Note: Descriptions are shown in the official language in which they were submitted.
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DEVELOPMENTS IN BIOLOGICALLY ACTIVE METHYLENE BLUE
DERIVATIVES (2)
FIELD OF THE INVENTION
This invention relates to biologically active photosensitisers which are
strongly
photocytotoxic and have application in the areas of photodynamic therapy
(PDT),
their compositions, their uses as medicaments particularly in the treatment of
cancer
and in the treatment and prevention of microbial infections, their uses in
diagnosis and
detection of medical conditions and related uses in photochemical
internalisation, in
the production of cancer vaccines and in photodisinfection or
photosterilisation.
The invention further relates to conjugates and composites of the
photosensitisers
which may be used in photodisinfection or photosterilisation.
BACKGROUND TO THE INVENTION
It is known that certain organic compounds ("photosensitisers") can induce
cell death
by absorption of light in the presence of oxygen. The cytotoxic effect
involves Type I
and/or Type II photooxidation. Such photosensitisers find use in the treatment
of
cancer and other diseases or infections with light (photodyhamzc therapy) and
in the
sterilisation (including disinfection) of surfaces and fluids by the light-
induced
destruction of microbes. In this context, the term sterilisation is taken to
mean the
reduction or elimination of microbes in a particular situation.
It is also known that certain coloured phenothiazinium compounds, (e.g.
methylene
blue) can take part in Type I and Type II photooxidation processes, but
compounds of
this type thus far have proved unsuitable or of low efficacy as sensitisers
for
photodynamic therapy, or have shown low photochemical antimicrobial activity,
or
have potential problems in use because they are Ames positive.
For application in PDT, a good sensitiser must have at least some and
preferably all of
the following properties:
~ it should cause the destruction of target cells (for example tumour cells or
bacterial cells) efficiently on exposure to light (preferably wavelengths ca.
600
- 8O0 nm)
~ it should show a high degree of selectivity between target and normal
tissues
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~ it should have relatively little dark toxicity
~ it should cause little or no skin photosensitivity in the patient
~ it should have short drug to light intervals for patient and hospital
convenience and to minimise treatment costs
~ it should be suitable for use in vivo, in particular it should not be
mutagenic.
~ For applications in photosterilisation, a good sensitiser must show a strong
phototoxic effect in a wide range of microrganisms, ideally using ambient
light, and should not photobleach readily.
In oncology, several different types of photosensitiser have been used to
treat both
solid tumours and thin tumours of hollow organs such as the oesophagus and
bladder.
However, the use of these photosensitisers has been restricted partly because
of lack
of selectivity between tumour and healthy tissue and partly because of the
prolonged
skin photosensitivity which can be caused. There is a need for new
photosensitisers
which cause little or no skin photosensitivity and which selectively destroy
tumour
cells.
Although PDT has previously been used in the treatment of tumours, it has not
yet
been used clinically against infections caused by bacteria and other
microorganisms.
The use of antibiotics to treat bacterial infections is becoming challenging
due to the
increasing resistance of many bacterial species to commonly used antibiotics,
such as
tetracyclines and beta-lactams. Hospital-acquired antibiotic resistant
infections such
as MRSA are especially problematic. Photodynamic antibacterial treatment is a
promising alternative to antibiotics for local treatment.
When developing antibacterial agents a major problem which must be overcome is
the uptake of the drug into the bacterial cell. Gram negative and Gram
positive
bacteria differ in the composition of their outer surface and respond
differently to
antimicrobial agents, especially in terms of uptake. Due to the high
negatively
charged surface of Gram negative bacteria they are relatively impermeable to
neutral
or anionic drugs, including most commonly used photosensitisers. Development
of
antimicrobial photosensitisers which are effective against Gram negative
bacteria, as
well as Gram positive bacteria would be highly beneficial to replace commonly
used
antibiotics and drugs which are becoming increasingly ineffective due to re
sistance.
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A number of different types of photosensitiser have been investigated in
bacteria.
These include phenothiazinium compounds, phthalocyanines, chlorins and
naturally
occurring photosensitisers. For uptake into Gram negative bacteria, it is
accepted that
the cationic derivatives are the most effective.
Phenothiazinium compounds are blue dyes with maximum absorption at wavelengths
between 600-700 nm. They have been studied for their non-photodynamic
antibacterial properties but few apart from methylene blue and toluidine blue
have
been investigated photodynamically.
Wainwright et al (1998) investigated the effect of a series of phenothiazinium
methylene blue derivatives in tumour cell lines and bacteria. New methylene
blue
(NMB) and di methyl methylene blue (DMMB) were effective at inactivating MRSA
and were shown to be more effective photosensitisers than methylene blue when
acting on pigmented melanoma cell lines. Wagner et al (1998) studied these
dyes and
in addition a hydrophobic derivative for the inactivation of enveloped
viruses.
The precise mode of antibacterial action of methylene blue is unknown, but one
hypothesis is that because of its stereochemistry it can intercalate into DNA,
and that
photodynamic action causes DNA damage. Methylene blue itself has been shown to
be ineffective as an anti-tumour agent. In addition, methylene blue is known
to be
susceptible to photobleaching, which could be a serious disadvantage in some
applications. Because of the recognised limitations of methylene blue, both
anti-
tumour PDT and antimicrobial PDT would benefit from development of new
phenothiazinium-based photosensitisers.
PCT application PCT/GB02/02278 describes certain phenothiaziniurn compounds
which are biologically active and suggests that in a series of N, N, N, N
tetra n-C i-6
alkyl derivatives that the tetra n-butyl derivative is the most active with
activity
decreasing rapidly as the number of carbon atoms in the chain increases.
Surprisingly further phenothiazinium compounds derivatives have now been found
which are biologically active and which are suitable for use as medicaments
particularly in the prevention of microbial infections and in the treatment of
cancer
and microbial infections.
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According the present invention there is provided a phenothiazinium compound
of
Formula (I) for use as an antimicrobial agent for the prevention of microbial
infections:
N~
R1 ~ ~ / ~ / ,R3 XP_
N S N
R2 + R4
_ P
(I)
wherein:
Rl, R2, R3 and R4 each independently is an optionally substituted linear,
branched
or cyclic hydrocarbon group, or Rl and R2 or R3 and R4 together with the N
atom to
which they are attached form an optionally substituted 5-, 6- or 7-membered
ring;
XP- is a counteranion; and
Pis l,2or3.
For prevention of wound infections the wound site is sterilised and in this
specification sterilisation means a significant reduction in bacterial load
on, in or
around a wound site which helps to promote efficient wound healing or which
minimises the chance that wound infection will occur.
The use of the compounds of Formula (I) for the prevention of infection is
preferably
by PDT in which the compound is applied to a wound site followed by the
application of light. Conventional antimicrobials suffer the disadvantage that
they
have a short lifetime for prevention of infection and need repeated
applications, such
as by swabbing, to maintain their effectiveness. The compounds of Formula (I)
have
improved properties over previously known and used antimicrobial agents
because
the prevention effect is prolonged and can be reactivated as necessary by
further
application of light without the need to re-administer the compound. The wound
site
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can be maintained in a sterile condition by continuous exposure to light of a
suitable
wavelength or by the intermittent use of light of a suitable wavelength when
needed.
According to a further feature of the present invention there is provided a
phenothiazinium compound of Formula (II) for use as an antiviral agent in
which the
compound of Formula (II) has the same structure as the compound of Formula (I)
but
wherein Rl, R2, R3 and R4 each independently is an optionally substituted
linear,
branched or cyclic hydrocarbon group, or Rl and R2 or R3 and R4 together with
the
N atom to which they are attached form an optionally substituted 5-, 6- or 7-
membered ring;
XP' is a counteranion; and
Pis l,2or3.
For uses to prevent microbial infection and as antivirals where the linear and
branched chain hydrocarbon groups represented by R1, R2, R3 and R4 preferably
contain from 1 to 12 carbon atoms. In compounds in which the hydrocarbon
groups
represented by R1, R2, R3 and R4 are the same it is preferred that each is
linear or
branched and contains 4 or 5 carbon atoms. In compounds in which Rl=R2 and
R3=R4 and in which Rl/R2 are different to R3/R4 it is preferred that each is
linear
or branched and contains from 1 to 6 carbon atoms, and further that the total
number
of carbon atoms in R1, R2, R3 and R4 is from 8 to 18
According to a further feature of the present invention there is provided a
phenothiazinium compound of Formula (III) for use as an antimicrobial agent in
the
treatment of a microbial infection in which the compound of Formula (III) has
the
same structure as the compound of Formula (I) but wherein:
i) Rl, R2, R3 and R4 each independently is selected from straight, branched or
cyclic C1_12-alkyl provided that at least one of Rl, R2, R3 and R4 is C7_12-
alkyl; or
ii) Rl, R2, R3 and R4 each independently is selected from straight, branched
or
cyclic C1_lz-alkyl in which at least one of R1, R2, R3 and R4 is branched or
cyclic;
or
iii) Rl, R2, R3 and R4 each independently is selected from straight, branched
or
cyclic C1_iz-alkyl in which Rl and R2 may be the same or different and R3 and
R4
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may be the same or different provided that at least one of Rl and R2 is not
the same
as at least one of R3 and R4; or
iv) Rl, R2, R3 and R4 each independently is selected from straight, branched
or
cyclic C1_12-alkyl in which R1 and R2 are different, or R3 and R4 are
different; or
v) Rl, R2, R3 and R4 each independently is selected from C1_12-alkyl and at
least one of R1 and R2, or R3 and R4 together with the N atom to which they
are
attached to form an optionally substituted 5-, 6- or 7-membered ring
According to a further feature of the present invention there is provided a
phenothiazinium compound of Formula (IV) for use as a medicament or for use as
an
anti cancer agent in which the compound of Formula (IV) has the same structure
as
the compound of Formula (I) but wherein:
i) Rl, R2, R3 and R4 each independently is selected from straight, branched or
cyclic C1_la-alkyl provided that at least one of R1, R2, R3 and R4 is C7_12-
alkyl; or
ii) Rl, R2, R3 and R4 each independently is selected from straight, branched
or
cyclic C1_12-alkyl in which at least one of R1, R2, R3 and R4 is branched or
cyclic;
or
iii) R1, R2, R3 and R4 each independently is selected from straight, branched
or
cyclic C1_12-alkyl in which Rl and R2 may be the same or different and R3 and
R4
may be the same or different provided that at least one of Rl and R2 is not
the same
as at least one of R3 and R4, except for the compound in which Rl and R2 are
both
HO(CH2)2- and R3 and R4 are both n-butyl or n-pentyl; or
iv) R1, R2, R3 and R4 each independently is selected from straight, branched
or
cyclic C1_12-alkyl in which R1 and R2 are different, or R3 and R4 are
different; or
v) Rl, R2, R3 and R4 each independently is selected from C1_i2-alkyl and at
least one of R1 and R2, or R3 and R4 together with the N atom to which they
are
attached to form an optionally substituted 5-, 6- or 7-membered ring except
for the
compound in which R1 and R2 together with the N atom to which they are
attached
form a morpholino ring and R3 and R4 are both n-butyl;
XP- is a counteranion; and
Pis l,2or3.
