PROMOTION DE LA CICATRISATION A L'AIDE DE STEROIDES AYANT DES EFFETS SECONDAIRES SYSTEMIQUES REDUITS COMPARATIVEMENT A CEUX HABITUELLEMENT ASSOCIES AUX GLUCOCORTICOIDES, AUX MINERALCORTICOIDES ET AUX STEROIDES SEXUELS

PROMOTION OF WOUND HEALING UTILIZING STEROIDS HAVING REDUCED DETERIOROUSSYSTEMIC SIDE EFFECTS TYPICAL OF GLUCOCORTICOIDS, MINERALOCORTICOIDS ANDSEX STEROIDS

Abrégé français

Composition pharmaceutique utilisée pour accroître la néovascularisation et l'angiogenèse durant la cicatrisation chez un mammifère par rapport au degré habituel de néovascularisation et d'angiogenèse en l'absence de traitement, cette composition contenant une quantité efficace de stéroïde angiostatique qui n'a que peu ou pas d'effets secondaires dommageables ou indésirables et une quantité efficace d'une forme d'hyaluronan comme l'acide hyaluronique et certains sels de cet acide qui ont une indication pharmaceutique.

Abrégé anglais

A pharmaceutical composition utilized for increasing
neovascularization and angiogenesis during wound healing in a mammal
beyond the level of neovascularization and angiogenesis which would occur at
the wound site without any treatment, said composition comprising an effective
amount of any angiostatic steroid which has reduced or no deteriorative or
detrimental side effects combined with an effective amount of a form of
hyaluronan such as hyaluronic acid and pharmaceutically acceptable salts
thereof.

Revendications

- 40 -
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE AS FOLLOWS:
1. A pharmaceutical composition for increasing neovascularization
and angiogenesis during wound healing in a mammal beyond the level of
neovascularization and angiogenesis which would occur at the wound site
without any treatment, said composition comprising an effective amount of an
angiostatic steroid combined with an effective amount of hyaluronan, such as
hyaluronic acid and a pharmaceutically acceptable salt thereof having a
molecular weight less than about 750,000 daltons, to promote neovascularization
and angiogenesis.
2. The composition of claim 1 wherein said composition also includes
a suitable diluent.
3. The composition of claim 1 wherein said composition is in a form
suitable to be administered nasally, vaginally, intraaurally, ocularly, rectally,
orally, locally or topically, intramuscularly, intermuscularly, transdermally and
intravenously.
4. The composition of claim 3 wherein the amounts of the steroid is
selected from between about .1-3mg of the steroid per kg of body weight and the
amount of hyaluronan is between about .1-20% by weight of the composition.
5. The composition of claim 4 wherein hyaluronan is 2.5% by weight
of the composition.
6. A method of treatment for increasing neovascularization and
angiogenesis during wound healing in a mammal beyond the level of

- 41 -
neovascularization and angiogenesis which would occur at the wound site
without any treatment, said method comprising application of a composition
including an effective amount of an angiostatic steroid combined with an
effective amount of a form of hyaluronic acid and/or pharmaceutically acceptablesalts thereof at the wound site, said application of the combination being for such
period as required wherein the molecular weight of the form of hyaluronic acid
has a molecular weight less than about 750,000 daltons.
7. The method of claim 6 wherein said composition also includes a
suitable diluent.
8. The method of claim 6 wherein the composition is applied or
administered nasally, rectally, vaginally, intraaurally, ocularly, orally, locally,
topically, intramuscularly, intermuscularly, transdermally and intravenously..
9. The method of claim 8 wherein the amounts of the steroid is
selected from between about .1-3mg of the steroid per kg of body weight and the
amount of hyaluronan is between about .1-20% by weight of the composition.
10. The method of claim 9 wherein hyaluronan is 2.5% by weight of the
composition.
11. The use of a pharmaceutical composition for increasing
neovascularization and angiogenesis during wound healing in a mammal
beyond the level of neovascularization and angiogenesis which would occur at
the wound site without any treatment, said use of said composition comprising
application of the composition containing an effective amount of an angiostatic
steroid combined with an effective amount of a form of hyaluronic acid and

- 42 -
pharmaceutically acceptable salts thereof, having a molecular weight less than
about 750,000 daltons at the wound site.
12. The use of claim 10 wherein said composition includes a suitable
diluent.
13. The use of claim 9 wherein the composition is applied or
administered nasally, vaginally, intraaurally, ocularly, rectally, orally, locally,
topically, intramuscularly, intermuscularly, transdermally and intravenously.
14. The use of claim 12 wherein the amounts of the steroid is selected
from between about .1-1mg of the steroid per kg of body weight and the amount
of hyaluronan is between about .1-20% by weight of the composition.
15. The use of claim 14 wherein the hyaluronan is 2.5% by weight of the
composition.
16. A composition for inducing angiogenesis in the healing of a
condition, such as a wound, said composition comprising hyaluronic acid in a
suitable form in combination with a suitable steroid known not to possess
detrimental systemic side effects such as bone mass loss, fluid retention,
infections and the like as is normally associated with glucocorticoids,
mineralocorticoids, and sex steroids, said suitable steroid being known not to
stimulate new blood vessel formation when utilized individually, wherein the
combination of hyaluronic acid and said suitable steroid (known to possess
reduced detrimental systemic side effects normally associated with
glucocorticoids, mineralocorticoids and sex steroids and which are normally
angiostatic and inhibit new blood vessel formation), stimulates new blood vesselformation and angiogenesis to accelerate scarless healing of a condition, the

- 43 -
molecular weight of the form of hyaluronic acid being less than about 750,000
daltons.
17. The composition of claim 16 wherein said suitable steroids are
angiostatic steroids.
18. The composition of claim 17 wherein said angiostatic steroids are
tetrahydro-steroids.
19. The composition of claim 16, 17, or 18 wherein the condition being
treated is a wound.
20. The composition of claim 17 wherein said suitable angiostatic
steroid is selected from tetrahydrocortisol (TH-F), tetrahydrocortisone (TH-E) and
tetrahydrocortexolone (TH-S) and combinations thereof.
21. The combination of an effective amount of a form of hyaluronic
acid selected from hyaluronic acid and pharmaceutically acceptable salts thereofwith a tetrahydro-steroid derivatives utilized for acceleration of wound healingby the stimulation of angiogenesis.
22. A process for the stimulation and/or induction of angiogenesis, in a
mammal (for example a human), the process comprising the steps of
administering at least one effective dosage amount of a pharmaceutical
composition for the stimulation and/or induction of angiogenesis to a site on/ina mammal in need of stimulation and/or induction of angiogenesis for the time
as required, each effective dosage amount of the composition comprising an
effective non-toxic dosage amount of a steroid known to have angiostatic
properties when utilized alone or in combination with known therapies and an

- 44 -
non-toxic effective dosage amount of hyaluronic acid and/or a pharmaceutically
acceptable salt thereof having a molecular weight less than about 750,000 daltons.
23. The process of claim 23 utilized for stimulation of blood vessel
growth in a condition such as a wound.
24. The process of claim 23 wherein said steroids known to have
angiostatic properties are angiostatic steroids.
25. The process of claim 24 wherein said angiostatic steroids are
tetrahydro-steroids.
26. The process of claim 24 wherein said angiostatic steroids are selected
from tetrahydrocortisone, tetrahydrocortisol or tetrahydrocortexolone.
27. The process of claim 22 wherein said effective non-toxic dosage
amount of hyaluronic acid is sodium hyaluronate.
28. The composition of claim 1 wherein the composition is in a form to
be administered topically in the form of a cream, lotion and/or gel for the
treatment of burns, ulcers, incisions or the like, nasally, vaginally, intraaurally,
ocularly or orally for the treatment of wounds therein or as a suppository for
hemorrhoids.
29. The method of claim 6 wherein the composition is administered in
a form topically as a cream, lotion and/or gel for the treatment of burns, ulcers,
incisions or the like, nasally, vaginally, intraaurally, ocularly or orally for the
treatment of wounds therein or as a suppository for hemorrhoids.

- 45 -
The use of claim 11 wherein the composition is administered in a
form topically as a cream, lotion and/or gel for the treatment of burns, ulcers,incisions or the like, nasally, vaginally, intraaurally, ocularly or orally for the
treatment of wounds therein or as a suppository for hemorrhoids.
31. The composition of claim 16 wherein the composition is
administered in a form topically as a cream, lotion and/or gel for the treatmentof burns, ulcers, incisions or the like, nasally, vaginally, intraaurally, ocularly or
orally for the treatment of wounds therein or as a suppository for hemorrhoids.
32. The process of claim 22 wherein the composition is administered in
a form topically as a cream, lotion and/or gel for the treatment of burns, ulcers,
incisions or the like, nasally, vaginally, intraaurally, ocularly or orally for the
treatment of wounds therein or as a suppository for hemorrhoids.
33. The use of a combination of a form of hyaluronic acid (for example,
hyaluronic acid and a pharmaceutically acceptable salt thereof, and a suitable
steroid known to have reduced systemic side effects, which are also known to be
normally angiostatic when administered in known compositions or solutions,
which in combination stimulate and induce angiogenesis and hence healing of a
condition such as a wound wherein the molecular weight of the form of
hyaluronic acid is less than about 750,000 daltons.
34. The use of claim 33 wherein said form of hyaluronic acid is sodium
hyaluronate.
35. The use of claim 33 wherein said suitable steroid is an angiostatic
steroids, for example tetrahydro-steroids.

- 46 -
36. The use of claim 35 wherein said angiostatic steroids are selected
from tetrahydrocortisol, tetrahydrocortisone and tetrahydrocortexolone and the
like known not to have the systemic side effects of glucocorticoids,
mineralocorticoids and sex steroids or the like.
37. The use of claim 33 wherein said composition is applied or
administered nasally, vaginally, intraaurally, ocularly, rectally, orally, locally or
topically, intramuscularly, intermuscularly, transdermally and intravenously..
38. An effective non-toxic dosage amount of a composition comprising
an effective non-toxic dosage amount of sodium hyaluronate having a molecular
weight less than about 750,000 daltons and a therapeutically effective non-toxicdosage amount of a known angiostatic steroid, used to stimulate and induce
angiogenesis.
39. The dosage amount of claim 38 wherein said angiostatic steroid is
selected from tetrahydro-steroids for example tetrahydrocortisol,
tetrahydrocortisone or tetrahydrocortexolone or the like.
40. The dosage of claim 38 wherein the amount of angiostatic steroid is
selected from between about .1-3mg of the steroid per kg of body weight and the
amount of hyaluronan is between about .1-20% by weight of the composition for
wounds in the order of 4 sq. cm..
41. A pharmaceutical composition (suitable for, topical application [on
the skin], rectally, vaginally, intraaurally, ocularly, locally, nasally, orally, on the
mucosa, etc.) (for example a multigram pharmaceutical composition for use
topically) effective for the stimulation and induction of angiogenesis, the
pharmaceutical composition containing a plurality of dosage amounts for

