U.S. patent application number 17/064126 was filed with the patent office on 2021-01-28 for treatment method and product for uterine fibroids using purified collagenase.
This patent application is currently assigned to BioSpecifics Technologies Corp.. The applicant listed for this patent is BioSpecifics Technologies Corp., Duke University. Invention is credited to Phyllis Carolyn Leppert, Thomas L. Wegman.
Application Number | 20210023014 17/064126 |
Document ID | / |
Family ID | 1000005137344 |
Filed Date | 2021-01-28 |
United States Patent
Application |
20210023014 |
Kind Code |
A1 |
Leppert; Phyllis Carolyn ;
et al. |
January 28, 2021 |
Treatment Method and Product for Uterine Fibroids using Purified
Collagenase
Abstract
The invention relates to compositions and methods for treating
uterine fibroids, wherein a uterine fibroid treatment agent
comprising collagenase in an amount effective to cause shrinkage of
uterine fibroids is injected or inserted into the uterine
fibroid.
Inventors: |
Leppert; Phyllis Carolyn;
(Salt Lake City, UT) ; Wegman; Thomas L.; (N.
Merrick, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioSpecifics Technologies Corp.
Duke University |
Wilmington
Durham |
DE
NC |
US
US |
|
|
Assignee: |
BioSpecifics Technologies
Corp.
Wilmington
DE
Duke University
Durham
NC
|
Family ID: |
1000005137344 |
Appl. No.: |
17/064126 |
Filed: |
October 6, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15666365 |
Aug 1, 2017 |
|
|
|
17064126 |
|
|
|
|
14213910 |
Mar 14, 2014 |
9744138 |
|
|
15666365 |
|
|
|
|
61790070 |
Mar 15, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 9/0034 20130101; A61K 9/1075 20130101; A61K 9/0019 20130101;
C12Y 304/24003 20130101; A61K 9/48 20130101; A61K 38/43 20130101;
A61K 38/4886 20130101 |
International
Class: |
A61K 9/48 20060101
A61K009/48; A61K 38/48 20060101 A61K038/48; A61K 45/06 20060101
A61K045/06; A61K 9/107 20060101 A61K009/107; A61K 38/43 20060101
A61K038/43; A61K 9/00 20060101 A61K009/00 |
Claims
1. A composition for treating a uterine fibroid in a patient,
comprising an amount of Clostridium histolyticum collagenase
effective to treat said uterine fibroid by injection through a
uterine fibroid capsule into said uterine fibroid in the patient,
wherein said collagenase is obtained by separately purifying
Clostridium histolyticum collagenase I and collagenase II and
recombining said collagenase I and collagenase II in an optimized
fixed mass ratio.
2. The composition of claim 1, wherein said composition can be
administered through a syringe fitted with a 10 gauge or smaller
needle.
3. A kit for providing at least one therapeutic dose of a
formulation of claim 1, said kit comprising in a unit package at
least one container containing a sterile thermosensitive hydrogel
fluid in an amount sufficient for at least one therapeutic dose;
and at least one second container containing an effective amount of
collagenase in lyophilized, powder form.
4. The composition of claim 1, wherein said composition further
comprises a chemical ablation agent, a non-steroidal
anti-inflammatory drug, an oral contraceptive, a GnRH agonist, an
antiprogestogen, or a selective progesterone receptor
modulator.
5. The composition of claim 4, wherein said composition further
comprises a chemical ablation agent.
6. The composition of claim 5, wherein said chemical ablation agent
is a salt.
7. The composition of claim 6, wherein said chemical ablation agent
is selected from an enzyme, an acid, a base and an oxidizing
agent.
8. The composition of claim 1, wherein said composition further
comprises one or more uterine fibroid treatment agents.
9. The composition of claim 1, wherein said composition comprises
0.25 mg/mL collagenase from Clostridium histolyticum.
10. The composition of claim 1, wherein said composition comprises
0.5 mg/mL collagenase from Clostridium histolyticum.
11. The composition of claim 1, wherein said composition comprises
1.0 mg/mL collagenase from Clostridium histolyticum.
12. The composition of claim 1, wherein said composition comprises
2.0 mg/mL collagenase from Clostridium histolyticum.
13. The composition of claim 1, wherein said composition comprises
collagenase I and collagenase II in a mass ratio of about
0.5:1.5.
14. The composition of claim 1, wherein said composition comprises
collagenase I and collagenase II in a mass ratio of about
0.6:1.3.
15. The composition of claim 1, wherein said composition comprises
collagenase I and collagenase II in a mass ratio of about 0.8:1.2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
pending U.S. patent application Ser. No. 15/666,365, filed Aug. 1,
2017, which is a continuation of U.S. patent application Ser. No.
14/213,910, filed Mar. 14, 2014 (now U.S. Pat. No. 9,744,138),
which claims priority to U.S. Provisional Application Ser. No.
61/790,070, filed on Mar. 15, 2013, all of which are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and products for
medical treatment designed to reduce, shrink change the
viscoelastic properties of, soften or eliminate unwanted tissue
such as uterine fibroid tissue.
BACKGROUND OF THE INVENTION
[0003] Uterine fibroid tumors (also referred to as "uterine
fibroids" or "leiomyomas") are non-cancerous smooth muscle tumors
of the uterine wall that occur in 20 to 50% of women, and have an
astonishingly high accumulative incidence. Current studies
demonstrate that by age 50, 70-80% of women have developed uterine
fibroids, with higher incidence in African-American women, who
commonly develop fibroids earlier than other racial groups. A
significant number of those with uterine fibroids suffer from
debilitating pelvic pain, heavy and prolonged bleeding (which may
lead to anemia and iron deficiency), bowel and bladder dysfunction
and infertility. Uterine fibroids also cause symptoms such as low
back pain, urinary frequency and urgency, pain during intercourse
(dyspareunia), and negative impact on fertility. They are
associated with high morbidity from uterine bleeding and pain along
with health care costs estimated to be between $2.1 and $34.4
billion annually in the United States alone. Therefore, uterine
fibroids have a significant impact on the health and well-being of
reproductive age women and on the economy. After menopause,
generally, fibroids shrink and only rarely cause problematic
symptoms.
[0004] The etiology of this disease remains unknown, therefore
there are no methods of preventing uterine fibroids. Several
treatments are available, but hysterectomy is the only treatment
which can permanently eliminate fibroids. The majority of the
hysterectomies performed in the United States each year are due to
uterine fibroids. It is obvious, but rarely stated in the
literature, that hysterectomies lead to irrevocable loss of
fertility. This invasive surgery also has a high cost, financially,
socially and otherwise. It is associated with lengthy recovery
times, potential for sometimes severe postoperative complications,
and physical discomfort. Thus, this solution is far from ideal.
[0005] Other surgical methods such as myomectomy (surgical removal
of the fibroid tissue leaving the remainder of the uterus intact)
is commonly used, but may not be suitable in cases where the
fibroids are too large or too numerous to leave enough normal
tissue behind. Further, the fibroids often recur. In addition,
about three-quarters of myomectomy surgeries are open surgeries
involving an abdominal incision. Therefore, this method also is
associated with complications, discomfort, long recovery, and
potentially loss of fertility as well. Myolysis and cryomyolysis,
in which uterine fibroids are burned or frozen via laparoscopic
surgery, can be used to cause the fibroids to shrink and die over
time. However, multiple punctures of the fibroids are needed to
treat the entire tumor, and the treatment may cause adhesions
post-surgery. MRI guided focused ultrasound also is used in the
treatment of uterine fibroids, but this procedure is very
expensive, and does not permanently eliminate the fibroids. Uterine
artery embolization, during which a catheter is inserted into a
femoral artery and guided to a uterine fibroid artery for injection
of small particles into the fibroid artery, blocks the supply of
blood, resulting in death of the fibroid tissue. Although this
procedure is less invasive than traditional surgery, post-surgical
pain is a frequent problem. In addition, this therapy, like
hysterectomy, is considered a standard treatment for women with no
desire for future fertility. Alternatively, MRgFUS provides
noninvasive fibroid-specific therapy utilizing high-intensity
ultrasonography through the abdominal wall to cause coagulative
necrosis in specific fibroids. Guidance and thermal monitoring is
provided by dynamic real-time magnetic resonance imaging. The
surgical procedures to destroy uterine fibroids while preserving
the uterus also have major drawbacks and often are not completely
successful, due to re-growth of the fibroid tumors.
[0006] Non-surgical, pharmaceutical-based medical therapies are
available. Fibroids often are treated by medications aimed at
treating the symptoms rather than the fibroid tumors themselves. In
the early stages, physicians employ a "wait-and-see" approach, with
no treatment or symptomatic treatment until the condition impacts
the ability of the patient to function in normal life. Most
fibroids are not treated unless they are causing symptoms. However,
even in the absence of hysterectomy, fibroids, particularly
subserosal fibroids, also can lead to infertility.
[0007] The pharmacotherapies which are aimed at shrinking fibroid
tumors or preventing increase in size have been disappointing and
often have significant side effects. Drugs have been studied and
sometimes are effective at shrinking uterine fibroids, but many of
these non-surgical therapies have been associated with systemic
side effects and therefore have not been approved for clinical use.
For example, selective progesterone receptor modulators (SPRM) have
not been approved by the FDA due to their effects on the
endometrium. Only one drug has been approved for use to shrink
uterine fibroids: leuprolide acetate. This drug is used as a
short-term treatment which suppresses ovarian function (and
therefore causes significant menopausal side effects), shrinking
fibroids prior to surgery. Other medical therapies have been
suggested in the recent past such as selective estrogen receptor
modulators (SERM), but clinical trial results have been
disappointing.
[0008] Current treatment options for uterine fibroids are
inadequate. Hence, there is a continuing need in the art for
alternative therapies for the treatment of uterine fibroids which
are not open procedures and which preserve the patient's uterus. In
particular, because treatment of uterine fibroids costs billions of
health care dollars each year, and yet this condition remains a
significant problem, there is a need for treatment methods that
reduce or eliminate symptoms, provide relief without highly
invasive procedures, and which preserve fertility.
SUMMARY OF THE INVENTION
[0009] The following brief summary is not intended to include all
features and aspects of the present invention, nor does it imply
that the invention must include all features and aspects discussed
in this summary.
[0010] Embodiments of the invention are designed to provide the
advantage of formulations, compositions and methods for treatment
of uterine fibroids which do not require open surgical procedures
and which preserve the patient's uterus. Another advantage of the
present invention is that injectable or insertable formulations are
provided, which display improved retention of agents within uterine
fibroid tissue, thereby improving delivery efficiency, while at the
same time minimizing adverse effects such as nonspecific damage and
systemic effects. These formulations, compositions and methods
include injectable, implantable or insertable formulations which
contain one or more uterine fibroid treatment agents, preferably at
least a purified collagenase in an amount effective to shrink or
eliminate fibroids that are exposed to the formulation.
