U.S. patent application number 11/207058 was filed with the patent office on 2006-02-16 for anti-angiogenic compositions and methods of use.
Invention is credited to A. Larry Arsenault, William L. Hunter, Lindsay S. Machan.
Application Number | 20060035832 11/207058 |
Document ID | / |
Family ID | 29253908 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060035832 |
Kind Code |
A1 |
Hunter; William L. ; et
al. |
February 16, 2006 |
Anti-angiogenic compositions and methods of use
Abstract
The present invention provides compositions comprising an
anti-angiogenic factor, and a polymeric carrier. Representative
examples of anti-angiogenic factors include Anti-Invasive Factor,
Retinoic acids and derivatives thereof, and taxol. Also provided
are methods for embolizing blood vessels, and eliminating biliary,
urethral, esophageal, and tracheal/bronchial obstructions.
Inventors: |
Hunter; William L.;
(Vancouver, CA) ; Machan; Lindsay S.; (Vancouver,
CA) ; Arsenault; A. Larry; (Paris, CA) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Family ID: |
29253908 |
Appl. No.: |
11/207058 |
Filed: |
August 19, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10962578 |
Oct 13, 2004 |
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11207058 |
Aug 19, 2005 |
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08486867 |
Jun 7, 1995 |
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10962578 |
Oct 13, 2004 |
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08417160 |
Apr 3, 1995 |
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08486867 |
Jun 7, 1995 |
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08094536 |
Jul 19, 1993 |
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08417160 |
Apr 3, 1995 |
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Current U.S.
Class: |
514/13.3 ;
514/19.3; 514/20.1; 514/559 |
Current CPC
Class: |
A61K 9/1658 20130101;
A61K 31/57 20130101; A61K 31/122 20130101; A61K 31/136 20130101;
A61K 31/203 20130101; Y10S 514/825 20130101; A61L 2300/606
20130101; A61L 31/16 20130101; A61L 31/145 20130101; A61K 47/6925
20170801; A61K 31/519 20130101; A61K 47/50 20170801; C08L 2312/00
20130101; A61K 31/337 20130101; A61K 9/167 20130101; Y10S 514/826
20130101; A61L 2300/416 20130101; A61K 9/1635 20130101; C08L 67/04
20130101; A61L 2300/622 20130101; A61K 47/34 20130101; A61K 9/0019
20130101; A61K 33/24 20130101; C08L 23/0815 20130101; C08L 23/0853
20130101; A61K 9/5153 20130101; A61K 47/32 20130101; A61K 31/335
20130101; A61L 31/10 20130101; A61L 2430/36 20130101; C07K 14/811
20130101; C08L 2203/02 20130101; A61K 9/7007 20130101; A61K 9/1647
20130101; A61K 45/06 20130101; A61K 31/185 20130101; A61K 31/57
20130101; A61K 2300/00 20130101; A61L 31/10 20130101; C08L 23/08
20130101; A61L 31/10 20130101; C08L 67/04 20130101; C08L 23/0853
20130101; C08L 2666/18 20130101; C08L 67/04 20130101; C08L 2666/06
20130101 |
Class at
Publication: |
514/012 ;
514/559 |
International
Class: |
A61K 38/54 20060101
A61K038/54; A61K 31/203 20060101 A61K031/203 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 19, 1994 |
WO |
PCT/CA94/00373 |
Claims
1. A composition comprising: (a) an anti-angiogenic factor; and (b)
a polymeric carrier.
2. The composition of claim 1, wherein said anti-angiogenic factor
is Anti-Invasive Factor.
3. The composition of claim 1, wherein said anti-angiogenic factor
is retinoic acid and derivatives thereof.
4. The composition of claim 1, wherein said anti-angiogenic factor
is selected from the group consisting of Suramin, Tissue Inhibitor
of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,
Plasminogen Activator Inhibitor-1 and Plasminogen Activator
Inhibitor-2.
5. A composition comprising: (a) taxol; and (b) a polymeric
carrier.
6. The composition of claim 1, wherein said composition has an
average size of 15 to 200 lm.
7. The composition of claim 1, wherein said polymeric carrier is
poly(ethylene-vinyl acetate) crosslinked with 40% vinyl
acetate.
8. The composition of claim 1, wherein said polymeric carrier is
poly(lactic-co-glycolic acid).
9. The composition of claim 1, wherein said polymeric carrier is
polycaprolactone.
10. The composition of claim 1, wherein said polymeric carrier is
polylactic acid.
11. The composition of claim 1, wherein said polymeric carrier is a
copolymer of poly(ethylene-vinyl acetate) crosslinked with 40%
vinyl acetate, and polylactic acid.
12. The composition of claim 1, wherein said polymeric carrier is a
copolymer of polylactic acid and polycaprolactone.
13. A method for embolizing a blood vessel, comprising delivering
into said vessel a therapeutically effective amount of composition
according to claims 1-12, such that said blood vessel is
effectively occluded.
14. The method claim 13 wherein said blood vessel nourishes a
tumor.
15. A stent, comprising a generally tubular structure, the surface
of which is coated with a composition according to claims 1-12.
16. A method for expanding the lumen of a body passageway,
comprising inserting a stent into the passageway, the stent having
a generally tubular structure, the surface of said structure being
coated with a composition according to claims 1-12, such that said
passageway is expanded.
17. A method for eliminating vascular obstructions, comprising
inserting a vascular stent into a vascular passageway, the stent
having a generally tubular structure, the surface of said structure
being coated with a composition according to claims 1-12, such that
said vascular obstruction is eliminated.
18. A method for eliminating biliary obstructions, comprising
inserting a biliary stent into a biliary passageway, the stent
having a generally tubular structure, the surface of said structure
being coated with a composition according to claims 1-12, such that
said biliary obstruction is eliminated.
19. A method for eliminating urethral obstructions, comprising
inserting a urethral stent into a urethra, the stent having a
generally tubular structure, the surface of said structure being
coated with a composition according to claims 1-12, such that said
urethral obstruction is eliminated.
20. A method for eliminating esophageal obstructions, comprising
inserting an esophageal stent into an esophagus, the stent having a
generally tubular structure, the surface of said structure being
coated with a composition according to claims 1-12, such that said
esophageal obstruction is eliminated.
21. A method for eliminating tracheal/bronchial obstructions,
comprising inserting a tracheal/bronchial stent into the trachea or
bronchi, the stent having a generally tubular structure, the
surface of which is coated with a composition according to claims
1-12, such that said tracheal/bronchial obstruction is
eliminated.
22. A method for treating a tumor excision site, comprising
administering a composition according to claims 1-12 to the
resection margin of a tumor subsequent to excision, such that the
local recurrence of cancer and the formation of new blood vessels
at said site is inhibited.
23. A method for treating corneal neovascularization, comprising
administering a therapeutically effective amount of a composition
according to claims 1-12 to the cornea, such that the formation of
blood vessels is inhibited.
24. The method of claim 23, wherein said composition further
comprises a topical corticosteroid.
25. A method for inhibiting angiogenesis in patients with
non-tumorigenic, angiogenesis-dependent diseases, comprising
administering a therapeutically effective amount of a composition
comprising taxol to a patient with a non-tumorigenic
angiogenesis-dependent disease, such that the formation of new
blood vessels is inhibited.
26. A method for embolizing a blood vessel in a non-tumorigenic,
angiogenesis-dependent diseases, comprising delivering to said
vessel a therapeutically effective amount of a composition
comprising taxol, such that said blood vessel is effectively
occluded.
27. A method for expanding the lumen of a body passageway,
comprising inserting a stent into the passageway, the stent having
a generally tubular structure, the surface of said structure being
coated with a composition comprising taxol, such that said
passageway is expanded.
28. A method for eliminating vascular obstructions, comprising
inserting a vascular stent into a vascular passageway, the stent
having a generally tubular structure, the surface of said structure
being coated with a composition comprising taxol, such that said
vascular obstruction is eliminated.
29. A method for eliminating biliary obstructions, comprising
inserting a biliary stent into a biliary passageway, the stent
having a generally tubular structure, the surface of said structure
being coated with a composition comprising taxol, such that said
biliary obstruction is eliminated.
30. A method for eliminating urethral obstructions, comprising
inserting a urethral stent into a urethra, the stent having a
generally tubular structure, the surface of said structure being
coated with a composition comprising taxol, such that said urethral
obstruction is eliminated.
31. A method for eliminating esophageal obstructions, comprising
inserting an esophageal stent into an esophagus, the stent having a
generally tubular structure, the surface of said structure being
coated with a composition comprising taxol, such that said
esophageal obstruction is eliminated.
32. A method for eliminating tracheal/bronchial obstructions,
comprising inserting a tracheal/bronchial stent into the trachea or
bronchi, the stent having a generally tubular structure, the
surface of said structure being coated with a composition
comprising taxol, such that said tracheal/bronchial obstruction is
eliminated.
33. A method for treating a tumor excision site, comprising
administering a composition comprising taxol to the resection
margin of a tumor subsequent to excision, such that the local
recurrence of cancer and the formation of new blood vessels at said
site is inhibited.
34. A method for treating corneal neovascularization, comprising
administering a therapeutically effective amount of a composition
comprising taxol to the cornea, such that the formation of new
vessels is inhibited.
35. A pharmaceutical product, comprising: (a) taxol, in a
container; and (b) a notice associated with said container in form
prescribed by a governmental agency regulating the manufacture,
use, or sale of pharmaceuticals, which notice is reflective of
approval by said agency of said taxol, for human or veterinary
administration to treat non-tumorigenic angiogenesis-dependent
diseases.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of co-pending PCT
application CA94/00373, filed Jul. 19, 1994. In addition, this
application is a continuation-in-part of pending U.S. patent
application Ser. No. 08/094,536, filed Jul. 19, 1993.
TECHNICAL FIELD
[0002] The present invention relates generally to compositions and
methods for treating cancer and other angiogenic-dependent
diseases, and more specifically, to compositions comprising
anti-angiogenic factors and polymeric carriers, stents which have
been coated with such compositions, as well as method for utilizing
these stents and compositions.
BACKGROUND OF THE INVENTION
[0003] Angiogenesis-dependent diseases (i.e., those diseases which
require or induce vascular growth) represent a significant portion
of all diseases for which medical treatment is sought. For example,
cancer is the second leading cause of death in the United States,
and accounts for over one-fifth of the total mortality. Briefly,
cancer is characterized by the uncontrolled division of a
population of cells which, most typically, leads to the formation
of one or more tumors. Such tumors are also characterized by the
ingrowth of vasculature which provide various factors that permit
continued tumor growth. Although cancer is generally more readily
diagnosed than in the past, many forms, even if detected early, are
still incurable.
[0004] A variety of methods are presently utilized to treat cancer,
including for example, various surgical procedures. If treated with
surgery alone however, many patients (particularly those with
certain types of cancer, such as breast, brain, colon and hepatic
cancer) will experience recurrence of the cancer. Therefore, in
addition to surgery, many cancers are also treated with a
combination of therapies involving cytotoxic chemotherapeutic drugs
(e.g., vincristine, vinblastine, cisplatin, methotrexate, 5-FU,
etc.) and/or radiation therapy. One difficulty with this approach,
however, is that radiotherapeutic and chemotherapeutic agents are
toxic to normal tissues, and often create life-threatening side
effects. In addition, these approaches often have extremely high
failure/remission rates.
[0005] In addition to surgical, chemo- and radiation therapies,
others have attempted to utilize an individual's own immune system
in order to eliminate cancerous cells. For example, some have
suggested the use of bacterial or viral components as adjuvants in
order to stimulate the immune system to destroy tumor cells. (See
generally "Principles of Cancer Biotherapy," Oldham (ed.), Raven
Press, New York, 1987.) Such agents have generally been useful as
adjuvants and as nonspecific stimulants in animal tumor models, but
have not as of yet proved to be generally effective in humans.
[0006] Lymphokines have also been utilized in the treatment of
cancer. Briefly, lymphokines are secreted by a variety of cells,
and generally have an effect on specific cells in the generation of
an immune response. Examples of lymphokines include Interleukins
(IL)-1, -2, -3, and -4, as well as colony stimulating factors such
as G-CSF, GM-CSF, and M-CSF. Recently, one group has utilized IL-2
to stimulate peripheral blood cells in order to expand and produce
large quantities of cells which are cytotoxic to tumor cells
(Rosenberg et al., N. Engl. J. Med. 313:1485-1492, 1.985).
[0007] Others have suggested the use of antibodies in the treatment
of cancer. Briefly, antibodies may be developed which recognize
certain cell surface antigens that are either unique, or more
prevalent on cancer cells compared to normal cells. These
antibodies, or "magic bullets," may be utilized either alone or
conjugated with a toxin in order to specifically target and kill
tumor cells (Dillman, "Antibody Therapy," Principles of Cancer
Biotherapy, Oldham (ed.), Raven Press, Ltd., New York, 1987).
However, one difficulty is that most monoclonal antibodies are of
murine origin, and thus hypersensitivity against the murine
antibody may limit its efficacy, particularly after repeated
therapies. Common side effects include fever, sweats and chills,
skin rashes, arthritis, and nerve palsies.
[0008] One additional difficulty of present methods is that local
recurrence and local disease control remains a major challenge in
the treatment of malignancy. In particular, a total of 630,000
patients annually (in the U.S.) have localized disease (no evidence
of distant metastatic spread) at the time of presentation; this
represents 64% of al those patients diagnosed with malignancy (this
does not include nonmelanoma skin cancer or carcinoma in situ). For
the vast majority of these patients, surgical resection of the
disease represents the greatest chance for a cure and indeed
428,000 will be cured after the initial treatment--428,000.
Unfortunately, 202,000 (or 32% of all patients with localized
disease) will relapse after the initial treatment. Of those who
relapse, the number who will relapse due to local recurrence of the
disease amounts to 133,000 patients annually (or 21% of all those
with localized disease). The number who will relapse due to distant
metastases of the disease is 68,000 patients annually (11% of all
those with localized disease). Another 102,139 patients annually
will die as a direct result of an inability to control the local
growth of the disease.
[0009] Nowhere is this problem more evident than in breast cancer,
which affects 186,000 women annually in the U.S. and whose
mortality rate has remained unchanged for 50 years. Surgical
resection of the disease through radical mastectomy, modified
radical mastectomy, or lumpectomy remains the mainstay of treatment
for this condition. Unfortunately, 39% of those treated with
lumpectomy alone will develop a recurrence of the disease, and
surprisingly, so will 25% of those in which the resection margin is
found to be clear of tumor histologically. As many as 90% of these
local recurrences will occur within 2 cm of the previous excision
site.
[0010] Similarly, in 1991, over 113,000 deaths and 238,600 new
cases of liver metastasis were reported in North America alone. The
mean survival time for patients with liver metastases is only 6.6
months once liver lesions have developed. Non-surgical treatment
for hepatic metastases include systemic chemotherapy, radiation,
chemoembolization, hepatic arterial chemotherapy, and intraarterial
radiation. However, despite evidence that such treatments can
transiently decrease the size of the hepatic lesions (e.g.,
systemic chemotherapy and hepatic arterial chemotherapy initially
reduces lesions in 15-20%, and 80% of patients, respectively), the
lesions invariably reoccur. Surgical resection of liver metastases
represents the only possibility for a cure, but such a procedure is
possible in only 5% of patients with metastases, and in only 15-20%
of patients with primary hepatic cancer.
[0011] One method that has been attempted for the treatment of
tumors with limited success is therapeutic embolization. Briefly,
blood vessels which nourish a tumor are deliberately blocked by
injection of an embolic material into the vessels. A variety of
materials have been attempted in this regard, including autologous
substances such as fat, blood clot, and chopped muscle fragments,
as well as artificial materials such as wool, cotton, steel balls,
plastic or glass beads, tantalum powder, silicone compounds,
radioactive particles, sterile absorbable gelatin sponge
(Sterispon, Gelfoam), oxidized cellulose (Oxycel), steel coils,
alcohol, lyophilized human dura mater (Lyodura), microfibrillar
collagen (Avitene), collagen fibrils (Tachotop), polyvinyl alcohol
sponge (PVA; Ivalon), Barium-impregnated silicon spheres (Biss) and
detachable balloons. The size of liver metastases may be
temporarily decreased utilizing such methods, but tumors typically
respond by causing the growth of new blood vessels into the
tumor.
[0012] A related problem to tumor formation is the development of
cancerous blockages which inhibit the flow of material through body
passageways, such as the bile ducts, trachea, esophagus,
vasculature and urethra. One device, the stent, has been developed
in order to hold open passageways which have been blocked by tumors
or other substances. Representative examples of common stents
include the Wallstent, Strecker stent, Gianturco stent, and the
Palmaz stent. The major problem with stents, however, is that they
do not prevent the ingrowth of tumor or inflammatory material
through the interstices of the stent. If this material reaches the
inside of a stent and compromises the stent lumen, it may result in
blockage of the body passageway into which it has been inserted. In
addition, presence of a stent in the body may induce reactive or
inflammatory tissue (e.g., blood vessels, fibroblasts, white blood
cells) to enter the stent lumen, resulting in partial or complete
closure of the stent.
[0013] The present invention provides compositions and methods
suitable for treating cancers, as well as other non-tumorigenic
angiogenesis-dependent diseases, and further provides other related
advantages.
SUMMARY OF THE INVENTION
[0014] Briefly stated, the present invention provides
anti-angiogenic compositions, as well as methods and devices which
utilize such compositions for the treatment of cancer and other
angiogenesis-dependent diseases. Within one aspect of the present
invention, compositions are provided (anti-angiogenic compositions)
comprising (a) an anti-angiogenic factor and (b) a polymeric
carrier. A wide variety of molecules may be utilized within the
scope of the present invention as anti-angiogenic factors,
including for example Anti-Invasive Factor, retinoic acids and
their derivatives, paclitaxel including analogues and derivatives
thereof, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue
Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1
and Plasminogen Activator Inhibitor-2, and lighter "d group"
transition metals. Similarly, a wide variety of polymeric carriers
may be utilized, representative examples of which include poly
(ethylene-vinyl acetate) (40% cross-linked), poly (D,L-lactic acid)
oligomers and polymers, poly (L-lactic acid) oligomers and
polymers, poly (glycolic acid), copolymers of lactic acid and
glycolic acid, poly (caprolactone), poly (valerolactone), poly
(anhydrides), copolymers of poly (caprolactone) or poly (lactic
acid) with polyethylene glycol, and blends thereof.
[0015] Within certain preferred embodiments, the compositions
comprise a compound which disrupts microtubule function, such as,
for example, paclitaxel, estramustine, colchicine, methotrexate,
curacin-A, epothilone, vinblastine or tBCEV. Within other preferred
embodiments, the compositions comprise a polymeric carrier and a
lighter d gropu transition metal (e.g., a vanadium species,
molybdenum species, tungsten species, titanium species, niobium
species or tantalum species) which inhibits the formation of new
blood vessels.
[0016] Within one embodiment of the invention, the composition has
an average size of 15 to 200 .mu.m, within other embodiments, the
polymeric carrier of the composition has a molecular weight ranging
from less than 1,000 daltons to greater than 200,000 to 300,000
daltons. Within yet other embodiments, the compositions provided
herein may be formed into films with a thickness of betweem 100
.mu.m and 2 mm, or thermologically active compositions which are
liquid at one temperature (e.g., above 45.degree. C.) and solid or
semi-solid at another (e.g., 37.degree. C.).
[0017] Within another aspect of the present invention methods for
embolizing a blood vessel are provided, comprising the step of
delivering into the vessel a therapeutically effective amount of an
anti-angiogenic composition (as described above), such that the
blood vessel is effectively occluded. Within one embodiment, the
anti-angiogenic composition is delivered to a blood vessel which
nourishes a tumor.
[0018] Within yet another aspect of the present invention, stents
are provided comprising a generally tubular structure, the surface
being coated with one or more anti-angiogenic compositions. Within
other aspects of the present invention, methods are provided for
expanding the lumen of a body passageway, comprising inserting a
stent into the passageway, the stent having a generally tubular
structure, the surface of the structure being coated with an
anti-angiogenic composition as described above, such that the
passageway is expanded. Within various embodiments of the
invention, methods are provided for eliminating biliary
obstructions, comprising inserting a biliary stent into a biliary
passageway; for eliminating urethral obstructions, comprising
inserting a urethral stent into a urethra; for eliminating
esophageal obstructions, comprising inserting an esophageal stent
into an esophagus; and for eliminating tracheal/bronchial
obstructions, comprising inserting a tracheal/bronchial stent into
the trachea or bronchi. In each of these embodiments, the stent has
a generally tubular structure, the surface of which is coated with
an anti-angiogenic composition as described above.
[0019] Within another aspect of the present invention, methods are
provided for treating tumor excision sites, comprising
administering an anti-angiogenic composition as described above to
the resection margins of a tumor subsequent to excision, such that
the local recurrence of cancer and the formation of new blood
vessels at the site is inhibited. Within yet another aspect of the
invention, methods for treating corneal neovascularization are
provided, comprising the step of administering to a patient a
therapeutically effective amount of an anti-angiogenic composition
as described above to the cornea, such that the formation of blood
vessels is inhibited. Within one embodiment, the anti-angiogenic
composition further comprises a topical corticosteroid.
[0020] Within another aspect of the present invention, methods are
provided for inhibiting angiogenesis in patients with
non-tumorigenic, angiogenesis-dependent diseases, comprising
administering to a patient a therapeutically effective amount of
paclitaxel to a patient with a non-tumorigenic
angiogenesis-dependent disease, such that the formation of new
blood vessels is inhibited. Within other aspects, methods are
provided for embolizing blood vessels in non-tumorigenic,
angiogenesis-dependent diseases, comprising delivering to the
vessel a therapeutically effective amount of a composition
comprising paclitaxel, such that the blood vessel is effectively
occluded.
[0021] Within yet other aspects of the present invention, methods
are provided for expanding the lumen of a body passageway,
comprising inserting a stent into the passageway, the stent having
a generally tubular structure, the surface of the structure being
coated with a composition comprising paclitaxel, such that the
passageway is expanded. Within various embodiments of the
invention, methods are provided for eliminating biliary
obstructions, comprising inserting a biliary stent into a biliary
passageway; for eliminating urethral obstructions, comprising
inserting a urethral stent into a urethra; for eliminating
esophageal obstructions, comprising inserting an esophageal stent
into an esophagus; and for eliminating tracheal/bronchial
obstructions, comprising inserting a tracheal/bronchial stent into
the trachea or bronchi. Within each of these embodiments the stent
has a generally tubular structure, the surface of the structure
being coated with a composition comprising paclitaxel.
[0022] Within another aspect of the present invention, methods are
provided for treating a tumor excision site, comprising
administering a composition comprising paclitaxel to the resection
margin of a tumor subsequent to excision, such that the local
recurrence of cancer and the formation of new blood vessels at the
site is inhibited. Within other aspects, methods are provided for
treating neovascular diseases of the eye, comprising administering
to a patient a therapeutically effective amount of an
anti-angiogenic factor (such as a compound which disrupts
microtubule function) to the eye, such that the formation of new
vessels is inhibited.
[0023] Within other aspects of the present invention, methods are
provided for treating inflammatory arthritis, comprising
administering to a patient a therapeutically effective amount of an
anti-angiogenic factor (such as a compound which disrupts
microtubule function), or a composition comprising an
anti-angiogenic factor and a polymeric carrier to a joint. Within
preferred embodiments, the anti-angiogenic factor may be a compound
which disrupts microtubule function such as paclitaxel, or an
element from the lighter `d group` transition metals, such as a
vanadium species.
[0024] Within yet another aspect of the invention, pharmaceutical
products are provided, comprising (a) a compound which disrupts
microtubule function, in a container, and (b) a notice associated
with the container in form prescribed by a governmental agency
regulating the manufacture, use, or sale of pharmaceuticals, which
notice is reflective of approval by the agency of a compound which
disrupts microtubule function, for human or veterinary
administration to treat non-tumorigenic angiogenesis-dependent
diseases such as, for example, inflammatory arthritis or
neovascular diseases of the eye. Briefly, Federal Law requires that
the use of a pharmaceutical agent in the therapy of humans be
approved by an agency of the Federal government. Responsibility for
enforcement (in the United States) is with the Food and Drug
Administration, which issues appropriate regulations for securing
such approval, detailed in 21 U.S.C. .sctn..sctn. 301-392.
Regulation for biological materials comprising products made from
the tissues of animals, is also provided under 42 U.S.C. .sctn.
262. Similar approval is required by most countries, although,
regulations may vary from country to country.
[0025] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings. In addition, various references are set forth
below which describe in more detail certain procedures, devices or
compositions, and are therefore incorporated by reference in their
entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1A is a photograph which shows a shell-less egg culture
on day 6. FIG. 1B is a digitized computer-displayed image taken
with a stereomicroscope of living, unstained capillaries
(1040.times.). FIG. 1C is a photograph of a corrosion casting which
shows CAM microvasculature that are fed by larger, underlying
vessels (arrows; 1300.times.). FIG. 1D is a photograph which
depicts a 0.5 mm thick plastic section cut transversely through the
CAM, and recorded at the light microscope level. This photograph
shows the composition of the CAM, including an outer double-layered
ectoderm (Ec), a mesoderm (M) containing capillaries (arrows) and
scattered adventitila cells, and a single layered endodemm (En)
(400.times.). FIG. 1E is a photograph at the electron microscope
level (3500.times.) wherein typical capillary structure is
presented showing thin-walled endothelial cells (arrowheads) and an
associated pericyte.
[0027] FIGS. 2A, 2B, 2C and 2D are a series of digitized images of
four different, unstained CAMs taken after a 48 hour exposure to
digitized images of four different living, unstained CAMs were
taken after a 48 h exposure to 10.mu. paclitaxel per 10 ml of
methylcellulose. The transparent methylcellulose disk (*)
containing paclitaxel is present on each CAM and is positioned over
a singular avascular zone (A) with surrounding blood islands (Is).
These avascular areas extend beyond the disk and typically have a
diameter of 6 mm. FIG. 2D illustrates the typical "elbowing" effect
(arrowheads) of both small and large vessels being redirected away
from the periphery of the avascular zone.
[0028] FIG. 3A is a photograph (Mag=400.times.) which shows just
peripheral to the avascular zone, that capillaries (arrowheads)
exhibit numerous endothelial cells arrested in mitosis. Ectoderm
(Ec); Mesoderm (M); Endoderm (En). FIG. 3B (Mag=400.times.) shows
that within the avascular zone proper the typical capillary
structure has been eliminated and there are numerous extravasated
blood cells (arrowheads). FIG. 3C (Mag=400.times.) shows that in
the central area of the avascular zone, red blood cells are
dispersed throughout the mesoderm.
[0029] FIGS. 3A, 3B and 3C are a series of photographs of 0.5 mm
thick plastic sections transversely cut through a
paclitaxel-treated CAM at three different locations within the
avascular zone.
[0030] FIGS. 4A, 4B and 4C are series of electron micrographs which
were taken from locations similar to that of FIGS. 3A, 3B and 3C
(respectively) above.
[0031] FIG. 4A (Mag=2,200.times.) shows a small capillary lying
subjacent to the ectodermal layer (Ec) possessing three endothelial
cells arrested in mitosis (*). Several other cell types in both the
ectoderm and mesoderm are also arrested in mitosis. FIG. 4B
(Mag=2,800.times.) shows the early avascular phase contains
extravasated blood cells subjacent to the ectoderm; these blood
cells are intermixed with presumptive endothelial cells (*) and
their processes. Degrative cellular vacuoles (arrowhead). FIG. 4C
(Mag=2,800.times.) shows that in response to paclitaxel, the
ecto-mesodermal interface has become populated with cells in
various stages of degradation containing dense vacuoles and
granules (arrowheads).
[0032] FIG. 5 is a bar graph which depicts the size distribution of
microspheres by number (5% poly (ethylene-vinyl acetate) with 10 mg
sodium suramin into 5% PVA).
[0033] FIG. 6 is a bar graph which depicts the size distribution of
microspheres by weight (5% poly (ethylene-vinyl acetate) with 10 mg
sodium suramin into 5% PVA).
[0034] FIG. 7 is a graph which depicts the weight of encapsulation
of Sodium Suramin in 50 mg poly (ethylene-vinyl acetate).
[0035] FIG. 8 is a graph which depicts the percent of encapsulation
of Sodium Suramin in 50 mg poly (ethylene-vinyl acetate).
[0036] FIG. 9 is a bar graph which depicts the size distribution by
weight of 5% ELVAX microspheres containing 10 mg sodium suramin
made in 5% PVA containing 10% NaCl.
[0037] FIG. 10 is a bar graph which depicts the size distribution
by weight of 5% microspheres containing 10 mg sodium suramin made
in 5% PVA containing 10% NaCl.
[0038] FIG. 11 is a bar graph which depicts the size distribution
by number of 5% microspheres containing 10 mg sodium suramin made
in 5% PVA containing 10% NaCl.
[0039] FIG. 12 is a line graph which depicts the time course of
sodium suramin release.
[0040] FIG. 13 is an illustration of a representative embodiment of
hepatic tumor embolization.
[0041] FIG. 14 is an illustration of the insertion of a
representative stent coated with an anti-angiogenic
composition.
[0042] FIG. 15A is a graph which shows the effect of the EVA:PLA
polymer blend ratio upon aggregation of microspheres. FIG. 15B is a
scanning electron micrograph which shows the size of "small"
microspheres. FIG. 15C (which includes a magnified inset--labelled
"15C-inset") is a scanning electron micrograph which shows the size
of "large" microspheres. FIG. 15D is a graph which depicts the time
course of in vitro paclitaxel release from 0.6% w/v
paclitaxel-loaded 50:50 EVA:PLA polymer blend microspheres into
phosphate buffered saline (pH 7.4) at 37.degree. C. Open circles
are "small" sized microspheres, and closed circles are "large"
sized microspheres. FIG. 15F is a photograph of a CAM which shows
the results of paclitaxel release by microspheres ("MS"). FIG. 15F
is a photograph similar to that of 15E at increased
magnification.
[0043] FIG. 16 is a graph which shows release rate profiles from
polycaprolactone microspheres containing 1%, 2%, 5% or 10%
paclitaxel into phosphate buffered saline at 37.degree. C. FIG. 16B
is a photograph which shows a CAM treated with control
microspheres. FIG. 16C is a photograph which shows a CAM treated
with 5% paclitaxel loaded microspheres.
[0044] FIGS. 17A and 17B, respectively, are two graphs which show
the release of paclitaxel from EVA films, and the percent
paclitaxel remaining in those same films over time. FIG. 17C is a
graph which shows the swelling of EVA/F127 films with no paclitaxel
over time. FIG. 17D is a graph which shows the swelling of EVA/Span
80 films with no paclitaxel over time. FIG. 17E is a graph which
depicts a stress vs. strain curve for various EVA/F127 blends.
[0045] FIGS. 18A and 18B are two graphs which show the melting
point of PCL/MePEG polymer blends as a function of % MePEG in the
formulation (18A), and the percent increase in time needed for PCL
paste at 60.degree. C. to being to solidify as a function of the
amount of MePEG in the formulation (18B). FIG. 18C is a graph which
depicts the softness of varying PCL/MePEG polymer blends. FIG. 18D
is a graph which shows the percent weight change over time for
polymer blends of various MePEG concentrations. FIG. 18E is a graph
which depicts the rate of paclitaxel release over time from various
polymer blends loaded with 1% paclitaxel. FIGS. 18F and 18G are
graphs which depict the effect of varying quantities of paclitaxel
on the total amount of paclitaxel released from a 20% MePEG/PCL
blend. FIG. 18H is a graph which depicts the effect of MePEG on the
tensile strength of a MePEG/PCL polymer.
[0046] FIG. 19A is a photograph which shows control (unloaded)
thermopaste on a CAM. Note that both large vessels and small
vessels (capillaries) are found immediately adjacent to the paste.
Blood flow in the area around and under the paste is unaffected.
FIG. 19B is a photograph of 20% paclitaxel-loaded thermopaste on a
CAM. Note the disruption of the vasculature when compared to the
surrounding tissues. The drug loaded paste has blocked the growth
of the capillaries, caused regression of the larger vessels, and
created a region of avascularity on the CAM assay. FIG. 19C is a
photograph of 0.5% paclitaxel-loaded thermopaste on a CAM
(Mag.-40.times.). Briefly, the paclitaxel-loaded thermopaste disk
induced an avascular zone measuring 6 mm in diameter on the CAM.
This avascular region was induced by blocking new capillary growth
and occluding, disrupting, and regressing the existing blood
vessels found within the treated region. FIG. 19D is a photograph
of control (unloaded) Thermopaste on a CAM. Briefly, after a 2 day
exposure, the blood vessel organization of the CAM (Mag=50.times.)
treated with the control paste shows normal blood vessel
organization. Functional vessels are located immediately adjacent
to the unloaded paste.
[0047] FIGS. 20A and 20B are two photographs of a CAM having a
tumor treated with control (unloaded) thermopaste. Briefly, in FIG.
20A the central white mass is the tumor tissue. Note the abundance
of blood vessels entering the tumor from the CAM in all directions.
The tumor induces the ingrowth of the host vasculature through the
production of "angiogenic factors." The tumor tissue expands
distally along the blood vessels which supply it. FIG. 20B is an
underside view of the CAM shown in 20A. Briefly, this view
demonstrates the radial appearance of the blood vessels which enter
the tumor like the spokes of a wheel.
[0048] Note that the blood vessel density is greater in the
vicinity of the tumor than it is in the surrounding normal CAM
tissue. FIGS. 20C and 20D are two photographs of a CAM having a
tumor treated with 20% paclitaxel-loaded thermopaste. Briefly, in
FIG. 20C the central white mass is the tumor tissue. Note the
paucity of blood vessels in the vicinity of the tumor tissue. The
sustained release of the angiogenesis inhibitor is capable of
overcoming the angiogenic stimulus produced by the tumor.
[0049] The tumor itself is poorly vascularized and is progressively
decreasing in size. FIG. 20D is taken from the underside of the CAM
shown in 20C, and demonstrates the disruption of blood flow into
the tumor when compared to control tumor tissue. Note that the
blood vessel density is reduced in the vicinity of the tumor and is
sparser than that of the normal surrounding CAM tissue.
[0050] FIG. 21A is a graph which shows the effect of paclitaxel/PCL
on tumor growth. FIGS. 21B and 21C are two photographs which show
the effect of control, 10%, and 20% paclitaxel-loaded thermopaste
on tumor growth.
[0051] FIG. 22A is a photograph of synovium from a PBS injected
joint. FIG. 22B is a photograph of synovium from a microsphere
injected joint. FIG. 22C is a photograph of cartilage from joints
injected with PBS, and FIG. 22D is a photograph of cartilage from
joints injected with microspheres.
[0052] FIG. 23A is a photograph of a 0.3% Paclitaxel Ophthalmic
Drop Suspension on a CAM (Mag.=32.times.). The plastic ring was
used to localize the drug treatment to the CAM. Note the lack of
blood vessels located within and immediately adjacent to the ring.
The functional blood vessels bordering the avascular zone are
defined by their "elbowing" morphology away form the drug source.
FIG. 23B is a photograph of a control (unloaded) Ophthalmic Drop
Suspension on a CAM (Mag=32.times.). Note the normal organization
of the CAM blood vessels and the abundance of functional vessels
located within the ring.
[0053] FIG. 24A is a photograph of a 2.5% Paclitaxel-Loaded Stent
Coating (Mag=26.times.). Briefly, the blood vessels surrounding the
avascular zone are morphologically redirected away from the
paclitaxel source; this produces an avascular zone which can
measure up to 6 mm in diameter. The disrupted vascular remnants
which represent vascular regression can be seen within the
avascular zone. FIG. 24B is a control (unloaded) Stent Coating
(Mag=26.times.). Briefly, the blood vessels of the CAM are found
immediately adjacent to the stent and do not illustrate any
morphological alterations.
