U.S. patent application number 12/176782 was filed with the patent office on 2009-01-22 for wound healing.
This patent application is currently assigned to Photopharmica Limited. Invention is credited to Stanley Beams Brown, Cassandra Clare O'Grady.
Application Number | 20090023715 12/176782 |
Document ID | / |
Family ID | 33306894 |
Filed Date | 2009-01-22 |
United States Patent
Application |
20090023715 |
Kind Code |
A1 |
Brown; Stanley Beams ; et
al. |
January 22, 2009 |
Wound Healing
Abstract
A phenothiazinium compound, compositions and medicaments for use
in promoting wound healing and a method for promoting wound healing
or cosmetic use by applying or administering a phenothiazinium
compound to a wound site or the skin, and optionally exposing the
wound site or skin to light.
Inventors: |
Brown; Stanley Beams;
(Burley-in-Wharfedale, GB) ; O'Grady; Cassandra
Clare; (Leeds, GB) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
Photopharmica Limited
Leeds
GB
|
Family ID: |
33306894 |
Appl. No.: |
12/176782 |
Filed: |
July 21, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11723523 |
Mar 20, 2007 |
7407953 |
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12176782 |
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PCT/GB2005/003536 |
Sep 14, 2005 |
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11723523 |
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Current U.S.
Class: |
514/225.2 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 19/08 20180101; A61P 9/00 20180101; C07D 279/28 20130101; A61P
1/02 20180101; A61P 17/16 20180101; A61P 17/02 20180101; A61P 41/00
20180101; A61P 35/00 20180101; A61P 17/00 20180101; A61P 3/10
20180101; C09B 5/60 20130101 |
Class at
Publication: |
514/225.2 |
International
Class: |
A61K 31/54 20060101
A61K031/54; A61P 17/00 20060101 A61P017/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 20, 2004 |
GB |
0420888.8 |
Claims
1-9. (canceled)
10. A method of cosmetic treatment comprising: a) applying or
administering to an area to be treated an effective amount of a
composition comprising a compound of Formula (I) ##STR00004##
wherein: R.sup.1-R.sup.6 each independently is selected from H,
optionally substituted C.sub.1-6-alkyl, F, Cl, Br and I; A and B
each independently is selected from: ##STR00005## wherein: Q is
selected from H and optionally substituted C.sub.1-4-alkyl; Z is
selected from C(R.sup.a).sub.2, O, S, SO.sub.2 and NR.sup.a in
which R.sup.a is selected from H and optionally substituted
C.sub.1-4-alkyl; R.sup.7 and R.sup.8 each independently is selected
from H and optionally substituted C.sub.1-6-alkyl; Y is a
counteranion; and p is 1, 2 or 3; and b) optionally exposing the
treated area to light.
11. The method according to claim 10, wherein said cosmetic
treatment is selected from skin rejuvenation, skin thickening,
reduction of scar formation and birth mark removal.
12. The method according to claim 10, wherein said cosmetic
treatment is skin rejuvenation.
13. The method according to claim 10, wherein said cosmetic
treatment is skin thickening.
14. The method according to claim 10, wherein said cosmetic
treatment is reduction of scar formation.
15. The method according to claim 10, wherein said cosmetic
treatment is birth mark removal.
16. The method according to claim 10, wherein said composition
further comprises a pharmaceutically acceptable diluent or
excipient.
17. The method according to claim 10, wherein R.sup.7 and R.sup.8
are independently selected from optionally-substituted C.sub.3-6
alkyl.
18. The method according to claim 10, wherein A and B are of
formula --NR.sup.7R.sup.8.
19. The method according to claim 10, wherein Y is a counterion
selected from F.sup.-, Br.sup.-, Cl.sup.-, I.sup.-, NO.sub.3.sup.-,
SCN.sup.-, ClO.sub.3.sup.-, ClO.sub.4.sup.-, IO.sub.3.sup.-,
BF.sub.4.sup.-, HSO.sub.4.sup.-, H.sub.2PO.sub.4.sup.-,
CH.sub.3SO.sub.4.sup.-, N.sub.3, SO.sub.4.sup.2-, HPO.sub.4.sup.2-,
PO.sub.4.sup.3-, acetate, lactate, citrate, tartrate, glycolate,
glycerate, glutamate, .beta.-hydroxyglutamate, glucouronate,
gluconate, malate and aspartate.
20. The method according to claim 10, wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5 and R.sup.6 each represents H, and A and
B represent --NR.sup.7R.sup.8 wherein R.sup.7 and R.sup.8 represent
butyl.