[0011] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The application file contains at least one drawing executed
in color. Copies of any patent or patent application publication
from this application containing color drawing(s) will be provided
by the Office upon request and payment of the necessary fee.
[0013] FIGS. 1A and 1B are electron micrographs (.times.31000)
showing collagen fibrils in a uterine fibroid (1A) and in
corresponding myometrium (1B).
[0014] FIG. 2 is a photograph of uterine tissue cubes.
[0015] FIGS. 3A and 3B are photographs of a uterine tissue cube
undergoing injection (FIG. 3A) and injected uterine tissue cubes
undergoing incubation (FIG. 3B).
[0016] FIGS. 4A and 4B are photographs of an excised uterus,
showing a uterine fibroid (FIG. 4A) and a uterine fibroid
undergoing injection (FIG. 4B).
[0017] FIG. 5 is a pair of photographs showing fibroid tissue cubes
injected with vehicle (control) or collagenase, after 48 hour
incubation.
[0018] FIGS. 6A-6C are a set of four micrographs. FIGS. 6A and 6B
show control tissue. FIGS. 6C and 6D show tissue that has been
degraded with collagenase.
[0019] FIG. 7 is a scanning electron micrograph of the BD 3D
OPLA.RTM. scaffold.
[0020] FIG. 8 is a micrograph showing H&E stain of fibroid
cells seeded onto an OPLA scaffold and cultured for 9 days (Zeiss
Axio Imager.RTM. widefield fluorescence microscopy).
[0021] FIGS. 9A and 9B are bright field images (FIG. 9A) with
fluorescent image overlay (FIG. 9B) showing primary cultures of
fibroid cells 8 days after static seeding (Zeiss Lumar.RTM.
stereoscopic image, stained with fluorescein-phalloidin for
f-Actin).
[0022] FIGS. 10A and 10B are micrographs showing primary fibroid
cells cultured on OPLA scaffolds and fixed in situ. Images FIG. 10A
and FIG. 10B were taken from the same field of vision every 10
micrometers.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Collagen is the major structural constituent of mammalian
organisms and makes up a large portion of the total protein content
of skin and other parts of the animal body. Various skin traumas
such as burns, surgery, infection and accident are often
characterized by the erratic accumulation of fibrous tissue rich in
collagen and having increased proteoglycan content. In addition to
the replacement of the normal tissue which has been damaged or
destroyed, excessive and disfiguring deposits of new tissue
sometimes form during the healing process. Some diseases and
conditions are associated with excess collagen deposition and the
erratic accumulation of fibrous tissue rich in collagen. Such
diseases and conditions are collectively referred to herein as
"collagen-mediated diseases".
[0024] It has now been found that uterine fibroids are a
collagen-mediated disease, associated with excess collagen
deposition and the erratic accumulation of fibrous tissue rich in
collagen. The considerable variation in growth rates over time of
individual fibroids, and microarray studies revealing that genes
encoding for ECM proteins or related to ECM synthesis and secretion
account for a large portion of changes in gene expression in
fibroids compared with myometrium make dysregulation of ECM
(extracellular matrix) a possible contributing factor to this
condition. Recent studies indicate that fibroids are formed by the
accumulation of extracellular matrix (ECM) as well as by cellular
proliferation. See FIG. 1, noting the disordered collagen fibrils
in the fibroid tissue. The appearance and spatial orientation of
collagen fibrils in uterine fibroids were shorter, randomly aligned
and widely dispersed compared with those of the myometrium. They
were non-aligned and not parallel whereas in the adjacent
myometrium the fibrils were well packed and parallel in orientation
to each other, a finding that is characteristic of collagen
containing tissue. Myofibroblast type cells (elongated appearance,
notched nucleus) also have been found in uterine fibroids. The
notched appearance of the fibroid cell nucleus represents folding
and envaginations of the nuclear membrane due to cell contraction
by stress fibers.
[0025] Therefore, the present invention takes advantage of
collagenase, an enzyme that has the specific ability to digest
collagen, to treat uterine fibroids. Degradation of the collagen
not only causes collagenolysis, it also reduces the increased cell
compression leading to mechanotransduction. Thereby, the cycle of
increased collagen secretion and enlargement of the uterine fibroid
is broken.
[0026] This specification describes embodiments of an invention for
treatment to reduce the symptoms of uterine fibroids, shrink
uterine fibroids, reduce the stiffness and mechanical stress of
fibroid tissue on the uterus and/or eliminate uterine fibroids by
local delivery of a purified collagenase composition to avoid
systemic side-effects and harm to other tissues. In general, some
of the preferred methods use a syringe and needle under ultrasound
or other visualization for guided injection of purified collagenase
directly into the uterine fibroid tissue to be treated. The
collagenase product preferably is in a vehicle for delivery, such
as a nanocarrier or other protective or sustained release
carrier.
[0027] Because the center of fibroids is more fibrotic and contains
smaller vascular capillary beds than the periphery, and due to a
dense vascular capsule which surrounds the fibroid tumor, systemic
therapy is not likely to provide therapeutic tissue levels of a
drug in the fibroid center while leaving the likely possibility of
systemic effects. Thus, pharmacotherapy has not been successful for
uterine fibroids. The local injection of a treatment agent under
imaging guidance allows for exact tissue placement of the drug and
greatly reduces the chance of systemic effects.
[0028] Uterine fibroids are classified into several types, based on
their location, including subserosal, intramural, submucosal,
pedunculated submucosal, fibroid in statu nascendi, and fibroid of
the broad ligament. Any and all of these uterine fibroids are
contemplated for treatment using the invention.
[0029] Myometrial Hyperplasia is a condition which can mimic
uterine fibroid symptoms and may be a precursor lesion of these
tumors. It is structural variation with irregular zones of
hypercellularity and increased nucleus/cell ratio, causing a
bulging, firm, enlarged uterus. The condition often leads to
hysterectomy. Deeper MMH has lower cellularity, and tends to have
increased collagen. Therefore, this condition also may be treated
using the methods and compositions of the invention.
[0030] The local treatment of uterine fibroids by injection of
collagenase can be conducted in an office or clinic visit under
ultrasound guidance with minimal chance for sequelae. This method
can be used to treat small to moderate size fibroids or
asymptomatic fibroids, which currently are not treated at all,
allowing the clinician to prevent potentially debilitating symptoms
and preservation of fertility in women of child-bearing years, and
also larger fibroids, eliminating the need for hysterectomy for
this disease. Thus, the methods of this invention are contemplated
to be useful to treat any stage or type of uterine fibroid
disease.
[0031] Collagenase for use according to the invention may be
obtained from any convenient source, including mammalian (e.g.,
human, porcine), crustacean (e.g., crab, shrimp), fungal, and
bacterial (e.g., from the fermentation of Clostridium,
Streptomyces, Pseudomonas, Vibrio or Achromobacter iophagus).
Collagenase can be isolated from a natural source or can be
genetically engineered/recombinant. One common source of crude
collagenase is from a bacterial fermentation process, specifically
the fermentation of Clostridium histolyticum. The crude collagenase
obtained from C. histolyticum can be purified using any of a number
of techniques known in the art of protein purification, including
chromatographic techniques. Collagenase compositions useful for the
invention also can be prepared using any commercially available or
isolated collagenase activity, or by mixing such activities. For
example, purified collagenase can be provided by Biospecifics
Technologies, Lynbrook, N.Y.
[0032] Preferred collagenases for use in the invention are from C.
histolyticum, i.e., collagenase class I and class II. A practical
advantage of using C. histolyticum for the production of
collagenases is that it can be cultured in large quantities in
simple liquid media, and it regularly produces amounts of
proteolytic enzymes which are secreted into the culture medium.
Bovine products have been used in culture media in the fermentation
of C. histolyticum, but these run the risk of contamination by
agents which cause transmissible spongiform encephalopathies (TSEs;
e.g., prions associated with bovine spongiform encephalopathy or
"mad cow disease"). Therefore, it is preferred to avoid such bovine
products. An animal-product-free system is preferred. The H4 strain
of Clostridium histolyticum, originally developed in 1956 can serve
as a source for cells for culture. This strain, and a strain
derived from the H4 strain, named the ABC Clostridium histolyticum
master cell bank (deposited as ATCC 21000) were developed using
animal products, but are suitable to use in the invention.
[0033] U.S. Pat. No. 7,811,560, which is incorporated herein by
reference in its entirety, discloses methods of producing
collagenases. Using soybean derived fermentation medium, the
methods described therein generated separately highly purified
collagenase I and II. This patent also discloses methods of
producing highly purified collagenases using culture media
containing porcine-derived products. Any of these methods are
suitable for use with the invention. U.S. Patent Publication
2010/0086971, which is also incorporated herein by reference in its
entirety, discloses numerous fermentation recipes which are based
on vegetable peptone, including soybean-derived peptone, or
vegetable-derived peptone plus fish gelatin. The methods described
in this publication are suitable to produce growth of Clostridium
and collagenase activities. These methods also are suitable and
contemplated for use with the invention, however any method known
in the art of producing collagenase enzyme activity may be
used.
[0034] In preferred culture methods, the peptone is from a plant
source selected from the group consisting of soy bean, broad bean,
pea, potato, and a mixture thereof. The peptone may be selected
from the group consisting of Oxoid VG100 Vegetable peptone No. 1
from pea (VG100), Oxoid VG200 Vegetable peptone phosphate broth
from Pea (VG200), Merck TSB CASO-Bouillion animal-free (TSB),
Invitrogen Soy bean peptone No 110 papainic digest (SP6), Fluka
Broad bean peptone (BP), Organotechnie Plant peptone E1 from potato
(E1P), BBL Phytone.TM. peptone and BD Difco Select Phytone.TM..
[0035] In a preferred embodiment of the invention, a single type of
peptone is present in the nutrient composition of the invention,
whereby the peptone is selected from the group consisting of BP,
E1P, Soy bean peptone E110, VG100, and VG200, and whereby the
concentration of the peptone in the composition is about 5% weight
by volume. In yet another very much preferred embodiment of the
invention, a single type of peptone is present in the nutrient
composition of the invention, whereby the peptone is BBL phytone
peptone or Difco Select Phytone.TM. UF, and whereby the
concentration of the peptone in the composition is about 10-13%
weight by volume.
