U.S. patent application number 09/735182 was filed with the patent office on 2001-05-24 for compound delivery using rapidly dissolving collagen film.
Invention is credited to DeVore, Dale P., Eiferman, Richard A., Keates, Edwin U..
Application Number | 20010001669 09/735182 |
Document ID | / |
Family ID | 22181375 |
Filed Date | 2001-05-24 |
United States Patent
Application |
20010001669 |
Kind Code |
A1 |
DeVore, Dale P. ; et
al. |
May 24, 2001 |
Compound delivery using rapidly dissolving collagen film
Abstract
Disclosed herein are collagen films which rapidly dissolve at
35.degree. C. Also disclosed are methods for the preparation of the
collagen films and their use as a vehicle for delivering a dose of
therapeutic compound to a specific tissue site.
Inventors: |
DeVore, Dale P.;
(Chelmsford, MA) ; Eiferman, Richard A.;
(Louisville, KY) ; Keates, Edwin U.; (Villanova,
PA) |
Correspondence
Address: |
Karen L. Elbing, Ph.D.
Clark & Elbing LLP
176 Federal Street
Boston
MA
02110
US
|
Family ID: |
22181375 |
Appl. No.: |
09/735182 |
Filed: |
December 12, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09735182 |
Dec 12, 2000 |
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09083899 |
May 22, 1998 |
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Current U.S.
Class: |
424/484 ;
530/356 |
Current CPC
Class: |
A61K 9/7007
20130101 |
Class at
Publication: |
424/484 ;
530/356 |
International
Class: |
A61K 009/14 |
Claims
What is claimed is:
1. A method of preparing a rapidly dissolving collagen film which
includes a therapeutic compound, said method comprising: a)
preparing a purified solution of monoreactive-amine modified
collagen; b) heating said collagen solution to about 35-45.degree.
C. for a time sufficient to reduce collagen viscosity; c) adding
said compound to the heated collagen solution; and d) casting said
solution into thin layers, wherein said solution dries and forms
said film.
2. The method of claim 1, wherein said collagen is modified by
glutaric anhydride derivatization.
3. A collagen film prepared by the method of claim 1.
4. A collagen film, wherein said film rapidly dissolves upon
exposure to about 35.degree. C.
5. The collagen film of claim 4, wherein said collagen film
dissolves within ten minutes upon exposure to about 35.degree.
C.
6. The collagen film of claim 4, wherein said collagen film
dissolves within five minutes upon exposure to about 35.degree.
C.
7. The collagen film of claim 4, wherein said collagen film
dissolves within two minutes upon exposure to about 35.degree.
C.
8. The collagen film of claim 4, wherein said collagen film
dissolves within one minute upon exposure to about 35.degree.
C.
9. The collagen film of claim 4, wherein said collagen film
dissolves within thirty seconds upon exposure to about 35.degree.
C.
10. The collagen film of claim 4, wherein said film includes a
compound which is an inhibitor of cell proliferation.
11. The collagen film of claim 10, wherein said compound is an
anti-metabolic antibiotic.
12. The collagen film of claim 11, wherein said compound is
mitomycin.
13. The collagen film of claim 4, wherein said film includes a
compound which is an anti-metabolite.
14. The collagen film of claim 13, wherein said compound is
5-fluorouracil.
15. The collagen film of claim 4, wherein said film includes a
compound which is an anti-fibrotic.
Description
BACKGROUND OF THE INVENTION
[0001] In general, the invention relates to rapidly dissolving
collagen films, methods of preparation, and the use of these films
for rapid compound delivery.
[0002] The ability to specifically deliver a compound to a
particular site in the human body is a desirable goal in many areas
of medicine. For example, in cancer therapy, administration of
chemotherapeutic agents to a tumor site with minimal exposure to
surrounding tissues would dramatically reduce undesirable side
effects to the surrounding tissues, or the body as a whole, while
facilitating delivery of potent doses to malignant cells.