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According to a further feature of the present invention there is provided a
phenothiazinium compound of Formula (V) in which the compound of Formula (V)
has the same structure as the compound of Formula (I) but wherein:
i) Rl, R2, R3 and R4 each independently is selected from straight, branched or
cyclic C1_i2-alkyl provided that at least one of Rl, R2, R3 and R4 is C7_la-
alkyl; or
ii) R1, R2, R3 and R4 each independently is selected from straight, branched
or
cyclic Ci_i2-alkyl in which at least one of R1, R2, R3 and R4 is branched or
cyclic;
or
iii) R1, R2, R3 and R4 each independently is selected from straight, branched
or
cyclic C1_12-alkyl in which R1 and R2 may be the same or different and R3 and
R4
may be the same or different provided that at least one of R1 and R2 is not
the same
as at least one of R3 and R4, except for the compound in which Rl and R2 are
both
HO(CH2)~- and R3 and R4 are both n-butyl or n-pentyl; or
iv) Rl, R2, R3 and R4 each independently is selected from straight, branched
or
cyclic C1_l~-alkyl in which Rl and R2 are different, or R3 and R4 are
different; or
v) Rl, R2, R3 and R4 each independently is selected from C1_i2-alkyl and at
least one of Rl and R2, or R3 and R4 together with the N atom to which they
are
attached to form an optionally substituted 5-, 6- or 7-membered ring except
for the
compound in which R1 and R2 together with the N atom to which they are
attached
form a morpholino ring and R3 and R4 are both n-butyl;
XP- is a counteranion; and
Pis l,2or3.
In general the linear and branched chain hydrocarbon groups represented by R1,
R2,
R3 and R4 in any one of the compounds of Formulae (I) to (V) may include one
or
more unsaturated links, for example one or more allcene groups, and may be
optionally substituted by a group selected from H, F, Cl, Br, I, -OH, -OC1_6-
alkyl,, -
CN, -OCOC1_6-alkyl or aryl, such as phenyl. These linear and branched chain
hydrocarbon groups are preferably unsubstituted and are preferably saturated
hydrocarbon groups.
The cyclic hydrocarbon groups represented by Rl, R2, R3 and R4 in any one of
the
compounds of Formulae (I) to (V) contain from 3 to 8 carbon atoms, preferably
from
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4 to 6 carbon atoms and more preferably 6 carbon atoms. These cyclic
hydrocarbon
groups may include one or more unsaturated links, they may be optionally
substituted
and may optionally include a heteroatom such as nitrogen.
Where Rl and R2 and/or R3 and R4 in any one of the compounds of Formulae (I)
to
(V) together with the N atom to which they are attached form an optionally
substituted 5-, 6- or 7-membered ring the ring may contain other heteroatoms
and
may be optionally substituted. The heteroatoms are preferably selected from N,
O or
S. Where the heteroatoms is S this may be substituted with O, where the
heteroatom
is N this may be substituted with H, -CO C1_6-alkyl or C1_6-alkyl which is
optionally
substituted by -OH, preferred substituted heteroatoms are selected from 502,
NH,
NCH3, NC2H5, NCH2CH20H and NCOCH3. The optional ring substituents may be
selected from -CI_6-alkyl, -OH, -OC1_6_ alkyl, -OC OCl_6_ alkyl. Examples
include:
-N z -N -N
U
in which Z is CH2, CH2-C1_6-alkyl, O, S, 502, NH, NCH3, NC2H5, NCH2CH20H,
or NCOCH3.
The counteranion represented by XP- in any one of the compounds of Formulae
(I) to
(V) may be an organic or inorganic counteranion and is preferably selected
from F-,
Br Cf, I-, N03-, SCN', C103-, C104 , I03-, BF4', HSO4 , H2P04 , CH3S04 , N3',
5042 , HPO42 , PO43 , acetate, lactate, citrate, tartrate, glycolate,
glycerate,
glutamate, (3-hydroxyglutamate, glucouronate, gluconate, malate and aspartate.
Preferably the counteranion is selected from the group comprising Cl', Br , I-
, F-,
N03-, HS04 , CH3C02-, 5042-, HPO42-, or P043' or from the group comprising Cl-
, Br
I-, acetate, lactate, citrate, tartrate, glycolate, glycerate, glutamate, (3-
hydroxyglutamate, glucouronate, gluconate, malate, aspartate, and more
preferably
from the group comprising Cl-, Br , I-.
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In a preferred sub-group of compounds of Formulae (I) to (V) R1, R2, R3 and R4
may be the same or different and the sum of the carbon atoms in the alkyl side
chains
represented by Rl and RZ is from 14 to 40, preferably from 16 to 36, and more
preferably from 18 to 30, and especially from 18 to 24.
In a further preferred sub group of compounds of Formulae (I) to (V) Rl, R2,
R3 and
R4 may be the same or different and the sum of the carbon atoms in the alkyl
side
chains represented by Rl and R2 is from 16 to 20 preferably from 18 to 20.
The phenothiazinium compounds of Formulae (I) to (V) may be synthesised as
follows:
1) Symmetrical phenothiazinium compounds where R1 and R2 = R3 and R4
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 R1R2NH (in which R1 and R2 are as defined above) or N-heterocycle in
chloroform. The solid formed is collected by filtration and purified for
example 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°C).
2) Unsymmetrical phenothiazinium compounds where Rl and R2 ~ R3 and R4,
or where Rl ~ R2 and/or R3 ~ R4.
a) Phenothiazine in chloroform is cooled to below 5 °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
an amine R1R2NH in methanol (in which Rl and R2 are as defined above). The
reaction mixture is stirred overnight, reduced by evaporation left to cool.
The solid
that formed is collected by filtration, washed with diethyl ether and dried.
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c) triethylamine in dichloromethane followed by a solution of a different
second
amine R3R4NH (in which R3 and R4 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 (MgS04). The majority of the solvent is
evaporation
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.
Compositions comprising a compound of Formula (V) together with one or more
pharmaceutically acceptable carriers, diluents or excipients (each selected
for certain
characteristics that permit optimal formulation of a pharmaceutical
composition)
form a further feature of the present invention. The compositions of the
present
invention also include those comprising any two or more compounds of Formulae
(I)
to (V) and those comprising any one or more compounds of Formulae (I) to (V)
with
one or more different therapeutic or active agents. The compositions include
liposomes, nanoparticles, colloidal suspensions, micelles, microemulsions,
vesicles
and nanospheres.
The compositions may also comprise further components such as conventional
delivery vehicles and excipients including solvents such as alcohols (for
example
ethanol, polyethylene glycol, glycerol or n-butanol), dimethyl sulphoxide,
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 or Solutol HS15 (Solutol is a trade mark of BASF AG) , isotonising
agents
such as urea, glycerol, aminoethanol, propylene glycol, pH regulators, dyes,
gelling
agents, thickeners, buffers, and combinations thereof.
Typically the compositions are prepared by mixing a compound of Formula (I)
with
one or more pharmaceutically acceptable carriers at an appropriate
temperature,
typically from 15° to 65°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.
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In compositions comprising any one or more compounds of Formulae (I) to (V)
the
concentration of the compounds in the compositions depends on the compound's
photosensitising ability and is preferably in the range from 0.0005 to 20% for
topical
use and from 100~,M to 30mM for intravenous use.
Dry compositions, which may be reconstituted before use, are also provided in
the
present invention. These may be prepared by dry mixing solid components of the
composition or preparing a liquid composition which is evaporated to dryness
generally iuider mild conditions under vacuum or in low temperature ovens.
The compounds of Formula(IV) and their compositions may be used as medicaments
in the treatment of a variety of conditions including infection and cancer;
the
treatment of dermatological, ophthalmic, cardiovascular, gynaecological and
dental
conditions; and in the prevention of infection. Preferably the use as
medicaments is
as anticancer agents, antibacterials, antifungals and antivirals. These uses
may be in
humans or animals.
In one embodiment of the present invention a compound of any one of Formulae
(I)
to (V) is used as a PDT agent or a photodiagnostic agent.
Examples of uses of the various compounds of Formulae (I) to (V) and their
compositions are as photosensitising drugs for PDT to treat cancer and pre-
cancerous
conditions including Barrett's oesophagus, vulval intraepithelial neoplasia
(VIII and
cervical intraepithelial neoplasia (CIN), bladder cancer, colon cancer, non-
melanoma
skin cancer, actinic keratoses, melanoma, brain-pituitary cancer, brain-
glioma,
pancreatic cancer, head and neck cancer, lung cancer, particularly non small
cell,
mesothelioma, oesophageal cancer, stomach cancer, cutaneous T-cell lymphoma;
to
treat systemic and local infections, for example for use as anti-microbial and
antifungal treatments for skin and wound infections such as burn wounds, in
treatment of ulcers particularly leg ulcers more particularly infected chronic
leg
ulcers, nail infections; for parasitic infection, stomach infection, malaria,
leprosy; for
bacterial and fungal spore inactivation; for treatment of prions and viral
infections
such as HIV; for ear, nose and throat infections, tuberculosis; for sexually
transmitted
diseases (STD's), herpes; for treatment of Candida localised infections for
example
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of hair, nails and epidermis, such as tinea pedis and candida vulvovaginitis;
and for
use as infection preventatives such as sterilisation of surgical wounds, skin
graft
sterilisation, stem cell sterilisation, graft versus host disease; to treat
ophthalmological conditions such as macular degeneration, occult choroidal
neovascularisation (CNV), CNV due to pathological myopia, occult with age
related
macular degeneration (AMD), diabetic macular oedema; for vascular problems
such
as cardiovascular disease, arteriosclerosis and restenosis; for autoimmune
diseases
such as rheumatoid arthritis; for skin diseases such as psoriasis, acne,
vitiligo and
eczema and other dermatological conditions such as hirsuitism, and sun damage,
other benign conditions such as endometriosis and menorrhagia; for the
treatment of
dental bacterial disease, such as gum abscesses, gum disease, gingivitis, and
removal,
deactivation or killing of plaque biofilms.
