Note: Descriptions are shown in the official language in which they were submitted.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
1
USE OF CYSTEATSIINE IN TREATING INFECTIONS CAUSED BY
YEASTS/MOULDS
FIELD OF THE INVENTION
The present invention relates to the use of cysteamine and derivatives thereof
in the
treatment and/or prevention of infection caused by yeasts and/or moulds.
BACKGROUND TO THE INVENTION
The frequency of invasive fungal infections has continued to increase over the
past two
decades, both in the general population and in immunosuppressed patients with
the vast
majority of infections caused by Aspergillus and Candida species (Pasqualotto,
A. C., and
Denning, D. W. (2005) Diagnosis of Invasive Fungal Infections ¨ Current
Limitations of
Classical and New Diagnostic Methods. Euro Oncol Rev). These infections carry
high
mortality rates and place significant burdens on health care systems. There
remains an
urgent need for more effective and safe therapeutic agents to treat and
prevent infections
by yeasts and mould (including e.g. Candida spp. or Aspergillus spp.).
STATEMENTS OF THE INVENTION
According to a first aspect of the invention, there is provided cysteamine or
a derivative
thereof (or a composition comprising cysteamine and/or a derivative thereof)
for use in the
treatment or prevention of an infection caused by yeasts and/or moulds.
The present invention is predicated on the surprising finding that cysteamine
and/or
derivatives thereof have particular utility in the treatment or prevention of
yeast and mould
infections, suitably fungal infections such as Aspergillus and/or Candida
infections for
example.
Suitably, the infection may be a fungal infection or a disease caused by a
fungal infection
such as e.g., a Candida infection and/or an Aspergillus infection. Suitably,
the infection
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
2
may be caused by yeast and/or moulds. Suitably, the infection may an infection
by one or
more of the group consisting of: Candida spp., (e.g. C.albicans),
Epidermophyton spp.,
Exophiala spp., Microsporum spp., Trichophyton spp., (e.g T.rubrum and T.
interdigitale),
Tinea spp., Aspergillus spp., Blastomyces spp., Blastoschizomyces spp.,
Coccidioides spp.,
Cryptococcus spp. (e.g. Cryptococcus neoformans), Histoplasma spp.,
Paracoccidiornyces
spp., Sporotrix spp., Absidia spp., Cladophialophora spp., Fonsecaea spp.,
Phialophora
spp., Lacazia spp., Arthrographis spp., Acremonium spp., Actinomadura spp.,
Apophysomyces spp., Emmonsia spp., Basidiobolus spp., Beauveria spp.,
Chrysosporium
spp., Conidiobolus spp., Cunninghamella spp., Fusarium spp., Geotrichum spp.,
Graphium spp., Leptosphaeria spp., Malassezia spp. (e.g Malassezia furfur),
Mucor spp.,
Neotestudina spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Phoma
spp.,
Piedraia spp., Pneumocystis spp., Pseudallescheria spp., Pyrenochaeta spp.,
Rhizomucor
spp., Rhizopus spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp.,
Scopulariopsis spp., Sporobolomyces spp., Syncephalastrum spp., Trichoderma
spp.,
Trichosporon spp., Ulocladium spp., Ustilago spp., Verticillium spp.,
Wangiella spp.
Suitably, the infection may be caused by a Candida spp. (e.g. Candida
albicans).
Suitably, the infection may be caused by Aspergillus spp. (e.g. Aspergillus
fumigatus).
Suitably, the infection may be caused by Exophiala spp. (e.g. Exophiala
dermatitidis).
Suitably, the infection may be caused by a Cyptococcus spp.
In a further aspect, the present invention provides cysteamine or a derivative
thereof or a
pharmaceutical composition comprising cysteamine and/or a derivative thereof
for use in
the prevention or treatment of any one or more of the group consisting of:
candidiasis
(including OPC), cystic fibrosis, aspergillosis (including bronchopulmonary
aspergillosis,
chronic pulmonary aspergillosis and aspergillomata), athlete's foot;
basidiodiabolomycosis; blastomycosis; coccidioidomycosis cryptoccocis; Chronic
obstructive pulmonary disease (COPD); basal meningitis; dermatophytosis;
onchomycosis;
demiatophytids; endothrix; exothrix; fungal meningitis, fungemia, heaves;
histoplasmosis,
mycosis, myrinogmycosis, paracoccidioidomycosis, penicilliosis, piedra,
pneumocytosis
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
3
pneumonia, sporptrichosis, tinea, zeospora and zygomycosis. COPD encompasses
lung
diseases including chronic bronchitis, emphysema and chronic obstructive
airways disease.
Suitably, the composition may be a pharmaceutical composition comprising a
pharmaceutically acceptable carrier, excipient or diluent.
Suitably, the composition may further comprise an antibiotic or antifungal.
Suitably, the
antibiotic may be selected from the group consisting of: Tobramycin, Colistin,
Gentamicin
or Ciprofloxacin. Suitably, the antibiotic may be tobramycin. The antifungal
may one or
more of Fluconazole, Itraconazole, Caspofungin and Amphotericin B.
Preferably, the composition further comprises an antifungal. Preferably still
the
composition is for use in treating an infection causd by Exophiala spp.
Suitably, the cysteamine may be used in combination a modified peptide.
As used herein the term "modified peptide" refers to a peptide comprising from
3 to 50
alpha, D and/or L amino acids wherein the amino acids are predominantly
arginine and
wherein the peptide, optionally farther comprises a modification which is
selected from
one or more of the group consisting of:
I) Incorporation of a histidine tag;
2) lipidation; and
3) pegylation
Suitably, the histidine tag may comprise at least two histidine residues.
In an additional or alternative aspect, the optional modified peptide of the
present
invention may be a lipidated peptide such that a fatty acid is conjugated to
the peptide.
Suitably, the fatty acid may be a C2 to C20 fatty acid. Preferably, the fatty
acid may be C3 to
C14.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
4
In another additional or alternative aspect, the optional modified peptide of
the present
invention may be PEGylated.
Suitably, the composition of the present invention may further comprise a
peptide
comprising from 3 ¨ 500 amino acids wherein the amino acids are predominantly
arginine.
In a further aspect the present invention provides a method of treating or
preventing an
infection caused by yeasts or moulds in a subject comprising administering a
pharmaceutically effective amount of a composition comprising cysteamine or a
derivative
thereof.
Suitably, the infection may be caused by one or more of the group consisting
of: Candida
spp., (e.g. C.albicans), Aspergillus spp., Epidermophyton spp., Exophiala
spp.,
Microsporum spp., Trichophyton spp., (e.g T.rubrum and T interdigitale), Tinea
spp.,
Blastomyces spp., Blastoschizomyces spp., Coccidioides spp., Cryptococcus spp.
(e.g.
Cryptococcus neoformans). Histoplasma spp., Paracoccidiomyces spp., Sporotrix
spp.,
Absidia spp, Cladophialophora spp., Fonsecaea spp., Phialophora spp., Lacazia
spp.,
Arthrographis spp., Acremonium spp., Actinomadura spp., Apophysomyces spp.,
Emmonsia spp., Basidiobolus spp., Beauveria spp., Chrysosporium spp.,
Conidiobolus
spp., Cunninghamella spp., Fusarium spp., Geotrichum spp., Graphium spp.,
Leptosphaeria spp., Malassezia spp. (e.g Malassezia furfur), Mucor spp.,
Neotestudina
spp., Nocardia spp., Nocardiopsis spp., Paecilomyces spp., Phoma spp.,
Piedraia spp.,
Pneumocystis spp., Pseudallescheria spp., Pyrenochaeta spp., Rhizomucor spp.,
Rhizopus
spp., Rhodotorula spp., Saccharomyces spp., Scedosporium spp., Scopulariopsis
spp.,
Sporobolomyces spp., Syncephalastrum spp., Trichoderma spp., Trichosporon
spp.,
Ulocladium spp., Ustilago spp., Verticillium spp., Wangiella spp.
Suitably, the infection may be caused by a Candida spp. or Aspergillus spp.
Suitably, the infection may be caused by Exophiala spp.
In another aspect, the present invention provides a method of preventing or
treating any
one or more of the group consisting of: candidiasis (including OPC), cystic
fibrosis,
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
aspergillosis (including bronchopulmonary aspergillosis, chronic pulmonary
aspergillosis
and aspergillomata), athlete's foot; basidiodiabolomycosis; blastomycosis;
coccidioidomycosis COPD; cryptoccocis; basal meningitis; dermatophytosis;
onchomycosis; dermatophytids; endothrix; exothrix; fungal meningitis,
fungemia, heaves;
5 histoplasmosis, mycosis, myrinogmycosis, paracoccidioidomycosis,
penicilliosis, piedra,
pneumocytosis pneumonia, sporptrichosis, tinea, zeospora and zygomycosis in a
patient
comprising administering a composition comprising a pharmaceutically effective
amount
of cysteamine or a derivative thereof to a patient.
Suitably compositions of the present invention may be pharmaceutical
compositions
comprising a phaimaceutically acceptable carrier, excipient or diluent
Suitably, the composition of the present invention may comprise an antibiotic,
such as one
or more selected from of the group consisting of: Tobramycin, Colistin,
Gentamicin or
Ciprofloxacin.
Suitably, the composition may comprise a peptide comprising from 3 to 50
alpha, D and/or
L amino acids wherein the amino acids are predominantly arginine and wherein
the
peptide, optionally further comprises a modification which is selected from
one or more of
the group consisting of:
1) Incorporation of a histidine tag;
2) lipidation; and
3) pegylation
Suitably, the composition may comprise a peptide comprising from 3 ¨ 500 amino
acids
wherein the amino acids are predominantly arginine.
