U.S. patent application number 09/973325 was filed with the patent office on 2002-05-23 for device and method for treating ophthalmic diseases.
Invention is credited to Baetge, E. Edward, Hammang, Joseph P., Spear, Peter D., Tsiarias, William G..
Application Number | 20020061327 09/973325 |
Document ID | / |
Family ID | 22553994 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020061327 |
Kind Code |
A1 |
Hammang, Joseph P. ; et
al. |
May 23, 2002 |
Device and method for treating ophthalmic diseases
Abstract
The invention provides a method for delivering biologically
active molecules to the eye by implanting biocompatible capsules
containing a cellular source of the biologically active molecule.
Also provided is a method of treating ophthalmic diseases using
biocompatible capsules.
Inventors: |
Hammang, Joseph P.;
(Barrington, RI) ; Baetge, E. Edward; (St.
Sulpice, CH) ; Tsiarias, William G.; (Barrington,
RI) ; Spear, Peter D.; (Boulder, CO) |
Correspondence
Address: |
Ivor R. Elrifi
Mintz Levin
One Financial Center
Boston
MA
02111
US
|
Family ID: |
22553994 |
Appl. No.: |
09/973325 |
Filed: |
October 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09973325 |
Oct 9, 2001 |
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09155066 |
Apr 27, 1999 |
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09155066 |
Apr 27, 1999 |
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PCT/US97/04701 |
Mar 24, 1997 |
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PCT/US97/04701 |
Mar 24, 1997 |
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08620982 |
Mar 22, 1996 |
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Current U.S.
Class: |
424/424 ;
424/130.1; 424/85.1; 424/94.1 |
Current CPC
Class: |
A61K 9/0051 20130101;
A61F 9/0017 20130101 |
Class at
Publication: |
424/424 ;
424/85.1; 424/94.1; 424/130.1 |
International
Class: |
A61K 039/395; A61K
038/43; A61K 038/19 |
Claims
We claim:
1. A method for delivering a biologically active molecule to the
eye comprising: implanting a capsule periocularly, the capsule
comprising a core containing a cellular source of the biologically
active molecule and a surrounding biocompatible jacket, the jacket
permitting diffusion of the biologically active molecule into the
eye.
2. The method of claim 1 wherein the jacket comprises a
permselective, immunoisolatory membrane.
3. The method of claim 1 wherein the capsule is implanted in the
sub-Tenon's space.
4. The method of claim 1 wherein the capsule is configured as a
hollow fiber or a flat sheet.
5. The method of claim 1 wherein the biologically active molecule
is selected from the group consisting of anti-angiogenic factors,
neurotrophic factors, growth factors, antibodies and antibody
fragments, neurotransmitters, hormones, enzymes, cytokines, and
lymphokines.
6. The method of claim 1 wherein the biologically active molecule
is selected from the group consisting of anti-angiogenic factors,
anti-inflammatory factors, neurotrophic factors, and combinations
thereof.
7. The method of claim 1 wherein the dosage of the biologically
active molecule delivered is between 50 pg to 1000 ng per eye per
patient per day.
8. The method of claim 1 wherein the number of capsules implanted
is between 1 to 5 capsules per eye.
9. The method of claim 1 wherein a second biologically active
molecule or peptide is co-delivered from the capsule to the
eye.
10. A method for delivering a biologically active molecule to the
eye comprising: implanting a capsule intraocularly, the capsule
comprising a core containing a cellular source of the biologically
active molecule and a surrounding biocompatible jacket, the jacket
permitting diffusion of the biologically active molecule into the
eye.
11. The method of claim 10 wherein the jacket comprises a
permselective, immunoisolatory membrane.
12. The method of claim 10 wherein the jacket comprises a
microporous membrane.
13. The method of claim 10 wherein the capsule is implanted in the
vitreous.
14. The method of claim 10 wherein the capsule is implanted in the
anterior chamber.
15. The method of claim 10 wherein the capsule is implanted in the
posterior chamber.
16. The method of claim 10 wherein the capsule is configured as a
hollow fiber or a flat sheet.
17. The method of claim 10 wherein the biologically active molecule
is selected from the group consisting of anti-angiogenic factors,
neurotrophic factors, growth factors, antibodies and antibody
fragments, neurotransmitters, hormones, enzymes, cytokines, and
lymphokines.
18. The method of claim 10 wherein the biologically active molecule
is selected from the group consisting of anti-angiogenic factors,
anti-inflammatory factors, neurotrophic factors, and combinations
thereof.
19. The method of claim 10 wherein the dosage of the biologically
active molecule delivered is between 50 pg to 500 ng per eye per
patient per day.
20. The method of claim 10 wherein the number of capsules implanted
is between 1 to 5 capsules per eye.
21. The method of claim 10 wherein a second biologically active
molecule or peptide is co-delivered from the capsule to the
eye.
22. A method for delivery of biologically active molecules to an
eye comprising: implanting a first capsule intraocularly, the first
capsule comprising a core containing a cellular source of a first
biologically active molecule and a surrounding biocompatible
jacket, the jacket permitting diffusion of the first biologically
active molecule into the eye; implanting a second capsule
periocularly, the second capsule comprising a core containing a
cellular source of a second biologically active molecule and a
surrounding biocompatible, immunoisolatory jacket, the jacket
permitting diffusion of the second biologically active molecule
into the eye.
23. The method of claim 22 wherein the first capsule is implanted
in the vitreous and the second capsule is implanted in the
sub-Tenon's space.
24. A method for treating ophthalmic disorders in a patient
suffering therefrom comprising: implanting into an eye of the
patient a biocompatible capsule, the capsule comprising a) a core
comprising a cellular source of a biologically active molecule, and
b) a jacket surrounding said core, the jacket comprising a
biocompatible material that permits diffusion of the biologically
active molecule to the eye in a therapeutically effective
amount.
25. The method of claim 24 wherein the biologically active molecule
is selected from the group consisting of anti-angiogenic factors,
neurotrophic factors, growth factors, antibodies and antibody
fragments, neurotransmitters, hormones, enzymes, cytokines, and
lymphokines.
26. The method of claim 24 wherein the biologically active molecule
is selected from the group consisting of anti-angiogenic factors,
anti-inflammatory factors, neurotrophic factors, and combinations
thereof.
27. The method of claim 24 wherein the ophthalmic disorder is
selected from the group consisting of angiogenic disorders,
inflammatory disorders, degenerative disorders, and combinations
thereof.
28. The method of claim 24 wherein the ophthalmic disorder is
selected from the group consisting of uveitis, retinitis
pigmentosa, age-related macular degeneration, and diabetic
retinopathy.
29. The method of claim 24 wherein the biologically active molecule
is selected from the group consisting of BDNF, TGF-.beta., GDNF,
NGF, CNTF, bFGF, aFGF, IL-1.beta., IL-10, IFN-.beta., IFN-.alpha.
and VEGF inhibitors.
30. The method of claim 24 wherein the capsule is
immunoisolatory.
31. The method of claim 24 wherein a second biologically active
molecule is co-delivered to the eye.
32. A device for delivery of a biologically active molecule to the
eye comprising a capsule, the capsule comprising: a core comprising
encapsulated cells that produce a biologically active molecule, a
biocompatible jacket surrounding said core, the jacket permitting
diffusion of the biologically active molecule into the eye; the
capsule configured as a hollow fiber, with an outer diameter of
less than or equal to 1 mm, and a length between 0.4 and 1.5
cm.
