Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Compositions of Clofazimine, Combinations Comprising Them, Processes for Their
Preparation, Uses and Methods Comprising Them
The present application claims priority to United States Provisional
Application
Number 62/722,048, filed August 23, 2018, and the present application also
claims
priority to United States Provisional Application Number 62/796,322, filed
January 25,
2019, the content of both of which are incorporated herein by reference.
Field of the invention
The present invention relates to pharmaceutical compositions for inhalation
comprising a therapeutically effective dose of clofazimine, wherein the
clofazimine is
provided in the form of a suspension; processes for their preparation; and
uses and
methods of treatment comprising them. Furthermore, the present invention
provides
pharmaceutical combinations comprising clofazimine in the form of an aerosol
for
pulmonary inhalation.
The combinations and compositions provided by the present invention may be
used
in the treatment and/or prophylaxis of pulmonary infections caused by
mycobacteria
and other gram-positive bacteria, and of pulmonary fungal infections.
.. Background of the invention
Clofazimine is an extremely hydrophobic riminophenazine antibiotic (Log P =
7.66)
with anti-mycobacterial and anti-inflammatory activities and was originally
described
in 1957. Its structural formula is as follows:
1
CA 03109572 2021-02-12
WO 2020/040818 PCT/US2019/025538
Ci
k
4
kLks,, HSCN,setc: . -CH3
4*Fkm)
i 1
i'
N' N
H
The exact mechanism through which clofazimine exerts its antimicrobial effect
is
unknown. However, it is known to bind preferentially to mycobacterial DNA,
thereby
inhibiting DNA replication and cell growth. Other suggested mechanisms of
action
include membrane damage/destabilization, generation of membrane-destabilizing
lysophospholipids, interference of potassium transport, and/or intracellular
redox
cycling. While impressively active against Mycobacterium tuberculosis (MTB) in
vitro,
including multidrug-resistant strains, clofazimine, until recently, was
generally
considered to be ineffective in the treatment of pulmonary tuberculosis (see,
for
example, Cholo M et al., J Antimicrob Chemother, 2012 Feb, 67(2):290-8).
Clofazimine is one of the three principal drugs recommended by the World
Health
Organization for the treatment of leprosy which is caused by Mycobacterium
leprae
and has been increasingly used for the treatment of other mycobacterial
infections
such as drug resistant tuberculosis and infections caused by nontuberculous
mycobacteria (NTM) in recent years.
Clofazimine has been classified as a Biopharmaceutics Classification System
(BCS)
class II drug as it is practically insoluble in water and shows high membrane
permeability.
To overcome the problems associated with poor oral absorption and poor
bioavailability of drugs, various strategies have been applied such as
micronization,
nanonization, supercritical fluid re-crystallization, spray freeze drying into
liquid, solid
dispersions and solutions in optimizing oral dosage forms.
2
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Being classified as a BCS class II drug, clofazimine is generally considered
an ideal
candidate for the formulation into solid dispersions for improvement of oral
bioavailability (see, for example, Bhusnure et al. IJRPC 2014, 4(4), 906-918).
In line with this, because of its lipophilicity, clofazimine is generally
administered as a
microcrystalline suspension in an oil-wax base to improve oral absorption. The
absorption in humans after oral administration varies considerably (45-62%).
Adverse
effects of clofazimine are dose related and primarily affect the skin, eyes,
gastrointestinal tract, and QT elongation Side effects include the development
of
reddish-brown discoloration of the skin and conjunctiva and are gradually
reversible
on cessation. They are the result of chronic systemic accumulation.
Mycobacterium is a genus Actinobacteria, with its own genus, Mycobacteriaceae.
Mycobacteria have characteristic rod-like shapes and waxy outer coats.
As such, Mycobacteria can be divided into three groups:
= Mycobacterium tuberculosis complex ¨ causative pathogen of tuberculosis
= Mycobacterium leprae¨ causative pathogen of leprosy
= Nontuberculous mycobacteria (NTM) which encompass all other mycobacteria
that are not M. tuberculosis or M. leprae, including Mycobacterium abscessus
complex (MABSC), Mycobacterium avium complex (MAC).
Tuberculosis (TB) is an infectious disease caused by Mycobacterium
tuberculosis
complex bacteria. As one of the oldest documented infectious agents in humans,
TB
remains a significant cause of mortality and morbidity worldwide, with an
estimated
10.4 million new cases of TB infection, and 1.4 million people killed by
active TB
.. disease in 2015 (see, for example, World Health Organization (WHO) Global
Tuberculosis Report 2016). In addition to the high prevalence and mortality
rates, the
incidence of multi-drug resistant tuberculosis (MDR-TB) is a growing concern,
with
580,000 patients presenting with a drug-resistant TB infection in 2015. Co-
morbidities, such as human immunodeficiency virus (HIV), complicate treatment,
and
were responsible for 1.2 million cases of TB in 2015.
To treat multi-drug resistant (MDR) infections, the WHO has recommended
implementing a 9 to 12 month treatment regimen of second-line anti-TB drugs.
These regimens, such as the 9 to 12 month Bangladesh regimen, treat MDR-TB
with
3
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
a combination of gatifloxacin, ethambutol, pyrazinamide, and clofazimine,
which led
to a relapse-free cure in 87.9% of patients (see, for example, Sotgiu, G, et
al.,
"Applicability of the shorter 'Bangladesh regimen' in high multidrug-resistant
tuberculosis settings", International Journal of Infectious Diseases (2017) 56
190-
193).
Other studies investigating shortened TB treatments demonstrated that
clofazimine
had no clinical benefit after two weeks of oral administration (see, for
example,
Diacon, A.H., et al., "Bactericidal Activity of Pyrazinamide and Clofazimine
Alone and
in Combinations with Pretomanid and Bedaquiline", American Journal of
Respiratory
and Critical Care Medicine (2015), 191 (8), 943-953). The lack of activity was
attributed to low bioavailability of the drug, as it was theorized to bind to
circulating
serum proteins with a high affinity. There, despite the fact that clofazimine
has been
empirically demonstrated to be effective for the treatment of MDR-TB, and
extensively-drug resistant TB (XDR-TB), its poor bioavailability after
systemic
adminstration appears to limit its biological activity over short duration
therapies (see,
for example, Swanson, R.V., et al., "Pharmacokinetics and Pharmacodynamics of
Clofazimine in a Mouse Model of Tuberculosis", Antimicrobial Agents and
Chemotherapy (2015), 59 (6), 3042-3051).
It is known that treatment of lung infections with inhaled antibiotics results
in higher
drug concentrations in the lungs and reduced adverse effects compared to
systemic
delivery (see, for example, Touw, D.J., et al., "Inhalation of antibiotics in
cystic
fibrosis", European Respiratory Journal (1995), 8, 1594-1604), which result in
.. increased biological activity and efficacy (see, for example, Hickey, A.J.,
"Inhaled
drug treatment for tuberculosis: Past progress and future prospects", Journal
of
Controlled Release, (2016), 240, 127-134).
In vivo mouse models have
demonstrated that aerosolized administration of clofazimine shows significant
improvement in bacilli clearance in TB-infection models compared to oral
administration of clofazimine only 28 days after treatment initiation (see,
for example,
Verma, R.K., et al., "Inhaled microparticles containing clofazimine are
efficacious in
treatment of experimental Tuberculosis in Mice", Antimicrobial Agents and
Chemotherapy (2013), 57 (2), 1050-1052). This improved efficacy over a short
duration is likely due to the direct delivery of clofazimine to the site of
infection in the
4
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
lungs resulting in higher clofazimine concentration in the pulmonary
macrophages
within the tuberculosis granulomas.
Accordingly, the use of an aerosolized administration of clofazimine in
patients with
MDR TB, or XDR-TB infections should further improve patient treatment
outcomes,
and may shorten the duration of current treatment regimens.
The group of nontuberculous mycobacteria (NTM), formerly called atypical or
ubiquitous mycobacteria, contains over 150 species. NTM can be found
ubiquitously
.. in nature and show a broad diversity. They can be detected in soil, ground
and
drinking water as well as in food like pasteurized milk or cheese. In general,
NTM are
considered to be less pathogenic. Nevertheless, they can cause severe illness
in
humans, especially in immune compromised persons or those who suffer from
previous pulmonary diseases. Currently NTM are classified according to their
growth
rate and are divided into slow-growing (SGM) and rapid-growing (RGM)
mycobacteria.
The slow growing Mycobacterium avium complex (MAC) comprises the species
Mycobacterium avium, Mycobacterium chimaera and Mycobacterium intracellulare
that are among the most important and most frequent pathogenic NTM. Just like
Mycobacterium kansasii, Mycobaceterium malmoense, Mycobacterium xenopi,
Mycobacterium. simiae, Mycobacterium abscessus, Mycobacterium gordonae,
Mycobacterium fortuitum, and Mycobacterium chelonae, they mostly cause
pulmonary infections. Mycobacterium marinum is responsible for skin and soft
tissue
.. infections like aquarium granuloma.
In particular, RGM cause serious, life-threatening chronic lung diseases and
are
responsible for disseminated and often fatal infections. Infections are
typically caused
by contaminated materials and invasive procedures involving catheters, non-
sterile
.. surgical procedures or injections and implantations of foreign bodies.
Exposure to
shower heads and jacuzzis has also been reported as risks for infections. NTM
typically cause opportunistic infections in patients with chronic pulmonary
diseases
such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF),
and
other immune compromised patients.
5
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
In recent years, the rapidly growing (RGM) Mycobacterium abscessus group
strains
(Mycobacterium abscessus complex, MABSC) comprising the subspecies
Mycobacterium abscessus subsp. abscessus (M. a. abscessus), Mycobacterium
abcessus bolletii. and Mycobacterium abscessus massiliense have emerged as
important human pathogens and are associated with significantly higher
fatality rates
than any other RGM.
Mycobacterium abscessus infection in CF patients are particularly problematic,
as it
results in enhanced pulmonary destruction and is often impossible to treat
with failure
rates as high as 60-66%. (see, for example, Obregon-Henao A et al,
Antimicrobial
Agents and Chemotherapy, November 2015, Vol 59, No 11, p. 6904-6912; Qvist,T.,
Pressler,T., Hesiby,N. and Katzenstein,TL., "Shifting paradigms of
nontuberculous
mycobacteria in cystic fibrosis", Respiratory Research (2014), 15(1):pp.41-
47).
Human infection with NTM became of greater relevance with the emergence of the
human acquired immune deficiency syndrome pandemic. Mycobacteria from
Mycobacterium avium complex (MAC) were identified as the major cause of
opportunistic infections in patients infected with the human immunodeficiency
virus
(HIV).