[0003] In addition, the inhibition of wound healing is beneficial
in certain circumstances, for example, following glaucoma
filtration surgery (otherwise known as trabeculectomy). The initial
stage in the process of wound healing is characterized by the
movement of intravascular components, such as plasma and blood
proteins, to the extravascular area (Peacock, In: Wound Repair,
491-492, 1984, ed. EE Peacock, WB Saunders Co, Philadelphia, Pa.).
Neutrophils and macrophages then migrate to the injury site,
functioning to prevent infection and promote fibroblast migration.
Subsequent phases of wound healing include fibroblast secretion of
collagen, collagen stabilization, angiogenesis, and wound closure
(Costa et al., Opth. Surgery 24: 152-170, 1993).
[0004] During surgery for the treatment of glaucoma, a fistula is
frequently created to allow for post-operative drainage of
intraopthalmic fluid from the eye. Accordingly, the inhibition of
fistula healing is beneficial in order to extend the drainage time
and reduce intraopthalmic pressure. Several therapies have been
adopted to inhibit fistula healing, including beta irradiation,
5-fluorouracil treatment, and mitomycin (also known as mitomycin-C
or mitomicin) treatment (Costa et al., Opth. Surgery 24: 152-170,
1993).
SUMMARY OF THE INVENTION
[0005] The present invention provides a method of preparing a
rapidly dissolving collagen film which includes a therapeutic
compound. The method involves (i) preparing a purified solution of
monoreactive-amine modified collagen, e.g., a glutaric anhydride
derivatized collagen, (ii) heating the collagen solution to about
35-45.degree. C. for a time sufficient to reduce collagen
viscosity, (iii) adding the compound to the heated collagen
solution, and iv) casting the solution into thin layers, wherein
the solution dries and forms the film.
[0006] The invention also includes a collagen film prepared by the
above described method and a collagen film which rapidly dissolves
upon exposure to about 35.degree. C. Preferably, the collagen film
dissolves within five to ten minutes upon exposure to about
35.degree. C. More preferably, the collagen film dissolves within
two minutes upon exposure to about 35.degree. C. . Most preferably,
the collagen film dissolves within one minute or 30 seconds upon
exposure to about 35.degree. C.
[0007] The therapeutic compound contained within the rapidly
dissolving collagen film may be an inhibitor of cell proliferation,
e.g., an anti-metabolic antibiotic, anti-metabolite, anti-fibrotic,
anti-viral compound, or angiostatic compound. Preferably, the
compound is an anti-metabolic antibiotic, e.g., mitomycin,
daunorubicin, mithramycin, bleomycin, or doxorubicin.
[0008] Alternatively, the therapeutic compound may be an
anti-metabolite. Examples of useful anti-metabolites include
5-fluorouracil, 5-fluorouridine-5'-monophosphate,
5-fluorodeoxyuridine, 5-fluorodeoxyuridine-5'-monophosphate, and
5-fluroorotate.
[0009] In yet other applications, the therapeutic compound
contained within the rapidly dissolving collagen film is an
anti-fibrotic. Examples of useful anti-fibrotics include inhibitors
of prolyl nydroxylase and lysyl hydroxylase, e.g., iron chelators,
.alpha.,.alpha.-dipyridyl, o-phenanthroline, proline analogs,
lysine analogs, and free radical inhibitors and scavengers;
inhibitors of collagen secretion, e.g., colchicine, vinblastin,
cytochalasin B, copper, zinc, and EGTA; inhibitors of collagen
secretion and maturation, e.g., BAPN, vincristine, and
D-penicillamine; and stimulators of collagen degradation, e.g.,
EDTA and colchicine.
[0010] As noted above, the therapeutic compound may also be an
anti-viral drug. Examples of anti-viral drugs that can be used in
the invention include vidarabine, acyclovir, AZT, and
amantadine.
[0011] Finally, angiostatic drugs, e.g., angiostatin, as well as
other miscellaneous anti-cell proliferative drugs, e.g., tissue
plasminogen activator (TPA), heparin, cytosine arabinoside, and
gamma-interferon, may also be used in the rapidly dissolving
collagen films described herein.