The compounds may also be used in photochemical internalisation (the use of
photosensitisers to assist the uptake and subcellular localisation of drugs)
through
their photosensitising properties and in non-therapeutic uses such as in
photodiagnosis through their fluorescence properties. The latter approach
takes
advantage of the fact that the photosensitiser concentrates more in tumours
than in
surrounding healthy tissue and when induced to fluoresce (by application of
blue
light), the tumour fluoresces more strongly than the surrounding tissue.
Examples of
applications areas include diagnoses for oral diseases and for diseases of the
bladder,
lung and skin.
The compounds of any of Formulae (I) to (V) and their compositions may be used
as
photosensitising drugs for PDT in veterinary applications, for example in
treatment
of cancers such as ear cancer in cats, as antifungal, antibacterial and
antviral
treatments, for sterilisation of wounds in animals and for ophthalmological
treatments in animals.
The use of any of the compounds of Formulae (I) to (V) and their compositions
is
preferably in treatments of localised andlor early cancer and/or pre-cancerous
lesions
in humans and in animals; or in the treatment and/or prevention of infections
in
wounds or skin in humans and animals.
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The compounds of Formulae (I) to (V) are particularly useful as
photosensitising
drugs for PDT of conditions where treatment requires removal, deactivation or
killing
of unwanted tissue or cells such as cancer, precancerous disease, ophthalmic
disease,
vascular disease, autoimmune disease, and proliferative conditions of the skin
and
other organs. Specific and unpredicted advantages of these materials relate to
their
ability to be photoactive against target tissues at different times after
systemic
administration (depending upon the particular sensitiser used) and therefore
their
ability to be targeted directly for example to the vasculature or tumour
cells. They
also have a low tendency to sensitise skin to ambient light when administered
systemically and a low tendency to colour skin.
In general terms any of the compounds of Formulae (I) and (V) and their
compositions may be used in the treatment of various conditions and diseases
described above by administration systemically, topically or locally, followed
by
application of light of an appropriate dose and wavelength or wavelength
range.
Where administered systemically the compounds may be delivered for example
intravenously, orally, sub-cutaneously, intramuscularly, directly into
affected tissues
and organs, intraperitoneally, directly into tumours, intradermally or via an
implant.
Where administered locally or topically the compounds may be delivered via a
variety of means for example via a spray, lotion, suspension, emulsion, gel,
ointment,
salves, sticks, soaps, liquid aerosols, powder aerosols, drops or paste.
For the present compounds activation is by light, including white light, of an
appropriate wavelength, usually in the range from 600 to 800 nm, preferred
wavelengths are from 630 nm to 700 nm.
The light source may be any appropriate light source such as a laser, laser
diode or
non-coherent light source.
The light dose administered during PDT can vary but preferably is from 1 to
200
J/cm2, more preferably from 20 to 100 J/cm2.
Light exposure may be given at any time after a drug is initially administered
or up to
48 hours after drug administration and the time may be tailored according to
the
condition being treated, the method of drug delivery and the specific compound
of
Formulae (I) to (V) used. Light exposure is preferably given at any time after
a drug
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is initially administered up to 3 hours, more preferably from the time after a
drug is
initially administered up to 1 hour, especially up to 10 minutes.
Increased intensity of the light dose generally reduces exposure times.
It is preferred that exposure to light is localised to the area/region to be
treated, and
where tumours are being treated more preferably localised to the tumour
itself.
In one embodiment of the present invention the compound of Formulae (I) to (V)
or
its composition is preferably administered to a subject in need of treatment
and the
light exposure is given up to 10 minutes after a drug is initially
administered.
In a further preferred embodiment of the invention, light exposure is given
within 1
minute after a drug is initially administered.
More preferably light exposure is given at the point of drug administration.
The compounds of the present invention have the advantage, compared with other
phenothiazinium photosensitisers, that they do not, in carrying out their cell-
destroying activity, target the nucleus of the cell so that there is a much
lower risk of
the cells undergoing mutagenic transformations.
Microbial Infections
The compounds of Formulae (I), (II) and (III) have a number of advantages for
the
prevention and treatment of microbial infections, including bacterial, fungal
and viral
infections:
~ Highly effective in deactivating a wide range 'of microorganisms, including
Gram
positive and Gram negative bacteria, MRSA and fungal infection.
~ Active against quiescent/stationary bacteria.
~ High selectivity for microorganisms with minimum damage to host tissue.
~ Unexpectedly low level of photobleaching.
Where a compound of Formula (I), (II) and (III) or its composition is used in
PDT as
a photoactivatable antimicrobial to prevent or treat a microbial infection,
including
bacterial, fungal and viral infections, treatments for skin and other local
infections,
for sterilisation of burn wounds and other lesions, treatments for ulcers, for
sterilisation of both recipient tissue and donated tissue during organ,
including skin,
transplantation and for the treatment of dental microbial disease, it is
administered
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systemically, locally or topically (by any of the means described above) by
applying
to the area to be treated a therapeutically effective amount of the compound
and
exposing the area to light to render active the compound.
The compounds of Formula (I) may be applied to prevent infection at any stage
including wound contamination, where non-replicating organisms are present in
a
wound; wound colonisation where replicating microorganisms are present in a
wound; and wound infection where replicating microorganisms are present that
cause
injury to the host. When there are >105 CFU/g tissue, it is more likely that
sepsis will
develop.
The concentration used for bacterial cell kill in vitro is in the range from
0.1 to 100
~,M, preferably from 1 to SO~,M and more preferably from S to 20pM, especially
l OwM.
In one embodiment the prevention of microbial infections preferably comprises
the
step of administering a compound according to Formula (I) in which R1, R2, R3
and
R4 may be the same or different and are selected independently from ethyl, n-
propyl,
n-butyl, n-pentyl, i-pentyl, 2-ethylpiperidino, 2-methylpyrrolidino and
cyclohexyl .
In one embodiment the treatment of microbial infections preferably comprises
the
step of administering a compound according to Formula (III) in which Rl, R2,
R3
and R4 may be the same or different and are selected independently from
methyl,
ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl, cyclohexyl,
MeO(CH2)a-
or HO (CH2)2- where at least one of R1 and R2 and/or R3 and R4 together with
the N
atom to which they are attached form a piperidino, 2-ethylpiperidino, 2
methylpyrrolidino or morpholino ring except for compounds in which Rl = R2 =
R3
= R4 = methyl, ethyl or n-hexyl and for the compound in which R1 = R2 = HO
(CH2)2- and R3 = R4 = n-butyl.
Preferred moieties for use in the prevention of microbial infections or for
use as
antivirals are as follows:
3,7-(tetra-n-butylamino)-phenothiazin-5-ium;
3,7-(tetra-n-pentylamino)-phenothiazin-5-ium;
3,7-(tetra-iso-butylamino)-phenothiazin-5-ium;
3,7-(tetra-iso-pentylamino)-phenothiazin-5-ium;
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3-(N,N-di-methylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-ethylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-n-butylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-n-butylamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;
3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium;
3-((N-ethyl-N-cyclohexyl) amino)-7((-N-ethyl)-N-cyclohexyl) amino-phenothiazin-
5-ium;
3, 7 -di(piperidino)-phenothiazin-5-ium;
3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;
3-(2-methylpyrrolidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;
3-morpholino-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-morpholino-7-(N,N-di-n-butylamino)-phenothiazin-5-ium;
3-morpholino-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;
3-(N,N-diethanolamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;
3-(N,N-dimethoxyethylamino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium; and
3,7-(tetra-benzylamino)-phenothiazin-5-ium. These compounds preferably include
a
halide as a counteranion which is preferably Cl-, Br or I-.
Especially preferred moieties for use in the prevention of microbial
infections or for
use as antivirals are as follows:
3,7-(tetra-n-butylamino)-phenothiazin-5-ium;
3,7-(tetra-n-pentylamino)-phenothiazin-5-ium;
3-(N,N-di-n-butylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; and
3-((N-ethyl-N-cyclohexyl) amino)-7((-N-ethyl)-N-cyclohexyl) amino-phenothiazin-
5-ium.
The compounds of Formula (I) and (III) preferably are used against bacteria,
more
preferably the compounds are used against antibiotic resistant bacteria.
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Anticancer
The compounds of Formula (IV) have a number of advantages for the treatment of
cancer:
~ Extremely strong photoactivity when compared with methylene and ethylene
blue.
~ Low absorption of light in the UV/blue region. This results in a lower
propensity
of the compounds to skin photosensitivity.
~ Rapid skin clearance.
~ High selectivity for tumours.
~ Low dark toxicity.
~ Low potential for DNA damage when compared with methylene blue.
~ Very short drug-to-light time interval compared with existing PDT drugs.
Where the compounds of the present invention are used as PDT agents for
mammalian cells and tumours they may be administered using the above described
compositions in a variety of ways, such as systemically, topically or locally
(by any
of the means described above) and may be used alone or as components or
mixtures
with other components and drugs.
The dose rates of the compounds of Formula (IV) for intravenous administration
to
humans for oncology treatments are in the range 0.01 to 10 ~,mol
(micromole)/kg,
preferably in the range 0.1 to 2.0 ~,mol (micromole) / kg. To achieve a dose
of say 2
~mol (micromole)/kg in a 70kg patient requires injection of 70m1 of a 2mM
solution,
or Sml at a concentration of 27mM (l6mg/ml) or 2.8m1 of a SOmM solution.
