# Patent

## Title
Galactoside inhibitor of galectin-3 and its use for treating pulmonary fibrosis

## Abstract

The present invention relates to a compound of the general formula (I) for pulmonary administration. The compound of formula (I) is suitable for treating pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a mammal. Furthermore the present invention concerns a method for treatment of pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a human subject.

## Background
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 fibrosis, such as Idiopathic pulmonary fibrosis in mammals. The invention also relates to pharmaceutical compositions comprising said novel compounds. Furthermore the present invention relates to pulmonary administration, in particular the use of nebulizers for providing optimal treatment of IPF.
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 median time to death from diagnosis is 2.5 years and the incidence and prevalence of IPF continues to rise. It remains one of the few respiratory conditions for which there are no effective therapies, and there are no reliable biomarkers to predict disease progression. The mechanisms resulting in pulmonary fibrosis are unclear but center 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 increased activation response to fibrogenic cytokines such as transforming growth factor-β1 (TGF-β1). 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. Bleomycin treatment causes oxidant-mediated DNA damage and induces initial lung inflammation followed by progressive fibrosis over 2-4 weeks. When administered during the later phase of the injury the anti-fibrotic potential of novel compounds can be assessed.
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 inhibitors have been tested in a bleomycin model. Some of the prior art galectin inhibitors have the following general formulas




as described in WO/2005/113568,

and





as described in WO/2005/113569, in which RI can be a D-galactose, and





as described in WO/2010/126435.

SUMMARY OF THE DISCLOSURE
Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies. The role of galectin-3 in bleomycin and TGF-β1-induced lung fibrosis in mice is examined, and its relevance in human IPF is established. Studies with galectin-3 are described in MacKinnon et al., “Regulation of TGF-β1 driven lung fibrosis by galectin-3”, Am. J. Respir. Crit. Care Med. 185: 537-546 (2012, originally available online on Nov. 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-β-induced β-catenin activation in vitro and attenuated the late stage progression of lung fibrosis following bleomycin in vivo.
The publications and other materials, including patents, used herein to illustrate the invention and, in particular, to provide additional details respecting the practice are incorporated by reference in their entirety.
Accordingly, provided is a compound of the general formula (I):




In a further aspect, provided is a composition, particularly, a pharmaceutical composition 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. The mode of administration is typically selected from oral, intra venous (i.v.), subcutaneous (s.c.), and pulmonary. In particular the pulmonary route has been shown to provide a considerably longer half-life than the i.v. or s.c. routes in mice. When treating pulmonary fibrosis, in particular IPF, it is important to obtain adequately high local concentrations of the therapeutic in the narrowest parts of the lung tissue, including the bronchioles and the alveoli. Further, it is important that the therapeutic obtains an adequate residence time at the site of action in the lung tissue. Furthermore, since the fibrosis in IPF patients is only located in the lung, it is preferable to obtain a high lung exposure with minimal or no systemic exposure and the use of nebulizers in particular electronic nebulizers of the ultrasonic type is effective. However, cough is a central symptom for patients with pulmonary fibrosis and in particular IPF—a symptom that is likely to be aggravated if an irritant is introduced into the lung. Hence, treatment with a dry powder, such as with a dry powder inhaler or similar, is not suitable for these patients. However, delivering the compound using a nebulizer, such as an electronic nebulizer, is particularly beneficial, since it allows delivery of the compound to the smallest compartments in the lung, without causing any irritation in the lung.
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).
In another aspect, provided is a process of preparing a compound of formula I comprising the step of reacting bis-(3-deoxy-3-azido-β-D-galactopyranosyl) 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.
In a further aspect, the present invention relates to a method for treatment of pulmonary fibrosis, such as idiopathic pulmonary fibrosis, in a human subject having a galectin-3 level indicative of pulmonary fibrosis or exacerbation of symptoms comprising administering 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, storing, or printing; or outputting to a user interface device, a computer readable storage medium, a local computer system or a remote computer system, information related to the likelihood of developing pulmonary fibrosis in the subject or for characterization of the degree of severity of the pulmonary fibrosis in said subject.
Accordingly, disclosed herein, inter alia are the following embodiments:
1. A compound of the general formula (I):