33. A device for delivery of a biologically active molecule to the
eye comprising a capsule, the capsule comprising: a core comprising
encapsulated cells that produce a biologically active molecule, a
biocompatible jacket surrounding said core, the jacket permitting
diffusion of the biologically active molecule into the eye; the
capsule configured as a flat sheet having a surface area less than
or equal to 25 mm.sup.2.
34. The device of claims 32 or 33 wherein the capsule further
comprises a tether adapted for securing the capsule to an ocular
structure.
35. An encapsulated cell system for periocular delivery of a
biologically active molecule to the eye comprising: at least one
capsule, each capsule comprising a core containing a cellular
source of a biologically active molecule and a surrounding
biocompatible jacket, the jacket permitting diffusion of the
biologically active molecule into the eye, the encapsulated cell
system delivering 50 pg to 1000 ng periocularly per eye per patient
per day of the biologically active molecule.
36. The system of claim 35 wherein the jacket comprises a
permselective, immunoisolatory membrane.
37. The system of claim 35 wherein the jacket comprises a
microporous membrane.
38. The system of claim 35 wherein the capsule is implanted in the
sub-Tenon's space.
39. The system of claim 35 wherein the capsule is configured as a
hollow fiber or a flat sheet.
40. The system of claim 35 wherein the biologically active molecule
is selected from the group consisting of anti-angiogenic factors,
neurotrophic factors, growth factors, antibodies and antibody
fragments, neurotransmitters, hormones, enzymes, cytokines, and
lymphokines.
41. The system of claim 35 wherein the biologically active molecule
is selected from the group consisting of anti-angiogenic factors,
anti-inflammatory factors, neurotrophic factors, and combinations
thereof.
42. The system of claim 35 wherein the number of capsules implanted
is between 1 to 5 capsules per eye.
43. The system of claim 35 wherein a second biologically active
molecule or peptide is co-delivered from the capsule to the
eye.
44. The system of claim 43 wherein the dosage of the second
biologically active molecule or peptide delivered is between 50 pg
to 1000 ng per eye per patient per day.
45. An encapsulated cell system for intraocular delivery of a
biologically active molecule to the eye comprising: at least one
capsule, each capsule comprising a core containing a cellular
source of a biologically active molecule and a surrounding
biocompatible jacket, the jacket permitting diffusion of the
biologically active molecule into the eye, the encapsulated cell
system delivering 50 pg to 500 ng intraocularly per eye per patient
per day of the biologically active molecule.
46. The system of claim 45 wherein the jacket comprises a
permselective, immunoisolatory membrane.
47. The system of claim 45 wherein the jacket comprises a
microporous membrane.
48. The system of claim 45 wherein the capsule is implanted in the
vitreous.
49. The system of claim 45 wherein the capsule is implanted in the
anterior chamber.
50. The system of claim 45 wherein the capsule is implanted in the
posterior chamber.
51. The system of claim 45 wherein the capsule is configured as a
hollow fiber or a flat sheet.
52. The system of claim 45 wherein the biologically active molecule
is selected from the group consisting of anti-angiogenic factors,
neurotrophic factors, growth factors, antibodies and antibody
fragments, neurotransmitters, hormones, enzymes, cytokines, and
lymphokines.
53. The system of claim 45 wherein the biologically active molecule
is selected from the group consisting of anti-angiogenic factors,
anti-inflammatory factors, neurotrophic factors, and combinations
thereof.
54. The system of claim 45 wherein the number of capsules implanted
is between 1 to 5 capsules per eye.
55. The system of claim 45 wherein a second biologically active
molecule or peptide is co-delivered from the capsule to the
eye.
56. The system of claim 55 wherein the dosage of the second
biologically active molecule or peptide delivered is between 50 pg
to 500 ng per eye per patient per day.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] This invention relates to devices and methods for treatment
of ophthalmic diseases and disorders using encapsulated cells for
intraocular and periocular delivery of biologically active
molecules.
BACKGROUND OF THE INVENTION
[0002] There are a number of vision-threatening disorders of the
eye for which there are presently no good therapies. One major
problem in treatment of such diseases is the inability to deliver
therapeutic agents into the eye and maintain them there at
therapeutically effective concentrations.
[0003] Oral ingestion of a drug or injection of a drug at a site
other than the eye can provide a drug systemically. However, such
systemic administration does not provide effective levels of the
drug specifically to the eye. In many ophthalmic disorders
involving the retina, posterior tract, and optic nerve, adequate
levels of drug cannot be achieved or maintained by oral or
parenteral routes of administration. Further, repeated
administration of the drug may be necessary to achieve these
concentrations. However, this may produce undesired systemic
toxicity. For example, subcutaneously or intramuscularly
administered alpha-interferon in adults may result in complications
such as flu-like symptoms with fatigue, anorexia, nausea, vomiting,
thrombocytopenia, and leukopenia.
[0004] Ophthalmic conditions have also been treated using drugs
applied directly to the eye in either liquid or ointment form. This
route of administration however is only effective in treating
problems involving the superficial surface of the eye and diseases
which involve the cornea and anterior segment of the eye. Topical
administration of drugs is ineffective in achieving adequate
concentrations of drug in the sclera, vitreous, or posterior
segment of the eye. In addition, topical eye drops may drain from
the eye through the nasolacrimal duct and into the systemic
circulation, further diluting the medication and risking unwanted
systemic side effects. Furthermore, the drug is administered
indiscriminately to all tissue compartments of the eye, including
those that may not need the medication and may in fact suffer
unwanted side effects to the drug.
[0005] Delivery of drugs in the form of topical eye drops is also
of little utility when the drug is a protein or peptide that lacks
the ability to cross the cornea and be made available to the
vitreous, retina, or other subretinal structures such as the
retinal pigment epithelium ("RPE") or choroidal vasculature. In
addition, many proteins or peptides are highly unstable and are
therefore not easily formulated for topical delivery.
[0006] Direct delivery of drugs into the eye by topical insert has
also been attempted. However, this method is not desirable. Topical
inserts require patient self-administration and thus education on
insertion and removal. This demands a certain degree of manual
dexterity, which can be problematic for geriatric patients. In many
instances such inserts may cause eye irritation. These devices are
prone to inadvertent loss due to lid laxity. In addition, these
devices provide drug only to the cornea and anterior chamber, and
do not provide any pharmacologic advantage over eye drops.
[0007] Another extraocular insert is a contact lens delivery system
that releases medication over an extended period. See, e.g., JAMA,
260:24, p. 3556 (1988). The lens generally only lasts for a matter
of hours or days before dissolving or releasing all of the
therapeutic compound. Continuous delivery of medication is
inconvenient, requiring frequent re-application. Again, these
contact lenses only provide drug to the cornea and anterior
chamber.
[0008] In rare cases, direct delivery of drugs has also been
accomplished using externalized tubes. This requires insertion of
one end of a tube into the comer of the patient's eye. The other
end of the tube is taped to the patient's forehead and terminates
in a septum, through which medication is delivered. This method is
undesirable, being both uncomfortable and inconvenient. Since
medication must be injected through the septum, the device is
incapable of continuous delivery of medication. Furthermore, such
tubes may become infected and in some cases ultimately threaten the
patient's vision.
[0009] Direct delivery of drugs can also be accomplished by the
intraocular injection of the drug, or microspheres that contain the
drug. However, microspheres tend to migrate within the eye, either
into the visual axis or into adjacent tissue sites.
[0010] Most previous intraocular inserts for direct delivery of
drugs into the eye have been unsuccessful either because they are
unsuitable for long-term use or are uncomfortable to use. For
example, the ocular device disclosed in U.S. Pat. No. 3,828,777 is
not anchored into position, thus causing pain, irritation, foreign
body sensation, retinal detachments, and watering when the device
moves. Other ocular inserts disclosed in patents do not disclose
sizes or shapes that would allow long-term retention of the insert.