Several species of NTM are known to form biofilms. Biofilms are microcolonies
of
bacteria embedded in the extracellular matrix that provide stability and
resistance to
human immune mechanisms. In recent years, some species of NTM have been
shown to form biofilms that enhance resistance to disinfectants and
antimicrobial
agents. Biofilm assembly proceeds through several phases, including reversible
attachment, irreversible attachment, biofilm formation via bacterial
aggregation,
organization, and signaling, and finally dispersion. During this process,
bacteria
develop a matrix containing extracellular polymeric substances (EPS), such as
polysaccharides, lipids and nucleic acids, to form a complex three-dimensional
structure (see, for example, Sousa S. et al., International Journal of
Mycobacteriology
4 (2015), 36-43). Specifically, mycobacterial EPS differ in nature from other
biofilms,
as mycobacteria do not produce exopolysaccharides (see, for example, Zambrano
MM, Kolter R. Mycobacterial biofilms: a greasy way to hold it together. Cell.
2005).
6
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Mycobacterial biofilms vary between species, but can contain mycolic acids,
glycopeptidolipids, mycolyl-diacylglycerols, lipooligosaccharides,
lipopeptides, and
extracellular DNA (Overview and original research from: Rose SJ, Babrak LM,
Bermudez LE (2015) Mycobacterium avium Possesses Extracellular DNA that
Contributes to Biofilm Formation, Structural Integrity, and Tolerance to
Antibiotics
PLoS ONE). The assembly in biofilms is known to enhance resistance to
antimicrobial agents (see, for example, Faria S. et al., Journal of Pathogens,
Vol
2015, Article ID 809014).
Delivery of aerosolized liposomal amikacin/inhaled amikacin solution nebulized
by a
jet nebulizer as a novel approach for treatment of NTM pulmonary infection has
been
suggested (Rose S. et al, 2014, PLoS ONE, Volume 9, Issue 9, e108703, and
Olivier
K. et al, Ann Am Thorac Soc Vol 11, No 1, pp. 30-35) as well as inhalation of
anti-TB
drugs dry powder microparticles for pulmonary delivery (Cholo M et al., J
Antimicrob
Chemother. 2012 Feb; 67(2):290-8 and Fourie B. and Nettey 0., 2015 Inhalation
Magazine, Verma 2013 Antimicrob Agents Chemother).
Multiple combination regimens with inhaled amikacin following initial
treatment with
parenteral aminoglycosides, tigecycline and other promising oral antibiotics
such as
linezolid, delamanid, and bedaquiline, and surgical intervention in selected
cases
have shown promising results in the treatment of NTM lung disease (Lu Ryu et
al.,
Tuberc Respir Dis 2016;79:74-84). However, the increasing incidence and
prevalence of NTM infections, in particular NTM lung disease and the limited
treatment options necessitate the development of novel dosage
forms/pharmaceutical formulation enhancing the bioavailability of the
currently used
antibiotics such as clofazimine. Inhalation may enhance efficacy and reduce
adverse
effects compared to oral and parenteral therapies.
Combinations of clofazimine and amikacin have been shown to act
synergistically in
vitro against both Mycobacterium abscessus and Mycobacterium avium (see, for
example, van Ingen, J., et al., In Vitro Synergy between Clofazimine and
Amikacin in
Treatment of Nontuberculous Mycobacterial Disease", Antimicrobial Agents and
Chemotherapy 56 (12), 6324-6327 (2012)). Further, synergy has been shown with
combinations of clofazimine and bedaquiline used against Mycobacterium
7
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
tuberculosis (see, for example, Cokol, M. et al., "Efficient Measurement and
factorization of high-order drug interactions in Mycobacterium tuberculosis",
Sciences
Advances 2017:3:e170881, 11 October 2017). Synergy has also been shown for a
clofazimine/bedaquiline combination against the nontuberculous bacterium
Myocbacterium abscessus (Ruth, M.M. et al., "A Bedaquiline/Clofazimine
Combination Regimen Might Add Activity to the Treatment of Clinically Relevant
Non-
Tuberculous Mycobacteria", Journal of Antimicrobial Chemotherapy (2019),
doi.org/10.1093/jac/dky526).
Fungal pathogens have emerged as a leading cause of human mortality. Current
estimates suggest death due to invasive fungal infections is on par with more
well-
known infectious diseases such as tuberculosis. Candida albicans, Cryptococus
neoformans, and Aspergiffis fumigatus represent the most prevalent fungal
pathogens of humans. Each of these species is responsible for hundreds of
thousands of infections annually with unacceptably high mortality rates due to
poor
diagnostics and limited treatment options. Clofazimine has been shown to
exhibit
efficacy as a combination agent against multiple fungi. (see, for example,
Robbins,
N., et al., An Antifungal Combination Matrix Identifies a Rich Pool of
Adjuvant
Molecules that Enhance Drug Activity against Diverse Fungal Pathogens", Cell
Reports 13, 1481-1492, November 17, 2015). Fungi also play a role as
commensals,
colonizers and/or pathogens in cystic fibrosis (see, for example, Chotirmall,
S.H. and
McElvaney, N.G., "Fungi in the cystic fibrosis lung: Bystanders or
pathogens?", The
International Journal of Biochemistry & Cell Biology 52 (2014), 161-173.
The low solubility of clofazimine in water results in low oral bioavailability
and high
microbial resistance and also requires specific techniques to solubilise and
stabilize
the drug for formulation in liquid aqueous carriers such as for aerosolization
by
nebulizers in order to obtain lower lung deposition of the aerosol particles.
Summary of the invention
In an embodiment of the invention, a pharmaceutical composition is provided
comprising:
(a) a therapeutically effective dose of clofazimine or a
pharmaceutically
8
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
acceptable derivative or salt thereof;
(b) a nonionic surfactant with an Hydrophilic-Lipophilic Balance value of
greater than 10; and
(c) an aqueous liquid carrier selected from water, isotonic saline,
buffered
saline and aqueous electrolyte solutions
wherein the clofazimine, or the pharmaceutically acceptable derivative or salt
thereof, is provided in the form of particles in a suspension,
and
wherein the particles of clofazimine, or the pharmaceutically acceptable
derivative or salt thereof, have a median size of less than 5 pm and a D90 of
less than 6 pm.
In another embodiment of the invention, the particles of clofazimine, or the
pharmaceutically acceptable derivative or salt thereof, have a mean size of
less than
2 pm and a D90 of less than 3 pm.
In another embodiment of the invention a pharmaceutical composition is
provided
comprising:
(a) a therapeutically effective dose of clofazimine;
(b) a nonionic surfactant with an Hydrophilic-Lipophilic Balance value of
greater than 10; and
(c) an aqueous liquid carrier selected from water, isotonic saline,
buffered
saline and aqueous electrolyte solutions
wherein the clofazimine is provided in the form of particles in a suspension,
and
wherein the particles of clofazimine have a median size of less than 5 pm and
a D90 of less than 6 pm.
In another embodiment of the invention the particles of clofazimine have a
median
size of less than 2 pm and a D90 of less than 3 pm.
The aerosolization of the compositions of the invention by an appropriate
nebulizer
provides significantly increased delivery of the aerosolized clofazimine into
the lower
lung (i.e. to the bronchi, bronchioli, and alveoli of the central and lower
peripheral
lungs), thereby substantially enhancing the therapeutic efficacy.
9
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
The inhalation device should, moreover, preferably be further adapted for
localized
pulmonary delivery of an aerosol having an optimal particle size distribution
for
homogenous deposition in the lower lung.
The invention therefore provides for an aerosol having aerosol particles of
sizes that
facilitate delivery to the alveoli and bronchiole. A suitable aerodynamic
particle size
for targeting the alveoli and bronchiole is between 1 and 5 pm. Particles
larger than
that are selectively deposited in the upper lungs, namely bronchi and trachea
and in
the mouth and throat, i.e. oropharyngeal area. Accordingly, the inhalation
device is
adapted to produce an aerosol having a mass median aerodynamic diameter
(MMAD) in the range from about 1 to about 5 pm, and preferably in the range
from
about 1 to about 3 pm. In a further embodiment, the particle size distribution
is
narrow and has a geometric standard deviation (GSD) of less than about 2.5.
Detailed description of the invention
The present invention is based on the unexpected discovery that by pulmonary
aerosol administration of clofazimine in the form of a suspension, lower (i.e.
deeper)
lung deposition of the active agent can be achieved, thereby significantly
increasing
the bioavailability of the extremely hydrophobic BCS class II agent, which
results in
significantly increased therapeutic efficacy coupled with reduced systemic
side
effects.
In another aspect, this finding leads to the provision of an improved
antibiotic therapy
for infections caused by mycobacteria and gram-positive bacteria, in
particular of
pulmonary infections with NTM, such as opportunistic infections in CF, COPD
and
immune compromised patients such as HIV patients.
The present invention, moreover, aims at overcoming systemic side effects of
established oral treatment regimens for pulmonary infections with gram
positive
bacteria, in particular TB and NTM infections of the lungs as well as at the
reduction
of dose and of duration of treatment with clofazimine.
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
It is understood by the person of skill in the art that the present
application also
discloses each and any combination of the individual features disclosed
herein.
Definitions
The term "pharmaceutically acceptable salt" refers to salts that retain the
biological
effectiveness and properties of the compounds of this invention and, which are
not
biologically or otherwise undesirable. In many cases, the compounds of this
invention
are capable of forming acid and/or base salts by virtue of the presence of
amino
and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable
acid
addition salts can be formed with inorganic acids and organic acids. Inorganic
acids
from which salts can be derived include, for example, hydrochloric acid,
hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids
from which
salts can be derived include, for example, acetic acid, propionic acid,
naphtoic acid,
oleic acid, palmitic acid, pamoic (emboic) acid, stearic acid, glycolic acid,
pyruvic
acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,
tartaric acid,
citric acid, ascorbic acid, glucoheptonic acid, glucuronic acid, lactic acid,
lactobionic
acid, tartaric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
Pharmaceutically acceptable base addition salts can be formed with inorganic
and
organic bases. Inorganic bases from which salts can be derived include, for
example,
sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum, and the like; particularly preferred are the ammonium,
potassium, sodium, calcium and magnesium salts. Organic bases from which salts
can be derived include, for example, primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted amines, cyclic
amines,
basic ion exchange resins, and the like, specifically such as isopropylamine,
trimethylamine, diethylamine, triethylamine, tripropylamine, histidine,
arginine, lysine,
benethamine, N-methyl-glucamine, and ethanolamine. Other acids include
dodecylsufuric acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic
acid,
and saccharin.
11
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
In accordance with the present invention, apart from the free base, the use of
the
methanesulfonic acid, maleic acid, isonicotinic acid, nicotinic acid, malonic
acid, and
salicylic acid salts, and in particular of clofazimine mesylate is preferred.
By the term "pharmaceutically acceptable derivative" as used herein, for
example,
compounds disclosed in US 9,540,336 are meant, the disclosure of US 9,540,336
is
incorporated herein in its entirety. In addition, derivatives are meant as
described in
Lu,Y., Zhen,M., Wang,B., Fu,L., Zhao,W., Li,P., Xu,J., Zhu,H., Jin,H., Yin,D.,
Huang,H., Upton,AM. and Ma,Z., "Clofazimine Analogs with Efficacy against
experimental Tuberculosis and reduced Potential for Accumulation"
Antimicrobial
Agents and Chemotherapy (2011), 55(11):pp.5185-5193. Additionally, The term,
"pharmaceutically acceptable derivative" of a compound is, for example, a
prodrug of
said compound. In general, a prodrug is a derivative of a compound which, upon
administration, is capable of providing the active form of the compound. Such
derivatives, for example, may be an ester or amide of a carboxyl group, a
carboxyl
ester of a hydroxyl group, or a phosphate ester of a hydroxyl group.