2. The compound of embodiment 1 selected from bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl) sulfane as the free form.
3. The compound according to any one of embodiments 1-2, for use as a medicament.
4. A pharmaceutical composition comprising the compound of any one of embodiments 1-3 and optionally a pharmaceutically acceptable additive, such as a carrier or an excipient.
5. The pharmaceutical composition of embodiment 4 wherein the composition is administered by the pulmonary route.
6. The pharmaceutical composition of embodiment 5 wherein administration by the pulmonary route is selected from a nebulizer such as an ultrasonic nebulizer or a jet nebulizer.
7. The compound of any one of the embodiments 1-3 for use in a method for treating pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a mammal.
8. The compound of embodiment 7, wherein the compound is administered by the pulmonary route.
9. The compound of embodiment 8, wherein administration by the pulmonary route is selected from a nebulizer such as an ultrasonic nebulizer or a jet nebulizer.
10. The compound of embodiment 7, 8 or 9 wherein said mammal is a human subject.
11. 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 any one of embodiments 1-3.
12. The method of embodiment 11, wherein the compound of any one of embodiments 1-3 is administered by the pulmonary route.
13. The method of embodiment 12, wherein administration by the pulmonary route is selected from a nebulizer such as an ultrasonic nebulizer or a jet nebulizer.
14. A process of preparing a compound of formula I comprising a step of reacting bis(3-deoxy-3-azido-β-D-galactopyranosyl) sulfane with 3-fluorophenylacetylene and an amine in a solvent, resulting in the compound of formula I.
15. The process of embodiment 14 wherein the amine is triethylamine, a catalyst is present, such as Cu(I), and the solvent is an organic solvent, such as N,N-dimethylformamide (DMF).
16. A nebulizer device for pulmonary administration comprising a compound of any one of embodiments 1-3.
17. The nebulizer device of embodiment 16, wherein the compound is bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl) sulfane as the free form.
18. The nebulizer of embodiment 16 or 17 which is selected from an ultrasonic nebulizer or a jet nebulizer.
19. A dry powder device for pulmonary administration comprising a compound of any one of embodiments 1-3.
20. The dry powder device of embodiment 19, wherein the compound is bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl) sulfane as the free form.
21. A method of diagnosing 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, 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.
22. The method of embodiment 21 wherein the indicative level of galectin-3 is at least 22 ng/ml, such as 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.
23. A method of predicting the prognosis of 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.
24. The method of embodiment 23 wherein the indicative level of galectin-3 is at least 22 ng/ml, such as 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.
25. 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.
26. The method of embodiment 25 wherein the time period between two measurements is independently selected from about 2 weeks to 2 years, such as 2 weeks, 4 weeks, 1 month, 2 months, 3 months 6 months, 1 year, or 2 years.
27. The method of embodiment 25 wherein when the indicative level of galectin-3 is below 22 ng/ml treatment of pulmonary fibrosis may be stopped, adjusted or put on hold.
28. The method of embodiment 25 wherein when the indicative level of galectin-3 is at least 22 ng/ml, such as 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 treatment of pulmonary fibrosis may be initiated or increased.
29. A method of monitoring or predicting 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 development or progression of exacerbation of symptoms, and c) repeating steps a) and b) to monitor or predict the development or progression of the exacerbation of symptoms in the human subject.
30. The method of embodiment 29 wherein when the indicative level of galectin-3 is below 22 ng/ml treatment of pulmonary fibrosis may be stopped, adjusted or put on hold.
31. The method of embodiment 29 wherein when the indicative level of galectin-3 is at least 22 ng/ml, such as 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 treatment of pulmonary fibrosis is initiated or increased.
32. The method of embodiment 29 wherein when the indicative level of galectin-3 is at least 50 ng/ml, at least 60 ng/ml, at least 70 ng/ml prophylactic treatment of exacerbation of symptoms is initiated or increased.
33. The method of any one of embodiments 17-28 wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.
34. The method of any one of embodiments 21-33 wherein the subject is diagnosed with mild, moderate or aggressive forms of pulmonary fibrosis according to the level of galectin-3.
35. The method of any one of embodiments 21-34 wherein 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.
36. The method of embodiment 35 wherein the bio-markers are selected from MMP7, perDLCO, KL-6, SP-A, MMP-7, CCL-18, IL13, CC-chemokines, IL10, IL1 receptor antagonist, CCL2, Calgranulin B (S100A9 or MRP14), macrophage migration inhibitory factor (MIF), pro-collagen, pro-collagen 3.
37. The method of embodiment 35 wherein the bio-markers are selected from analysis of the presence and frequency of certain cell types in body fluids from said human subject.
38. The method of embodiment 36 wherein the bio-markers are selected from analysis of the presence and frequency of fibrocytes and T-cell subpopulations in body fluids from said human subject.
39. The method of any one of embodiments 21-38 wherein the predetermined reference level for galectin-3 is in the range from about 10.0 ng/mL to about 25.0 ng/mL, such as in the range from about 13.0 ng/mL to about 19.2 ng/mL.
40. The method of any one of embodiments 21-39 wherein the body sample is selected from blood, serum, plasma, broncho-alveolar lavage fluid, lung tissue.
41. The method of any one of embodiments 21-40 wherein the suitable test method is selected from an immunoassay, an immunohistochemical assay, a colorimetric assay, a turbidimetric assay, and flow cytometry.
42. The method of embodiment 17 or 29, wherein the subject has a galectin-3 blood concentration determined to be within a target range.
43. The method of embodiment 42, wherein the target range is from about 10 ng/ml to about 70 ng/ml.
44. A method for treatment of pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a human subject having a galectin-3 level indicative of pulmonary fibrosis or exacerbation of symptoms comprising administering to a human subject a therapeutically effective amount of a galectin-3 inhibitor.
45. The method of embodiment 40 wherein the galectin-3 inhibitor is selected from the compound of any one of embodiments 1-3.
46. The method of embodiment 44 wherein the indicative level of galectin-3 is at least 22 ng/ml, such as at least about 25 ng/ml, such as at least about 30 ng/ml, at least about 40 ng/ml, at least about 50 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml.
47. The method of embodiment 44 comprising the additional step of monitoring the subject's galectin-3 blood level after the therapy is initiated.
Accordingly, also disclosed herein, inter alia are the following embodiments:
1. A compound of the general formula (I):




