U.S. patent application number 10/969759 was filed with the patent office on 2005-09-01 for compositions and methods for treating or preventing diseases of body passageways.
This patent application is currently assigned to ANGIOTECH INTERNATIONAL AG. Invention is credited to Hunter, William L., Machan, Lindsay S..
Application Number | 20050192235 10/969759 |
Document ID | / |
Family ID | 24619925 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050192235 |
Kind Code |
A1 |
Hunter, William L. ; et
al. |
September 1, 2005 |
Compositions and methods for treating or preventing diseases of
body passageways
Abstract
The present invention provides methods for treating or
preventing diseases associated with body passageways, comprising
the step of delivering to an external portion of the body
passageway a therapeutic agent. Representative examples of
therapeutic agents include anti-angiogenic factors,
anti-proliferative agents, anti-inflammatory agents, and
antibiotics.
Inventors: |
Hunter, William L.;
(Vancouver, CA) ; Machan, Lindsay S.; (Vancouver,
CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVENYUE, SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
ANGIOTECH INTERNATIONAL AG
Vancouver
CA
THE UNIVERSITY OF BRITISH COLUMBIA
|
Family ID: |
24619925 |
Appl. No.: |
10/969759 |
Filed: |
October 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10969759 |
Oct 19, 2004 |
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10671327 |
Sep 25, 2003 |
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10671327 |
Sep 25, 2003 |
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09933652 |
Aug 20, 2001 |
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6759431 |
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09933652 |
Aug 20, 2001 |
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08653207 |
May 24, 1996 |
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Current U.S.
Class: |
514/34 ; 514/182;
514/49; 514/492 |
Current CPC
Class: |
A61K 33/24 20130101;
A61K 9/12 20130101; A61K 31/00 20130101; A61K 31/28 20130101; A61K
9/0024 20130101; A61K 9/1075 20130101; A61K 9/5138 20130101; A61P
27/02 20180101; A61K 9/1635 20130101; A61P 35/00 20180101; A61K
9/7007 20130101; A61P 31/04 20180101; A61K 31/337 20130101; A61K
31/335 20130101; A61P 1/00 20180101; A61P 9/00 20180101; A61K 9/70
20130101; A61P 13/08 20180101; A61P 13/00 20180101; A61K 9/10
20130101; A61P 1/04 20180101; A61P 15/00 20180101; A61P 31/06
20180101; A61K 9/5031 20130101; A61K 9/5153 20130101; A61K 47/34
20130101; A61P 13/02 20180101; A61K 9/167 20130101; A61K 9/1652
20130101; A61K 9/5192 20130101; A61K 9/1647 20130101; A61P 11/00
20180101; A61K 9/5146 20130101; A61P 11/08 20180101; A61P 9/10
20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/034 ;
514/049; 514/182; 514/492 |
International
Class: |
A61K 031/7072; A61K
031/704; A61K 031/28; A61K 031/56 |
Claims
We claim:
1. A method for treating or preventing a vascular disease
associated with graft anastomosis, comprising delivering to an
external portion of the site of graft anastomosis a therapeutically
effective amount of a therapeutic agent or a composition comprising
a therapeutic agent, such that the vascular disease is treated.
2. The method of claim 1 wherein the vascular disease is
stenosis.
3. The method of claim 1 wherein the vascular disease is
restenosis.
4. The method of claim 1 wherein the vascular disease is
atherosclerosis.
5. The method of claim 1 wherein the site of graft anastomosis is
an artery.
6. The method of claim 5 wherein the artery is a carotid
artery.
7. The method of claim 1 wherein the site of graft anastomosis is a
vein.
8. The method of claim 1 wherein the graft anastomosis a distal
anastamosis.
9. The method of claim 1 wherein the graft anastomosis a proximal
anastamosis.
10. The method of claim 1 wherein the therapeutic agent is an
anti-angiogenic factor.
11. The method of claim 1 wherein the therapeutic agent is a
compound which disrupts microtubule function.
12. The method of claim 1 wherein the therapeutic agent is an
inhibitor of platelet adhesion or aggregation.
13. The method of claim 1 wherein the therapeutic agent is a
vasodilator.
14. The method of claim 1 wherein the therapeutic agent is an
anti-inflammatory agent.
15. The method of claim 1 wherein the therapeutic agent is an
immunosuppressive agent.
16. The method of claim 1 wherein the therapeutic agent is a growth
factor inhibitor.
17. The method of claim 1 wherein the therapeutic agent is a
promoter of re-endothelialization.
18. The method of claim 1 wherein the therapeutic agent is
mitoxantrone.
19. The method of claim 1 wherein the therapeutic agent is a
metalloproteinase inhibitor.
20. The method of claim 1 wherein the therapeutic agent is
angiostatin.
21. The method of claim 1 wherein the therapeutic agent is an
anthracycline.
22. The method of claim 1 wherein the therapeutic agent is
estradiol.
23. The method of claim 1 wherein the therapeutic agent is
carboplatin.
24. The method of claim 1 wherein the therapeutic agent is
doxorubicin.
25. The method of claim 1 wherein the therapeutic agent is
5-fluorouracil.
26. The method of claim 1 wherein the composition is
biodegradable.
27. The method of claim 1 wherein the composition is
non-biodegradable.
28. The method of claim 1 wherein the composition further comprises
a polymer.
29. The method of claim 28 wherein the polymer is
biodegradable.
30. The method of claim 28 wherein the polymer is
non-biodegradable.
31. The method of claim 1 wherein the composition further comprises
a copolymer of lactic acid and glycolic acid.
32. The method of claim 1 wherein the composition further comprises
a poly(caprolactone).
33. The method of claim 1 wherein the composition further comprises
a poly(lactic acid).
34. The method of claim 1 wherein the composition further comprises
a copolymer of poly(lactic acid) and poly(caprolactone).
35. The method of claim 1 wherein the composition further comprises
a poly(ethylene-vinyl acetate).
36. The method of claim 1 wherein the composition further comprises
a polyester.
37. The method of claim 1 wherein the composition further comprises
a polyurethane.
38. The method of claim 1 wherein the composition further comprises
a polyanhydride.
39. The method of claim 1 wherein the composition further comprises
a gelatin.
40. The method of claim 1 wherein the composition is in the form of
a paste.
41. The method of claim 1 wherein the composition is in the form of
a film.
42. The method of claim 1 wherein the composition is in the form of
a spray.
43. The method of claim 1 wherein the composition comprises
microspheres having an average size ranging from about 0.5 .mu.m to
200 .mu.m.
44. The method of claim 1 wherein the graft is a PTFE graft.
45. The method of claim 1 wherein the therapeutic agent or the
composition comprising the therapeutic agent is administered
percutaneously to the exterior surface of the site of graft
anastomosis.
46. The method of claim 1 wherein the therapeutic agent or the
composition comprising the therapeutic agent is applied to the
adventitial surface of the site of graft anastomosis.
47. The method of claim 46 wherein the composition is in the form
of a film.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 10/671,327, filed Sep. 25, 2003, which is a
continuation U.S. patent application Ser. No. 09/933,652, filed
Aug. 20, 2001, now patented as U.S. Pat. No. 6,759,431, on Jul. 6,
2004; which is a continuation of U.S. patent application Ser. No.
08/653,207, filed May 24, 1996, now abandoned, which applications
are incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to compositions and
methods for treating or preventing diseases of body passageways,
and more specifically, to compositions comprising therapeutic
agents which may be delivered to the external walls of body
passageways.
BACKGROUND OF THE INVENTION
[0003] There are many passageways within the body which allow the
flow of essential materials. These include, for example, arteries
and veins, the esophagus, stomach, small and large intestine,
biliary tract, ureter, bladder, urethra, nasal passageways, trachea
and other airways, and the male and female reproductive tract.
Injury, various surgical procedures, or disease can result in the
narrowing, weakening and/or obstruction of such body passageways,
resulting in serious complications and/or even death.
[0004] For example, many types of tumors (both benign and
malignant) can result in damage to the wall of a body passageway or
obstruction of the lumen, thereby slowing or preventing the flow of
materials through the passageway. In 1996 alone, it has been
estimated that over 11,200 deaths will occurr due to esophageal
cancer, over 51,000 deaths due to large and small intestine cancer
and nearly 17,000 deaths due to rectal cancer in the United States.
Obstruction in body passageways that are affected by cancer are not
only in and of themselves life-threatening, they also limit the
quality of a patient's life.
[0005] The primary treatment for the majority of tumors which cause
neoplastic obstruction is surgical removal and/or chemotherapy,
radiation therapy or laser therapy. Unfortunately, by the time a
tumor causes an obstruction in a body passageway it is frequently
inoperable and generally will not responded to traditional
therapies. One approach to this problem has been the insertion of
endoluminal stents. Briefly, stents are devices placed into the
lumen of a body passageway to physically hold open a passageway
that has been blocked by a tumor or other tissues/substances.
Representative examples of commonly deployed stents include the
Wallstent, Stecker stent, Gianturco stent and Palmaz stent (see
e.g., U.S. Pat. Nos. 5,102,417, 5,195,984, 5,176,626, 5,147,370,
5,141,516, 4,776,337). A significant drawback however to the use of
stents in neoplastic obstruction is that the tumor is often able to
grow into the lumen through the interstices of the stent. In
addition, the presence of a stent in the lumen can induce the
ingrowth of reactive or inflammatory tissue (e.g., blood vessels,
fibroblasts and white blood cells) onto the surface of the stent.
If this ingrowth (composed of tumor cells and/or inflammatory
cells) reaches the inner surface of the stent and compromises the
lumen, the result is re-blockage of the body passageway which the
stent was inserted to correct.
[0006] Other diseases, which although not neoplastic nevertheless
involve proliferation, can likewise obstruct body passageways. For
example, narrowing of the prostatic urethra due to benign prostatic
hyperplasia is a serious problem affecting 60% of all men over the
age of 60 years of age and 100% of all men over the age of 80 years
of age. Present pharmacological treatments, such as
5-alphareductase inhibitors (e.g., Finasteride), or
alpha-adrenergic blockers (e.g., Terazozan) are generally only
effective in a limited population of patients.
[0007] Moreover, of the surgical procedures that can be performed
(e.g., transurethral resection of the prostate (TURPs); open
prostatectomy, or endo-urologic procedures such as laser
prostatectomy, use of microwaves, hypothermia, cryosurgery or
stenting), numerous complications such as bleeding, infection,
incontinence, impotence, and recurrent disease, typically
result.
[0008] In addition to neoplastic or proliferative diseases, other
diseases such vascular disease can result in the narrowing,
weakening and/or obstruction of body passageways. According to 1993
estimates (source--U.S. Heart and Stroke Foundation homepage), over
60 million Americans have one or more forms of cardiovascular
disease. These diseases claimed 954,138 lives in the same year (41%
of all deaths in the United States).
[0009] Balloon angioplasty (with or without stenting) is one of the
most widely used treatments for vascular disease; other options
such as laser angioplasty are also available. While this is the
treatment of choice in many cases of severe narrowing of the
vasculature, about one-third of patients undergoing balloon
angioplasty (source Heart and Stoke Foundation homepage) have
renewed narrowing of the treated arteries (restenosis) within 6
months of the initial procedure; often serious enough to
necessitate further interventions.
[0010] Such vascular diseases (including for example, restenosis)
are due at least in part to intimal thickening secondary to
vascular smooth muscle cell (VSMC) migration, VSMC proliferation,
and extra-cellular matrix deposition. Briefly, vascular endothelium
acts as a nonthrombogenic surface over which blood can flow
smoothly and as a barrier which separates the blood components from
the tissues comprising the vessel wall. Endothelial cells also
release heparin sulphate, prostacyclin, EDRF and other factors
that, inhibit platelet and white cell adhesion, VSMC contraction,
VSMC migration and VSMC proliferation. Any loss or damage to the
endothelium, such as occurs during balloon angioplasty,
atherectomy, or stent insertion, can result in platelet adhesion,
platelet aggregation and thrombus formation. Activated platelets
can release substances that produce vasoconstriction (serotonin and
thromboxane) and/or promote VSMC migration and proliferation (PDGF,
epidermal growth factor, TGF-.beta., and heparinase). Tissue
factors released by the arteries stimulates clot formation
resulting in a fibrin matrix into which smooth muscle cells can
migrate and proliferate.
[0011] This cascade of events leads to the transformation of
vascular smooth muscle cells from a contractile to a secretory
phenotype. Angioplasty induced cell lysis and matrix destruction
results in local release of basic fibroblast growth factor (bFGF)
which in turn stimulates VSMC proliferation directly and indirectly
through the induction of PDGF production. In addition to PDGF and
bFGF, VSMC proliferation is also stimulated by platelet released
EGF and insulin growth factor-1.
[0012] Vascular smooth muscle cells are also induced to migrate
into the media and intima of the vessel. This is enabled by release
and activation of matrix metalloproteases which degrade a pathway
for the VSMC through the extra-cellular matrix and internal elastic
lamina of the vessel wall. After migration and proliferation the
vascular smooth muscle cells then deposit an extra-cellular matrix
consisting of gylcosaminoglycans, elastin and collagen which
comprises the largest part of intimal thickening. A significant
portion of the restenosis process may be due to remodeling of the
vascular wall leading to changes in the overall size of the artery;
at least some of which is secondary to proliferation within the
adventitia (in addition to the media). The net result of these
processes is a recurrence of the narrowing of the vascular wall
which is often severe enough to require a repeat intervention.
[0013] In summary, virtually any forceful manipulation within the
lumen of a blood vessel will damage or denude its endothelial
lining. Thus, treatment options for vascular diseases themselves
and for restenosis following therapeutic interventions continue to
be major problems with respect to longterm outcomes for such
conditions.
[0014] In addition to neoplastic obstructions and vascular disease,
there are also a number of acute and chronic inflammatory diseases
which result in obstructions of body passages. These include, for
example, vasculitis, gastrointestinal tract diseases (e.g., Crohn's
disease, ulcerative colitis) and respiratory tract diseases (e.g.
asthma, chronic obstructive pulmonary disease).
[0015] Each of these diseases can be treated, to varying degrees of
success, with medications such as anti-inflammatories or
immunosuppressants. Current regimens however are often ineffective
at slowing the progression of disease, and can result in systemic
toxicity and undesirable side effects. Surgcal procedures can also
be utilized instead of or in addition to medication regimens. Such
surgical procedures however have a high rate of local recurrence to
due to scar formation, and can under certain conditions (e.g.,
through the use of balloon catheters), result in benign reactive
overgrowth.
[0016] Other diseases that can also obstruct body passageways
include infectious diseases. Briefly, there are a number of acute
and chronic infectious processes that can result in the obstruction
of body passageways including for example, urethritis, prostatitis
and other diseases of the male reproductive tract, various diseases
of the female reproductive tract, cystitis and urethritis (diseases
of the urinary tract), chronic bronchitis, tuberculosis and other
mycobacteria infections and other respiratory problems and certain
cardiovascular diseases.
[0017] Such diseases are presently treated either by a a variety of
different therapeutic regimens and/or by surgical procedures. As
above however, such therapeutic regimens have the difficulty of
associated systemic toxicity that can result in undesired side
effects. In addition, as discussed above surgical procedures can
result in local recurrence due to scar formation, and in certain
procedures (e.g., insertion of commercially available stents), may
result in benign reactive overgrowth.
[0018] The existing treatments for the above diseases and
conditions for the most part share the same limitations. The use of
therapeutic agents have not resulted in the reversal of these
conditions and whenever an intervention is used to treat the
conditions, there is a risk to the patient as a result of the
body's response to the intervention. The present invention provides
compositions and methods suitable for treating the conditions and
diseases which are generally discussed above. These compositions
and methods address the problems associated with the existing
procedures, offer significant advantages when compared to existing
procedures, and in addition, provide other, related advantages.
SUMMARY OF THE INVENTION
[0019] Briefly stated, the present invention provides methods for
treating or preventing diseases associated with body passageways,
comprising the step of delivering to an external portion of the
body passageway a therapeutic agent. Within a related aspect,
methods for treating or preventing diseases associated with body
passageways are provided comprising the step of delivering to
smooth muscle cells of said body passageway, via the adventia, a
therapeutic agent. By delivering the therapeutic compound locally
to the site of disease, systemic and unwanted side effects can be
avoided and total dosages can potentially be reduced. Delivery
quadrantically or circumferentially around diseased passageway also
avoids many of the disadvantages of endoluminal manipulations
including damage to the epithelial lining of the tissue. For
example damage to the endothelium can result in thrombosis, changes
to laminar flow patterns and/or a foreign body reaction to an
endoluminal device, any of which can initiate the restenosis
cascade. In the case of prostatic disease, avoiding instrumentation
of the urethra can reduce the likelihood of strictures and preserve
continence and potency.
[0020] A wide variety of therapeutic agents may be utilized within
the scope of the present invention, including for example
anti-angiogenic agents, anti-proliferative agents,
anti-inflammatory agents, and antibiotics.
[0021] Within certain embodiments of the invention, the therapeutic
agents may further comprise a carrier (either polymeric or
non-polymeric), such as, for example, poly(ethylene-vinyl acetate)
(40% crosslinked), copolymers of lactic acid and glycolic acid,
poly(caprolactone), poly(lactic acid), copolymers of poly(lactic
acid) and poly (caprolactone), gelatin, hyaluronic acid, collagen
matrices, and albumen.
[0022] The therapeutic agents may be utilized to treat or prevent a
wide variety of diseases, including for example, vascular diseases,
neoplastic obstructions, inflammatory diseases and infectious
diseases. Representative body passageways which may be treated
include, for example, arteries, the esophagus, the stomach, the
duodenum, the small intestine, the large intestine, biliary tracts,
the ureter, the bladder, the urethra, lacrimal ducts, the trachea,
bronchi, bronchioles, nasal airways, eustachian tubes, the external
auditory canal, uterus and fallopian tubes.
[0023] Within one particularly preferred embodiment of the
invention, the therapeutic agent is delivered to an artery by
direct injection via an outer wall of the artery into the
adventia.
[0024] 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
[0025] FIG. 1 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.
[0026] FIG. 2 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
[0027] FIG. 3 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
[0028] FIG. 4 is a graph which shows the effect of precoating
plasma+/-2% pluronic F127 on the chemilumiesenece response of
neutrophils (5.times.10.sup.6 cells/ml) to PLA microspheres
[0029] FIG. 5 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
[0030] FIG. 6 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.
[0031] FIG. 7 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.
[0032] FIG. 8 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.
[0033] FIG. 9 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.
[0034] FIG. 10A is a graph which shows the effect of the EVA:PLA
polymer blend ratio upon aggregation of microspheres. FIG. 10B is a
scanning electron micrograph which shows the size of "small"
microspheres. FIG. 10C (which includes a magnified inset--labelled
"10C-inset") is a scanning electron micrograph which shows the size
of "large" microspheres. FIG. 10D 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. 10E is a photograph of a CAM which shows
the results of paclitaxel release by microspheres ("MS"). FIG. 10F
is a photograph similar to that of 10E at increased
magnification.
[0035] FIG. 11A 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. 11B
is a photograph which shows a CAM treated with control
microspheres. FIG. 11C is a photograph which shows a CAM treated
with 5% paclitaxel loaded microspheres.
[0036] FIGS. 12A and 12B, 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. 12C is a
graph which shows the swelling of EVA/F127 films with no paclitaxel
over time. FIG. 12D is a graph which shows the swelling of EVA/Span
80 films with no paclitaxel over time. FIG. 12E is a graph which
depicts a stress vs. strain curve for various EVA/F127 blends.
[0037] FIGS. 13A and 13B are two graphs which show the melting
point of PCL/MePEG polymer blends as a function of % MePEG in the
formulation (13A), 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 (13B). FIG. 13C is a graph which
depicts the softness of varying PCL/MePEG polymer blends. FIG. 13D
is a graph which shows the percent weight change over time for
polymer blends of various MePEG concentrations. FIG. 13E is a graph
which depicts the rate of paclitaxel release over time from various
polymer blends loaded with 1% paclitaxel. FIGS. 13F and 13G 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. 13H is a graph which depicts the effect of MePEG on the
tensile strength of a MePEG/PCL polymer.
[0038] FIG. 14 is a graph which shows paclitaxel release from
various polymeric formulations.
[0039] FIG. 15 is a graph which depicts, over a time course the
release of paclitaxel from PCL pastes into PBS at 37.degree. C. The
PCL pastes contain microparticles of paclitaxel and various
additives prepared using mesh #140. The error bars represent the
standard deviation of 3 samples.
[0040] FIG. 16 is a graph which depicts time courses of paclitaxel
release from paclitaxel-gelatin-PCL pastes into PBS at 37.degree.
C. This graph shows the effects of gelatin concentration (mesh
#140) and the size of paclitaxel-gelatin (1:1) microparticles
prepared using mesh #140 or mesh #60. The error bars represent the
standard deviation of 3 samples.
[0041] FIGS. 17A and 17B are graphs which depict the effect of
additives (17A; mesh #140) and the size of microparticles (17B;
mesh #140 or #60) and the proportion of the additive (mesh #140) on
the swelling behavior of PCL pastes containing 20% paclitaxel
following suspension in distilled water at 37.degree. C.
Measurements for the paste prepared with 270 .mu.m microparticles
in paclitaxel-gelatin and paste containing 30% gelatin were
discontinued after 4 hours due to disintegration of the matrix. The
error bars represent the standard deviation of 3 samples.
[0042] FIGS. 18A, 18B, 18C and 18D are representative scanning
electron micrographs of paclitaxel-gelatin-PCL (20:20:60) pastes
before (18A) and after (18B) suspending in distilled water at
37.degree. C. for 6 hours. Micrographs 18C and 18D are higher
magnifications of 18B, showing intimate association of paclitaxel
(rod shaped) and gelatin matrix.
[0043] FIGS. 19A and 19B are representative photomicrographs of
CAMs treated with gelatin-PCL (19A) and paclitaxel-gelatin-PCL
(20:20:60; 19B) pastes showing zones of avascularity in the
paclitaxel treated CAM.
[0044] FIG. 20 is a table which shows the effect of peri-tumoral
injection of paclitaxel-gelatin-PCL paste into mice with
established tumors.
[0045] FIG. 21 is a table which shows the melting temperature,
enthalpy, molecular weight, polydispersity and intrinsic viscosity
of PDLLA-PEG-PDLLA compositions.
[0046] FIG. 22 is a graph which depicts DSC thermograns of
PDLLA-PEG-PDLLA and PEG. The heating rate was 110.degree. C./min.
See FIG. 21 for melting temperatures and enthalpies.
[0047] FIG. 23 is a graph which depicts the cumulative release of
paclitaxel from 20% paclitaxel loaded PDLLA-PEG-PDLLA cylinders
into PBS albumin buffer at 37.degree. C. The error bars represent
the standard deviation of 4 samples. Cylinders of 40% PEG were
discontinued at 4 days due to disintegration.
[0048] FIGS. 24A, 24B and 24C are graphs which depict the change in
dimensions, length (A), diameter (B) and wet weight (C) of 20%
paclitaxel laded PDLLA-PEG-PDLLA cylinders during the in vitro
release of paclitaxel at 37.degree. C.
[0049] FIG. 25 is a graph which shows gel permeation chromatograms
of PDLLA-PEG-PDLLA cylinders (20% PEG, 1 mm diameter) loaded with
20% paclitaxel during the release in PBS albumin buffer at
37.degree. C.
[0050] FIG. 26 is a table which shows the mass loss and polymer
composition change of PDLLA-PEG-PDLLA cylinders (loaded with 20%
paclitaxel) during the release into PBS albumin buffer at
37.degree. C.
[0051] FIGS. 27A, 27B, 27C and 27D are SEMs of dried
PDLLA-PEG-PDLLA cylinders (loaded with 20% paclitaxel, 1 mm in
diameter) before and during paclitaxel release. A: 20% PEG, day 0;
B: 30% PEG, day 0; C: 20% PEG, day 69; D: 30% PEG, day 69.
[0052] FIG. 28 is a graph which depicts the cumulative release of
paclitaxel from 20% paclitaxel loaded PDLLA:PCL blends and PCL into
PBS albumin buffer at 37.degree. C. The error bars represent the
standard deviations of 4 samples.