Typical
injections volumes are in the range 0.1 to 100m1, preferably from 5 to SOmI.
In one embodiment the method fox treatment of cancer comprises the step of
administering a compound according to Formula (IV) where R1, R2, R3 and R4 are
selected independently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-
pentyl, n
hexyl, 2-ethylpiperidino, 2-methylpyrrolidino, cyclohexyl, benzyl and
HO(CH2)2,
preferably where Rl, R2, R3 and R4 are selected independently from ethyl, n-
propyl,
n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl, 2-ethylpiperidino, 2-
methylpyrrolidino
and benzyl.
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In the phenothiazinium compounds of Formula (IV) for use as a medicament or
for
use as an anti cancer agent each one of Rl, R2, R3 and R4 is preferably
selected
independently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-
hexyl, 2-
ethylpiperidino, 2-methylpyrrolidino, cyclohexyl, benzyl and HO(CH~)2, more
preferably where R1, R2, R3 and R4 are selected independently from ethyl, n-
propyl,
n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl, 2-ethylpiperidino, 2-
methylpyrrolidino
and benzyl.
Sterilisation/Disinfection of Articles/Surfaces
According to a further feature of the present invention compounds of Formula
(V)
may be used as photoactivated antimicrobial agents, including antibacterial,
antifungal and antiviral agents for general sterilisation of surfaces and
fluids, for
example they may be used to sterilise surgical implants and stems,
particularly where
these are coated or impregnated, to sterilise textiles such as bandages and
dressings,
IV lines and catheters, for sterilisation of water, air, blood, blood
products, and food
and food packaging to prevent transfer of infection, and for general
household,
hospital and office cleaning. The compounds may be applied directly to or
contacted
with the surfaces and fluids and activating the compound by exposure to light.
Additionally the surface to be sterilised may be immersed in a mixture or
solution of
the compound or the fluid to be sterilised may be mixed with the compound or a
solution or mixture containing the compound.
Specific advantages of these compounds over existing known antimicrobial
photosensitisers are their high photocytotoxicity at low light levels,
combined with a
low tendency to undergo photobleaching.
The present invention further provides a conjugate or composite formed between
a
compound of Formula (V) and a polymer. The term composite as used herein
refers
to the situation wherein a compound of the invention is embedded in a polymer
or
physically occluded within or adsorbed onto a matrix or substrate. The polymer
may
be a biological polymer such as a peptide or a protein. Preferred polymers
include
those having anhydride and/or ester groups. Preferred compounds of Formula (V)
which form a conjugate or composite with a polymer are those in which at least
one
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of R1 and R2 and/or R3 and R4 together with the N atom to which they are
attached
form a piperazinyl group.
The present invention further provides a compound formed by the reaction
between a
compound of Formula (V) and a chlorotriazine derivative. The chlorotriazine
derivative may be a polymer having chlorotriazine groups attached thereto.
Appropriate compounds of Formula (V) may be attached directly to a surface of
an
article, particularly a polymeric surface or via a conjugate or composite
formed
between a compound of Formula (V) and a polymer or via a chlorotriazine
derivative, permanently by covalent bonds or reversibly by intermolecular
interactions. This provides a surface that can be sterilised whenever required
by the
application of light and is particularly useful, for example, with intravenous
lines in
patients and in sutures and catheters and intravenous lines, where maintaining
long
term sterility of the relevant surfaces is problematical. The resistance of
the
compounds to photobleaching is an advantage in such applications, where
prolonged
colour stability is required.
Accordingly the present invention further provides an article having at least
one
surface to which is attached a compound of Formula (V).
Preferably the article is a medical device such as a venous, urinary or
balloon
catheter, suture, orthopaedic or artificial implant, heart valve, surgical
screw or pin,
pacemaker lead, feeding or breathing tube, vascular stmt, intraocular lens, or
small
joint replacement. The article may also be of use in wound care and for
packaging
materials for medical use, for example, materials for medical equipment.
A compound of Formula (V) may be applied to or contacted with walls, floors
and
ceilings of hospitals, clinical surfaces such as operating tables, abattoirs,
clean rooms
in scientific laboratories, fibres which may be converted into woven, knitted
or non
woven textile articles such as cleaning cloths, wipes, surgical gowns, bed
linen,
wound dressings and bandages. The compound may be applied directly or via
attachment to a polymeric species.
Where the compound is to be applied to walls, floors, ceilings, and work
surfaces, it
is envisaged that it will be used as a component of a paint or lacquer, which
comprises the compound, film forming polymers, which may or may not be cross
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linkable, and an appropriate solvent, optionally with drying agents and other
colorants. The surface coating may take the form of a solution or water-based
dispersion.
Alternatively the article is one for use in the food and beverage industry and
may be
an item of packaging, a wrapper or storage carton or a piece of processing
equipment.
The article may be a refrigerator, vending machine, ice making machine, a
piece of
restaurant equipment or other kitchen appliance.
The present invention further provides a use of a compound of Formula (V) for
sterilising a surface or a fluid comprising contacting or applying the
compound of
Formula (V) to said surface or fluid and activating said compound by means of
light.
The compound of Formula (V) may be contacted or applied by any means, for
example as a spray, liquid, solution, suspension, foam, cream, gel or
emulsion.
According to a further feature of the present invention there is provided a
use a
compound of Formula (I) to (V) for sterilising fluids in which the fluid is
contacted
with any one of a compound of Formulae (I) to (V) or with a conjugate or
composite
formed between any one of a compound of Formulae (I) to (V) and a polymer
whilst
the compound or the conjugate or composite is illuminated.
The fluid may be a liquid or a gas or a vapour. The method may for example be
applied to sterilisation of liquids, for example for sterilisation of water,
or liquids
used medically such as parenteral liquids for example saline or glucose and
particularly for sterilisation of biological liquids such as blood, blood
products, red
cells, bone marrow cells, and stem cells. The method may also be applied to
sterilisation of gases such as air, particularly air used in air conditioning
systems, and
oxygen used medically. This method is particularly useful for sterilising
materials
which cannot be easily sterilised by filtration methods.
The method is used preferably for sterilisation of water, or liquids used
medically
such as parenteral liquids such as saline or glucose and for sterilisation of
biological
liquids such as bone marrow cells and stem cells.
Any of the compounds of Formulae (I) to (V) and their conjugates or composites
may
be used as is, preferably with its surface area maximised such as in a finely
divided
form or in the form of beads or plates, or it may be used on or associated
with any
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support material which provides a large surface area such as glass, glass
wool,
ceramics, plastics, metals and metal oxides. The support material is
preferably
transparent to light or allows light to pass through it. Where a support
material is
used this is arranged to maximise the surface area covered by the conjugate or
composite and may be in the form of beads, plates, large surface areas in
columns or
tubes, foams or fibres.
Any one of the compounds of Formulae (I) to (V) or their conjugates or
composites is
preferably continuously illuminated at the wavelengths and at the light doses
described above.
The preferred compounds of Formula (I) to (V) are those preferred in this
sterilisation
method.
In a particular embodiment of this aspect of the invention any one of the
compound s
of Formulae (I) to (V) or their conjugates or composites either alone or on a
support
material is packed into a column, typically made of a material which is
transparent to
light, such as silica glass or synthetic fibres. The fluid requiring
sterilisation is
passed into one end of the column, the whole column is continuously
illuminated and
sterilised material flows out from the other end of the column.
Certain novel moieties of the present include:
3,7-(N,N-tetra- iso-butylamino)-phenothiazin-5-ium;
3,7-(N,N-tetra- iso-pentylamino)-phenothiazin-5-ium;
3-(N,N-di-methylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-ethylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-n-butylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium;
3-(N,N-di-methylamino)-7-(N,N-di-n-octylamino)-phenothiazin-5-ium;
3-((N-ethyl-N-cyclohexyl) amino)-7((-N-ethyl)-N-cyclohexyl) amino-phenothiazin-
5-ium;
3,7 di-(piperidino)-phenothiazin-5-ium;
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3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;
3-(2-methylpyrrolidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;
3-(morpholino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;
3-(morpholino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium;
3-(morpholino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;
3-(N,N-diethanolamino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium;
3-(N,N-diethanolamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;
3-(N,N-dimethoxyethylamino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium; and
3,7-(N,N-tetra- benzylamino)-phenothiazin-5-ium.
These compounds preferably include a halide as a counteranion which is
preferably
Cl-, Bi or I-.
The novel moieties of the present invention exhibit unexpected advantages over
compounds described in PCT/GB02/02778.
For example:
3,7-(N,N-tetra-iso-butylamino)-phenothiazin-5-ium when compared with the n-
butyl
analogue surprisingly causes minimal tissue damage when used as an anticancer
agent and is Ames negative;
3,7-(N,N-tetra-iso-pentylamino)-phenothiazin-5-ium when compared with the n-
pentyl analogue surprisingly is effective at a shorter drug to light interval;
3-(N,N-di-methylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium has
improved antibacterial activity when compared with both the tetramethyl and
tetra n-
propyl derivatives;
3-(N,N-di-ethylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium has improved
antibacterial activity when compared with both the tetraethyl and tetra n-
propyl
derivatives;
3-(N,N-di-n-butylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium has better
antibacterial activity and better Ames performance when compared with tetra n-
propyl derivative and surprisingly is equivalent to the antibacterial activity
and Ames
performance of the tetra n-butyl derivative when expected to be somewhat
worse;
3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium has
better
antibacterial activity and better Ames performance when compared with tetra n-
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propyl derivative and surprisingly is equivalent to the antibacterial activity
and Ames
performance of the tetra n-pentyl derivative when expected to be somewhat
worse;
3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium has better
antibacterial activity compared with both the tetra n-hexyl and tetra n-propyl
derivatives, and has better performance than the tetra n-hexyl derivative as
an
anticancer agent and has equivalent performance as an anticancer agent at half
the
dose rate of the tetra n-propyl derivative;
3-(N,N-di-n-butylamino)-7-(N,N-di-pentylamino)-phenothiazin-5-ium has better
activity against Candida albicans than the tetra n-pentyl derivative and
almost the
same activity as the tetra n-butyl when expected to be somewhat worse;
3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium has
better
activity against Candida albicans than both the tetra n-butyl and the tetra n-
pentyl
derivatives;
3-(N,N-di-methylamino)-7-(N,N-di-n-octylamino)-phenothiazin-5-ium has better
anti
tumour activity than the tetra methyl derivative and causes minimal normal
tissue
damage when used as an anticancer agent;
3-((N-ethyl-N-cyclohexyl) amino)-7((-N-ethyl)-N-cyclohexyl) amino-phenothiazin-
5-ium has better anti tumour activity than the tetra ethyl derivative and has
better
antibacterial activity compared with both the tetra ethyl and tetra n-hexyl
derivatives;
3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium when
compared
with the tetra n-pentyl derivative is effective at a shorter drug to light
interval;
3-(2-methylpyrrolidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium when
compared with the tetra n-pentyl derivative is effective at a shorter drug to
light
interval;
and
3,7-(N,N-tetra- benzylamino)-phenothiazin-5-ium has better anti tumour
activity than
the tetra methyl derivative.