2. The compound of embodiment 1 selected from bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl) sulfane as the free form.
3. A composition comprising the compound of embodiment 1 and optionally a pharmaceutically acceptable additive.
4. A method for treatment of pulmonary fibrosis comprising administering to a mammal in need thereof an amount of the compound of embodiment 1 effective to treat said pulmonary fibrosis.
5. The method of embodiment 4, wherein said compound is administered by the pulmonary route.
6. A process of preparing a compound of formula I comprising a step of reacting bis-(3-deoxy-3-azido-β-D-galactopyranosyl) sulfane with 3-fluorophenylacetylene and an amine in a solvent, resulting in the compound of formula I.
7. The process of embodiment 5, wherein the amine is triethylamine, a catalyst is present, such as Cu(I), and the solvent is an organic solvent, such as N,N-dimethylformamide (DMF).
8. A device for pulmonary administration, wherein said device is a nebulizer or dry powder device comprising the compound of embodiment 1.
9. The device of embodiment 8, wherein said device is a nebulizer which is selected from an ultrasonic nebulizer or a jet nebulizer.
10. A method of detecting the presence and/or extent of pulmonary fibrosis to diagnose pulmonary fibrosis and/or predict the prognosis of 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.
11. The method of embodiment 10, wherein the indicative level of galectin-3 is at least about 22 ng/ml.
12. A method of monitoring development or progression of or exacerbation of symptoms 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.
13. The method of embodiment 12, wherein the time period between two measurements is independently selected from about 2 weeks to about 2 years.
14. The method of embodiment 12, wherein when the level of galectin-3 is below about 22 ng/ml treatment of pulmonary fibrosis is stopped, adjusted or put on hold.
15. The method of embodiment 12, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.
16. The method of embodiment 12, wherein the subject is diagnosed with mild, moderate or aggressive forms of pulmonary fibrosis according to the level of galectin-3.
17. The method embodiment 12, wherein in step a) further bio-markers are measured, where said markers are linked to Galectin-3 levels.
18. The method of embodiment 17, wherein the bio-markers are selected from MMP7, perDLCO, KL-6, SP-A, MMP-7, CCL-18, IL13, CC-chemokines, IL10, IL1 receptor antagonist, CCL2, Calgranulin B (S100A9 or MRP14), macrophage migration inhibitory factor (MIF), pro-collagen, pro-collagen 3.
19. The method of embodiment 17, wherein the bio-markers are selected from analysis of the presence and frequency of certain cell types in body fluids from said human subject.
20. The method of embodiment 17, wherein the bio-markers are selected from analysis of the presence and frequency of fibrocytes and T-cell subpopulations in body fluids from said human subject.
21. The method of embodiment 12, wherein the predetermined reference level for galectin-3 is in the range from about 10.0 ng/mL to about 25.0 ng/mL.
22. The method of embodiment 12, wherein the body sample is selected from blood, serum, plasma, broncho-alveolar lavage fluid, lung tissue.
23. The method of embodiment 10, wherein the suitable test method is selected from an immunoassay, an immunohistochemical assay, a colorimetric assay, a turbidimetric assay, and flow cytometry.
24. The method of embodiment 10, wherein the subject has a galectin-3 blood concentration determined to be within a target range from about 10 ng/ml to about 70 ng/ml.
25. The method of embodiment 12, wherein the subject has a galectin-3 blood concentration determined to be target range is from about 10 ng/ml to about 70 ng/ml.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows (A) Total lung collagen measured by Sircol assay; (B) Fibrosis score; and (C) Inflammatory score, Results represent the mean and SEM (A) or box and whiskers (median, interquartile range, minimum to maximum, and C) of n=8 mice per group (n=7 bleo). ***P<0.005, **P<0.01, *P<0.05. FIG. (1D) shows the results of Western Blot analysis of cells lysed and analyzed for active β-catenin, total β-catenin and β-actin, FIG. (1E) shows studies of beta-catenin activation in vivo by scoring sections of bleomycin treated mouse lung (control and 10 ug TD139 treated) stained with an anti-active beta catenin.