[0054] FIG. 25 is a photograph of a control stent. Briefly, this
image shows the longitudinal orientation of a nylon stent
incorporated within gliosarcoma tissue of the rat liver. Ingrowth
within the nylon stent is evident.
[0055] FIG. 26 is a photograph of a control stent. Briefly, this
image also illustrates tumor ingrowth within the lumen of the nylon
stent.
[0056] FIG. 27 is a photograph of a lung. Briefly, in addition to
large liver tumors, metastasis to the lung is common. Such
metastases are evident by the presence of small white lobules seen
throughout the lung.
[0057] FIG. 28A is a photograph of Suramin and Cortisone Acetate on
a CAM (Mag=8.times.). Briefly, this image shows an avascular zone
treated with 20 .mu.g of surarnin and 70 .mu.g of cortisone acetate
in 0.5% methylcellulose. Note the blood vessels located at the
periphery of the avascular zone which are being redirected away
from the drug source. FIG. 28B is a photograph which shows the
vascular detail of the effected region at a higher magnification
(Mag=20.times.). Note the avascular regions and the typical
"elbowing" effect of the blood vessels bordering the avascular
zone.
[0058] FIG. 29A is a graph which shows the chemiluminescence
response of neutrophils (5.times.10.sup.6 cells/ml) to plasma
opsonized CPPD crystals (50 mg/ml). Effect of paclitaxel at
(.smallcircle.) no paclitaxel, (.cndot.) 4.5 .mu.M, (.DELTA.) 14
.mu.M, (.tangle-solidup.) 28 .mu.M, (.quadrature.) 46 .mu.M; n=3.
FIG. 29B is a graph which shows the time course concentration
dependence of paclitaxel inhibition of plasma opsonized CPPD
crystal induced neutrophil chemiluminescence.
[0059] FIG. 30A is a graph which shows superoxide anion production
by neutrophils (5.times.10.sup.6 cells/ml) in response to plasma
opsonized CPPD crystals (50 mg/ml). Effect of paclitaxel at
(.smallcircle.) no paclitaxel, (.cndot.) 28 .mu.M, (.DELTA.)
Control (cells alone); n=3. FIG. 30B is a graphic which shows the
time course concentration dependence of paclitaxel inhibition of
plasma opsonized CPPD crystal induced neutrophil superoxide anion
production; n 3.
[0060] FIG. 31A is a graph which shows the chemiluminescence
response of neutrophils (5.times.10.sup.6 cells/ml) in response to
plasma opsonized zymozan (1 mg/ml). Effect of paclitaxel at
(.smallcircle.) no drug, (.cndot.) 28 .mu.M; n=3. FIG. 31B is a
graph which shows plasma opsonized zymosan induced neutrophil
superoxide anion production.
[0061] Effect of paclitaxel at (.smallcircle.) no paclitaxel,
(.cndot.) 28 .mu.M, (.DELTA.) Control (cells alone); n 3.
[0062] FIG. 32A is a graph which shows myeloperoxidase release from
neutrophils (5.times.10.sup.6 cells/ml) in response to plasma
opsonized CPPD crystals (50 mg/ml). Effect of paclitaxel at
(.smallcircle.) no paclitaxel, (.cndot.) 28 .mu.M. (.DELTA.)
Control (cells alone), (.tangle-solidup.) Control (cells with
paclitaxel at 28 .mu.M); n=3.
[0063] FIG. 32B is a graph which shows the concentration dependence
of paclitaxel inhibition of myeloperoxidase release from
neutrophils in response to plasma opsonized CPPD crystals; n 3.
[0064] FIG. 33 is a graph which shows lysozyme release from
neutrophils (5.times.10.sup.6/ml) in response to plasma opsonized
CPPD crystals (50 mg/ml). Effect of paclitaxel at (.smallcircle.)
no paclitaxel, (.cndot.) 28 .mu.M, (.DELTA.) Control (cells alone),
(.tangle-solidup.) Control (cells and paclitaxel at 28 .mu.M); n
3.
[0065] FIG. 34 is a graph which depicts proliferation of
synoviocytes at various concentrations of paclitaxel.
[0066] FIG. 35 is a bar graph which depicts the cytotoxicity of
paclitaxel at various concentrations to proliferating
synoviocytes.
[0067] FIGS. 36A, 36B, and 36C are photographs of a series of gels
which show the effect of various concentrations of paclitaxel on
c-FOS expression.
[0068] FIGS. 37A and 37B are photographs of a series of gels which
show the effect of various concentrations of paclitaxel on
collagenase expression.
[0069] FIG. 38 is a bar graph which depicts the effects of
paclitaxel on viability of normal chondrocytes in vitro.
[0070] FIG. 39 is a graph which shows the percentage of paclitaxel
release based upon gelatinized-paclitaxel of either a large (7200
.mu.m) or small (2100 .mu.m) size.
[0071] FIG. 40 is a graph which shows the effect of gelatin and/or
sodium chloride on the release of paclitaxel from PCL.
[0072] FIG. 41 is a graph which shows the release of paclitaxel
from PDLLA-PEG-PDLLA cylinders containing 20% paclitaxel.
[0073] FIG. 42A is a graph which depicts the time course of
paclitaxel release from 2.5 mg pellets of PCL. FIG. 42B is a graph
which shows the percent paclitaxel remaining in the pellet, over
time.
[0074] FIG. 43A is a graph which shows the effect of MePEG on
paclitaxel release from PCL paste leaded with 20% paclitaxel. FIG.
43B is a graph which shows the percent paclitaxel remaining in the
pellet, over time.
[0075] FIGS. 44A and 44B are graphs which show the effect of
various concentrations of MePEG in PCL in terms of melting point
(44A) and time to solidify (44B).
[0076] FIG. 45 is a graph which shows the effect of MePEG
incorporation into PCL on the tensile strength and time to fail of
the polymer.
[0077] FIG. 46 is a graph which shows the effect of irradiation on
paclitaxel release.
[0078] FIGS. 47A, B, C, D and E show the effect of MTX release from
PCL over time.
[0079] FIG. 48 is a graph of particle diameter (.mu.m) determined
by a Coulter.RTM. LS 130 Particle Size Analysis.
[0080] FIG. 49 is a graph of particle diameter (.mu.m) determined
by a Coulter.RTM. LS130 Particle Size Analysis.
[0081] FIG. 50 is a graph which shows paclitaxel release from
various polymeric formulations.
[0082] FIG. 51 is a graph which shows the effect of plasma
opsonization of polymeric microspheres on the chemiluminescence
response of neutrophils (20 mg/ml microspheres in 0.5 ml of cells
(conc. 5.times.10.sup.6 cells/ml) to PCL microspheres.
[0083] FIG. 52 is a graph which shows the effect of precoating
plasma +/-2% pluronic F127 on the chemiluminescence response of
neutrophils (5.times.10.sup.6 cells/ml) to PCL microspheres
[0084] FIG. 53 is a graph which shows the effect of precoating
plasma +/-2% pluronic F127 on the chemiluminescence response of
neutrophils (5.times.10.sup.6 cells/ml) to PMMA microspheres
[0085] FIG. 54 is a graph which shows the effect of precoating
plasma +/-2% pluronic F127 on the chemiluminescence response of
neutrophils (5.times.10.sup.6 cells/ml) to PLA microspheres
[0086] FIG. 55 is a graph which shows the effect of precoating
plasma +/-2% pluronic F127 on the chemiluminescence response of
neutrophils (5.times.10.sup.6 cells/ml) to EVA:PLA microspheres
[0087] FIG. 56 is a graph which shows the effect of precoating IgG
(2 mg/ml), or 2% pluronic F127 then IgG (2 mg/ml) on the
chemiluminescence response of neutrophils to PCL microspheres.
[0088] FIG. 57 is a graph which shows the effect of precoating IgG
(2 mg/ml), or 2% pluronic F127 then IgG (2 mg/ml) on the
chemiluminescence response of neutrophils to PMMA microspheres.
[0089] FIG. 58 is a graph which shows the effect of precoating IgG
(2 mg/ml), or 2% pluronic F127 then IgG (2 mg/ml) on the
chemiluminescence response of neutrophils to PVA microspheres.
[0090] FIG. 59 is a graph which shows the effect of precoating IgG
(2 mg/ml), or 2% pluronic F127 then IgG (2 mg/ml) on the
chemiluminescence response of neutrophils to EVA:PLA
microspheres.
[0091] FIG. 60 is a photograph of 10% methotrexate ("MTX") loaded
microspheres made from a 50:50 ratio of PLA:GA (IV 0.78).
[0092] FIG. 61 is a graph which depicts the release of 10% loaded
vanadyl sulfate from PCL.
[0093] FIG. 62 is a photograph of hyaluronic acid microspheres
containing vanadium sulfate.
[0094] FIG. 63A is a graph which depicts the release of organic
vanadate from PCL. FIG. 63B depicts the percentage of organic
vanadate remaining over a time course.
[0095] FIG. 64 is a photograph showing poly D,L, lactic acid
microspheres containing organic vanadate.
[0096] FIGS. 65A and 65B are photographs of control (uncoated)
stents which show typical epithelial ingrowth seen at both 8 weeks
(A) and at 16 weeks (B). Indentations of the stent tines (t) and
narrowing of the lumen (lu) are shown. There is progressive
epithelial overgrowth of the stent surface over this time by
fibrous and inflammatory tissue.
[0097] FIGS. 66A, 66B, 66C, and 66D are a series of photographs
which show control and paclitaxel-coated biliary stents. FIG. 66A
illustrates the obliteration of the stent lumen by the process of
benign epithelial overgrowth. At higher magnification (66B), the
fibrous and inflammatory tissue is evident with little luminal
space remaining. The paclitaxel-treated biliary duct remains patent
(66C). At higher magnification, normal biliary tract epithelium is
present with only minimal alteration of the mucosal lining by the
coated stent tines (t).
DETAILED DESCRIPTION OF THE INVENTION
[0098] As noted above, the present invention provides methods and
compositions which utilize anti-angiogenic factors. Briefly, within
the context of the present invention, anti-angiogenic factors
should be understood to include any protein, peptide, chemical, or
other molecule, which acts to inhibit vascular growth. A variety of
methods may be readily utilized to determine the anti-angiogenic
activity of a given factor, including for example, chick
chorioallantoic membrane ("CAM") assays. Briefly, as described in
more detail below in Examples 2A and 2C, a portion of the shell
from a freshly fertilized chicken egg is removed, and a methyl
cellulose disk containing a sample of the anti-angiogenic factor to
be tested is placed on the membrane. After several days (e.g., 48
hours), inhibition of vascular growth by the sample to be tested
may be readily determined by visualization of the chick
chorioallantoic membrane in the region surrounding the methyl
cellulose disk. Inhibition of vascular growth may also be
determined quantitatively, for example, by determining the number
and size of blood vessels surrounding the methyl cellulose disk, as
compared to a control methyl cellulose disk. Although
anti-angiogenic factors as described herein are considered to
inhibit the formation of new blood vessels if they do so in merely
a statistically significant manner, as compared to a control,
within preferred aspects such anti-angiogenic factors will
completely inhibit the formation of new blood vessels, as well as
reduce the size and number of previously existing vessels.
[0099] In addition to the CAM assay described above, a variety of
other assays may also be utilized to determine the efficacy of
anti-angiogenic factors in vivo, including for example, mouse
models which have been developed for this purpose (see Roberston et
al., Cancer. Res. 51:1339-1344, 1991). In addition, a variety of
representative in vivo assays relating to various aspects of the
inventions described herein have also been described in more detail
below in Examples 5 to 7, and 17 to 19.
[0100] As noted above, the present invention provides compositions
comprising an anti-angiogenic factor, and a polymeric carrier.
Briefly, a wide variety of anti-angiogenic factors may be readily
utilized within the context of the present invention.
Representative examples include Anti-Invasive Factor, retinoic acid
and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of
Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,
Plasminogen Activator Inhibitor-1, Plasminogen Activator
Inhibitor-2, and various forms of the lighter "d group" transition
metals. These and other anti-angiogenic factors will be discussed
in more detail below.
[0101] Briefly, Anti-Invasive Factor, or "AIF" which is prepared
from extracts of cartilage, contains constituents which are
responsible for inhibiting the growth of new blood vessels. These
constituents comprise a family of 7 low molecular weight proteins
(<50,000 daltons) (Kuettner and Pauli, "Inhibition of
neovascularization by a cartilage factor" in Development of the
Vascular System, Pitman Books (CIBA Foundation Symposium 100), pp.
163-173, 1983), including a variety of proteins which have
inhibitory effects against a variety of proteases (Eisentein et al,
Am. J. Pathol. 81:337-346, 1975; Langer et al., Science 193:70-72,
1976; and Horton et al., Science 199:1342-1345, 1978). AIF suitable
for use within the present invention may be readily prepared
utilizing techniques known in the art (e.g., Eisentein et al,
supra; Kuettner and Pauli, supra; and Langer et al., supra).
Purified constituents of AIF such as Cartilage-Derived Inhibitor
("CDI") (see Moses et al., Science 248:1408-1410, 1990) may also be
readily prepared and utilized within the context of the present
invention.
[0102] Retinoic acids alter the metabolism of extracellular matrix
components, resulting in the inhibition of angiogenesis. Addition
of proline analogs, angiostatic steroids, or heparin may be
utilized in order to synergistically increase the anti-angiogenic
effect of transretinoic acid. Retinoic acid, as well as derivatives
thereof which may also be utilized in the context of the present
invention, may be readily obtained from commercial sources,
including for example, Sigma Chemical Co. (# R2625).
[0103] Paclitaxel is a highly derivatized diterpenoid (Wani et al.,
J. Am. Chem. Soc. 93:2325, 1971) which has been obtained from the
harvested and dried bark of Taxus brevifolia (Pacific Yew.) and
Taxomyces Andreanae and Endophytic Fungus of the Pacific Yew
(Stierle et al., Science 60:214-216, 1993). Generally, paclitaxel
acts to stabilize microtubular structures by binding tubulin to
form abnormal mitotic spindles. "Paclitaxel" (which should be
understood herein to include analogues and derivatives such as, for
example, TAXOL.RTM., TAXOTERE.RTM. 10-desacetyl analogues of
paclitaxel and 3'N-desbenzoyl-3'N-t-butoxy carbonyl analogues of
paclitaxel) may be readily prepared utilizing techniques known to
those skilled in the art (see also WO 94/07882, WO 94/07881, WO
94/07880, WO 94/07876, WO 93/23555, WO 93/10076, U.S. Pat. Nos.
5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534,
5,229,529, and EP 590267), or obtained from a variety of commercial
sources, including for example, Sigma Chemical Co., St. Louis, Mo.
(T7402--from Taxus brevifolia).
[0104] Suramin is a polysulfonated naphthylurea compound that is
typically used as a trypanocidal agent. Briefly, Suramin blocks the
specific cell surface binding of various growth factors such as
platelet derived growth factor ("PDGF"), epidermal growth factor
("EGF"), transforming growth factor ("TGF-.beta."), insulin-like
growth factor ("IGF-1"), and fibroblast growth factor
(".beta.FGF"). Suramin may be prepared in accordance with known
techniques, or readily obtained from a variety of commercial
sources, including for example Mobay Chemical Co., New York. (see
Gagliardi et al., Cancer Res. 52:5073-5075, 1992; and Coffey, Jr.,
et al., J. of Cell. Phys. 132:143-148, 1987).
[0105] Tissue Inhibitor of Metalloproteinases-1 ("TIMP") is
secreted by endothelial cells which also secrete MTPases. TIMP is
glycosylated and has a molecular weight of 28.5 kDa. TIMP-1
regulates angiogenesis by binding to activated metalloproteinases,
thereby suppressing the invasion of blood vessels into the
extracellular matrix. Tissue Inhibitor of Metalloproteinases-2
("TIMP-2") may also be utilized to inhibit angiogenesis. Briefly,
TIMP-2 is a 21 kDa nonglycosylated protein which binds to
metalloproteinases in both the active and latent, proenzyme forms.
Both TIMP-1 and TIMP-2 may be obtained from commercial sources such
as Synergen, Boulder, Colo.
[0106] Plasminogen Activator Inhibitor--1 (PA) is a 50 kDa
glycoprotein which is present in blood platelets, and can also be
synthesized by endothelial cells and muscle cells. PAI-1 inhibits
t-PA and urokinase plasminogen activator at the basolateral site of
the endothelium, and additionally regulates the fibrinolysis
process. Plasminogen Activator Inhibitor-2 (PAI-2) is generally
found only in the blood under certain circumstances such as in
pregnancy, and in the presence of tumors. Briefly, PAI-2 is a 56
kDa protein which is secreted by monocytes and macrophages. It is
believed to regulate fibrinolytic activity, and in particular
inhibits urokinase plasminogen activator and tissue plasminogen
activator, thereby preventing fibrinolysis.
[0107] Lighter "d group" transition metals include, for example,
vanadium, molybdenum, tungsten, titanium, niobium, and tantalum
species. Such transition metal species may form transition metal
complexes. Suitable complexes of the above-mentioned transition
metal species include oxo transition metal complexes.
[0108] Representative examples of vanadium complexes include oxo
vanadium complexes such as vanadate and vanadyl complexes. Suitable
vanadate complexes include metavanadate (i.e., VO.sub.3.sup.-) and
orthovanadate (i.e., VO.sub.4.sup.3-) complexes such as, for
example, ammonium metavanadate (i.e., NH.sub.4VO.sub.3), sodium
metavanadate (i.e., NaVO.sub.3), and sodium orthovanadate (i.e.,
Na.sub.3VO.sub.4). Suitable vanadyl (i.e., VO.sup.2+) complexes
include, for example, vanadyl acetylacetonate and vanadyl sulfate
including vanadyl sulfate hydrates such as vanadyl sulfate mono-
and trihydrates.
[0109] Representative examples of tungsten and molybdenum complexes
also include oxo complexes. Suitable oxo tungsten complexes include
tungstate and tungsten oxide complexes. Suitable tungstate (i.e.,
WO.sub.4.sup.2-) complexes include ammonium tungstate (i.e.,
(NH.sub.4).sub.2WO.sub.4), calcium tungstate (i.e., CaWO.sub.4),
sodium tungstate dihydrate (i.e., Na.sub.2WO.sub.4.2H.sub.2O), and
tungstic acid (i.e., H.sub.2WO.sub.4). Suitable tungsten oxides
include tungsten (IV) oxide (i.e., WO.sub.2) and tungsten (VI)
oxide (i.e., WO.sub.3). Suitable oxo molybdenum complexes include
molybdate, molybdenum oxide, and molybdenyl complexes. Suitable
molybdate (i.e., MoO.sub.4.sup.2-) complexes include ammonium
molybdate (i.e., (NH.sub.4).sub.2MoO.sub.4) and its hydrates,
sodium molybdate (i.e., Na.sub.2MoO.sub.4) and its hydrates, and
potassium molybdate (i.e., K.sub.2MoO.sub.4) and its hydrates.
Suitable molybdenum oxides include molybdenum (VI) oxide (i.e.,
MoO.sub.2), molybdenum (VI) oxide (i.e., MoO.sub.3), and molybdic
acid. Suitable molybdenyl (i.e., MoO.sub.2.sup.2+) complexes
include, for example, molybdenyl acetylacetonate. Other suitable
tungsten and molybdenum complexes include hydroxo derivatives
derived from, for example, glycerol, tartaric acid, and sugars.
[0110] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include Platelet Factor 4 (Sigma Chemical
Co., #F1385); Protamine Sulphate (Clupeine) (Sigma Chemical Co.,
#P4505); Sulphated Chitin Derivatives (prepared from queen crab
shells), (Sigma Chemical Co., #C3641; Murata et al., Cancer Res.
51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex
(SP-PG) (the function of this compound may be enhanced by the
presence of steroids such as estrogen, and tamoxifen citrate);
Staurosporine (Sigma Chemical Co., #S4400); Modulators of Matrix
Metabolism, including for example, proline analogs
{[(L-azetidine-2-carboxylic acid (LACA) (Sigma Chemical Co.,
#A0760)), cishydroxyproline, d,L-3,4-ehydroproline (Sigma Chemical
Co., #D0265), Thiaproline (Sigma Chemical Co., #T0631)],
.alpha.,.alpha.-dipyridyl (Sigma Chemical Co., #D7505),
.beta.-aminopropionitrile fumarate (Sigma Chemical Co., #A3134)]};
MDL 27032 (4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Merion Merrel
Dow Research Institute); Methotrexate (Sigma Chemical Co., #A6770;
Hirata et al., Arthritis and Rheumatism 32:1065-1073, 1989);
Mitoxantrone (Polyerini and Novak, Biochem. Biophys. Res. Comm.
140:901-907); Heparin (Folkman, Bio. Phar. 34:905-909, 1985; Sigma
Chemical Co., #P8754); Interferons (e.g., Sigma Chemical Co.,
#13265); 2 Macroglobulin-serum (Sigma Chemical Co., #M7151);
ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992);
Chymostatin (Sigma Chemical Co., #C7268; Tomkinson et al., Biochem
J. 286:475-480, 1992); .beta.-Cyclodextrin Tetradecasulfate (Sigma
Chemical Co., #C4767); Eponemycin; Camptothecin; Fumagillin (Sigma
Chemical Co., #F6771; Canadian Patent No. 2,024,306; Ingber et al.,
Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Sigma:G4022; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446,
1987); (D-Penicillamine ("CDPT"; Sigma Chemical Co., #P4875 or
P5000(HCl)); .beta.-1-anticollagenase-serum; .alpha.2-antiplasmin
(Sigma Chem. Co.:A0914; Holmes et al., J. Biol. Chem.
262(4):1659-1664, 1987); Bisantrene (National Cancer Institute);
Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid
disodium or "CCA"; Takeuchi et al., Agents Actions 36:312-316,
1992); Thalidomide; Angostatic steroid; AGM-1470;
carboxynaminolmidazole; metalloproteinase inhibitors such as BB94
and the peptide CDPGYIGSR-NH.sub.2 (SEQUENCE ID NO. 1) (Iwaki
Glass, Tokyo, Japan).
[0111] Although the above anti-angiogenic factors have been
provided for the purposes of illustration, it should be understood
that the present invention is not so limited. In particular,
although certain anti-angiogenic factors are specifically referred
to above, the present invention should be understood to include
analogues, derivatives and conjugates of such anti-angiogenic
factors. For example, paclitaxel should be understood to refer to
not only the common chemically available form of paclitaxel, but
analogues (e.g., taxotere, as noted above) and paclitaxel
conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or
paclitaxel-xylos).
[0112] Anti-angiogenic compositions of the present invention may
additionally comprise a wide variety of compounds in addition to
the anti-angiogenic factor and polymeric carrier. For example,
anti-angiogenic compositions of the present invention may also,
within certain embodiments of the invention, also comprise one or
more antibiotics, anti-inflammatories, anti-viral agents,
anti-fungal agents and/or anti-protozoal agents. Representative
examples of antibiotics included within the compositions described
herein include: penicillins; cephalosporins such as cefadroxil,
cefazolin, cefaclor; aminoglycosides such as gentamycin and
tobramycin; sulfonamides such as sulfamethoxazole; and
metronidazole. Representative examples of anti-inflammatories
include: steroids such as prednisone, prednisolone, hydrocortisone,
adrenocorticotropic hormone, and sulfasalazine; and non-steroidal
anti-inflammatory drugs ("NSAIDS") such as aspirin, ibuprofen,
naproxen, fenoprofen, indomethacin, and phenylbutazone.
Representative examples of antiviral agents include acyclovir,
ganciclovir, zidovudine. Representative examples of antifungal
agents include: nystatin, ketoconazole, griseofulvin, flucytosine,
miconazole, clotrimazole. Representative examples of antiprotozoal
agents include: pentamidine isethionate, quinine, chloroquine, and
mefloquine.
[0113] Anti-angiogenic compositions of the present invention may
also contain one or more hormones such as thyroid hormone,
estrogen, progesterone, cortisone and/or growth hormone, other
biologically active molecules such as insulin, as well as T.sub.H1
(e.g., Interleukins-2, -12, and -15, gamma interferon) or T.sub.H2
(e.g., Interleukins-4 and -10) cytokines.
[0114] Within certain preferred embodiments of the invention,
anti-angiogenic compositions are provided which contain one or more
compounds which disrupt microtubule function. Representative
examples of such compounds include paclitaxel (discussed above),
estramustine (available from Sigma; Wang and Stearns Cancer Res.
48:6262-6271, 1988), epothilone, curacin-A, colchicine,
methotrexate, vinblastine and 4-tert-butyl-[3-(2-chloroethyl)
ureido] benzene ("tBCEU").
[0115] Anti-angiogenic compositions of the present invention may
also contain a wide variety of other compounds, including for
example: .alpha.-adrenergic blocking agents, angiotensin II
receptor antagonists and receptor antagonists for histamine,
serotonin, endothelin; inhibitors of the sodium/hydrogen antiporter
(e.g., amiloride and its derivatives); agents that modulate
intracellular Ca.sup.2+ transport such as L-type (e.g., diltiazem,
nifedipine, verapamil) or T-type Ca.sup.2+ channel blockers (e.g.,
amiloride), calmodulin antagonists (e.g., H.sub.7) and inhibitors
of the sodium/calcium antiporter (e.g., amiloride); ap-1 inhibitors
(for tyrosine kinases, protein kinase C, myosin light chain kinase,
Ca.sup.2+/calmodulin kinase II, casein kinase I); anti-depressants
(e.g. amytriptyline, fluoxetine, LUVOX.RTM. and PAXII.RTM.);
cytokine and/or growth factors, as well as their respective
receptors, (e.g., the interleukins, .alpha., .beta., or
.gamma.-IFN, GM-CSF, G-CSF, epidermal growth factor, transforming
growth factors alpha and beta, TNF, and antagonists of vascular
epithelial growth factor, endothelial growth factor, acidic or
basic fibroblast growth factors, and platelet dervived growth
factor); inhibitors of the IP.sub.3 receptor (e.g., heparin);
protease and collagenase inhibitors (e.g., TIMPs, discussed above);
nitrovasodilators (e.g., isosorbide dinitrate); anti-mitotic agents
(e.g., colchicine, anthracyclines and other antibiotics, folate
antagonists and other anti-metabolites, vinca alkaloids,
nitrosoureas, DNA alkylating agents, topoisomerase inhibitors,
purine antagonists and analogs, pyrimidine antagonists and analogs,
alkyl sulfonates); immunosuppressive agents (e.g.,
adrenocorticosteroids, cyclosporine); sense or antisense
oligonucleotides (e.g., DNA, RNA, nucleic acid analogues (e.g.,
peptide nucleic acids) or any combinations of these); and
inhibitors of transcription factor activity (e.g., lighter d group
transition metals).
[0116] Anti-angiogenic compositions of the present invention may
also comprise additional ingredients such as surfactants (either
hydrophilic or hydrophobic; see Example 13), anti-neoplastic or
chemotherapeutic agents (e.g., 5-fluorouracil, vincristine,
vinblastine, cisplatin, doxyrubicin, adriamycin, or tamocifen),
radioactive agents (e.g., Cu-64, Ga-67, Ga-68, Zr-89, Ru-97,
Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I-131, Re-186,
Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212) or
toxins (e.g., ricin, abrin, diphtheria toxin, cholera toxin,
gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and
Pseudomonas exotoxin A).
[0117] As noted above, anti-angiogenic compositions of the present
invention comprise an anti-angiogenic factor and a polymeric
carrier. In addition to the wide array of anti-angiogenic factors
and other compounds discussed above, anti-angiogenic compositions
of the present invention are provided in a wide variety of
polymeric carriers, including for example both biodegradable and
non-biodegradable compositions. Representative examples of
biodegradable compositions include albumin, gelatin, starch,
cellulose, dextrans, polysaccharides, fibrinogen, poly (D,L
lactide), poly (D,L-lactide-co-glycolide), poly (glycolide), poly
(hydroxybutyrate), poly (alkylcarbonate) and poly (orthoesters)
(see generally, Illum, L., Davids, S. S. (eds.) "Polymers in
controlled Drug Delivery" Wright, Bristol, 1987; Arshady, J.
Controlled Release 17:1-22, 1991; Pitt, Int. J. Phar. 59:173-196,
1990; Holland et al., J. Controlled Release 4:155-0180, 1986).
Representative examples of nondegradable polymers include EVA
copolymers, silicone rubber and poly (methylmethacrylate).
Particularly preferred polymeric carriers include poly
(ethylene-vinyl acetate)(40% cross-linked), poly (D,L-lactic acid)
oligomers and polymers, poly (L-lactic acid) oligomers and
polymers, poly (glycolic acid), copolymers of lactic acid and
glycolic acid, poly (caprolactone), poly (valerolactone),
polyanhydrides, copolymers of poly (caprolactone) or poly (lactic
acid) with polyethylene glycol and blends thereof.
[0118] Polymeric carriers may be fashioned in a variety of forms,
including for example, rod-shaped devices, pellets, slabs, or
capsules (see, e.g., Goodell et al., Am. J. Hosp. Pharm.
43:1454-1461, 1986; Langer et al., "Controlled release of
macromolecules from polymers", in Biomedical polymers, Polymeric
materials and pharmaceuticals for biomedical use, Goldberg, E. P.,
Nakagim, A. (eds.) Academic Press, pp. 113-137, 1980; Rhine et al.,
J. Pharm. Sci. 69:265-270, 1980; Brown et al., J. Pharm. Sci.
72:1181-1185, 1983; and Bawa et al., J. Controlled Release
1:259-267, 1985). Anti-angiogenic factors may be linked by
occlusion in the matrices of the polymer, bound by covalent
linkages, or encapsulated in microcapsules. Within certain
preferred embodiments of the invention, anti-angiogenic
compositions are provided in non-capsular formulations such as
microspheres (ranging from nanometers to micrometers in size),
pastes, threads of various size, films and sprays.
[0119] Preferably, anti-angiogenic compositions of the present
invention (which comprise one or more anti-angiogenic factors, and
a polymeric carrier) are fashioned in a manner appropriate to the
intended use. Within certain aspects of the present invention, the
anti-angiogenic composition should be biocompatible, and release
one or more anti-angiogenic factors over a period of several days
to months. For example, "quick release" or "burst" anti-angiogenic
compositions are provided that release greater than 10%, 20%, or
25% (w/v) of an anti-angiogenic factor (e.g., paclitaxel) over a
period of 7 to 10 days. Such "quick release" compositions should,
within certain embodiments, be capable of releasing
chemotherapeutic levels (where applicable) of a desired
anti-angiogenic factor. Within other embodiments, "low release"
anti-angiogenic compositions are provided that release less than 1%
(w/v) of an anti-angiogenic factor over a period of 7 to 10 days.
Further, anti-angiogenic compositions of the present invention
should preferably be stable for several months and capable of being
produced and maintained under sterile conditions.
[0120] Within certain aspects of the present invention,
anti-angiogenic compositions may be fashioned in any size ranging
from 50 nm to 500 .mu.m, depending upon the particular use. For
example, when used for the purpose of tumor embolization (as
discussed below), it is generally preferable to fashion the
anti-angiogenic composition in microspheres of between 15 and 500
.mu.m, preferably between 15 and 200 .mu.m, and most preferably,
between 25 and 150 .mu.m. Alternatively, such compositions may also
be readily applied as a "spray", which solidifies into a film or
coating. Such sprays may be prepared from microspheres of a wide
array of sizes, including for example, from 0.1 .mu.m to 3 .mu.m,
from 10 .mu.m to 30 .mu.m, and from 30 .mu.m to 100 .mu.m (see
Example 8).
[0121] Anti-angiogenic compositions may also be prepared, given the
disclosure provided herein, for a variety of other applications.
For example, for administration to the cornea, the anti-angiogenic
factors of the present invention may be incorporated into
muco-adhesive polymers (e.g., polyacrylic acids such as
(CARBOPOL.RTM., dextron, polymethacrylate, or starch (see LeYung
and Robinson, J. of Controlled Rel. 5:223, 1988)), or
nanometer-sized microspheres (see generally, Kreuter J. Controlled
Release 16:169-176, 1991; Couvreur and Vauthier, J. Controlled
Release 17:187-198, 1991).
[0122] Anti-angiogenic compositions of the present invention may
also be prepared in a variety of "paste" or gel forms. For example,
within one embodiment of the invention, anti-angiogenic
compositions are provided which are liquid at one temperature
(e.g., temperature greater than 37.degree. C., such as 40.degree.
C., 45.degree. C., 50.degree. C., 55.degree. C. or 60.degree. C.),
and solid or semi-solid at another temperature (e.g., ambient body
temperature, or any temperature lower than 37.degree. C.). Such
"thermopastes" may be readily made given the disclosure provided
herein (see, e.g., Examples 10 and 14).
[0123] Within yet other aspects of the invention, the
anti-angiogenic compositions of the present invention may be formed
as a film. Preferably, such films are generally less than 5, 4, 3,
2, or 1, mm thick, more preferably less than 0.75 mm or 0.5 mm
thick, and most preferably less than 500 .mu.m to 100 .mu.m thick.
Such films are preferably flexible with a good tensile strength
(e.g., greater than 50, preferably greater than 100, and more
preferably greater than 150 or 200 N/cm.sup.2), good adhesive
properties (i.e., readily adheres to moist or wet surfaces), and
has controlled permeability. Representative examples of such films
are set forth below in the Examples (see e.g., Example 13).
[0124] Representative examples of the incorporation of
anti-angiogenic factors such as those described above into a
polymeric carriers is described in more detail below in Examples 3,
4 and 8-15.
Polymeric Carriers for the Release of Hydrophobic Compounds
[0125] Within further aspects of the present invention, polymeric
carriers are provided which are adapted to contain and release a
hydrophobic compound, the carrier containing the hydrophobic
compound in combination with a carbohydrate, protein or
polypeptide. Within certain embodiments, the polymeric carrier
contains or comprises regions, pockets, or granules of one or more
hydrophobic compounds. For example, within one embodiment of the
invention, hydrophobic compounds may be incorporated within a
matrix which contains the hydrophobic compound, followed by
incorporation of the matrix within the polymeric carrier. A variety
of matrices can be utilized in this regard, including for example,
carbohydrates and polysaccharides such as starch, cellulose,
dextran, methylcellulose, and hyaluronic acid, proteins or
polypeptides such as albumin, collagen and gelatin (see e.g.,
Example 31). Within alternative embodiments, hydrophobic compounds
may be contained within a hydrophobic core, and this core contained
within a hydrophilic shell. For example, as described in Example
38, paclitaxel may be incorporated into a hydrophobic core (e.g.,
of the poly D,L lactic acid-PEG or MePEG aggregate) which has a
hydrophilic shell.
[0126] A wide variety of hydrophobic compounds may be released from
the polymeric carriers described above, including for example:
certain hydrophobic compounds which disrupt microtubule function
such as paclitaxel and estramustine; hydrophobic proteins such as
myelin basic protein, proteolipid proteins of CNS myelin,
hydrophobic cell wall protein, porins, membrane proteins (EMBO J.
12(9):3409-3415, 1993), myelin oligodendrocyte glycoprotein ("MOG")
(Biochem. and Mol. Biol. Int. 30(5):945-958, 1993, P27 Cancer Res.
53(17):4096-4101, 1913, bacterioopsin, human surfactant protein
("HSB"; J. Biol. Chem. 268(15): 11160-11166, 1993), and SP-B or
SP-C (Biochimica et Biophysica Acta 1105(1):161-169, 1992).
