U.S. patent application number 10/464001 was filed with the patent office on 2003-11-13 for facilitation of wound healing with cm101/gbs toxin.
Invention is credited to Abramovitch, Rinat, Hellerqvist, Carl G., Neeman, Michal, Wamil, Barbara D..
Application Number | 20030212041 10/464001 |
Document ID | / |
Family ID | 29736032 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030212041 |
Kind Code |
A1 |
Hellerqvist, Carl G. ; et
al. |
November 13, 2003 |
Facilitation of wound healing with CM101/GBS toxin
Abstract
The method of the present invention provides a means of treating
a patient having a wound, especially by minimizing scarring and
accelerating wound healing, by administering CM101 or GBS toxin
isolated from Group B .beta.-hemolytic Streptococcus bacteria.
Types of wounds that may be treated include surface and internal
wounds. The method of the present invention also includes
administration of CM101 or GBS toxin to surgery patients having
tumors in order to facilitate wound healing and minimize the
likelihood of tumor progression.
Inventors: |
Hellerqvist, Carl G.;
(Brentwood, TN) ; Neeman, Michal; (Mazkeret Batya,
IL) ; Wamil, Barbara D.; (Nashville, TN) ;
Abramovitch, Rinat; (Mordechai, IL) |
Correspondence
Address: |
COOLEY GODWARD, LLP
3000 EL CAMINO REAL
5 PALO ALTO SQUARE
PALO ALTO
CA
94306
US
|
Family ID: |
29736032 |
Appl. No.: |
10/464001 |
Filed: |
June 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10464001 |
Jun 17, 2003 |
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09363968 |
Jul 29, 1999 |
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09363968 |
Jul 29, 1999 |
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PCT/US98/01853 |
Jan 29, 1998 |
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09363968 |
Jul 29, 1999 |
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08791763 |
Jan 29, 1997 |
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5858991 |
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Current U.S.
Class: |
514/54 |
Current CPC
Class: |
A61K 31/715 20130101;
A01N 1/021 20130101 |
Class at
Publication: |
514/54 |
International
Class: |
A61K 031/739 |
Claims
What is claimed is:
1. A method for treating a patient having a wound, which method
comprises: administering a group B .beta.-hemolytic Streptococcus
(GBS) toxin to the patient in a quantity sufficient to reduce
scarring at a site of the wound.
2. A method of treating a patient having a wound, which method
comprises: administering a group B .beta.-hemolytic Streptococcus
(GBS) toxin to the patient in an amount sufficient to accelerate
wound healing.
3. The method of claim 1 wherein healing of the wound is
accelerated.
4. The method of claim 2 where the wound healing is achieved with
minimal scarring.
5. The method of claim 1 or 2 wherein the GBS toxin is CM101.
6. The method of claim 5 wherein the CM101 is substantially
pure.
7. The method of claim 6 wherein the CM101 has a purity of at least
approximately 90%.
8. The method of claim 1 or 2 wherein the GBS toxin is administered
to the patient in a quantity sufficient to provide reduced vessel
density at the site of the wound with GBS toxin treatment relative
to vessel density at the site of a comparable wound without GBS
treatment.
9. The method of claim 8 wherein the reduced vessel density is
within the range of 0.0 to 0.2 (1-AC).
10. The method of claim 1 or 2 wherein the GBS toxin is
administered to the patient in a quantity sufficient to provide
improved tensile strength at the site of the wound with GBS toxin
treatment relative to tensile strength at the site of the wound
without GBS treatment.
11. The method of claim 1 or 2 wherein the GBS toxin is
administered to the patient parenterally.
12. The method of claim 11 wherein the GBS toxin is administered to
the patient intravenously.
13. The method of claim 1 or 2 wherein the GBS toxin is
administered proximate to the site of the wound.
14. The method of claim 1 or 2 wherein the GBS toxin is
administered to the patient at a dosage in the range of 1 to 100
.mu.g/kg body weight.
15. The method of claim 14 wherein the GBS toxin is administered to
the patient at a dosage in the range of 1 to 25 .mu.g/kg body
weight.
16. The method of claim 14 wherein the dosage is administered in a
single infusion of thirty minute duration or less.
17. The method of claim 14 wherein the dosage is administered once
weekly.
18. The method of claim 1 or 2 wherein the GBS toxin is
administered to the patient in a quantity sufficient to reduce
vascularization at the site of the wound.
19. The method of claim 18 wherein the GBS toxin is administered to
the patient in a quantity sufficient to reduce hypoxia-induced,
VEGF-driven neovascularization at the site of the wound.
20. The method of claim 1 or 2 wherein the patient has a tumor.
20.
21. The method of claim 1 or 2 wherein the patient has no known
tumor.
22. The method of claim 1 or 2 wherein the patient is at least four
days old.
23. The method of claim 22 wherein the patient is at least seven
days old.
24. The method of claim 1 or 2 wherein the wound is the result of
trauma.
25. The method of claim 1 or 2 wherein the wound is the result of
surgery.
26. The method of claim 1 or 2 wherein the wound is within a blood
vessel.
27. A method of treating a patient having a keloid, which method
comprises: (a) excising the keloid to create a wound, and (b)
administering a group B .beta.-hemolytic Streptococcus (GBS) toxin
to the patient in a quantity sufficient to reduce scarring at a
site of the wound.
28. The method of claim 27 wherein the GBS toxin is CM101.
29. The method of claim 28 wherein the CM101 has a purity of at
least approximately 90%.