DETAILED DESCRIPTION
In a broad aspect, provided is a compound of the general formula (I):




In a further aspect the present invention concerns a composition for pulmonary administration, wherein said composition comprises a compound of formula (I).
The compound of formula (I) has the chemical name (IUPAC) bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-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-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl) sulfane as the free form.
In a further embodiment the compound of formula (I) is bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl) sulfane as the free form without any solvate, such as anhydrated.
In a further embodiment the compound of formula (I) is bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl) sulfane on amorphous form.
In a further embodiment the compound of formula (I) is bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl) sulfane on a crystalline form.
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 embodiment the pulmonary administration is carried out by a nebulizer, such as a nebulizer which is selected from an ultrasonic nebulizer or a jet nebulizer. When the administration is carried out by a nebulizer the compound of formula (I) may be in solution, in suspension, such as a nanosuspension, or in a liposome formulation.
In a further embodiment the pulmonary administration is carried out by a dry powder inhaler. When the administration is carried out by a dry powder inhaler the compound of formula (I) may be present as nanoparticles or as a liposome formulation.
In a still further embodiment the pulmonary administration is carried out by a pressurized metered-dose inhaler. When the administration is carried out by a pressurized metered-dose inhaler the compound of formula (I) may be present in solution or in suspension, such as a nanosuspension.
In a further embodiment the composition for pulmonary administration is for use in a method for treatment of pulmonary fibrosis, such as IPF.
When the compound of formula (I) is in suspension or nanosuspension or a liposome formulation it may be present in a suitable particle size selected from particles from a mean mass aerodynamic diameter between 0.1 and 20 μm, such as a MMAD between 0.5 and 10 μm, such as between 1 and 5 μm. The selected ranges does not exclude the presence of particles sizes outside these ranges, but the selected ranges are those that provide the desired effect as described herein.
In a still further embodiment the composition for pulmonary administration is a pharmaceutical composition.
A pharmaceutical composition comprises a pharmaceutically acceptable additive, such as an excipient and/or carrier, which is known to the person skilled in the art.
In a further aspect the present invention concerns a device for pulmonary administration comprising the composition for pulmonary administration. Typically, such device is a nebulizer, such as a nebulizer selected from an ultrasonic nebulizer or a jet nebulizer; or dry powder inhaler.
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 therapeutically 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 administered 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 the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate 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 understood 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 adequate 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 dosage 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 physician 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 additive, 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 invention in order to make a pharmaceutical composition.
The adjuvants, diluents, excipients and/or carriers that may be used in the composition 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 composition, 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.
As mentioned above, the compositions and particularly pharmaceutical compositions as herein disclosed may, in addition to the compounds herein disclosed, further comprise 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 purposes 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 pharmaceutical composition may be in the form of, for example, tablets, capsules, powders, nanoparticles, crystals, amorphous substances, solutions, transdermal patches or suppositories.
Thus, in a still further aspect provided is a composition, particularly a pharmaceutical composition for intrapulmonary administration. Typically, such composition is delivered by a nebulizer or inhaler, preferably a nebulizer.
Boehringer Ingelheim provided a new technology in 1997 named Raspimat which is a mechanical nebulizer of the soft mist inhaler type. This mechanical nebulizer is operated by hand without any need for a gas propellant and no need for electrical power. Another mechanical nebulizer is a human powered nebulizer developed by a team from Marquette University. This nebulizer can by operated by an electrical compressor, but it is also suitable for simple mechanical pumps in order to provide a mist into the lungs of patients. Further nebulizers of the electrical type are ultrasonic nebulizers based on the vibrating mesh technology developed by inter alia PARI, Respironics, Omron, Beurer, Aerogen, or ultrasonic nebulizers based on an electronic oscillator that generate a high frequency ultrasonic wave developed by inter alia Omron and Beurer. A further electrical nebulizer is a jet nebulizer also known as atomizers. In a further embodiment the nebulizer is selected from a mechanical nebulizer, such as a soft mist inhaler or a human powered nebulizer. In another embodiment the nebulizer is selected from an electrical nebulizer, such as a nebulizer based on ultrasonic vibrating mesh technology, a jet nebulizer, or an ultrasonic wave nebulizer. Particular suitable nebulizers are based on vibrating mesh technology such as eFlow from PARI. When treating pulmonary fibrosis, in particular IPF, it is important to obtain adequately high local concentrations of the therapeutic in the narrowest parts of the lung tissue, including the bronchioles and the alveoli. Further, it is important that the therapeutic obtains an adequate residence time at the site of action in the lung tissue. However, cough is a central symptom for patients with pulmonary fibrosis and in particular IPF—a symptom that is likely to be aggravated if an irritant is introduced into the lung. Hence, treatment with a dry powder, such as with a dry powder inhaler or similar, is not suitable for these patients. However, delivering the compound using a nebulizer, such as an electronic nebulizer, is particularly beneficial, since it allows delivery of the compound to the smallest compartments in the lung, without causing any irritation in the lung. Such relevant nebulizer systems are described in published patent applications US20040089295, US20050056274, US20060054166, US20060097068, US20060102172, US20080060640, US20110155768, and US20120167877, all of which are incorporated herein by reference. Other suitable nebulizers are Tyvaso inhalation system from United Therapeutics, Allera nebulizer system from Gilead, Bronchitol inhaler from Pharmaxis, Diskhaler from GSK, jet and ultrasonic nebulizers from Actelion and Profile Pharma.
The following characteristics are required for the pulmonary 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 immediately 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 aerosol 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 respiratory tract upon inhalation.
To the person skilled in the art it is well known that particles with a mean mass aerodynamic diameter (MMAD) between 0.1 and 20 μm, such as between 0.5 and 10 μm, e.g. between 1 and 5 μm (micro meter) have an increased probability of depositing in the terminal bronchial and alveolar regions. This particle size range is ideal for many indications in pulmonary drug delivery, since a portion of the material will still deposit in the upper airways as well. (Cf. Controlled Pulmonary Drug Delivery, Smith and Hickey, Editors, Springer 2011, chapter 13).