[0036] Preferred methods of isolating collagenase avoid undesirable
contaminating proteases such as clostripain. Clostripain, a
cysteine protease, is believed to be a major cause of collagenase
degradation and instability, and is present in Clostridium culture.
When such proteases are present in a crude collagenase mixture, one
must take extra precautions to neutralize the proteases, including
using protease inhibitors, such as leupeptin, and performing all of
the purification steps in specially designed cold rooms with
chilled solutions to reduce protease activity. Preferred methods of
isolation therefore take advantage of one of two approaches to
avoid clostripain: remove clostripain as early as possible in the
purification method or reduce clostripain production during the
fermentation stage.
[0037] Preferred collagenase compositions are produced by
fermenting C. histolyticum in medium free of animal
material-derived ingredients and are substantially free of
clostripain, and thus are highly stable. "Substantially free"
indicates that the collagenase contains less than 10 U clostripain
per mg total collagenase, more preferably less than 5 U/mg, and
most preferably about 1 U/mg or less, and/or that no visible band
appears representing clostripain and/or degraded collagenase on
SDS-PAGE gel compared to a reference standard.
[0038] Preferred methods for purifying collagenase involve using a
"low glucose" medium as described herein, which contains less than
about 5 g/L glucose, more preferably less than about 1 g/L, even
more preferably less than about 0.5 g/L glucose, or is
glucose-free, for culture of C. histolyticum. High salt
concentrations in the growth media can reduce the amount of
clostripain produced in culture, thus preferred media for C.
histolyticum culture contain greater than about 5 g/L (or 0.5% w/v)
total salt, more preferably greater than about 7.5 g/L (or 7.5%)
total salt, and more preferably about 9 g/L (or 9%) or more. It is
contemplated that any salt known to be suitable for use in
microbiological fermentation media may be used in the current
invention. In a preferred embodiment, chloride, phosphate or
sulfate salts may be used. In a more preferred embodiment, the
salts may be sodium chloride, potassium chloride, monosodium
phosphate, disodium phosphate, tribasic sodium phosphate, potassium
monophosphate, potassium diphosphate, tripotassium phosphate,
calcium chloride, magnesium sulfate or various combinations
thereof. In certain embodiments, potassium diphosphate may be about
0.1-0.3%, potassium phosphate may be about 0.75% to 0.175%, sodium
phosphate may be about 0.2-0.5%, and/or sodium chloride may be
about 0.15-0.35%. Preferably, the medium further comprises
magnesium sulfate and vitamins, including, riboflavin, niacin,
calcium pantothenate, pimelic acid, pyridoxine and thiamine.
[0039] In another preferred embodiment, the nutrient composition
may contain 0.5-5% yeast extract, more preferably about 1-4%, and
most preferably about 1.5-2.5%. Yeast extract is available from a
variety of suppliers, including Cole Parmer (Vernon Hills, Ill.)
and Fisher Scientific (Pittsburgh, Pa.).
[0040] In yet a preferred embodiment of the invention, the pH of
the media is between pH 7 and pH 8. Even more preferred is a pH
between about pH 7.2 and about pH 7.7, most preferably about
7.4.
[0041] The collagenase contemplated for use with the invention can
be any collagenase which is active under the necessary conditions.
However, preferred compositions contain a mass ratio of collagenase
I and collagenase II which is modified or optimized to produce a
desired or even a maximal synergistic effect. Preferably,
collagenase I and collagenase II are purified separately from the
crude collagenase mixture produced in culture, and the collagenase
I and collagenase II are recombined in an optimized fixed mass
ratio. Preferred embodiments contain a collagenase I to collagenase
II mass ratio of about 0.5 to 1.5, more preferably 0.6 to 1.3, even
more preferably 0.8 to 1.2, and most preferably, 1 to 1, however
any combination or any single collagenase activity may be used.
[0042] A preferred method of producing collagenase which is
contemplated for use with the invention involves fermenting C.
histolyticum in a non-mammalian or non-animal medium, wherein the
culture supernatant is substantially clostripain-free. The
collagenases so produced can be isolated, purified, and combined to
provide a composition for use in the invention which comprises a
mixture of collagenase I and collagenase II in an optimized fixed
mass ratio which is substantially clostripain-free. The crude
collagenase obtained from fermentation of C. histolyticum may be
purified by a variety of methods known to those skilled in the art,
including dye ligand affinity chromatography, heparin affinity
chromatography, ammonium sulfate precipitation, hydroxylapatite
chromatography, size exclusion chromatography, ion exchange
chromatography, and/or metal chelation chromatography.
Additionally, purification methods for collagenases are known, such
as, for example, those described in U.S. Pat. No. 7,811,560, which
is hereby incorporated by reference in its entirety.
[0043] Both collagenase I and collagenase II are metalloproteases
and require tightly bound zinc and loosely bound calcium for their.
Both collagenases have broad specificity toward all types of
collagen. Collagenase I and Collagenase II digest collagen by
hydrolyzing the triple-helical region of collagen under
physiological conditions. Each collagenase shows different
specificity (e.g. each have a different preferred target amino
sequence for cleavage), and together they have synergistic activity
toward collagen. Collagenase II has a higher activity towards all
kinds of synthetic peptide substrates than collagenase I as
reported for class II and class I collagenase in the
literatures.
[0044] The preferred collagenase consists of two microbial
collagenases, referred to as Collagenase ABC I and Collagenase ABC
II. The terms "Collagenase I", "ABC I", and "collagenase ABC I"
mean the same and can be used interchangeably. Similarly, the terms
"Collagenase II", "ABC II", and "collagenase ABC II" refer to the
same enzyme and can also be used interchangeably. These
collagenases are secreted by bacterial cells. Preferably, they are
isolated and purified from Clostridium histolyticum culture
supernatant by chromatographic methods. Both collagenases are
special proteases and share the same EC number (E.C 3.4.24.3).
However, a collagenase or a combination of collagenases from other
sources are contemplated for use with the invention. Collagenase
ABC I has a single polypeptide chain consisting of approximately
1000 amino acids with a molecular weight of 115 kDa. Collagenase
ABC II has also a single polypeptide chain consisting of about 1000
amino acids with a molecular weight of 110 kDa.
[0045] Preferably, the collagenase product is at least 95% pure
collagenase(s) and is substantially free of any contaminating
proteases. More preferably, the collagenase product is 97% pure and
most preferably 98% pure or more as determined by one or more of
the following: sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE); high performance liquid chromatography
(HPLC); reverse-phase HPLC; or by enzymatic assays. The preferred
collagenase product is essentially clostripain-free, and the
purification preferably is performed in the absence of leupeptin.
The preferred collagenase product for use with the invention has at
least one specification selected from Table 1 below.
TABLE-US-00001 TABLE 1 Preferred Specifications for Collagenase
Products Specification Test ABC-I ABC-II Appearance Clear colorless
and essentially free from particulate matter Endotoxin <10 EU/mL
Identity (and purity) by Major collagenase Major collagenase
SDS-PAGE (Reduced band between 98- band between 97- conditions,
Coomasie) 188 kDa 200 kDa .gtoreq.95% .gtoreq.95% SRC assay (ABC-I)
1967-3327 SRC NA units/mg GPA assay (ABC-II) NA81934-119522 GPA
units/mg Analysis of Proteins .gtoreq.98% main peak; .ltoreq.2%
aggregates by area HPLC System (Aggregation by size exclusion
chromatography) Identity and purity by Major peak (ABC I or ABC
II), .gtoreq.95% by reverse phase liquid area; Retention times of
ABC-I and ABC-II chromatography) within 5% of reference Clostripain
assay (BAEE .ltoreq.1 U/mg assay) Bioburden <1 cfu/mL
[0046] The collagenase products described for use herein are useful
for the treatment of collagen-mediated disease, including uterine
fibroids. Examples of other collagen mediated-diseases that may be
treated by the compositions of the invention include but are not
limited to: Dupuytren's disease; Peyronie's disease; frozen
shoulder (adhesive capsulitis), keloids; tennis elbow (lateral
epicondylitis); scarred tendon; glaucoma; herniated discs; adjunct
to vitrectomy; hypertrophic scars; depressed scars such as those
resulting from inflammatory acne; post-surgical adhesions; acne
vulgaris; lipomas, and disfiguring conditions such as wrinkling,
cellulite formation and neoplastic fibrosis.
[0047] In addition to its use in treating specific
collagen-mediated diseases, the compositions of the invention also
are useful for the dissociation of tissue into individual cells and
cell clusters as is useful in a wide variety of laboratory,
diagnostic and therapeutic applications. These applications involve
the isolation of many types of cells for various uses, including
microvascular endothelial cells for small diameter synthetic
vascular graft seeding, hepatocytes for gene therapy, drug
toxicology screening and extracorporeal liver assist devices,
chondrocytes for cartilage regeneration, and islets of Langerhans
for the treatment of insulin-dependent diabetes mellitus. Enzyme
treatment works to fragment extracellular matrix proteins and
proteins which maintain cell-to-cell contact. In general, the
compositions of the present invention are useful for any
application where the removal of cells or the modification of an
extracellular matrix, are desired.
[0048] The collagenase compositions according this invention are
designed to administer to a patient in need thereof a
therapeutically effective amount of a collagenase composition as
described, or a therapeutically effective amount of a
pharmaceutical collagenase formulation as described. A
"therapeutically effective amount" of a compound, composition or
formulation is an amount of the compound which confers a
therapeutic effect on the treated subject, at a reasonable
benefit/risk ratio applicable to any medical treatment. A
therapeutic effect includes but is not limited to a shrinkage or
reduction in the size of one or more uterine fibroids (including
elimination of the fibroid), liquification, partial liquification,
or reduction in stiffness (increase in softness) or pressure in or
around a uterine fibroid, a change in viscoelastic properties, or
reduction in symptoms such as pain, hemorrhage and the like.
[0049] The therapeutic effect may be objective (i.e., measurable by
some test or marker) or subjective (i.e., subject gives an
indication of or feels an effect), and may be determined by the
clinician or by the patient. Effective doses will also vary
depending on route of administration, as well as the possibility of
co-usage with other agents. It will be understood, however, that
the total daily usage of the compositions of the present invention
will be decided by the attending physician within the scope of
sound medical judgment. The specific therapeutically effective dose
level for any particular patient will depend upon a variety of
factors including the disorder being treated and the severity of
the disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general
health, and diet of the patient; the time of administration, route
of administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or contemporaneously with the specific compound employed; and like
factors well known in the medical arts.
[0050] The term "patient" or "patient in need" encompasses any
mammal having a uterus and uterine fibroids or symptoms thereof.