30. A method of reducing the likelihood of metastatic tumor
implantation proximate to a site of surgery in a surgery patient,
which method comprises: administering a group B .beta.-hemolytic
Streptococcus (GBS) toxin to the surgery patient in a quantity
sufficient to reduce scarring or accelerate wound healing at the
site of surgery.
31. The method of claim 30 wherein the GBS toxin is CM101.
32. The method of claim 31 wherein the CM101 has a purity of at
least approximately 90%.
33. The method of claim 30 wherein the GBS toxin is administered to
the patient intravenously.
34. A method of reducing the likelihood of tumor proliferation in a
surgery patient, which method comprises: administering a group B
.beta.-hemolytic Streptococcus (GBS) toxin to the surgery patient
in a quantity sufficient to reduce scarring or accelerate wound
healing at a site of surgery.
35. The method of claim 34 wherein the GBS toxin is CM101.
36. The method of claim 35 wherein the CM101 has a purity of at
least approximately 90%.
37. The method of claim 34 wherein the GBS toxin is administered to
the patient intravenously.
38. A method of protecting against reperfusion injury in a patient,
which method comprises: administering a group B .beta.-hemolytic
Streptococcus (GBS) toxin to the patient prior to reperfusion of a
blood vessel.
39. The method of claim 38 wherein the GBS toxin is administered
prior to occlusion of the blood vessel.
40. An article of manufacture comprising: (a) a pharmaceutical
composition having (i) a group B .beta.-hemolytic Streptococcus
(GBS) toxin, and (ii) a pharmaceutically acceptable carrier, and
(b) instructions for administering the pharmaceutical composition
to a patient having a wound.
41. The article of claim 40 wherein the GBS toxin is CM101.
42. The article of claim 41 wherein the CM101 has a purity of at
least approximately 90%.
43. The article of claim 40 wherein the instructions describe
administration of the pharmaceutical composition to the patient in
a quantity sufficient to reduce scarring at a site of the
wound.
44. The article of claim 40 wherein the instructions describe
administration of the pharmaceutical composition to the patient in
a quantity sufficient to accelerate wound healing.
45. The article of claim 40 wherein the instructions describe
administration of the pharmaceutical composition to the patient to
treat a keloid by excising the keloid and administering the
pharmaceutical composition in a quantity sufficient to reduce
scarring at a site of the wound.
46. The article of claim 40 wherein the instructions describe
administration of the pharmaceutical composition to the patient to
reduce the likelihood of metastatic tumor implantation proximate to
a site of surgery in the patient by administering the
pharmaceutical composition in a quantity sufficient to reduce
scarring or accelerate wound healing at the site of surgery.
47. The article of claim 40 wherein the instructions describe
administration of the pharmaceutical composition to the patient to
reduce the likelihood of tumor proliferation in the patient by
administering the pharmaceutical composition in a quantity
sufficient to reduce scarring or accelerate wound healing at a site
of surgery.
48. A method of making an article of manufacture, which method
comprises: combining (a) a container including a pharmaceutical
composition comprising (i) a group B .beta.-hemolytic Streptococcus
(GBS) toxin, and (ii) a pharmaceutically acceptable carrier, and
(b) labelling instructions for treating a patient having a wound by
administering the pharmaceutical composition to the patient.
49. The method of claim 48 wherein the instructions describe
administration of the pharmaceutical composition to the patient in
a quantity sufficient to reduce scarring at a site of the
wound.
50. The method of claim 48 wherein the instructions describe
administration of the pharmaceutical composition to the patient in
a quantity sufficient to accelerate wound healing.
51. The method of claim 48 wherein the instructions describe
administration of the pharmaceutical composition to the patient to
treat a keloid by excising the keloid and administrating the
pharmaceutical composition in a quantity sufficient to reduce
scarring at a site of the wound.
52. The method of claim 48 wherein the instructions describe
administration of the pharmaceutical composition to the patient to
reduce the likelihood of metastatic tumor implantation proximate to
a site of surgery in the patient by administering the
pharmaceutical composition in a quantity sufficient to reduce
scarring or accelerate wound healing at the site of surgery.
53. The method of claim 48 wherein the instructions describe
administration of the pharmaceutical composition to the patient to
reduce the likelihood of tumor proliferation in the patient by
administering the pharmaceutical composition in a quantity
sufficient to reduce scarring or accelerate wound healing at a site
of surgery.
Description
TECHNICAL FIELD
[0001] This invention relates to the facilitation of wound healing
in patients, by minimizing scarring and accelerating healing. This
invention also relates to the reduction of wound-related tumor
progression.
BACKGROUND
[0002] The normal process of healing a skin wound that has been
surgically induced or is the result of trauma involves formation of
a blood clot and, often, a scab. More particularly, first
intention, or primary healing, generally occurs at clean incisions,
whereas second intention, or secondary healing, occurs where wound
edges are far apart. The protein fibrin holds the edges of the skin
surrounding the wound together and the scab seals the wound and
staves off infection. While an inflammatory response brings
increased numbers of blood cells to the area to aid in the repair
process, epithelial tissue regenerates and capillaries grow from
blood vessels at the edges of the wound. The capillaries
revascularize the area of the wound and contribute to the formation
of granulation tissue which, in turn, causes scarring.
[0003] Granulation tissue begins to form in the wound site and
fills the site approximately five days after wound induction.