In accordance with Controlled Pulmonary Drug Delivery, Smith and Hickey, Editors, Springer 2011 in particular chapters 13, 14 and 15 the skilled person will know how to formulate compounds, such as the compound of formula (I) for pulmonary drug delivery. Typically, liposomes, nanoparticles and microparticles are suitable for suspension as well as for dry powders.
A variety of inhalation nebulizers are known. EP 0 170 715 A1 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 compressed-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 liquid storage container, a perforate membrane and a vibrator. The vibrator is operable to vibrate the membrane such that it dispenses an aerosol from a liquid through holes provided in the membrane.
U.S. Pat. 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 representative of continuously operating inhalation nebulizers, in which the aerosol generator produces an aerosol not only during inhalation but also while the patient exhales. The aerosol produced by the aerosol generator is actually inhaled by the patient only in the inhalation phase, while any aerosol produced at other times is lost.
Dry powder inhalers (DPI), 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.
Current designs include pre-metered and device-metered DPIs, both of which can be driven by patient inspiration alone or with power-assistance of some type. Pre-metered DPIs contain previously measured doses or dose fractions in some type of units (e.g., single or multiple presentations in blisters, capsules, or other cavities) that are subsequently inserted into the device during manufacture or by the patient before use. Thereafter, the dose may be inhaled directly from the pre-metered unit or it may be transferred to a chamber before being inhaled by the patient. Device-metered DPIs have an internal reservoir containing sufficient formulation for multiple doses that are metered by the device itself during actuation by the patient. The wide array of DPI designs, many with characteristics unique to the design, will present challenges in developing information in support of an application. Regardless of the DPI design, the most crucial attributes are the reproducibility of the dose and particle size distribution. Maintaining these qualities through the expiration dating period and ensuring the functionality of the device through its lifetime under patient-use conditions will probably present the most formidable challenge.
Pressurized Metered-Dose Inhalers (pMDI) may also be suitable delivery devices for the present compound of formula (I) and are described in Controlled Pulmonary Drug Delivery, Smith and Hickey, Editors, Springer 2011, chapter 8.
Several types of non-aerosol, breath actuated dry powder inhalers have therefore been provided. For example, U.S. Pat. No. 5,503,144 to Bacon, shows a breath-actuated dry-powder inhaler. The device includes a dry powder reservoir for containing a dry powdered medicament, a metering chamber for removal of the powdered medicament from the reservoir in discrete amounts, and an air inlet for entraining the removed powdered medicament through a mouthpiece upon patient inhalation.
U.S. Pat. No. 5,458,135 discloses a method and apparatus for producing an aerosolized dose of a medicament for subsequent inhalation by a patient. The method comprises 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.
U.S. Pat. No. 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 conduit 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 importance 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 dispensed. It has therefore been found preferably to provide breath-actuated dry powder inhalers with means for breaking down the agglomerates of medicament or medicament and carrier before inhalation of the medicament.
The composition and particularly pharmaceutical composition may optionally comprise 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 individual 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 section below.
Accordingly, provided is a process of preparing a compound of formula I comprising the step of reacting bis-(3-deoxy-3-azido-β-D-galactopyranosyl) 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. 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 experimental section. Moreover, the present invention relates to a compound of formula (I) obtainable by the step of reacting bis-(3-deoxy-3-azido-β-D-galactopyranosyl) 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.
Typically, the pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF).
In a further embodiment the human subject is diagnosed with mild, moderate or aggressive forms of pulmonary fibrosis according to the level of galectin-3.
In a further aspect the present invention relates to a method for treatment of pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a human subject having a galectin-3 level indicative of pulmonary fibrosis or exacerbation of symptoms comprising administering to a human subject a therapeutically effective amount of a galectin-3 inhibitor. In a particular 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 about 22 ng/ml, such as at least about 25 ng/ml, such as at least about 30 ng/ml, at least about 40 ng/ml, at least about 50 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml.
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 present, and detecting or measuring the amount of any immunospecific binding by the antibody. Any suitable immunoassay can be used, including, without limitation, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, 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.
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 techniques 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 preferred enzyme-linked immunosorbent assay kit (ELISA) for detecting galectin-3 is commercially available (BG Medicine, Waltham, Mass.).
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 Dekker, New York (1984) and Harlow et al. Antibodies, A Laboratory Approach, ed. Cold Spring Harbor Laboratory (1988).
Further embodiments of the process are described in the experimental section herein, and each individual process as well as each starting material constitutes embodiments that may form part of embodiments.
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 individually and specifically indicated to be incorporated by reference and was set forth in its entirety 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 contradicted by context.
The terms “a” and “an” and “the” and similar referents as used in the context of describing 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 otherwise 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 otherwise 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 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 invention 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 composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).
This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable 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 combination thereof, be material for realizing the invention in diverse forms thereof.
EXPERIMENTAL
Synthesis of bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-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 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 recorded 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, 60 Å, 35-70 μm, Grace Amicon). Acetonitrile was distilled from calcium hydride and stored over 4 Å molecular sieves. DMF was distilled from 4 Å molecular sieves and stored over 4 Å molecular sieves.
Bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl) sulfane (TD139) was prepared in accordance with the reaction scheme 1 below







