Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
1
Treatment of Wounds
This invention relates to the use of cyclic guanosine 3', 5'-monophosphate
type
five (cGMP PDES) inhibitors (hereinafter PDE5 inhibitors), including in
particular
the compound sildenafil, for the treatment of chronic wounds of a non-diabetic
origin including in particular chronic venous ulcers, chronic decubitus
(pressure
sores) and arterial ulcers; and acute wounds.
Chronic wounds, by definition, take a long time to heal. Part of the process
of
repair requires a good blood supply and a pro-healing environment that allow
the
healing process to occur. Typical phases in the healing of a wound include
haemostasis, inflammation, repair and regeneration and finally re-modeling. In
a
chronic wound, one or more of these mechanisms is impaired.
The method of treating a wound depends on its type.
Chronic venous ulcers, also known as venous leg ulcers or venous stasis
ulcers,
common in patients with venous insufficiency, are characterised by increased
2o healing time and resistance to treatment. They are treated by simply
applying the
appropriate dressing and applying a compressive bandage.
Chronic arterial ulcers are caused typically by plaque in the arteries which
lead to
blockage and impaired blood supply. They heal slowly because of poor oxygen
supply and nutrition. Treatment requires support and re-vascularistion if
possible.
Chronic decubitus ulcers or pressure sores are caused by exerting pressure on
an area of the body for extended periods, typically longer than 3 hours.
Decubitus ulcers are treated by dressing the wound and removing the pressure.
If the sore is small enough then the sores can be closed surgically.
Acute wounds, e.g. cuts and grazes to the skin, are treated by simply keeping
the
wound clean and dry. In young, healthy individuals the rate of healing is
rapid.
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
2
However, in the elderly or immunocompromised healing can be prolonged.
Healing will also be prolonged if the wound becomes infected.
There is some suggestion in the literature that nitric oxide improves the rate
of
wound healing.
It is known that cGMP PDE5 inhibitors increase intracellular concentrations of
nitric oxide derived cGMP, thereby enhancing the effect of nitric oxide, which
is
responsible for the efficacy of sildenafil in the treatment of male erectile
7 0 dysfunction.
We have found elevated levels of the enzyme cGMP PDE5 in wounded tissue. In
particular, where the tissue is inflamed or scarred. Myofibroblasts in healing
wounds i.e skin and areas of organising infarction in, for example, cardiac
tissue
from patients with ischaemic heart disease express PDE 5 whereas fibroblasts
populating those areas in non-pathological conditions demonstrate no PDE 5
expression. Myofibroblasts from granulation tissue in normally healing wounds
temporarily express a smooth muscle phenotype whereas myofibroblasts with a
smooth muscle phenotype persist in abnormally healing wounds and fibro-
proliferative conditions. cGMP inhibits smooth muscle cell proliferation and
thus
potentiation of cGMP levels potentially leads to improved wound healing.
Without wishing to be bound by theory, it is believed that the wound healing
effect is due to improved blood supply to the wound region. PDE 5 inhibition
at
an appropriate stage in the wound-healing cycle in conjunction with an
appropriate signal such as NO-mediated smooth muscle relaxation results in
vasodilation leading to wound healing. Other factors may also be involved.
No therapeutic agent is currently commercially available which increases the
rate
of healing of these wound types.
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
3
According to a first aspect, the invention provides a method of treating
wounds in
a patient which comprises treating the patient with an effective amount of a
cGMP PDE5 inhibitor, or a pharmaceutical composition thereof, wherein the
wound type is selected from: chronic venous ulcers, chronic arterial ulcers,
chronic decubitus and acute wounds.
According to a second aspect, the invention provides the use of a cGMP PDE5
inhibitor for the manufacture of a medicament for the treatment of wounds,
selected from the following types: chronic venous ulcers, chronic arterial
ulcers,
chronic decubitus and acute wounds.
By PDE5 inhibitors it is meant a compound which is a potent and selective
inhibitor of the cGMP PDE5 isoenzyme.
Suitable PDE5 inhibitors for use in the pharmaceutical combinatiions according
to the present invention are the cGMP PDE5 inhibitors hereinafter detailed.
Particularly preferred for use herein are potent and selective cGMP PDE5
inhibitors.