Granulation tissue contains new collagen, fibroblasts, new blood
vessels and inflammatory cells, especially macrophages (E. Rubin
and J. L. Farber, Pathology, Lippincott, publ., pp. 85-95 (1994)).
After seven to ten days, the wound has regained only 10% of the
tissue's original strength.
[0004] Secondary healing causes a greater inflammatory response and
more granulation tissue is formed. In addition, contraction of the
wound, resulting from contraction of the fibroblasts of the
granulation tissue, brings the edges of the wound together to speed
the healing process, but sometimes contributes to disfiguring and
debilitating scars. Additionally, excessive deposition of
extracellular matrix leads to the formation of keloids, or
hypertrophic scars, which are irregularly-shaped, elevated scars
that tend toward progressive enlargement.
[0005] Angiogenesis is generally believed to be a necessary feature
of repair (Kovacs, E. et al., Fibrogenic cytokines and connective
tissue production, FASEB J., 8:854-861 (1994). Numerous growth
factors and cytokines, secreted first by platelets in response to
coagulation and then by macrophages in response to hypoxia and
lactic acidosis, stimulate angiogenesis (Shah, M. et al., The
Lancet, 339:213-214 (1992)). Angiogenesis generally becomes visible
at a microscopic level about four days after injury but begins two
or three days earlier when new capillaries sprout out of
preexisting venules and grow toward the injury in response to
chemoattractants released by platelets and macrophages. In
primarily closed wounds, sprouting vessels soon meet counterparts
migrating from the other side of the wound and blood flow across
the wound is reestablished. In unclosed wounds, or those not well
closed, the new capillaries fuse only with neighbors migrating in
the same direction, and a large amount of granulation tissue is
formed instead.
[0006] In normal wound healing, the tissue surrounding a wound
undergoes a degree of hypoxia and a concomitant increase in
secretion of vascular endothelial growth factor, or VEGF, typically
occurring one to two days following injury (Brown, L. F. et al.,
Expression of VPF (VEGF) by epidermal keratinocytes during wound
healing, J. Exp. Med., 176:1375-79 (1992)). VEGF stimulates the
rapid proliferation of blood vessel endothelial cells which results
in the formation of densely sprouting capillaries. This rapid
hypoxia-induced, VEGF-driven capillary formation stimulates
infiltration of inflammatory cells and leads eventually to
scarring.
[0007] While inflammation causes scarring, inflammation is also
beneficial. Inflammatory cells release growth signals and lytic
enzymes that are very important for repair. In fact, patients who
receive anti-inflammatory agents often experience impaired healing
due to inadequate inflammation at the site of a wound.
[0008] An important aspect of wound repair is the time involved.
The rate at which a wound heals has implications for the prevention
of infection and improvement of the overall health of the patient.
Rapid, even healing without excessive contraction is a desirable
result from a medical and cosmetic standpoint.
[0009] Furthermore, it is a recognized clinical phenomenon that
surgery in a tumor patient may lead to tumor progression if the
site of the surgical incision is in proximity to the site of the
tumor. In addition, the surgical incisions show high susceptibility
to metastatic implantation. (Murthy et al., Cancer, 64:2035-2044
(1989); Murthy et al., Cancer, 68:1724-1730 (1991); Schackert, H.
K. et al., Int. J. Cancer, 44:177-81 (1989)). The stimulatory
effect of wounds on tumors is manifested as accelerated growth of
residual tumor near the site of surgical intervention, as well as
an increased probability of metastatic implantation at the site of
surgery. Furthermore, wounds located at the site of a tumor
regularly fail to heal (Gatenby, R. A. et al., Suppression of wound
healing in tumor bearing animals, Cancer Research, 50:7997-8001
(1990)). Persistent wounds that continuously accelerate tumor
progression may be a frequent side effect of surgical interventions
associated with cancer therapy. Therefore, deciding whether to
operate on a tumor patient is often a difficult decision in which
the benefits of surgery must be compared to the risks of worsening
a cancer patient's overall condition.
[0010] It is an object, therefore, of the present invention to
provide a method of preventing or minimizing scar formation during
the wound healing process.
[0011] Another object of the present invention is to provide a
method of accelerating the rate at which a wound heals.
[0012] A further object of the present invention is to provide a
method of facilitating wound healing in tumor patients and
minimizing the likelihood of tumor progression.
SUMMARY OF THE INVENTION
[0013] The method of the present invention provides for treating a
patient having a wound by administering CM101, a generally nontoxic
polysaccharide isolated from group B .beta.-hemolytic Streptococcus
(GBS) bacteria, to minimize scarring and to accelerate wound
healing. The invention finds use in treatment of surface as well as
internal wounds.
[0014] Another aspect of the present invention is a method for
treating a keloid by excising the keloid and administering
CM101.
[0015] The present invention also provides a method of minimizing
the likelihood of tumor progression, i.e. wound-induced tumor
proliferation or metastatic implantation by administration of CM101
before, during and/or after surgery or other induction of a wound
in a tumor patient.
[0016] An article of manufacture including GBS toxin, and
particularly CM101, along with instructions for treatment, and a
method of making the article are also disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 graphs vessel density around wounds in tumor-free
mice treated with CM101 or saline solution.
[0018] FIG. 2 graphs vessel density around wounds in tumor-bearing
mice treated with CM101 or saline solution.
[0019] FIG. 3 presents a graphic representation of the effect of
CM101 on the recovery of skin strength after wounding.