Arterial Embolization
[0127] In addition to the compositions described above, the present
invention also provides a variety of methods which utilize the
above-described anti-angiogenic compositions. In particular, within
one aspect of the present invention methods are provided for
embolizing a blood vessel, comprising the step of delivering into
the vessel a therapeutically effective amount of an anti-angiogenic
composition (as described above), such that the blood vessel is
effectively occluded. Therapeutically effective amounts suitable
for occluding blood vessels may be readily determined given the
disclosure provided below, and as described in Example 6. Within a
particularly preferred embodiment, the anti-angiogenic composition
is delivered to a blood vessel which nourishes a tumor (see FIG.
13).
[0128] Briefly, there are a number of clinical situations (e.g.,
bleeding, tumor development) where it is desirable to reduce or
abolish the blood supply to an organ or region. As described in
greater detail below, this may be accomplished by injecting
anti-angiogenic compositions of the present invention into a
desired blood vessel through a selectively positioned catheter (see
FIG. 13). The composition travels via the blood stream until it
becomes wedged in the vasculature, thereby physically (or
chemically) occluding the blood vessel. The reduced or abolished
blood flow to the selected area results in infarction (cell death
due to an inadequate supply of oxygen and nutrients) or reduced
blood loss from a damaged vessel.
[0129] For use in embolization therapy, anti-angiogenic
compositions of the present invention are preferably non-toxic,
thrombogenic, easy to inject down vascular catheters, radio-opaque,
rapid and permanent in effect, sterile, and readily available in
different shapes or sizes at the time of the procedure. In
addition, the compositions preferably result in the slow (ideally,
over a period of several weeks to months) release of an
anti-angiogenic factor. Particularly preferred anti-angiogenic
compositions should have a predictable size of 15-200 .mu.m after
being injected into the vascular system. Preferably, they should
not clump into larger particles either in solution or once
injected. In addition, preferable compositions should not change
shape or physical properties during storage prior to use.
[0130] Embolization therapy may be utilized in at least three
principal ways to assist in the management of neoplasms: (1)
definitive treatment of tumors (usually benign); (2) for
preoperative embolization; and (3) for palliative embolization.
Briefly, benign tumors may sometimes be successfully treated by
embolization therapy alone. Examples of such tumors include simple
tumors of vascular origin (e.g., haemangiomas), endocrine tumors
such as parathyroid adenomas, and benign bone tumors.
[0131] For other tumors, (e.g., renal adenocarcinoma), preoperative
embolization may be employed hours or days before surgical
resection in order to reduce operative blood loss, shorten the
duration of the operation, and reduce the risk of dissemination of
viable malignant cells by surgical manipulation of the tumor. Many
tumors may be successfully embolized preoperatively, including for
example nasopharyngeal tumors, glomus jugular tumors, meningiomas,
chemodectomas, and vagal neuromas.
[0132] Embolization may also be utilized as a primary mode of
treatment for inoperable malignancies, in order to extend the
survival time of patients with advanced disease. Embolization may
produce a marked improvement in the quality of life of patients
with malignant tumors by alleviating unpleasant symptoms such as
bleeding, venous obstruction and tracheal compression. The greatest
benefit from palliative tumor embolization, however, may be seen in
patients suffering from the humoral effects of malignant endocrine
tumors, wherein metastases from carcinoid tumors and other
endocrine neoplasms such as insulinomas and glucagonomas may be
slow growing, and yet cause great distress by virtue of the
endocrine syndromes which they produce.
[0133] In general, embolization therapy utilizing anti-angiogenic
compositions of the present invention is typically performed in a
similar manner, regardless of the site. Briefly, angiography (a
road map of the blood vessels) of the area to be embolized is first
performed by injecting radiopaque contrast through a catheter
inserted into an artery or vein (depending on the site to be
embolized) as an X-ray is taken. The catheter may be inserted
either percutaneously or by surgery. The blood vessel is then
embolized by refluxing anti-angiogenic compositions of the present
invention through the catheter, until flow is observed to cease.
Occlusion may be confirmed by repeating the angiogram.
[0134] Embolization therapy generally results in the distribution
of compositions containing anti-angiogenic factors throughout the
interstices of the tumor or vascular mass to be treated. The
physical bulk of the embolic particles clogging the arterial lumen
results in the occlusion of the blood supply. In addition to this
effect, the presence of an anti-angiogenic factor(s) prevents the
formation of new blood vessels to supply the tumor or vascular
mass, enhancing the devitalizing effect of cutting off the blood
supply.
[0135] Therefore, it should be evident that a wide variety of
tumors may be embolized utilizing the compositions of the present
invention. Briefly, tumors are typically divided into two classes:
benign and malignant. In a benign tumor the cells retain their
differentiated features and do not divide in a completely
uncontrolled manner. In addition, the tumor is localized and
nonmetastatic. In a malignant tumor, the cells become
undifferentiated, do not respond to the body's growth and hormonal
signals, and multiply in an uncontrolled manner; the tumor is
invasive and capable of spreading to distant sites
(metastasizing).
[0136] Within one aspect of the present invention, metastases
(secondary tumors) of the liver may be treated utilizing
embolization therapy. Briefly, a catheter is inserted via the
femoral or brachial artery and advanced into the hepatic artery by
steering it through the arterial system under fluoroscopic
guidance. The catheter is advanced into the hepatic arterial tree
as far as necessary to allow complete blockage of the blood vessels
supplying the tumor(s), while sparing as many of the arterial
branches supplying normal structures as possible. Ideally this will
be a segmental branch of the hepatic artery, but it could be that
the entire hepatic artery distal to the origin of the
gastroduodenal artery, or even multiple separate arteries, will
need to be locked depending on the extent of tumor and its
individual blood supply. Once the desired catheter position is
achieved, the artery is embolized by injecting anti-angiogenic
compositions (as described above) through the arterial catheter
until flow in the artery to be blocked ceases, preferably even
after observation for 5 minutes. Occlusion of the artery may be
confirmed by injecting radiopaque contrast through the catheter and
demonstrating by fluoroscopy or X-ray film that the vessel which
previously filled with contrast no longer does so. The same
procedure may be repeated with each feeding artery to be
occluded.
[0137] As noted above, both benign and malignant tumors may be
embolized utilizing compositions of the present invention.
Representative examples of benign hepatic tumors include
Hepatocellular Adenoma, Cavernous Haemangioma, and Focal Nodular
Hyperplasia. Other benign tumors, which are more rare and often do
not have clinical manifestations, may also be treated. These
include Bile Duct Adenomas, Bile Duct Cystadenomas, Fibromas,
Lipomas, Leiomyomas, Mesotheliomas, Teratomas, Myxomas, and Nodular
Regenerative Hyperplasia.
[0138] Malignant Hepatic Tumors are generally subdivided into two
categories: primary and secondary. Primary tumors arise directly
from the tissue in which they are found. Thus, a primary liver
tumor is derived originally from the cells which make up the liver
tissue (such as hepatocytes and biliary cells). Representative
examples of primary hepatic malignancies which may be treated by
arterial embolization include Hepatocellularcarcinoma,
Cholangiocarcinoma, Angiosarcoma, Cystadenocarcinoma, Squamous Cell
Carcinoma, and Hepatoblastoma.
[0139] A secondary tumor, or metastasis, is a tumor which
originated elsewhere in the body but has now spread to a distant
organ. The common routes for metastasis are direct growth into
adjacent structures, spread through the vascular or lymphatic
systems, and tracking along tissue planes and body spaces
(peritoneal fluid, cerebrospinal fluid, etc.). Secondary hepatic
tumors are one of the most common causes of death in the cancer
patient. and are by far and away the most common form of liver
tumor. Although virtually any malignancy can metastasize to the
liver, tumors which are most likely to spread to the liver include:
cancer of the stomach, colon, and pancreas; melanoma; tumors of the
lung, oropharynx, and bladder; Hodgkin's and non-Hodgkin's
lymphoma; tumors of the breast, ovary, and prostate. Each one of
the above-named primary tumors has numerous different tumor types
which may be treated by arterial embolization (for example, there
are over 32 different types of ovarian cancer).
[0140] As noted above, embolization therapy utilizing
anti-angiogenic compositions of the present invention may also be
applied to a variety of other clinical situations where it is
desired to occlude blood vessels. Within one aspect of the present
invention, arteriovenous malformation may be treated by
administration of one of the above-described compositions. Briefly,
arteriovenous malformations (vascular malformations) refers to a
group of diseases wherein at least one (and most typically, many)
abnormal communications between arteries and veins occur, resulting
in a local tumor-like mass composed predominantly of blood vessels.
Such disease may be either congenital or acquired.
[0141] Within one embodiment of the invention, an arteriovenous
malformation may be treated by inserting a catheter via the femoral
or brachial artery, and advancing it into the feeding artery under
fluoroscopic guidance. The catheter is preferably advanced as far
as necessary to allow complete blockage of the blood vessels
supplying the vascular malformation, while sparing as many of the
arterial branches supplying normal structures as possible (ideally
this will be a single artery, but most often multiple separate
arteries may need to be occluded, depending on the extent of the
vascular malformation and its individual blood supply). Once the
desired catheter position is achieved, each artery may be embolized
utilizing anti-angiogenic compositions of the present
invention.
[0142] Within another aspect of the invention, embolization may be
accomplished in order to treat conditions of excessive bleeding.
For example, menorrhagia (excessive bleeding with menstruation) may
be readily treated by embolization of uterine arteries. Briefly,
the uterine arteries are branches of the internal iliac arteries
bilaterally. Within one embodiment of the invention, a catheter may
be inserted via the femoral or brachial artery, and advanced into
each uterine artery by steering it through the arterial system
under fluoroscopic guidance. The catheter should be advanced as far
as necessary to allow complete blockage of the blood vessels to the
uterus, while sparing as many arterial branches that arise from the
uterine artery and supply normal structures as possible. Ideally a
single uterine artery on each side may be embolized, but
occasionally multiple separate arteries may need to be blocked
depending on the individual blood supply. Once the desired catheter
position is achieved, each artery may be embolized by
administration of the anti-angiogenic compositions as described
above.
[0143] In a like manner, arterial embolization may be accomplished
in a variety of other conditions, including for example, for acute
bleeding, vascular abnormalities, central nervous system disorders,
and hypersplenism.
Use of Anti-Angiogenic Compositions as Coatings for Stents
[0144] As noted above, the present invention also provides stents,
comprising a generally tubular structure (which includes for
example, spiral shapes), the surface of which is coated with a
composition as described above. Briefly, a stent is a scaffolding,
usually cylindrical in shape, that may be inserted into a body
passageway (e.g., bile ducts) or a portion of a body passageway,
which has been narrowed, irregularly contured, obstructed, or
occluded by a disease process (e.g., ingrowth by a tumor) in order
to prevent closure or reclosure of the passageway. Stents act by
physically holding open the walls of the body passage into which
they are inserted.
[0145] A variety of stents may be utilized within the context of
the present invention, including for example, esophageal stents,
vascular stents, biliary stents, pancreatic stents, ureteric and
urethral stents, lacrimal stents, Eustachian tube stents, fallopian
tube stents and tracheal/bronchial stents.
[0146] Stents may be readily obtained from commercial sources, or
constructed in accordance with well-known techniques.
Representative examples of stents include those described in U.S.
Pat. No. 4,768,523, entitled "Hydrogel Adhesive;" U.S. Pat. No.
4,776,337, entitled "Expandable Intraluminal Graft, and Method and
Apparatus for Implanting and Expandable Intraluminal Graft;" U.S.
Pat. No. 5,041,126 entitled "Endovascular Stent and Delivery
System;" U.S. Pat. No. 5,052,998 entitled "Indwelling Stent and
Method of Use;" U.S. Pat. No. 5,064,435 entitled "Self-Expanding
Prosthesis Having Stable Axial Length;" U.S. Pat. No. 5,089,606,
entitled "Water-insoluble Polysaccharide Hydrogel Foam for Medical
Applications;" U.S. Pat. No. 5,147,370, entitled "Nitinol Stent for
Hollow Body Conduits;" U.S. Pat. No. 5,176,626, entitled
"Indwelling Stent;" U.S. Pat. No. 5,213,580, entitled
"Biodegradable polymeric Endoluminal Sealing Process;" and U.S.
Pat. No. 5,328,471, entitled "Method and Apparatus for Treatment of
Focal Disease in Hollow Tubular Organs and Other Tissue
Lumens."
[0147] Stents may be coated with anti-angiogenic compositions or
anti-angiogenic factors of the present invention in a variety of
manners, including for example: (a) by directly affixing to the
stent an anti-angiogenic composition (e.g., by either spraying the
stent with a polymer/drug film, or by dipping the stent into a
polymer/drug solution), (b) by coating the stent with a substance
such as a hydrogel which will in turn absorb the anti-angiogenic
composition (or anti-angiogenic factor above), (c) by interweaving
anti-angiogenic composition coated thread (or the polymer itself
formed into a thread) into the stent structure, (d) by inserting
the stent into a sleeve or mesh which is comprised of or coated
with an anti-angiogenic composition, or (e) constructing the stent
itself with an anti-angiogenic composition. Within preferred
embodiments of the invention, the composition should firmly adhere
to the stent during storage and at the time of insertion, and
should not be dislodged from the stent when the diameter is
expanded from its collapsed size to its full expansion size. The
anti-angiogenic composition should also preferably not degrade
during storage, prior to insertion, or when warmed to body
temperature after expansion inside the body. In addition, it should
preferably coat the stent smoothly and evenly, with a uniform
distribution of angiogenesis inhibitor, while not changing the
stent contour. Within preferred embodiments of the invention, the
anti-angiogenic composition should provide a uniform, predictable,
prolonged release of the anti-angiogenic factor into the tissue
surrounding the stent once it has been deployed. For vascular
stents, in addition to the above properties, the composition should
not render the stent thrombogenic (causing blood clots to form), or
cause significant turbulence in blood flow (more than the stent
itself would be expected to cause if it was uncoated).
[0148] Within another aspect of the present invention, methods are
provided for expanding the lumen of a body passageway, comprising
inserting a stent into the passageway, the stent having a generally
tubular structure, the surface of the structure being coated with
an anti-angiogenic composition (or, an anti-angiogenic factor
alone), such that the passageway is expanded. A variety of
embodiments are described below wherein the lumen of a body
passageway is expanded in order to eliminate a biliary, esophageal,
tracheal/bronchial, urethral or vascular obstruction. In addition,
a representative example is described in more detail below in
Example 7.
[0149] Generally, stents are inserted in a similar fashion
regardless of the site or the disease being treated. Briefly, a
preinsertion examination, usually a diagnostic imaging procedure,
endoscopy, or direct visualization at the time of surgery, is
generally first performed in order to determine the appropriate
positioning for stent insertion. A guidewire is then advanced
through the lesion or proposed site of insertion, and over this is
passed a delivery catheter which allows a stent in its collapsed
form to be inserted. Typically, stents are capable of being
compressed, so that they can be inserted through tiny cavities via
small catheters, and then expanded to a larger diameter once they
are at the desired location. Once expanded, the stent physically
forces the walls of the passageway apart and holds them open. As
such, they are capable of insertion via a small opening, and yet
are still able to hold open a large diameter cavity or passageway.
The stent may be self-expanding (e.g., the Wallstent and Gianturco
stents), balloon expandable (e.g., the Palmaz stent and Strecker
stent), or implanted by a change in temperature (e.g., the Nitinol
stent).
[0150] Stents are typically maneuvered into place under radiologic
or direct visual control, taking particular care to place the stent
precisely across the narrowing in the organ being treated. The
delivery catheter is then removed, leaving the stent standing on
its own as a scaffold. A post insertion examination, usually an
x-ray, is often utilized to confirm appropriate positioning.
[0151] Within a preferred embodiment of the invention, methods are
provided for eliminating biliary obstructions, comprising inserting
a biliary stent into a biliary passageway, the stent having a
generally tubular structure, the surface of the structure being
coated with a composition as described above, such that the biliary
obstruction is eliminated. Briefly, tumor overgrowth of the common
bile duct results in progressive cholestatic jaundice which is
incompatible with life. Generally, the biliary system which drains
bile from the liver into the duodenum is most often obstructed by
(1) a tumor composed of bile duct cells (cholangiocarcinoma), (2) a
tumor which invades the bile duct (e.g., pancreatic carcinoma), or
(3) a tumor which exerts extrinsic pressure and compresses the bile
duct (e.g., enlarged lymph nodes).
[0152] Both primary biliary tumors, as well as other tumors which
cause compression of the biliary tree may be treated utilizing the
stents described herein. One example of primary biliary tumors are
adenocarcinomas (which are also called Klatskin tumors when found
at the bifurcation of the common hepatic duct). These tumors are
also referred to as biliary carcinomas, choledocholangiocarcinomas,
or adenocarcinomas of the biliary system. Benign tumors which
affect the bile duct (e.g., adenoma of the biliary system), and, in
rare cases, squamous cell carcinomas of the bile duct and
adenocarcinomas of the gallbladder, may also cause compression of
the biliary tree and therefore, result in biliary obstruction.
[0153] Compression of the biliary tree is most commonly due to
tumors of the liver and pancreas which compress and therefore
obstruct the ducts. Most of the tumors from the pancreas arise from
cells of the pancreatic ducts. This is a highly fatal form of
cancer (5% of all cancer deaths; 26,000 new cases per year in the
U.S.) with an average of 6 months survival and a 1 year survival
rate of only 10%. When these tumors are located in the head of the
pancreas they frequently cause biliary obstruction, and this
detracts significantly from the quality of life of the patient.
While all types of pancreatic tumors are generally referred to as
"carcinoma of the pancreas" there are histologic subtypes
including: adenocarcinoma, adenosquamous carcinoma,
cystadeno-carcinoma, and acinar cell carcinoma. Hepatic tumors, as
discussed above, may also cause compression of the biliary tree,
and therefore cause obstruction of the biliary ducts.
[0154] Within one embodiment of the invention, a biliary stent is
first inserted into a biliary passageway in one of several ways:
from the top end by inserting a needle through the abdominal wall
and through the liver (a percutaneous transhepatic cholangiogram or
"PTC"); from the bottom end by cannulating the bile duct through an
endoscope inserted through the mouth, stomach, and duodenum (an
endoscopic retrograde cholangiogram or "ERCP"); or by direct
incision during a surgical procedure. A preinsertion examination,
PTC, ERCP, or direct visualization at the time of surgery should
generally be performed to determine the appropriate position for
stent insertion. A guidewire is then advanced through the lesion,
and over this a delivery catheter is passed to allow the stent to
be inserted in its collapsed form. If the diagnostic exam was a
PTC, the guidewire and delivery catheter is inserted via the
abdominal wall, while if the original exam was an ERCP the stent
may be placed via the mouth. The stent is then positioned under
radiologic, endoscopic, or direct visual control taking particular
care to place it precisely across the narrowing in the bile duct.
The delivery catheter is then removed leaving the stent standing as
a scaffolding which holds the bile duct open. A further
cholangiogram may be performed to document that the stent is
appropriately positioned.
[0155] Within yet another embodiment of the invention, methods are
provided for eliminating esophageal obstructions, comprising
inserting an esophageal stent into an esophagus, the stent having a
generally tubular structure, the surface of the structure being
coated with an anti-angiogenic composition as described above, such
that the esophageal obstruction is eliminated. Briefly, the
esophagus is the hollow tube which transports food and liquids from
the mouth to the stomach. Cancer of the esophagus or invasion by
cancer arising in adjacent organs (e.g., cancer of the stomach or
lung) results in the inability to swallow food or saliva. Within
this embodiment, a preinsertion examination, usually a barium
swallow or endoscopy should generally be performed in order to
determine the appropriate position for stent insertion. A catheter
or endoscope may then be positioned through the mouth, and a
guidewire is advanced through the blockage. A stent delivery
catheter is passed over the guidewire under radiologic or
endoscopic control, and a stent is placed precisely across the
narrowing in the esophagus. A post insertion examination, usually a
barium swallow x-ray, may be utilized to confirm appropriate
positioning.
[0156] Within other embodiments of the invention, methods are
provided for eliminating tracheal/bronchial obstructions,
comprising inserting a tracheal/bronchial stent into the trachea or
bronchi, the stent having a generally tubular structure, the
surface of which is coated with an anti-angiogenic composition as
described above, such that the tracheal/bronchial obstruction is
eliminated. Briefly, the trachea and bronchi are tubes which carry
air from the mouth and nose to the lungs. Blockage of the trachea
by cancer, invasion by cancer arising in adjacent organs (e.g.,
cancer of the lung), or collapse of the trachea or bronchi due to
chondromalacia (weakening of the cartilage rings) results in
inability to breathe. Within this embodiment of the invention,
preinsertion examination, usually an endoscopy, should generally be
performed in order to determine the appropriate position for stent
insertion. A catheter or endoscope is then positioned through the
mouth, and a guidewire advanced through the blockage. A delivery
catheter is then passed over the guidewire in order to allow a
collapsed stent to be inserted. The stent is placed under
radiologic or endoscopic control in order to place it precisely
across the narrowing. The delivery catheter may then be removed
leaving the stent standing as a scaffold on its own. A post
insertion examination, usually a bronchoscopy may be utilized to
confirm appropriate positioning.
[0157] Within another embodiment of the invention, methods are
provided for eliminating urethral obstructions, comprising
inserting a urethral stent into a urethra, the stent having a
generally tubular structure, the surface of the structure being
coated with an anti-angiogenic composition as described above, such
that the urethral obstruction is eliminated. Briefly, the urethra
is the tube which drains the bladder through the penis. Extrinsic
narrowing of the urethra as it passes through the prostate, due to
hypertrophy of the prostate, occurs in virtually every man over the
age of 60 and causes progressive difficulty with urination. Within
this embodiment, a preinsertion examination, usually an endoscopy
or urethrogram should generally first be performed in order to
determine the appropriate position for stent insertion, which is
above the external urinary sphincter at the lower end, and close to
flush with the bladder neck at the upper end. An endoscope or
catheter is then positioned through the penile opening and a
guidewire advanced into the bladder. A delivery catheter is then
passed over the guidewire in order to allow stent insertion. The
delivery catheter is then removed, and the stent expanded into
place. A post insertion examination, usually endoscopy or
retrograde urethrogram, may be utilized to confirm appropriate
position.
[0158] Within another embodiment of the invention, methods are
provided for eliminating vascular obstructions, comprising
inserting a vascular stent into a blood vessel, the stent having a
generally tubular structure, the surface of the structure being
coated with an anti-angiogenic composition as described above, such
that the vascular obstruction is eliminated. Briefly, stents may be
placed in a wide array of blood vessels, both arteries and veins,
to prevent recurrent stenosis at the site of failed angioplasties,
to treat narrowings that would likely fail if treated with
angioplasty, and to treat post surgical narrowings (e.g., dialysis
graft stenosis). Representative examples of suitable sites include
the iliac, renal, and coronary arteries, the superior vena cava,
and in dialysis grafts. Within one embodiment, angiography is first
performed in order to localize the site for placement of the stent.
This is typically accomplished by injecting radiopaque contrast
through a catheter inserted into an artery or vein as an x-ray is
taken. A catheter may then be inserted either percutaneously or by
surgery into the femoral artery, brachial artery, femoral vein, or
brachial vein, and advanced into the appropriate blood vessel by
steering it through the vascular system under fluoroscopic
guidance. A stent may then be positioned across the vascular
stenosis. A post insertion angiogram may also be utilized in order
to confirm appropriate positioning.
Use of Anti-Angiogenic Compositions in Surgical Procedures
[0159] As noted above, anti-angiogenic compositions may be utilized
in a wide variety of surgical procedures. For example, within one
aspect of the present invention an anti-angiogenic compositions (in
the form of, for example, a spray or film) may be utilized to coat
or spray an area prior to removal of a tumor, in order to isolate
normal surrounding tissues from malignant tissue, and/or to prevent
the spread of disease to surrounding tissues. Within other aspects
of the present invention, anti-angiogenic compositions (e.g., in
the form of a spray) may be delivered via endoscopic procedures in
order to coat tumors, or inhibit angiogenesis in a desired locale.
Within yet other aspects of the present invention, surgical meshes
which have been coated with anti-angiogenic compositions of the
present invention may be utilized in any procedure wherein a
surgical mesh might be utilized. For example, within one embodiment
of the invention a surgical mesh ladened with an anti-angiogenic
composition may be utilized during abdominal cancer resection
surgery (e.g., subsequent to colon resection) in order to provide
support to the structure, and to release an amount of the
anti-angiogenic factor.
[0160] Within further aspects of the present invention, methods are
provided for treating tumor excision sites, comprising
administering an anti-angiogenic composition as described above to
the resection margins of a tumor subsequent to excision, such that
the local recurrence of cancer and the formation of new blood
vessels at the site is inhibited. Within one embodiment of the
invention, the anti-angiogenic composition(s) (or anti-angiogenic
factor(s) alone) are administered directly to the tumor excision
site (e.g., applied by swabbing, brushing or otherwise coating the
resection margins of the tumor with the anti-angiogenic
composition(s) or factor(s)). Alternatively, the anti-angiogenic
composition(s) or factor(s) may be incorporated into known surgical
pastes prior to administration. Within particularly preferred
embodiments of the invention, the anti-angiogenic compositions are
applied after hepatic resections for malignancy, and after
neurosurgical operations.
[0161] Within one aspect of the present invention, anti-angiogenic
compositions (as described above) may be administered to the
resection margin of a wide variety of tumors, including for
example, breast, colon, brain and hepatic tumors. For example,
within one embodiment of the invention, anti-angiogenic
compositions may be administered to the site of a neurological
tumor subsequent to excision, such that the formation of new blood
vessels at the site are inhibited. Briefly, the brain is highly
functionally localized; i.e., each specific anatomical region is
specialized to carry out a specific function. Therefore it is the
location of brain pathology that is often more important than the
type. A relatively small lesion in a key area can be far more
devastating than a much larger lesion in a less important area.
Similarly, a lesion on the surface of the brain may be easy to
resect surgically, while the same tumor located deep in the brain
may not (one would have to cut through too many vital structures to
reach it). Also, even benign tumors can be dangerous for several
reasons: they may grow in a key area and cause significant damage;
even though they would be cured by surgical resection this may not
be possible; and finally, if left unchecked they can cause
increased intracranial pressure. The skull is an enclosed space
incapable of expansion. Therefore, if something is growing in one
location, something else must be being compressed in another
location--the result is increased pressure in the skull or
increased intracranial pressure. If such a condition is left
untreated, vital structures can be compressed, resulting in death.
The incidence of CNS (central nervous system) malignancies is 8-16
per 100,000. The prognosis of primary malignancy of the brain is
dismal, with a median survival of less than one year, even
following surgical resection. These tumors, especially gliomas, are
predominantly a local disease which recur within 2 centimeters of
the original focus of disease after surgical removal.
[0162] Representative examples of brain tumors which may be treated
utilizing the compositions and methods described herein include
Glial Tumors (such as Anaplastic Astrocytoma, Glioblastoma
Multiform, Pilocytic Astrocytoma, Oligodendroglioma, Ependymoma,
Myxopapillary Ependymoma, Subependymoma, Choroid Plexus Papilloma);
Neuron Tumors (e.g., Neuroblastoma, Ganglioneuroblastoma,
Ganglioneuroma, and Medulloblastoma); Pineal Gland Tumors (e.g.,
Pineoblastoma and Pineocytoma); Menigeal Tumors (e.g., Meningioma,
Meningeal Hemangiopericytoma, Meningeal Sarcoma); Tumors of Nerve
Sheath Cells (e.g., Schwannoma (Neurolemmoma) and Neurofibroma);
Lymphomas (e.g., Hodgkin's and Non-Hodgkin's Lymphoma (including
numerous subtypes, both primary and secondary); Malformative Tumors
(e.g., Craniopharyngioma, Epidermoid Cysts, Dermoid Cysts and
Colloid Cysts); and Metastatic Tumors (which can be derived from
virtually any tumor, the most common being from lung, breast,
melanoma, kidney, and gastrointestinal tract tumors).
Inflammatory Arthritis
[0163] Inflammatory arthritis is a serious health problems in
developed countries, particularly given the increasing number of
aged individuals. For example, one form of inflammatory arthritis,
rheumatoid arthritis (RA) is a multisystem chronic, relapsing,
inflammatory disease of unknown cause. Although many organs can be
affected, RA is basically a severe form of chronic synovitis that
sometimes leads to destruction and ankylosis of affected joints
(taken from Robbins Pathological Basis of Disease, by R. S. Cotran,
V. Kumar, and S. L. Robbins, W.B. Saunders Co., 1989).
Pathologically the disease is characterized by a marked thickening
of the synovial membrane which forms villous projections that
extend into the joint space, multilayering of the synoviocyte
lining (synoviocyte proliferation), infiltration of the synovial
membrane with white blood cells (macrophages, lymphocytes, plasma
cells, and lymphoid follicles; called an "inflammatory synovitis"),
and deposition of fibrin with cellular necrosis within the
synovium. The tissue formed as a result of this process is called
pannus and eventually the pannus grows to fill the joint space. The
pannus develops an extensive network of new blood vessels through
the process of angiogenesis which is essential to the evolution of
the synovitis. Release of digestive enzymes [matrix
metalloproteinases (e.g., collagenase, stromelysin)] and other
mediators of the inflammatory process (e.g., hydrogen peroxide,
superoxides, lysosomal enzymes, and products of arachadonic acid
metabolism) from the cells of the pannus tissue leads to the
progressive destruction of the cartilage tissue. The pannus invades
the articular cartilage leading to erosions and fragmentation of
the cartilage tissue. Eventually there is erosion of the
subchondral bone with fibrous ankylosis and ultimately bony
ankylosis, of the involved joint.
[0164] It is generally believed, but not conclusively proven, that
RA is an autoimmune disease, and that many different arthriogenic
stimuli activate the immune response in the immunogenetically
susceptible host. Both exogenous infectious agents (Ebstein-Barr
Virus, Rubella virus, Cytomegalovirus, Herpes Virus, Human T-cell
Lymphotropic Virus, Mycoplasma, and others) and endogenous proteins
(collagen, proteoglycans, altered immunoglobulins) have been
implicated as the causative agent which triggers an inappropriate
host immune response. Regardless of the inciting agent,
autoimmunity plays a role in the progression of the disease. In
particular, the relevant antigen is ingested by antigen-presenting
cells (macrophages or dendritic cells in the synovial membrane),
processed, and presented to T lymphocytes. The T cells initiate a
cellular immune response and stimulate the proliferation and
differentiation of B lymphocytes into plasma cells. The end result
is the production of an excessive inappropriate immune response
directed against the host tissues [e.g., antibodies directed
against Type II collagen, antibodies directed against the Fc
portion of autologous IgG (called "Rheumatoid Factor")]. This
further amplifies the immune response and hastens the destruction
of the cartilage tissue. Once this cascade is initiated numerous
mediators of cartilage destruction are responsible for the
progression of rheumatoid arthritis.
[0165] Thus, within one aspect of the present invention, methods
are provided for treating or preventing inflammatory arthritis
(e.g., rheumatoid arthritis) comprising the step of administering
to a patient a therapeutically effective amount of an
anti-angiogenic factor or anti-angiogenic composition to a joint.
Within a preferred embodiment of the invention, anti-angiogenic
factors (including anti-angiogenic compositions, as described
above) may be administered directly by intra-articular injection,
as a surgical paste, or as an oral agent (e.g., containing the
anti-angiogenic factor thalidomide). One representative example of
such a method is set forth in more detail below in Example 19.
[0166] As utilized within the context of the present invention, it
should be understood that efficatious administration of the
anti-angiogenic factors and compositions described herein may be
assessed in several ways, including: (1) by preventing or lessening
the pathological and/or clinical symptoms associated with
rheumatoid arthritis; (2) by downregulating the white blood cell
response which initiates the inflammatory cascade and results in
synovitis, swelling, pain, and tissue destruction; (3) by
inhibiting the "tumor-like" proliferation of synoviocytes that
leads to the development of a locally invasive and destructive
pannus tissue; (4) by decreasing the production/activity of matrix
metalloproteinases produced by white blood cells, synoviocytes,
chondrocytes, and endothelial cells, which degrade the cartilage
matrix and result in irreversible destruction of the articular
cartilage; and (5) by inhibiting blood vessel formation which
provides the framework and nutrients necessary for the growth and
development of the pannus tissue. Furthermore, the anti-angiogenic
factors or compositions should not be toxic to normal chondrocytes
at therapeutic levels. Each of these aspects will be discussed in
more detail below.
[0167] A. Inflammatory Response
[0168] Neutrophils are found in abundance in the synovial fluid,
but only in small numbers in the synovial membrane itself. It is
estimated that more than 1 billion neutrophils enter a moderately
inflamed rheumatoid knee joint each day (Hollingsworth et al.,
1967) and remain there because no pathway exists by which they can
leave the joint. These cells release reactive free radicals and
lysosomal enzymes which degrade the cartilage tissue. Other PMN
products such as prostaglandins and leukotrienes augment the
inflammatory response and recruit more inflammatory cells into the
joint tissue.
[0169] Lymphocytes, particularly T cells, are present in abundance
in the diseased synovial tissue. Activated T cells produce a
variety of lymphokines and cooperate with B cells to produce
autoantibodies. T cells products result in the activation
macrophages, a cell which is thought to have an important role in
the pathology of the disease. The macrophages produce a variety
destructive lysosomal enzymes, prostaglandins, and monokines and
are also capable of stimulating angiogenesis. One of the more
important monokines secreted by macrophages is IL-1. Briefly, IL-1
is known to: stimulate synthesis and release of collagenase by
synoviocytes and synovial fibroblasts, inhibit proteoglycan
synthesis by chondrocytes, activate osteoclasts, induce changes in
the endothelium of the synovial vasculature [stimulation of
endothelial production of plasminogen activator and colony
stimulating factor, expression of leukocyte adhesion molecules,
promotion of procoagulant activity (Wider et al., 1991)], and act
as a chemoattractant for lymphocytes and neutrophils.
[0170] Within one embodiment, downregulation of the white blood
cell response, or inhibition of the inflammatory response, may be
assessed by determination of the effect of the anti-angiogenic
factor or anti-angiogenic composition on the response of
neutrophils stimulated with opsonized CPPD crystals or opsonized
zyrosan. Such methods are illustrated in more detail below in
Example 22.
[0171] B. Synoviocyte Hyperplasia
[0172] During the development of RA, the synovial lining cells
become activated by products of inflammation or through
phagocytosis of immune complexes. Several subtypes of synovial
lining cells have been identified and all of them become intensely
activated and undergo excessive hyperplasia and growth when
stimulated. As the synovial tissue organizes to form a pannus, the
number of synoviocytes, blood vessels, connective tissue elements,
and inflammatory cells increases to form a mass 100 times its
original size. In many ways, the synovitis in rheumatoid arthritis
behaves much like a localized neoplasia (Harris, 1990). In fact,
cultured rheumatoid synovial cells develop the phenotypic
characteristics of anchorage-independent growth usually associated
with neoplastic cells if they given sufficient plateletderived
growth factor (Lafyatis et al., 1989). In addition, the
synoviocytes also produce large amounts of collagenase,
stromelysin, prostaglandins, and Interleukin-1.
[0173] The tumor-like proliferation of the cells of the synovial
connective tissue stroma (synoviocytes, fibroblast-like cells and
neovascular tissue) produces a pannus with many features of a
localized malignancy. Supporting this tumor analogy are several
findings: the pannus expresses high levels of oncoproteins such as
c-myc and c-fos, produces metalloproteinases to facilitate
surrounding tissue invasion, express cytoskeletal markers
characteristic of poorly differentiated mesenchymal tissue (e.g.,
vimentin); synoviocytes in vitro grow rapidly, do not contact
inhibit, form foci, and can be grown under anchorage-independent
conditions in soft agarose; and pannus tissue is capable of
inducing the growth of a supporting vasculature (i.e.
angiogenesis). All these findings are suggestive of a tissue in
which normal growth regulation as been lost.
