus of
pegaptanib bringing the vitreal concentration to about 0.3 mg. In
another particular embodiment the regimen further comprises the
step of administering a second pharmaceutical formulation
comprising pegaptanib at a time of four weeks post after the second
burst, during which time the polymeric metabolites are cleared. In
this embodiment, the administration essentially gains an additional
month of efficacy.
[0065] One advantage of such a regimen includes the reduction of
multiple intravitreous injections of polymer encapsulated
biologically active agents. Another advantage includes limiting any
possible risk of having the clearance pathways of the eye hindered
by a buildup of polymeric breakdown products causing detrimental
effects on ocular function.
[0066] The microparticles of the present invention can be used for
any purpose. In a particular embodiment, they are administered to a
patient. They may be administered to patients in a single or
multiple dose. The microparticles may also be administered in a
single dose form that functions to release a biologically active
agent over a prolonged period of time, eliminating the need for
multiple administrations.
[0067] In one aspect, the invention provides a method of treating
or inhibiting an ocular disease state in a mammal in need thereof
using any of the microsphere compositions described herein. The
method includes administering the microparticles to a mammal in
amounts sufficient to treat or inhibit the disease. In one
embodiment, in order to treat an ocular disorder, the
microparticles are injected into the vitreous of the eye
(intravitreous injection). In another embodiment, in order to treat
an ocular disorder, the microparticles are disposed upon the outer
surface of the sclera (sub-conjunctival injection). In such a
system, once the biologically active agent is released out of the
microparticle, the biologically active agent traverses the sclera
to exert its effect, for example, reduce or inhibit the activity of
a VEGF receptor, within the eye.
[0068] The microparticles may be used to treat a variety of ocular
disorders including, for example, optic disc neovascularization,
iris neovascularization, retinal neovascularization, choroidal
neovascularization, corneal neovascularization, vitreal
neovascularization, glaucoma, pannus, pterygium, macular edema,
vascular retinopathy, retinal degeneration, uveitis, inflammatory
diseases of the retina, and proliferative vitreoretinopathy. The
corneal neovascularization to be treated or inhibited may be caused
by trauma, chemical burns and corneal transplantation. The iris
neovascularization to be treated or inhibited may be associated
with diabetic retinopathy, vein occlusion, ocular tumor and retinal
detachment. The retinal neovascularization to be treated or
inhibited may be associated with diabetic retinopathy, vein
occlusion, sickle cell retinopathy, retinopathy of prematurity,
retinal detachment, ocular ischemia and trauma. The intravitreous
neovascularization to be treated or inhibited may be associated
with diabetic retinopathy, vein occlusion, sickle cell retinopathy,
retinopathy of prematurity, retinal s detachment, ocular ischemia
and trauma. The choroidal neovascularization to be treated or:
inhibited may be associated with retinal or subretinal disorders,
such as, age-related macular degeneration, presumed ocular
histoplasmosis syndrome, myopic degeneration, angioid streaks and
ocular trauma.
[0069] Ophthalmic solutions are sterile solutions, essentially free
from foreign particles, suitably compounded and packaged for
instillation or injection into the eye. Preparation of an
ophthalmic solution requires careful consideration of such factors
as the inherent toxicity of the drug itself, isotonicity value, the
need for buffering agents, the need for a preservative (and, if
needed, its selection), sterilization, and proper packaging.
[0070] While specific reference has been made to the use of the
formulations of the present invention to administer biologically
active agent to the eye, it is to be understood that the present
invention can be used to deliver a biologically active agent to any
desired site, including, but not limited to, intraorbital,
intraocular, intraaural, intratympanic, intrathecal, intracavitary,
peritumoral, intratumoral, intraspinal, epidural, intracranial, and
intracardial. While referring to the eye, the formulations of the
present invention may be administered intravitreously or
periocularly (retrobulbarly, sub-tenonly, sub-conjunctivaly).
[0071] According to one embodiment, the microsphere formulations of
the present invention are suitable for local application for the
treatment of various cancers. According to this embodiment, the
microsphere formulations are injected locally at the tumor site or
post-operatively at the tumor site after tumor resection. According
to this embodiment, microsphere formulations and related methods of
use are provided for treating various difficult to treat cancers
such as glioblastoma multiforme, ovarian cancer, and head or neck
cancer, for example.
[0072] A formulation of the invention may be used in the treatment
of any eye disease. A formulation of the invention may also be used
to direct a biologically active agent to a particular eye tissue,
e.g., the retina or the choroid. The biologically active agent or
combination of agents will be chosen based on the disease,
disorder, or condition being treated. In addition to a biologically
active agent for a particular condition, other compounds may be
included for secondary effects, for example, an antibiotic to
prevent microbial growth. The amount and frequency of the dosage
will depend on the disease, disorder, or condition being treated
and the biologically active agent employed. One skilled in the art
can make this determination.
[0073] By "treating" is meant the medical management of a patient
with the intent that a cure, amelioration, stasis or prevention of
a disease, pathological condition, or disorder will result. This
term includes active treatment, that is, treatment directed
specifically toward improvement of a disease, pathological
condition, or disorder, and also includes causal treatment, that
is, treatment directed toward removal of the cause of the disease,
pathological condition, or disorder. In addition, this term
includes palliative treatment, that is, treatment designed for the
relief of symptoms rather than the curing of the disease,
pathological condition, or disorder; preventive treatment, that is,
treatment directed to prevention of the disease, pathological
condition, or disorder; and supportive treatment, that is,
treatment employed to supplement another specific therapy directed
toward the improvement of the disease, pathological condition, or
disorder. The term "treating" also includes symptomatic treatment,
that is, treatment directed toward constitutional symptoms of the
disease, pathological condition, or disorder.
[0074] In one embodiment, the method of the invention provides a
means for suppressing or treating an ocular neovascular disorder.
In some embodiments, ocular neovascular disorders amenable to
treatment or suppression by the method of the invention include
ischemic retinopathy, iris neovascularization, intraocular
neovascularization, age-related macular degeneration, corneal
neovascularization, retinal neovascularization, choroidal
neovascularization, retinopathy of prematurity, retinal vein
occlusion, diabetic retinal ischemia, diabetic macular edema, or
proliferative diabetic retinopathy. In still another embodiment,
the method of the invention provides a means for suppressing or
treating psoriasis or rheumatoid arthritis in a patient in need
thereof or a patient diagnosed with or at risk for developing such
a disorder.
[0075] As used herein, the terms "neovascularization" and
"angiogenesis" are used interchangeably. Neovascularization and
angiogenesis refer to the generation of new blood vessels into
cells, tissue, or organs. The control of angiogenesis is typically
altered in certain disease states and, in many cases, the
pathological damage associated with the disease is related to
altered, unregulated, or uncontrolled angiogenesis. Persistent,
unregulated angiogenesis occurs in a multiplicity of disease
states, including those characterized by the abnormal growth by
endothelial cells, and supports the pathological damage seen in
these conditions including leakage and permeability of blood
vessels.
[0076] By "ocular neovascular disorder" is meant a disorder
characterized by altered or unregulated angiogenesis in the eye of
a patient. Exemplary ocular neovascular disorders include optic
disc neovascularization, iris neovascularization, retinal
neovascularization, choroidal neovascularization, corneal
neovascularization, vitreal neovascularization, glaucoma, pannus,
pterygium, macular edema, diabetic retinopathy, diabetic macular
edema, vascular retinopathy, retinal degeneration, uveitis,
inflammatory diseases of the retina, and proliferative
vitreoretinopathy.
[0077] In addition to treating pre-existing neovascular disorders,
the therapy that includes a biologically active agent can be
administered prophylactically in order to prevent or slow the onset
of these disorders. In prophylactic applications, the biologically
active agent is administered to a patient susceptible to or
otherwise at risk of a particular neovascular disorder. The precise
timing of the administration and amounts that are administered
depend on various factors such as the patient's state of health,
weight, etc.
[0078] A "biologically active agent", "biologically active moiety"
or "biologically active molecule" can be any substance which can
affect any physical or biochemical properties of a biological
organism, including but not limited to, viruses, bacteria, fungi,
plants, animals, and humans. Biologically active molecules can
include any substance intended for diagnosis, cure mitigation,
treatment, or prevention of disease in humans or other animals, or
to otherwise enhance physical or mental well-being of humans or
animals.
[0079] Examples of biologically active agents include, but are not
limited to, nucleic acids, nucleosides, oligonucleotides, antisense
oligonucleotides, RNA, DNA, siRNA, RNAi, aptamers, antibodies,
peptides, proteins, enzymes, fusion proteins, porphyrins, and small
molecule drugs. Other biologically active agents include, but are
not limited to, dyes, lipids, cells, viruses, liposomes,
microparticles and micelles. Examples of antibodies include, but
are not limited to, anti-VEGF antibodies bevacizumab (Avastin.TM.)
and ranizumab (Lucentis.TM.). Examples of aptamers include, but are
not limited to, pegaptanib (Macugen.RTM.). Examples of porphyrins
include, but are not limited to, verteporfin (Visudine.RTM.).
Examples of steroids include, but are not limited to, anecortave
acetate (Retaane.RTM.). Examples of fusion proteins include, but
are not limited to, VEGF Trap.TM. (Regeneron Pharmaceuticals, Inc.
Tarrytown, N.Y.). Examples of RNAi include, but are not limited to,
Direct RNAi.TM. (Alnylam Pharmaceuticals, Cambridge, Mass.).
[0080] Classes of biologically active agents that are suitable for
use with the invention include, but are not limited to,
antibiotics, fungicides, anti-viral agents, anti-infective agents,
anti-inflammatory agents, anti-tumor agents, anti-tubulin agents,
cardiovascular agents, anti-anxiety agents, hormones, growth
factors, steroidal agents, and the like.