DETAILED DESCRIPTION OF THE INVENTION
[0020] This invention is based in part on the discovery that GBS
toxin, and particularly CM101, facilitates wound healing by
promoting rapid healing with minimal scarring. CM101 presumably
produces these beneficial effects by diminishing the rapid
hypoxia-related VEGF-driven neovascularization that contributes to
the scarring associated with wound healing. This mechanism of
action also contributes to rapid, even healing of wounds in tumor
patients, thus reducing the likelihood of tumor proliferation or
metastatic implantation at the site of surgery or other
wounding.
[0021] CM101, a GBS toxin, is a polysaccharide molecule isolated
from group B .beta.-hemolytic Streptococcus (GBS) bacteria.
Specifically, pathogenic group B .beta.-hemolytic streptococcus
produces a polysaccharide exotoxin. This exotoxin is the putative
agent for GBS pneumonia or "early onset disease" in neonatal
humans. These newborn infants may suffer from sepsis,
granulocytopenia, and respiratory distress, i.e. pulmonary
hypertension and proteinaceous pulmonary edema (Hellerqvist, C. G.
et al., Studies on group B .beta.-hemolytic streptococcus I.
Isolation and partial characterization of an extra-cellular toxin.,
Pediatr. Res., 12:892-898 (1981)). It is believed that receptors
for CM101 are present primarily on the lungs of newborns, making
them susceptible to early onset disease, but that lung cells lose
CM101 receptors approximately four to seven days after birth. Thus,
despite the harmful effects on neonates exposed to GBS, CM101 is
not known to cause toxicity in older humans.
[0022] Isolated CM101 has been shown to have toxic effects on sheep
experimental models that mimic GBS infant pneumonia (Hellerqvist,
C. G. et al., Studies on group B .beta.-hemolytic streptococcus I.
Isolation and partial characterization of an extra-cellular toxin.,
Pediatr. Res., 12:892-898 (1981)). In the sheep model for neonatal
early onset disease, GBS toxin causes pulmonary hypertension,
increased pulmonary vascular permeability, granulocytopenia, and
pulmonary sequestration of granulocytes.
[0023] CM101 has a molecular weight of approximately 300,000
Daltons and comprises N-acetyl-galactosamine, N-acetyl-glucosamine,
glucose, galactose, and mannose residues, in an approximate
1:1:1:3:1 ratio. Carboxylic acid residues are also believed to be
integral parts of the molecule. Repeating active epitopes most
likely play an important role in the pathophysiological response to
CM101 by crosslinking receptors on target endothelium (Hellerqvist,
C. G. et al., Early Results of a Phase I Trial of CM101 in Cancer
Patients., Proceedings of the American Association of Cancer
Research Annual Meeting, 36:224 (1995)).
[0024] A method of preparation of a GBS toxin is provided in U.S.
Pat. No. 5,010,062. Preferably, however, the CM101 is purified
according to the method taught in International Application No.
PCT/US97/17535, incorporated herein by reference.
[0025] Starting material for isolating CM101 for use in the method
of the present invention may be obtained by culturing strains of
Group B .beta.-hemolytic Streptococcus bacteria that have recently
infected or are capable of infecting newborn infants. Isolates of
such strains may be obtained from the blood or cerebrospinal fluid
of infected infants.
[0026] GBS toxin as used herein is defined as any fraction or
component isolated from natural or lysed GBS bacteria, or derived
from media supernatants of lysed and/or autoclaved GBS bacteria,
and which has a biological activity evidenced by induction of
respiratory distress in the sheep assay (Hellerqvist, C. G. et al.,
Studies on group B .beta.-hemolytic streptococcus I. Isolation and
partial characterization of an extra-cellular toxin., Pediatr.
Res., 12:892-898 (1981)) or activation of complement and binding to
neovasculature as demonstrated by a peroxidase-antiperoxidase (PAP)
assay of a tumor tissue specimen (Hellerqvist, C. G. et al.,
Anti-tumor effects of GBS toxin: a polysaccharide exotoxin from
group B .beta.-hemolytic streptococcus, J. Canc. Res. Clin. Oncol.,
120:63-70 (1993); and Hellerqvist, C. G. et al., Early Results of a
Phase I Trial of CM101 in Cancer Patients., Proceedings of the
American Association of Cancer Research Annual Meeting, 36:224
(1995)). GBS toxin also means any natural or synthetic
polysaccharide with the same structure or function as any
GBS-derived molecule with the aforementioned activity.
[0027] Substantially pure GBS toxin means a preparation in which
GBS toxin is greater than 40% pure (e.g., present in a
concentration of at least about 40% by weight), preferably at least
approximately 60% pure, more preferably at least approximately 90%
pure, and most preferably at least approximately 95% pure. The
purity of GBS toxin is discussed in greater detail in International
Application No. PCT/US97/17535. The dosages described herein are
for 95% pure GBS toxin. Dosages of lower purity GBS toxin should be
altered accordingly.
[0028] One aspect of the present invention is a method of treating
a patient having a wound by administering a GBS toxin, e.g. CM101,
in an amount sufficient to reduce scarring and/or to accelerate
wound healing. Determination of the reduction of scarring and/or
the acceleration of healing may be performed by a variety of
methods including, but not limited to, visual observation,
measurement of vessel density at the site of the wound, e.g., by
magnetic resonance imaging, measurement of the amount and/or rate
at which granulation tissue is formed, and measurement of skin
tensile strength at the site of the wound.