Suitable cGMP PDE5 inhibitors for the use according to the present invention
include:
the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in EP-A-0463756; the pyrazolo
[4,3-d]pyrimidin-7-ones disclosed in EP-A-0526004; the pyrazolo [4,3-
d]pyrimidin-
7-ones disclosed in published international patent application WO 93/06104;
the
isomeric pyrazolo [3,4-d]pyrimidin-4-ones disclosed in published international
patent application WO 93/07149; the quinazolin-4-ones disclosed in published
international patent application WO 93/12095; the pyrido [3,2-d]pyrimidin-4-
ones
disclosed in published international patent application WO 94/05661; the purin-
6-
ones disclosed in published international patent application WO 94/00453; the
pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published international patent
application WO 98/49166; the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
4
published international patent application WO 99/54333; the pyrazolo [4,3-
d]pyrimidin-4-ones disclosed in EP-A-0995751; the pyrazolo [4,3-d]pyrimidin-7-
ones disclosed in published international patent application WO 00/24745; the
pyrazolo [4,3-d]pyrimidin-4-ones disclosed in EP-A-0995750; the compounds
disclosed in published international application W095/19978; the compounds
disclosed in published international application WO 99/24433 and the
compounds disclosed in published international application WO 93/07124.
The pyrazolo [4,3-d]pyrimidin-7-ones disclosed in published international
application WO 01/27112; the pyrazolo [4,3-d]pyrimidin-7-ones disclosed in
published international application WO 01/27113; the compounds disclosed in
EP-A-1092718 and the compounds disclosed in EP-A-1092719.
Preferred type V phosphodiesterase inhibitors for the use according to the
present invention include:
5-[2-ethoxy-5-(4-methyl-1-piperazinylsulphonyl)phenyl]-1-methyl-3-n-propyl-1,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil) also known as 1-[[3-
(6,7-
dihydro-1-methyl-7-oxo-3-propyl-1 H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-
ethoxyphenyl]sulphonyl]-4-methylpiperazine (see EP-A-0463756);
5-(2-ethoxy-5-morpholinoacetylphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (see EP-A-0526004);
3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-n-propoxyphenyl]-2-(pyridin-2-
yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see W098/49166);
3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-methoxyethoxy)pyridin-3-yl]-
2-
(pyridin-2-yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see
W099/54333);
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
(+)-3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-methoxy-1 (R)-
methylethoxy)pyridin-3-yl]-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-
one, also known as 3-ethyl-5-~5-(4-ethylpiperazin-1-ylsulphonyl]-2-(((1 R)-2-
methoxy-1-methylethyl]oxy)pyrid in-3-yl}-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-
d]
5 pyrimidin-7-one (see W099/54333);
5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyrid in-3-yl]-3-ethyl-2-[2-
methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, also known as 1-
{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3-
d]pyrimidin-5-yl]-3-pyridylsulphonyl~-4-ethylpiperazine (see WO 01127113,
Example 8);
5-[2-iso-Butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-(1-
methylpiperidin-4-yl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO
01 /27113, Example 15);
5-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-phenyl-
2,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (see WO 01/27113, Example 66);
5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-
dihydro-
7H-pyrazolo[4,3-d]pyrimidin-7-one (seeWO 01/27112, Example 124);
5-(5-Acetyl-2-butoxy-3-pyrid i nyl)-3-ethyl-2-(1-ethyl-3-azetid inyl)-2,6-d
ihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (see WO 01/27112, Example 132);
(6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl) -
pyrazino[2',1':6,1 ]pyrido[3,4-b]indole-1,4-dione (IC-351 ), i.e. the compound
of
examples 78 and 95 of published international application W095/19978, as well
as the compound of examples 1, 3, 7 and 8;
2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl]-5-methyl-7-propyl-
3H-
imidazo[5,1-f][1,2,4]triazin-4-one (vardenafil) also known as 1-[[3-(3,4-
dihydro-5-
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
6
methyl-4-oxo-7-propylimidazo[5,1-f]-as-triazin-2-yl)-4-ethoxyphenyl]sulphonyl]-
4-
ethylpiperazine, i.e. the compound of examples 20, 19, 337 and 336 of
published
international application W099/24433; and
the compound of example 11 of published international application W093/07124
(EISAI); and
compounds 3 and 14 from Rotella D P, J. Med. Chem., 2000, 43, 1257.