Compound (1) (cf. reaction scheme above) is obtained commercially from Carbosynth Limited 8 & 9 Old Station Business Park-Compton-Berkshire-RG20 6NE-UK or synthesized 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-O-acetyl-4,6-O-benzylidene-1-thio-3-O-trifluoromethanesulfonyl-β-D-galactopyranoside (2)
Compound 1 (10.5 g, 29.2 mmol) was dissolved in dried pyridine (4.73 mL, 58.4 mmol) and dried CH2Cl2 (132 mL). The reaction mixture was cooled, under stirring, until −20° C. (Ice and NaCl bath 3:1). Slowly and under N2 atmosphere, Tf2O (5.68 mL, 33.6 mmol) was added. The reaction mixture was monitored by TLC (heptane:EtOAc, 1:1 and toluene:acetone, 10:1). When the reaction was complete, AcCl (2.29 mL, 32.1 mmol) was added and keeping stirring, the temperature was increased to room temperature. This mixture was monitored by TLC too (heptane:EtOAc, 1:1 and toluene:acetone, 10:1). When it was complete, it was quenched with CH2Cl2 and washed with 5% HCl, NaHCO3 (saturated—hereafter sat) and NaCl (sat). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure.
Phenyl 2-O-acetyl-4,6-O-benzyliden-1-thio-β-D-gulopyranoside (3)
Tetrabutylammonium nitrite (25.3 g, 87.7 mmol) was added to a solution of compound 2 (15.6 g, 29.2 mmol) in DMF (110 mL) and was kept stirring, under N2 atmosphere, at 50° C. (The reaction started being purple and turned garnet). The reaction was monitored by TLC (heptane:EtOAc, 1:1 and toluene:acetone, 10:1) and quenched with CH2Cl2. The mixture was washed with 5% HCl, NaHCO3 (sat) and NaCl (sat). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure followed by purification by flash chromatography (Eluent heptane:EtOAc, 1:1 and heptane:EtOAc, 1:2) and recrystallized from a mixture of EtOAc and Heptane (1:3). 1H NMR in CDCl3 δ 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, 1H, 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-O-acetyl-4,6-O-benzylidene-1-thio-3-O-trifluoromethanesulfonyl-β-D-gulopyranoside (4)
Compound 3 (1.00 g, 2.48 mmol) was dissolved in dried CH2Cl2 (12.5 mL) and dried pyridine (0.40 mL, 4.96 mmol). The reaction mixture was cooled, under stirring, until −20° C. (Ice and NaCl bath 3:1). Slowly and under N2 atmosphere, Tf2O (0.48 mL, 2.85 mmol) was added. The reaction mixture was monitored by TLC (heptane:EtOAc, 1:1 and toluene:acetone, 10:1) and when it was complete, it was quenched with CH2Cl2 and washed with 5% HCl, Na—HCO3 (sat) and NaCl (sat). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure until being dry.
Phenyl 2-O-acetyl-3-azido-4,6-O-benzylidene-3-deoxy-1-thio-β-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 atmosphere, 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:EtOAc, 2:1 and heptane:EtOAc, 1:1). 1H NMR in CDCl3 δ 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, J=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-O-acetyl-3-azido-3-deoxy-1-thio-β-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:EtOAc, 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-1-thio-β-D-galactopyranoside (7)
Acetic anhydride (30 mL) was added to a solution of compound 6 (373 mg, 1.1 mmol) in dry pyridine (30 mL). The reaction was monitored by TLC (heptane:EtOAc, 1:1) and when it was complete, it was concentrated under reduced pressure. 1H NMR in CDCl3 δ 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−2), 4.73 (d, 1H, H−1), 4.15 (d, 2H, H−6, H−6), 3.94 (dt, 1H, 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-O-acetyl-3-azido-3-deoxy-α-D-galactopyranosyl bromide (8)