DETAILED DESCRIPTION
The present invention provides cysteamine or a derivative thereof (or a
composition
comprising cysteamine and/or a derivative thereof) for use in the treatment or
prevention
of an infection caused by yeasts and/or moulds.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
6
The term "composition comprising cysteamine and/or a derivative thereof'
encompasses
the use of cysteamine and/or a derivative thereof optionally in combination
with other
undefined ingredients. The teini "composition comprising" where used may
optionally be
substituted with "consisting essentially of' or "consisting",
Examples of cysteamine derivatives include: 2-methylthio ethylamine
(cinnamate), 2-
methyl thio ethylurea, N-(2-methylthio ethyl) p-acetamido benzamide, 2-
aminoethanethiol,
N-(2-methylthio ethyl)p-acetamido benzenesulfonamide,N-(2-propylthioethyl)-p-
methoxy
benzamide, N-(butylthio ethyl) nicotinamide, N-(2-dodecylthio ethyl) p-
butoxybenzamide,
N-(2-methylthio ethyl) p-toluenesulfonamide, N-(2-isopropylthio ethyl)
propionamide, N-
(2-octylthio ethyl) acetamide, N-(2-butylthio ethyl) methanesulfonamide, N-(2-
isopentylthio
ethyl)butane,
bis 1,4-(2-acetamido ethylthio), 2 ,3-butanediol, 2-hexadecylthio ethylamine
hydrochloride,
2-allylthio ethylamine malate,9-octadecene 2-ylthio ethylamine hydrochloride,
2-
dodecylthio ethylamine hydrochloride, 2-isopentylthio ethylamine mandelate, 2-
octadecylthio ethylamine salicylate, 2-.beta.-hydroxyethyl thio ethylurea,
2-.beta.-hydroxyethylthio ethylamine hydrochloride, 2-
(2,3-dihydroxy
propylthio)ethylamine p-toluenesulfonate, 2-(2-hydroxy propylthio)ethylamine
oxalate, N-(2-methylthio ethyl)phenylacetamide, 2-(2,2-dimethoxy ethylthio)
ethylamine
hydrochloride, 2-(2,2-dimethoxy ethylthio) ethylamine undecylenate,
2-(2,2-diethoxy ethylthio) ethylamine undecylenate, 2-(2,2-diethoxy
ethylthio)ethylamine
acetate,
2-undecenylthio ethylamine, 2-.beta.-ureidoethylthio ethylamine hydrochloride,
2-.beta.-
acetamidoethylthio ethylamine tropate, 2,2'-thio diethylamine fumarate, 2,2'-
thio
diethylurea, 3-.beta.-aminoethylthio propylamine hydrochloride, S-.beta.-
ureidoethyl
thiocarbamate,
2-ethoxycarbonylthio ethylamine hydrochloride, 2-dimethylamino carbonylthio
ethylamine
sulfate, 2-butoxycarbonyl methylthio ethylurea, 2-ethyloxycarbonylmethylthio
ethylamine
hydrochloride, 6-.beta.-aminoethylthio hexanoate of methyl hydrochloride, 5-
.beta.-
aminoethylthio pentanoic
acid,
2-phenylthio ethylamine dihydrogen phosphate, 2-p-t-butylphenylthio ethylamine
trichloracetate, 2-p-methoxyphenylthio ethylamine ditartrate, 2-tolylthio
ethylamine
hydrobromide, 2-(1-biphenyl thio) ethylamine
hydrochloride,
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
7
2-N-pentachlorophenylthio ethyl acetamide, 2-benzylthio ethylamine malate,
2-benzylthio ethylamine nicotinate, 2-benzylthio 2-methyl propylamine
hydrochloride, 2-benzylthio propylamine lactate, N-(2-benzylthio
ethyl)nicotinamide
hydrochloride, N-(2-benzylthio ethyl) 10-undecene amide, N-(2-benzylthio
ethyl)
hexadecanamide, S-.beta.-aminoethyl mercaptobutyric acid, N-(2-benzylthio
ethyl)formamide, N-(2-benzylthio
ethyl)phenylacetamide, N-[2-(2,6-dimethyl
phenyl)ethyl] hexanamide, 2-o-aminophenylthio ethylamine succinate, N-(2-
benzylthio
ethyl) glutamine, S-.beta.-aminoethyl mercapto acetic acid (3-S-.beta.-
aminoethyl)
mercapto propionic acid, (3-S-.gamma.-amino propyl) mercapto acetic acid, S(2-
p-
methoxybenzamido ethyl) mercapto 2-(2-naphtyl methylthio) ethylamine
hydrochloride, 2-
(2-naphtyl methylthio) ethylamine disuccinate, (2-thenyl) 2-thio ethylamine
hydrobromide,
2-N-acetyl (2-thenylthio- ethylamine, 2-o-chlorobenzylthio ethylamine
hydrochloride, 2-p-
chlorobenzylthio ethylamine glycolate, 2-o-fluorobenzylthio ethylamine
hydrochloride, 2-
furfurylthio ethylamine hydrochloride, 2-tetrahydrofurfurylthio ethylamine p-
amino-
benzoate, 2-.beta.-phenylethylthio ethylamine glutamate, 2-diphenylmethylthio
ethylamine
hydrochloride, 2-triphenyl methylthio ethylamine hydrochloride hemihydrate, 2-
(2-pyridyl
ethylthio)ethylamine hydrochloride, 2-(2-p-toluene sulfonamido ethylthio)
pyridine N-
oxide, 2-.beta.-aminoethylthiomethyl pyridine N-oxide dihydrochloride, 2-
.beta.-
aminoethylthio pyridine N-oxide hydrochloride, 2,4-dichloro 2-benzylthio
ethylamine
aspartate, N-[2-(3,4-dichloro benzylthio)ethyl] butyramide, N-[2-(2,6-dichloro
benzylthio)ethyl] dodecanamide, N-[2-(3,5-dichloro benzylthio)ethyl]
trifluoroacetamide
hydrochloride, 2-p-ethoxybenzylthio ethylamine hydrochloride, N-[2-m-
fluorobenzylthio
ethyl] chloroacetamide, 2-p-bromobenzylthio ethylamine succinate, 2-(3,4-
dimethoxy
benzylthio)ethylamine malate, 2-(3 ,4-methylenedioxy
benzylthio)ethylamine
hydrochloride, 2-(2,4-dichloro cetylthio)ethylamine, 2 (3,4,5-trimethoxy
benzylthio)ethylamine hydrocinnamate, 2-p-methoxy benzylthio ethylamine
salicylate, 2-o-
methylbenzylthio ethylamine phenyl-acetate, N-[2-p-dimethylaminobenzylthio
ethyl]
methane-sulfonamide,2-p-phenoxybenzylthio ethylamine hydrochloride, 2-.beta.-
aminoethylthio pyridine
hydrochloride,
2-benzylthio ethylamine citrate, N[2-benzylthio ethyl] 2,4-dihydroxy
3,3-dimethyl butyramide, N-(2-benzylthio ethyl) 6,8-dihydroxy 7,7-dirnethyl 5-
oxo 4-aza
octanamide, N-[2-(2-pyridyl thio)ethyl] propionamide, 2-(2-pyridyl
methylthio)ethylamine
dihydrochloride, 2-benzylthio ethylamine
pantothenate,
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
8
S -(.beta. -acetamidoethyl)mercapto acetate of
beta.-morpholinoethyl, S -(.beta.-
phenylacetamidoethyl)mercaptoacetate N'-methyl 2-piperazino
ethyl,
S-(.beta.-ureidoethyl)mercaptoacetate of beta.-pyrrolidino-ethy, S-
(.beta.-
trifluoroacetamidoethyl)-.beta.mercapto-propionate
of .beta.-dimethylaminoethyl,
2-p-nitrobenzylthio ethylamine crotonate, 2-.beta.-morpholinocarbonyl
ethylthio
ethylamine hydrochloride, N,N-di(hydroxyethyl)S-(.beta.-benzamido-ethyl)
mercapto-
acetamido, N[2-N'-methyl piperazino carbonylthio ethyl] acetamide, 2-(1-
naphthyl
thio)ethylamine hydrochloride, N-(3-.beta.-ureidoethylthio propyl) succinamic
acid, 3-allylthio propylamine, 3-
(2,2'-dimethoxy ethylthio)propylamine,
3-(2,2'-dimethoxy ethylthio)propylamine sulfate, S-.beta.-aminoethylmercapto
acetic acid,
the hydrochloride of S-.beta.-aminoethyl mercapto
acetic acid,
N-(2-benzylthioethyl)acetamide, N-(2-
benzylthioethyl)propionami de, N-(2-
benzylthioethyl)butyramide, N-(2-benzylthioethyl)methanesulfonamide, N-
(2-
benzylthioethyl)ethanesulfonamide, N-
(2-benzylthioethyl-propanesulfonamide,
N-(2-benzylthio ethyl)butane sulfonamide, S-(2-p-acetamidobenzenesulfonamido
ethyl)
mercapto acetic acid, S-(2-p-
acetamidobenzamido ethyl) mercapto
acetic acid, N-(2-thenylthioethyl)acetamide, 2-benzylthio propylamine, 2-
benzylthio 2-
methyl propylamine, 2-(2-p-toluenesulfonamido ethylthio) pyridine N-oxide,
S-(2-p-butoxybenzamidoethyl)mercapto acetic acid, 2-t-butylthio ethylamine
hydrochloride, 2-methoxycarbonyl methylthio
ethylamine
hydrochloride, 2-ethoxycarbonylmethylthio ethylamine
hydrochloride,
2-propoxycarbonylmethyl thio ethylamine hydrochloride, 2-
butoxycarbonylmethylthio
ethylamine hydrochloride, 2,2'-thio diethylamine
dihydrochloride,
3 -(2-aminoethylthio)alanine hydrochloride, 2-benzylthio ethylammonium diacid
phosphate, 2-methylthio ethylamine, N-(methylthioethyl) p-acetamidobenzamide,
N-(2-methylthioethyl)nicotinamide, N-(2-
methylthioethypbenzamide, N-(2-
methylthioethyl) p-butoxybenzamide, N-(2-methylthioethyl)
butyramide,
N-(2-methylthioethyl) propionamide, N-(2-methylthioethyl) acetamide, N-(2-
methylthioethyl) butanesulfonamide, N-(2-octylthioethyl) methanesulfonamide, 2-
cetylthio
ethylamine hydrochloride, 2-(2-hydroxyethylthio) ethylamine hydrochloride,
2-methylthio ethylamine phenylacetatesnd 2-methylthio ethylamine undecylenate
OPTIONAL MODIFIED PEPTIDE
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
9
The compositions of the present invention may further comprise a modified
peptide
comprising from 3 to 50 D and/or L amino acids wherein the amino acids are
predominantly arginine and wherein the peptide comprises a modification which
is
selected from one or more of the group consisting of:
1) Incorporation of a histidine tag;
2) lipidation; and
3) pegylation
INCORPORATION OF A HISTIDINE TAG
In one aspect, the modified peptide when used preferably comprises a histidine
tag at either
the N terminus or C terminus. Advantageously, the presence of a histidine may
enhance the
effectiveness of the peptide against fungal infections such as Candida. This
is extremely
unexpected given that the cationic charge may not be significantly changed at
such a pH
range when compared to an equivalent peptide without the presence of a
histidine tag.
Suitably, the histidine tag may comprise at least two histidine residues.
Preferably, the
number of histidine residues may be up to 10. For example the histidine tag
may consist of
1 to 10 histidine residues, preferably 2 to 6. In one embodiment, the
histidine tag may
consist of two histidine residues
Advantageously, the presence of a histidine tag may be particularly useful for
treating
fungal infections of the mouth such as oropharyngeal candidiasis.