[0174] Within one embodiment, inhibition of synoviocyte
proliferation may be determined by, for example, analysis of
.sup.3H-thymidine incorporation into synoviocytes, or in vitro
synoviocyte proliferation. Such methods are illustrated in more
detail below in Example 23.
[0175] C. Matrix Metalloproteinases (MMP)
[0176] Irreparable degradation of the cartilage extracellular
matrix is believed to be largely due to the enzymatic action of
matrix metalloproteinases on the components of the cartilage
matrix. Although numerous other enzymes are likely involved in the
development of RA, collagenase (MMP-1) and stromelysin (MMP-3) play
an important role (Vincetti et al., 1994) in disease progression.
These enzymes are capable of degrading type 11 collagen and
proteoglycans respectively; the 2 major extracellular components of
cartilage tissue. Cytokines such as IL-1, epidermal growth factor
(EGF), platelet-derived growth factor, and tumor necrosis factor
are all potent stimulators of collagenase and stromelysin
production. As described above, numerous cell types found in the
arthritic joint (white blood cells, synoviocytes, endothelial
cells, and chondrocytes) are capable of synthesizing and secreting
MMPS.
[0177] In proliferating rheumatoid synovial tissue, collagenase and
stromelysin become the major gene products of the pannus and may
comprise as much as 2% of the messenger RNAs produced by the
synovial fibroblasts (Brinkerhoff and Auble, 1990). Increased
levels of collagenase and stromelysin are present in the cartilage
of patients with RA and the level of enzyme activity in the joint
correlates well with the severity of the lesion (Martel-Pelletier
et al., 1993; Walakovitis et al., 1992). Because these enzymes are
fundamental to the pathology of RA and result in irreversible
cartilage damage, many therapeutic strategies have been devised to
inhibit their effects.
[0178] Numerous naturally present inhibitors of MMP activity have
been identified and named "TIMPS" for Tissue Inhibitors of
Metalloproteinases. Many of these protein inhibitors bind with the
active MMPs to form 1:1 noncovalent complexes which inactivate the
MMP enzymes. The TIMPs are produced locally by chondrocytes and
synovial fibroblasts and are likely responsible for the normal
regulation of connective tissue degradation. It is thought that
much of the damage to the cartilage matrix is due to a local
imbalance between MMP and TIMP activity. This is probably due to
increased production of metalloproteinases while the production of
TIMPs remains at a normal or constant level (Vincetti et al.,
1994). To overcome this, therapeutic strategies have been designed
to add exogenous TIMPs (e.g., the chemically modified tetracycline
molecules, collagen substrate analogues) or to upregulate TIMP
production (retinoids, transforming growth factor .beta., IL-6,
IL-11, oncostatin M) in an effort to restore the enzymatic balance.
However this approach has yet to translate into significant
clinical results.
[0179] An alternative approach is to inhibit or downregulate the
production of the MMPs to restore a normal balance of activity.
Naturally occurring compounds (TNF.beta., all-trans retinoic acid)
and synthetic compounds (retinoids, glucocorticoid hormones) have
been demonstrated to inhibit MMP activity by suppressing
transcription and synthesis of these proteins. A
post-transcriptional method of blocking MMP release could also be
expected to result in a decrease in the amount of MMP produced and
an improved balance between MMP and TIMP activity in the joint.
[0180] Within one embodiment, a decrease in the production or
activity of MMP's may be determined by, for example, analysis of
IL-1 induced collagenase expression. One such method is illustrated
in more detail below in Example 24.
[0181] D. Angiogenesis
[0182] The development of an extensive network of new blood vessels
is essential to the development of the synovitis present in
rheumatoid arthritis (Harris, 1990; Folkman et al., 1989; Sano et
al., 1990). Several local mediators such as plateletderived growth
factor (PDGF), TGF-.beta., and fibroblast growth factor (FGF) are
likely responsible for the induction and perpetuation of
neovascularization within the synovium. Pannus tissue composed of
new capillaries and synovial connective tissue invades and destroys
the articular cartilage. The migrating angiogenic vessels
themselves produce and secrete increased levels of
metalloproteinases such as collagenase and stromelysin capable of
degrading the cartilage matrix (Case et al., 1989). The newly
formed vessels are also quite "leaky" with gaps present between the
microvascular endothelial cells. This facilitates the exudation of
plasma proteins into the synovium (which increases swelling),
enhances WBCs movement from the circulation into the pannus tissue
(which increases inflammation), and leads to the perivascular
accumulation of mononuclear inflammatory cells (Wilder et al.,
1991).
[0183] In summary, the endothelial tissue plays an important role
in the development of this disease by expressing the necessary
surface receptors to allow inflammatory cells to leave the
circulation and enter the developing pannus, secreting proteolytic
enzymes capable of degrading the cartilage matrix, and
proliferating to form the new vessels (angiogenesis) required for
the pannus tissue to increase in size and invade adjacent
tissues.
[0184] Within one embodiment, inhibition of new blood vessel
formation may be readily determined in a variety of asays,
including the CAM assay described above and within Example 2.
Neovascular Diseases of the Eye
[0185] As noted above, the present invention also provides methods
for treating neovascular diseases of the eye, including for
example, corneal neovascularization, neovascular glaucoma,
proliferative diabetic retinopathy, retrolental fibroblasia and
macular degeneration.
[0186] Briefly, corneal neovascularization as a result of injury to
the anterior segment is a significant cause of decreased visual
acuity and blindness, and a major risk factor for rejection of
corneal allografts. As described by Burger et al., Lab. Invest.
48:169-180, 1983, corneal angiogenesis involves three phases: a
pre-vascular latent period, active neovascularization, and vascular
maturation and regression. The identity and mechanism of various
angiogenic factors, including elements of the inflammatory
response, such as leukocytes, platelets, cytokines, and
eicosanoids, or unidentified plasma constituents have yet to be
revealed.
[0187] Currently no clinically satisfactory therapy exists for
inhibition of corneal neovascularization or regression of existing
corneal new vessels. Topical corticosteroids appear to have some
clinical utility, presumably by limiting stromal inflammation.
[0188] Thus, within one aspect of the present invention methods are
provided for treating neovascular diseases of the eye such as
corneal neovascularization (including corneal graft
neovascularization), comprising the step of administering to a
patient a therapeutically effective amount of an anti-angiogenic
composition (as described above) to the cornea, such that the
formation of blood vessels is inhibited. Briefly, the cornea is a
tissue which normally lacks blood vessels. In certain pathological
conditions however, capillaries may extend into the cornea from the
pericorneal vascular plexus of the limbus. When the cornea becomes
vascularized, it also becomes clouded, resulting in a decline in
the patient's visual acuity. Visual loss may become complete if the
cornea completely opacitates.
[0189] Blood vessels can enter the cornea in a variety of patterns
and depths, depending upon the process which incites the
neovascularization. These patterns have been traditionally defined
by ophthalmologists in the following types: pannus trachomatosus,
pannus leprosus, pannus phylctenulosus, pannus degenerativus, and
glaucomatous pannus. The corneal stroma may also be invaded by
branches of the anterior ciliary artery (called interstitial
vascularization) which causes several distinct clinical lesions:
terminal loops, a "brush-like" pattern, an umbel form, a lattice
form, interstitial arcades (from episcleral vessels), and aberrant
irregular vessels.
[0190] A wide variety of disorders can result in corneal
neovascularization, including for example, corneal infections
(e.g., trachoma, herpes simplex keratitis, leishmaniasis and
onchocerciasis), immunological processes (e.g., graft rejection and
Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of
any cause), toxic and nutritional deficiency states, and as a
complication of wearing contact lenses.
[0191] While the cause of corneal neovascularization may vary, the
response of the cornea to the insult and the subsequent vascular
ingrowth is similar regardless of the cause. Briefly, the location
of the injury appears to be of importance as only those lesions
situated within a critical distance of the limbus will incite an
angiogenic response. This is likely due to the fact that the
angiogenic factors responsible for eliciting the vascular invasion
are created at the site of the lesion, and must diffuse to the site
of the nearest blood vessels (the limbus) in order to exert their
effect. Past a certain distance from the limbus, this would no
longer be possible and the limbic endothelium would not be induced
to grow into the cornea. Several angiogenic factors are likely
involved in this process, many of which are products of the
inflammatory response. Indeed, neovascularization of the cornea
appears to only occur in association with an inflammatory cell
infiltrate, and the degree of angiogenesis is proportional to the
extent of the inflammatory reaction. Corneal edema further
facilitates blood vessel ingrowth by loosening the corneal stromal
framework and providing a pathway of "least resistance" through
which the capillaries can grow.
[0192] Following the initial inflammatory reaction, capillary
growth into the cornea proceeds in the same manner as it occurs in
other tissues. The normally quiescent endothelial cells of the
limbic capillaries' and venules are stimulated to divide and
migrate. The endothelial cells project away from their vessels of
origin, digest the surrounding basement membrane and the tissue
through which they will travel, and migrate towards the source of
the angiogenic stimulus. The blind ended sprouts acquire a lumen
and then anastomose together to form capillary loops. The end
result is the establishment of a vascular plexus within the corneal
stroma.
[0193] Anti-angiogenic factors and compositions of the present
invention are useful by blocking the stimulatory effects of
angiogenesis promoters, reducing endothelial cell division,
decreasing endothelial cell migration, and impairing the activity
of the proteolytic enzymes secreted by the endothelium.
[0194] Within particularly preferred embodiments of the invention,
an anti-angiogenic factor may be prepared for topical
administration in saline (combined with any of the preservatives
and antimicrobial agents commonly used in ocular preparations), and
administered in eyedrop form. The anti-angiogenic factor solution
or suspension may be prepared in its pure form and administered
several times daily. Alternatively, anti-angiogenic compositions,
prepared as described above, may also be administered directly to
the cornea. Within preferred embodiments, the anti-angiogenic
composition is prepared with a muco-adhesive polymer which binds to
cornea. Within further embodiments, the anti-angiogenic factors or
anti-angiogenic compositions may be utilized as an adjunct to
conventional steroid therapy.
[0195] Topical therapy may also be useful prophylactically in
corneal lesions which are known to have a high probability of
inducing an angiogenic response (such as chemical burns). In these
instances the treatment, likely in combination with steroids, may
be instituted immediately to help prevent subsequent
complications.
[0196] Within other embodiments, the anti-angiogenic compositions
described above may be injected directly into the corneal stroma by
an ophthalmologist under microscopic guidance. The preferred site
of injection may vary with the morphology of the individual lesion,
but the goal of the administration would be to place the
composition at the advancing front of the vasculature (i.e.,
interspersed between the blood vessels and the normal cornea). In
most cases this would involve perilimbic corneal injection to
"protect" the cornea from the advancing blood vessels. This method
may also be utilized shortly after a corneal insult in order to
prophylactically prevent corneal neovascularization. In this
situation the material could be injected in the perilimbic cornea
interspersed between the corneal lesion and its undesired potential
limbic blood supply. Such methods may also be utilized in a similar
fashion to prevent capillary invasion of transplanted corneas. In a
sustained-release form injections might only be required 2-3 times
per year. A steroid could also be added to the injection solution
to reduce inflammation resulting from the injection itself.
[0197] Within another aspect of the present invention, methods are
provided for treating neovascular glaucoma, comprising the step of
administering to a patient a therapeutically effective amount of an
anti-angiogenic composition to the eye, such that the formation of
blood vessels is inhibited.
[0198] Briefly, neovascular glaucoma is a pathological condition
wherein new capillaries develop in the iris of the eye. The
angiogenesis usually originates from vessels located at the
pupillary margin, and progresses across the root of the iris and
into the trabecular meshwork. Fibroblasts and other connective
tissue elements are associated with the capillary growth and a
fibrovascular membrane develops which spreads across the anterior
surface of the iris. Eventually this tissue reaches the anterior
chamber angle where it forms synechiae. These synechiae in turn
coalesce, scar, and contract to ultimately close off the anterior
chamber angle. The scar formation prevents adequate drainage of
aqueous humor through the angle and into the trabecular meshwork,
resulting in an increase in intraocular pressure that may result in
blindness.
[0199] Neovascular glaucoma generally occurs as a complication of
diseases in which retinal ischemia is predominant. In particular,
about one third of the patients with this disorder have diabetic
retinopathy and 28% have central retinal vein occlusion. Other
causes include chronic retinal detachment, end-stage glaucoma,
carotid artery obstructive disease, retrolental fibroplasia,
sickle-cell anemia, intraocular tumors, and carotid cavernous
fistulas. In its early stages, neovascular glaucoma may be
diagnosed by high magnification slitlamp biomicroscopy, where it
reveals small, dilated, disorganized capillaries (which leak
fluorescein) on the surface of the iris. Later gonioscopy
demonstrates progressive obliteration of the anterior chamber angle
by fibrovascular bands. While the anterior chamber angle is still
open, conservative therapies may be of assistance. However, once
the angle closes surgical intervention is required in order to
alleviate the pressure.
[0200] Therefore, within one embodiment of the invention
anti-angiogenic factors (either alone or in an anti-angiogenic
composition, as described above) may be administered topically to
the eye in order to treat early forms of neovascular glaucoma.
[0201] Within other embodiments of the invention, anti-angiogenic
compositions may be implanted by injection of the composition into
the region of the anterior chamber angle. This provides a sustained
localized increase of anti-angiogenic factor, and prevents blood
vessel growth into the area. Implanted or injected anti-angiogenic
compositions which are placed between the advancing capillaries of
the iris and the anterior chamber angle can "defend" the open angle
from neovascularization. As capillaries will not grow within a
significant radius of the anti-angiogenic composition, patency of
the angle could be maintained. Within other embodiments, the
anti-angiogenic composition may also be placed in any location such
that the anti-angiogenic factor is continuously released into the
aqueous humor. This would increase the anti-angiogenic factor
concentration within the humor, which in turn bathes the surface of
the iris and its abnormal capillaries, thereby providing another
mechanism by which to deliver the medication. These therapeutic
modalities may also be useful prophylactically and in combination
with existing treatments.
[0202] Within another aspect of the present invention, methods are
provided for treating proliferative diabetic retinopathy,
comprising the step of administering to a patient a therapeutically
effective amount of an anti-angiogenic composition to the eyes,
such that the formation of blood vessels is inhibited.
[0203] Briefly, the pathology of diabetic retinopathy is thought to
be similar to that described above for neovascular glaucoma. In
particular, background diabetic retinopathy is believed to convert
to proliferative diabetic retinopathy under the influence of
retinal hypoxia. Generally, neovascular tissue sprouts from the
optic nerve (usually within 10 mm of the edge), and from the
surface of the retina in regions where tissue perfusion is poor.
Initially the capillaries grow between the inner limiting membrane
of the retina and the posterior surface of the vitreous.
[0204] Eventually, the vessels grow into the vitreous and through
the inner limiting membrane. As the vitreous contracts, traction is
applied to the vessels, often resulting in shearing of the vessels
and blinding of the vitreous due to hemorrhage. Fibrous traction
from scarring in the retina may also produce retinal
detachment.
[0205] The conventional therapy of choice is panretinal
photocoagulation to decrease retinal tissue, and thereby decrease
retinal oxygen demands. Although initially effective, there is a
high relapse rate with new lesions forming in other parts of the
retina. Complications of this therapy include a decrease in
peripheral vision of up to 50% of patients, mechanical abrasions of
the cornea, laser-induced cataract formation, acute glaucoma, and
stimulation of subretinal neovascular growth (which can result in
loss of vision). As a result, this procedure is performed only when
several risk factors are present, and the risk-benefit ratio is
clearly in favor of intervention.
[0206] Therefore, within particularly preferred embodiments of the
invention, proliferative diabetic retinopathy may be treated by
injection of an anti-angiogenic factor(s) (or anti-angiogenic
composition) into the aqueous humor or the vitreous, in order to
increase the local concentration of anti-angiogenic factor in the
retina. Preferably, this treatment should be initiated prior to the
acquisition of severe disease requiring photocoagulation. Within
other embodiments of the invention, arteries which feed the
neovascular lesions may be embolized (utilizing anti-angiogenic
compositions, as described above)
[0207] Within another aspect of the present invention, methods are
provided for treating retrolental fibroblasia, comprising the step
of administering to a patient a therapeutically effective amount of
an anti-angiogenic factor (or anti-angiogenic composition) to the
eye, such that the formation of blood vessels is inhibited.
[0208] Briefly, retrolental fibroblasia is a condition occurring in
premature infants who receive oxygen therapy. The peripheral
retinal vasculature, particularly on the temporal side, does not
become fully formed until the end of fetal life. Excessive oxygen
(even levels which would be physiologic at term) and the formation
of oxygen free radicals are thought to be important by causing
damage to the blood vessels of the immature retina. These vessels
constrict, and then become structurally obliterated on exposure to
oxygen. As a result, the peripheral retina fails to vascularize and
retinal ischemia ensues. In response to the ischemia,
neovascularization is induced at the junction of the normal and the
ischemic retina.
[0209] In 75% of the cases these vessels regress spontaneously.
However, in the remaining 25% there is continued capillary growth,
contraction of the fibrovascular component, and traction on both
the vessels and the retina. This results in vitreous hemorrhage
and/or retinal detachment which can lead to blindness. Neovascular
angle-closure glaucoma is also a complication of this
condition.
[0210] As it is often impossible to determine which cases will
spontaneously resolve and which will progress in severity,
conventional treatment (i.e., surgery) is generally initiated only
in patients with established disease and a well developed
pathology. This "wait and see" approach precludes early
intervention, and allows the progression of disease in the 25% who
follow a complicated course. Therefore, within one embodiment of
the invention, topical administration of anti-angiogenic factors
(or anti-angiogenic compositions, as described above) may be
accomplished in infants which are at high risk for developing this
condition in an attempt to cut down on the incidence of progression
of retrolental fibroplasia. Within other embodiments, intravitreous
injections and/or intraocular implants of an anti-angiogenic
composition may be utilized. Such methods are particularly
preferred in cases of established disease, in order to reduce the
need for surgery.
Other Therapeutic Uses of Anti-Angiogenic Compositions
[0211] Anti-angiogenic factors and compositions of the present
invention may be utilized in a variety of additional methods in
order to therapeutically treat a cancer or tumor. For example,
anti-angiogenic factors or compositions described herein may be
formulated for topical delivery, in order to treat cancers such as
skin cancer, head and neck tumors, breast tumors, and Kaposi's
sarcoma. Within yet other aspects, the anti-angiogenic factors or
compositions provided herein may be utilized to treat superficial
forms of bladder cancer by, for example, intravesical
administration.
[0212] In addition to cancer, however, numerous other
non-tumorigenic angiogenesis-dependent diseases which are
characterized by the abnormal growth of blood vessels may also be
treated with the anti-angiogenic factors or compositions of the
present invention. Representative examples of such non-tumorigenic
angiogenesis-dependent diseases include hypertrophic scars and
keloids, proliferative diabetic retinopathy (discussed above),
rheumatoid arthritis (discussed above), arteriovenous malformations
(discussed above), atherosclerotic plaques, delayed wound healing,
hemophilic joints, nonunion fractures, Osler-Weber syndrome,
psoriasis, pyogenic granuloma, scleroderma, tracoma, menorrhagia
(discussed above) and vascular adhesions.
[0213] For example, within one aspect of the present invention
methods are provided for treating hypertrophic scars and keloids,
comprising the step of administering one of the above-described
anti-angiogenic compositions to a hypertrophic scar or keloid.
[0214] Briefly, healing of wounds and scar formation occurs in
three phases: inflammation, proliferation, and maturation. The
first phase, inflammation, occurs in response to an injury which is
severe enough to break the skin. During this phase, which lasts 3
to 4 days, blood and tissue fluid form an adhesive coagulum and
fibrinous network which serves to bind the wound surfaces together.
This is then followed by a proliferative phase in which there is
ingrowth of capillaries and connective tissue from the wound edges,
and closure of the skin defect. Finally, once capillary and
fibroblastic proliferation has ceased, the maturation process
begins wherein the scar contracts and becomes less cellular, less
vascular, and appears flat and white. This final phase may take
between 6 and 12 months.
[0215] If too much connective tissue is produced and the wound
remains persistently cellular, the scar may become red and raised.
If the scar remains within the boundaries of the original wound it
is referred to as a hypertrophic scar, but if it extends beyond the
original scar and into the surrounding tissue, the lesion is
referred to as a keloid. Hypertrophic scars and keloids are
produced during the second and third phases of scar formation.
Several wounds are particularly prone to excessive endothelial and
fibroblastic proliferation, including burns, open wounds, and
infected wounds. With hypertrophic scars, some degree of maturation
occurs and gradual improvement occurs. In the case of keloids
however, an actual tumor is produced which can become quite large.
Spontaneous improvement in such cases rarely occurs.
[0216] Therefore, within one embodiment of the present invention
either anti-angiogenic factors alone, or anti-angiogenic
compositions as described above, are directly injected into a
hypertrophic scar or keloid, in order to prevent the progression of
these lesions. The frequency of injections will depend upon the
release kinetics of the polymer used (if present), and the clinical
response. This therapy is of particular value in the prophylactic
treatment of conditions which are known to result in the
development of hypertrophic scars and keloids (e.g., burns), and is
preferably initiated after the proliferative phase has had time to
progress (approximately 14 days after the initial injury), but
before hypertrophic scar or keloid development.
[0217] As noted above, within yet another aspect of the present
invention, vascular grafts are provided comprising a synthetic
tube, the surface of which is coated with an anti-angiogenic
composition as described above. Briefly, vascular grafts are
synthetic tubes, usually made of Dacron or Gortex, inserted
surgically to bypass arterial blockages, most frequently from the
aorta to the femoral, or the femoral to the popliteal artery. A
major problem which particularly complicates femoral-popliteal
bypass grafts is the formation of a subendothelial scar-like
reaction in the blood vessel wall called neointimal hyperplasia,
which narrows the lumen within and adjacent to either end of the
graft, and which can be progressive. A graft coated with or
containing anti-angiogenic factors (or anti-angiogenic
compositions, as described above) may be utilized to limit the
formation of neointimal hyperplasia at either end of the graft. The
graft may then be surgically placed by conventional bypass
techniques.
[0218] Anti-angiogenic compositions of the present invention may
also be utilized in a variety of other manners. For example, they
may be incorporated into surgical sutures in order to prevent
stitch granulomas, implanted in the uterus (in the same manner as
an IUD) for the treatment of menorrhagia or as a form of female
birth control, administered as either a peritoneal lavage fluid or
for peritoneal implantation in the treatment of endometriosis,
attached to a monoclonal antibody directed against activated
endothelial cells as a form of systemic chemotherapy, or utilized
in diagnostic imaging when attached to a radioactively labeled
monoclonal antibody which recognizes activated endothelial
cells.
Formulation and Administration
[0219] As noted above, anti-angiogenic compositions of the present
invention may be formulated in a variety of forms (e.g.,
microspheres, pastes, films or sprays). Further, the compositions
of the present invention may be formulated to contain more than one
anti-angiogenic factor, to contain a variety of additional
compounds, to have certain physical properties (e.g., elasticity, a
particular melting point, or a specified release rate). Within
certain embodiments of the invention, compositions may be combined
in order to achieve a desired effect (e.g., several preparations of
microspheres may be combined in order to achieve both a quick and a
slow or prolonged release of one or more anti-angiogenic
factor).
[0220] Anti-angiogenic factors and compositions of the present
invention may be administered either alone, or in combination with
pharmaceutically or physiologically acceptable carrier, excipients
or diluents. Generally, such carriers should be nontoxic to
recipients at the dosages and concentrations employed. Ordinarily,
the preparation of such compositions entails combining the
therapeutic agent with buffers, antioxidants such as ascorbic acid,
low molecular weight (less than about 10 residues) polypeptides,
proteins, amino acids, carbohydrates including glucose, sucrose or
dextrins, chelating agents such as EDTA, glutathione and other
stabilizers and excipients. Neutral buffered saline or saline mixed
with nonspecific serum albumin are exemplary appropriate
diluents.
[0221] As noted above, anti-angiogenic factors, anti-angiogenic
compositions, or pharmaceutical compositions provided herein may be
prepared for administration by a variety of different routes,
including for example intrarticularly, intraocularly, intranasally,
intradermally, sublingually, orally, topically, intravesically,
intrathecally, topically, intravenously, intraperitoneally,
intracranially, intramuscularly, subcutaneously, or even directly
into a tumor or disease site. Other representative routes of
administration include gastroscopy, ECRP and colonoscopy, which do
not require full operating procedures and hospitalization, but may
require the presence of medical personnel.
[0222] The anti-angiogenic factors, anti-angiogenic compositions
and pharmaceutical compositions provided herein may be placed
within containers, along with packaging material which provides
instructions regarding the use of such materials. Generally, such
instructions will include a tangible expression describing the
reagent concentration, as well as within certain embodiments,
relative amounts of excipient ingredients or diluents (e.g., water,
saline or PBS) which may be necessary to reconstitute the
anti-angiogenic factor, anti-angiogenic composition, or
pharmaceutical composition.
[0223] The following examples are offered by way of illustration,
and not by way of limitation.
EXAMPLES
Example 1
Preparation of Anti-Invasive Factor
[0224] The shoulder girdle and skull from a dogfish is excised,
then scraped with a scalpel in order to remove all muscle and
associated connective tissue from the cartilage. The cartilage is
then homogenized with a tissue grinder, and extracted by continuous
stirring at room temperature for 2 to 5 days in a solution
containing 2.0 M guanidium hydrochloride and 0.02 M MES at pH
6.0.
[0225] After 2 to 5 days, the cartilage extract is passed through
gauze netting in order to remove the larger constituents. The
filtrate is then passed through an Amicon ultrafiltration unit
which utilizes spiral-wound cartridges, with a molecular weight
cutoff of 100,000. The filtrate (containing proteins with a
molecular weight of less than 100,000 daltons) is then dialyzed
against 0.02 M MES buffer (pH 6) with an Amicon ultrafiltration
unit which retains proteins with a molecular weight of greater than
3,000 daltons. Utilizing this method, low molecular weight proteins
and constituents are removed, as well as excessive amounts of
guanidium HCl. The dialysate is concentrated to a final
concentration 9 mg/ml.
Example 2
Analysis of Various Agents for Anti-Angiogenic Activity
[0226] A. Chick Chorioallantoic Membrane ("Cam") Assays
[0227] Fertilized, domestic chick embryos were incubated for 3 days
prior to shell-less culturing. In this procedure, the egg contents
were emptied by removing the shell located around the air space.
The interior shell membrane was then severed and the opposite end
of the shell was perforated to allow the contents of the egg to
gently slide out from the blunted end. The egg contents were
emptied into round-bottom sterilized glass bowls and covered with
petri dish covers. These were then placed into an incubator at 90%
relative humidity and 3% CO.sub.2 and incubated for 3 days.
[0228] Paclitaxel (Sigma, St. Louis, Mich.) was mixed at
concentrations of 1, 5, 10, 30 .mu.g per 10 ml aliquot of 0.5%
aqueous methylcellulose. Since paclitaxel is insoluble in water,
glass beads were used to produce fine particles. Ten microliter
aliquots of this solution were dried on parafilm for 1 hour forming
disks 2 mm in diameter. The dried disks containing paclitaxel were
then carefully placed at the growing edge of each CAM at day 6 of
incubation. Controls were obtained by placing paclitaxel-free
methylcellulose disks on the CAMs over the same time course. After
a 2 day exposure (day 8 of incubation) the vasculature was examined
with the aid of a stereomicroscope. Liposyn II, a white opaque
solution, was injected into the CAM to increase the visibility of
the vascular details. The vasculature of unstained, living embryos
were imaged using a Zeiss stereomicroscope which was interfaced
with a video camera (Dage-MTI Inc., Michigan City, Ind.). These
video signals were then displayed at 160 times magnification and
captured using an image analysis system (Vidas, Kontron; Etching,
Germany). Image negatives were then made on a graphics recorder
(Model 3000; Matrix Instruments, Orangeburg, N.Y.).
[0229] The membranes of the 8 day-old shell-less embryo were
flooded with 2% glutaraldehyde in 0.1M Na cacodylate buffer;
additional fixative was injected under the CAM. After 10 minutes in
situ, the CAM was removed and placed into fresh fixative for 2
hours at room temperature. The tissue was then washed overnight in
cacodylate buffer containing 6% sucrose. The areas of interest were
postfixed in 1% osmium tetroxide for 1.5 hours at 4.degree. C. The
tissues were then dehydrated in a graded series of ethanols,
solvent exchanged with propylene oxide, and embedded in Spurr
resin. Thin sections were cut with a diamond knife, placed on
copper grids, stained, and examined in a Joel 1200EX electron
microscope. Similarly, 0.5 mm sections were cut and stained with
toluene blue for light microscopy.
[0230] At day 11 of development, chick embryos were used for the
corrosion casting technique. Mercox resin (Ted Pella, Inc.,
Redding, Calif.) was injected into the CAM vasculature using a
30-gauge hypodermic needle. The casting material consisted of 2.5
grams of Mercox CL-2B polymer and 0.05 grams of catalyst (55%
benzoyl peroxide) having a 5 minute polymerization time. After
injection, the plastic was allowed to sit in situ for an hour at
room temperature and then overnight in an oven at 65.degree. C. The
CAM was then placed in 50% aqueous solution of sodium hydroxide to
digest all organic components. The plastic casts were washed
extensively in distilled water, air-dried, coated with
gold/palladium, and viewed with the Philips 501B scanning electron
microscope.
[0231] Results of the above experiments are shown in FIGS. 1-4.
Briefly, the general features of the normal chick shell-less egg
culture are shown in FIG. 1A. At day 6 of incubation, the embryo is
centrally positioned to a radially expanding network of blood
vessels; the CAM develops adjacent to the embryo. These growing
vessels lie close to the surface and are readily visible making
this system an idealized model for the study of angiogenesis.
Living, unstained capillary networks of the CAM can be imaged
noninvasively with a stereomicroscope. FIG. 1B illustrates such a
vascular area in which the cellular blood elements within
capillaries were recorded with the use of a video/computer
interface. The 3-dimensional architecture of such CAM capillary
networks is shown by the corrosion casting method and viewed in the
scanning electron microscope (FIG. 1C). These castings revealed
underlying vessels which project toward the CAM surface where they
form a single layer of anastomotic capillaries.
[0232] Transverse sections through the CAM show an outer ectoderm
consisting of a double cell layer, a broader mesodermal layer
containing capillaries which lie subjacent to the ectoderm,
adventitial cells, and an inner, single endodermal cell layer (FIG.
1D). At the electron microscopic level, the typical structural
details of the CAM capillaries are demonstrated. Typically, these
vessels lie in close association with the inner cell layer of
ectoderm (FIG. 1E)
[0233] After 48 hours exposure to paclitaxel at concentrations of
0.25, 0.5, 1, 5, 10, or 30 ug, each CAM was examined under living
conditions with a stereomicroscope equipped with a video/computer
interface in order to evaluate the effects on angiogenesis. This
imaging setup was used at a magnification of 160 times which
permitted the direct visualization of blood cells within the
capillaries; thereby blood flow in areas of interest could be
easily assessed and recorded. For this study, the inhibition of
angiogenesis was defined as an area of the CAM lacking a capillary
network and vascular blood flow. Throughout the experiments,
avascular zones were assessed on a 4 point avascular gradient
(Table I). This scale represents the degree of overall inhibition
with maximal inhibition represented as a 3 on the avascular
gradient scale. Paclitaxel was very consistent and induced a
maximal avascular zone (6 mm in diameter or a 3 on the avasculare
gradient scale) within 48 hours depending on its concentration.
TABLE-US-00001 TABLE I AVASCULAR GRADIENT 0 normal vascularity 1
lacking some microvascular movement 2* small avascular zone
approximately 2 mm in diameter 3* avascularity extending beyond the
disk (6 mm in diameter)
[0234] The dose-dependent, experimental data of the effects of
paclitaxel at different concentrations are shown in Table II.
TABLE-US-00002 TABLE II Angiogenic Inhibition by Paclitaxel
Paclitaxel in Methylcellulose Disks Embryos Evaluated (.mu.g)
positive/total) % Inhibition 0.25 2/11 18 0.5 6/11 54 1 6/15 40 5
20/27 76 10 16/21 76 30 31/31 100 0 (control) 0/40 0
[0235] TABLE-US-00003 TABLE III Angiogenic Inhibition of
Paclitaxel-Loaded Thermopaste Paclitaxel-loaded Thermopaste (%)
Embryos Evaluated (positive/n) 0.25 4/4 0.5 4/4 1 8/8 5 4/4 10 5/5
20 6/6 0 (control) 0/30
[0236] Typical paclitaxel-treated CAMs are also shown with the
transparent methylcellulose disk centrally positioned over the
avascular zone measuring 6 mm in diameter. At a slightly higher
magnification, the periphery of such avascular zones is clearly
evident (FIG. 2C); the surrounding functional vessels were often
redirected away from the source of paclitaxel (FIGS. 2C and 2D).
Such angular redirecting of blood flow was never observed under
normal conditions. Another feature of the effects of paclitaxel was
the formation of blood islands within the avascular zone
representing the aggregation of blood cells.
[0237] The associated morphological alterations of the
paclitaxel-treated CAM are readily apparent at both the light and
electron microscopic levels. For the convenience of presentation,
three distinct phases of general transition from the normal to the
avascular state are shown. Near the periphery of the avascular zone
the CAM is hallmarked by an abundance of mitotic cells within all
three germ layers (FIGS. 3A and 4A). This enhanced mitotic division
was also a consistent observation for capillary endothelial cells.
However, the endothelial cells remained junctionally intact with no
extravasation of blood cells. With further degradation, the CAM is
characterized by the breakdown and dissolution of capillaries
(FIGS. 3B and 4B). The presumptive endothelial cells, typically
arrested in mitosis, still maintain a close spatial relationship
with blood cells and lie subjacent to the ectoderm; however, these
cells are not junctionally linked. The most central portion of the
avascular zone was characterized by a thickened ectodermal and
endodermal layer (FIGS. 3C and 4C). Although these layers were
thickened, the cellular junctions remained intact and the layers
maintained their structural characteristics. Within the mesoderm,
scattered mitotically arrested cells were abundant; these cells did
not exhibit the endothelial cell polarization observed in the
former phase. Also, throughout this avascular region, degenerating
cells were common as noted by the electron dense vacuoles and
cellular debris (FIG. 4C).
[0238] In summary, this study demonstrated that 48 hours after
paclitaxel application to the CAM, angiogenesis was inhibited. The
blood vessel inhibition formed an avascular zone which was
represented by three transitional phases of paclitaxel's effect.
The central, most affected area of the avascular zone contained
disrupted capillaries with extravasated red blood cells; this
indicated that intercellular junctions between endothelial cells
were absent. The cells of the endoderm and ectoderm maintained
their intercellular junctions and therefore these germ layers
remained intact; however, they were slightly thickened. As the
normal vascular area was approached, the blood vessels retained
their junctional complexes and therefore also remained intact. At
the periphery of the paclitaxel-treated zone, further blood, vessel
growth was inhibited which was evident by the typical redirecting
or "elbowing" effect of the blood vessels (FIG. 2D).
[0239] Paclitaxel-treated avascular zones also revealed an
abundance of cells arrested in mitosis in all three germ layers of
the CAM; this was unique to paclitaxel since no previous study has
illustrated such an event. By being arrested in mitosis,
endothelial cells could not undergo their normal metabolic
functions involved in angiogenesis. In comparison, the avascular
zone formed by suramin and cortisone acetate do not produce
mitotically arrested cells in the CAM; they only prevented further
blood vessel growth into the treated area. Therefore, even though
these agents are anti-angiogenic, there are many points in which
the angiogenesis process may be targeted.