- 47 -
stimulating and inducing angiogenesis, each of said dosage amounts comprising
a therapeutically effective non-toxic (to the patient) dosage amount of a steroid
known to be angiostatic (for example tetrahydro-steroids) and an effective
non-toxic dosage amount of the hyaluronic acid and/or pharmaceutically acceptable
salts thereof to stimulate and induce angiogenesis, the molecular weight of the
form of hyaluronic acid being less than about 750,000 daltons.
42. The composition of claim 1, 16, or 41 further comprising suitable
excipients depending upon the route of administration for example excipients to
make a gel, lotion, spray, ointment, suppository, or cream (topical
administration).
43. The method of claim 6 wherein said composition further comprises
suitable excipients depending upon the route of administration for example
excipients to make a gel, lotion, spray, ointment, suppository, or cream (topical
administration).
44. The use of claim 11 or 33 wherein said composition further
comprises suitable excipients depending upon the route of administration for
example excipients to make a gel, lotion, spray, ointment, suppository, or cream(topical administration).
45. The process of claim 22 wherein said composition further comprises
suitable excipients depending upon the route of administration for example
excipients to make a gel, lotion, spray, ointment, suppository, or cream (topical
administration).
46. The dosage of claim 38 wherein said composition further comprises
suitable excipients depending upon the route of administration for example

- 48 -
excipients to make a gel, lotion, spray, ointment, suppository, or cream (topical
administration).
47. A dosage amount of a pharmaceutical composition for stimulating
and inducing angiogenesis at a wound site, the composition comprising:
(1) a known angiostatic steroid agent known not to have
systemic side effects for example tetrahydro-steroids; and
(2) hyaluronic acid and/or pharmaceutically acceptable salts thereof (for
example sodium hyaluronate) having a molecular weight less than about 750,000
daltons characterized in that said composition:
(a) is in a dosage form (e.g. in a cream, lotion, gel, ointment,
spray, or solution, etc.) which is suitable for administration at the wound site;
and
(b) is in such an amount and in such form that each of
component (1) and (2) are in an effective dosage amount together to
stimulate and induce angiogenesis (for example in scarless wound healing).
48. The dosage form of claim 47 wherein the pharmaceutical
composition may further comprise a plurality of dosage amounts.
49. The use of
(1) a known angiostatic steroid agent known not to have
systemic side effects typical of glucocorticoids, mineralocorticoids or sex steroids
for example tetrahydro-steroids; and
(2) hyaluronic acid and/or pharmaceutically acceptable salts
thereof (for example sodium hyaluronate) having a molecular weight less than
about 750,000 daltons,
in the manufacture of a pharmaceutical composition for use to stimulate and
induce angiogenesis in mammals (for example in humans) at a wound site,

- 49 -
wherein dosage amounts may be taken from the composition and each dosage
amount taken comprises:
a therapeutically effective non-toxic dosage amount of each of
components (1) and (2) to stimulate and induce angiogenesis at said wound site.
50. A method of treatment comprising forms of hyaluronic acid (for
example sodium hyaluronate) having a molecular weight less than about 750,000
daltons with suitable angiostatic steroids known to have reduced systemic side
effects typical of glucocorticoids, mineralocorticoids and sex steroids.
51. A pharmaceutical composition comprising forms of hyaluronic acid
(for example sodium hyaluronate) having a molecular weight less than about
750,000 daltons with suitable angiostatic steroids known to have reduced
systemic side effects typical of glucocorticoids, mineralocorticoids and sex
steroids.
52. A dosage amount of a composition comprising forms of hyaluronic
acid (for example sodium hyaluronate) having a molecular weight less than
about 750,000 daltons with suitable angiostatic steroids known to have reduced
systemic side effects typical of glucocorticoids, mineralocorticoids and sex
steroids.
53. The use of a composition comprising forms of hyaluronic acid (for
example sodium hyaluronate) having a molecular weight less than about 750,000
daltons with suitable angiostatic steroids known to have reduced systemic side
effects typical of glucocorticoids, mineralocorticoids and sex steroids.
54. The dosage of any previous dosage claim wherein the angiostatic
steroid is selected from:

- 50 -
tetrahydrocortisol 5-pregnane-3,11,17,21-tetrol-20-one
tetrahydrocortisone 5-pregnane-3,17,21-triol-11,20-dione
17-hydroxyprogesterone 4-pregnene-17-ol-3,20-dione
medroxyprogesterone 6-methyl-5-pregnene-17-ol-3,20-dione
6,6'-dehydromedroxyprogesterone 6-methylene-5-pregnene-17-ol-3,20-dione
megestrol 17-hydroxy-6-methyl-4,6-pregnadiene-
3,20-dione
1-dehydro-megestrol 17-hydroxy-6-methyl-1,4,6-
pregnatriene-3,20-dione
melengestrol 17-methylene-6-methyl-4,6-pregnadiene-
3,20-dione
1-dehydro-melengestrol 17-methylene-6-methyl-1,4,6-
pregnatriene-3,20-dione
2-methoxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol-2-
methyl ether
2-hydroxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one
4-methoxyoestradiol 1,3,5(10)-oestratriene-3,4,17-triol-4-
methyl ether
2-methoxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-2,3-
dimethyl ether
2-methoxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one-
2-methyl ether
2-hydroxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol
2-methoxyoestriol 1,3,5(10)-oestratriene-2,3,16,17-tetrol-
2-methyl ether
2-hydroxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-3-
methyl ether

- 51 -
Definite angiogenesis action was obtained utilizing tetrahydrocortisol where R1
= -OH and tetrahydrocortisone where R1 = =O
<IMG>
The following chart indicates the various predictable forms that steroids
should take:
R1 R2 R3 R4 R5 R6
Tetrahydrocortexolone <IMG>
17 Hydroxyprogesterone <IMG>
<IMG>
Further positive results are also predicted for acetate derivatives
<IMG>
possessing either
<IMG>
or
or hemissuccinate derivatives
<IMG>

-52-
<IMG>
The following chart indicates further acetate derivatives which may be
effective:
R1 R2 R3
tetrahydrocortisol 17-acetate <IMG>
tetrahydrocortisone 17-acetate <IMG>
tetrahydrocortisol 17 <IMG>
hemisuccinate
tetrahydrocortisone 17 <IMG>
hemisuccinate
<IMG>
tetrahydrocortisol 21 acetate <IMG>
tetrahydrocortisone 21 acetate <IMG>
tetrahydrocortisone 21 <IMG>
hemisuccinate
tetrahydrocortisol 21 <IMG>
hemisuccinate

- 53 -
<IMG>
The following chart indicates further various predictable forms for
applicable steroids:
R1 R2 R3 R4 R5 R6 R7
Medroxyprogesterone <IMG>
6:6'- dehydro- <IMG>
medroxyprogesterone
Megestrol <IMG>
1-dehydro- <IMG>
medroxyprogesterone
Melengestrol <IMG>
1-dehydro melengestrol <IMG>
<IMG>
and also 17-OH or 17-H or the following derivatives:
20~CH2OH
or

- 54 -
<IMG>
or <IMG>
or <IMG>
All steroids conjugated at the 20 position (R1) were applicable (i.e. not the
oestrogens)
<IMG>
or
<IMG>
All steroids conjugated at the 3 position (R1) with

- 55 -
<IMG>
heparin
adipic
hydrazide
or
<IMG>
heparin
hydrazide
The following chart indicates further various other acceptable steroidsutilized:
R1 R2 R3 R4 R5
2-methoxyoestradiol <IMG>
2-hydroxyoestrone <IMG>
4-methoxyoestradiol <IMG>
2-methoxyoestradiol <IMG>
3-methyl ether
2-methoxyoestrone <IMG>

- 56 -
2-hydroxyoestradiol <IMG>
2-methoxyoestriol <IMG>
2 Hydroxyoestradiol <IMG>
3-methyl ether
<IMG>
Known Structure of angiostatic steroids.
TETRAHYDROCORTISOL (5.beta.-pregnane-3.alpha., 11.beta., 17.alpha., 21-tetrol-20-one;
Tetrahydro-F)
<IMG>

- 57 -
TETRAHYDROCORTISONE (5.beta.-pregnane-3.alpha., 17.alpha., 21-triol-11, 20-dione;
Tetrahydro-E)
<IMG>
TETRAHYDROXYCORTEXOLONE (5.beta.-pregnane-3.alpha., 17.alpha., 21-triol-20-one;
Tetrahydro-S; 11-deoxytetrahydrocortisol)
<IMG>
55. The use of any previous use claim wherein the angiostatic steroid is
selected from:
tetrahydrocortisol 5-pregnane-3,11,17,21 -tetrol-20-one
tetrahydrocortisone 5-pregnane-3,17,21 -triol- 11,20-dione
17-hydroxyprogesterone 4-pregnene- 17 -ol- 3,20-dione
medroxyprogesterone 6-methyl-5-pregnene-17-ol-3,20-dione
6,6'-dehydromedroxyprogesterone 6-methylene-5-pregnene-17-ol-3,20-
dione
megestrol 17-hydroxy-6-methyl-4,6-pregnadiene-
3,20-dione
1-dehydro-megestrol 17-hydroxy-6-methyl-1,4,6-
pregnatriene-3,20-dione

- 58 -
melengestrol 17-methylene-6-methyl-4,6-pregnadiene-
3,20-dione
1-dehydro-melengestrol 17-methylene-6-methyl-1,4,6-
pregnatriene-3,20-dione
2-methoxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol-2-
methyl ether
2-hydroxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one
4-methoxyoestradiol 1,3,5(10)-oestratriene-3,4,17-triol-4-
methyl ether
2-methoxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-2,3-
dimethyl ether
2-methoxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one-
2-methyl ether
2-hydroxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol
2-methoxyoestriol 1,3,5(10)-oestratriene-2,3,16,17-tetrol-
2-methyl ether
2-hydroxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-3-
methyl ether
Definite angiogenesis action was obtained utilizing tetrahydrocortisol where R1
= -OH and tetrahydrocortisone where R1 = =O
<IMG>

- 59 -
The following chart indicates the various predictable forms that steroids
should take:
R1 R2 R3 R4 R5 R6
Tetrahydrocortexolone <IMG>
17 Hydroxyprogesterone <IMG>
<IMG>
Further positive results are also predicted for acetate derivatives
<IMG>
possessing either
or <IMG>
or hemissuccinate derivatives
<IMG>
<IMG>
The following chart indicates further acetate derivatives which may be
effective:

- 60 -
R1 R2 R3
tetrahydrocortisol 17-acetate <IMG>
tetrahydrocortisone 17-acetate <IMG>
tetrahydrocortisol 17 <IMG>
hemisuccinate
tetrahydrocortisone 17 <IMG>
hemisuccinate
tetrahydrocortisol 21 acetate <IMG>
tetrahydrocortisone 21 acetate <IMG>
tetrahydrocortisone 21 <IMG>
hemisuccinate
tetrahydrocortisol 21 <IMG>
<IMG>
The following chart indicates further various predictable forms for
applicable steroids:
R1 R2 R3 R4 R5 R6 R7
Medroxyprogesterone <IMG>
6:6-dehydro- <IMG>

- 61 -
medroxyprogesterone
Megestrol <IMG>
1-dehydro-
medroxyprogesterone <IMG>
Melengestrol <IMG>
1-dehydro melengestrol <IMG> =CH2
<IMG>
and also 17-OH or 17-H or the following derivatives:
<IMG>
or
<IMG>
or
<IMG>
or
<IMG>
All steroids conjugated at the 20 position (R1) were applicable (i.e. not the
oestrogens)

- 62 -
<IMG>
or
<IMG>
All steroids conjugated at the 3 position (R1) with
<IMG>
heparin
adipic
hydrazide
or

- 63 -
<IMG>
The following chart indicates further various other acceptable steroids
utilized:
R1 R2 R3 R4 R5
2-methoxyoestradiol <IMG>
2-hydroxyoestrone <IMG>
4-methoxyoestradiol <IMG>
2-methoxyoestradiol <IMG>
3-methyl ether
2-methoxyoestrone <IMG>
2-hydroxyoestradiol <IMG>
2-methoxyoestriol <IMG>
2 Hydroxyoestradiol <IMG>
3-methyl ether
<IMG>
Known Structure of angiostatic steroids.