[0053] FIG. 29 is a table which shows the efficacy of paclitaxel
loaded surgicalpaste formulations applied locally tosubcutaneous
tumor in mice.
[0054] FIG. 30A is a graph which depicts the time course of
paclitaxel release from 2.5 mg pellets of PCL. FIG. 30B is a graph
which shows the percent paclitaxel remaining in the pellet, over
time.
[0055] FIG. 31A is a graph which shows the effect of MePEG on
paclitaxel release from PCL paste leaded with 20% paclitaxel. FIG.
31B is a graph which shows the percent paclitaxel remaining in the
pellet, over time.
[0056] FIGS. 32A and 32B are graphs which show the effect of
various concentrations of MePEG in PCL in terms of melting point
(32A) and time to solidify (32B).
[0057] FIG. 33 is a graph which shows the effect of MePEG
incorporation into PCL on the tensile strength and time to fail of
the polymer.
[0058] FIG. 34 is a graph which shows the effect of irradiation on
paclitaxel release.
[0059] FIG. 35 is a graph which depicts the range of particle sizes
for control microspheres (PLLA:GA--85:15).
[0060] FIG. 36 is a graph which depicts the range of particle sizes
for 20% paclitaxel loaded microspheres (PLLA:GA--85-15).
[0061] FIG. 37 is a graph which depicts the range of particle sizes
for control microspheres (PLLA:GA--85-15).
[0062] FIG. 38 is a graph which depicts the range of particle sizes
for 20% paclitaxel loaded microspheres (PLLA:GA--85-15).
[0063] FIGS. 39A, 39B and 39C are graphs which depict the range of
particle sizes for various ratios of PLLA and GA.
[0064] FIGS. 40A and 40B are graphs which depict the range of
particle sizes for various ratios of PLLA and GA
[0065] FIGS. 41A, 41B and 41C are graphs which depict the range of
particle sizes for various ratios of PLLA and GA.
[0066] FIGS. 42A and 42B are graphs which depict the range of
particle sizes for various ratios of PLLA and GA
[0067] FIG. 43 is a table which shows the molecular weights, CMCs
and maximum paclitaxel loadings of selected diblock copolymers.
[0068] FIGS. 44A and 44B are graphs which depict the solubilization
of paclitaxel crystals in water (37.degree. C.) by the copolymers
and Cremophor EL. 44A; effect of the concentration of copolymer on
Cremophor (20 hours incubation); 44B: effect of time (copolymer or
Cremophor concentration 0.5%).
[0069] FIGS. 45A and 45B are graphs which depict the turbidity
(uv-vis absorbance at 450 nm) of micellar paclitaxel solutions at
room temperature (22.degree. C.). Paclitaxel concentration was 2
mg/ml in water. Paclitaxel loading was 10% except MePEG 5000-30/70
where the loading was 5%.
[0070] FIG. 46 is a graph which depicts paclitaxel release from
paclitaxel-nylon microcapsules.
[0071] FIG. 47 is a graph which plots the observed pseudo first
order kinetic degradation of paclitaxel (20 .mu.g ml.sup.-1 in 10%
HP.beta.CD and 10% HP.gamma.CD solutions at 37.degree. C. and pH of
3.7 and 4.9, respectively.
[0072] FIG. 48 is a graph which shows the phase solubility for
cyclodextrins and paclitaxel in water at 37.degree. C.
[0073] FIG. 49 is a graph which shows second order plots of the
complexation of paclitaxel and .gamma.CD, HP.beta.CD or HP.gamma.CD
at 37.degree. C.
[0074] FIG. 50 is a graph which shows the phase solubility for
paclitaxel at 37.degree. C. and hydroxypropyl-.beta.-cyclodextrin
in 50:50 water:ethanol solutions.
[0075] FIG. 51 is a graph which shows dissolution rate profiles of
paclitaxel in 0, 5, 10 or 20% HP.gamma.CD solutions at 37.degree.
C.
[0076] FIGS. 51A and 51B are two photographs of a CAM having a
tumor treated with control (unloaded) thermopaste. Briefly, in FIG.
51A 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. 51B is an
underside view of the CAM shown in 51A. Briefly, this view
demonstrates the radial appearance of the blood vessels which enter
the tumor like the spokes of a wheel. Note that the blood vessel
density is greater in the vicinity of the tumor than it is in the
surrounding normal CAM tissue. FIGS. 51C and 51D are two
photographs of a CAM having a tumor treated with 20%
paclitaxel-loaded thermopaste. Briefly, in FIG. 51C 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. The tumor itself is poorly
vascularized and is progressively decreasing in size. FIG. 51D is
taken from the underside of the CAM shown in 51C, 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.
[0077] FIG. 52A is a graph which shows the effect of paclitaxel/PCL
on tumor growth. FIGS. 52B and 52C are two photographs which show
the effect of control, 10%, and 20% paclitaxel-loaded thermopaste
on tumor growth.
[0078] FIG. 53 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).
[0079] FIG. 54 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).
[0080] FIG. 55 is a graph which depicts the weight of encapsulation
of Sodium Suramin in 50 mg poly(ethylene-vinyl acetate).
[0081] FIG. 56 is a graph which depicts the percent of
encapsulation of Sodium Suramin in 50 mg poly(ethylene-vinyl
acetate).
[0082] FIG. 57 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.
[0083] FIG. 58 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.
[0084] FIG. 59 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.
[0085] FIG. 60A 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 suramin 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. 60B 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.
[0086] FIGS. 61A, B, C, D and E show the effect of MTX release from
PCL over time.
[0087] FIG. 62 is a photograph of 10% methotrexate-loaded
microspheres made from PLA:GA (50:50); Inherent Viscosity
"IV"=0.78.
[0088] FIG. 63 is a graph which depicts the release of 10% loaded
vanadyl sulfate from PCL.
[0089] FIG. 64 is a photograph of hyaluronic acid microspheres
containing vanadium sulfate.
[0090] FIG. 65A is a graph which depicts the release of organic
vanadate from PCL. FIG. 65B depicts the percentage of organic
vanadate remaining over a time course.
[0091] FIG. 66 is a photograph showing poly D,L, lactic acid
microspheres containing organic vanadate.
[0092] FIGS. 67A and 67B are graphs which show the time course of
BMOV release from PCL (150 mg slabs). (A) .mu.g drug released or
(B) % of drug remaining in slab. Initial loading of BMOV in PCL
given by (.largecircle.), 5%; (.circle-solid.), 10%; (.DELTA.),
15%; (.sigma.) 20%; ( ), 30% and (.gradient.), 35%.
[0093] FIGS. 68A and 68B are graphs which show the time course of
BMOV release from 150 mg slabs of PCL:MEPEG (80:20, w:w) expressed
as (A) .mu.g drug released or (B) % drug remaining in slab. Initial
loading of BMOV in PCL:MEPEG given by (.largecircle.), 5%;
(.circle-solid.), 10%; (.DELTA.), 15%; (.sigma.), 20%.
[0094] FIGS. 69A, 69B and 69C are Scanning electron micrographs of
(69A: top), BMOV crystals; (69B: middle) surface morphology of the
PCL slab containing 20% BMOV at the start of the drug release
experiment and (69C: bottom), surface morphology of the PCL slab
containing 20% BMOV at the end of the drug release experiment (72
days in PBS).
[0095] FIGS. 70A and 70B are two graphs which show the effect of
increasing concentration of BMOV on cell survival using 1 hour
exposure of cells to BMOV (70A), or, continuous exposure to BMOV
(70B). Cells described by (.largecircle.), HT-29 colon cells;
(.circle-solid.), MCF-7 breast cells; (.DELTA.), Skmes-1 non-small
lung cells and (.sigma.), normal bone marrow cells.
[0096] FIG. 71 is a table which shows the effect of BMOV loaded
paste on the weights of MDAY-D2 tumors grown in mice. Briefly, PCL
paste (150 mg) containing either 25%, 30%, or 35% BMOV was injected
subcutaneously into mice bearing MDAY-2 tumors. Tumor weights were
determined after 10 days treatment. This table shows the results
from 2 separate experiments using (top table) 25% BMOV and (bottom
table) 30% or 35% BMOV. Control data describes mice treated with
PCL, containing no BMOV.
[0097] FIG. 72 is a table which sets forth the effects of BMOV
loaded PCL:MePEG paste on the weights of RIF-1 tumors grown in
mice. Briefly, RIF-1 tumors were grown in mice for 5 days at which
time 90% of the tumor was surgically removed and the resection site
treated with 150 mg of PCL:MePEG (80:20, w:w) paste containing
either no BMOV (control) or 5% BMOV. Tumor regrowth was determined
on days 4, 5 and 6 following this treatment.
[0098] FIGS. 73A and 73B are two graphs. FIG. 73A shows the effect
of increased loading of BEMOV in PCL thermopaste (150 mg pellet) on
the time course of BEMOV released into 15 mL PBS/ALB. FIG. 73B also
shows the effect of increase loading of BEMOV in PCL thermopaste
(150 mg pellet) on the time course of BEMOV released into 15 mL
PBS/ALB. Drug release is expressed as the % of BEMOV remaining in
the pellet.
[0099] FIGS. 74A and 74B are two graphs. FIG. 74A shows the effect
of increased loading of V5 in PCL thermopaste (150 mg pellet) on
the time course of V5 released into 15 mL PBS/ALB. FIG. 74B also
shows the effect of increase loading of V5 in PCL thermopaste (150
mg pellet) on the time course of V5 released into 15 mL PBS/ALB.
Drug release is expressed as the % of V5 remaining in the
pellet.
[0100] FIGS. 75A and 75B are two graphs. FIG. 75A shows the effect
of increased loading of PRC-V in PCL thermopaste (150 mg pellet) on
the time course of PRC-V released into 15 mL PBS/ALB. FIG. 75B also
shows the effect of increase loading of PRC-V in PCL thermopaste
(150 mg pellet) on the time course of PRC-V released into 15 mL
PBS/ALB. Drug release is expressed as the % of PRC-V remaining in
the pellet.
[0101] FIGS. 76A, 76B, 76C and 76D are a series of graphs which
show the effect of loading different concentrations of MePEG in PCL
thermopaste (150 mg pellet) with 5% BMOV (76A), 10% BMOV (76B), 15%
BMOV (76C), and 20% BMOV (76D) on the time course of BMOV released
into 15 mL PBS/ALB.
[0102] FIGS. 77A, 77B, 77C and 77D are a series of graphs which
show the effect of loading different concentrations of MePEG in PCL
thermopaste (150 mg pellet) with 0% MePEG (77A), 5% MePEG (77B),
10% MePEG (77C), and 15% MePEG (77D) on the time course of BMOV
released into 15 mL PBS/ALB. Drug release is expressed as the % of
BMOV remaining in the pellet.
[0103] FIGS. 78A and 78B are photographs of fibronectin coated PLLA
microspheres on bladder tissue (78A), and poly(L-lysine)
microspheres on bladder tissue.
DETAILED DESCRIPTION OF THE INVENTION
[0104] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
that will be used hereinafter.
[0105] "Body passageway" as used herein refers to any of number of
passageways, tubes, pipes, tracts, canals, sinuses or conduits
which have an inner lumen and allow the flow of materials within
the body. Representative examples of body passageways include
arteries and veins, lacrimal ducts, the trachea, bronchi,
bronchiole, nasal passages (including the sinuses) and other
airways, eustachian tubes, the external auditory canal, oral
cavities, the esophagus, the stomach, the duodenum, the small
intestine, the large intestine, biliary tracts, the ureter, the
bladder, the urethra, the fallopian tubes, uterus, vagina and other
passageways of the female reproductive tract, the vasdeferens and
other passageways of the male reproductive tract, and the
ventricular system (cerebrospinal fluid) of the brain and the
spinal cord.
[0106] "Therapeutic agent" as used herein refers to those agents
which can mitigate, treat, cure, or prevent a given disease or
condition. Representative examples of therapeutic agents are
discussed in more detail below, and include, for example,
anti-angiogenic agents, anti-proliferative agents,
anti-inflammatory agents, and antibiotics.
[0107] As noted above, the present invention provides methods for
treating or preventing diseases associated with body passageways,
comprising the step of delivering to an external portion of the
body passageway (i.e., a non-luminal surface), a composition
comprising a therapeutic agent, and within preferred embodiments, a
compositions comprising a therapeutic agent and a polymeric
carrier. Briefly, delivery of a therapeutic agent to an external
portion of a body passageway (e.g., quadrantically or
circumferentially) avoids many of the disadvantages of traditional
approaches which involve endoluminal manipulation. In addition,
delivery of a therapeutic agent as described herein allows the
administration of greater quantities of the therapeutic agent with
less constraint upon the volume to be delivered.
[0108] As discussed in more detail below, a wide variety of
therapeutic agents may be delivered to external portions of body
passageways, either with or without a carrier (e.g., polymeric), in
order to treat or prevent a disease associated with the body
passageway. Each of these aspects is discussed in more detail
below.
Therapeutic Agents
[0109] As noted above, the present invention provides methods and
compositions which utilize a wide variety of therapeutic agents.
Within one aspect of the invention, the therapeutic agent is an
anti-angiogenic factor. 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, 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 completely inhibits the formation of new
blood vessels, as well as reduce the size and number of previously
existing vessels.
[0110] 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).
[0111] 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, Suramin, Tissue Inhibitor of
Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,
Plasminogen Activator Inhibitor-1, Plasminogen Activator
Inhibitor-2, compounds which disrupt microtubule function, and
various forms of the lighter "d group" transition metals. These and
other anti-angiogenic factors will be discussed in more detail
below.
[0112] 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.
[0113] 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).
[0114] 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).
[0115] Tissue Inhibitor of Metalloproteinases-1 ("TIMP") is
secreted by endothelial cells which also secrete MMPases. 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.
[0116] 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.
[0117] Therapeutic agents of the present invention also include
compounds which disrupt microtubule function. Representative
examples of such compounds include estramustine (available from
Sigma; Wang and Stearns Cancer Res. 48:6262-6271, 1988),
epothilone, curacin-A, colchicine, methotrexate, and paclitaxel,
vinblastine, vincristine, D.sub.2O and
4-tert-butyl-[3-(2-chloroethyl)ureido]benzene ("tBCEU"). Briefly,
such compounds can act in several different manners. For example,
compounds such as colchicine and vinblastine act by depolymerizing
microtubules.
[0118] Within one preferred embodiment of the invention, the
therapeutic agent is paclitaxel, a compound which disrupts
microtubule formation by binding to tubulin to form abnormal
mitotic spindles. Briefly, 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). "Paclitaxel" (which should be understood herein to include
prodrugs, 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 WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO
93/23555, WO 93/10076, WO94/00156, WO 93/24476, EP 590267, WO
94/20089; 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, 5,254,580, 5,412,092,
5,395,850, 5,380,751, 5,350,866, 4,857,653, 5,272,171, 5,411,984,
5,248,796, 5,248,796, 5,422,364, 5,300,638, 5,294,637, 5,362,831,
5,440,056, 4,814,470, 5,278,324, 5,352,805, 5,411,984, 5,059,699,
4,942,184; Tetrahedron Letters 35(52):9709-9712, 1994; J. Med.
Chem. 35:4230-4237, 1992; J. Med. Chem. 34:992-998, 1991; J.
Natural Prod. 57(10):1404-1410, 1994; J. Natural Prod.
57(11):1580-1583, 1994; J. Am. Chem. Soc. 110:6558-6560, 1988), or
obtained from a variety of commercial sources, including for
example, Sigma Chemical Co., St. Louis, Mo. (T7402--from Taxus
brevifolia).
[0119] Representative examples of such paclitaxel derivatives or
analogues include 7-deoxy-docetaxol, 7,8-Cyclopropataxanes,
N-Substituted 2-Azetidones, 6,7-Epoxy Paclitaxels, 6,7-Modified
Paclitaxels, 10-Desacetoxytaxol, 10-Deacetyltaxol (from
10-deacetylbaccatin III), Phosphonooxy and Carbonate Derivatives of
Taxol, Taxol 2',7-di(sodium 1,2-benzenedicarboxylate,
10-desacetoxy-11,12-dihydrotaxol-10,12(18)-dien- e derivatives,
10-desacetoxytaxol, Protaxol (2'- and/or 7-O-ester derivatives),
(2'- and/or 7-Ocarbonate derivatives), Asymmetric Synthesis of
Taxol Side Chain, Fluoro Taxols, 9-deoxotaxane,
(13-acetyl-9-deoxobaccatine III, 9-deoxotaxol,
7-deoxy-9-deoxotaxol, 10-desacetoxy-7-deoxy-9-deoxotaxol,
Derivatives containing hydrogen or acetyl group and a hydroxy and
tert-butoxycarbonylamino, sulfonated 2'-acryloyltaxol and
sulfonated 2'-O-acyl acid taxol derivatives, succinyltaxol,
2'-.gamma.-aminobutyryltaxol formate, 2'-acetyl taxol, 7-acetyl
taxol, 7-glycine carbamate taxol, 2'-OH-7-PEG(5000) carbamate
taxol, 2'-benzoyl and 2',7-dibenzoyl taxol derivatives, other
prodrugs (2'-acetyltaxol; 2',7-diacetyltaxol; 2'succinyltaxol;
2'-(beta-alanyl)-taxol); 2'gamma-aminobutyryltaxol formate;
ethylene glycol derivatives of 2'-succinyltaxol; 2'-glutaryltaxol;
2'-(N,N-dimethylglycyl) taxol;
2'-[2-(N,N-dimethylamino)propionyl]taxol; 2' orthocarboxybenzoyl
taxol; 2' aliphatic carboxylic acid derivatives of taxol, Prodrugs
{2'(N,N-diethylaminopropionyl)taxol, 2' (N,N-dimethylglycyl)taxol,
7(N,N-dimethylglycyl)taxol, 2',7-di-(N,N-dimethylglycyl)taxol,
7(N,N-diethylaminopropionyl)taxol,
2',7-di(N,N-diethylaminopropionyl)taxol, 2'-(L-glycyl)taxol,
7-(L-glycyl)taxol, 2',7-di(L-glycyl)taxol, 2'-(L-alanyl)taxol,
7-(L-alanyl)taxol, 2',7-di(L-alanyl)taxol, 2'-(L-leucyl)taxol,
7-(L-leucyl)taxol, 2',7-di(L-leucyl)taxol, 2'-(L-isoleucyl)taxol,
7-(L-isoleucyl)taxol, 2',7-di(L-isoleucyl)taxol, 2'-(L-valyl)taxol,
7-(L-valyl)taxol, 2'7-di(L-valyl)taxol, 2'-(L-phenylalanyl)taxol,
7-(L-phenylalanyl)taxol-2',7-di(L-phenylalanyl)taxol,
2'-(L-prolyl)taxol, 7-(L-prolyl)taxol, 2',7-di(L-prolyl)taxol,
2'-(L-lysyl)taxol, 7-(L-lysyl)taxol, 2',7-di(L-lysyl)taxol,
2'-(L-glutamyl)taxol, 7-(L-glutamyl)taxol,
2',7-di(L-glutamyl)taxol, 2'-(L-arginyl)taxol, 7-(L-arginyl)taxol,
2',7-di(L-arginyl)taxol}, Taxol analogs with modified
phenylisoserine side chains, taxotere,
(N-debenzoyl-N-tert-(butoxycaronyl- )-10-deacetyltaxol, and taxanes
(e.g., baccatin III, cephalomannine, 10-deacetylbaccatin III,
brevifoliol, yunantaxusin and taxusin).
[0120] Other therapeutic agents which may be utilized within the
present invention include lighter "d group" transition metals, such
as, 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.
[0121] 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, Bis[maltolato(oxovanadium)- ](IV)] ("BMOV"),
Bis[(ethylmaltolato)oxovanadium](IV) ("BEOV"), and Bis(cysteine,
amide N-octyl)oxovanadium(IV) ("naglivan").
[0122] 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.
[0123] 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-dehydroproline (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)-oxazol- one; Merion
Merrel Dow Research Institute); Methotrexate (Sigma Chemical Co.,
#A6770; Hirata et al., Arthritis and Rheumatism 32:1065-1073,
1989); Mitoxantrone (Polverini 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 and
derivatives (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-seru- m; .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; and
metalloproteinase inhibitors such as BB94, estrogen and estrogen
analogues, antiestrogens, antioxidants, bioflavonoids (Pycnogenol),
ether lipids (s-phosphonate, ET-18-OCH.sub.3), tyrosine kinase
inhibitors (genisteine, erbstatin, herbamycin A, lavendustine-c,
hydroxycinnamates), .alpha. chemokines [Human interferon-inducible
protein 10 (IP-10)], -C-X-C-Chemokines (Gro-beta), Nitric Oxide,
Antifungal Agents (Radicicol), 15-deoxyspergualin, Metal Complexes
(Titanocene dichloride--cyclopentadienyl titanium dichloride),
Triphenylmethane Derivatives (aurintricarboxylic acid), Linomide,
Thalidomide, IL-12, Heparinase, Angiostatin, Antimicrobial Agents
(Minocycline), Plasma Proteins (Apolipoprotein E), Anthracyclines
(TAN-1120), Proliferin-Related Protein, FR-111142, Saponin of Panax
ginseng (Ginsenoside-Rb2), Pentosan polysulfate.
[0124] Compositions of the present invention may also contain a
wide variety of other therapeutic agents, 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 II); anti-depressants
(e.g., amytriptyline, fluoxetine, LUVOX.RTM. and PAXIL.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); antimitotic 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).
[0125] Other therapeutic agents that can be utilized within the
present invention include a wide variety of antibiotics, including
antibacterial, antimicrobial, antiviral, antiprotozoal and
antifungal agents. Representative examples of such agents include
systemic antibiotics such as aminoglycosides (e.g., streptomycin,
amikacin, gentamicin, netilmicin, tobramycin); 1st, 2nd, and 3rd
generation cephalosporins (e.g., cephalothin, cefazolin,
cephapirin, cephradine, cephalexin, cefadroxil, cefaclor,
cefamandole, cefuroxime, cefuroxime axetil, cefonicid, ceforamide,
cefoxitin, cefotaxime, cefotetan, ceftizoxime, cefoperazone,
ceftazidime, ceftriaxone, moxalactam, other semisynthetic
cephalosporins such as cefixime and cefpodoxime proxetil);
penicillins (e.g., penicillin G (benzathine and procaine salts),
cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin,
penicillin V, ampicillin, amoxicillin, bacampicillin, cyclacillin,
carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin,
amdinocillin, and penicillins combined with clavulanic acid);
quinolones (e.g., cinoxacin, ciprofloxacin, nalidixic acid,
norfloxacin, pipemidic acid, perloxacin, fleroxacin, enoxacin,
ofloxacin, tosufloxacin, lomefloxacin, stereoisomers of the
quinolones); sulfonamides (e.g., sulfacytine, sulfamethizole,
sulfamethoxazole, sufisoxazole, sulfasalazine, and trimethoprim
plus sulfamethoxazole combinations); tetracyclines (e.g.,
doxycycline, demeclocycline, methacycline, minocycline,
oxytetracycline, tetracycline); macrolides (e.g., erythromycins,
other semisythetic macrolides such as azithromycin and
clarithromycin); monobactams (new synthetic class) (e.g.,
aztreonam, loracarbef); and miscellaneous agents such as
actinomycin D, doxorubicin, mitomycin C, novobiocin, plicamycin,
rifampin, bleomycin, chloramphenicol, clindamycin, oleandomycin,
kanamycin, lincomycin, neomycin, paromomycin, spectinomycin,
troleandomycin, amphotericin B, colistin, nystatin, polymyxin B,
griseofulvin, aztreonam, cycloserine, clindamycin, colistimethate,
imipenem-cilastatin, methenamine, metronidazole, nitrofurantoin,
rifabutan, spectinomycin, trimethoprim, bacitracin, vancomycin,
other .beta.-lactam antibiotics.
[0126] Further therapeutic agents that can be utilized within the
present invention include topical antibiotics such as bacitracin,
zinc, neomycin, mupirocin, clindamcin; antipathogenic polypeptides
such as cecropionins, mangainins; and Antitubercular agents such as
sulfadimethoxine, sulfisoxazole, sulfisomidine, ethambutor
hydrochloride, isoniazide, calcium paraminosalicylate.