[0012] In addition to methods of collagen film preparation, the
invention also provides a method of rapidly delivering a compound
dose to a specific tissue site in a mammal. The method involves
administering a collagen film containing the compound dose to the
tissue site, wherein the collagen film rapidly dissolves upon
exposure to the mammalian tissue site. Using this method to deliver
toxic compounds, the toxic side effects are essentially restricted
to the specific tissue site of compound delivery.
[0013] In a related aspect, the invention also includes a method of
treating a mammal to inhibit cellular proliferation, e.g., wound
healing or tumor growth, at a specific tissue site. The method
involves administering a collagen film comprising an inhibitor of
cell proliferation, e.g., an anti-metabolic antibiotic,
anti-metabolite, anti-fibrotic, anti-viral compound, or angiostatic
compound, to the tissue site, wherein the collagen film rapidly
dissolves upon exposure to the tissue and delivers a dose of the
compound sufficient to inhibit cell proliferation at the tissue
site.
[0014] In preferred embodiments, the cell proliferation inhibitor
is mitomycin, 5-fluorouracil, or an anti-fibrotic. In addition, in
other preferred embodiments, the collagen film dissolves within
five to ten minutes upon exposure to the mammalian tissue site,
more preferably, within two minutes, and, most preferably, within
one minute or 30 seconds. In addition, the mammal is preferably a
human.
[0015] This method can be used, for example, in treating a mammal
undergoing surgery for glaucoma. In this application, the collagen
film is administered to the trabeculectomy-created fistula in the
mammal, wherein the dose of cell proliferation inhibitor is
sufficient to inhibit closure of the fistula. Preferably, the cell
proliferation inhibitor used is mitomycin at a dose of 200-400
.mu.g and may be administered in a 4.times.4 mm collagen film
patch. Most preferably, the mitomycin dose is 400 .mu.g.
[0016] Use of this treatment results in reduced post-operative
intraocular pressure. Preferably, post-operative intraocular
pressure as a result of this method is less than 16 mmHg, more
preferably, less than 12 mmHg, and, most preferably, less than 6
mmHg.
[0017] As used herein, by "mono-reactive amine-modified" is meant
reacted with a monoreactive amine-modifying agent, also known as a
monoacylating or sulfonating agent. Useful agents include, without
limitation, anhydrides, acid halides, sulfonyl halides, and active
esters. The modifying agent is preferably a compound or combination
of compounds which contains an acidic, carboxylic, or sulfonide
group, or generates an acidic, carboxylic, or sulfonic group during
reaction.
[0018] By "inhibitor of cell proliferation" is meant an inhibitor
of an increase in the number of cells located at a particular site.
Such inhibition may occur by inhibition of cell migration or
attachment, cell replication, cell survival, or angiogenesis.
[0019] By "specific tissue site" is meant the area of tissue
directly in contact with the collagen film administered to the
tissue.
[0020] By "rapidly dissolves" is meant dissolves, or melts, in
approximately 30 minutes or less.
[0021] The present invention provides a number of advantages. For
example, the present techniques and collagen film compositions
facilitate an improved approach for delivering a compound in
situations where both a precise dose and accurate placement are
required. The dose can be adjusted to any desired amount, i.e., by
modifying the concentration of compound in the film or the size of
the film, and the solid nature of the film allows its placement at
any site in the body which can be reached by surgical techniques.
In addition, the invention provides for the rapid dissolution of
the collagen film upon exposure to normal body temperature. Taken
together, these features ensure that a delivered compound achieves
a certain concentration at a specific site, reducing possible
inaccuracy due to mistaken dose or improper placement.
[0022] For delivery of mitomycin or 5-fluorouracil to a
post-trabeculectomy fistula, the present invention represents an
improvement over current empirical techniques employed, which
typically involve placing a sponge wetted with compound on the
fistula site for 3-5 minutes.