The oral cavity has a pH between 5.5 and 7 in disease states whereas the
normal pH of the
mouth of a healthy oral cavity is around pH 7 when not feeding. However, pH
influences
the charge of AMPs. Furthermore, secreted saliva also contains proteases that
aid the
breakdown of peptides.
The present inventors have surprisingly found that peptide of the invention
modified to
comprise a histidine tag are particularly adept at overcoming the pH and
protease
challenges associated which oral administration.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
Accordingly, modified peptides comprising a histidine tag may be comprised in
pharmaceutical foimulations adapted for oral administration.
Suitably, the peptide used in the pharmaceutical compositions of the present
invention,
5 method of treatment or prevention of the present invention and second
medical uses of the
present invention may comprises a histidine tag when the route of
administration or
intended route of administration is oral administration.
Preferably, the pH of the pharmaceutical compositions of the present invention
is in the
10 region of pH 5.5 to 6.5.
LIPIDATION
In one aspect, the modified peptides when used in the composition of the
present invention
are lipidated. For example, a lipid may be conjugated to a peptide comprising
from 3 to 50
D and/or L amino acids wherein the amino acids are predominantly arginine.
The present invention has surprisingly found that lipidation of the peptides
can
advantageously broaden the spectrum of activity of the peptides against
microbes and/or
enhance the activity of the peptides against some microbial infection.
Suitably, lipidated peptides of the present invention may be used in the
treatment or
prevention of yeast and mould infections (preferably such as Candida and/or
Aspergillus
infections, preferably Aspergillus infections). It has been surprisingly found
that lipidation
of the peptides claimed can confer potent activity on such lipidated peptides.
Accordingly, the modified peptides of the present invention may comprise a
lipid which
may be at either the C teiminus, N terminus or flanked with amino acid
residues.
Suitably the peptides of the present invention may comprise a C3 to C20 fatty
acid,
preferably a C4 to C14 fatty acid, preferably a Cs to C14 fatty acid,
preferably a C12 fatty
acid.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
11
Suitably the modified peptides of the present invention may comprise 3 to 50
amino acids
and a C3 to C20 fatty acid, preferably a C4 to C14 fatty acid, preferably a C8
to C14 fatty acid,
preferably a C12 fatty acid. Preferably, the modified peptides of the present
invention may
comprise 6 to 50 amino acids and a C3 to C20 fatty acid, preferably a C4 to
C14 fatty acid,
preferably a C8 to C14 fatty acid, preferably a C12 fatty acid.
In one aspect, the fatty acid may be flanked on either side by amino acid
residues. It has
surprisingly been found that the flanking of the fatty acid can lead to a
reduction in
haemolytic activity.
In another aspect, the fatty acid may be located on the terminus of the
peptide. It has
surprisingly been found that this may increase the antimicrobial effects of
the peptide in
terms of lower MIC.
In one preferable embodiment the fatty acid is a C12 fatty acid.
Advantageously, this length
of fatty acids exhibits both good antimicrobial effects and additionally has
low cytotoxicity
and haemolytic activity.
PEGYLATED PEPTIDES
In one aspect the modified peptide of the present invention is a PEGylated
peptide.
Advantageously, such PEGylated peptides have enhanced stability whilst still
providing
antimicrobial effects.
Suitably, the size of the PEG component may be approx. 300Da to approx. 40KDa
AMINO ACID RESIDUES
The peptide if used in the composition of the present invention may comprise
from 3 to 50
(preferably contiguous) amino acids.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
12
Suitably the peptide may comprise at least 3 or at least 4 or at least 5 or at
least 6 or at least
7 or at least 8 or at least 9 or at least 10 or at least 12 or at least 15 or
at least 20 or at least
25 or at least 30 or at least 35 or at least 40 or at least 45 amino acids.
Suitably, the peptide may comprise less than 50 or less than 45 or less than
40 or less than
35 or less than 30 or less than 25 or less than 0 or less than 15 amino acids.
In one aspect the number of amino acid residues referred to in the ranges
above does not
include the histidine tag residues. Thus, in one aspect, histidine residues at
either end of the
peptide are discounted when deteunining the numbering of amino acids in the
modified
peptide. In another aspect, all amino acid residues are counted including
those making up a
histidine tag.
In a preferred aspect of the invention the peptide comprises 3 to 20
(preferably
contiguous) amino acids, for example 3 to 16 amino acids. Preferably still the
peptide
comprises 5 to 14 amino acids. In some aspects, the peptide may comprise 12
(preferably
contiguous) amino acids.
As known to the skilled person, amino acids can be placed into different
classes depending
primarily upon the chemical and physical properties of the amino acid side
chain. For
example, some amino acids are generally considered to be hydrophilic or polar
amino acids
and others are considered to be hydrophobic or non-polar amino acids.
Hydrophobic
amino acid may be selected from the group of hydrophobic amino acids
consisting of
glycine, leucine, phenylalanine, proline, alanine, tryptophan, valine,
isoleucine, methionine,
tyrosine and threonine; cationic amino acids may be selected from the group
consisting of
omithine, histidine, arginine and lysine. As used herein, the temis
"hydrophobic" and
"cationic" may refer to amino acids having a hydrophobicity that is greater
than or equal to
¨1.10 and/or a net charge that is greater than or equal to 0 as described in
Fauchere and
Pliska Eur. J. Med Chem. 10:39 (1983). A hydrophobic or non-polar amino acid
may also
refer to an amino acid having a side chain that is uncharged at physiological
pH, is not
polar and that is generally repelled by aqueous solution. The amino acids may
be naturally
occurring or synthetic.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
13
Suitably, the arginine residue is the predominant amino acid in the peptide.
Suitably, at
least 50% of the amino acid residues are arginine residues, preferably at
least 60% or at
least 70% or at least 80% of the amino acids in the peptide are arginine.
Preferably, at least
90% are arginine residues. In some embodiments all the amino acids in the
peptide are
arginine residues (optionally with the exception of a histidine tag).
Suitably, the peptide may comprise amino acids other than arginine is non-
predominant
amounts. For example, histidine, ornithine and lysine could be used.
Suitably, 3 to 50 (preferably contiguous) D and/or L amino acids consist of
arginine or a
combination of arginine and lysine residues except for 0, 1, or 2
substitutions to an amino
acid residues other than arginine or lysine. Preferably, such substitutions
(if present) are
with another cationic amino acids selected from the group consisting of
histidine, ornithine
and lysine. Preferably the substations are with lysine.
Suitably, the peptide may be substituted with 0, 1, 2, 3, 4, 5, 6, 7 or 8
substitutions
provided that the arginine make up at least 60%, preferably at least 75% of
the peptide.
Preferably, the amino acids are L-amino acids.
In a preferred aspect of the invention, at least 90%, for example at least 95%
such as 97-
99% or even 100%, of the amino acids in the peptide are L-amino acids.
The invention also includes known isomers (structural, stereo-, confoimational
&
configurational), peptidomimetics, structural analogues of the above amino
acids, and
those modified either naturally (e.g. post-translational modification) or
chemically,
including, but not exclusively, phosphorylation, glycosylation, sulfonylation
and/or
hydroxylation.
In general, the peptide of the invention does not include the amino acids
aspartic acid,
glutamic acid, asparagine, glutamine or serine, but certain peptides of the
invention may
have activity even though these amino acids are present.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
14
One or more of the residues of the peptide can be exchanged for another to
alter, enhance
or preserve the biological activity of the peptide. Such a variant can have,
for example, at
least about 10% of the biological activity of the corresponding non-variant
peptide.
Conservative amino acids are often utilised, i.e. substitutions of amino acids
with similar
chemical and physical properties as described above. Hence, for example,
conservative
amino acid substitutions may involve exchanging lysine for arginine, ornithine
or histidine;
or exchanging arginine for lysine or isoleucine, ornithine for histidine; or
exchanging one
hydrophobic amino acid for another. After the substitutions are introduced,
the variants are
screened for biological activity.
PEPTIDES
The term "peptide" as used herein means, in general terms, a plurality of
amino acid
residues joined together by peptide bonds. It is used interchangeably and
means the same
as polypeptide and protein.
The term "modified peptide" refers to a peptide comprising 3 to 50 amino acid
residues
predominantly arginine further comprising: a histidine tag; and/or a fatty
acid and/or a
pegylated peptide. Suitably, the modified peptides of the present invention,
may be linear
peptides.
Preferably, the modified peptides of the present invention may consist of:
I) 3 to 50 amino acid residues predominantly arginine and a histidine tag;
2) 3 to 50 amino acid residues predominantly arginine and one or more fatty
acids;
3) 3 to 50 amino acid residues predominantly arginine, a histidine tag and one
or more
fatty acids;
4) a PEGylated peptide of 3 to 50 amino acid residues predominantly arginine
and a
histidine tag;
5) a PEGylated peptide of 3 to 50 amino acid residues predominantly arginine
and one
or more fatty acids; or
6) a PEGylated peptide of 3 to 50 amino acid residues predominantly arginine,
a
histidine tag and one or more fatty acids.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
The peptides may generally be synthetic peptides. The peptides may be
isolated, purified
peptides or variants thereof, which can be synthesised in vitro, for example,
by a solid
phase peptide synthetic method, by enzyme catalysed peptide synthesis or with
the aid of
recombinant DNA technology.
5
To identify active peptides that have little or no undesired toxicity for
mammalian cells,
individual peptides, or libraries of peptides, can be made and the individual
peptides or
peptides from those libraries can be screened for antimicrobial activity and
toxicity,
including, but not limited to, antifungal, antibacterial, antiviral,
antiprotozoal, anti-parasitic
10 activity and toxicity.
The peptides of the invention can exist in different forms, such as free
acids, free bases,
esters and other prodrugs, salts and tautomers, for example, and the invention
includes all
variant forms of the compounds.
Thus, the invention encompasses the salt or pro-drug of a peptide or peptide
variant of the
invention.
ADMINSTRATION
The composition of the invention may be administered in the form of a
pharmaceutically
acceptable salt. The pharmaceutically acceptable salts of the present
invention can be
synthesized from the parent peptide which contains a basic or acidic moiety by
conventional chemical methods. Generally, such salts can be prepared by
reacting the free
acid or base ft:inns of the peptide with a stoichiometric amount of the
appropriate base or
acid in water or in an organic solvent, or in a mixture of the two; generally,
nonaqueous
media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are
preferred. Lists of
suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed.,
Mack
Publishing Company, Easton, Pa., US, 1985, p. 1418, the disclosure of which is
hereby
incorporated by reference; see also Stahl et al, Eds, "Handbook of
Pharmaceutical Salts
Properties Selection and Use", Verlag Helvetica Chimica Acta and Wiley-VCH,
2002.