21. A method of cosmetic treatment comprising: a) applying or
administering to an area to be treated an effective amount of a
composition comprising a compound of Formula (1) ##STR00006##
wherein: R.sup.1-R.sup.6 each independently is selected from H,
optionally substituted C.sub.1-6-alkyl, F, Cl, Br and I; A and B
each independently is selected from: ##STR00007## wherein: Q is
selected from H and optionally substituted C.sub.1-4-alkyl; Z is
selected from C(R.sup.a).sub.2, O, S, SO.sub.2, NR.sup.a in which
R.sup.a is selected from H and optionally substituted
C.sub.1-4-alkyl; R.sup.7 and R.sup.8 each independently is selected
from optionally substituted C.sub.3-6-alkyl; Y is a counteranion;
and p is 1, 2 or 3; and b) optionally exposing the treated area to
light.
Description
FIELD OF THE INVENTION
[0001] This invention relates to wound healing, the use of certain
heterocyclic compounds and compositions and medicaments containing
them in a wound healing process and for cosmetic uses.
BACKGROUND INFORMATION
[0002] The rate of chronic and acute wound healing can be delayed
or impaired by a number of factors (exogenous and endogenous) and a
variety of medical conditions. Examples include infection,
ulceration particularly through diabetes, circulation problems
associated with vascular disease, malnutrition, stress, cancer
radiotherapy and/or chemotherapy, compromise of the immune system
or simply due to the normal aging process. At present there is a
clear need for therapeutic and cosmetic approaches that promote
wound healing processes.
[0003] The literature describes the PDT use of various
photosensitisers in attempts to improve wound healing. For example,
Photofrin (a porphyrin, Photofrin is a trade mark of Johnson &
Johnson) showed delayed wound healing in rat skin flap
reconstruction (Kubler et al Lasers in surgery and Medicine (1996),
18(4), 397-405. Parekh et al concluded in Lasers in surgery and
Medicine (1999), 24(5), 375-381 that a benzoporphyrin and a
phthalocyanine didn't alter wound healing. Lambrects et al
concluded in Photochemical & Photobiological Sciences (2005),
4(7), 503-509 that a porphyrin delayed wound healing. Hamblin
concluded in Journal of infectious diseases (2003), 187(11),
1717-25 that for a photosensitiser (Poly-lysine-chlorin e6) topical
PDT did not accelerate wound healing, compared with that in
untreated wounds. Belmont et al in the Laryngoscope (1999), 109(6),
886-90 using Photofrin concluded that PDT has been shown to delay
wound healing. Heckenkamp et al in Journal of vascular surgery,
(2000), 31(6), 1168-77 concludes that local photodynamic action of
methylene blue exerts a retarding effect on intimal hyperplasia
(one of the natural wound healing processes).
[0004] Compounds, methods and compositions/medicaments have now
been identified which address these problems and provide a basis
for promoting the wound healing process.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] The following figures represent exemplary embodiments of the
invention and are not intended to be limiting.
[0006] FIGS. 1A and 1B depict the results involving chronic leg
ulcer. The wound had shown no response to conventional treatments
for 18 months prior to treatment with
3,7-bis(N,N-dibutylamino)phenothiazin-5-ium bromide and PDT.
[0007] FIGS. 2A and 2B depict results involving chronic leg ulcer.
The wound had shown no response to conventional treatments for 7
months prior to treatment with
3,7-bis(N,N-dibutylamino)phenothiazin-5-ium bromide and PDT
[0008] FIGS. 3A and 3B depict results involving chronic leg ulcer.
The wound had shown no response to conventional treatments for 14
months prior to treatment with 3,7-bis(N,N-dibutylamino)
phenothiazin-5-ium bromide and PDT.
[0009] FIGS. 4A and 4B depict results involving chronic leg ulcer.
The wound had shown no response to conventional treatments for 3
months prior to treatment with 3,7-bis(N,N-dibutylamino)
phenothiazin-5-ium bromide and PDT.
SUMMARY OF THE INVENTION
[0010] According to the present invention there is provided a
phenothiazinium compound of Formula (1) for use in promoting wound
healing, in which the phenothiazinium compound is of Formula
(1):
##STR00001##
wherein: R.sup.1-R.sup.6 each independently is selected from H,
optionally substituted C.sub.1-8-alkyl, F, Cl, Br and I; A and B
each independently is selected from:
##STR00002##
in which:
[0011] Q is selected from H and optionally substituted
C.sub.1-8-alkyl;
[0012] Z is selected from C(R.sup.a).sub.2, O, S, SO.sub.2,
NR.sup.a in which each R.sup.a independently is selected from H and
optionally substituted C.sub.1-8-alkyl;
R.sup.7 and R.sup.8 each independently is selected from H and
optionally substituted C.sub.1-8-alkyl; Y is a counteranion; and p
is 1, 2 or 3. Where any one of R.sup.1-R.sup.8, R.sup.a or Q is
optionally substituted alkyl the optional substituents are
preferably selected from aryl, particularly Ph; F; Cl; Br; I; OH;
OC.sub.1-4-alkyl, particularly OCH.sub.3, OC.sub.2H.sub.5,
OC.sub.3H.sub.7; CN; OCOC.sub.1-4-alkyl, particularly OCOCH.sub.3;
optionally substituted C.sub.3-6-cycloalkyl particularly
cyclohexyl, methyl-substituted cyclohexyl, cyclopentyl, and
methyl-substituted cyclopentyl; COOH; COOC.sub.1-4-alkyl;
SO.sub.3H;
[0013] The alkyl groups represented by R.sup.1-R.sup.8, R.sup.a or
Q may be straight or branched chain and may optionally include C--C
double or triple bonds. R.sup.1-R.sup.6 each independently is
preferably selected from H, CH.sub.3, F, Cl, Br and I.