[0240] The effects of paclitaxel over the 48 hour duration were
also observed. During this period of observation it was noticed
that inhibition of angiogenesis occurs as early as 9 hours after
application. Histological sections revealed a similar morphology as
seen in the first transition phase of the avascular zone at 48
hours illustrated in FIGS. 3A and 4A. Also, we observed in the
revascularization process into the avascular zone previously
observed. It has been found that the avascular zone formed by
heparin and angiostatic steroids became revascularized 60 hours
after application. In one study, paclitaxel-treated avascular zones
did not revascularize for at least 7 days after application
implying a more potent long-term effect.
Example 3
Encapsulation of Suramin
[0241] One milliliter of 5% ELVAX (poly(ethylene-vinyl acetate)
cross-linked with 5% vinyl acetate) in dichloromethane ("DCM") is
mixed with a fixed weight of sub-micron ground sodium suramin. This
mixture is injected into 5 ml of 5% Polyvinyl Alcohol ("PVA") in
water in a 30 ml flat bottomed test tube. Tubes containing
different weights of the drug are then suspended in a multi-sample
water bath at 40.degree. for 90 minutes with automated stirring.
The mixtures are removed, and microsphere samples taken for size
analysis. Tubes are centrifuged at 1000 g for 5 min. The PVA
supernatant is removed and saved for analysis (nonencapsulated
drug). The microspheres are then washed (vortexed) in 5 ml of water
and recentrifuged. The 5 ml wash is saved for analysis (surface
bound drug). Microspheres are then wetted in 50 ul of methanol, and
vortexed in 1 ml of DCM to dissolve the ELVAX. The microspheres are
then warmed to 40.degree. C., and 5 ml of 50.degree. C. water is
slowly added with stirring. This procedure results in the immediate
evaporation of DCM, thereby causing the release of sodium suramin
into the 5 ml of water.
[0242] All samples were assayed for drug content by quantification
of fluorescence. Briefly, sodium suramin absorbs uv/vis with a
lambda max of 312 nm. This absorption is linear in the 0 to 100
ug/ml range in both water and 5% PVA. Sodium suramin also
fluoresces strongly with an excitation maximum at 312 nm, and
emission maximum at 400 nm. This fluorescence is quantifiable in
the 0 to 25 ug/ml range.
[0243] The results of these experiments is shown in FIGS. 5-11.
Results are shown in FIGS. 5-10. Briefly, the size distribution of
microspheres by number (FIG. 5) or by weight (FIG. 6) appears to be
unaffected by inclusion of the drug in the DCM. Good yields of
microspheres in the 20 to 60 .mu.m range may be obtained.
[0244] The encapsulation of suramin is very low (<1%) (see FIG.
8). However as the weight of drug is increased in the DCM the total
amount of drug encapsulated increased although the % encapsulation
decreased. As is shown in FIG. 7, 50 ug of drug may be encapsulated
in 50 mg of ELVAX. Encapsulation of sodium suramin in 2.5% PVA
containing 10% NaCl is shown in FIG. 9 (size distribution by
weight). Encapsulation of sodium suramin in 5% PVA containing 10%
NaCl is shown in FIGS. 10 and 11 (size distribution by weight, and
number, respectively).
[0245] To assess suramin and cortisone acetate as potential
anti-angiogenic agents, each agent was mixed with 0.5%
methylcellulose and applied the dried disks containing the agent
onto the developing blood vessels of the 6-day old CAM. A
combination treatment of suramin (70 .mu.g) with cortisone acetate
(20 .mu.g) was successful in inhibiting angiogenesis when tested on
the CAM for 48 hours. The resulting avascular region measured 6 mm
in diameter and revealed an absence of blood flow and the
appearance of sparse blood islands (FIGS. 28A and 28B).
Example 4
Encapsulation of Paclitaxel
[0246] Five hundred micrograms of either paclitaxel or baccatin (a
paclitaxel analog, available from Inflazyme Pharmaceuticals Inc.,
Vancouver, British Columbia, Canada) are dissolved in 1 ml of a
50:50 ELVAX:poly-1-lactic acid mixture in dcm. Microspheres are
then prepared in a dissolution machine (Six-spindle dissolution
tester, VanderKanp, Van Kell Industries Inc., U.S.A.) in triplicate
at 200 rpm, 42.degree. C., for 3 hours. Microspheres so prepared
are washed twice in water and sized on the microscope.
[0247] Determination of paclitaxel encapsulation is undertaken in a
uv/vis assay (uv/vis lambda max. at 237 nm, fluorescence assay at
excitation 237, emission at 325 nm; Fluorescence results are
presented in square brackets [ ]). Utilizing the procedures
described above, 58 .mu.g (+/-12 .mu.g) [75 .mu.g (+/-25 .mu.g)] of
paclitaxel may be encapsulated from a total 500 .mu.g of starting
material. This represents 12% (+/-2.4%) [15% (+/-5%)] of the
original weight, or 1.2% (+/-0.25%) [1.5% (+/-0.5%)] by weight of
the polymer. After 18 hours of tumbling in an oven at 37.degree.
C., 10.3% (+/-10%) [6% (+/-5.6%)] of the total paclitaxel had been
released from the microspheres.
[0248] For baccatin, 100+/-15 .mu.g [83+/-231 g] of baccatin can be
encapsulated from a total of 500 .mu.g starting material. This
represents a 20% (+/-3%) [17% (+/-5%) of the original weight of
baccatin, and 2% (+/-0.3%) [1.7% (+/-0.5%)] by weight of the
polymer. After 18 hours of tumbling in an oven at 37.degree. C.,
55% (+/-13%) [60% (+/-23%)] of the baccatin is released from the
microspheres.
Example 5
Analysis of Surgical Paste Containing Anti-Angiogenic
Compositions
[0249] Fisher rats weighing approximately 300 grams are
anesthetized, and a 1 cm transverse upper abdominal incision is
made. Two-tenths of a milliliter of saline containing
1.times.10.sup.6 live 9 L gliosarcoma cells (eluted immediately
prior to use from tissue culture) are injected into 2 of the 5
hepatic lobes by piercing a 27 gauge needle 1 cm through the liver
capsule. The abdominal wound is closed with 6.0 resorptible suture
and skin clips and the GA terminated.
[0250] After 2 weeks, the tumor deposits will measure approximately
1 cm. At this time, both hepatic tumors are resected and the bare
margin of the liver is packed with a hemostatic agent. The rats are
divided into two groups: half is administered polymeric carrier
alone, and the other half receives an anti-angiogenic
composition.
[0251] Rats are sacrificed 2, 7, 14, 21 and 84 days post hepatic
resection. In particular, the rats are euthanized by injecting
Euthanyl into the dorsal vein of the tail. The liver, spleen, and
both lungs are removed, and histologic analysis is performed in
order to study the tumors for evidence of anti-angiogenic
activity.
Example 6
Embolization of Rat Arteries
[0252] Fisher rats weighing approximately 300 grams are
anesthetized. Utilizing aseptic procedures, a 1 cm transverse upper
abdominal incision is made, and the liver identified. Two-tenths of
a milliliter of saline containing 1 million live 9 L gliosarcoma
cells (eluted immediately prior from tissue culture) is injected
into each of the 5 hepatic lobes by piercing a 27 gauge needle 1 cm
through the liver capsule. One-tenth of a milliliter of normal
saline is injected into the needle as it is withdrawn to ensure
that there is no spillage of cells into the peritoneal cavity. A
pledget of gelfoam is placed on each of the puncture sites to
ensure hemostasis. The abdominal wound is closed with 6.0
resorptible suture with skin clips, and the anesthetic terminated.
The rat is returned to the animal care facility to have a standard
diet for 14 days, at which time each tumor deposit will measure 1
cm in diameter. The same procedure is repeated using Westar rats
and a Colon Cancer cell line (Radiologic Oncology Lab, M. D.
Anderson, Houston, Tex.). In this instance, 3 weeks are required
post-injection for the tumor deposits to measure 1 cm in diameter
each.
[0253] After 2 or 3 weeks, depending on the rat species, the same
general anesthetic procedure is followed and a midline abdominal
incision is performed. The duodenum is flipped and the
gastroduodenal artery is identified and mobilized. Ties are placed
above and below a cutdown site on the midportion of the
gastroduodenal artery (GDA), and 0.038 inch polyethylene tubing is
introduced in a retrograde fashion into the artery using an
operating microscope. The tie below the insertion point will ligate
the artery, while the one above will fix the catheter in place.
Angiography is performed by injecting 0.5 ml of 60% radiopaque
contrast material through the catheter as an x-ray is taken. The
hepatic artery is then embolized by refluxing particles measuring
15-200 .mu.m through the gastroduodenal artery catheter until flow,
observed via the operating microscope, is seen to cease for at
least 30 seconds. Occlusion of the hepatic artery is confirmed by
repeating an angiogram through the GDA catheter. Utilizing this
procedure, one-half of the rats receive 15-200 .mu.m particles of
polymer alone, and the other half receive 15-200 .mu.m particles of
the polymer-anti-angiogenic factor composition. The upper GDA
ligature is tightened to occlude the GDA as the catheter is
withdrawn to ensure hemostasis, and the hepatic artery (although
embolized) is left intact. The abdomen is closed with 6.0
absorbable suture and surgical clips.
[0254] The rats are subsequently sacrificed at 2, 7, 14, 21 and 84
days post-embolization in order to determine efficacy of the
anti-angiogenic factor. Briefly, general anesthetic is given, and
utilizing aseptic precautions, a midline incision performed. The
GDA is mobilized again, and after placing a ligature near the
junction of the GDA and the hepatic artery (i.e., well above the
site of the previous cutdown), a 0.038-inch polyethylene tubing is
inserted via cutdown of the vessel and angiography is performed.
The rat is then euthanized by injecting Euthanyl into the dorsal
vein of the tail. Once euthanasia is confirmed, the liver is
removed en bloc along with the stomach, spleen and both lungs.
[0255] Histologic analysis is performed on a prepared slide stained
with hematoxylin and eosin ("H and E") stain. Briefly, the lungs
are sectioned at 1 cm intervals to assess passage of embolic
material through the hepatic veins and into the right side of
circulation. The stomach and spleen are also sectioned in order to
assess inadvertent immobilization from reflux of particles into the
celiac access of the collateral circulation.
Example 7
Transplantation of Biliary Stents in Rats
[0256] General anesthetic is administered to 300 gram Fisher rats.
A 1 cm transverse incision is then made in the upper abdomen, and
the liver identified. In the most superficial lobe, 0.2 ml of
saline containing 1 million cells of 9 L gliosarcoma cells (eluted
from tissue culture immediately prior to use) is injected via a 27
gauge needle to a depth of 1 cm into the liver capsule. Hemostasis
is achieved after removal of the needle by placing a pledget of
gelfoam at the puncture sites. Saline is injected as the needle is
removed to ensure no spillage of cells into the peritoneal cavity
or along the needle track. The general anesthetic is terminated,
and the animal returned to the animal care center and placed on a
normal diet.
[0257] Two weeks later, general anesthetic is administered, and
utilizing aseptic precautions, the hepatic lobe containing the
tumor is identified through a midline incision. A 16 gauge
angiographic needle is then inserted through the hepatic capsule
into the tumor, a 0.038-inch guidewire passed through the needle,
and the needle withdrawn over the guidewire. A number 5 French
dilator is passed over the guide into the tumor and withdrawn. A
number 5 French delivery catheter is then passed over the wire
containing a self-expanding stainless steel Wallstent (5 mm in
diameter and 1 cm long). The stent is deployed into the tumor and
the guidewire delivery catheter is removed. One-third of the rats
have a conventional stainless steel stent inserted into the tumor,
one-third a stainless steel stent coated with polymer, and one
third a stent coated with the polymer-anti-angiogenic factor
compound. The general anesthetic is terminated and the rat returned
to the animal care facility.
[0258] A plain abdominal X-ray is performed at 2 days in order to
assess the degree of stent opening. Rats are sacrificed at 2, 7,
14, 28 and 56 days post-stent insertion by injecting Euthanyl, and
their livers removed en bloc once euthanasia is confirmed. After
fixation in formaldehyde for 48 hours, the liver is sectioned at
0.5 mm intervals; including severing the stent transversely using a
fresh blade for each slice. Histologic sections stained with H and
E are then analyzed to assess the degree of tumor ingrowth into the
stent lumen.
Example 8
Manufacture of Microspheres
[0259] Equipment which is preferred for the manufacture of
microspheres described below include: 200 ml water jacketed beaker
(Kimax or Pyrex), Haake circulating water bath, overhead stirrer
and controller with 2 inch diameter (4 blade, propeller type
stainless steel stirrer--Fisher brand), 500 ml glass beaker, hot
plate/stirrer (Corning brand), 4.times.50 ml polypropylene
centrifuge tubes (Nalgene), glass scintillation vials with plastic
insert caps, table top centrifuge (GPR Beckman), high speed
centrifuge-floor model (JS 21 Beckman), Mettler analytical balance
(AJ 100, 0.1 mg), Mettler digital top loading balance (AE 163, 0.01
mg), automatic pipetter (Gilson). Reagents include Polycaprolactone
("PCL"--mol wt 10,000 to 20,000; Polysciences, Warrington Pa.,
USA), "washed" (see later method of "washing") Ethylene Vinyl
Acetate ("EVA"), Poly(DL)lactic acid ("PLA"--mol wt 15,000 to
25,000; Polysciences), Polyvinyl Alcohol ("PVA"--mol wt 124,000 to
186,000; 99% hydrolyzed; Aldrich Chemical Co., Milwaukee Wis.,
USA), Dichloromethane ("DCM" or "methylene chloride"; HPLC grade
Fisher scientific), and distilled water.
[0260] A. Preparation of 5% (w/v) Polymer Solutions
[0261] Depending on the polymer solution being prepared, 1.00 g of
PCL or PLA, or 0.50 g each of PLA and washed EVA is weighed
directly into a 20 ml glass scintillation vial. Twenty milliliters
of DCM is then added, and the vial tightly capped. The vial is
stored at room temperature (25.degree. C.) for one hour (occasional
shaking may be used), or until all the polymer has dissolved (the
solution should be clear). The solution may be stored at room
temperature for at least two weeks.
[0262] B. Preparation of 5% (w/v) Stock Solution of PVA
[0263] Twenty-five grams of PVA is weighed directly into a 600 ml
glass beaker. Five hundred milliliters of distilled water is added,
along with a 3 inch Teflon coated stir bar. The beaker is covered
with glass to decrease evaporation losses, and placed into a 2000
ml glass beaker containing 300 ml of water (which acts as a water
bath). The PVA is stirred at 300 rpm at 85.degree. C. (Corning hot
plate/stirrer) for 2 hours or until fully dissolved. Dissolution of
the PVA may be determined by a visual check; the solution should be
clear. The solution is then transferred to a glass screw top
storage container and stored at 4.degree. C. for a maximum of two
months. The solution, however should be warmed to room temperature
before use or dilution.
[0264] C. Procedure for Producing Microspheres
[0265] Based on the size of microspheres being made (see Table 1),
100 ml of the PVA solution (concentrations given in Table IV) is
placed into the 200 ml water jacketed beaker. Haake circulating
water bath is connected to this beaker and the contents are allowed
to equilibrate at 27.degree. C. (+/-10.degree. C.) for 10 minutes.
Based on the size of microspheres being made (see Table IV), the
start speed of the overhead stirrer is set, and the blade of the
overhead stirrer placed half way down in the PVA solution. The
stirrer is then started, and 10 ml of polymer solution (polymer
solution used based on type of microspheres being produced) is then
dripped into the stirring PVA over a period of 2 minutes using a 5
ml automatic pipetter. After 3 minutes the stir speed is adjusted
(see Table IV), and the solution stirred for an additional 2.5
hours. The stirring blade is then removed from the microsphere
preparation, and rinsed with 10 ml of distilled water so that the
rinse solution drains into the microsphere preparation. The
microsphere preparation is then poured into a 500 ml beaker, and
the jacketed water bath washed with 70 ml of distilled water, which
is also allowed to drain into the microsphere preparation. The 180
ml microsphere preparation is then stirred with a glass rod, and
equal amounts are poured into four polypropylene 50 ml centrifuge
tubes. The tubes are then capped, and centrifuged for 10 minutes
(force given in Table III). A 5 ml automatic pipetter or vacuum
suction is then utilized to draw 45 ml of the PVA solution off of
each microsphere pellet. TABLE-US-00004 TABLE III PVA
concentrations, stir speeds, and centrifugal force requirements for
each diameter range of microspheres. PRODUCTION MICROSPHERE
DIAMETER RANGES STAGE 30 .mu.m to 100 .mu.m 10 .mu.m to 30 .mu.m
0.1 .mu.m to 3 .mu.m PVA 2.5% (w/v) (i.e.,) 5% (w/v) (i.e., 3.5%
(w/v) (i.e., concentration dilute 5% stock undiluted stock) dilute
5% stock with distilled water with distilled water Starting Stir
500 rpm +/- 50 rpm 500 rpm +/- 50 rpm 3000 rpm +/- 200 rpm Speed
Adjusted Stir Speed 500 rpm +/- 50 rpm 500 rpm +/- 50 rpm 2500 rpm
+/- 200 rpm Centrifuge Force 1000 g +/- 100 g 1000 g +/- 100 g 10
000 g +/- 1000 g (Table top model) (Table top model) (High speed
model)
[0266] Five milliliters of distilled water is then added to each
centrifuge tube, which is then vortexed to resuspend the
microspheres. The four microsphere suspensions are then pooled into
one centrifuge tube along with 20 ml of distilled water, and
centrifuged for another 10 minutes (force given in Table 1). This
process is repeated two additional times for a total of three
washes. The microspheres are then centrifuged a final time, and
resuspended in 10 ml of distilled water. After the final wash, the
microsphere preparation is transferred into a preweighed glass
scintillation vial. The vial is capped, and left overnight at room
temperature (25.degree. C.) in order to allow the microspheres to
sediment out under gravity. Microspheres which fall in the size
range of 0.1 um to 3 um do not sediment out under gravity, so they
are left in the 10 ml suspension.
[0267] D. Drying of 10 .mu.m to 30 .mu.m or 30 .mu.m to 100 .mu.m
Diameter Microspheres
[0268] After the microspheres have sat at room temperature
overnight, a 5 ml automatic pipetter or vacuum suction is used to
draw the supernatant off of the sedimented microspheres. The
microspheres are allowed to dry in the uncapped vial in a drawer
for a period of one week or until they are fully dry (vial at
constant weight). Faster drying may be accomplished by leaving the
uncapped vial under a slow stream of nitrogen gas (flow approx. 10
ml/min.) in the fume hood. When fully dry (vial at constant
weight), the vial is weighed and capped. The labeled, capped vial
is stored at room temperature in a drawer. Microspheres are
normally stored no longer than 3 months.
[0269] E. Drying of 0.1 .mu.m to 3 .mu.m Diameter Microspheres
[0270] This size range of microspheres will not sediment out, so
they are left in suspension at 4.degree. C. for a maximum of four
weeks. To determine the concentration of microspheres in the 10 ml
suspension, a 200 .mu.l sample of the suspension is pipetted into a
1.5 ml preweighed microfuge tube. The tube is then centrifuged at
10,000 g (Eppendorf table top microfuge), the supernatant removed,
and the tube allowed to dry at 50.degree. C. overnight. The tube is
then reweighed in order to determine the weight of dried
microspheres within the tube.
[0271] F. Manufacture of Paclitaxel Loaded Microsphere
[0272] In order to prepare paclitaxel containing microspheres, an
appropriate amount of weighed paclitaxel (based upon the percentage
of paclitaxel to be encapsulated) is placed directly into a 20 ml
glass scintillation vial. Ten milliliters of an appropriate polymer
solution is then added to the vial containing the paclitaxel, which
is then vortexed until the paclitaxel has dissolved.
[0273] Microspheres containing paclitaxel may then be produced
essentially as described above in steps (C) through (E).
Example 9
Manufacture of Stent Coating
[0274] Reagents and equipment which are utilized within the
following experiments include (medical grade stents obtained
commercially from a variety of manufacturers; e.g., the "Strecker"
stent) and holding apparatus, 20 ml glass scintillation vial with
cap (plastic insert type), TLC atomizer, Nitrogen gas tank, glass
test tubes (various sizes from 1 ml and up), glass beakers (various
sizes), Pasteur pipette, tweezers, Polycaprolactone ("PCL"--mol wt
10,000 to 20,000; Polysciences), Paclitaxel (Sigma Chemical Co.,
St. Louis, Mo., 95% purity), Ethylene vinyl acetate
("EVA"--washed--see previous), Poly(DL)lactic acid ("PLA"--mol wt
15,000 to 25,000; Polysciences), dichloromethane ("DCM"--HPLC
grade, Fisher Scientific).
[0275] A. Procedure for Sprayed Stents
[0276] The following describes a typical method using a 3 mm
crimped diameter interleaving metal wire stent of approximately 3
cm length. For larger diameter stents, larger volumes of
polymer/drug solution are used.
[0277] Briefly, a sufficient quantity of polymer is weighed
directly into a 20 ml glass scintillation vial, and sufficient DCM
added in order to achieve a 2% w/v solution. The vial is then
capped and mixed by hand in order to dissolve the polymer. The
stent is then assembled in a vertical orientation, tying the stent
to a retort stand with nylon. Position this stent holding apparatus
6 to 12 inches above the fume hood floor on a suitable support
(e.g., inverted 2000 ml glass beaker) to enable horizontal
spraying. Using an automatic pipette, a suitable volume (minimum 5
ml) of the 2% polymer solution is transferred to a separate 20 ml
glass scintillation vial. An appropriate amount of paclitaxel is
then added to the solution and dissolved by hand shaking.
[0278] To prepare for spraying, remove the cap of this vial and dip
the barrel (only) of an TLC atomizer into the polymer solution.
Note that the reservoir of the atomizer need not be used in this
procedure: the 20 ml glass vial acts as a reservoir. Connect the
nitrogen tank to the gas inlet of the atomizer. Gradually increase
the pressure until atomization and spraying begins. Note the
pressure and use this pressure throughout the procedure. To spray
the stent use 5 second oscillating sprays with a 15 second dry time
between sprays. After 5 sprays, rotate the stent 90.degree. and
spray that portion of the stent. Repeat until all sides of the
stent have been sprayed. During the dry time, finger crimp the gas
line to avoid wastage of the spray. Spraying is continued until a
suitable amount of polymer is deposited on the stents. The amount
may be based on the specific stent application in vivo. To
determine the amount, weigh the stent after spraying has been
completed and the stent has dried. Subtract the original weight of
the stent from the finished weight and this produces the amount of
polymer (plus paclitaxel) applied to the stent. Store the coated
stent in a sealed container.
[0279] B. Procedure for Dipped Stents
[0280] The following describes a typical method using a 3 mm
crimped diameter interleaving metal wire stent of approximately 3
cm length. For larger diameter stents, larger volumes of
polymer/drug solution are used in larger sized test tubes.
[0281] Weigh 2 g of EVA into a 20 ml glass scintillation vial and
add 20 ml of DCM. Cap the vial and leave it for 2 hours to dissolve
(hand shake the vial frequently to assist the dissolving process).
Weigh a known weight of paclitaxel directly into a 1 ml glass test
tube and add 0.5 ml of the polymer solution. Using a glass Pasteur
pipette, dissolve the paclitaxel by gently pumping the polymer
solution. Once the paclitaxel is dissolved, hold the test tube in a
near horizontal position (the sticky polymer solution will not flow
out). Using tweezers, insert the stent into the tube all the way to
the bottom. Allow the polymer solution to flow almost to the mouth
of the test tube by angling the mouth below horizontal and then
restoring the test tube to an angle slightly above the horizontal.
While slowly rotating the stent in the tube, slowly remove the
stent (approximately 30 seconds).
[0282] Hold the stent in a vertical position to dry. Some of the
sealed perforations may pop so that a hole exists in the continuous
sheet of polymer. This may be remedied by repeating the previous
dipping procedure, however repetition of the procedure can also
lead to further popping and a general uneven build up of polymer.
Generally, it is better to dip the stent just once and to cut out a
section of stent that has no popped perforations. Store the dipped
stent in a sealed container.
Example 10
Manufacture of Surgical "Pastes"
[0283] As noted above, the present invention provides a variety of
polymeric-containing drug compositions that may be utilized within
a variety of clinical situations. For example, compositions may be
produced: (1) as a "thermopaste" that is applied to a desired site
as a fluid, and hardens to a solid of the desired shape at a
specified temperature (e.g., body temperature); (2) as a spray
(i.e., "nanospray") which may delivered to a desired site either
directly or through a specialized apparatus (e.g., endoscopy), and
which subsequently hardens to a solid which adheres to the tissue
to which it is applied; (3) as an adherent, pliable, resilient,
angiogeneis inhibitor-polymer film applied to a desired site either
directly or through a specialized apparatus, and which preferably
adheres to the site to which it is applied; and (4) as a fluid
composed of a suspension of microspheres in an appropriate carrier
medium, which is applied to a desired site either directly or via a
specialized apparatus, and which leaves a layer of microspheres at
the application site. Representative examples of each of the above
embodiments is set forth in more detail below.
[0284] A. Procedure for Producing Thermopaste
[0285] Reagents and equipment which are utilized within the
following experiments include a sterile glass syringe (1 ml),
Corning hot plate/stirrer, 20 ml glass scintillation vial, moulds
(e.g., 50 .mu.l DSC pan or 50 ml centrifuge tube cap inner
portion), scalpel and tweezers, Polycaprolactone ("PCL"--mol wt
10,000 to 20,000; Polysciences, Warrington, Pa. USA), and
Paclitaxel (Sigma grade 95% purity minimum).
[0286] Weigh 5.00 g of polycaprolactone directly into a 20 ml glass
scintillation vial. Place the vial in a 600 ml beaker containing 50
ml of water. Gently heat the beaker to 65.degree. C. and hold it at
that temperature for 20 minutes. This allows the polymer to melt.
Thoroughly mix a known weight of paclitaxel, or other angiogenesis
inhibitor into the melted polymer at 65.degree. C. Pour the melted
polymer into a prewarmed (60.degree. C. oven) mould. Use a spatula
to assist with the pouring process. Allow the mould to cool so the
polymer solidifies. Cut or break the polymer into small pieces
(approximately 2 mm by 2 mm in size). These pieces must fit into a
1 ml glass syringe. Remove the plunger from the 1 ml glass syringe
(do not remove the cap from the tip) and place it on a balance.
Zero the balance.
[0287] Weigh 0.5 g of the pieces directly into the open end of the
syringe. Place the glass syringe upright (capped tip downwards)
into a 500 ml glass beaker containing distilled water at 65.degree.
C. (coming hot plate) so that no water enters the barrel. The
polymer melts completely within 10 minutes in this apparatus. When
the polymer pieces have melted, remove the barrel from the water
bath, hold it horizontally and remove the cap. Insert the plunger
into the barrel and compress the melted polymer into a sticky mass
at the tip end of the barrel. Cap the syringe and allow it to cool
to room temperature.
[0288] For application, the syringe may be reheated to 60.degree.
C. and administered as a liquid which solidifies when cooled to
body temperature.
[0289] B. Procedure for Producing Nanospray
[0290] Nanospray is a suspension of small microspheres in saline.
If the microspheres are very small (i.e., under 1 .mu.m in
diameter) they form a colloid so that the suspension will not
sediment under gravity. As is described in more detail below, a
suspension of 0.1 .mu.m to 1 .mu.m microparticles may be created
suitable for deposition onto tissue through a finger pumped
aerosol. Equipment and materials which may be utilized to produce
nanospray include 200 ml water jacketed beaker (Kimax or Pyrex),
Haake circulating water bath, overhead stirrer and controller with
2 inch diameter (4 blade, propeller type stainless steel stirrer;
Fisher brand), 500 ml glass beaker, hot plate/stirrer (Corning
brand), 4.times.50 ml polypropylene centrifuge tubes (Nalgene),
glass scintillation vials with plastic insert caps, table top
centrifuge (Beckman), high speed centrifuge--floor model (JS 21
Beckman), Mettler analytical balance (AJ 100, 0.1 mg), Mettler
digital top loading balance (AE 163, 0.01 mg), automatic pipetter
(Gilson), sterile pipette tips, pump action aerosol (Pfeiffer
pharmaceuticals) 20 ml, laminar flow hood, Polycaprolactone
("PCL"--mol wt 10,000 to 20,000; Polysciences, Warrington, Pa.
USA), "washed" (see previous) Ethylene Vinyl Acetate ("EVA"),
Poly(DL)lactic acid ("PLA" mol wt 15,000 to 25,000; Polysciences),
Polyvinyl Alcohol ("PVA"--mol wt 124,000 to 186,000; 99%
hydrolyzed; Aldrich Chemical Co., Milwaukee, Wis. USA),
Dichloromethane ("DCM" or "methylene chloride;" HPLC grade Fisher
scientific), Distilled water, sterile saline (Becton and Dickenson
or equivalent)
[0291] 1. Preparation of 5% (w/v) Polymer Solutions
[0292] Depending on the polymer solution being prepared, weigh 1.00
g of PCL or PLA or 0.50 g each of PLA and washed EVA directly into
a 20 ml glass scintillation vial. Using a measuring cylinder, add
20 ml of DCM and tightly cap the vial. Leave the vial at room
temperature (25.degree. C.) for one hour or until all the polymer
has dissolved (occasional hand shaking may be used). Dissolving of
the polymer can be determined by a visual check; the solution
should be clear. Label the vial with the name of the solution and
the date it was produced. Store the solutions at room temperature
and use within two weeks.
[0293] 2. Preparation of 3.5% (w/v) Stock Solution of PVA
[0294] The solution can be prepared by following the procedure
given below, or by diluting the 5% (w/v) PVA stock solution
prepared for production of microspheres (see Example 8). Briefly,
17.5 g of PVA is weighed directly into a 600 ml glass beaker, and
500 ml of distilled water is added. Place a 3 inch Teflon coated
stir bar in the beaker. Cover the beaker with a cover glass to
reduce evaporation losses. Place the beaker in a 2000 ml glass
beaker containing 300 ml of water. This will act as a water bath.
Stir the PVA at 300 rpm at 85.degree. C. (Corning hot
plate/stirrer) for 2 hours or until fully dissolved. Dissolving of
the PVA can be determined by a visual check; the solution should be
clear. Use a pipette to transfer the solution to a glass screw top
storage container and store at 4.degree. C. for a maximum of two
months. This solution should be warmed to room temperature before
use or dilution.
[0295] 3. Procedure for Producing Nanospray
[0296] Place the stirring assembly in a fume hood. Place 100 ml of
the 3.5% PVA solution in the 200 ml water jacketed beaker. Connect
the Haake water bath to this beaker and allow the contents to
equilibrate at 27.degree. C. (+/-1.degree. C.) for 10 minutes. Set
the start speed of the overhead stirrer at 3000 rpm (+/-200 rpm).
Place the blade of the overhead stirrer half way down in the PVA
solution and start the stirrer. Drip 10 ml of polymer solution
(polymer solution used based on type of nanospray being produced)
into the stirring PVA over a period of 2 minutes using a 5 ml
automatic pipetter. After 3 minutes, adjust the stir speed to 2500
rpm (+/-200 rpm) and leave the assembly for 2.5 hours. After 2.5
hours, remove the stirring blade from the nanospray preparation and
rinse with 10 ml of distilled water. Allow the rinse solution to go
into the nanospray preparation.
[0297] Pour the microsphere preparation into a 500 ml beaker. Wash
the jacketed water bath with 70 ml of distilled water. Allow the 70
ml rinse solution to go into the microsphere preparation. Stir the
180 ml microsphere preparation with a glass rod and pour equal
amounts of it into four polypropylene 50 ml centrifuge tubes. Cap
the tubes. Centrifuge the capped tubes at 10 000 g (+/-1000 g) for
10 minutes. Using a 5 ml automatic pipetter or vacuum suction, draw
45 ml of the PVA solution off of each microsphere pellet and
discard it. Add 5 ml of distilled water to each centrifuge tube and
use a vortex to resuspend the microspheres in each tube. Using 20
ml of distilled water, pool the four microsphere suspensions into
one centrifuge tube. To wash the microspheres, centrifuge the
nanospray preparation for 10 minutes at 10 000 g (+/-1000 g). Draw
the supernatant off of the microsphere pellet. Add 40 ml of
distilled water and use a vortex to resuspend the microspheres.
Repeat this process two more times for a total of three washes. Do
a fourth wash but use only 10 ml (not 40 ml) of distilled water
when resuspending the microspheres. After the fourth wash, transfer
the microsphere preparation into a preweighed glass scintillation
vial.
[0298] Cap the vial and let it to sit for 1 hour at room
temperature (25.degree. C.) to allow the 2 .mu.m and 3 .mu.m
diameter microspheres to sediment out under gravity. After 1 hour,
draw off the top 9 ml of suspension using a 5 ml automatic
pipetter. Place the 9 ml into a sterile capped 50 ml centrifuge
tube. Centrifuge the suspension at 10 000 g (+/-1000 g) for 10
minutes. Discard the supernatant and resuspend the pellet in 20 ml
of sterile saline. Centrifuge the suspension at 10 000 g (+/-1000
g) for 10 minutes. Discard the supernatant and resuspend the pellet
in sterile saline. The quantity of saline used is dependent on the
final required suspension concentration (usually 10% w/v).
Thoroughly rinse the aerosol apparatus in sterile saline and add
the nanospray suspension to the aerosol.
[0299] C. Manufacture of Paclitaxel Loaded Nanospray
[0300] To manufacture nanospray containing paclitaxel, use
Paclitaxel (Sigma grade 95% purity). To prepare the polymer drug
stock solution, weigh the appropriate amount of paclitaxel directly
into a 20 ml glass scintillation vial. The appropriate amount is
determined based on the percentage of paclitaxel to be in the
nanospray.
[0301] For example, if nanospray containing 5% paclitaxel was
required, then the amount of paclitaxel weighed would be 25 mg
since the amount of polymer added is 10 ml of a 5% polymer in DCM
solution (see next step).
[0302] Add 10 ml of the appropriate 5% polymer solution to the vial
containing the paclitaxel. Cap the vial and vortex or hand swirl it
to dissolve the paclitaxel (visual check to ensure paclitaxel
dissolved). Label the vial with the date it was produced. This is
to be used the day it is produced.
[0303] Follow the procedures as described above, except that
polymer/drug (e.g., paclitaxel) stock solution is substituted for
the polymer solution.
[0304] D. Procedure for Producing Film
[0305] The term film refers to a polymer formed into one of many
geometric shapes. The film may be a thin, elastic sheet of polymer
or a 2 mm thick disc of polymer. This film is designed to be placed
on exposed tissue so that any encapsulated drug is released from
the polymer over a long period of time at the tissue site. Films
may be made by several processes, including for example, by
casting, and by spraying.
[0306] In the casting technique, polymer is either melted and
poured into a shape or dissolved in dichloromethane and poured into
a shape. The polymer then either solidifies as it cools or
solidifies as the solvent evaporates, respectively. In the spraying
technique, the polymer is dissolved in solvent and sprayed onto
glass, as the solvent evaporates the polymer solidifies on the
glass. Repeated spraying enables a build up of polymer into a film
that can be peeled from the glass.
[0307] Reagents and equipment which were utilized within these
experiments include a small beaker, Corning hot plate stirrer,
casting moulds (e.g., 50 ml centrifuge tube caps) and mould holding
apparatus, 20 ml glass scintillation vial with cap (Plastic insert
type), TLC atomizer, Nitrogen gas tank, Polycaprolactone
("PCL"--mol wt 10,000 to 20,000; Polysciences), Paclitaxel (Sigma
95% purity), Ethanol, "washed" (see previous) Ethylene vinyl
acetate ("EVA"), Poly(DL)lactic acid ("PLA"--mol wt 15,000 to
25,000; Polysciences), Dichloromethane (HPLC grade Fisher
Scientific).