Examples
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1) General synthesis of symmetrical phenothiazinium bromides of Formula (I)
where R1=R2=R3=R4, or Rland R2, and/or R3 and R4 together with the N atom to
which they are attached form an N-heterocycle; P =1, Xp- = Br ).
a) Preparation of 3,7-dibromophenothiazin-5-ium bromide
To a solution of phenothiazine (2.00g, 0.01 mol) (Note 1)in oxygen- free,
glacial
acetic acid (150 cm3) was added, in one portion and with vigorous stirring, a
solution
of bromine in oxygen-free, glacial acetic acid (100 cm3, 10% v/v Bra). The
reaction
mixture became dark with the formation of a dark solid. Stirring was continued
for
one minute and water (400 cm3) was then added, when the suspension took on a
red
appearance. The reaction mixture was vacuum filtered to produce a dark solid
and a
brown filtrate. The solid was washed with ether and dried under vacuum
(40°C, 50
mmHg) for one hour to yield a brick red product. Mass of solid = 3.63g Yield =
83%.
b) Preparation of the symmetrical phenothiazinium bromides
To a solution of the appropriate amine R1R2NH or N-heterocycle (32.4 mmol) in
chloroform (200cm3) under nitrogen and with vigorous stirring was added, in
one
portion, 3,7-dibromophenothiazin-5-ium bromide (2.0g, 4.6 mmol). The reaction
mixture became blue in colour and was stirred under nitrogen for 3 hours. The
chloroform solution was washed successively with HBr (2% aq., 2 x SOcm3) and
water (2 x SOcm3), and then dried over MgS04. After filtration, the majority
of the
solvent was removed by rotary evaporation, an excess of diethyl ether was
added and
the reaction mixture then left to stand. After some time, a large amount of
colourless
solid was deposited. This material was removed by filtration. The filtrate was
evaporated to dryness and the residual crude product was purified by flash
column
chromatography over silica gel 60, employing sequentially a mobile phase of
chloroform, chloroform/methanol (98/2) and finally chloroform/methanol
(90/10).
The relevant blue chromatographic fractions were combined and the solvent
removed
by rotary evaporation. The dark blue product was taken up in a minimum volume
of
dichloromethane (1 Ocm~) and the final product precipitated in crystalline
form by the
addition of an excess of petroleum ether (b.p. 60-80°C). The solid was
collected by
filtration, washed with ether and air dried.
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The purity of each product was confirmed by thin layer chromatography (showing
a
single detectable blue spot), and the structure was confirmed by electrospray
mass
spectrometry and UV/visible absorption spectroscopy).
2) General synthesis of unsymmetrical phenothiazinium iodides of Formula (I)
where (R1R2N ~ R3R4N or Rland R2, and/or R3 and R4 together with the N atom
to which they are attached form an N-heterocycle, P = l, Xp- = I-).
a) Preparation of phenothiazin-5-ium tetraiodide hydrate
To a stirred solution of phenothiazine (10 mmole) in chloroform (100 cm3)
cooled to
below 5 °G in an ice bath was added, over 1.5 hours, a solution of
iodine (33 mmole)
in chloroform (400 cm3). The mixture was stirred for 30 minutes and the
resultant
precipitate was collected by filtration, washed with chloroform until free of
iodine
and then kept at room temperature under vacuum overnight to give the product.
b) Preparation of the unsymmetrical phenothiazin-5-ium iodides
To a stirred solution of phenothiazin-5-ium tetraiodide hydrate (1.4 mmole) in
methanol (300 cm3) was added, dropwise, over a period of 60 minutes a solution
of
the appropriate amine R1R2NH (3.6 mmole) in methanol (50 cm3). The reaction
mixture was stirred overnight. The volume of the reaction mixture was then
reduced
by evaporation and the hot solution left to cool. The solid that formed was
collected
by filtration, washed with diethyl ether and dried.
c) To a solution of this solid (0.34 mmol) in dichloromethane (100 cm3) was
added a solution of triethylamine (0.40 mmol) in dichloromethane (5 cm3)
followed
by a solution of a different second amine R3R4NH (1.4 mmol) in dichloromethane
(50 cm3) over 60 minutes. The reaction mixture was stirred overnight. The
organic
layer was then washed with dilute hydrochloric acid (4 x 25cm3) followed by
water
(2 x 25 cm3). T'he organic layer was then dried (MgS04). The majority of the
solvent
was removed by rotary evaporation and an excess of diethyl ether added to
precipitate
the solid. The solid was collected by filtration, washed with diethyl ether
and dried.
Further purification of the compound, if necessary, was by flash column
chromatography.
CA 02547556 2006-05-26
WO 2005/054217 PCT/GB2004/004918
The purity of each product was confirmed by thin layer chromatography (a
single
detectable blue spot). Structures were confirmed by electrospray mass
spectrometry
and UV/visible absorption spectroscopy.
The following specific compounds were prepared by the above methods:
N \
Rl ~ \ ~ ~ / ,R3
N S N
R2 + R4
Compound 1 R1- R4 = n- C3H7 : tetra-n-propyl
Compound 2 Rl - R4 = n- C4H9 : tetra-n-butyl
Compound 3 Rl - R4 = n- CSH11 : tetra-n-pentyl
Compound 4 Rl - R4 = n- C6H13 : tetra-n-hexyl
Methylene blue (R1 - R4 = n-CH3) Compound 5 and ethylene blue (R1 - R4 = n-
CaHS) Compound 1 - 6 were examined for comparative purposes. Compounds 1 to 6
have iodide counteranions.
Compounds 7, 7a, 8, 8a, 8b and 14 - 29 were made by analogous methods.
Compound 9 3-(N,lV dimethylamino)-7-(N,1V dipropylamino)-phenothiazin-5-
ium iodide (20%)
This compound was obtained following isolation of 3-(N,N dipropylamino)-
phenothiazin-5-ium triiodide and subsequent treatment with dimethylamine
hydrochloride. Precipitation from dichloromethane by addition of diethyl ether
yielded purple lustrous crystals. Mass spectrometry: C2pH26N30S requires m/z =
340;
found m/z = 340 (I- not detected by mass spectrometry).
Compound 10 - 3-(N,1V diethylamino)-7-(N,1V dipropylamino)-phenothiazin-5-
ium iodide (15%)
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This compound was obtained following isolation of 3-(N,N dipropylamino)
phenothiazin-5-ium triiodide and subsequent treatment with diethylamine.
Precipitation from dichloromethane by addition of diethyl ether yielded purple
lustrous crystals. Mass spectrometry: C22H3oN34S requires m/z = 368; found m/z
=
368 (I- not detected by mass spectrometry).
Compound 11 3-(N,N dibutylamino)-7-(N,N dipropylamino)-phenothiazin-5-
ium iodide (19%)
This compound was obtained following isolation of 3-(N,N dipropylamino)
phenothiazin-5-ium triiodide and subsequent treatment with dibutylamine.
Precipitation from dichloromethane by addition of diethyl ether yielded purple
lustrous crystals. Mass spectrometry: C26H38N3OS requires m/z = 424; found m/z
=
424 (I- not detected by mass spectrometry).
Compound 12 3-(N,N dipentylamino)-7-(N,N dipropylamino)-phenothiazin-5-
ium iodide (20%)
This compound was obtained following isolation of 3-(N,N dipropylamino)-
phenothiazin-5-ium triiodide and subsequent treatment with dipentylamine.
Precipitation from dichloromethane by addition of diethyl ether yielded purple
lustrous crystals. Mass spectrometry: C2gH42N3OS requires m/z = 452; found m/z
=
452 (I- not detected by mass spectrometry).
Compound 13 3-(N,N dihexylamino)-7-(N,N dipropylamino)-phenothiazin-5-
ium iodide (22%)
This compound was obtained following isolation of 3-(N,N dipropylamino)-
phenothiazin-5-ium triiodide and subsequent treatment with dihexylamine.
Precipitation from dichloromethane by addition of diethyl ether yielded purple
lustrous crystals. Mass spectrometry: C3oH46N30S requires m/z = 480; found m/z
=
480 (I- not detected by mass spectrometry).
Compound 28 3-(N,N diethanolamino)-7-(N,1V dipentylamino)-phenothiazin-5-
ium iodide (23%)
This compound was obtained following isolation of 3-(N,N dipentylamino)
phenothiazin-5-ium triiodide and subsequent treatment with diethanolamine.
Precipitation from dichloromethane by addition of diethyl ether yielded purple
27
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WO 2005/054217 PCT/GB2004/004918
lustrous crystals. Mass spectrometry: C26H38N30~,S requires m/z = 456; found
mlz =
456 (I- not detected by mass spectrometry).
Further compounds have been synthesised and these are summarised in Table A.
Compounds 5 and 6 axe not compounds of the present invention and are included
for
comparative purposes.
Stock solutions of photosensitisers were made up in water and/or DMSO and
stored
in the dark until required. Test solutions were made up in buffer or solvent
or
biological medium as required.