The invention thus includes pharmaceutically-acceptable salts of the
composition of the
invention wherein the parent compound is modified by making acid or base salts
thereof
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
16
for example the conventional non-toxic salts or the quaternary ammonium salts
which are
formed, e.g., from inorganic or organic acids or bases. Examples of such acid
addition
salts include acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate,
butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,
digluconate,
dodecylsulfate, ethanesulfonate, fumarate,
glucoheptano ate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-
hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-
naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, 3 -
phenylpropionate, picrate, pivalate, propionate, succinate, tartrate,
thiocyanate, tosylate,
and undecanoate. Base salts include ammonium salts, alkali metal salts such as
sodium
and potassium salts, alkaline earth metal salts such as calcium and magnesium
salts, salts
with organic bases such as dicyclohexylamine salts, N-methyl-D-glutamine, and
salts with
amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-
containing
groups may be quaternized with such agents as lower alkyl halides, such as
methyl, ethyl,
propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like
dimethy 1, diethyl,
dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl,
myristyl and stearyl
chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl
bromides and
others.
Salts of carboxyl groups of a peptide or peptide variant of the invention may
be prepared in
the usual manner by contacting the peptide with one or more equivalents of a
desired base
such as, for example, a metallic hydroxide base, e.g. sodium hydroxide; a
metal carbonate
or bicarbonate such as, for example, sodium carbonate or bicarbonate; or an
amine base
such as, for example, triethylamine, triethanolamine and the like.
The invention includes prodrugs for the active pharmaceutical species of the
described
peptide, for example in which one or more functional groups are protected or
derivatised
but can be converted in vivo to the functional group, as in the case of esters
of carboxylic
acids convertible in vivo to the free acid, or in the case of protected
amines, to the free
amino group. The term "prodrug," as used herein, represents in particular
structures which
are rapidly transformed in vivo to the parent structure, for example, by
hydrolysis in blood.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
17
A further aspect of the invention provides a pharmaceutical composition
comprising a
pharmaceutically effective amount of cysteamine and/or a derivative thereof.
When a
modified peptide is used this may form part of the composition or may be
administered
separately.
The composition may also include a pharmaceutically acceptable carrier,
excipient or
diluent. The phrase "pharmaceutically acceptable" is employed herein to refer
to those
compounds, materials, compositions, and/or dosage forms which are, within the
scope of
sound medical judgment, suitable for use in contact with the tissues of human
beings or, as
the case may be, an animal without excessive toxicity, irritation, allergic
response, or other
problem or complication, commensurate with a reasonable benefit/risk ratio.
In a preferred use according to the invention the fungal pathogen is of the
genera Candida
spp. or Aspergillus spp. For example the fungal pathogen may be Candida
albicans, or
Aspergillus fumigatus.
The fungal infection may be a systemic, topical, subcutaneous, cutaneous or
mucosal
infection. Preferably, the fungal infection may be a systemic or mucosal
infection.
The compositions of the invention are potent antifungals for a wide variety of
pathogenic
yeast and moulds. However, the compositions of the invention may also be
useful in the
treatment of other conditions including, but not limited to, conditions
associated with
mucosal infections, for example, cystic fibrosis, gastrointestinal,
urogenital, urinary (e.g
kidney infection or cystitis) or respiratory infections.
In one embodiment, the compositions of the invention are useful in treating
the symptoms
of respiratory/lung disease, particulary in cystic fibrosis or COPD.
The Win). "treatment" relates to the effects of the peptides described herein
that in
imparting a benefit to patients afflicted with an (infectious) disease,
including an
improvement in the condition of the patient or delay in disease progression.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
18
In a further aspect, the invention provides a method of treating or preventing
a microbial
infection in a subject comprising administering to said subject a
therapeutically effective
amount of a composition according to the invention.
In a preferred method of the invention, the microbial infection is a fungal
infection. In the
method of the invention the peptide is preferably administered orally.
Mammals, birds and other animals may be treated by the peptides, compositions
or
methods described herein. Such mammals and birds include humans, dogs, cats
and
livestock, such as horses, cattle, sheep, goats, chickens and turkeys and the
like. Moreover,
plants may also be treated by the peptides, compositions or methods of the
invention.
Where the subject is an animal, the method of the invention may be applied to
nail-like
features, including, but not exclusive to, hooves, claws and trotters.
To achieve the desired effect(s), the composition, a variant thereof or a
combination
thereof, may be administered as single or divided dosages, for example, of at
least about
0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about
300 to 500
mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1
mg/kg to
about 50 to 100 mg/kg of body weight or at least about 1 mg/kg to about 20
mg/kg of body
weight, although other dosages may provide beneficial results. The amount
administered
will vary depending on various factors including, but not limited to, the
peptide chosen and
its clinical effects, the disease, the weight, the physical condition, the
health, the age of the
mammal, whether prevention or treatment is to be achieved, and if the peptide
is
chemically modified.
Administration of the therapeutic agents in accordance with the present
invention may be
in a single dose, in multiple doses, in a continuous or intermittent manner,
depending, for
example, upon the recipient's physiological condition, whether the purpose of
the
administration is therapeutic or prophylactic, and other factors known to
skilled
practitioners. The administration of the peptides of the invention may be
essentially
continuous over a pre-selected period of time or may be in a series of spaced
doses. Both
local and systemic administration is contemplated.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
19
To prepare the composition, cysteamine and/or a derivative thereof is
synthesized or
otherwise obtained, purified as necessary or desired, and then lyophilized and
stabilized.
The composition can then be adjusted to the appropriate concentration and
optionally
combined with other agents. The absolute weight of a given peptide included in
a unit dose
can vary widely. For example, about 0.01 mg to about 2 g or about 0.01 mg to
about 500
mg, of at least one peptide of the invention, or a plurality of peptides
specific for a
particular cell type can be administered. Alternatively, the unit dosage can
vary from about
0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to
about 25 g, from
about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to
about 4 g, or
from about 0.5 g to about 2 g.
Thus, one or more suitable unit dosage forms comprising the therapeutic
composition of
the invention can be administered by a variety of routes including oral,
parenteral
(including subcutaneous, intravenous, intramuscular and intraperitoneal),
rectal, deinial,
transdermal, intrathoracic, intrapulmonary and intranasal (respiratory)
routes. The
therapeutic peptides may also be formulated in a lipid folinulation or for
sustained release
(for example, using microencapsulation, see WO 94/07529, and US Patent No.
4,962,091).
The formulations may, where appropriate, be conveniently presented in discrete
unit
dosage forms and may be prepared by any of the methods well-known to the
pharmaceutical arts. Such methods may include the step of mixing the
therapeutic agent
with liquid carriers, solid matrices, semi-solid carriers, finely divided
solid carriers or
combinations thereof, and then, if necessary, introducing or shaping the
product into the
desired delivery system.
When the therapeutic compositions of the invention are prepared for oral
administration,
they are generally combined with a pharmaceutically acceptable carrier,
diluent or
excipient to form a pharmaceutical formulation, or unit dosage form. For oral
administration, the peptides may be present as a powder, a granular formation,
a solution, a
suspension, an emulsion or in a natural or synthetic polymer or resin for
ingestion of the
active ingredients from a chewing gum. The active ingredients may also be
presented as a
bolus, electuary or paste. Orally administered therapeutic compositions of the
invention
can also be foimulated for sustained release, e.g., cysteamine can be coated,
micro-
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
encapsulated, or otherwise placed within a sustained delivery device. The
total active
ingredients in such formulations comprise from 0.1% to 99.9% by weight of the
formulation.
5 Pharmaceutical formulations containing the therapeutic composition of the
invention can
be prepared by procedures known in the art using well-known and readily
available
ingredients. For example, the peptide can be formulated with common
excipients, diluents,
or carriers, and formed into tablets, capsules, solutions, suspensions,
powders, aerosols and
the like. Examples of excipients, diluents, and carriers that are suitable for
such
10 formulations include buffers, as well as fillers and extenders such as
starch, cellulose,
sugars, mannitol, and silicic derivatives. Binding agents can also be included
such as
carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose
and
other cellulose derivatives, alginates, gelatine, and polyvinyl-pyrrolidone.
Moisturizing
agents can be included such as glycerol, disintegrating agents such as calcium
carbonate
15 and sodium bicarbonate. Agents for retarding dissolution can also be
included such as
paraffin. Resorption accelerators such as quaternary ammonium compounds can
also be
included. Surface active agents such as cetyl alcohol and glycerol
rnonostearate can be
included. Adsorptive carriers such as kaolin and bentonite can be added.
Lubricants such
as talc, calcium and magnesium stearate, and solid polyethyl glycols can also
be included.
20 Preservatives may also be added. The compositions of the invention can
also contain
thickening agents such as cellulose and/or cellulose derivatives. They may
also contain
gums such as xanthan, guar or carbo gum or gum arabic, or alternatively
polyethylene
glycols, bentones and montmorillonites, and the like.
For example, tablets or caplets containing the composition of the invention
can include
buffering agents such as calcium carbonate, magnesium oxide and magnesium
carbonate.
Suitable buffering agents may also include acetic acid in a salt, citric acid
in a salt, boric
acid in a salt and phosphoric acid in a salt. Caplets and tablets can also
include inactive
ingredients such as cellulose, pregelatinized starch, silicon dioxide,
hydroxyl propyl methyl
cellulose, magnesium stearate, microcrystalline cellulose, starch, talc,
titanium dioxide,
benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol,
sodium phosphate,
zinc stearate, and the like. Hard or soft gelatine capsules containing at
least one peptide of
the invention can contain inactive ingredients such as gelatine,
microcrystalline cellulose,
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
21
sodium lauryl sulphate, starch, talc, and titanium dioxide, and the like, as
well as liquid
vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover,
enteric-coated
caplets or tablets containing one or more peptides of the invention are
designed to resist
disintegration in the stomach and dissolve in the more neutral to alkaline
environment of
the duodenum.
The therapeutic composition of the invention can also be formulated as elixirs
or solutions
for convenient oral administration or as solutions appropriate for parenteral
administration,
for instance by intramuscular, subcutaneous, intraperitoneal or intravenous
routes. The
pharmaceutical formulations of the invention can also take the form of an
aqueous or
anhydrous solution or dispersion, or alternatively the fotin of an emulsion or
suspension or
salve.
Thus, the therapeutic compositions may be formulated for parenteral
administration (e.g.
by injection, for example, bolus injection or continuous infusion) and may be
presented in
unit dose form in ampules, pre-filled syringes, small volume infusion
containers or in
multi-dose containers. The active ingredients may form suspensions, solutions,
or
emulsions in oily or aqueous vehicles, and may contain formulatory agents such
as
suspending, stabilizing and/or dispersing agents. Alternatively, the active
ingredients may
be in powder faun, obtained by aseptic isolation of sterile solid or by
lyophilization from
solution for constitution with a suitable vehicle, e.g. sterile, pyrogen-free
water before use.