[0127] Other therapeutic agents that can be utilized within the
present invention include antibiotics such as iodine, povidone
iodine, boric acid, sodium borate, oxydale, potassium permanganate,
ethanol, isopropanol, formalin, cresol, dimazole, siccanin,
phenyliodoundecynoate, hexachlorophene, resorcin, benzethonin
chloride, sodium lauryl sulfate, mercuric chloride, mercurochrome,
silver sulfadiazine and other inorganic and organic silver and zinc
salts, salts of mono- and divalent cations, chlorhexidine
gluconate, alkylpolyaminoethylglycine hydrochloride, benzalkonium
chloride, nitrofurazone, nystatin, acesulfamin, clotrimazole,
sulfamethizole, sulfacetamide, diolamine, tolnaftate, pyrrolnitrin,
undecylenic acid, microazole, variotin, haloprogin, and dimazole,
(meclocycline, trichomycin and pentamycin), penicillins. Antifungal
agents include flucytosine, fluconazole, griseofluvin, ketoconazole
and miconazole. Antiviral and AIDS agents include acyclovir,
amantadine, didanosine (formerly ddI), griseofulvin, flucytosine,
foscarnet, ganciclovir, idoxuridine, miconazole, clotrimazole,
pyrimethamine, ribavirin, rimantadine, stavudine (formerly d4T),
trifluridine, trisulfapyrimidine, valacyclovir, vidarabine,
zalcitabine (formerly ddC) and zidovudine (formerly AZT). Adjunct
therapeutic agents for AIDS (e.g., erythropoietin; fluconazole
(antifungal); interferon alpha-2a and -2b (Kaposi's sarcoma);
atovaquone, pentamidine and trimetrexate (antiprotozoal);
megestraol acetate (appetite enhancer); rifabutin
(antimycobacterial). Representative examples of antiprotozoal
agents include: pentamidine isethionate, quinine, chloroquine, and
mefloquine.
[0128] Other therapeutic agents that can be utilized within the
present invention include anti-proliferative, anti-neoplastic or
chemotherapeutic agents. Representative examples of such agents
include androgen inhibitors, antiestrogens and hormones such as
flutamide, leuprolide, tamoxifen, estradiol, estramustine,
megestrol, diethylstilbestrol, testolactone, goserelin,
medroxyprogesterone; Cytotoxic agents such as altretamine,
bleomycin, busulfan, carboplatin, carmustine (BiCNU), cisplantin,
cladribine, dacarbazine, dactinomycin, daunorubicin, doxorubicin,
estramustine, etoposide, lomustine, cyclophosphamide, cytarabine,
hydroxyurea, idarubicin, interferon alpha-2a and -2b, ifosfamide,
mitoxantrone, mitomycin, paclitaxel, streptozocin, teniposide,
thiotepa, vinblastine, vincristine, vinorelbine; Antimetabolites
and antimitotic agents such as floxuridine, 5-fluorouracil,
fluarabine, interferon alpha-2a and -2b, leucovorin,
mercaptopurine, methotrexate, mitotane, plicamycin, thioguanine,
colchicine, anthracyclines and other antibiotics, folate
antagonists and other anti-metabolites, vinca alkaloids,
nitrosoureas, DNA alkylating agents, purine antagonists and
analogs, pyrimidine antagonists and analogs, alkyl solfonates;
enzymes such as asparaginase, pegaspargase; 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), toxins (e.g., ricin, abrin,
diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral
protein, tritin, Shigella toxin, and Pseudomonas exotoxin A),
adjunct therapeutic agents such as granisetron and ondansetron
(antinauseants, antiemetics), dexrazoxane (cardiomyopathy), gallium
nitrate (hypercalcemia), GCSF and GMSCF (chemotherapy and BMT),
IL-1 alpha, IL-2, IL-3, IL-4, levamisole, pilocarpine (saliva
generation in radiation therapy setting), strontium 89 (bone
tumors).
[0129] Further therapeutic agents that can be utilized within the
present invention include Cardiovascular agents; Antihypertensive
agents; Adrenergic blockers and stimulators (e.g., doxazosin,
guanadrel, guanethidine, pheoxybenzamine, prazosin plus
polythiazide, terazosin, methyldopa, clonidine, guanabenz,
guanfacine); Alpha-/betaadrenergic blockers (e.g., Labetalol);
angiotensin converting enzyme (ACE) inhibitors (e.g., benazepril,
catopril, enalapril, enalaprilat, fosinopril, lisinopril,
moexipril, quinapril, ramipril, and combinations with calcium
channel blockers and diuretics; ACE-receptor antagonists (e.g.,
losartan); Beta blockers (e.g., acebutolol, atenolol, betaxolol,
bisoprolol, carteolol, esmolol, fimolol, pindolol, propranolol,
penbatolol, metoprolol, nadolol, sotalol); Calcium channel blockers
(e.g., Amiloride, amlodipine, bepridil, diltiazem, isradipine,
nifedipine, verapamil, felodipine, nicardipine, nimodipine);
Antiarrythmics, groups I-IV (e.g., bretylium, disopyramide,
encainide, flecainide, lidocaine, mexiletine, moricizine,
propafenone, procainamide, quinidine, tocainide, esmolol,
propranolol, acebutolol, amiodarone, sotalol, verapamil, diltiazem,
pindolol, bupranolol hydrochloride, trichlormethiazide, furosemide,
prazosin hydrochloride, metoprolol tartrate, carteolol
hydrochloride, oxprenolol hydrochloride, and propranolol
hydrochloride); and miscellaneous antiarrythmics and cardiotonics
(e.g., adenosine, digoxin; metildigoxin, caffeine, dopamine
hydrochloride, dobutamine hydrochloride, octopamine hydrochloride,
diprophylline, ubidecarenon, digitalis).
[0130] Other therapeutic agents that can be utilized within the
present invention include diuretics (e.g., acetazolamide,
amiloride, triamterene plus hydrochlorothiazide combinations,
spironolactone plus hydrochlorothiazide combinations, torsemide,
furosemide, ethacrynate, bumetanide, triamterene,
methylchorothizide, hydrochlorothiazide, metdazone, chlorthalidone,
hydroflumethiazide, metolazone, methyclothiazide, polythiazide,
quinithazone, trichlormethiazide, benroflumethiazide,
benzthiazide); hypotensive diuretics (e.g., mefruside,
penflutizide, bumetamide, hydrothiazide, bentroflumethiazide,
reserpine); Inotropic agents (e.g., digoxin, digitoxin, dobutamine,
amrinone, milrinone); vasodilators (e.g., papaverine, isosorbide
mono- and dinitrates, nitroglycerin, dizoxide, hydralazine,
minoxidil, nitroprusside, prazosin, terazosin,
1,2,3-propanetriolmononitrate, 1,2,3-propanetriolnitrate and their
ester derivatives, pentaerythritol tetranitrate, hepronicate,
molsidomine, nicomol, simfibrate, diltiazem hydrochloride,
cinnarizine, dipyridamole, trapidil, trimetazidine hydrochloride,
carbocromene, prenylamine lactate, dilazep dihydrochloride);
vasopressors (e.g., metaraminol, isoproterenol, phenylephrine,
methaxamine); anticoagulant and thrombolytic agents (e.g., tissue
plasminogen activator (TPA), urokinase, streptokinase,
pro-urokinase, urokinase, heparin, warfarin); Calmodulin
antagonists (e.g., H.sub.7); inhibitors of the sodium/calcium
antiporter (e.g., Amiloride); and inhibitors of the ryanodine
receptor (e.g., Ryanodine); inhibitors of the IP.sub.3 receptor
(e.g., Heparin).
[0131] Other therapeutic agents that can be utilized within the
present invention include anti-inflammatory agents. Representative
examples of such agents include nonsteroidal agents ("NSAIDS") such
as salicylates (e.g., salsalate, mesalamine, diflunisal, choline
magnesium trisalicylate), diclofenac, diflunisal, etodolac,
fenoprofen, flurbiprofen, ibuprofen, indomethacin, mefenamic acid,
nabumetone, naproxen, piroxicam, phenylbutazone, ketoprofen,
S-ketoprofen, ketorolac tromethamine, sulindac, tolmetin). Other
anti-inflammatory drugs include steroidal agents such as
beclomethasone, betamethasone, cortisone, dexamethasone,
fluocinolone, flunisolide, fluticasone proprionate,
fluorinated-corticoids, triamcinolone-diacetate, hydorcortisone,
prednisolone, methylprednisolone and prednisone. Immunosuppressive
agents (e.g., adenocorticosteroids, cyclosporin); and
antihistamines and decongestants (e.g., astemizole (histamine
H1-receptor antagonist), azatidine, brompheniramine, clemastine,
chlorpheniramine, cromolyn, cyproheptadine, diphenylimidazole,
diphenhydramine hydrochloride, hydroxyzine, glycyrrhetic acid,
homochlorocyclizine hydrochloride, ketotifen, loratadine,
naphazoline, phenindamine, pheniramine, promethazine, terfenadine,
trimeprazine, tripelennamine, tranilast, and the decongestants
phenylpropanolamine and pseudoephedrine.
[0132] Further therapeutic agents that can be utilized within the
present invention include central nervous system agents.
Representative examples of such agents include anti-depressants
(e.g., Prozac, Paxil, Luvox, Mannerex and Effexor); CNS stimulants
(e.g., pemoline, methamphetamine, dextroamphetamine); hypnotic
agents (e.g., pentobarbital, estazolam, ethchlorynol, flurazepam,
propofol, secobarbital, temazepam, triazolam, quazeparn, zolpidem
tartrate); antimanic agents (e.g., lithium); sedatives and
anticonvulsant barbiturates (e.g., pentobarbitol, phenobarbital,
secobarbital, mephobarbital, butabarbital primidone, amobarbital);
non-barbiturate sedatives (e.g., diphehydramine, doxylamine,
midazolam, diazepam, promethazine, lorazepam, temazepam); and other
miscellaneous hypnotics and sedatives (e.g., methaqualone,
glutethimide, flurazepam, bromovalerylurea, flurazepam,
hydrochloride, haloxazolam, triazolam, phenobarbital, chloral
hydrate, nimetazepam, estazolam).
[0133] Other therapeutic agents that can be utilized within the
present invention include Alzheimer's agents such as tacrine
(reversible cholinesterase inhibitor); Parkinson's disease agents
such as amantadine, bromocriptine mesylate, biperiden, benztropine
mesylate, carbidopa-levodopa, diphenhydramine, hyoscyamine,
levodopa, pergolide mesylate, procyclidine, selegiline HCl,
trihexyphenidyl HCl; and other miscellaneous CNS agents such as
fluphenazine, flutazolam, phenobarbital, methylphenobarbital,
thioridazine, diazepam, benzbromarone, clocapramine hydrochloride,
clotiazepam, chlorpromazine, haloperidol, lithium carbonate.
[0134] Further therapeutic agents that can be utilized within the
present invention include anti-migraine agents (e.g., ergotamine,
methylsergide, propranolol, dihydroergotamine, Sertroline and
Immitrex); Post-cerebral embolism agents (e.g., nicardipine
hydrochloride, cinepazide maleate, pentoxifylline, ifenprodil
tartrate); local anesthetics (e.g., lidocaine, benzocaine, ethyl
aminobenzoate, procaine hydrochloride, dibucaine, procaine;
antiulcer/antireflux agents (e.g., Losec (Omeprazole), aceglutamide
aluminum, cetraxate hydrochloride, pirenzepine hydrochloride,
cimetidine, famotidine, metoclopramide, ranitidine, L-glutamine,
gefarnate, and any stereoisomer of these compounds, and the
pharmaceutically acceptable salts of these compounds, such compound
used singly or in combination of more than one compound, properly
chosen); protease inhibitors (e.g., serine protease,
metalloendoproteases and aspartyl proteases (such as HIV protease,
renin and cathepsin) and thiol protease inhibitors (e.g.,
benzyloxycarbonyl-leu-norleucinal (calpeptin) and
acetyl-leu-leu-norleucinal); phosphodiesterase inhibitors (e.g.,
isobutyl methylxanthine); Phenothiazines; growth factor receptor
antagonists (e.g., platelet-derived growth factor (PDGF), epidermal
growth factor, interleukins, transforming growth factors alpha and
beta, and acidic or basic fibroblast growth factors); antisense
oligonucleotides (e.g., sequences complementary to portions of mRNA
encoding DPGF or other growth factors); and protein kinase
inhibitors (e.g., For tyrosine kinases, protein kinase C, myosin
light chain kinase, Ca.sup.2+/calmodulin kinase II, casein kinase
II);
[0135] Other therapeutic agents that can be utilized within the
present invention include anti-tissue damage agents. Representative
examples of such agents include Superoxide dismutase; Immune
Modulators (e.g., lymphokines, monokines, interferon .alpha.,
.beta., .tau.-1b, .alpha.-n3, .alpha.-2b, .alpha.-2b; Growth
Regulators (e.g., IL-2, tumor necrosis factor, epithelial growth
factor, somatrem, fibronectin, GM-CSF, CSF, platelet derived growth
factor, somatotropin, rG-CSF, epidermal growth factor, IGF-1).
[0136] Other therapeutic agents that can be utilized within the
present invention include monoclonal and polyclonal antibodies
(e.g., those active against: venoms, toxins, tumor necrosis factor,
bacteria); hormones (e.g., estrogen, progestin, testosterone, human
growth hormone, epinephrine, levarterenol, thyroxine,
thyroglobulin, oxytocin, vasopressin, ACTH, somatropin,
thyrotropin, insulin, parathyrin, calcitonin); vitamins (e.g.,
vitamins A, B and its subvitamins, C, D, E, F, G, J, K, N, P, PP,
T, U and their subspecies); amino acids such as arginine,
histidine, proline, lysine, methionine, alanine, phenylalanine,
aspartic acid, glutamic acid, glutamine, threonine, tryptophan,
glycine, isoleucine, leucine, valine; Prostaglandins (e.g.,
E.sub.1, E.sub.2, F.sub.2.alpha., I.sub.2); enzymes such as pepsin,
pancreatin, rennin, papain, trypsin, pancrelipase, chymopapain,
bromelain, chymotrypsin, streptokinase, urokinase, tissue
plasminogen activator, fibrinolysin, desoxyribonuclease, sutilains,
collagenase, asparaginase, heparinase; buffers and salts (e.g.,
NaCl, cations including: Na.sup.+, K.sup.+, Ca.sup.++, Mg.sup.++,
Zn.sup.++, NH.sub.4.sup.+ triethanolamine, anions including:
phosphate, sulfate, chloride, citrate, ascorbate, acetate, borate,
carbonate ions); preservatives (e.g., benzalkonium chloride, Na or
K bisulfite, Na or K thiosulfate, parabans); antigout agents (e.g.,
allopurinol, cochicine, probenicid, sulfinpyrazone); antidepressant
agents such as amitriptyline, amoxapine, desipramine, doxepin,
imipramine, nortriptyline, protriptyline, trimipramine;
contraceptives (e.g., norethindrone combinations, such as with
ethinyl estradiol or with mestranol); and antinauseants/antiemetic
agents (e.g., dimenhydrinate, hydroxyzine, meclizine,
metoclopramide, prochlorperazine, promethazine, scopolamine,
thiethylperazine, triethobenzamide).
[0137] Other therapeutic agents that can likewise be utilized
within the present invention include antiasthmatic agents,
antipsychotic agents, bronchodilators, gold compounds, hypoglycemic
agents, hypolipedemic agents, anesthetics, vaccines, agents which
affect bone metabolism, anti-diarrhetics, fertility agents, muscle
relaxants, appetite suppressants, 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 -1.5, gamma interferon) or T.sub.H2
(e.g., Interleukins-4 and -10) cytokines.
[0138] Although the above therapeutic agents have been provided for
the purposes of illustration, it should be understood that the
present invention is not so limited. For example, although agents
are specifically referred to above, the present invention should be
understood to include analogues, derivatives and conjugates of such
agents. As an illustration, 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). In addition, as will be evident to one of skill
in the art, although the agents set forth above may be noted within
the context of one class, many of the agents listed in fact have
multiple biological activities. Further, more than one therapeutic
agent may be utilized at a time (i.e., in combination), or
delivered sequentially.
Polymeric Carriers
[0139] As noted above, therapeutic compositions of the present
invention may additionally comprise a polymeric carrier. A wide
variety of polymeric carriers may be utilized to contain and or
delivery one or more of the therapeutic agents discussed above,
including for example both biodegradable and non-biodegradable
compositions. Representative examples of biodegradable compositions
include albumin, collagen, gelatin; starch, cellulose
(methylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose
acetate phthalate, cellulose acetate succinate,
hydroxypropylmethylcellulose phthalate), casein, dextrans,
polysaccharides, fibrinogen, poly(D,L lactide),
poly(D,L-lactide-co-glycolide), poly(glycolide),
poly(hydroxybutyrate), poly(alkylcarbonate) and poly(orthoesters),
polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethylene
terephthalate), poly(malic acid), poly(tartronic acid),
polyanhydrides, polyphosphazenes, poly(amino acids and their
copolymers (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, acrylic polymers (polyacrylic acid,
polymethylacrylic acid, polymethylmethacrylate,
polyalkylcynoacrylate), polyethylene, polyproplene, polyamides
(nylon 6,6), polyurathane, poly(ester urathanes), poly(ether
urathanes), poly(ester-urea), polyethers (poly(ethylene oxide),
poly(propylene oxide), pluronics, poly(tetramethylene glycol))xxx,
silicone rubbers and vinyl polymers [polyvinylpyrrolidone,
poly(vinyl alcohol; poly(vinyl acetate phthalate. Polymers may also
be developed which are either anionic (e.g., alginate, carrageenin,
caboxymethyl cellulose and poly(acrylic acid), or cationic (e.g.,
Chitosan, poly-1-lysine, polyethylenimine, and poly (allyl amine))
(see generally, Dunn et al., J. Applied Polymer Sci. 50:353-365,
1993; Cascone et al., J. Materials Sci.: Materials in Medicine
5:770-774, 1994; Shiraishi et al., Biol. Pharm. Bull.
16(1):1164-1168, 1993; Thacharodi and Rao, Int'l J. Pharm.
120:115-118, 1995; Miyazaki et al., Int'l J. Pharm. 118:257-263,
1995). 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.
[0140] Polymeric carriers can be fashioned in a variety of forms,
with desired release characteristics and/or with specific desired
properties. For example, polymeric carriers may be fashioned to
release a therapeutic agent upon exposure to a specific triggering
event such as pH (see, e.g., Heller et al., "Chemically
Self-Regulated Drug Delivery Systems," in Polymers in Medicine III,
Elsevier Science Publishers B.V., Amsterdam, 1988, pp. 175-188;
Kang et al., J. Applied Polymer Sci. 48:343-354, 1993; Dong et al.,
J. Controlled Release 19:171-178, 1992; Dong and Hoffman, J.
Controlled Release 15:141-152, 1991; Kim et al., J. Controlled
Release 28:143-152, 1994; Cornejo-Bravo et al., J. Controlled
Release 33:223-229, 1995; Wu and Lee, Pharm. Res. 10(10):1544-1547,
1993; Serres et al., Pharm. Res. 13(2):196-201, 1996; Peppas,
"Fundamentals of pH- and Temperature-Sensitive Delivery Systems,"
in Gurny et al. (eds.), Pulsatile Drug Delivery, Wissenschaftliche
Verlagsgesellschaft mbH, Stuttgart, 1993, pp. 41-55; Doelker,
"Cellulose Derivatives," 1993, in Peppas and Langer (eds.),
Biopolymers I, Springer-Verlag, Berlin). Representative examples of
pH-senstive polymers include poly(acrylic acid) and its derivatives
(including for example, homopolymers such as poly(aminocarboxylic
acid); poly(acrylic acid); poly(methyl acrylic acid)), copolymers
of such homopolymers, and copolymers of poly(acrylic acid) and
acrylmonomers such as those discussed above. Other pH sensitive
polymers include polysaccharides such as cellulose acetate
phthalate; hydroxypropylmethylcellulose phthalate;
hydroxypropylmethylcellulose acetate succinate; cellulose acetate
trimellilate; and chitosan. Yet other pH sensitive polymers include
any mixture of a pH sensitive polymer and a water soluble
polymer.
[0141] Likewise, polymeric carriers can be fashioned which are
temperature sensitive (see, e.g., Chen et al., "Novel Hydrogels of
a Temperature-Sensitive Pluronic Grafted to a Bioadhesive
Polyacrylic Acid Backbone for Vaginal Drug Delivery," in Proceed.
Intern. Symp. Control. Rel. Bioact. Mater. 22:167-168, Controlled
Release Society, Inc., 1995; Okano, "Molecular Design of
Stimuli-Responsive Hydrogels for Temporal Controlled Drug
Delivery," in Proceed. Intern. Symp. Control. Rel. Bioact. Mater.
22:111-112, Controlled Release Society, Inc., 1995; Johnston et
al., Pharm. Res. 9(3):425-433, 1992; Tung, Int'l J. Pharm.
107:85-90, 1994; Harsh and Gehrke, J. Controlled Release
17:175-186, 1991; Bae et al., Pharm. Res. 8(4):531-537, 1991;
Dinarvand and D'Emanuele, J. Controlled Release 36:221-227, 1995;
Yu and Grainger, "Novel Thermo-sensitive Amphiphilic Gels: Poly
N-isopropylacrylamide-co-s- odium acrylate-co-n-N-alkylacrylamide
Network Synthesis and Physicochemical Characterization," Dept. of
Chemical & Bioligal Sci., Oregon Graduate Institute of Science
& Technology, Beaverton, Oreg., pp. 820-821; Zhou and Smid,
"Physical Hydrogels of Associative Star Polymers," Polymer Research
Institute, Dept. of Chemistry, College of Environmental Science and
Forestry, State Univ. of New York, Syracuse, N.Y., pp. 822-823;
Hoffman et al., "Characterizing Pore Sizes and Water `Structure` in
Stimuli-Responsive Hydrogels," Center for Bioengineering, Univ. of
Washington, Seattle, Wash., p. 828; Yu and Grainger,
"Thermo-sensitive Swelling Behavior in Crosslinked
N-isopropylacrylamide Networks: Cationic, Anionic and Ampholytic
Hydrogels," Dept. of Chemical & Biological Sci., Oregon
Graduate Institute of Science & Technology, Beaverton, Oreg.,
pp. 829-830; Kim et al., Pharm. Res. 9(3):283-290, 1992; Bae et
al., Pharm. Res. 8(5):624-628, 1991; Kono et al., J. Controlled
Release 30:69-75, 1994; Yoshida et al., J. Controlled Release
32:97-102, 1994; Okano et al., J. Controlled Release 36:125-133,
1995; Chun and Kim, J. Controlled Release 38:39-47, 1996;
D'Emanuele and Dinarvand, Int'l J. Pharm. 118:237-242, 1995; Katono
et al., J. Controlled Release 16:215-228, 1991; Hoffman, "Thermally
Reversible Hydrogels Containing Biologically Active Species," in
Migliaresi et al. (eds.), Polymers in Medicine III, Elsevier
Science Publishers B.V., Amsterdam, 1988, pp. 161-167; Hoffman,
"Applications of Thermally Reversible Polymers and Hydrogels in
Therapeutics and Diagnostics," in Third International Symposium on
Recent Advances in Drug Delivery Systems, Salt Lake City, Utah,
Feb. 24-27, 1987, pp. 297-305; Gutowska et al., J. Controlled
Release 22:95-104, 1992; -Palasis and Gehrke, J. Controlled Release
18:1-12, 1992; Paavola et al., Pharm. Res. 12(12):1997-2002,
1995).