Spectral and physical properties of the phenothiazinium compounds
Spectral data of the phenothiazinium compounds in methanol (Table 1) show that
all
of the compounds have absorption peaks in the 650 - 700 nm region, but that
there is
considerable variability in the precise peak position.
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Table 1
Rl RZ R3 R4 ~,max in
methanol
(nm)
1 n-Pr n-Pr n-Pr n-Pr 656
2 n-Bu n-Bu n-Bu n-Bu 661
3 n-Pent n-Pent n-Pent n-Pent 665
4 n-Hex n-Hex n-Hex n-Hex 668
n-Me n-Me n-Me n-Me 669
6 n-Et n-Et n-Et n-Et 669
7 i-Bu i-Bu i-Bu i-Bu 668
8 i-Pent i-Pent i-Pent i-Pent 662
9 Me Me n-Pr n-Pr 659
Et Et n-Pr n-Pr 661
11 n-Bu n-Bu n-Pr n-Pr 665
12 n-Pent n-Pent n-Pr n-Pr 665
13 n-Hex n-Hex n-Pr n-Pr 666
14 n-Bu n-Bu n-Pent n-Pent 660
n-Bu n-Bu i-Pent i-Pent 661
16 Et Et n-Hept n-Hept 661
17 Me n-Oct Me n-Oct 655
18 Et Cyclohex Et Cyclohex668
19 piperidino piperidino 667
2-ethylpiperidino n-Pent n-Pent 668
21 2-methyl n-Pent n-Pent 663
pyrrolidino
22 Morpholino Morpholino 660
23 Morpholino n-Pr n-Pr 663
24 Morpholino n-Bu n-Bu 661
Morpholino n-Pent n-Pent 663
26 HO(CH~)2 HO(CHa)2 n-Pr n-Pr 663
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WO 2005/054217 PCT/GB2004/004918
Rl RZ R3 R4 ~,max in
methanol
(nm)
27 HO(CH2)2 HO(CH2)2 n-Bu n-Bu 660
28 HO(CHZ)Z HO(CHa)2 n-Pent n-Pent 663
29 Me0(CHa)2 Me0(CH2)2 n-Bu n-Bu
30 PhCH~ PhCH2 PhCH~, PhCH2 649
Me, Et, Pr, Bu, Pent, Hex, Hept, Oct in the above table and throughout this
specification represent methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl
and octyl
respectively, and that n- and i- indicate normal and iso alkyl chains
respectively.
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Cancer Treatment
Compounds of Formula (I) were assessed for PDT efficacy in RIF-1 marine
fibrosarcoma cells in culture. Cells were incubated with the phenothiazinium
for 1h,
then washed 3 times with PBS and fresh culture medium added. Cells were then
illuminated with 18 J/cmz (lOmW/cm2) 665nm light using a diode laser. Dark
toxicity was measured in parallel. The MTT assay was used to assess cell
viability
24h after treatment. Subcellular localisation was also measured in RIF-1 cells
following 1h incubation with the phenothiaziniums using fluorescence
microscopy.
Localisation was measured before and after exposure to light. Sample data for
these
compounds are shown in Table 2.
Table 2 Phototoxicity, dark toxicity and subcellular localisation of example
phenothiaziniums in RIF-1 cells
PDT LDso Ratio PDT Subcellular
: localisation
(~M) dark LDso - light + light
Tetra methyl 13.1 9 Lysosomal Nuclear
Dimethyl dipropyl0.22 119 Lysosomal Lysosomal
Diethyl dipropyl0.11 159 Lysosomal Lysosomal
Dibutyl dipropyl0.09 54 Lysosomal Mitochondrial
Dipentyl dipropyl0.12 40 Lysosomal Mitochondrial
Dihexyl dipropyl0.19 47 Lysosomal Mitochondrial
These data show that several asymmetric phenothiazinium derivatives (where Rl=
R2
~ R3 = R4) have superior properties as photosensitisers to methylene blue,
both in
terms of absolute activity and in terms of the light to dark toxicity ratio.
In addition,
unlike methylene blue these compounds are excluded from the cell nucleus.
Anti-tumour efficacy in vivo
Tumour destruction was assessed in CBA/gy mice bearing subcutaneous CaNT
tumours. Photosensitiser was administered intravenously at doses up to 16.7
wmol/kg. The dose was reduced to 8.35 ~mol/kg if high levels of morbidity or
31
CA 02547556 2006-05-26
WO 2005/054217 PCT/GB2004/004918
mortality were observed or if solubility was limited. At various times after
photosensitiser administration, the tumour was illuminated superficially with
60
J/cm2, 50 mW/cm2 light from a Paterson lamp using a 660 ~ l5nm filter. Drug -
light intervals ranged from Oh (in practice, 1-2 minutes) up to 96h. 72h after
illumination a cross sectional slice was removed from the centre of the tumour
parallel to the incident light, an image of this was captured and the
macroscopic
necrotic area quantified using image analysis software. Necrosis was expressed
as
area of the total tumour slice. % tumour necrosis in control tumours was
generally
<10%. Antitumour activity was categorized: None 0 - 10% tumour necrosis, Low
11
- 39% tumour necrosis, Medium 40 - 69% tumour necrosis, High 70 -100% tumour
necrosis.
Antitumour activity at the optimal dose and drug - light interval for each
compound
is shown in Table 4.
Due to the close proximity of the subcutaneous tumour to internal organs such
as
kidney and liver in the mouse, damage to these organs is often observed after
PDT in
this model. Damage to internal organs following PDT was scored as shown in
Table
3.
Table 3: Scoring system for damage to internal organs
Score -___ Description of damage
0 None
_ _
1 Minimal: Minimal damage evident on post mortem involving
kidney /
liver
2 Moderate: Moderate damage evident on post mortem /
transient weight
loss / transient reduced activity: involving kidney
/ liver / quadriceps
muscle
3 Severe: Moribund, re uire killin
The following compounds showed relatively high antitumour activity with no
normal
tissue damage (average score = 0): Tetrahexyl, dipentyl diethanolamine. The
following compounds showed relatively high antitumour activity with minimal
normal tissue damage (average score < 1): Tetra isobutyl, dihexyl dipropyl,
bis
methyl octyl.
32
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WO 2005/054217 PCT/GB2004/004918
O ~ ~ N
_w
4-~ ~ '!J
° ~ "" ~~ D O
O ~ 4O ~ ,Z~, ''~'' ~ ~ 00
E~ eke ~ .~ ~ F~., ~-' n WI ~°
a
... ,s,
i
w w w ~ z z z
-°a ~ ~ ~ ~ ~ ~ 0 6
+~ ~ :p o a ,.''7, 0 0 0
:G U a~
e'~i
.Y
o ~ ~~ o ~ oo~c°r,,c~'~i o0
~ N. ~ ~ ~ ~ 0 0 0 0 0 0 0
V ~ ~ V ~~11 N 0~1 r N O V~7
Yr ~ O O O et V7 O N
M
i
y ~ ~~ .-~''-i ~ ~O O ~ O O
. ~ ~° A ., .~
O
w
O
N ..~.~ ~ ~ O ~' l~ l~ l'~ V1 t~ ~n I~
'O \O ~O M ~O M
A ~ x ,~ ,~ r,
x"
~ a
y F~ O '~ ~ N M M l0 ~ M
-H -ii 'N -H +i ~ -N
o, N ~ o'~, o°°o i"
~ v~ V ,.s,~-,, CI .d.
o ~ ~ o
U
O
a~ a~ .s~
° ~ .~ '.G o 0
Z
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E-~ o o ~ "" U '"' r '' pq 4 ~q ' '' 0.
cue, a b
U
b
CB N O
--, N m d-
~O U
h
(V O
H a, '~; U
~n
33
CA 02547556 2006-05-26
WO 2005/054217 PCT/GB2004/004918
N ~ N ~ ,.N.i
0
w
.~ a
ni
H
z ~ w ~ z z ~ w w ~ z r~
...., a off,, ,°° o
o ~ ,...,
o ° ~ o
Lj~'~V ~ ~o~o
U ~~ ,~ e~i ~' v~ c~i
-, ,-, ,.-. ,-. .. ,-, ~ .-, ,-,
b ~ CI ~ M 'et et o 00 eh 00 o r-I ~! O M
w-IV1~--IMNM~MO~~O
C C O O O C O C C O C C
GA ~ w 'r a a wr a wr ws a wr 'r wr
00 N ~ ~D O N V7 r!' N M r!' h
1n 00 O 00 ~ ~O N 00 00 N h h
U ~ H .~ M N M d' <F <l' M N O C eW-~I
oyo ~ 0 0 0 0 0 0 ~ o .-a o 0 0
A
-, ~n t~ t~ t~ ~n ~n ~ n r h
~O ~D ~D M M ~O ~ ~D ~O
CD
v
0
~,
G ~'j~ ~CV,~-, fV~M~MM_'_-'d_'O_
-H ~ ~'~ ~ -H ~ -li ~-~ 'N 'H 'H
p ~ m ~ ~ M ~ ~ ~ N ~O due' c~r1
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x a ~ x a 'x~
s.
b ,
0
~ i-, .-., ~ ~ ~ ~ i-,
U
~n
b
0
~ a,
0
U
34
CA 02547556 2006-05-26
WO 2005/054217 PCT/GB2004/004918
0 0 0
~ .~ N O v~
O
..
b ~ a sa
40i ~ O M
yd ~ bA "~ D
-~~
ca ~. x a s.
d d a a ~ ~i d
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O ~ f.~~ N
a b ~ ~ ~ O vD00 N
O w N O M M N C
O O O O O O
U"~~' ~ ~ N ~ ov oo ~
~. .~ M cri. ,-r~ .~ . o ,-i .-i
nn as o 0 0 0 ~ -~ 0 0 0
A
;r~
r v~ r; t~ t~~ t~ t~ ~n t~ v,
y M M O
D ~ ~ ~D ~O~ ~D ~ ~O
a O
H ~ 000, ~ ~ '~ ~ M n
a
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a~
0
',G ~ ~.;~ X0.7~~-1~ ~~-1a O
z z x
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N N N N N cV N N N N M
O .r
U w
CA 02547556 2006-05-26
WO 2005/054217 PCT/GB2004/004918
The present compounds have a number of advantages over currently available
compounds such as Photofrin (trade mark, Axcan Pharma PDT Inc) j d Foscan
(trade mark, Bioscience Technology Investment Holdings Limited). For example
the
compounds of the present invention are single isomer free compounds produced
by
relatively simple processes, whereas Photofrin is a complex mixture of
porphyrin
derivatives. A short drug administration to light interval is desirable both
in terms of
patient convenience and time in hospital during treatment and associated
costs.