- 64 -
TETRAHYDROCORTISOL (5.beta.-pregnane-3.alpha., 11.beta., 17.alpha., 21-tetrol-20-one;
Tetrahydro-F)
<IMG>
TETRAHYDROCORTISONE (5.beta.-pregnane-3.alpha., 17.alpha., 21-triol-11,
20-dione; Tetrahydro-E)
<IMG>
TETRAHYDROXYCORTEXOLONE (5.beta.-pregnane-3.alpha., 17.alpha., 21-triol-20-one;
Tetrahydro-S; 11-deoxytetrahydrocortisol)
<IMG>
56. The composition of any previous composition claim wherein the
angiostatic steroid is selected from:
tetrahydrocortisol 5-pregnane-3,11,17,21-tetrol-20-one

- 65 -
tetrahydrocortisone 5-pregnane-3,17,21-triol-11,20-dione
17-hydroxyprogesterone 4-pregnene-17-ol-3,20-dione
medroxyprogesterone 6-methyl-5-pregnene-17-ol-3,20-dione
6,6'-dehydromedroxyprogesterone 6-methylene-5-pregnene-17-ol-3,20-
dione
megestrol 17-hydroxy-6-methyl-4,6-pregnadiene-
3,20-dione
1-dehydro-megestrol 17-hydroxy-6-methyl-1,4,6-
pregnatriene-3,20-dione
melengestrol 17-methylene-6-methyl-4,6-pregnadiene-
3,20-dione
1-dehydro-melengestrol 17-methylene-6-methyl-1,4,6-
pregnatriene-3,20-dione
2-methoxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol-2-
methyl ether
2-hydroxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one
4-methoxyoestradiol 1,3,5(10)-oestratriene-3,4,17-triol-4-
methyl ether
2-methoxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-2,3-
dimethyl ether
2-methoxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one-
2-methyl ether
2-hydroxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol
2-methoxyoestriol 1,3,5 (10)-oestratriene-2,3,16,17-tetrol-
2-methyl ether
2-hydroxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-3-
methyl ether

- 66 -
Definite angiogenesis action was obtained utilizing tetrahydrocortisol where R1
= -OH and tetrahydrocortisone where R1 = =O
<IMG>
The following chart indicates the various predictable forms that steroids
should take:
R1 R2 R3 R4 R5 R6
Tetrahydrocortexolone <IMG>
17 Hydroxyprogesterone <IMG>
<IMG>
Further positive results are also predicted for acetate derivatives
<IMG>
possessing either
<IMG>
or
or hemissuccinate derivatives
<IMG>

- 67 -
<IMG>
The following chart indicates further acetate derivatives which may be
effective:
R1 R2 R3
tetrahydrocortisol 17-acetate <IMG>
tetrahydrocortisone 17-acetate <IMG>
tetrahydrocortisol 17 <IMG>
hemisuccinate
tetrahydrocortisone 17 <IMG>
hemisuccinate
tetrahydrocortisol 21 acetate <IMG>
tetrahydrocortisone 21 acetate <IMG>
tetrahydrocortisone 21 <IMG>
hemisuccinate
tetrahydrocortisol 21 <IMG>
hernisuccinate
<IMG>

- 68 -
The following chart indicates further various predictable forms for
applicable steroids:
R1 R2 R3 R4 R5 R6 R7
Medroxyprogesterone <IMG>
6:6'-dehydro- <IMG>
medroxyprogesterone
<IMG>
Megestrol
1-dehydro- <IMG>
medroxyprogesterone
Melengestrol <IMG> =CH~
1-dehydro melengestrol <IMG> =CH~
<IMG>
and also 17-OH or 17-H or the following derivatives:
20~CH2OH
or
<IMG>
or
<IMG>
or

- 69 -
<IMG>
All steroids conjugated at the 20 position (R1) were applicable (i.e. not the
oestrogens)
<IMG>
or
<IMG>
All steroids conjugated at the 3 position (R1) with

- 70 -
<IMG>
or
<IMG>
The following chart indicates further various other acceptable steroidsutilized:
R1 R2 R3 R4 R5
2-methoxyoe stradiol <IMG>
2-hydroxyoestrone <IMG>
4-methoxyoestradiol <IMG>
2-methoxyoestradiol <IMG>
3-methyl ether
2-methoxyoestrone <IMG>

- 71 -
2-hydroxyoestradiol <IMG>
2-methoxyoestriol <IMG>
2 Hydroxyoestradiol <IMG>
3-methyl ether
<IMG>
Known Structure of angiostatic steroids.
TETRAHYDROCORTISOL (5.beta.-pregnane-3.alpha., 11.beta., 17.alpha., 21-tetrol-20-one;
Tetrahydro-F)
<IMG>
TETRAHYDROCORTISONE (5.beta.-pregnane-3.alpha., 17.alpha., 21-triol-11,
20-dione; Tetrahydro-E)
<IMG>

- 72 -
TETRAHYDROXYCORTEXOLONE (5.beta.-pregnane-3.alpha., 17.alpha., 21-triol-20-one;
Tetrahydro-S; 11-deoxytetrahydrocortisol)
<IMG>
57. The method of any previous method claim wherein the angiostatic
steroid is selected from:
tetrahydrocortisol 5-pregnane-3,11,17,21-tetrol-20-one
tetrahydrocortisone 5-pregnane-3,17,21-triol-11,20-dione
17-hydroxyprogesterone 4-pregnene-17-ol-3,20-dione
medroxyprogesterone 6-methyl-5-pregnene-17-ol-3,20-dione
6,6'-dehydromedroxyprogesterone 6-methylene-5-pregnene-17-ol-3,20-
dione
megestrol 17-hydroxy-6-methyl-4,6-pregnadiene-
3,20-dione
1 -dehydro-megestrol 17-hydroxy-6-methyl-1,4,6-
pregnatriene-3 ,20-dione
melengestrol 17-methylene-6-methyl-4,6-pregnadiene-
3,20-dione
1 -dehydro-melengestrol 17-methylene-6-methyl-1,4,6-
pregnatriene-3,20-dione
2-methoxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol-2-
methyl ether
2-hydroxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one

- 73 -
4-methoxyoestradiol 1,3,5(10)-oestratriene-3,4,17-triol-4-
methyl ether
2-methoxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-2,3-
dimethyl ether
2-methoxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one-
2-methyl ether
2-hydroxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol
2-methoxyoestriol 1,3,5(10)-oestratriene-2,3,16,17-tetrol-
2-methyl ether
2-hydroxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-3-
methyl ether
Definite angiogenesis action was obtained utilizing tetrahydrocortisol where R1
= -OH and tetrahydrocortisone where R1 = =O
<IMG>
The following chart indicates the various predictable forms that steroids
should take:
R1 R2 R3 R4 R5 R6
Tetrahydrocortexolone <IMG>
17 Hydroxyprogesterone <IMG>

- 74 -
<IMG>
Further positive results are also predicted for acetate derivatives
possessing either <IMG>
or <IMG>
or hemissuccinate derivatives
<IMG>
<IMG>
The following chart indicates further acetate derivatives which may be
effective:
R1 R2 R3
<IMG>
tetrahydrocortisol 17-acetate
tetrahydrocortisone 17-acetate <IMG>
tetrahydrocortisol 17 <IMG>
hemisuccinate
tetrahydrocortisone 17 <IMG>
hemisuccinate

- 75 -
tetrahydrocortisol 21 acetate <IMG>
tetrahydrocortisone 21 acetate <IMG>
tetrahydrocortisone 21 <IMG>
hemisuccinate
tetrahydrocortisol 21 <IMG>
hemisuccinate
<IMG>
The following chart indicates further various predictable forms for
applicable steroids:
R1 R2 R3 R4 R5 R6 R7
Medroxyprogesterone <IMG>
6:6-dehydro- <IMG>
medroxyprogesterone
<IMG>
Megestrol
1-dehydro- <IMG>
medroxyprogesterone
Melengestrol <IMG>
1-dehydro melengestrol <IMG>

- 76 -
<IMG>
and also 17-OH or 17-H or the following derivatives:
<IMG>
or
<IMG>
or
<IMG>
or
<IMG>
All steroids conjugated at the 20 position (R1) were applicable (i.e. not the
oestrogens)
<IMG>
or

- 77 -
<IMG>
All steroids conjugated at the 3 position (R1) with
<IMG>
heparin
adipic
hydrazide
or
<IMG>
heparin
hydrazide

- 78 -
The following chart indicates further various other acceptable steroids
utilized:
R1 R2 R3 R4 R5
2-methoxyoestradiol <IMG>
2-hydroxyoestrone <IMG>
4-methoxyoe stradiol <IMG>
2-methoxyoestradiol <IMG>
3-methyl ether
2-methoxyoestrone <IMG>
2-hydroxyoestradiol <IMG>
2-methoxyoestriol <IMG>
2 Hydroxyoestradiol <IMG>
3-methyl ether
<IMG>
Known Structure of angiostatic steroids.
TETRAHYDROCORTISOL (5.beta.-pregnane-3.alpha., 11.beta., 17.alpha., 21-tetrol-20-one;
Tetrahydro-F)
<IMG>

- 79 -
TETRAHYDROCORTISONE (5.beta.-pregnane-3.alpha., 17.alpha., 21-triol-11,
20-dione; Tetrahydro-E)
<IMG>
TETRAHYDROXYCORTEXOLONE (5.beta.-pregnane-3.alpha., 17.alpha., 21-triol-20-one;
Tetrahydro-S; 11-deoxytetrahydrocortisol)
<IMG>
58. The process of any previous process claim wherein the angiostatic
steroid is selected from:
tetrahydrocortisol 5-pregnane-3,11,17,21-tetrol-20-one
tetrahydrocortisone 5-pregnane-3,17,21-triol-11,20-dione
17-hydroxyprogesterone 4-pregnene-17-ol-3,20-dione
medroxyprogesterone 6-methyl-5-pregnene-17-ol-3,20-dione
6,6'-dehydromedroxyprogesterone 6-methylene-5-pregnene-17-ol-3,20-
dione
megestrol 17-hydroxy-6-methyl-4,6-pregnadiene-
3,20-dione

- 80 -
1-dehydro-megestrol 17-hydroxy-6-methyl-1,4,6-
pregnatriene-3,20-dione
melengestrol 17-methylene-6-methyl-4,6-pregnadiene-
3,20-dione
1-dehydro-melengestrol 17-methylene-6-methyl-1,4,6-
pregnatriene-3,20-dione
2-methoxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol-2-
methyl ether
2-hydroxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one
4-methoxyoestradiol 1,3,5(10)-oestratriene-3,4,17-triol-4-
methyl ether
2-methoxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-2,3-
dimethyl ether
2-methoxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one-
2-methyl ether
2-hydroxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol
2-methoxyoestriol 1,3,5(10)-oestratriene-2,3,16,17-tetrol-
2-methyl ether
2-hydroxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-3-
methyl ether
Definite angiogenesis action was obtained utilizing tetrahydrocortisol where R1
= -OH and tetrahydrocortisone where R1 = =O
<IMG>