[0142] Representative examples of thermogelling polymers, and their
gelatin temperature (LCST (.degree. C.)) include homopolymers such
as poly(N-methyl-N-n-propylacrylamide), 19.8;
poly(N-n-propylacrylamide), 21.5;
poly(N-methyl-N-isopropylacrylamide), 22.3;
poly(N-n-propylmethacry- lamide), 28.0;
poly(N-isopropylacrylamide), 30.9; poly(N, n-diethylacrylamide),
32.0; poly(N-isopropylmethacrylamide), 44.0;
poly(N-cyclopropylacrylamide), 45.5; poly(N-ethylmethyacrylamide),
50.0; poly(N-methyl-N-ethylacrylamide), 56.0;
poly(N-cyclopropylmethacrylamide)- , 59.0; poly(N-ethylacrylamide),
72.0. Moreover thermogelling polymers may be made by preparing
copolymers between (among) monomers of the above, or by combining
such homopolymers with other water soluble polymers such as
acrylmonomers (e.g., acrylic acid and derivatives thereof such as
methylacrylic acid, acrylate and derivatives thereof such as butyl
methacrylate, acrylamide, and N-n-butyl acrylamide).
[0143] Other representative examples of thermogelling polymers
include cellulose ether derivatives such as hydroxypropyl
cellulose, 41.degree. C.; methyl cellulose, 55.degree. C.;
hydroxypropylmethyl cellulose, 66.degree. C.; and ethylhydroxyethyl
cellulose, and pluronics such as F-127, 10-15.degree. C.; L-122,
19.degree. C.; L-92, 26.degree. C.; L-81, 20.degree. C.; and L-61,
24.degree. C.
[0144] A wide variety of forms may be fashioned by the polymeric
carriers of the present invention, 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). Therapeutic agents 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, therapeutic 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.
[0145] Preferably, therapeutic compositions of the present
invention are fashioned in a manner appropriate to the intended
use. Within certain aspects of the present invention, the
therapeutic composition should be biocompatible, and release one or
more therapeutic agents over a period of several days to months.
For example, "quick release" or "burst" therapeutic compositions
are provided that release greater than 10%, 20%, or 25% (w/v) of a
therapeutic agent (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 agent. Within other embodiments, "low
release" therapeutic compositions are provided that release less
than 1% (w/v) of a therapeutic agent over a period of 7 to 10 days.
Further, therapeutic compositions of the present invention should
preferably be stable for several months and capable of being
produced and maintained under sterile conditions.
[0146] Within certain aspects of the present invention, therapeutic
compositions may be fashioned in any size ranging from 50 nm to 500
.mu.m, depending upon the particular use. 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.
[0147] Therapeutic 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, therapeutic 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.
[0148] Within yet other aspects of the invention, the therapeutic
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 have controlled
permeability.
[0149] Within certain embodiments of the invention, the therapeutic
compositions may also comprise additional ingredients such as
surfactants (e.g. pluronics such as F-127, L-122, L-92, L-81, and
L-61).
[0150] 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. Within
alternative embodiments, hydrophobic compounds may be contained
within a hydrophobic core, and this core contained within a
hydrophilic shell. For example, as described within the Examples,
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.
[0151] 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).
[0152] Representative examples of the incorporation of therapeutic
agents such as those described above into a polymeric carriers to
form a therapeutic composition, is described in more detail below
in the Examples.
Other Carriers
[0153] Other carriers that may likewise be utilized to contain and
deliver the therapeutic agents described herein include:
hydroxypropyl .beta. cyclodextrin (Cserhati and Hollo, Int. J.
Pharm. 108:69-75, 1994), liposomes (see e.g., Sharma et al., Cancer
Res. 53:5877-5881, 1993; Sharma and Straubinger, Pharm. Res.
11(60):889-896, 1994; WO 93/18751; U.S. Pat. No. 5,242,073),
liposome/gel (WO 94/26254), nanocapsules (Bartoli et al., J.
Microencapsulation 7(2):191-197, 1990), micelles (Alkan-Onyuksel et
al., Pharm. Res. 11(2):206-212, 1994), implants (Jampel et al.,
Invest. Ophthalm. Vis. Science 34(11):3076-3083, 1993; Walter et
al., Cancer Res. 54:22017-2212, 1994) nanoparticles (Violante and
Lanzafame PAACR), nanoparticles--modified (U.S. Pat. No.
5,145,684), nanoparticles (surface modified) (U.S. Pat. No.
5,399,363), taxol emulsion/solution (U.S. Pat. No. 5,407,683),
micelle (surfactant) (U.S. Pat. No. 5,403,858), synthetic
phospholipid compounds (U.S. Pat. No. 4,534,899), gas borne
dispersion (U.S. Pat. No. 5,301,664), liquid emulsions, foam spray,
gel lotion cream, ointment, dispersed vesicles, particles or
droplets solid- or liquid-aerosols, microemulsions (U.S. Pat. No.
5,330,756), polymeric shell (nano- and micro-capsule) (U.S. Pat.
No. 5,439,686), taxoid-based compositions in a surface-active agent
(U.S. Pat. No. 5,438,072), emulsion (Tarr et al., Pharm Res. 4:
62-165, 1987), nanospheres (Hagan et al., Proc. Intern. Symp.
Control Rel. Bioact. Mater. 22, 1995; Kwon et al., Pharm Res.
12(2):192-195; Kwon et al., Pharm Res. 10(7):970-974; Yokoyama et
al., J. Contr. Rel. 32:269-277, 1994; Gref et al., Science
263:1600-1603, 1994; Bazile et al., J. Pharm. Sci. 84:493-498,
1994) and implants (U.S. Pat. No. 4,882,168).
[0154] As discussed in more detail below, therapeutic agents of the
present invention, which are optionally incorporated within one of
the carriers described herein to form a therapeutic composition,
may be prepared and utilized to treat or prevent a wide variety of
diseases.
Treatment or Prevention of Disease
[0155] As noted above, the present invention provides methods for
treating or preventing a wide variety of diseases associated with
the obstruction of body passageways, including for example,
vascular diseases, neoplastic obstructions, inflammatory diseases,
and infectious diseases.
[0156] For example, within one aspect of the present invention a
wide variety of therapeutic compositions as described herein may be
utilized to treat vascular diseases that cause obstruction of the
vascular system. Representative examples of such diseases include
artherosclerosis of all vessels (around any artery, vein or graft)
including, but not restricted to: the coronary arteries, aorta,
iliac arteries, carotid arteries, common femoral arteries,
superficial femoral arteries, popliteal arteries, and at the site
of graft anastomosis; vasospasms (e.g, coronary vasospasms and
Raynaud's Disease); restenosis (obstruction of a vessel at the site
of a previous intervention such as balloon angioplasty, bypass
surgery, stent insertion and graft insertion); inflammatory and
autoimmune conditions (e.g. Temporal Arteritis, vasculitis).
[0157] Briefly, in vascular diseases such as atherosclerosis, white
cells, specifically monocytes and T lymphocytes adhere to
endothelial cells, especially at locations of arterial branching.
After adhering to the endothelium, leukocytes migrate across the
endothelial cell lining in response to chemostatic stimuli, and
accumulate in the intima of the arterial wall, along with smooth
muscle cells. This initial lesion of athersosclerosis development
is known as the "fatty streak". Monocytes within the fatty streak
differentiate into macrophages; and the macrophages and smooth
muscle cells progressively take up lipids and lipoprotein to become
foam cells.
[0158] As macrophages accumulate, the overlying endothelium becomes
mechanically disrupted and chemically altered by oxidized lipid,
oxygen-derived free radicals and proteases which are released by
macrophages. Foam cells erode through the endothelial surface
causing micro-ulcerations of the vascular wall. Exposure of
potentially thrombogenic subendothelial tissues (such as collagen
and other proteins) to components of the bloodstream results in
adherence of platelets to regions of disrupted endothelium.
Platelet adherence and other events triggers the elaboration and
release of growth factors into this mileau, including
platelet-derived growth factor (PDGF), platelet activating factor
(PAF), and interleukins 1 and 6 (IL-1, IL-6). These paracrine
factors are thought to stimulate vascular smooth muscle cell (VSMC)
migration and proliferation.
[0159] In the normal (non-diseased) blood vessel wall, vascular
smooth muscle cells have a contractile phenotype and low index of
mitotic activity. However, under the influence of cytokines and
growth factors released by platelets, macrophages and endothelial
cells, VSMC undergo phenotypic alteration from mature contractile
cells to immature secretory cells. The transformed VSMC proliferate
in the media of the blood vessel wall, migrate into the intima,
continue to proliferate in the intima and generate large quantities
of extracellular matrix. This transforms the evolving vascular
lesion into a fibrous plaque. The extracellular matrix elaborated
by secretory VSMC includes collagen, elastin, glycoprotein and
glycosaminoglycans, with collagen comprising the major
extracellular matrix component of the atherosclerotic plaque.
Elastin and glycosaminoglycans bind lipoproteins and also
contribute to lesion growth. The fibrous plaque consists of a
fibrous cap of dense connective tissue of varying thickness
containing smooth muscle cells and overlying macrophages, T cells
and extracellular material.
[0160] In addition to PDGF, IL-1 and IL-6, other mitogenic factors
are produced by cells which infiltrate the vessel wall including:
transforming growth factor beta (TGF-.beta.), fibroblast growth
factor (FGF), thrombospondin, serotonin, thromboxane A.sub.2,
norepenephrine, and angiotension II. This results in the
recruitment of more cells, elaboration of further extracellular
matrix and the accumulation of additional lipid. This progressively
enlarges the atherosclerotic lesion until it significantly
encroaches upon the vascular lumen. Initially, obstructed blood
flow through the vascular tube causes ischemia of the tissues
distal to the atherosclerotic plaque only when increased flow is
required--later as the lesion further blocks the artery, ischemia
occurs at rest.
[0161] Macrophages in the enlarging atherosclerotic plaque release
oxidized lipid, free radicals, elastases, and collageneses that
cause cell injury and necrosis of neighbouring tissues. The lesion
develops a necrotic core and is transformed into a complex plaque.
Complex plaques are unstable lesions that can: break off causing
embolization; local hemorrhage (secondary to rupture of the vasa
vasora supplying the plaque which results in lumen obstruction due
to rapid expansion of the lesion); or ulceration and fissure
formation (this exposes the thrombogenic necrotic core to the blood
stream producing local thrombosis or distal embolization). Even
should none of the above sequela occur, the adherent thrombus may
become organized and incorporated into the plaque, thereby
accelerating its growth. Furthermore, as the local concentrations
of fibrinogen and thrombin increase, proliferation of vascular
smooth muscle cells within the media and intima is stimulated; a
process which also ultimately leads to additional narrowing of the
vessel.
[0162] The intima and media of normal arteries are oxygenated and
supplied with nutrition from the lumen of the artery or from the
vasa vasorum in the adventitia. With the development of
atherosclerotic plaque, microvessels arising from the adventitial
vasa vasorum extend into the thickened intima and media. This
vascular network becomes more extensive as the plaque worsens and
diminishes with plaque regression.
[0163] Hemorrhage from these microvessels may precipitate sudden
expansion and rupture of plaque in association with arterial
dissection, ulceration, or thrombosis. It has also been postulated
that the leakage of plasma proteins from these microvessels may
attract inflammatory infiltrates into the region and these
inflammatory cells may contribute to the rapid growth of
atherosclerotic plaque and to associated complications (through
local edema and inflammation).
[0164] In order to treat vascular diseases, such as those discussed
above, a wide variety of therapeutic agents (either with or without
a carrier) may be delivered to the external portion of the body
passageway, or to smooth muscle cells via the adventia of the body
passageway. Particularly preferred therapeutic agents in this
regard include anti-angiogenic factors, inhibitors of platelet
adhesion/aggregation (e.g., aspirin, dipyridamole, thromboxane
synthesis inhibitors, fish oils that result in production of
thromboxane AE rather than the more potent thromboxane A2,
antibodies against the platelet IIb/IIIa receptors that binds
fibrinogen and prostacyclin), vasodilators (e.g., calcium entry
blockers, and the nitric oxide donors nitroglycerine,
nitroprusside, and molsidomine) and anthithrombotics and thrombin
antagonists (e.g., heparin (low-molecular-weight heparins, warfarin
andudin). Other therapeutics which may be utilized include
anti-inflammatory agents (e.g., glucorticoids, dexamethasone and
methylprednisolone), growth factor inhibitors (e.g., PDGF
antagonist such as trapidil; receptor inhibitors (e.g., inhibitors
of the receptors for FGF, VEGF, PDGF and TNF), including inhibitors
of tyrosine kinase and promoters of tyrosine phosphatase;
somatostatin analogs, including angiopeptin; angiotensin converting
enzyme inhibitors; and 5HT.sub.2 serotenergic receptor antagonists
such as ketanserin). Yet other therapeutic agents include
anti-proliferative agents (e.g., colchicine, heparin, beta (e.g.,
P-32) or gamma emitters (e.g., Ir-192), calcium-entry blockers such
as verapamil, diltiazem and nifedipine, cholesterol-lowering HMB
Co-A reductase inhibitors such as lovastatin, compounds which
disrupt microtubule function such as paclitaxel and nitric oxide
donors as discussed above), and promoters of re-endothelialization
(e.g., bFGF and vascular endothelial cell growth factor).
[0165] Within other aspects of the invention, the therapeutic
agents or compositions described herein may be utilized to treat
neoplastic obstructions. Briefly, as utilized herein, a "neoplastic
obstruction" should be understood to include any neoplastic (benign
or malignant) obstruction of a bodily tube regardless of tube
location or histological type of malignancy present. Representative
examples include gastrointestinal diseases (e.g., oral-pharyngeal
carcinoma (adenocarcinoma, esophageal carcinoma (squamous cell,
adenocarcinoma, lymphoma, melanoma), gastric carcinoma
(adenocarcinoma, linitis plastica, lymphoma, leiomyosarcoma), small
bowel tumors (adenomas, leiomyomas, lipomas, adenocarcinomas,
lymphomas, carcinoid tumors), colon cancer (adenocarcinoma) and
anorectal cancer); biliary tract diseases (e.g., neoplasms
resulting in biliary obstruction such as pancreatic carcinoma
(ductal adenocarcinoma, islet cell tumors, cystadenocarcinoma),
cholangiocarcinoma and hepatocellular carcinoma); pulmonary
diseases (e.g., carcinoma of the lung and/or tracheal/bronchial
passageways (small cell lung cancer, non-small cell lung cancer);
female reproductive diseases (e.g., malignancies of the fallopian
tubes, uterine cancer, cervical cancer, vaginal cancer); male
reproductive diseases (e.g, testicular cancer, cancer of the
epididymus, tumors of the vas deferens, prostatic cancer, benign
prostatic hypertrophy); and urinary tract diseases (e.g., renal
cell carcinoma, tumors of the renal pelvis, tumors of the urinary
collection system such as transitional cell carcinoma, bladder
carcinoma, and urethral obstructions due to benign strictures, or
malignancy).
[0166] As an example, benign prostatic hyperplasia (BPH) is the
enlargement of the prostate, particularly the central portion of
the gland which surrounds the urethra, which occurs in response to
prolonged androgenic stimulation. It affects more than 80% of the
men over 50 years of age. This enlargement can result in
compression of the portion of the urethra which runs through the
prostate, resulting in bladder outflow tract obstruction, i.e., an
abnormally high bladder pressure is required to generate urinary
flow. In 1980, 367,000 transurethral resections of the prostate
were performed in the United States as treatment for BPH. Other
treatments include medication, transurethral sphincterotomy,
transurethral laser or microwave, transurethral hyperthermia,
transurethral ultrasound, transrectal microwave, transrectal
hyperthermia, transrectal ultrasound and surgical removal. All have
disadvantages including interruption of the sphincter mechanism
resulting in incontinence and stricture formation.
[0167] In order to treat neoplastic diseases, such as those
discussed above, a wide variety of therapeutic agents (either with
or without a polymeric carrier) may be delivered to the external
portion of the body passageway, or to smooth muscle cells via the
adventia of the body passageway. Particularly preferred therapeutic
agents in this regard include anti-angiogenic, anti-proliferative
or anti-neoplastic agents discussed above, including for example,
compounds which disrupt microtuble function, such as paclitaxel and
derivatives or analogues thereof.
[0168] For example, within one preferred embodiment a needle or
catheter is guided into the prostate gland adjacent to the urethra
via the transrectal route (or alternatively transperineally) under
ultrasound guidance and through this deliver a therapeutic agent,
preferably in several quadrants of the gland, particularly around
the urethra. The needle or catheter can also be placed under direct
palpation or under endoscopic, fluoroscopic, CT or MRI guidance,
and administered at intervals. As an alternative, the placement of
pellets via a catheter or trocar can also be accomplished. The
above procedures can be accomplished alone or in conjunction with a
stent placed in the prostatic urethra. By avoiding urethral
instrumentation or damage to the urethra, the sphincter mechanism
would be left intact, avoiding incontinence, and a stricture is
less likely.
[0169] Within other aspects of the invention, methods are provided
for preventing or treating inflammatory diseases which affect or
cause the obstruction of a body passageway. Inflammatory diseases
include both acute and chronic inflammation which result in
obstruction of a variety of body tubes. Representative examples
include vasculitis (e.g., Giant cell arteritis (temporal arteritis,
Takayasu's arteritis), polyarteritis nodosa, allergic angiitis and
granulomatosis (Churg-Strauss disease), polyangiitis overlap
syndrome, hypersensitivity vasculitis (Henoch-Schonlein purpura),
serum sickness, drug-induced vasculitis, infectious vasculitis,
neoplastic vasculitis, vasculitis associated with connective tissue
disorders, vasculitis associated with congenital deficiencies of
the complement system), Wegener's granulomatosis, Kawasaki's
disease, vasculitis of the central nervous system, Buerger's
disease and systemic sclerosis); gastrointestinal tract diseases
(e.g., pancreatitis, Crohn's Disease, Ulcerative Colitis,
Ulcerative Proctitis, Primary Sclerosing Cholangitis, benign
strictures of any cause including ideopathic (e.g., strictures of
bile ducts, esophagus, duodenum, small bowel or colon));
respiratory tract diseases (e.g, asthma, hypersensitivity
pneumonitis, asbestosis, silicosis, and other forms of
pneumoconiosis, chronic bronchitis and chronic obstructive airway
disease); nasolacrimal duct diseases (e.g., strictures of all
causes including ideopathic); and eustachean tube diseases (e.g.,
strictures of all causes including ideopathic).
[0170] In order to treat inflammatory diseases, such as those
discussed above, a wide variety of therapeutic agents (either with
or without a carrier) may be delivered to the external portion of
the body passageway, or to smooth muscle cells via the adventia of
the body passageway. Particularly preferred therapeutic agents in
this regard include both nonsteroidal agents ("NSAIDS") and
steroidal agents, as well as the anti-angiogenic factors discussed
above. Other agents which may also be utilized include a wide
variety of anti-angiogenic facts, including for example compounds
which disrupt microtubule function, such as paclitaxel, and lighter
"d" group transition metals.
[0171] Within yet other aspects of the present invention, methods
are provided for treating or preventing infectious diseases that
are associated with, or causative of, the obstruction of a body
passageway. Briefly, infectious diseases include several acute and
chronic infectious processes can result in obstruction of body
passageways including for example, obstructions of the male
reproductive tract (e.g., strictures due to urethritis,
epididymitis, prostatitis); obstructions of the female reproductive
tract (e.g., vaginitis, cervicitis, pelvic inflammatory disease
(e.g., tuberculosis, gonococcus, chlamydia, enterococcus and
syphilis); urinary tract obstructions (e.g., cystitis, urethritis);
respiratory tract obstructions (e.g., chronic bronchitis,
tuberculosis, other mycobacterial infections (MAI, etc.), anaerobic
infections, fungal infections and parasitic infections) and
cardiovascular obstructions (e.g., mycotic aneurysms and infective
endocarditis).
[0172] In order to treat infectious diseases, such as those
discussed above, a wide variety of therapeutic agents (either with
or without a carrier) may be delivered to the external portion of
the body passageway, or to smooth muscle cells via the adventia of
the body passageway. Particularly preferred therapeutic agents in
this regard include a wide variety of antibiotics as discussed
above.
Formulation and Administration
[0173] As noted above, therapeutic 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
therapeutic agents, 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).
[0174] Therapeutic agents 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.
[0175] As noted above, therapeutic agents, therapeutic
compositions, or pharmaceutical compositions provided herein may be
prepared for administration by a variety of different routes,
including for example, directly to a body passageway under direct
vision (e.g., at the time of surgery or via endoscopic procedures)
or via percutaneous drug delivery to the exterior (adventitial)
surface of the body passageway (e.g., perivascular delivery). 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.
[0176] Briefly, perivascular drug delivery involves percutaneous
administration of localized (often sustained release) therapeutic
formulations using a needle or catheter directed via ultrasound,
CT, fluoroscopic, MRI or endoscopic guidance to the disease site.
Alternatively the procedure can be performed intra-operatively
under direct vision or with additional imaging guidance. Such a
procedure can also be performed in conjunction with endovascular
procedures such as angioplasty, atherectomy, or stenting or in
association with an operative arterial procedure such as
endarterectomy, vessel or graft repair or graft insertion.
[0177] For example, within one embodiment, polymeric paclitaxel
formulations can be injected into the vascular wall or applied to
the adventitial surface allowing drug concentrations to remain
highest in regions where biological activity is most needed. This
has the potential to reduce local "washout" of the drug that can be
accentuated by continuous blood flow over the surface of an
endovascular drug delivery device (such as a drug-coated stent).
Administration of effective therapeutic agents to the external
surface of the vascular tube can reduce obstruction of the tube and
reduce the risk of complications associated with intravascular
manipulations (such as restenosis, embolization, thrombosis, plaque
rupture, and systemic drug toxicity).
[0178] For example, in a patient with narrowing of the superficial
femoral artery, balloon angioplasty would be performed in the usual
manner (i.e., passing a balloon angioplasty catheter down the
artery over a guide wire and inflating the balloon across the
lesion). Prior to, at the time of, or after angioplasty, a needle
would be inserted through the skin under ultrasound, fluoroscopic,
or CT guidance and a therapeutic agent (e.g., paclitaxel
impregnated into a slow release polymer) would be infiltrated
through the needle or catheter in a circumferential manner directly
around the area of narrowing in the artery. This could be performed
around any artery, vein or graft, but ideal candidates for this
intervention include diseases of the carotid, coronary, iliac,
common femoral, superficial femoral and popliteal arteries and at
the site of graft anastomosis. Logical venous sites include
infiltration around veins in which indwelling catheters are
inserted.
[0179] The therapeutic agents, therapeutic 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 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.
[0180] The following examples are offered by way of illustration,
and not by way of limitation.
EXAMPLES
Example 1
Manufacture of "Pastes"
[0181] 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,
drug-loaded-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.
[0182] A. Procedure for Producing Thermopaste
[0183] 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).
[0184] 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.
[0185] 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.
[0186] For application, the syringe may be reheated to 60.degree.
C. and administered as a liquid which solidifies when cooled to
body temperature.
[0187] B. Procedure for Producing Nanospray
[0188] 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).
[0189] 1. Preparation of 5% (w/v) Polymer Solutions
[0190] 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.
[0191] 2. Preparation of 3.5% (w/v) Stock Solution of PVA
[0192] 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 2). 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.
[0193] 3. Procedure for Producing Nanospray
[0194] 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.
[0195] 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.
[0196] 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.
[0197] C. Manufacture of Paclitaxel Loaded Nanospray
[0198] 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. 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).
[0199] 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.
[0200] Follow the procedures as described above, except that
polymer/drug (e.g., paclitaxel) stock solution is substituted for
the polymer solution.
[0201] D. Procedure for Producing Film
[0202] 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.
[0203] 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.
[0204] 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).
[0205] 1. Procedure for Producing Films--Melt Casting
[0206] 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.
[0207] 2. Procedure for Producing Films--Solvent Casting
[0208] 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.
[0209] 3. Procedure for Producing Films--Sprayed
[0210] 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.
[0211] 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.
[0212] E. Procedure for Producing Nanopaste
[0213] 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.
[0214] 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).
[0215] 1. Preparation of 5% w/v Carbopol Gel
[0216] 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 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.
[0217] 2. Procedure for Producing Nanopaste
[0218] 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 2
Manufacture of Microspheres
[0219] 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.
[0220] A. Preparation of 5% (w/v) Polymer Solutions
[0221] 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.
[0222] B. Preparation of 5% (w/v) Stock Solution of PVA
[0223] 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.