[0029] The CM101 or other GBS toxin is preferably combined with a
pharmaceutically acceptable carrier and administered to a patient
systemically. The carrier is preferably one that is readily mixed
with CM101 to form a composition that is administrable by
intravenous (IV) means. Thus, the carrier is preferably saline,
which may have other pharmaceutically acceptable excipients
included to ensure its suitability for intravenous administration.
The resulting composition will be sterile and will have acceptable
osmotic properties. In general, a suitable IV formulation is
prepared in accordance with standard techniques known to one of
skill in the art. For example, Chapter 85 entitled "Intravenous
Admixtures" by Salvatore J. Turco in the Eighteenth Edition of
Remington's Pharmaceutical Sciences, Mach Publishing Co. (1990),
incorporated herein by reference, provides standard techniques for
preparing a pharmaceutically acceptable IV composition useful in
accordance with this invention. Other dosage forms to administer
CM101 may also be used. As an alternative to systemic
administration, CM101 may be administered locally to a wound site.
Administration of CM101 to the patient may occur before, during,
and/or after infliction of a wound by surgery or trauma.
Preferably, CM101 is administered within an appropriate temporal
window following the wounding. Administration of CM101 soon after
infliction of the wound is most preferred. For example,
administration within 1 day, or preferably within six hours is
best.
[0030] The amount of CM101 that is administered to a patient to
reduce scarring or accelerate the rate of wound healing is an
amount that is sufficient to reduce the amount or rate of
granulation tissue formation at a wound site, or that is sufficient
to reduce vascularization, and particularly vessel density, at a
wound site. A preferred dosage range is 1 to 100 .mu.g/kg body
weight. A more preferred dosage range, however, is 1 .mu.g/kg to 50
.mu.g/kg body weight, and most preferred is a dosage in the range
of 1 .mu.g/kg to 25 .mu.g/kg. It will be understood, however, that
the specific dose level for any particular patient will depend on a
variety of factors including the age, body weight, general health,
sex, diet, and severity of the wound. Each dosage is preferably
administered in an infusion of up to 120 minutes, with 5 to 60
minutes being the preferred duration range, and 5 to 30 minutes
being the most preferred dosage range. Once weekly treatment is
preferred, and is likely to be all that is necessary for evidence
of results.
[0031] CM101 treatment inhibits scarring and accelerates healing of
wounds in various types of tissues and is appropriate for wounds of
various depths. The treatment of the present invention is
preferably used in conjunction with suturing of muscle tissue or
some other known tissue manipulation for wounds which are deep or
in which the edges of the tissue are not close together. Scarring
at the level of the epidermis as well as internal scarring is
reduced by the method of the present invention. A scar as used
herein is an irregularity of the skin or other tissue formed from
connective tissue replacement of tissue, especially tissue damaged
by a wound process. An adhesion may also be considered a scar in
this context. A wound as used herein is injury or damage wherein
the skin or other tissue is adversely affected, particularly
wherein the skin or other tissue is torn, pierced, cut, or
broken.
[0032] Additionally, the present invention may be used to treat
reperfusion injuries due to surgical wounds or traumatized wounds.
For instance, blood flow to a part of the body may be temporarily
occluded, as by clamping, during a surgical procedure. After
removal of the occlusion, reperfusion into a previously ischemic
area may result in a reperfusion injury to the tissue, especially
inside the blood vessel. Reperfusion injury is a major contributor
to organ failure and organ rejection in transplant surgery. CM101
may be administered to the patient before occlusion to minimize
damage caused by ischemia and reperfusion. CM101 may also be
administered after the occlusion has occurred. Restenosis, often
resulting from scarring within a blood vessel that leads to
stricture of the vessel lumen, is also advantageously treated by
the method of the present invention. Further, the invention may be
used to minimize the likelihood of formation of adhesions, such as
the type which sometimes occurs as a result of abdominal surgery.
Clearly, the method may be used in specific types of surgery, such
as plastic or cosmetic surgery, implantation, reconstruction,
transplantation, bypass operations, and balloon angioplasty where
improved wound healing is critical and scarring is particularly
problematic. Many of these procedures are believed to involve
hypoxia-induced neovascularization or angiogenesis.
[0033] CM101 is especially useful in the treatment of patients
having keloids. Normally, surgery to excise these overgrown scars
is unsuccessful, because they continually return in a larger and
more unsightly form. The present invention teaches a method of
treatment whereby surgery to excise the keloid and create a fresh
wound is immediately followed by administration of CM101 to allow
for healing with minimal scarring.
[0034] As stated above, the stimulatory effect of wounds on tumors
is a recognized clinical phenomenon that is manifested as
accelerated growth of the residual tumor near the site of surgical
intervention, as well as an increased probability of metastatic
implantation at the site of surgery. The present invention also
includes administration of CM101 to surgery patients with tumors in
order to facilitate wound healing and to minimize metastatic
implantation and tumor proliferation. Treatment for wound healing
in surgery patients with tumors includes prevention or reduction of
the likelihood of occurrence, and reduction of any tumor that has
moved to the wound site.
[0035] Clearly, administration of CM101 pre-surgery to reduce
scarring and accelerate wound healing has great utility and will
favorably impact the medical community. Similarly, the
administration of CM101 to a patient having a wound resulting from
unexpected injury has great utility in facilitating healing of the
wound.