Photofrin requires a long drug administration to light interval, typically of
48 hours,
as unacceptable damage to normal tissues surrounding the tumour occurs at
short
drug-to-light intervals. The compounds of the present invention are active at
short
drug-light intervals without any damage to normal tissue surrounding the
tumour. For
example the dibutyl dipropyl derivative causes 98% tumour necrosis where
illumination is immediately after administration.
Comparison of damage to skin overlying the tumour (assessed by scab formation)
shows that with all of the present compounds no scab formation is observed at
any
drug to light interval whereas Photofrin gave up to 25% scab formation at
short drug
to light intervals (0 - 3 hours), longer drug to light intervals of 48 hours
gave no scab
formation but tumour necrosis was only 50%.
For another available compound, Foscan, there is a delay of 4 days, to allow
time for
accumulation in the cancer cells, between injection into the bloodstream and
activation with laser light. Administration of Foscan results in patients
becoming
highly sensitive to light, with a period of sensitivity of approximately 15
days.
Photo-antimicrobial activity
1) General Methods
Method for microbial bacterial photoinactivation experiment
a) Stahdard preparation of photosehsitsiers
Stock solutions of the photosensitisers were made up to 5 mM in
dimethylsulfoxide
(DMSO). The 5 mM stock was further diluted in DMSO to a working concentration
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CA 02547556 2006-05-26
WO 2005/054217 PCT/GB2004/004918
of 1 mM. All photosensitisers were stored in foil covered vials at room
temperature
until required.
b) Standard preparation of microorganisms
The standard protocol outlined below was modified as appropriate to study
variation
of various experimental parameters.
A single bacterial colony from an agar plate was used to aseptically inoculate
100 ml
of nutrient media (0.5 % yeast extract: 1.0 % tryptone w/v) in a 1 1 conical
flask. For
C. albicahs a single fungal colony was used to inoculate 100 ml of sabouraud
dextrose media in a 11 conical flask. The culture was incubated in a shaking
incubator
overnight, at 37 °C. The incubator was set to 250 strokes per minute
and a rotary
motion of a 2.Scm circle..This culture was used for the stationary phase
experiments.
For the log phase bacterial experiments the overnight culture was used to
inoculate
200 ml of nutrient media (in a 2 1 bevelled flask), for C. albica~s 200 ml of
sabouraud dextrose media was inoculated, both were to an optical density of
0.1 at
600 nm. The microorganisms were grown until in the mid-logarithmic phase and
then
harvested and resuspended.
c) P~epa~~atioh of mic~oo~gahisms for PDT
The log or stationary phase cells were collected by centrifugation and washed
twice
in 0.1 M potassium phosphate buffer (pH 7.0). Following washing, the cells
were
resuspended in the same buffer to an absorbance of 0.87 at 650 nm. This
absorbance
was equivalent to 3.5x10$ CFU/ml or 8.5 logloCFU/ml for E. coli, S aureus,
MRSA
and P. aerugihosa. For C. albicahs this correlated to 1.0x107 CFU/ml or 7.0
logloCFUlmI. For photoinactivation of E. coli cells in media, the bacteria
were
resuspended in nutrient media at this stage.
Microbial cell photoihactivatio~ experiments
Sta~da~d incubation with photosehsitise~
25m1 of the prepared cell suspension was incubated with 0.25m1s of a 1mM stock
solution of photosensitiser (giving a final concentration of 10 ~M) in a 250m1
sterile
foil covered conical flask. The suspension was incubated for 30 minutes in the
dark
in a 37°C shaking incubator at 250rpm.
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Illumination from a white light source
After incubation with 10 ~,M phenothiazinium compound, the suspension was
irradiated by a SOOW halogen lamp, from a distance of 75cm, for 60 minutes,
the
power of the lamp was 1.3mW/cm2 giving 4.68J/cm2 over the hour illumination.
Illumination f-om a 665nm laser
After incubation with 10 ~,M phenothiazinium compound, 10 rnl of the bacterial
culture was aseptically traxisferred to a sterile cell. This consisted of a
sealed vial with
a sealed capillary tube inserted, into which the optical fibre could be
placed.
Illumination was carried out with a Ceram Optec diode laser (665 nm) which
used an
optical fibre with a 3cm diffusing tip, at 100mW. For experiments comparing
the
phenothiazinium compound series in E. coli, samples were illuminated for 4
min.
This equated to a fluence rate of 5.3 mW/cm2 assuming that the area of the
illuminated cylinder was 18.86 cm2. After a 4 min illumination the total
fluence was
1.3 J/cm2. Other experiments used a 10 min illumination and 5 0 ~.1 samples
were
removed for CFU analysis after 0,1,2,4,8 and 10 min illumination. These
illumination times are equivalent to the following fluences respectively: 0
J/cm2; 0.32
J/cm2; 0.64 Jlcm~; 1.3 J/cm2; 2.5 J/cm~ or 3.2 J/cmz. Oxygen electrode traces
showed oxygen was not limiting during the illumination period. For experiments
comparing the effect of tetra-n-pentyl-3,7-diaminophenothiazin-5-ium compound
on
different bacteria illumination used the laser set up but for 10 minutes
giving a light
dose of 3.2J/cm2. This light dose was also used for experiments comparing
compounds 17-29. Results are tabulated above in Table 5 and below in Table 5.
Bacterial and yeast survival analysis
SOmI of the illuminated and non-illuminated samples of the suspension were
removed and diluted in 0.1 M pH 7.0 potassium phosphate buffer. 501 of the
diluted suspension was then plated on nutrient agar (0.5% yeast extract, 1.0%
tryptone, 2.0% agar w/v) for bacteria, or sabouraud dextrose agar for C.
albicans. The
plates were incubated overnight at 37°C to give a number of colony
forming units
between 30-300. Cell inactivation was then measured.
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Control studies involving plating out of bacteria before and after the 30
minute
incubation step with no phenothiazinium compound but 0.25m1s DMSO showed no
change in CFU/ml. Illumination of the bacterial culture alone with no
phenothiazinium compound but 0.25m1s DMSO also demonstrated no change in
CFU. For illumination in nutrient media, control tests showed a loglo increase
in
CFU/ml of 0.2 during the hour illumination.
Determi~atioh of the effect of phenothiazi~ium compounds ova bacterial cell
growth
200m1 of nutrient media (0.5% yeast extract 1.0% tryptone wlv) in foil covered
250m1 conical flasks was aseptically innoculated with l Oml of a fully grown
bacterial
culture (E. coli). In addition the media contained 1.0 ml of a 1mM stock
solution of
phenothiazinium compounds with a final concentration of lOwM, apart from the
control which contained no phenothiazinium compounds but 1.0 ml DMSO.
The suspension was incubated at 37°C and 250rpm in a shaking incubator
in the dark.
1m1 samples were taken every hour for 6 hours and turbidity based on apparent
optical density at SSOnm caused by light scattering was measured. Control
studies
show this wavelength is out of the region of photosensitiser absorption.
Following
optical density readings the l.Oml sample was spun in a MSE Micro-Centaur
centrifuge (10 OOOg x 5 minutes) and the absorbance spectra of the supernatant
read
spectrophotometrically.
For the tetra-n-butyl derivative only, similar experiments were carried out
where the
bacteria were allowed to grow without photosensitiser for 3 hours, after which
time
the phenothiazinium compounds was added. Subsequent growth was monitored as a
function of time, both for exposure to light and in the dark.
Uptake of the photosensitises i~tto E. coli
Following incubation of the bacteria with photosensitiser, 2 ml of the non-
illuminated bacterial culture was sedimented using a Benchtop Centaur 5
centrifuge
(1500 g x 10 min). The bacterial pellet was washed twice with 0.1 M potassium
phosphate buffer (pH 7.0) to remove extracellular and loosely bound
photosensitiser.
Finally the pellet was resuspended and vortexed in 1 ml of 0.1 M NaOH 2 %
(w/v)
SDS and left at room temperature, in the dark, for at least 24 h. Fluorescence
readings were taken using a Kontron SFM-25 spectrofluorimeter. The
concentration
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WO 2005/054217 PCT/GB2004/004918
of phenothiazinium compound in the cellular samples was determined from
interpolation of the standard curves.
Photobleaching 0.25m1s of a 1mM solution of photosensitiser, 0.25m1s of IOmM
tryptophan was added to 25m1s of 60% methanol, 40% potassium phosphate buffer
(pH7.0). Experiments were also carried out in the absence of tryptophan where
this
was replaced by 0.25m1s of the 60% methanol, 40% potassium phosphate buffer
(pH7.0).
The mixture was illuminated as in the cell inactivation experiments above
(l.3mW/cm2) for 60 minutes, samples were taken every 15 minutes and spectra
recorded on a UV-Visible spectrophotometer between SOOnm and 700nm. For high
light dose, illumination was at 9mW/cm2 for 60 minutes.
Results
Ahtibacte~ial properties of phehothiazihium derivatives
Many antibiotics are only poorly effective against non-growing or stationary
bacteria
and it is important to assess the ability of the phenothiazinium compounds to
inactivate stationary phase bacteria. During the stationary period the cell
has a thicker
peptidoglyan cell wall and differences in protein metabolism and therefore
might be
less susceptible to the photodynamic effect.
Inactivation of bacteria may be more challenging in a therapeutic environment,
because the sensitiser may bind preferentially to extracellular proteins
rather than the
bacterial lipopolysaccharide membrane. This was tested by resuspending the
bacteria
in nutrient medium containing many factors which might compete with bacterial
cells
for photosensitiser binding.