Such "patients" or "patients in need" include humans or any mammal,
including farm animals such as horses and pigs, companion animals
such as dogs and cats, and experimental animals such as mice, rats
and rabbits. Preferred patients are human females of child-bearing
age.
[0051] The pharmaceutical compositions of this invention preferably
are administered by injection, insertion or implantation directly
into or onto the uterine fibroid tissue to be treated, i.e. local
administration to the tissue to be treated. Other modes of
administration contemplated included, but are not limited to
transvaginal instillation or application onto the affected tissues,
instillation or application during surgery (such as laparoscopy or
hysteroscopy) onto the affected tissues, i.e. topical
administration to the fibroid tissue, by spray or other application
of a liquid, fluid or gel formulation.
[0052] Formulations of the present invention are injected/inserted
into uterine tissue in a variety of forms, by a variety of routes,
using a variety of apparatuses. In some embodiments, the
formulation is injected/inserted using an apparatus consisting of a
simple needle (e.g., a 10 gauge or smaller needle) and sample
pusher (e.g., a mandrel or modified obturator). For example,
according to one embodiment, a formulation (e.g., a rod-shaped or
other shaped solid or semi-solid formulation, beads, suspension,
gel, polymer or the like) is placed in the needle or in a syringe
or other chamber affixed to the needle. Once the needle is placed
at the desired depth and location in the tissue, the pusher is used
to push the sample from the needle and into the tissue. In some
embodiments, the sample pusher is provided with a holding clip or
it is provided with a hollow end to secure the sample up to the
time of delivery.
[0053] In still other embodiments, formulations in accordance with
the present invention are injected/inserted via jet injection
without a physical delivery channel such as a needle, as is known
in the art. Typically, a compression system (e.g., a mechanical
system or a gas, such as helium, nitrogen, carbon dioxide, etc.) is
used to accelerate the formulations to a high enough velocity so
that the formulation can penetrate the tissue to a desired depth.
Jet injector devices can be, for example, disposable, or reusable
with medication cartridges that are prefilled or non-prefilled
medication cartridges. Examples of jet injectors include
Biojector.RTM. from Bioject, N.J., USA and the PowderJect.RTM.
System from PowderJect, UK. In other embodiments, a device is
employed that cores out a section of the fibroid (e.g., a biopsy
device or tissue morcellator or laser radiation), thereby leaving
behind a void for insertion of a dosage form.
[0054] The formulations for collagenase delivery to a patient
generally are contemplated to comprise injectable or implantable
formulations, or any fluid, liquid, solid, semi-solid, gel, or
other composition which is suitable to administer the collagenase
to the tissue to be treated as described herein. Formulations in
accordance with the present invention may be formulated by any
method known in the pharmaceutical arts. Thus, any injectable or
implantable formulation known in the art and consistent with
collagenase activity may be used. Formulations which create a depot
or extended release of the active collagenase agent are
contemplated. In particular, injectable extended or sustained
release compositions are preferred, however any implantable
formulation can be used. Such compositions produce or form a depot
effect, where active agent is present in the tissue where
administered and release active agent over a period of time to
continuously treat the tissue. Immediate release injectable
formulations, where the active agent is immediately released for
activity upon administration, also are contemplated for use with
the invention. These formulations are known in the art and can be
adapted for use with the present invention by any person of
skill.
[0055] In some embodiments, the injectable or insertable
formulations of the present invention are solids, semi-solids or
high-viscosity fluids. This improves dosage retention in the
tissue, thereby improving delivery efficiency of the treatment
agents and/or minimizing the adverse effects such as unintended,
nonspecific tissue damage. "High viscosity" and other such terms
are used herein to describe fluids having viscosities greater than
1000 cps as measured by any of a number of standard techniques,
including, for example, a Brookfield Kinematic Viscometer, model
HBDV-II+CP with a CPE-40 cone spindle, set at 37.degree. C. and
using a 0.5 rpm speed setting. "Low viscosity" fluids have
viscosities less than this value.
[0056] In some embodiments, a formulation in accordance with the
present invention is injected into a patient in a fluid state,
whereupon it converts (or is converted) in vivo into a more readily
retained form, for example, into a solid form (including conversion
of an injected liquid into a solid, conversion of an injected
semi-solid into a solid and conversion of a liquid into a gel),
into a semi-solid form (including conversion of an injected liquid
into a semi-solid, conversion of an injected semi-solid into a
semi-solid having increased yield stress and/or viscosity and
conversion of a liquid into a gel), or into a high-viscosity fluid
(including conversion of a low-viscosity fluid into a
high-viscosity fluid, and conversion of a high-viscosity fluid into
a higher-viscosity fluid).
[0057] Preferred formulations for injection into a uterine fibroid
use a carrier or nanocarrier. Appropriate carriers include solid or
semi-solid pellets, beads or gel-forming polymers, high-viscosity
liquids and the like to maintain the active collagenase in the
tissue, protecting the active enzyme from action of the tissue or
tissue components which could inactivate the collagenase, and allow
steady release of the enzyme to the tissue for treatment. Any
injectable dosage form which can protect and contain the active
compound(s) in place may be used. In mammals, C. histolyticum
collagenase is inhibited rapidly in the blood stream by serum.
Therefore, systemic administration, or administration under
conditions where the collagenase can be deactivated, or orally,
where the collagenase can be degraded by digestive enzymes, is
problematic.
[0058] Nanocarriers are designed to deliver and protect drug
therapeutics (e.g. proteins, for example) from degradation. A
nanocarrier formulation also is preferred because this method
impedes diffusion and distribution of the drug away from the
injected fibroid, prolongs release, delays inactivation, and
therefore reduces the frequency of repeat injections. Any such
nanocarrier known in the art can be used with the invention. Some
of these nanocarriers also are referred to as thermoresponsive
delivery systems.
[0059] Atrigel.RTM. comprises a water-insoluble biodegradable
polymer (e.g., poly(lactic-co-glycolic acid, PLGA) dissolved in a
bio-compatible, water-miscible organic solvent (e.g.,
N-methyl-2-pyrrolidone, NMP). In use, collagenase is added to form
a solution or suspension. Both the PLGA molecular weight and
lactide-glycolide molar ratio (L:G ratio) governs drug delivery.
Using an L:G ratio of from 50:50 to 85:15 and a polymer
concentration of from 34 to 50%, clinical studies have demonstrated
a depot which was maintained for more than 3 months.
[0060] ReGel.RTM. is a 4000 Da triblock copolymer formed from PLGA
and polyethylene glycol (PEG, 1000 Da or 1450 Da) in repetitions of
PLGA-PEG-PLGA or PEG-PLGA-PEG. ReGel.RTM. is formulated as a 23 wt
% copolymer solution in aqueous media. A drug is added to the
solution and upon temperature elevation to 37.degree. C. the whole
system gels. Degradation of ReGel.RTM. to final products of lactic
acid, glycolic acid and PEG occurs over 1-6 weeks depending on
copolymer molar composition. Chemically distinct drugs like porcine
growth hormone and glucagon-like peptide-1 (GLP-1) may be
incorporated, one at a time, and released from ReGel.RTM..
[0061] LiquoGel.TM. can work by mechanistically independent drug
delivery routes: entrapment and covalent linkage. Two or more drugs
can be delivered to the tumor site using this carrier. LiquoGel.TM.
is a tetrameric copolymer of thermogelling N-isopropylacrylamide;
biodegrading macromer of poly(lactic acid) and 2-hydroxyethyl
methacrylate; hydrophilic acrylic acid (to maintain solubility of
decomposition products); and multi-functional hyperbranched
polyglycerol to covalently attach drugs. LiquoGel.TM. generally is
formulated as a 16.9 wt % copolymer solution in aqueous media. The
solution gels under physiological conditions and degrades to
release drug contents within 1-6 days.
[0062] Any of the above carriers can be used as a nanocarrier with
the invention. A preferred nanocarrier, however, contains
hyperbranched polyglycerols (HPG), which have many desirable
features. HPGs grow by imperfect generations of branched units and
are produced in a convenient single step reaction. Previous
problems of large polydispersities in molecular weight in their
production have been overcome. The resulting polymers contain a
large number of modifiable surface functional groups as well as
internal cavities for drug interaction. Other polymer approaches
cannot easily provide these properties without significant
increases in the number of synthetic steps and, consequently, cost.
HPG polymers are based on glycerol and because of structural
similarity with polyethylene glycol, is biocompatible.
[0063] Additional components optionally can be added to the
polymer, therefore, modified HPG polymers and co-polymers of HPG
are contemplated. These additional components or monomers can
include, for example, crosslinks, biodegradable moieties, and
thermoresponsive moieties. For example, thermally responsive
hydrogels are attractive for injection therapy since it is possible
to inject the necessary fluid volume from a syringe maintained
below body temperature and upon warming, the mechanical properties
are increased, thereby restraining the material at the injection
site. Poly(N-isopropylacrylamide) (poly-NIPAAm) is a thermally
responsive polymer with a lower critical solution temperature
(LCST) of approximately 32.degree. C. Copolymers of HPG with NIPAAm
are therefore contemplated for use with the invention, and are
preferred. This nanocarrier has a versatile mesh size and can be
customized to entrap small drug molecules, large proteins, or a
mixture of components, and gels at body temperature to permit slow
release as the nanocarrier biodegrades.
[0064] In preferred embodiments of the invention, formulations
exist as a liquid at temperatures below body temperature and as a
gel at body temperature. The temperature at which a transition from
liquid to gel occurs is sometimes referred to as the LCST, and it
can be a small temperature range as opposed to a specific
temperature. Materials possessing an LCST are referred to as LCST
materials. Typical LCST's for the practice of the present invention
range, for example, from 10 to 37.degree. C. As a result, a
formulation injected below the LCST warms within the body to a
temperature that is at or above the LCST, thereby undergoing a
transition from a liquid to a gel.
[0065] Suitable LCST materials for use with the invention include
polyoxyethylene-polyoxypropylene (PEO-PPO) block copolymers. Two
acceptable compounds are Pluronic acid F127 and F108, which are
PEO-PPO block copolymers with molecular weights of 12,600 and
14,600, respectively. Each of these compounds is available from
BASF (Mount Olive, N.J.). Pluronic acid F108 at 20-28%
concentration concentration, in phosphate buffered Saline (PBS) is
an example of a suitable LCST material. One beneficial preparation
is 22.5% Pluronic acid F108 in PBS. A preparation of 22% Pluronic
acid F108 in PBS has an LCST of 37.degree. C. Pluronic acid F127 at
20-35% concentration in PBS is another example of a suitable LCST
material. A preparation of 20% Pluronic acid F127 in PBS has an
LCST of 37.degree. C. Typical molecular weights are between 5,000
and 25,000, and, for the two specific compounds identified above
are 12,600 and 14,600. More generally, materials, including other
PEO-PPO block copolymers, which are biodisintegrable, and which
exist as a gel at body temperature and as a liquid below body
temperature can also be used according to the present invention.