[0308] 1. Procedure for Producing Films--Melt Casting
[0309] Weigh a known weight of PCL directly into a small glass
beaker. Place the beaker in a larger beaker containing water (to
act as a water bath) and put it on the hot plate at 70.degree. C.
for 15 minutes or until the polymer has fully melted. Add a known
weight of drug to the melted polymer and stir the mixture
thoroughly. To aid dispersion of the drug in the melted PCL, the
drug may be suspended/dissolved in a small volume (<10% of the
volume of the melted PCL) of 100% ethanol. This ethanol suspension
is then mixed into the melted polymer. Pour the melted polymer into
a mould and let it to cool. After cooling, store the film in a
container.
[0310] 2. Procedure for Producing Films--Solvent Casting
[0311] Weigh a known weight of PCL directly into a 20 ml glass
scintillation vial and add sufficient DCM to achieve a 10% w/v
solution. Cap the vial and mix the solution. Add sufficient
paclitaxel to the solution to achieve the desired final paclitaxel
concentration. Use hand shaking or vortexing to dissolve the
paclitaxel in the solution. Let the solution sit for one hour (to
diminish the presence of air bubbles) and then pour it slowly into
a mould. The mould used is based on the shape required. Place the
mould in the fume hood overnight. This will allow the DCM to
evaporate. Either leave the film in the mould to store it or peel
it out and store it in a sealed container.
[0312] 3. Procedure for Producing Films--Sprayed
[0313] Weigh sufficient polymer directly into a 20 ml glass
scintillation vial and add sufficient DCM to achieve a 2% w/v
solution. Cap the vial and mix the solution to dissolve the polymer
(hand shaking). Assemble the moulds in a vertical orientation in a
suitable mould holding apparatus in the fume hood. Position this
mould holding apparatus 6 to 12 inches above the fume hood floor on
a suitable support (e.g., inverted 2000 ml glass beaker) to enable
horizontal spraying. Using an automatic pipette, transfer a
suitable volume (minimum 5 ml) of the 2% polymer solution to a
separate 20 ml glass scintillation vial. Add sufficient paclitaxel
to the solution and dissolve it by hand shaking the capped vial. To
prepare for spraying, remove the cap of this vial and dip the
barrel (only) of an TLC atomizer into the polymer solution. Note:
the reservoir of the atomizer is not used in this procedure--the 20
ml glass vial acts as a reservoir.
[0314] Connect the nitrogen tank to the gas inlet of the atomizer.
Gradually increase the pressure until atomization and spraying
begins. Note the pressure and use this pressure throughout the
procedure. To spray the moulds use 5 second oscillating sprays with
a 15 second dry time between sprays. During the dry time, finger
crimp the gas line to avoid wastage of the spray. Spraying is
continued until a suitable thickness of polymer is deposited on the
mould. The thickness is based on the request. Leave the sprayed
films attached to the moulds and store in sealed containers.
[0315] E. Procedure for Producing Nanopaste
[0316] Nanopaste is a suspension of microspheres suspended in a
hydrophilic gel. Within one aspect of the invention, the gel or
paste can be smeared over tissue as a method of locating drug
loaded microspheres close to the target tissue. Being water based,
the paste will soon become diluted with bodily fluids causing a
decrease in the stickiness of the paste and a tendency of the
microspheres to be deposited on nearby tissue. A pool of
microsphere encapsulated drug is thereby located close to the
target tissue.
[0317] Reagents and equipment which were utilized within these
experiments include glass beakers, Carbopol 925 (pharmaceutical
grade, Goodyear Chemical Co.), distilled water, sodium hydroxide (1
M) in water solution, sodium hydroxide solution (5 M) in water
solution, microspheres in the 0.1 lm to 3 lm size range suspended
in water at 20% w/v (See previous).
[0318] 1. Preparation of 5% w/v Carbopol Gel
[0319] Add a sufficient amount of carbopol to 1 M sodium hydroxide
to achieve a 5% w/v solution. To dissolve the carbopol in the 1 M
sodium hydroxide, allow the mixture to sit for approximately one
hour. During this time period, stir the 35 mixture using a glass
rod. After one hour, take the pH of the mixture. A low pH indicates
that the carbopol is not fully dissolved. The pH you want to
achieve is 7.4. Use 5 M sodium hydroxide to adjust the pH. This is
accomplished by slowly adding drops of 5 M sodium hydroxide to the
mixture, stirring the mixture and taking the pH of the mixture. It
usually takes approximately one hour to adjust the pH to 7.4. Once
a pH of 7.4 is achieved, cover the gel and let it sit for 2 to 3
hours. After this time period, check the pH to ensure it is still
at 7.4. If it has changed, adjust back to pH 7.4 using 5 M sodium
hydroxide. Allow the gel to sit for a few hours to ensure the pH is
stable at 7.4. Repeat the process until the desired pH is achieved
and is stable. Label the container with the name of the gel and the
date. The gel is to be used to make nanopaste within the next
week.
[0320] 2. Procedure for Producing Nanopaste
[0321] Add sufficient 0.1 .mu.m to 3 .mu.m microspheres to water to
produce a 20% suspension of the microspheres. Put 8 ml of the 5%
w/v carbopol gel in a glass beaker. Add 2 ml of the 20% microsphere
suspension to the beaker. Using a glass rod or a mixing spatula,
stir the mixture to thoroughly disperse the microspheres throughout
the gel. This usually takes 30 minutes. Once the microspheres are
dispersed in the gel, place the mixture in a storage jar. Store the
jar at 4.degree. C. It must be used within a one month period.
Example 11
Controlled Delivery of Paclitaxel from Microspheres Composed of a
Blend of Ethylene-Vinyl-Acetate Copolymer and Poly (D,L Lactic
Acid). In Vivo Testing of the Microspheres on the Cam Assay
[0322] This example describes the preparation of paclitaxel-loaded
microspheres composed of a blend of biodegradable poly (d,l-lactic
acid) (PLA) polymer and nondegradable ethylene-vinyl acetate (EVA)
copolymer. In addition, the in vitro release rate and
anti-angiogenic activity of paclitaxel released from microspheres
placed on a CAM are demonstrated.
[0323] Reagents which were utilized in these experiments include
paclitaxel, which is purchased from Sigma Chemical Co. (St. Louis,
Mo.); PLA (molecular weight 15,000-25,000) and EVA (60% vinyl
acetate) (purchased from Polysciences (Warrington, Pa.); polyvinyl
alcohol (PVA) (molecular weight 124,000-186,000, 99% hydrolysed,
purchased from Aldrich Chemical Co. (Milwaukee, Wis.)) and
Dichloromethane (DCM) (HPLC grade, obtained from Fisher Scientific
Co).
[0324] Distilled water is used throughout.
[0325] A. Preparation of Microspheres
[0326] Microspheres are prepared essentially as described in
Example 8 utilizing the solvent evaporation method. Briefly, 5% w/v
polymer solutions in 20 mL DCM are prepared using blends of EVA:PLA
between 35:65 to 90:10. To 5 mL of 2.5% w/v PVA in water in a 20 mL
glass vial is added 1 mL of the polymer solution dropwise with
stirring. Six similar vials are assembled in a six position
overhead stirrer, dissolution testing apparatus (Vanderkamp) and
stirred at 200 rpm. The temperature of the vials is increased from
room temperature to 40.degree. C. over 15 min and held at
40.degree. C. for 2 hours. Vials are centrifuged at 500.times.g and
the microspheres washed three times in water. At some EVA:PLA
polymer blends, the microsphere samples aggregated during the
washing stage due to the removal of the dispersing or emulsifying
agent, PVA. This aggregation effect could be analyzed
semi-quantitatively since aggregated microspheres fused and the
fused polymer mass floated on the surface of the wash water. This
surface polymer layer is discarded during the wash treatments and
the remaining, pelleted microspheres are weighed. The % aggregation
is determined from % .times. .times. aggregation = 1 - ( weight
.times. .times. of .times. .times. pelleted .times. .times.
microspheres ) .times. 100 initial .times. .times. polymer .times.
.times. weight ##EQU1##
[0327] Paclitaxel loaded microspheres (0.6% w/w paclitaxel) are
prepared by dissolving the paclitaxel in the 5% w/v polymer
solution in DCM. The polymer blend used is 50:50 EVA:PLA. A "large"
size fraction and "small" size fraction of microspheres are
produced by adding the paclitaxel/polymer solution dropwise into
2.5% w/v PVA and 5% w/v PVA, respectively. The dispersions are
stirred at 40.degree. C. at 200 rpm for 2 hours, centrifuged and
washed 3 times in water as described previously. Microspheres are
air dried and samples are sized using an optical microscope with a
stage micrometer. Over 300 microspheres are counted per sample.
Control microspheres (paclitaxel absent) are prepared and sized as
described previously.
[0328] B. Encapsulation Efficiency
[0329] Known weights of paclitaxel-loaded microspheres are
dissolved in 1 mL DCM, 20 mL of 40% acetonitrile in water at
50.degree. C. are added and vortexed until the DCM had been
evaporated. The concentration of paclitaxel in the 40% acetonitrile
is determined by HPLC using a mobile phase of
water:methanol:acetonitrile (37:5:58) at a flow rate of 1 mL/min
(Beckman isocratic pump), a C8 reverse phase column (Beckman) and
UV detection at 232 nm. To determine the recovery efficiency of
this extraction procedure, known weights of paclitaxel from
100-1000 .mu.g are dissolved in 1 mL of DCM and subjected to the
same extraction procedure in triplicate as described previously.
Recoveries are always greater than 85% and the values of
encapsulation efficiency are corrected appropriately.
[0330] C. Drug Release Studies
[0331] In 15 mL glass, screw capped tubes are placed 10 mL of 10 mM
phosphate buffered saline (PBS), pH 7.4 and 35 mg paclitaxel-loaded
microspheres. The tubes are tumbled at 37.degree. C. and at given
time intervals, centrifuged at 1500.times.g for 5 min and the
supernatant saved for analysis. Microsphere pellets are resuspended
in fresh PBS (10 mL) at 37.degree. C. and reincubated. Paclitaxel
concentrations are determined by extraction into 1 mL DCM followed
by evaporation to dryness under a stream of nitrogen,
reconstitution in 1 mL of 40% acetonitrile in water and analysis
using HPLC as previously described.
[0332] D. Scanning Electron Microscopy (SEM)
[0333] Microspheres are placed on sample holders, sputter coated
with gold and micrographs obtained using a Philips 501B SEM
operating at 15 kV.
[0334] E. CAM Studies
[0335] Fertilized, domestic chick embryos are incubated for 4 days
prior to shell-less culturing. The egg contents are incubated at
90% relative humidity and 3% CO.sub.2 for 2 days. On day 6 of
incubation, 1 mg aliquots of 0.6% paclitaxel loaded or control
(paclitaxel free) microspheres are placed directly on the CAM
surface. After a 2 day exposure the vasculature is examined using a
stereomicroscope interfaced with a video camera; the video signals
are then displayed on a computer and video printed.
[0336] F. Results
[0337] Microspheres prepared from 100% EVA are freely suspended in
solutions of PVA but aggregated and coalesced or fused extensively
on subsequent washing in water to remove the PVA. Blending EVA with
an increasing proportion of PLA produced microspheres showing a
decreased tendency to aggregate and coalesce when washed in water,
as described in FIG. 15A. A 50:50 blend of EVA:PLA formed
microspheres with good physical stability, that is the microspheres
remained discrete and well suspended with negligible aggregation
and coalescence.
[0338] The size range for the "small" size fraction microspheres is
determined to be >95% of the microsphere sample (by weight)
between 10-30 mm and for the "large" size fraction, >95% of the
sample (by weight) between 30-100 mm. Representative scanning
electron micrographs of paclitaxel loaded 50:50 EVA:PLA
microspheres in the "small" and "large" size ranges are shown in
FIGS. 15B and 15C, respectively. The microspheres are spherical
with a smooth surface and with no evidence of solid drug on the
surface of the microspheres. The efficiency of loading 50:50
EVA:PLA microspheres with paclitaxel is between 95-100% at initial
paclitaxel concentrations of between 100-1000 mg paclitaxel per 50
mg polymer. There is no significant difference (Student t-test,
p<0.05) between the encapsulation efficiencies for either
"small" or "large" microspheres.
[0339] The time course of paclitaxel release from 0.6% w/v loaded
50:50 EVA:PLA microspheres is shown in FIG. 15D for "small" size
(open circles) and "large" size (closed circles) microspheres. The
release rate studies are carried out in triplicate tubes in 3
separate experiments. The release profiles are biphasic with an
initial rapid release of paclitaxel or "burst" phase occurring over
the first 4 days from both size range microspheres. This is
followed by a phase of much slower release. There is no significant
difference between the release rates from "small" or "large"
microspheres. Between 10-13% of the total paclitaxel content of the
microspheres is released in 50 days.
[0340] The paclitaxel loaded microspheres (0.6% w/v loading) are
tested using the CAM assay and the results are shown in FIG. 15E.
The paclitaxel microspheres released sufficient drug to produce a
zone of avascularity in the surrounding tissue (FIG. 15F). Note
that immediately adjacent to the microspheres ("MS" in FIGS. 15E
and 15F) is an area in which blood vessels are completely absent
(Zone 1); further from the microspheres is an area of disrupted,
non-functioning capillaries (Zone 2); it is only at a distance of
approximately 6 mm from the microspheres that the capillaries
return to normal. In CAMs treated with control microspheres
(paclitaxel absent) there is a normal capillary network
architecture (figure not shown.)
Discussion
[0341] Arterial chemoembolization is an invasive surgical
technique. Therefore, ideally, a chemoembolic formulation of an
anti-angiogenic drug such as paclitaxel would release the drug at
the tumor site at concentrations sufficient for activity for a
prolonged period of time, of the order of several months. EVA is a
tissue compatible nondegradable polymer which has been used
extensively for the controlled delivery of macromolecules over long
time periods (>100 days).
[0342] EVA is initially selected as a polymeric biomaterial for
preparing microspheres with paclitaxel dispersed in the polymer
matrix. However, microspheres prepared with 100% EVA aggregated and
coalesced almost completely during the washing procedure.
[0343] Polymers and copolymers based on lactic acid and glycolic
acid are physiologically inert and biocompatible and degrade by
hydrolysis to toxicologically acceptable products. Copolymers of
lactic acid and glycolic acids have faster degradation rates than
PLA and drug loaded microspheres prepared using these copolymers
are unsuitable for prolonged, controlled release over several
months. Dollinger and Sawan blended PLA with EVA and showed that
the degradation lifetime of PLA is increased as the proportion of
EVA in the blend is increased. They suggested that blends of EVA
and PLA should provide a polymer matrix with better mechanical
stability and control of drug release rates than PLA.
[0344] FIG. 15A shows that increasing the proportion of PLA in a
EVA:PLA blend decreased the extent of aggregation of the
microsphere suspensions. Blends of 50% or less EVA in the EVA:PLA
matrix produced physically stable microsphere suspensions in water
or PBS. A blend of 50:50 EVA:PLA is selected for all subsequent
studies.
[0345] Different size range fractions of microspheres could be
prepared by changing the concentration of the emulsifier, PVA, in
the aqueous phase. "Small" microspheres are produced at the higher
PVA concentration of 5% w/v whereas "large" microspheres are
produced at 2.5% w/v PVA. All other production variables are the
same for both microsphere size fractions. The higher concentration
of emulsifier gave a more viscous aqueous dispersion medium and
produced smaller droplets of polymer/paclitaxel/DCM emulsified in
the aqueous phase and thus smaller microspheres. The paclitaxel
loaded microspheres contained between 95-100% of the initial
paclitaxel added to the organic phase encapsulated within the solid
microspheres. The low water solubility of paclitaxel favoured
partitioning into the organic phase containing the polymer.
[0346] Release rates of paclitaxel from the 50:50 EVA:PLA
microspheres are very slow with less than 15% of the loaded
paclitaxel being released in 50 days. The initial burst phase of
drug release may be due to diffusion of drug from the superficial
region of the microspheres (close to the microsphere surface).
[0347] The mechanism of drug release from nondegradable polymeric
matrices such as EVA is thought to involve the diffusion of water
through the dispersed drug phase within the polymer, dissolution of
the drug and diffusion of solute through a series of
interconnecting, fluid filled pores. Blends of EVA and PLA have
been shown to be immiscible or bicontinuous over a range of 30 to
70% EVA in PLA. In degradation studies in PBS buffer at 37.degree.
C., following an induction or lag period, PLA hydrolytically
degraded and eroded from the EVA:PLA polymer blend matrix leaving
an inactive sponge-like skeleton. Although the induction period and
rate of PLA degradation and erosion from the blended matrices
depended on the proportion of PLA in the matrix and on process
history, there is consistently little or no loss of PLA until after
40-50 days.
[0348] Although some erosion of PLA from the 50:50 EVA:PLA
microspheres may have occurred within the 50 days of the in vitro
release rate study (FIG. 15C), it is likely that the primary
mechanism of drug release from the polymer blend is diffusion of
solute through a pore network in the polymer matrix.
[0349] At the conclusion of the release rate study, the
microspheres are analyzed from the amount of drug remaining. The
values for the percent of paclitaxel remaining in the 50 day
incubation microsphere samples are 94%+/-9% and 89%+/-12% for
"large" and "small" size fraction microspheres, respectively.
[0350] Microspheres loaded with 6 mg per mg of polymer. (0.6%)
provided extensive inhibition of angiogenesis when placed on the
CAM of the embryonic chick (FIGS. 15E and 15F).
Example 12
Paclitaxel Encapsulation in Poly(E-Caprolactone) Microspheres.
Inhibition of Angiogenesis on the Cam Assay by Paclitaxel-Loaded
Microspheres
[0351] This example evaluates the in vitro release rate profile of
paclitaxel from biodegradable microspheres of poly(e-caprolactone)
and demonstrates the anti-angiogenic activity of paclitaxel
released from these microspheres when placed on the CAM.
[0352] Reagents which were utilized in these experiments include:
poly(e-caprolactone) ("PCL") (molecular weight 35,000-45,000;
purchased from Polysciences (Warrington, Pa.)); dichloromethane
("DCM") from Fisher Scientific Co., Canada; polyvinyl alcohol (PVP)
(molecular weight 12,00-18,000, 99% hydrolysed) from Aldrich
Chemical Co. (Milwaukee, Wis.), and paclitaxel from Sigma Chemical
Co. (St. Louis, Mo.). Unless otherwise stated all chemicals and
reagents are used as supplied. Distilled water is used
throughout.
[0353] A. Preparation of Microspheres
[0354] Microspheres are prepared essentially as described in
Example 8 utilizing the solvent evaporation method. Briefly, 5% w/w
paclitaxel loaded microspheres are prepared by dissolving 10 mg of
paclitaxel and 190 mg of PCL in 2 ml of DCM, adding to 100 ml of 1%
PVP aqueous solution and stirring at 1000 rpm at 25.degree. C. for
2 hours. The suspension of microspheres is centrifuged at
1000.times.g for 10 minutes (Beckman GPR), the supernatant removed
and the microspheres washed three times with water. The washed
microspheres are air-dried overnight and stored at room
temperature. Control microspheres (paclitaxel absent) are prepared
as described above. Microspheres containing 1% and 2% paclitaxel
are also prepared. Microspheres are sized using an optical
microscope with a stage micrometer.
[0355] B. Encapsulation Efficiency
[0356] A known weight of drug-loaded microspheres (about 5 mg) is
dissolved in 8 ml of acetonitrile and 2 ml distilled water is added
to precipitate the polymer. The mixture is centrifuged at 1000 g
for 10 minutes and the amount of paclitaxel encapsulated is
calculated from the absorbance of the supernatant measured in a UV
spectrophotometer (Hewlett-Packard 8452A Diode Array
Spectrophotometer) at 232 nm.
[0357] C. Drug Release Studies
[0358] About 10 mg of paclitaxel-loaded microspheres are suspended
in 20 ml of 10 mM phosphate buffered saline, pH 7.4 (PBS) in
screw-capped tubes. The tubes are tumbled end-over-end at
37.degree. C. and at given time intervals 19.5 ml of supernatant is
removed (after allowing the microspheres to settle at the bottom),
filtered through a 0.45 um membrane filter and retained for
paclitaxel analysis. An equal volume of PBS is replaced in each
tube to maintain sink conditions throughout the study. The
filtrates are extracted with 3.times.1 ml DCM, the DCM extracts
evaporated to dryness under a stream of nitrogen, redissolved in 1
ml acetonitrile and analyzed by HPLC using a mobile phase of
water:methanol:acetonitrile (37:5:58) at a flow rate of 1 ml
min.sup.-1 (Beckman Isocratic Pump), a C8 reverse phase column
(Beckman), and UV detection (Shimadzu SPD A) at 232 nm.
[0359] D. CAM Studies
[0360] Fertilized, domestic chick embryos are incubated for 4 days
prior to shell-less culturing. On day 6 of incubation, 1 mg
aliquots of 5% paclitaxel-loaded or control (paclitaxel-free)
microspheres are placed directly on the CAM surface. After a 2-day
exposure the vasculature is examined using a stereomicroscope
interfaced with a video camera; the video signals are then
displayed on a computer and video printed.
[0361] E. Scanning Electron Microscopy
[0362] Microspheres are placed on sample holders, sputter-coated
with gold and then placed in a Philips 501B Scanning Electron
Microscope operating at 15 kV.
[0363] F. Results
[0364] The size range for the microsphere samples is between 30-100
um, although there is evidence in all paclitaxel-loaded or control
microsphere batches of some microspheres falling outside this
range. The efficiency of loading PCL microspheres with paclitaxel
is always greater than 95% for all drug loadings studied. Scanning
electron microscopy demonstrated that the microspheres are all
spherical and many showed a rough or pitted surface morphology.
There appeared to be no evidence of solid drug on the surface of
the microspheres.
[0365] The time courses of paclitaxel release from 1%, 2% and 5%
loaded PCL microspheres are shown in FIG. 16A. The release rate
profiles are bi-phasic. There is an initial rapid release of
paclitaxel or "burst phase" at all drug loadings. The burst phase
occurred over 1-2 days at 1% and 2% paclitaxel loading and over 3-4
days for 5% loaded microspheres. The initial phase of rapid release
is followed by a phase of significantly slower drug release. For
microspheres containing 1% or 2% paclitaxel there is no further
drug release after 21 days. At 5% paclitaxel loading, the
microspheres had released about 20% of the total drug content after
21 days.
[0366] FIG. 16B shows CAMs treated with control PCL microspheres,
and FIG. 16C shows treatment with 5% paclitaxel loaded
microspheres. The CAM with the control microspheres shows a normal
capillary network architecture. The CAM treated with paclitaxel-PCL
microspheres shows marked vascular regression and zones which are
devoid of a capillary network.
[0367] G. Discussion
[0368] The solvent evaporation method of manufacturing
paclitaxel-loaded microspheres produced very high paclitaxel
encapsulation efficiencies of between 95-100%. This is due to the
poor water solubility of paclitaxel and its hydrophobic nature
favouring partitioning in the organic solvent phase containing the
polymer.
[0369] The biphasic release profile for paclitaxel is typical of
the release pattern for many drugs from biodegradable polymer
matrices. Poly(e-caprolactone) is an aliphatic polyester which can
be degraded by hydrolysis under physiological conditions and it is
non-toxic and tissue compatible. The degradation of PCL is
significantly slower than that of the extensively investigated
polymers and copolymers of lactic and glycolic acids and is
therefore suitable for the design of long-term drug delivery
systems. The initial rapid or burst phase of paclitaxel release is
thought to be due to diffusional release of the drug from the
superficial region of the microspheres (close to the microsphere
surface). Release of paclitaxel in the second (slower) phase of the
release profiles is not likely due to degradation or erosion of PCL
because studies have shown that under in vitro conditions in water
there is no significant weight loss or surface erosion of PCL over
a 7.5-week period. The slower phase of paclitaxel release is
probably due to dissolution of the drug within fluid-filled pores
in the polymer matrix and diffusion through the pores. The greater
release rate at higher paclitaxel loading is probably a result of a
more extensive pore network within the polymer matrix.
[0370] Paclitaxel microspheres with 5% loading have been shown to
release sufficient drug to produce extensive inhibition of
angiogenesis when placed on the CAM. The inhibition of blood vessel
growth resulted in an avascular zone as shown in FIG. 16C.
Example 13
Paclitaxel-Loaded Polymeric Films Composed of Ethylene Vinyl
Acetate and a Surfactant
[0371] Two types of films are investigated within this example:
pure EVA films loaded with paclitaxel and EVA/surfactant blend
films loaded with paclitaxel.
[0372] The surfactants being examined are two hydrophobic
surfactants (Span 80 and Pluronic L101) and one hydrophilic
surfactant (Pluronic F127). The pluronic surfactants are themselves
polymers, which is an attractive property since they can be blended
with EVA to optimize various drug delivery properties. Span 80 is a
smaller molecule which is in some manner dispersed in the polymer
matrix, and does not form a blend.
[0373] Surfactants is useful in modulating the release rates of
paclitaxel from films and optimizing certain physical parameters of
the films. One aspect of the surfactant blend films which indicates
that drug release rates can be controlled is the ability to vary
the rate and extent to which the compound will swell in water.
Diffusion of water into a polymer-drug matrix is critical to the
release of drug from the carrier. FIGS. 17C and 17D show the degree
of swelling of the films as the level of surfactant in the blend is
altered. Pure EVA films do not swell to any significant extent in
over 2 months. However, by increasing the level of surfactant added
to the EVA it is possible to increase the degree of swelling of the
compound, and by increasing hydrophilicity swelling can also be
increased.
[0374] Results of experiments with these films are shown below in
FIGS. 17A-E. Briefly, FIG. 17A shows paclitaxel release (in mg)
over time from pure EVA films. FIG. 17B shows the percentage of
drug remaining for the same films. As can be seen from these two
figures, as paclitaxel loading increases (i.e., percentage of
paclitaxel by weight is increased), drug release rates increase,
showing the expected concentration dependence. As paclitaxel
loading is increased, the percent paclitaxel remaining in the film
also increases, indicating that higher loading may be more
attractive for long-term release formulations.
[0375] Physical strength and elasticity of the films is assessed in
FIG. 17E. Briefly, FIG. 17E shows stress/strain curves for pure EVA
and EVA-Surfactant blend films. This crude measurement of stress
demonstrates that the elasticity of films is increased with the
addition of Pluronic F127, and that the tensile strength (stress on
breaking) is increased in a concentration dependent manner with the
addition of Pluronic F127. Elasticity and strength are important
considerations in designing a film which can be manipulated for
particular clinical applications without causing permanent
deformation of the compound.
[0376] The above data demonstrates the ability of certain
surfactant additives to control drug release rates and to alter the
physical characteristics of the vehicle.
Example 14
Incorporating Methoxypolyethylene Glycol 350 (MePEG) into
Poly(e-Caprolactone) to Develop a Formulation for the Controlled
Delivery of Paclitaxel from a Paste
[0377] Reagents and equipment which were utilized within these
experiments include methoxypolyethylene glycol 350 ("MePEG"--Union
Carbide, Danbury, Conn.). MePEG is liquid at room temperature, and
has a freezing point of 10.degree. to -5.degree. C.
[0378] A. Preparation of a MePEG/PCL Paclitaxel-Containing Paste
MePEG/PCL paste is prepared by first dissolving a quantity of
paclitaxel into MePEG, and then incorporating this into melted PCL.
One advantage with this method is that no DCM is required.
[0379] B. Analysis of Melting Point
[0380] The melting point of PCL/MePEG polymer blends may be
determined by differential scanning calorimetry from 30.degree. C.
to 70.degree. C. at a heating rate of 2.5.degree. C. per minute.
Results of this experiment are shown in FIGS. 18A and 18B. Briefly,
as shown in FIG. 18A the melting point of the polymer blend (as
determined by thermal analysis) is decreased by MePEG in a
concentration dependent manner. The melting point of the polymer
blends as a function of MePEG concentration is shown in FIG. 18A.
This lower melting point also translates into an increased time for
the polymer blends to solidify from melt as shown in FIG. 18B. A
30:70 blend of MePEG:PCL takes more than twice as long to solidify
from the fluid melt than does PCL alone.
[0381] C. Measurement of Brittleness
[0382] Incorporation of MePEG into PCL appears to produce a less
brittle solid, as compared to PCL alone. As a "rough" way of
quantitating this, a weighted needle is dropped from an equal
height into polymer blends containing from 0% to 30% MePEG in PCL,
and the distance that the needle penetrates into the solid is then
measured. The resulting graph is shown as FIG. 18C. Points are
given as the average of four measurements +/-1 S.D.
[0383] For purposes of comparison, a sample of paraffin wax is also
tested and the needle penetrated into this a distance of 7.25
mm+/0.3 mm.
[0384] D. Measurement of Paclitaxel Release
[0385] Pellets of polymer (PCL containing 0%, 5%, 10% or 20% MePEG)
are incubated in phosphate buffered saline (PBS, pH 7.4) at
37.degree. C., and % change in polymer weight is measured over
time. As can be seen in FIG. 18D, the amount of weight lost
increases with the concentration of MePEG originally present in the
blend. It is likely that this weight loss is due to the release of
MePEG from the polymer matrix into the incubating fluid. This would
indicate that paclitaxel will readily be released from a MePEG/PCL
blend since paclitaxel is first dissolved in MePEG before
incorporation into PCL.
[0386] E. Effect of Varying Quantities of MePEG on Paclitaxel
Release
[0387] Thermopastes are made up containing between 0.8% and 20%
MePEG in PCL. These are loaded with 1% paclitaxel. The release of
paclitaxel over time from 10 mg pellets in PBS buffer at 37.degree.
C. is monitored using HPLC. As is shown in FIG. 18E, the amount of
MePEG in the formulation does not affect the amount of paclitaxel
that is released.
[0388] F. Effect of Varying Quantities of Paclitaxel on the Total
Amount of Paclitaxel Released From a 20% MePEG/PCL Blend
[0389] Thermopastes are made up containing 20% MePEG in PCL and
loaded with between 0.2% and 10% paclitaxel. The release of
paclitaxel over time is measured as described above. As shown in
FIG. 18F, the amount of paclitaxel released over time increases
with increased paclitaxel loading. When plotted as the percent
total paclitaxel released, however, the order is reversed (FIG.
18G). This gives information about the residual paclitaxel
remaining in the paste and allows for a projection of the period of
time over which paclitaxel may be released from the 20% MePEG
Thermopaste.
[0390] G. Strength Analysis of Various MePEG/PCL Blends
[0391] A CT-40 mechanical strength tester is used to measure the
strength of solid polymer "tablets" of diameter 0.88 cm and an
average thickness of 0.560 cm. The polymer tablets are blends of
MePEG at concentrations of 0%, 5%, 10% or 20% in PCL.
[0392] Results of this test are shown in FIG. 18H, where both the
tensile strength and the time to failure are plotted as a function
of % MePEG in the blend. Single variable ANOVA indicated that the
tablet thicknesses within each group are not different. As can be
seen from FIG. 18H, the addition of MePEG into PCL decreased the
hardness of the resulting solid.
EXAMPLE 15
Effect of Paclitaxel-Loaded Thermopaste on Angiogenesis In Vivo
[0393] Fertilized, domestic chick embryos were incubated for 4 days
prior to shell-less culturing as described in Example 2. The egg
contents are removed from the shell and emptied into roundbottom
sterilized glass bowls and covered with petri dish covers.
[0394] Paclitaxel is incorporated into thermopaste at
concentrations of 0.05%, 0.1%, 0.25%, 0.5%, 1.0%, 5%, 10%, and 20%
(w/v) essentially as described above (see Example 10), and used in
the following experiments on the CAM. Dried thermopaste disks
weighing 3 mg were made by heating the paste to 60.degree. C.,
forming drop size aliquots, and then allowing it to cool.
[0395] In addition, unloaded thermopaste and thermopaste containing
20% paclitaxel were also heated to 60.degree. C. and placed
directly on the growing edge of each CAM at day 6 of incubation;
two animals each were treated in this manner. There was no
observable difference in the results obtained using the different
methods of administration indicating that the temperature of the
paste at the time of application was not a factor in the
outcome.
[0396] Each concentration of paclitaxel-loaded thermopaste (0.05%,
0.1%, 0.25%, 0.5%, 1.0%, 5%, 10%, and 20%) was tested-on 4 to 9
embryos at day 6 of development (see Table III). After a 2 day
exposure (day 8 of incubation) the vasculature was examined with
the aid of a stereomicroscope. Liposyn II, a white opaque solution,
was injected into the CAM which increases the visibility of the
vascular details.
[0397] The 20% paclitaxel-loaded thermopaste induced an extensive
area of avascularity (see FIG. 19B) in all 6 of the CAMs receiving
this treatment. The highest degree of inhibition was defined as a
region of avascularity covering an area of 6 mm in diameter. All of
the CAMs treated with 20% paclitaxel-loaded thermopaste displayed
this degree of angiogenesis inhibition.
[0398] In the animals treated with 5% paclitaxel-loaded paste, 4
animals demonstrated maximum inhibition of angiogenesis. Of the
animals treated with 10% paclitaxel-loaded thermopaste, only 5
illustrated maximal inhibition.
[0399] The results of this study also show that paclitaxel-loaded
thermopaste, as low as 0.25%, can release a significant amount of
drug to induce angiogenesis inhibition on the CAM. (Table IV, FIGS.
19C and 19D).
[0400] By comparison, the control (unloaded) thermopaste did not
inhibit angiogenesis on the CAM (see FIG. 19A); this higher
magnification view (note that the edge of the paste is seen at the
top of the image) demonstrates that the vessels adjacent to the
paste are unaffected by the thermopaste. This suggests that the
avascular effect observed is due to the sustained release of
paclitaxel and is not due to the polymer itself or due to a
secondary pressure effect of the paste on the developing
vasculature.
[0401] This study also demonstrates that thermopaste releases
sufficient quantities of angiogenesis inhibitor (in this case
paclitaxel) to inhibit the normal development of the CAM
vasculature. TABLE-US-00005 TABLE IV Angiogenic Inhibition of
Paclitaxel-Loaded Thermopaste Paclitaxel-loaded Thermopaste (%)
Embryos Evaluated (positive/n) 0.05 0/9 0.1 1/8 0.25 4/4 0.5 4/4 1
8/8 5 4/4 10 5/5 20 6/6 0 (control) 0/30
Example 16
Effect of Paclitaxel-Loaded Thermopaste on Tumor Growth and Tumor
Angiogenesis In Vivo
[0402] Fertilized domestic chick embryos are incubated for 3 days
prior to having their shells removed. The egg contents are emptied
by removing the shell located around the airspace, severing the
interior shell membrane, perforating the opposite end of the shell
and allowing the egg contents to gently slide out from the blunted
end. The contents are emptied into round-bottom sterilized glass
bowls, covered with petri dish covers and incubated at 90% relative
humidity and 3% carbon dioxide (see Example 2).
[0403] MDAY-D2 cells (a murine lymphoid tumor) is injected into
mice and allowed to grow into tumors weighing 0.5-1.0 g. The mice
are sacrificed, the tumor sites wiped with alcohol, excised, placed
in sterile tissue culture media, and diced into 1 mm pieces under a
laminar flow hood. Prior to placing the dissected tumors onto the
9-day old chick embryos, CAM surfaces are gently scraped with a 30
gauge needle to insure tumor implantation. The tumors are then
placed on the CAMs after 8 days of incubation (4 days after
deshelling), and allowed to grow on the CAM for four days to
establish a vascular supply. Four embryos are prepared utilizing
this method, each embryo receiving 3 tumors. For these embryos, one
tumor receives 20% paclitaxel-loaded thermopaste, the second tumor
unloaded thermopaste, and the third tumor no treatment. The
treatments are continued for two days before the results were
recorded.