[0014] Where A and B are both NR.sup.7R.sup.8, R.sup.7 and R.sup.8
each independently preferably is selected from H and
C.sub.1-6-alkyl optionally substituted by Ph, F, Cl, Br, I, OH,
OCH.sub.3, OC.sub.2H.sub.5, OC.sub.3H.sub.7, CN, OCOCH.sub.3,
cyclohexyl, methyl-substituted cyclohexyl, cyclopentyl, and
methyl-substituted cyclopentyl.
Q is preferably H or CH.sub.3. Z is preferably selected from
CH.sub.2, O, S, SO.sub.2, NH, NCH.sub.3,
NC.sub.2H.sub.5NCH.sub.2CH.sub.2OH and NCOCH.sub.3. R.sup.a is
preferably selected from H, CH.sub.3, C.sub.2H.sub.5,
CH.sub.2CH.sub.2OH and COCH.sub.3. Y may be an organic or inorganic
counteranion is preferably selected from F.sup.-, Br.sup.-,
Cl.sup.-, I.sup.-, NO.sub.3.sup.-, SCN.sup.-, ClO.sub.3.sup.-,
ClO.sub.4.sup.-, IO.sub.3.sup.-, BF.sub.4.sup.-, HSO.sub.4.sup.-,
H.sub.2PO.sub.4.sup.-, CH.sub.3SO.sub.4.sup.-, N.sub.3,
SO.sub.4.sup.2-, HPO.sub.4.sup.2-, PO.sub.4.sup.3-, acetate,
lactate, citrate, tartrate, glycolate, glycerate, glutamate,
.beta.-hydroxyglutamate, glucouronate, gluconate, malate and
aspartate.
[0015] In compounds of Formula (1) where one or both of A and B
is
##STR00003##
one or both of R.sup.7 and R.sup.8 together with the nitrogen atom
to which they are attached may form an optionally substituted 5-,
6- or 7-membered ring. In compounds of Formula (1) the groups
R.sup.1 and R.sup.2 and/or R.sup.4 and R.sup.5 together with the
carbon atoms to which they are attached may form an optionally
substituted 5-, 6- or 7-membered ring.
[0016] The rings formed from one or both of R.sup.7 and R.sup.8
together with the nitrogen atom to which they are attached and from
the groups R.sup.1 and R.sup.2 and/or R.sup.4 and R.sup.5 together
with the carbon atoms to which they are attached may be saturated
or unsaturated.
[0017] The optional substituents for the optionally substituted 5-,
6- or 7-membered rings formed from R.sup.7 and R.sup.8 together
with the nitrogen atom to which they are attached, and formed from
the groups R.sup.1 and R.sup.2 and/or R.sup.4 and R.sup.5 together
with the carbon atoms to which they are attached may be selected
from any of those substituents described above for R.sup.1.
[0018] Preferably methylene blue and ring substituted derivatives
thereof and toluidine blue are excluded from the compounds of
Formula (1). According to a feature of the present invention there
is provided a compound of Formula (1) for use as a wound healing
agent.
[0019] According to a feature of the present invention there is
provided a wound healing agent comprising a compound of Formula (1)
and a pharmaceutically acceptable diluent or excipient.
[0020] The present invention also provides a composition,
preferably a pharmaceutical composition, for use in promoting wound
healing that comprises a phenothiazinium compound of Formula (1)
together with a diluent or excipient.
[0021] According to a further feature of the present invention
there is provided a use of a compound according to Formula (1) in
the manufacture of a medicament for promoting wound healing.
DETAILED DESCRIPTION
[0022] The medicament may be in a form for topical or systemic use
and is preferably in a form for topical use.
[0023] The medicaments are preferably administered topically more
preferably administered as a cream, suspension, emulsion, gel,
ointment, salve, stick, soap, paste, or via an antimicrobial
dressing such as a bandage impregnated with a compound of Formula
(1) or a composition or medicament comprising a compound of Formula
(1).