[0036] Without limitation to a particular theory, it is believed
that GBS toxin, and specifically CM101, plays an important role in
allowing wounds to heal at an accelerated rate and with minimal
scarring because it interferes with the hypoxia-induced,
VEGF-driven angiogenesis that results in tissue granulation and
scarring, but not with physiological blood vessel repair processes
which are necessary for the wound to heal. Angiogenesis is believed
to involve dedifferentiation of endothelial cells in response to
upregulation of VEGF under hypoxic conditions, ultimately leading
to the rapid formation of densely-sprouting capillaries. On the
other hand, physiological neovascularization is believed to be a
basic repair mechanism involving proliferation of existing
endothelial cells after the disruption of contact inhibition
resulting from infliction of a wound. Because CM101 allows
physiological repair mechanisms to proceed, but interferes with
pathological angiogenesis, it is an extremely useful compound for
the beneficial treatment of wounds. The further suggestion is that
CM101 opsonizes, by complement C3 activation, the budding capillary
sprout thereby inhibiting inflammatory angiogenesis necessary for
scarring. VEGF-driven angiogenesis is also believed to be at work
in reperfusion injury-type wounds, thus administration of CM101 is
effective in preventing such injuries.
[0037] Previous work by some of the inventors of the present
application utilized GBS toxin as an anticancer agent in the
treatment of tumors. Particularly, U.S. Pat. No. 5,010,062 to
Hellerqvist and the corresponding European Patent No. EP 0 445 280
B1 teach a method of at least partially inhibiting vascularization
of a developing solid tumor by parenterally administering to a
patient GBS toxin in an amount effective for inhibition.
[0038] The previous work led to investigation of the effect of GBS
toxin, and particularly CM101, on wound site vasculature. The
conclusion of the early investigation was that CM101 had no effect
on wound healing. Specifically, Quinn, T. E. et al., J. Cancer Res.
Clin. Oncol. 121:253-6 (1995) utilized a polyvinyl alcohol (PVA)
sponge implantation technique in mice as a model for wound healing
and a carmine dye infusion method to measure new vessel formation.
In that study, there was no significant difference in the level of
vasculature exhibited by mice treated with CM101 or control
Dextran. This was true for both normal and tumor-bearing mice.
These results indicated that CM101 had no significant effects on
the neovasculature of healing wounds as measured by the sponge
model. Thus, early studies of the effects of CM101 on wound healing
lead one away from the present invention.
[0039] Magnetic resonance imaging (MRI) is useful for visualizing
vascularization and providing a relative measure of vessel density.
This technique has been used to visualize wound-tumor interactions
in vivo in nude mice (Abramovitch R., Meir G. and Neeman M.,
Neovascularization induced growth of implanted C6 glioma
multicellular spheroids: magnetic resonance microimaging, Cancer
Res., 55:1956-1962 (1995)) and to demonstrate that wounds influence
tumor progression indirectly by stimulating tumor
neovascularization and directly by inducing tumor cell
proliferation. In the course of these observations, wounds located
at the site of a tumor did not heal.
[0040] Most antiangiogenic or antineovasculature therapies
(Broadley. K. N. et al., Lab. Investigation, 61(5):571-575 (1989);
Stout, A. J. et al., Int. J. Exp. Pathol., 74(1):79-85 (1993);
Pierce, G. F., Annu. Rev. Med., 46:467-481(1995)), with the
exception of CM101, inhibit wound healing and therefore cannot be
used to inhibit the stimulatory effect of surgery on tumor growth.
It was previously demonstrated that CM101 inhibits tumor
neovascularization, (U.S. Pat. No. 5,010,062) and previous
investigators concluded that CM101 has no significant effect on
neovascularization of wounds (Quinn T. E., Thurman G. B., Sundell
A. K., Zhang M., and Hellerqvist C. G., CM101, a polysaccharide
antitumor agent, does not inhibit wound healing in murine models,
J. Cancer Res. Clin. Oncol., 121:253-256 (1995)). The present
invention, by contrast, teaches that CM101 increases the rate of
wound healing, decreases scarring and further, in cancer patients,
acts to inhibit the proliferative effect of wounds on tumors.
[0041] Another aspect of the present invention is an article of
manufacture, such as a kit, and a method for making the article of
manufacture. The article includes a pharmaceutical composition
comprising a GBS toxin, and particularly CM101, and a
pharmaceutically acceptable carrier. The pharmaceutical composition
may be placed in a suitable container, as is well known in the art.
Also included are instructions for treatment of patients according
to the methods of the present invention.
[0042] The invention now being generally described may be better
understood by reference to the following examples, which are
presented for illustration only and are not to be construed as
limitations on the scope or spirit of the present invention.
EXAMPLES
Example 1
CM101 Facilitates Wound Healing in Tumor-Free Subjects
[0043] The effect of CM101 on wound healing was determined in CD-1
nude mice lacking tumors. The wounds were produced by fine surgical
scissors and consisted of 4 mm full thickness skin incisions. A
sterile adhesive bandage (Tegaderm.TM., USA) was used to cover each
wound.
[0044] On day 0 of the experiment, mice were intravenously
administered 240 .mu.g/kg CM101 or saline and wounded. New vessel
formation at each wound site was assessed using magnetic resonance
microimaging (MRI). Magnetic resonance images were obtained on days
0, 1, 2, 3, and 5.