[0224] C. Procedure for Producing Microspheres
[0225] Based on the size of microspheres being made (see Table I),
100 ml of the PVA solution (concentrations given in Table I) 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. (+/-1.degree. C.) for 10 minutes.
Based on the size of microspheres being made (see Table I), 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 I), 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 I). A 5 ml automatic pipetter or vacuum
suction is then utilized to draw 45 ml of the PVA solution off of
each microsphere pellet.
1TABLE I PVA concentrations, stir speeds, and centrifugal force
requirements for each diameter range of microspheres. PRODUC- TION
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., concen- dilute 5% stock undiluted stock)
dilute 5% stock tration with distilled water with distilled water
Starting 500 rpm .+-. 500 rpm .+-. 3000 rpm .+-. Stir Speed 50 rpm
50 rpm 200 rpm Adjusted 500 rpm .+-. 500 rpm .+-. 2500 rpm .+-.
Stir Speed 50 rpm 50 rpm 200 rpm Centrifuge 1000 g .+-. 1000 g .+-.
10 000 g .+-. Force 100 g 100 g 1000 g (Table top model) (Table top
model) (High speed model)
[0226] 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 I). 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.
[0227] D. Drying of 10 .mu.m to 30 .mu.m or 30 .mu.m to 100 .mu.m
Diameter Microspheres
[0228] 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.
[0229] E. Drying of 0.1 .mu.m to 3 .mu.m Diameter Microspheres
[0230] 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.
[0231] F. Manufacture of Paclitaxel Loaded Microsphere
[0232] 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.
[0233] Microspheres containing paclitaxel may then be produced
essentially as described above in steps (C) through (E).
Example 3
Surfactant Coated Microspheres
[0234] A. Materials and Methods
[0235] 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 2. Size ranged from 10 to 100 um with a mean diameter 45
um.
[0236] 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").
[0237] 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.
[0238] 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.
[0239] B. Results
[0240] FIG. 1 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. 1). 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.
[0241] FIGS. 2-5 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.
[0242] 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.
[0243] FIGS. 6-9 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.
[0244] 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 4
Encapsulation of Paclitaxel
[0245] 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.
[0246] 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.
[0247] For baccatin, 100+/-15 .mu.g [83+/-23 .mu.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
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
[0248] 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.
[0249] 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). Distilled water is used throughout.
[0250] A. Preparation of Microspheres
[0251] Microspheres are prepared essentially as described in
Example 2 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.
[0252] The % aggregation is determined from 1 % aggregation = 1 - (
weight of pelleted microspheres ) .times. 100 initial polymer
weight
[0253] 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.
[0254] B. Encapsulation Efficiency
[0255] 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.
[0256] C. Drug Release Studies
[0257] 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.
[0258] D. Scanning Electron Microscopy (SEM)
[0259] Microspheres are placed on sample holders, sputter coated
with gold and micrographs obtained using a Philips 501B SEM
operating at 15 kV.
[0260] E. CAM Studies
[0261] 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.
[0262] F. Results
[0263] 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. 10A. 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.
[0264] 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. 10B and 10C, 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.
[0265] The time course of paclitaxel release from 0.6% w/v loaded
50:50 EVA:PLA microspheres is shown in FIG. 10D 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.
[0266] The paclitaxel loaded microspheres (0.6% w/v loading) are
tested using the CAM assay and the results are shown in FIG. 10E.
The paclitaxel microspheres released sufficient drug to produce a
zone of avascularity in the surrounding tissue (FIG. 10F). Note
that immediately adjacent to the microspheres ("MS" in FIGS. 10E
and 10F) 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.)
[0267] Discussion
[0268] Peritubular drug administration is a mildly invasive
surgical technique. Therefore, ideally, a perivascular formulation
of an anti-proliferative drug such as paclitaxel would release the
drug at the tumor or disease 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).
[0269] 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.
[0270] 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.
[0271] FIG. 10A 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.
[0272] 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.
[0273] 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).
[0274] 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.
[0275] 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. 10C), 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.
[0276] 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.
[0277] 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. 10E and 10F).
Example 6
Therapeutic Agent Encapsulation in poly(.epsilon.-caprolactone)
Microspheres. Inhibition of Angiogenesis on the CAM Assay by
Paclitaxel-Loaded Microspheres
[0278] This example evaluates the in vitro release rate profile of
paclitaxel from biodegradable microspheres of
poly(.epsilon.-caprolactone- ) and demonstrates the in vivo
anti-angiogenic activity of paclitaxel released from these
microspheres when placed on the CAM.
[0279] Reagents which were utilized in these experiments include:
poly(.epsilon.-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 12,000-18,000, 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.
[0280] A. Preparation of Microspheres
[0281] Microspheres are prepared essentially as described in
Example 2 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.
[0282] B. Encapsulation Efficiency
[0283] 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.
[0284] C. Drug Release Studies
[0285] 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.
[0286] D. CAM Studies
[0287] 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.
[0288] E. Scanning Electron Microscopy
[0289] Microspheres are placed on sample holders, sputter-coated
with gold and then placed in a Philips 501B Scanning Electron
Microscope operating at 15 kV.
[0290] F. Results The size range for the microsphere samples is
between 30-100 um, although there is evidence in all
paclitaxel-laded 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.
[0291] The time courses of paclitaxel release from 1%, 2% and 5%
loaded PCL microspheres are shown in FIG. 11A. 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.
[0292] FIG. 11B shows CAMs treated with control PCL microspheres,
and FIG. 11C 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.
[0293] G. Discussion
[0294] 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.
[0295] The biphasic release profile for paclitaxel is typical of
the release pattern for many drugs from biodegradable polymer
matrices. Poly(.epsilon.-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 peritubular 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.
[0296] 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. 11C.
Example 7
Therapeutic Agent-Loaded Peritubular Polymeric Films Composed of
Ethylene Vinyl Acetate and a Surfactant
[0297] Two types of films are investigated within this example:
pure EVA films loaded with paclitaxel and EVA/surfactant blend
films loaded with paclitaxel.
[0298] 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.
[0299] 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. 12C and 12D 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.
[0300] Results of experiments with these films are shown below in
FIGS. 12A-E. Briefly, FIG. 12A shows paclitaxel release (in mg)
over time from pure EVA films. FIG. 12B 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.
[0301] Physical strength and elasticity of the films is assessed in
FIG. 12E. Briefly, FIG. 12E 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 peritubular clinical applications without causing
permanent deformation of the compound.
[0302] 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 8
Incorporating Methoxypolyethylene Glycol 350 (MePEG) into
poly(.epsilon.-caprolactone) to Develop a Formulation for the
Controlled Delivery of Therapeutic Agents from a Paste
[0303] 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. C. to -5.degree. C.
[0304] A. Preparation of a MePEG/PCL Paclitaxel-Containing
Paste
[0305] 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.
[0306] B. Analysis of Melting Point
[0307] 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. 13A and 13B. Briefly,
as shown in FIG. 13A 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. 13A.
This lower melting point also translates into an increased time for
the polymer blends to solidify from melt as shown in FIG. 13B. A
30:70 blend of MePEG:PCL takes more than twice as long to solidify
from the fluid melt than does PCL alone.
[0308] C. Measurement of Brittleness
[0309] 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. 13C. Points are
given as the average of four measurements+/-1 S.D.
[0310] 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.
[0311] D. Measurement of Paclitaxel Release
[0312] 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. 13D, 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.
[0313] E. Effect of Varying Quantities of MePEG on Paclitaxel
Release
[0314] 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. 13E, the amount of
MePEG in the formulation does not affect the amount of paclitaxel
that is released.
[0315] F. Effect of Varying Quantities of Paclitaxel on the Total
Amount of Paclitaxel Released from a 20% MePEG/PCL Blend
[0316] 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. 13F, 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.
13G). 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.
[0317] G. Strength Analysis of Various MePEG/PCL Blends
[0318] 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.
[0319] Results of this test are shown in FIG. 13H, 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. 13H, the addition of MePEG into PCL decreased the
hardness of the resulting solid.
Example 9
Alteration of Therapeutic Agent Release from Thermopaste Using Low
Molecular Weight poly(d,l, Lactic Acid)
[0320] 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.
[0321] 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).
[0322] A. Experimental Materials
[0323] 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.
[0324] B. Synthesis of Low Molecular Weight PDLLA
[0325] 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.
[0326] C. Formulation of Paclitaxel Thermopastes
[0327] Paclitaxel was loaded, at 20%, into the following materials
by hand mixing at a temperature about 60.degree. C.
[0328] 1. low molecular weight PDLLA with polymerization time of 40
min.
[0329] 2. low molecular weight PDLLA with polymerization time of
120 min.
[0330] 3. low mol. wt PDLLA with polymerization time of 160
min.
[0331] 4. a mixture of 50:50 high molecular weight PDLLA and low
molecular weight PDLLA 40 min.
[0332] 5. a mixture of 50:50 high molecular weight PLGA and low
molecular weight PDLLA 40 min.
[0333] 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.
[0334] Mixtures of high molecular weight PDLLA or PLGA with low
molecular weight. PDLLA were obtained by dissolving the materials
in acetone followed by drying.
[0335] D. Release Study
[0336] The release of paclitaxel into PBS albumin buffer at
37.degree. C. was measured as described above with HPLC at various
times.
[0337] E. Results
[0338] 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.
[0339] 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).
[0340] The release of paclitaxel from formulations 2-5 were shown
in FIG. 14. 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.
[0341] 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-hydroxybutyric acid), and their copolymers.
Example 10
Preparation of Polymeric Compositions Containing Water Soluble
Additives and Paclitaxel
[0342] A. Preparation of Polymeric Compositions
[0343] Microparticles of co-precipitates of paclitaxel/additive
were prepared and subsequently added to PCL to form pastes.
Briefly, paclitaxel (100 mg) was dissolved in 0.5 ml of ethanol
(95%) and mixed with the additive (100 mg) previously dissolved or
dispersed in 1.0 ml of distilled water. The mixture was triturated
until a smooth paste was formed. The paste was spread on a Petri
dish and air-dried overnight at 37.degree. C. The dried mass was
pulverized using a mortar and pestle and passed through a mesh #140
(106 .mu.m) sieve (Endecotts Test Sieves Ltd, London, England). The
microparticles (40%) were then incorporated into molten PCL (60%)
at 65.degree. C. corresponding to a 20% loading of paclitaxel. The
additives used in the study were gelatin (Type B, 100 bloom, Fisher
Scientific), methylcellulose, (British Drug Houses), dextran, T500
(Pharmacia, Sweden), albumin (Fisher Scientific), and sodium
chloride (Fisher Scientific). Microparticles of paclitaxel and
gelatin or albumin were prepared as described above but were passed
through a mesh # 60 (270 .mu.m) sieve (Endecotts Test Sieves Ltd,
London, England) to evaluate the effect of microparticle size on
the release of paclitaxel from the paste. Pastes were also prepared
to contain 10, 20 or 30% gelatin and 20% paclitaxel in PCL to study
the effect of the proportion of the additive on drug release.
Unless otherwise specified, pastes containing 20% paclitaxel
dispersed in PCL were prepared to serve as controls for the release
rate studies.
[0344] B. Drug Release Studies
[0345] Approximately 2.5 mg pellet of paclitaxel-loaded paste was
suspended in 50 ml of 10 mM phosphate buffered saline, pH 7.4 (PBS)
in screw-capped tubes. The tubes were tumbled end-over-end at
37.degree. C. and at given time intervals 49.5 ml of supernatant
was removed, filtered through a 0.45 .mu.m membrane filter and
retained for paclitaxel analysis. An equal volume of PBS was
replaced in each tube to maintain sink conditions throughout the
study. For analysis, the filtrates were extracted with 3.times.1 ml
dichloromethane (DCM), the DCM extracts evaporated to dryness under
a stream of nitrogen and redissolved in 1 ml acetonitrile. The
analysis was 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 C18 reverse phase column
(Beckman), and UV detection (Shimadzu SPD A) at 232 nm.
[0346] C. Swelling Studies
[0347] Paclitaxel/additive/PCL pastes, prepared using
paclitaxel-additive microparticles of mesh size # 140 (and #60 for
gelatin only), were extruded to form cylinders, pieces were cut,
weighed and the diameter and length of each piece were measured
using a micrometer (Mitutoyo Digimatic). The pieces were suspended
in distilled water (10 ml) at 37.degree. C. and at predetermined
intervals the water was discarded and the diameter and the length
of the cylindrical pieces were measured and the samples weighed.
The morphology of the samples (before and after suspending in
water) was examined using scanning electron microscopy (SEM)
(Hitachi F-2300). The samples were coated with 60% Au and 40% Pd
(thickness 10-15 nm) using a Hummer Instrument (Technics, USA).
[0348] D. Chick Embryo Chorioallantoic Membrane (CAM) Studies
[0349] Fertilized, domestic chick embryos were incubated for 4 days
prior to shell-less culturing. The egg contents were incubated at
90% relative humidity and 3% CO.sub.2 and on day 6 of incubation, 1
mg pieces of the paclitaxel-loaded paste containing 6% paclitaxel,
24% gelatin and 70% PCL) or control (30% gelatin in PCL) pastes
were placed directly on the CAM surface. After a 2-day exposure the
vasculature was examined using a stereomicroscope interfaced with a
video camera; the video signals were then displayed on a computer
and video printed.
[0350] E. In Vivo Anti-Tumor Activity
[0351] Pastes, prepared as described above (using mesh size 140
fractions of the paclitaxel-gelatin microparticles) containing 20%
paclitaxel, 20% gelatin and 60% PCL were filled into 8.times.1 ml
syringes (BD Insulin Syringe, 1/2 cc) each syringe containing 150
mg of the paste (equivalent to 30 mg of paclitaxel). Ten week old
DBA/2j female mice (16) weighing 18-20 g were acclimatized for 4
days after arrival and each mouse was injected in the
posteriolateral flank with MDAY-D2 tumor cells, (10.times.10.sup.6
ml.sup.-1) in 100 .mu.l of phosphate buffered saline on day 1. On
day 6, the mice were divided into two groups of eight, the tumor
site opened under anesthesia and 150 mg of the paste, previously
heated to about 60.degree. C. was extruded at the tumor site and
the wound closed. One group was implanted with the
paclitaxel-loaded paste and the other group with control paste
containing gelatin and PCL only. On day 16, the mice were
sacrificed and the weight of the mice and the excised tumor were
measured.
[0352] F. Results and Discussion
[0353] Microparticles of co-precipitated paclitaxel and gelatin or
albumin were hard and brittle and were readily incorporated into
PCL while the other additives produced soft particles which showed
a tendency to break up during the preparation of the paste.
[0354] FIG. 15 shows the time courses of paclitaxel release from
pastes containing 20% paclitaxel in PCL or 20% paclitaxel, 20%
additive and 60% PCL. The release of paclitaxel from PCL with or
without additives followed a bi-phasic release pattern; initially,
there was a faster drug release rate followed by a slower drug
release of the drug. The initial period of faster release rate of
paclitaxel from the pastes was thought to be due to dissolution of
paclitaxel located on the surface or diffusion of paclitaxel from
the superficial regions of the paste. The subsequent slower phase
of the release profiles may be attributed to a decrease in the
effective surface area of the drug particles in contact with the
solvent, a slow ingress of the solvent into the polymer matrix or
an increase in the mean diffusion paths of the drug through the
polymer matrix.
[0355] Both phases of the release profiles of paclitaxel from PCL
increased in the presence of the hydrophilic additives with
gelatin, albumin and methylcellulose producing the greatest
increase in drug release rates (FIG. 15). There were further
increases in the release of paclitaxel from the polymer matrix when
larger paclitaxel-additive particles (270 .mu.m) were used to
prepare the paste compared with the smaller paclitaxel-additive
particles (106 .mu.m) were used (FIG. 16). Increases in the amount
of the additive (e.g., gelatin) produced a corresponding increase
in drug release (FIG. 16). FIG. 17A shows the swelling behavior of
pastes containing 20% paclitaxel, 20% additive and 60% PCL. The
rate of swelling followed the order gelatin>albumin>me-
thylcellulose>dextran>sodium chloride. In addition, the rate
of swelling increased when a higher proportion of the water-soluble
polymer was added to the paste (FIG. 17B). The pastes containing
gelatin or albumin swelled rapidly within the first 8-10 hours and
subsequently the rate of swelling decreased when the change in the
volume of the sample was greater than 40%. The paste prepared using
the larger (270 .mu.m) paclitaxel-gelatin particles swelled at a
faster rate than those prepared with the smaller (106 .mu.m)
paclitaxel-gelatin particles. All pastes disintegrated when the
volume increases were greater than 50%. The SEM studies showed that
the swelling of the pastes was accompanied by the cracking of the
matrix (FIG. 18). At high magnifications (FIGS. 18C and 18D) there
was evidence of needle or rod shaped paclitaxel crystals on the
surface of the paste and in close association with gelatin
following swelling (FIGS. 18C and 18D).
[0356] Osmotic or swellable, hydrophilic agents embedded as
discrete particles in the hydrophobic polymer result in drug
release by a combination of the erosion of the matrix, diffusion of
drug through the polymer matrix, and/or diffusion and/or convective
flow through pores created in the matrix by the dissolution of the
water soluble additives. Osmotic agents and swellable polymers
dispersed in a hydrophobic polymer would imbibe water (acting as
wicking agents), dissolve or swell and exert a turgor pressure
which could rupture the septa (the polymer layer) between adjacent
particles, creating microchannels and thus facilitate the escape of
the drug molecules into the surrounding media by diffusion or
convective flow. The swelling and cracking of the paste matrix
(FIG. 18) likely resulted in the formation of microchannels
throughout the interior of the matrix. The different rates and
extent of swelling of the polymers (FIG. 17) may account for the
differences in the observed paclitaxel release rates (FIGS. 15 and
16).
[0357] FIG. 19 shows CAMs treated with control gelatin-PCL paste
(FIG. 19A) and 20% paclitaxel-gelatin-PCL paste (FIG. 19B). The
paste on the surface of the CAMs are shown by the arrows in the
figures. The CAM with the control paste shows a normal capillary
network architecture. The CAMs treated with paclitaxel-PCL paste
consistently showed vascular regression and zones which were devoid
of a capillary network. Incorporation of additives in the paste
markedly increased the diameter of the zone of avascularity (FIG.
19).
[0358] The results of the in vivo study are shown in FIG. 20.
Briefly, peri-tumoral injection of paclitaxel-gelatin-PCL paste
into mice with established and palpable tumors showed that this
preparation produced a mean reduction of 63% in tumor mass compared
with controls. In addition, there was no significant effect on the
weights of the mice following treatment. Paclitaxel-PCL pastes
(without additives) did not produce any significant reduction in
tumor mass.
[0359] This study showed that the in vitro release of paclitaxel
from PCL could be increased by the incorporation of
paclitaxel/hydrophilic polymer microparticles into PCL matrix. In
vivo studies evaluating the efficacy of the formulation in treating
subcutaneous tumors in mice also showed that the
paclitaxel/gelatin/PCL paste significantly reduced the tumor mass.
Factors such as the type of water soluble agent, the microparticle
size and the proportion of the additives were shown to influence
the release characteristics of the drug.
[0360] Peritubular injection of a chemotherapeutic paste into the
adventitia of a tube obstructed by malignant overgrowth can reduce
local tumor growth and could relieve symptoms of obstruction
without invasive surgical procedures.
Example 11
Modification of Paclitaxel Release from Thermopaste Using
PDLLA-PEG-PDLLA and Low Molecular Weight Poly(D,L, Lactic Acid)
[0361] A. Preparation of PDLLA-PEG-PDLLA and Low Molecular Weight
PDLLA
[0362] DL-lactide was purchased from Aldrich. Polyethylene glycol
(PEG) with molecular weight 8,000, stannous octoate, and DL-lactic
acid were obtained from Sigma. Poly-.epsilon.-caprolactone (PCL)
with molecular weight 20,000 was obtained from Birmingham Polymers
(Birmingham, Ala.). Paclitaxel was purchased from Hauser Chemicals
(Boulder, Colo.). Polystyrene standards with narrow molecular
weight distributions were purchased from Polysciences (Warrington,
Pa.). Acetonitrile and methylene chloride were HPLC grade (Fisher
Scientific).
[0363] The triblock copolymer of PDLLA-PEG-PDLLA was synthesized by
a ring opening polymerization. Monomers of DL-lactide and PEG in
different ratios were mixed and 0.5 wt % stannous octoate was
added. The polymerization was carried out at 150.degree. C. for 3.5
hours. Low molecular weight PDLLA was synthesized through
polycondensation of DL-lactic acid. The reaction was performed in a
glass flask under the conditions of gentle nitrogen purge,
mechanical stirring, and heating at 180.degree. C. for 1.5 hours.
The PDLLA molecular weight was about 800 measured by titrating the
carboxylic acid end groups.
[0364] B. Manufacture of Paste Formulations
[0365] Paclitaxel at loadings of 20% or 30% was thoroughly mixed
into either the PDLLA-PEG-PDLLA copolymers or blends of PDLLA:PCL
90:10, 80:20 and 70:30 melted at about 60.degree. C. The paclitaxel
loaded pastes were weighed into 1 ml syringes and stored at
4.degree. C.
[0366] C. Characterization of PDLLA-PEG-PDLLA and the Paste
Blends
[0367] The molecular weights and distributions of the
PDLLA-PEG-PDLLA copolymers were determined at ambient temperature
by GPC using a Shimadzu LC-10AD HPLC pump and a Shimadzu RID-6A
refractive index detector (Kyoto, Japan) coupled to a 10.sup.4
.ANG. Hewlett Packard Plgel column. The mobile phase was chloroform
with a flow rate of 1 ml/min. The injection volume of the sample
was 20 .mu.l at a polymer concentration of 0.2% (w/v). The
molecular weights of the polymers were determined relative to
polystyrene standards. The intrinsic viscosity of PDLLA-PEG-PDLLA
in CHCl.sub.3 at 25.degree. C. was measured with a Cannon-Fenske
viscometer.
[0368] Thermal analysis of the copolymers was carried out by
differential scanning calorimetry (DSC) using a TA Instruments 2000
controller and DuPont 910S DSC (Newcastle, Del.). The heating rate
was 10.degree. C./min and the copolymer and paclitaxel/copolymer
matrix samples were weighed (3-5 mg) into crimped open aluminum
sample pans.
[0369] .sup.1H Nuclear magnetic resonance (NMR) was used to
determine the chemical composition of the polymer. .sup.1H NMR
spectra of paclitaxel loaded PDLLA-PEG-PDLLA were obtained in
CDCl.sub.3 using an NMR instrument (Bruker, AC-200E) at 200 MHz.
The concentration of the polymer was 1-2%.
[0370] The morphology of the paclitaxel/PDLLA-PEG-PDLLA paste was
investigated using scanning electron microscopy (SEM) (Hitachi
F-2300). The sample was coated with 60% Au and 40% Pd (thickness
10-15 nm) using a Hummer instrument (Technics, USA).
[0371] D. In vitro Release of Paclitaxel
[0372] A small pellet of 20% paclitaxel loaded PDLLA:PCL paste
(about 2 mg) or a cylinder (made by extrusion melten paste through
a syringe without needle) of 20% paclitaxel loaded PDLLA-PEG-PDLLA
paste were put into capped 14 ml glass tubes containing 10 ml
phosphate buffered saline (PBS, pH 7.4) with 0.4 g/L albumin. The
tube was incubated at 37.degree. C. with gentle rotational mixing.
The supernatant was withdrawn periodically for paclitaxel analysis
and replaced with fresh PBS/albumin buffer. The supernatant (10 ml)
was extracted with 1 ml methylene chloride. The water phase was
decanted and the methylene chloride phase was dried under a stream
of nitrogen at 60.degree. C. The dried residue was reconstituted in
a 40:60 water:acetonitrile mixture and centrifuged at 10,000 g for
about 1 min. The amount of the paclitaxel in the supernatant was
then analyzed by HPLC. HPLC analysis was performed using a 110A
pump and C-8 ultrasphere column (Beckman), and a SPD-6A uv detector
set at 232 nm, a SIL-9A autoinjector and a C-R3A integrator
(Shimadzu). The injection volume was 20 .mu.l and the flow rate was
1 ml/min. The mobile phase was 58% acetonitrile, 5% methanol, and
37% distilled water.