- 81 -
The following chart indicates the various predictable forms that steroids
should take:
R1 R2 R3 R4 R5 R6
Tetrahydrocortexolone <IMG>
17 Hydroxyprogesterone <IMG>
<IMG>
Further positive results are also predicted for acetate derivatives
<IMG>
possessing either
<IMG>
or
or hemissuccinate derivatives
<IMG>
<IMG>
The following chart indicates further acetate derivatives which may be
effective:

- 82 -
R1 R2 R3
tetrahydrocortisol 17-acetate <IMG>
tetrahydrocortisone 17-acetate <IMG>
tetrahydrocortisol 17 <IMG>
hemisuccinate
tetrahydrocortisone 17 <IMG>
hemisuccinate
tetrahydrocortisol 21 acetate <IMG>
tetrahydrocortisone 21 acetate <IMG>
tetrahydrocortisone 21 <IMG>
hemisuccinate
tetrahydrocortisol 21 <IMG>
hemisuccinate
<IMG>
The following chart indicates further various predictable forms for
applicable steroids:
<IMG>

- 83 -
medroxyprogesterone
Megestrol <IMG>
1-dehydro- <IMG>
medroxyprogesterone
Melengestrol <IMG>
1-dehydro melengestrol <IMG>
<IMG>
and also 17-OH or 17-H or the following derivatives:
20~CH2OH
or
<IMG>
or
<IMG>
or
<IMG>
All steroids conjugated at the 20 position (R1) were applicable (i.e. not the
oestrogens)

- 84 -
<IMG>
or
<IMG>
All steroids conjugated at the 3 position (R1) with
<IMG>
or

- 85 -
<IMG>
The following chart indicates further various other acceptable steroids
utilized:
<IMG>

- 86-
<IMG>
Known Structure of angiostatic steroids.
TETRAHYDROCORTISOL (5.beta.-pregnane-3.alpha., 11.beta., 17.alpha., 21-tetrol-20-one;
Tetrahydro-F)
<IMG>
TETRAHYDROCORTISONE (5.beta.-pregnane-3.alpha., 17.alpha., 21-triol-11,
20-dione; Tetrahydro-E)
<IMG>

- 87 -
TETRAHYDROXYCORTEXOLONE (5.beta.-pregnane-3.alpha., 17.alpha., 21-triol-20-one;
Tetrahydro-S; 11-deoxytetrahydrocortisol)
<IMG>

Description

CA 02208916 1997-07-03
TITLE OF INVENTION
Promotion of wound healing utilizing steroids having reduced
deteriorous systemic side effects typical of glucocorticoids, mineralocorticoids and
sex steroids.
5 FIELD OF INVENTION
This invention relates to the use of angiostatic steroids, which are
known to have reduced or no systemic side effects typical of glucocorticoids,
mineralocorticoids and sex steroids, for the promotion of angiogenesis and thus
wound healing when used in combination with hyaluronan (for example
10 hyaluronan and pharmaceutically acceptable salts thereof). This invention in
one particular application may be used in creams, lotions, sprays, suppositories,
and gels and the like for application in wound healing for delivery of for
example an effective amount of tetrahydro steroids in combination with an
effective amount of hyaluronic acid to the wound site, for example to treat
15 incisions, burns, skin lesions, and ulcers. Equally appropriate applications would
be administration of the invention intradermally, transdermally,
intermuscularly, intramuscularly and intravenously when appropriate.
It is well documented that tetrahydro steroids such as
tetrahydrocortisol (TH-F), tetrahydrocortisone (TH-E) and tetrahydrocortexolone
20 (TH-S) are known not to possess the detrimental systemic side effects normally
associated with glucocorticoids, mineralocorticoids and sex steroids since they do
not bind to appropriate receptors such as do known glucocorticoids,
mineralocorticoids and sex steroids. Typically, the continued use of
glucocorticoids, mineralocorticoids and sex steroids for chronic ailments may
25 result in a number of well known documented side effects such as bone wasting,
fluid retention and infections. Therefore, these glucocorticoid steroids,
mineralocorticoids and sex steroids are normally not recommended for a chronic
program of treatment for patients. Thus for wound healing for chronic type
wounds such as dermatitis, skin ulcers, hemorrhoids, and the like although they

CA 02208916 1997-07-03
--2 -
would benefit from the use of these aforementioned steroids, the expected
systemic side effects would materialize.
The tetrahydra steroids are themselves angiostatic steroids. Other
steroids are also angiostatic or anti-angiogenic steroids. They are known not to5 possess detrimental systemic side effects normally associated with
glucocorticoids, mineralocorticoids and sex steroids. These angiostatic steroidshave therefore very much reduced detrimental side effects (and in some
instances are known not to possess these side effects). These angiostatic steroids
are also normally associated with the inhibition of new blood vessel formation.
10 For a discussion of the various research and use of angiostatic steroids, the reader
is referred to the following listing of patents, patent applications and technical
articles which are representative only and are not in any way implied to be an
exhaustive listing. Throughout this disclosure, "angiostatic steroids" or the like
are to be assumed to mean those steroids not possessing significant angiogenetic15 properties.

CA 02208916 1997-07-03
.,
- 3 -
Patents and Patent Applications
Country Patent No. Title of Invention Inventors Assignee
US 4,771,042 Inhibition of John M. Braughler; The Upjohn
Angiogenesis Edward D. Hall, both Company,
Involving the of Portage; John M. K~l~m~oo,
Coadministration of McCall, Kalamazoo; Mich.
Steroids with Wendell Wierenga,
Heparin or Heparin Oshtemo Township,
Fragments Kalamazoo County,
Mich.; Judah
Folkman, Brookline,
Mass.
US 4,975,537 Angiostatic Steroids Paul A. Aristoff, The Upjohn
Portage; Harvey I. Company,
Skulnick; Wendell ~ m~zoo,
Wierenga, both of Mich.
m~i700, all of
Mich.
US 5,336,767 Total or Partial Francesco della Fidia, S.p.A.,
Esters of Hyaluronic Valle, Padova; AbanoTerme,
Acid Aurelio Romeo, Italy
Rome, both of Italy

CA 02208916 1997-07-03
US 5,506,354 Imidazolylpipera- John M. McCall; The Upjohn
zinylSteroids Donald E. Ayer, Company,
both of ~alama700; Kalamazoo,
E. Jon Jacobsen, Mich.
Plainwell; Frederick
J. VanDoornik,
Hamilton; John R.
Palmer; Harold A.
Karnes, both of
Kalamazoo, all of
Mich.
PCT WO Tetrahydro Paul Aristoff, TheUpjohn
87/02672 Angiostatic Steroids Harvey I. Skulnick, Company Wendell Wierenga
PCT WO Topical Anti- J. Holland, Duane B. The Upjohn
90/12577 Angiogenic as Hair Lakings Company
Growth Inhibitors
PCT WO SuraminType Paul A. Aristoff, The Upjohn
90/15816 Compounds and Mark A. Mitchell, Company
Angiostatic Steroids John W. Wilks
to ~hibit
Angiogenesis
PCT WO Steroids which John Wilks, Thomas The Upjohn
91/19731 lnhibitAngiogenesis FrarlkDekorling, Company
Paul Adrian
Aristoff

CA 02208916 1997-07-03
PCT WO Pharmaceutical Alberto Perbellini,
94/17~40 Compositions Riccardo Gabriele
Comprising a Ferretti, Franco
Spongy Material Dorigatti, Lanfranco
Consisting of Ester Callegaro
Derivatives of
Hyaluronic Acid
Combined with
Other Pharmacolo-
gically Active
Substances
EP 0 221 705 Tetrahydro Paul A. Aristoff, The Upjohn
Angiostatic Steroids Harvey I. Skulnick, Company
Wendell Wierenga
Journal Articles
1. Opal Ka, C.J. et al., Synthesis (1995): 766-8.
2. Cockerill, G.W. et al., International Review of Cytology (1995) 159: 113-60.
3. Diaz-Flores, L. et al., Histology and Histopathology (1994 Oct) 9(4): 807-43.
4. Sipos, E.P. et al, Annals of the New York Academy of Sciences (1994 Sept 6);
732: 263-72.
5. Thorpe, P.E. et al., Cancer Research (1993 Jul 1) 53(13): 3000-7.
6. Folkman, J. and Ingber, D., Seminars in Cancer Biology (1992 Apr) 3(2): 89-96.
7. Ribatti, D. et al., Haematologica (1991 Jul-Aug) 76(4): 311-20.
8. Tobelem, G., Blood Coagulation and Fibrinolysis (Dec 1990) 1(6): 703-5.
9. Wilks, J.W. et al., International Journal of Radiation Biology, (1991 Jul-Aug)
60(1-2): 73-7.
10. Folkman, J. et al., Science (1989 Mar 17) 243(4897): 1490-3.

CA 02208916 1997-07-03
11. Cariou, R. et al., Cell Biology International Reports (1988 Dec) 12(12): 1037-47.
12. Folkman, J. and Ingber, D.E., Annals of Surgery (1987 Sep) 206(3): 374-83.
13. Ingber, D.E. et al, Endocrinology (1986 Oct) 119(4): 1768-75.
14. Folkman, J., Cancer Research (1986 Feb) 46(2): 467-73.
15. Crum, R. et al., Science (1985 Dec 20) 230(4732): 1375-8.
16. Folkman, J., Pediatrics (1984 Nov) 74(5): 850-6.
17. Blei, F. et al., Journal of Cellular Physiology (1993 Jun) 155(3): 568-78.
18. Folkman, J. and Shing Y., Advances in Experimental Medicine and Biology
(1992) 313: 355-64.
10 19. Yamamoto, T., Terada, N., Nishizawa, Y. and Petrow, V., Angiostatic
Activities of Medroxyprogesterone Acetate and its Analogues (1994): 56, 393-
399.
20. Colville-Nash, P., Alam, C., Appleton, I., Brown, J., Seed, M. and
Willoughby, D., The Pharmacological Modulation of Angiogenesis in
Chronic Granulomatous Inflammation (1995) JPET 274: 1463-1472.
Since wound healing depends on angiogenesis, (the reader is directed to article 3
above at page 811 for a discussion of angiogenesis) it would be desirable to induce
angiogenesis utilizing safe compounds and which compounds do not have
systemic side effects typical of glucocorticoids, mineralocorticoids and sex
steroids, or at least substantially reduced side effects. However, the angiostatic
steroids have not previously been recognized to stimulate new blood vessel
growth. See for example, article 4 and page 265 therein. Specific reference is
made to article 19 above to Nishizawa which verifies that angiostatic steroids do
not bind glucocorticoid, mineralocorticoid and sex steroid receptors.
Applicants have in their PCT Application W094/23725 published
on October 27, 1994 by Professor Willoughby et al., utilized an NSAID in
combination with a form of hyaluronic acid for controlling, inhibiting, and/or

CA 02208916 1997-07-03
--7-
regressing angiogenesis for the treatment of tumours and other diseases and
conditions.
PCT Application W 094/17840 to Fidia published August 18, 1994
discusses a spongy material consisting of total or partial ester derivatives of
hyaluronic acid utilized for the administration of hyaluronic acid solutions to
enhance the recovery of patients suffering from decubitus ulcers, wounds and
burns. This document purports to describe the capability of hyaluronic acid to
induce a rapid and complete tissue repair process. It is discussed further that
anti-inflammatory agents may be contained in the solutions of hyaluronic acid.
10 Specifically, the reference purports to teach a combination of hyaluronic acid in
solution with, for example, corticosteroids which include glucocorticoids and
mineralocorticoids. These steroids are known to have undesirable systemic side
effects.
It is therefore an object of this invention to provide a novel
15 treatment for promoting the formation of blood vessel growth and proliferation
(angiogenesis) for wound healing.
It would also be advantageous to be able to utilize substantially
reduced levels of medicines for wound healing in relation to the levels normallyrequired when the treatment involves the use of corticosteroids. Typically when
20 corticosteroids are used for a topical treatment, formulations .1 to 1%
incorporating corticosteroids are used. It would also be advantageous if
corticosteroids are used to reduce the amount used. Furthermore, it is an objectof this invention to utilize other compounds heretofore not known for
promotion of angiogenesis to stimulate angiogenesis.
Further and other objects of the invention will become apparent to
those skilled in the art from the following summary of the invention and the
more detailed description of embodiments thereof.