[0045] MRI experiments were performed on a horizontal 4.7 T Biospec
spectrometer (Bruker, Germany) spectrometer using a 2 cm surface
radiofrequency coil. Gradient echo images (slice thickness of 0.5
mm, TR 100 ms, 256.times.256 pixels, in plane resolution of 110
.mu.m) were acquired with echo times of 10.5 and 20 ms. Growth of
the capillary bed was reflected by reduction of the mean intensity
at a region of interest of 1 mm surrounding the incision.
Angiogenic contrast (AC) was defined as the ratio of the mean
intensity at a region of interest of 1 mm surrounding the incision
to the mean intensity of a distant tissue. Apparent vessel density
is given as 1-AC. Each anesthetized mouse was then placed supine on
an MRI device with the incision site located at the center of the
surface coil, and MRI images were recorded for 1 hour.
[0046] FIG. 1 shows the vessel density measurements by MRI over the
time course of the experiment. As shown in FIG. 1, wounds in
saline-treated control mice exhibited intense neovascularization
near the wound. This neovascularization peaks on the second day
after injury. In comparison, CM101-treated mice have a severely
diminished level of neovascularization around the wounds.
Example 2
CM101 Facilitates Wound-Healing in Tumor-Bearing Subjects
[0047] The effect of CM101 on neovascularization of wounds was
tested using mice implanted with glioma spheroids. A single C6
glioma spheroid (about 800 .mu.m in diameter) was implanted
subcutaneously to the lower back of each of 6 male CD1-nude mice
(Abramovitch R., Meir G. and Neeman M., Neovascularization induced
growth of implanted C6 glioma multicellular spheroids: magnetic
resonance microimaging, Cancer Res., 55:1956-1962 (1995)).
[0048] Eight days later, on day 0 of the experiment, the mice were
anesthetized with a intraperitoneal injection of 75 .mu.g/g
Ketamine and 3 .mu.g/g Xylazine. The mice were injected through the
tail vein with physiological saline containing 0, 60, 120, or 240
.mu.g/kg CM101. Neovascularization at the wound site was assessed
using magnetic resonance microimaging (MRI), as described in
Example 1. Each anesthetized mouse was placed supine on the MRI
device with the tumor located at the center of the surface coil,
and MRI images were recorded for one hour.
[0049] After 1 hour of observation and MRI data recording, the mice
were wounded by a 4 mm cutaneous incision 5-10 mm from the tumor.
As before, the wounds were made through 4 mm full thickness of
tissue by fine surgical scissors and were covered with a sterile
adhesive bandage (Tegaderm.TM., USA). Subsequent MRI images of
neovascularization were taken on days 2, 5, 7, and 13.
[0050] Wound healing in mice with gliomas was improved by a single
injection of CM101. Mice treated with CM101 displayed approximately
six-fold reduced vessel density at the edges of the wound 2 days
after injury, as seen in FIG. 2. The initial wave of massive
neovascularization following wounding was inhibited in
CM101-treated mice, but this inhibition did not impair wound
healing. In fact, at 24 and 48 hours after injury, wounds were
almost undetectable externally in mice treated with CM101, whereas
control mice more clearly showed the wound. Furthermore, compared
to saline-treated control mice, CM101 treated, tumorbearing mice
had almost no detectable scar 13 days after the injury.
[0051] Thus, in tumor-bearing subjects treatment with CM101
severely diminishes the massive neovascularization that surrounds
the wound on days 1-2 after injury, accelerates wound healing, and
minimizes scarring.
Example 3
CM101 Treatment Promotes Improved Incision Healing
[0052] Visual observation of wounds also provides evidence of the
benefits of CM101 treatment. Two mice were wounded as in the
previous examples. CM101 was then administered intravenously at a
240 .mu.g/kg dosage in saline to one mouse. Saline was administered
to the control mouse using the same procedure.
[0053] Visual observation of the wound site 48 hours post-incision
and post-CM101 treatment confirmed that the wound site is barely
detectable. By contrast, a comparable wound site in the control
animal viewed at the same time point shows clear evidence of
wounding. The experimental animal also presented little evidence of
scarring as compared to the control animal at a later time
point.
Example 4
CM101 Accelerates Wound Healing
[0054] Wound disruption strength, or tissue tensile strength across
a wound, was evaluated with CD1-nude mice. Full thickness skin
incisions were made on the right lumbar region followed by
intravenous injection with CM101 at 30 and 60 .mu.g/kg or saline.
The force needed to disrupt the wound was measured at 40 hours and
7 days after incision. Animals treated with CM101 demonstrated
significant increases relative to controls in the strength of the
healing wounds.
[0055] Six millimeter incisions were made on day 0. Tensile
strength was measured day 2 and day 7 by applying vacuum
(Dimensional Analysis Systems, Inc.) over the wound and registering
the movement of two small dots of reflective material applied on
each side of the wound by a video camera emitting infrared light.
FIG. 3 shows that by 2 days, there is a measurable difference
between CM101 treated and control wounds. By 7 days, there is no
visible scar tissue in the CM101 treated mice and the tissue has
the strength of uninjured tissue. In contrast, untreated wounds
were 50% less strong than CM101 treated wounds.
Example 5
CM101 Reduces the Effect of Wound-Induced Tumor Progression
[0056] Twenty-four CD-1 nude mice carrying subcutaneous C6 glioma
tumors of at least 5 mm diameter are prepared.