1 o Still other type cGMP PDE5 inhibitors useful in conjunction with the
present
invention include:4-bromo-5-(pyridylmethylamino)-6-[3-(4-chlorophenyl)-
propoxy]-
3(2H)pyridazinone; 1-[4-[(1,3-benzodioxol-5- ylmethyl)amiono]-6-chloro-2-
quinozolinyl]-4-piperidine-carboxylic acid, monosodium salt; (+)-cis-
5,6a,7,9,9,9a-
hexahydro-2-[4-(trifluoromethyl)-phenylmethyl-5-methyl-cyclopent-
4,5]imidazo[2,1-b]purin-4(3H)one; furazlocillin; cis-2-hexyl-5-methyl-
3,4,5,6a,7,8,9,9a- octahydrocyclopent[4,5]-imidazo[2,1-b]purin-4-one; 3-acetyl-
1-
(2-chlorobenzyl)-2-propylindole-6- carboxylate; 3-acetyl-1-(2-chlorobenzyl)-2-
propylindole-6-carboxylate; 4-bromo-5-(3-pyridylmethylamino)-6-(3-(4-
chlorophenyl) propoxy)-3- (2H)pyridazinone; I-methyl-5(5-morpholinoacetyl-2-n-
propoxyphenyl)-3-n-propyl-1,6-dihydro- 7H-pyrazolo(4,3-d)pyrimidin-7-one; 1-[4-
[(1,3-benzodioxol-5-ylmethyl)arnino]-6-chloro-2- quinazolinyl]-4-
piperidinecarboxylic acid, monosodium salt; Pharmaprojects No. 4516 (Glaxo
Wellcome); Pharmaprojects No. 5051 (Bayer); Pharmaprojects No. 5064 (Kyowa
Hakko; see WO 96/26940); Pharmaprojects No. 5069 (Schering Plough); GF-
196960 (Glaxo Wellcome); E-8010 and E-4010 (Eisai); Bay-38-3045 & 38-9456
(Bayer) and Sch-51866.
According to a third aspect of the invention there is provided a
pharmaceutical
pack comprising: a pharmaceutical composition comprising a PDE5 inhibitor,
directions relating to the use of the composition for treating wounds, and a
container.
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
7
For the avoidance of doubt, the PDE 5 inhibiting compounds referred to above
which are described in detail in the referenced published patent
specifications
mentioned above specifically form a part of this disclosure and represent a
part
of the inventive subject matter of this application.
The suitability of any particular cGMP PDE5 inhibitor can be readily
determined
by evaluation of its potency and selectivity using literature methods followed
by
evaluation of its toxicity, absorption, metabolism, pharmacokinetics, etc in
accordance with standard pharmaceutical practice.
Preferably, the cGMP PDES inhibitors have an IC50 for PDE5 at less than 100
nanomolar, more preferably, at less than 50 nanomolar, more preferably still
at
less than 10 nanomolar.
IC50 values for the cGMP PDE5 inhibitors may be determined using established
literature methodology, for example as described in EP0463756-B1 and
EP0526004-A1.
Preferably the cGMP PDE5 inhibitors used in the invention are selective for
the
PDE5 enzyme. Preferably they are selective over PDE3, more preferably over
PDE3 and PDE4. Preferably, the cGMP PDE5 inhibitors of the invention have a
selectivity ratio greater than 100 more preferably greater than 300, over PDE3
and more preferably over PDE3 and PDE4.
Selectivity ratios may readily be determined by the skilled person. 1C50
values for
the PDE3 and PDE4 enzyme may be determined using established literature
methodology, see S A Ballard et al, Journal of Urology, 1998, vol. 159, pages
2164-2171.
To be effective as a treatment, the compounds of the invention are preferably
orally bioavailable. Oral bioavailablity refers to the proportion of an orally
administered drug that reaches the systemic circulation. The factors that
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
8
determine oral bioavailability of a drug are dissolution, membrane
permeability
and metabolic stability. Typically, a screening cascade of firstly in vitro
and then
in vivo techniques is used to determine oral bioavailablity.
Dissolution, the solubilisation of the drug by the aqueous contents of the
gastro-
intestinal tract (GIT), can be predicted from in vitro solubility experiments
conducted at appropriate pH to mimic the GIT. Preferably the compounds of the
invention have a minimum solubility of 50 mcg/ml. Solubility can be determined
by standard procedures known in the art such as described in Adv. Drug Deliv.
Rev. 23, 3-25, 1997.