CA 02208916 1997-07-03
SUMMARY OF THE INVENTION
According to one aspect of this invention, we have provided
compositions including known steroids such as angiostatic steroids which have
limited or no angiogenic effect in combination with a form of hyaluronic acid
5 which in combination results in accelerated scarless wound healing and the
stimulation of new blood vessel formation (angiogenesis).
According to another aspect of the invention, angiogenesis is
promoted in wound healing treatment by, for example topical application, by
utilizing significantly lower dosages of agents such as angiostatic anti-
10 inflammatory agents such as known angiostatic steroids having significantlyreduced or no systemic side effects, (when compared to glucocorticoids,
mineralocorticoids and sex steroids), in combination with a form of hyaluronic
acid to foster and induce angiogenesis when utilized in combination with the
form of hyaluronic acid in wound healing and for the stimulation of new blood
15 vessels in the acceleration of scarless wound healing.
According to another aspect of the invention, the compositions and
treatment include steroids known not to possess detrimental systemic side effects
typical of glucocorticoid, mineralocorticoid and sex steroids in combination with
a form of hyaluronic acid (such as hyaluronan and pharmaceutically acceptable
20 salts thereof) for topical application for wound healing and which combination
induces angiogenesis and accelerates scarless wound healing.
Therefore, according to another aspect of the invention there is
provided a pharmaceutical composition utilized for increasing
neovascularization and angiogenesis during wound healing in a mammal, for
25 example a human, beyond the level of neovascularization and angiogenesis
which would occur at the wound site without any treatment, said composition
comprising an effective amount of any angiostatic steroid combined with an
effective amount of a form of hyaluronan such as hyaluronic acid and

CA 02208916 1997-07-03
pharmaceutically acceptable salts thereof. Preferably, said composition includes a
suitable diluent such as purified water or the like. The composition may be
applied or administered nasally, vaginally, intraaurally, ocularily, rectally, orally,
locally or topically at preferred levels of .1 - 3mg of said steroid per kg of body
5 weight of the patient preferably within the order of about 2.5% hyaluronic acid by
weight in the formulation. In various embodiments, .1%-20% hyaluronic acid by
weight of the formulation may be utilized and preferably .3%-10% may be
utilized. Suitable compositions may also be formulated for application of the
compositions of the invention transdermally, intermuscularly, intramuscularly
10 and intravenously when appropriate.
According to yet another aspect of the invention there is provided a
method of treatment for increasing neovascularization and angiogenesis during
wound healing in a mammal, for example a human, beyond the level of
neovascularization and angiogenesis which would occur at the wound site
15 without any treatment, said method comprising application of a composition
including an effective amount of any angiostatic steroid combined with an
effective amount of a form of hyaluronic acid (such as hyaluronan and
pharmaceutically acceptable slats thereof) at the wound site. Preferably said
composition includes a suitable diluent such as purified water or the like. The
20 composition may be applied or administered nasally, vaginally, intraaurally,
ocularly, rectally, orally, locally or topically at preferred levels of .1-3mg of the
steroid per kilogram of body weight of the patient in a composition comprising
in the order of about preferably 2.5% hyaluronic acid by weight. Alternatively,
formulations containing .1%-20% hyaluronic acid by weight may be utilized and
25 preferably .3%-10% may be utilized together with an angiostatic steroid which is
in the order of .1-1 mg of the steroid per kilogram of body weight. Suitable
compositions can be formulated for administration transdermally,
intermuscularly, intramuscularly and intravenously when appropriate.

CA 02208916 1997-07-03
- 10 -
According to yet another aspect of the invention there is provided
the use of a pharmaceutical composition for increasing neovascularization and
angiogenesis during wound healing in a mammal beyond the level of
neovascularization and angiogenesis which would occur at the wound sight
5 without any treatment, said use of said composition comprising application of
the composition containing an effective amount of an angiostatic steroid
combined with an effective amount of a form of hyaluronan such as hyaluronic
acid and a pharmaceutically acceptable salt thereof at the wound site. Preferably
said composition also includes a suitable diluent such as purified water or the
10 like. The composition may be applied or administered nasally, vaginally,
intraaurally, ocularly, rectally, orally, locally or topically at preferred levels of .1-
3mg of said steroid per kg of body weight of the patient with for example 2.5%
hyaluronic acid by weight of the composition. Alternatively .1%-20% hyaluronic
acid may be utilized by weight in the formulation, and more preferably .3%-10%
15 by weight of the composition may be a form of hyaluronic acid. Equally
appropriate would be the application of the invention transdermally,
intermuscularly, intramuscularly and intravenously when appropriate.
According to yet another aspect of the invention, there is provided a
composition for inducing angiogenesis in the healing of a condition, such as a
20 wound, for example for topical application, said composition comprising a form
of hyaluronic acid in a suitable form and effective amount in combination with
an effective amount of a suitable steroid, such as angiostatic steroids, for example
tetrahydro-steroids, that are known not to possess detrimental systemic side
effects or to possess significantly reduced detrimental side effects such as bone
25 mass loss, fluid retention, infections and the like as is normally associated with
glucocorticoids, mineralocorticoids, and sex steroids, said suitable steroid, such as
an angiostatic steroid, (for example tetrahydro-steroids known not to stimulate
new blood vessel formation when utilized individually) wherein the

CA 02208916 1997-07-03
combination of hyaluronic acid and said suitable steroid (known to possess
reduced detrimental side effects, or not to possess detrimental systemic side
effects, normally associated with glucocorticoids, mineralocorticoids and sex
steroids and which are normally angiostatic and inhibit new blood vessel
5 formation), stimulates new blood vessel formation and angiogenesis to accelerate
scarless healing of a condition such as a wound. Preferably, the steroids such as
tetrahydrocortisol (THF), tetrahydrocortisone (THE) and tetrahydrocortexolone
(THS) known not to possess detrimental systemic side effects and known to be
angiostatic are the agents combined with the form of hyaluronic acid. The reader10 is referred to the previous prior art listing for a partial listing of typical angiostatic
steroids. For example, a combination of the form of hyaluronic acid in
combination with tetrahydro-steroid derivatives would be beneficial for
acceleration of wound healing by the stimulation of angiogenesis.
According to another aspect of the invention, there is provided a
15 process for the stimulation and/or induction of angiogenesis, for example,
stimulation of blood vessel growth in a condition such as a wound in a mammal
(for example a human), the process comprising the step of administering an
effective dosage amount of a pharmaceutical composition for the stimulation
and/or induction of angiogenesis to a site on/in a mammal in need of
20 stimulation and/or induction of angiogenesis such as at a wound site, and
repeating the administration for such time as required, each effective dosage
amount of the composition comprising an effective non-toxic dosage amount of
a steroid known to have angiostatic properties when utilized alone or in
combination with known therapies, examples of which angiostatic steroids
25 include tetrahydro-steroids, for example tetrahydrocortisone, tetrahydrocortisol
or tetrahydrocortexolone or the like (for example, in solution in the
composition), and an effective non-toxic dosage amount of a form of hyaluronic
acid and/or a pharmaceutically acceptable salt thereof (for example, sodium

CA 02208916 1997-07-03
hyaluronate) preferably sodium hyaluronate. Preferably, the composition may be
administered topically in the form of a cream, lotion and/or gel for the treatment
of burns, ulcers, incisions or the like, nasally, vaginally, intraurally, ocularly, or
orally for the treatment of wounds therein or as a suppository for hemorrhoids.
5 Administration may also comprise application transdermally, intermuscularly,
intramuscularly and intravenously when appropriate.
According to another aspect of the invention, the use of a
combination of a form of hyaluronic acid (for example, hyaluronic acid and a
pharmaceutically acceptable salt thereof) for example, sodium hyaluronate, and a10 suitable steroid known not to have systemic side effects, such as angiostatic steroids, for example tetrahydro-steroids such as tetrahydrocortisol,
tetrahydrocortisone or tetrahydrocortexolone and the like (among others also
known not to have the systemic side effects of glucocorticoids, mineralocorticoids
and sex steroids or the like,) which are also known to be normally angiostatic
15 when administered in known compositions or solutions is provided for
angiogenesis. Surprisingly and unexpectedly, this combination of the form of
hyaluronic acid with the specified agent stimulates and induces angiogenesis andhence healing of a condition such as a wound. The composition may be applied
or administered nasally, vaginally, intraaurally, ocularly, rectally, orally, locally
20 or topically at preferred levels of .1-3mg of said steroid per kilogram of the patient
in combination with .1%-20% by weight of the form of hyaluronic acid in the
composition, ~refeLably .3%-10% by weight of the form of hyaluronic acid may be
used in the formulation. Equally appropriate would be the application of the
compositions transdermally, intermuscularly, intramuscularly and
25 intravenously when appropriate.
Thus, an effective non-toxic dosage amount of a composition
comprising an effective non-toxic dosage amount of sodium hyaluronate and a
therapeutically effective non-toxic dosage amount of a known angiostatic steroid,

CA 02208916 1997-07-03
- 13-
such as tetrahydro-steroids for example tetrahydrocortisol, tetrahydrocortisone or
tetrahydrocortexolone or the like is provided and may be used to stimulate and
induce angiogenesis. The amount of angiostatic steroid (for example,
tetrahydrocortisol, tetrahydrocortisone or tetrahydrocortexolone or the like)
5 administered in one example may be about .1-3mg/kg of body weight of the
patient for application topically in a composition which comprises preferably
2.5% hyaluronic acid or salt thereof by weight for wounds in the order of 4 sq. cm.
Alternatively, the composition may comprise about .1%-20% hyaluronic acid,
preferably .3%-10% by weight of the composition may be utilized.
According to another aspect of the invention, a pharmaceutical
composition (suitable for, topical application [on the skin], rectally, vaginally,
intraaurally, ocularly, locally, nasally, orally, on the mucosa, etc.) is provided (for
example a multigram pharmaceutical composition for use topically) effective for
the stimulation and induction of angiogenesis, the pharmaceutical composition
15 containing a plurality of dosage amounts for stimulating and inducing
angiogenesis, each of said dosage amounts comprising a therapeutically effectivenon-toxic (to the patient) dosage amount of a steroid known to be angiostatic (for
example tetrahydro-steroids) as described above and in the literature and an
effective non-toxic dosage amount of the form of hyaluronic acid such as
20 hyaluronan and/or pharmaceutically acceptable salts thereof effective to
stimulate and induce angiogenesis. Equally appropriate would be the
application of the composition transdermally, intermuscularly and
intramuscularly when appropriate.
The pharmaceutical composition may comprise suitable excipients
25 depending upon the route of administration for example excipients to make a
gel, lotion, spray, ointment, suppository, or cream (topical administration).