[0081] The term "anti-VEGF agent" refers to any biologically active
agent where the primary mode of action is to (a) impair binding of
any VEGF isoform to its receptor, or (b) block signaling of VEGF
receptors R1 and R2.
[0082] It will be understood that pharmaceutically acceptable salts
of the biologically active molecule disclosed herein are also
included in the present invention and can be used in the
compositions and methods disclosed herein.
[0083] The term "oligonucleotide" refers to an oligomer or polymer
of nucleotide or nucleoside monomers consisting of naturally
occurring bases, sugars and inter-sugar (backbone) linkages. The
term also includes modified or substituted oligomers comprising
non-naturally occurring monomers or portions thereof, which
function similarly. Incorporation of substituted oligomers is based
on factors including enhanced cellular uptake, or increased
nuclease resistance and are chosen as is known in the art. The
entire oligonucleotide or only portions thereof may contain the
substituted oligomers.
[0084] As used herein, the term "aptamer" means any polynucleotide,
or salt thereof, having selective binding affinity for a
non-polynucleotide molecule via non-covalent physical interactions.
An aptamer can be a polynucleotide that binds to a ligand in a
manner analogous to the binding of an antibody to its epitope. The
target molecule can be any molecule of interest. An example of a
non-polynucleotide molecule is a protein. An aptamer can be used to
bind to a ligand-binding domain of a protein, thereby preventing
interaction of the naturally occurring ligand with the protein.
[0085] "Anti-VEGF aptamers" or "VEGF aptamers" are meant to
encompass polynucleotide aptamers that bind to, and inhibit the
activity of, VEGF. Such anti-VEGF aptamers may be RNA aptamers, DNA
aptamers or aptamers having a mixed (i.e., both RNA and DNA)
composition. Such aptamers can be identified using known methods.
For example, Systematic Evolution of Ligands by Exponential
enrichment, or SELEX, methods can be used as described in U.S. Pat.
Nos. 5,475,096 and 5,270,163, each of which are incorporated herein
by reference in its entirety. Anti-VEGF aptamers include the
sequences described in U.S. Pat. Nos. 6,168,778, 6,051,698,
5,859,228, and 6,426,335, each of which are incorporated herein by
reference in its entirety. The sequences can be modified to include
5'-5' and/or 3'-3' inverted caps. (See Adamis, A. P. et al.,
published application No. WO 2005/014814, which is hereby
incorporated by reference in its entirety).
[0086] For ophthalmic drug delivery applications, exemplary disease
states include macular degeneration (dry and wet), diabetic
retinopathy, glaucoma, optic disc neovascularization, iris
neovascularization, retinal neovascularization, choroidal
neovascularization, pannus, pterygium, macular edema, vascular
retinopathy, retinal vein occlusion, histoplasmosis, ischemic
retinal disease, retinal degeneration, uveitis, inflammatory
diseases of the retina, keratitis, cytomegalovirus retinitis, an
infection, conjunctivitis, cystoid macular edema, cancer, and
proliferative vitreoretinopathy.
[0087] Classes of biologically active agents include
anti-infectives including, without limitation, antibiotics,
antivirals, and antifungals; analgesics; antiallergenic agents;
mast cell stabilizers; steroidal and non-steroidal
anti-inflammatory agents; decongestants; anti-glaucoma agents
including, without limitation, adrenergics, beta-adrenergic
blocking agents, alpha-adrenergic blocking agonists,
parasympathomimetic agents, cholinesterase inhibitors, carbonic
anhydrase inhibitors, and protaglandins; antioxidants; nutritional
supplements; angiogenesis inhibitors; antimetabolites;
fibrinolytics; wound modulating agents; neuroprotective drugs;
angiostatic steroids; mydriatics; cyclopegic mydriatics; miotics;
vasoconstrictors; vasodilators; anticlotting agents; anticancer
agents; immunomodulatory agents; VEGF antagonists;
immunosuppressant agents; and combinations and prodrugs
thereof.
[0088] The biologically active agent may be conjugated to non-toxic
long-chain polymers such as poly(ethylene glycol) (PEG). Such
polymers may increase blood circulation lifetimes, improve efficacy
and safety, and increase stability. Competitive binding studies of
aptamers utilizing this strategy revealed that the PEG unit
actively assists in the inhibitory potency of Macugen, specifically
with its ability to prevent VEGF binding to the Flt-1 receptor. (US
Patent Application Publication No. US 2005/0260651, which is hereby
incorporated herein by reference in its entirety) While the
clinical relevance is uncertain, strong evidence indicates that
inhibition of Flt-1 binding is a major contributor to the
anti-inflammatory properties of Macugen (Usui, T. et al. (2004)
"VEGF164(165) as the Pathological Isoform: Differential Leukocyte
and Endothelial Responses through VEGFR1 and VEGFR2." I.O.V.S.
45(2), 368).
[0089] Specific biologically active agents include MACUGEN.RTM.
(pegaptanib sodium injection) as described in U.S. Pat. No.
6,051,698, herein incorporated in its entirety by reference.
Pegaptanib is also referred to as EYE001 (previously referred to as
NX1838).
[0090] Pegaptanib is a covalent conjugate of an oligonucleotide of
twenty-eight nucleotides in length that terminates in a pentylamino
linker, to which two 20-kilodalton (kDa) monomethoxypolyethylene
glycol (PEG) units are covalently attached via the two amino groups
on a lysine residue. Pegaptanib is optionally provided in the form
of a pharmaceutically acceptable salt. In one embodiment,
pegaptanib is provided as pegaptanib sodium. The molecular formula
for pegaptanib sodium is
C.sub.294H.sub.342F.sub.13N.sub.107Na.sub.28O.sub.188P.sub.28(C-
.sub.2H.sub.4O), (where n is approximately 900) and the molecular
weight is approximately 50 kDa.
[0091] The chemical name for pegaptanib sodium is as follows: RNA,
((2'-deoxy-2'-fluoro)C-G.sub.m-G.sub.m-AA-(2'-deoxy-2'-fluoro)U-(2'-deoxy-
-2'-fluoro)C-A.sub.m-G.sub.m-(2'-deoxy-2'-fluoro)U-G.sub.m-A.sub.m-A.sub.m-
-(2'-deoxy-2'-fluoro)U-G.sub.m-(2'-deoxy-2'-fluoro)C-(2'-deoxy-2'-fluoro)U-
-(2'-deoxy-2'-fluoro)U-A.sub.m-(2'-deoxy-2'-fluoro)U-A.sub.m-(2'-deoxy-2'--
fluoro)C-A.sub.m-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)C-(2'-deoxy-2'--
fluoro)C-G.sub.m-(3.fwdarw.3')-dT), 5'-ester with
.alpha.,.alpha.'[4,12-dioxo-6-[[[5-(phosphoonoxy)pentyl]amino]carbonyl]-3-
,13-dioxa-5,11-diaza-1,15-pentadecanediyl]bis[.omega.-methoxypoly(oxy-1,2--
ethanediyl)], sodium salt.
[0092] Dosage levels of pegaptanib sodium on the order of about 1
.mu.g/kg to 100 mg/kg of body weight per administration are useful
in the treatment of neovascular disorders. Examples of formulations
are found in WO 03/039404, which is hereby incorporated by
reference in its entirety. In some embodiments, pegaptanib sodium
is administered at a dosage of about 0.1 mg to about 1.0 mg locally
into the eye, wherein the treatment is effective to treat occult,
minimally classic, and predominantly classic forms of wet macular
degeneration. When administered directly to the eye, the dosage
range is about 0.3 mg to about 3 mg per eye, in some embodiments
the dosage range is about 0.1 mg to about 1.0 mg per eye. In one
embodiment, pegaptanib sodium is administered in a therapeutically
effective amount of about 0.1-3.0 mg, 0.1-1.0 mg, or about 0.3 mg.
In one embodiment, pegaptanib sodium is present in an ophthalmic
injection solution formulation at a concentration ranging from 0.1
to 3.0 mg/mL. According to one embodiment, the carrier comprises
sodium phosphate and sodium chloride. According to one specific
embodiment the carrier comprises 10 mM sodium phosphate and 0.9%
sodium chloride.
[0093] According to one embodiment, the dose is effective to
achieve a vitreous concentration of the anti-VEGF aptamer of about
10-30 ng/mL. According to another embodiment, the dose is effective
to maintain a vitreous concentration of the anti-VEGF aptamer of
about 10-30 ng/mL throughout the administration period.
[0094] In alternative embodiments, the anti-VEGF agent is an
anti-VEGF aptamer and is administered at a dosage of less than 0.3
mg to about 0.003 mg locally into the eye. In some embodiments, the
anti-VEGF aptamer is administered at a dosage less than about 0.30
mg. Examples of such formulations are found in U.S. Patent
Application Ser. No. 60/692,727; which is incorporated herein by
reference in its entirety.
[0095] As used herein, "microparticles" refers to particles having
a diameter of typically less than 1.0 mm, and more typically
between 1.0 and 250 microns.
[0096] The microparticles of the present invention include, but are
not limited to, microspheres, microcapsules, microsponges,
microgranules and particles in general, with an internal structure
comprising a matrix of agent and excipient. Microparticles may also
include nanoparticles.
[0097] Microspheres are typically solid spherical microparticles.
Microcapsules are typically spherical microparticles typically
having a core of a different polymer, drug, or composition.
[0098] As used herein, the term "nanoparticles" refers to particles
having a diameter of typically between about 20 nanometers (nm) and
about 2.0 microns (.mu.m), typically between about 100 nm and about
1.0 .mu.m.
[0099] An "injection" is a preparation intended for parenteral
administration. Injections include, but are not limited to, liquid
preparations that are drug substances or solutions or suspensions
thereof.
[0100] The grammatically correct and preferred term "intravitreous"
is used herein and in the art. The term "intravitreal" is used
colloquially as an alternative to the term "intravitreous" for
injections into the eye's vitreous humor between the lens and the
retina.
[0101] The term "controlled release" refers to control of the rate
and/or quantity of biologically active molecules delivered
according to the drug delivery formulations of the invention. The
controlled release kinetics can be continuous, discontinuous,
variable, linear or non-linear. This can be accomplished using one
or more types of polymer compositions, drug loadings, inclusion of
excipients or degradation enhancers, or other modifiers,
administered alone, in combination or sequentially to produce the
desired effect. "Controlled release" microparticles include, but
are not limited to, "sustained release" microparticles and "delayed
release" microparticles.
[0102] The term "sustained release" refers to releasing a
biologically active agent into the body steadily, over an extended
period of time. Sustained release formulations offer the ability to
provide a subject with a biologically active agent over a time
period greater than that achieved by a typical bolus administration
of the biologically active agent. Sustained release microparticles
may advantageously reduce the dosing frequency of a biologically
active agent.
[0103] "Zero-order" or "linear release" is generally construed to
mean that the amount of the biologically active molecule released
over time remains relatively constant as a function of amount/unit
time during the desired time frame. Multi-phasic is generally
construed to mean that release occurs in more than one "burst".
[0104] The term "packed bed apparatus" refers to any vessel
containing packing material capable of creating an emulsion upon
contact with two immiscible fluids.
[0105] The term "biodegradable" or "bioerodible," as used herein,
refer to polymers that dissolve or degrade within a period that is
acceptable in the desired application (usually in vivo therapy),
typically less than about five years, and more preferably less than
about one year, once exposed to a physiological solution of pH
ranging from about 6 to about 9 and at a temperature of ranging
from about 25 C to about 38 C.
[0106] A variety of biodegradable polymers used for controlled
release formulations are well known in the art. Suitable polymers
for example include, but are not limited to, poly(hydroxy acids),
poly(lactic acid), poly(glycolic acid), poly(lactic
acid-co-glycolic acid), polycaprolactones, polyanhydrides,
polycarbonates, polyamides, polyesters, polyorthoesters,
polyhydroxybutryate, certain types of protein and polysaccharide
polymers, and blends, copolymers or mixtures thereof.
[0107] The biodegradable polymers are optionally capped or
un-capped. Capped polymers include, but are not limited to, those
having esterified or amidated end groups. Un-capped polymers
include free hydroxyl or carboxyl end-groups. In one embodiment,
the microparticles comprise free-acid poly(lactic acid-co-glycolic
acid). In another embodiment, the microparticles comprises lauryl
or N-capped poly(lactic acid-co-glycolic acid).
[0108] Preferred polymers include poly(hydroxy acids). In one
embodiment the polymer is poly(lactic acid-co-glycolic acid)
("PLGA") that degrade by hydrolysis following exposure to the
aqueous environment of the body. The polymer is then hydrolyzed to
yield lactic and glycolic acid monomers, which are normal
byproducts of cellular metabolism. The rate of polymer
disintegration can vary from several weeks to periods of greater
than one year, depending on several factors including polymer
molecular weight, ratio of lactide to glycolide monomers in the
polymer chain, and stereoregularity of the monomer subunits
(mixtures of L and D stereoisomers disrupt the polymer
crystallinity enhancing polymer breakdown). Microparticles may
contain blends of two and more biodegradable polymers, of different
molecular weight and/or monomer ratio.
[0109] PLGA may have any suitable monomer ratio of
lactide:glycolide. In one embodiment the amount of lactide ranges
from 40-100%. In another embodiment the amount of glycolide ranges
from 0-60%. In one embodiment, the PLGA has a monomer ratio of
lactide:glycolide in the range of about 40:60 to 100:0. In another
embodiment, the PLGA has a monomer ratio of lactide:glycolide in
the range from about 45:55 to 100:0. In one particular embodiment,
the PLGA has a monomer ratio of lactide:glycolide of about 50:50.
In another particular embodiment, the PLGA has a monomer ratio of
lactide:glycolide of about 65:35. In another particular embodiment,
the PLGA has a monomer ratio of lactide:glycolide of about 75:25.
The particular ratio of the polymers may be determined based on
pharmacokinetic evaluations.
[0110] The microparticles may release a biologically active agent
by any suitable means to allow for a controlled release of the
biologically active agent. While not wishing to be bound by theory,
the microparticles can release the biologically active agent by
bulk erosion, diffusion or a combination of both.
[0111] A surfactant is optionally used in order to provide
formulations that have the required syringability. In one
embodiment, a surfactant is used for providing a stable emulsion
during the process of forming the microparticles of the present
invention. In another embodiment, a surfactant is used for
preventing agglomeration during lyophilization during the process
of forming the microparticles. In another embodiment, a surfactant
is used for preventing agglomeration within the injection vehicle
during the process of delivering the microparticles. Without
wishing to be bound by theory, surfactants may provide
batch-to-batch consistency of microparticles by forming a thin
layer of material around the microparticles that helps prevent
clumping. Any suitable surfactant may be used. Suitable surfactants
include, but are not limited to, cationic, anionic, and nonionic
compounds such as poly(vinyl alcohol), carboxymethyl cellulose,
lecithin, gelatin, poly(vinyl pyrrolidone), polyoxyethylenesorbitan
fatty acid ester (Tween 80, Tween 60, Tween 20), sodium dodecyl
sulfate (SDS), mannitol and the like.
[0112] In one embodiment, the microparticles are formed using an
emulsion comprising poly(vinyl alcohol). In a particular
embodiment, the microparticles are formed using an emulsion
comprising 1.0% poly(vinyl alcohol). The concentration of
surfactant in the process medium is established to be an amount
sufficient to stabilize the emulsion.
[0113] In one embodiment, the microparticles are lyophilized in a
solution comprising SDS, Tween 20 or mannitol. In one particular
embodiment, the microparticles are lyophilized in a solution
comprising 7.8% SDS.
[0114] In one embodiment, the microparticles are suspended in an
injection solution comprising SDS, Tween 20 or mannitol. In one
particular embodiment, the microparticles are suspended in an
injection solution comprising 0.5% SDS.
[0115] In one embodiment, the inherent viscosity of the
biodegradable polymer may be in the range 0.1 to 2.0 dL/g. In
another embodiment, the inherent viscosity of the biodegradable
polymer ranges from about 0.1 to about 1.0 dL/g. In another
embodiment, the inherent viscosity of the biodegradable polymer is
about 0.16 dL/g, 0.35 dL/g or 0.61 dL/g.
[0116] Derivatized biodegradable polymers are also suitable for use
in the present invention, including hydrophilic polymers attached
to PLGA and the like. To form microparticles, in particular, a
variety of techniques known in the art can be used. These include,
for example, single or double emulsion steps followed by solvent
removal. Solvent removal may be accomplished by extraction,
evaporation or spray drying among other methods.
[0117] In a typical solvent extraction method, a polymer is
dissolved in a continuous phase (e.g., an organic solvent) that is
at least partially soluble in a discontinuous phase (e.g., an
extraction solvent such as water). A biologically active molecule,
either in soluble form or dispersed as fine particles, is then
added to the polymer solution, and the mixture is dispersed into an
aqueous phase that contains a surface-active agent such as
poly(vinyl alcohol). The resulting emulsion is added to a larger
volume of water where the organic solvent is removed by extraction
or evaporation from the polymer/biologically active agent to form
hardened microparticles.
[0118] Microparticles of the present invention may be prepared
using any suitable method. In one embodiment, the microparticles
are prepared using an emulsion process. A general scheme
illustrating the process is shown in FIG. 8. In general, the
methods combine a first phase and a second phase and pass the
combination though an emulsifier. After an emulsion is formed, the
solvent of the first phase is removed from the emulsion in an
extractor, producing hardened microparticles. The microparticles
are then sieved and dried.
[0119] In one embodiment, the methods have a first phase consisting
of an organic solvent, a polymer and a biological or chemical agent
dissolved or dispersed in the first solvent. The second phase
comprises water and a stabilizer and, optionally, the first
solvent. The first and the second phases are emulsified and, after
an emulsion is formed, the first solvent is removed from the
emulsion, producing hardened microparticles.
[0120] In another embodiment, the methods use a non-aqueous oil-in
oil (o/o) process for making microparticles. In general an o/o
method includes the steps of: a) dissolving a biocompatible polymer
in a solvent to form a solution; b) combining a biologically active
agent with the solution to produce a mixture; c) optionally
combining the mixture of step (b) with a coacervating agent
(optionally, while homogenizing the solution); and d) permitting
the biocompatible polymer to form microparticles containing the
biologically active agent. (See PCT Publication No. WO 03/092665
and K. G. Carrasquillo et al., "Controlled Delivery of the
Anti-VEGF Aptamer EYE001 with Poly(lactic-co-glycolic) Acid
Microspheres," I.O.V.S. (2003) 44(1), 290, each of which are hereby
incorporated herein by reference in their entirety.)
[0121] In another embodiment, the methods use a
water-in-oil-in-water (w/o/w) process for making microparticles.
Such microparticles can be prepared by forming a primary
water-in-oil emulsion comprising a water soluble
molecule-containing solution as the inner aqueous phase and a
polymer-containing solution as the oil phase. This primary emulsion
can then be dispersed in an outer aqueous phase to form the final
water-in-oil-in-water emulsion. The w/o/w tri-phasic emulsion can
then be allowed to stir for a set period of time to promote
extraction and evaporation of the organic solvent in which the
solvent of the oil phase is removed and results in formation of
hardened microparticles. Examples of w/o/w emulsion processes are
described in U.S. Pat. Nos. 4,954,298; 5,330,767; 5,851,451 and
5,902,834, each of which are hereby incorporated herein by
reference in their entirety. FIG. 5 illustrates an exemplary w/o/w
process for forming microparticles of the present invention.
Microparticles of the present invention, formed by a w/o/w process,
are described in Example 2.
[0122] Emulsions may be formed by any suitable method. In one
embodiment, a batch device for mixing the first and second phases
under turbulent conditions such as with a stirrer as disclosed in
U.S. Pat. No. 5,407,609, which is hereby incorporated herein by
reference in its entirety. Other batch processes may employ a
homogenizer or a sonicator. In another embodiment, an emulsion is
formed by continuously mixing the first phase and second phase,
in-line, using turbulent flow conditions, as in the use of an
in-line dynamic mixer or an in-line static mixer such as described
in U.S. Pat. No. 5,654,008, which is hereby incorporated herein by
reference in its entirety.
[0123] In one embodiment, the microparticles are prepared according
to the process disclosed in PCT publication No. WO 2005/003180,
which is hereby incorporated by reference in its entirety, which
discloses an emulsion-based technique employing a packed bed system
that uses laminar flow conditions to produce an emulsion that
results in microparticles containing biological or chemical agents
after solvent removal.
[0124] The packed bed apparatus for the production of
microparticles through an emulsion-based technique may be a vessel
of any shape capable of being filled with packing material that
allows liquid to flow through it (See FIG. 6). The apparatus of the
present invention may further provide a material capable of
insertion into both ends for enclosure of materials in such
apparatus. FIG. 6 illustrates an exemplary apparatus according to
one embodiment of the present invention. In this embodiment, a tube
(1) is filled with beads as packing material (2).
[0125] Microparticle morphology is observed by Scanning Electron
Microscopy (SEM) analysis. Microparticles are sputter coated with
gold using an Anatech LTD Hummer 6.2 system. Scanning electron
microscopic images were taken using a JEOL JSM-5600 scanning
electron microscope and accompanying software at an accelerating
voltage of 5-10 keV. For selected samples, SEM analysis of the
internal microsphere structure was made after embedding
microparticles in L.R. White Resin and then splitting the
preparation after the resin hardened. Sample images are shown in
FIGS. 1 and 2.
[0126] The microparticles of the present invention can be stored as
a dry material such as a sterile lyophilized (or freeze dried)
powder. In the instance of administration to a patient, prior to
such use, the dry microparticles can be suspended in any
pharmaceutically acceptable vehicle. Upon suspension, the
microparticles may then be injected into the patient or otherwise
utilized. Suitable pharmaceutically acceptable vehicles include,
but are not limited to, a liquid vehicle, a suspension vehicle or
an injection vehicle. The vehicle may include a surfactant, such as
SDS, Tween 20 or mannitol.
[0127] Microparticles may be present in any suitable formulation.
Methods well known in the art for making formulations are found,
for example, in Remington: The Science and Practice of Pharmacy
(20th ed., A. R. Gennaro ed., Lippincott: Philadelphia, 2000).
Microparticles may be administered to humans, domestic pets,
livestock, or other animals with a pharmaceutically acceptable
diluent, carrier, or excipient. In one embodiment, the
microparticles may be present in any suitable formulation for
delivery to the eye.
[0128] Conventional pharmaceutical practice may be employed to
provide suitable formulations or compositions to administer the
identified compound to patients suffering from a disease, disorder,
or condition of the eye. Administration may begin before, during,
or after the patient is symptomatic.
[0129] In one embodiment, the microparticles are suspended in an
acceptable pharmaceutical liquid vehicle, such as a 2.5 wt. %
solution of carboxymethyl cellulose in water. In another
embodiment, pegaptanib microparticles are suspended in an aqueous
solution comprising 10 mM Sodium Phosphate, 136.9 mM Sodium
Chloride, 2.7 mM Potassium Chloride, 0.05% Tween 20, and pH 7.4
(Filtered 0.2 .mu.m). In another embodiment, pegaptanib
microparticles are formulated for a dose of 1 mg of pegaptanib per
100 .mu.L of microparticle solution. In another embodiment,
pegaptanib microparticles are suspended in a solution comprising
pegaptanib. In a particular embodiment, pegaptanib microparticles
are suspended in a solution comprising pegaptanib formulated at
3.47 mg/mL, measured as the free acid form of the oligonucleotide,
sodium chloride, monobasic sodium phosphate monohydrate, dibasic
sodium phosphate heptahydrate, hydrochloric acid, and/or sodium
hydroxide to adjust the pH and water for injection.
[0130] The volume of injection will depend on the route of
administration. A typical intravitreous injection requires a 27
gauge needle or narrower and a delivered volume less than or equal
to about 150 .mu.L. A typical subconjunctival injection can
accommodate a 23 gauge needle and a volume of up to 750 .mu.L.
[0131] Sustained release formulations can be advantageous by
reducing intravitreous (IVT) dosing frequencies of therapy
involving biologically active agents. Applicants evaluated the in
vivo release properties of pegaptanib-loaded microparticles as set
forth in Example 9 and thereby demonstrated the feasibility of
delivering pegaptanib over a period of approximately one month or
more from a poly(lactide-co-glycolide) (PLGA) polymer based
sustained release formulation.
[0132] Elimination of pegaptanib from the eye into systemic
circulation is considered to be the rate-limiting step of
pegaptanib's plasma pharmacokinetics. Therefore the plasma
pharmacokinetics of pegaptanib should mirror the in vivo release of
pegaptanib from the sustained release formulation. Following
bilateral intravitreous (IVT) administration of liquid pegaptanib
(pegaptanib sodium in phosphate buffered saline solution), plasma
concentrations declined slowly overtime. The terminal phase rate
constant, in plasma, reflected the slow absorption of pegaptanib
from the eye into the systemic circulation after an IVT injection
(see FIG. 12). Since it is expected that pegaptanib will be
released in a sustained fashion from PLGA formulations into the
vitreous, it is expected that the pegaptanib released into the
vitreous will have distribution and clearance properties similar to
those of pegaptanib administered by a phosphate buffered saline
solution.
[0133] Plasma concentrations resulting from a 28 day in vivo ocular
distribution study in rabbits dosed intravitreously with 5 mg of
PLGA microparticles containing 15% weight percent pegaptanib are
shown in FIG. 12. The results show that the microparticles have a
low burst release. A large burst release, common to PLGA
formulations containing water soluble or hydrophilic compounds, is
absent. In addition, plasma pegaptanib concentration levels were
measured at a relatively constant level between 0.05-0.4 nM over
the 28 day study period relative to an equivalent IVT liquid
pegaptanib dose, indicating a sustained release of pegaptanib in
the vitreous was achieved.
[0134] It is known in the art that modifying the polymer
composition of a sustained release microparticle formulation
affects the rate of polymer decomposition in vivo and therefore
effects the release characteristics of the microparticle
formulation. Therefore demonstrating a release duration of one
month from a microparticle formulation in vivo indicates that a
release duration of greater than one month from a microparticle
formulation in vivo is feasible.
EXAMPLES
[0135] The following examples serve to illustrate certain useful
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof. Alternative materials and
methods can be utilized to obtain similar results.
Example 1
Preparation of Microparticles
Oil-in-Water
[0136] Formulations were prepared via an oil-in-water solvent
extraction/evaporation method. Macugen.RTM.; pegaptanib sodium
((OSI) Eyetech, Inc., NY, N.Y.) and PLGA were dissolved in
methylene chloride and an emulsion was formed according to the
process disclosed in PCT publication No. WO 2005/003180, which is
incorporated herein by reference in its entirety. Following solvent
extraction from the emulsion particles, the hardened microparticles
were sieved through a 45 .mu.m screen. Microparticles .ltoreq.45
.mu.m were collected by centrifugation and dried by
lyophilization.
Example 2
Preparation of Pegaptanib Microparticles
Water-in-Oil-in-Water
[0137] A batch size of 200 milligrams dry microspheres containing
pegaptanib was prepared according to the following procedure:
Step 1.
[0138] Preparation of primary aqueous phase [0139] a. 30 mg
Pegaptanib [0140] b. 300 .mu.L water [0141] c. The mixture was
vortexed to dissolve components
Step 2.
[0142] Preparation of organic phase [0143] a. 200 mg PLGA (i.e.
50:50 lactide:glycolide, IV=0.37 dL/g) [0144] b. 7 ml methylene
chloride (CH.sub.2Cl.sub.2) [0145] c. The mixture was vortexed to
dissolve components
Step 3.
[0146] Preparation of secondary aqueous phase/quench solution
[0147] b. 10.2 g polyvinyl alcohol [0148] c. 104 g sucrose [0149]
d. 1.25 mL 1M Tris, pH 8.0 [0150] e. 1 mL 0.5M EDTA, pH 8.0 [0151]
f. All components were dissolved in .about.800 mL water. The pH was
adjusted to 7.4 and the final volume to was brought to 1 L.
Step 4.
[0152] The organic solution was homogenized at 20000 RPM for a
total of 2 minutes using a Virtis homogenizer. While homogenizing,
the primary aqueous (drug containing) solution was slowly injected
through 21G needle over 20 seconds to form primary water-oil
emulsion.
Step 5.
[0153] The secondary aqueous phase (35 mL) was homogenized at 20000
RPM for 1 minute. The primary emulsion was immediately transferred
into a beaker containing homogenized secondary aqueous phase to
form a secondary water-oil-water emulsion.
Step 6.
[0154] The secondary emulsion was poured into a 250 mL beaker
containing 100 mL quench solution (stirring speed=4) to extract
CH.sub.2Cl.sub.2. Allowed to stir at room temperature for 3.5
hours.
Step 7.
[0155] The material was transferred to a collection vessel for
centrifugation and washing. [0156] a. Centrifuged at 1500 RPM for
15 minutes and pour off supernatant. [0157] b. Repeated
centrifugation and decanting of supernatant two more times.
Step 8.
[0158] The final pellet was re-suspended in DI water and lyophilize
for about 96 hours (4 days). During first 24 hours on lyophilizer,
the vacuum on system was purged every 2 hours.
Example 3
Morphology Analysis of Microparticles
[0159] Microparticle morphology was observed by Scanning Electron
Microscopy (SEM) analysis. Microparticles were sputter coated with
gold using an Anatech LTD Hummer 6.2 system. Scanning electron
microscopic images were taken using a JEOL JSM-5600 scanning
electron microscope and accompanying software at an accelerating
voltage of 5-10 keV. For selected samples, SEM analysis of the
internal microsphere structure was made after embedding
microparticles in L.R. White Resin and then splitting the
preparation after the resin hardened.
[0160] Scanning electron micrograph images of microparticles formed
by the process as set forth in Example 1 are shown in FIGS. 1 and
2. The images of FIG. 1 show that the microparticles have a smooth
external morphology. The image of FIG. 2 shows that the
microparticles have a monolithic internal morphology.
[0161] A scanning electron micrograph image of microparticles
formed by the water-in-oil-in water process as set forth in Example
2 are shown in FIG. 3. The image shows that the microparticles have
a particle size of less than about 10 .mu.m and a smooth external
morphology.
[0162] The in vitro and in vivo release analysis methods as set
forth in Example 7 and Example 8 below indicate that the smooth,
monolithic morphology of the microparticles release pegaptanib in a
sustained release manner with a low burst.
Example 4
Analysis of Pegaptanib Coreload and Purity
[0163] Pegaptanib microspheres were prepared as set forth in
Example 1 above. The microspheres were analyzed to determine if
pegaptanib was degraded during their preparation. The microsphere
preparations were characterized for Pegaptanib content and purity
by HPLC. Approximately 6.5 mg of formulation was accurately weighed
into a 2 mL microcentrifuge tube. The formulation was dissolved in
1.0 mL of 5% (water)/95% (acetonitrile), and the polymer
precipitated by the addition of 1.0 mL 10-mM Na-phosphate pH 2.5.
The resulting cloudy suspension was vortexed and clarified by
centrifugation at 14,000 rpm for 2 minutes. Clear supernatant was
assayed by HPLC. Samples were assayed against a 670.8 .mu.g/mL
EYE-001 (equivalent to 126.5 .mu.g/mL oligomer) standard solution
prepared in water and stored at -80.degree. C.
[0164] FIG. 8 shows a RP-HPLC chromatogram used to measure the core
load and purity of pegaptanib extracted from the microspheres. The
chromatogram shows a high core load (12.3%) and that the purity of
pegaptanib (99.18%) is unchanged by the formulation process.
Example 5
Analysis of Surfactant Effect on Lyophilization
[0165] Microparticles were prepared with 50:50-2.5 A (Alkermes)
polymer and a 15% initial load of pegaptanib as set forth in
Example 1. Following solvent extraction the microspheres were
sieved through a 45 .mu.m screen and collected on a 25 .mu.m
screen. The microspheres were washed with water and the batch was
split in three. One fraction was lyophilized in water. The second
fraction was lyophilized in SDS, such that the dried product was
7.8% (w/w) SDS. The third fraction was lyophilized in Tween20, such
that the dried product was 0.24% (w/w) Tween20. In order to gauge
suspendability and syringability, 100 .mu.l vehicle was added to 20
mg pegaptanib microspheres in a 1.5 ml centrifuge tube. The
following vehicles were tested: PBS; PBS with 0.5% SDS; PBS with
0.02% Tween20; PBS with 0.5% CMC; and PBS with 0.5% CMC and 0.2%
Tween20. The mixture was suspended by tapping to determine ease of
suspendability. The mixture was then vortexed for several seconds.
The mixture was examined for uniformity of suspension and
syringability. The results are shown in Table 1.
TABLE-US-00001 TABLE 1 077-141 077-141 077-151 077-141 0.24%
Tween20 7.8% SDS 2% Mannitol PBS Suspend easily Suspend OK Visibly
Clumpy Inject OK, slight Some sticks to side Sticks to side
clogging Inject OK, slight Injects OK at first, Little force
required clogging worst with time after settling Little force
required after settling PBS + Difficult to Suspend easily 0.02%
Tween 20 suspend Injects well Inject Fine Little force required
Settle quickly after settling PBS + 0.5% SDS Suspend easily Injects
well Little force required after settling Foamy PBS + 0.5% CMC
Suspend OK Suspend OK Injects with some Inject OK clogging Even
after settling PBS + 0.5% CMC + Very difficult to Suspend easily
Injects OK 0.02% Tween20 suspend Injects well Even with settling
Clog initially then Stays in suspension OK well
Example 6
Analysis of Suspendability and Syringability
[0166] Aliquots of 10, 20 or 30 mg of pegaptanib microspheres,
prepared by the method as set forth in Example 1, were added to 100
.mu.L vehicle (0.5% CMC and 0.05% SDS in PBS). The mixture was
examined for uniformity of suspension. The solution was then drawn
into a 1 cc syringe through a 1/2 inch, 27 gauge needle and held
for a time before pushing the sample out through the same needle.
The procedure was repeated for a 1/2 inch, 29 gauge needle. The
test was repeated after allowing microspheres to settle toward the
syringe hub for 30 seconds. The following vehicles were tested:
Saline; PBS; PBS with 0.05 or 0.5% SDS; PBS with 0.02% Tween20; PBS
with 1% Mannitol; PBS with 0.5% SDS and 0.5% CMC; and PBS with
0.02% Tween20 and 0.5% CMC. The results are shown in Tables 2 and
3.
TABLE-US-00002 TABLE 2 200 mg micro- spheres/ 100 mg
microspheres/ml 150 mg/ml ml Saline Visibly clumpy Sticks to tube
Injects fine PBS Sticks to tube Injects fine PBS +0.1% Sticks to
tube SDS Injects fine Force required after settling PBS + 0.5%
Injects fine Difficult to SDS Injects fine after settling suspend
Foamy Settles quickly Clogs PBS + 0.02% Clogged initially, then
Difficult Difficult to Tween.sub.20 injected fine to suspend
Injects fine after settling suspend Withdraws Clogs/ nicely
pancakes Pancakes on injection PBS + 1% Sticks to tube Mannitol
Injects fine initially, then visible clumping PBS + 0.5% Difficult
to suspend CMC Sticks to tube Injects fine Injects fine after
settling PBS + 0.5% Difficult to suspend CMC + 0.5% Injects fine
SDS Injects fine after settling PBS + 0.5% Difficult to suspend
Difficult CMC + 0.02% Clogged on withdrawal to suspend Tween.sub.20
Pancake formed on injection Pancakes on injection
TABLE-US-00003 TABLE 3 >45 .mu.m <25 .mu.m (Lot 45-38 .mu.m
38-25 .mu.m (Lot 077- 077- (Lot 077- (Lot 077- 110) 132) 132) 132)
100 200 100 100 200 100 200 mg/ mg/ mg/ml mg/ml mg/ml mg/ml mg/ml
ml ml PBS clumps clumps PBS clumps clumps 0.05% SDS PBS Can Injects
Injects Injects In- Injects 0.5% not well well well jects well SDS
draw Even Even Even well Even into w/ w/ w/ w/ syringe settling
settling settling settling PBS, Injects Injects 0.02% well well
Tween20 PBS clumps 1% Mannitol
Example 7
In Vitro Release of Pegaptanib Microspheres
[0167] The microspheres were analyzed to determine the in vitro
release profile of pegaptanib. In vitro release of pegaptanib from
microsphere formulations was determined in PBS (pH 7.4) containing
0.02% Tween-20 and 0.05% sodium azide. Typically 10 mg of
microspheres were added to 1 mL buffer in a capped tube and placed
in a shaking (150 cycles per minute) water bath incubator at
37.degree. C. The release medium supernatant was sampled
periodically and assayed for conjugate quantity and approximate
purity by the reverse phase HPLC. The "burst release" was
determined by the percentage of drug that was released in the first
three hours of incubation.
[0168] The release profiles were characterized by a low initial
burst release of pegaptanib in the first 24 hours of release
followed by a period of sustained release ranging from 40 days to
greater than 200 days dependent on the composition and inherent
viscosity of the PLGA polymer used to prepare the formulation.
Example 8
In Vivo Release of Pegaptanib Microspheres
[0169] The in vivo duration of release for microsphere formulations
produced according to Example 1 were assessed in New Zealand
rabbits by monitoring blood plasma concentration after bilateral
intravitreous dosing of the test formulations.
[0170] Microspheres containing pegaptanib were suspended in PBS
injection vehicle containing 0.02% surfactant at a concentration of
100 mg microspheres per milliliter. A volume of 50 .mu.L of the
test formulation suspension was injected intravitreously into the
eye using a 300 .mu.L insulin syringe fitted with a 29G, half-inch
needle to provide a 5 mg dose of test formulation
[0171] Blood plasma samples were harvested at specified intervals
and stored at -20.degree. C. until analysis. Samples were analyzed
for pegaptanib concentration via Dual Hybridization-PCR Assay.
Animals
[0172] Adult male New Zealand White rabbits, weighing approximately
2.5-4.0 kg were used.
Experimental Methodology
[0173] Animals were weighed and anesthetized with xylazine (5 to 10
mg/kg administered subcutaneously) followed by ketamine (35 to 50
mg/kg administered intramuscularly). One to two drops of
Tropicamide.RTM. were administered to each eye prior to ophthalmic
examination and dosing.
[0174] The intravitreal injections were administered on Study Day 1
by a board certified veterinary ophthalmologist (DACVO). For each
of the intravitreal injections, the rabbit was placed in a lateral
recumbent position and the eye was topically anesthetized with 1 to
2 drops of 0.1% proparacaine solution. The dose volume (for each
eye) was 50 .mu.L containing 5 mg of PLGA formulation. The test
article was injected intravitreously using a 300 .mu.L insulin
syringe fitted with a 29G, half-inch needle, or other appropriately
sized needle. The needle was inserted 1 to 2 mm posterior to the
limbus in the superotemporal quadrant. The bevel was kept in an
anterior position and the needle was advanced into the
mid-vitreous. Antibiotic ointment (triple antibiotic or equivalent)
was administered to the eye, following injection. The procedure was
repeated on the opposite eye. The first day of dosing was
designated as Study Day 1. Following dose administration on Study
Day 1, the animals were observed for the duration of the study.
[0175] At the designated time point 2 mL of whole blood was
collected from the lateral ear vein using potassium EDTA as the
anti-coagulant. All samples were analyzed for pegaptanib
concentrations via a dual hybridization-RT PCR Assay. The trapezoid
rule method was used to calculate AUC and assess the relative
bioavailability of pegaptanib release from PLGA microspheres. After
the final blood collection, all animals were euthanized via
anesthesia by ketamine:xylazine mixture followed by an overdose of
sodium pentobarbital.
Statistical Analysis
[0176] Statistical analysis of dual hybridization-RT PCR data was
performed using Graph Pad Prism.RTM. (GraphPad Software, Inc., San
Diego, Calif.). Standard curve sample concentrations was calculated
by a 4 parameter curve fit meeting appropriate statistical
parameters.
[0177] Previous studies have demonstrated that after bilateral IVT
administration of pegaptanib sodium in a phosphate buffered saline
solution, pegaptanib vitreous and plasma concentrations decline in
a well defined and predictable manner. The terminal phase rate
constant was equivalent in both vitreous humor and plasma and
reflects the slow absorption of pegaptanib from the eye into the
systemic circulation after an IVT injection. Thus blood plasma
levels were used as a marker for vitreous levels after
intravitreous dosing of pegaptanib sodium in phosphate buffered
saline.
[0178] It was expected that pegaptanib would be released in a
sustained fashion from the test formulations into the vitreous and
that the pegaptanib released into the vitreous would have
distribution and clearance properties similar to the properties of
pegaptanib dosed in a phosphate buffered saline solution. Thus, the
plasma pharmacokinetics of pegaptanib reflected the in vivo release
of pegaptanib from the sustained release formulation as elimination
from the eye into systemic circulation. This was considered to be
the rate-limiting step determining pegaptanib's plasma
pharmacokinetics.
Example 9
Release Profiles of Pegaptanib Microspheres
(Formulations 093-063, 093-003-1, and 093-059)
[0179] Pegaptanib microspheres having a core load between about 12
and 15 w/w % were prepared as set forth in Example 1. Formulations
093-063, 093-003-1, and 093-059 are summarized in Table 4. The
formulations were sieved to provide microspheres having a particle
size of less than 45 .mu.m.
TABLE-US-00004 TABLE 4 Drug Coreload 1-Day Burst Lot No. (wt. %)
PLGA Polymer (%) 093-033-1 14.3 50:50 4A 8.1 093-059 12.9 75:25 2A
13.5 093-063 14.4 50:50 3A 6.1
[0180] The in vitro release profile of the microspheres were
analyzed as set forth in Example 7. The results of the in vitro
release analysis are depicted in FIG. 13. The results shown in FIG.
13 demonstrate the sustained release properties of the
microparticles of the present invention. The results also
demonstrate that the microparticles of the present invention can be
selectively designed to control the release of a biologically
active agent over a desired time period. Preparing microspheres
having various polymers demonstrated that the in vitro release of
pegaptanib can be extended.
[0181] The in vivo release profile of the microspheres were
analyzed as set forth in Example 8. The animal groups and blood
collection schedule are summarized in Table 5.
Group Identification and Sampling Schedule
TABLE-US-00005 [0182] TABLE 5 Pegaptanib Number Number of Total
Content of Eyes/ Animals/ Number of Group Test Article (w/w %)
Route Timepoints Timepoint Timepoint Animals Group Pegaptanib 14.4%
Intravitreous 2 and 6 12 6 6 1M Microspheres hours post 093-063
dose; and Study Days 2, 4, 9, 16, 23, 30, 37, 44, 51, 58, 65, 72,
79, and 86 Group Pegaptanib 14.3% Intravitreous 2 and 6 12 6 6 2M
Microspheres hours post 093-003-1 dose; and Study Days 2, 4, 9, 16,
23, 30, 37, 44, 51, 58, 65, 72, 79, and 86 Group Pegaptanib 13.9%
Intravitreous 2 and 6 12 6 6 3M Microspheres hours post 093-059
dose; and Study Days 2, 4, 9, 16, 23, 30, 37, 44, 51, 58, 65, 72,
79, and 86
[0183] The in vivo release profile of the microspheres are
illustrated in Table 6 and FIGS. 14 and 15. Pharmacokinetic data is
presented in Table 7.
TABLE-US-00006 TABLE 6 093-003-1 093-059 093-063 Formulation
50:50-4A 75:25-2A 50:50-3A Polymer 14.2% 12.9% 14.4% Drug Content
134 122 136 API Dose (ug) Ave Ave Ave Day (ng/mL) Std Dev (ng/mL)
Std Dev (ng/mL) Std Dev 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.08
0.67 0.17 0.85 1.06 0.72 0.66 0.25 1.99 0.82 3.57 2.67 1.61 1.04 2
3.38 1.26 5.00 1.42 4.57 1.91 4 4.72 1.44 3.41 1.65 5.41 2.65 9
2.24 0.56 2.14 1.30 2.67 0.98 16 1.08 0.32 1.39 0.69 0.94 0.32 23
0.50 0.30 0.60 0.47 0.55 0.13 30 4.81 1.71 4.60 1.73 1.90 0.38 37
4.15 2.09 5.61 1.69 5.94 1.22 44 1.93 0.85 2.62 0.90 5.52 2.16 51
0.74 0.50 0.84 0.53 3.29 1.80 58 0.20 0.21 0.28 0.19 1.14 0.95 65
0.00 0.00 0.03 0.08 0.42 0.31 72 0.00 0.00 0.00 0.00 0.17 0.24 79
0.00 0.00 0.00 0.00 0.04 0.09 86 0.00 0.00 0.00 0.00 0.00 0.00
[0184] FIGS. 14 and 15 show plasma concentrations resulting from an
86 day in vivo ocular study in rabbits dosed intravitreously with 5
mg of PLGA microparticles containing 15% weight percent pegaptanib
are shown in FIGS. 14 and 15. FIG. 14 illustrates that three tested
formulations all had low in vivo burst release followed by a period
maintenance of blood plasma levels for 60-70 days. The in vitro
release analysis of these formulations indicated similar release
profiles for the three test formulations which was predictive of
the in vivo performance.
[0185] FIG. 14 illustrates that the three tested formulations had
in vivo release profiles that are well correlated with the in vitro
release profiles for ranking burst and duration of release.
[0186] The plasma concentration curves demonstrate a controlled
burst release that was well predicted by in vitro release analysis.
In addition, a typical PLGA microsphere release profile is observed
with a lag phase followed by polymer controlled secondary release
phase resulting in a sustained plasma concentration above what is
achieved by intravitreous dosing of the same dose of liquid
pegaptanib sodium in rabbits. The bioanalytical analysis revealed
that the blood plasma concentration of pegaptanib was sustained
over a period of several weeks.
TABLE-US-00007 TABLE 7 Dose AUC.sub.tot In vitro In vivo
Formulation Description (ug) ng*hr/mL F.sub.rel Burst Burst Liquid
141 4427 100% 093-003-1 50:50-4A 134 3173 75% 8.08% 7.20% 093-059
75:25-2A 122 3589 94% 13.54% 12.93% 093-063 50:50-3A 136 4429 104%
6.06% 5.83%
Example 10
Release Profiles of Pegaptanib Microspheres
(Formulations 093-023, 093-051, 077-189 and 093-041)
[0187] Pegaptanib microspheres having a core load between about 13
and 17 w/w % were prepared as set forth in Example 1. Formulations
093-023, 093-051, 077-189 and 093-041 are set forth in Table 8. The
formulations were sieved to provide microspheres having a particle
size of less than 45 .mu.m.
TABLE-US-00008 TABLE 8 Drug Coreload 1-Day Burst Lot No. (wt. %)
PLGA Polymer (%) 093-023 13.6 50:50 5A 32.8 093-051 14.1 65:35 3A
3.4 077-189 16.1 75:25 4A 5.2 093-041 13.6 PLA 0.36 dL/g IV 1.6
[0188] The in vitro release profile of the microspheres were
analyzed as set forth in Example 7. The results of the in vitro
release analysis of are depicted in FIG. 16. The results shown in
FIG. 16 demonstrate the sustained release properties of the
microparticles of the present invention. The results also
demonstrate that the microparticles of the present invention can be
selectively designed to control the release of a biologically
active agent over a desired time period. Preparing microspheres
having various polymers demonstrated that the in vitro release of
pegaptanib can be extended.
[0189] The in vivo release profile of the microspheres were
analyzed as set forth in Example 8. The in vivo release profile of
the microspheres are illustrated in Table 9 and FIG. 17.
Pharmacokinetic data is presented in Table 10.