[0024] The wound healing agents, compositions and medicaments
provided by the present invention may comprise mixtures of two or
more different compounds of Formula (1); they may also further
comprise one or more different therapeutic or active agents.
[0025] According to a further feature of the present invention
there is provided a method for promoting wound healing in both
chronic and acute wounds by applying or administering a
phenothiazinium compound of Formula (1) to a wound site, and
optionally exposing the wound site to light.
[0026] More specifically the method for promoting wound healing
includes the following steps:
a) preparing a composition or medicament comprising a compound of
Formula (1); b) applying or administering an effective amount of
the composition or medicament to a wound area to be treated: and c)
optionally exposing the wound area to light.
[0027] The compounds, compositions, wound healing agents and
medicaments of the present invention may be used to treat any
condition where the integrity of tissue is damaged, and thus covers
chronic and acute wounds, wounds in connective tissue and wounds in
muscle, bone and nerve tissue.
[0028] The wounds may include, but are not limited to the
following: surgical wounds; bites; burns; acid and alkali burns;
cold burn (frostbite), sun burn, minor cuts, major cuts, abrasions,
lacerations, wounds caused by gunshot or knife injury; wounds
caused by congenital disorders; wounds following dental surgery;
periodontal disease; wounds following trauma; tumour associated
wounds, which can be classified as malignant cutaneous ulcers
related to the primary tumour or metastases; ulcers, leg ulcers;
foot ulcers; pressure sores and corneal wounds.
[0029] Wounds can be classified by having either an acute or
chronic etiology. Acute wounds are caused by external damage to
intact skin and include surgical wounds, bites, burns, cuts and
abrasions, as well as more traumatic wounds such as lacerations and
those caused by crush or gun shot injuries.
[0030] Chronic wounds are most frequently caused by endogenous
mechanisms associated with a predisposing condition that ultimately
compromises the integrity of dermal or epithelial tissue.
Pathophysiological abnormalities that may dispose to the formation
of chronic wounds such as leg ulcers, foot ulcers, and pressure
sores include compromised tissue perfusion as a consequence of
impaired arterial supply (peripheral vascular disease) or impaired
venous drainage (venous hypertension) and diseases such as diabetes
mellitus. Advancing age, obesity, smoking, poor nutrition and
immunosuppression associated with disease (e.g., AIDS) or drugs
(e.g. chemotherapy or radiation therapy) may also exacerbate
chronic ulceration. Pressure or decubitis ulcers have a different
etiology from other chronic wounds in that they are caused by
sustained external skin pressure, most commonly in the buttocks,
sacrum, and heels. All chronic wounds heal slowly and in an
unpredictable manner (Bowler et al., Clinical Microbiology Reviews,
14 (2) 244). For example, in a study of diabetic foot ulcers, with
20 weeks of good wound care only 31% of the ulcers healed
(Margolis, et al. Diabetes Care, 22 (5), 692).
[0031] The present compounds, compositions, wound healing agents
and medicaments, and method for promoting wound healing may be used
any time from wound identification to treat chronic and acute
wounds. For wounds that are associated with surgical interventions,
such times include pre-, during or post surgery, prior to surgical
repair, and post surgical repair, to aid wound healing, preferably
at a time from 0 to 7 days after, more preferably from 2 to 6 days
after applying or administering the compound of Formula (1). After
application or administration of the compound, composition, wound
healing agent or medicament to the area to be treated, the area to
be treated is optionally exposed to light, and where exposed to
light, the time between application or administration of the
compound and exposure to light is as defined below. They may also
be used any time pre-, during or post in surgical procedures for
example: tissue welding, skin grafting and tissue grafting
including grafting used in transplant surgery. They may be used to
heal inflammatory disorders of the skin that result in itching,
crusting, scaling or blisters, such as eczema, psoriasis and acne.
The method invention may also be used to treat forms of eczema for
example those that result from a weakened immune response (allergy)
such as atopic dermatitis.
[0032] Application or administration of the compounds,
compositions, wound healing agents and medicaments may need to be
repeated and typically from 1 to 20 treatments may be applied, more
preferably from 1 to 5 treatments to promote wound healing.
[0033] The compounds, compositions, wound healing agents,
medicaments and methods described herein are preferably used for
treating epithelial wounds, more preferably for treating skin
wounds.
[0034] According to a further feature of the present invention
there is provided a cosmetic agent for example for uses in skin
rejuvenation and thickening, reducing scar formation and birth mark
removal.