CA 02208916 1997-07-03
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According to yet another aspect of the invention, a dosage amount
of a pharmaceutical composition is provided for stimulating and inducing
angiogenesis at a wound site, the composition comprising:
(1) a known angiostatic steroid agent known not to have
5 systemic side effects typical of glucocorticoids, mineralocorticoids and sex
steroids, for example tetrahydro-steroids; and
(2) a form of hyaluronic acid and/or pharmaceutically acceptable salts thereof
(for example sodium hyaluronate) characterized in that said composition:
(a) is in a dosage form (e.g. in a cream, lotion, gel, ointment,
10 spray, or solution, etc.) which is suitable for administration at the wound site;
and
(b) is in such an amount and in such form that
component (1) is in an effective dosage amount together with
component (2) to stimulate and induce angiogenesis (for example in scarless
15 wound healing). Preferably, the pharmaceutical composition may further
comprise a plurality of dosage amounts.
According to still another aspect of the invention there is provided
the use of
(1) a known angiostatic steroid agent known not to have
2 0 systemic side effects typical of glucocorticoids, mineralocorticoids and sex steroids, for example tetrahydro-steroids; and
(2) hyaluronic acid and/or pharmaceutically acceptable salts
thereof (for example sodium hyaluronate)
in the manufacture of a pharmaceutical composition for use to stimulate and
25 induce angiogenesis in mammals (for example in humans) at a wound site,
wherein dosage amounts may be taken from the composition and each dosage
amount taken comprises:

CA 02208916 1997-07-03
a therapeutically effective non-toxic dosage amount of each of
components (1) and (2) to stimulate and induce angiogenesis at said wound site.
The composition containing the form of hyaluronic acid and
known angiostatic steroid provides significantly greater stimulation of
5 angiogenesis than a composition comprising a form of hyaluronic acid (for
example sodium hyaluronate) only. Thus, according to another aspect of the
invention Applicants have provided similar methods of treatment,
pharmaceutical compositions, dosage amounts and uses comprising forms of
hyaluronic acid (for example sodium hyaluronate having a molecular weight
less than about 750,000 daltons) with the angiostatic steroids known not to havesystemic side effects typical of glucocorticoids, mineralocorticoids and sex
steroids.
The amount of the form of hyaluronic acid (for example sodium
hyaluronate) per dosage amount may vary. A concentration of .1%-20%
15 hyaluronic acid may be utilized in the composition by weight and preferably .3%-
10% may be utilized. Preferably the form of hyaluronic acid (for example sodium
hyaluronate) administered, has a molecular weight less than about 750,00 daltons(for example about 150,00 to about 225,000 daltons). While higher molecular
weights of the hyaluronic acid and forms thereof may be used in the
2 0 compositions to stimulate angiogenesis, where the molecular weight of the
hyaluronic acid chosen for use is very large, the form of hyaluronic acid is
autoclaved, to break down the form of hyaluronic acid to fragments of lesser
molecular weight and if feasible diluted to permit administration and ensure no
coagulation (whatever the route of administration). Where the molecular
25 weight of the form of hyaluronic acid being employed is at the upper range, the
concentration of the form of the hyaluronic acid in the composition may be
adjusted, for example be reduced (for example to less than about 1%) dependent
on the molecular weight.

CA 02208916 1997-07-03
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Many forms of hyaluronan may be suitable although those
preferred are those discussed hereinafter (identified Molecular Weights were
determined using the Protein Standard):
One form of hyaluronic acid and/or pharmaceutically acceptable
5 salts thereof (for example sodium salt) suitable for use with our invention is an
amount having the following specifications/characteristics:
TESTS SPECIFICATIONS RESULTS
pH 5.0 to 7.0 at 25 degrees C. 6.0
Specific Gravity 0.990 to 1.010 at 25 degrees C. 1.004
Intrinsic Viscosity 4.5 to 11.0 dL/g. 7.07
MolecularWeight 178,000 to 562,000 daltons (protein 319,378 daltons
standard)
Sodium Hyaluronate 9.0 to 11.0 mg/mL. Positive 9.9 mg/ML
Assay and Identification Positive
Another such amount may comprise:
TESTS SPECIFICATIONS
1. Description White or cream odourless powder
2. Identification (IR Spectrum) Conforms to Ref. Std. Spectrum
3. pH (1% solution) 5.0 to 7.0
4. Loss onDrying NMT 10%
5. Residue on Ignition 15.0% to 19.0%
6. Protein Content NMT 0.1%
7. Heavy Metals NMT 20 ppm
8. Arsenic NMT 2 ppm
9. Residual Solvents
a) Formaldehyde NMT 100 ppm
b) Acetone NMT 0.1%
c) Ethanol NMT 2.0%

CA 02208916 1997-07-03
10. Sodium Hyaluronate Assay 97.0 to 102.0%
(dried basis)
11. Intrinsic Viscosity 10.0to 14.5dL/g
12. Molecular Weight 500,000 to 800,000 daltons
13. Total Aerobic Microbial Count NMT 50 microorganisms/g
(USP 23)
14. Escherichia coli (USP 23) Absent
15. Yeasts and Moulds (USP 23) NMT 50 microorganisms/g
16. Bacterial Endotoxins (LAL) NMT 0.07 EU/mg
(USP 23)
Another such amount is available from Hyal Pharmaceuticals
Limited and comes in a 15 ml vial of Sodium hyaluronate 20mg/ml (300mg/vial
- Lot 2F3). The sodium hyaluronate amount is a 2% solution with a mean
average molecular weight of about 225,000. The amount also contains water q.s.
which is triple distilled and sterile in accordance with the U.S.P. for injection
formulations. The vials of hyaluronic acid and/or salts thereof may be carried in
a Type 1 borosilicate glass vial closed by a butyl stopper which does not react with
the contents of the vial.
The amount of hyaluronic acid and/or salts thereof (for example
sodium salt) may also comprise the following characteristics:
a purified, substantially pyrogen-free amount of hyaluronic acid
obtained from a natural source having at least one characteristic selected from
the group (and preferably all characteristics) consisting of the following:
i) a molecular weight within the range of 150,000-225,000;
ii) less than about 1.25% sulphated mucopoly-saccharides on a
total weight basis;
iii) less than about 0.6% protein on a total weight basis;
iv) less than about 150 ppm iron on a total weight basis;

CA 02208916 1997-07-03
- 18 -
v) less than about 15 ppm lead on a total weight basis;
vi) less than 0.0025% glucosamine;
vii) less than 0.025% glucuronic acid;
viii) less than 0.025% N-acetylglucosamine;
ix) less than 0.0025% amino acids;
x) a W extinction coefficient at 257 nm of less than about 0.275;
xi) a W extinction coefficient at 280 nm of less than about 0.25;
and
xii) a pH within the range of 7.3-7.9. Preferably, the hyaluronic acid
10 is mixed with sterile water and the amount of hyaluronic acid has a mean
average molecular weight within the range of 150,000-225,000 daltons (protein
standard). More preferably, the amount of hyaluronic acid comprises at least onecharacteristic selected from the group (and preferably all characteristics)
consisting of the following characteristics:
i) less than about 1% sulphated mucopolysaccharides on a total
weight basis;
ii) less than about 0.4% protein on a total weight basis;
iii) less than about 100 ppm iron on a total weight basis;
iv) less than about 10 ppm lead on a total weight basis;
v) less than 0.00166% glucosamine;
vi) less than 0.0166% glucuronic acid;
vii) less than 0.0166% N-acetylglucosamine;
viii) less than 0.00166% amino acids;
x) a W extinction coefficient at 257 nm of less than about 0.23;
xi) a W extinction coefficient at 280 nm of less than 0.19; and
xii) a pH within the range of 7.5-7.7
Applicants may also use sodium hyaluronate produced and
supplied by LifeCoreTM Biomedical, Inc., having the following specifications:

CA 02208916 1997-07-03
- 19 -
Characteristics Specification
Appearance White to cream
colored particles
Odor No perceptible odor
Viscosity Average < 750,000 Daltons
Molecular Weight
UV/Vis Scan, 190-820nm Matches reference scan
OD, 260nm < 0.25 OD units
Hyaluronidase Sensitivity Positive response
0 IR Scan Matches reference
pH, 10mg/g solution 6.2 - 7.8
Water 8% maximum
Protein < 0.3 mcg/mg NaHy
Acetate < 10.0 mcg/mg NaHy
Heavy Metals, maximum ppm
As Cd Cr Co Cu Fe Pb Hg N i
2.0 5.0 5.0 10.0 10.0 25.0 10.0 10.0 5.0
Microbial Bioburden None observed
Endotoxin < 0.07EU/mg NaHy
2 0 Biological Safety Testing Passes Rabbit Ocular
Toxicity Test
Another amount of sodium hyaluronate proposed to be used is sold
under the name Hyaluronan HA-M5070 by Skymart Enterprises, Inc. having the
following specifications:
Specifications' Test Results
Lot No. HG1004
pH 6.12
Condroitin Sulfate not detected

CA 02208916 1997-07-03
- 20 -
Protein 0.05%
Heavy Metals Not more than 20 ppm
Arsenic Not more than 2 ppm
Loss on Drying 2.07%
Residue on Ignition 16.69%
Intrinsic Viscosity 12.75 dl/s (XW: 679,000)
Nitrogen 3.14%
Assay 104.1%
Microbiological Counts 80/g
E. coli Negative
Mold and Yeast Not more than 50/g
Other forms of hyaluronic acid and/or its salts may be chosen from
other suppliers and those described in prior art documents provided they are
suitable.
The following references teach hyaluronic acid, sources thereof, and
processes for the manufacture and recovery thereof which may be suitable.
As there is no toxicity of the form of hyaluronic acid, the form of
hyaluronic acid may be administered in doses in excess of 12mg/kg of body
weight, for example, in excess of 1000mg/70kg person and even up to amounts of
3000mg/70kg person without adverse toxic effects. Lower amounts may include
10-50mg of hyaluronan. Example amounts of Hyaluronan used may be 3-lOmg
of HA/kg of body weight of the patient wherein the molecular weight (protein
standard) is less than 750,000 daltons.
Suitable forms of hyaluronan may have molecular weights of forms
of hyaluronan between about 150,000 daltons and about 750,000 daltons (protein
standard) in sterile water prepared having a viscosity for intravenous
administration.

CA 02208916 1997-07-03
One specific form of pharmaceutical grade is a 1% sterile sodium
hyaluronate solution (50 ml vials) provided by Hyal Pharmaceutical Corporation
which has the following characteristics:
Tests Specifications
1. Container Description 150 mL Flint glass vial with
a red or gray rubber stopper
and an aluminum seal, 20
mm in size.
2. Product Description A clear, colourless, odourless,
transparent, slightly viscous
liquid.
3. Fill Volume 50.0 to 52.0 mL
4. pH 5.0 to 7.0 at 25 degrees C.
5. Specific Gravity 0.990 to 1.010 at 25 degrees C.
6. IntrinsicViscosity 4.5 to 11.0 dL/g
7. Molecular Weight 178,000 to 562,000 daltons
8. Sodium Hyaluronate Assay 9.0 to 11.0 mg/mL. Positive
and Identification
9. Particulate Matter No visible Particulate Matter
2 0 10. Sterility Meets Requirements for Sterility,
USP 23
11. Bacterial Endotoxins (LAL) Meets Requirements for Bacterial
Endotoxins, USP 23.
This pharmaceutical grade 1% sterile solution of hyaluronan may be
25 made from granules/powder having the following characteristics:

CA 02208916 1997-07-03
Tests Specifications
1. Description White or cream-coloured granules or
powder, odourless
2. Identification (IR Spectrum) Must conform with the Reference
Standard Spectrum.
3. pH (1% Solution) Between 5.0 and 7.0 at 25 degrees C.
4. Loss on Drying NMT 10.0% at 102 degrees C. for 6
hours.
5. Residue on Ignition Between 15.0 and 19.0%
6. Protein Content NMT 0.10%
7. Heavy Metals NMT 20 ppm (as per USP 23 p. 1727).
8. Arsenic NMT 2 ppm (as per USP 23, p. 1724).
9. Residual Solvents a) Acetone: NMT 0.1%
b) Ethanol: NMT 2.0%
c) Formaldehyde: NMT 100 ppm
10. Sodium Hyaluronate Assay 97.0 to 102.0% (dried basis)
11. Intrinsic Viscosity Between 10.0 to 14.5 deciliters per gram.
12. Molecular Weight Between 500,000 to 800,000 daltons
lc~ t~cl usir~ the Laurent Fonnula) (based on intrincis viscosity).
13.TotalAerobicMicrobialCount NMT 50 microorganism/gram (as per
USP 23, p. 1684).
14. Test for Escherichia coli Escherichia coli is absent (as per USP
23, p. 1685).
15. Yeasts & Molds NMT 50 microorganisms/gram (as per
USP 23, p. 1686).
16. Endotoxins (LAL) NMT 0.07 EU/mg (as per USP 23, p.
1696).