By "therapeutically effective amount", "therapeutically effective dose", or
"pharmaceutically effective amount" is meant an amount of clofazimine, or a
pharmaceutically acceptable salt or derivative thereof, as disclosed for this
invention,
which has a therapeutic effect. The doses of clofazimine which are useful in
treatment are therapeutically effective amounts. Thus, as used herein, a
therapeutically effective amount means those amounts of clofazimine which
produce
the desired therapeutic effect as judged by clinical trial results and/or
model animal
infection studies.
The amount of the clofazimine and daily dose can be routinely determined by
one of
skill in the art, and will vary, depending on several factors, such as the
particular
microbial strain involved. This amount can further depend upon the patient's
height,
weight, sex, age and medical history. For prophylactic treatments, a
therapeutically
effective amount is that amount which would be effective to prevent a
microbial
infection.
12
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
A "therapeutic effect" relieves, to some extent, one or more of the symptoms
of the
infection, and includes curing an infection. "Curing" means that the symptoms
of
active infection are eliminated, including the total or substantial
elimination of
excessive members of viable microbe of those involved in the infection to a
point at
or below the threshold of detection by traditional measurements. However,
certain
long-term or permanent effects of the infection may exist even after a cure is
obtained (such as extensive tissue damage). As used herein, a "therapeutic
effect" is
defined as a statistically significant reduction in bacterial load in a host,
emergence of
resistance, or improvement in infection symptoms as measured by human clinical
results or animal studies.
"Treat", "treatment", or "treating" as used herein refers to administering a
pharmaceutical composition/combination for prophylactic and/or therapeutic
purposes.
The term "prophylactic treatment" refers to treating a patient who is not yet
infected,
but who is susceptible to, or otherwise at risk of, a particular infection.
The term
"therapeutic treatment" refers to administering treatment to a patient already
suffering
from an infection. Thus, in preferred embodiments, treating is the
administration to a
mammal (either for therapeutic or prophylactic purposes) of therapeutically
effective
amounts of clofazimine.
Unless stated otherwise herein, the term "inhalation" is meant to refer to
pulmonary
inhalation.
Unless stated otherwise herein, the term "infection" as used herein is meant
to refer
to pulmonary infections.
Unless otherwise stated, the term "substantially" when used to refer to the
purity of a
compound, indicates a purity of compound of 95% or greater purity.
Unless otherwise stated, the term "appropriate particle size" refers to a
particle size
of clofazimine in a composition, or a composition that provides the desired
therapeutic effect when administered to a patient.
13
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Unless otherwise stated, the term "appropriate concentration" refers to a
concentration of a component in a composition or combination which provides a
pharmaceutically acceptable composition or combination.
Pharmaceutical compositions and combinations
The following water grades are particularly applicable to the present
invention: sterile
purified water, sterile water for injection, sterile water for irrigation,
sterile water for
inhalation (USP) and corresponding water grades in accordance with e.g.
European
Pharmacopoeia or National Formulary.
Aqueous electrolyte solutions as used in accordance with the present invention
as
the aqueous liquid carrier may further comprise sodium chloride, potassium
chloride,
lithium chloride, magnesium chloride, calcium chloride or mixtures thereof.
The aqueous liquid carrier is preferably isotonic saline solution (0.9% NaCI
corresponding to about/approximately 150 mM NaCI, preferably 154 mM NaCI).
Clofazimine has been shown to exist in at least four polymorphic forms (see,
for
example, Bannigan, et al., "Investigation into the Solid and Solution
Properties of
Known and Novel Polymorphs of the Antimicrobial Molecule Clofazimine", Cryst.
Growth Des. 2016, 16 (12), pp. 7240-7250). Clofazimine can exist in a
triclinic form
Fl, a monoclinic form FII, and an orthorhombic form Fill. A further form FIV
has also
been seen only at high temperatures.
Accordingly, in a further embodiment of the invention a pharmaceutical
composition
is provided comprising:
(a) a therapeutically effective dose of clofazimine;
(b) a nonionic surfactant with an Hydrophilic-Lipophilic Balance value of
greater than 10; and
(c) an aqueous liquid carrier selected from water, isotonic
saline, buffered
saline and aqueous electrolyte solutions
wherein the clofazimine is provided in the form of particles in a suspension,
14
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
and
wherein the particles of clofazimine have a median size of less than 5 pm
and a D90 of less than 6 pm, preferably a median size of less than 2 pm and a
D90
of less than 3 pm, and wherein the clofazimine is provided in a polymorphic
form or
forms selected from triclinic form Fl, monoclinic form Fll and orthorhombic
form Fill
and mixtures of such forms. In another embodiment, the clofazimine is provided
substantially in orthorhombic form Fill.
In a further embodiment of the invention a pharmaceutical composition
according to
any of the composition embodiments herein described is provided wherein the
nonionic surfactant is selected from polysorbate 20 (for example Tweee 20,
polysorbate 60 (for example Tweee 60) , polysorbate 80 (for example Tweee 80),
stearyl alcohol, a polyethylene glycol derivative of hydrogenated castor oil
with an
Hydrophilic-Lipophilic Balance value of 14 to 16 (for example Cremophor RH
40) , a
polyethylene glycol derivative of hydrogenated castor oil with an Hydrophilic-
Lipophilic Balance value of 15 to 17 (for example Cremophor RH 60), sorbitan
monolaurate (for example Span 20), sorbitan monopalmitate (for example Span
40), sorbitan monostearate (for example Span 60), polyoxyethylene (20) oleyl
ether
(for example Brij 020), polyoxyethylene (20) cetyl ether (for example Brij
58),
polyoxyethylene (10) cetyl ether (for example Brij C10), polyoxyethylene
(10) oleyl
ether (for example Brij 010), polyoxyethylene (100) stearyl ether (for
example Brij
S100), polyoxyethylene (10) stearyl ether (for example Brij S10),
polyoxyethylene
(20) stearyl ether (for example Brij S20), polyoxyethylene (4) lauryl ether
(for
example Brij L4), polyoxyethylene (20) cetyl ether (for example Brij 93),
polyoxyethylene (2) cetyl ether (for example Brij S2), caprylocaproyl
polyoxy1-8
glyceride (for example Labrason, polyethylene glycol (20) stearate (for
example
MyrjTM 49), polyethylene glycol (40) stearate (for example MyrjTM S40),
polyethylene
glycol (100) stearate (for example MyrjTM S100), polyethylene glycol (8)
stearate (for
example Myrj TM S8), and polyoxyl 40 stearate (for example MyrjTM 52), and
mixtures
thereof.
In another embodiment of the invention, a pharmaceutical composition according
to
any of the composition embodiments described herein is provided, wherein the
non-
ionic surfactant is polysorbate 80, and wherein the aqueous liquid carrier is
distilled
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
water, hypertonic saline or isotonic saline. In another embodiment of the
invention, a
pharmaceutical composition is provided wherein the hypertonic saline is from
1% to
7% (w/v) sodium chloride.
In a further embodiment of the invention, a
pharmaceutical composition is provided wherein the non-ionic surfactant is
ultrapure
polysorbate 80 (for example NOF Corporation Polysorbate 80 (Hx2)), and wherein
the aqueous liquid carrier is isotonic saline.
In another embodiment of the invention, a pharmaceutical composition according
to
any one of the composition embodiments described herein is provided wherein
the
osmolality of the composition is in the range of 200-700 mOsm/kg. In a further
embodiment, the osmolality of the composition is in the range of 300-400
mOsm/kg.
In a further embodiment of the invention, a pharmaceutical composition
according to
any one of the composition embodiments described herein, is provided wherein
the
nonionic surfactant is in the range of 0.001% to 5% (v/v) of the total
composition and
the amount of clofazimine is in the range of 0.1% to 20% (w/v) of the total
composition.
In another embodiment of the invention, a pharmaceutical composition according
to
any one of the composition embodiments described herein is provided, wherein
the
pharmaceutical composition is prepared by a process comprising the following
steps:
(1) homogenization of a suspension of clofazimine, the nonionic
surfactant
and water to obtain a suspension comprising clofazimine of an
appropriate particle size,
(2)
adjusting the pH of the suspension resulting from (1) to a pH of between
pH 5.5 and pH 7.5, and
(3) adjusting the sodium chloride concentration to an appropriate
concentration and
(4) adjusting the osmolality to an appropriate level.
In a further embodiment, the pH is adjusted to 7.4, and the sodium chloride
concentration is adjusted to 154 mM sodium chloride. In another embodiment,
the
homogenization in step (1) is carried out by high pressure homogenization,
high
shear homogenization, wet milling, ultrasonic homogenization, or a combination
of
16
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
such processes. In another aspect, the homogenization of clofazimine is
carried out
in multiple steps of homogenization. In another embodiment, the appropriate
particle
size of the clofazimine are particles having a mean size of less than 5 pm and
D90 of
less than 6 [tm. In a further embodiment, the appropriate particle size of
clofazimine
are particles having a mean size of less than 2 [tm and D90 of less than 3 pm.
In a further embodiment of the invention, a pharmaceutical composition
according to
any one of the composition embodiments described herein is provided, wherein
the
pharmaceutical composition is prepared by a process comprising the following
steps:
(1)
homogenization of a suspension of clofazimine and a non-aqueous
liquid to obtain a suspension comprising clofazimine of an appropriate
particle size,
(2) isolation of the clofazimine,
(3) addition of the clofazimine to the nonionic surfactant and water,
(4)
adjusting the pH of the suspension resulting from (3) to a pH of between
pH 5.5 and pH 7.5, and
(5) adjusting the sodium chloride concentration to an appropriate
concentration.
In a further embodiment, the pH is adjusted to 7.4, and the sodium chloride
concentration is adjusted to 154 mM sodium chloride. In a further embodiment,
the
homogenization in step (1) is carried out by high pressure homogenization,
high
shear homogenization, wet milling, ultrasonic homogenization, or a combination
of
such processes. In another embodiment, the homogenization of clofazimine is
carried
out in multiple steps of homogenization. In another embodiment, the
appropriate
particle size of the clofazimine are particles having a mean size of less than
5 pm and
D90 of less than 6 pm. In a further embodiment, the appropriate particle size
of
clofazimine are particles having a mean size of less than 2 pm and D90 of less
than 3
pm.
In a further embodiment, a pharmaceutical composition according to any one of
the
composition embodiments described herein is provided, wherein the composition
is
prepared by a process comprising the following steps:
(1) micronization of clofazimine to obtain clofazimine of an
appropriate
17
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
particle size,
(2) addition of the clofazimine to the nonionic surfactant and water,
(3) adjusting the pH of the suspension resulting from (2) to a pH of
between
pH 5.5 and pH 7.5, and
(4) adjusting the sodium chloride concentration to an appropriate
concentration.
In a further embodiment, the pH is adjusted to 7.4, and the sodium chloride
concentration is adjusted to 154 mM sodium chloride.