Further information regarding LCST materials can be found in U.S.
Pat. Nos. 6,565,530 B2 and 6,544,227 B2, each of which is hereby
incorporated by reference.
[0066] Pharmaceutical formulations of the collagenase compounds for
the invention include a collagenase composition formulated together
with one or more pharmaceutically acceptable vehicles or
excipients. As used herein, the term "pharmaceutically acceptable
carrier or excipient" means a non-toxic, inert, solid, semi-solid
or liquid filler, diluent, encapsulating material, vehicle,
solvent, or formulation auxiliary of any type, and may be made
available in individual dosage forms or in bulk. Other dosage forms
designed to create a depot of the active compound also are
contemplated for use with the invention. Dosage forms for
collagenase suitable for use with the invention include, but are
not limited to lyophilized or other dried powder for reconstitution
prior to injection, in multiple or single dose amounts, individual
dosage units ready for injection (which preferably also include one
or more preservatives), frozen unit dosage forms, or any mode of
preparation known in the art. The formulations also may be provided
in the form of a kit, which can contain the collagenase in solid
form, liquid or solvent for reconstitution and injection, and any
equipment necessary for administration, such as a syringe and
needle, particularly a specialized syringe and/or needle for
administration to a uterine fibroid. Preferably, the formulations
are sterile. The products may be sterilized by any method known in
the art, such as by filtration through a bacterial-retaining filter
or are produced under aseptic conditions. Other methods include
exposing the formulation or components thereof to heat, radiation
or ethylene oxide gas.
[0067] Some examples of materials which can serve as
pharmaceutically acceptable carriers are solvents for injection as
known in the art. Examples include, but are not limited to sterile
water, buffering solutions, saline solutions such as normal saline
or Ringer's solution, pyrogen-free water, ethyl alcohol, non-toxic
oils, and the like, or any solvent compatible with injection or
other forms of administration as described herein for use with the
invention.
[0068] In addition, any solid excipients known in the art for use
in pharmaceutical products can be used with the invention as a
vehicle or filler, for example. Sugars such as lactose, glucose and
sucrose; starches such as corn starch and potato starch; cellulose
and its derivatives such as microcrystalline cellulose, sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; gums; talc; glycols such as
propylene glycol; esters such as ethyl oleate and ethyl laurate;
agar, and the like can be used. Buffering agents compatible with
the active compounds and the methods of use are contemplated for
use, including acid or alkali compounds, such as magnesium
hydroxide and aluminum hydroxide, citric acid, phosphate or
carbonate salts and the like. Non-toxic compatible excipients such
as lubricants, emulsifiers, wetting agents, suspending agents,
binders, disintegrants, preservatives or antibacterial agents,
antioxidants, sustained release excipients, coating agents and the
like (e.g., sodium lauryl sulfate and magnesium stearate) also may
be used, as well as coloring agents, perfuming agents, viscosity
enhancing agents, bioadhesives, and the like, according to the
judgment of the formulator.
[0069] For example, one or more biodisintegrable binders may be
included in the formulations of the present invention, typically in
connection with dosage forms having solid characteristics. Where
employed, a wide range of biodisintegrable binder concentrations
may be utilized, with the amounts varying based, for example, on
the desired physical characteristics of the resulting dosage form
and on the characteristics of the uterine fibroid treatment agent
that is selected (e.g., the degree of dilution, release delay, etc.
that is desired/tolerated), among other considerations. The
concentration of biodisintegrable binder typically ranges are from
about 1 to 80 wt % of biodisintegrable binder, more typically about
5 to 50 wt %. A "biodisintegrable" material is one that, once
placed in tissue such as uterine tissue, undergoes dissolution,
degradation, resorption and/or other disintegration processes.
Where such materials are included, formulations in accordance with
the present invention will typically undergo at least a 10%
reduction in weight after residing in tissue such as uterine tissue
for a period of 7 days, more typically a 50-100% reduction in
weight after residing in the tissue for a period of 4 days.
Suitable biodisintegrable binders for use in connection with the
present invention include, but are not limited to biodisintegrable
organic compounds, such as glycerine, and biodisintegrable
polymers, or any known disintegrant compound known in the art of
pharmaceutics.
[0070] Where used, viscosity adjusting agent(s) are typically
present in an amount effective to provide the formulation with the
desired viscosity, for example, by rendering the formulation highly
viscous, for example, in an amount effective to provide a viscosity
between about 5,000 and 200,000 cps, more typically between about
10,000 and 100,000 cps, and even more typically between about
20,000 and 40,000 cps. By providing formulations having viscosities
within these ranges, the formulations can be injected into tissue,
such as uterine tissue, using conventional injection equipment
(e.g., syringes). However, due to their elevated viscosities, the
formulations have improved retention within the tissue at the
injection site. The concentration of the viscosity adjusting
agent(s) that is (are) used can vary widely. Commonly, the overall
concentration of the viscosity adjusting agent(s) is between about
1 and 20 wt %. In many embodiments, the viscosity adjusting agents
are polymers, which may be of natural or synthetic origin and are
typically biodisintegrable. The polymers are also typically water
soluble and/or hydrophilic. However, in some embodiments, for
instance where an organic solvent such as dimethylsulfoxide (DMSO)
is used as a liquid component, the viscosity adjusting agent can be
relatively hydrophobic. The polymeric viscosity adjusting agents
include homopolymers, copolymers and polymer blends.
[0071] Examples of viscosity adjusting agents for the practice of
the present invention include, but are not limited to the
following: cellulosic polymers and copolymers, for example,
cellulose ethers such as methylcellulose (MC),
hydroxyethylcellulose (HEC), hydroxypropyl cellulose (HPC),
hydroxypropyl methyl cellulose (HPMC), methylhydroxyethylcellulose
(MHEC), methylhydroxypropylcellulose (MHPC), carboxymethyl
cellulose (CMC) and its various salts, including, e.g., the sodium
salt, hydroxyethylcarboxymethylcellulose (HECMC) and its various
salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various
salts, other polysaccharides and polysaccharide derivatives such as
starch, hydroxyethyl starch (HES), dextran, dextran derivatives,
chitosan, and alginic acid and its various salts, carrageenan,
various gums, including xanthan gum, guar gum, gum arabic, gum
karaya, gum ghatti, konjac and gum tragacanth, glycosaminoglycans
and proteoglycans such as hyaluronic acid and its salts, heparin,
heparin sulfate, dermatan sulfate, proteins such as gelatin,
collagen, albumin, and fibrin, other polymers, for example,
carboxyvinyl polymers and their salts (e.g., carbomer),
polyvinylpyrrolidone (PVP), polyacrylic acid and its salts,
polyacrylamide, polyacrylic acid/acrylamide copolymer, polyalkylene
oxides such as polyethylene oxide, polypropylene oxide and
poly(ethylene oxide-propylene oxide) (e.g., Pluronic acid),
polyoxyethylene (polyethylene glycol), polyethyleneamine and
polypyrridine, poly-metaphosphate (Kurrol salts), polyvinyl
alcohol, additional salts and copolymers beyond those specifically
set forth above, and blends of the foregoing (including mixtures of
polymers containing the same monomers, but having different
molecular weights), and so forth. Many of these species are also
useful as binders.
[0072] In other embodiments of the invention, formulations or
carriers are crosslinked, either prior to use or in vivo.
Crosslinking is advantageous, for example, in that it acts to
improve formulation retention (e.g., by providing a more
rigid/viscous material and/or by rendering the polymer less soluble
in a particular environment). Where the formulation is crosslinked
in vivo, a crosslinking agent is commonly injected into tissue
either before or after the injection or insertion of a formulation
in accordance with the present invention. Depending on the nature
of the formulation and the crosslinking agent, the formulation may
be converted, for example, into a solid, into a semi-solid, or into
a high-viscosity fluid.
[0073] Crosslinking agents suitable for use in the present
invention include, any non-toxic crosslinking agent, including
ionic and covalent crosslinking agents. For example, in some
embodiments, polymers are included within the formulations of the
present invention, which are ionically crosslinked, for instance,
with polyvalent metal ions. Suitable crosslinking ions include
polyvalent cations selected from the group consisting of calcium,
magnesium, barium, strontium, boron, beryllium, aluminum, iron,
copper, cobalt, lead and silver cations ions. Polyvalent anions
include phosphate, citrate, borate, succinate, maleate, adipate and
oxalate anions. More broadly, crosslinking anions are commonly
derived from polybasic organic or inorganic acids. Ionic
crosslinking may be carried out by methods known in the art, for
example, by contacting ionically crosslinkable polymers with an
aqueous solution containing dissolved ions.
[0074] In some embodiments, polymers are included, which are
covalently crosslinkable, for example, using a polyfunctional
crosslinking agent that is reactive with functional groups in the
polymer structure. The polyfunctional crosslinking agent can be any
compound having at least two functional groups that react with
functional groups in the polymer. Various polymers described herein
can be both covalently and ionically crosslinked.
[0075] Suitable polymers for ionic and/or covalent crosslinking can
be selected, for example, from the non-limiting list of the
following: polyacrylates; poly(acrylic acid); poly(methacrylic
acid); polyacrylamides; poly(N-alkylacrylamides); polyalkylene
oxides; poly(ethylene oxide); poly(propylene oxide); poly(vinyl
alcohol); poly(vinyl aromatics); poly(vinylpyrrolidone);
poly(ethylene imine); poly(ethylene amine); polyacrylonitrile;
poly(vinyl sulfonic acid); polyamides; poly(L-lysine); hydrophilic
polyurethanes; maleic anhydride polymers; proteins; collagen;
cellulosic polymers; methyl cellulose; carboxymethyl cellulose;
dextran; carboxymethyl dextran; modified dextran; alginates;
alginic acid; pectinic acid; hyaluronic acid; chitin; pullulan;
gelatin; gellan; xanthan; carboxymethyl starch; hydroxyethyl
starch; chondroitin sulfate; guar; starch; and salts, copolymers,
mixtures and derivatives thereof.