[0035] According to a further feature of the present invention
there is provided a method that may be used for cosmetic purposes,
including, but not limited to, skin rejuvenation and thickening,
reducing scar formation and birth mark removal. The cosmetic method
includes the following steps:
a) preparing a composition comprising a compound of Formula (1); b)
applying or administering an effective amount of the composition to
an area to be treated: and c) optionally exposing the wound area to
light.
[0036] Where the wound is an internal wound, such as a stomach
ulcer, the compound, composition, wound healing agent or medicament
may be applied via an endoscope and where it is desirable to expose
the wound site to light this may be achieved by use of an
endoscopic light source.
[0037] The compound of Formula (1) may be applied directly to the
wound site or in a pharmaceutically acceptable composition, wound
healing agent or medicament. The compound, composition, wound
healing agent or medicament may be administered locally or
topically and delivered via a variety of means, for example via a
spray, local injection, local infusion, cream, lotion, suspension,
emulsion, gel, ointment, salve, stick, soap, liquid aerosol, powder
aerosol, drops, paste, endoscopically or antimicrobial dressings
such as bandages. Preferably administration is topical, more
preferably as a cream, suspension, emulsion, gel, ointment, salve,
stick, soap, paste, or via an antimicrobial dressing, and
especially as a cream, suspension, emulsion, gel or ointment.
[0038] The compounds of Formula (1) may be formulated into a
variety of pharmaceutical compositions, wound healing agents or
medicaments which contain the compounds and pharmaceutically
acceptable carriers, excipients, adjuvants (each selected for
certain characteristics that permit optimal formulation); these may
include liposomes, nanoparticles, colloidal suspensions, micelles,
microemulsions, vesicles and nanospheres.
[0039] The compositions, wound healing agents or medicaments may
also comprise further components such as conventional delivery
vehicles and excipients including organic liquids such as alcohols
(for example ethanol, propanol and isopropanol), dimethyl
sulphoxide, glycols such as propylene glycol and polyethylene
glycol, liquid, semi-solid and solid paraffins, water, saline,
solubilisers such as castor oil derivatives for example ethoxylated
castor oils like Cremophor EL (trade mark BASF AG) or Tween (trade
mark, ICI Americas Inc.) types, Unguentum (trade mark, Merck KGaA),
Solutol (trade mark, BASF AG), isotonising agents such as urea,
glycerol, aminoethanol, propylene glycol, pH regulators, dyes,
gelling agents, thickeners, buffers, hydrocarbon waxes and
combinations thereof. The compositions of the invention may also
optionally include other carriers, stabilizers, preservatives or
adjuvants. For typical examples of these classes of compounds, see
Remington: The Science and Practice of Pharmacy, Lippincott,
Williams & Wilkins (2005), which is incorporated by reference
in its entirety.
[0040] The composition, wound healing agents or medicaments may
further comprise a mild reducing agent for example a sugar such as
glucose or dextrose; ascorbic acid; sodium metabisulphite and
nicotinamide adenine dinucleotide (NADH).
[0041] Typically the compositions, wound healing agents or
medicaments are prepared by mixing a compound of Formula (1) with
one or more pharmaceutically acceptable carriers at an appropriate
temperature, typically from 15.degree. to 40.degree. C. at an
appropriate pH, typically from pH 3 to 9 and preferably at a
physiologically acceptable pH such as from pH 6.5 to 7.5.
[0042] The concentration of the compounds of Formula (1) in the
compositions, wound healing agents or medicaments depends on the
compound's photosensitising ability and is typically in the range
from 0.001 to 20%.
[0043] Dry compositions, wound healing agents or medicaments which
may be reconstituted before use, are also provided in the present
invention. These may be prepared by dry mixing solid components or
preparing a liquid composition which is evaporated to dryness
generally under mild conditions under vacuum or in low temperature
ovens, freeze drying is a suitable drying technique.
[0044] The compounds of Formula (1) may also be administered
systemically by any convenient means, for example intravenously,
orally, sub-cutaneously, intramuscularly, directly into affected
tissues and organs or intraperitoneally.
[0045] Application or administration by any of the means described
above may need to be repeated and typically from 1 to 20 treatments
may be applied, more preferably from 1 to 5 treatments to promote
wound healing.
[0046] The dose rate of the compounds of Formula (1) for topical,
direct or systemic administration is preferably in the range from
0.1 to 2000 .mu.mol, preferably in the range 10 to 1000 .mu.mol,
more preferably from 50 to 500 .mu.mol. For systemic administration
such as by injection typical injections volumes are in the range
0.1 to 100 ml, preferably from 5 to 50 ml.
[0047] Where wound healing is promoted on exposure to light the
light is preferably at wavelengths of 600-800 nm, more preferably
at wavelengths from 630 nm to 700 nm
[0048] The light source may be any appropriate light source such as
light emitting diode (LED), a laser or laser diode, a broad
spectrum halogen lamp, or filtered lamp.