See, e.g., U.S. Pat. Nos. 4,343,787; 4,730,013; 4,164,559. Even in
patents asserting an improved retention and prolonged period of
use, the contemplated period is measured in days, such as 7 to 14
days. See, e.g., U.S. Pat. No. 5,395,618.
[0011] One intraocular insert is currently available for delivery
of ganciclovir to the eye. Known as Vitrasert, the device consists
of a nonerodible, polymer-based, sustained-release package
containing ganciclovir, a non-proteinaceous nucleoside analog. The
device is surgically implanted in the vitreous humor of the eye to
treat cytomegalovirus retinitis. See, e.g., Anand, R., et al.,
Arch. Ophthalmol., 111, pp. 223-227 (1993).
[0012] Another intraocular insert is disclosed by U.S. Pat. No.
5,466,233. This tack-shaped device is surgically implanted so that
the head of the tack is external to the eye, abutting the scleral
surface. The post of the tack crosses the sclera and extends into
the vitreous humor, where it provides for vitreal drug release.
[0013] However, release of proteins from such devices (or other
erodible or nonerodible polymers) can be sustained for only short
periods of time due to protein instability. Such devices are
unsuitable for long-term delivery of most, if not all, protein
molecules.
[0014] Clinical treatment for retinal and choroidal
neovascularization includes destruction of new vessels using
photocoagulation or cryotherapy. However, side effects are numerous
and include failure to control neovascularization, destruction of
macula and central vision, and decrease in peripheral vision. See,
e.g., Aiello, L. P., et al., PNAS, 92, pp. 10457-10461 (1995).
[0015] A number of growth factors show promise in the treatment of
ocular disease. For example, BDNF and CNTF have been shown to slow
degeneration of retinal ganglion cells and photoreceptors in
various animal models. See, e.g., Genetic Technology News, vol. 13,
no. 1 (January 1993). Nerve growth factor has been shown to enhance
retinal ganglion cell survival after optic nerve section and has
also been shown to promote recovery of retinal neurons after
ischemia. See, e.g., Siliprandi, et al., Invest. Ophthalmol. &
Vis. Sci., 34, pp. 3232-3245 (1993).
[0016] Direct injection of neurotrophic factors to the vitreous
humor of the eye has been shown to promote the survival of retinal
neurons and photoreceptors in a variety of experimentally induced
injuries as well as inherited models of retinal diseases. See,
e.g., Faktorovich et al., Nature, vol. 347 at 83 (Sep. 6, 1990);
Siliprandi et al., Investigative Ophthalmology and Visual Science,
34, p. 3222 (1993); LaVail et al., PNAS, 89, p. 11249 (1992);
Faktorovich et al., Nature, 347, pp. 83-86 (1990).
[0017] However, previous methods of delivery of such
neurotransmitters, growth factors, and neurotrophic factors have
significant drawbacks. Some problems stem from the fact that growth
factors do not cross the blood-brain barrier well and are readily
degraded in the bloodstream. Further, problems arise with direct
injection into the vitreous. For example, direct injection of bFGF
resulted in an increased incidence of retinal macrophages and
cataracts. See LaVail, PNAS, 89, p. 11249 (1992).
[0018] Accordingly, delivery of biologically active molecules to
the eye without adverse effects remains a major challenge.
SUMMARY OF THE INVENTION
[0019] This invention provides a novel method of treating
ophthalmic diseases and disorders by intraocular and periocular
delivery of a continuously-produced source of a suitable
biologically active molecule ("BAM").
[0020] A capsule containing a cellular source of the BAM is
surgically placed in the desired location in the eye.
[0021] The capsule jacket comprises a membrane surrounding the
encapsulated cells and interposes a physical barrier between the
cells and the patient. The capsule may be retrieved from the
patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a schematic diagram of a horizontal cross section
of the eye, indicating a macrocapsule implanted in the vitreous.
The diagram is not to scale, and for the sake of clarity shows the
capsule in an approximate placement--when actually placed in the
human eye, the preferred vitreous placement is in the anterior
portion of the vitreous. The letter "A" refers to the sclera, "B"
refers to Tenon's capsule, and "C" refers to the conjunctiva.
[0023] FIG. 2 is a schematic diagram of a side view of the eye
showing an implanted capsule beneath Tenon's capsule.
[0024] FIG. 3A shows a device with frangible hub assembly for
loading. FIG. 3B depicts the device after detachment of the
frangible hub. The device has an eyelet for tethering the device in
the eye.
[0025] FIG. 4A shows a device with frangible hub assembly for
loading. FIG. 4B depicts the device after detachment of the
frangible hub. The device has a disk for tethering the device.
DETAILED DESCRIPTION OF THE INVENTION
[0026] This invention relates to delivery of biologically active
molecules ("BAMs") intraocularly (e.g., in the anterior chamber,
posterior chamber, or vitreous) or periocularly (e.g., within or
beneath Tenon's capsule), or both. The invention may be useful in
providing controlled and sustained release of BAMs effective in
treating various ophthalmic disorders, ophthalmic diseases, or
diseases which have ocular effects.
[0027] The devices and techniques of this invention provide
numerous advantages over other delivery routes:
[0028] Drug can be delivered to the eye directly, reducing or
minimizing unwanted peripheral side effects; very small doses of
drug (nanogram or low nicrogram quantities rather than milligrams)
can be delivered compared with topical applications, also
potentially lessening side effects; the viable cells of the devices
continuously produce newly synthesized product, avoiding the
fluctuation in drug dose that characterizes injection delivery of
drugs; and the devices and methods of this invention are less
invasive than many of the prior art devices and surgical
techniques, which result in a large number of retinal
detachments.
[0029] Most, if not all, ophthalmic diseases and disorders are
associated with one or more of three types of indications: (1)
angiogenesis, (2) inflammation, and (3) degeneration. To treat
these disorders, the devices of the present invention permit
delivery of anti-angiogenic actors; anti-inflammatory factors;
factors that retard cell degeneration, promote cell sparing, or
promote cell growth; and combinations of the foregoing. Based on
the indications of a particular disorder, one of ordinary skill in
the art can administer any suitable molecule or combination of
molecules from the three groups at the dosages specified below.
[0030] Diabetic retinopathy, for example, is characterized by
angiogenesis. This invention contemplates treating diabetic
retinopathy by implanting devices delivering one or more
anti-angiogenic factors either intraocularly, preferably in the
vitreous, or periocularly, preferably in the sub-Tenon's region. We
most prefer delivery into the vitreous for this indication. It is
also desirable to co-deliver one or more neurotrophic factors,
either intraocularly or periocularly, preferably intraocularly, and
most preferably intravitreally.
[0031] Uveitis involves inflammation. This invention contemplates
treating uveitis by intraocular, preferably vitreal or anterior
chamber, implantation of devices secreting one or more
anti-inflammatory factors.
[0032] Retinitis pigmentosa, by comparison, is characterized by
retinal degeneration. This invention contemplates treating
retinitis pigmentosa by intraocular, preferably vitreal, placement
of devices secreting one or more neurotrophic factors.
[0033] Age-related macular degeneration involves both angiogenesis
and retinal degeneration. This invention contemplates treating this
disorder by using the inventive devices to deliver one or more
neurotrophic factors intraocularly, preferably to the vitreous,
and/or one or more anti-angiogenic factors intraocularly or
periocularly, preferably periocularly, most preferably to the
sub-Tenon's region.