[0373] E. In Vivo Animal Studies
[0374] Ten week old DBA/2j female mice were acclimatized for 3-4
days after arrival. Each mouse was injected subcutaneously in the
posterior lateral flank with 10.times.10.sup.5 MDAY-D2 tumor cells
in 100 .mu.l of PBS on day 1. On day 6, the mice were randomly
divided into two groups. Group 1 were implanted with paste alone
(control), and group 2 were implanted with paste loaded with
paclitaxel. A subcutaneous pocket near the tumor was surgically
formed under anaesthesia and approximately 100 mg of molten paste
(warmed to 50.degree. C.-60.degree. C.) was placed in the pocket
and the wound closed. On day 16, the mice were sacrificed, and the
tumors were removed and weighed. Day 16 was selected to allow the
tumor growing into a easily measurable size within the ethical
limit.
[0375] F. Results and Discussion
[0376] The molecular weight and molecular weight distribution of
PDLLA-PEG-PDLLA, relative to polystyrene standards, were measured
by GPC (FIG. 21). The intrinsic viscosity of the copolymer in
CHCl.sub.3 at 25.degree. C. was determined using a Canon-Fenske
viscometer. The molecular weight and intrinsic viscosity decreased
with increasing PEG content. The polydispersities of
PDLLA-PEG-PDLLA with PEG contents of 10%-40% were from 2.4 to 3.5.
However, the copolymer with 70% PEG had a narrow molecular weight
distribution with a polydispersity of 1.21. This might be because a
high PEG content reduced the chance of side reactions such as
transesterfication which results in a wide distribution of polymer
molecular weight. Alternatively, a coiled structure of the
hydrophobic-hydrophilic block copolymers may result in an
artificial low polydispersity value.
[0377] DSC scans of pure PEG and PDLLA-PEG-PDLLA copolymers are
given in FIGS. 21 and 22. The PEG and PDLLA-PEG-PDLLA with PEG
contents of 70% and 40% showed endothermic peaks with decreasing
enthalpy and temperature as the PEG content of the copolymer
decreased. The endothermic peaks in the copolymers of 40% and 70%
PEG were probably due to the melting of the PEG region, indicating
the occurrence of phase separation. While pure PEG had a sharp
melting peak, the copolymers of both 70% and 40% PEG showed broad
peaks with a distinct shoulder in the case of 70% PEG. The broad
melting peaks may have resulted from the interference of PDLLA with
the crystallization of PEG. The shoulder in the case of 70% PEG
might represent the glass transition of the PDLLA region. No
thermal changes occurred in the copolymers with PEG contents of
10%, 20% and 30% in a temperature range of 10-250.degree. C.,
indicating that no significant crystallization (therefore may be
the phase separation) had occurred.
[0378] DSC thermograms of PDLLA:PCL (70:30, 80:20, 90:10) blends
without paclitaxel or with 20% paclitaxel showed an endothermic
peak at about 0.60.degree. C., resulting from the melting of PCL.
Due to the amorphous nature of the PDLLA and its low molecular
weight (800), melting and glass transitions of PDLLA were not
observed. No thermal changes due to the recrystallization or
melting of paclitaxel was observed.
[0379] PDLLA-PEG-PDLLA copolymers of 20% and 30% PEG content were
selected as optimum formulation materials for the paste for the
following reasons. PDLLA-PEG-PDLLA of 10% PEG could not be melted
at a temperature of about 60.degree. C. The copolymers of 40% and
70% PEG were readily melted at 60.degree. C., and the 20% and 30%
PEG copolymer became a viscous liquid between 50.degree. C. to
60.degree. C. The swelling of 40% and 70% PEG copolymers in water
was very high resulting in rapid dispersion of the pastes in
water.
[0380] The in vitro release profiles of paclitaxel from
PDLLA-PEG-PDLLA cylinders are shown in FIG. 23. The experiment
measuring release from the 40% PEG cylinders was terminated since
the cylinders had a very high degree of swelling (about 200% water
uptake within one day) and disintegrated in a few days. The
released fraction of paclitaxel from the 30% PEG cylinders
gradually increased over 70 days. The released fraction from the
20% PEG cylinders slowly increased up to 30 days and then abruptly
increased, followed by another period of gradual increase. A
significant difference existed in the extent to which each
individual cylinder (20% PEG content) showed the abrupt change in
paclitaxel release. Before the abrupt increase, the release
fraction of paclitaxel was lower for copolymers of lower PEG
content at the same cylinder diameter (1 mm). The 40% and 30% PEG
cylinders showed much higher paclitaxel release rates than the 20%
PEG cylinders. For example, the cylinder of 30% PEG released 17%
paclitaxel in 30 days compared to a 2% release from the 20% PEG
cylinder. The cylinders with smaller diameters resulted in faster
release rates, e.g., in 30 days, the 30% PEG cylinders with 0.65 mm
and 1 mm diameters released 26% and 17% paclitaxel, respectively
(FIG. 23).
[0381] The above observations may be explained by the release
mechanisms of paclitaxel from the cylinders. Paclitaxel was
dispersed in the polymer as crystals as observed by optical
microscopy. The crystals began dissolving in the copolymer matrix
at 170.degree. C. and completely dissolved at 180.degree. C. as
observed by hot stage microscope. DSC thermograms of 20% paclitaxel
loaded PDLLA-PEG-PDLLA (30% PEG) paste revealed a small
recrystallization exotherm (16 J/g, 190.degree. C.) and a melting
endotherm (6 J/g, 212.degree. C.) for paclitaxel (FIG. 21)
indicating the recrystallization of paclitaxel from the copolymer
melt after 180.degree. C. In this type of drug/polymer matrix,
paclitaxel could be released via diffusion and/or polymer
erosion.
[0382] In the diffusional controlled case, drug may be released by
molecular diffusion in the polymer and/or through open channels
formed by connected drug particles. Therefore at 20% loading, some
particles of paclitaxel were isolated and paclitaxel may be
released by dissolution in the copolymer followed by diffusion.
Other particles of paclitaxel could form clusters connecting to the
surface and be released through channel diffusion. In both cases,
the cylinders with smaller dimension gave a faster drug release due
to the shorter diffusion path (FIG. 23).
[0383] The dimension changes and water uptake of the cylinders were
recorded during the release (FIG. 24). The changes in length,
diameter and wet weight of the 30% PEG cylinders increased rapidly
to a maximum within 2 days, remained unchanged for about 15 days,
then decreased gradually. The initial diameter of the cylinder did
not affect the swelling behavior. For the cylinder of 20% PEG, the
length decreased by 10% in one day and leveled off, while the
diameter and water uptake gradually increased over time. Since more
PEG in the copolymer uptaken more water to facilitate the diffusion
of paclitaxel, a faster release was observed (FIG. 23).
[0384] The copolymer molecular weight degradation of
PDLLA-PEG-PDLLA paste was monitored by GPC. For the 20% PEG
cylinder, the elution volume at the peak position increased with
time indicating a reduced polymer molecular weight during the
course of the release experiment (FIG. 25). A biphasic molecular
weight distribution was observed at day 69. Polymer molecular
weight was also decreased for 30% PEG cylinders (1 mm and 0.65 mm).
However no biphasic distribution was observed.
[0385] NMR spectra revealed a PEG peak at 3.6 ppm and PDLLA peaks
at 1.65 ppm and 5.1 ppm. The peak area of PEG relative to PDLLA in
the copolymer decreased significantly after 69 days (FIG. 26),
indicating the dissolution of PEG after its dissociation from
PDLLA. The dry mass loss of the cylinders was also recorded (FIG.
26) and shows a degradation rate decreasing in the order 30%
PEG-0.65 mm>30% PEG-1 mm>20% PEG-1 mm.
[0386] The morphological changes of the dried cylinders before and
during paclitaxel release were observed using SEM (FIG. 27).
Briefly, solid paclitaxel crystals and non-porous polymer matrices
were seen before the release (FIGS. 27A and 27B). After 69 days of
release, no paclitaxel crystals were observed and the matrices
contained many pores due to polymer degradation and water uptake
(FIGS. 27C and 27D).
[0387] The 30% PEG cylinders showed extensive swelling after only
two days in water (FIG. 24) and therefore the hindrance to
diffusion of the detached water soluble PEG block and degraded
PDLLA (i.e., DL-lactic acid oligomers) was reduced. Since the mass
loss and degradation of the 30% PEG cylinders was continuous, the
contribution of erosion release gradually increased resulting in a
sustained release of paclitaxel without any abrupt change (FIG.
23). vFor the 20% PEG cylinders, the swelling was low initially
(FIG. 24) resulting in a slow diffusion of the degradation
products. Therefore the degradation products in the interior region
are primarily retained while there are much less degradation
products in the outer region due to the short diffusion path. The
degradation products accelerated the degradation rate since the
carboxylic acid end groups of the oligomers catalyzed the
hydrolytic degradation. This results in a high molecular weight
shell and a low molecular weight interior as indicated by the
biphasic copolymer molecular weight distribution (FIG. 25, day 69).
Since the shell rupture was dependent on factors such as the
strength, thickness and defects of the shell and interior
degradation products, the onset and the extent of the loss of
interior degradation products are very variable. Because the shell
rupture is not consistent and the drug in the polymer is not
microscopically homogenous, the time point for the release burst
and the extent of the burst were different for the 4 samples tested
(FIG. 23).
[0388] The release of paclitaxel from PDLLA and PCL blends and pure
PCL are shown in FIG. 28. Briefly, the released fraction increased
with PDLLA content in the blend. For example, within 10 days, the
released paclitaxel from 80:20, 70:30, and 0:100 PDLLA:PCL were
17%, 11%, and 6%, respectively. After an initial burst in one day,
approximately constant release was obtained from 80:20 PDLLA:PCL
paste. No significant degree of swelling was observed during the
release. For the PDLLA:PCL blends, since PDLLA had a very low
molecular weight of about 800, it was hydrolyzed rapidly into water
soluble products without a long delay in mass loss. PCL served as
the "holding" material to keep the paste from rapidly
disintegrating. Therefore the release rate increased with PDLLA
content in the blend due to the enhanced degradation. The
continuous erosion of the PDLLA controlled the release of
paclitaxel and resulted in a constant release. The release of
paclitaxel from pure PCL was probably diffusion controlled due to
the slow degradation rate (in 1-2 years) of PCL.
[0389] Difficulties were encountered in the release study for 20%
paclitaxel loaded 90:10 PDLLA:PCL paste due to the disintegration
of the paste pellet within 24 hours of incubation. Briefly, during
the first 12 hours of incubation, samples were taken every hour in
order to ensure sink conditions for paclitaxel release. The
released paclitaxel from the 90:10 paste was 25-35% within 10
hours.
[0390] The efficacy of the paste formulations for regressing tumor
growth in mice were evaluated (FIG. 29). Briefly, pastes examined
were PCL.+-.20% paclitaxel, 80:20 PDLLA:PCL+20% paclitaxel, 90:10
PDLLA:PCL+20% paclitaxel and PDLLA-PEG-PDLLA (30% PEG).+-.20%
paclitaxel. The paste formulations, 90:10 PDLLA:PCL and
PDLLA-PEG-PDLLA, containing paclitaxel reduced tumor growth in vivo
by 54 and 40%, respectively. In contrast, the paste formulations,
PCL and 80:20 PDLLA:PCL, containing paclitaxel had little or no
effect on tumor growth. All control pastes (drug absent) had no
significant effect on tumor growth. The paste formulations with
faster release rates of paclitaxel (90:10 PDLLA:PCL and
PDLLA-PEG-PDLLA) were also more effective in reducing tumor growth,
suggesting that a critical local concentration of paclitaxel is
required at the tumor site for tumor growth inhibition. Paste
formulations releasing paclitaxel slowly, such as PCL and 80:20
PDLLA:PCL, were not effective. All of the paste formulations
examined had no significant effect on the body weights of mice,
indicating that the paclitaxel loaded paste was well tolerated in
vivo.
[0391] These data suggest that local application of paclitaxel at
the tumor site is an effective therapeutic strategy to inhibit
local tumor growth without increasing systemic toxicity. The
inability to the paclitaxel loaded formulations to completely
inhibit tumor growth is most likely due to insufficient release of
paclitaxel from the polymer and rapid tumor growth of MDAY-D2
tumors. The ability of 90:10 PDLLA:PCL paste containing 30%
paclitaxel, which released more paclitaxel than 90:10 PDLLA:PCL
paste containing 20% paclitaxel, and which inhibited tumor growth
more effectively is consistent in this regard. Thus, modulation of
the release rate of paclitaxel, which is regulated by the
properties of the polymer and chemotherapeutic agents as well as
the site of administration, is in important step in the development
of local therapy for inhibiting tumor growth.
Example 12
Manufacture of Polymeric Compositions Containing PCL and MePEG
[0392] A. Paclitaxel Release from PCL
[0393] Polycaprolactone containing various concentrations of
paclitaxel was prepared as described in Example 1. The release of
paclitaxel over time was measured by HPLC essentially as described
above. Results are shown in FIG. 30.
[0394] B. Effect of MePEG on Paclitaxel Release
[0395] MePEG at various concentrations was formulated into PCL
paste containing 20% paclitaxel, utilizing the methods described in
Example 1. The release of paclitaxel over time was measured by HPLC
essentially as described above. Results of this study are shown in
FIG. 31.
[0396] C. Effect of MePEG on the Melting Point of PCL
[0397] 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. 32A (melting point vs. % MePEG) and 32B (percent
increase in time to solidify vs. % MePEG).
[0398] D. Tensile Strength of MePEG Containing PCL
[0399] 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. 33.
[0400] E. Effect of .gamma.-Irradiation or the Release of
Paclitaxel
[0401] 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. 34.
[0402] 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 13
Preparation if PCL Microspheres: Scale Up Studies
[0403] Microspheres (50 g) were prepared using PCL (nominal
molecular weight 80,000) using the solvent evaporation method
described below.
[0404] A. Method
[0405] A preparation of 500 ml of 10% PCL in methylene chloride and
a 4000 ml solution of 1% PVA (mol. Wt 13,000-23,000; 99%
hydrolyzed) were emulsified using the Homo Mixer controlled with a
rheostat at 40 setting for 10 hours. The mixture was strained using
sieve #140 until the microspheres settled at the bottom then
supernatant was decanted. The preparation was then washed 3.times.
with distilled water (using the sedimentation followed by decanting
method) and then re-suspended in 250 ml of distilled water and
filtered. The microspheres were then air-dried overnight at
37.degree. C.
[0406] B. Results
[0407] Microsphere yields were as follows:
2 Initial wt of PCL = 50.1 g Wt. Of microspheres obtained = 41.2 g
% yield = (43.2/50.0) .times. 100 = 86.4
[0408] Yield (10-50 .mu.m) about 72%
[0409] Mean size 21.4 .mu.m, median 22.0 .mu.m mode 24.7 .mu.m.
[0410] Narrower size ranges (20-40 .mu.m) can be obtained by
sieving or by separation using the sedimentation method.
Example 14
Manufacture of PLGA Microspheres
[0411] Microspheres were manufactured from (PLLA) lactic
acid-glycolic acid (GA) copolymers.
[0412] A. Method:
[0413] Microspheres were manufactured in the size ranges 0.5 to 10
.mu.m, 10-20 .mu.m and 30-100 .mu.m using standard methods (polymer
was dissolved in dichloromethane and emulsified in a polyvinyl
alcohol solution with stirring as previously described in PCL or
PDLLA microspheres manufacture methods). Various ratio's of PLLA to
GA were used as the polymers with different molecular weights
[given as Intrinsic Viscosity (I.V.)].
[0414] B. Result:
[0415] Microspheres were manufactured successfully from the
following starting polymers:
3 PLLA GA I.V. 50 50 0.74 50 50 0.78 50 50 1.06 65 35 0.55 75 25
0.55 85 15 0.56
[0416] Paclitaxel at 10% or 20% loadings was successfully
incorporated into all these microspheres. Examples of size
distributions for one starting polymer (85:15, IV=0.56) are given
in FIGS. 35-38. Paclitaxel release experiments were performed using
microspheres of various sizes and various compositions. Release
rates are shown in FIGS. 39-42.
Example 15
Di-Block Copolymers
[0417] Diblock copolymers of poly(DL-lactide)-block-methoxy
polyethylene glycol (PDLLA-MePEG), polycaprolactone-block-methoxy
polyethylene glycol (PCL-MePEG) and
poly(DL-lactide-co-caprolactone)-block-methoxy polyethylene glycol
(PDLLACL-MePEG) were synthesized using a bulk melt polymerization
procedure. Briefly, given amounts of monomers DL-lactide,
caprolactone, and methoxy polyethylene glycols with different
molecular weights were heated (130.degree. C.) to melt under the
bubbling of nitrogen and stirring. Catalyst stannous octoate (0.2%
w/w) was added to the molten monomers. The polymerization was
carried out for 4 hours. The molecular weights, critical micelle
concentrations, and the maximum paclitaxel loadings were measured
with GPC, fluorescence, and solubilization testing, respectively
(FIG. 43). High paclitaxel carrying capacities were obtained. The
ability of solubilizing paclitaxel depends on the compositions and
concentrations of the copolymers (FIGS. 43 and 44). PDLLA-MePEG
gave the most stable solubilized paclitaxel (FIGS. 44 and 45).
Example 16
Encapsulation of Paclitaxel in Nylon Microcapsules
[0418] A. Preparation of Paclitaxel-Loaded Microcapsules
[0419] Paclitaxel was encapsulated into nylon microcapsules using
the interfacial polymerization techniques. Briefly, 100 mg of
Paclitaxel and 100 mg of Pluronic F-127 was dissolved in 1 ml of
dichloromethane (DCM) and 0.4 ml (about 500 mg) of adipoyl chloride
(ADC) was added. This solution was homogenized into 2% PVA solution
using the Polytron homogenizer (1 setting) for 15 seconds. A
solution of 1,6-hexane-diamine (HMD) in 5 ml of distilled water was
added dropwise while homogenizing. The mixture was homogenized for
a further 10 seconds after the addition of HMD solution. The
mixture was transferred to a beaker and stirred with a magnetic
stirrer for 3 hours. The mixture was centrifuged, collected and
resuspended in 1 ml distilled water.
[0420] B. Encapsulation Efficiency/Paclitaxel-Loading
[0421] About 0.5 ml of the suspension was filtered and the
microspheres were dried. About 2.5 mg of the microcapsules was
weighed and suspended in 10 ml of acetonitrile for 24 hours. The
supernatant analyzed for paclitaxel and the result was expressed as
a percentage of paclitaxel. Preliminary studies have shown that
paclitaxel could be encapsulated in nylon microcapsules at a high
loading (up to 60%) and high encapsulation efficiency (greater than
80%).
[0422] C. Paclitaxel Release Studies
[0423] About 2.5 mg of the paclitaxel-nylon microspheres were
suspended in 50 ml water containing 1M each of sodium chloride and
urea and analyzed periodically. Release of paclitaxel from the
microcapsule was fast with more than 95% of the drug released after
72 hours. (FIG. 46).
Example 17
Complexation of Paclitaxel with Cyclodextrins
[0424] A. Materials
[0425] Paclitaxel was obtained from Hauser Chemicals Inc., Boulder,
Colo. Disodium phosphate (Fisher), citric acid (British Drug
Houses), Hydroxypropyl-.beta.-cyclodextrin (HP.beta.CD),
.gamma.-cyclodextrin (.gamma.-CD) and
hydroxypropyl-.gamma.-cyclodextrin (HP.gamma.CD) were obtained from
American Maize-Products Company (Hammond, Ind.) and were used as
received.
[0426] B. Methods
[0427] 1. Solubility Studies
[0428] Excess amounts of paclitaxel (5 mg) were added to aqueous
solutions containing various concentrations of .gamma.-CD,
HP.gamma.-CD, or HP.beta.-CD and tumbled gently for about 24 hours
at 37.degree. C. After equilibration, aliquots of the suspension
were filtered through a 0.45 .mu.m membrane filter (Millipore),
suitably diluted and analyzed using HPLC. The mobile phase was
composed of a mixture of acetonitrile, methanol and water (58:5:37)
at a flow rate of 1.0 ml min.sup.-1. The solubility of paclitaxel
in a solvent composed of 50:50 water and ethanol (95%) containing
various concentrations, up to 10%, of HP.beta.-CD was also
investigated. In addition, dissolution rate profiles of paclitaxel
were investigated by adding 2 mg of paclitaxel (as received) to 0,
5, 10 or 20% HP.gamma.-CD solutions or 2 mg of previously hydrated
paclitaxel (by suspending in water for 7 days) to pure water and
tumbling gently at 37.degree. C. Aliquots were taken at various
time intervals and assayed for paclitaxel.
[0429] 2. Stability Studies
[0430] The solutions containing 20% HP.beta.CD or HP.gamma.CD had
pH values of 3.9 and 5.2, respectively. The stability of paclitaxel
in cyclodextrin solutions was investigated by assaying paclitaxel
in solutions (20 .mu.g ml.sup.-1) containing 10 or 20% HP.gamma.-CD
or HP.beta.-CD in either water or a 50:50 water-ethanol mixture at
37.degree. C. or 55.degree. C. at various time intervals. In
addition, stability of paclitaxel in solutions (1 .mu.g/ml)
containing 1%, 2% or 5% HP.beta.CD at 55.degree. C. were
determined.
[0431] C. Results
[0432] 1. Solubility Studies
[0433] The solubility of paclitaxel increased over the entire CD
concentration range studied; HP.beta.CD producing the greatest
increase in the solubility of paclitaxel (FIG. 47). The shape of
the solubility curves suggests that the stoichiometries were of
higher order than a 1:1 complex. Paclitaxel formed Type A.sub.P
curves with both HP.beta.CD and HP.gamma.CD and Type A.sub.N curves
with .gamma.CD. The solubility of paclitaxel in a 50% solution of
HP.beta.CD in water was 3.2 mg ml.sup.-1 at 37.degree. C. which was
about a 2000-fold increase over the solubility of paclitaxel in
water. The estimated stability constants (from FIG. 48) for first
order complexes of paclitaxel-cyclodextrins were 3.1, 5.8 and 7.2
M.sup.-1 for .gamma.-CD, HP.gamma.CD and HP.beta.CD and those for
second order complexes were 0.785.times.10.sup.3,
1.886.times.10.sup.3 and 7.965.times.10.sup.3 M.sup.-1 for
.gamma.-CD, HP.gamma.CD and HP.beta.CD respectively. The values of
the observed stability constants suggested that the inclusion
complexes formed by paclitaxel with cyclodextrins were
predominantly second order complexes.
[0434] The solubility of paclitaxel in 50:50 water:ethanol mixture
increased with an increase in the cyclodextrin concentration (FIG.
49) as observed for complexation in pure water. The apparent
stability constant for the complexation of paclitaxel and
HP.beta.CD in the presence of 50% ethanol (26.57 M.sup.-1) was
significantly lower (about 300 times) than the stability constant
in the absence of ethanol. The lower stability constant may be
attributed to a change in the dielectric constant or the polarity
of the solvent in the presence of ethanol.
[0435] The dissolution profiles of paclitaxel in 0, 5, 10 and 20%
.gamma.CD solutions (FIG. 50) illustrates the formation of a
metastable solution of paclitaxel in pure water or the cyclodextrin
solutions; the amount of paclitaxel in solution gradually
increased, reached a maximum and subsequently decreased.
Dissolution studies using paclitaxel samples which were previously
hydrated by suspending in water for 48 hours did not show the
formation of the metastable solution. In addition, DSC analysis of
the hydrated paclitaxel (dried in a vacuum oven at room
temperature) showed two broad endothermic peaks between 60 and
110.degree. C. These peaks were accompanied by about 4.5% weight
loss (determined by thermogravimetric analysis) indicating the
presence of hydrate(s). A loss in weight of about 2.1% would
suggest the formation of a paclitaxel monohydrate. Therefore, the
occurrence of the DSC peaks between 60.degree. C. and 110.degree.