[0049] The light dose administered during PDT can vary but
preferably is from 1 to 200 J/cm.sup.2, more preferably from 5 to
100 J/cm.sup.2, especially from 5 to 75 and ideally from 5 to 25
J/cm.sup.2.
[0050] Wound healing promotion may be achieved by applying or
administering the drug alone or by applying or administering the
drug and exposing to light.
[0051] Generally, where light exposure is given this may be at the
time of drug administration or up to 12 hours after drug
administration and the time may be tailored according to the wound
being treated, the method of drug delivery and the specific
compound of Formula (I) used. Light exposure is preferably given at
any time from the time of drug administration up to 3 hours, more
preferably from the time of drug administration up to 1 hour. Where
the phenothiazinium compound is administered topically the wound is
preferably left occluded in the dark for up to 1 hour, more
preferably from 1 to 20 minutes, and especially from 8 to 12
minutes before exposure to light.
[0052] Wound healing promotion may also be achieved by applying or
administering the drug and exposing to light over many hours, for
example where the drug is applied to a wound and a light source is
included in a wound covering or dressing applied to the wound.
Where light is applied over many hours this typically is a lower
intensity light. The period where light is applied may be from 1
second to 100 hours.
[0053] Increasing the intensity (fluence rate) of the light dose
generally reduces light exposure times.
[0054] It is preferred that exposure to light is localised to the
area/region to be treated, and where wounds are being treated more
preferably localised to the wound itself.
[0055] After applying or administering the drug the wound may also
be exposed intermittently to light, for example light exposures of
1 second up to 72 hours may be used with intermittent periods of 1
second up to 72 hours where there is no light exposure
[0056] The compounds of Formula (1) and compositions, wound healing
agents and medicaments comprising compounds of Formula (1) may be
used in the dark or on exposure to light to promote wound healing.
In the dark it is the compound of Formula (1) alone that promotes
wound healing.
[0057] In preferred embodiments of the present inventions wound
healing promotion is achieved by applying or administering the drug
and exposing to light.
[0058] The phenothiazinium salts of Formula (1) may be synthesised
as follows:
1) Symmetrical phenothiazinium compounds where A=B a) Phenothiazine
is firstly brominated with bromine in glacial acetic acid to give
3,7-dibromophenothiazin-5-ium bromide, the suspension formed is
collected by filtration. b) the 3,7-dibromophenothiazin-5-ium
bromide is added to an amine represented by R.sup.7R.sup.8NH (in
which R.sup.7 and R.sup.8 are as defined above) or an N-heterocycle
in chloroform. The solid formed is collected by filtration and
purified by any suitable means such as by flash column
chromatography over silica gel 60, using chloroform,
chloroform/methanol (98/2) and then chloroform/methanol (90/10).
The product may be further purified by precipitation from
chloroform with petroleum ether (b.p. 60-80.degree. C.). 2)
Unsymmetrical phenothiazinium compounds where A.noteq.B a)
Phenothiazine in chloroform is cooled to below 5.degree. C. and a
solution of iodine in chloroform added. The solid formed is
collected by filtration, washed with chloroform until free of
iodine and then kept at room temperature under vacuum overnight to
give phenothiazin-5-ium tetraiodide hydrate. b) the
phenothiazin-5-ium tetraiodide hydrate in methanol is added to a
solution of an amine R.sup.7R.sup.8NH (in which R.sup.7 and R.sup.8
are as defined above). The reaction mixture is stirred overnight,
reduced by evaporation and left to cool. The solid that formed is
collected by filtration, washed with diethyl ether and dried. c)
triethylamine in dichloromethane followed by a solution of a
different second amine R.sup.7R.sup.8NH (in which R.sup.7 and
R.sup.8 are as defined above) in dichloromethane is added to a
solution of the solid from b) above in dichloromethane. The
reaction mixture is stirred overnight, the organic layer washed
with dilute hydrochloric acid and water, separated and dried
(MgSO.sub.4). The majority of the solvent is evaporated and diethyl
ether added to precipitate the product which is collected by
filtration, washed with diethyl ether and dried. Further
purification of the product, if necessary, may be by flash column
chromatography as described in 1b) above.
EXAMPLES
Example 1
A Study to Investigate the Effect of 3,7-bis(N,N-dibutylamino)
phenothiazin-5-ium bromide PDT on Wound Healing in Spontaneous
Diabetic Mutation (Strain c57BLKs/Bom db/db Obtainable from
Taconic, Denmark) Mice
Background
[0059] When wounds are created on non diabetic (db/+) mice they
heal quickly. When wounds are created on (db/db) diabetic mice the
wounds heal more slowly compared to the non diabetic mice. This
delayed healing may be caused by a lack of vasculature to feed the
wound. The delayed healing model is representative of a chronic
wound, and wound healing effects of drugs can be more closely
monitored and more easily measured.