These formulations can contain pharmaceutically acceptable carriers, vehicles
and
adjuvants that are well-known in the art. It is possible, for example, to
prepare solutions
using one or more organic solvent(s) that is/are acceptable from the
physiological
standpoint, chosen, in addition to water, from solvents such as acetone,
acetic acid,
ethanol, isopropyl alcohol, dimethyl sulphoxide, glycol ethers such as the
products sold
under the name "Dowanol", polyglycols and polyethylene glycols, C l-C4 alkyl
esters of
short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such
as the products
marketed under the name "Miglyol", isopropyl mytrisate, animal, mineral and
vegetable
oils and polysiloxanes.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
22
Solvents or diluents comprising the peptides of the invention may include acid
solutions,
dimethylsulphone, N-(2-mercaptopropionyl) glycine, 2-n-nonyl-1,3-dioxolane and
ethyl
alcohol. Preferably the solvent/diluent is an acidic solvent, for example,
acetic acid, citric
acid, boric acid, lactic acid, propionic acid, phosphoric acid, benzoic acid,
butyric acid,
malic acid, malonic acid, oxalic acid, succinic acid or tartaric acid.
Also contemplated are combination products that include one or more peptides
of the
present invention and one or more other antimicrobial or antifungal agents,
for example,
polyenes such as amphotericin B, amphotericin B lipid complex (ABCD),
liposomal
amphotericin B (L-AMB), and liposomal nystatin, azoles and triazoles such as
voriconazole, fluconazole, ketoconazole, itraconazole, pozaconazole and the
like; glucan
synthase inhibitors such as caspofungin, micafungin (FK463), and V-
echinocandin
(LY303366); griseofulvin; allylamines such as terbinafine; flucytosine or
other antifungal
agents, including those described herein. In addition, it is contemplated that
the peptides
might be combined with topical antifungal agents such as ciclopirox olamine,
haloprogin,
tolnaftate, undecylenate, topical nysatin, amorolfine, butenafine, naftifine,
terbinafine, and
other topical agents.
Additionally, the compositions may be foimulated as sustained release dosage
forms and
the like. The formulations can be so constituted that they release the active
peptide, for
example, in a particular part of the intestinal or respiratory tract, possibly
over a period of
time. Coatings, envelopes, and protective matrices may be made, for example,
from
polymeric substances, such as polylactide-glycolates, liposomes,
microemulsions,
microparticles, nanoparticles, or waxes. These coatings, envelopes, and
protective matrices
are useful to coat indwelling devices, e.g. stents, catheters, peritoneal
dialysis tubing,
draining devices and the like.
For topical administration, the active agents may be foirnulated as is known
in the art for
direct application to a target area. Forms chiefly conditioned for topical
application take
the form, for example, of creams, milks, gels, powders, dispersion or
microemulsions,
lotions thickened to a greater or lesser extent, impregnated pads, ointments
or sticks,
aerosol formulations (e.g. sprays or foams), soaps, detergents, lotions or
cakes of soap.
Other conventional foinis for this purpose include wound dressings, coated
bandages or
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
23
other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays,
and aerosols.
Thus, the therapeutic peptides of the invention can be delivered via patches
or bandages for
dennal administration. Alternatively, the composition can be foimulated to be
part of an
adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer.
For long-term
applications it might be desirable to use microporous and/or breathable
backing laminates,
so hydration or maceration of the skin can be minimized. The backing layer can
be any
appropriate thickness that will provide the desired protective and support
functions. A
suitable thickness will generally be from about 10 microns to about 200
microns.
Topical administration may be in the form of a nail coating or lacquer. For
example, the
antifungal peptides can be foimulated in a solution for topical administration
that contains
ethyle acetate (NF), isopropyl alcohol (USP), and butyl monoester of
poly[methylvinyl
ether/maleic acid] in isopropyl alcohol.
Pharmaceutical formulations for topical administration may comprise, for
example, a
physiologically acceptable buffered saline solution containing between about
0.001 mg/ml
and about 100 mg/ml, for example between 0.1 mg/ml and 10 mg/ml, of one or
more of the
peptides of the present invention specific for the indication or disease to be
treated.
Ointments and creams may, for example, be formulated with an aqueous or oily
base with
the addition of suitable thickening and/or gelling agents. Lotions may be
formulated with
an aqueous or oily base and will in general also contain one or more
emulsifying agents,
stabilizing agents, dispersing agents, suspending agents, thickening agents,
or coloring
agents. The active peptides can also be delivered via iontophoresis, e.g., as
disclosed in US
Patent Nos. 4,140,122; 4,383,529; or 4,051,842. The percentage by weight of a
therapeutic
agent of the invention present in a topical formulation will depend on various
factors, but
generally will be from 0.01% to 95% of the total weight of the fommlation, and
typically
0.1-85% by weight.
Drops, such as eye drops or nose drops, may be formulated with one or more of
the
therapeutic peptides in an aqueous or non-aqueous base also comprising one or
more
dispersing agents, solubilizing agents or suspending agents. Liquid sprays can
be pumped,
or are conveniently delivered from pressurized packs. Drops can be delivered
via a simple
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
24
eye dropper-capped bottle, via a plastic bottle adapted to deliver liquid
contents drop-wise,
or via a specially shaped closure.
The therapeutic peptide may further be formulated for topical administration
in the mouth
or throat. For example, the active ingredients may be formulated as a lozenge
further
comprising a flavoured base, usually sucrose and acacia or tragacanth;
pastilles comprising
the composition in an inert base such as gelatine and glycerine or sucrose and
acacia; and
mouthwashes comprising the composition of the present invention in a suitable
liquid
carrier. Alternatively, the active ingredients may be fonnulated as a film
strip or buccal
tablet, which may or may not be dissolvable.
Specific non-limiting examples of the carriers and/or diluents that are useful
in the
pharmaceutical foimulations of the present invention include water and
physiologically
acceptable buffered saline solutions such as phosphate buffered saline
solutions pH 7.0-
8Ø
The compositions of the invention can also be administered to the respiratory
tract For
administration by inhalation or insufflation, the composition may take the
faun of a dry
powder, for example, a powder mix of the therapeutic agent and a suitable
powder base
such as lactose or starch. Therapeutic peptides of the present invention can
also be
administered in an aqueous solution when administered in an aerosol or inhaled
form.
Thus, other aerosol pharmaceutical fotmulations may comprise, for example, a
physiologically acceptable buffered saline solution containing between about
0.001 mg/ml
and about 100 mg/ml for example between 0.1 and 100 mg/ml, such as 0.5-50
mg/ml, 0.5-
20 mg/ml, 0.5-10 mg/ml, 0.5-5 mg/ml or 1-5 mg/ml of one or more of the
peptides of the
present invention specific for the indication or disease to be treated.
Antibiotic/Antifungal Agent
The compositions of the present invention may further comprise an antibiotic.
The term
"antibiotic" is used to refer to antibacterial agents that may be derived from
bacterial
sources. Antibiotic agents may be bactericidal and/or bacteriostatic.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
Generally the antibiotic agent is of the group consisting of aminoglycosides,
ansamycins,
carbacephem, carbapenems, cephalosporins (including first, second, third,
fourth and fifth
generation cephalosporins), lincosamides, macrolides, monobactams,
nitrofurans,
quinolones, penicillin, sulfonamides, polypeptides and tetracyclins.
Alternatively or
5 additionally the antibiotic agent may be effective against mycobacteria.
According to one embodiment, the antibiotic agent may be an aminoglycoside
such as
Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin or
Paromomycin.
10 According to one embodiment, the antibiotic agent may be an such as
Geldanamycin and
Herbimycin
Alternatively the antibiotic agent may be a carbacephem such as Loracarbef.
15 According to a further embodiment, the antibiotic agent is a carbapenem
such as
Ertapenem, Doripenem, Imipenem/Cilastatin or Meropenem.
Alternatively the antibiotic agent may be a cephalosporins (first generation)
such as
Cefadroxil, Cefazolin, Cefalexin, Cefalotin or Cefalothin, or alternatively a
Cephalosporins
20 (second generation) such as Cefaclor, Cefamandole, Cefoxitin, Cefprozil
or Cefuroxime.
Alternatively the antibiotic agent may be a Cephalosporins (third generation)
such as
Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime,
Ceftibuten,
Ceftizoxime and Ceftriaxone or a Cephalosporins (fourth generation) such as
Cefepime
and Ceftobiprole.
The antibiotic agent may be a lincosamides such as Clindamycin and
Azithromycin, or a
macrolide such as Azithromycin, Clarithromycin, Dirithromycin, Erythromycin,
Roxithromycin, Troleandomycin, Telithromycin and Spectinomycin.
Alternatively the antibiotic agent may be a monobactams such as Aztreonam, or
a
nitrofuran such as Furazolidone or Nitrofurantoin.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
26
The antibiotic agent may be a penicillin such as Amoxicillin, Ampicillin,
Azlocillin,
Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin,
Nafcillin, Oxacillin,
Penicillin G or V. Piperacillin, Temocillin and Ticarcillin.
The antibiotic agent may be a sulfonamide such as Mafenide,
Sulfonamidochrysoidine,
Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfamethizole,
Sulfamethoxazole,
Sulfasalazine, Sulfisoxazole, Trimethoprim, and Trimethoprim-
S ulfamethoxazole (Co-trimoxazole) (TMP-SMX).
The antibiotic agent may be a quinolone such as Ciprofloxacin, Enoxacin,
Gatifloxacin,
Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin,
Ofloxacin,
Trovafloxacin, Grepafloxacin, Sparfloxacin and Temafloxacin.
According to one embodiment, the antibiotic agent may be a polypeptide such as
Bacitracin, Colistin and Polymyxin B.
Alternatively, the antibiotic agent may be a tetracycline such as
Demeclocycline,
Doxycycline, Minocycline and Oxytetracycline
Alternatively or additionally the antibiotic agent may be effective against
mycobacteria.
In particular the antibiotic agent may be Clofazimine, Lamprene, Dapsone,
Capreomycin,
Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin,
Rifabutin,
Rifapentine or Streptomycin.
Generally the antibiotic agent is active in the treatment or prophylaxis of
infections caused
by gram-negative or gram-positive bacteria, such as Escherichia coli and
Klebsiella
particularly Pseudomonas aeruginosa.
The ratio of cysteamine and/or a derivative thereof to antibiotic in the
products of the
invention may be from 1:10 to 10:1; generally at least 2:1 for example at
least 3:1 or 4:1.
Alternatively, the ratio of the antibiotic agent to the second agent in the
products of the
invention may be from 1:100 1:2000, for example from 1:500 to 1:1000.
According to one
embodiment, the ratio of the antibiotic agent to the second agent is
approximately 1:1.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
27
Preferably the first antibiotic agent is a non-peptide antibiotic and the
second agent is
cysteamine and the product contains these components at a ratio from 2:1 up to
4:1.