The potential advantages of a laser source are increased accuracy of light
doses and
shorter illumination times.
Uptake of the photosensitisers into bacterial cells is clearly important in
determining
photo-activity.
S.au~eus is a Gram positive organism which differs from Gram negative
organisms in
that it has a thick outer peptidoglycan layer and no external
lipopolysaccharide _ The
bacterial structure is the same as in MRSA (Methicillin resistant S.au~eus)
which is
resistant to almost all commonly used antibiotics. The data show that after
only a 1
CA 02547556 2006-05-26
WO 2005/054217 PCT/GB2004/004918
minute illumination almost 99% of the bacteria are inactivated and that after
10
minutes there is almost 5 logs of cell kill, illustrating the very high
photoactivity of
the tetra-n-butyl derivative against this Gram positive organism.
It is important to determine if the photosensitiser would also be active
against the
antibiotic resistant form, MRSA, as this would have major health and
industrial
applications.
Anti fungal properties of phenothiazihium derivatives
In order to test the ability of the compounds of Formula (I) to kill fungal
cells in the
light, the photosensitiser was incubated with cells of Ca~dida albicahs and
the
culture was subjected to laser light as described above. . This
photosensitiser is
therefore also highly photoactive against this fungal organism which is
responsible
for many common infections e.g. thrush.
Selectivity fog bacterial cells versus mammalian tissues
It is clearly important for therapeutic purposes that there is minimal damage
to host
tissues while microorganisms are being destroyed. This was tested by applying
a
solution of the compound of Formula (I) to the ears of experimental mice and
illuminating, under conditions in which the total dose was almost 20 times
that
needed for bacterial or fungal elimination. The possible effects on the host
tissue
were assessed by measuring any increase in ear thickness. This is a standard
model
for detecting photodynamic reactions in the skin.
Comparison with results from intravenous administration of PHP, a drug
equivalent
to Photofrin which is known to cause prolonged skin reaction. The reaction
from
PHP is very strong, as expected, there is little or no reaction from the
compound of
Formula (I), suggesting that mammalian tissues would not be damaged during
antimicrobial treatment.
Photobleachihg
Photobleaching removes detectable colour from the photosensitiser, rendering
it
inactive and is the result of its instability to light and reduction or
oxidation. Such
photobleaching may have advantages or disadvantages depending on the potential
application. For example, photobleaching is undesirable in the coating of
lines and
catheters. Two sets of experiments were carried out; one at a high light dose
41
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(9.OmW/cm2 ) and one at a low light dose (l.3mW/cm2) with and without
tryptophan
as described above. Absorption spectra at low light dose, with and without
tryptophan, showed no changes for any of the phenothiazinium compounds
demonstrating they are stable at this dose. At the high light dose, spectral
changes
were observed for the methylene blue, indicating photobleaching. The maximum
absorbance decreased and the wavelength peak shifted over the one hour
illumination. These changes occurred to the same extent with and without
tryptophan. However, none of the other phenothiazinium compounds showed this
degradation and remained stable to photobleaching at the high light dose.
The antibacterial properties of tetra-n-pentyl-3,7-diaminophenothiazin-5-ium
are
tabulated below in Table 6:
Photosensitiser Bacteria Growth Cell kill Standard
/Yeast phase (log error of
reduction the mean
in
CFU/ml)
Tetra-n-pentyl-3,7-P.ae~uginosaLog 3.88 0.27
diaminophenothiazin- Stationary 1.28 0.22
5-ium S. aureus Log 4.21 0.22
Stationary 3.17 0.04
MRSA Log 3.80 0.34
Stationary 1.85 0.16
~'. albicavcsLog 3.61 0.36
Table 6 Photoinactivation of bacteria and yeast in the log and stationary
growth
phase, following incubation with 10~,M photosensitiser and illumination with
665nm
laser light at a fluence rate of 3.2 J/cm2.
The data in the table above show the log reduction in CFU/ml of bacteria or
yeast
incubated with 10~,M photosensitiser, and illuminated using a 665nm laser for
lOmin, at a fluence of 3.2 J/cm2.
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The susceptibility of bacteria to phenothiazinium mediated PDT can depend on
if the
bacteria are Gram-positive or Gram-negative. Gram-positive bacteria (S.
aureus,
MRSA) have a highly cross linked peptidoglycan cell wall approximately 25nm in
thickness. Gram negative bacteria (E. coli, P. aet~ugi~cosa) have a thinner
Snm cell
wall and a unique lipopolysaccharide outer membrane. The presence of the outer
membrane gives an increased resistance of Gram negative bacteria to many
antibacterial agents.
Following illumination the tetra-n-pentyl-3,7-diaminophenothiazin-5-ium
compound
led to >3 log reduction in CFUImI for both log phase, Gram negative (E. coli,
P.
ae~ugi~osa) and Gram positive bacteria (S. aureus, MRSA).
Many antibiotics have a low activity against bacteria in the stationary growth
phase.
Bacteria in the two growth phases differ in their physiology and morphology.
Stationary phase cells are less active and more resistant to environmental
stress,
therefore, may be resistant to phenothiazinium mediated PDT. The above table
shows
that the effectiveness of the tetra-~-pentyl-3,7-diaminophenothiazin-5-ium
compound
is only slightly reduced against stationary phase cells compared to log phase
cells.
MRSA, an antibiotic resistant strain of S aureus is a major cause of nosocomal
infection. MRSA and S. au~eus are equally susceptible to tetra-~-pentyl-3,7-
diaminophenothiazin-5-ium compound mediated anti microbial PDT. There was a
log reduction of 3.80 logloCFU/ml using the tetra-~-pentyl-3,7-
diaminophenothiazin-
5-ium compound against log phase MRSA.
Ames Testing
Ames testing was carried out (using a lcit from Discovery Partners
International) in S.
typhimurium mixed strains (TA7001, TA7002, TA7003, TA7004, TA7005 and
TA7006) which detect base pair substitution mutagens at both GC and AT sites,
and
strain TA98 which detects frame shift mutagens (Gee et al, Proc Natl Acad Sci,
91,
11606-11610, 1994). Approximately 107 bacteria in medium containing sufficient
histidine for 2 cell divisions were incubated (in triplicate) at 37°C,
250rpm for 90
min with 6 concentrations of the test agent, solvent control and positive
control.
Incubations were carried out in both light and dark conditions and with and
without
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metabolic activation with S9 rat liver extract (4.5%). The light source was a
bank of
seven Sylvania Grolux 30W light tubes. A 2-fold dilution series of the test
agent was
used with the top concentration being a toxic concentration (i.e. a
concentration
causing a visible reduction in cell number in a prescreen) or the maximum
soluble
concentration. In the absence of S9, the positive control was a mixture of 4-
nitroquinoline-N-oxide (500 ng/ml) and 2-nitrofluorene (2 ~g/ml). In the
presence of
S9, the positive control was 2-aminoanthracene (10 ~,g/ml). After the 90 min
incubation, bacteria were diluted with pH indicator medium lacking histidine
and
transferred to 384 well plates to give 48 wells per concentration in
triplicate. The
plates were incubated at 37°C for 48h, then positive wells (wells in
which the growth
of his+ reverse mutants has reduced the pH, producing a colour change from
purple
to yellow) were counted. Results were expressed as positive wells per 48 (mean
~
SD). A positive response was defined as a concentration related increase in
the
number of positive wells and a significant increase in the number of positive
wells at
one or more test agent concentrations compared to negative control, with
statistical
significance assessed using an unpaired two-tailed Student's t-test. The
extent of the
response was assessed by calculating the fold increase in the background
mutation
rate at the optimal test agent concentration (with a fold increase of <10
classified as a
weak positive response).
In the mixed strains, all compounds tested were negative under all four
conditions (~
light, t S9). In strain TA98, all compounds tested were negative in the
absence of S9
(~ light). In the presence of S9 (~ light), some compounds showed a positive
response in strain TA98, indicating that they are metabolically activated to a
frame
shift mutagen (intercalating agent). Results in strain TA98 (+S9, - light) are
shown in
Table 5 above.
Compounds of Formula (I) suitable for inclusion in polymers or attachment to,
or adsorption on, polymer surfaces
(a) Inclusion within polymers
Example
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This may be illustrated by adding O.Olg of a compound of Formula (V), such as
3,7-
(N,N-tetra- iso-butylamino)-phenothiazin-5-ium, to a clear solution of
cellulose
triacetate (0.5 g) in dichloromethane (10 cm3) and stirring until the compound
dissolves completely. Casting the solution on a glass plate and drying slowly,
gives a
clear film. The film shows typical singlet oxygen generating properties on
exposure
to light for example an aerated red solution of tetraphenylcyclopentadienone
(a
characteristic singlet oxygen detector) in toluene containing the film is
rapidly
bleached on exposure to light from a 40 w tungsten filament lamp. An identical
solution showed no bleaching when irradiated for the same period of time in
the
absence of the film.
(b) Adsorption on polymers
This may be illustrated by a phenothiazinium compound (Ia) which may be made
according to the following reaction scheme:
j1 ~ ~ ~NH
R2N I ~ S ~ N RZN ~ S ~ N J
/ / C
N N / N /
H
in which both R groups = n-pentyl.
The compound will be extremely basic and readily protonated in dilute acids to
give
(IIa) below, which could be adsorbed strongly on polymeric surfaces, e.g.
polyamides, polyacrylates, polyesters, polycarbonates, polyurethanes, and
strongly
resisted removal by water or solvents. Alternatively Ia could be adsorbed
directly
onto acidic surfaces to give their corresponding cationic salts directly.
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XO
O
O O ~NHz
R2N ~ S ~ N J
N
IIa; R = n-pentyl
REFERENCES
Wainwright M, Phoenix DA, Laycock SL, Wareing DRA, Wright PA. (1998).
Photobactericidal activity of phenothiazinium dyes against methicillin-
resistant
strains of Staphylococcus aureus. FEMS Microbiology Letters 160, 177-181.
Wagner SJ, Skripchenko A, Robinette D, Foley JW, Cincotta L (1998). Factors
affecting virus photoinactivation by a series of phenothiazine dyes.
Photochemistry
and Photobiology 67, 343-349.
46