In another embodiment, the micronization of the clofazimine is carried out by
jet
milling, spray drying, ball milling, or super critical fluids processing. In
another
embodiment, the micronization of clofazimine is carried out in multiple steps
of
micronization. In another embodiment, the appropriate particle size of the
clofazimine
are particles having a mean size of less than 5 pm and D90 of less than 6 pm.
In a
further embodiment, the appropriate particle size of clofazimine are particles
having a
mean size of less than 2 [tm and D90 of less than 3 pm.
In a further embodiment, a pharmaceutical composition according to any one of
the
composition embodiments described herein is provided, wherein the composition
is
prepared by a process comprising homogenization of a suspension of clofazimine
in
the nonionic surfactant, water containing an appropriate concentration of
sodium
chloride, and which has been adjusted to a pH of between pH 5.5 and pH 7.5, to
obtain clofazimine of an appropriate particle size. In a further embodiment,
the pH is
adjusted to 7.4, and the sodium chloride concentration is adjusted to 154 mM
sodium
chloride. In a further embodiment, the homogenization is carried out by high
pressure
homogenization, high shear homogenization, wet milling, ultrasonic
homogenization,
or a combination of such processes. In another embodiment, the homogenization
of
clofazimine is carried out in multiple steps of homogenization. In another
embodiment, the appropriate particle size of the clofazimine are particles
having a
mean size of less than 5 pm and D90 of less than 6 pm. In a further
embodiment, the
appropriate particle size of clofazimine are particles having a mean size of
less than
2 pm and D90 of less than 3 pm.
In another embodiment, a process for the preparation of a pharmaceutical
composition according to any of the composition embodiments described herein
is
18
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
provided, comprising the following steps:
(1) homogenization of a suspension of clofazimine, the non-ionic surfactant
and water to obtain a suspension comprising clofazimine of an appropriate
particle
size,
(2) adjusting the pH of the suspension resulting from (1) to a pH of between
pH 5.5 and pH 7.5, and
(3) adjusting the sodium chloride concentration to an appropriate
concentration, and
(4) adjusting the osmality to an appropriate level.
In another embodiment, the pH is adjusted to 7.4, and the sodium chloride
concentration is adjusted to 154 mM sodium chloride. In a further embodiment,
the
homogenization is carried out by high pressure homogenization, wet milling,
ultrasonic homogenization, or a combination of such processes. In a further
embodiment, the homogenization of clofazimine is carried out in multiple steps
of
homogenization. In a further embodiment, the appropriate particle size of
clofazimine
are particles having a mean size of less than 5 [tm and a D90 of less than 6
[tm. In
another embodiment, the appropriate particle size of clofazimine are particles
having
a mean size of 2 [tm and a D90 of less than 3 [tm.
In another embodiment, a process for the preparation of any of the
pharmaceutical
composition embodiments as described herein is provided, wherein
(1) homogenization of a suspension of clofazimine and a non-aqueous
liquid to obtain a suspension comprising clofazimine of the appropriate
particle size,
(2) isolation of the clofazimine,
(3) addition of the clofazimine to the nonionic surfactant and water,
(4) adjusting the pH of the suspension resulting from (3) to a pH of
between
pH 5.5 and pH 7.5, and
(5) adjusting the sodium chloride concentration to an appropriate
concentration.
In another embodiment, the pH is adjusted to 7.4, and the sodium chloride
concentration is adjusted to 154 mM sodium chloride. In a further embodiment,
the
homogenization is carried out by high pressure homogenization, wet milling,
19
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
ultrasonic homogenization, or a combination of such processes. In a further
embodiment, the homogenization of clofazimine is carried out in multiple steps
of
homogenization. In a further embodiment, the appropriate particle size of
clofazimine
are particles having a mean size of less than 5 [tm and a D90 of less than 6
[tm. In
another embodiment, the appropriate particle size of clofazimine are particles
having
a mean size of 2 [tm and a D90 of less than 3 [tm.
In a further embodiment, a process for the preparation of a pharmaceutical
composition according to any one of the pharmaceutical composition embodiments
as described herein is provided, comprising the following steps:
(1) micronization of clofazimine to obtain clofazimine of an appropriate
particle size,
(2) addition of the clofazimine to the nonionic surfactant and water,
(3) adjusting the pH of the suspension resulting from (2) to a pH of
between
pH 5.5 and pH 7.5, and
(4) adjusting the sodium chloride concentration to an appropriate
concentration.
In another embodiment, the pH is adjusted to 7.4, and the sodium chloride
concentration is adjusted to 154 mM sodium chloride. In a further embodiment,
the
micronization of the clofazimine is carried out by jet milling, spray drying,
ball milling,
or super critical fluids processing. In a further embodiment, the
micronization of
clofazimine is carried out in multiple steps of micronization. In a further
embodiment,
the appropriate particle size of clofazimine are particles having a mean size
of less
than 5 [tm and a D90 of less than 6 [tm. In another embodiment, the
appropriate
particle size of clofazimine are particles having a mean size of 2 [tm and a
D90 of
less than 3 [tm.
In another embodiment, a process for the preparation of a pharmaceutical
composition according to any one of pharmaceutical composition embodiment
described herein is provided, comprising homogenization of a suspension of
clofazimine in the nonionic surfactant, water containing an appropriate
concentration
of sodium chloride, and which has been adjusted to a pH of between pH 5.5 and
pH
7.5, to obtain clofazimine of an appropriate particle size. In another
embodiment, the
pH is 7.4, and the appropriate concentration of sodium chloride is 154 mM
sodium
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
chloride. In a further embodiment, the homogenization is carried out by high
pressure
homogenization, wet milling, ultrasonic homogenization, or a combination of
such
processes. In a further embodiment, the homogenization of clofazimine is
carried out
in multiple steps of homogenization. In a further embodiment, the appropriate
particle
size of clofazimine are particles having a mean size of less than 5 [tm and a
D90 of
less than 6 [tm. In another embodiment, the appropriate particle size of
clofazimine
are particles having a mean size of 2 [tm and a D90 of less than 3 [tm.
In a further embodiment a process for the preparation of a pharmaceutical
composition according to any one of composition embodiments described herein,
is
provided, comprising the following steps: (a) homogenization of a suspension
of
clofazimine, the non-ionic surfactant and water to obtain a suspension
comprising
clofazimine of an appropriate particle size; (b) adjusting the pH of the
resulting
suspension a pH of between pH 5.5 and pH 7.5; (c) adjusting the sodium
chloride
concentration to an appropriate concentration, and (d) adjusting the osmality
to an
appropriate level; and wherein steps (b), (c) and (d), may occur in the order
of (b),
(c), (d); (b), (d), (c); (c), (b), (d); (c), (d), (b); (d), (b), (c); or (d),
(c), (b).
In another embodiment, a process for the preparation of a pharmaceutical
composition according to any one of the composition embodiments described
herein,
is provided comprising the following steps: (a) homogenization of a suspension
of
clofazimine and a non-aqueous liquid to obtain a suspension comprising
clofazimine
of the appropriate particle size; (b) isolation of the clofazimine; (c)
addition of the
clofazimine to the nonionic surfactant and water; (d) adjusting the pH of to
resulting
suspension to a pH of between pH 5.5 and pH 7.5; and (e) adjusting the sodium
chloride concentration to an appropriate concentration; and wherein steps (d)
and (e)
may occur in the order of (d), (e); or (e), (d).
In another embodiment, a process for the preparation of a pharmaceutical
composition according to any one or the composition embodiments described
herein,
is provided, comprising the following steps: (a) micronization of clofazimine
to obtain
clofazimine of an appropriate particle size, and (b) addition of the
clofazimine to the
nonionic surfactant, water containing an appropriate concentration of sodium
chloride, and which has been adjusted to a pH of between between pH 5.5 and
7.5.
21
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
In another embodiment of the present invention, a pharmaceutical combination
in the
form of an aerosol for inhalation is provided, prepared by aerosolization of
the
composition according to any one of the composition embodiments described
herein,
by a nebulizing device selected from an ultrasonic nebulizer, an electron
spray
nebulizer, a vibrating membrane nebulizer, a jet nebulizer and a mechanical
soft mist
inhaler, and
wherein the aerosol particles produced by the nebulizing device have a mass
median
aerodynamic diameter of 1 to 5 pm. In a further embodiment, the aerosol for
inhalation is for lower lung deposition. In another embodiment, the nebulizing
device
exhibits an output rate of 0.1 to 1.0 ml/min. In another embodiment, the total
inhalation volume is between 1 ml and 5 ml.
In another embodiment, a pharmaceutical composition according to any one of
the
composition embodiments described herein is provided which is for use in
combination with an agent for dispersing and/or destruction of biofilm, with
mucolytic
and/or mucoactive agents, and/or agents that reduce biofilm formation selected
from
nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate,
sodium
bicarbonate, tromethamine, silver nano particles, bismuth thiols, ethylene
diamine
tetraacetic acid, gentamicin loaded phosphatidylcholine-decorated gold
nanoparticles, chelators, cis-2-decenoic acid, D-amino acids, D-enantiomeric
peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin,
patulin,
penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic
acid, fatty
acids, host defense peptides, and antimicrobial peptides. In another
embodiment,
the composition for the use is administered before, simultaneously, or
subsequently
to the administration of an agent selected from bedaquiline or a
pharmaceutically
acceptable salt or derivative thereof, cefoxitine, am ikacin, clarithromycin,
pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-amino salicylate,
and
mixtures thereof.
In another embodiment, a pharmaceutical combination according to any of the
combination embodiments described herein is provided which is for use in
combination with an agent for dispersing and/or destruction of biofilm, with
mucolytic
22
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
and/or mucoactive agents, and/or agents that reduce biofilm formation selected
from
nebulized 4-7% hypertonic saline, metaperiodate, sodium dodecyl sulfate,
sodium
bicarbonate, tromethamine, silver nano particles, bismuth thiols, ethylene
diamine
tetraacetic acid, gentamicin loaded phosphatidylcholine-decorated gold
nanoparticles, chelators, cis-2-decenoic acid, D-amino acids, D-enantiomeric
peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX, curcumin,
patulin,
penicillic acid, baicalein, naringenin, ursolic acid, asiatic acid, corosolic
acid, fatty
acids, host defense peptides, and antimicrobial peptides. In another
embodiment, the
combination for the use is used to administer a composition of the present
invention
before, simultaneously, or subsequently to the administration of an agent
selected
from bedaquiline or a pharmaceutically acceptable salt or derivative thereof,
cefoxitine, amikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin,
levofloxacin, and para-amino salicylate, and mixtures thereof. In another
embodiment, the composition is administered before, simultaneously or
subsequently
to the administration of an agent selected from bedaquiline or a
pharmaceutically
acceptable salt or derivative thereof, and amikacin, and mixtures thereof. In
a further
embodiment, the composition is administered before, simultaneously or
subsequently
to the administration of bedaquiline or a pharmaceutically acceptable salt or
derivative thereof.