C. and the loss in weight of about 4.5% suggests the presence of a
dihydrate. There was no evidence of endothermic peak(s) between
60.degree. C. and 110.degree. C. (DSC results) or a weight loss
(TGA results) for paclitaxel samples as received. Therefore, (as
received) paclitaxel was anhydrous and on suspension in water it
dissolved to form a supersaturated solution which recrystallized as
a hydrate of lower solubility (FIG. 50).
[0436] 2. Stability Studies
[0437] Paclitaxel degradation depended on the concentration of the
cyclodextrin and followed pseudo-first order degradation kinetics
(e.g., FIG. 51). The rate of degradation of paclitaxel in solutions
(1 .mu.g/ml paclitaxel) containing 1% HP.beta.CD at 55.degree. C.
faster (k=3.38.times.10.sup.-3 h.sup.-1) than the rate at higher
cyclodextrin concentrations. Degradation rate constants of
1.78.times.10.sup.-3 h.sup.-1 and 0.96.times.10.sup.-3 h.sup.-1
were observed for paclitaxel in 10% HP.beta.CD and HP.gamma.CD,
respectively. Paclitaxel solutions (1 .mu.g/ml) containing 2, 4, 6
or 8% HP.beta.CD did not show any significant difference in the
rate of degradation from that obtained with the 10 or 20%
HP.beta.CD solutions (20 .mu.g/ml). The presence of ethanol did not
adversely affect the stability of paclitaxel in the cyclodextrin
solutions.
[0438] D. Conclusion
[0439] This study showed that the solubility of paclitaxel could be
increased by complexation with cyclodextrins. These aqueous based
cyclodextrin formulations may have potential for peritubular in the
treatment of various cancers.
Example 18
Polymeric Compositions with Increased Concentrations of
Paclitaxel
[0440] 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".
[0441] A. Materials
[0442] 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).
[0443] For synthesizing PDLLA-MePEG, a mixture of
DL-lactide/MePEG/stannou- s 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 II and III. In
all the cases, the amount of stannous octoate was 0.5%-0.7%.
[0444] B. Methods
[0445] 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 II.
4TABLE II 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)
[0446] In the case of PDLLA-PEG-PDLLA (Table III), 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.
5TABLE III 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.
[0447] C. Results
[0448] 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 19
Analysis of Drug Release
[0449] 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.
[0450] 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.
[0451] 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 nm. For Vanadium containing compounds the liquid
buffer is analyzed directly using a UV/VIS spectrometer in the 200
to 300 nm range.
Example 20
Effect of Paclitaxel-Loaded Thermopaste on Tumor Growth and Tumor
Angiogenesis In Vivo
[0452] 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.
[0453] 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.
[0454] 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.
[0455] 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. 51C and 51D), 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 (FIGS. 51A and 51B).
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.
[0456] 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. 51A and
51B). 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.
[0457] 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.
[0458] This study demonstrates that thermopaste releases sufficient
quantities of anti-proliferative agent (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 21
Effect of Therapeutic Agent-Loaded Thermopaste on Tumor Growth In
Vivo in a Murine Tumor Model
[0459] The murine MDAY-D2 tumor model may be used to examine the
effect of local slow release of an anti-proliferative compound such
as paclitaxel on tumor growth, tumor metastasis, and animal
survival. Briefly, 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.
[0460] 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.
[0461] 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.
[0462] The weights of the tumors for each animal is shown in Table
IV below:
6TABLE IV Tumor Weights (gm) Animal No. Control Control 10%
Paclitaxel 20% (empty) (PCL) Thermopaste Thermopaste Paclitaxel 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
[0463] 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. 52A). Therefore, thermopaste containing 20%
paclitaxel was more effective in reducing tumor growth than
thermopaste containing 10% paclitaxel (see FIG. 52C; see also FIG.
52B).
[0464] 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 thermopaste did not appear to
affect the integrity or cellularity of the skin or tissues
surrounding the tumor. Grossly, wound healing was unaffected.
Example 22
Use of Paclitaxel Loaded Surgical Paste to Delay Regrowth of
Partially Resected RIF-1 Tumors in Mice
[0465] The effectiveness of a biodegradable polymeric sustained
release surgical paste formulation of paclitaxel in delaying
regrowth of partially resected RIF-1 tumors in C3H/HeJ mice was
investigated.
[0466] A. Methods
[0467] Paclitaxel (20%) was incorporated into a 4:1 blend of
poly(.epsilon.-caprolactone) and methoxypolyethylene glycol. The in
vitro release profile for this formulation in phosphate buffered
saline containing albumin (0.4 mg/mL) at 37.degree. C. was
investigated using an HPLC assay for paclitaxel. Briefly, seventeen
mice were injected in the right flank with 100 .mu.l of a RIF-1
cell suspension (1.0.times.10.sup.6 cells) in Hanks Buffer. The
tumors were allowed to grow for 5 days following which more than
70% of each tumor was surgically resected and the remaining tumor
mass was left untreated or coated with 20-30 .mu.L of either 20%
paclitaxel in surgical paste or surgical paste alone (no drug).
This was day 0. Dimensions of the visible tumor mass beneath the
skin were measured on days 4 through 7 and day 9. The area of this
visible cylindrical tumor surface was shown to be correlated to
tumor volume (r=0.812).
[0468] B. Results The in vitro release curve of paclitaxel was
characterized by an initial 1 day burst phase followed by a long
period of slow sustained release. The areas of the visible
cylindrical tumor surfaces from each mouse from days 4 through 9
are shown in Table V. All the mice except 1 in each of the two
groups which did not receive paclitaxel showed extensive tumor
regrowth by day 4. One mouse which received polymer only showed
delayed tumor regrowth while one mouse which received no treatment
showed no regrowth. In contrast all but one of the mice treated
with paclitaxel surgical paste showed no tumor regrowth until at
least day 5 and two mice still did not show regrowth until day
6.
[0469] C. Conclusion
[0470] Paclitaxel loaded paste significantly inhibited tumor
regrowth between days 1 to 5. Regrowth occurred after day 6 due to
the aggressiveness of the cell line.
7TABLE V RIF-1 cell tumor size (expressed as area of visible tumor
mass beneath the skin in mm.sup.2) measured on days following tumor
resection surgery Tumor sizes (mm.sup.2) Treatment day 4 day 5 day
6 day 7 day 9 20% paclitaxel in 0.0 0.0 41.3 58.8 * polymer 20%
paclitaxel in 30.7 35.3 49.0 67.9 * polymer 20% paclitaxel in 0.0
0.0 0.0 * 42.4 polymer 20% paclitaxel in 0.0 47.2 55.4 47.8 *
polymer 20% paclitaxel in 0.0 25.5 41.9 57.1 * polymer 20%
paclitaxel in 0.0 0.0 0.0 * 46.6 polymer polymer only 44.2 52.8 * *
* polymer only 36.3 39.0 32.7 * 38.5 polymer only 36.9 44.2 51.5
42.7 * polymer only 0.0 12.9 14.9 * 29.2 polymer only 37.4 39.6
40.7 54.1 * polymer only 40.7 24.2 40.7 * 36.9 no treatment 0.0 0.0
0.0 * * no treatment 52.2 77.8 * * * no treatment 39.0 45.4 43.6 *
* no treatment 22.1 21.2 31.2 * 26.9 no treatment 21.2 30.7 36.3 *
* * Tumor not measured or animal already sacrificed
Example 23
Encapsulation of Suramin
[0471] 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. C. 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.
[0472] 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.
[0473] The results of these experiments is shown in FIGS. 53-59.
Briefly, the size distribution of microspheres by number (FIG. 53)
or by weight (FIG. 54) 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.
[0474] The encapsulation of suramin is very low (<1%) (see FIG.
56). 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. 55, 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. 57 (size
distribution by weight). Encapsulation of sodium suramin in 5% PVA
containing 10% NaCl is shown in FIGS. 58 and 59 (size distribution
by weight, and number, respectively).
[0475] 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. 60A and 60B).
Example 24
Methotrexate-Loaded Paste
[0476] A. Manufacture of Methotrexate-Loaded Paste
[0477] 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.
[0478] B. Results
[0479] Results are shown in FIGS. 61A-E. Briefly, FIG. 61A shows
MTX release from PCL discs containing 20% MePEG and various
concentrations of MTX. FIG. 61B shows a similar experiment for
paste which does not contain MePEG. FIGS. 61C, D, and E show the
amount of MTX remaining in the disk.
[0480] 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 25
Manufacture of Microspheres Containing Methotrexate
[0481] A. Microspheres with MTX Alone
[0482] 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.
[0483] 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.
[0484] Using this method MTX loaded microspheres can be
reproducibly manufactured in the 30 to 160 micron range (see FIG.
62).
[0485] B. Microspheres with MTX and Hyaluronic Acid
[0486] 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 26
Manufacture of Polymeric Compositions Containing Vanadium
Compounds
[0487] A. Polymeric Paste Containing Vanadyl Sulfate
[0488] 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.
[0489] 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.
[0490] Results are shown in FIG. 63. 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.
[0491] B. Polymeric Microspheres Containing Vanadyl Sulfate
[0492] Vanadyl sulfate was incorporated into microspheres of
polylactic acid or hyaluronic acid essentially as described in
Example 25. Results are shown in FIG. 64.
[0493] C. Polymeric Paste Containing Organic Vanadate
[0494] Organic vanadate is loaded into a PCL paste essentially as
described above in Example 24. Vanadate release from the
microspheres was determined as described above. Results are shown
in FIGS. 65A and 65B.
[0495] D. Organic Vanadate Containing Microspheres
[0496] Organic vanadate may also be loaded into microspheres
essentially as described in Example 25. Such microspheres are shown
in FIG. 66 for poly D,L lactic acid (M.W. 500,000;
Polysciences).
Example 27
Polymeric Composition Containing bis(maltolato) Oxovanadium
(BMOV)
[0497] A. Manufacture of bis(maltolato) Oxovanadium Loaded
Paste
[0498] Poly(.epsilon.-caprolactone) (Molecular weight 20000) (BPI
Birmingham Ala.) and BMOV were weighed directly into a glass beaker
in the appropriate proportions. In some formulations
methoxypolyethylene glycol (MEPEG) (molecular weight 350) (Union
Carbide, Danbury Conn.) was also added to the PCL and BMOV. The
beaker and contents were warmed to 55.degree. C. with gentle
stirring for 5 minutes until the BMOV was thoroughly dispersed in
the molten polymer. The molten mix was then drawn into a prewarmed
syringe and stored at 4.degree. C. until use.
[0499] B. Drug Release Studies
[0500] To tubes containing 15 ml of 10 mM phosphate buffered saline
(PBS pH 7.4) and 100 ug/ml bovine serum albumin (fraction 5
Boehringer Mannheim, Germany) were added 150 mg disc-shaped slabs
of PCL-BMOV paste. The tubes were sealed and tumbled end over end
at 30 rpm at 37.degree. C. At appropriate times the PCL-BMOV slab
was allowed to settle under gravity for 5 minutes and all the
supernatant was removed. The BMOV concentration was determined in
the supernatants by measuring the absorbance at 256 nm (A256) and
276 nm (A276). The supernatant was replaced with 15 ml of fresh PBS
and the tubes were retumbled. A linear calibration curve of BMOV
concentration vs. A256 or A276 was obtained using BMOV standards in
the 0 to 25 ug/ml range. The absorbance values at 256 nm or 276 nm
of these standards were shown to be unaffected by storage in sealed
tubes at 37.degree. C. for 2 to 3 days (the same conditions used
for drug release studies).
[0501] At the end of the drug release experiments samples of the
PCL-BMOV matrix were assayed for residual drug content by
dissolution of a known dried weight of the matrix in 0.5 ml of
dichloromethane (DCM) (Fisher). To this solution was added 50 ml of
warm water (50.degree. C.) with mixing to evaporate the DCM leaving
the BMOV in water for spectrophotometric analysis (A256).
[0502] C. Scanning Electron Microscopy (SEM)
[0503] Samples of the PCL-BMOV matrix that had been used in the 2
month drug release experiments were examined using SEM. These
samples were compared with freshly prepared control samples.
Polymer samples were coated (60:40 gold:palladium) (Hummer
instruments, Technics, USA) and examined using a Hitachi (model
F-2300) scanning electron microscope with a IBM data collection
system.
[0504] D. Results
[0505] The release of BMOV from the PCL matrix is shown in FIGS.
67A and 67B. Increasing the loading of BMOV from 5% to 35% in the
PCL matrix increased the rate of drug release over the two month
period (FIG. 67A). At 35% BMOV loading the release rate increased
dramatically. The release profiles for the 20% to 30% BMOV loadings
showed an initial more rapid phase of drug release in the first two
days followed by a controlled, almost zero order release over the
next 2 months.
[0506] The drug release profiles are also expressed in terms of the
% of drug remaining in the pellet (FIG. 67B). Briefly, the % of
BMOV remaining in the PCL matrix was almost identical at all time
points for all the BMOV loadings up to (and including) 30% so that
between 65% and 80% of the original BMOV was still present in the
matrix after two months. (Only the data for 25% and 30% BMOV
loadings are shown in FIG. 67B for clarity). The percentage of drug
remaining in the matrix decreased more rapidly at a loading 35%
BMOV so that a little over 50% of the original BMOV was present in
the matrix after one month. In order to verify the cumulative drug
release data, samples of the PCL-BMOV matrix were assayed for
remaining drug content at the end of each drug release experiment.
The % drug remaining at 70 days as determined by this residual
assay was as follows: 67%+/-10% (5% BMOV), 56%+/-10% (10% BMOV),
80%+/-20% (15% BMOV), 84%+/-20% (20% BMOV), 85%+/-12% (25% BMOV),
77%+/-14% (30% BMOV) and 57%+/-15% (35% BMOV).
[0507] FIGS. 68A and 68B show the effect of adding 20% MEPEG to the
PCL matrix on the drug release profiles for various loading
concentrations of BMOV. The addition of MEPEG to the matrix
increases the release rate of BMOV dramatically compared to the
release of BMOV from the PCL alone (FIG. 67). Greater than 50% of
the BMOV was released from the polymer matrix within 7 days 1 week
at all BMOV loading concentrations. Residual analysis of the
PCL-BMOV-MEPEG pellets gave the following values for the % of BMOV
remaining at 7 days in the pellets: 24% (+/-9%), 5% BMOV; 25%
(+/-8%), 10% BMOV; 22% (+/-4%), 15% BMOV; 27% (+/-7%), 20%
BMOV.
[0508] FIG. 69A shows the morphology of the BMOV crystals under
high magnification. FIGS. 69B and 69C show the morphology of the
polymer-drug matrices both before and after the two month drug
release study in aqueous buffer. The PCL-BMOV matrices for 15%, 20%
and 30% BMOV loadings were typically smooth on their external faces
before the release study and a representative SEM is shown in FIG.
69B. Following incubation in the aqueous buffer for two months the
external surfaces were rough and pitted.
[0509] E. Discussion
[0510] This compound was found to release very slowly from the PCL
matrix with almost ideal release characteristics for the
maintenance of sustained concentrations of vanadium. These
characteristics included only a small burst effect of drug release
in the first few days followed by almost zero order release
kinetics at most drug loadings (FIG. 67). BMOV is less water
soluble than vanadyl sulphate or sodium orthovanadate and the
hydrophobicity of the molecule probably increases the affinity of
the BMOV molecules for the hydrophobic PCL matrix and decreases the
rate of drug release into an aqueous incubation medium.
[0511] The addition of 20% MEPEG to PCL has been shown to improve
the thermal flow properties of the paste by reducing the viscosity
of the matrix and the temperature at which the polymer solidifies.
These properties are important in applying the paste to a
peritubular tumor site as better coverage of the site is obtained
under these conditions. The addition of MEPEG to the BMOV-PCL paste
matrix has been shown to enhance the release rates of BMOV in vitro
(FIG. 68) at all BMOV loading concentrations (5% to 20%) relative
to the release rates from BMOV-PCL (no MEPEG) (FIG. 67). The 5%
BMOV loaded PCL-MEPEG paste was shown to release between 500 an
1000 ug of BMOV per day (which was similar to the release rate from
35% BMOV loaded PCL).
Example 28
In Vitro and In Vivo Efficacy of bis(maltolato)Oxovanadium Loaded
Thermopaste
[0512] BMOV loaded polymeric PCL thermopaste was prepared as
described previously and was tested for its efficacy against tumor
cell lines both in vitro and in vivo.
[0513] A. Human Tumor Cell Lines
[0514] The HT-29 colon, MCF-7 breast and SKMES1 non-small cell lung
human tumor cell lines were obtained from the American Type Culture
Collection. The HT-29 colon cell line was cultured in RPMI 1640
with 10% heat inactivated fetal bovine serum (HIFBS), the MCF-7
breast cell line in Iscove's modified Eagles medium with 5% HIFBS
plus 10-9 M insulin, and the SKMES1 lung cell line in Eagle's
minimal essential medium with 10% non-heat inactivated FBS.
[0515] B. Normal Human Marrow Cells
[0516] Normal human bone marrow (histologically negative for tumor
cells) was obtained from patients who were to have bone marrow
transplants for their solid tumors but died before the marrow was
used. After centrifugation, the buffy coat was removed and cells
were treated with lysis buffer and washed twice with and then
resuspended in RPMI 1640 with 20% HIFBS. The cells were drawn
through a 25 g needle and counted.
[0517] C. Radiometric (Bactec) System
[0518] The Bactec system (Johnston Laboratories, Towson, Md.) is
based on a clinical instrument which was developed to detect
bacteria in blood cultures and has been used to screen for new
antineoplastic agents. This radiometric system is a rapid,
semiautomated system which utilizes the inhibition of the
conversion of .sup.14C glucose to .sup.14CO.sub.2 as an index of
cytotoxicity. The Bactec machine automatically flushes out the
.sup.14CO.sub.2 into an ion chamber where the signal of the
radiolabelled CO.sub.2 is changed into a proportional electrical
signal or growth index value on a scale of 1 to 1000. For the
continuous exposure, the tumor cells or normal marrow cells were
added to 2 ml of the appropriate growth medium containing 2 uC of
.sup.14C glucose plus BMOV at final concentrations of 0.01, 0.1, 1,
10, 25 and 50 uM and injected into 20 ml rubber stoppered serum
vials which contained a mixture of 5% CO.sub.2 and air, and
incubated at 37.degree. C. for 24 days. For one more hour exposure,
cells and BMOV at the same final concentrations were incubated in
15 ml polypropylene conicals in a 37.degree. C. water bath for one
hour. The cells were then centrifuged and washed in medium, then
resuspended in 2 ml of the appropriate growth medium containing 2
uCi of .sup.14C glucose and injected into 20 ml rubber stoppered
serum vials which contained a mixture of 5% CO.sub.2 and air, and
incubated at 37.degree. C. for 24 days at days 6, 9 and 12 for
tumor cell lines and days 6, 15 and 24 for marrow cells the vials
were removed and inserted into the Bactec instrument for
determination of the amount of .sup.14CO.sub.2 produced by the
cells upon metabolizing the .sup.14C glucose. The growth index
values of BMOV treated cells were compared to the growth index
values of non-treated cells and the % survival compared to
untreated controls was calculated.
[0519] D. Resected Tumor Studies
[0520] Seven week old, male C3H/HeJ mice were used in these
studies. RIF-1 (murine radiation induced fibrosarcoma) cells were
cultured in alpha-MEM media containing 10% FBS (Gibco Canada).
Cells were suspended in 1% Hanks buffered salt solution (HBSS pH
7.4) (Gibco Canada) at a concentration of 1.times.10.sup.7
cells/ml. One hundred microliters of these cells (1.times.10.sup.6
cells) was injected into the right flank of each mouse. The tumors
were allowed to grow for 5 days (at which time the tumors ranged
from 6 to 8 mm in diameter). At day 5 the mice were anesthetized
with a Ketamine:Rompom (70 mg/kg: 10 mg/kg) combination (0.02
ml/g). An incision was made 5 mm from the tumor edge and
approximately 90% of each tumor was removed and 150 mg of molten
(50.degree. C.) PCL-BMOV or PCL alone (control) was extruded from a
500 ul syringe onto the entire surface of the resected tumor site.
The PCL solidified within 30 seconds and the area was closed with
5-0 prolene sutures. The mice were examined on days 4, 5, 6 and 7.
On each day tumors were measured (long and short diameters) and
images taken. When the tumors reached a maximum diameter of 9 mm
the mice were sacrificed and the tumor area was excised for future
histological studies.
[0521] Results are shown in FIG. 72.
[0522] E. Tumor Inhibition Studies
[0523] The MDAY-D2 haematopoetic cell line (obtained from Dr. J
Dennis, Mount Sinai Hospital, Toronto) was plated or grown in
suspension in DMEM containing 5% FBS (Gibco Canada). Each mouse was
injected subcutaneously on the posterior lateral side with
4.times.10.sup.5 cells in 100 ul of PBS. After 5 days tumor growth,
150 mg of the PCL or PCL-BMOV molten paste was implanted in an area
adjacent to the tumor site of each mouse. After 15 days the mice
were sacrificed, weighed and the tumors dissected and weighed.
Results are shown in FIG. 71.
[0524] F. Results and Discussion
[0525] Against the normal human bone marrow cells, the one hour
exposure BMOV also had very little effect, even at a concentration
of 50 .mu.M. Using a continuous exposure, the effect of BMOV on the
marrow cells was still not very pronounced, with sensitivity (49%
survival) observed at the 50 .mu.M concentration only. Thus the
BMOV compound did not appear to be very myleosuppressive at the
concentrations and exposures tested.
[0526] In this study, the antineoplastic effect of BMOV have been
shown in vitro against three human cancer cell lines under
conditions that ensure continuous exposure to the drug. However, in
vivo, the efficacy may depend on the continuous exposure of the
tumor cells to BMOV. The main objective of this study was to design
and test (in vivo) a biodegradable polymeric delivery system that
might provide a continuous supply of vanadium (BMOV form) at low
concentrations.
[0527] In vivo experiments showed that single administration of
PCL-BMOV paste subcutaneously inhibited MDAY D2 tumor growth. FIG.
71 shows the data for tumor weights from control mice (PCL-no BMOV)
and mice treated with 25%, 30% and 35% BMOV loaded PCL. There was a
54% inhibition of tumor growth for 25% BMOV loaded PCL (significant
at p<0.05). The 30% and 35% BMOV loadings produced 76% and 80%
inhibition of tumor growth respectively and one of the six mice in
the 35% BMOV group showed complete eradication of the tumor.
[0528] Interestingly, the in vivo drug release profiles showed that
paste consisting of 25% and 30% BMOV released approximately 500 ug
of BMOV per day which has previously shown efficacy. However, 35%
BMOV loaded PCL paste which was the most effective in reducing
tumor growth, released approximately twice this amount of drug in
vitro. These data demonstrate that sustained release of low
quantities of vanadium compounds will be an equally or more
effective antineoplastic regime compared to a daily vanadate
administration regime.
[0529] Although, PCL-BMOV paste was equally effective in inhibiting
tumor growth, the mice showed no signs of stress and weight loss.
Increased stress and weight loss commonly observed with daily
injections of high doses of vanadate is most likely related to
toxicity induced by the high vanadate levels in the plasma
immediately following administration. Using PCL-BMOV paste to
provide sustained release of vanadate for long periods may reduce
large fluctuations in plasma vanadate concentrations and prevent
vanadate induced toxicity. These data are consistent with our
hypothesis that a slow sustained release of BMOV is equally or more
effective in reducing tumor growth and prevents vanadate induced
toxicity.
Example 29
Effect of BMOV Microspheres on Tumor Growth in Mice
[0530] The objective of this study was to test the ability of
BMOV-loaded PLLA microspheres (20%) to regress tumor growth in
mice.
[0531] Twenty of twenty-four mice were injected subcutaneously with
100 ml of MDAY-D2 cells with density of 10.times.10.sup.6/ml. On
day 6 the mice were divided into 6 groups. Group 1, empty control,
group 2, tumor control, group 3, were injected subcutaneously with
0.25 mg/100 ul BMOV, twice a day. Group 4 was injected IP with 20
mg PLLA microspheres containing 5 mg of BMOV. Group 5 was injected
IP with 10 mg of BMOV microspheres on day 6 and day 9 respectively.