Compound 7 (237.4 mg, 560 μmol) was dissolved in dry CH2Cl2 (2 mL), and bromine (32 μl, 620 μmol) was added. The reaction was monitored by TLC (heptane:EtOAc, 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: 500 mL heptane:EtOAc, 2:1).
2,4,6-tri-O-acetyl-3-azido-3-deoxy-α-D-galactopyranose-1-isothiouronium bromide (9)
The sensitive bromide 8 (70.6 mg, 180 μmol) was immediately dissolved in dry acetonitrile (1.7 mL) and refluxed with thiourea (13.7 mg, 180 μmol) under N2 for 4 hours. The reaction was monitored by TLC (heptane:EtOAc, 1:1) and when it was complete, the mixture was cooled.
Bis-(2,4,6-tri-O-acetyl-3-azido-3-deoxy-b-D-galactopyranosyl)-sulfane (10)
The sensitive bromide 8 (77.0 mg, 196 μmol) and Et3N (60 μl, 430 μmol) was added to the last mixture (9). The reaction was monitored by TLC (heptane:EtOAc, 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:EtOAc, 1:1). 1H NMR in CDCl3 δ 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, 2OAc), 2.15 (s, 6H, 2OAc), 2.18 (s, 6H, 2OAc).
Bis-(3-azido-3-deoxy-β-D-galactopyranosyl)-sulfane (11)
Compound 10 (160 mg, 0.00024 mol) was dissolved in dry MeOH (2.6 mL) and dry CH2Cl2 (1.6 mL), and NaOMe (1M, 24 μL, 24 μmol) was added. The reaction was monitored by TLC (heptane:EtOAc 1:1 and D:M 5:1). When the reaction was complete, the mixture was neutralized with Duolite C436 until pH 7, filtered and washed with MeOH. The filtered solution was concentrated under reduced pressure. The residue was purified by flash chromatography (Eluent: CH2Cl2:MeOH, 5:1) to give pure 11 (74.1 mg, 75%). 1H NMR in CDCl3 δ 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-[4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl]-β-D-galactopyranosyl}sulfane (Named TD139)
TD139 was synthesized at ambient temperature by Cu(I)-catalyzed cycloaddition between bis-(3-azido-3-deoxy-β-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 tlc until complete, concentrated and first purified by flash chromatography (Eluent: CH2Cl2:MeOH, 8:1), followed by final purification by preparative hplc to give TD139 in 76% yield as a white amorphous solid. 1H-NMR (CD3OD, 400 MHz) d 8.59 (s, 2H, triazole-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, H1), 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 m/z calcd for C28H30F2N6NaO8S (M+Na+), 671.1712. found, 671.1705.
Model of Bleomycin-Induced Lung Fibrosis
Female C57/Bl6 mice (10-14 weeks old) were anaesthetized with halothane, and bleomycin or saline was administered intratracheally (33 μg in 50 μl 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 administered intratracheally on days 18, 20, 22 and 24. Lungs were harvested on day 26.
FIG. 1 shows (A) Total lung collagen measured by Sircol assay; (B) Fibrosis 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. FIG. 1E) Beta-catenin activation in vivo was assessed by scoring sections of bleomycin treated mouse lung (control and 10 ug TD139 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-β1 in the presence or absence of 10 μM TD139. FIG. 1D) Cells were lysed and analyzed for active β-catenin, total β-catenin and β-actin by western blot.
In conclusion TD139 is a galectin-3 inhibitor and blocked TGF-β-induced β-catenin activation 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 processed for immunohistochemistry and the following primary antibodies used: mouse anti-active (ABC) beta-catenin (Millipore) and sections visualized and quantified.