Membrane permeability refers to the passage of the compound through the cells
of the GIT. Lipophilicity is a key property in predicting this and is defined
by in
vitro Log D,.4 measurements using organic solvents and buffer. Preferably the
compounds of the invention have a Log D,.4 of -2 to +4, more preferably -1 to
+2.
The log D can be determined by standard procedures known in the art such as
described in J. Pharm. Pharmacol. 1990, 42:144.
Cell monolayer assays such as caco-2 add substantially to prediction of
2o favourable membrane permeability in the presence of efflux transporters
such as
p-glycoprotein, so-called caco-2 flux. Preferably, compounds of the invention
have a caco-2 flux of greater than 2x10-scms-', more preferably greater than
5x10-scms'. The caco flux value can be determined by standard procedures
known in the art such as described in J. Pharm. Sci, 1990, 79, 595-600
Metabolic stability addresses the ability of the GIT or the liver to
metabolise
compounds during the absorption process: the first pass effect. Assay systems
such as microsomes, hepatocytes etc are predictive of metabolic liability:
Preferably the compounds of the Examples show metabolic stability in the assay
system that is commensurate with an hepatic extraction of less then 0.5.
Examples of assay systems and data manipulation are described in Curr. Opin.
Drug Disc. Devel., 201, 4, 36-44, Drug Met. Disp.,2000, 28, 1518-1523
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
9
Because of the interplay of the above processes further support that a drug
will
be orally bioavailable in humans can be gained by in vivo experiments in
animals.
Absolute bioavailability is determined in these studies by administering the
compound separately or in mixtures by the oral route. For absolute
determinations (% absorbed) the intravenous route is also employed. Examples
of the assessment of oral bioavaiiability in animals can be found in Drug Met.
Disp.,2001, 29, 82-87; J. Med Chem , 1997, 40, 827-829, Drug Met. Disp.,1999,
27, 221-226
Preferably the cGMP PDES inhibitor is Sildenafil.
The cGMP PDE5 inhibitors can be administered alone but, in human therapy will
generally be administered in admixture with a suitable pharmaceutical
excipient
diluent or carrier selected with regard to the intended route of
administration and
standard pharmaceutical practice.
For example, the cGMP PDE5 inhibitors can be administered orally, buccally or
sublingually in the form of tablets, capsules, ovules, elixirs, solutions or
suspensions, which may contain flavouring or colouring agents, for immediate-,
delayed-, modified-, or controlled-release applications.
Such tablets may contain excipients such as microcrystalline cellulose,
lactose,
sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine,
disintegrants such as starch (preferably corn, potato or tapioca starch),
sodium
starch glycollate, croscarmellose sodium and certain complex silicates, and
granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl
cellulose,
hydroxypropylcellulose, sucrose, gelatin and acacia. Additionally, lubricating
agents such as magnesium stearate, stearic acid, glyceryl behenate and talc
may be included.
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
Solid compositions of a similar type may also be employed as fillers in
gelatin
capsules. Preferred excipients in this regard include lactose, starch, a
cellulose,
milk sugar or high molecular weight polyethylene glycols. For aqueous
suspensions and/or elixirs, the cGMP PDE5 inhibitors of the invention may be
5 combined with various sweetening or flavouring agents, colouring matter or
dyes,
with emulsifying andlor suspending agents and with diluents such as water,
ethanol, propylene glycol and glycerin, and combinations thereof.
The cGMP PDE5 inhibitors can also be administered parenterally, for example,
1 o intravenously, intra-arterially, intraperitoneally, intramuscularly or
subcutaneously, or they may be administered by infusion techniques. For such
parenteral administration they are best used in the form of a sterile aqueous
solution which may contain other substances, for example, enough salts or
glucose to make the solution isotonic with blood. The aqueous solutions should
be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The
preparation of suitable parenteral formulations under sterile conditions is
readily
accomplished by standard pharmaceutical techniques well-known to those skilled
in the art.
The following dosage levels and other dosage levels herein are for the average
human subject having a weight range of about 65 to 70 kg. The skilled person
will readily be able to determine the dosage levels required for a subject
whose
weight falls outside this range, such as children and the elderly.
The dosage of cGMP PDE5 inhibitor in such formulations will depend on its
potency, but can be expected to be in the range of from 1 to 500 mg for
administration up to three times a day. For oral and parenteral administration
to
human patients, the daily dosage level of the cGMP PDE5 inhibitor will usually
be
from 5 to 500 mg (in single or divided doses). In the case of sildenafil, a
preferred
dose is in the range 10 to 100 mg (e.g. 10, 25, 50 and 100 mg) which can be
administered once, twice or three times a day (preferably once). However the
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
11
precise dose will be as determined by the prescribing physician and will
depend
on the age and weight of the patient and severity of the symptoms.