[0076] In one preferred embodiment, the collagenase is formulated
as a lyophilized injectable composition formulated with lactose,
sucrose or any suitable sugar. One preferred collagenase
composition is a lyophilized injectable composition formulated with
sucrose, Tris at a pH level of about 8.0. Most preferably, 1.0 mg
of the drug substance of the invention is formulated in 60 mM
sucrose, 10 mM Tris, at a pH of about 8.0 (e.g., about 20.5 mg/mL
of sucrose and 1.21 mg/mL of Tris in the formulation buffer).
[0077] Preferred collagenase compositions for use in the invention
comprise a mixture of collagenase I and collagenase II has a
specific activity of at least about 700 SRC units/mg, such as at
least about 1000 SRC units/mg, more preferably at least about 1500
SRC units/mg. One SRC unit will solubilize rat tail collagen into
ninhydrin reaction material equivalent to 1 nanomole of leucine per
minute, at 25.degree. C., pH 7.4. Collagenase has been described in
ABC units as well. This potency assay of collagenase is based on
the digestion of undenatured collagen (from bovine tendon) at pH
7.2 and 37.degree. C. for 20-24 hours. The number of peptide bonds
cleaved are measured by reaction with ninhydrin. Amino groups
released by a trypsin digestion control are subtracted. One net ABC
unit of collagenase will solubilize ninhydrin reactive material
equivalent to 1.09 nanomoles of leucine per minute. One SRC unit
equal approximate 6.3 ABC unit or 18.5 GPA unit. In one embodiment,
each milligram of collagenase for injection will contain
approximately 2800 SRC units.
[0078] Doses contemplated for administration by direct injection to
the uterine fibroid tissue will vary depending on the size of the
tissue to be treated and the discretion of the treating physician.
However, doses generally are about 0.06 mg collagenase to about 1
mg collagenase per cm.sup.3 of tissue to be treated or about 0.1 mg
collagenase to about 0.8 mg collagenase per cm.sup.3 of tissue to
be treated, or about 0.2 mg collagenase to about 0.6 mg collagenase
per cm.sup.3 of tissue to be treated.
[0079] Formulations that contain an additional active agent or
medication also are contemplated. Optional additional agents which
can be included in the formulation for concomitant, simultaneous or
separate administration include, for example, any pharmaceutical
known in the art for shrinkage, treatment or elimination of uterine
fibroids or their symptoms, or to assist in performance of the
present treatment methods. For example, one or more fibroid
treatment agents such as aromatase inhibitors (e.g., letrozole,
anastrozole, and exemestande), progesterone receptor agonists and
modulators (e.g., progesterone, progestins, mifepristone,
levonoergestrel, norgestrel, asoprisnil, ulipristal and ulipristal
acetate, telepristone), selective estrogen receptor modulators
(SERMs) (e.g., benzopyran, benzothiophenes, chromane, indoles,
naphtalenes, tri-phenylethylene compounds, arzoxifene, EM-652, CP
336,156, raloxifene, 4-hydroxytamoxifen and tamoxifen),
gonadotrophin-releasing hormone analogs (GnRHa) (e.g., GnRH agonist
peptides or analogs with D-amino acid alterations in position 6
and/or ethyl-amide substitutions for carboxyl-terminal Gly10-amide
such as triptorelin or GnRH antagonists such as cetrorelix,
ganirelix, degarelix and ozarelix), growth factor modulators (e.g.,
TGFb neutralizing antibodies), leuprolide acetate, non-steroidal
anti-inflammatory drugs, inhibitors of the mTOR pathway, inhibitors
of the WNT signaling pathway, vitamin D, vitamin D metabolites,
vitamin D modulators, and/or an additional anti-fibrotic compound
(e.g., pirfenidone and halofuginone) may be co-administered with
collagenase in the same or a separate administration.
[0080] Chemical ablation agents also can be included in the
formulations of the present invention. In effective amounts, such
compounds cause tissue necrosis or shrinkage upon exposure. Any
known ablation agent can be used according to the art, in
concentrations as appropriate to the conditions while avoiding
inactivation of the collagenase, with the amounts employed being
readily determined by those of ordinary skill in the art. Typical
concentration ranges are from about 1 to 95 wt % of ablation agent,
more typically about 5 to 80 wt %. Ablation agents suitable for use
with the invention include, but are not limited to
osmotic-stress-generating agents (e.g., a salt, such as sodium
chloride or potassium chloride), organic compounds (e.g., ethanol),
basic agents (e.g., sodium hydroxide and potassium hydroxide),
acidic agents (e.g., acetic acid and formic acid), enzymes (e.g.,
hyaluronidase, pronase, and papain), free-radical generating agents
(e.g., hydrogen peroxide and potassium peroxide), oxidizing agents
(e.g., sodium hypochlorite, hydrogen peroxide and potassium
peroxide), tissue fixing agents (e.g., formaldehyde, acetaldehyde
or glutaraldehyde), and/or coagulants (e.g., gengpin). These agents
may be combined with collagenase in the same formulation so long as
they do not negatively affect the enzymatic activity of the
collagenase, or they may be administered separately, at the same
time or at different times.
[0081] The methods according to the invention may be used in
conjunction with any known treatments to control symptoms caused by
fibroids. For example, NSAIDs or other analgesics can be used to
reduce painful menses, oral contraceptive pills are may be
prescribed to reduce uterine bleeding, and iron supplementation may
be given to treat anemia. A levonorgestrel intrauterine device can
be used to reduce hemorrhage and other symptoms if the condition of
the uterus does not result in expulsion of the device.
[0082] The ability to non-invasively image regions where the
formulations of the present invention are being introduced and
where they have been introduced is a valuable diagnostic tool for
the practice of the present invention. Therefore, in addition to a
uterine fibroid treatment agent and any of the various optional
components discussed above, the uterine fibroid formulations of the
present invention also optionally include one or more imaging
contrast agents to assist with guiding the clinician to administer
the collagenase compound to the fibroid or tissue to be treated or
to determine that administration has been correctly located.
Non-non-invasive imaging techniques include magnetic resonance
imaging (MRI), ultrasonic imaging, x-ray fluoroscopy, nuclear
medicine, and others. Any contrast agent suitable for use with such
techniques and known in the art can be used as part of the
inventive compositions and formulations.
[0083] Any real-time imaging technology can be used to guide
injection or insertion in the invention. For example, X-ray based
fluoroscopy is a diagnostic imaging technique that allows real-time
patient monitoring of motion within a patient. To be
fluoroscopically visible, formulations are typically rendered more
X-ray absorptive than the surrounding tissue. In various
embodiments of the invention, this is accomplished by the use of
contrast agents. Examples of contrast agents for use in connection
with X-ray fluoroscopy include metals, metal salts and oxides
(particularly bismuth salts and oxides), and iodinated compounds.
More specific examples of such contrast agents include tungsten,
platinum, tantalum, iridium, gold, or other dense metal, barium
sulfate, bismuth subcarbonate, bismuth trioxide, bismuth
oxychloride, metrizamide, iopamidol, iothalamate sodium, iodomide
sodium, and meglumine.
[0084] Ultrasound and magnetic resonance imaging can provide two-
and/or three-dimensional images of a portion of the body.
Ultrasound and MRI are advantageous, inter alia, because they do
not expose the patient or medical practitioner to harmful radiation
and they can provide detailed images of the observed area. These
detailed images are valuable diagnostic aids to medical
practitioners and can be used to more precisely control the
quantity and location of the formulations of the present
invention.
[0085] Suitable ultrasonic imaging contrast agents for use in
connection with the present invention include solid particles
ranging from about 0.01 to 50 microns in largest dimension (e.g.,
the diameter, where spherical particles are used), more typically
about 0.5 to 20 microns. Both inorganic and organic particles can
be used. Examples include microparticles/microspheres of calcium
carbonate, hydroxyapatite, silica, poly(lactic acid), and
poly(glycolic acid). Microbubbles can also be used as ultrasonic
imaging contrast agents, as is known in the imaging art. The
ultrasonic imaging contrast agents for use in connection with the
present invention are preferably biocompatible and stable in the
formulation. Concentrations of the ultrasonic imaging contrast
agents typically range from about 0.01 wt % to 10 wt % of the
formulation, more typically about 0.05 to 2 wt %, where solid
particles are used.
[0086] For contrast-enhanced MRI, a suitable contrast agent has a
large magnetic moment, with a relatively long electronic relaxation
time. Based upon these criteria, contrast agents such as Gd(III),
Mn(II) and Fe(III) can be used. Gadolinium(III) has the largest
magnetic moment among these three and is, therefore, a widely-used
paramagnetic species to enhance contrast in MRI. Chelates of
paramagnetic ions such as Gd-DTPA (gadolinium ion chelated with the
ligand diethylenetriaminepentaacetic acid) also are suitable.
Further information can be found, for example, in U.S. Patent
Application No. 2003-0100830 entitled "Implantable or insertable
medical devices visible under magnetic resonance imaging," the
disclosure of which is incorporated herein by reference.
[0087] The collagenase formulations described here preferably are
injected into one or more individual uterine fibroid tumors using a
hollow delivery channel, such as a hollow needle or cannula. For
instance, administration can be performed using a needle in
association with a conventional or specially designed syringe,
cannula, catheter, and the like. A source of manual, mechanical,
hydraulic, pneumatic or other means to apply pressure (e.g., a
conventional syringe plunger, a pump, aerosol, etc.) can be used to
inject the formulation into the fibroid. Alternatively, the
formulations can be administered during surgery, for example via a
trocar during laparoscopic surgery and during hysteroscopic
treatment.
[0088] Injection routes include, for example, transabdominal,
transcervical and transvaginal routes. Where the formulations have
fluid attributes, the injection volume will vary, depending, for
example, on the size of the fibroid, the type and concentration of
treatment agent, and so forth, and will typically range from 1.0 to
10.0 ml per injection. Similarly, where formulations having solid
attributes (e.g., pellets or powders) are used, the amount of
formulation injected/inserted will also depend, for example, on the
size of the fibroid, the type and concentration treatment agent
utilized, etc. Multiple pellets or doses of collagenase composition
can be administered at a single injection site. Regardless of the
physical attributes of the formulation, multiple
injection/insertion sites may be established within a single
fibroid, with the number of injections depending on the size and
shape of the fibroid as well as the type and/or concentration of
the treatment agent that is used. Multiple fibroids or a single
fibroid can be treated.
[0089] In various embodiments, the injection/insertion device is
guided to the fibroid site under image guidance. Image guidance can
include, for example, direct visual guidance (e.g., laparoscopic
guidance in trans-abdominal procedures and hysteroscopic guidance
in trans-vaginal procedures) and non-direct visual guidance (e.g.,
ultrasound guidance, fluoroscopic guidance, and/or MRI
guidance).