According to a further embodiment the ratio may be approximately 1:1.
The antifungal may sleeted from the group consisting of Fluconazole,
Itraconazole,
Caspofungin and Amphotericin B, for example, one or more of Fluconazole,
Itraconazole
and Caspofungin.
The active agents may be administered simultaneously, sequentially or
separately. The
active agents may be provided as a combination package. The combination
package may
contain the product of the invention together with instructions for
simultaneous, separate or
sequential administration of each of the active agents. For sequential
administration, the
active agents can be administered in any order.
Throughout the description and claims of this specification, the words
"comprise" and
"contain" and variations of the words, for example "comprising" and
"comprises", means
"including but not limited to", and is not intended to (and does not) exclude
other moieties,
additives, components, integers or steps.
Throughout the description and claims of this specification, the singular
encompasses the
plural unless the context otherwise requires. In particular, where the
indefinite article is
used, the specification is to be understood as contemplating plurality as well
as singularity,
unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups
described in
conjunction with a particular aspect, embodiment or example of the invention
are to be
understood to be applicable to any other aspect, embodiment or example
described herein
unless incompatible therewith.
FIGURES
Figure 1 shows the effects of cysteamine (Lynovex, NM001) on Candida albi cans
73/025
after 24 hours.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
28
Figure 2 shows the effects of cysteamine (Lynovex, NM001) on Candida albicans
73/025
after 48 hours.
Figures 3A-B shows the effect of cysteamine (NM001) on four Aspergillus
fumigatus
strains (AF20, AF22 and NCPF2939 in Figure 3A and AF 2002/0066 in Figure 3B).
Figures 4 A-D show petri dishes with Aspergillus growth isolated from the
sputum of 4
patients with cystic fibrosis.
Figures 5 A-B shows the microbial load of the sputum of 10 cystic fibrosis
patients at 4
hours (Figure 5A) and 24 hours (Figure 5B) of treatment with cysteamine
(Lynovex),
Tobramycin or a combination thereof.
EXAMPLES
The following Example illustrates the invention.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
29
EXAMPLE 1
Minimum inhibitory concentration test of cysteamine (Lynovex) (NM001) against
a
vaginal isolate of C. albicans.
Aim:
The aim of the experiment was to determine the MIC of Lynovex (NM001) against
vulvovaginal isolate Candida albicans 73/025. Plates were prepared which
contain a serial
doubling dilution of Lynovex (NM001) at 2 x final concentration. The cultures
were then
prepared in 2 x RPMI-1640, according to the CLSI M27-A3 protocol, and mixed in
equal
volumes in the plate resulting in 1 x RPMI-1640, the concentration of compound
mentioned below, and the CLSI standard inoculum for C. albicans. These were
incubated
at 35 C and read at 24 h and 48 h on the BioTek plate reader.
Compounds for antifungal testing
Lynovex (NM001)
Method:
Culture preparation
1. Culture of Candida albicans grown overnight at 30 C
2. Culture removed from incubator. Add 2 ml sterile dH20 to the overnight
culture
and wash the cells from the surface of the agar. Cells placed in fresh bijou
tube.
3. Serial doubling dilution carried out and measured against same volume of
MacFarland Standard
a. 100 pl culture added to well Al of microtitre plate.
b. 100 pl sterile dH20 added to wells A2 ¨ Al 1.
c. 100 IA culture added to well A2 of microtitre plate and mixed well
resulting
in a 1 in 2 dilution.
d. 100 p1 taken from A2 and added to A3 on plate and mixed well resulting in
a 1 in 4 dilution.
e. This is continued to All where 100 pl is removed and discarded.
f. Well Al2 contains 100 ul 0.5 MacFarland standard which had been mixed
well.
a Plate read at OD530 on BioTek
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
h. Concentration of cells required will be the one that is the equivalent to
the
0.5 MacFarland Standard
4. Dilute cells to the equivalent of the 0.5 MacFarland Standard (for
example a 1 in 16
dilution may be required) in sterile dH20
5 5. Culture then further diluted 1 in 50 into water (which is 100 IA
culture + 4900 pi
sterile water).
6. Culture then further diluted 1 in 20 into 2 x RPMI-1640 (which is 1 ml
culture + 19
ml 2 x RPMI-1640).
10 Plate preparation
1. The highest final concentration on the plate will be as described in
layouts at the
end of the protocol. To allow for the addition of the cells this is prepared
at 2 x
final concentration described e.g 20,000 [ig/m1 is prepared, and so when cells
are
added this halves to 10,000 g/ml.
15 2. Solutions of each of the compounds are prepared in a sterile bijoux
giving a final
volume of 5 ml (5000 11).
3. Wells A1-A3, A4- A6, A7-A9 and A10-Al2 are prepared by the addition of 100
I
highest concentration of compound to the well (except Clotrimazole plate where
the
compounds go in A1-3 and A7-9 only).
20 4. All other wells contain 50 1 sterile dH20.
5. Serial doubling dilutions are carried out from Al ¨ A3 to B1 ¨ B3 whereby
50 Ill is
removed from Al- A3 and placed into B1 ¨ B3 and mixed well. Then take 50 IA
B 1 -B3 and place in C1-C3 and mix well. Take 50 1 from C 1 -C3 and add to D1-
D3 and mix well. This is repeated down to Fl ¨ F3. At Fl ¨ F3 50 I is removed
25 and discarded. G1 ¨ G3 and H1 ¨ H3 contain water alone and will be
untreated and
uninoculated controls.
6. The same procedure is repeated for the wells in columns A4-A6, A7-A9 and
A10-
Al2. See plate layout at bottom of page.
30 Inoculating plates with Candida
1. Add 50 1 of the prepared Candida culture to the all wells on the plate
except wells
Hl-H12 (bottom row) which will contain water and 2 x RPMI-1640 alone,
resulting
in an uninoculated control.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
31
2. The plate is read in the BioTek at OD530.
3. Plates are then placed in a plastic box which contains either a Petri
dish of water or
a bed of damp tissue. The box is sealed and placed in the 35 C. The presence
of
the water in the dish or on the tissue provides and environment of relatively
high
humidity.
4. Plates are read at 24 h and 48 h and MIC graphs plotted.
Plate Layout
Lynovex Plate
1 2 3 4 5 6 7 8 9 10 11
12
A 10,000 39.0625
B 5,000 19.53125
C 2,500 9.765625
D 1250 4.8828125
E 625 2.44140625
F 312.5 1.220703125
G 156.25 0
H 78.125 No cells
Results
As can be seen Cysteamine inhibited the growth of Candida albicans.
EXAMPLE 2
Mimimum inhibitory concentration test of Lynovex (cysteamine) (NM001) against
Aspergillus fumigatus.
Aim:
The aim of the experiment was to determine the MIC of Lynovex (NM001) against
various
Aspergillus fumigatus strains. Microtitre plates were prepared which contained
a serial
doubling dilution of Lynovex (NM001) at 2 x final concentration. The cultures
were then
prepared in 2 x RPMI-1640, according to the CLSI M38-A2 protocol, and mixed in
equal
SUBSTITUTE SHEET (RULE 26)
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
32
volumes in the plate resulting in 1 x RPMI-1640, the concentration of compound
mentioned below, and the CLSI standard inoculum for A. fumigatus.
These were
incubated at 37 C and read at 24 h and 48 h on the BioTek plate reader.
Compounds for antifungal testing
Lynovex (NM001)
Method:
Culture preparation
1. Cultures of A. fumigatus 2002/0066, AF20, AF22 and NCPF2939 grown at 37 C
on Potato Dextrose Agar (PDA) slopes for >72 h to allow for optimal spore
production).
2. Culture removed from incubator and 1 ml sterile 0.85% saline added to the
culture
on the slope to wash the culture from the surface of the agar. Spores were
collected
in a fresh tube.
3. Serial doubling dilution of the spores were carried out and measured at
an optical
density of 530 nm. The cell density was then adjusted to and OD 530 nm of 0.09
to
0.13, as specified in the CLSI document M38A.
4. The spores were then further diluted 1 in 50 into 2 x RPMI-1640.
5. The diluted spores were then added in equal volumes to the Lynovex in the
microtitre plates (50 ul cells added to 50 ul Lynovex in the microtitre
plate).
Plate preparation
1. A stock solution of Lynovex was prepared at 10 mg/ml. The plate layout at
the end
of this protocol is the final concentration in the plate following the
addition of the
spores, therefore the plate has to be prepared at 2x final concentration to
allow the
dilution when the cells are added. Therefore the Lynovex prepared in the plate
is
10 mg/ml (10,000 p g/m1) and the final concentration in the plate following
addition
of cells is 5 mg/ml (5,0001,tg/m1).
2. 100 pi of Lynovex at 10 mg/ml is added to wells A1-A3 and A7-A9.
3. All other wells contain 50 ul sterile dH20.
4. Serial doubling dilutions are carried out from Al ¨ A3 to B1 ¨ B3 whereby
50 pl is
removed from Al- A3 and placed into B1 ¨ B3 and mixed well. Then take 50 ul
CA 02959003 2017-02-22
WO 2016/046524
PCT/GB2015/052714
33
B1-B3 and place in C1-C3 and mix well. Take 50 I from CI-C3 and add to D1-
D3 and mix well. This is repeated down to 1-11 ¨ H3. At HI ¨ H3 50 I is
removed
and added to A4-6 and the dilutions carried down to F4-6. G4-6 and H4-6
contain
water alone and will be untreated and uninoculated controls.
5. The same procedure is repeated for the wells in the second half of the
plate with
Lynovex being added to A7-9 and dilutions being carried out to H7-9 and then
continuing from A10-12.
Inoculating plates with A. fumigatus
1. Add 50 IA of the prepared A. fumigatus spores to the all wells on the plate
except
wells H4-6 and H10-H12 which will contain water and 2 x RPMI-1640 alone,
resulting in an uninoculated control.
2. G4-6 and G10-12 are the inoculated controls and have spores but no Lynovex.
3. The plate is read in the BioTek at 0D530.
4. Plates are then placed in a plastic box which contains either a Petri dish
of water or
a bed of damp tissue. The box is sealed and placed in the 37 C. The presence
of
the water in the dish or on the tissue provides and environment of relatively
high
humidity.
5. Plates are read at 24 h and 48 h and MIC graphs plotted.
Plate Layout
Final Lynovex Plate following addition of spores.