Thus, for example, tablets or capsules of the cGMP PDES inhibitor may contain
from 5 to 250 mg (e.g. 10 to 100 mg) of active compound for administration
singly or two or more at a time, as appropriate. The physician in any event
will
determine the actual dosage which will be most suitable for any individual
patient
and it will vary with the age, weight and response of the particular patient.
The
above dosages are exemplary of the average case. There can, of course, be
1 o individual instances where higher or lower dosage ranges are merited and
such
are within the scope of this invention.
The cGMP PDE5 inhibitors can also be administered intranasally or by
inhalation
and are conveniently delivered in the form of a dry powder inhaler or an
aerosol
spray presentation from a pressurised container, pump, spray or nebuliser with
the use of a suitable propellant, e.g. dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as
1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane, carbon dioxide
or
other suitable gas. In the case of a pressurised aerosol, the dosage unit may
be
2o determined by providing a valve to deliver a metered amount. The
pressurised
container, pump, spray or nebuliser may contain a solution or suspension of
the
cGMP PDES inhibitor, e.g. using a mixture of ethanol and the propellant as the
solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
Capsules and cartridges (made, for example, from gelatin) for use in an
inhaler or
insufflator may be formulated to contain a powder mix of the cGMP PDE5
inhibitor and a suitable powder base such as lactose or starch.
Aerosol or dry powder formulations are preferably arranged so that each
metered
dose or "puff' contains from 1 to 50 mg of the cGMP PDE5 inhibitor, for
delivery
to the patient. The overall daily dose with an aerosol will be in the range of
from
1 to 50 mg which may be administered in a single dose or, more usually, in
divided doses throughout the day.
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
12
Alternatively, the cGMP PDES inhibitors can be administered in the form of a
suppository or pessary.
The cGMP PDE5 inhibitor may be applied topically in the form of a gel,
hydrogel,
lotion, solution, cream, ointment or dusting powder. The cGMP PDES inhibitors
may also be dermally or transdermally administered, for example, by the use of
a
skin patch.
Since ulcers occur on the skin surface, topical administration is a preferred
route
of administration.
For application topically to the skin, the cGMP PDE5 inhibitors can be
formulated
as a suitable ointment containing the inhibitor suspended or dissolved in, for
example, a mixture with one or more of the following: mineral oil, liquid
petrolatum, white petrolatum, propylene glycol, polyoxyethylene
polyoxypropylene compound, emulsifying wax and water. Alternatively, they can
be formulated as a suitable lotion or cream, suspended or dissolved in, for
example, a mixture of one or more of the following: mineral oil, sorbitan
2o monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl
esters
wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
The cGMP PDE5 inhibitors may also be used in combination with a cyclodextrin.
Cyclodextrins are known to form inclusion and non-inclusion complexes with
drug
molecules. Formation of a drug-cyclodextrin complex may modify the solubility,
dissolution rate, bioavailability andlor stability property of a drug
molecule. Drug-
cyclodextrin complexes are generally useful for most dosage forms and
administration routes. As an alternative to direct complexation with the drug
the
cyclodextrin may be used as an auxiliary additive, e.g. as a carrier, diluent
or
solubiliser. Alpha-, beta- and gamma-cyclodextrins are most commonly used and
suitable examples are described in WO-A-91/11172, WO-A-94/02518 and WO-A-
98155148.
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
13
Generally, in humans, oral administration of the cGMP PDE5 inhibitors is the
preferred route, being the most convenient. In circumstances where the
recipient
suffers from a swallowing disorder or from impairment of drug absorption after
oral administration, the drug may be administered parenterally, sublingually
or
buccally.