[0090] As a specific example, visual guidance of the
injection/insertion device is conducted laparoscopically using a
scope that is positioned in the abdomen (e.g., by insertion through
a trocar). In this way, a device (e.g., a delivery needle or
canula) can be inserted percutaneously into the abdomen and guided
under laparoscopic vision to the uterine fibroid. Once the fibroid
is reached, fluoroscopy, MRI or ultrasound (e.g., trans-vaginal
ultrasound, trans-abdominal ultrasound, intra-abdominal ultrasound,
etc.) preferably is used to guide the tip of the delivery needle to
a desired position within the fibroid, at which point the
formulation is injected or inserted into the fibroid. To the extent
that there is sufficient contrast between the formulation and the
surrounding tissue, the location of the formulation within the
fibroid will also be viewed.
[0091] The compositions and processes of the present invention will
be better understood in connection with the following examples,
which are intended as an illustration only and not limiting of the
scope of the invention. Various changes and modifications to the
disclosed embodiments will be apparent to those skilled in the art
and such changes and modifications including, without limitation,
those relating to the processes, formulations and/or methods of the
invention may be made without departing from the spirit of the
invention and the scope of the appended claims.
EXAMPLES
Example 1. General Collagenase Production
[0092] To prepare an animal-material-free clostridia cell bank,
Clostridium histolyticum cells are suspended in a medium containing
a vegetable peptone and optionally yeast extract. For example, one
general method for accomplishing this is as follows.
TABLE-US-00002 TABLE 2 General Method to Produce Clostridium Cell
Bank. Step 1 Starting cells: any Clostridium histolyticum culture
which is convenient and available, for example Clostridium
histolyticum ATCC 21000, strain 004 Step 2 Inoculate 1 mL of step 1
into 300 mL of media containing 15.45 g Phytone, 2.55 g yeast
extract, and water sufficient to produce 0.3 L (M#1); step 2 for 24
hours at 37.degree. C. (1.sup.st culture); Step 4 Transfer 3 mL of
step 3 (1.sup.st culture) to 1000 mL of M#1; Step 5 Incubate step 4
for 16 hours at 37.degree. C. (2.sup.nd culture); Step 6 Centrifuge
the 2.sup.nd culture; Step 7 Re-suspend the pellet with the 5 mL of
media #1 and 5 mL of 20% glycerol; Step 8 Freeze the aliquot of
cells gradually; Step 9 Store the aliquot at -80.degree. C.
[0093] Once an animal material-free cell bank is established, the
cells can be grown or fermented in convenient media known in the
art, preferably non-animal-derived medium. The medium can
optionally contain yeast extract. Exemplary, non-limiting examples
of such media are M#1, M#2, M#3, and M#4 as described in Table 3,
below. In addition, see Table 4 for an exemplary, non-limiting
general example of the steps of the fermentation process.
TABLE-US-00003 TABLE 3 Media recipes and preparation. M #1 M #2 M
#3 M #4 Phytone 15.45 g 103 g Veggitone 15.45 g 103 g Yeast extract
2.55 g 2.55 g 17 g 17 g KH.sub.2PO.sub.4 1.92 g 1.92 g
K.sub.2HPO.sub.4 1.25 g 1.25 g Na.sub.2HPO.sub.4 3.5 g 3.5 g NaCl
2.5 g 2.5 g vol of water 0.3 L 0.3 L 1 L 1 L
TABLE-US-00004 TABLE 4 Fermentation Process. Step 1 Starting cells:
Animal material free Clostridia cell bank Step 2 Inoculate 1 mL of
step 1 into the 300 mL of M#1; Step 3 Incubate step 2 for 16 to 24
hours at 37.degree. C. (1.sup.st culture); Step 4 Transfer 10 mL of
step 3 (1.sup.st culture) and 10 mL Vitamin/Mg solution* to 1000 mL
of M#3, or 4 respectively; Step 5 Incubate step 4 for about 22
hours at 37.degree. C.(2.sup.nd culture); Step 6 Use 2.sup.nd
culture for downstream isolation and purification. *Prepared
separately by dissolving 8 g MgSCU, 1.2 g ferrous sulfate, 0.05 g
riboflavin, 0.1 g Niacin, 0.1 g Calcium pantothenate, 0.1 g pimelic
acid, 0.1 g pyridoxine, and 0.1 g thiamine in 1100 mL water,
followed by sterilization by 0.22 urn filtration.
[0094] After preparation of "2.sup.nd culture," the collagenase I
and collagenase II can be isolated and purified using any method
capable of producing each enzyme separately to at least 95% purity.
The method may combine one or more of the steps of ammonium sulfate
precipitation, dialysis, hydroxyapatite (HA) chromatography, gel
filtration and ion-exchange, for example, preferably in that order.
The gel filtration is preferably G75 gel filtration. The
ion-exchange is preferably anion-exchange: Q-Sepharose
chromatography. In addition, when the Clostridia have been cultured
in medium containing less glucose and more salt compared to the
majority of known bacterial culture, as preferred, protease
inhibitors such as leupeptin are not required.
Example 2. Preparation of Animal Material Free Clostridium Cell
Bank
[0095] The starter cell culture was Clostridium histolyticum ATCC
21000, strain 004 which was originally created with bovine-derived
materials. The cells were first grown in animal material free
medium (M #1, Table 3). Briefly, the recipe includes: phytone, 51.5
g, yeast extract 8.5 g, 1000 mL water. The pH was adjusted to 7.30
with NaOH, and the medium sterilized at 121.degree. C. for 20
minutes. One milliliter of the starting material was then
inoculated into 300 mL of M#1 and incubated for 24 hours at
37.degree. C. (1st culture). Three milliliters of the 1st culture
was transferred to 1000 mL of M#1 and incubated for 16 hours (2nd
culture). The 2nd culture was then centrifuged aseptically. The
pellet was re-suspended in 5 mL M#1 with 5 mL 20% glycerol. The
aliquots of cell suspension were frozen gradually and stored at
-80.degree. C.
Example 3. Fermentation Process
[0096] Clostridium histolyticum ATCC 21000, strain 004 was
inoculated into the starting culture with M#1 or M#2 and incubated
at 37.degree. C. for 16 hours. Ten milliliters of the starting
culture (M#1 or M#2) and 10 mL Mg/vitamin solution (prepared
separately by dissolving 8 g MgSO4, 1.2 g ferrous sulfate, 0.05 g
riboflavin, 0.1 g Niacin, 0.1 g Calcium pantothenate, 0.1 g pimelic
acid, 0.1 g pyridoxine, and 0.1 g thiamine in 1100 mL water,
followed by sterilization by 0.22 .mu.m filtration) was then
transferred to each liter of M#3 or M#4 (or a variation thereof),
and incubated for 22 hours. Clostridium histolyticum grew well with
the OD600 reaching >2.5.
Example 4. General Procedure for Isolation and Purification of
Collagenase I and Collagenase II
TABLE-US-00005 [0097] TABLE 5 General Exemplary, Non-Limiting
Isolation and Purification Procedure for Collagenase I and
Collagenase II. Stages of Product Operations Fermentation broth
Centrifugation or 1.0 .mu.m filtration; Clarified fermentation
broth Add ammonium sulfate (590 g/liter); centrifugation; Crude
collagenase precipitate Dissolve crude collagenase precipitate by
adding purified water; Crude collagenase solution Dialyze crude
collagenase solution against purified water overnight (store at
-20.degree. C.) with 10 kDa pore size dialysis membrane; Dialyzed
crude collagenase Clarify the dialyzed crude collagenase solution
with either centrifugation or filtration or the combination of
both; Clarified solution Add potassium phosphate buffer, pH 6.7 to
a final conc, of 0.1M; Collagenase in phosphate Load collagenase
solution to hydroxylapatite column and elute column buffer with
gradient of increasing K.sub.2PO.sub.4 conc, at ambient temp.
(20.degree. C.); Collagenase HA eluate Concentrate the eluate with
ultrafiltration (30 kDa of pore size); Concentrated collagenase
Load the concentrate onto a G75 gel filtration column at ambient
temperature (20.degree. C.) and elute with 20 mM Tris/150 mM NaCl;
Collagenase G75 eluate Dialyze the eluate against a buffer (10 mM
Tris, 3 mM calcium chloride (CaCl.sub.2), pH 8.0) overnight;
Dialyzed G75 eluate Load dialyzed eluate on to a Q-Sepharose
anion-exchange column at ambient temperature (20.degree. C.); elute
using a gradient of 10 mM Tris HCl, 3 mM CaCl.sub.2 , pH 8.0 buffer
and 10 mM Tris HCl, 3 mM CaCl.sub.2, 1M NaCl, pH 8.0 buffer;
Collagenase class I and class Store separately at -20.degree. C. II
fractions
Example 5. Ex Vivo Treatment of Uterine Fibroid Tissue
[0098] Samples of fibroid tissue and myometrium were obtained
post-hysterectomy from women with consent and identified by
evaluation by a surgical pathologist. The tissue samples were
transported to the laboratory and cut into 1 cm.sup.3 cubes. See
FIG. 2. These cubes were injected with purified collagenase (0.06
or 0.2 mg in 100 .mu.L) dissolved in media or serum and then
incubated for 24, 48, 72, or 96 hours at 37.degree. C. See FIG. 3.
Each treatment was carried out in tissues from three different
patients with two tissue samples per treatment because fibroid
tissue is extremely variable. Control fibroid and myometrium cubes
were injected with vehicle or sham injected. At the end of the
incubation, the tissue samples were photographed to document gross
appearance. Degree of liquefaction and softening was observed and
documented using a 4-point subjective scale.
[0099] Samples were frozen for biomechanical assessment
(compression analysis). Samples were fixed in formalin for
histology and Masson trichrome and picrosirius red staining. They
were analyzed by light microscopy for the presence or absence of
collagen and assessed using computer morphometry to determine the
extent of degradation. In the case of picrosirius red staining,
polarized light microscopy was performed to determine collagen
fiber orientation. Samples were fixed in glutaraldehyde and
postfixed with osmium tetraoxide for electron microscopy to
determine collagen fibril orientation and evidence of fibril
degradation. Additional injections were done at a dose of 0.58
mg/injection (250 ul of 2.3 mg/ml).
[0100] These ex-vivo studies have shown the efficacy of purified
collagenase in softening and partial liquefaction of
post-hysterectomy fibroid specimens, as well as a decrease in the
collagen content. Treated fibroid-specimens were grossly softer and
had partially liquefied centers. Masson trichrome and picrosirius
red stains of theses tissues showed a dramatic subjective decrease
in collagen content compared to fibroid tissue injected with
vehicle.