[0404] The explanted MDAY-D2 tumors secrete angiogenic factors
which induce the ingrowth of capillaries (derived from the CAM)
into the tumor mass and allow it to continue to grow in size. Since
all the vessels of the tumor are derived from the CAM, while all
the tumor cells are derived from the explant, it is possible to
assess the effect of therapeutic interventions on these two
processes independently. This assay has been used to determine the
effectiveness of paclitaxel-loaded thermopaste on: (a) inhibiting
the vascularization of the tumor and (b) inhibiting the growth of
the tumor cells themselves.
[0405] Direct in vivo stereomicroscopic evaluation and histological
examination of fixed tissues from this study demonstrated the
following. In the tumors treated with 20% paclitaxel-loaded
thermopaste, there was a reduction in the number of the blood
vessels which supplied the tumor (see FIGS. 20C and 20D), a
reduction in the number of blood vessels within the tumor, and a
reduction in the number of blood vessels in the periphery of the
tumor (the area which is typically the most highly vascularized in
a solid tumor) when compared to control tumors. The tumors began to
decrease in size and mass during the two days the study was
conducted. Additionally, numerous endothelial cells were seen to be
arrested in cell division indicating that endothelial cell
proliferation had been affected. Tumor cells were also frequently
seen arrested in mitosis. All 4 embryos showed a consistent pattern
with the 20% paclitaxel-loaded thermopaste suppressing tumor
vascularity while the unloaded thermopaste had no effect.
[0406] By comparison, in CAMs treated with unloaded thermopaste,
the tumors were well vascularized with an increase in the number
and density of vessels when compared to that of the normal
surrounding tissue, and dramatically more vessels than were
observed in the tumors treated with paclitaxel-loaded paste. The
newly formed vessels entered the tumor from all angles appearing
like spokes attached to the center of a wheel (see FIGS. 20A and
20B). The control tumors continued to increase in size and mass
during the course of the study. Histologically, numerous dilated
thin-walled capillaries were seen in the periphery of the tumor and
few endothelial were seen to be in cell division. The tumor tissue
was well vascularized and viable throughout.
[0407] As an example, in two similarly-sized (initially, at the
time of explantation) tumors placed on the same CAM the following
data was obtained. For the tumor treated with 20% paclitaxel-loaded
thermopaste the tumor measured 330 mm.times.597 mm; the immediate
periphery of the tumor has 14 blood vessels, while the tumor mass
has only 3-4 small capillaries. For the tumor treated with unloaded
thermopaste the tumor size was 623 mm.times.678 mm; the immediate
periphery of the tumor has 54 blood vessels, while the tumor mass
has 12-14 small blood vessels. In addition, the surrounding CAM
itself contained many more blood vessels as compared to the area
surrounding the paclitaxel-treated tumor.
[0408] This study demonstrates that thermopaste releases sufficient
quantities of angiogenesis inhibitor (in this case paclitaxel) to
inhibit the pathological angiogenesis which accompanies tumor
growth and development. Under these conditions angiogenesis is
maximally stimulated by the tumor cells which produce angiogenic
factors capable of inducing the ingrowth of capillaries from the
surrounding tissue into the tumor mass. The 20% paclitaxel-loaded
thermopaste is capable of blocking this process and limiting the
ability of the tumor tissue to maintain an adequate blood supply.
This results in a decrease in the tumor mass both through a
cytotoxic effect of the drug on the tumor cells themselves and by
depriving the tissue of the nutrients required for growth and
expansion.
Example 17
Effect of Angiogenesis Inhibitor-Loaded Thermopaste on Tumor Growth
In Vivo in a Murine Tumor Model
[0409] The murine MDAY-D2 tumor model may be used to examine the
effect of local slow release of the chemotherapeutic and
anti-angiogenic compounds such as paclitaxel on tumor growth, tumor
metastasis, and animal survival. The MDAY-D2 tumor cell line is
grown in a cell suspension consisting of 5% Fetal Calf Serum in
alpha mem media. The cells are incubated at 37.degree. C. in a
humidified atmosphere supplemented with 5% carbon dioxide, and are
diluted by a factor of 15 every 3 days until a sufficient number of
cells are obtained. Following the incubation period the cells are
examined by light microscopy for viability and then are centrifuged
at 1500 rpm for 5 minutes. PBS is added to the cells to achieve a
dilution of 1,000,000 cells per ml.
[0410] Ten week old DBA/2j female mice are acclimatized for 3-4
days after arrival. Each mouse is then injected subcutaneously in
the posteriolateral flank with 100,000 MDAY-D2 cells in 100 ml of
PBS. Previous studies have shown that this procedure produces a
visible tumor at the injection site in 3-4 days, reach a size of
1.0-1.7 g by 14 days, and produces visible metastases in the liver
19-25 days post-injection. Depending upon the objective of the
study a therapeutic intervention can be instituted at any point in
the progression of the disease.
[0411] Using the above animal model, 20 mice are injected with
140,000 MDAY-D2 cells s.c. and the tumors allowed to grow. On day 5
the mice are divided into groups of 5. The tumor site was
surgically opened under anesthesia, the local region treated with
the drug-loaded thermopaste or control thermopaste without
disturbing the existing tumor tissue, and the wound was closed. The
groups of 5 received either no treatment (wound merely closed),
polymer (PCL) alone, 10% paclitaxel-loaded thermopaste, or 20%
paclitaxel-loaded thermopaste (only 4 animals injected) implanted
adjacent to the tumor site. On day 16, the mice were sacrificed,
the tumors were dissected and examined (grossly and histologically)
for tumor growth, tumor metastasis, local and systemic toxicity
resulting from the treatment, effect on wound healing, effect on
tumor vascularity, and condition of the paste remaining at the
incision site.
[0412] The weights of the tumors for each animal is shown in the
table below: TABLE-US-00006 TABLE V Tumor Weights (gm) Control
Control 10% Paclitaxel 20% Paclitaxel Animal No. (empty) (PCL)
Thermopaste Thermopaste 1 1.387 1.137 0.487 0.114 2 0.589 0.763
0.589 0.192 3 0.461 0.525 0.447 0.071 4 0.606 0.282 0.274 0.042 5
0.353 0.277 0.362 Mean 0.6808 0.6040 0.4318 0.1048 Std. Deviation
0.4078 0.3761 0.1202 0.0653 P Value 0.7647 0.358 0.036
Thermopaste loaded with 20% paclitaxel reduced tumor growth by over
85% (average weight 0.105) as compared to control animals (average
weight 0.681). Animals treated with thermopaste alone or
thermopaste containing 10% paclitaxel had only modest effects on
tumor growth; tumor weights were reduced by only 10% and 35%
respectively (FIG. 21A). Therefore, thermopaste containing 20%
paclitaxel was more effective in reducing tumor growth than
thermopaste containing 10% paclitaxel (see FIG. 21C; see also FIG.
21B).
[0413] Thermopaste was detected in some of the animals at the site
of administration. Polymer varying in weight between 0.026 g to
0.078 g was detected in 8 of 15 mice. Every animal in the group
containing 20% paclitaxel-loaded thermopaste contained some
residual polymer suggesting that it was less susceptible to
dissolution. Histologically, the tumors treated with
paclitaxel-loaded thermopaste contained lower cellularity and more
tissue necrosis than control tumors. The vasculature was reduced
and endothelial cells were frequently seen to be arrested in cell
division. The paclitaxel-loaded themmopaste did not appear to
affect the integrity or cellularity of the skin or tissues
surrounding the tumor. Grossly, wound healing was unaffected.
Example 18
The Use of Angiogenesis-Inhibitor Loaded Surgical Films in the
Prevention of Iatrogenic Metastatic Seeding of Tumor Cells During
Cancer Resection Surgery
[0414] As discussed above, sterile, pliable, stretchable
drug-polymer compounds (e.g., films) may be utilized in accordance
with the methods described herein in order to isolate normal
surrounding tissues from malignant tissue during resection of
cancer. Such material prevents iatrogenic spread of the disease to
adjacent organs through inadvertent contamination by cancer cells.
Such polymers may be particularly useful if placed around the liver
and/or other abdominal contents during bowel cancer resection
surgery in order to prevent intraperitoneal spread of the disease
to the liver.
[0415] A. Materials and Methods
[0416] Preparation of Surgical Film. Surgical films are prepared as
described in Example 10. Thin films measuring approximately 1
cm.times.1 cm are prepared containing either polymer alone (PCL) or
PCL loaded with 5% paclitaxel.
[0417] Rat Hepatic Tumor Model. In an initial study Wistar rats
weighing approximately 300 g underwent general anesthesia and a 3-5
cm abdominal incision is made along the midline. In the largest
hepatic lobe, a 1 cm incision is made in the hepatic parenchyma and
part of the liver edge is resected. A concentration of 1 million
live 9 L Glioma tumor cells (eluted from tissue culture immediately
prior to the procedure) suspended in 100 ml of phosphate buffered
saline are deposited onto the cut liver edge with a 30 gauge
needle. The surgical is then placed over the cut liver edge
containing the tumor cells and affixed in place with Gelfoam. Two
animals received PCL films containing 5% paclitaxel and two animals
received films containing PCL alone. The abdominal wall is closed
with 3.0 Dexon and skin clips. The general anesthetic is terminated
and the animal is allowed to recover. Ten days later the animals
are sacrificed and the livers examined histologically.
[0418] B. Results
[0419] Local tumour growth is seen in the 2 livers treated with
polymer alone. Both livers treated with polymer plus paclitaxel are
completely free of tumour when examined histologically. Also of
importance, the liver capsule had regenerated and grown completely
over the polymeric film and the cut surface of the liver indicating
that there is no significant effect on wound healing. There is no
evidence of local hepatic toxicity surrounding any (drug-loaded or
drug-free) of the surgical films.
[0420] C. Discussion
[0421] This study indicates that surgical films placed around
normal tissues and incision sites during surgery may be capable of
decreasing the incidence of accidental implantation of tumor cells
into normal surrounding tissue during resection of malignant
tumors.
Example 19
Intra-Articular Injection of Angiogenesis-Inhibitor-Loaded
Biodegradable Microspheres in the Treatment of Arthritis
[0422] Articular damage in arthritis is due to a combination of
inflammation (including WBCs and WBC products) and pannus tissue
development (a tissue composed on neovascular tissue, connective
tissue, and inflammatory cells). Paclitaxel has been chosen for the
initial studies because it is a potent inhibitor of
neovascularization. In this manner, paclitaxel in high local
concentrations will prove to be a disease modifying agent in
arthritis.
[0423] In order to determine whether microspheres have a
deleterious effect on joints, the following experiments are
conducted. Briefly, plain PCL and paclitaxel-loaded microspheres
are prepared as described previously in Example 8. Three rabbits
are injected intra-articularly with 0.5-5.0 .mu.m, 10-30 .mu.m, or
30-80 .mu.m microspheres in a total volume of 0.2 mls (containing
0.5 mg of microspheres). The joints are assessed visually
(clinically) on a daily basis. After two weeks the animals are
sacrificed and the joints examined histologically for evidence of
inflammation and depletion of proteoglycans.
[0424] The rabbit inflammatory arthritis and osteoarthritis models
may be utilized in order to evaluate the use of microspheres in
reducing synovitis and cartilage degradation. Briefly, degenerative
arthritis is induced by a partial tear of the cruciate ligament and
meniscus of the knee. After 4 to 6 weeks, the rabbits develop
erosions in the cartilage similar to that observed in human
osteoarthritis. Inflammatory arthritis is induced by immunizing
rabbits with bovine serum albumen (BSA) in Complete Freund's
Adjuvant (CFA). After 3 weeks, rabbits containing a high titer of
anti-BSA antibody receive an intra-articular injection of BSA (5
mg). Joint swelling and pronounced synovitis is apparent by seven
days, a proteoglycan depletion is observed by 7 to 14 days, and
cartilage erosions are observed by 4 to 6 weeks.
[0425] Inflammatory arthritis is induced as described above. After
4 days, the joints are injected with microspheres containing 5%
paclitaxel or vehicle. One group of animals is sacrificed on day 14
and another on day 28. The joints are examined histologically for
inflammation and cartilage degradation. The experiment is designed
to determine if paclitaxel microspheres can affect joint
inflammation and cartilage matrix degradation.
[0426] Angiogenesis-inhibitor microspheres may be further examined
in an osteoarthritis model. Briefly, degenerative arthritis is
induced in rabbits as described above, and the joints receive an
intra-articular injection of microspheres (5% paclitaxel or vehicle
only) on day 4. The animals are sacrificed on day 21 and day 42 and
the joints examined histologically for evidence of cartilage
degradation.
Results
[0427] Unloaded PCL microspheres of differing sizes (0.5-5.0 .mu.m,
10-30 .mu.m, or 30-80 .mu.m) are injected intra-articularly into
the rabbit knee joint. Results of these experiments are shown in
FIGS. 22A to 22D. Briefly, FIG. 22A is a photograph of synovium
from PBS injected joints. FIG. 22B is a photograph of joints
injected with microspheres. FIG. 22C is a photograph of cartilage
from joints injected with PBS, and FIG. 22D is a photograph of
cartilage from joints injected with microspheres.
[0428] As can be seen from these photographs, histologically, there
is no difference between joints receiving a microsphere injection
and those which did not. Clinically, there was no evidence of joint
inflammation during the 14 days the experiment was conducted.
Grossly, there is no evidence of joint inflammation or cartilage
damage in joints where microspheres are injected, as compared to
untreated normal joints.
CONCLUSIONS
[0429] Microspheres can be injected intra-articularly without
causing any discernible changes to the joint surface. This
indicates that this method may be an effective means of delivering
a targeted, sustained-release of disease-modifying agents to
diseased joints, while minimizing the toxicity which could be
associated with the systemic administration of such biologically
active compounds.
[0430] As discussed above, microspheres can be formulated into
specific sizes with defined drug release kinetics. It has also been
demonstrated that paclitaxel is a potent inhibitor of angiogenesis
and that it is released from microspheres in quantities sufficient
to block neovascularization on the CAM assay. Therefore,
angiogenesis-inhibitor-loaded (e.g., paclitaxel-loaded)
microspheres may be administered intra-articularly in order to
block the neovascularization that occurs in diseases such as
rheumatoid arthritis. In this manner the drug-loaded microspheres
can act as a "chondroprotective" agent which protects the cartilage
from irreversible destruction from invading neovascular pannus
tissue.
Example 20
The Anti-Angiogenic Effects of Paclitaxel in an Ophthalmic
Suspension
[0431] In order to test whether paclitaxel would inhibit the
pathogenesis of corneal neovascularization, an ophthalmic
suspension of 0.3% paclitaxel and a 10% paclitaxel microsphere
suspension was first prepared and tested on the CAM in order to
determine whether sufficient quantities of paclitaxel could be
released to inhibit angiogenesis.
[0432] Briefly, fertilized, chick eggs were incubated for 4 days
prior to shell-less culturing as described previously in Example 2.
The egg contents are removed from the shell and emptied into
round-bottom sterilized glass bowls and covered with petri dish
covers.
[0433] On day 6 of development, the ophthalmic drops were tested on
the CAM. To deliver the ophthalmic suspensions, a 0.5 mL syringe
was sliced into rings measuring 1 mm thick. These rings, which
formed wells when placed onto the CAM were used to localize a 15
.mu.L aliquot of ophthalmic suspension to the CAM's blood vessels.
The paclitaxel (0.3%) suspension was tested on 11 embryos, whereas
the 10% paclitaxel-loaded microsphere suspension was tested on 4
other embryos. The control (unloaded) ophthalmic suspension was
tested on the remaining 5 CAMs. After a 48 hour period, Liposyn II,
a white opaque solution, was injected into the CAM which increased
the visibility of the vascular details when observed with a
stereomicroscope.
[0434] Within 48 hours, the 0.3% paclitaxel suspension inhibited
angiogenesis on 11/11 CAMs tested and the 10% paclitaxel-loaded
microsphere suspension inhibited angiogenesis on 4/4 of the embryos
tested. This was evident by the presence of avascular zones
measuring 6 mm in diameter in the vicinity of the treated area
(FIG. 23A); in many cases the avascular zone exceeded the size of
the application ring. This avascular zone was defined as a region
containing disrupted blood vessel fragments and discontinuous blood
flow. The functional vessels adjacent to the avascular zone were
modified in such a way to redirect the blood flow away from the
drug source; these vessels possessed an angular architecture which
was not evident in the control (unloaded) thermopaste treated
CAMs.
[0435] In comparison, the control (unloaded) ophthalmic vehicle did
not inhibit angiogenesis on any of the 5 CAMs tested; this was
evident by the functional vessels visible within the center of the
application ring (FIG. 23B). In some cases, there was some
reduction in the amount of microvessels located in the control
treated CAMs although this was only due to the aqueous suspension
vehicle in which paclitaxel was administered.
[0436] In summary, paclitaxel was sufficiently released from the
ophthalmic drop suspension to inhibit angiogenesis on the CAM.
Therefore, since paclitaxel can be released from this vehicle
system, it may likewise be utilized in the treatment of neovascular
disease of the eye, such as corneal neovascularization.
Example 21
The Anti-Angiogenic Effects of Paclitaxel-Loaded Stent Coating
[0437] A. Testing of Paclitaxel-loaded Stents on a CAM
[0438] As noted above, stents are currently used for the prevention
of luminal closure induced by a disease process, such as biliary
tumor ingrowth. Although stents prevent tumor ingrowth temporarily,
tumor ingrowth eventually recurs. In this study, strecker stents
were coated with an EVA polymer containing paclitaxel at
concentrations of 33%, 10%, and 2.5% and were tested for their
ability to inhibit angiogenesis on the CAM.
[0439] Briefly, paclitaxel-coated stent tynes (3 mm in size) were
placed onto the growing vessels of the CAM at day 6 of development.
Of these CAMs, 3 received 33% paclitaxel-loaded stent coating, 5
CAMs received 2.5% paclitaxel, and 1 CAM received 10%
paclitaxel-loaded stent coating. In addition, control stents,
coated with unloaded EVA, were tested on a total of 6 CAMs. After
48 hours, Liposyn II was injected within the CAM to increase the
vascular details during observations.
[0440] All 3 different paclitaxel concentrations of the stent
coating inhibited angiogenesis on the CAM within the 48 hour
period. The CAMs were maximally inhibited which was characterized
by the induction of avascular zones measuring 6 mm in diameter
(FIG. 24A).
[0441] B. Testing of Control Stents in Tumors
[0442] Similar to above, control stents were prepared as described
above and placed into established gliosarcoma tumors of the rat
liver. After a 7 day period, these rats were sedated and perfused
with a 2.0% glutaraldehyde in sodium cacodylate solution. The
livers were excised and the stents were dissected away from the
surrounding tissue. Images of the gross anatomy revealed that the
nylon stents had become incorporated into the tumor and tumor
ingrowth had been established within the lumen of the stent (FIGS.
25 and 26). FIG. 27 shows that metastasis had occurred within the
lung.
[0443] Unlike the paclitaxel-loaded stent coating, the control
coating did not inhibit angiogenesis and maintained its normal
architecture in all of the 6 CAMs which were tested (FIG. 24B).
[0444] In summary, since paclitaxel coated stents have the
capability of releasing sufficient drug to inhibit angiogenesis on
the CAM, paclitaxel coated stents may likewise be utilized for a
variety of applications in order to prevent tumor ingrowth within
the binary lumen.
Example 22
Effect of Paclitaxel on Neutrophil Activity
[0445] The example describes the effect of paclitaxel on the
response of neutrophils stimulated with opsonized CPPD crystals or
opsonized zymosan. As shown by experiments set forth below,
paclitaxel is a strong inhibitor of particulate inducted neutrophil
activation as measured by chemiluminescence, superoxide anion
production and degranulation in response to plasma opsonized
microcrystals or zymosan.
[0446] A. Materials and Methods
[0447] Hanks buffered salt solution (HBSS) pH 7.4 was used
throughout this study. All chemicals were purchased from Sigma
Chemical Co (St. Louis,) unless otherwise stated. All experiments
were performed at 37.degree. C. unless otherwise stated.
[0448] 1. Preparation and Characterization of Crystals
[0449] CPPD (triclinic) crystals were prepared. The size
distribution of the crystals was approximately 33% less than 10
.mu.m, 58% between 10 and 20 .mu.m and 9% greater than 20 .mu.m.
Crystals prepared under the above conditions are pyrogen free and
crystals produced under sterile, pyrogen free conditions produced
the same magnitude of neutrophil response as crystals prepared
under normal, non-sterile laboratory conditions.
[0450] 2. Opsonization of Crystals and Zymosan
[0451] All experiments that studied neutrophil responses to
crystals or zymosan in the presence of paclitaxel were performed
using plasma opsonized CPPD or zymosan. Opsonization of crystals or
zymosan was done with 50% heparinized plasma at a concentration of
75 mg of CPPD or 12 mg of zymosan per ml of 50% plasma. Crystals or
zymosan were incubated with plasma for 30 min. at 37.degree. C. and
then washed in excess HBSS.
[0452] 3. Neutrophil Preparation
[0453] Neutrophils were prepared from freshly collected human
citrated whole blood. Briefly, 400 ml of blood were mixed with 80
ml of 4% dextran T500 (Phamacia LKB, Biotechnology AB Uppsala,
Sweden) in HBSS and allowed to settle for 1 h. Plasma was collected
continuously and 5 ml applied to 5 ml of Ficoll Paque (Pharmacia)
in 15 ml polypropylene tubes (Corning, N.Y.). Following
centrifugation at 500.times.g for 30 min, the neutrophil pellets
were washed free of erythrocytes by 20 s of hypotonic shock.
Neutrophils were resuspended in HBSS, kept on ice and used for
experiments within 3 h. Neutrophil viability and purity was always
greater than 90%.
[0454] 4. Incubation of Neutrophils with Paclitaxel
[0455] A stock solution of paclitaxel at 12 mM in DMSO was freshly
prepared before each experiment. This stock solution was diluted in
DMSO to give solutions of paclitaxel in the 1 to 10 mM
concentration range. Equal volumes of these diluted paclitaxel
solutions was added to neutrophils at 5,000,000 cells per ml under
mild vortexing to achieve concentrations of 0 to 50 .mu.M with a
final DMSO concentration of 0.5%. Cells were incubated for 20
minutes at 33.degree. C. then for 10 minutes at 37.degree. C.
before addition to crystals or zymosan.
[0456] 5. Chemiluminescence Assay
[0457] All chemiluminescence studies were performed at a cell
concentration of 5,000,000 cells/ml in HBSS with CPPD (50 mg/ml).
In all experiments 0.5 ml of cells was added to 25 mg of CPPD or
0.5 mg of zymosan in 1.5 ml capped Eppendorf tubes. 10 .mu.l of
luminol dissolved in 25% DMSO in HBSS was added to a final
concentration of 1 .mu.M and the samples were mixed to initiate
neutrophil activation by the crystals or zymosan. Chemiluminescence
was monitored using an LKB Luminometer (Model 1250.) at 37.degree.
C. with shaking immediately prior to measurements to resuspend the
crystals or zymosan. Control tubes contained cells, drug and
luminol (crystals absent).
[0458] 6. Superoxide Anion Generation
[0459] Superoxide anion concentrations were measured using the
superoxide dismutase inhibitable reduction of cytochrome c assay.
Briefly, 25 mg of crystals or 0.5 mg of zymosan was placed in a 1.5
ml capped Eppendorf tube and warmed to 37.degree. C. 0.5 ml of
cells at 370C were added together with ferricytochrome c (final
concentration 1.2 mg/ml) and the cells were activated by shaking
the capped tubes. At appropriate times tubes were centrifuged at
10,000 g for 10 seconds and the supernatant collected for assay be
measuring the absorbance of 550 nm. Control tubes were set up under
the same conditions with the inclusion of superoxide dismutase at
600 units per ml.
[0460] 7. Neutrophil Degranulation Assay
[0461] One and a half milliliter Eppendorf tubes containing either
25 mg of CPPD or 1 mg of zymosan were preheated to 37.degree. C.
0.5 ml of cells at 37.degree. C. were added followed by vigorous
shaking to initiate the reactions. At appropriate times, tubes were
centrifuged at 10,000.times.g for 10 seconds and 0.4 ml of
supernatant was stored at -20.degree. C. for later assay.
[0462] Lysozyme was measured by the decrease in absorbance at 450
nm of a Micrococcus lysodeikticus suspension. Briefly, Micrococcus
lysodeikticus was suspended at 0.1 mg/ml in 65 mM potassium
phosphate buffer, pH 6.2 and the absorbance at 450 nm was adjusted
to 0.7 units by dilution. The crystal (or zymosan) and cell
supernatant (100 .mu.l) was added to 2.5 ml of the Micrococcus
suspension and the decrease in absorbance was monitored. Lysozyme
standards (chicken egg white) in the 0 to 2000 units/ml range were
prepared and a calibration graph of lyzozyme concentration against
the rate of decrease in the absorbance at 450 nm was obtained.
[0463] Myeloperoxidase (MPO) activity was measured by the increase
in absorbance at 450 nm that accompanies the oxidation of
dianisidine. 7.8 mg of dianisidine was dissolved in 100 ml of 0.1 M
citrate buffer, pH 5.5 at 3.2 mM by sonication. To a 1 ml cuvette,
0.89 mL of the dianisidine solution was added, followed by 50 .mu.l
of 1% Triton.times.100, 10 .mu.l of a 0.05% hydrogen peroxide in
water solution and 50 ul of crystal-cell supernatant. MPO activity
was determined from the change in absorbance (450 nm) per minute,
Delta A 450, using the following equation: Dianisidine oxidation
(nmol/min)=50.times.Delta A 450
[0464] 8. Neutrophil Viability
[0465] To determine the effect of paclitaxel on neutrophil
viability the release of the cytoplasmic marker enzyme, Lactate
Dehydrogenase (LDH) was measured. Control tubes containing cells
with drug (crystals absent) from degranulation experiments were
also assayed for LDH.
[0466] B. Results
[0467] In all experiments statistical significance was determined
using Students' t-test and significance was claimed at p<0.05.
Where error bars are shown they describe one standard deviation
about the mean value for the n number given.
[0468] 1. Neutrophil Viability
[0469] Neutrophils treated with paclitaxel at 46 .mu.M for one hour
at 37.degree. C. did not show any increased level of LDH release
(always less than 5% of total) above controls indicating that
paclitaxel did not cause cell death.
[0470] 2. Chemiluminescence
[0471] Paclitaxel at 28 .mu.M produced strong inhibition of both
plasma opsonized CPPD and plasma opsonised zymosan induced
neutrophil chemiluminescence as shown in FIGS. 29A, 29B and 33A
respectively. The inhibition of the peak chemiluminescence response
was 52% (+/-12%) and 45% (+/-11%) for CPPD and zymosan
respectively. The inhibition by paclitaxel at 28 .mu.M of both
plasma opsonized CPPD and plasma opsonized zymosan induced
chemiluminescence was significant at all times from 3 to 16 minutes
(FIGS. 29A, 29B and 33A). FIGS. 29A and 29B show the concentration
dependence of paclitaxel inhibition of plasma opsonized CPPD
induced neutrophil chemiluminescence. In all experiments control
samples never produced chemiluminescence values of greater than 5
mV and the addition of paclitaxel at all concentrations used in
this study had no effect on the chemiluminescence values of
controls.
[0472] 3. Superoxide Generation
[0473] The time course of plasma opsonised CPPD crystal induced
superoxide anion production, as measured by the superoxide
dismutase (SOD) inhibitable reduction of cytochrome c, is shown in
FIG. 2. Treatment of the cells with paclitaxel at 28 .mu.M produced
a decrease in the amount of superoxide generated at all times. This
decrease was significant at all times shown in FIG. 30A. The
concentration dependence of this inhibition is shown in FIG. 30B.
Stimulation of superoxide anion production by opsonised zymosan
(FIG. 31B) showed a similar time course to CPPD induced activation.
The inhibition of zymosan induced superoxide anion production by
paclitaxel at 28 .mu.M was less dramatic than the inhibition of
CPPD activation but was significant at all times shown in FIG.
31B.
[0474] 4. Neutrophil Degranulation
[0475] Neutrophil degranulation was monitored by the plasma
opsonized CPPD crystal induced release of myeloperoxidase and
lysozyme or the plasma opsonized zymosan induced release of
myeloperoxidase. It has been shown that sufficient amounts of these
two enzymes are released into the extracellular media when plasma
coated CPPD crystals are used to stimulate neutrophils without the
need for the addition of cytochalasin B to the cells. FIGS. 32 and
33 show the time course of the release of MPO and lysozyme
respectively, from neutrophils stimulated by plasma coated CPPD.
FIG. 32A shows that paclitaxel inhibits myeloperoxidase release
from plasma opsonized CPPD activated neutrophils in the first 9
minutes of the crystal-cell incubation. Paclitaxel significantly
inhibited CPPD induced myeloperoxidase release at all times as
shown in FIG. 32A. FIG. 32B shows the concentration dependence of
paclitaxel inhibition of CPPD induced myeloperoxidase release.
[0476] Paclitaxel at 28 .mu.M reduced lysozyme release and this
inhibition of degranulation was significant at all times as shown
in FIG. 33.
[0477] Only minor amounts of MPO and lysozyme were released when
neutrophils were stimulated with opsonized zymosan. Despite these
low levels it was possible to monitor 50% inhibition of MPO release
after 9 minutes incubation in the presence of paclitaxel at 28
.mu.M that was statistically significant (p<0.05) (data not
shown).
[0478] C. Discussion
[0479] These experiments demonstrate that paclitaxel is a strong
inhibitor of crystal induced neutrophil activation. In addition, by
showing similar levels of inhibition in neutrophil responses to
another form of particulate activator, opsonized zymozan, it is
evident that the inhibitory activity of paclitaxel is not limited
to neutrophil responses to crystals.
Example 23
Effect of Paclitaxel on Synoviocyte Proliferation
[0480] Two experiments were conducted in order to assess the effect
of differing concentrations of paclitaxel on tritiated thymidine
incorporation (a measurement of synoviocyte DNA synthesis) and
synoviocyte proliferation in vitro.
[0481] A. Materials and Methods
[0482] 1. .sup.3H-Thymidine Incorporation into Synoviocytes
[0483] Synoviocytes were incubated with different concentrations of
paclitaxel (10.sup.-5 M, 10.sup.-6 M, 10.sup.-7 M, and 10.sup.-8 M)
continuously for 6 or 24 hours in vitro. At these times,
1.times.10.sup.-6 cpm of .sup.3H-thymidine was added to the cell
culture and incubated for 2 hours at 37.degree. C. The cells were
placed through a cell harvester, washed through a filter, the
filters were cut, and the amount of radiation contained in the
filter sections determined. Once the amount of thymidine
incorporated into the cells was ascertained, it was used to
determine the rate of cell proliferation. This experiment was
repeated three times and the data collated together.
[0484] 2. Synoviocyte Proliferation
[0485] Bovine synovial fibroblasts were grown in the presence and
absence of differing concentrations (10.sup.-5 M, 10.sup.-6 M,
10.sup.-7 M, and 10.sup.-8 M) of paclitaxel for 24 hours. At the
end of this time period the total number of viable synoviocyte
cells was determined visually by dye exclusion counting using
Trypan blue staining. This experiment was conducted 4 times and the
data collated.
B. Results
[0486] 1. .sup.3H-Thymidine Incorporation into Synoviocytes
[0487] This study demonstrated that paclitaxel at low
concentrations inhibits the incorporation of 3H-thymidine (and by
extension DNA synthesis) in synoviocytes at concentrations as low
as 10.sup.-8 M. At six hours there was no significant difference in
the between the degree of inhibition produced by the higher versus
the lower concentrations of paclitaxel (see FIG. 34). However, by
24 hours some of the effect as lost at lower concentrations of the
drug (10.sup.-8 M), but was still substantially lower than that
seen in control animals.
[0488] 2. Synoviocyte Proliferation
[0489] This study demonstrated that paclitaxel was cytotoxic to
proliferating synovial fibroblasts in a concentration dependent
manner. Paclitaxel at concentrations as low as 10.sup.-7 M is
capable of inhibiting proliferation of the synoviocytes (see FIG.
35). At higher concentrations of paclitaxel (10.sup.-6 M and
10.sup.-5 M) the drug was toxic to the synovial fibroblasts in
vitro.
C. Discussion
[0490] The above study demonstrates that paclitaxel is capable of
inhibiting the proliferation of synovial fibroblasts at relatively
low concentrations in vitro. Therefore, given the role of these
cells in the development of pannus tissue and their growth during
the pathogenesis of rheumatoid arthritis, blocking synoviocyte
proliferation can be expected to favorably affect the outcome of
the disease in vivo.
Example 24
Effect of Paclitaxel on Collagenase Expression
[0491] As noted above, collagenase production by a variety of
tissues (synovial fibroblasts, endothelial cells, chondrocytes, and
white blood cells) plays an critical role in the development of the
pathology of arthritis. Degradation of the cartilage matrix by
proteolytic enzymes represents an irreversible step in the
development of the disease resulting in irreparable damage to the
articular cartilage. Numerous attempts have been made to restore
the balance between the enzymes which degrade connective tissue
(matrix metalloproteinases--MMPs; collagenase is an important
member of this family) and those which inhibit degradation (tissue
inhibitors of metalloproteinases--TIMPs). Evidence suggests that
the imbalance of proteolytic versus inhibitory activity which
results in cartilage destruction is due to an excess of MMP
activity as opposed to a paucity of TIMP activity. Treatment that
decreases the amount of MMP activity may thus favorably influence
the outcome of the disease.
[0492] C-fos is an oncogene transcription factor shown to be
involved and required for the induction of genes involved in cell
proliferation and coliagenase expression. In cultured chondrocytes,
both interleukin-1 (IL-1) and tumor necrosis factor (TNF) have been
shown to stimulate c-fos expression and produce all of the signals
necessary to induce the expression of collagenase. When IL-1 is
administered to chondrocytes in vitro there is a transient increase
in fos mRNA levels which peak 30-60 minutes later, while
collagenase mRNA is detected 9 hours later and continues to
increase up to 12 hours (data not shown) after IL-1 stimulation.
The fos and collagenase mRNA can be detected using the respective
cDNA probes and analyzed by Northern blot analysis. This allows the
determination of agents capable of inhibiting collagenase
production and an approximation of the step in the collagenase
synthetic pathway that is affected by the treatment.
A. Materials and Methods
[0493] 1. Effect of Paclitaxel on c-fos Expression
[0494] Chondrocytes were treated with different concentrations of
paclitaxel (10.sup.-6 M, 10.sup.-7 M, and 10.sup.-8 M) for 2 hours
and then treated with TNFa (Sigma Chemical Co., St. Louis, Mo.) at
30 ng/ml for 1 hour. Human recombinant TNF.alpha. was dissolved in
phosphate buffered saline (PBS) with 0.1% bovine serum albumin
(BSA). Total RNA from bovine articular chondrocytes was isolated by
the acidified guanidine isothiocyanate method and the levels of
c-fos mRNA determined by Northern blot analysis. Denatured RNA
samples (12 .mu.g) were analyzed by gel electrophoresis in a
denaturing 1% agarose gel, transferred to a nylon membrane
(Bio-Rad), cross-linked with an ultraviolet cross-linker
(Stratagene UV stratalinker 1800), and hybridized with
.sup.32P-labeled rat c-fos DNA. mRNA for tubulin and total RNA were
used as controls. To determine tubulin mRNA, the blots described
above were subsequently stripped of DNA and re-probed with
.sup.32P-labeled rat P-tubulin cDNA. This experimented was
conducted three times and the data collated.