The cGMP PDE5 inhibitors of the invention can also be administered in
combination with one or more of the following:
a, -Adrenergic receptor antagonist compounds also known as cc
adrenoceptors or a-receptors or a-blockers. Suitable compounds for use
herein include: the a-adrenergic receptors as described in PCT application
W099/30697 published on 14th June 1998, the disclosures of which
relating to a-adrenergic receptors are incorporated herein by reference
and include, selective cc,-adrenoceptors or a.~ adrenoceptors and non-
selective adrenoceptors, suitable oc,-adrenoceptors include: phentolamine,
phentolamine mesylate, trazodone, alfuzosin, indoramin, naftopidil,
tamsulosin, dapiprazole, phenoxybenzamine, idazoxan, efaraxan,
2o yohimbine, rauwolfa alkaloids, Recordati 15/2739, SNAP 1069, SNAP
5089, RS17053, SL 89.0591, doxazosin, terazosin, abanoquil and
prazosin; a,~ blockers from US 6,037,346 [14th March 2000] dibenarnine,
tolazoline, trimazosin and dibenarnine; cc-adrenergic receptors as
described in US patents: 4,188,390; 4,026,894; 3,511,836; 4,315,007;
3,527,761; 3,997,666; 2,503,059; 4,703,063; 3,381,009; 4,252,721 and
2,599,000 each of which is incorporated herein by reference; a,2
Adrenoceptors include: clonidine, papaverine, papaverine hydrochloride,
optionally in the presence of a cariotonic agent such as pirxamine;
ii) NO-donor (NO-agonist) compounds. Suitable NO-donor compounds for
use herein include organic nitrates, such as mono- di or tri-nitrates or
organic nitrate esters including glyceryl brinitrate (also known as
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
14
nitroglycerin), isosorbide 5-mononitrate, isosorbide dinitrate,
pentaerythritol tetranitrate, erythrityl tetranitrate, sodium nitroprusside
(SNP), 3-morpholinosydnonimine molsidomine, S-nitroso- N-acetyl
penicilliamine (SNAP) S-nitroso-N-glutathione (SNO-GLU), N-hydroxy - L-
arginine, amylnitrate, linsidomine, linsidomine chlorohydrate, (SIN-1) S-
nitroso - N-cysteine, diazenium diolates,(NONOates), 1,5-pentanedinitrate,
L-arginene, ginseng, zizphi fructus, molsidomine, Re - 2047, nitrosylated
maxisylyte derivatives such as NMI-678-11 and NMI-937 as described in
published PCT application WO 0012075;
iii) Vasodilator agents. Suitable vasodilator agents for use herein include
nimodepine, pinacidil, cyclandelate, isoxsuprine, chloroprumazine, halo
peridol, Rec 15!2739, trazodone, pentoxifylline;
iv) Thromboxane A2 agonists;
v) Substrates for NO-synthase, such as L-arginine;
vi) Calcium channel blockers such as amlodipine;
vii) Steroidal or non-steroidal anti-inflammatory agents;
viii) Matrix metalloprotease inhibitors (MMP), particularly MMP-3, MMP-12 and
MMP-13; and
ix) Urokinase type plasminogen activator inhibitors (uPA).
Particularly preferred agents for use in combination with the PDES inhibitors
of
the invention for treating wounds include: MMP inhibitors (particularly
inhibitors of
3o MMP-3, MMP-12 and MMP-13); uPA inhibitors; and vasodilator agents
(particularly pentoxyfyline).
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
Preferably the MMP inhibitor is a MMP-3 and/or MMP-13 inhibitor such as those
specifically and generically disclosed in W099/35124, EP 931788, W099/29667
or WO00/74681. Especially preferred MMP inhibitors are those of the Examples
of W099/35124, EP 931788, W099129667 and WO00/74681.
5
Preferably the uPA inhibitor is selected from those specicially and
generically
disclosed in W099/20608, EP 1044967 or WO00/05214. Especially preferred
uPA inhibitors are those of the Examples of W099/20608, EP 1044967 and
WO00/05214.
It is to be appreciated that all references herein to treatment include
curative,
palliative and prophylactic treatment.
The utility of the present invention is illustrated by the following figures
in which:
Figure 1 is a photomicrograph of a paraffin section of skin at 10 x
magnification;
Figure 2 is a photomicrograph of a paraffin section of skin at 20 x
magnification;
Figure 3 is a photomicrograph of a paraffin section of skin at 20 x
magnification;
Figure 4 is a photomicrograph of a paraffin section of skin at 40 x
magnification;
Figure 5 is a photomicrograph of a paraffin section of skin at 60 x
magnification;
2o and
Figure 6 is a photomicrograph of a paraffin section of skin at 60 x
magnification.
Anti-human polyclonal antiserum was raised in rabbits and affinity purified
against the LIP-1 [MERAGPSFGQQR] peptide n accordance with the method of
Fawcett et al (Proc Natl Acod Sci USA 2000; 97:3702-3707), corresponding to
amino acid residues 1-12 of human PDE5A1. LIP-1 is specific for PDE5 A1.