TABLE I







Treat-

Dosing
Admin-


Group
Induction
ment
Dose
days
istration





















1
Control
Vehicle


N/A



2
Bleomycin
Vehicle


18, 20, 22
Intratracheal







and 24


3
Bleomycin
TD139
10
ug
18, 20, 22
Intratracheal







and 24


4
Bleomycin
TD139
3
ug
18, 20, 22
Intratracheal







and 24


5
Bleomycin
TD139
1
ug
18, 20, 22
Intratracheal







and 24


6
Bleomycin
TD139
0.1
ug
18, 20, 22
Intratracheal







and 24


7
Bleomycin
Pirfen-
200
mg/kg
b.i.d. from
oral




idone


day 18









Determination of Lung Fibrosis and Inflammation

Histological lung inflammation and fibrosis score were carried out in Masson's trichrome stained sections. Inflammation (peribronchiolar, perivascular, and alveolar wall 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%, 3=<50%, 4=>50%). Only fields where the majority of the field was composed of alveoli were scored.
Determination of Lung Collagen by Sircol Assay
Collagen content in the left lung lobe was determined by sircol assay as per manufacturer'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 4° C. for 24 h. Cleared lung extract (0.2 ml) was incubated with 0.8 ml sircol reagent for 1 h at room temperature and precipitated collagen centrifuged at 10,000 g for 5 min at 4° C. Pellets solubilised in 1 ml 1 M NaOH and absorbance measured at 570 nm alongside collagen standards.
Primary Type II Alveolar Epithelial Cell Isolation
Treated and control mouse type II lung alveolar epithelial cells (AECs) were extracted following a standard method. Briefly, 1 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 containing 50 μg/ml DNAse I (Sigma-Aldrich, UK). The cell suspension was passed through a 100-μm 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 μg/ml collagen (AMS Biotechnology) and 10 μg/ml fibronectin (Sigma-Aldrich), Cells were washed three times in PBS before treatment. Epithelial cells were either incubated in DMEM containing 10% FCS, 50 U/ml penicillin, 50 μg/ml streptomycin and 5 μg/ml L-glutamine or transferred to complete mouse media (DMEM/F-12 containing 0.25% BSA, 10 nM hydrocortisone, 5 μg/ml Insulin-Transferrin-Sodium-Selenite (ITS) and supplemented with 0.1 mg/ml sodium succinate, 75 μg/ml succinic acid and 1.8 μg/ml choline bitartrate).
Western Blotting
Cells were lysed in 25 mM HEPES pH 7.4, 0.3 M NaCl, 1.5 mM MgCl2, 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 resolved 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 12-24 h prior to embedding in paraffin wax for sectioning. Sum sections were cut and transferred 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 retrieval 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 peroxide 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, Calif., USA) for 30 minutes followed by liquid diaminobenzidine (DAB) (Liquid DAB+Substrate Chromogen System, K3468, Dako UK Ltd, Cambridgeshire) in the dark for 10 minutes.
Slides were rinsed 3 times in PBS, counterstained for 30 seconds with Mayers haematoxylin (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 microplate 6 with 100 μL per well of the diluted Capture Antibody. 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 (400 μL) 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 μL of Reagent Diluent to each well. Incubate at room temperature 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 μL 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 μL 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 μL 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 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.

8. Add 50 μL 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 correction 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
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 patients (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 exacerbation 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).

## Claims

1. A composition for pulmonary administration, wherein said composition comprises a compound of formula (I).

2. The composition of claim 1, the compound of formula (I) is bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl) sulfane as the free form.

3. The composition of claim 1, wherein the compound of formula (I) is in crystalline form.

4. The composition of claim 1, wherein the compound of formula (I) is in solution.

5. The composition of claim 1, wherein the compound of formula (I) is in suspension.

6. The composition of claim 1, wherein the compound of formula (I) is present as nanoparticles.

7. The composition of claim 1, wherein the compound of formula (I) is a liposome formulation.

8. The composition of claim 1, wherein the compound of formula (I) is present in a suitable particle size selected from particles having from a mean mass aerodynamic diameter between 0.1 and 20 μm.

9. The composition of claim 1, wherein the composition is a pharmaceutical composition.

10. The composition of claim 9, comprising a pharmaceutically acceptable additive.

11. A device for pulmonary administration comprising the composition of claim 1.

12. The device of claim 11, wherein said device is a nebulizer or dry powder inhaler.

13. The device of claim 12, wherein said device is a nebulizer which is selected from an ultrasonic nebulizer or a jet nebulizer.

14. The device of claim 11, wherein said device is a pressurized metered-dose inhaler.

15. A method for treating pulmonary fibrosis comprising administering an amount of the composition of claim 1 effective to treat said pulmonary fibrosis.

16. The method according to claim 15, wherein said pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF).

17. The composition of claim 5, wherein the compound of formula (I) is in a nanosuspension.

18. The composition of claim 8, wherein the compound of formula (I) is present in a mean mass aerodynamic diameter (MMAD) between 0.5 and 10 μm.

19. The composition of claim 8, wherein the compound of formula (I) is present in a mean mass aerodynamic diameter (MMAD) between 1 and 5 μm.