1 2 3 4 5 6 7 8 9 10 11
12
A 5,000 19.53125 5.000 19.53125
B 2,500 9.765625 2,500
9.765625
= 1250 4.8828125 - 1250
4.8828125
625 2.44140625 625 2.44140625
E 312.5 1.220703125 312.5
1.220703125
F 156.25 0.610365625 156.25 0.610365625
G 78.125 0 78.125 0
H 39.0625 No cells 39.0625 No cells
SUBSTITUTE SHEET (RULE 26)
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
34
EXAMPLE 3
Freshly produced sputum samples were obtained from CF patients and samples of
this were
exposed to a single dose of 1mg/m1 cysteamine of 1mg/m1 cysteamine and
tobramycin (10-
100 g/m1) before being plated out and incubated for 4 h and 24 h before
assessment of the
resulting number of colony forming units of microbes as compared to those from
untreated
sputum samples. Results are shown in Figure 5.
EXAMPLE 4
Minimum inhibitory concentration test of Itraconazole, Fluconazole and
Caspofungin
against a clinical isolate of E. dennatitidis.
Aim:
The aim of the experiment was to determine the MIC of Itraconazole,
Fluconazole and
Caspofungin against the clinical isolate Exophiala dermatitidis CA01. Two
Candida spp.
strains (C. krusei ATCC6258 & C. parapsilosis ATCC22019) and Exophiala
jeansehnei
reference strain NCPF2377 were also tested. These acted as quality control
strains.
Flat-bottomed Nunc Plates were prepared containing a serial doubling dilution
of
antifungals at 2 x final concentration. Cultures were then prepared in 2 x
RPMI-1640,
according to the CLSI M38-A2 protocol, and mixed in equal volumes in the plate
resulting
in 1 x RPMI-1640, the concentration of compound mentioned below, and the CLSI
standard inoculum. These were incubated at 35 C and read at 24 h and 48 h
(Candida
spp.) or 120 h (Exophiala spp.) on a BioTek plate reader at 530nm.
Compounds for antifungal testing
Itraconazole
Fluconazole
Caspofungin
Dimethylsulfoxide (DMSO; Vehicle Control)
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
Method:
Culture preparation:
7. Culture of Candida spp. grown on Sabouraud dextrose agar slope overnight at
30 C
8. Exophiala cultures grown on potato dextrose agar slope for 6 days at 35 C
5 9.
Cultures were removed from incubator and 2 ml sterile 0.15M NaC1 was added to
the overnight culture and spore and hyphal suspensions were harvested from the
surface of the agar and transferred to a sterile plastic bijou tube.
a. Exophiala spp. cultures were allowed to rest for up to 10 mm, allowing
hyphae to settle to the bottom. The top layers of spores were transferred to a
10 sterile plastic bijou tube.
10. Two-fold, serial dilutions were carried out and optical density measured
spectrophotometrically to attain the 0.5 McFarland Standard
a. 100 1 culture added to Al of microtitre plate.
b. 100 1 sterile 0.15M NaCl added to wells A2 ¨ Al2.
15 c.
100 I culture added to well A2 of microtitre plate and mixed by pipetting.
This results in a 1 in 2 dilution.
d. 100 1 taken from A2 and added to A3 on plate and mixed by pipetting (1
in 4 dilution).
e. This is continued to Al2 where 100 1 is removed and discarded.
20 f. This dilution series was repeated in rows B-D for the remaining 3
cultures
g. 0.5 McFarland Standard added to wells El ¨ E3
h. Plate read at 0D530 on BioTek.
i. Concentration of cells required will be the one that is the equivalent
to the
0.5 McFarland Standard
25 11.
Dilute cells to the equivalent of the 0.5 McFarland Standard (for example a 1
in 32
dilution may be required) in sterile 0.15M NaC1
12. Culture then further diluted 1 in 50 into 2 xRPMI-1640 (100 I culture +
4900 .1 2
x RPMI-1640).
30 Plate preparation
7. The highest final concentration on the plate will be as described in
layouts at the
end of the protocol. To allow for the addition of the cells this is prepared
at 2 x
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
36
final concentration described e. g. 2000 ig/m1 Fluconazole is prepared, and so
when cells are added this halves to 1000 pg/ml.
8. Solutions of each of the test compounds are prepared in a sterile plastic
bijou tube
giving a final volume of 5 ml.
9. Wells A1-A3 and A7-A9 are prepared by the addition of 200111 highest
concentration of compound to the wells.
10. All other wells contain 100 ill sterile dH20.
11. Serial dilutions are carried out from Al ¨ A3 to B1 ¨ B3 whereby 100 pl is
removed from Al- A3 and placed into B1 ¨ B3 and mixed well by pipetting. Then
take 100 1 Bl-B3 and place in Cl-C3 and mix well. Take 100 pl from Cl-C3 and
add to Dl-D3 and mix well. This is repeated down to H1 ¨ H3. At H1 ¨ H3100 iI
is removed and added to A4 ¨ A6. The dilution series is carried down the plate
in
the same manner to wells G4 ¨ G6 where a final 100 Ill is removed and
discarded.
H4 ¨ H6 contain water alone and will be untreated controls.
12. The same procedure is repeated for the wells in columns A7-A9 and Al 0-
Al2. See
plate layout at bottom of page.
Inoculating plates with Candida spp./Exophiala spp.
5. Add 100 ill of the prepared cultures to the all wells on the plate except
wells H10-
H12 (bottom row) which will contain water and 2 x RPMI-1640 alone, resulting
in
an uninoculated control.
6. Plates are read in the BioTek at 0D530,,,,,.
7. Plates are then placed in a plastic box which contains either a Petri dish
of water.
The box is sealed and incubated at 35 C (Exophiala spp.) or 30 C (Candida
spp.).
The presence of the water in the dish or on the tissue provides and
environment of
relatively high humidity.
8. The 0D530 of the Candida spp. plates are read after 24 h and 48 h, while
the
slower growing Exophiala spp. plates are read after 96 h and 120 h.
9. Raw BioTek data is transformed using Gen5 software and represented
graphically
using GraphPad Prism 4 software and MICs determined.
Plate Layouts
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
37
Exophiala FLUCONAZOLE ITRACONAZOLE
Plate 1 1 2 3 4 5 6 7 8 9 10 11 12
A 1000 3.91 32 0.125
B 500 1.95 16 0.063
C 250 0.98 8 0.031
D 125 0.49 4 0.016
E 62.5 0.24 2 0.008
F 31.25 0.12 1 0.004
G 15.63 0.06 0.5 0.002
H 7.81 0 0.25 No
Cells
Candida FLUCONAZOLE ITRACONAZOLE
Plate 1 1 2 3 4 5 6 7 8 9 10 11 12
A 500 1.95 32 0.125
B 250 0.98 16 0.063
C 125 0.49 8 0.031
D 62.5 0.24 4 0.016
E 31.25 0.12 2 0.008
F 15.63 0.06 1 0.004
G 7.81 0.03 0.5 0.002
H 3.91 0 0.25 No
Cells
All Strains, CASPOFUNGIN DMSO (Y())
Plate 2 1 2 3 4 5 6 7 8 9 10 11
12
A 64 0.25 0.5 0.002
B 32 0.1 0.25
0.001
C 16 0.06 0.125 0.000
D 8 0.031 0.063
0.000
E 4 0.016 0.031
0.000
F 2 0.008 0.016 0.000
G 1 0.004 0.008
0.000
H 0.5 0 0.004 No Cells
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
38
Antifungal activity of Antifungal drugs used in combination with Lynovex
(NM001)
against Exophiala dermatitidis CA01.
Aim:
The aim of the experiment was to determine the effect of Lynovex (NM001) on
the
antifungal drug susceptibility of Exophiala dermatitidis CA01. Microtitre
plates were
prepared following CLSI recommendations for broth microdilution chequerboard
method.
These plates contained a serial doubling dilution of Lynovex (NM001) combined
with
serial doubling dilutions of antifungals drugs; Fluconazole, Itraconazole,
Caspofungin and
Amphotericin B. Lynovex (NM001) and antifungals were initially prepared at 4x
final
concentration which were diluted to 2 x final concentration following their
combination.
The cultures were then prepared in 2 x RPMI-1640, and mixed in equal volumes
in the
plate resulting in 1 x RPMI-1640, and the 1 x concentration of compound
mentioned
below. These were incubated at 35 C and read at 72 h, 96 h, 120 h and 144 h on
the
BioTek plate reader.
Compounds for antifungal testing
Lynovex (NM001) in combination with Fluconazole, Itraconazole, Caspofungin and
Amphotericin B
Method:
Culture preparation
6. Three independent E. dermatitidis CA01 cultures were grown on PDA slope for
6
days at 35 C
7. Cultures were removed from incubator. 2 ml sterile 0.15M NaC1 added to the
overnight culture and cells harvested from the surface of the agar. Cells
placed in
fresh bijou tube.
8. The cultures were allowed to rest for up to 10 minutes, allowing any hyphae
to
settle to the bottom. The top layer of conidia was removed into fresh bijou.
Hyphae
were discarded.
9. Two-fold serial dilutions carried out with each culture and optical density
measured spectrophotometrically to attain the 0.5 McFarland Standard
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
39
a. 100 IA culture added to Fl of microtitre plate.
b. 100 1 sterile 0.15M NaC1 added to wells F2 ¨ F12.
c. 100 I culture added to well F2 of microtitre plate and mixed by
pipetting.
This results in a 1 in 2 dilution.
d. 100 1 taken from F2 and added to F3 on plate and mixed by pipetting. (1
in 4 dilution.)
e. This is continued to F12 where 100 1 is removed and discarded.
f. This dilution series was repeated in rows G-H for the remaining 2 cultures
g. Plate read at 0D530 on BioTek
h. Concentration of cells required will be the one that is the equivalent to
the
0.5 McFarland Standard, or 0D530 at 0.12-0.15
10. Dilute cells to the equivalent of the 0.5 McFarland Standard (for example
a 1 in 128
dilution may be required) in sterile 0.15M NaC1
11. Culture then further diluted 1 in 50 into 2 xRPMI-1640 (1.1 ml culture +
53.9 ml 2
xRPMI-1640).
Plate preparation
This preparation step occurs in two parts. First, Lynovex (NM001) and
antifungals are
serially diluted, down and across microtitre plates, respectively. Equal
volumes of
Lynovex and antifungal are then combined in a fresh microtitre plate, prior to
incoculation.
Step 1: Antifungal Plate Preparation
6. Stock solutions of Lynovex and antifungals were prepared at 4 x desired
concentrations. The plate layout at the end of this protocol is the final
concentration in the plate following the combination of Lynovex and
antifungals
and the addition of the inocula. For example, Lynovex is prepared initially at
10
mg/ml and the final concentration in the plate following addition of cells is
2.5
mg/ml.
7. 3001.11 of Lynovex at 10 mg/ml is added to each well in row A.
8. All other wells contain 150 p 1 sterile dH20.
9. Serial doubling dilutions are carried out from A to B whereby 150 IA is
removed
from A and placed into B and mixed well. Then take 150 pl B and place in C and
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
mix well. This is repeated down to row G where the final 150 pi is discarded.