[0023] The advantage of delivering essentially all compound to a
specific site also provides for limited compound delivery to
tissues surrounding the delivery site. This advantage is especially
relevant when the compound to be delivered has toxic effects. By
restricting delivery to the targeted tissues, any unintentional or
unnecessary toxic damage to surrounding tissues is reduced.
[0024] Furthermore, compounds, such as mitomycin, exhibit increased
stability in the collagen film as compared to stability in
solution. Thus, one collagen film sample preparation can be
subdivided and used for several applications over the course of
several weeks. This feature provides the advantages of reducing
experimental variation when administered over several days and
eliminating the need for daily pre-surgical sample preparation.
[0025] Other features and advantages of the invention will be
apparent from the following detailed description thereof, and from
the claims.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Described herein are methods of preparing collagen films
containing therapeutic compounds that readily dissolve upon
exposure to normal human body temperature (35-37.degree. C.). These
collagen films can be used for the rapid and accurate delivery of
compounds to specific tissue sites.
[0027] For the purposes of this invention, collagen can be
collected, solubilized, subjected to modification by mono-reactive,
amine-modifying agents, and re-precipitated by any standard
technique, e.g., those provided in DeVore et al. (U.S. Pat. No.
4,713,446), herein incorporated by reference. The following example
is provided as an illustration and is in no way intended to limit
the scope of the invention.
[0028] Preparation of Collagen
[0029] As a first step toward producing rapidly dissolving films,
soluble collagen was prepared by standard procedures. Young calf
hide was washed thoroughly with reagent alcohol and with deionized,
pyrogen-free water, cut into approximately 1 cm.sup.2 sections, and
stirred overnight in 40 volumes of 0.5 M acetic acid. The mixture
was then supplemented with pepsin (3% hide wet weight) and stirred
for 72 hours. The digested, solubilized collagen was filtered
through cheesecloth and precipitated by increasing the NaCl
concentration to 0.8 M. The collagen was then cycled twice through
steps of redissolution, in 0.5 M acetic acid, and reprecipitated.
The collagen precipitate was then redissolved in 0.1 N acetic acid,
dialyzed against 0.1 M acetic acid, filtered (0.45 .mu.m), and
refiltered (0.22 .mu.m).
[0030] The purified, telopeptide-poor collagen was derivatized with
glutaric anhydride as previously described (U.S. Pat. Nos.
5,631,243 and 5,492,135). Briefly, the collagen solution
(approximately 3 mg/ml) was adjusted to pH 9.0 with 10 N and 1 N
NaOH. While stirring the solution, glutaric anhydride was added at
10% (weight of collagen). For twenty minutes, the stirring
continued, and the pH was maintained.
[0031] The pH of the solution was adjusted to 4.3 with 6 N and 1 N
HCl to precipitate the derivatized collagen. The precipitate was
centrifuged at 3500 rpm for 30 minutes. The pellet was washed three
times in pyrogen-free deionized water and then redissolved in
phosphate buffered glycerol (2% glycerol in 0.004 M phosphate
buffer, pH 7.4) to achieve a final concentration of approximately
10 mg/ml.
[0032] Preparation of Collagen Films Containing Mitomycin
[0033] To prepare collagen films containing mitomycin, the collagen
solution, described above, was heated in a 35.degree. C. water bath
for 30 minutes to reduce viscosity. Mitomycin (e.g.,
Mutamycin.RTM., Bristol Myers Squibb, Princeton, N.J.), also known
as mitomicin C, was added to the heated collagen. The collagen
solution was then poured into petri dishes in a thin layer and
allowed to air dry under a laminar-flow hood.
[0034] Collagen film melting time at 35.degree. C. was measured
after placing the films in 0.8% saline in a 35.degree. C. water
bath. Pre-heated collagen films melted in approximately one minute.
In contrast, collagen films poured into petri dishes without
pre-heating melted at 35.degree. C. in approximately 30
minutes.