In another embodiment, a pharmaceutical composition according to any one of
the
composition embodiments as described herein is provided for use in the
treatment
and/or prophylaxis of a pulmonary infection caused by mycobacteria or other
gram
positive bacteria. In a further embodiment, the infection is caused by a
species of the
genus mycobacterium selected from nontuberculous mycobacteria and
Mycobacterium tuberculosis complex, and a combination thereof. In a further
embodiment, the nontuberculous mycobacteria is selected from Mycobacterium
avium, Mycobacterium intracellulare, Mycobacterium abscessus, and
Mycobacterium
leprae, and a combination thereof. In another embodiment, the infection is an
opportunistic infection, selected from MAC pulmonary disease and
nontuberculous
infection, in a patient with cystic fibrosis, chronic obstructive pulmonary or
acquired
immune deficiency syndrome. In another embodiment, the infection is an
opportunistic nontuberculous mycobacteria infection in patients with cystic
fibrosis. In
another embodiment, the composition for the use is administered before,
23
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
simultaneously, or subsequently to the administration of an agent selected
from
bedaquiline or a pharmaceutically acceptable salt or derivative thereof,
cefoxitine,
am ikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin,
and
para-amino salicylate, and mixtures thereof. In another embodiment, the
composition
.. is administered before, simultaneously or subsequently to the
administration of an
agent selected from bedaquiline or a pharmaceutically acceptable salt or
derivative
thereof, and amikacin, and mixtures thereof. In a further embodiment, the
composition is administered before, simultaneously or subsequently to the
administration of bedaquiline or a pharmaceutically acceptable salt or
derivative
thereof.
In another embodiment a pharmaceutical combination according to any of the
combination embodiments as described herein is provided for use in the
treatment
and/or prophylaxis of a pulmonary infection caused by mycobacteria or other
gram
positive bacteria. In a further embodiment, the infection is caused by a
species of the
genus mycobacterium selected from nontuberculous mycobacteria and
Mycobacterium tuberculosis complex, and a combination thereof. In a further
embodiment, the nontuberculous mycobacteria is selected from Mycobacterium
avium, Mycobacterium intracellulare, Mycobacterium abscessus, and
Mycobacterium
leprae, and a combination thereof. In another embodiment, the infection is an
opportunistic infection, selected from MAC pulmonary disease and
nontuberculous
infection, in a patient with cystic fibrosis, chronic obstructive pulmonary or
acquired
immune deficiency syndrome. In another embodiment, the infection is an
opportunistic nontuberculous mycobacteria infection in patients with cystic
fibrosis. In
another embodiment, the combination for the use is used to administer a
composition
of the present invention before, simultaneously, or subsequently to the
administration
of an agent selected from bedaquiline or a pharmaceutically acceptable salt or
derivative thereof, cefoxitine, amikacin, clarithromycin, pyrazinamide,
rifampin,
moxifloxacin, levofloxacin, and para-amino salicylate, and mixtures thereof.
In
another embodiment, the combination for the use is used to administer a
composition
of the present invention before, simultaneously or subsequently to the
administration
of an agent selected from bedaquiline or a pharmaceutically acceptable salt or
derivative thereof, and amikacin, and mixtures thereof. In another embodiment,
the
combination for the use is used to administer a composition of the present
invention
24
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
before, simultaneously or subsequently to the administration of bedaquiline or
a
pharmaceutically acceptable salt or derivative thereof.
In another embodiment, a system for use in providing antibiotic activity when
treating
or providing prophylaxis against a pulmonary infection caused by mycobacteria
or
other gram-positive bacteria is provided wherein the system comprises:
1) a nebulized pharmaceutical combination comprising:
(a) a therapeutically effective dose of clofazimine;
(b) a nonionic surfactant with an Hydrophilic-Lipophilic Balance
value of greater than 10; and
(c) an aqueous liquid carrier selected from water, isotonic
saline, buffered saline and aqueous electrolyte solutions
and
2) a nebulizer,
wherein the clofazimine is present in the form of a suspension,
and
wherein the aerosol particles produced by the system have a mass
median aerodynamic diameter of 1 to 5 pm.
In a further embodiment, a pharmaceutical composition according to any one of
composition embodiments described herein is provided, for use in the treatment
and/or prophylaxis of pulmonary fungal infections or clostridium difficile, or
a
combination thereof. In another embodiment, a pharmaceutical composition
according to any one of composition embodiments described herein is provided,
for
use in the treatment and/or prophylaxis of pulmonary fungal infections. In a
further
embodiment, the pulmonary fungal infection is candida albicans or aspergilus
fumigatus, or a combination thereof.
In a further embodiment, a pharmaceutical combination according to any one of
the
combination embodiments described herein is provided, for use in the treatment
and/or prophylaxis of pulmonary fungal infections or clostridium difficile, or
a
combination thereof. a pharmaceutical combinations according to any one of
combinations embodiments described herein is provided, for use in the
treatment
and/or prophylaxis of pulmonary fungal infections. In a further embodiment,
the
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
pulmonary fungal infection is candida albicans or aspergilus fumigatus, or a
combination thereof.
In another embodiment, a method of treatment or prophylaxis of a pulmonary
infection is provided, in a patient in need thereof, comprising administering
by
inhalation a composition according to any one the composition embodiments
described herein. In another embodiment, the infection is caused by a species
of the
genus mycobacterium selected from nontuberculous mycobacteria and
Mycobacterium tuberculosis complex, and a combination thereof. In a further
embodiment, the nontuberculous mycobacterium is selected from Mycobacterium
avium, Mycobacterium intracellulare, Mycobacterium abscessus, and
Mycobacterium
leprae, and a combination thereof. In a further embodiment, the infection is
an
opportunistic infection, selected from MAC pulmonary disease and
nontuberculous
infection, in a patient with cystic fibrosis, chronic obstructive pulmonary
disease or
acquired immune deficiency syndrome. In another embodiment, the infection is
an
opportunistic nontuberculous mycobacteria infection in a patient with cystic
fibrosis.
In a further embodiment, a method of treatment or prophylaxis of a pulmonary
infection is provided caused by mycobacteria or other gram positive bacteria,
in a
patient in need thereof, comprising administering by inhalation a composition
according to any one of the composition embodiments described herein, before,
simultaneously, or subsequently to the administration of an agent selected
from
bedaquiline, or a pharmaceutically acceptable salt of derivative thereof,
cefoxitine,
am ikacin, clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin,
and
para-amino salicylate, and mixtures thereof. In another embodiment, the agent
is
bedaquiline or am ikacin. In a further embodiment, the agent is bedaquiline.
Particle Size and Distribution
The therapeutic effect of aerosolized therapies is dependent upon the dose
deposited
and its distribution. Aerosol particle size is one of the important variables
in defining
the dose deposited and the distribution of drug aerosol in the lung.
26
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Generally, inhaled aerosol particles are subject to deposition by one of two
mechanisms: impaction, which usually predominates for larger aerosol
particles, and
sedimentation, which is prevalent for smaller aerosol particles. Impaction
occurs
when the momentum of an inhaled aerosol particle is large enough that the
particle
does not follow the air stream and encounters a physiological surface. In
contrast,
sedimentation occurs primarily in the lower lung when very small aerosol
particles
which have traveled with the inhaled air stream encounter physiological
surfaces as a
result of gravitational settling.
Pulmonary drug delivery may be accomplished by inhalation of an aerosol
through
the mouth and throat. Aerosol particles having an aerodynamic diameter of
greater
than about 5 pm generally do not reach the lung; instead, they tend to impact
the
back of the throat and are swallowed and possibly orally absorbed. Aerosol
particles
having diameters of about 3 to about 5 pm are small enough to reach the upper-
to
mid-pulmonary region (conducting airways), but are too large to reach the
alveoli.
Smaller aerosol particles, i.e. about 0.5 to about 3 pm, are capable of
reaching the
alveolar region. Aerosol particles having diameters smaller than about 0.5 pm
tend to
be exhaled during tidal breathing, but can also be deposited in the alveolar
region by
a breath hold.
Aerosols used in pulmonary drug delivery are made up of a wide range of
aerosol
particle sizes, so statistical descriptors are used. Aerosols used in
pulmonary drug
delivery are typically described by their mass median diameter (MMD), that is,
half of
the mass is contained in aerosol particles larger than the MMD, and half the
mass is
contained in aerosol particles smaller than the MMD. For particles with
uniform
density, the volume median diameter (VMD) can be used interchangeably with the
MMD. Determinations of the VMD and MMD are made by laser diffraction. The
width
of the distribution is described by the geometric standard deviation (GSD).
However,
the deposition of an aerosol particle in the respiratory tract is more
accurately
described by the particle's aerodynamic diameter, thus, the mass median
aerodynamic diameter is typically used. MMAD determinations are made by
inertial
impaction or time of flight measurements. For aqueous particles, VMD, MMD and
MMAD should be the same. However, if humidity is not controlled as the aerosol
transits the impactor, MMAD determinations will be smaller than MMD and VMD
due
27
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
to dehydration. For the purposes of this description, VMD, MMD and MMAD
measurements are considered to be under controlled conditions such that
descriptions of VMD, MMD and MMAD will be comparable.
Nonetheless, for the purpose of the description, the aerosol particle size of
the
aerosol particles will be given as MMAD as determined by measurement at room
temperature with a Next Generation Impactor (NG!) in accordance with US
Pharmacopeial Convention. In Process Revision <601> Aerosols, Nasal Sprays,
Metered-Dose Inhalers, and Dry Powder Inhalers, Pharmacopeial Forum (2003),
Volume Number 29, pages 1176-1210 also disclosed in Jolyon Mitchell, Mark
Nagel
"Particle Size Analysis of Aerosols from Medicinal Inhalers", KONA Powder and
Particle Journal (2004), Volume 22, pages 32-65.
In accordance with the present invention, the particle size of the aerosol is
optimized
to maximize the deposition of clofazimine at the site of infection and to
maximize
tolerability. Aerosol particle size may be expressed in terms of the mass
median
aerodynamic diameter (MMAD). Large particles (e.g., MMAD > 5 pm) tend to
deposit
in the extrathoracic and upper airways because they are too large to navigate
bends
in the airways. Intolerability (e.g., cough and bronchospasm) may occur from
upper
airway deposition of large particles.
Thus, in accordance with a preferred embodiment, the MMAD of the aerosol
should
be less than about 5 pm, preferably between about 1 and 5 pm, more preferably
below 3 pm (<3 pm).
However, a guided breathing maneuver can be used to allow larger particles to
pass
through the extrathoracic and upper airways and deeper into the lungs than
during
tidal breathing which will increase the central and lower lung deposition of
the
aerosol. A guided breathing maneuver may be as slow as 100 ml/min. Thus, when
used with a guided breathing maneuver, the preferred MMAD of the aerosol
should
be less than about 10 pm.
Another equally important factor (in addition to aerosol particle size) is the
particle
size and size distribution of the solid particles, in this case clofazimine
particle size
and distribution. The size of a solid particle in a given aerosol particle
must be
28
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
smaller than the aerosol particle in which it is contained. A larger aerosol
particle
may contain one or more solid particles.
Further, when dealing with dilute
suspensions, a majority of aerosol particles may contain no solid particle.