Materials and Methods
[0060] 18 animals were included in this study. 6 control non
diabetic mice (db/+), 6 control diabetic (db/db) mice and 6 (db/db)
PDT treated diabetic mice.
[0061] The 3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide was
formulated in ethanol and Unguentum M as described in Example 2
below at a concentration of 0.266 mg/mL.
Procedure
[0062] On day 0, the animals were anaesthetised and a single
standardised full thickness wound (7.5 mm.times.7.5 mm) was created
in the flank skin of each experimental animal. Wounds were then
dressed with a 1.5.times.1.5 cm pad of encapsulated gauze moistened
with a fixed volume of sterile saline solution. Wounds were allowed
to heal without intervention for a period of 4 days, other than the
replacement of saline moistened gauze and film dressings as
required.
[0063] On day 4 dressings were removed. For the PDT treated group
100 mg of 0.266 mg/mL 3,7-bis(N,N-dibutylamino) phenothiazin-5-ium
bromide cream was applied to the wound The wound was then covered
and left for 6 minutes and 40 seconds. Following this, the wound
was illuminated with 688 nm light, at a fluence rate of 125
mW/cm.sup.2 to give a light dose of 25 J/cm.sup.2. No drug or light
was given to the control animals.
[0064] Dressings were reapplied immediately after treatment and
subsequently on post-wounding days 7 & 10.
[0065] Immediately after wounding (day 0) and subsequently on days
4 and 7 all the wounds were digitally photographed together with a
calibration/identity plate.
[0066] Image Pro image analysis software (version 4.1.0.0, obtained
from Media Cybernetics, USA) was used to calculate wound closure
from wound images in each of the experimental groups over time. For
each wound at each time point--open wound area was measured and
expressed in terms of % wound area relative to day 4 (treatment
day).
TABLE-US-00001 Results % wound area 3 days post Treatment day
treatment Control non diabetic mice 100 48 Control diabetic mice
100 71 PDT treated diabetic mice 100 46 (500 .mu.M 3,7-bis(N,N-
dibutylamino) phenothiazin-5- ium bromide in Unguentum M and 25
J/cm.sup.2) Vehicle and light treated 100 67 diabetic mice
(Unguentum M and 50 J/cm.sup.2) 6 animals were included in each
group.
[0067] Thus in combination with light 3,7-bis(N,N-dibutylamino)
phenothiazin-5-ium bromide provides a substantial increase in the
rate of wound healing when the control diabetic mice and treated
diabetic mice are compared: 67% wound area remaining vs. 46%
remaining respectively. In addition the wound healing in treated
diabetic mice was improved and was comparable to the wound healing
in the control non diabetic mice: 46% wound area remaining vs. 48%
remaining respectively.
Example 2
A Study to Investigate the Effect of 3,7-bis(N,N-dibutylamino)
phenothiazin-5-ium bromide PDT on Wound Healing in Wistar Rat
[0068] 3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide was
solubilised in ethanol to give a concentration of 10 mg/ml. This
was diluted in Unguentum M: 80% water, to give creams with final
concentrations of 100 .mu.M or 500 .mu.M of
3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide.
Treatment Method
[0069] The above creams were used on the closure of full thickness
excisional wounds in Wistar rats with exposure to light. The effect
of topical application of 50 mg of 3,7-bis(N,N-dibutylamino)
phenothiazin-5-ium bromide (at concentrations of 100 .mu.M and 500
.mu.M) followed by photo-activation with 688 nm laser light was
investigated and compared to that of application of the drug
delivery vehicle (Unguentum M) followed by 688 nm photo-activation.
The light dose used was 50 J/cm.sup.2. Wound repair was measured in
terms of the closure of standardised full-thickness excisional
wounds created in the flank skin of adult Wistar rats.
[0070] At the lower dose (100 .mu.M) wounds in receipt of light
treatment closed more rapidly than similar wounds in receipt of the
3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide alone.
[0071] At the higher dose of 3,7-bis(N,N-dibutylamino)
phenothiazin-5-ium bromide (i.e. 500 .mu.M) wounds closed more
rapidly than similar wounds in receipt of the lower dose. From
gross examination of wound and wound margin tissue--no adverse
tissue responses were noted consequent to any treatment regime
followed in this study.
Example 3
A Clinical Study to Investigate the Effect of
3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide PDT on the
Wound Healing of Chronic Ulcers
Materials and Methods
[0072] After ethical review board approval and written informed
participant consents were obtained, patients were recruited,
provided they satisfied the ethically approved inclusion criteria.
MHRA approval had been obtained.