[0495] 2. Effect of Paclitaxel on Collagenase Expression
[0496] Chondrocytes were treated with different concentrations of
paclitaxel (10.sup.-6 M and 10.sup.-7M,) for 2 hours prior to the
addition of IL-1 (20 ng/ml). The cells were then incubated for a
further 16 hours. Total RNA from bovine articular chondrocytes was
isolated by the acidified guanidine isothiocyanate method and the
collagenase mRNA determined by Northern blot analysis. The RNA
samples were prepared as described above using .sup.32P-labeled rat
collagenase cDNA.
B. Results
[0497] 1. Effect of Paclitaxel on c-fos Expression
[0498] This experiment demonstrates that paclitaxel does not alter
c-fos expression at any concentration (see FIG. 36). Comparable
levels of c-fos mRNA were detectable in the controls and all of the
experimental groups regardless of the paclitaxel concentration
present. Total RNA and tubulin expression was similarly
unaffected.
[0499] 2. Effect of Paclitaxel on Collagenase Expression
[0500] This experiment demonstrates that paclitaxel at a
concentration of 10.sup.-6 M completely inhibited IL-1 induced
collagenase expression. Collagenase mRNA was not detectable above
background at this concentration of paclitaxel in vitro (see FIG.
37).
C. Discussion
[0501] Paclitaxel is capable of inhibiting collagenase production
by chondrocytes in vitro at concentrations of 10.sup.-6M. This
inhibition occurs downstream from the transcription factor activity
of c-fos, but still represents a secondary gene response, as
collagenase mRNA production is affected. As such, paclitaxel
inhibition of collagenase production is not strictly due to
interruption of the microtubules involved in the protein secretory
pathway (which is dependent upon microtubular function for the
movement of secretory granules), but acts at the level of the gene
response to stimulation of collagenase production. Regardless of
the mechanism of action, paclitaxel is capable of inhibiting
collagenase production by at least one cell type known to produce
this enzyme in the arthritic disease process.
Example 25
Effect of Paclitaxel on Chondrocyte Viability
[0502] While it is important that a disease modifying agent be
capable of strongly inhibiting a variety of inappropriate cellular
activities (proliferation, inflammation, proteolytic enzyme
production) which occur in excess during the development of RA, it
must not be toxic to the normal joint tissue. It is particularly
critical that normal chondrocytes not be damaged, as this would
hasten the destruction of the articular cartilage and lead to
progression of the disease. In this example, the effect of
paclitaxel on normal chondrocyte viability in vitro was
examined.
[0503] Briefly, chondrocytes were incubated in the presence
(10.sup.-5 M, 10.sup.-7 M, and 10.sup.-9 M) or absence (control) of
paclitaxel for 72 hours. At the end of this time period, the total
number of viable chondrocytes was determined visually by dye
exclusion counting using Trypan blue staining. This experiment was
conducted 4 times and the data collated.
[0504] Results of this experiment are shown in FIG. 38. Briefly, as
is evident from FIG. 38, paclitaxel does not affect the viability
of normal chondrocytes in vitro even at high concentrations
(10.sup.-5 M) of paclitaxel. More specifically, even at drug
concentrations sufficient to block the pathological processes
described in the preceding examples, there is no cytotoxicity to
normal chondrocytes.
Example 26
Ophthalmic Drops Containing Paclitaxel or Prednisolone Acetate
[0505] Three formulations containing 0.3% paclitaxel for ophthalmic
use were prepared. The particle size distribution of paclitaxel as
received from a supplier was not within acceptable limits for
ophthalmic use. In particular, for ophthalmic drops at least 90% of
the particles should preferably be below 10 .mu.m, with no particle
above 20 .mu.m. Two methods were used to reduce the particle size.
The first method involved precipitating paclitaxel from its
solution in acetone. Briefly, 150 mg of paclitaxel was dissolved in
5 ml of acetone. This solution was added in a gentle stream, with
stirring, to 20 ml of Sterile water USP to precipitate the drug.
The suspension was homogenized with the Dounce homogenizer until
about 90% of the drug was under 10 .mu.m. The suspension was
allowed to stand for about 1 hour. The larger particles settled and
were separated from the smaller ones by decantation. The larger
particles were again reduced until all particles were under 20
.mu.m (see FIG. 48). This suspension was added to the one
previously decanted and the acetone was evaporated by heating at
50.degree. C. for 2 hours and then in a vacuum oven at 30.degree.
C. and 25 torr overnight to remove the residual acetone. Sodium
chloride (0.45 g) was dissolved in 5 ml Sterile water USP. This
solution and 20 ml of 5% PVA solution were mixed with the
paclitaxel suspension, made up to 50 ml with sterile water and
bottled.
[0506] Paclitaxel suspension (0.3%) was also prepared by adding 150
mg of paclitaxel to 10 ml of sterile water and comminuted using the
Fritsch Pulverizer for 15 minutes. It was not possible to produce
particles lower than 60 .mu.m with this method probably because the
solid paclitaxel was not hard and brittle (see FIG. 49). The
suspension was mixed with 5 ml sterile water containing 0.45 g of
NaCl and 20 ml of 5% PVA solution and made up to 50 ml with sterile
water.
[0507] Paclitaxel microspheres containing 10% paclitaxel in PCL
were also prepared. Briefly, paclitaxel (60 mg) and PCL (540 mg)
were dissolved in 3 ml of DCM, 20 ml of 3% PVA solution was added
and homogenized with the Polytron homogenizer at point 3 setting
for about 1 minute. The emulsion was poured into a 30 ml beaker and
stirred until the microspheres were formed (about 3 hours). This
suspension was placed in a vacuum oven, at 30.degree. C. and 25
torr, overnight to remove residual DCM. The small microsphere
suspension (15 ml) was decanted and evaporated under vacuum to
about 5 ml and assayed for paclitaxel. This suspension was mixed
with 2 ml solution of NaCl (0.45 g) solution and made up to 10 ml
with sterile water.
[0508] Prednisolone acetate suspension containing 1% drug was
prepared by homogenizing the appropriate amount of the drug (as
received) in 20 ml of 5% PVA, NaCl solution was added and made up
to volume with sterile water.
Example 27
Paclitaxel in an Animal Model of Corneal Neovascularization
Induction of Corneal Neovascularization
[0509] Corneal angiogenesis is induced in male New Zealand white
rabbits (2.5 to 3.0 kg) essentially as described by Scroggs et al.,
Invest. Ophthalmol. Vis. Sci. 32:2105-2111, 1991. Briefly, rabbits
are anesthetized with a subcutaneous injection of 0.15 cc of a 1:1
mixture of ketamine (80 mg/ml) and xylazine (4 mg/ml), and the eyes
cauterized by applying the tip of a new silver-potassium nitrate
applicator (75% silver nitrate: 25% potassium nitrate;
Graham-Field, Hauppage, N.Y.) 3 to 4 millimeters from the
corneo-scleral limbus.
[0510] Immediately following chemical cauterization, one drop of
the study solution (e.g., the study solutions may be vehicle alone,
prednisolone acetate 1%, or 0.3% paclitaxel in suspension) is
applied to the cauterized eyes. Gentamicin ophthalmic ointment is
then applied to the treated eyes. Over the next two weeks, one drop
of the study solution is applied four times daily.
[0511] In a second study, 0.5 ml aliquots of a 10%
paclitaxel-loaded microsphere suspension and a 20%
paclitaxel-loaded thermopaste is administered via subconjuctival
injection to the experimental animals.
[0512] On the eighth day and fourteenth following cauterization of
the corneas, all animals are re-anesthetized as described above,
and the corneas photographed using a Nikon biomicroscope and Kodak
ASA 180 tungsten film under microscope incandescent illumination.
The highest magnification that incorporates the entire cornea is
used.
[0513] The photographs are randomly presented to a masked observer
who grades the corneal vessels based upon a 0 to 4 scale of vessel
density, and who measures the total extent in clock hours of
circumferential corneal neovascularization. Vessel density grade is
based on two standard photographs obtained from pilot experiments
that had been assigned grades 2 (moderate vessel density) and 4
(severe vessel density) respectively. Grades 1 and 3 were
established be interpolation; grade 0 is applied to corneas that
demonstrate a central cautery scar, but the absence of new vessel
growth.
[0514] Differences in both corneal vessel density and extent, in
terms of clock hours of involvement, is analyzed using non-paired
Student's t tests. Tests are two-tailed, with a p value of
.ltoreq.0.05 considered significant. Measures are reported as mean
.+-.standard deviation.
Example 28
Modification of Paclitaxel Release from Thermopaste Using Low
Molecular Weight Poly(D,L, Lactic Acid)
[0515] As discussed above, depending on the desired therapeutic
effect, either quick release or slow release polymeric carriers may
be desired. For example, polycaprolactone (PCL) and mixtures of PCL
with poly(ethylene glycol) (PEG) produce compositions which release
paclitaxel over a period of several months. In particular, the
diffusion of paclitaxel in the polymers is very slow due to its
large molecular size and extreme hydrophobicity.
[0516] On the other hand, low molecular weight poly(DL-lactic acid)
(PDLLA) gives fast degradation, ranging from one day to a few
months depending on its initial molecular weight. The release of
paclitaxel, in this case, is dominated by polymer degradation.
Another feature of low molecular weight PDLLA is its low melting
temperature, (i.e., 40.degree. C.-60.degree. C.), which makes it
suitable material for making Thermopaste. As described in more
detail below, several different methods can be utilized in order to
control the polymer degradation rate, including, for example, by
changing molecular weight of the PDLLA, and/or by mixing it with
high mol wt. PCL, PDLLA, or poly(lactide-co-glyocide) (PLGA).
A. Experimental Materials
[0517] D,L-lactic acid was purchased from Sigma Chemical Co., St.
Louis, Mo. PCL (molecular weight 10-20,000) was obtained from
Polysciences, Warrington, Pa. High molecular weight PDLLA
(intrinsic viscosity 0.60 dl/g) and PLGA (50:50 composition,
viscosity 0.58 dl/g) were from Birmingham Polymers.
B. Synthesis of Low Molecular weight PDLLA
[0518] Low molecular weight PDLLA was synthesized from DL-lactic
acid through polycondensation. Briefly, DL-lactic acid was heated
in a glass beaker at 200.degree. C. with nitrogen purge and
magnetic stirring for a desired time. The viscosity increased
during the polymerization, due to the increase of molecular weight.
Three batches were obtained with different polymerization times,
i.e., 40 min (molecular weight 800), 120 min, 160 min.
C. Formulation of Paclitaxel Thermopastes
[0519] Paclitaxel was loaded, at 20%, into the following materials
by hand mixing at a temperature about 60.degree. C.
[0520] 1. low molecular weight PDLLA with polymerization time of 40
min.
[0521] 2. low molecular weight PDLLA with polymerization time of
120 min.
[0522] 3. low mol. wt PDLLA with polymerization time of 160
min.
[0523] 4. a mixture of 50:50 high molecular weight PDLLA and low
molecular weight PDLLA 40 min.
[0524] 5. a mixture of 50:50 high molecular weight PLGA and low
molecular weight PDLLA 40 min.
[0525] 6. mixtures of high molecular weight PCL and low molecular
weight. PDLLA 40 min with PCL:PDLLA of 10:90, 20:80, 40:60, 60:40,
and 20:80.
Mixtures of high molecular weight PDLLA or PLGA with low molecular
weight. PDLLA were obtained by dissolving the materials in acetone
followed by drying.
D. Release Study
[0526] The release of paclitaxel into PBS albumin buffer at
37.degree. C. was measured as described above with HPLC at various
times.
E. Results
[0527] Low molecular weight PDLLA 40 min was a soft material with
light yellow color. The color is perhaps due to the oxidation
during the polycondensation. Low molecular weight PDLLA 120 min
(yellow) and 160 min (brown) were brittle solids at room
temperature. They all become melts at 60.degree. C. Mixtures of
50:50 high molecular weight PDLLA or PLGA with low molecular weight
PDLLA 40 min also melted about 60.degree. C.
[0528] During the release, low molecular weight PDLLA 40 min and
120 min broke up into fragments within one day, other materials
were intact up to this writing (3 days).
[0529] The release of paclitaxel from formulations 2-5 were shown
in FIG. 50. Low molecular weight PDLLA 40 min and 120 min gave the
fastest release due to the break up of the paste. The release was
perhaps solubility limited. Low molecular weight PDLLA 160 min.
also gave a fast release yet maintained an intact pellet. For
example, 10% of loaded paclitaxel was released with one day. The
50:50 mixtures of high molecular weight PDLLA or PLGA with low
molecular weight PDLLA 40 min were slower, i.e., 3.4% and 2.2%
release within one day.
[0530] Although not specifically set forth above, a wide variety of
other polymeric carriers may be manufactured, including for
example, (1) low molecular weight (500-10,000) poly(D,L-lactic
acid), poly(L-lactic acid), poly(glycolic acid),
poly(6-hydroxycaproic acid), poly(5-hydroxyvaleric acid),
poly(4-hydroxybutyric acid), and their copolymers; (2) blends of
above (#1) above; (3) blends of (#1) above with high molecular
weight poly(DL-lactic acid), poly(L-lactic acid), poly(glycolic
acid), poly(6-hydroxycaproic acid), poly(5-hydroxyvaleric acid),
poly(4-hydroxybutyric acid), and their copolymers; and (4)
copolymers of poly(ethylene glycol) and pluronics with
poly(D,L-lactic acid), poly(L-lactic acid), poly(glycolic acid),
poly(6-hydroxycaproic acid), poly(5-hydroxyvaleric acid),
poly(4-ydroxybutyric acid), and their copolymers.
Example 29
Surfactant Coated Microspheres
A. Materials and Methods
[0531] Microspheres were manufactured from Poly (DL) lactic acid
(PLA), poly methylmethacrylate (PMMA), polycaprolactone (PCL) and
50:50 Ethylene vinyl acetate (EVA):PLA essentially as described in
Example 8. Size ranged from 10 to 100 um with a mean diameter 45
um.
[0532] Human blood was obtained from healthy volunteers.
Neutrophils (white blood cells) were separated from the blood using
dextran sedimentation and Ficoll Hypaque centrifugation techniques.
Neutrophils were suspended at 5 million cells per ml in Hanks
Buffered Salt Solution ("HBSS").
[0533] Neutrophil activation levels were determined by the
generation of reactive oxygen species as determined by
chemiluminescence. In particular, chemiluminescence was determined
by using an LKB luminometer with 1 uM luminol enhancer. Plasma
precoating (or opsonization) of microspheres was performed by
suspending 10 mg of microspheres in 0.5 ml of plasma and tumbling
at 37.degree. C. for 30 min.
[0534] Microspheres were then washed in 1 ml of HBSS and the
centrifuged microsphere pellet added to the neutrophil suspension
at 37.degree. C. at time t=0. Microsphere surfaces were modified
using a surfactant called Pluronic F127 (BASF) by suspending 10 mg
of microspheres in 0.5 ml of 2% w/w solution of F127 in HBSS for 30
min at 37.degree. C. Microspheres were then washed twice in 1 ml of
HBSS before adding to neutrophils or to plasma for further
precoating.
B. Results
[0535] FIG. 51 shows that the untreated microspheres give
chemiluminescence values less than 50 mV. These values represent
low levels of neutrophil activation. By way of comparison,
inflammatory microcrystals might give values close to 1000 mV,
soluble chemical activators might give values close to 5000 mV.
However, when the microspheres are precoated with plasma, all
chemiluminescence values are amplified to the 100 to 300 mV range
(see FIG. 51). These levels of neutrophil response or activation
can be considered mildly inflammatory. PMMA gave the biggest
response and could be regarded as the most inflammatory. PLA and
PCL both become three to four times more potent in activating
neutrophils after plasma pretreatment (or opsonization) but there
is little difference between the two polymers in this regard.
EVA:PLA is not likely to be used in angiogenesis formulations since
the microspheres are difficult to dry and resuspend in aqueous
buffer. This effect of plasma is termed opsonization and results
from the adsorption of antibodies or complement molecules onto the
surface. These adsorbed species interact with receptors on white
blood cells and cause an amplified cell activation.
[0536] FIGS. 52-55 describe the effects of plasma precoating of
PCL, PMMA, PLA and EVA:PLA respectively as well as showing the
effect of pluronic F127 precoating prior to plasma precoating of
microspheres. These figures all show the same effect: (1) plasma
precoating amplifies the response; (2) Pluronic F127 precoating has
no effect on its own; (3) the amplified neutrophil response caused
by plasma precoating can be strongly inhibited by pretreating the
microsphere surface with 2% pluronic F127.
[0537] The nature of the adsorbed protein species from plasma was
also studied by electrophoresis. Using this method, it was shown
that pretreating the polymeric surface with Pluronic F127 inhibited
the adsorption of antibodies to the polymeric surface.
[0538] FIGS. 56-59 likewise show the effect of precoating PCL,
PMMA, PLA or EVA:PLA microspheres (respectively) with either IgG (2
mg/ml) or 2% pluronic F127 then IgG (2 mg/ml). As can be seen from
these figures, the amplified response caused by precoating
microspheres with IgG can be inhibited by treatment with pluronic
F127.
[0539] This result shows that by pretreating the polymeric surface
of all four types of microspheres with Pluronic F127, the
"inflammatory" response of neutrophils to microspheres may be
inhibited.
Example 30
Preparation of Low Molecular Weight Poly(D,L-Lactic Acid)
[0540] Five hundred grams of D,L-lactic acid (Sigma Chemical Co.,
St. Louis, Mo.) was heated in a heating mantle at 190.degree. C.
for 90 minutes under a stream of nitrogen gas. This process
produced 400 g of poly(D,L-lactic acid) with a molecular weight of
700-800 as determined by end group titration and gel permeation
chromatography (Fukusaki et al, Eur. Polym. J 25(10):1019-1026,
1989).
Example 31
Preparation of Polymeric Compositions Containing Gelatinized
Paclitaxel
A. Preparation of Polymers
[0541] Two hundred milligrams of gelatin (Type B, bloom strength
225, Fisher Scientific) 200 mg of NaCl, or 100 mg of gelatin and
100 mg of NaCl were dissolved in 0.5 mL of water. Next, 200 mg of
paclitaxel was dissolved in 0.5 mL of ethanol. The dissolved
gelatin, salt, or gelatin and salt were then added to the
paclitaxel and triturated on a petri dish incubating in a water
bath at 80.degree. C., until dry. The precipitate was then ground
in a mortar and pestle and sieved through either no. 60 or no. 140
mesh (Endecott, London, England). (No. 60 mesh produces larger
granules and no. 140 mesh produces smaller granules.)
[0542] Polycaprolactone was then heated to 60.degree. C., and
granules added to a final ratio of 40:60 (w/w). The polymeric
composition was placed into a 1 ml syringe and extruded.
B. Analysis of Paclitaxel Release
[0543] A measured amount of the cylindrical polymeric composition
is then added into an albumin buffered solution, and, over a time
course, aliquots are removed and paclitaxel extracted with DCM. The
extracts are then analyzed by HPLC. Results of these experiments
are shown in FIGS. 39 and 40. Briefly, FIG. 38 shows a greater
percentage of paclitaxel released when large gelatinized particles
(>200 .mu.m) are utilized. FIG. 39 shows that addition of NaCl
is not preferred when higher amounts of paclitaxel release is
desired.
Example 32
Copolymerization of Poly(D,L-Lactic Acid) and Polyethylene
Glycol
[0544] D,L,lactide (Aldrich Chemical Co.) was added to polyethylene
glycol (molecular weight 8,000; Sigma Chemical Co., St. Louis, Mo.)
in a tube and heated with 0.5% stannous octoate (Sigma Chemical
Co.) for 4 hours at 150.degree. C. in an oven.
[0545] This process produces a copolymer of poly(D,L-lactic acid)
with polyethylene glycol as a triblock polymer (i.e.,
PDLLA-PEG-PDLLA). Paclitaxel release from this polymer is shown in
FIG. 41.
Example 33
Analysis of Drug Release
[0546] A known weight of a polymer (typically a 2.5 mg pellet) is
added to a 15 ml test tube containing 14 ml of a buffer containing
10 mm Na.sub.2HPO.sub.4--NaH.sub.2PO.sub.4, 0.145 m NaCl and 0.4
g/l bovine serum albumin. The tubes are capped and tumbled at
37.degree. C. At specific times all the 14 ml of the liquid buffer
are removed and replaced with fresh liquid buffer.
[0547] The liquid buffer is added to 1 milliliter of methylene
chloride and shaken for 1 minute to extract all the paclitaxel into
the methylene chloride. The aqueous phase is then removed and the
methylene chloride phase is dried under nitrogen. The residue is
then dissolved in 60% acetonitrile: 40% water and the solution is
injected on to a HPLC system using the following conditions: C8
column (Beckman Instruments USA), mobile phase of 58%:5%:37%
acetonitrile: methanol: water at a flow rate of 1 minute per
minute.
[0548] For paclitaxel the collected buffer is then analyzed at 232
nm. For MTX the collected buffer is applied directly to the HPLC
column with no need for extraction in methylene chloride. MTX is
analyzed at 302 mm. For Vanadium containing compounds the liquid
buffer is analyzed directly using a UV/VIS spectrometer in the 200
to 300 nm range.
Example 34
Manufacture of Polymeric Compositions Containing PCL and MePEG
A. Paclitaxel Release from PCL
[0549] Polycaprolactone containing various concentrations of
paclitaxel was prepared as described in Example 10. The release of
paclitaxel over time was measured by HPLC essentially as described
above. Results are shown in FIG. 42.
B. Effect of MePEG on Paclitaxel Release
[0550] MePEG at various concentrations was formulated into PCL
paste containing 20% paclitaxel, utilizing the methods described in
Example 10. The release of paclitaxel over time was measured by
HPLC essentially as described above. Results of this study are
shown in FIG. 43.
C. Effect of MePEG on the Melting Point of PCL
[0551] MePEG at various concentrations (formulated into PCL paste
containing 20% paclitaxel) was analyzed for melting point using DSC
analysis at a heating rate of 2.5.degree. C. per minute. Results
are shown in FIGS. 44A (melting point vs. % MePEG) and 44B (percent
increase in time to solidify vs. % MePEG).
D. Tensile Strength of MePEG Containing PCL
[0552] PCL containing MePEG at various concentrations was tested
for tensile strength and time to fail by a CT-40 Mechanical
Strength Tester. Results are shown in FIG. 45.
E. Effect of .gamma.-Irradiation or the Release of Paclitaxel
[0553] PCL:MePEG (80:20) paste loaded with 20% paclitaxel was
.gamma.-irradiated and analyzed for paclitaxel release over time.
Results are set forth in FIG. 46.
[0554] In summary, based on the above experiments it can be
concluded that the addition of MePEG makes the polymer less brittle
and more wax like, reduces the melting point and increases the
solidification time of the polymer. All these factors improve the
application properties of the paste. At low concentrations (20%)
MePEG has no effect on the release of paclitaxel from PCL.
Gamma-irradiation appears to have little effect on paclitaxel
release.
Example 35
Methotrexate-Loaded Paste
A. Manufacture of Methotrexate-Loaded Paste
[0555] Methotrexate ("MTX"; Sigma Chemical Co.) is ground in a
pestle and mortar to reduce the particle size to below 5 microns.
It is then mixed as a dry powder with polycaprolactone (molecular
wt 18000 Birmingham Polymers, AL USA). The mixture is heated to
65.degree. C. for 5 minutes and the molten polymer/methotrexate
mixture is stirred into a smooth paste for 5 minutes. The molten
paste is then taken into a 1 mL syringe, and extruded as
desired.
B. Results
[0556] Results are shown in FIGS. 47A-E. Briefly, FIG. 47A shows
MTX release from PCL discs containing 20% MePEG and various
concentrations of MTX. FIG. 47B shows a similar experiment for
paste which does not contain MePEG. FIGS. 47C, D, and E show the
amount of MTX remaining in the disk.
[0557] As can be seen by the above results, substantial amounts of
MTX can be released from the polymer when high MePEG concentrations
are utilized.
Example 36
Manufacture of Microspheres Containing Methotrexate
A. Microspheres With MTX Alone
[0558] Methotrexate (Sigma) was ground in a pestle and mortar to
reduce the particle size to below 5 microns. One hundred
milliliters of a 2.5% PVA (w/v) (Aldrich or Sigma) in water was
stirred for 15 minutes with 500 mg of unground MTX at 25.degree. C.
to saturate the solution with MTX. This solution was then
centrifuged at 2000 rpm to remove undissolved MTX and the
supernatant used in the manufacture of microspheres.
[0559] Briefly, 10 ml of a 5% w/v solution of poly(DL) lactic acid
(molecular weight 500,000; Polysciences), Polylactic:glycolic acid
(50:50 IV 0.78 polysciences) or polycaprolactone (molecular weight
18,000, BPI) containing 10:90 w/w MTX(ground):POLYMER were slowly
dripped into 100 mL of the MTX saturated 2.5% w/v solution of PVA
(Aldrich or Sigma) with stirring at 600 rpm. The mixture was
stirred at 25.degree. C. for 2 hours and the resulting microspheres
were washed and dried.
[0560] Using this method MTX loaded microspheres can be
reproducibly manufactured in the 30 to 160 micron size range.
[0561] FIG. 60 depicts the results for 10% methotrexate-loaded
microspheres made from PLA:GA (50:50); Inherent Viscosity
"IV"=0.78.
B. Microspheres with MTX and Hyaluronic Acid
[0562] MTX loaded microspheres can be made using hyaluronic acid
("HA") as the carrier by a water in oil emulsion manufacture
method, essentially as described below. Briefly, 50 ml of Parafin
oil (light oil; Fisher Scientific) is warmed to 60.degree. C. with
stirring at 200 rpm. A 5 mL solution of sodium hyaluronate (20/mL);
source=rooster comb; Sigma) in water containing various amounts MTX
is added dropwise into the Parafin oil. The mixture is stirred at
200 rpm for 5 hours, centrifuged at 500.times.g for 5 minutes. The
resulting microspheres are washed in hexane four times, and allowed
to dry.
Example 37
Manufacture of Polymeric Compositions Containing Vanadium
Compounds
A. Polymeric Paste Containing Vanadyl Sulfate
[0563] Vanadyl Sulfate (Fisher Scientific) is first ground in a
pestle and mortar to reduce the particle size, then dispersed into
melted PCL as described above for MTX. It is then taken up into a
syringe to solidify and is ready for use.
[0564] Drug release was determined essentially as described above
in Example 33, except that a 65 mg pellet of a 10% w/w
VOSO.sub.4:PCL was suspended in 10 ml of water and the supernatant
analyzed for released Vanadyl Sulphate using UV/Vis absorbance
spectroscopy of the peak in the 200 to 300 nm range.
[0565] Results are shown in FIG. 61. Briefly, from a polymeric
composition containing 10% VOSO.sub.4, 1 mg of VOSO.sub.4 was
released in 6 hours, 3 mg after 2 days and 5 mg by day 6.
B. Polymeric Microspheres Containing Vanadyl Sulfate
[0566] Vanadyl sulfate was incorporated into microspheres of
polylactic acid or hyaluronic acid essentially as described in
Example 36B. Results are shown in FIG. 62.
C. Polymeric Paste Containing Organic Vanadate
[0567] Organic vanadate is loaded into a PCL paste essentially as
described above in Example 35. Vanadate release from the
microspheres was determined as described above and in Example 33.
Results are shown in FIGS. 63A and 63B.
D. Organic Vanadate Containing Microspheres
[0568] Organic vanadate may also be loaded into microspheres
essentially as described in Example 36A. Such microspheres are
shown in FIG. 64 for poly D,L lactic acid (M.W. 500,000;
Polysciences).
Example 38
Polymeric Compositions with Increased Concentrations of
Paclitaxel
[0569] PDLLA-MePEG and PDLLA-PEG-PDLLA are block copolymers with
hydrophobic (PDLLA) and hydrophilic (PEG or MePEG) regions. At
appropriate molecular weights and chemical composition, they may
form tiny aggregates of hydrophobic PDLLA core and hydrophilic
MePEG shell. Paclitaxel can be loaded into the hydrophobic core,
thereby providing paclitaxel with an increased "solubility".
A. Materials
[0570] D,L-lactide was purchased from Aldrich, Stannous octoate,
poly (ethylene glycol) (mol. wt. 8,000), MePEG (mol. wt. 2,000 and
5,000) were from Sigma. MePEG (mol. wt. 750) was from Union
Carbide. The copolymers were synthesized by a ring opening
polymerization procedure using stannous octoate as a catalyst (Deng
et al, J. Polym. Sci., Polym, Lett. 28:411-416, 1990; Cohn et al,
J. Biomed, Mater. Res. 22: 993-1009, 1988).
[0571] For synthesizing PDLLA-MePEG, a mixture of
DL-lactide/MePEG/stannous octoate was added to a 10 milliliter
glass ampoule. The ampoule was connected to a vacuum and sealed
with flame. Polymerization was accomplished by incubating the
ampoule in a 150.degree. C. oil bath for 3 hours. For synthesizing
PDLLA-PEG-PDLLA, a mixture of D,L-lactide/PEG/stannous octoate was
transferred into a glass flask, sealed with a rubber stopper, and
heated for 3 hours in a 150.degree. C. oven. The starting
compositions of the copolymers are given in Tables V and VI. In all
the cases, the amount of stannous octoate was 0.5%-0.7%.
B. Methods
[0572] The polymers were dissolved in acetonitrile and centrifuged
at 10,000 g for 5 minutes to discard any non-dissolvable
impurities. Paclitaxel acetonitrile solution was then added to each
polymer solution to give a solution with paclitaxel
(paclitaxel+polymer) of 10%-wt. The solvent acetonitrile was then
removed to obtain a clear paclitaxel/PDLLA-MePEG matrix, under a
stream of nitrogen and 60.degree. C. warming. Distilled water, 0.9%
NaCl saline, or 5% dextrose was added at four times weight of the
matrix. The matrix was finally "dissolved" with the help of vortex
mixing and periodic warming at 60.degree. C. Clear solutions were
obtained in all the cases. The particle sizes were all below 50 nm
as determined by a submicron particle sizer, NICOMP Model 270. The
formulations are given in Table V. TABLE-US-00007 TABLE V
Formulations of Paclitaxel/PDLLA-MePEG* Paclitaxel Loading (final
PDLLA-MePEG Dissolving Media paclitaxel concentrate) 2000/50/50
water 10% (20 mg/ml) 2000/40/60 water 10% (20 mg/ml) 2000/50/50
0.9% saline 5% (10 mg/ml) 2000/50/50 0.9% saline 10% (20 mg/ml)
2000/50/50 5% dextrose 10% (10 mg/ml) 2000/50/50 5% dextrose 10%
(20 mg/ml)
[0573] In the case of PDLLA-PEG-PDLLA, since the copolymers cannot
dissolve in water, paclitaxel and the polymer were co-dissolved in
acetone. Water or a mixture of water/acetone was gradually added to
this paclitaxel polymer solution to induce the formation of
paclitaxel/polymer spheres. TABLE-US-00008 TABLE VI Composition of
PDLLA-PEG-PDLLA Copolymer Name Wt. of PEG (g) Wt. of DL-lactide (g)
PDLLA-PEG-PDLLA 1 9 90/10 PDLLA-PEG-PDLLA 2 8 80/20 PDLLA-PEG-PDLLA
3 7 70/30 PDLLA-PEG-PDLLA 4 6 60/40 PDLLA-PEG-PDLLA 14 6 30-/70 *
PEG molecular weight. 8,000.
C. Results
[0574] Many of the PDLLA-MePEG compositions form clear solutions in
water, 0.9% saline, or 5% dextrose, indicating the formation of
tiny aggregates in the range of nanometers. Paclitaxel was loaded
into PDLLA-MePEG nanoparticles successfully. For example, at %
loading (this represents 10 mg paclitaxel in 1 ml
paclitaxel/PDLLA-MePEG/aqueous system), a clear solution was
obtained from 2000-50/50 and 2000-40/60. The particle size was
about 20 nm.
Example 39
Insertion of Control and Paclitaxel Coated Stents into
Microswine
[0575] As discussed above, various tubes within the body can be
occluded by disease processes. One method for treating such
occlusion is to insert an endoluminal stent within the tube in
order to relieve the obstruction. Unfortunately, the stents
themselves are often overgrown by epithelial cells, thus limiting
the duration and effectiveness of the treatment. As described in
more detail below, stainless steel stents were coated with
paclitaxel-loaded EVA polymer and placed into the biliary duct of
microswine in order to assess prevention of benign epithelial
overgrowth.
[0576] A. Materials and Methods
[0577] Yucatan microswine were placed under general anesthetic and
a 5 cm transverse upper abdominal incision performed. The
gallbladder was grasped and sewn to the anterior abdominal wall and
a tiny incision was made in the gallbladder fundus. A 5F catheter
was inserted into the gallbladder and radiopaque contrast injected
to outline the biliary tree. A hydrophilic guidewire was advanced
through the cystic duct into the common bile duct, and over this a
7F (purpose--built, reusable) delivery catheter containing a
stainless steel (5 mm diameter.times.4.2 cm long) was advanced and
deployed in the common bile duct. The delivery catheter was
withdrawn into the gallbladder and a repeat cholangiogram
performed. The gallbladder incision was closed, a radiopaque staple
was fixed at the incision site, and then the abdominal incision was
closed. The swine was randomized into groups of receiving uncoated
stents, polymer coated stents, and paclitaxel-loaded (33%) polymer
coated stents. Tantalum (strecker) stainless steel stents and
stainless steel Wallstents were used for each of these studies.
Swines from each group were sacrificed at 14, 28, 56, and 112 days
post-stent insertion by injecting Euthanyl. After sacrifice, the
gallbladder was cannulated percutaneously under X-ray by puncturing
at the staple and radiopaque contrast injected to outline the
biliary tree. X-rays were taken and analyzed for narrowing at or
adjacent to the stent. The liver and biliary tree were removed en
bloc. The portion of bile duct containing the stent was sectioned
transversely at 1 cm intervals and the histologic sections were
used to assess the degree of overgrowth of the stent. The liver was
also examined histologically for signs of chronic obstruction or
inflammation.
[0578] B. Results
[0579] Control, uncoated stainless steel stents were inserted into
microswine as described above, and sacrificed at various times. At
two weeks, the bile mucosa appeared normal in 2 of the sacrificed
pigs, while one presented a small non-obstructive bile concretion
within the biliary lumen, and a slight indentation in the bile duct
mucosa at the site of the tines. At 4 weeks, of the 3 pigs which
were sacrificed a small bile concretion was present on the distal
stent, as well as mucosal indentations of the stent tine within the
bile duct mucosa. At 8 weeks, the bile duct mucosa at the site of
stent insertion in the sacrificed pigs partially overgrew the stent
tines in a crescentic manner over approximately 25-30% of the
radius of the stent (FIGS. 65A, 66A, and 66B). In addition, one pig
contained thick bile containing inflammatory cells within the
lumen. At 16 weeks, pigs which were sacrificed presented a stent
which was completely overgrown distally by fibrous tissue, and no
evidence of a lumen (FIG. 65B). Histologically, the tissue was
uniformly fibrous. Surprisingly, the liver biopsy of all of the
control treated swines were normal and there was no evidence of
obstructive changes.
[0580] In another group of microswine, stents coated with ethylene
vinyl acetate and 33% paclitaxel were inserted into the biliary
duct. After an 8 week exposure, one pig was sacrificed and showed a
slight indentation of the bile duct mucosa at the site of the stent
tines and no indication of overgrowth (FIGS. 66C and 66D). The
underlying mucosa was normal apart from some inflammatory cell
infiltration. Non-obstructive bile concretions were noted in the
lumen of the stent. The liver biopsy was normal, with no evidence
of obstructive changes.
[0581] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
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