Because of this, it is desirable to have solid drug particles that are
significantly
smaller than the MMAD of the aerosol particles. For example, if MMAD of the
aerosol particles is 3 pm, than a desired solid particle would be 1 pm, or
smaller.
Another consideration, for example when using a vibrating mesh nebulizer, the
formulation is pumped through orifices in a plate, which breaks up the
suspension
into droplets. It follows, then, that the solid particles must also be smaller
than these
orifices, in order to pass through.
Solid particle size in the suspension may be given by the mean size of the
particles,
and also by the distribution of the particles. D90 values indicate that 90% of
the
particles within the suspension are of the mean size or smaller.
Nebulizer
For aqueous and other non-pressurized liquid systems, a variety of nebulizers
(including small volume nebulizers) are available to aerosolize the
formulations.
Compressor-driven nebulizers incorporate jet technology and use compressed air
to
generate the liquid aerosol. Such devices are commercially available from, for
example, Healthdyne Technologies, Inc.; Invacare, Inc.; Mountain Medical
Equipment, Inc.; Pan i Respiratory, Inc.; Mada Medical, Inc.; Puritan-Bennet;
Schuco,
Inc., DeVilbiss Health Care, Inc.; and Hospitak, Inc. Ultrasonic nebulizers
rely on
mechanical energy in the form of vibration of a piezoelectric crystal to
generate
respirable liquid droplets and are commercially available from, for example,
Omron
Heathcare, Inc. and DeVilbiss Health Care, Inc. Vibrating mesh nebulizers rely
upon
either piezoelectric or mechanical pulses to respirable liquid droplets
generate. Other
examples of nebulizers for use with clofazimine described herein are described
in
U.S. Patent Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911;
4,510,929;
4,624,251; 5,164,740; 5,586,550; 5,758,637; 6,644,304; 6,338,443; 5,906,202;
5,934,272; 5,960,792; 5,971,951; 6,070,575; 6,192,876; 6,230,706; 6,349,719;
29
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
6,367,470; 6,543,442; 6,584,971; 6,601,581; 4,263,907; 5,709,202; 5,823,179;
6,192,876; 6,644,304; 5,549,102; 6,083,922; 6,161,536; 6,264,922;6,557,549;
and
6,612,303, all of which are hereby incorporated by reference in their
entirety.
Commercial examples of nebulizers that can be used with the clofazimine
compositions described herein include Respirgard II, Aeroneb, Aeroneb Pro, and
Aeroneb Go produced by Aerogen; AERe and AERx EssenceTM produced by
Aradigm; Porta-Nee, Freeway Freedom TM, Sidestream, Ventstream and I-neb
produced by Respironics, Inc.; and PARI LCPlue, PARI LC-Star, and e-Flow7m
produced by PARI, GmbH. Further non-limiting examples are disclosed in
US 6,196,219.
In accordance with the present invention, the pharmaceutical composition may
be
preferably aerosolized using a nebulising device selected from an ultrasonic
nebulizer, an electron spray nebulizer, a vibrating membrane nebulizer, a jet
nebulizer or a mechanical soft mist inhaler.
It is preferred that the device controls the patient's inhalation flow rate
either by an
electrical or mechanical process.
In a further preferred embodiment, the aerosol production by the device is
triggered
by the patient's inhalation, such as with an AKITA device.
Preferred (commercially available) examples of the above nebulizers/devices to
be
used in accordance with the present invention are Vectura fox, Pan i eFlow,
Pan i Trek S, Philips Innospire mini, Philips InnoSpire Go, Medspray device,
Aeroneb
Go, Aerogen Ultra, Respironics Aeroneb, Akita, Medspray Ecomyst and Respimat.
Use in treatment and/or prophylaxis
The pharmaceutical compositions and pharmaceutical combinations (aerosols,
aerosolized formulations) and systems according to the present invention are
intended for the use in the treatment and/or prophylaxis of pulmonary
infections
caused by mycobacteria or other clofazimine susceptible bacteria, such as
Staphylococcus aureus (including methicillin-resistant and vancomycin
intermediate-
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
resistant strains), Streptococcus pneumoniae, and Enterococcus spp.
The
pharmaceutical compositions and pharmaceutical combinations of the present
invention may also be used for the treatment and/or prophylaxis of pulmonary
fungal
infections.
Dosing of clofazimine
In accordance with the present invention, the pharmaceutical composition is
delivered by nebulization in about 1-5 ml, preferably 1-2 ml of the
pharmaceutical
composition of the invention.
Thus, the target fill dose is about 1-5 ml corresponding to 20-100 mg
clofazimine,
based on a clofazimine concentration in the pharmaceutical composition of
about 20
mg/ml.
The daily lung dose (i.e. the dose deposited in the lung) of clofazimine to be
administered in accordance with the present invention is about 5-10 mg, which
corresponds to a nominal dose of 15-30 mg (device dose) in the case of M.
abscessus infections.
It is understood that the person of skill in the art will routinely adjust the
lung dose of
clofazimine to be administered (and thus the fill/nominal dose/the volume to
be
nebulized) based on the minimum inhibitory concentration (MIC) of clofazimine
for
the respective bacteria strain well established in the art.
Depending on the dosing frequency, once or twice per day, the daily lung dose
will be
split accordingly.
In accordance with the present invention, clofazimine is to be administered
once or
twice daily with a resulting total daily lung dose of about 5 to 10 mg.
It will be obvious to a person skilled in the art that the above amounts
relate to
clofazimine free base, the dosage amounts for derivatives, and salts will have
to be
adjusted accordingly based on the MIC of the respective compound and strain.
31
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Mucolytic agents/biofilm modifying agents
In order to reduce sputum viscosity during aerosol treatment and to destroy
existing
biofilm, the treatment and/or prophylaxis according with the present invention
can
involve additional administration of mucolytic and/or biofilm destructing
agents.
These agents can be prepared in fixed combination or be administered
simultaneously or subsequently to the pharmaceutical composition/aerosol
combination comprising clofazimine in accordance with the present invention.
Agents for dispersing/destruction of the biofilm, mucolytic and/or mucoactive
agents
and/or agents that reduce biofilm formation to be used in accordance with the
present
invention are selected from nebulized 4-7% hypertonic saline, metaperiodate,
sodium
dodecyl sulfate, sodium bicarbonate, tromethamine, silver nano particles,
bismuth
thiols, ethylene diamine tetraacetic acid, gentamicin loaded
phosphatidylcholine-
decorated gold nanoparticles, chelators, cis-2-decenoic acid, D-amino acids, D-
enantiomeric peptides, gallium mesoporphyrin IX, gallium protoporphyrin IX,
curcumin, patulin, penicillic acid, baicalein, naringenin, ursolic acid,
asiatic acid,
corosolic acid, fatty acids, host defense peptides, and antimicrobial
peptides.
Furthermore, also other pharmaceutically active agents may be used in
combination
with the pharmaceutical compositions/aerosol combinations in accordance with
the
present invention. Such active agents may be selected from bedaquiline or a
pharmaceutically acceptable salt or derivative thereof, cefoxitine, amikacin,
clarithromycin, pyrazinamide, rifampin, moxifloxacin, levofloxacin, and para-
amino
salicylate, and mixtures thereof.
These agents can be prepared in fixed combination or be administered prior to,
simultaneously or subsequently to the pharmaceutical composition/aerosol
combination comprising clofazimine in accordance with the present invention.
Examples
32
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
The following examples serve to more fully describe the manner of using the
above-
described invention, as well as to set forth the best modes contemplated for
carrying
out various aspects of the invention. The Examples according to the invention
are
.. those falling within the scope of the claims herein.
Experimental
The exemplary compositions and combinations below have been prepared in
accordance with the processes described herein.
Example 1
200 mg of clofazimine (as triclinic form I), 90 mg of sodium chloride, and 9.5
ml of
water were mixed in an Ultra-Turrax homogenizer two times at 10,000 rpm for 5
minutes. 0.5 ml of polysorbate 80 (NOF Hx2) was added. This mixture was
treated
with an ultrasonic probe (Branson Digital SonifierTM 250D with Bandelin
Sonoplus
Probe MS73) seven times, 3 minutes each, with an amplitude of 70%. The volume
was adjusted to 10 ml with water. This suspension was filtered through VWR
folded
.. qualitative filter paper (303, particle retention 5-13 pm, Size: 150 mm),
to give the
Composition of Example 1. The composition of Example 1 had a median particle
size of clofazimine of 3.9 pm, with a D90 of 6.7 pm. The concentration of
clofazimine
was determined by ultraviolet/visible spectroscopy at 280 nm, calibrating with
a stock
solution of 1 mg/ml of clofazimine diluted in the mobile phase, and determined
to be
7.16 mg/ml.
The composition of Example 1 is shown in Table 1
Clofazimine Form Fl 71.6 mg
Polysorbate 80 (NOF Hx2) 0.5 ml
Sodium chloride 90.0 mg
Water (V/V) 9.5 ml
Table 1
33
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Preparation of Clofazimine of Orthorhombic Form III
A slurry of clofazimine (10 g) in toluene (20 ml) was stirred at 40 C in an
oil bath for
72 hours using a magnetic stirrer at 800 rpm. The solid portion of the slurry
was
collected by filtration through a crucible and dried at a maximum temperature
of 40 C
under vacuum in an oven. This yielded 8.64 g of clofazimine as substantially
pure
(98`)/0) orthorhombic form III.
Example 2
A suspension containing 6g of clofazimine of orthorhombic form III in 100 ml
of water
containing 0.5% polysorbate 80 (NOF Hx2) and 0.6% sodium chloride was pre-
micronized for approximately 40 seconds at 10,000 rpm using an Ultra-Turrax0.
The
pre-formulation was prepared by adding 0.6% sodium chloride in water to give a
volume of 300 ml. 300 ml of this suspension was added into the inlet of the
homogenizer, a M-110EH-30 microfluidizer (Microfluidics, Westwood, MA, USA)
and
a pre-homogenization step was performed for 15 minutes by circulation of the
suspension through the H3OZ chamber at 5,000 psi. Subsequently, the second
H1OZ
chamber was installed in series with the first chamber and the suspension was
further homogenized for 23 minutes at 25,000 psi. Particle size analysis was
performed with a HORIBA LA 950 indicating a median particle size of 0.83 [tm
with a
D90 value of 1.17 [tm. A concentration of clofazimine of 16.05mg/m1 was
determined
by ultraviolet/visible spectroscopy at 280 nm, calibrating with a stock
solution of 1
mg/ml of clofazimine diluted in the mobile phase.
The composition of Example 2 is shown in Table 2
Composition of Example 2
Clofazimine form III 4.81g (16.05 mg/ml)
Polysorbate 80 (NOF Hx2) 1.5m1 (0.5% v/v)
Sodium Chloride 1.8g (0.6% w/v)
Water 298.5 ml
Table 2
Example 3
34
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
A suspension of clofazimine (crystal modification orthorhombic Form III) in a
solution
of water, sodium chloride and Polysorbate 80, was treated using a
M-110EH-30 Microfluidizer Processor (chambers: H3OZ and G10Z) operated for 30
minutes at a pressure of 28,250 psi, with the H30Z-G1OZ configuration to
produce
the Compostion of Example 3, with the resulting particles of clofazimine
having a
median particle size of 1.28 [tm and a D90 below 2 [tm.