Group 6 was injected intramuscularly with 10 mg of BMOV
microspheres on day 6 and day 9. On day 16, the mice were
sacrificed and tumors were dissected.
[0532] The results of these experiments are shown in Tables VI and
VII below.
8TABLE VI Body weights of mice in control and treated groups BMOV
BMOV BMOV BMOV Solution beads, IP beads, IP beads, IM Tumor 0.25 mg
5 mg/ 2 mg 2 mg Control Control x1 once x2 x2 1 19 23.9 18 18.8
19.2 18.2 2 20.2 21 17.8 19.3 18.9 22.6 3 18.4 20 18.4 21.6 17.2
20.1 4 22.2 24.1 17.3 19.1 22.2 Average 19.95 22.25 17.87 19.9 18.6
20.78
[0533]
9TABLE VII Tumor weight of control and treated groups BMOV BMOV
BMOV BMOV Tumor solution beads, IP beads, IP beads, Im control s.c.
5 mg/once 2 mg x2 2 mg x2 1 3.8 0.4 0.2 0.12 2.0 2 1.2 0.12 0.9 0.4
3.3 3 1.3 0.07 0.26 0.3 1.1 4 1.5 0.04 died 1.2 1.6 Average 1.95
0.15 0.45 0.50 1.6
Example 30
Controlled-Release Polymeric Drug Delivery System: Comparative
Study of the In Vitro Drug Release Profiles of Organic Vanadium
Complexes from poly(.epsilon.-caprolactone) (PCL) Thermopastes and
PCL-Methoxypolyethylene Glycol (PCL-MePEG) Thermopastes
[0534] This study was conducted to investigate the encapsulation
and in vitro release kinetics of the four organic complex forms of
vanadium (BMOV, BEMOV, V5, PRC-V) in poly(.epsilon.-caprolactone)
(PCL) thermopastes and/or PCL-methoxypolyethylene glycol
(PCL-MePEG) thermopastes.
[0535] A. Method
[0536] Quantitative analysis (UV/VIS spectrophometric analysis) was
done with different concentrations of vanadium solutions to obtain
the wavelengths of peak absorbance and the calibration equations.
Solubility of vanadium was studies in 10 mM phosphate buffered
saline with albumin (PBS/ALB) (pH 7.4). 1%, 5%, 10%, and 20% (%
vanadium complex in PCL) of each organic complex form of vanadium
were encapsulated in a biodegradable polymer PCL and/or in blends
of PLC with MePEG (MW 350) to produce a polymeric drug delivery
product called a "thermopaste". In vitro release studies of the
various forms of vanadium from thermopastes were carried out at
37.degree. C. in PBS/ALB with vanadium release measured by UV/VIS
absorbance spectroscopy.
[0537] B. Results Results are shown in FIGS. 73 to 77. Briefly, the
peak absorbance of BMOV, BEMOV, and V5 occurred at 276 nm and that
of PRC-V was at 266 nm. The rate and extent of solubility of each
form of vanadium was in the order of V5>BMOV &
BEMOV>PRC-V. In vitro release studies showed that the rate of
drug released from the thermopastes increased with (1) increasing
drug solubility, (2) increasing drug concentration, and (3)
increasing amount of MePEG incorporated into the thermopastes.
[0538] C. Conclusion A controlled dose of vanadium may be released
from PCL thermopastes by using a different form of vanadium (as the
drug), by changing the % loading of the particular form of vanadium
in the PCL, or by including MePEG into the PCL. Whereas changing
the % loading of a particular form of vanadium may control the
release rate in the short term, the delivery of a controlled dose
may involve the implantation of a huge (and potentially toxic)
depot of the drug (in PCL) into the animal. Therefore, the use of
an alternative form of vanadium or the inclusion of MePEG into the
PCL may offer an alternative method delivering a controlled dose of
vanadium.
Example 31
Encapsulation of Camptothecin into PCL Thermopaste and
Anti-Angiogenesis Analysis
[0539] A. Incorporation of Camptothecin into PCL
[0540] Camptothecin was ground with a pestle and mortar to reduce
the particle size to below 5 microns. It was then mixed as a dry
powder with polycaprolactone (molecular wt. 18,000 Birmingham
Polymers, AL USA). The mixture is heated to 65.degree. C. for 5
minutes and the molten polymer/agent mixture is stirred into a
smooth paste for 5 minutes. The molten paste is then taken into a 1
ml syringe and extruded to form 3 mg pellets. These pellets were
then placed onto the CAM to assess their anti-angiogenic
properties.
[0541] B. Results
[0542] Camptothecin-loaded thermopaste was effective at inhibiting
angiogenesis when compared to control PCL pellets. At 5% drug
loading, 4/5 of the CAMs tested showed potent angiogenesis
inhibition. In addition, at 1% and 0.25% loading, 2/3 and 1/4 of
the CAMs showed angiogenesis inhibition respectively. Therefore, it
is evident from these results that camptothecin was sufficiently
released from the PCL thermopaste and it has therapeutic
anti-angiogenic efficacy.
Example 32
Manufacture of S-Phosphonate-Loaded Thermopaste
[0543] In this study, we demonstrated that s-phosphonate, an ether
lipid with antineoplastic activity, was successfully incorporated
into PCL thermopaste and showed efficacy in the CAM assay.
[0544] A. Manufacture of S-Phosphonate Loaded Paste
[0545] Polycaprolactone (Birmingham Polymers, Birmingham, Ala.) and
s-phosphonate were levigated in the appropriate proportions at
55.degree. C. for 2 minutes. The molten mixture was then pipetted
into 3 mg semi-spherical pellets and allowed to set at 4.degree.
C.
[0546] B. CAM Bioassay
[0547] Fertilized, domestic chick embryos (Fitzsimmons Consulting
& Research Services Ltd., B.C.) were incubated for 4 days and
then windowed. Briefly, a small hole (measuring approximately 2 cm
in diameter) was formed by removing the shell and inner shell
membrane from the blunt end of the egg (air space site) and then
the exposed area was sealed with sterilized Parafilm wax. The egg
was then placed into an incubator at 37.degree. C. for an
additional 2 days with the window upright. On day 6 of incubation,
3 mg pellets of s-phosphonate-loaded polymer or control (no drug)
polymer was placed on the surface of the growing CAM vessels. After
a 2 day exposure (day 8 of incubation), the vasculature was
examined using a stereomicroscope fitted with a Contax camera
system. To increase the contrast of the vessels and to mask any
background information, the CAM was injected with 1 ml of
intralipid solution (Abbott Laboratories) prior to imaging.
[0548] C. Results
[0549] PCL pellets loaded with s-phosphonate at 0%, 1%, 2%, 4% and
8% induced a dose-dependent anti-angiogenic reaction in the CAM.
The anti-angiogenic reaction is characterized by the absence of
blood vessels in the region directly below the s-phosphonate loaded
pellet. The normal growth of the dense capillary network seen in
the control CAM has clearly been inhibited in the s-phosphonate
treated CAM's. At higher concentrations (4% and 8%), the treated
CAMs were structurally altered in the vicinity of the drug/polymer
pellet. This alteration included a pronounced thickening of the CAM
immediately adjacent to the s-phosphonate/polymer pellet and
membrane thinning subjacent to the pellet. In all of the CAMs
treated with s-phosphonate, an avascular zone was apparent after a
two day exposure; this was defined as an area devoid of a capillary
network measuring approximately 3 mm.sup.2 in area.
Example 33
Encapsulation of Tyrosine Kinase Inhibitors into PCL Thermopaste
and Analysis Using the CAM Assay
[0550] Tyrosine kinase inhibitors were ground with a pestle and
mortar to reduce the particle size to below 5 microns. They were
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/agent mixture is
stirred into a smooth paste for 5 minutes. The molten paste is then
taken into a 1 ml syringe, and extruded to form 3 mg pellets. These
pellets were then tested in the CAM assay. The tyrosine kinase
inhibitors that were tested in the CAM assay include,
lavendustine-c, erbstatin, herbimysin, and genistein. When
comparing the anti-angiogenic inhibition effects of these agents,
herbimysin (2% in PCL) was the most potent inducing an avascular
zone in 4/4 of the CAMs tested.
Example 34
Encapsulation of Vinca Alkaloids Into PCL Thermopaste and Analysis
Using the Cam Assay
[0551] A. Incorporation of Inhibitors into PCL Thermopaste
[0552] Vinca alkaloids (vinblastine and vincristine) were ground
with a pestle and mortar to reduce the particle size to below 5
microns. They were 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/agent
mixture is stirred into a smooth paste for 5 minutes. The molten
paste is then taken into a 1 ml syringe and extruded to form 3 mg
pellets. These pellets were then tested in the CAM assay.
[0553] B. Results
[0554] When testing the formulations on the CAM, it was evident
that the agents were being released from the PCL pellet in
sufficient amounts to induce a biological effect. Both vinblastine
and vincristine induced anti-angiogenic effects in the CAM assay
when compared to control PCL thermopaste pellets.
[0555] At concentrations of 0.5% and 0.1% drug loading, vincristine
induced angiogenesiss inhibition in all of the CAMs tested. When
concentrations exceeding 2% were tested, toxic drug levels were
achieved and unexpected embryonic death occurred.
[0556] Vinblastine was also effective in inhibiting angiogenesis on
the CAM at concentrations of 0.25%, 0.5% and 1%. However, at
concentrations exceeding 2%, vinblastine was also toxic to the
embryo.
Example 35
Bioadhesive Microspheres
[0557] A. Preparation of Bioadhesive Microspheres
[0558] Microspheres were made from 100 k g/mol PLLA with a particle
diameter range of 10-60 .mu.m. The microspheres were incubated in a
sodium hydroxide solution to produce carboxylic acid groups on the
surface by hydrolysis of the polyester. The reaction was
characterized with respect to sodium hydroxide concentration and
incubation time by measuring surface charge. The reaction reached
completion after 45 minutes of incubation in 0.1 M sodium
hydroxide. Following base treatment, the microspheres were coated
with dimethylaminoproylcarbodiimi- de (DEC), a cross-linking agent
by suspending the microspheres in an alcoholic solution of DEC and
allowing the mixture to dry into a dispersible powder. The weight
ratio of microspheres to DEC was 9:1. After the microspheres ere
dried, they were dispersed with stirring into a 2% w/v solution of
poly (acrylic acid) and the DEC allowed to react with PAA to
produce a water insoluble network of cross-linked PAA on the
microspheres surface. Scanning electron microscopy was used to
confirm the presence of PAA on the surface of the microspheres.
[0559] Differential scanning calorimetry of the microspheres before
and after treatment with base revealed that no changes in bulk
thermal properties (Tg, melting, and degree of crystallinity) were
observed by scanning electron microscopy.
[0560] B. In Vitro Paclitaxel Release Rates
[0561] Paclitaxel loaded microspheres, (10% and 30% w/w loadings)
with the same particle diameter size range were manufactured and in
vitro release profiles for 10 days release in phosphate buffered
saline. Release was proportional to drug loading, with 400 .mu.g of
Paclitaxel released from 5 mg of 30% loaded microspheres in 10 days
and 150 .mu.g released from 10% loaded microspheres in the same
period. The efficiency of encapsulation was about 80%. The
Paclitaxel loaded microspheres were incubated in 0.1M sodium
hydroxide for 45 minutes and the zeta potential measured before and
after incubation in sodium hydroxide. The surface charge of
Paclitaxel loaded microspheres was lower than microspheres with no
Paclitaxel both before and after treatment with base.
[0562] C. Preparation and In Vitro Evaluation of PLLA Coated with
Either Poly-lysine or Fibronectin
[0563] PLLA microspheres were prepared containing 1% sudan black
(to color the microspheres). These spheres were suspended in a 2%
(w/volume) solution of either polylysine (Sigma
chemicals--Hydrobromell form) or Fibronectin (Sigma) for 10
minutes. The microspheres were wasted in buffer once and placed on
the inner surface of freshly prepared bladders from rats. The
bladder were left for 10 minutes then washed three times in buffer.
Residual (bound) microspheres were present on the bladder wall
after the process therefore showing bioadhesion had occurred (FIGS.
78A and 78B) for both Fibronectin and poly-1-lysine coated
microspheres.
Example 36
Synthesis of poly(N-isopropylacrylamide)
[0564] Polyacrylamide and its derivatives may be readily
synthesized through free radical polymerization and irradiation
induced polymerization. The following is an example of synthesizing
poly(N-isopropylacrylamide), wherein the monomer
N-isopropylacrylamide is purified by recrystallization from an
organic solvent such as hexane and toluene.
[0565] Briefly, N-isopropylacrylamide is dissolved in toluene at a
temperature such as 60.degree. C. This solution is cooled down to a
lower temperature (e.g. 4.degree. C.) to allow the
recrystallization of the monomer. The monomer is then collected by
filtration and dried under vacuum. For synthesis, the purified
monomer (20 g) is dissolved in distilled water (180 ml) in a round
bottomed glass flask. The solution is purged with nitrogen for 30
minutes to replace dissolved oxygen. The temperature is then raised
to 65.degree. C. and a small amount (0.1 g) of initiator such as
ammonium persulfate and 2,2'-azobis-isobutyronitrile is added to
start the polymerization. The polymerization is completed within 10
hours and is under the protection of nitrogen and stirring.
Finally, poly(N-isopropylacrylamide) is precipitated by adding
ethanol. The polymer is dried and stored.
Example 37
Synthesis of Poly(acrylic acid) Derivatives
[0566] Poly(acrylic acid) and its derivatives can be synthesized
through free radical polymerization and irradiation induced
polymerization. The following is an example of synthesizing
poly(acrylic acid), wherein monomer acrylic acid is purified by
distillation (e.g., 40.degree. C.) under a reduced pressure (e.g.,
10 mmHg).
[0567] Briefly, the purified monomer (20 g) is dissolved in dioxane
(180 ml) in a round bottomed glass flask. The solution is purged
with nitrogen for 30 minutes to replace the dissolved oxygen. The
temperature is then raised to 65.degree. C. and a small amount (0.1
g) of initiator 2,2'-azobis-isobutyronitrile is added to start the
polymerization. The polymerization is completed within 24 hours and
is under the protection of nitrogen and stirring. Finally,
poly(acrylic acid) is precipitated by adding n-hexane. The polymer
is dried and stored.
Example 38
Preparation of Micellular Paclitaxel
[0568] A. Synthesis of Diblock Copolymer of PDLLA-MePEG
[0569] Monomers of methoxy polyethylene glycol (e.g., M.W. 2000,
150 g) and DL-lactide (100 g) are added to a round bottomed flask
and the temperature is raised to 130-150.degree. C. After the
melting of the monomers, 0.6 g catalyst stannous octoate is added.
The polymerization is finished within 0 hours. The reaction is
under protection of N.sub.2 and with stirring.
[0570] B. Preparation of Micellar Paclitaxel
[0571] The PDLLA-MePEG copolymer is dissolved in acetonitrile or
50:50 ethanol:acetone (polymer conc.<40%). The polymer solution
is centrifuged (14000 rpm) for 5 min. The supernatant (insoluble
polymer discarded) is transferred to a glass test tube. Paclitaxel
is dissolved in acetonitrile or 50:50 ethanol:acetone and is added
to the purified polymer solution. After vortex mixing, the solvent
is evaporated at 60.degree. C. under a stream of nitrogen (normally
takes 2 hours for a typical 40 mg paclitaxel batch). The residual
solvent can be removed by applying vacuum and heat. The matrix is
heated at about 60.degree. C. until it becomes a transparent gel.
Then a certain volume of water (>4 times of matrix weight) is
added to the matrix. This is followed immediately by vortex mixing
until the solubilization of the paclitaxel/polymer matrix.
[0572] C. Preparation of Delivery Systems of Micellar
Paclitaxel/Thermogelling Polymers
[0573] A thermogelling polymer such as poly(N-isopropylacrylamide),
is dissolved in distilled water. Separately, water is added to a
micellar paclitaxel/polymer matrix, e.g., 10% paclitaxel loaded
PDLLA-MePEG 2000-40/60, to form a micellar paclitaxel solution.
Both solutions are cooled (e.g., 4.degree. C.) and mixed to form
the thermogelling delivery system.
Example 39
Perivascular Administration of Paclitaxel
[0574] WISTAR rats weighing 250-300 g are anesthetized by the
intramuscular injection of Innovar (0.33 ml/kg). Once sedated they
are then placed under Halothane anesthesia. After general
anesthesia is established, fur over the neck region is shaved, the
skin clamped and swabbed with betadine. A vertical incision is made
over the left carotid artery and the external carotid artery
exposed. Two ligatures are placed around the external carotid
artery and a transverse arteriotomy is made. A number 2 FRENCH
FOGART balloon catheter is then introduced into the carotid artery
and passed into the left common carotid artery and the balloon is
inflated with saline. The catheter is passed up and down the
carotid artery three times. The catheter is then removed and the
ligature is tied off on the left external carotid artery.
[0575] Paclitaxel (33%) in ethelyne vinyl acetate (EVA) is then
injected in a circumferential fashion around the common carotid
artery in ten rats. EVA alone is injected around the common carotid
artery in ten additional rats. Five rats from each group are
sacrificed at 14 days and the final five at 28 days. The rats are
observed for weight loss or other signs of systemic illness. After
14 or 28 days the animals are anesthetized and the left carotid
artery is exposed in the manner of the initial experiment. The
carotid artery is isolated, fixed at 10% buffered formaldehyde and
examined for histology.
Example 40
Treatment of Artherosclerosis
[0576] A. Atherosclerosis
[0577] Atherosclerotic lesions are created in New Zealand white
rabbits by diet only. Briefly, New Zealand white rabbits weighing
approximately 1.6 kg are placed on a powdered chow supplemented by
0.25% cholesterol by weight. Total plasma cholesterol is measured
on a weekly basis by taking samples from a marginal ear vein after
an injection of Innovar (0.1 ml/kg) to dilate blood vessels.
Samples are mixed with EDTA to achieve a 0.15% concentration in the
sample and placed on ice until separation of plasma by low speed
centrifugation.
[0578] One week after initiation of the full cholesterol diet, the
animals are randomized into 3 groups of 10. After anaesthetic
induction with Ketamine 35 mg/kg and Xylazine 7 mg/kg, and then
general anesthesia via intubation, the fur is shaved and the skin
sterilized over the abdomen. A laparotomy is performed and the
abdominal aorta isolated. Using a 22 g needle, ethylene vinyl
acetate paste, ethylene vinyl acetate paste containing 5%
paclitaxel, or ethylene vinyl acetate paste containing 33%
paclitaxel is placed in a circumferential manner around the
proximal half of the infrarenal abdominal aorta. The distal half of
the aorta extending to the aortic bifurcation is not treated. In 10
control rabbits, the infrarenal abdominal aorta is isolated, but
nothing is injected around it.
[0579] The atherogenic chow is continued for 24 weeks. At that
time, the animals are anesthetized with an injection of Ketamine
(350 mg/kg), and xylazine (7 mg/kg) intramuscularly and then
sacrificed with an intravenous overdose of Euthanol (240 mg/ml; 2
ml/4.5 kg). The animals are then perfusion fixed at 100 mm mercury
via the left ventricle by perfusing Hanks' balanced salt solution
with 0.15 mmol/litre N-2-hydroxyethylpaparazine-N'-2-ethanesulfonic
acid (ph 7.4) containing Heparin (1 IU/ml) for ten minutes followed
by dilute Kamovsky's fixative for 15 minutes. The thoracic and
abdominal aorta and iliac arteries are removed en bloc and are
placed in a similar solution for a further 30 minutes.
[0580] Serial thin sections are then performed through the thoracic
aorta and particularly through the infrarenal abdominal aorta.
Movat, H&E, and Masson stains are performed and histologic
analysis made to examined the degree of luminal compromise, the
degree of atherosclerotic lesion development, and any perilumenl
reaction to the circumferential arterial medications.
Example 41
Treatment of Restenosis
[0581] WISTAR rats weighing 250-300 g are anesthetized by the
intramuscular injection of innovar (0.33 ml/kg). Once they are
sedated they are placed under Halothane anesthesia. After general
anesthesia is established, the fur over the neck region is shaved
and the skin cleansed with betadine. A vertical incision is made
over the left carotid artery and the external carotid artery
exposed. Two ligatures are placed around the external carotid
artery and a transverse arteriotomy is made between them. A 2 Fr
Fogarty balloon catheter is introduced into the external carotid
artery and passed into the left common carotid artery and the
balloon is inflated with saline. The catheter is passed up and down
the carotid artery three times to denude the endothelium. The
catheter is removed and the ligatures tied off on the left external
carotid artery.
[0582] The animals are randomized into groups of 5. Subgroups of 5
rats are control, carrier polymer alone, carrier polymer plus 1, 5,
10, 20, and 33% paclitaxel is delivered. There are two carrier
polymers to be investigated; EVA and EVA/PLA blend. The polymer
mixture is placed in a circumferential manner around the carotid
artery. The wound is then closed. Rats in each group are sacrificed
at 14 and 28 days. In the interim, the rats are observed for weight
loss or other signs of systemic. After 14 or 28 days, the animals
are sacrificed by initial sedation with intramuscular Innovar (0.33
ml/kg). The arteries are then examined for histology.
Example 42
Intimal Hyperplasia Causing Graft Stenosis
[0583] A. Animal Studies
[0584] General anaesthesia is induced into domestic swine. The neck
region is shaved and the skin sterilized with cleansing solution.
Vertical incisions are made on each side of the neck and the
carotid artery is exposed and 8 mm PTFE graft is inserted by 2 end
to side anastomoses, the proximal anastomosis on the common carotid
artery and the distal anastomosis on the internal carotid artery
bilaterally. The intervening bypassed artery is ligated. The
animals are randomized into groups of 10 pigs receiving carrier
polymer alone, 10 pigs receiving carrier polymer plus 5%
paclitaxel, and 10 pigs receiving carrier polymer plus 33%
paclitaxel adjacent to each surgical created anastamosis on the
left side only. The right sided grafts will serve as a control in
each pig. The wounds are closed and the pigs recovered.
[0585] A second group of pigs are studied. The grafts are created
in a similar manner. No vasoactive agent is placed next to the
anastamotic sites at the time of operation. The animals are
recovered. Two weeks after the graft has been performed, a second
general anaesthetic is administered and the left carotid artery is
reexplored. Adjacent to the proximal and distal anastamoses, 10
pigs each receive carrier alone, carrier polymer plus 5% paclitaxel
and carrier polymer plus 33% paclitaxel in a circumferential manner
adjacent to both proximal and distal anastamoses. The wounds are
closed and the pigs recovered. Opposite the right sided graft
serves as a control.
[0586] At 3 months, all pigs undergo general anesthetic. A cutdown
is made on the femoral artery and a pigtail catheter is inserted in
the ascending thoracic aorta under fluoroscopic guidance. Arch
injection with imaging of the carotid vasculature is performed.
Specifically, the degree of stenoses of the proximal and distal
grafts and the artery immediately distal to the distal anastamosis
of the graft is measured and the % stenosis calculated. If
necessary, selective injections of the common carotid arteries are
performed.
[0587] Five pigs in each group are sacrificed. The animals are then
perfusion fixed at 100 mm mercury via the left ventricle by
perfusing Hanks' balanced salt solution with 0.15 mmol/litre
N-2-hydroxyethylpaparazine-N'-2-ethanesulfonic acid (ph 7.4)
containing Heparin (1 IU/ml) for ten minutes followed be dilute
Kamovsky's fixative for 15 minutes. The thoracic and abdominal
aorta and carotid arteries are removed en bloc and are placed in a
similar solution for a further 30 minutes.
[0588] Histological sections through the carotid artery immediately
proximal to the proximal anastamosis, at the proximal anastamosis,
at the distal anastamosis and the carotid artery immediately distal
to the distal anastamosis are made. The sections are stained with
Movat and H&E and Masson stains. Histologic analysis of intimal
and advantitial reaction as well as perivascular reaction are
noted. Morphometric analysis with degree of luminal narrowing is
calculated.
[0589] The remaining pigs are studied at 6 months and a similar
angiography sacrifice procedures is performed.
[0590] 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.
* * * * *