CA 02208916 1997-07-03
- 23 -
A topical grade of hyaluronan may, in certain circumstances be
suitable and may be made from the following granules/powder which have the
following characteristics:
Tests Specifications
1. Description White or cream-coloured granules or
powder, odourless
2. Identification (IR Spectrum) Must conform to the Reference Standard
Spectrum.
3. pH (1% Solution) Between 6.0 and 8.0 at 25 degrees C.
4. Loss on Drying NMT 10.0% at 102 degrees C. for 6
hours.
5. Residue on Ignition Between 15.0 and 19.0%
6. ProteinContent NMT 0.40%
7. Heavy Metals NMT 20 ppm (as per USP 23 p. 1727).
8. Arsenic NMT 2 ppm (as per USP 23, p. 1724).
9. ResidualSolvents a) Acetone: NMT 0.1%
b) Ethanol: NMT 2.0%
c) Formaldehyde: NMT 100 ppm
10. Sodium Hyaluronate Assay 97.0 to 102.0% (dried basis)
11. Intrinsic Viscosity Between 11.5 to 14.5 deciliters per gram.
12. Molecular Weight Between 600,000 to 800,000 daltons
t ~cl using the Laurent Formula) (based on intrinsic viscosity).
13. Total Aerobic Microbial Count NMT 100 microorganism/gram (as per
USP 23, p. 1684).
14. Test for Staphylococcus aureus Staphylococcus aureus is absent (as per
USP 23, p. 1684).
15. Test for Pseudomonas aeruginosa Pseudomonas aeruginosa is absent (as
per USP 23, p. 1684).

CA 02208916 1997-07-03
- 24 -
~6. Yeasts & Molds NMT 200 CFU/gram (as per USP 23, p.
1686).
This topical grade may then be sterilized.
Other forms of hyaluronic acid and/or its salts may be chosen from
5 other suppliers, for example those described in prior art documents disclosingforms of hyaluronic acid having lower molecular weights between about 150,000
daltons and 750,000 daltons being prepared as for example, 1-2% solutions in
sterile water for intravenous administration.
The following references teach hyaluronic acid, sources thereof and
10 processes of the manufacture and recovery thereof.
Canadian Letters Patent 1,205,031 (which refers to United States
Patent 4,141,973 as prior art) refers to hyaluronic acid fractions having average
molecular weights of from 50,000 to 100,000; 250,000 to 350,000; and 500,000 to
730,000 and discusses processes of their manufacture
Where high molecular weight hyaluronic acid (or salts or other
forms thereof) is used, it must, prior to use, be diluted to permit administration
and ensure no intramuscular coagulation. Recently, it has been found that large
molecular weight hyaluronic acid having a molecular weight exceeding about
1,000,000 daltons self-aggregates and thus, does not interact very well with HA
20 receptors. Thus, the larger molecular weight hyaluronic acid should be avoided.
The invention will now be illustrated by reference to the following
Figures and description of embodiments.

CA 02208916 1997-07-03
- 25 -
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graph showing the effect of two angiostatic steroids
applied topically in hyaluronan to mice with granulomatous air pouches against
a control.
Figure 2 is a graph showing the increase of angiogenesis over a
control of varying concentrations of topical tetrahydrocortisol applied daily inhyaluronan to murine granulomatous tissue.
Figure 3 is a graph illustrating typical activity of an angiostatic
steroid alone and the predictable decrease in angiogenesis as the dose of this
steroid is increased.
DETAILED DESCRIPTION OF EMBODIMENTS
The invention will now be illustrated with reference to the
following experimental data and tests performed with respect to embodiments.
The angiostatic steroid tetrahydrocortisol (TH-F),
tetrahydrocortisone (TH-E) or tetrahydro-cortexolone (TH-S) and the like s.c at 1
mg/kg of body weight of a mammal is capable of inhibiting angiogenesis in the
murine chronic granulomatous air pouch (1). The same effect is obtained with 1
mg/kg s.c cortisone in combination with 1000U p.o heparin. The cortisone
heparin combination also induces regression of the existing neo-vasculature (2).2 o We investigated the use of topical application of angiostatic steroids in
combination with hyaluronan (in these examples at a concentration of 2.5% by
weight of the composition). Reference is made to article 20 listed previously.
Methods
Chronic granulomatous air pouches were raised in mice by known
methods (the reader is referred to PCT/CA94/00207 at page 16 for an
experimental method in this regard) utilizing 0.5 ml Freund's Complete
adjuvant with 0.1% croton oil injected into 1 day old air pouches. Animals were
dosed with saline/0.01% DMSO either subcutaneously (s.c.) into the nape of the

CA 02208916 1997-07-03
- 26 -
neck, or with saline applied topically in 0.1 ml 2.5% hyaluronan. Vascular castsincorporating carmine in gelatin were formed at termination, the tissues
weighed dry (mg), papain digested, and the carmine content (~g) determined by
spectrophotometric assay. Vascularity was calculated as the Vascularity Index
5 (,ug/mg). Results are expressed as means + s.e.m..
Results
Investigation of the action of tetrahydrocortisol, tetrahydrocortisone
and tetrahydrocortexolone and the like on inflammation-induced angiogenesis
in the murine chronic granulomatous air pouch was pursued. Groups of mice
10 (n=8-13) were dosed with tetrahydrocortisol, tetrahydrocortisone or
tetrahydrocortexolone or the like (0.1- 3.0 mg/kg in 0.1 ml saline / 0.01% DMSO,s.c) daily from induction (day 0) to termination on day 6. Controls were dosed
with 0.1 ml saline/0.01% DMSO subcutaneously.
Table 1
DRUG VASCULAR INDEX (,ug/mg)
Control 8.31 + 1.46
Cortisone 1 mg/kg s.c. 8.23 + 0.62
Heparin 1000U p.o. 7.93 + 0.83
Heparin & Cortison 6.03 + 1.12
TH-F lmg/kg s.c. 5.29 + 0.61
TH-F & Heparin 7.11 + 0.85
The angiostatic glucocorticoid cortisone requires simultaneous dosing with
heparin (Table 1) to be angiostatic, being inactive when administered alone at the
sub-antiinflammatory dose of 1 mg/kg, whereas tetrahydrocortisol etc. may not
require heparin. In addition, the co-administration of heparin reduces
20 effectiveness of the tetrahydrosteroid (Table 1).

CA 02208916 1997-07-03
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Table 2
Investigation of the action of angiostatic and anti-inflammatory
steroids on inflammation-induced angiogenesis in the murine chronic
granulomatous air pouch was pursued. Groups of mice (n=8-13) were treated
5 with the steroids at 1.0 mg/kg (0.1 ml saline / 0.01% DMSO, s.c), except of
dexamethasone at (0.2 mg/kg), daily from the day of induction (day 0) to
termination on day 6. Controls were given 0.1 ml saline / 0.01% DMSO s.c. (Key:
Tetrahydrocortisol (TH-F), Tetrahydrocortisone (TH-E).
Treatment Vascular Index
~g/mg
Saline/DMSO 15.774 + 1.152
TH-F 13.088 + 1.630
TH-E 18.860 + 1.830
Table 3
Investigation of the action of tetrahydrocortisol, tetrahydrocortisone
and tetrahydrocortexolone and the like on the established 7-day old vasculature
of the murine chronic granulomatous air pouch was pursued. Groups of mice
15 were dosed with tetrahydrocortisol, tetrahydrocortisone or tetrahydrocortexolone
or the like (0.1 - 3.0 mg/kg in 0.1 ml saline / 0.01% DMSO, s.c) daily from day 7 to
termination at day 14 or day 21. Controls were dosed with 0.1 ml saline (0.01%
DMSO alone).
Day/ Vascular Index
Treatment ,ug/mg
Day 7 Saline 10.638 + 0.883
Day 14 Saline 17.97 + 2.10
0.1 TH-E 18.18 + 1.97
0.3 TH-E 20.38 + 1.91

CA 02208916 1997-07-03
- 28 -
1.0 TH-E 22.49 + 3.77
3.0TH-E 18.87 1.29
Day 21 Saline29.98 + 2.66
0.1 TH-E 18.59 _ 1.84
0.3 TH-E 31.33 2.91
1.0 TH-E 25.75 2.40
3.0 TH-E 25.91 _ 1.85
Table 4
Investigation of the action of topical tetrahydrocortisol,
tetrahydrocortisone, tetrahydrocortexolone and the like on inflammatory-
5 induced angiogenesis in the murine chronic granulomatous air pouch waspursued. Groups of mice (n=8-13) were depilated and the inflammation induced
4 days later. Tetrahydrocortisol (TH-F) and tetrahydrocortisone (TH-E) were
applied topically (1 mg/kg) in 2.5% hyaluronan (0.1 ml) daily from the day of
induction (day 0) to termination (day 6). Controls were dosed with 0.1 ml 2.5%
10 hyaluronan alone.
Treatment Vascular Index
~lg/mg
HA Control 11.180 1.980
HA + TH-E 21.118 _ 1.927
HA + TH-F 29.394 + 2.213
Table 5
Investigation of the action of topical tetrahydrocortisol,
15 tetrahydrocortisone and tetrahydrocortexolone and the like on inflammatory-
induced angiogenesis in the murine chronic granulomatous air pouch was
pursued. Groups of mice (n=8-13) were depilated and the inflammation induced

CA 02208916 1997-07-03
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4 days later. Tetrahydrocortisol (TH-F) was applied topically with 2.5%
hyaluronan (0.1 ml of composition 0.1 - 1.0 mg of tetrahydrocortisol /kg) by
weight daily from the day of induction (day 0) to termination (day 6). Controls
were dosed with 0.1 ml 2.5% hyaluronan alone.
Treatment Vascular Index
~lg/mg
HA 11.180 _ 1.98
HA + TH-F 11.800 + 1.920
HA + TH-F 12.250 0.230
HA + TH-F 15.73 + 3.218
Conclusions
The data supports the conclusion that the angiostatic action of
tetrahydrocortisol, tetrahydrocortisone and tetrahydrocortexolone and the like on
subcutaneous administration in the absence of heparin is unique amongst the
angiostatic steroids. The angiostatic glucocorticoid cortisone requires
simultaneous dosing with heparin (Table 1) to be angiostatic, being inactive
when administered alone at the sub-antiinflammatory dose of 1 mg/kg, whereas
tetrahydrocortisol etc. may not require heparin. In addition, the co-
administration of heparin reduces effectiveness of the tetrahydrosteroid (Table 1).
Another tetrahydrosteroid (TH-E) was without effect in the absence of heparin
whether dosed prophylactically (Table 2) or therapeutically (Table 3). In this
instance, tetrahydrocortisol, tetrahydrocortisone and tetrahydrocortexolone and
the like induced a decrease in vascularity (Table 1).
2 0 Contrary to this, the topical application of tetrahydrocortisol,tetrahydrocortisone and tetrahydrocortexolone and the like in 2.5% hyaluronan
resulted in significant acceleration of angiogenesis (Table 4), which in the case of
tetrahydrocortisol, tetrahydrocortisone and tetrahydrocortexolone and the like,