The composition of Example 3 is shown in Table 3.
Composition of Example 3
Clofazimine (Form FIII) 20 mg/ml
Polysorbate 80 (NOF Hx2) 0.5% (v/v)
Sodium Chloride 0.9% (w/v)
Water 99.5% (v/v)
Table 3
Viscosity Measurements
The viscosity of the Composition of Example 3 was tested using a STRESSTECH
Rheometer in stress control mode. A double gap geometry was utilized and the
spindle was continuously rotated to ensure the particulates remained in
suspension
during temperature points. Viscosity was measured across 0.01, 0.05, and 0.1
Pa
stress each at 20 C, 25 C, and 30 C. Two separate loadings were performed to
obtain the average viscosities shown in Table 4 below.
Viscosity Measurements
Temperature ( C) Viscosity Pa=s
20 1.146E-03
1.049E-03
9.831E-04
Table 4
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Animal models and efficacy testing
Compositions of the present invention have been tested for their ability to
inhibit
growth of clinical NTM species in an acute in vivo pulmonary infection mouse
model
to obtain preliminary data to establish clofazimine concentration levels in
lung tissue
after direct respiratory delivery as opposed to systemic administration. Two
separate
mouse models are used in order to investigate pulmonary NTM infection,
dependent
on the bacterial species of interest. For testing, Mycobacterium avium 2285,
and
Mycobacterium abscessus 103 bacterial strains have been used (Strain details
can
be found in "Phylogenetic analysis of Mycobacterial species using whole genome
sequences". Hazbon M.H., Riojas M.A., Damon A.M., Alalade R., Cantwell B.J.,
Monaco A., King S., Sohrabi A. Submitted (SEP-2014) to the EMBL/GenBank/DDBJ
databases). These two species have been previously used in literature as
models of
NTM infection (Obregon-Henao etal. 2015 Antimicrob Agents Chemother; and Chan
et al. Animal Models of Non-Tuberculous Mycobacterial Infections, Mycobact Dis
2016).
In vivo safety study in Balb/C mice:
For in vivo safety and tolerability, 6-8 week old Balb/C female mice are
obtained from
Charles River. The mice are rested for one week before dosing. For each dose
of
clofazimine, three healthy mice are given a total of three doses every other
day.
Mice were dosed at 10.0, 5.01, and 2.51 mg/kg of clofazimine in the
composition of
Example 1. The compounds were given to 3 healthy mice for a total of three
doses,
every other day, by Microsprayer@ aerosol intratracheal administration.
Clofazimine was found to be safe at 20 mg/kg (gavage, 200 pl). The composition
of
Example 1 showed no toxicity at the highest dose tested (10.0 mg/kg; 0.2506
mg/dose in 35 pl intratracheally). Accordingly, the composition of Formula I
was
considered safe and well tolerated at 10.0 mg/kg.
Determination of Minimum Inhibitory Concentration
36
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Minimum inhibitory concentration (MIC) testing was performed by microbroth
dilution
method using MOeller Hinton (MH) broth (Cation Adjusted) to the calcium and
magnesium ion concentration recommended in the CLSI standard M7-A7 (Becton
Dickinson). MIC testing also was performed by microbroth dilution method using
7H9
broth (Sigma-Aldrich). The justification for use of both MH and 7H9 broth for
compound screening is that antimycobacterial compounds have been shown to
display different MIC activity depending on the broth that is used in the MIC
assay.
M. abscessus was grown on 7H11 agar plates (Sigma-Aldrich) for 3 days at 35-37
C
in ambient air (depending on bacterial strain), and M. avium was grown on agar
7H11
plates (Sigma-Aldrich) for 21-30 days at 37 C in ambient air.
The colony forming units (CFUs) are taken from the agar plates and placed in
either
MH or 7H9 broth with 0.05% tween-80 and grown at 35-37 C in ambient air until
the
optical density (OD) absorbance taken after 3 days (M. abscessus) or 12 (M.
avium)
of growth is an (OD) 0.08 -0.1 (0.5 McFarland Standard). The bacterial cell
suspensions are then confirmed by preparing them in saline, matching the (OD)
0.08
- 0.1 (0.5 McFarland Standard). Compound stock solutions were made by
suspending the compounds in DMSO at a concentration of 1.28 mg/ml, and used
immediately for test range 64-0.062 pg/m I. Following this, 180 pl of broth
(either MH
or 7H9) was added to the first column in the 96 well plates, and 100 pl of
broth to the
remaining columns in the 96 well plate. 20 pl of compound stock solution was
added
to the first column of wells, and serially diluted. Finally, 100 pl NTM cell
suspension
was added in all the wells except the media only control wells. QC agents
specific for
each organism 1) bacteria only negative control 2) media only negative control
3)
clarithromycin positive drug controls.
M. abscessus ODs were assayed on day 3, and M. avium on day 12. Following
these
measurements, the plate was assayed by using the Resazurin Microtiter Assay
Plate
method. Briefly, the method uses the addition of resazurin (7-Hydroxy-3H-
phenoxazin-3-one 10-oxide) to the 96 well plate. Resazurin is a blue dye,
itself
weakly fluorescent until it is irreversibly reduced to the pink colored and
highly red
fluorescent resorufin. It is used as an oxidation-reduction indicator to
determine
bacterial cell viability in MIC assays.
37
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Assays were done in triplicate. Assay #1 was performed after storage of the
Composition of Example 1 at 4 C for 2 months, Assay #2 was performed at 4
months, and Assay #3 at five months.
Minimum Inhibitory Concentrations in the Presence and Absence of CF Sputum
Minimum inhibitory concentrations assays were performed as described above.
To investigate the effect of cystic fibrosis (CF) patient sputum on
antimicrobial activity
of clofazimine (CFZ) and the Composition of Example 1, sputum was collected
from
patients who had not received antibiotics for the previous 48 hours, and their
sputum
was sterilized by exposure to UV light to eliminate endogenous bacteria.
Following
sterilization, M. abscessus , M. avium, M. intracellulare, and M. Chimaera
were
incubated in 10% CF sputum before undergoing MIC testing. The MIC of the
Composition of Example 1 was measured following the same CLSI protocol as
described above, in the presence and absence of cystic fibrosis patient
sputum. All
studies were performed in duplicate.
MIC values of clofazimine and the Composition of Example 1 in the presence and
absence of sputum are shown in Table 5.
38
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Species Identification CFZ Composition CFZ +
Composition
Number of Ex. 1 SP of Ex. 1 +
SP
(p.g/m1)
(pg/m1) (Kg/m1)
(pg/m1)
M. avium ATCC 700898 0.25 0.125 >2 >2
M. intracellulare DSM 43223 1 0.125 >2 >2
M. Chimaera CIP 107892 0.5 0.125 >2 >2
M. avium B18101968 0.5 0.125 >2 >2
M. abscessus CIP104536 0.25 0.125 >2 >2
M. abscessus B18104072 1 0.25 >2 >2
M. abscessus B15029863 2 2 >2 >2
M.abscessus B12052284 2 0.5 >2 >2
Table 5
The results presented in Table 5 indicate consistent MICs of both clofazimine
and the
Composition of Example 1, against a range of nontuberculous mycobacterial
species.
These data indicate that the Composition of Example 1 demonstrates potent in
vitro
activity against both M. abscessus and M. avium, and is stable at least over
this time
period.
Mouse model of M. abscessus in the SCID mouse
6-8 week old SCID female mice were ordered from Charles River. Mice were
rested
one week before infection.
Working stocks of M. abscessus strain 103 were frozen in 1 ml aliquots and
sotred at
-80 C before use. For infection an aliquot was thawed, disrupted 20 times
with a 1
ml luer-lok syringe fitted with a 26 g needle, and dluted in sterile lx PBS.
The acute SCID mouse model received a non-invasive intratracheal instillation
pulmonary infection with 1x106 CFU/mouse (M. abscessus strain 103).
Three mice were sacrificed day 1 post-infection to determine bacterial uptake.
Whole
lungs, spleens, and livers are extracted, homogenized in 4.5 ml of lx PBS.
39
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Homogenates were serially diluted in 1:10 dilutions and dilutions (0-1-2-3-4-5-
6-7)
plated on 7H11 agar plates. The plates are placed in 32 C dry-air incubator
(strain
dependent) for 7 days.
The Composition of Example 110.0 mg/kg was administered by a Microsprayee (35
pl) through the pulmonary route, and clofazimine (gavage), amikacin
(subcutaneous)
in a volume of 200 pl per mouse which begins day 2 post-infection and
continued
every other day for 8 consecutive days.
Mice were sacrificed 2 days after administration of the last dose of the
compounds.
Six mice of all groups (untreated control, clofazimine (gavage), composition
of
Example 1, and amikacin treated mice) were sacrificed and bacterial loads were
determined. Plating of lung homogenate at 0-1-2-3-4-5-6-7, spleen at 0-1-2-3-4-
5-6-7
and liver at 0-1-2-3-4-5-6-7.
Log 10 protection values of at least 0.60 indicate activity is statistically
significant.
Statistical analysis was performed by first converting CFU to logarithms,
which were
then evaluated by a one-way ANOVA followed by a multiple comparison analysis
of
variance by a one-way Tukey test (GraphPad Prism analysis software).
Differences
are considered significant at the 95% level of confidence.
Table 6 shows the average Logi CFU data and standard error of mean (SEM)
following SCID mouse M. abscessus infection, where "n" is the total number of
animals in group at time of sacrifice.
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Lung Spleen Liver
Group Logio CFU Logio CFU Logio CFU
SEM SEM SEM
Day 1 (Pre-treatment Control)
5.08 0.03 4.42 0.04 5.00 0.11
(n=3)
Treatment End
Saline Control (IT) (n=6)
5.51 0.06 6.33 0.85 6.45 0.34
Amikacin (SQ) 150 mg/kg (n=6)
3.94 0.11 4.37 0.31 3.07 0.10
Clofazimine (Oral) 20 mg/kg
3.33 0.17 3.79 0.10 4.78 0.18
(n=6)
Composition of Example 1 (IT,
aerosol) 10.0 mg/kg (n=6) 2.66 0.28 3.29 0.25 4.57 0.14
Table 6
The data in Table 6 indicate that treatment with the composition of Example 1
led to
the greatest reduction in bacterial recovery in the lungs and spleen of
animals
infected with M. abscessus. This bacterial reduction was statistically
improved over
treatment with am ikacin, or oral clofazimine.
Mouse model of M. avium infection in the Beige mouse
6-8 week old Beige female mice were ordered from Charles River. Mice were
rested
one week before infection.
The acute Beige mouse model received a non-invasive aerosol exposure pulmonary
infection with 1x108 colony forming units (CFU)/m1 (M. avium strain 2285
rough).
Working stocks of M. avium strain 2285 rough were frozen in 1 ml aliquots and
stored
at -80 C before use. For infection an aliquot was thawed, disrupted 20 times
with a 1
ml luer-lok syringe fitted with a 26 g needle, and diluted in sterile 1 x
phosphate
buffered saline (PBS).