Example 6. Treatment of Whole Uterine Fibroids Ex Vivo
[0101] Donated tissue was obtained from four female adult patients
18 years of age or older who can give legally effective consent and
who were planning to undergo definitive treatment for fibroids by
hysterectomy. After the removal of the hysterectomy specimen, the
uterus was observed grossly by standard procedures by a surgical
pathologist. Complete fibroids (submucosal (abutting the
endometrium), intramural (within the myometrium), and subserosal
(abutting the uterine serosa) fibroids, or pedunculated fibroids
(attached to the uterus by a stalk) if they are present) from 1 to
4 cm (including the capsule) along with 1.5 cm of the surrounding
adjacent myometrium and, if available, a 0.5 cm section of
endometrium were dissected free from the specimen and placed in
normal saline.
[0102] Tissues were brought to the laboratory immediately, washed
and injected with purified Clostridium histolyticum collagenase
(PCHC) (0.1 mg/100 .mu.l/cm.sup.3). Optionally, a higher
concentration of the collagenase was used to decrease the volume of
the injection. Purified collagenase was diluted in 0.3 mg/mL
calcium chloride dihydrate in 0.9% sodium chloride, optionally
combined with 1% methylene blue as a marker to visually assess the
area of distribution of the injected material within the fibroid
and uterus. Fibroids were injected with PCHC or vehicle in the
center of the obtained specimen. See FIGS. 4A and 4B. The amount of
collagenase injected depended on the size of the fibroids (1-4 cm).
Generally, about 818 .mu.L of material was injected into a fibroid
with a diameter of about 2.5 cm. If injecting the entire treatment
volume centrally was not feasible due to tissue resistance to the
injection or other factors, multiple locations were injected within
the fibroid. The fibroid tissue then was incubated in DMEM/F12
culture medium at 37.degree. C. for 24 hours. At least one fibroid
with attached myometrium served as the control. This specimen
received an injection of 1% methylene blue in vehicle without
collagenase as a non-randomized placebo injection, centrally into
the fibroid.
[0103] Color photographs were taken of the uterus and of the
fibroid and myometrial pieces pre- and post-injection. Fibroid
diameters were measured with a metric ruler.
[0104] At the end of the incubation, the samples were reassessed
grossly for size, consistency and firmness, and color photographs
were obtained, as well as optional video recording to record
fibroid manual distensibility and any liquefied portions upon
sectioning. The degree of liquefaction and softening were observed
and documented using a 4-point subjective scale.
[0105] Whether the collagenase can penetrate the capsule and affect
the nearby myometrium was determined. Samples were obtained,
including tissue from the injected fibroid and adjacent tissue,
plus a section that included fibroid and adjacent myometrium and/or
endometrium still attached, and myometrium alone. Samples were
fixed in formalin for histology and Masson trichrome, picrosirius
red, and hematoxylin-eosin staining. The samples were analyzed by
light microscopy for the presence or absence of collagen and using
computer morphometry to assess the extent of degradation.
Picrosirius red staining was used with polarized light microscopy
to determine collagen fiber orientation.
[0106] Exemplary treatment schemes for each patient:
[0107] fibroid 1: inject 818 .mu.L 1 mg/mL collagenase;
[0108] fibroid 2: inject 818 .mu.L 1 mg/mL collagenase;
[0109] fibroid 3: inject 818 .mu.L control vehicle;
[0110] Injections were given through the fibroid capsule into the
center of the fibroid, through the myometrium into the center of
the fibroid, or through the endometrium into the center of the
fibroid, simulating in vivo injection routes. The fibroids here
were liquefied in the same manner as shown in FIG. 5 (see
below).
Example 7. Biomechanical Evaluation of Human Uterine Fibroids after
Injection with Purified Clostridial Collagenase
[0111] The two collagenases isolated from Clostridium histolyticum
(ABC I and ABC II) were combined in a 1:1 mass ratio. Both
collagenases are metalloproteases and have a broad hydrolyzing
reactivity and degrade type I and Ill collagens. The biomechanical
properties of uterine fibroid tissue were analyzed by rheometry in
control and collagenase-treated specimens.
[0112] Fibroid tissue was obtained after surgery (hysterectomy or
myomectomy) from 4 different patients and cut into cubes (1
cm.sup.3; n=43). Tissue cubes were injected into the center with
100 .mu.L of purified collagenase (0, 0.25, 0.5, 1.0, 2.0 mg/mL;
n=4-14 per dose) and incubated at 37.degree. C. for 24, 48, or 96
hours. At the end of the incubation period, cubes were cut in half
and snap-frozen in liquid nitrogen. Different degrees of softening
and liquefaction at the center were noted. An AR-G2 rheometer was
used to measure the sample stiffness dynamically (complex shear
modulus (Pa) at 10 rad/sec), taking into account both the viscous
and elastic behavior of the material. At least 2 specimens (5 mm
diameter punch) from each tissue cube were measured. Data were
analyzed by 2-way ANOVA and Dunnett's multiple comparisons
test.
[0113] Overall, stiffness in control fibroid cubes (6585.+-.707 Pa;
n=13) was greater than in treated cubes (2003.+-.275 Pa; n=30;
p.ltoreq.0.0001). More specifically, stiffness in fibroid tissues
was reduced in a time and dose dependent manner. At 48 hours,
treatment with 0.25 mg/mL did not reduce stiffness (5032.+-.1796
Pa), but treatment with 0.5 mg/mL did (2014.+-.1331 Pa;
p.ltoreq.0.05). At 96 hours, both the 0.25 and the 0.5 doses were
effective (1720.+-.377 and 1072.+-.160 Pa; p.ltoreq.0.01). The 1.0
and 2.0 mg/mL treatments reduced stiffness at 24 hours, but not
significantly (2177.+-.37 and 2480.+-.984 Pa; n=4). However, doses
of 1.0 and 2.0 mg/mL were effective at 48 hours (3588.+-.637;
p.ltoreq.0.05 and 1254.+-.445 Pa; p.ltoreq.0.01; n=6;) and at 96
hours (921.+-.305 and 1350.+-.571 Pa; p.ltoreq.0.0001; n=10).
[0114] Using a torsional rheometer, tissue stiffness was
quantitated over a wide range (very firm to liquefied). Our data
indicate that treatment of the fibroid tissue with defined doses of
purified clostridial collagenase significantly decreased the
stiffness (modulus) of the tissue. See FIG. 5, which shows
collagenolysis in fibroid tissue after 48 hour incubation. The left
photograph is tissue that was injected with vehicle (control) and
the right photograph is tissue that was injected with collagenase.
FIG. 6 shows micrographs of control (FIGS. 6A and 6B) and
collagenase-treated (FIGS. 6C and 6D) tissue. Mason stain in
Figures A and C (left) shows that collagen is decreased. Picrosirus
red stain visualized under polarized light (FIG. 6D) clearly shows
in the bottom right that collagen fibers are degraded.
Example 8. Treatment of Human Uterine Fibroids in Nude Mouse
Model
[0115] The xenograft mouse model, in which three-dimensional
organotypic cultures of human uterine fibroid cells are implanted
under the skin of female nude mice, has been successfully employed
to study keloids, a fibrotic skin disorder with biology similar to
fibroids. This model is used to demonstrate effects of PCHC
injection, in an HPG nanocarrier formulation, on fibroid tissue in
vivo.
[0116] Polylactic acid sponges, other synthetic polylactic acid
scaffolds, or any suitable commercially available scaffold is
inoculated with human uterine fibroid cells to produce an
organotypic 3-D culture of uterine fibroid cells that can be
implanted into nude mice. These 3-dimensional organotypic cultures
(3D-fibroids) are representative of human fibroids and produce and
contain extracellular matrix.
[0117] OPLA sponges (Open-Cell Polylactic Acid, BD Biosciences;
FIG. 7) are synthetic polymer scaffolds that are synthesized from
D,D-L,L polylactic acid. This material has a facetted architecture
which is effective for culturing high density cell suspensions. The
cells will be seeded onto the 3D sponge-like scaffolds under
dynamic conditions, leading to uniform cell population throughout
the sponges and higher cell numbers per sponge than static seeding.
Post-sterilization, the molecular weight of the OPLA is 100-135 kD.
They have an approximate size of 5 mm.times.3 mm (0.04 cm.sup.3)
with an average pore size of 100-200 .mu.m.
[0118] Cells and scaffolds are placed into cell culture chambers of
a bioreactor consisting of a fluid (culture media)-filled, rotating
chamber that allows for constant floating of cells while minimizing
shearing forces and gravitational settlement of cells and/or
scaffolds (Synthecon, Inc.). Cells inside the rotating bioreactor
chamber are suspended in virtual weightlessness.
[0119] Primary human fibroid cells from specimens obtained at
hysterectomy are seeded statically or dynamically into OPLA sponges
and grown for 30 days to allow for production and assembly of
extracellular matrix. Cells grow throughout the scaffold and can be
formalin fixed, paraffin embedded and thin sectioned for
observation, optionally with staining for multiple markers. See
FIG. 8, which shows the formation of the cell lattice following the
outlines of the sponge-like scaffold.
[0120] FIG. 9 shows primary cultures of fibroid cells after static
seeding. The cells are fixed on the scaffold and observed in situ.
Scaffolds containing cells were fixed and were unstained (FIG. 9A)
or stained for f-actin with fluorescent phalloidin (FIG. 9B). Cells
were evenly distributed throughout the scaffold. The imaged
scaffolds are >1 mm thick and therefore not all cells are in
focus, indicating that the cells are growing not only on the
surface, but also deep inside the scaffolds. FIG. 10 shows the
population of cells throughout the sponge-like scaffolds using
confocal microscopy (FIGS. 10A and 10B).
[0121] High quality RNA is extracted from the 3D-cultures of
fibroid cells on OPLA sponges and used to verify the expression of
two genes of interest. Versican and TGF.beta.3 are known to be
highly expressed in fibroid tissue and cells. Results in Table 6
show that both a fibroid cell line and primary cultures of fibroid
cells in this 3D-culture system express these two genes in high
amounts.
TABLE-US-00006 TABLE 6 Real Time PCR Assay Results cDNA (ng) per
Threshold Cycle Ct (mean .+-. SEM) reaction Versican
TGF.beta..sub.3 Fibroid 50 22.1 .+-. 0.07 26.8 .+-. 0.07 Cell Line
Primary 25 22.2 .+-. 0.21 24.0 .+-. 0.04 Fibroid Cells
[0122] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. All other
published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.
[0123] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *