Note: Descriptions are shown in the official language in which they were submitted.
CA 02795753 2012-11-15
10021CA01
1
NOVEL GALACTOSIDE INHIBITOR OF GALECTINS
Technical field
The present invention relates to novel compounds, the use of said compounds as
medicament and for the manufacture of a medicament for the treatment of
pulmonary fibro-
sis, such as Idiopathic pulmonary fibrosis in mammals. The invention also
relates to phar-
maceutical compositions comprising said novel compounds. The present invention
also re-
lates to a method of monitoring development or progression of pulmonary
fibrosis as well
as a method of monitoring or predicting exacerbation of symptoms.
Background Art
Idiopathic pulmonary fibrosis (IPF) represents a massive worldwide health
burden.
It is a chronic condition of unknown etiology in which repeated acute lung
injury causes
progressive fibrosis resulting in destruction of lung architecture,
deteriorating lung function
with consequent respiratory failure and death. Although idiopathic pulmonary
fibrosis (IPF)
is the archetypal and most common cause of lung fibrosis, numerous respiratory
diseases
can progress to pulmonary fibrosis, and this usually signifies a worse
prognosis. The me-
dian time to death from diagnosis is 2.5 years and the incidence and
prevalence of IPF con-
tinues to rise. It remains one of the few respiratory conditions for which
there are no effec-
tive therapies, and there are no reliable biomarkers to predict disease
progression. The
mechanisms resulting in pulmonary fibrosis are unclear but centre around
aberrant wound
healing as a consequence of repetitive epithelial injury from an as yet
unknown cause. IPF
is characterized by fibroblastic foci containing fibroblasts/ myofibroblasts
which show in-
creased activation response to fibrogenic cytokines such as transforming
growth factor-131
(TGF-131). Given the non-responsiveness of many cases of IPF to current anti-
inflammatory
treatments the myofibroblasts within fibroblastic foci represent a potential
novel therapeutic
target. There is a big unmet need for drugs for treatment of Idiopathic
pulmonary fibrosis.
The bleomycin model of pulmonary fibrosis is the best characterized rodent
model
and is the industry standard model. It causes oxidant-mediated DNA damage and
induces
initial lung inflammation followed by progressive fibrosis over 2 ¨ 4 weeks.
When admin-
istered during the later phase of the injury the anti-fibrotic potential of
novel compounds
can be assessed.
CA 02795753 2012-11-15
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Galectin inhibitors, in particular Gal-3 inhibitors have been described by the
some of
the present inventors in earlier published patent applications. None of these
galectin inhibi-
tors have been tested in a bleomycin model. Some of the prior art galectin
inhibitors have
the following general formulas
HO OH
R". R' X 10 0 Rix
Rvo Rv
oRvit
Rffi
as described in WO/2005/113568,
and
R11--Y HO oF1
\
NN õ
'N HO
as described in WO/2005/113569, in which RI can be a D-galactose,
and
OH 01-1
X
OH
as described in WO/2010/126435.
Furthermore there is a big need for bio-markers for making it less complicated
to
perform clinical trials in patients with pulmonary fibrosis. No biomarkers
exist that are suit-
able for detection of patients with pulmonary fibrosis or specifically
idiopathic pulmonary
fibrosis. Similarly, no biomarkers are suitable for prediction of the
prognosis for patients
with pulmonary fibrosis, for identification of patients with mild or
aggressive forms of the
disease, for identification of patients with ongoing or preeminent
exacerbations, and for
tracking the development of the patient's disease level. This makes it very
complicated and
costly to perform clinical trials of novel treatments in these patients.
CA 02795753 2012-11-15
3
Summary of the Disclosure
Galectin-3 is a 13-galactoside binding lectin that is highly expressed in
fibrotic tissue
of diverse etiologies. The role of galectin-3 in bleomycin and TGF-131-induced
lung fibro-
sis in mice is examined, and its relevance in human IPF is established.
Studies with galec-
tin-3 are described in MacKinnon et al., "Regulation of TGF-131 driven lung
fibrosis by ga-
lectin-3", Am. J. Respir. Crit. Care Med. 185: 537-546 (2012, originally
available online on
November 17, 2011). In particular, it is shown that galectin-3 inhibition may
represent a
novel therapeutic strategy for treatment of lung fibrosis. A novel compound
has been tested
and shown to be an inhibitor of galectin-3, in particular, this compound
blocked TGF-(3-
induced 13-catenin activation in vitro and attenuated the late stage
progression of lung fibro-
sis following bleomycin in vivo.
Accordingly, provided is a compound of the general formula (I):
HO OH
OH
N
HO H
N
N
In a further aspect, provided is a composition, particularly, a pharmaceutical
compo-
sition comprising the compound of formula (I) and optionally a
pharmaceutically acceptable
additive, such as carrier or excipient.
The compound of formula (I) is suitable for use in a method for treating
pulmonary
fibrosis, such as Idiopathic pulmonary fibrosis in a mammal. Typically, such
mammal is a
human subject.
Moreover, in a still further aspect provided is a method for treatment of
pulmonary
fibrosis, such as Idiopathic pulmonary fibrosis comprising administering to a
mammal in
need thereof a therapeutically effective amount of the compound of formula
(I).
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4
In another aspect, provided is a process of preparing a compound of formula I
com-
prising the step of reacting bis-(3-deoxy-3-azido-3-D-ga1actopyranosy1)
sulfane with 3-
fluorophenylacetylene and an amine, such as triethylamine, optionally in the
presence of a
catalyst, such as Cu(I), in a solvent, such as N,N-dimethylformamide (DMF),
resulting in
the compound of formula I.
Moreover, the present inventors have discovered that concentrations of the
human
protein galectin-3 in body fluids or tissues of a human subject can be used to
for instance
predict or monitor disease progression or therapeutic efficacy in human
subjects with pul-
monary fibrosis.
Measurement of galectin-3 levels in lung tissue, broncho-alveolar lavage (BAL)
flu-
id, blood, serum or plasma can be used to identify patients with lung fibrosis
and to assess
the severity of the disease. Longitudinal studies of the levels of galectin-3
in lung tissue,
broncho-alveolar lavage fluid, blood, serum or plasma can be used to predict
exacerbations
and to follow the development of the disease. Hence, measurement of galectin-3
levels in
lung tissue, broncho-alveolar lavage fluid, blood, serum or plasma before,
during and after
completion of a treatment for pulmonary fibrosis can be used to assess the
effect of such
treatment.
The results of the present inventors demonstrate that serum galectin-3 levels
may
help distinguish patients with usual interstitial pneumonia (UIP) from
patients with non-
specific interstitial pneumonia (NS1P) and identify patients undergoing an
acute exacerba-
tion.
In a further aspect the present invention relates to a method of diagnosing
pulmo-
nary fibrosis in a human subject comprising a) measuring a galectin-3 level
(e.g. concentra-
tion) in a body sample from the human subject using a suitable test method, b)
comparing
the galectin-3 level to a predetermined reference level, and c) determining
whether the ga-
lectin-3 level is indicative of diagnosing the subject with pulmonary
fibrosis.
In a still further aspect the present invention relates to a method of
predicting the
prognosis pulmonary fibrosis in a human subject comprising a) measuring a
galectin-3 level
(e.g. concentration) in a body sample from the human subject using a suitable
test method,
and b) determining whether the galectin-3 level is indicative of a poor
prognosis or not for
the human subject.
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In a further aspect the present invention relates to a method of monitoring
develop-
ment or progression of pulmonary fibrosis in a human subject, comprising a)
measuring a
galectin-3 level in a body sample from the subject at least two times with
sufficient inter-
val(s) to measure a clinically relevant change, b) comparing the galectin-3
level to a prede-
5 termined reference level, and repeating steps a) and b) one or more times
to monitor the
development or progression of pulmonary fibrosis in the human subject.
In a still further aspect the present invention relates to a method of
monitoring or
predicting exacerbation of symptoms in a human subject with pulmonary fibrosis
compris-
ing a) measuring a galectin-3 level (e.g. concentration) in a body sample from
the human
subject using a suitable test method, b) comparing the galectin-3 level to a
predetermined
reference level, and c) determine the presence or absence of a galectin-3
level indicative of
the development or progression of exacerbation of symptoms..
In a further aspect the present invention relates to a method for treatment of
pulmo-
nary fibrosis, such as Idiopathic pulmonary fibrosis, in a human subject
having a galectin-3
level indicative of pulmonary fibrosis or exacerbation of symptoms comprising
administer-
ing to a human subject a therapeutically effective amount of a galectin-3
inhibitor.
Any one of the above methods can include the step of transmitting, displaying,
stor-
ing, or printing; or outputting to a user interface device, a computer
readable storage me-
dium, a local computer system or a remote computer system, information related
to the like-
lihood of developing pulmonary fibrosis in the subject or for characterization
of the degree
of severity of the pulmonary fibrosis in said subject.
Detailed Description
In a broad aspect, provided is a compound of the general formula (I):
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6
HO OH
NN OH
= N HO 196-197'7j
OH
N"
F
(I)
The compound of formula (I) has the chemical name (IUPAC) bis (3-deoxy-3-(3-
fluoropheny1-1H-1,2,3-triazol-1-y1)-p-D-galactopyranosyl) sulfane, and as used
herein is
intended to cover the compound of formula (I) in any possible form, such as
solid or liquid,
a salt, a solvate, or in free form.
Typically, the compound of formula (I) is bis (3-deoxy-3-(3-fluoropheny1-1H-
1,2,3-
triazol-1-y1)-0-D-galactopyranosy1) sulfane as the free form. In a further
embodiment the
compound of formula (I) is bis (3-deoxy-3-(3-fluoropheny1-1H-1,2,3-triazol-1-
y1)-3-D-
galactopyranosyl) sulfane as the free form without any solvate, such as
anhydrated.
In a still further embodiment, the compound of formula (I) is useful for
treating
pulmonary fibrosis, and therefore is suitable for use as a medicament.
In a further aspect, provided is a compound of formula (I) for use in a method
for
treating pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a
mammal. Such a
mammal is typically a human subject, preferably a human subject diagnosed with
IPF.
In a still further aspect, provided is a method for treatment of pulmonary
fibrosis,
such as Idiopathic pulmonary fibrosis comprising administering to a mammal a
therapeuti-
cally effective amount of a compound of formula (I).
When the compounds and pharmaceutical compositions herein disclosed are used
for the above treatment, a therapeutically effective amount of at least one
compound is ad-
ministered to a mammal in need of said treatment.
The term "treatment- and -treating" as used herein means the management and
care
of a patient for the purpose of combating a condition, such as a disease or a
disorder. The
term is intended to include the full spectrum of treatments for a given
condition from which
CA 02795753 2012-11-15
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the patient is suffering, such as administration of the active compound to
alleviate the symp-
toms or complications, to delay the progression of the disease, disorder or
condition, to al-
leviate or relief the symptoms and complications, and/or to cure or eliminate
the disease,
disorder or condition as well as to prevent the condition, wherein prevention
is to be under-
stood as the management and care of a patient for the purpose of combating the
disease,
condition, or disorder and includes the administration of the active compounds
to prevent
the onset of the symptoms or complications. The treatment may either be
performed in an
acute or in a chronic way. The patient to be treated is preferably a mammal;
in particular a
human being, but it may also include animals, such as dogs, cats, cows, sheep
and pigs.
The term "a therapeutically effective amount" of a compound of formula (I) of
the
present invention as used herein means an amount sufficient to cure, alleviate
or partially
arrest the clinical manifestations of a given disease and its complications.
An amount ade-
quate to accomplish this is defined as "therapeutically effective amount".
Effective amounts
for each purpose will depend on the severity of the disease or injury as well
as the weight
and general state of the subject. It will be understood that determining an
appropriate dos-
age may be achieved using routine experimentation, by constructing a matrix of
values and
testing different points in the matrix, which is all within the ordinary
skills of a trained phy-
sician or veterinary.
In a still further aspect the present invention relates to a pharmaceutical
composition
comprising the compound of formula (I) and optionally a pharmaceutically
acceptable addi-
tive, such as a carrier or an excipient.
As used herein -pharmaceutically acceptable additive- is intended without
limitation
to include carriers, excipients, diluents, adjuvant, colorings, aroma,
preservatives etc. that
the skilled person would consider using when formulating a compound of the
present inven-
tion in order to make a pharmaceutical composition.
The adjuvants, diluents, excipients and/or carriers that may be used in the
composi-
tion of the invention must be pharmaceutically acceptable in the sense of
being compatible
with the compound of formula (I) and the other ingredients of the
pharmaceutical composi-
tion, and not deleterious to the recipient thereof. It is preferred that the
compositions shall
not contain any material that may cause an adverse reaction, such as an
allergic reaction.
The adjuvants, diluents, excipients and carriers that may be used in the
pharmaceutical
composition of the invention are well known to a person within the art.
CA 02795753 2012-11-15
8
As mentioned above, the compositions and particularly pharmaceutical composi-
tions as herein disclosed may, in addition to the compounds herein disclosed,
further com-
prise at least one pharmaceutically acceptable adjuvant, diluent, excipient
and/or carrier. In
some embodiments, the pharmaceutical compositions comprise from 1 to 99 weight
% of
said at least one pharmaceutically acceptable adjuvant, diluent, excipient
and/or carrier and
from 1 to 99 weight % of a compound as herein disclosed. The combined amount
of the
active ingredient and of the pharmaceutically acceptable adjuvant, diluent,
excipient and/or
carrier may not constitute more than 100% by weight of the composition,
particularly the
pharmaceutical composition.
In some embodiments, only one compound as herein disclosed is used for the pur-
poses discussed above.
In some embodiments, two or more of the compound as herein disclosed are used
in
combination for the purposes discussed above.
The composition, particularly pharmaceutical composition comprising a compound
set forth herein may be adapted for oral, intravenous, topical,
intraperitoneal, nasal, buccal,
sublingual, or subcutaneous administration, or for administration via the
respiratory tract in
the form of, for example, an aerosol or an air-suspended fine powder.
Therefore, the phar-
maceutical composition may be in the form of, for example, tablets, capsules,
powders, na-
noparticles, crystals, amorphous substances, solutions, transdermal patches or
suppositories.
Thus, in a still further aspect provided is a composition, particularly a
pharmaceuti-
cal composition for intrapulmonary administration. Typically, such composition
is delivered
by a nebulizer or inhaler, preferably a nebulizer.
The following characteristics are required for the delivery device: It should
be able
to provide a specific dose accurately and repeatedly. It should be able to
provide 2 or more
different dose levels, for instance through repeated dosing or by adjusting
the dose provide
to the patient. The device should ensure that the drug is delivered to the
bronchiolar space
or preferably to the bronchiolar and the alveolar space of the lung preferably
uniformly over
the lung tissue. Hence, the device should generate aerosols or dry powder of
an adequately
small size to ensure this delivery, while not delivering particles so small
that they are im-
mediately exhaled and thus not remaining in the lung tissue.
Inhalation nebulizers deliver therapeutically effective amounts of
pharmaceuticals
by forming an aerosol which includes particles of a size that can easily be
inhaled. The aer-
CA 02795753 2012-11-15
9
osol can be used, for example, by a patient within the bounds of an inhalation
therapy,
whereby the therapeutically effective pharmaceutical or drug reaches the
patient's respira-
tory tract upon inhalation.
A variety of inhalation nebulizers are known. EP 0 170 715 Al uses a
compressed
gas flow to form an aerosol. A nozzle is arranged as an aerosol generator in
an atomizer
chamber of the inhalation nebulizer and has two suction ducts arranged
adjacent a com-
pressed-gas channel. When compressed air flows through the compressed-gas
channel, the
liquid to be nebulized is drawn in through the suction ducts from a liquid
storage container.
EP 0 432 992 A discloses a nebulizer comprising an aerosol generator having a
liq-
uid storage container, a perforate membrane and a vibrator. The vibrator is
operable to vi-
brate the membrane such that it dispenses an aerosol from a liquid through
holes provided
in the membrane.
US Patent No. 5,918,593 relates to ultrasonic nebulizers generating an aerosol
by
interaction between an amount of liquid and a piezo electric element. Droplets
of various
sizes are expelled from a surface of a liquid bulk when vibrational energy is
transferred
from the piezo element to the liquid. The droplets thus generated are filtered
in an atomizer
chamber since oversized droplets have to be removed from the droplets expelled
from the
surface in order to generate an aerosol for inhalation by a patient. This
nebulizer is repre-
sentative of continuously operating inhalation nebulizers, in which the
aerosol generator
produces an aerosol not only during inhalation but also while the patient
exhales. The aero-
sol produced by the aerosol generator is actually inhaled by the patient only
in the inhala-
tion phase, while any aerosol produced at other times is lost.
Dry powder inhalers, such as metered dose medicament inhalers are well known
for
dispensing medicament to the lungs of a patient. Some previous inhalers have
comprised a
pressurized aerosol dispensing container, wherein the aerosols contain gas
propellants in
which the powdered medicament is suspended. Upon actuation, the aerosol
contents are
expelled, through a metering valve, and into the lungs of the patient.
Several types of non-aerosol, breath actuated dry powder inhalers have
therefore
been provided. For example, U.S. Patent No. 5,503,144 to Baconõ shows a breath-
actuated
dry-powder inhaler. The device includes a dry powder reservoir for containing
a dry pow-
dered medicament, a metering chamber for removal of the powdered medicament
from the
CA 02795753 2012-11-15
reservoir in discrete amounts, and an air inlet for entraining the removed
powdered me-
dicament through a mouthpiece upon patient inhalation.
U.S. Patent No. 5,458,135 discloses a method and apparatus for producing an
aero-
solized dose of a medicament for subsequent inhalation by a patient. The
method comprises
5 first dispersing a preselected amount of the medicament in a
predetermined volume of gas,
usually air. The dispersion may be formed from a liquid or a dry powder. The
method relies
on flowing substantially the entire aerosolized dose into a chamber that is
initially filled
with air and open through a mouthpiece to the ambient. After the aerosolized
medicament
has been transferred to the chamber, the patient will inhale the entire dose
in a single breath.
10 US 6,065,472 discloses a powder inhalation device comprising a housing
containing
a pharmacologically active compound, a conduit with an outlet extending into
the housing
through which a user can inhale to create an airflow through the conduit, a
dosing unit for
delivering a dose of the compound to the conduit and baffles arranged within
the said con-
duit to aid disintegration of powder agglomerates entrained in said airflow.
Regardless of whether an aerosol or non-aerosol inhaler is used, it is of
utmost im-
portance that particles of the dispensed dry powder medicament be small enough
to ensure
the adequate penetration of the medicament into the bronchial region of a
patient's lungs
during inhalation. However, because the dry powder medicament is composed of
very small
particles, and often provided in a composition including a carrier such as
lactose, non-
defined agglomerates or aggregates of the medicament form at random prior to
being dis-
pensed. It has therefore been found preferably to provide breath-actuated dry
powder inhal-
ers with means for breaking down the agglomerates of medicament or medicament
and car-
rier before inhalation of the medicament.
The composition and particularly pharmaceutical composition may optionally com-
prise two or more compounds of the present invention. The composition may also
be used
together with other medicaments within the art for the treatment of related
disorders.
The typical dosages of the compounds set forth herein vary within a wide range
and
depend on many factors, such as the route of administration, the requirement
of the individ-
ual in need of treatment, the individual's body weight, age and general
condition.
The compound of formula (I) may be prepared as described in the experimental
sec-
tion below.
Accordingly, provided is a process of preparing a compound of formula I
compris-
ing the step of reacting bis-(3-deoxy-3-azido-3-D-galactopyranosyl) sulfane
with 3-
CA 02795753 2012-11-15
11
fluorophenylacetylene and an amine, such as triethylamine, optionally in the
presence of a
catalyst, such as Cu(I), in a solvent, such as N,N-dimethylformamide (DMF),
resulting in
the compound of formula I. In a particular embodiment, provided is a process
of preparing a
compound of formula I comprising the steps as described in the scheme 1 in the
experimen-
tal section. Moreover, the present invention relates to a compound of formula
(I) obtainable
by the step of reacting bis-(3-deoxy-3-azido-3-D-ga1actopyranosy1) sulfane
with 3-
fluorophenylacetylene and an amine, such as triethylamine, optionally in the
presence of a
catalyst, such as Cu(I), in a solvent, such as N,N-dimethylformamide (DMF),
resulting in
the compound of formula I, such as obtainable by the steps as described in the
scheme 1 in
the experimental section.
The present invention also relates to a method of diagnosing pulmonary
fibrosis in a
human subject comprising a) measuring a galectin-3 level in a body sample from
the human
subject using a suitable test method, b) comparing the galectin-3 level to a
predetermined
reference level, and c) determining whether the galectin-3 level is indicative
of diagnosing
the subject with pulmonary fibrosis. Such galectin-3 level is typically the
galectin-3 concen-
tration measured in ng/ml in a body sample such as body fluid, e.g. blood,
plasma, or se-
rum.
The term "a predetermined reference level- as used herein means a galectin-3
level
which is determined through analysis of a large group of human subject which
are not suf-
fering from pulmonary fibrosis. Such determination of the predetermined
reference level
have been investigated in several publications, such as US20120220671 and
MacKinnon et
al., -Regulation of TGF-131 driven lung fibrosis by galectin-3-, Am. J.
Respir. Crit. Care
Med. 185: 537-546, Journal of the American College of Cardiology Vol. xx, No.
x. 2012,
Ho et al., title: "Galectin-3, a Marker of Cardiac Fibrosis, Predicts Incident
Heart Failure in
the Community" and Clin Res Cardiol (2010) 99:323-328, Lok et al., title:
"Prognostic val-
ue of galectin-3, a novel marker of fibrosis, in patients with chronic heart
failure: data from
the DEAL-HF study-. Base on the studies the levels have been determined to be
within a
concentration range from about 10.0 ng/mL to about 25.0 ng/mL galectin-3. In
some popu-
lations said range may be from about 13.0 ng/mL to about 19.2 ng/mL galectin-
3.
The indicative level is the level of galectin-3, which to the person skilled
in the art,
such as a physician, provides such person with a tool to set a diagnosis.
Typically, the in-
dicative level of galectin-3 is at least 22 ng/ml. In a further embodiment the
indicative level
CA 02795753 2012-11-15
12
of galectin-3 is at least 25 ng/ml, such as at least 30 ng/ml, at least 40
ng/ml, at least 50
ng/ml, at least 60 ng/ml, at least 70 ng/ml.
Furthermore, the present invention relates to a method of predicting a
prognosis of
pulmonary fibrosis in a human subject comprising a) measuring a galectin-3
level (e.g. con-
centration) in a body sample from the human subject using a suitable test
method, and b)
determining whether the galectin-3 level is indicative of a poor prognosis or
not for the hu-
man subject.
The indicative level is the level of galectin-3, which to the person skilled
in the art,
such as a physician, provides such person with a tool to predict the prognosis
of the subject.
Typically, the indicative level of galectin-3 is at least 22 ng/ml. In a
further embodiment the
indicative level of galectin-3 is at least 25 ng/ml, such as at least 30
ng/ml, at least 40
ng/ml, at least 50 ng/ml, at least 60 ng/ml, at least 70 ng/ml.
As the tracking of the development of the human subject's disease level is
desired, it
is of great importance to develop a method of monitoring the development, such
as im-
provement or deterioration, or progression of pulmonary fibrosis, e.g. IPF. It
is generally
very complicated and costly to perform clinical trials of novel treatments in
these patients.
Accordingly, the present invention relates to a method of monitoring
development or
progression of pulmonary fibrosis in a human subject, comprising a) measuring
a galectin-3
level in a body sample from the subject at least two times with sufficient
interval(s) to
measure a clinically relevant change, b) comparing the galectin-3 level to a
predetermined
reference level, and repeating steps a) and b) one or more times to monitor
the development
or progression of pulmonary fibrosis in the human subject.
Whether a change is clinically relevant will be determined by a person skilled
in the
art, in particular a physician. The time period between two measurements (that
is the suffi-
cient interval) is independently selected from 2 weeks to 2 years. In
individual embodi-
ments such time period between two measurements is selected from 2 weeks, 4
weeks, 1
month, 2 months, 3 months 6 months, I year, or 2 years.
When the indicative level of galectin-3 is below 22 ng/ml treatment of
pulmonary
fibrosis may be stopped, adjusted or put on hold. This is typically determined
by the physi-
cian.
When the indicative level of galectin-3 is at least 22 ng/ml treatment of
pulmonary
fibrosis may be initiated or increased. This is typically determined by the
physician. In fur-
CA 02795753 2012-11-15
13
ther embodiments the galectin-3 level is at least 25 ng/ml, such as at least
30 ng/ml, at least
40 ng/ml, at least 50 ng/ml, at least 60 ng/ml, or at least 70 ng/ml. In
another embodiment
the subject has a galectin-3 blood concentration determined to be within a
target range. Typ-
ically, such target range is from 10 ng/ml to 70 ng/ml. In a selected or
treated human sub-
ject, the blood concentration of galectin-3 may be determined to be above a
minimum
threshold, below a maximum threshold or within a target range defined by a
minimum and a
maximum threshold. The minimum threshold may be, for example, more than 10
ng/ml;
between 10 and 15 ng/ml; between 15 and 20 ng/ml; between 20 and 25 ng/ml;
between 25
and 30 ng/ml; or be more than 30 ng/ml. The maximum threshold may be, for
example,
below 70 ng/ml; below 60 ng/ml; below 40 ng/ml; between 30 and 40 ng/ml;
between 25
and 30 ng/ml; between 20 and 25 ng/ml; or between 15 and 20 ng/ml.
In a further aspect the present invention relates to a method of monitoring or
predict-
ing exacerbation of symptoms in a human subject with pulmonary fibrosis
comprising a)
measuring a galectin-3 level (e.g. concentration) in a body sample from the
human subject
using a suitable test method, b) comparing the galectin-3 level to a
predetermined reference
level, b) determine the presence or absence of a galectin-3 level indicative
of the develop-
ment or progression of exacerbation of symptoms, and if deemed necessary c)
repeating
steps a) and b) to monitor or predict the development or progression of the
exacerbation of
symptoms in the human subject.
When the indicative level of galectin-3 is at least 22 ng/ml treatment of
pulmonary
fibrosis may be initiated or increased or it may be decided to monitor the
patient more
closely to counter the effect of the ongoing or eminent exacerbation, if
possible. This is typ-
ically determined by the physician. In further embodiments the galectin-3
level is at least 25
ng/ml, such as at least 30 ng/ml, at least 40 ng/ml, at least 50 ng/ml, at
least 60 ng/ml, or at
least 70 ng/ml. In another embodiment the subject has a galectin-3 blood
concentration de-
termined to be within a target range. Typically, such target range is from 10
ng/ml to 70
ng/ml. In a selected or treated human subject, the blood concentration of
galectin-3 may be
determined to be above a minimum threshold, below a maximum threshold or
within a tar-
get range defined by a minimum and a maximum threshold. The minimum threshold
may
be, for example, more than 10 ng/ml; between 10 and 15 ng/ml; between 15 and
20 ng/ml;
between 20 and 25 ng/ml; between 25 and 30 ng/ml; or be more than 30 ng/ml.
The maxi-
mum threshold may be, for example, below 70 ng/ml; below 60 ng/ml; below 40
ng/ml;
CA 02795753 2012-11-15
14
between 30 and 40 ng/ml; between 25 and 30 ng/ml; between 20 and 25 ng/ml; or
between
15 and 20 ng/ml.
As it is desired to prevent or reduce exacerbation of symptoms such
prophylactic
treatment should be initiated in good time before the level of galectin-3
reaches 70 ng/ml,
thus it is preferred to initiate or increase prophylactic treatment of
exacerbation of symp-
toms at a galectin level of least 50 ng/ml, such as at least 60 ng/ml, e.g. at
least 70 ng/ml.
Typically, the pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF).
In a further embodiment the human subject is diagnosed with mild, moderate or
ag-
gressive forms of pulmonary fibrosis according to the level of galectin-3.
Multimarker analysis can be used to improve the accuracy of diagnosis and
monitor-
ing. Expression of markers, such as MMP7 and perDLCO has been linked with
pulmonary
fibrosis (Am J Respir Crit Care Med 185;2012:A6241) When measuring the levels
of the
above markers, corrections for age, gender and concomitant morbidity may be
incorporated
to improve the accuracy of diagnosis.
Thus, in a further embodiment in step a) further bio-markers are measured
which
markers are relevant for pulmonary fibrosis, including markers linked to
Galectin-3 levels,
leading to a more accurate diagnosis, prognosis, and/or monitoring. Typically,
such bio-
markers are selected from MMP7, perDLCO, KL-6, SP-A, MMP-7, CCL-18, IL13, CC-
chemokines, IL 10, IL 1 receptor antagonist, CCL2, Calgranulin B (S100A9 or
MRP14),
macrophage migration inhibitory factor (MIF), pro-collagen, or pro-collagen 3
or the pres-
ence and frequency of certain cell types in the body sample, such as
fibrocytes and T-cell
subpopulations.
The term -a body sample" as used herein means a sample obtained and isolated
from
a human subject. The body sample may be obtained by various known means, such
as by
biopsy tools, such as a needle biopsy tool or a bronchoscope, or by using a
syringe.
In a further embodiment the body sample is selected from blood, serum, plasma,
broncho-
alveolar lavage fluid, and lung tissue.
As explained below several suitable test methods exists and such test methods
are
typically selected from an immunoassay, an immunohistochemical assay, a
colorimetric
assay, a turbidimetric assay, and flow cytometry.
In a further aspect the present invention relates to a method for treatment of
pulmo-
nary fibrosis, such as Idiopathic pulmonary fibrosis in a human subject having
a galectin-3
CA 02795753 2012-11-15
level indicative of pulmonary fibrosis or exacerbation of symptoms comprising
administer-
ing to a human subject a therapeutically effective amount of a galectin-3
inhibitor. In a par-
ticular embodiment the galectin-3 inhibitor is selected from the compound of
formula (I).
In an embodiment the indicative level of galectin-3 is at least 22 ng/ml, such
as at
5 least 25 ng/ml, such as at least 30 ng/ml, at least 40 ng/ml, at least 50
ng/ml, at least 60
ng/ml, at least 70 ng/ml.
In a further embodiment an additional step of monitoring the subject's
galectin-3
blood level after the therapy is initiated. In particular such monitoring is
made in accor-
dance with the invention as described herein.
10 The present invention provides methods for identification and evaluation
of patients
with pulmonary fibrosis by measuring the levels of markers such as galectin-3,
optionally in
combination with one or more other markers. Many methods for detecting of a
protein of
interest, with or without quantitation, are well known and can be used in the
practice of the
present invention. Such test methods are termed -a suitable test method-
herein and several
15 useful methods of testing are described below.
Examples of such assays are described below and can include, for example, immu-
noassays, chromatographic methods, and mass spectroscopy. Such assays can be
performed
on any biological sample including, among others, blood, plasma, and serum.
Accordingly,
multiple assays can be used to detect galectin-3, and samples can be analyzed
from one or
more sources.
Markers can be detected or quantified in a sample with the help of one or more
sepa-
ration methods. For example, suitable separation methods may include a mass
spectrometry
method, such as electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS,
ESI-
MS/(MS)n (n is an integer greater than zero), matrix-assisted laser desorption
ionization
time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorp-
tion/ionization time-of-flight mass spectrometry (SELDI-TOF-MS),
desorption/ionization
on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-
flight (Q-
TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS),
APCI-
MS/MS, APCI-(MS) or atmospheric pressure photoionization mass spectrometry
(APPI-
MS), APPI-MS/MS, and APPI-(MS) Other mass spectrometry methods may include,
inter
alia, quadrupole, fourier transform mass spectrometry (FTMS) and ion trap.
Spectrometric
techniques that can also be used include resonance spectroscopy and optical
spectroscopy.
CA 02795753 2012-11-15
16
Other suitable separation methods include chemical extraction partitioning,
column
chromatography, ion exchange chromatography, hydrophobic (reverse phase)
liquid chro-
matography, isoelectric focusing, one-dimensional polyacrylamide gel
electrophoresis
(PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), or other
chro-
matographic techniques, such as thin-layer, gas or liquid chromatography, or
any combina-
tion thereof. In one embodiment, the biological sample to be assayed may be
fractionated
prior to application of the separation method.
Markers can may be detected or quantified by methods that do not require
physical
separation of the markers themselves. For example, nuclear magnetic resonance
(NMR)
spectroscopy may be used to resolve a profile of a marker from a complex
mixture of mole-
cules.
A marker in a sample also may be detected or quantified, for example, by
combining
the marker with a binding moiety capable of specifically binding the marker.
The binding
moiety may include, for example, a member of a ligand-receptor pair, i.e., a
pair of mole-
cules capable of having a specific binding interaction. The binding moiety may
also include,
for example, a member of a specific binding pair, such as antibody-antigen,
enzyme-
substrate, nucleic acid-nucleic acid, protein-nucleic acid, protein-protein,
or other specific
binding pairs known in the art. Binding proteins may be designed which have
enhanced
affinity for a target. Optionally, the binding moiety may be linked with a
detectable label,
such as an enzymatic, fluorescent, radioactive, phosphorescent or colored
particle label. The
labeled complex may be detected, e.g., visually or with the aid of a
spectrophotometer or
other detector, or may be quantified.
Galectin-3 levels can be quantitated by performing an immunoassay. A galectin-
3
immunoassay involves contacting a sample from a subject to be tested with an
appropriate
antibody under conditions such that immunospecific binding can occur if
galectin-3 is pre-
sent, and detecting or measuring the amount of any immunospecific binding by
the anti-
body. Any suitable immunoassay can be used, including, without limitation,
competitive
and non-competitive assay systems using techniques such as Western blots,
radioimmuno-
assays, immunohistochemistry, ELISA (enzyme linked immunosorbent assay),
"sandwich"
immunoassays, immunoprecipitation assays, immunodiffusion assays,
agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent immunoassays
and
protein A immunoassays.
CA 02795753 2012-11-15
17
In a "sandwich" assay, two molecules ("binding moieties") such as monoclonal
anti-
bodies that specifically bind to non-overlapping sites (epitopes) on galectin-
3 are used. Typ-
ically, one binding moiety is immobilized on a solid surface where it binds
with and cap-
tures galectin-3. This first binding moiety is therefore also referred to as
the capture binding
moiety. A second binding moiety is detectably labeled, for example, with a
fluorophore,
enzyme, or colored particle, such that binding of the second binding moiety to
the galectin-
3-complex indicates that galectin-3 has been captured. The intensity of the
signal is propor-
tional to the concentration of galectin-3 in the sample. The second binding
moiety is there-
fore also referred to as the detection binding moiety or label binding moiety.
A binding
moiety can be any type of molecule, as long as it specifically binds to a
portion of the N-
terminus of galectin-3. In a preferred embodiment, the binding moieties used
are mono-
clonal anti-galectin-3 antibodies, i.e., monoclonals raised against or
otherwise selected to
bind to separate portions of galectin-3.
Such assay procedures can be referred to as two-site immunometric assay
methods,
"sandwich" methods or (when antibodies are the binders) "sandwich
immunoassays." As is
known in the art, the capture and detection antibodies can be contacted with
the test sample
simultaneously or sequentially. Sequential methods can be accomplished by
incubating the
capture antibody with the sample, and adding the labeled detection antibody at
a predeter-
mined time thereafter (sometimes referred to as the "forward" method).
Alternatively, the
labeled detection antibody can be incubated with the sample first and then the
sample can
be exposed to the capture antibody (sometimes referred to as the "reverse"
method). After
any necessary incubation(s), which may be of short duration, to complete the
assay, the la-
bel is measured. Such assays may be implemented in many specific formats known
to those
of skill in the art, including through use of various high throughput clinical
laboratory ana-
lyzers or with a point of care or home testing device.
In one embodiment, a lateral flow device may be used in the sandwich format
wherein the presence of galectin-3 above a baseline sensitivity level in a
biological sample
will permit formation of a sandwich interaction upstream of or at the capture
zone in the
lateral flow assay. See, for example, U.S. Pat. No. 6,485,982. The capture
zone may contain
capture binding moieties such as antibody molecules, suitable for capturing
galectin-3, or
immobilized avidin or the like for capture of a biotinylated complex. See, for
example, U.S.
Pat. No. 6,319,676. The device may also incorporate a luminescent label
suitable for cap-
CA 02795753 2012-11-15
18
ture in the capture zone, the concentration of galectin-3 being proportional
to the intensity
of the signal at the capture site. Suitable labels include fluorescent labels
immobilized on
polystyrene microspheres. Colored particles also may be used.
Other assay formats that may be used in the methods of the invention include,
but
are not limited to, flow-through devices. See, for example, U.S. Pat. No.
4,632,901. In a
flow-through assay, one binding moiety (for example, an antibody) is
immobilized to a de-
fined area on a membrane surface. This membrane is then overlaid on an
absorbent layer
that acts as a reservoir to pump sample volume through the device. Following
immobiliza-
tion, the remaining protein-binding sites on the membrane are blocked to
minimize non-
specific interactions. In operation, a biological sample is added to the
membrane and filters
through the matrix, allowing any analyte specific to the antibody in the
sample to bind to
the immobilized antibody. In a second step, a labeled secondary antibody may
be added or
released that reacts with captured marker to complete the sandwich.
Alternatively, the sec-
ondary antibody can be mixed with the sample and added in a single step. If
galectin-3 is
present, a colored spot develops on the surface of the membrane.
The most common enzyme immunoassay is the "Enzyme-Linked Immunosorbent
Assay (ELISA)." ELISA is a technique for detecting and measuring the
concentration of an
antigen using a labeled (e.g., enzyme-linked) form of the antibody. There are
different
forms of ELISA, which are well known to those skilled in the art. Standard
ELISA tech-
niques are described in "Methods in Immunodiagnosis". 2nd Edition, Rose and
Bigazzi, eds.
John Wiley & Sons, 1980; Campbell et al., "Methods and Immunology", W. A.
Benjamin,
Inc., 1964; and Oellerich. M. (1984), J. Clin. Chem. Clin. Biochem. 22:895-
904. A pre-
ferred enzyme-linked immunosorbent assay kit (ELISA) for detecting galectin-3
is com-
mercially available (BG Medicine, Waltham, Mass.).
In a "sandwich ELISA," an antibody (e.g., anti-galectin-3) is linked to a
solid phase
(i.e., a microtiter plate) and exposed to a biological sample containing
antigen (e.g., galec-
tin-3). The solid phase is then washed to remove unbound antigen. A labeled
antibody (e.g.,
enzyme linked) is then bound to the bound-antigen (if present) forming an
antibody-
antigen-antibody sandwich. Examples of enzymes that can be linked to the
antibody are
alkaline phosphatase, horseradish peroxidase, luciferase, urease, and .beta.-
galactosidase.
The enzyme linked antibody reacts with a substrate to generate a colored
reaction product
that can be measured. Any of the immunoassays described herein suitable for
use with the
CA 02795753 2012-11-15
19
kits and methods of the present invention can also use any binding moiety in
the place of an
antibody.
A detailed review of immunological assay design, theory and protocols can be
found
in numerous texts in the art, including Butt, W. R., Practical Immunology, ed.
Marcel Dek-
ker, New York (1984) and Harlow et al. Antibodies, A Laboratory Approach, ed.
Cold
Spring Harbor Laboratory (1988).
In general, immunoassay design considerations include preparation of
antibodies
(e.g., monoclonal or polyclonal antibodies) having sufficiently high binding
specificity for
the target to form a complex that can be distinguished reliably from products
of nonspecific
interactions. As used herein, the term "antibody" is understood to mean
binding proteins, for
example, antibodies or other proteins comprising an immunoglobulin variable
region-like
binding domain, having the appropriate binding affinities and specificities
for the target.
The higher the antibody binding specificity, the lower the target
concentration that can be
detected. As used herein, the terms "specific binding" or "binding
specifically" are under-
stood to mean that the binding moiety, for example, a binding protein, has a
binding affinity
for the target of greater than about 105 M-1, more preferably greater than
about 107 Mi.
Antibodies to an isolated target marker which are useful in assays for
detecting heart
failure in an individual may be generated using standard immunological
procedures well
known and described in the art. See, for example Practical Immunology, supra.
Briefly, an
isolated marker is used to raise antibodies in a xenogeneic host, such as a
mouse, goat or
other suitable mammal. The marker is combined with a suitable adjuvant capable
of en-
hancing antibody production in the host, and is injected into the host, for
example, by in-
traperitoneal administration. Any adjuvant suitable for stimulating the host's
immune re-
sponse may be used. A commonly used adjuvant is Freund's complete adjuvant (an
emul-
sion comprising killed and dried microbial cells and available from, for
example, Calbio-
chem Corp., San Diego, or Gibco, Grand Island, N.Y.). Where multiple antigen
injections
are desired, the subsequent injections may comprise the antigen in combination
with an
incomplete adjuvant (e.g., cell-free emulsion). Polyclonal antibodies may be
isolated from
the antibody-producing host by extracting serum containing antibodies to the
protein of
interest. Monoclonal antibodies may be produced by isolating host cells that
produce the
desired antibody, fusing these cells with myeloma cells using standard
procedures known in
CA 02795753 2012-11-15
the immunology art, and screening for hybrid cells (hybridomas) that react
specifically with
the target and have the desired binding affinity.
Exemplary epitopes from the N-terminus of galectin-3 include, but are not
limited
to, MADNFSLHDALS (SEQ ID NO:1); MADNFSLHDALSGS (SEQ ID NO:2);
5 WGNQPAGAGG (SEQ ID NO:3); YPGAPGAYPGAPAPGV (SEQ ID NO:4);
GNPNPQGWPGA (SEQ ID NO:5); YPSSGQPSATGA (SEQ ID NO:6); YP-
GQAPPGAYPGQAPPGA (SEQ ID NO:7); YPGAPAPGVYPGPPSGPGA (SEQ ID
NO:8); and YPSSGQPSATGA (SEQ ID NO:9). Other galectin-3 epitopes, including
non-
linear epitopes, can also be used as targets for detection by an anti-galectin-
3 antibody. Ex-
10 emplary antibodies are discussed in U.S. Patent Publication No.
2010/014954, the entire
contents of which are incorporated herein by reference.
Antibody binding domains also may be produced biosynthetically and the amino
ac-
id sequence of the binding domain manipulated to enhance binding affinity with
a preferred
epitope on the target. Specific antibody methodologies are well understood and
described in
15 the literature. A more detailed description of their preparation can be
found, for example, in
Practical Immunology, (supra).
In addition, genetically engineered biosynthetic antibody binding sites, also
known
in the art as BABS or sFv's, may be used to determine if a sample contains a
marker. Meth-
ods for making and using BABS comprising (i) non-covalently associated or
disulfide
20 bonded synthetic VH and VL dimers, (ii) covalently linked
VH-VL sin-
gle chain binding sites, (iii) individual VH or VL domains, or (iv)
single chain
antibody binding sites are disclosed, for example, in U.S. Pat. Nos.
5,091,513; 5,132,405;
4,704,692; and 4,946,778. Furthermore, BABS having requisite specificity for
the marker
can be derived by phage antibody cloning from combinatorial gene libraries
(see, for exam-
ple, Clackson et al. Nature 352: 624-628 (1991)). Briefly, phages, each
expressing on their
coat surfaces BABS having immunoglobulin variable regions encoded by variable
region
gene sequences derived from mice pre-immunized with an isolated marker, or a
fragment
thereof, are screened for binding activity against the immobilized marker.
Phages which
bind to the immobilized marker are harvested and the gene encoding the BABS is
se-
quenced. The resulting nucleic acid sequences encoding the BABS of interest
then may be
expressed in conventional expression systems to produce the BABS protein.
CA 02795753 2012-11-15
21
Further embodiments of the process are described in the experimental section
here-
in, and each individual process as well as each starting material constitutes
embodiments
that may form part of claims.
The above embodiments should be seen as referring to any one of the aspects
(such as
'method for treatment', 'pharmaceutical composition', 'compound for use as a
medicament',
or 'compound for use in a method') described herein as well as any one of the
embodiments
described herein unless it is specified that an embodiment relates to a
certain aspect or aspects
of the present invention.
All references, including publications, patent applications and patents, cited
herein
are hereby incorporated by reference to the same extent as if each reference
was individu-
ally and specifically indicated to be incorporated by reference and was set
forth in its en-
tirety herein.
All headings and sub-headings are used herein for convenience only and should
not
be construed as limiting the invention in any way.
Any combination of the above-described elements in all possible variations
thereof
is encompassed by the invention unless otherwise indicated herein or otherwise
clearly con-
tradicted by context.
The terms -a- and -an" and -the- and similar referents as used in the context
of de-
scribing the invention are to be construed to cover both the singular and the
plural, unless
otherwise indicated herein or clearly contradicted by context.
Recitation of ranges of values herein are merely intended to serve as a
shorthand
method of referring individually to each separate value falling within the
range, unless oth-
er-wise indicated herein, and each separate value is incorporated into the
specification as if
it were individually recited herein. Unless otherwise stated, all exact values
provided herein
are representative of corresponding approximate values (e.g., all exact
exemplary values
provided with respect to a particular factor or measurement can be considered
to also pro-
vide a corresponding approximate measurement, modified by "about," where
appropriate).
All methods described herein can be performed in any suitable order unless
other-
wise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (e.g., -such as")
provided
herein, is intended merely to better illuminate the invention and does not
pose a limitation
on the scope of the invention unless otherwise indicated. No language in the
specification
CA 02795753 2012-11-15
22
should be construed as indicating any element is essential to the practice of
the invention
unless as much is explicitly stated.
The citation and incorporation of patent documents herein is done for
convenience
only and does not reflect any view of the validity, patentability and/or
enforceability of such
patent documents.
The description herein of any aspect or embodiment of the invention using
terms
such as -comprising", -having", -including" or -containing" with reference to
an element
or elements is intended to provide support for a similar aspect or embodiment
of the inven-
tion that "consists of", -consists essentially of', or "substantially
comprises" that particular
element or elements, unless otherwise stated or clearly contradicted by
context (e.g., a com-
position described herein as comprising a particular element should be
understood as also
describing a composition consisting of that element, unless otherwise stated
or clearly con-
tradicted by context).
This invention includes all modifications and equivalents of the subject
matter re-
cited in the aspects or claims presented herein to the maximum extent
permitted by applica-
ble law.
The present invention is further illustrated by the following examples that,
however,
are not to be construed as limiting the scope of protection. The features
disclosed in the
foregoing description and in the following examples may, both separately and
in any com-
bination thereof, be material for realizing the invention in diverse forms
thereof.
Experimental
Synthesis of bis (3-deoxy-3-(3-fluoropheny1-1H-1,2,3-triazol-1-y1)13-D-
galactopyranosyl) sulfane.
General Methods.
Melting points were recorded on a Kofler apparatus (Reichert) and are
uncorrected.
Proton nuclear magnetic resonance (1H) spectra were recorded on a Bruker DRX
400 (400
MHz) or a Bruker ARX 300 (300 MHz) spectrometer; multiplicities are quoted as
singlet
(s), doublet (d), doublet of doublets (dd), triplet (t), apparent triplet (at)
or apparent triplet of
doublets (atd). Carbon nuclear magnetic resonance (13C) spectra were recorded
on a Bruker
CA 02795753 2012-11-15
23
DRX 400 (100.6 MHz) spectrometer. Spectra were assigned using COSY, HMQC and
DEPT experiments. All chemical shifts are quoted on the d-scale in parts per
million (ppm).
Low- and high-resolution (FAB-HRMS) fast atom bombardment mass spectra were re-
corded using a JEOL SX-120 instrument and low- and high- resolution (ES-HRMS)
were
recorded on a Micromass Q-TOF instrument. Optical rotations were measured on a
Perkin-
Elmer 341 polarimeter with a path length of 1 dm; concentrations are given in
g per 100
mL. Thin layer chromatography (TLC) was carried out on Merck Kieselgel sheets,
pre-
coated with 60F254 silica. Plates were developed using 10% sulfuric acid.
Flash column
chromatography was carried out on silica (Matrex, 60A, 35-70gm, Grace Amicon).
Ace-
tonitrile was distilled from calcium hydride and stored over 4A molecular
sieves. DMF was
distilled from 4A molecular sieves and stored over 4A molecular sieves.
Bis (3-deoxy-3-(3-fluoropheny1-1H-1,2,3-triazol-1-y1)-fi-D-ga1actopyranosyl)
sulfane
(TD139) was prepared in accordance with the reaction scheme 1 below:
CA 027 95753 2012-11-15
24
Ph PhPh Ph
Bu4NP-NO2-
'Tf20 DMF
\O pyridine \'10 60% over 2 \C) Tf20 L\o
pyridine \
0 -20 C o steps when o
'' -20 C 04.____
HO--.& AcCI
Tf0--... ...\_--SPh )\......4.\,-SPh SPh
OH OAc OAc OAc
OH OTf
(1) (2) (3) (4)
Ph
Bu4N+N3-
DMF 0 AcOH HO OH Ac20
Ac0 OAc
59% over 0 (80%) 0 pyridine 0
2 steps 60 C
______),,
N3 SPh N3 SPh
N3--.10. ....\õ-SPh
OAc OAc
OAc (6) (7)
(5)
Br2
CH2C12 AcCi OAc
68% over MeCN
_), ..
3 steps Et3N Ac0 OAc
N3 50-60% Ac0
over 2 steps 0
Br
Ac0
(8) Ac0
OAc
Na_
Thiourea a0Me
11
MeCN
reflux
HO OH Me0H (10)
75% Ac
OH
Ac0 OAc
N3 S
HO
N3 S OH
OAc
./ Br
_¨ F NH2 (11) N3 OH
(9) H2N
, 3-fluorophenylacetylene
I Cul
+
fa 1 triethylamine
l'1tF
HO OH ''6
HO
N / N_--S
N HO
HO
F
/ NN
\ OH
TD139 * N
(Scheme 1)
Compound (1) (cf. reaction scheme above) is commercial from Carbosynth Limited
5 8 & 9 Old Station Business Park - Compton - Berkshire - RG20 6NE ¨ UK or
synthesized
CA 02795753 2012-11-15
in three near-quantitative steps from D-galactose, (cf e.g. Li, Z. and
Gildersleeve, J. C. J.
Am. Chem. Soc. 2006, 128, 11612-11619)
Phenyl 2-0-acety1-4,6-0-benzylidene-1-thio-3-0-trifluoromethanesulfonyl*D-
5 galactopyranoside (2)
Compound 1 (10.5 g, 29.2 mmol) was dissolved in dried pyridine (4.73 mL, 58.4
mmol) and dried CH2C12 (132 mL ). The reaction mixture was cooled, under
stirring, until -
20 C (Ice and NaC1 bath 3:1). Slowly and under N2 atmosphere, Tf20 (5.68 mL,
33.6
mmol) was added. The reaction mixture was monitored by TLC (heptane:Et0Ac, 1:1
and
10 toluene:acetone, 10:1). When the reaction was complete, AcC1 (2.29 mL,
32.1 mmol) was
added and keeping stirring, the temperature was increased to room temperature.
This mix-
ture was monitored by TLC too (heptane:Et0Ac, 1:1 and toluene:acetone, 10:1).
When it
was complete, it was quenched with CH2Cl2 and washed with 5 % HC1, NaHCO3
(saturated
¨ hereafter sat) and NaC1 (sat). The organic layer was dried over MgSO4,
filtered and con-
15 centrated under reduced pressure.
Phenyl 2-0-acetyl-4,6-0-benzyliden-1-thio*D-gulopyranoside (3)
Tetrabutylammonium nitrite (25.3 g, 87.7 mmol) was added to a solution of com-
pound 2 (15.6 g, 29.2 mmol) in DMF (110 mL ) and was kept stirring, under N2
atmos-
20 phere, at 50 C. (The reaction started being purple and turned garnet).
The reaction was
monitored by TLC (heptane:Et0Ac, 1:1 and toluene:acetone, 10:1) and quenched
with
CH2C12 . The mixture was washed with 5 % FIC1, NaHCO3 (sat) and NaC1 (sat).
The or-
ganic layer was dried over MgSO4, filtered and concentrated under reduced
pressure fol-
lowed by purification by flash chromatography (Eluent heptane:Et0Ac, 1:1 and
hep-
25 tane:Et0Ac, 1:2) and recrystallized from a mixture of Et0Ac and Heptane
(1:3). 1H NMR
in CDC13 6. 7.60-7.57 (m, 2H, Ar), 7.43-7.40 (m, 2H, Ar), 7.37-7.34 (m, 3H,
Ar), 7.29-7.25
(m, 3H, Ar), 5.50 (s, 1H, PhCH), 5.15 (d, 1H, J=10.29 Hz, H-1), 5.10 (dd, 1H,
J=10.27 Hz,
2.85 Hz, H-2), 4.36 (dd, I H, J= 12.49 Hz,1.4 Hz, H-6), 4.18 (br s, 1H, H-3),
4.08 (dd, 1H,
J= 3.59 Hz, 1.04 Hz, H-6), 4.03 (dd, 1H, J= 12.53 Hz, 1.75 Hz, H-4), 3.88 (s,
2H, H-5 +
OH), 2.12 ( s, 3H, OAc).
Phenyl 2-0-acetyl-4,6-0-benzylidene-1-thio-3-0-trifluoromethanesulfonyl-p-D-
CA 02795753 2012-11-15
26
gulopyranoside (4)
Compound 3 (1.00 g, 2.48 mmol) was dissolved in dried CH2C12 (12.5 mL) and
dried pyridine (0.40 mL, 4.96 mmol). The reaction mixture was cooled, under
stirring, until
-20 C (Ice and NaC1 bath 3:1). Slowly and under N2 atmosphere, Tf20 (0.48 mL,
2.85
mmol) was added. The reaction mixture was monitored by TLC (heptane:Et0Ac, 1:1
and
toluene:acetone, 10:1) and when it was complete, it was quenched with CH2C12
and washed
with 5 % HC1, NaHCO3 (sat) and NaC1 (sat). The organic layer was dried over
MgSO4, fil-
tered and concentrated under reduced pressure until being dry.
Phenyl 2-0-acety1-3-azido-4,6-0-benzylidene-3-deoxy-1-thio-p-D-
galactopyranoside
(5)
Tetrabutylammonium azide (2.12 g, 7.44 mmol) was added carefully to a solution
of
compound 4 (1.3256 g, 2.48 mmol) in DMF (10 mL ) and was kept stirring, under
N2 at-
mosphere, at 50 C. The reaction was monitored by TLC (E:H, 1:1) and
concentrated under
reduced pressure followed by purification by flash chromatography (Eluent
heptane:Et0Ac,
2:1 and heptane:Et0Ac, 1:1). 1H NMR in CDC13 6 7.61-7.58 (m, 2H, Ar), 7.44-
7.41 (m, 2H,
Ar), 7.39-7.36 (m, 3H, Ar), 7.30-7.24 (m, 3H, Ar), 5.59 (s, 1H, PhCH), 5.35
(t, 1H, J= 9.95
Hz, H-2), 4.73 (d, 1H, J= 9.63 Hz, H-1), 4.44 (dd, 1H, .1= 6.24 Hz, 1.60 Hz, H-
6), 4.35-4.34
(dd, 1H, J= 3.33 Hz, 0.88 Hz, H-4), 4.11 (dd, 1H, J= 12.48 Hz, 1.67 Hz, H-6),
3.57 (d, 1H,
J= 1.15 Hz, H-5), 3.44 (dd, 1H, J= 10.21 Hz, 3.29 Hz, H-3), 2.17 (s, 3H, OAc).
Phenyl 2-0-acetyl-3-azido-3-deoxy-l-thio-P-D-galactopyranoside (6)
Compound 5 (470 mg, 1.1 mmol) was dissolved in 80% acetic acid (75 mL ) and
the
mixture was heated at 60 C. The reaction was monitored by TLC (heptane:Et0Ac,
1:1).
When the reaction was complete, the mixture was concentrated under reduced
pressure and
heating.
Phenyl 2,4,6-tri-O-acetyl-3-azido-3-deoxy-l-thio-13-D-galactopyranoside (7)
Acetic anhydride (30 mL) was added to a solution of compound 6 (373 mg,
1.1mmol) in dry pyridine (30 mL). The reaction was monitored by TLC
(heptane:Et0Ac,
1:1) and when it was complete, it was concentrated under reduced pressure. 1H
NMR in
CDC13 6 7.54-7.51 (m, 2H, Ar), 7.35-7.30 (m, 3H, Ar), 5.46 (dd, 1H, H-4), 5.23
(t, 1H, H-
CA 02795753 2012-11-15
27
2), 4.73 (d, 1H, H-1), 4.15 (d, 2H, H-6, H-6), 3.94 (dt, I H, H-5), 3.68 (dd,
1H, H-3), 2.18 (s,
3H, OAc), 2.15 (s, 3H, OAc), 2.06 (s, 3H, OAc).
2,4,6-tri-0-acetyl-3-azido-3-deoxy-a-D-galactopyranosyl bromide (8)
Compound 7 (237.4 mg, 560 gmol) was dissolved in dry CH2Cl2 (2 mL), and bro-
mine (32 I, 620 mot) was added. The reaction was monitored by TLC
(heptane:Et0Ac,
1:1). When the reaction was complete, a small amount of cyclopentene was added
to the
reaction mixture to remove the rests of Br2. The mixture was concentrated
under reduced
pressure and purified by quick Flash chromatography (Eluent: 500mL
heptane:Et0Ac,
2:1).
2,4,6-tri-O-acety1-3-azido-3-deoxy-a-D-galactopyranose-1-isothiouronium
bromide (9)
The sensitive bromide 8 (70.6 mg, 180 mop was immediately dissolved in dry
ace-
tonitrile (1.7 mL) and refluxed with thiourea (13.7 mg, 180 mop under N2 for
4 hours. The
reaction was monitored by TLC (heptane:Et0Ac, 1:1) and when it was complete,
the mix-
ture was cooled.
Bis-(2,4,6-tri-0-acety1-3-azido-3-deoxy-b-D-galactopyranosyl)-sulfane (10)
The sensitive bromide 8 (77.0 mg, 196 mop and Et3N (60 1, 430 mot) was
added
to the last mixture (9). The reaction was monitored by TLC (heptane:Et0Ac,
1:1). When it
was complete, the reaction mixture was concentrated under reduced pressure and
without
heating. The residue was purified by flash chromatography (Eluent:
heptane:Et0Ac, 1:1).
H NMR in CDCI3 6 5.50 (dd, 2H, H-4,), 5.23 (t, 2H, H-2, H-2'), 4.83 (d, 2H, H-
1, H-1'),
4.15 (dd, 4H, H-6, H-6, H-6', H-6'), 3.89 (dt, 2H, H-5, H-5'), 3.70 (dd, 2H, H-
3, H-3'), 2.19
(s, 6H, 20Ac), 2.15 (s, 6H, 20Ac), 2.18 (s, 6H, 20Ac).
Bis-(3-azido-3-deoxy-p-D-galactopyranosyl)-su1fane (11)
Compound 10 (160 mg, 0.00024 mol) was dissolved in dry Me0H (2.6 mL) and dry
CH2C12 (1.6 mL), and Na0Me (1M, 24 jtL, 24 mol) was added. The reaction was
moni-
tored by TLC (heptane:Et0Ac 1:1 and D:M 5:1). When the reaction was complete,
the mix-
ture was neutralized with Duolite C436 until pH 7, filtered and washed with
Me0H. The
filtered solution was concentrated under reduced pressure. The residue was
purified by flash
CA 02795753 2012-11-15
28
chromatography (Eluent: CH2C12:Me0H, 5:1) to give pure 11 (74.1 mg, 75%). 1H
NMR in
CDC13 6 4.72 (d, 2H, J=9.7 Hz, H-1, H-1'), 3.95 (br s, 2H, H-4, H-4'), 3.84
(t, 2H, J= 9.8
Hz, H-2, H-2'), 3.74 (dd, 2H, J= 11.47 Hz, 7.23 Hz, H-6, H-6'), 3.64 (dd, 2H,
J= 11.48 Hz,
4.72 Hz, H-6, H-6'), 3.60-3.55 (ddd, 2H, 7.15 Hz, 4.67 Hz, 0.93 Hz, H-5, H-
5'), 3.36 (dd,
2H, J= 10 Hz, 3.05 Hz, H-3, H-3').
Bis-{3-deoxy-3-14-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl]-13-D-
galactopyranosyl} sul-
fane (Named TD139)
TD139 was synthesized at ambient temperature by Cu(1)-catalyzed cycloaddition
between bis-(3-azido-3-deoxy-13-D-galactopyranosyl)-sulfane (11) and 3-
fluorophenylacetylene (3 eq.) with Cu(I) (0.2 eq), triethylamine (2 eq.) in
N,N-
dimethylformamide (DMF, 100 mL/mmol sulfane). The reaction was monitored with
tic
until complete, concentrated and first purified by flash chromatography
(Eluent:
CH2C12:Me0H, 8:1), followed by final purification by preparative hplc to give
TD139 in
76% yield as a white amorphous solid. 1H-NMR (CD30D, 400 MHz) d 8.59 (s, 2H,
tria-
zole-H), 7.63 (br d, 2H, 7.6 Hz, Ar-H), 7.57 (br d, 2H, 8.4 Hz, Ar-H), 7.41
(dt, 2H, 6,0 and
8.0 Hz, Ar-H), 7.05 (br dt, 2H, 2.4 and 6.4 Hz, Ar-H), 4.93 (dd, 2H, 2,4 and
10.4 Hz, H3),
4.92 (d, 2H, 10.4 Hz, HI), 4.84 (2H, 10.4 Hz, H2), 4.18 (d, 2H, 2.4 Hz, H4),
3.92 (dd, 2H,
4.2 and 7.6 Hz, H5), 3.84 (dd, 2H, 7.6 and 11.4 Hz, H6), 3.73 (dd, 2H, 4.2 and
11.4 Hz,
H6): FAB-HRMS miz calcd for C28F130F2N6Na08S (M+Na+), 671.1712; found,
671.1705.
Model of bleomycin-induced lung fibrosis
Female C57/B16 mice (10-14 weeks old) were anaesthetized with halothane, and
bleomycin or saline was administered intratracheally (33 lig in 50 1 of
saline) and lungs
were harvested on day 26. TD139 was instilled into the lungs of mice on days
18, 20, 22
and 24 of bleomycin induced lung injury. Fibrosis was assessed by histological
score of
collagen stained lung sections and by total collagen content by Sircol assay.
Mice were treated with bleomycin (bleo) or saline (control) and bleomycin
treated
mice were treated with 200 mg/kg pirfenidone twice daily on days 18-24. TD139
was ad-
ministered intratracheally on days 18, 20, 22 and 24. Lungs were harvested on
day 26.
Figure 1 shows (A) Total lung collagen measured by Sircol assay; (B) Fibrosis
CA 02795753 2012-11-15
29
score; and (C) Inflammatory score. Results represent the mean and SEM (A) or
box and
whiskers (median, interquartile range, minimum to maximum, B and C) of n=8
mice per
group (n=7 bleo). ***P<0.005, "P<0.01, *P<0.05. Figure 1E) Beta-catenin
activation in
vivo was assessed by scoring sections of bleomycin treated mouse lung (control
and 10 ug
TD treated) stained with an anti-active beta catenin.
Effect on alveolar epithelial cells
Primary alveolar epithelial cells from WT mice were plated and treated with
TGF-i31 in the
presence or absence of 10 .1.1\4 TD139. Figure ID) Cells were lysed and
analyzed for active
P-catenin, total f3-catenin and f3-actin by western blot.
In conclusion TD139 is a galectin-3 inhibitor and blocked TGF-E3-induced [3-
catenin activa-
tion in vitro and bleomycin induced lung fibrosis in vivo and is believed to
represent a novel
therapeutic strategy for treatment of lung fibrosis in mammals, in particular
humans.
Drug treatment
Mice were divided into the following groups set forth in Table I:
Immunohistochemistry
Paraffin-embedded sections of mouse tissue were stained with Masson's
trichrome
and haemotoxylin and eosin (H&E) as per manufacturer's instructions. Sections
were proc-
essed for immunohistochemistry and the following primary antibodies used:
mouse anti-
active (ABC) beta-catenin (Millipore) and sections visualized and quantified.
Table I
CA 02795753 2012-11-15
Dosing
Group Induction Treatment Dose Administration
days
1 Control Vehicle N/A
18 20 22
2 Bleomycin Vehicle , , Intratracheal
and 24
22 20,
3 Bleomycin TD139 10 ug 18, Intratracheal
and 24
22 20,
4 Bleomycin TD139 3 ug 18, Intratracheal
and 24
22 20,
5 Bleomycin TD139 1 ug 18, Intratracheal
and 24
18, 20, 22
6 Bleomycin TD139 0.1 ug and 24 Intratracheal
200 b.i.d. from
7 Bleomycin Pirfenidone oral
mg/kg day 18
Determination of lung fibrosis and inflammation
Histological lung inflammation and fibrosis score were carried out in Masson's
tri-
chrome stained sections. Inflammation (peribronchiolar, perivascular, and
alveolar wall
5 thickness) scored in > 5 random fields at magnification X630 using the
following system
(peribronchiolar and perivascular, 1 = no cells. 2 = <20 cells, 3 = 20 ¨ 100
cells, 4 => 100
cells; alveolar wall thickness, 1 = no cells, 2 = 2 ¨ 3 cells thick, 3 = 4 ¨ 5
cells thick, 4 = > 5
cells thick). The combined inflammatory score was the sum of these scores.
Fibrosis score
was evaluated as the area of the section positively stained for collagen (1 =
none. 2 = <10%.
10 3 = < 50%, 4 => 50%). Only fields where the majority of the field was
composed of alve-
oli were scored.
Determination of lung collagen by sircol assay
Collagen content in the left lung lobe was determined by sircol assay as per
manu-
15 facturer's instructions. The left lobe was minced in 5 ml of 3 mg/ml
pepsin in 0.5 M acetic
acid and incubated with shaking at 4oC for 24 h. Cleared lung extract (0.2 ml)
was incu-
bated with 0.8 ml sircol reagent for 1 h at room temperature and precipitated
collagen cen-
trifuged at 10,000g for 5 min at 4oC. Pellets solubilised in 1 ml 1 M NaOH and
absorbance
measured at 570 nm alongside collagen standards.
Primary Type 11 alveolar epithelial cell isolation
CA 02795753 2012-11-15
31
Treated and control mouse type II lung alveolar epithelial cells (AECs) were
ex-
tracted following a standard method. Briefly, I ml of 50 U/ml dispase (BD
Biosciences)
was administered intratracheally into perfused lungs followed by instillation
of 0.5 ml of
1% low melting point agarose. The agarose within the upper airways was allowed
to set on
ice for 2 minutes and the lungs were placed in 4 ml 50 U/ml dispase for 45 min
at room
temperature. The lung lobes minus the upper airways were then dispersed in
DMEM con-
taining 50 ug/m1 DNAse I (Sigma-Aldrich, UK). The cell suspension was passed
through a
100-p,m cell strainer and the cells washed in DMEM followed by resuspension in
DMEM
containing 10% FCS. The cell suspension was plated onto tissue culture plastic
for 1 h to
allow any contaminated fibroblasts and macrophages to adhere. Non-adherent
epithelial
cells were counted and cultured for 2 days on tissue culture plastic or cover-
slips pre-coated
with 5 ug/m1 collagen (AMS Biotechnology) and 10 ug/m1 fibronectin (Sigma-
Aldrich),
Cells were washed three times in PBS before treatment. Epithelial cells were
either incu-
bated in DMEM containing 10% FCS, 50 U/ml penicillin, 50 g/ml streptomycin
and 5
[Egiml L-glutamine or transferred to complete mouse media (DMEM/F-12
containing 0.25%
BSA, 10 nM hydrocortisone, 5 ug/m1 Insulin-Transferrin-Sodium-Selenite (ITS)
and sup-
plemented with 0.1 mg/ml sodium succinate, 75 ug/m1 succinic acid and 1.8
ug/m1 choline
bitartrate).
Western Blotting
Cells were lysed in 25 mM HEPES pH 7.4, 0.3 M NaC1, 1.5 mM MgC12, 0.2 mM
EDTA, 0.5% triton X-100, 0.5 mM dithiothreitol, 1 mM sodium orthovanadate and
protease
inhibitors (Boehringer Mannheim, Sussex, UK; prepared as per manufacturers
instructions).
Lysates equilibrated for protein using Pierce BCA protein assay reagent
(Pierce) and re-
solved on 12% SDS-PAGE gels. Western blot analysis undertaken using the
following
primary antibodies; rabbit anti beta-catenin, (BD Biosciences), rabbit
polyclonal anti-beta-
actin antibody (Sigma, UK), mouse anti-active (ABC) beta-catenin (Millipore).
Example 1 Measurement of Galectin-3 levels in human lung biopsies:
Biopsies were sampled from patients with usual interstitial pneumonia (UIP),
the
most common cause of IPF. Biopsies were fixed in neutral buffered formalin for
12-24h
prior to embedding in paraffin wax for sectioning. Sum sections were cut and
transferred
CA 02795753 2012-11-15
32
onto glass slides. Sections were dewaxed in xylene for 10 mins and rehydrated
by placing
slides for 2 min each in graded ethanol (100%-95%-80%-70%-50%-water) Antigen
re-
trieval was performed by microwaving sections in 0.01M citrate pH 6.0 for 15
min. After
cooling in running tap water peroxidase was blocked by incubating in 1%
hydrogen perox-
ide solution for 15 mins. Slides were rinsed in phosphate buffered saline
(PBS) and non
specific binding was blocked using serum free protein block and avidin/biotin
blocking kit
(Vector Laboratories, USA). The sections were incubated with mouse monoclonal
anti-
human galectin-3 clone 9C4 from Novocastra. (diluted to 1:100 in antibody
diluent, DAKO,
UK) overnight at 4 C. After 3 washes with PBS, sections were incubated with
biotinylated
rabbit anti-mouse IgG (H+L) secondary antibody (diluted 1:200 in antibody
diluent) for 30
minutes at room temperature. Slides were rinsed 3 times with PBS and incubated
with 3
drops of avidin:biotinylated enzyme complex (R.T.U. Vectastain Elite ABC
Reagent, PK-
7100, Vector Labs, Burlingame, CA, USA) for 30 minutes followed by liquid
diaminoben-
zidine (DAB) (Liquid DAB+Substrate Chromogen System, K3468, Dako UK Ltd, Cam-
bridgeshire) in the dark for 10 minutes.
Slides were rinsed 3 times in PBS, counterstained for 30 seconds with Mayers
hae-
matoxylin (ThermoShandon, UK) and 30 seconds in Scotts tap water (83 mM MgSO4,
7.1
mM NaHCO3 in tap water), dehydrated through graded ethanol (70%, 90%, 100% 2
min
each), and cleared in xylene. Slides were mounted using Pertex mounting
solution (CellPath
Hemel Hempstead, UK).
Sections were visualized by light microscopy.
Galectin-3 is markedly up-regulated in fibroproliferative areas in the lung of
patients
with UIP.
Example 2 Method for measurement of Galectin-3 levels in human serum or human
broncho-alveolar lavage fluid:
1. Dilute the Capture Antibody to the working concentration in PBS without
carrier protein.
Immediately coat a 96-well microplate6 with 100 pl per well of the diluted
Capture Anti-
body. Seal the plate and incubate overnight at room temperature.
2. Aspirate each well and wash with Wash Buffer, repeating the process two
times for a
total of three washes. Wash by filling each well with Wash Buffer
CA 02795753 2012-11-15
33
(400 [IL) using a squirt bottle, manifold dispenser, or autowasher. Complete
removal of
liquid at each step is essential for good performance. After the
last wash, remove any remaining Wash Buffer by aspirating or by inverting the
plate and
blotting it against clean paper towels.
3. Block plates by adding 300 IA of Reagent Diluent to each well. Incubate at
room tem-
perature for a minimum of 1 hour.
4. Repeat the aspiration/wash as in step 2. The plates are now ready for
sample addition.
Assay Procedure
1. Add 100 f.tL of sample or standards in Reagent Diluent, or an appropriate
diluent, per
well. Cover with an adhesive strip and incubate 2 hours at
room temperature.
2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
3. Add 100 [IL of the Detection Antibody, diluted in Reagent Diluent, to each
well. Cover
with a new adhesive strip and incubate 2 hours at room
temperature.
4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
5. Add 100 [IL of the working dilution of Streptavidin-HRP to each well. Cover
the plate
and incubate for 20 minutes at room temperature. Avoid placing the plate in
direct light.
6. Repeat the aspiration/wash as in step 2.
7. Add 100 [IL of Substrate Solution to each well. Incubate for 20 minutes at
room tempera-
ture. Avoid placing the plate in direct light.
8. Add 50 0_, of Stop Solution to each well. Gently tap the plate to ensure
thorough mixing.
9. Determine the optical density of each well immediately, using a microplate
reader set to
450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If
wavelength cor-
rection is not available, subtract readings at 540 nm or 570 nm from the
readings at 450 nm.
This subtraction will correct for optical imperfections in the plate. Readings
made directly
at 450 nm without
correction may be higher and less accurate.
Example 3 Measurement of galectin-3 levels in serum from patients and
controls:
CA 02795753 2012-11-15
34
Serum was sampled from patients with UIP, patients with non-specific
interstitial
pneumonia (NSIP) and aged matched controls. Galectin-3 levels were measured
using the
ELISA method described in example 2. Serum was collected and stored at -80 C
prior to
assay. Samples were normally diluted 1:10 in PBS prior to assay. ELISA was
carried out
as described in the manufacturers protocol:
Galectin-3 was measured serially (on 2-5 occasions) in the serum of 6 patients
with
stable IPF (UIP). Stable IPF was defined as no significant change in exercise
tolerance,
breathlessness or lung function. Galectin-3 was elevated in the serum of
patients with IPF
(control 17.9 0.95 ng/ml n=8, IPF 26.7 4.7 ng/ml n = 6, P < 0.05) but not
in patients
with non-specific interstitial pneumonia (NSIP) (serum concentration 14.57
0.84 ng/ml
(n=10)).
The serum level of galectin-3 remains remarkably constant over time in these
pa-
tients (serum galectin-3 25.5 0.8 ng/ml n=23). We tested 5 serum samples
from patients
undergoing an acute exacerbation of IPF. These patients were defined as having
an acute
exacerbation by decreased exercise to tolerance, decreased lung function and
increased
breathlessness. In these patients there was a dramatic rise in serum galectin-
3, 73.8 12.2
ng/ml. Furthermore, we identified 2 patients who had serial galectin-3
measurements prior
and during an acute exacerbation of their IPF. Both patients show stable
galectin-3 serum
levels during the period while their lung function was stable. However, during
an acute ex-
acerbation when lung function declined there was a sharp rise in serum
galectin-3.
Example 4 Measurement of galectin-3 levels in BAL fluid from patients and
controls:
Broncho-alveolar lavage (BAL) fluid was sampled from IPF patients and age
matched controls using a standard technique. Briefly, a bronchoscope was
passed through
the mouth or nose into the lungs and a small lung section was flushed with a
specified
amount of saline. The BAL fluid was collected and stored at -80 C. The level
of Galectin-3
was measured using the ELISA method described in Example 2.
Galectin-3 levels were significantly elevated in BAL samples from IPF patients
compared to age matched controls (control 18.8 3.6 ng/ml n = 16, IPF 39.7 +
3.7 ng/ml n
= 15, P< 0.01).