[0073] Patients were recruited who had chronic non healing leg
ulcers or diabetic foot ulcers. These patients had shown little or
no wound healing for a number of weeks prior to treatment.
Procedure
[0074] For the treatment 3,7-bis(N,N-dibutylamino)
phenothiazin-5-ium bromide, formulated in ethanol and Unguentum M
as described in Example 2 above at 500 .mu.M, was applied topically
to the ulcer surface and surrounding ulcer margin (5 mm) giving a
cream depth of up to 5 mm. The ulcer was then left, not occluded,
but in minimal lighting, for 15 minutes. After this pre-incubation
time, the cream was rubbed into the ulcer so underlying skin/tissue
could be seen through the cream. If this was not possible excess
cream was removed. The ulcer was then illuminated using a broad
band CureLight lamp (obtainable from PhotoCure) at a fluence rate
of 20-50 mW/cm.sup.2 giving a total light dose of 50
J/cm.sup.2.
[0075] The ulcer area was measured pre treatment and after 1 week
and one month.
Results
[0076] There was a reduction in the area of the treated ulcers
following the photodynamic treatment. This is shown in Table 1.
TABLE-US-00002 TABLE 1 Percentage reduction in ulcer area after 1
week and 1 month compared to pre treatment. Percentage reduction at
1 Percentage reduction at 1 Patient week month 1 0 10 2 Not
measured 50 3 23 34 4 8 13 5 14 8
Example 4
A Study to Investigate the Effect of 100 .mu.M and 500 .mu.M
3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide on Wound
Healing in the Wistar Rat
Materials and Methods
[0077] On day 0, animals were anaesthetised and a single
standardised full thickness wound (7.5 mm.times.7.5 mm) was created
in the flank skin of each experimental animal. Wounds were dressed
with a 1.5.times.1.5 cm pad of encapsulated gauze moistened with a
fixed volume of sterile saline. Wounds were allowed to heal without
intervention for a period of 4 days, other than the replacement of
saline moistened gauze and film dressings, as required.
[0078] On day 4 standard secondary dressings were removed. 100 mg
of 100 .mu.M or 500 .mu.M 3,7-bis(N,N-dibutylamino)
phenothiazin-5-ium bromide cream was applied to the wound. The
wounds were not illuminated. The area of the wounds were measured
prior to treatment (day 4) and on day 6, 8 and 10.
TABLE-US-00003 Results % Wound area remaining, no light 2 days post
4 days post 6 days post treatment treatment treatment 100 .mu.M 62
33 25 500 .mu.M 62 30 19
[0079] In control tests the cream vehicle used alone had no effect
on wound healing. In general terms the wounds heal faster at a 500
.mu.M concentration than at 100 .mu.M.
Example 5
Phase IIa Randomised, Placebo Controlled Trial to Investigate Wound
Healing Using Photodynamic Therapy in Chronic Leg Ulcers
Trial Design
[0080] 16 patients with chronic leg ulcers were recruited for the
study. All patients had ulcers that had been present for at least 3
months prior to treatment. After randomisation, 8 patients were
treated with 3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide
PDT and 8 patients were treated with placebo. Ulcer area was
measured before treatment and at 1 week, 1 month, 2 months and 3
months post treatment using a Visitrak system. Photographs were
taken of the ulcers before treatment and at 24 h, 1 week, 1, 2 and
3 months post treatment.
Methodology
[0081] Placebo cream was Unguentum Merck:water (1:2).
3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide cream was 500
.mu.M 3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide in
Unguentum Merck:water (1:2). For the treatment a 1 mm depth of
cream was spread over the surface of the ulcer. The ulcer was then
left, occluded under film, shielded from light for 15 minutes. At
the end of the 15 minute incubation, excess cream was removed and
the ulcer was illuminated using a CureLight 01 (obtainable from
Photocure) at a light dose of 50 J/cm.sup.2.
Results: Chronic Leg Ulcers
[0082] Better healing, as measured by the size of the wound, was
found in patients with chronic leg ulcers treated with
3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide compared to
those treated with placebo. At 2 months, 2 out of 8 (25%) patients
who received 3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide
showed complete wound healing compared to none of those who
received placebo. At 3 months, 4 out of 8 (50%) patients who
received 3,7-bis(N,N-dibutylamino) phenothiazin-5-ium bromide
showed complete wound healing compared to 1 out of 8 (12.5%)
patients who received placebo.
[0083] Although any methods and materials, similar or equivalent to
those described herein, can be used in the practice or testing of
the present invention, the preferred methods and materials are
described herein. All publications, patents and patent applications
cited herein are incorporated herein by reference for the purpose
of disclosing and describing specific aspects of the invention for
which the publication is cited.
* * * * *