[0035] Mitomycin-containing collagen films had a final mitomycin
concentration of 400 .mu.g per 16 mm.sup.2. 6 mm diameter discs
were cut from the film and applied to human subconjunctival
fibroblasts derived from Tenon's membrane layered in 96 well plates
(CSM supplemented with 10% fetal bovine serum). After 72 hours,
mitomycin-induced inhibition of cell division was assessed by
measuring the reduction in fluorescence intensity (RFU) using a
fluorogenic CalceinAM assay (see, for example, Decherchi et al., J.
Neurosci. Meth. 71: 205 (1997); Sugita, Pflitgers Arch. 429: 555
(1995); Padanilam et al., Ann. NY Acad. Sci. 720: 111 (1994);
Lichtenfels et al., J. Immunol. Meth. 172: 227 (1994); and Wang et
al., Human Immunol. 37: 264 (1993)). The mitomycin-containing
collagen films inhibited approximately 91% of the cell division
demonstrated in control cells.
[0036] Mitomycin-containing films may be stored for later use. For
example, mitomycin activity in the collagen films described above
was maintained for at least 6 weeks after preparation of the films
(stored at 4.degree. C.). Administration of mitomycin-collagen
films, 2, 4, and 6 weeks old, demonstrated 91%, 90%, and 92%
inhibition of cell division, respectively, compared to
mitomycin-free controls. These values were comparable to the % cell
death inhibition elicited by administration of a freshly prepared
mitomycin solution (0.4 mg/ml solution, dissolved in USP sterile
water).
[0037] In contrast to the stability of mitomycin in the collagen
film, HPLC analysis of the mitomycin solution determined that
stability was maintained for only 4 days following storage at
ambient temperature and 4.degree. C. in the dark. Dissolution and
storage in 0.9% saline or phosphate buffer (pH 7.4) is not
recommended due to degradation and precipitation.
[0038] Use
[0039] Rapidly dissolving collagen films containing therapeutic
compounds are useful for various treatments. For example, the
collagen-mitomycin film may be administered to the external opening
of the fistula created during glaucoma filtering surgery
(trabeculectomy). Immediately following surgery, a collagen film,
e.g., a 4.times.4 mm patch, containing 100-1000 .mu.g mitomycin
(preferably 400 .mu.g), is directly applied to the external opening
of the fistula prior to replacing the scleral flap. Administration
of the mitomycin increases the duration of fistula patency,
increasing filtration from the eye and reducing intraocular
pressure.
[0040] Other compounds may also be administered to the
trabeculectomy-created fistula to increase filtration during
recovery. For example, 5-fluorouracil-containing films may be
administered in the same fashion to deliver a 5-fluorouracil dose
of 25-250 .mu.g (preferably 100 .mu.g). Other alternative compounds
that are effective for this treatment are anti-fibrotic,
angiostatic, and anti-viral compounds.
[0041] Administration of the rapidly dissolving collagen films
containing inhibitors of cell proliferation are also useful for
treatment during recovery from other surgical procedures where
prevention of wound healing is beneficial.
[0042] In addition, the collagen films of the invention may be
administered to reduce cellular proliferation in specific tissue
sites, such as for the localized inhibition of neoplastic or
non-neoplastic cell growth. For this application, any
chemotherapeutic compound may be dissolved in the collagen matrix
in concentrations appropriate for inhibiting cell growth.
OTHER EMBODIMENTS
[0043] While the treatment regimens described herein are preferably
applied to human patients, they also find use in the treatment of
other animals, such as domestic pets or livestock.
[0044] Moreover, while the invention has been described in
connection with specific embodiments thereof, it will be understood
that it is capable of further modifications and this application is
intended to cover any variations, uses, or adaptations of the
invention following, in general, the principles of the invention
and including such departures from the present disclosure come
within known or customary practice within the art to which the
invention pertains and may be applied to the essential features
hereinbefore set forth, and follows in the scope of the appended
claims.
* * * * *