Three mice were sacrificed on day 1 and day 7 post-infection to determine
bacterial
uptake. Whole lungs, spleens, and livers were extracted, homogenized in 4.5 ml
of lx
41
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
PBS and diluted 1:10. Dilutions (0-1-2-3-4-5-6-7) are plated on 7H11/0ADC, TSA
and charcoal agar plates and incubated at 32 C in a dry-air incubator (strain
dependent) for 30 days.
The composition of Example 1, 10.0 mg/kg was administered by a Microsprayer
(35
pl) though the pulmonary route and clofazimine (gavage) in a volume of 200 pl
per
mouse which begins on day 7 post-infection and continued every other day for
10
consecutive days.
Mice were sacrificed 5 days after administration of the last dose of the
compounds.
Six mice of all groups (untreated control, clofazimine (gavage), and the
composition
of Example 1) were sacrificed and bacterial loads were determined. Plating of
lung
homogenate at 0-1-2-3-4-5-6-7, spleen at 0-1-2-3-4-5-6-7 and liver at 0-1-2-3-
4-5-6-
7.
Log 10 protection values of at least 0.60 indicate activity is statistically
significant.
Statistical analysis was performed by first converting CFU to logarithms,
which were
then evaluated by a one-way ANOVA followed by a multiple comparison analysis
of
variance by a one-way Tukey test (SigmaStat software program). Differences are
considered significant at the 95% level of confidence.
Table 7 shows the average Logi CFU data following Beige mouse M. avium
infection.
42
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Lung Spleen Liver
Group Logio CFU Logio CFU Logio CFU
SEM SEM SEM
Day 1 (Pre-treatment Control)
4.74 0.01 0.00 0.00 0.00 0.00
(n=3)
Day 7 (Pre-treatment Control)
5.19 0.04 0.00 0.00 0.00 0.00
(n=3)
Treatment End
Saline Control (IT) (n=6)
5.20 0.03 3.24 0.08 3.63 0.34
Clofazimine (Oral) 20 mg/kg
3.95 0.03 3.14 0.03 3.01 0.03
(n=6)
Composition of Example 1 (Sus.)
3.43 0.18 2.93 0.25 3.10 0.02
(IT, aerosol) 10.0 mg/kg (n=6)
Table 7
The data in Table 7 indicate that treatment with the composition of Example 1
led to
the greater reduction in bacterial recovery in the lungs and spleen of animals
infected
with M. avium.
Chronic Beige Mouse Model
6 to 8 week-old Beige mice were rested one week before infection. Mice
received a
pulmonary infection of 1x108 CFU of M. avium 2285 rough on Day 0. Three mice
were sacrificed on Day 1, and six mice on Day 27 to determine bacterial uptake
and
pre-treatment bacterial loads. Whole lungs, spleens, and livers were
extracted,
homogenized in 4.5 ml of 1xPBS and plated at (0-1-2-3-4-5-6-7) dilutions on
7H11
and charcoal agar plates. The plates were placed in a 37 C dry-air incubator
for 25
to 30 days.
The remaining infected Beige mice were treated every other day, starting on
Day 28,
for a total of 14 treatments. Animals received one of the following
treatments: Saline
(Microsprayer0, 35 [1,1); Clofazimine (oral gavage, 20 mg/kg, 200 [1,1);
Composition of
Example 1 (IT, Microsprayer0, 10 mg/kg, 35 [1,1).
Mice were sacrificed on Day 57, two days after the final treatment. Plates
were
placed in a 37 C dry-air incubator for 30 days.
Statistical analysis was performed by first converting CFU to logarithms,
which were
then evaluated by a one-way ANOVA followed by a multiple comparison analysis
of
43
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
variance by a one-way Tukey test. Differences are considered significant at
the 95%
level of confidence.
Average Logio CFU data following Beige mouse M. avium chronic infection are
shown in Table 8
Lung Spleen Liver
Group Logio CFU Logio CFU Logio CFU
SEM SEM SEM
Day 1 (Pre-treatment Control)
5.24 0.05 0.00 0.00 0.00 0.00
(n=3)
Day 27 (Pre-treatment Control)
6.02 0.07 5.37 0.35 6.52 0.12
(n=6)
Treatment End (Day 57
Saline Control (IT) (n=6)
6.40 0.06 6.15 0.16 6.69 0.14
Clofazimine (Oral) 20 mg/kg
5.94 0.12 3.08 0.04 4.07 0.04
(n=6)
Composition of Example 1 (Sus.)
2.45 0.09 4.83 0.22 3.89 0.011
(IT, aerosol) 10.0 mg/kg (n=6)
Table 8
These data suggest that clofazimine has a difficult time penetrating the
granuloma-
like structures formed by established, "chronic" animal NTM infection models.
It
appears that the composition of the present invention does not have the same
issues, and is able to maintain antimycobacterial activity even after the
infection has
become well-established.
Effect of the Composition of Example 3 on Barrier Integrity and Inflammation
Following Exposure to Pulmonary Epithelial Cells in vitro
Cell Viability
Three different cell types under two in vitro conditions were used to assess
pulmonary epithelial cell viability: Calu-3; A549; and hAELVi cells. Cells
were either
treated under "submerged conditions" (i.e. in cell culture media on
TranswellTm
plates) or "air-liquid interface" mimicking conditions (ALI), which had cell
culture
media removed from the apical side of the cells. In "submerged conditions",
Calu-3
cells were exposed to three doses of the Composition of Example 3 (10%, 50%,
or
44
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
100%) for four hours. To estimate cell viability, cells were stained using
acridine
orange/propidium iodide (AO/PI) staining to differentiate live/dead cells. Red
fluorescence signaled cell death.
Macrophage Uptake
THP-1 cells were differentiated to macrophage-like cells following incubation
with
124 ng/mlphorbol 12-myristate 13 acetate (PMA) for 3 days. Once the cells were
matured, they were exposed to the Composition of Example 3 (diluted 1:200 in
Hank's Buffered Salt Solution (HBSS)) for four hours. Cells were stained via
AO/PI,
as described above, to determine cell viability following exposure.
Transepithelial Electrical Resistance (TEER) Measurements
Calu-3 cells were seeded at 1x105 cells/well on a TranswellTm 3460, and left
for 12
days to grow to confluence. TEER measurements were performed using an EVOM2
(World Precision Instruments, Friedberg, Germany) according to the
manufacture's
instructions. Following seeding, Calu-3 cells were exposed to either saline
(negative
control) or the Composition of Example 3 (concentrations: 20 mg/ml, 10 mg/ml,
or
2 mg/ml). The cells were exposed from 2 to 4 hours, before measuring TEER.
Inflammatory Cytokine Production
Differentiated THP-1 cells (dTHP-1) were exposed to the Composition of Example
3
for 4 hours or 24 hours (1:200 HBSS dilution). HBSS exposure alone was used as
a
negative control, and lipopolysaccharide (LPS) (100 ng/ml) was administered as
a
positive control.
Following the incubation, supernatant was removed from the cells t=4 hours or
24 hours, and the pooled. An enzyme linked immunosorbent assay (ELISA) was
performed on the pooled supernatant samples. Individual ELISA kits for TNF-a,
IL-6,
IL-8, and IL-10 were used, according to the manufacturer's instructions.
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Statistical analysis was performed by one-way analysis of variance (ANOVA)
followed by Tukey post-hoc test. Statistical significance was determined at a
probability value < 0.05.
Results
Under "submerged" conditions the Composition of Example 3 led to no visual
reduction in cell viability over four-hour incubation at any of the
concentrations
administered.
Under "ALI" conditions, three different cell types (Calu-3, A549, and HAELVi
cells)
were investigated over three different time points (five hours, two days, and
seven
days). The was little to no cytotoxicity observed at four hours in any of the
cells, or at
day 2 in the Calu-3 cells. Some toxicity was seen at day 2 and day 7 in A549
cells,
and day 7 in Calu-3 cells. Technical limitations did not permit quantification
of cell
death.
With regard to macrophage uptake, differentiated THP-1 cells were incubated at
1:200 HBSS for four hours to determine macrophage cell viability after
exposure. The
Composition of Example 3 did not induce cell death, but did show clofazimine
uptake
by the macrophages.
With regard to TEER measurements, Calu-3 cells were exposed to HBSS or three
concentrations of the Composition of Example 3 for four hours, and TEER
measurements were sampled at various time points throughout the exposure. A
reduction in TEER of 50% compared to controls at any given time point was
considered a significant loss in barrier integrity.
Exposure to the Composition of Example 3 to Calu-3 cells had no effect on
barrier
.. integrity after one-hour exposure. Exposure at 20 mg/m I led to significant
(i.e. 50%)
reduction after two hours. At a concentration of 10 mg/ml showed a slight
reduction
(i.e. 25-35%) at all time points after two hours. Exposure at 2 mg/m I did not
show
any reduction in barrier function over the full study duration.
46
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Inflammatory Cytokine Production
The positive control LPS behaved as expected in this model. The Composition of
Example 3 demonstrated no significant changes in cytokine at any timepoint
investigated.
Results are shown in Table 9.
Cytokine production following dTHP-1 cell exposure
TNF-a IL-8 IL-10 IL-
6
(4 hr) (4 hr) (4hr)
(24 hr)
Control 603.0 23.0 398.3 12.0 16.5
7.9 0.3
LPS 2130.7 23.0* 2596.8 84.0*
65.1 7.0* 22.6 0.7*
Composition of Ex. 3 441.9 48.3 978.9 395.4 18.9 7.1
6.5 1.4
Table 9 (*p<0.05)
In vivo Safety and Tolerability
6-8-week-pld Balb/C female mice were given a total of three doses every other
day.
Mice were dosed at 10.0, 5.01, and 2.51 mg/kg using the Composition of Example
1.
The composition was given via Microsprayer0 aerosol intratracheal (IT)
administration, at volumes of 35 [1,1/mouse. Following instillation, the mice
were
observed at 10 minutes, 1, 2 and 4 hours after dosing, and then daily
afterwards.
Table 10 shows gross observations following administration. "BAR" indicates
the
animals were bright, active and responsive.
47
CA 03109572 2021-02-12
WO 2020/040818
PCT/US2019/025538
Treatment Group Day 1 Day 2 Day 3
Group 1 BAR BAR BAR
10.0 mg/kg
Group 2 BAR BAR BAR
5.01 mg/kg
Group 3 BAR BAR BAR
2.51 mg/kg
Table 10
Table 11 shows weights of the animals over the three days tested.
Mouse Dose Weight (grams)
Day 1 Day 2 Day 3
1 10 mg/kg 20.1 20.2 22.1
2 21 21.2 22
3 21.1 20.2 22.2
1 5.01 mg/kg 22.3 22.3 23.1
2 22.1 22 23.2
3 22 21.9 23.2
1 2.51 mg/kg 22 22 23.3
2 22.2 22.2 22.8
3 22.4 22 23.1
Table 11
These data indicate that there was no statistically significant change in body
weight
over the three treatment days.
These results indicate that compositions of the present invention are well
tolerated at
the doses tested.
48