[0057] The effects of CM101 on tumor proliferation and metastatic
implantation is tested using mice implanted with glioma spheroids.
A single C6 glioma spheroid (at least 5 mm in diameter) is
implanted subcutaneously to the lower back of each of 24 male
CD1-nude mice (Abramovitch R., Meir G. and Neeman M.,
Neovascularization induced growth of implanted C6 glioma
multicellular spheroids: magnetic resonance microimaging, Cancer
Res., 55:1956-1962 (1995)).
[0058] Eight days later, on day 0 of the experiment, one group of
mice (n=8) receive a 4 mm cutaneous incisional injury proximal to
the tumor (within 3 mm of the tumor's edge), a second group of mice
(n=8) receive a 4 mm cutaneous incisional injury at a location
remote from the tumor (more than 10 mm from the tumor's edge), and
a control group of mice (n=8) are left without a wound. On the same
day, the mice receive treatments of 0, 60, 120 or 240 .mu.g/kg
CM101 (in each group described above, two mice receive a particular
concentration of CM101).
[0059] For two weeks, tumor growth in situ, tumor implantation at
the site of wound, and neovascularization at the wound site are
assessed using magnetic resonance microimaging (MRI), as described
in Example 1. At the end of two weeks, the mice are sacrificed and
the tissue surrounding the tumor and wound are formalin-fixed for
histological analysis of wound healing and tumor morphology.
[0060] In animals with wounds proximal to tumors, CM101 treatment
reduces tumor proliferation into the site of the wound. Similarly,
CM101 inhibits metastatic tumor implantation at the wound site in
animals with tumors distant from wounds.
Example 6
CM101 is Effective Against Reperfusion Injury
[0061] The ability of CM101 to guard against reperfusion injury was
tested using established animal models (Han et al., Sialyl Lewis
Oligosaccharide Reduces Ischemia-Reperfusion Injury in the Rabbit
Ear, J. of Immunol., 155(8):4011-4015 (1995), Missawa et al., Role
of Sialyl Lewis.sup.x in Total Hepatic Ischemia and Reperfusion, J.
of the American College of Surgeons, 182:251-256 (1996),
Lopez-Neblina et al., Mechanism of Protection of Verapamil by
Preventing Neutrophil Infiltration in the Ischemic Rat Kidney, J.
Surg. Res., 61(2):469472 (1996). In both the ear model and the
kidney model, the CM101-treated organ exhibited significantly less
damage than did the untreated organ.
[0062] Mouse Ear Model: Balb/c mice were injected intravenously
with either phosphate-buffered saline (n=10) or 60 .mu.g/kg CM101
prepared in phosphatebuffered saline (n=10). Following injection,
in each mouse, the artery supplying blood to one ear was clamped
for a period of 60 minutes. At the end of the time period, the
clamped ear had lost color, showing the effectiveness of the
occlusion. After removal of the clamp, the ear of each mouse was
allowed to reperfuse normally. The mice were maintained at a stable
ambient temperature of 24.degree. C. throughout the experiment.
After six hours, the ears of all mice were visually examined
directly and under a microscope (100.times.). Measurements of
swelling were also performed using standard calipers.
[0063] The ear that had been subject to clamping was examined in
each saline-treated mouse and showed swelling and significant
vascular leakage indicated by hemorrhage and inflammatory
infiltration as compared to the other ear of the mouse which had
not been subject to clamping. By contrast, the reperfused ear of
each CM101-treated mouse showed no significant difference from the
unclamped control ear of the same mouse.
[0064] Mouse Kidney Model: Anesthetized Balb/c mice were opened and
the blood flow to one kidney was blocked by clamping. After
clamping of the one kidney, either phosphate-buffered saline
(Subgroup A) or 60 .mu.g/kg CM101 in phosphate-buffered saline
(Subgroup B) was infused intravenously through the tail vein of
each mouse. Five minutes after the infusion, the blood flow to the
other kidney of each animal was clamped. Both clamps were removed
from each mouse simultaneously after the designated occlusion time
period and reperfusion was allowed to proceed normally. A stable
ambient temperature of 24.degree. C. was maintained. After the
designated reperfusion period, each mouse was sacrificed and
examined. The first group of mice {6 saline-treated mice (Group 1A)
and 6 CM101-treated mice (Group 1B)} had the blood flow to their
kidneys occluded by clamping for 30 minutes and reperfusion was
allowed to proceed for 2 hours. The second group {6 saline-treated
mice (Group 2A) and 6 CM101-treated mice (Group 2B)} were subject
to a 45 minute occlusion period and a 5 hour reperfusion period.
The third group {6 saline-treated mice (Group 3A) and 6
CM101-treated mice (Group 3B)} was subject to occlusion for 60
minutes followed by reperfusion for 6 hours.
[0065] In the saline-treated mice, histological examination of the
saline-treated kidneys revealed signs of reperfusion damage,
showing greater injury with increased reperfusion time. Thus,
damage to capillaries was evident after 2 hours of reperfusion
(Group 1A) and evidence of angiogenesis, hemorrhage, and
inflammation was observed in the 6-hour reperfusion mice (Group
3A). Additionally, cell death was observed by 4 hours (Group 2A).
By contrast, at all time points the CM101-treated mice showed minor
dilation of blood vessels, but no hemorrhaging between blood
vessels, inflammation, or cell death. This demonstrates the
protective role of CM101 in ischemia and reperfusion injury.
[0066] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0067] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
* * * * *