[0034] Glaucoma is characterized by increased ocular pressure and
loss of retinal ganglion cells. Treatments for glaucoma
contemplated in this invention include delivery of one or more
neuroprotective agents that protect cells from excitotoxic damage.
Such agents include N-methyl-D-aspartate (NMDA) antagonists,
cytokines, and neurotrophic factors, delivered intraocularly,
preferably intravitreally.
[0035] Any suitable BAM may be delivered according to the devices,
systems, and methods of this invention. Such molecules include
neurotransmitters, cytokines, lymphokines, neuroprotective agents,
neurotrophic factors, hormones, enzymes, antibodies, and active
fragments thereof. Three preferred types of BAMs are contemplated
for delivery using the devices of the present invention: (1)
anti-angiogenic factors, (2) anti-inflammatory factors, and (3)
factors that retard cell degeneration, promote cell sparing, or
promote cell growth.
[0036] The anti-angiogenic factors contemplated for use include
vasculostatin, angiostatin, endostatin, anti-integrins, vascular
endothelial growth factor inhibitors (VEGF-inhibitors), platelet
factor 4, heparinase, and bFGF-binding molecules. The VEGF
receptors Flt and Flk are also contemplated. When delivered in the
soluble form these molecules compete with the VEGF receptors on
vascular endothelial cells to inhibit endothelial cell growth.
[0037] VEGF inhibitors may include VEGF-neutralizing chimeric
proteins such as soluble VEGF receptors. See Aiello, PNAS, 92,
10457 (1995). In particular, they may be VEGF-receptor-IgG chimeric
proteins. Another VEGF inhibitor contemplated for use in the
present invention is antisense phosphorothiotac
oligodeoxynucleotides (PS-ODNs).
[0038] Intraocularly, preferably in the vitreous, we contemplate
delivery of an anti-angiogenic factor in a dosage range of 50 pg to
500 ng, preferably 100 pg to 100 ng, and most preferably 1 ng to 50
ng per eye per patient per day. For periocular delivery, preferably
in the sub-Tenon's space or region, slightly higher dosage ranges
are contemplated of up to 1 .mu.g per patient per day.
[0039] The anti-inflammatory factors contemplated for use in the
present invention include antiflammins (see, e.g.. U.S. Pat. No.
5,266,562, incorporated herein by reference), beta-interferon
(IFN-.beta.), alpha-interferon (IFN-.alpha.), TGF-beta,
interleukin-10 (IL-10), and glucocorticoids and mineralocorticoids
from adrenal cortical cells. It should be noted that certain BAMs
may have more than one activity. For example, it is believed that
IFN-.alpha. and IFN-.beta. may have activities as both
anti-inflammatory molecules and as anti-angiogenic molecules.
[0040] Intraocularly, preferably in the vitreous, we contemplate
delivery of an anti-inflammatory factor in a dosage range of 50 pg
to 500 ng, preferably 100 pg to 100 ng, and most preferably 1 ng to
50 ng per eye per patient per day. For periocular delivery,
preferably in the sub-Tenon's space or region, slightly higher
dosage ranges are contemplated of up to 1 .mu.g per patient per
day.
[0041] The factors contemplated for use in retarding cell
degeneration, promoting cell sparing, or promoting new cell growth
are collectively referred to herein as "neurotrophic factors". The
neurotrophic factors contemplated include neurotrophin 4/5 (NT4/5),
cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), glial
cell line derived neurotrophic factor (GDNF), nerve growth factor
(NGF), insulin-like growth factor-1 (IGF-1), neurotrophin 3 (NT-3),
brain-derived neurotrophic factor (BDNF), PDGF, neurturin, acidic
fibroblast growth factor (aFGF), basic fibroblast growth factor
(bFGF), EGF, neuregulins, heregulins, TGF-alpha, bone morphogenic
proteins (BMP-1, BMP-2, BMP-7, etc.), the hedgehog family (sonic
hedgehog, indian hedgehog, and desert hedgehog, etc.), the family
of transforming growth factors (including, e.g., TGF.beta.-1,
TGF.beta.-2, and TGF.beta.-3), interleukin 1-B (IL1-.beta.), and
such cytokines as interleukin-6 (IL-6), IL-10, CDF/LIF, and
beta-interferon (IFN-.beta.). The preferred neurotrophic factors
are GDNF, BDNF, NT-4/5, neurturin, CNTF, and CT-1.
[0042] Intraocularly, preferably in the vitreous, we contemplate
delivery of a neurotrophic factor in a dosage range of 50 pg to 500
ng, preferably 100 pg to 100 ng, and most preferably 1 ng to 50 ng
per eye per patient per day. For periocular delivery, preferably in
the sub-Tenon's space or region, slightly higher dosage ranges are
contemplated of up to 1 .mu.g per patient per day.
[0043] Modified, truncated, and mutein forms of the above-mentioned
molecules are also contemplated. Further, active fragments of these
growth factors (i.e., those fragments of growth factors having
biological activity sufficient to achieve a therapeutic effect) are
also contemplated. Also contemplated are growth factor molecules
modified by attachment of one or more polyethylene glycol (PEG) or
other repeating polymeric moieties. Combinations of these proteins
and polycistronic versions thereof are also contemplated.
[0044] A gene of interest (i.e., a gene that encodes a suitable
BAM) can be inserted into a cloning site of a suitable expression
vector by using standard techniques. The nucleic acid and amino
acid sequences of the human (and other mammalian) genes encoding
the above identified BAMs are known. See, e.g., U.S. Pat. Nos.
4,997,929; 5,141,856; 5,364,769; 5,453,361; WO 93106116; WO
95/30686, incorporated herein by reference.
[0045] The expression vector containing the gene of interest may
then be used to transfect the desired cell line. Standard
transfection techniques such as calcium phosphate co-precipitation,
DEAE-dextran transfection or electroporation may be utilized.
Commercially available mammalian transfection kits may be purchased
from e.g., Stratagene. Transgenic-mouse-derived cell lines can also
be used. See, e.g., Hammang et al., Methods in Neurosci., 21, p.
281 (1994).
[0046] A wide variety of host/expression vector combinations may be
used to express the gene encoding the growth factor, or other
BAM(s) of interest.
[0047] Suitable promoters include, for example, the early and late
promoters of SV40 or adenovirus and other known non-retroviral
promoters capable of controlling gene expression.
[0048] Useful expression vectors, for example, may consist of
segments of chromosomal, non-chromosomal, and synthetic DNA
sequences, such as various known derivatives of SV40 and known
bacterial plasmids, e.g., pUC, pBlueScript.TM. plasmids from E.
coli including pBR322, PCR1, pMB9, and their derivatives.
[0049] Expression vectors containing the geneticin (G418) or
hygromycin drug selection genes (Southern, P. J., In Vitro, 18, p.
315 (1981), Southern, P. J. and Berg, P., J. Mol. Appl. Genet., 1,
p. 327 (1982)) are also useful. These vectors can employ a variety
of different enhancer/promoter regions to drive the expression of
both a biologic gene of interest (e.g., NGF) and/or a gene
conferring resistance to selection with toxin such as G418 or
hygromycin B. A variety of different mammalian promoters can be
employed to direct the expression of the genes for G418 and
hygromycin B and/or the biologic gene of interest.
[0050] Examples of expression vectors that can be employed are the
commercially available pRC/CMV, pRC/RSV, and pCDNA1NEO
(InVitrogen).
[0051] If cells of a CNS origin are used, preferably the promoter
is selected from the following group:
[0052] promoters of hDBH (human dopamine beta hydroxylase) (Mercer
et al., Neuron, 7, pp. 703-716, (1991)), hTH (human tyrosine
hydroxylase) (Kaneda, et al., Neuron, 6, pp. 583-594 (1991)), hPNMT
(human phenylethanolamine N-methyltransferase) (Baetge et al.,
PNAS, 85, pp. 3648-3652 (1988)), mGFAP (mouse glial fibrillary
acidic protein) (Besnard et al., J. Biol. Chem., 266, pp.
18877-18883 (1991)), myelin basic protein (MBP), MNF-L (mouse
neurofilament-light subunit) (Nakahira et al., J. Biol. Chem., 265,
pp. 19786-19791 (1990)), hPo (human P.sub.0, the promoter for the
gene encoding the major myelin glycoprotein in the peripheral
nervous system) (Lemke et al., Neuron, 1, pp. 73-83 (1988)), mMt-1
(mouse metallothionein I), rNSE (rat neuron-specific enolase)
(Sakimura, et al., Gene, 60, pp. 103-113 (1987)), and the like.
[0053] In one preferred embodiment, the phosphoglycerate kinase
(PGK) promoter is used. See, e.g., Adra et al., Gene, 60, pp. 65-74
(1987). The pPI vector is one preferred expression vector using the
PGK promoter to drive the expression of the gene of interest (i.e.
the gene encoding the BAM). This vector also uses the SV40 early
promoter to drive expression of neo phosphotransferase, a
selectable marker. One may optimize or enhance expression of a BAM
from the pPI vector by inserting the Kozak sequence and/or the Ig
signal peptide. The pPI vector also contains a mutant DHFR gene
suitable for MTX amplification.
[0054] In another embodiment, the pNUT expression vector, which
contains the cDNA of the mutant DHFR and the entire pUC18 sequence
including the polylinker, can be used. See, e.g., Aebischer, P., et
al., Transplantation, 58, pp. 1275-1277 (1994); Baetge et al.,
PNAS, 83, pp. 5454-58 (1986). The pNUT expression vector can be
modified such that the DHFR coding sequence is replaced by the
coding sequence for G418 or hygromycin drug resistance. The SV40
promoter within the pNUT expression vector can also be replaced
with any suitable constitutively expressed mammalian promoter, such
as those discussed above.
[0055] Increased expression can be achieved by increasing or
amplifying the copy number of the transgene encoding the desired
molecule, using amplification methods well known in the art. Such
amplification methods include, e.g., DHFR amplification (see, e.g.,
Kaufman et al., U.S. Pat. No. 4,470,461) or glutamine synthetase
("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464, and
European published application EP 338,841).
[0056] A wide variety of cells may be used. These include well
known, publicly available immortalized cell lines as well as
dividing primary cell cultures. Examples of suitable publicly
available cell lines include, Chinese hamster ovary (CHO), mouse
fibroblast (L-M), NIH Swiss mouse embryo (NIH/3T3), African green
monkey cell lines (including COS-1, COS-7, BSC-1, BSC40, BMT-10,
and Vero), rat adrenal pheochromocytoma (PC12 and PC12A), AT3, rat
glial tumor (C6), astrocytes, and other fibroblast cell lines.
Primary cells that may be used include EGF-responsive neural stem
cells and their differentiated progeny (Reynolds and Weiss,
Science, 255, pp. 1707-1710 (1992)), bFGF-responsive neural
progenitor stem cells derived from the CNS of mammals (Richards et
al., PNAS 89, pp. 8591-8595 (1992), Ray et al., PNAS 90, pp.
3602-3606 (1993)), CNS neural stem cells that are both
EGF-responsive and bFGF-responsive, primary fibroblasts, Schwann
cells, .beta.-TC cells, Hep-G2 cells, oligodendrocytes and their
precursors, myoblasts (including L6 and C.sub.2C.sub.12 cells),
chondrocytes or chondroblasts, and the like.
[0057] Conditionally-immortalized cells may also be used. Such
cells include cells with temperature sensitive oncogenes, or cells
engineered with chimeric genes composed of an oncogene under the
direction of an inducible promoter element.
[0058] One preferred cell type chosen for the gene transfer
technique is the baby hamster kidney (BHK) cell. BHK cells are
particularly amenable to MTX amplification, most likely because
they do not express a highly functional DHFR gene.
[0059] The suitable cell types include cells from allogeneic and
xenogeneic sources. A particular advantage to using xenogeneic
cells is that in the unlikely event of membrane or device failure,
such cells are more likely to be targeted for destruction by the
immune system.
[0060] For delivery in the eye, it may be particularly beneficial
to employ primary cells (including primary cells that can be
induced to divide using mitogens such as EGF or bFGF or the like)
or cell lines, conditionally-immortalized or otherwise, derived
from various regions of the eye. Potentially useful cell types
include lens epithelial cells, glial and neuronal elements of the
neural retina, photoreceptor cells, retinal pigmented epithelial
cells, Schwann cells and other ciliary body cells, and the like.
Such cells can be allogeneic or xenogeneic.
[0061] As used herein "a biocompatible capsule" means that the
capsule, upon implantation in a host mammal, does not elicit a
detrimental host response sufficient to result in the rejection of
the capsule or to render it inoperable, for example through
degradation.
[0062] As used herein "an immunoisolatory capsule" means that the
capsule upon implantation into a mammalian host minimizes the
deleterious effects of the host's immune system on the cells within
its core. To be immunoisolatory, the capsule should provide a
physical barrier sufficient to prevent detrimental immunological
contact between the isolated cells and the host's immune system.
The thickness of this physical barrier can vary, but it will always
be sufficiently thick to prevent direct contact between the cells
and/or substances on either side of the barrier. The thickness of
this region generally ranges between 5 and 200 microns; thicknesses
of 10 to 100 microns are preferred, and thickness of 20 to 75
microns are particularly preferred.
[0063] The exclusion of IgG from the core of the vehicle is not the
touchstone of immunoisolation, because in most cases IgG alone is
insufficient to produce cytolysis of the target cells or tissues.
Thus, for immunoisolatory capsules, jacket nominal molecular weight
cutoff (MWCO) values between 50-2000 kD are contemplated.
Preferably, the MWCO is between 50-700 kD. Most preferably, the
MWCO is between 70-300 kD. See, e.g., WO 92/19195. If
immunoisolation is not required, the jacket can be microporous.
See, e.g., U.S. Pat. Nos. 4,968,733; 4,976,859; and 4,629,563; all
incorporated herein by reference.
[0064] A variety of biocompatible capsules are suitable for
delivery of molecules according to this invention. Useful
biocompatible polymer capsules comprise (a) a core which contains a
cell or cells, either suspended in a liquid medium or immobilized
within a biocompatible matrix, and (b) a surrounding jacket
comprising a membrane which does not contain isolated cells, which
is biocompatible, and permits diffusion of the cell-produced BAM
into the eye.
[0065] Many transformed cells or cell lines are advantageously
isolated within a capsule having a liquid core, comprising, e.g., a
nutrient medium, and optionally containing a source of additional
factors to sustain cell viability and function.
[0066] Alternatively, the core may comprise a biocompatible matrix
of a hydrogel or other biocompatible matrix material which
stabilizes the position of the cells. The term "hydrogel" herein
refers to a three dimensional network of cross-linked hydrophilic
polymers. The network is in the form of a gel, substantially
composed of water, preferably gels being greater than 90%
water.
[0067] Any suitable matrix or spacer may be employed within the
core, including precipitated chitosan, synthetic polymers and
polymer blends, microcarriers, and the like, depending upon the
growth characteristics of the cells to be encapsulated.
Alternatively, the capsule may have an internal scaffold. The
scaffold may prevent cells from aggregating and improve cellular
distribution within the device. See PCT publication no. WO
96/02646.
[0068] Preferably, for implant sites that are not immunologically
privileged, such as periocular sites, the capsules are
immunoisolatory.
[0069] The capsule can be any suitable configuration, including
cylindrical, rectangular, disk-shaped, patch-shaped, ovoid,
stellate, or spherical. Configurations that tend to lead to
migration of the capsules from the site of implantation, such as
spherical, are not preferred. For implantations in the vitreous,
flat sheets may not be preferred because they may block the visual
path to the retina.
[0070] Preferably the device has a tether that aids in maintaining
device placement during implant and aids in retrieval. Such a
tether may have any suitable shape that is adapted to secure the
capsule in place. In one embodiment, the tether is shaped like an
eyelet, so that suture may be used to secure the tether (and thus
the capsule) to the sclera, or other suitable ocular structure. In
another embodiment, the tether is continuous with the capsule at
one end, and forms a pre-threaded suture needle at the other end.
The capsules contemplated here have a minimum core volume of about
1 to 20 .mu.l, most preferably about 1 to 10 .mu.l.
[0071] In a hollow fiber configuration, the fiber will have an
inside diameter of less than 1000 microns, preferably less than 950
microns. In one series of embodiments, the device is configured to
have an 870 .mu.m inner diameter and a length of about 8.5 mm. In
another series of embodiments, the device is configured to have a
500 .mu.m inner diameter and a length of 10.5 mm. For implantation
in the eye, in a hollow fiber configuration the capsule will
preferably be between 0.4 cm to 1.5 cm in length, most preferably
between 0.5 to 1.0 cm in length. Longer devices may be accommodated
in the eye, however, a curved or arcuate shape may be required for
secure and appropriate placement. The hollow fiber configuration is
preferred for intraocular placement.
[0072] For periocular placement, either a hollow fiber
configuration (with dimensions substantially as above) or a flat
sheet configuration is contemplated. The upper limit contemplated
for a flat sheet is approximately 5 mm.times.5 mm--assuming a
square shape. Other shapes with approximately the same surface area
are also contemplated
[0073] The hydraulic permeability will typically be in the range of
1-100 mls/min/M.sup.2/mmHg, preferably in the range of 25 to 70
mls/min/M.sup.2/mmHg. The glucose mass transfer coefficient of the
capsule, defined, measured, and calculated as described by Dionne
et al., ASAIO Abstracts, p. 99 (1993), and Colton et al., The
Kidneys, Brenner B M and Rector F C, pp. 2425-89 (1981) will be
greater than 10.sup.-6 cm/sec, preferably greater than 10.sup.-4
cm/sec.
[0074] The capsule jacket may be manufactured from various polymers
and polymer blends including polyacrylates (including acrylic
copolymers), polyvinylidenes, polyvinyl chloride copolymers,
polyurethanes, polystyrenes, polyamides, cellulose acetates,
cellulose nitrates, polysulfones (including polyether sulfones),
polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyl
chloride), as well as derivatives, copolymers, and mixtures thereof
Capsules manufactured from such materials are described, e.g., in
U.S. Pat. Nos. 5,284,761 and 5,158,881, incorporated herein by
reference. Capsules formed from a polyether sulfone (PES) fiber,
such as those described in U.S. Pat. Nos. 4,976,859 and 4,968,733,
incorporated herein by reference, may also be used.
[0075] Depending on the outer surface morphology, capsules have
been categorized as Type 1 (T1), Type 2 (T2), Type 1/2 (T1/2), or
Type 4 (T4). Such membranes are described, e.g., in Lacy et al.,
"Maintenance Of Normoglycemia In Diabetic Mice By Subcutaneous
Xenografts Of Encapsulated Islets," Science, 254, pp. 1782-84
(1991), Dionne et al., WO 92119195 and Baetge, WO 95/05452. We
prefer a smooth outer surface morphology.
[0076] Capsule jackets with permselective immunoisolatory membranes
are preferable for sites that are not immunologically privileged.
In contrast, microporous membranes or permselective membranes may
be suitable for immunologically privileged sites. For implantation
into immunologically privileged sites, we prefer capsules made from
the PES membranes.
[0077] Any suitable method of sealing the capsules may be used,
including the employment of polymer adhesives and/or crimping,
knotting, and heat sealing. These sealing techniques are known in
the art. In addition, any suitable "dry" sealing method can also be
used. In such methods, a substantially non-porous fitting is
provided through which the cell-containing solution is introduced.
Subsequent to filling, the capsule is sealed. Such a method is
described in copending U.S. application Ser. No. 08/082,407, herein
incorporated by reference (see also PCT/US94/0701 5). That
application describes the frangible hub assembly shown
diagrammatically in FIGS. 3 and 4 that can be used to conveniently
load and seal the devices of this invention.
[0078] We contemplate use of the present invention to treat a wide
variety of ophthalmic diseases and disorders characterized by but
not limited to angiogenesis, inflammation, degeneration, or some
combination thereof.
[0079] Some examples of ophthalmic disorders that may be treated by
various embodiments of the present invention include uveitis,
retinitis pigmentosa, age-related macular degeneration and other
acquired disorders, retinopathy, retinal vascular diseases and
other vascular anomalies, endophthalmitis, infectious diseases,
inflammatory but non-infectious diseases, ocular ischemia syndrome,
peripheral retinal degenerations, retinal degenerations and tumors,
choroidal disorders and tumors, vitreous disorders, retinal
detachment, non-penetrating and penetrating trauma, post-cataract
complications, and inflammatory optic neuropathies.
[0080] Age-related macular degeneration includes but is not limited
to dry age-related macular degeneration, exudative age-related
macular degeneration, and myopic degeneration.
[0081] Retinopathy includes but is not limited to diabetic
retinopathy, proliferative vitreoretinopathy, and toxic
retinopathy.
[0082] The present invention may be useful for the treatment of
ocular neovascularization, a condition associated with many ocular
diseases and disorders and accounting for a majority of severe
visual loss. For example, we contemplate treatment of retinal
ischemia-associated ocular neovascularization, a major cause of
blindness in diabetes and many other diseases; corneal
neovascularization, which predisposes patients to corneal graft
failure; and neovascularization associated with diabetic
retinopathy, central retinal vein occlusion, and possibly
age-related macular degeneration.
[0083] The present invention may also be used to treat ocular
symptoms resulting from diseases or conditions that have both
ocular and non-ocular symptoms. Some examples include AIDS-related
disorders such as cytomegalovirus retinitis and disorders of the
vitreous; pregnancy-related disorders such as hypertensive changes
in the retina; and ocular effects of various infectious diseases
such as tuberculosis, syphilis, lyme disease, parasitic disease,
toxocara canis, ophthalmonyiasis, cyst cercosis, and fungal
infections.
[0084] In one embodiment of the present invention, living cells are
encapsulated and surgically inserted (under retrobulbar anesthesia)
into the vitreous of the eye. For vitreal placement, the device may
be implanted through the sclera, with a portion of the device
protruding through the sclera. Most preferably, the entire body of
the device is implanted in the vitreous, with no portion of the
device protruding into or through the sclera. Preferably the device
is tethered to the sclera (or other suitable ocular structure). The
tether may comprise a suture eyelet (FIG. 3), or disk (FIG. 4), or
any other suitable anchoring means. The device can remain in the
vitreous as long as necessary to achieve the desired prophylaxis or
therapy. Such therapies for example include promotion of neuron or
photoreceptor survival or repair, or inhibition and/or reversal of
retinal or choroidal neovascularization, as well as inhibition of
uveal, retinal, and optic nerve inflammation. This embodiment is
preferable for delivering the BAM to the retina.
[0085] With vitreal placement, the BAM, preferably a trophic
factor, may be delivered to the retina or the RPE. In addition,
retinal neovascularization may be best treated by delivering an
anti-angiogenic factor to the vitreous.
[0086] In another embodiment, cell-loaded devices are implanted
periocularly, within or beneath the space known as Tenon's capsule.
This embodiment is less invasive than implantation into the
vitreous and thus is generally preferred. This route of
administration also permits delivery of BAMs (e.g., trophic factors
and the like) to the RPE or the retina. This embodiment is
especially preferred for treating choroidal neovascularization and
inflammation of the optic nerve and uveal tract. In general,
delivery from this implantation site will permit circulation of the
desired BAM to the choroidal vasculature, the retinal vasculature,
and the optic nerve.
[0087] According to this embodiment we prefer periocular delivery
(implanting beneath Tenon's capsule) of anti-angiogenic molecules,
anti-inflammatory molecules (such as cytokines and hormones), and
neurotrophic factors to the choroidal vasculature to treat macular
degeneration (choroidal neovascularization).
[0088] Delivery of anti-angiogenic factors directly to the
choroidal vasculature (periocularly) or to the vitreous
(intraocularly) using the devices and methods of this invention may
reduce the above-mentioned problems and may permit the treatment of
poorly defined or occult choroidal neovascularization. It may also
provide a way of reducing or preventing recurrent choroidal
neovascularization via adjunctive or maintenance therapy.
[0089] In a preferred embodiment, the pNUT vector carrying the
desired gene or genes is transfected into baby hamster kidney (BHK)
cells or C.sub.2C.sub.12 myoblast cells using a standard calcium
phosphate transfection procedure and selected with increasing
concentrations of methotrexate (1 .mu.M to a maximum of 200 .mu.M)
over 8 weeks to produce stable, amplified cell lines. Following
this selection, the engineered cells may be maintained in vitro in
50-200 .mu.M methotrexate, prior to encapsulation.
[0090] The present invention contemplates co-delivery of different
factors. One of ordinary skill in the art may deliver one or more
anti-angiogenic factors, anti-inflammatory factors or factors
retarding cell degeneration, promoting cell sparing, or promoting
cell growth, depending on the indications of the particular
ophthalmic disorder. For example, it may be preferable to deliver
one or more neurotrophic factors together with one or more
anti-angiogenic factors, or one or more anti-inflammatory
molecules.
[0091] One example is co-delivery of NT4/5 with endostatin. In this
situation, the neurotrophic factor can promote photoreceptor
survival while the heparinase would act as an anti-angiogenic
factor.
[0092] Co-delivery can be accomplished in a number of ways. First,
cells may be transfected with separate constructs containing the
genes encoding the described molecules. Second, cells may be
transfected with a single construct containing two or more genes
and the necessary control elements. We prefer multiple gene
expression from a single transcript over expression from multiple
transcription units. See, e.g., Macejak, Nature, 353, pp. 90-94
(1991); WO 94/24870; Mountford and Smith, Trends Genet., 11, pp.
179-84 (1995); Dirks et al., Gene, 128, pp. 247-49 (1993);
Martinez-Salas et al., J. Virology, 67, pp. 3748-55 (1993) and
Mountford et al., Proc. Natl. Acad. Sci. USA, 91, pp. 4303-07
(1994).
[0093] Third, either two or more separately engineered cell lines
can be co-encapsulated, or more than one device can be implanted at
the site of interest. And fourth, devices may be implanted in two
or more different sites in the eye concurrently, to deliver the
same or different BAMs. For example, it may be desirable to deliver
a neurotrophic factor to the vitreous to supply the neural retina
(ganglion cells to the RPE) and to deliver an anti-angiogenic
factor via the sub-Tenon's space to supply the choroidal
vasculature. While treatment using more than one device is
contemplated and up to five devices per eye, we prefer implantation
of three devices or less per eye.
[0094] Dosage can be varied by any suitable method known in the
art. This includes changing (1) the number of cells per device, (2)
the number of devices per eye, or (3) the level of BAM production
per cell. Cellular production can be varied by changing, for
example, the copy number of the gene for the BAM in the transduced
cell, or the efficiency of the promoter driving expression of the
BAM. We prefer use of 10.sup.3 to 10.sup.8 cells per device, more
preferably 5.times.10.sup.4 to 5.times.10.sup.6 cells per
device.
[0095] This invention also contemplates use of different cell types
during the course of the treatment regime. For example, a patient
may be implanted with a capsule device containing a first cell type
(e.g., BHK cells). If after time, the patient develops an immune
response to that cell type, the capsule can be retrieved, or
explanted, and a second capsule can be implanted containing a
second cell type (e.g., CHO cells). In this manner, continuous
provision of the therapeutic molecule is possible, even if the
patient develops an immune response to one of the encapsulated cell
types.
[0096] Alternatively, capsules with a lower MWCO may be used to
further prevent interaction of molecules of the patient's immune
system with the encapsulated cells.
[0097] The methods and devices of this invention are intended for
use in a primate, preferably human host, recipient, patient,
subject or individual.
EXAMPLES
Example 1
Preparation and Encapsulation of Cells
[0098] BHK-hNGF cells (Winn et al., PNAS, 1994) were produced as
follows:
[0099] The human NGF (hNGF) gene with the rat insulin intron, as
described by Hoyle et al., was inserted between the BamHI and SmaI
sites of pNUT to be driven by the metallothionein I promoter. The
pNUT-hNGF construct was introduced into BHK cells by using a
standard calcium phosphate-mediated transfection method. BHK cells
were grown in Dulbecco's modified Eagle's medium/10% fetal bovine
serum/antibiotic/antimycotic/L-glutamine (GIBCO) in 5% CO.sub.2/95%
air and at 37.degree. C. Transfected BHK cells were selected in
medium containing 200 .mu.M methotrexate (Sigma) for 3-4 weeks, and
resistant cells were maintained as a polyclonal population either
with or without 200 .mu.M methotrexate.
[0100] The cells were maintained in DMEM with 10% FBS, L-glutamine
with 50 .mu.M methotrexate prior to these experiments. The cells
were passaged 1 to 2 times per week in the presence of
methotrexate. The BHK-hNGF cells and BHK control cells were washed
with Hank's buffer, then trypsinized and mixed with Zyderm.RTM.
collagen matrix. The cell lines and matrix were loaded into
separate Hamilton syringes that were equipped with blunted,
25-gauge needles.
[0101] The encapsulation procedure was as follows: The hollow
fibers were fabricated from polyether sulfone (PES) with an
approximate outside diameter of 720 .mu.m and a wall thickness of
approximately 100 .mu.m (AKZO-Nobel Wuppertal, Germany). These
fibers are described in U.S. Pat. Nos. 4,976,859 and 4,968,733,
herein incorporated by reference.
[0102] The devices comprise:
[0103] a semipermeable poly (ether sulfone) hollow fiber membrane
fabricated by AKZO Nobel Faser AG;
[0104] a hub membrane segment;
[0105] a light cured methacrylate (LCM) resin leading end; and
[0106] a silicone tether. The devices had a septal fixture at the
proximal end for cellular loading access and were sealed at the
distal end. BHK cells were prepared as a single-cell suspension and
infused into the septal port at a density of 15K cells per .mu.l
after mixing 1:1 with physiologic collagen (Vitrogen: PC-1). After
infusing 1.5 .mu.l of the cellular suspension, the septum was
removed, and the access port was scaled with LCM 23 resin.