CA 02208916 1997-07-03
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was dose related. The application of angiostatic steroids with hyaluronan
resulted in a potentiation of the ability of tetrahydrocortisol, tetrahydrocortisone
and tetrahydrocortexolone and the like to raise vascularity, whilst the angiostatic
action of tetrahydrocortisol, tetrahydrocortisone and tetrahydrocortexolone and
5 the like was converted into profound stimulation of angiogenesis (compare
Tables 2, 4 & 5). We have previously investigated that the angiostatic
glucocorticoid cortisone, given topically at 1 mg/kg of body weight, is angiostatic
in combination with 2.5% hyaluronan. There may therefore be a structural basis
to the diverse pharmacological actions noted with this steroid series, in their
10 interaction with hyaluronan. The 3-ketone combination with ~4 double bond
may result in an angiostatic profile in conjunction with hyaluronan. These
groups are required for glucocorticoid activity, their reduction leading to the
absence of glucocorticoid activity in the tetrahydro-derivatives and stimulationof angiogenesis in the presence of HA.
It should be noted that proprietary formulations of topical
hydrocortisone are typically 0.1-1.0% whilst the formulations used in these
studies contain a maximum of 0.02% angiostatic steroid in 2.5% hyaluronan.
From these studies, topical applications of tetrahydro-derivatives of
corticosteroids devoid of glucocorticoid activity in hyaluronan would be
2 0 beneficial for the acceleration of wound healing via the stimulation of
angiogenesis. Inherent in this observation is that they could be contra-indicated
for conditions dependent on neovascularisation, such as superficial carcinomas.
The reader is referred to, the British Journal of Pharm. 107:259P,1992, 1st
International Workshop Hyaluronan Delivery, Royal Soc. Med. Round Table
series 33:21-31, 1994 and 2nd International Workshop Hyaluronan Delivery,
Royal Soc. Med. Round Table series 1995.
The following represents typical chemical structures of basic
angiostatic steroids which may be utilized in combination with hyaluronic acid

CA 02208916 1997-07-03
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to induce angiogenesis and foster wound healing. The list is not considered to be
exhaustive but are believed merely representative.
tetrahydrocortisol 5-pregnane-3,11,17,21-tetrol-20-one
tetrahydrocortisone 5-pregnane-3,17,21-triol-11,20-dione
17-hydroxyprogesterone 4-pregnene-17-ol-3,20-dione
medroxyprogesterone 6-methyl-5-pregnene-17-ol-3,20-dione
6,6'-dehydromedroxyprogesterone 6-methylene-5-pregnene-17-ol-3,20-dione
megestrol 17-hydroxy-6-methyl-4,6-pregnadiene-
3,20-dione
l-dehydro-megestrol 17-hydroxy-6-methyl-1,4,6-pregnatriene-
3,20-dione
melengestrol 17-methylene-6-methyl-4,6-pregnadiene-
3,20-dione
l-dehydro-melengestrol 17-methylene-6-methyl-1,4,6-
pregnatriene-3,20-dione
2-methoxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol-2-methyl
ether
2-hydroxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one
4-methoxyoestradiol 1,3,5(10)-oestratriene-3,4,17-triol-4-methyl
ether
2-methoxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-2,3-
dimethyl ether
2-methoxyoestrone 1,3,5(10)-oestratriene-2,3-diol-17-one-2-
methyl ether
2-hydroxyoestradiol 1,3,5(10)-oestratriene-2,3,17-triol
2-methoxyoestriol 1,3,5(10)-oestratriene-2,3,16,17-tetrol-2-
methyl ether

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2-hydroxyoestradiol 3-methyl ether 1,3,5(10)-oestratriene-2,3,17-triol-3-methyl
ether
Definite angiogenesis action was obtained utilizing tetrahydrocortisol where Rl =
5 -OH and tetrahydrocortisone where Rl = =O
O~,~CH20H
CH3 1~ OH
Rl~
The following chart indicates the various predictable forms that steroids shouldtake:
Rl R2 R3 R4 R5 R6
Tetrahydrocortexolone ~ --H --H --H --OH --OH
17 Hydroxyprogesterone ~~ --H --OH --H
O~,~CH2R6
R4~Rs
~1 >
~ ~/
RlJ~R2' 3 J
Further positive results are also predicted for acetate derivatives
o\\
17--O--C--CH3
possessmg elther
or 21--O--C--CH3

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- 33 -
or hemissuccinate derivatives
O\\ ,O
17--O--C--(C2H4)C_oH
21--O--C--(C2H4)C_ OH
The following chart indicates further acetate derivatives which may be effective:
Rl R2 R3
o~
tetrahydrocortisol 17-acetate --OH O--C--CH3 --H
O~
lo tetrahydrocortisone 17-acetate ~ --o--c--CH3 --H
0~ ,0
tetrahydrocortisol 17 --OH --O--C--(C2H4)C- OH --H
hemisuccinate
o~ ,o
tetrahydrocortisone 17 ~ --o-c--(C2H4)c-oH --H
hemisuccinate
tetrahydrocortisol 21 acetate --OH --H --C--CH3
tetrahydrocortisone 21 acetate ~ --H "
tetrahydrocortisone 21 --OH --H --C(C2H4)COOH
2 0 hemisuccinate
tetrahydrocortisol 21 ~ --H --C(C2H4)COOH
hemisuccinate

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- 34 -
~CH20 R3
Rl~--~R2
HO
The following chart indicates further various predictable forms for applicable
steroids:
Rl R2 R3 R4 R5 R6 R7
Medroxyprogesterone --H --H --CH3 _ H --H
6:6-dehydro- --H --H =CH2 --H --H
medroxyprogesterone
Megestrol --H --H . CH3 --H
l-dehydro- CH3 --H
medroxyprogesterone
Melengestrol --H --H : CH3 = CH~
I
l-dehydro melengestrol - CH3 =CH2
o~H3
CH3 \\
~ ~0--C--CH
CH3 l R7
R ,R
O~R3' 4~R,R6
and also 17-OH or 17-H or the following derivatives:
20--CH2OH

CA 02208916 1997-07-03
or
20--CH2--O--//C--CH3
or
17--O--C(C2H4)COOH
or
21--O--//C(C2H4)COOH
All steroids conjugated at the 20 position (Rl) were applicable (i.e. not the
oestrogens)
H 1~l H
N \~\ ,N~+ "R
- CH20SO3 CO - O
\~ ~Y' n= 1-50
NSO3 OH
or
H H
/N--N =R
CH20SO3 O=C
\0~0_~
- HNSO3- OH n=1-50
15 All steroids conjugated at the 3 position (Rl) with

CA 02208916 1997-07-03
- 36 -
H 1~l H
N \~\ ,N~+ ~R
- CH2Oso3 CO ~ O H
\ ~ 0~\ n= 1-50
HNSO3 OH
heparin
adipic
hydrazide
or
H H
/N--N =R
CH20SO3 0= lC
\o~LO~\
- HNSO3 OH - n= 1-50
heparin
hydrazide
The following chart indicates further various other acceptable steroids
utilized:
Rl R2 R3 R4 R5
2-methoxyoestradiol --OCH3 --OH --H --OH --H
2-hydroxyoestrone --OH --OH --H = O --H
4-methoxyoestradiol --H --OH --OCH3 --OH --H
2-methoxyoestradiol --OCH3 --OCH3 --H --OH --H
3-methyl ether

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- 37 -
2-methoxyoestrone --OCH3 --OH --H --OH --H
2-hydroxyoestradiol --OH --OH --H --OH --H
2-methoxyoestriol --OCH3 --OH --H --OH --OH
2 Hydroxyoestradiol --OH --OCH3 --H --OH --H
3-methyl ether
CH3
R
R¦~R5
Known Structure of angiostatic steroids.
TETRAHYDROCORTISOL (5~-pregnane-3a, 11~, 17a, 21-tetrol-20-one;
Tetrahydro-F)
O~CH20H
HO ~l ' OH
HO I ~J

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TETRAHYDROCORTISONE (5~-pregnane-3a, 17a, 21-triol-11, 20-dione;
Tetrahydro-E)
O~CH20H
O~'OH
HO I ~
TETRAHYDROXYCORTEXOLONE (5~-pregnane-3a, 17cc, 21-triol-20-one;
Tetrahydro-S; 11-deoxytetrahydrocortisol)
O~CH20H
OH
o~
Referring now to Figure 1, it may be observed that firstly different
angiostatic steroids have varying effects in terms of their percentage increase in
angiogenesis over a control. Significant, however, is the fact that all angiostatic
steroids tested provided a marked increase in angiogenesis over either the
15 control, the angiostatic steroid alone, or HA alone. This is verified when
reviewing the tables described above.
Referring now to Figure 2, it may be seen for tetrahydrocortisol, and
it is expected for the other angiostatic steroids listed above as well, that within the
range of .1-1 mg of the angiostatic steroid administered/day, which is
20 significantly less than the amount of steroid used for wound healing when
utilizing glucocorticoid or mineralocorticoid steroids such as cortisone, which is

CA 02208916 1997-07-03
- 39 -
up to 3 mg/day when utilized alone, that a significant increase in
neovascularization can be produced in utilizing the combination of hyaluronan
(HA) plus an angiostatic steroid in substantially reduced amounts to limit the
impact of the consumption of steroids on the individual, and yet realize the
objectives of fostering angiogenesis and accelerating wound healing.
Figure 3 illustrates typical activity of an angiostatic steroid alone
with the predictable decrease in angiogenesis as the dose is increased.
All of the aforementioned data supports the conclusion of HA and
steroids of the type listed above, in combination, acting synergistically to induce
10 angiogenesis in wound healing for example leg ulcers and pressure ulcers
developed during various stages of diabetes. Also, the trend is such that the form
of HA and the angiostatic steroid taken from the listed alternatives above
induces angiogenesis much more than HA alone, or for that matter any of the
listed angiostatic steroids alone. The results of the tests and experiments firmly
15 establish that forms of hyaluronic acid (for example sodium hyaluronate having
a molecular weight less than 750,000 daltons - e.g. 225,000 daltons) in
combination with known angiostatic steroids act to induce angiogenesis and
neovascularization in wound healing without the known systemic side effects of
angiogenic glucocorticoid or mineralocorticoid steroids.
It will also be appreciated by those skilled in the art that the
processes, uses, compositions and dosage forms according to aspects of the
invention may be applied to induce angiogenesis in other instances where
angiogenesis is desired. It is therefore clear that many uses can be made of
embodiments and aspects of this invention without departing from the scope
25 thereof. It is therefore intended that all material contained herein be interpreted
as illustrative of the invention and not in a limiting sense.