TABLE-US-00009 TABLE 9 Formulation 093-023 093-051 077-189 093-041
Polymer 50:50-5A 65:35-3A 75:25-4A PLA (0.36) Drug Content 13.6%
14.1% 16.1% 13.6% API Dose (ug) 128 133 152 128 Day Ave (ng/mL) Std
Dev Ave (ng/mL) Std Dev Ave (ng/mL) Std Dev Ave (ng/mL) Std Dev 0
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.08 0.79 0.40 0.15 0.30
0.69 0.97 0.25 0.33 0.25 2.59 1.13 0.60 0.26 1.78 1.12 1.45 0.96 1
5.83 3.67 1.91 0.25 2.81 0.96 3.78 2.41 3 3.23 1.84 1.20 0.32 2.38
1.09 2.73 1.38 7 2.55 0.61 1.02 0.28 2.47 1.36 1.86 0.73 14 1.64
0.65 1.45 0.39 1.23 0.31 1.21 0.10 21 0.99 0.35 0.73 0.22 0.72 0.31
0.60 0.17 28 3.43 0.92 0.46 0.11 0.46 0.04 0.22 0.18 35 7.28 2.10
3.24 2.22 0.25 0.05 0.00 0.00 42 3.06 0.94 6.96 2.45 0.25 0.07 0.00
0.00 49 1.50 0.52 5.63 1.38 0.38 0.09 0.00 0.00 56 0.60 0.17 2.63
1.03 1.25 0.50 0.00 0.00 63 0.11 0.12 0.56 0.18 2.75 0.82 0.00 0.00
70 0.03 0.06 0.26 0.14 3.64 1.33 0.00 0.00 77 0.00 0.00 0.00 0.00
3.16 1.54 0.00 0.00 84 0.00 0.00 0.00 0.00 1.53 0.83 0.00 0.00 91
0.00 0.00 0.00 0.00 0.78 0.35 0.00 0.00 98 0.00 0.00 0.00 0.00 0.35
0.16 0.00 0.00 105 0.00 0.00 0.00 0.00 0.14 0.22 0.00 0.00 112 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 119 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 126 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 133 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 140 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 147 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 154 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 161 0.00 0.00 0.00 0.00 0.00
0.00 0.03 0.08 168 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.13 175 0.00
0.00 0.00 0.00 0.00 0.00 0.18 0.28 182 0.00 0.00 0.00 0.00 0.00
0.00 0.47 0.53 196 1.44 0.34 210 1.71 0.72 217 1.25 0.48 224 0.93
0.47 231 0.62 0.24 238 0.31 0.15 245 0.18 0.16 252 0.12 0.15 259
0.00 0.00
[0190] FIG. 17 depicts plasma concentrations resulting from an
8-month in vivo ocular study in rabbits dosed intravitreously with
5 mg of PLGA microparticles containing 15% weight percent
pegaptanib. Of note is the absence of a large burst release common
to PLGA formulations containing water soluble or hydrophilic
compounds. Blood plasma levels were monitored as a surrogate marker
for vitreous concentrations based on the established
pharmacokinetics of Pegaptanib Sodium.
[0191] FIG. 17 illustrates that the four tested formulations all
had low in vivo burst release followed by a period maintenance of
blood plasma levels. The in vitro release analysis of these
formulations indicated similar release profiles for the three test
formulations which was predictive of the in vivo performance. FIG.
17 illustrates that the four tested formulations had in vivo
release profiles that are well correlated with the in vitro release
profiles for ranking burst and duration of release.
[0192] The plasma concentration curves demonstrate a controlled
burst release that was well predicted by in vitro release analysis.
The bioanalytical analysis revealed that the blood plasma
concentration of pegaptanib was sustained over a period of several
months.
TABLE-US-00010 TABLE 10 Dose AUC.sub.tot In vitro In vivo
Formulation Description (ug) ng*hr/mL F.sub.rel Burst Burst Liquid
141 4427 100% 093-023 5050-5A 128 3924 98% 11.9% 21.1% 093-051
6535-3A 133 3974 96% 3.4% 6.9% 077-189 7525-4A 151 3449 73% 5.2%
10.2% 093-041 PLA 0.36 128 2573 64% 16.2% 13.7%
Example 11
In Vivo Release of Pegaptanib-Loaded Microparticles
(Formulations 079-089 and 079-102)
[0193] Applicants evaluated the in vivo release properties of
pegaptanib-loaded microparticles. Applicants administered
pegaptanib-loaded poly(lactide-co-glycolide) (PLGA) microspheres,
formed by the process as set forth in Example 1, intravitreously in
New Zealand White rabbits and plasma samples were collected at
various time points from 2 hours to 28 days as set forth in Table
11. This evaluation demonstrated the feasibility of delivering
pegaptanib over a period of approximately one month or more from a
(PLGA) polymer based sustained release formulation.
TABLE-US-00011 TABLE 11 Number of Number of animals/time Total
Number of Group Time points point animals Intravitreous Injection 6
3 18 of Microspheres Control Intravitreous 2 3 6 Injection of Blank
Microspheres Control Intravitreous 2 3 6 Injection of 50 .mu.L 0.9%
Sodium Chloride Total Number of 30 Animals
Materials and Methods
[0194] PLGA microsphere formulations 079-089 and 079-102 were
prepared as set forth in Example 1 above and were blended to
provide sufficient material for animal dosing. Microspheres
containing pegaptanib 15% on a weight percent basis were suspended
in PBS injection vehicle containing 0.02% surfactant at a
concentration of 100 mg microspheres per milliliter. The
Pegaptanib-loaded PLGA microsphere formulations were administered
by intravitreous injection (IVT) in New Zealand White rabbits.
Placebo microspheres were dosed in an identical manner in control.
Blood Plasma samples were collected at various time points, from 2
hours to 28 days as set forth in Table 10. Samples were stored at
-20.degree. C. until analysis. Samples were analyzed for pegaptanib
concentration via a Dual Hybridization-PCR assay as set forth in
Example 14. (see Patent Application Publication No. WO 2006/012468,
which is incorporated herein by reference in its entirety).
Animals
[0195] Adult female New Zealand White rabbits, weighing
approximately 2.5-4.0 kg were used. The groups are summarized in
Table 11. The treatment schedule is summarized in Table 12.
Treatment Schedule
TABLE-US-00012 [0196] TABLE 12 Number Number of of Eyes/ Animals/
Total Number Group Test Article Route Timepoints Timepoint
Timepoint of Animals Group 1 Pegaptanib Intravitreous 2 hour 1, 5,
6 3 18 Microspheres 9, 14, 28 day Group 2 Blank Intravitreous 1, 9
day 6 3 6 Microspheres
Experimental Methodology
[0197] Intraocular pressure was measured in each animal prior to
anesthesia using a hand-held applanation tonometer (Tonopen.TM.).
Animals were weighed and anesthetized with ketamine/xylazine
administered intramuscularly. Tropicamide.RTM. were administered to
each eye prior to ophthalmic examination. With the rabbit in right
lateral recumbency, the left eye was topically anesthetized with
0.1% proparacaine solution.
[0198] A volume of 50 .mu.L of the test article was injected
intravitreously, using a 500 .mu.L insulin syringe fitted with a
29G, half-inch needle, or other appropriately sized device. For
Intravitreous injections, the needle was inserted 1-2 mm posterior
to the limbus in the superotemporal quadrant. The bevel was kept in
an anterior position and the needle was advanced into the
mid-vitreous. Antibiotic ointment (triple antibiotic or equivalent)
was administered to the eye following injection. The procedure was
repeated on the right eye. Observations were recorded, including,
but not limited to, leakage of test material from the injection
site.
[0199] Whole blood (500 .mu.L) was collected from the lateral ear
vein. For animals in Group 1 (Day 28 time point), blood samples
were collected at the following time points: 2 and 6 hours, 1, 3,
5, 9, 14, and 21 days, in addition to the terminal time point.
Statistical Analysis
[0200] Statistical analysis was performed using Graph Pad Prism.
Samples are analyzed by 4 parameter curve fit meeting appropriate
statistical parameters.
Results
[0201] Plasma concentrations resulting from a 28 day in vivo ocular
distribution study in rabbits dosed intravitreously with 5 mg of
PLGA microparticles containing 15% weight percent pegaptanib are
shown in Table 13 and FIG. 12. The results show that the
microparticles have a low burst release. A large burst release,
common to PLGA formulations containing water soluble or hydrophilic
compounds, is absent. In addition plasma pegaptanib concentration
levels are measured at a relatively constant level between 0.05-0.4
nM over the 28 day study period relative to an equivalent IVT
liquid pegaptanib dose, indicating a sustained release of
pegaptanib in the vitreous was achieved.
[0202] It is known in the art that modifying the polymer
composition of a sustained release microparticle formulation
affects the rate of polymer decomposition in vivo and therefore
effects the release characteristics of the microparticle
formulation. Therefore demonstrating a release duration of one
month from a microsphere formulation in vivo indicates that a
release duration of greater than one month from a microsphere
formulation in vivo is feasible.
TABLE-US-00013 TABLE 13 Pegaptanib Plasma Concentration Following
Intravitreous Injection Pegaptanib Microsphere Pegaptanib Liquid
Days Conc (nM) SD Days Conc (nM) StDev 0.08 0.055 0.078 0.08 0.051
0.05 0.25 0.142 0.200 0.25 0.436 0.12 1 0.126 0.065 1 1.861 0.06 4
0.137 0.067 5 1.476 0.26 7 0.080 0.014 8 0.941 0.04 11 0.051 0.005
11 0.539 0.21 16 0.069 0.016 15 0.451 0.13 19 0.138 0.070 19 0.241
0.06 23 0.362 0.203 23 0.219 0.09 26 0.423 0.273 28 0.088 0.02 28
0.415 0.271
Example 12
Release Profile of Pegaptanib Microparticles
(Water-in-Oil-in-Water)
[0203] Table 14 shows the properties of pegaptanib microparticles
that were prepared as set forth in Example 2. The in vitro release
profiles of the pegaptanib microparticles were determined by the
process as set forth in Example 7
[0204] The release profiles of the microparticles from Table 14 are
shown in FIG. 10. FIG. 10 is a graph depicting in vitro dissolution
rate profiles demonstrating control release kinetics from
Pegaptanib-PLGA microspheres.