[0107] The components of the device are commercially available. The
LCM glue is available from Ablestik Laboratories (Newark, DE);
Luxtrak Adhesives LCM23 and LCM24).
Example 2
Implantation of Encapsulated Cells into the Sub-Tenon's Space
(Under Tenon's Capsule)
[0108] The patient is prepared and draped in the usual fashion
after a retrobulbar injection of 3 cc 2% xylocaine is given to the
eye. A speculum is inserted beneath the upper and lower lids. The
operating microscope is brought into position. A perpendicular
incision is made through both conjunctiva and Tenon's capsule in
the superotemporal quadrant approximately 4 mm back from the
limbus. The incision is extended approximately 4-5 mm back from the
limbus. At that point, a blunt-tipped scissor is inserted through
the incision and is used to bluntly dissect back an additional 5 mm
or so on the scleral surface. At that point, a membrane device as
described in Example 1 is placed in position through this incision
to come to rest on the surface of the sclera. The end of the device
that is closest to the limbus has a small loop that is attached to
the cell-loaded device. At this point, a #10-0 nylon suture is
passed through this loop and sutured into the superficial sclera to
anchor the membrane to the sclera. At that point, both Tenon's
capsule and the conjunctiva are closed with #6-0 plain gut sutures.
The speculum is removed and the procedure is concluded.
Example 3
Implantation of Encapsulated Cells into the Vitreous
[0109] The patient is prepared and draped in the usual fashion
after a retrobulbar injection of 2% xylocaine is given to the eye.
At that point, a speculum is inserted into the upper and lower lids
and the microscope is brought into position. A small incision is
made through both the conjunctiva and Tenon's capsule parallel to
and approximately 4 mm from the limbus in the supranasal quadrant.
The area exposed is cauterized with a wet-field cautery apparatus.
A 3 mm incision is then made perpendicular to the limbus
approximately 4 mm back from the limbus. The incision is made
through the sclera and into the vitreous cavity with a #65 blade.
Any of the vitreous which presents itself in the incision is cut
away and removed. At this point, a membrane device as described in
Example 1 is inserted through the incision into the vitreous
cavity. At the end of the membrane, there is a small 2 mm loop that
is attached to the membrane. The loop remains outside the sclera.
The sclera is closed with interrupted #9-0 nylon sutures. The #9-0
nylon sutures are also used to anchor this loop of the device to
the sclera. The conjunctiva is closed with #6-0 plain gut
sutures.
Example 4
Delivery of Interferon-.alpha. (IFN .alpha.-2A or IFN .alpha.-2B)
in the Treatment of Age-Related Macular Degeneration
[0110] Candidate cell lines are genetically engineered to express
the interferon molecules. Various interferons may be used; however,
we prefer to use IFN .alpha.-2A or .alpha.-2B. More than one
interferon molecule may be delivered at one time. Various cell
lines can also be utilized, we prefer BHK cells
[0111] Cell lines will be encapsulated in pre-assembled devices
substantially according to example 1. Following the device
manufacture, a tether is applied. This tether contains an eyelet
through which suture material can be passed. The tether is then
used to anchor the device in place to avoid device drift or loss.
The cell-loaded devices will be held for a standard period to
assure device sterility. The capsule is implanted beneath the
Tenon's capsule according to example 2.
[0112] Patients that have been diagnosed with angiographically
proven subfoveal choroidal neovascularization involving any part of
the foveal avascular zone are to be selected for this therapy.
[0113] The effects of IFN .alpha.-2a therapy are assessed by visual
acuity, clinical appearance, and fluorescein angiographic
appearance. The clinical appearance of the fundus is assessed
subjectively with particular reference to macular elevation by
subretinal fluid and the presence of intraretinal hemorrhage.
[0114] Devices will be removed using the same preparation and
surgical procedure as described above. The device will be placed in
vitro and assayed for 24 hours for release of IFN-.alpha.. After
the assay period, the device will be submitted for routine
histological analysis to determine the extent of cell survival.
Example 5
Delivery of hNGF to Neonatal Feline Eyes via Encapsulated BHK-hNGF
Cell Line
[0115] BHK-hNGF clone 36 cells were produced according to example
1. The cells were then encapsulated into 4 mm LCM 24 light-cured
capsules made from AKZO microporous 10/10 membranes according to
example 1. The capsules were implanted in neonatal feline eyes
substantially according to example 4 for 1 month.
[0116] Results
[0117] In vitro tests for NGF-induced neurite outgrowth were
performed before and after implantation in the feline eyes.
Conditioned medium (CM) from unencapsulated BHK-control and
BHK-hNGF cells was passed through a 0.2 .mu.m filter and added to
cultures of a PC12 cell subclone, PC12A, grown on 6- or 24-well
plates at a density of 200,000 cells per ml to test for the
presence of hNGF. Encapsulated cells in the polymeric devices were
also tested for their ability to release bioactive hNGF by placing
the devices in individual wells of a 24-well plate and allowing
them to equilibrate for 1-2 days in serum-free defined PC1 medium
(Hycor, Portland, Me.); the medium was then removed and replaced
with 1 ml of fresh PC1 for an additional 24 hour. This CM was
collected, placed on the PC12A cells, and evaluated. Neurite
processes that were equal to or greater than three times the length
of the cell-body diameter were scored as positive. In addition, the
rate of neurite induction and the stability of the neurites was
examined.
[0118] The level of NGF secretion was also tested by ELISA.
Quantitation of HNGF released from both encapsulated and
unencapsulated BHK-hNGF cells was performed by a two-site enzyme
immunoassay. The protocol was a modification of that described by
Boehringer Mannheim using Nunc-Immuno Maxisorp ELISA plates. After
color development (30 min.), the samples were analyzed on a plate
reader and measured against recombinant mouse NGF protein
standards.
[0119] The results were as follows:
1 ELISA ELISA ELISA Capsule Capsule Pre-1* Pre-2* Post Histology
No. BAM pg/24 h. pg/24 hr Explant Cell Survival 1 NGF 152 329 268
(+) 2 NGF 271 162 156 (+) 7 Control nd** nd 0 (+) 8 Control nd nd 0
(+) *Devices were assayed twice prior to implantation, once prior
to shipment to the collaborators' laboratory, and a second time
immediately prior to implantation, with a 48-hour time interval
between the two assays. "Pre-1" refers to the results of the first
assay, and "Pre-2" refers to the results of the second assay.
**"nd" is an abbreviation for "not detected."
[0120] In a post explant NGF bioactivity assay, robust neurite
outgrowth was seen for devices 1 and 2 (NGF), but not for devices 3
and 4 (control).
[0121] A second similar experiment was conducted. The results are
as follows:
2 ELISA Capsule Capsule ELISA Pre Post Histology No. BAM pg/24 h.
Explant Cell Survival 5 NGF 1800 nd* (-) 6 NGF 3900 291 (+) 18
Control nd nd (-) 19 Control nd nd (-) *"nd" is an abbreviation for
"not detected."
[0122] In further experiments, BHK cells that secreted hCNTF or
NT4/5 were produced and encapsulated substantially according to
Example 1. However, we experienced difficulties mainly related to
shipping and handling of these devices, leading to poor cell
survival in the capsules. Thus no data for these capsules is
presented here. The shipping difficulties included desiccation,
kinking, breakage, and long exposure to low temperatures.
[0123] Continuing experiments are in progress to deliver various
BAMs into normal and transgenic pig eyes. Pig models are considered
one of the most appropriate animal models for the human eye, based
on size and vasculature.
* * * * *