4~.m sections of formalin-fixed paraffin embedded tissue were cut and picked
up
on to APES (3-aminopropyltriethoxysilane) coated slides and dried at
60°C for 1
3o hour. Sections were de-waxed and rehydrated followed by proteolytic antigen
retrieval in 0.1 % trypsin in 0.1 % calcium chloride [pH7-6] at 37°C
for 8 minutes.
Following a brief water wash, endogenous peroxidase activity was blocked by
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
16
incubation in 9m1 H~Oa made up to 100m1 with distilled water for 10 minutes.
Sections were washed in tap water then transferred to PBS. Excess buffer was
removed from the slide and test sections were incubated in LIP-1 antibody
diluted 1:600 in PBS for 1 hour at room temperature. Negative controls were
included by omission of the primary antibody. Positive control tissue used was
human corpus cavernosum. Immunodetection was carried out using DAKO
Rabbit Envision TM system with 3-amino-9-ethylcarbazole (3AEC) as a substrate
chromogen (red/brown staining).
Figure 1 illustrates a section of reactive but non-inflamed skin at the edge
of a
skin wound. The positive staining of the smooth muscle cells within the media
of
the venules and negative fibroblasts indicates the expression of PDES in the
healing wound. Hyperplastic but intact squamous epithelium 1 is negative. The
underlying dermis contains mature scar tissue with small and large venules 2.
Note the positive dark staining of the smooth muscle cells within the media of
the
venules (Original mag. x 10).
Figure 2 is a paraffin section taken from the border between a healing ulcer
of 14
days (left) and intact epithelium (right). Again, the positive staining of the
smooth
muscle cells within the media of the venules (right) and the spindle cells
(myofibroblasts) within the base of the ulcer (left) indicates PDES
expression.
Hyperplastic but intact squamous epithelium (right) and necrotic inflammatory
exudate 3 is negative. Note the positive dark staining of the smooth muscle
cells
within the media of the venules 4 and of spindle cells within the base of the
ulcer
5 (original mag. x20).
Figure 3 is a paraffin section taken from the healed ulcer base where
fascicles of
young scar tissue have replaced normal dermal structures. Positive staining of
some of the spindle cells (myofibroblasts) (8) and of some vascular structures
is
again indicative of PDE 5 expression. (Original mag x20).
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
17
Figure 4 is a higher power view of the paraffin section of skin of Figure 3.
The
section is taken from the healed ulcer base where fascicles of young scar
tissue
have replaced normal dermal structures. PDE 5 expression is illustrated by the
positive staining of some of the spindle cells (myofibroblasts) (9) and of
some of
the microvessels which have thin media (10). (Original mag x40).
Figure 5 is a higher powered view of Figure 4 and shows a section taken from
the healed ulcer base of Figure 4 where fascicles of young scar tissue have
replaced normal dermal structures. There is positive staining of some of the
spindle cells (myofibroblasts) (11) which are present in acellular collagen.
The
immunolocalisation in the cytoplasm of some of these spindle cells has a
patchy
distribution. Positive staining of the medial smooth muscle cells within a
small
arteriole (12) indicates PDE 5 expression. There is negative staining of the
lining
endothelial cells (13) indicating the absence of PDE 5. (Original mag. x60).
2o Figure 6 is also a higher powered view of Figure 4 showing a section from
the
healed ulcer base in an area of relatively young scar tissue. Again, positive
staining of some of the spindle cells (myofibroblasts) (14) and medial smooth
muscle cells within the small arteriole (centre) (15) is indicative of PDE 5.
In
some of these spindle cells the immunolocalisation has a patchy distribution.
(Original mag. x60).
The following formulation examples are illustrative only and are not intended
to
limit the scope of the invention. Active ingredient means a cGMP PDE5
inhibitor.
Formulation 1:
A tablet is prepared using the following ingredients
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
18
Sildenafil citrate (50 mg) is blended with cellulose (microcrystalline),
silicon
dioxide, stearic acid (fumed) and the mixture is compressed to form tablets.
CA 02415791 2003-O1-16
WO 02/15893 PCT/IBO1/01470
19
Formulation 2
An intravenous formulation may be prepared by combining the active ingredient
(100 mg) with isotonic saline (1000 ml).