Row
H contains water alone and will be antifungal only as well as untreated and
uninoculated controls when combined.
10. The same procedure is repeated for the other 4 antifungals, carrying the
dilutions
5 across the plate i.e. Column 1 to 2. At the final dilution in column 10,
the final 150
i.t1 is discarded. Columns 11 and 12 contain water alone and will be Lynovex
only
as well as untreated and uninoculated controls when combined.
Step 2: Challenge Plate Preparation
10 1. 50 IA Lynovex dilution from Al combined with 50 ul antifungal
dilution from Al
into Al of fresh microtitre plate
2. This step is repeated until all wells have been combined, halving the
Lynovex/antifungal concentration to a total volume of 1004
3. Wells H11 and H12 contain 100 [11 of sterile dH20 and will become a no
treatment
15 and uninoculated control respectively.
Inoculating plates with E. dermatitidis CA01
6. Add 100 ul (total volume 200 pi) of the prepared E. dermatitidis CA01
culture to
the all wells on the plate except wells H12. One culture was tested against
all
20 combinations, this yielded 3 replicates of each combination
7. H12 will contain 100 1 water and 100 ul 2 x RPMI-1640 alone, constituting
an
uninoculated control.
8. The plate is read in the BioTek at 0D530.
9. Plates are then placed in a plastic box containing a Petri dish of water,
sealed and
25 incubated at 35 C. The presence of the water in the dish or on the
tissue provides
and environment of relatively high humidity.
10. 0D530 is then read at 72 h, 96 h, 120 h and 144 h and raw data processed
using
Microsoft Excel.
11. Fractional Inhibitory Concentration (FIC) Index values are generated using
MICI oo
30 for 72 h data and MIC50for later time points using the following
formula:
MIC Drug A in Combination MIC Drug B in Combination
FICI ______________________________
MIC Drug A Alone MIC Drug B Alone
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
41
Plate Layout
Step 1 Layouts:
1 2 3 4 5 6 7 8 9 10 11 12
A 10,000 ug/mlLynovex
5,000 ug/mlLynovex
2,500 g/mlLynovex
1,250 pg/m1Lynovex
625 ng/m1Lynovex
312.5 ug/mlLynovex
156.25 ug/mlLynovex
0 g/mlLynovex
1 2 3 4 5 6 7 8 9 10 11
12
A
cdbOto to
Sto0o
C ¨7j1,0' Th¨tcl Mit
D -61 b r= 1 b.') ti3
<
FA t---5-0
bO
t_
00
H
Where x is 120 jig/m1 for Amphotericin B, 500 ug/rn1 for Caspofungin, 8000
jig/m1 for
Fluconazole and 64 jig/m1 for Itraconazole.
Step 2 Layouts:
Antifungal (jig/m1)
x x/2 x/4 x/8 x/16 x/32 x/64 x/128 x/256 x/512 0 0
1 2 3 4 5 6 7 8
9 10 11 12
2500 A
¨E 1250 B
625 C
=?--=-= 312.5 D
(1) 156.25 E
2 78.125 F
39.0625 G
0 H
Where x is 30 jig/m1 for Amphotericin B, 125 jig/m1 for Caspofungin, 2000
jig/ml for
Fluconazole and 16 jig/ml for Itraconazole.
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
42
Results
Table 1: In vitro minimum inhibitory concentrations (MICs) of Exophiala
dermatitidis
CA01 against the clinical antifungals Fluconazole, Itraconazole and
Caspofungin using
CLSI M3 8A2* broth microdilution procedure. CLSI endorsed Candida spp. QC
strains
provide MIC values within the accepted range.
(jighni) Fluconazole Itraconazole
Caspofungin
Species Strain I miCioo MICK, 1VIICso MICioo mIC80 IvtICso MICIoo
M1C80 MICso
ATCC
Candida krusei 62.5 62.5 31.25
0.25 0.125 0.004 1 1 0.5
6258
ATCC
Candida parapsilosis
22019 3.9 1 0.5 0.125 0.016 0.008
64 2 1
NCPF
Exophiala jeanselmei 1000 500 250 0.25 0.25 0.125 >64 64 0.125
2377
Exophiala dermatitidis CA01 500 500 250 >32 >32 2
32 32 16
* - CLSI (2008) Reference Method for Broth Dilution Antifungal Susceptibility
Testing of
Filamentous Fungi; Approved Standard - Second Edition. (M38-A2).
Table 2: Broth microdilution chequerboard analysis of Lynovex (NM001) used in
combination with clinical antifugals. In vitro MICioo data of Lynovex and
antifungals both
in isolation and in combination were generated following 72 b incubation at 35
C and
analysed for FICI. FICI values for each combination were identified and
averages are
presented, where <0.5; Synergy, 0.5-1; Additive, >1; Indifference. (Burkhart
et al 2002)
NM001 MICioo
ANTIFUNGAL STAT NM001 MICso in Combination Antifungal
Antifungal MICIoo FICI
MICioo in Combination (n) (n)
ITO
Itraconazole MEAN 1510 203 4.7 0.24 0.19
(5)
GEOMEAN 1250 179 4.5 0.14
0.18
(5)
Range 312.5-2500 78 -312.5 (5) 4.0 -
8.0 0.03 -0.5 (5)
Fluconazole MEAN 1250 365 250 34
0.43
(3)
GEOMEAN 1250 312.5 250 25
0.42
(3)
Range 1250 156 - 625 (3) 250
7.81 -62.5 (3)
Amphotericin B MEAN 667 39 0.86 0.23
0.53
(1)
GEOMEAN 461 39 0.59 0.23
0.53
(1)
Range 125 - 1250 39 (1) 0.23 - 1.88 0.23
(1)
Caspofungin MEAN 1458 78 26 2.0 0.25
(1)
GEOMEAN 1250 78 25 2.0
0.25
(1)
Range 625 - 2500 78(1) 15.625 - 31.25
2.0(1)
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
43
Table 3: Broth microdilution chequerboard analysis of Lynovex (NM001) used in
combination with clinical antifugals. In vitro MIC5o data of Lynovex and
antifungals both
in isolation and in combination were generated following 96 h incubation at 35
C and
analysed for FICI. FICI values for each combination were identified and
averages are
presented, where <0.5; Synergy, 0.5-1; Additive, >1; Indifference. (Burkhart
et al 2002)
NM001 MICso in Antifungal Antifungal
MICso
ANTIFIJNGAL STAT NM001 MiCso
FICI (n)
Combination (n) MICso in Combination
(n)
Itraconazole MEAN 1518 89 4.0 0.74 0.32 (7)
GEOMEAN 1132 78 4.0 0.37 0.27
(7)
Range 312.5 - 2500 39.06- 156.25 (7) 4.0
0.06 - 2 (7)
Fluconazole MEAN 885 176 229 51 0.45
(6)
GEOMEAN 787 139 223 35 0.44
(6)
Range 312.5 - 1250 39.06- 156.25 (6) 125 -250
7.81 - 125 (6)
Amphotericin B MEAN 885 59 0.47 0.18 0.64
(2)
GEOMEAN 625 55 0.47 0.17 0.63
(2)
Range 156.25 - 1250 39.0625 - 78.125 (2)
0.47 0A2 -0.23 (2)
Caspofungin MEAN 1531 102 31 14.84 0.58(4)
GEOMEAN 1088 90 31 11.84
0.48(4)
Range 156.25 -2500 39.0625 - 156.25 (4)
31.25 3.91 -31.25 (4)
Table 4: Broth microdilution chequerboard analysis of Lynovex (NM001) used in
combination with clinical antifugals. In vitro MICso data of Lynovex and
antifungals both
in isolation and in combination were generated following 120 h incubation at
35 C and
analysed for FICI. FICI values for each combination were identified and
averages are
presented, where <0.5; Synergy, 0.5-1; Additive, >1; Indifference. (Burkhart
et al 2002)
ANTIFUNGAL STAT NM001 MICso NM001
MIC5oin Antifungal Antifungal MIC50 no (n)
Combination (n) MIC5o in Combination
(n)
Itraconazole MEAN 1500 156.25 4.8 0.33
0.20 (5)
GEOMEAN 1250 136 4.6 0.19
0.18 (5)
Range 625 - 2500 78.13 - 312.5 (5) 4 - 8
0.06- 1(5)
Fluconazole MEAN 1875 312.5 219 59
0.42(4)
GEOMEAN 1768 263 210 44
0.41 (4)
Range 1250 - 2500 156.25 - 625 (4) 125-250
15.63 - 125 (4)
Amphotericin B MEAN 977 221 0.47 0.20
0.59 (3)
GEOMEAN 743 156 0.47 0.19
0.58 (3)
Range 156.25- 1250 39.06 - 312.5 (3) 0.5 0.12 -
0.23 (3)
Caspofungin MEAN 1750 148 22 4.20
0.27 (4)
GEOMEAN 1436 118 21 1.95
0.24(4)
Range 625 -2500 39.06 - 312.5 (4) 15.63 -
31.25 0.49 - 15.625 (4)
CA 02959003 2017-02-22
WO 2016/046524 PCT/GB2015/052714
44
Table 5: Broth microdilution chequerboard analysis of Lynovex (NM001) used in
combination with clinical antifugals. In vitro MIC5o data of Lynovex and
antifungals both
in isolation and in combination were generated following 144 h incubation at
35 C and
analysed for FICI. FICI values for each combination were identified and
averages are
presented, where <0.5; Synergy, 0.5-1; Additive, >1; Indifference. (Burkhart
et al 2002)
NM001 NM001 MICso in Antifungal Antifungal
MICso
ANTIFUNGAL STAT
FICI (n)
MICso Combination (n) MICso in Combination
(n)
Itraconazole MEAN 2031 208 8 0.27
0.19(3)
GEOMEAN 1768 197 6.7 0.20
0.18 (3)
Range 625 - 2500 156.25 -312.5 (3) 4 - >16 0.06 -
0.5 (3)
Fluconazole MEAN 1719 400 375 98
0.55 (4)
GEOMEAN 1487 263 354 63
0.53 (4)
Range 625 - 2500 39.0625 - 625 (4) 250 - 500
15.63 - 250 (4)
Amphotericin B MEAN 1302 469 0.63 0.23
0.75 (2)
GEOMEAN 787 442 0.59 0.23
0.75 (2)
Range 156.25 -2500 312.5 -625 (2) 0.47 - 0.94
0.23 (2)
Caspofungin MEAN 1875 417 26 6.51
0.52 (3)
GEOMEAN 1575 394 25 3.91
0.41 (3)
Range 625 - 2500 312.5 - 625 (3) 0.47 - 0.94
0.23(3)