TABLE-US-00014 TABLE 14 EYE001 Core Load % Encapsulation Particle
Surface Description Yield (% w/w) Efficiency Size Morphology EYE001
50:50 PLGA 65.78% 7.09 .+-. 0.84 46.14 .+-. 5.5 <10 .mu.m Smooth
EYE001 50:50 PLGA 66.02% 7.05 .+-. 0.07 47.09 .+-. 0.45 <10
.mu.m Smooth EYE001 50:50 PLGA 66.28% 8.22 .+-. 0.09 52.8 .+-. 0.54
NT NT Placebo 50:50 PLGA 64.05% n/a n/a <10 .mu.m Smooth EYE001
50:50 PLGA 56.49% 9.02 .+-. 0.11 59.08 .+-. 0.69 <10 .mu.m
Smooth Placebo 50:50 PLGA 64.10% n/a n/a <10 .mu.m Smooth EYE001
50:50 PLGA 87.01% 7.70 .+-. 0.46 51.06 .+-. 3.04 <10 .mu.m
Smooth
[0205] The results of the in vitro release analysis of are depicted
in FIG. 10. FIG. 10 shows the release properties of microparticles
formed by the w/o/w process as set forth in Example 2. The results
shown in FIG. 10 demonstrate the sustained release properties of
the microparticles of the present invention. The results also
demonstrate that the microparticles of the present invention can be
selectively designed to control the release of a biologically
active agent over a desired time period. Preparing microspheres
having various polymers demonstrated that the in vitro release of
pegaptanib can be extended.
Example 13
Pegaptanib Inhibition of VEGF Induced Tissue Factor Expression in
HUVEC Cells
[0206] Pegaptanib microspheres were prepared as set forth in
Example 1 above. The microspheres were analyzed to determine if
pegaptanib maintained its efficacy following release from the
microspheres.
[0207] HUVEC cells were plated at 1.5.times.10.sup.5 cells/well in
complete medium (Cascade Biologics Medium 200, supplemented with
Low Serum Growth Supplement and Penicillin, Streptomycin, and
Amphotericin B (PSA)) in 24 well plates and allowed to attach
overnight in a 37.degree. C./5% CO.sub.2 incubator. Sixteen hours
later, complete media was removed and cells are washed once with 1%
medium (Cascade Biologics Medium 200, supplemented with 1% Fetal
Bovine Serum and PSA). Cells were then starved in the 1% medium for
4 hours in a 37.degree. C./5% CO.sub.2 incubator. During
starvation, assay controls and samples were prepared. Assay
controls included 1% medium alone (zero control), 1% medium with
12.5 ng/mL VEGF (VEGF induction control), and 1% medium with
Pegaptanib at 10 nM (Pegaptanib control). The test microsphere
samples were prepared at a concentration of 10 nM in 1% medium with
VEGF (12.5 ng/mL). After 4 hours of starvation, media was removed
and the prepared assay samples were added to respective wells. All
controls and test samples were done in duplicate (2 wells each).
Cells were treated for 1 hour in a 37.degree. C./5% CO.sub.2
incubator. After the 1 hour incubation, media was removed and cells
were washed with sterile 1.times.PBS. Cells were then lysed with
RLT/.beta.ME lysis buffer (Qiagen). Lysed cells were collected in
sterile tubes and stored at -80.degree. C. or used immediately for
total RNA isolation. RNA isolation was performed using the Qiagen
RNeasy.RTM. Protocol. cDNA was made and the Tissue Factor gene was
quantitated using a typical Taqman.RTM. Real Time PCR protocol.
[0208] The results of cell proliferation assays of human umbilical
vein endothelial cells (HUVEC) incubated with EYE001 formulations
after release from PLGA microparticles are shown in FIG. 11. The
figure illustrates that addition of VEGF to the media increased
expression of Tissue Factor by 9-fold. In the presence of 10 nM
pegaptanib, Tissue Factor expression was reduced to basal levels.
Pegaptanib released from PLGA microspheres had a similar effect on
Tissue Factor expression as native pegaptanib. The results thereby
demonstrated that the in vitro bioactivity of pegaptanib was
unchanged by the microsphere fabrication process and subsequent in
vitro release.
Example 14
Dual Hybridization-PCR Analysis of In Vivo Pegaptanib Release
Vitreous Digestion
[0209] Rabbit microsphere Vitreous samples are placed in 15 mL and
50 mL tubes and incubated overnight at 37.degree. C. Following the
incubation, Proteinase K (20 .mu.L/sample) is added and the samples
are incubated for 2 hours at 65.degree. C. After 2 hours the
samples are cooled to room temperature and then put in ice. The
cooled samples are ready for dual hybridization assay. Plasma does
not require digestion. 1.times.PBS and SDS is not added to the
Vitreous digestion
[0210] The digestion cocktail is prepared (per sample) as
follows:
TABLE-US-00015 1x PBS 500 .mu.L Hyaluronidase cocktail 20 .mu.L
Blendzyme III 20 .mu.L SDS 5 .mu.L DNAse 10 .mu.L
[0211] Hyaluronidase cocktail is a mixture of 4 hyaluronidases in
equal parts 5 .mu.L. Each Hyaluronidase cocktail is a 1 mg/mL
solution in 1.times.PBS and 1 xBSA. Blendzyme (Roche Diagnostics
Corporation Indianapolis, Ind.); Liberase Blendzymes are mixtures
of highly purified collagenase and neutral protease enzymes,
formulated for efficient, gentle, and reproducible dissociation of
tissue from a wide variety of sources) is made as 7 mg/ml solution
in 1.times.PBS. Proteinase K is made as 20 mg/ml solution in
1.times.PBS.
Dual Hybridization Assay
(Plasma)
[0212] Standards were prepared in 1.times. tissue (control tissue
diluted 1:10). 165 .mu.L of primer mix was added per sample into
0.2 PCR tubes. Primer mix was prepared by adding 1 .mu.M detection
primer to Hybridization buffer so that the final concentration of
the detection primer in the required mixture was 1 nM. Standards or
sample (25 .mu.L) were added. Capture beads (5 .mu.L) were added.
The PCR tubes were then placed in a Thermal Cycler and programmed
to run 75.degree. C. for 15 minutes, 37.degree. C. for 1 hour and
then down to 25.degree. C. Then the samples and standards were
transferred from the PCR tubes into 96 well plates and 180 .mu.L
were transferred from each.
[0213] The plate was then run in a Kingfisher.RTM. 96 magnetic
particle processor.
The following 96-well plates were prepared: [0214] 1-96 well plate
(100 .mu.L of water in each well) [0215] 5-96 well plates (200
.mu.L of 1.times. wash buffer in each well)
[0216] The magnetic comb picked up the beads and moved them from
plate to plate. The beads were washed in each plate leaving behind
anything that was not specifically bound to the beads. The beads
were deposited in the water plate at the end of the run. At this
point the plate was ready to be run in Taqman.RTM. RT PCR.
Taqman.RTM. RT PCR
[0217] A 384-well plate was used. Total volume in a well was 10
.mu.L. (Sample=4 .mu.L; Cocktail=6 .mu.L). A duplicate was prepared
for each sample in the 96 well plate.
[0218] The following cocktail was prepared per 384 well:
TABLE-US-00016 H20 0.4 .mu.L 2x PCR mix 5 .mu.L Forward primer 0.05
.mu.L Reverse primer 0.05 .mu.L Probe 0.5 .mu.L
[0219] First the cocktail was added and then the samples from the
96 well plate were transferred. The 384 well plate was spun in a
centrifuge (1000 rpm) for less than a minute. The plate was then
run in Taqman: 50.degree. C. for 2 min, 95.degree. C. for 10 min,
and then 40 cycles of 95.degree. C. 15 seconds to 60.degree. C. for
1 min.
Data Analysis
[0220] At the end of the run, data was available for the run from
the SDS software. These data were then plugged in Microsoft Excel
and Prism and the concentrations of the samples were then
extrapolated from a standard curve.
[0221] The results of a 28 day in vivo release study of pegaptanib
from PLGA microparticles are discussed in Example 11. FIG. 12 is a
graph showing pegaptanib concentration in rabbits plasma samples
dosed intravitreous or subconjunctival with 5 mg of PLGA
microparticles containing 15% weight percent pegaptanib.
Example 15
Delayed Release Microparticle Pegaptanib Dosing Regimen
[0222] Step 1.
[0223] Administer a 100 .mu.L pharmaceutical formulation comprising
a bolus of about 0.3 mg free pegaptanib in solution and delayed
release PLGA microparticles encapsulating about 35 mg
pegaptanib.
[0224] Step 2.
[0225] The microspheres will have an initial burst of about 5-30%
of pegaptanib and then will release at some rate constant over a
predefined period.
[0226] Step 3.
[0227] At the end of the microsphere release profile, a second
burst will occur releasing a second bolus of pegaptanib bringing
the vitreal concentration to about 0.3 mg.
[0228] Step 4.
[0229] Four weeks post burst, during which time the polymeric
metabolites are cleared, a new pegaptanib/microparticle injection
would be administered as described in Step 1.
INCORPORATION BY REFERENCE
[0230] The patent and scientific literature referred to herein
establishes knowledge that is available to those of skill in the
art. All patents, patent applications, and published references
cited herein are hereby incorporated by reference in their
entirety.
EQUIVALENTS
[0231] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific embodiments described specifically
herein. Such equivalents are intended to be encompassed in the
scope of the following claims.
* * * * *